I really like this whole paragraph, it is a great explanation of Burr's character by comparing him to Hamilton, and you did a great job at describing how Burr's feelings of doubt can be universal.

Well said— giving very Orwellian

Very good point about how all this digesting of news can get your body to feel overloaded. So when something important in your life actually happens, you don't hve any space left. You are already filled to the top.

We need to allow ourselves space to be partially empty. So we can be more observant. To allow ourselves the opportunity to be absorbant. Instead of constantly full all the time.

Hmmm, this is an interesting point. When you read other people's reactions to news on twitter and other social networks, you get only clips. And it's seemingly infinite.

I've long supported the idea of people commenting. Of people digesting something, and reacting with their own words. Alas, that never quite worked for me, because there aren't many people who read my words. Like really, I'm not sure if anyone I know regularly reads my blog posts, and those are just once a week now. Usually just 300-350 words.

Yeah, it's mostly because there's simply too much to read. Too many people commenting about other stuff online.

I like this notion of reading mostly in print. It's a time to focus. You don't get swept away with all the emotions of the general public. Although it begs the question of how do people get their work into newspapers. You'd think I would know that after working in the newspaper business for 24+ years. Lol. Especially at a syndicate. It's mostly just timing and luck, and skill at writing.

Almost every social media site should be moderated, specifically for posts, unless they are advertised as unmoderated. I didn't know about quality control. I kind of thought that even if the video was boring or random, it would still be shown to people. I'm also curious about other legal concerns social media follows. Of course, safety concern videos should be moderated unless it's just a video of someone trying something dumb and getting hurt; that is more educational for the person watching. I don't think potentially offensive videos should be moderated, only hurtful posts against a specific kind of people should be deleted.

I feel like every social media app should have some sort of moderation, whether it's trained or not. Especially Twitter, he can still have the app as real as he wants but should have some sort of moderation around cyberbullying and if he already has it, I think it needs more of it.

Setting boundaries on social media isn't just about control, but about keeping conversations constructive and respectful. This balance reflects the ethical side of moderation—aiming for harmony both within ourselves and in our interactions. It’s a great reminder that the virtues we aim for personally can have meaningful applications even in something as modern as online content moderation.

The authors argue that a more comprehensive approach is needed. Training for school professionals shouldn’t just be a one-time session but a continuous and evolving part of school policy, making it clear that Title IX obligations are about creating safe, equitable environments for all students. This isn’t just about compliance with federal laws; it’s about fostering a school culture that acknowledges and actively counters biases, harassment, and discrimination. Schools that implement ongoing programs, such as weekly discussions and anti-bias training, are shown to be more inclusive and supportive.

Welcome back and in this demo lesson you're going to experience the difference that EFS can make to our WordPress application architecture.

Now this demo lesson has three main components.

First we're going to deploy some infrastructure automatically using the one-click deployments.

Then I'm going to step through the CloudFormation template and explain exactly how this architecture is built.

And then right at the end you're going to have the opportunity to see exactly what benefits EFS provides.

So to get started make sure that you're currently logged in to the general AWS account, so the management account of the organization, and as always you need to have the Northern Virginia region selected.

Now this lesson actually has two one-click deployments.

The first deploys the base infrastructure and the second deploys a WordPress EC2 instance, which has been enhanced to utilize EFS.

So you need to apply both of these templates in order and wait for the first one to finish before applying the second.

So we're going to start with the base VPC RDS EFS template first.

So this deploys the base VPC, the Elastic File System and an RDS instance.

Now everything should be pre-populated.

The stack should be called EFS demo -vpc -rds -efs.

Just scroll all the way down to the bottom, check the capabilities box and click on create stack.

While that's going let's switch over to the CloudFormation template and just step through exactly what it does.

So this is the template that you're deploying using the one-click deployment.

It's deploying the Base Animals for Life VPC, an EFS file system as well as mount targets and an Aurora database cluster.

So if we just scroll down we can see all of the VPC and networking resources used by the Base Animals for Life VPC.

Continue scrolling down we'll see the subnets that this VPC contains IP version 6 information.

We'll see an RDS security group, a database subnet group.

We've got the database instance.

Then we've got an instance security group which controls access to all the resources in the VPC that we use that security group on.

Then we have a rule which allows anything with that security group attached to it to communicate with anything else.

We have a rule that the WordPress instance will use and note that this includes permissions on the Elastic File System.

Then we have the instance profile that that instance uses.

Then we have the CloudWatch agent configuration and this is all automated.

And if we just continue scrolling down here we can see the Elastic File System.

So we create an EFS file system and then we create a file system mount target in each application subnet.

So we've got mount target zero which is in application subnet A which is in US East 1A.

We've got mount target one which is an application subnet B which logically is in US East 1B.

And then finally target two which is in subnet app C which is in availability zone 1C.

So we create the VPC, the database and the Elastic File System in this first one click deployment.

Now we need this to be in a create complete state before we continue with the demo lessons.

So go ahead and pause the video, wait for this to move into a create complete status and then we can use the second one click deployment.

Okay that stacks now finished creating which means we can move on to the second one click deployment.

Now there are actually two WordPress one click deployments which are attached to this lesson.

We're going to use them both but for now I want you to use the WordPress one one click deployment.

So go ahead and click on that link this will create a stack called EFS demo hyphen WordPress one.

Everything should be pre-populated just go ahead and click on create stack.

Now this is going to use the infrastructure provided by that first one click deployment.

So it's going to use EFS demo hyphen VPC hyphen RDS hyphen EFS and let's quickly step through exactly what this is doing while it's provisioning.

So this is the cloud formation template that is being used and we can skip past most of this.

What I want to focus on is the resource that's being created so that's WordPress EC2.

So this is using cross stack references to import a lot of the resources created in that first cloud formation stack.

So it's importing the instance profile to use it's importing the web a subnet so it knows where to place this instance.

And it's importing the instance security group that's created in that previous cloud formation stack.

Now in addition to this if we look through the user data for this WordPress instance one major difference is that it's mounting the EFS file system into this folder.

So forward slash var forward slash w w w forward slash HTML forward slash WP hyphen content.

Now if you remember from earlier demo lessons this is the folder which WordPress users to store its media.

So now instead of this folder being on the local EC2 file system this is now the EFS file system.

The EFS file system is mapped into this folder on this WordPress instance.

Other than that everything else is the same WordPress is installed.

It's configured to you as the RDS instance the cow say custom login banner is displayed.

It automatically configures the cloud watch agent and then it signals cloud formation that it's finished provisioning this instance.

Now what we'll end up with when this stack has finished creating is an EC2 instance which will use the services provided by this original stack.

So let's just refresh this.

It's still in progress so go ahead and pause the video and wait for this stack to move into a create complete state and then we good to continue.

So this stacks now finished creating and if we move across to the EC2 console so click on services locate EC2 right click and open that in a new tab.

Then click on instances running and you'll see that we have this A4L WordPress instance.

Now if we select that copy the IP address into your clipboard and then open that in a new tab we need to perform the WordPress installation.

So go ahead and enter the site title the best cats and add some exclamation points.

For username we need to use admin then for the password go back to the cloud formation stack and click on parameters and we're going to use the DB password.

So copy that into your clipboard then go back paste it into the password box and then put test at test.com for the email address and click install WordPress.

Then as before we need to log in so click on login admin for username reenter that password and click on login.

Then we need to go to posts we need to click on trash below hello world to delete that post then click on add new close down this dialogue.

For title put the best cats ever and some exclamation points then click on the plus click gallery click upload.

There's a link attached to this lesson with four cat images so go ahead and download that link and extract it locate those four images select them and click on open.

And then once you've done that click on publish and publish again and then click on view post.

Now what that's doing in the background is it's adding these images to the WP hyphen content folder which is on the EC two instance but now we have that folder mounted using EFS and so the images are being stored on the elastic file system rather than the local instance file system.

The cat pictures are there but what we're going to do to validate this is to go back to instances right click on this a four L hyphen WordPress instance and click on connect and then connect to this instance using EC two instance connect.

Now once we connected to the instance you CD space forward slash VAR forward slash WWW forward slash HTML and then do an LS space hyphen LA to do a full listing you'll see that we have this WP hyphen content folder.

So type CD space WP hyphen content and press enter then we'll clear the screen and do an LS space hyphen LA and then inside this folder we have plugins themes and uploads go into the uploads folder do an LS space hyphen LA depending on when you do this demo lesson you should see a folder representing the year so move into that folder then a folder representing the month again this will vary depending on when you do the demo lesson.

Move into that folder and then you should see all four of my cat images and if you do a DF space hyphen K you'll be able to see that this folder so forward slash VAR forward slash WWW forward slash HTML WP hyphen content this is actually mounted using EFS so this is an EFS file system.

Now this means the local instance file system is no longer critical it no longer stores the actual media that we upload to these posts so what we can do is we can go back to cloud formation go to stacks select the EFS demo hyphen WordPress one stack and then click on delete and delete that stack so that's going to terminate the EC two instance that we've just used to upload that media.

We need to wait for that stack to fully delete before continuing so go ahead and pause the video and wait for this stack to disappear so that stacks disappeared and now there's a second WordPress one click deployment link attached to this lesson remember there are two so now go ahead and click on the second one this one should create a stack called EFS demo hyphen WordPress two scroll to the bottom and click on create stack that's going to create a new stack and a new EC two instance.

So while we're doing this just close down all of these additional tabs at the top of the screen close them all down apart from the cloud formation one.

We're going to need to wait for this to finish provisioning and move into the create complete state so again pause the video wait for this to change into create complete and then we go to to continue.

After a few minutes the WordPress two stack has moved into a create complete state click on services open the EC two console in a new tab click on instances running you'll see a new A4L hyphen WordPress instance this is a brand new instance which has been provisioned using the one click deployment link that you've just used so the WordPress two one click deployment link.

If we select this copy the public IP address into your clipboard and open that in a new tab it again loads our WordPress blog if we open the blog post.

Now we can see these images because they're being loaded from EFS from the file system that EFS provides so no longer are we limited to only operating from a single EC two instance for our WordPress application because now there's nothing which gets stored specifically on that EC two instance.

Instead everything stored on EFS and accessible from any EC two instance that we decide to give permissions to know what we can do to demonstrate this if we go back to cloud formation.

Now remember attached to this lesson are two WordPress one click deployments we initially applied number one then we deleted that and applied number two so now I want you to reapply number one.

So again click on the WordPress one one click deployment this again will create a new stack this time called EFS demo hyphen WordPress one click on create stack you need to wait for this to move into a create complete state so pause the video and resume it once the stack changes to create complete after a few minutes this stack also moves into create complete.

Let's click on resources we can see it's provisioned a single EC two instance so let's click on this to move directly to this new instance select it copy this instance is IP address into your clipboard and open that in a new tab and again we have our WordPress blog and if we click on the post it loads those images so now we have a number of EC two instances we have to EC two instances both with WordPress installed both using the same RDS data.

And both using the shared file system provided by EFS and it means that if any posts are edited or any images uploaded on either of these two EC two instances then those updates will be reflected on all other EC two instances and this means that we've now implemented this architecture that's on screen now and this is what's going to support us when we evolve this architecture more and add scalability in an upcoming section of the core.

For now though we've just been focused on the shared file system now all that remains at this point is for us to tidy up the infrastructure that we've used in this demo lesson so close down all of these tabs we need to be at the cloud formation console we need to start by deleting EFS demo WordPress one and WordPress two so pick either of those click delete and then delete stack then select the other delete and then delete stack.

Now we need both of these to finish deleting and then we can delete this last stack so go ahead and pause the video wait for both of these to disappear and then we can resume both of those have deleted so now we can click the final stack EFS demo hyphen VPC hyphen RDS hyphen EFS so select that delete and then delete stack and that's everything that you need to do in this demo lesson and once that stacks finished deleting the account will be in the same state as it was at the start of this.

Now I hope you've enjoyed this demo lesson and that it's been useful what you've implemented in this demo is one more supportive step towards us moving this architecture from being a monolith through to being fully elastic.

Now the application is in this state where we have a single shared RDS database for all of our application instances and we're also using a shared file system provided by EFS and this means that we can have one single EC2 instance we could have two EC2 instances or even 200 all of them sharing the same database and the same shared file system provided by EFS.

Now in an upcoming section of this course we're going to extend this further by creating a launch template which automatically builds EC2 instances as part of this application architecture.

We're going to utilize auto scaling groups together with application load balancers to implement an architecture which is fully elastic and resilient and this has been one more supportive step towards that objective.

At this point though that's everything that you needed to do in this demo lesson so go ahead complete this video and when you're ready I look forward to you joining me in the next.

Welcome back, this is part two of this lesson.

We're going to continue immediately from the end of part one, so let's get started.

Okay, so all three of these mount targets are now in an available state and that means we can connect into this EFS file system from any of the availability zones within the Animals for Life VPC.

So what we need to do is test out this process and we're going to interact with this file system from our EC2 instances.

So move back to the tab where we have the EC2 console open.

And at this point I want you to either, and this depends on your browser, I'll either want you to right click and duplicate this tab to open another identical copy.

If you can't do this in your browser then just open a new tab and copy and paste this URL into that tab.

You'll end up with two separate tabs open to the same EC2 screen.

So on the first tab we're going to connect to A4L-EFS instance A.

So right click and then select connect.

We're going to use instance connect.

So make sure the username is EC2-user and then click on connect.

Now right now this instance is not connected to this EFS file system and we can verify that by running a DF space-k and press enter.

You'll see that nowhere here is listed this EFS file system.

These are all volumes directly attached to the EC2 instance and of course the boot volume is provided by EBS.

Now within Linux all devices or all file systems are mounted into a folder.

So the first thing that we need to do to interact with EFS is to create a folder for the EFS file system to be mounted into.

And we can do that using this command so shudu space mkdir space-p space/efs/wp-content.

Now the hyphen p option just means that everything in this path will be created if it's not already.

So this will create forward/EFS if it doesn't already exist.

So press enter to create that folder.

So I'm going to clear the screen to keep this easy to see.

And the next thing I need to do is to install a package of tools which allows this instance or specifically the operating system to interact with the EFS product.

Now the command I'm going to use to install these tools is shudu to give us admin permissions and then DNF which is the package manager for this operating system.

And then a space hyphen y to automatically acknowledge any prompts and then a space and then install because I want to install a package and then a space.

And then the name of the tools that I want to install is amazon hyphen EFS hyphen utils.

So this is a set of tools which allows this operating system to interact with EFS.

So go ahead and press enter and that will install these tools and then we can configure the interaction between this operating system and EFS.

Again I'm going to clear the screen to keep this easy to see and I want to mount this EFS file system in that folder that we've just created.

But specifically I want it to mount every time the instance is restarted.

So of course that means we need to add it to the FSTAB file.

Now if you remember this file from elsewhere in the course it's contained within the forward/ETC folder.

So we need to move into that folder cd///ETC and then the file is called FSTAB.

So we need to run shudu to give us admin permissions and then nano which is a text editor and then the name of the file which is FSTAB.

So press enter and the file will likely have only one or two lines which is the root and/or boot volume of this instance.

So let's just move to the end because we're going to add a new line and this is contained within the lesson commands document but we're going to paste in this line.

So this line tells us that we want to mount this file system ID so file system ID colon forward/.

We want to mount that into this folder so forward/efs forward/wp-content.

We tell it that the file system type is EFS.

Remember EFS is actually based on NFS which is the network file system but this is one provided by AWS as a service and so we use a specific AWS file system which is EFS.

And the support for this has been installed by that tools package which we just installed.

Now the exact functionality of this is beyond the scope of this course but if you do want to research further then go ahead and investigate exactly what these options do.

What we need to do though is to point it at our specific EFS file system.

So this is this component of the line all the way from the start here to this forward/.

So to get the file system ID we need to go back to the EFS console and we need to copy down this full file system ID and yours will be different so make sure you copy your own file system ID into the clipboard.

Then go back here and select the colon and then delete all the way through to the start of this line.

And once you've done that paste in your file system ID what it should look like is the file system ID then a colon and then a forward/.

So at this point we need to save this file so control O to save and then enter and control X to exit.

Again I'm going to clear the screen to make it easier to see.

Then I'll run a DF space -K and this is what the file systems currently attached to this instance look like.

Then we're going to mount the EFS file system into the folder that we've created and the way that we do this is with this command.

So shudu mount and then we specify the name of the folder that we want to mount.

Now the way that this works is that this uses what we've just defined in the FSTAB file.

So we're going to mount into this folder whatever file system is defined in that file.

So that's the EFS file system and if we press enter after a few moments it should return back to the prompt and that's mounted that file system.

There we go we back at the prompt and if we do a DF space -K again we'll see that now we've got this extra line at the bottom.

So this is the EFS file system mounted into this folder.

Now to show you that this is in fact a network file system let's go ahead and move into that folder using this command.

And now that we're in that folder we're going to create a file.

So we're going to use shudu so that you have admin privileges and then we're going to use the command touch which if you remember from earlier in the course just creates an empty file.

And we're going to call this file amazing test file dot txt.

Go ahead and press enter and then do an LS space -LA and you'll see that we now have this file created within this folder.

And while we're creating it on this EC2 instance it's actually put this file on a network file system.

Now to verify that let's move back to the other tab that we have open to the EC2 console the one that's still on this running instances screen.

And now let's go ahead and connect to instance B.

So right click on instance B select connect again instance connect verify the username is as it should be and click on connect.

So now we're on instance B.

Let's do a DF space -K to verify that we don't currently have any EFS file system mounted.

Next we need to install the EFS tools package so that we can mount this file system.

So let's go ahead and install that package clear the screen to make it easier to see then we need to create the folder that we're going to be mounting this file system into.

We'll use the same command as on instance A.

Then we need to edit the FSTAB file to add this file system configuration.

So we'll do that using this command so shudu space nano space forward slash ETC forward slash FSTAB press enter.

Remember this is instance B so it won't have the line that we added on instance A.

So we need to go down to the bottom paste in this placeholder and then we need to replace the file system ID at the start with the actual file system ID.

So delete this leaving the colon and forward slash go back to the EFS console copy the file system ID into your clipboard.

Move back to this instance paste that in everything looks good.

Save that file with control O press enter exit with control X then we back at the prompt clear the screen.

We'll use the shudu mount forward slash EFS forward slash WP hyphen content command again to mount the EFS file system onto this instance and again it's using the configuration that we've just defined in the FSTAB file press enter.

After a few moments you'll be placed back at the prompt we can verify whether this is mounted with DF space hyphen K.

It has mounted by the looks of things it's at the bottom.

So now if we move into that folder so CD forward slash EFS forward slash WP hyphen content forward slash and press enter.

We now in that folder and if we do a listing so LS space hyphen LA what we'll see is the amazing test file dot txt which was created on instance A.

So this proves that this is a shared network file system where any files added on one instance are visible to all other instances.

So EFS is a multi user network based file system that can be mounted on both EC2 Linux instances as well as on premises physical or virtual servers running Linux.

Now this is a simple example of how to use EFS for now we've done everything that we need to do in this demo lesson so we just need to clean up all of the infrastructure that we've used to do that.

Go back to the EFS console we're going to go ahead and delete this file system so we should already have it selected just select delete you'll need to confirm that process by pasting in the file system ID.

So go ahead and put your file system ID and then select confirm.

Now that can take some time to delete and you'll need to wait for this process to complete.

Once it has completed we're going to go ahead and move across to the cloud formation console.

You should still have this open in a tab if you don't just type cloud formation in the search box at the top and then move to the cloud formation console.

You should still have the stack name of implementing EFS which is the stack you created at the start with the one click deployment.

Go ahead and select this stack then click on delete and confirm that deletion and once that finishes deleting that's all of the infrastructure gone that we've created in this demo lesson.

So I hope this has been a fun and enjoyable demo lesson where you've gained some practical experience of working with EFS at this point though that is everything that you need to do in this demo lesson.

So go ahead and complete the video and when you're ready I'll look forward to you joining me in the next.

Welcome back and in this demo lesson I want to give you some abstract practical experience of using the Elastic File System or EFS.

Now we're going to need some infrastructure.

Before we apply that as always make sure that you're logged into the general AWS account, so the management account of the organization and you'll need the Northern Virginia region selected.

Now attached to this lesson is a one-click deployment link so go ahead and click that.

This is going to provision some infrastructure.

It's going to take you to the quick create stack screen and everything should be pre-populated.

You'll just need to scroll to the bottom, check the box beneath capabilities and then click on create stack.

You're also going to be typing some commands within this demo lesson so also attached to this lesson is a lesson commands document.

Go ahead and open that in a new tab.

So this is just a list of the commands that we're going to be using during the demo lesson and there are some placeholders such as file system ID that you'll need to replace as we go but make sure you've got this open for reference.

Now we're going to need this stack to be in a create complete state before we continue with the demo lesson so go ahead pause the video and resume it once your stack moves into a create complete state.

Okay so the stacks now moved into a create complete status and what this has actually done is create the animals for life base VPC as well as a number of EC2 instances.

So if we go to the EC2 console and click on instances running you'll note that we've created a for L - EFS instance A and a for L - EFS instance B and we're going to be creating an EFS file system and mount points and then mounting that on both of these instances and interacting with the data stored on that file system.

We're going to get you the experience of working with a network shared file system so let's go ahead and do that.

So to get started we need to move to the EFS console so in the search box at the top just type EFS and then open that in a brand new tab.

We're going to leave this tab open to the instances part of the EC2 console because we're going to come back to this very shortly.

So let's move across to the EFS console that we have open in a separate tab and the first step is to create a file system so a file system is the base entity of the elastic file system product and that's what we're going to create.

Now you've got two options for setting up an EFS file system you can use this simple dialogue or you can click on customize to customize it further.

So if we're using the simple dialogue we'd start by naming the file system so let's say we use A4L - EFS and then you'd need to pick a VPC for this file system to be provisioned into and of course we'd want to select the animals for life VPC.

Now we want to customize this further we don't want to just accept these high-level defaults so we need to click on customize.

This is going to move us to this user interface which has many more options so we've still got the A4L - EFS name for this file system.

Now for the storage class we're going to pick standard which means the data is replicated across multiple availability zones.

If you're doing this in a test or development environment or you're storing data which is not important then you can choose to use one zone which stores data redundantly but only within a single AZ.

Now again in this demonstration we are going to be using multiple availability zones so make sure that you pick standard for storage class.

You're able to configure automatic backups of this file system using AWS backup and if you're taking an appropriate certification course this is something which I'll be covering in much more detail.

You can either enable this or disable it obviously for a production usage you'd want to enable it but for this demonstration we're going to disable it.

Now EFS as I mentioned in the theory lesson comes with different classes of storage and you can configure lifecycle management to move files between those different storage classes so if you want to configure lifecycle management to move any files not accessed for 30 days you can move those into the infrequent access storage class and you can also transition out of infrequent access when anything is accessed so go ahead and select on first access for transition out of IA.

So in many ways this is like S3 with the different classes of storage for different use cases.

When you're creating a file system you're able to set different performance and throughput modes.

For throughput mode you can choose between bursting and enhanced.

If you pick enhanced you're able to select between elastic and provisioned.

I've talked more about these in the theory lesson.

We're going to pick bursting.

Now for performance you can choose between general purpose and max I/O.

General purpose is the default and rightfully so and you should use this for almost all situations.

Only use max I/O if you want to scale to really high levels of aggregate throughput and input output operations per second so only select it if you absolutely know that you need this option.

You've also got the ability to encrypt the data on the file system and if you do encrypt it it uses KMS and you need to pick a KMS key to use.

Of course this means that in order to interact with objects on this file system permissions are needed both on the EFS service itself as well as the KMS key that's used for the encryption operation.

Now this is something that you will absolutely need to use for production usage but for this demonstration we're going to switch it off.

We won't be setting any tags for this file system so let's go ahead and click on next.

You need to configure the network settings for this file system so specifically the mount targets that will be created to access this file system.

Now best practice is that any availability zones within a VPC where you're consuming the services provided by EFS you should be creating a mount target so in our case that's US - East - 1A, 1B and 1C.

So we're going to go through and configure this so first let's delete all of these default security group assignments.

Every mount target that you create will have an associated security group so we'll be setting these specifically.

For now though we need to choose the application subnet in each of these availability zones so in the top drop-down which is US - East - 1A I'm looking for app A so go ahead and do the same.

In US - East - 1B I want to select the app B subnet and then in US - East - 1C logically I'll be selecting the app C subnet so that's app A, app B and app C.

Now for security groups the CloudFormation 1 click deployment has provisioned this instance security group and by default this security group allows all connections from any entities which have this attached so this is a really easy way that we can allow our instances to connect to these mount targets so for each of these lines go ahead and select the instance security group you'll need to do that for each of the mount targets so we'll do the second one and then we'll do the third one and that's all of the network configuration options that we need to worry about so click on next it's here where you can define any policies on the file system so you can prevent root access by default you can enforce read only access by default you can prevent anonymous access or you can enforce encryption in transit for all clients connected to this EFS file system so any clients that connect to the mount targets to access the file system you can ensure that that uses encryption in transit and if you're using this in production you might want to select at least this last option to improve security for this demo lesson we're not going to use any of these policy options nor are we going to define a custom policy in the policy editor instead we'll just click on next at this point we just need to review everything's to our satisfaction everything looks good so we're going to scroll down to the bottom and just click on create now in order to continue with this demo lesson we're going to need both the file system and all of its mount targets so go into the file system click on network and you'll see three mount targets being created all three of these need to be ready before we can continue the demo lesson so this seems like a great time to end part one of this demo lesson go ahead and finish this video and then when all of these mount targets are ready to go you can start part two.

This passage talks about the "coming-out" experience for LGBTQ+ people and how it is shown in American movies and media. Many non-LGBTQ+ people think of coming out as a big event that happens once, but for many LGBTQ+ youth, it is something that happens over and over again throughout their lives. Ngo mentions that you might come out every time you meet someone new, based on his experiences in middle and high school. He observes that his classmates often hinted at being queer and came out at different times to different people. He shares a personal story about coming out to his mother multiple times, saying, "I told my Mom, and she didn't believe me... I told her two years later, and she didn't believe me." In his book, Mayo discusses how LGBTQ+ youth often don't get support from their families or schools. This lack of support can include not having role models in schools or having tough relationships between families and schools. This all connects back to the idea that coming out is not just a one-time thing but a continuous process for many LGBTQ+ individuals.

To [[Matt Webb]] the project using GPT3 extracting data from web pages saved him 4d of work (compared to 20 mins coding up the GPT-3 instructions, and ignoring GPT-3 then ran overnight). Saying that's about 10yrs of Moore's law happening to him all at once. 'computers got 10yrs better' an enticing thought and framing. It depends on the use case probably, others will lose 10 yrs of their time making sense of generated nonsense. (Vgl the #pke24 experiments I did w text generation, none of it was usable bc enough was wrong to not be able to trust anything). Sticking to specific niches probably true : [[Waar AI al redelijk goed in is 20201226155259]], turning the issue into the time needed to spot those niches for yourself.

Its sad but its true. These challenges come from anything nd anywhere and it just questions what are they being taught or for why do they do things to fit in. It's hard seeing how damaging these situations really are where kids will forever remember how they were treated growing up. it's even worse when teachers don't put a stop to they way kids talk.

I understand what you're trying to say here, but the grammar is getting in your way a bit - it's a bit jarring to go from saying that it is more than a song in the previous sentence to referring to it as "this song" in the next sentence

I know that you probably just copy/pasted this text over from a Word doc, but moving forward please make sure that it's not all bolded :-)

There's nothing wrong with this kind of transition, but it's generally not needed - just the phrase ""Love Wins All" is more than just a song" already signals to your reader that you're wrapping up your discussion

It's grammatically a bit unclear what "these dark moments" is referring to here - is it what was discussed at the beginning of the paragraph (Sulli's death) or what was just discussed in the previous couple of sentences (body shaming of female idols, etc.)?

I know that you probably just copy/pasted this text over from a Word doc, but moving forward please make sure that it's not all bolded :-)

I know that you probably just copy/pasted this text over from a Word doc, but moving forward please make sure that it's not all bolded :-)

I know that you probably just copy/pasted this text over from a Word doc, but moving forward please make sure that it's not all bolded :-)

for - sensory substitution - citizen science - David Eagleman

for - Deep Humanity - BEing journey - example - umwelt - visual to audio app - The Voice - David Eagleman - to - search - Google - android app "The Voice" translates images into audio signal - https://hyp.is/OJKKmJ1MEe-TAp_w_0SK_Q/www.google.com/search?q=android+app+%22The+Voice%22+translates+images+into+audio+signal&sca_esv=6fa4053b1bfce2fa&sxsrf=ADLYWIK_UqZZZ9OCRCwH4D6FoSaykbMTpQ:1731013461104&ei=VSstZ4eCBqi8xc8P5KP_kAU&ved=0ahUKEwjHgM3Tj8uJAxUoXvEDHeTRH1IQ4dUDCA8&uact=5&oq=android+app+%22The+Voice%22+translates+images+into+audio+signal&gs_lp=Egxnd3Mtd2l6LXNlcnAiO2FuZHJvaWQgYXBwICJUaGUgVm9pY2UiIHRyYW5zbGF0ZXMgaW1hZ2VzIGludG8gYXVkaW8gc2lnbmFsMggQABiABBiiBDIIEAAYgAQYogQyCBAAGIAEGKIEMggQABiABBiiBDIIEAAYgAQYogRI2xdQpglYjRJwAXgCkAEAmAGZA6ABmQOqAQM0LTG4AQPIAQD4AQGYAgOgAqADwgIKEAAYsAMY1gQYR8ICBBAAGEeYAwDiAwUSATEgQIgGAZAGCJIHBTIuNC0xoAewBA&sclient=gws-wiz-serp

it's easier to just say we're fucked out of fear that our elites will never be able to meet the Earth's demands

Salvage the Bones passage

This is true! You shouldn't be basign your self-esteem, or worse, self-worth on where you are compared to other people. While I think it's good to know where you are relative to other people (bc when you are better at a skill than others, you can use that as an advantage), it's also important to remain humble. I always remind myself that anyone can become as good as or even better than you--it's just a matter of time and effort. (This applies to you too!)

Seems it's just setting flags for page faults based on flags param

It's crazy that just the phones from which the tweets were posted was enough to tell all of this. I feel like this really reenforces the importance of being careful about what you put out there, as even the smallest details are enough to tell a lot.

Alkene Protonation Step: In this context, "alkene protonation" means adding a proton (H⁺) to an alkene (a molecule with a carbon-carbon double bond). This addition creates a carbocation (a positively charged carbon atom) as an intermediate.

Endergonic Reaction: An endergonic reaction is one that requires energy input to proceed; it’s not energetically favorable on its own. When an alkene is protonated, forming a carbocation intermediate, energy is required to reach this unstable, high-energy state.

Carbocation Stability and Transition State: When forming a carbocation, the reaction passes through a transition state, which is a high-energy state that comes just before the carbocation actually forms. The energy level of this transition state largely depends on how stable the resulting carbocation will be.

More Substituted Carbocations: A carbocation is more stable when it's more substituted (i.e., when the positively charged carbon is bonded to more alkyl groups). This is because alkyl groups help stabilize the positive charge through electron-donating effects.

Linking Stability to Transition State: Because a more substituted carbocation is more stable, the transition state leading to its formation is also more stable. This means it requires slightly less energy to reach this transition state compared to forming a less substituted, less stable carbocation.

So, in summary: Protonating an alkene to form a carbocation is an energy-requiring (endergonic) step. However, if the carbocation formed is highly substituted and stable, the transition state (which precedes the carbocation) will also be relatively stable, making it easier for the reaction to proceed in that direction.

IMP

@bgates ..,.. "uvrin?"

ringing the idea ... the metaphorical idea; where's the universal mandate for a "guild of programmer's that program away the need to fight?"

have you played starcraft galactica?

do you think it's just the Bureau or .. the KGB or Courtney Cox herself; who has it?

This is PERFECT!!

I know it's supposed to be a diary, so it is hard to like actually introduce the character, but the reader has no idea who that is. You could maybe say like My husband has been acting strange. Earl (something he does)... This might just contextualize it better.

Welcome back and in this demo lesson you're going to get some experience of migrating a snapshot which you've previously taken from Aurora in provisioned mode and migrate this into Aurora running in serverless mode.

Now before we begin as always make sure that you're logged in to the general AWS account, so the management account of the organization and you'll need to have Northern Virginia selected.

Now attached to this lesson is a one-click deployment link and I'll need you to go ahead and open that to start the process.

Now once you've got this open we're going to need the Aurora snapshot name that you created in a previous demo.

So click on the services drop down and locate RDS.

It's probably going to be in the recently visited section if not you can search in the box at the top but go ahead and open that in a new tab.

Go to that tab and once it's loaded click on snapshots and you should have these two snapshots in your account.

The first snapshot is a4l wordpress -with -cat - post -mySQL57.

The other snapshot the one that we're going to use is this one so a4l wordpress -aura -with -cat -post.

Go ahead and select that entire snapshot name and copy that into your clipboard because we're restoring an Aurora provisioned snapshot into Aurora serverless.

We're not performing a migration we're performing a restore and so we don't need the snapshot ARN we need the snapshot name.

So go back to the cloud formation stack everything should be pre-populated but there's a box that you need to paste in the snapshot name that you'll be restoring so that's this one paste that in check the acknowledgement box at the bottom under capabilities and then create stack.

Now that process can take up to 45 minutes to complete sometimes it can be a little bit quicker and while that's working we're going to follow the same process through manually but we're going to stop before provisioning the Aurora serverless cluster.

So go back to the RDS tab make sure that you still have this snapshot selected then click on actions and then restore snapshot and I want to step through the options available when restoring an Aurora provisioned snapshot into Aurora serverless.

So these are the options you'll have when you're restoring an Aurora provisioned snapshot you'll see a list of compatible engines so anything compatible with the snapshot that you're restoring in our case it's only my SQL compatibility then you'll have to select your capacity type now it defaults to provisioned but we want to restore to a serverless cluster so we'll select serverless.

You need to select the version of Aurora serverless that you're restoring to and again it's only going to show you compatible versions in this case only 2.07.1 and that's why I was so precise with the version numbers when doing the demos earlier in this section.

Now under database identifier it's here where we would need to provide a unique identifier within this region inside this account for what we're restoring so we might use a4l wordpress-serveless we then need to provide connectivity information so we'd click in the VPC drop-down and make sure we select the animals for life VPC we'd still need to provide a database subnet group to use now currently there isn't one that exists in the account because the cloud formation template is still provisioning but we'd need to choose a relevant subnet group in this box we'd also need to choose a VPC security group which controls access to this database cluster then we have additional configuration and this is a feature which I'm going to be talking about in a dedicated lesson if you're doing the developer or sysops associate courses and this is an API which can be provisioned to give access to the data within this Aurora serverless cluster and it can do so in a way which is very lightweight and this makes it ideal for use with things like serverless applications which prefer a connectionless architecture so this is something that you will use if you want to use for example Aurora serverless with a serverless application based on lambda now something unique to Aurora serverless is the concept of capacity units and I've talked about these in the theory lesson where I talk about Aurora serverless these are the units of database service which the Aurora serverless cluster can make use of and you're able to set a minimum capacity unit and a maximum capacity unit and this provides a range of resources that this cluster can move between based on load on the cluster so as I've talked about in the theory lesson it will automatically provision more capacity or less capacity between these two values based on load now you have additional options for scaling and one that I'll be demonstrating a little bit later on in this demo lesson is how you can actually pause the compute capacity after a consecutive number of minutes of inactivity and this as long as your application supports it can actually reduce the amount of cost that you have running a database platform down to almost zero so you won't have any compute capacity build when the Aurora serverless cluster isn't in use and again I'll be demonstrating that very shortly in this demo lesson you're able to set encryption options just like with other forms of RDS and then under additional configuration you can also configure backup options now these options are obviously based on restoring a snapshot and you have a similar yet more extensive set of options if you're creating an Aurora serverless cluster from scratch so if we select Amazon Aurora and then we go down and select the serverless capacity type then obviously we can select from different versions and we have a wider range of options that we can set so the cluster identifier the admin username and password we've still got the capacity settings we still need to define connectivity options we've got additional configuration options around creating a database controlling the parameter group customizing backup options encryption and enabling deletion protection so whether you're restoring a snapshot or creating an Aurora serverless cluster from scratch these options are similar but you have access to slightly more configuration if you're creating a brand new cluster because when you're restoring a snapshot many of these configuration items are taken from that snapshot at this point we're not going to actually create the cluster manual so I'm going to cancel out of that and I'm going to refresh and as you can see we already have our Aurora serverless DB cluster and it's in an available state so let's go back to our cloud formation stack and refresh it's still in a create in progress state for the stack itself and in order to continue with this demo lesson we're going to need this to be in a create complete state so go ahead pause the video wait for your stack to move into a create complete state and then we can continue so this stacks now moved into a create complete state and we're good to continue so the first thing that I want to draw your attention to if we move back to the RDS console and then let's just refresh you'll see that this cluster is currently using two Aurora capacity units let's go inside the cluster we'll be able to see that it's available it's currently using two capacity units but otherwise it looks very similar to a provisioned Aurora cluster now what we're going to do is click on services open the EC2 console in a new tab go to instances running you should see a single WordPress instance so select that copy the public IP version 4 address into your clipboard making sure not to use this open address and open that in a new tab you'll see that it loads up the WordPress application and it still has the post within it that you created in the previous demo lesson the best cats ever and if you open this post you'll see that it doesn't have any of the attached images because remember they're not stored in the database they're stored on the local instance file system and that's something that we're going to rectify in an upcoming section of the course either called advanced storage or network storage depending on what course you're currently taking but I just wanted to demonstrate that all we've done is restore an Aurora provision snapshot into an Aurora serverless cluster and it still operates in the same way as Aurora provisioned but this is where things change if we go back to the RDS console we know that this Aurora serverless cluster makes use of Aurora capacity units or ACUs and currently it's set to move between one and two Aurora capacity units and the reason it's currently set to two is because we've just used it we've just restored an existing snapshot into this cluster and that operation comes with a relatively high amount of overhead so it needs to go to the two capacity units maximum in order to give us the best performance now what we're going to see over the next few minutes if we just sit here and keep refreshing this screen what should happen is that because we're not using our application first we're going to see it drop down from two capacity units to one capacity unit and that will of course reduce the costs for running this Aurora serverless cluster after a certain amount of time it's going to go from one capacity unit to zero capacity units because it's going to pause the cluster because of no usage we've got this configured if I click on the configuration tab to pause the compute capacity after a number of consecutive minutes of inactivity and it's set to five minutes so after five minutes of no usage on this database it's actually going to pause the compute capacity and we won't be incurring any costs for the compute side of this Aurora serverless cluster so that's one of the real benefits of Aurora serverless versus all of the other types of RDS database engine so let's just go ahead and refresh this and see if it's already changed from two capacity units it's currently still on two so let's select logs and events and refresh we don't see any events currently so this means that we've had no scaling events on this database but if we click on monitoring you'll see how the CPU utilization has decreased from around 25% to just over 5% and the database connection count has reduced from the one when we just accessed the application back down to zero after a few refreshers we'll see that it either decreases from two capacity units down to one or it will go straight to zero if we reach this five minute timer before it performs that scaling event to reduce from two to one so in our case we've skipped the point of having one capacity unit we've reached that five minute threshold where it pauses the compute capacity and so it's gone straight down to zero so your experience might vary it might go from two down to one and then pause or it might go from two straight down to zero but in a case for me my database is currently running at zero capacity units because this time frame has been reached with no activity and the compute has been paused so this means I have no costs for the compute side of Aurora serverless now if I go back to the application and do a refresh you'll see that we don't get a refresh straight away there's a pause and this is because now that the database cluster experiences some incoming load it's unpausing that compute it's resuming the compute part of the cluster and this isn't an immediate process so it's important to understand that when you implement an application and use this functionality the application does need to be able to tolerate lengthier connection times now sometimes in the case of WordPress you will see an error page when you attempt to do a refresh because a timeout value within WordPress is reached before the cluster can resume in the case of this demo lesson that didn't happen it was able to resume the cluster straight away and if we go back to the RDS console and then refresh this page we'll be able to see just how many capacity units this cluster is now operating with and it's operating with two capacity units now in production usage you could be a lot more granular and customize this based on the needs of your application in my case my minimum is one and my maximum is two and my pause time frame is a relatively low five minutes because I wanted to keep it simple for this demo lesson in production usage you might have a larger range between minimum and maximum you might have a higher minimum to be able to cope with a certain level of base load and the time frame between the last access and the pausing of the compute might be significantly longer than five minutes but this demonstration lesson is just that a demo and it's just designed to highlight this at a really high level so that when it comes to you using this in production you understand the architecture now that's everything that I wanted to cover in this demo lesson it's just been a brief bit of experience of using Aurora serverless now to tidy up to return the account into the same state as it was at the start of the demo lesson just go ahead and close down all of these tabs we need to go back to the cloud formation console make sure the Aurora serverless stack is selected and then just go ahead and click on delete and then delete stack and that will remove all of those resources returning the account into the same state as it was at the start of the demo now this whole section of the course has been around trying to improve the database part of our application so we've moved from having a database running on the same server as the application we've split that off we've moved it into rds and we've evolved that from my sequel rds through to Aurora provisioned and now to Aurora serverless we still have one major limitation with our application and that's that for any posts you make on the blog the media for those posts are stored locally on the instance file system and that's something that we're going to start tackling next in the course and we're going to be using the elastic file system product or EFS at this point though that's everything that I wanted to cover in this demo lesson go ahead and complete this video and when you're ready I look forward to you joining me in the next.

Welcome back.

This is part two of this lesson.

We're going to continue immediately from the end of part one.

So let's get started.

Now the next thing that I want to demonstrate is how we can restore RDS if we have data corruption.

The way that we're going to simulate this is to go back to our WordPress blog and we're going to corrupt part of this data.

So we're going to change the title of this blog post from the best cats ever to not the best cats ever, which is clearly untrue.

But we're going to change this and this is going to be our simulation of data corruption of this application.

So go ahead and click on update to update the blog post with this new obviously incorrect data.

Now let's assume that we need to restore this database from an earlier snapshot.

Now let's ignore the automatic backup feature of RDS and just look at manual snapshots.

Well, let's move back to the RDS console and click on snapshots and we'll be able to see the snapshot that we created at the start of this demo lesson.

Remember, this does have the blog post contained within it in its original correct form.

Now to do a restore, we need to select this snapshot, click on actions and then restore snapshot.

Now I mentioned this in the theory lesson about backups and restores within RDS.

Restoring a snapshot actually creates a brand new database instance.

It doesn't restore to the existing one using normal RDS.

So we have to restore a snapshot.

Obviously the engine set to MySQL community and we're provided with an entry box for a brand new database identifier.

And we're going to use a4lwordpress-restore.

So this allows us to more easily distinguish between this and the original database instance.

We also need to decide on the deployment option.

So go ahead and select single DB instance.

This is only a demo, so we don't need to select multi-AZ DB instance.

We need to pick the type of instance that we're going to restore to.

And again, because this is a new instance, we're not limited to the previous free tier restrictions.

So we're able to select from any of the available instance types.

So go ahead and select burstable classes and then pick either t2 or t3.micro.

We'll leave storage as default.

We'll need to provide the VPC to provision this new database instance into.

So we'll make sure that a4l-vpc1 is selected and we'll use the same subnet group that was created by the one-click deployment, which you used at the start of this demo.

You're allowed to choose between public access yes or no.

We'll choose no.

You'll have to pick a VPC security group to use for this RDS instance.

Now the one-click deployment did create one, so click in the drop-down and select the RDS multi-AZ snap RDS security group.

So not the instance security group, but the RDS security group.

Once you've selected that, then delete default, scroll down.

You can specify database authentication and encryption settings.

And again, if applicable in the course that you're studying, I'll be covering these in a separate lesson.

We'll leave all of that as default and click on restore DB instance.

Now this is going to begin the process of restoring a brand new database instance from that snapshot.

Now the important thing that you need to understand is this is a brand new instance.

We're not restoring the snapshot to the same database instance.

Instead, it's creating a brand new one.

Now when this finishes restoring, when it's available for use, if we want our application to make use of it, and the restored non-corrupted data, then we're going to need to change the application to point at this newly restored database.

So at this point, go ahead and pause the video because for the next step, which is to adjust the WordPress configuration, we need this database to be in an available state.

So pause the video, wait for the status to change from creating all the way through to available, and then we're good to continue.

Okay, so the snapshot restore is now completed and we have a brand new database instance, A4LWordPress-Restore.

And in my case, it took about 10 minutes to perform that restoration.

Now just to reiterate this concept, because it's really important, it features all the time in the exams, and you'll need this if you operate in the real world using AWS.

If we go into the original RDS instance, just pay attention to this endpoint DNS name.

So we have a standard part, which is the region, and then .rds, and then .amazonaws.com.

Before this, though, we have this random part.

Now this represents the name of the database instance as well as some random.

If we go back to the databases list and then go into the restored version, now we can see that we have A4LWordPress-Restore.

And this is different than that original database endpoint name for the original database.

So the really important, the critical thing to understand is that a restore with a normal RDS will create a brand new database instance.

It will have a brand new database endpoint DNS name, the CNAME, and you will need to update any application configuration to use this brand new database.

So go ahead and just leave this open in this tab because we'll be needing it very shortly.

Click on Services, find EC2, and open that in a new tab.

So as a reminder, if we go back to the WordPress tab and just hit Refresh, we can see that we still have the corrupt data.

Now what we want to do is point WordPress at the restored correct database.

So to do that, go to the EC2 tab that you just opened, right click on the A4LWordPress instance, select Connect.

We're going to use Instance Connect, so choose that to make sure the username is EC2-user and then connect to the instance.

This process should be familiar by now because we're going to edit the WordPress configuration file.

So cd/var/www/html, then we'll do a listing ls-la, and we want to edit the configuration file which is wp-config.php, so shudu, space, nano which is the text editor, space, wp-config.php.

Once we're in this file, just scroll down and again we're looking for the dbhost configuration which is here.

Now this DNS name you'll recognize is pointing at the existing database with the corrupt data.

So we need to delete all of this just to leave the two single quotes.

Make sure your cursor's over the second quote.

Go back to the RDS console and we need to locate the DNS name for the A4LWordPress-Restore instance.

Remember this is the one with the correct data.

So copy that into your clipboard, go back to EC2 and paste that in, and then Ctrl+O and Enter to save, and Ctrl+X to exit.

That's all of the configuration changes that we need.

If we go back to the WordPress application and hit refresh, we'll see that it's now showing the correct post, the best cats ever, because we're now pointing at this restored database instance.

So the key part about this demo lesson really is to understand that when you're restoring a normal RDS snapshot, you're restoring it to a brand new database instance, its own instance with its own data and its own DNS endpoint name.

So you have to update your application configuration to point at this new database instance.

With normal RDS, it's not possible to restore in place.

You have to restore to a brand new database instance.

Now this is different with a feature of Aurora which I'll be covering later in this section, but for normal RDS, you have to restore to a brand new instance.

So those are the features which I wanted to demonstrate in this demo lesson.

I wanted to give you a practical understanding of the types of recovery options and resilience options that you have available using the normal RDS version, so MySQL.

Now different versions of RDS such as Microsoft SQL, PostgreSQL, Oracle, and even AWS specific versions such as Aurora and Aurora Serverless, they all have their own collections of features.

For the exam and for most production usage, you just need to be familiar with a small subset of those.

Generally, you'll either be using Oracle, MSSQL, or one of the open source or community versions, so you'll only have to know the feature set of a small subset of the wider RDS product.

So I do recommend experimenting with all of the different features and depending on the course that you're taking, I will be going into much more depth on those specific features elsewhere in this section.

For now though, that is everything that I wanted to talk about, so all that remains is for us to tidy up the infrastructure that we've used in this demo lesson.

So go to databases.

I want you to select the A4L WordPress -Restore instance because we're going to delete this fully.

We're not going to be using this anymore in this section of the course, so select it, click on the Actions drop down, and then select Delete.

Don't create a final snapshot.

We don't need that.

Don't retain automated backups and because we don't choose either of these, we need to acknowledge our understanding of this and type Delete Me into this box.

So do that and then click on Delete.

Now that's going to delete that instance as well as any snapshots created as part of that instance.

So if we go to Snapshots, we only have the one manual snapshot.

If we go to System Snapshots, we can see that we have one snapshot for this Restore database, and if you're deleting a database instance, then any system created snapshots for that database instance will also be deleted either immediately or after the retention period expires.

So those will be automatically cleared up as part of this deletion process.

We're not going to delete the manual snapshot that we created at the very start of this lesson with the catpost in because we're going to be using this elsewhere in the course.

So leave this in place.

Click on Databases again.

We're going to need to wait for this Restored Database instance to finish deleting before we can continue.

So go ahead and pause the video, wait for this to disappear from the list, and then we can continue.

Okay, so that Restored Database instance has completed deleting.

So now all that remains is to move back to the CloudFormation console.

You should still have a tab open.

Select the stack deployed as part of the one-click deployment.

It should be called RDS Multi-AZ Snap.

Select Delete and then confirm that deletion, and that will clear up all of the infrastructure that we've used in this demo lesson.

It will return the account into the same state as it was at the start of this demo with one exception.

And that one exception is the snapshot that we created of the RDS instance as part of this deployment.

So that's everything you need to do in this demo lesson.

I hope you've enjoyed it.

I know it's been a fairly long one where you've been waiting a lot of the time in the demo for things to happen, but it's important for the exam and real-world usage that you get the practical experience of working with all of these different features.

So you should leave this demo lesson with some good experience of the resilience and recovery features available as part of the normal RDS product.

Now at this point, that's everything you need to do, so go ahead and complete this video, and when you're ready, I look forward to you joining me in the next.

Welcome back and in this demo lesson we're going to continue implementing this architecture.

So in the previous demo lesson you migrated a database from a self-managed MariaDB running on EC2 into RDS.

In this demo lesson you're going to get the experience working with RDS's multi-availability zone mode as well as creating snapshots, restoring those snapshots and experimenting with RDS failover.

Now in order to complete this demo lesson you're going to need some infrastructure.

So let's move across to our AWS console.

You need to be logged in to the general AWS account.

So that's the management account of the organization and as always make sure that you have the Northern Virginia region selected.

Now attached to this lesson is a one-click deployment link so go ahead and open that.

This will take you to a quick create stack page and everything should be pre-populated and ready to go.

So the stack name is RDS multi-AZ snap.

All of the parameters have default values.

Multi-AZ is currently set to false so leave that at false, check the capabilities box at the bottom and then click on create stack.

Now this infrastructure will take about 15 minutes to apply and we need it to be in a create complete state before we continue.

So go ahead, pause the video and resume it once CloudFormation has moved into a create complete state.

Okay so now that this stack has moved into a create complete state we need to complete the installation of WordPress and add our test blog post because we're going to be using those throughout this demo lesson.

Now this is something that you've done a number of times before so we can speed through this.

So click on the services drop down, move to the EC2 console.

We need to go to running instances and we'll need the public IP version 4 address of A4L-WordPress.

So go ahead and copy the public IP version 4 address into your clipboard.

Don't use this open address.

Open that in a new tab.

We'll be calling the site as always the best cats for username put admin for the password.

We'll be using the animals for life strong password and then as always test at test.com for the email address.

Enter all of that and click on install WordPress.

Then you'll need to log in admin for the username and to the password click on login.

Once we logged in go to posts click on trash under hello world to delete the existing post and then add a new post.

Close down this dialogue for the title of the post the best cats ever click on the plus select gallery.

At this point go ahead and click the link that's attached to this lesson to download the blog images.

Once downloaded extract that zip file and you'll get four images.

Once you've got those images ready click on upload locate those images select them and click on open and that will add those to the post.

Once they're fully loaded in we can go ahead and click on publish and then publish again and that will publish this post to our blog.

And as a reminder that stores these images on the local instance file system and adds the post metadata to the database and that's now running within RDS.

Now I want to step through a few pieces of functionality of RDS and I want you for a second to imagine that this blog post is actually a production enterprise application.

Maybe a content management system and I want to view all of the actions that we perform in this demo lesson through the lens of this being a production application.

So go ahead and return to the AWS console click on services and we're going to move back to RDS.

The first thing that we're going to do is to take a snapshot of this RDS instance.

So just close down any additional dialogues that you see go to databases.

Then I want you to select the database that's been created by the one click deployment link that you used at the start of this demo lesson.

Then select actions and then we're going to take a snapshot.

Now a snapshot is a point in time copy of the database.

When you first do a snapshot it takes a full copy of that database so it consumes all of the capacity of the data that's being used by the RDS instance.

So this initial snapshot is a full snapshot containing all of the data within that database instance.

Now we're going to take a snapshot and we're going to call it a four L wordpress hyphen with hyphen cat hyphen post hyphen mySQL hyphen and then the version number without any dots or spaces.

Now depending on when you're watching this video doing this lesson you might have been using a different version of SQL.

And so in the lesson description for this lesson I've included the name of the snapshot that you need to use.

So go ahead and check that now and include that in this box.

So that informs us what it is, what it contains and the version number that this snapshot refers to.

So go ahead and enter that and then click on take snapshot and that's going to begin the process of creating this snapshot.

Now the process takes a variable amount of time.

It depends on the speed of AWS on that particular day.

It depends on the amount of data contained within the database and it also depends on whether this is the first snapshot or a subsequent snapshot.

Now the way that snapshots work within AWS is the first snapshot contains a full copy of all of the data of the thing being snapshotted and any subsequent snapshot only contains the blocks of data which have changed from that last previous successful snapshot.

So of course the first snapshot always takes the longest and everything else only takes the amount of time required to copy the changed data.

So if we just give this a few minutes let's keep refreshing.

Mine's still reporting at 0% complete so we need to allow this to complete before we move on.

So go ahead and pause the video and resume it once your snapshot has completed.

And there we go our snapshots now moved into an available status and the progress has completed.

And in my case that took about five minutes to complete from start to finish.

So again just to reiterate this snapshot has been taken.

It's a copy of an RDS MySQL database of a particular version and it contains our WordPress database together with the cat post that we just added.

And that's important to keep in mind as we move on with the demo lesson.

Now you could go ahead and take another snapshot and this one would be much quicker to complete.

It would only contain any data changed between the point that you take it and when you took this previous snapshot.

I'm not going to demonstrate that in this video but you can do that.

And for production usage you may use snapshots in addition to the normal automated backups provided by RDS.

Snapshots that you take manually live past the life cycle of the RDS instance.

And if you want to tidy them up you have to do that manually or by using scripts that you create.

So snapshots that are taken manually are not managed by RDS in any way.

And that's important to understand from a DR and the cost management perspective.

Now the next thing that I want to demonstrate is the multi AZ mode of RDS.

So if we go back to the RDS console just expand this menu and go to databases.

Currently this database is using a single RDS instance.

So this RDS instance is not resilient to the failure of an availability zone within this region.

Now to change that process we can provision a standby replica in another availability zone and that's known as multi AZ.

Now it's worth noting that this is not included within the AWS free tier.

So there will be a small charge to do this optional step to enable multi AZ mode.

Make sure that you have the database instance selected and then click on modify.

Now it's on this screen that we can change a lot of the options which relate to this entire RDS instance.

We've got the option to adjust the database identifier, provide a new database admin password.

We can change the DB instance size or type if we want.

We can adjust the amount of storage available to the database instance, even enable storage auto scaling.

But what we're looking for specifically is adjusting the availability and durability settings.

Currently this is set to do not create a standby instance and we're going to modify this.

We're going to change it to create a standby instance and this is something that's recommended for any production usage.

This creates a standby replica in a different availability zone.

So it picks another availability zone, specifically another subnet that's available within the database subnet group that was created by the one click deployment.

So we're going to set that option and scroll down and then select continue.

Now because we have a maintenance window defined on this RDS instance, we have two different options of when to apply this change.

We can either apply the change during the next scheduled maintenance window.

Remember, this is a definable value that you can set when you create an RDS instance or you modify its settings.

Or we can specify that we want to apply immediately the change that we're making.

And for this demo lesson, that's what we're going to do.

Now it does warn you that any changes could cause a performance impact and even an outage.

So it's really important that if you are applying changes immediately, you understand the impact of those changes.

So make sure that you have apply immediately selected and then click on modify DB instance.

Now a multi AZ deployment is essentially an automatic standby replica in a separate availability zone.

What happens behind the scenes is that the primary database instance is synchronously replicated into this standby replica inside a different availability zone.

Now this provides a few benefits.

It provides data redundancy benefits.

It means that any operations which can interrupt IO such as system backups will occur from the standby replica.

So won't impact the primary database and that provides a real advantage for production RDS deployments.

But the main reason beyond performance is that it helps protect any databases in the primary instance against failure of an availability zone.

So if the availability zone of the primary instance fails and then the C name of the database will be changed to point at the standby replica.

And that will minimize any disruption to your application and its users.

Now if we just hit refresh, we can see the status is modifying and what's happening behind the scenes is AWS are taking a snapshot of the primary DB instance.

It's restoring that snapshot into the standby replica, which is in a different availability zone.

And then it's setting up synchronous replication between the primary and the standby replica.

So this is a process which happens behind the scenes.

But it does mean that we need to wait for this process to be complete until the process is complete.

This is not a multi AZ deployment.

So go ahead and pause the video and wait for the status to change away from modifying.

We need this to be in an available state in order to continue with the demo.

So go ahead and pause the video and resume it once this modification has completed.

Okay, so the status has now changed to available.

And in my case, it took about 10 minutes to enable multi AZ mode.

So that's the provisioning of a standby replica in another availability zone.

Now, the likelihood of an AZ failure happening while I'm recording this demo lesson is relatively small, but we can simulate a failure to do that.

If we have the database instance selected and then select the actions drop down and then reboot, we can use the option reboot with failover.

If we choose this option, then part of the process is that a simulated failover occurs.

So the C name, the database endpoint, that's moved so that it now points at the standby replica and then the old primary instance is restarted.

So that's what we're going to do to simulate this process.

So go ahead and select to reboot the database instance.

Make sure that you have reboot with failover selected and then click on confirm.

And this will begin the process of rebooting the database instance.

Now, if we go back to the WordPress blog and we click on view post, you'll see that right away it's not immediately loading.

And that's because the failover from the primary to the standby isn't immediate.

Failover times are typically 60 to 120 seconds.

So that's important to keep in mind if you're deploying RDS in a business critical situation.

It doesn't offer immediate failover.

So let's just stop this and hit reload again.

And now we can see that the page is starting to load because the C name for the database has been moved from pointing at the primary to pointing at the standby replica, which is the new primary.

Okay, so this is the end of part one of this lesson.

It was getting a little bit on the long side and so I wanted to add a break.

It's an opportunity just to take a rest or grab a coffee.

Part two will be continuing immediately from the end of part one.

So go ahead, complete the video and when you're ready, join me in part two.

Welcome back.

This is part two of this lesson.

We're going to continue immediately from the end of part one.

So let's get started.

Okay, so the instance is now in an available state.

Let's just close down this informational dialogue at the top.

And let's just minimize this menu on the left.

Let's maximize the amount of screen space that we have for this specific purpose.

So I just want us to go inside this database instance and explore together the information that we have available.

So I talked in the theory lesson how every RDS instance is given an endpoint name and an endpoint port.

So this is the information that we'll use to connect to this RDS instance.

Networking wise, this instance has been provisioned in US-EAST-1A.

It's in the Animals for Life VPC and it's used our A4L subnet group that we created at the start of this demo.

And that means that it's currently utilizing all three database subnets in the Animals for Life VPC.

But it's chosen because we only deployed one instance to use US-EAST-1A.

Now this is the VPC security group that we're going to need to configure.

So right click on this and open it in a new tab and move to that tab.

This is the security group which controls access to this RDS instance.

So let's expand this at the bottom.

So currently it has my IP address being the only source allowed to connect into this RDS instance.

So the only inbound rule on the security group protecting this RDS instance is allowing my IP address.

So we're going to click on Edit and then click on Add Rule.

And we're going to add a rule which allows our other instances to connect to this RDS instance.

So first in the type drop down click and then type mySQL to get the same option as the line above and then click to select.

Next go ahead and type instance into the source box and then select the migrate to RDS-instance security group.

Now this is the security group that's used by any instances deployed by our one click deployment.

And this allows those instances to connect to our RDS instance and that's what we want.

So go ahead and select that and then click on Save Rules.

And this means now that our WordPress instance can communicate with RDS.

So now let's move back to the RDS tab and then make sure we're inside the A4L WordPress database instance.

So that's the connectivity and the security tab.

We also have the monitoring tab and it's here where you can see various CloudWatch provided metrics about the database instance.

You also have logs and events related to this instance.

So if we go and have a look at recent events we can see all of the events such as when the database instance was created, when its first backup was created.

And you can explore those because they might be different in your environment.

You can click on the Configuration tab and see the current configuration of the RDS instance.

The Maintenance and Backups tab is where you can configure the maintenance and backup processes and then of course you can tag the RDS instance.

Now in other lessons in this section of the course and depending on what course you're taking I will be talking about many of these options, what you can modify and which actions you can perform on RDS instances.

But for now we're just going to move on with this demo.

So the next step is that we need to migrate our existing data into this RDS instance.

So what we're going to do is to click on the Connectivity and Security tab and we're going to leave this open.

We're going to need this endpoint name and port very shortly.

You should still have a tab open to the EC2 console.

If you don't you can reach that by going on Services and then opening EC2 in a new tab.

But I want you to select the A4L-WordPress instance and then right click and connect to it using Instance Connect.

So go ahead and do that.

Once you've done that we're going to start referring to the lesson commands document.

So make sure you've got that open.

We're going to use this command to take a backup of the existing MariaDB database.

So we need to replace a placeholder.

What we need to do is delete this and replace it with the private IP address of the MariaDB EC2 instance.

So go back to the EC2 console, select the DB-WordPress instance and copy the private IP version 4 address into your clipboard.

And then let's move back to the WordPress instance and paste that in.

Go ahead and press Enter and it will prompt you for the password.

And the password is the same Animals for Life strong password that we've been using everywhere.

Copy that into your clipboard.

So this is the password for the A4L WordPress user on the MariaDB EC2 instance.

So paste that in and press Enter and then LS-LA to confirm that we now have this A4L WordPress.SQL database backup file.

And we do, so that's good.

So as we did in the previous demo lesson, we're going to take this backup file and we're going to import it into the new destination database, which is going to be the RDS instance.

To do that, we'll use this command, but we're going to need to replace the placeholder hostname with the CNAME of the RDS instance.

So go ahead and delete this placeholder, then go back to the RDS console and I'll want you to copy the endpoint name into your clipboard.

So select it, right click and then copy.

We won't need the port number because this is the standard MySQL port and if you don't specify it, most applications will assume this default.

So just make sure that you have the endpoint DNS name or endpoint CNAME in your clipboard.

And then back on the WordPress EC2 instance, go ahead and paste this database name into this command and press Enter.

And again, you'll be asked for the password and that's the same Animals for Life strong password.

So copy that into your clipboard, paste that in and press Enter.

And that's imported this A4LWordPress.SQL file into the RDS instance.

So now we need to follow the same process and change WordPress so that it points at the RDS instance.

And we do that by moving to where the WordPress configuration file is.

So cd space forward slash var forward slash ww w forward slash html and press Enter.

And then shudu.

So we have admin privileges, nano, which is a text editor and then wp-config.php and press Enter.

Then we need to scroll down and we're looking for where it says DB host and currently it has a host name here.

Now if you go back to the EC2 console and you look at the A4L-DB-WordPress instance, you'll see that its private IP version for DNS name is what's listed inside this configuration item.

So it's currently pointing at this dedicated database instance.

What we need to do is replace that and we're going to replace it with the RDS database DNS name or the CNAME of this RDS instance.

So copy that into your clipboard and then go ahead and delete this private DNS name for the MariaDB EC2 instance and then paste in the RDS endpoint name, also known as the RDS CNAME.

Once you've done that, control O and Enter to save and control X to exit.

And now our WordPress instance is pointing at the RDS instance for its database.

Now we can verify that by checking WordPress, move back to instances, select the WordPress instance, copy the public IP version for addressing to your clipboard.

Don't use this open address link.

Open that in a new tab.

Go ahead and just click on the best cats ever to verify the functionality and it does look as though it's working.

And to verify that, if we go back to the EC2 console, select the A4L-DB-WordPress instance and right click and then stop that instance.

Now the original database that was providing database services to WordPress is going to move into a stopped state.

And if our WordPress blog continues functioning, we know that it's using the RDS instance.

So let's keep refreshing and wait for this to change into a stopped state.

There we go.

It's stopped.

And if we go back to our WordPress page and refresh, it still loads.

And so we know that it's now using RDS for its database services.

So at this point, that's everything that I wanted you to do in this demo lesson.

You've stepped through the process of provisioning an RDS instance.

So you've created a subnet group, provisioned the instance itself, explored the functionality of the instance, including how to provide access to it by selecting a security group.

And then editing that security group to allow access.

You've performed a database migration and you've explored how the RDS instance is presented in the console.

So that's everything that you need to do within this demo lesson.

And don't worry, we're going to be exploring much more of the advanced functionality of RDS as we move through this section of the course.

For now, though, I want us to clear up the infrastructure that we've created as part of this demo lesson.

Now, because we've provisioned RDS manually outside of CloudFormation, unfortunately, there is a little bit more manual work involved in the cleanup.

So I want you to go to the RDS console, move to databases, select this database, click on actions, and then select delete.

Now it will prompt you to create a final snapshot and we're not going to do that.

We're not going to retain automated backups and so you'll need to acknowledge that upon instance deletion, automated backups including any system snapshots and pointing time recoveries will no longer be available.

And don't worry, I'll be talking about backups and recovery in another lesson in this section of the course.

For now, just acknowledge that and then type delete me into this box and confirm the deletion.

Now this deletion is going to take a few minutes.

It's not an immediate process.

It will start in a deleting state and we need to wait for this process to be completed before we continue the cleanup.

So go ahead and pause this video and wait for this instance to fully delete before continuing.

Now that the instance has been deleted, it vanishes from this list.

Next, we need to delete the subnet group that we created earlier.

So click on subnet groups, select the subnet group and then delete it.

You'll need to confirm that deletion.

Once done, it too should vanish from that list.

Next, go to the tab you've got open to the VPC console, scroll down and select security groups.

Now look through this list and locate the security group that you created as part of provisioning the RDS instance.

It should be called a4LVPC-RDS-SG.

Select that, click on actions and then delete security group and you'll need to confirm that process as well.

Once that's deleted, the final step is to go to the cloud formation console and then you'll need to delete the cloud formation stack that was created using the one-click deployment at the start of the demo.

It should be called migrate to RDS.

Select it, click on delete and confirm that deletion.

And once deleted, the account will be returned into the same state as it was at the start of the demo lesson.

So all of the infrastructure that we've used will be removed from the account and the account will be in the same state as at the start of the demo.

Now I hope you've enjoyed this demo and that we're repeating the same WordPress installation and then the creation of the blog post over and over again.

But I want you to get used to different parts of this process over and over again.

You need to know why not to use a database on EC2.

You need to know why not to perform a lot of these processes manually.

From this point onward in the course, we're going to be using RDS to evolve our WordPress design into something that is truly elastic.

And so all of these processes, the things I'm having you repeat are really useful to aid in your understanding of all of these different components.

So from this point onward, we're going to be automating the creation of RDS and focusing on the specific pieces of functionality that you need to understand.

But at this point, that's everything that you need to do in this demo.

So go ahead, complete the video and when you're ready, I look forward to you joining me in the next.

Welcome back and in this demo lesson you're going to get some experience of how to provision an RDS instance and how to migrate a database from an existing self-managed MariaDB database instance through to RDS.

So over the next few demo lessons in this section of the course, you're going to be evolving your database architecture.

We're going to start with a single database instance, then we're going to add multi-AZ capability as well as talking about backups and restores.

But in this demo lesson specifically, we're going to focus on provisioning an RDS instance and migrating data into it.

Now in order to get started with this demo lesson, as always make sure that you're logged into the general AWS account, so the management account of the organization and you need to have the Northern Virginia region selected.

Now attached to this lesson is a one-click deployment link that you'll need to use to provision this demo lesson's infrastructure.

So go ahead and click on that link now.

That's going to move you to a quick create stack screen.

The stack name should be pre-populated with migrate to RDS.

Scrolling down all of the parameter values will be pre-populated.

All you need to do is to click on the capabilities checkbox and then create stack.

There's also a lesson commands document linked to this lesson and I'd suggest you go ahead and open that in a new tab because you'll be referencing it as you move through this demo lesson.

Now you'll notice that this will look similar to the previous demo lesson's lesson commands document, but it has one small difference.

The initial command way of doing the backup of the source database, because that source database is going to be stored on a separate MariaDB database running on a separate EC2 instance, instead of taking the backup from the local instance, in this case it's connecting to a separate EC2 instance.

Otherwise, most of these commands are similar to the ones you used in the previous demo lesson.

Now you're going to need to wait for this stack to move into a create complete state before you continue the demo.

So go ahead and pause the video, wait for your stack to change to create complete and then you're good to continue.

Okay, so that cloud formation stack has now moved into a create complete state and it's created a familiar set of infrastructure.

Let's go ahead and click on the services drop down and then move to the EC2 console and just take a look.

So if we click on instances, you'll see that we have the same two instances as you saw in the previous demo lesson.

So we have A4L-WordPress, which is running the Apache web server and the WordPress application.

And then we have A4L-DB-WordPress and this is running the separate MariaDB database instance.

So what we need to do in order to perform this migration is first create the WordPress blog itself and the sample blog post.

And this is the same thing that we did in the previous demo.

So we should be able to go through this pretty quickly.

So go ahead and select the A4L-WordPress instance and copy its public IP version for address into your clipboard and then open that in a new tab.

And again, make sure not to use the open address because this uses HTTPS.

So copy the public IP version for address and then open that in a new tab.

Again, we're going to call the site the best cats.

We're going to use admin for the username.

And then for the password, let's go back to the CloudFormation tab.

Make sure you've got the migrate to RDS stack selected and then click on parameters.

We're going to use the same database password.

So copy that into your clipboard and replace the automatically generated one with the animals for live complex password.

And then enter test@test.com into the email box and click on install WordPress.

Once installed, click on login.

You'll need to use the admin username and the same password.

Click on login.

Then we're going to go to posts.

We're going to select the existing Hello World post.

Select trash this time.

Then click on add new.

Close down this dialog for title.

We're going to use the best cats ever.

Click on the plus.

Select gallery.

At this point, go ahead and click the link that's attached to this lesson to download the blog images.

Once downloaded, extract that zip file and you'll get four images.

Once you've got those images ready, click on upload, locate those images, select them and click on open.

Wait for them to load in.

Select publish and publish again.

And that saved the images onto the application instance and added the data for this post onto the separate MariaDB database.

So now we have this simple working blog.

Let's go ahead and look at how we can provision an RDS instance and how we can migrate the data into that RDS instance.

So move back to the AWS console.

Click on the services drop down and type RDS into the search box and open that in a new tab.

Now, as I've mentioned in the theory parts of this section, RDS is a managed database server as a service product from AWS.

It allows you to create database instances and those instances can contain databases that your applications can make use of.

Now to provision an RDS instance, the first thing that we need to do is to create a subnet group.

Now a subnet group is how we inform RDS which subnets within a VPC we want to use for our database instance.

So first we need to create a subnet group.

So select subnet groups on the menu on the left and then create a DB subnet group.

Now we're going to use a4lsn group, so animals for life subnet group for both the name and for the description.

And then select the VPC drop down and we're going to select the a4l-vpc1vpc.

So this is the animals for life VPC which has been created by the one click deployment that you used at the start of this demo.

Now once we've selected a name and a description and a VPC for this subnet group, then what we need to do is select the subnets that this database will be going into.

So we're going to select the database subnets in US East 1A, US East 1B and US East 1C.

So click on the availability zone drop down and pick those three availability zones.

So 1A, 1B and 1C.

Once we've selected the availability zones that this subnet group is going to use, next we pick the subnets.

So click on the drop down.

Now we want to pick the database subnets within the animals for life VPC and all we can see here are the IP address ranges.

So to help us with this click on the services drop down, type VPC and then open that in another new tab.

Once that loads, go ahead and click on subnets, sort the subnets by name and then locate sn-dba, dbb and dbc.

And just move your cursor across to the right hand side and note what the IP address ranges are for those different database subnets.

So 16, 80 and 144.

Go back to the RDS console, click on the subnets drop down and we need to pick each of those three subnets.

So 16, 80 and 144.

So these represent the database subnets in availability zone 1A, 1B and 1C.

And then once we've configured all of that information, we can go ahead and click on create to create this subnet group.

So this subnet group is something that we use when we're provisioning an RDS instance.

And as I mentioned moments ago, it's how RDS determines which subnets to place database instances into.

Now when we're only using a single database instance, then that decision is fairly easy.

But RDS deployments can scale up to use multiple replicas in multiple different availability zones.

You can have multi-AZ instances, read replicas.

Aurora has a cluster architecture which we'll talk about later in this section.

And so subnet groups are essential to inform RDS which subnets to place things into.

So now that we've configured that subnet group, let's go ahead and provision our RDS database instance.

So to do that, click on databases and then we're going to create a database.

So click on create database.

Now when you're creating a database, you have the option of using standard create where you have visibility of all of the different options and then easy create which applies some best practice configurations.

Now I want you to get the maximum experience possible, so we're going to use standard create.

Now when you're creating an RDS database instance, you have the ability to pick from many different engines.

So some of these are commercial like Oracle or Microsoft SQL Server.

And with some of these, you have the option of either paying for a license included with RDS or you can bring your own license.

For other database engines, there isn't a commercial price to pay for their usage and so they're much cheaper to use.

But you should select the engine type which is compatible with your application.

Now we're going to be talking about Amazon Aurora in dedicated lessons later in this section of the course.

Amazon Aurora is an AWS designed database product which has compatibility with MySQL and PostgreSQL.

For this demo lesson, we're going to use MySQL.

So go ahead and select MySQL and it's going to be using MySQL Community Edition.

So now let's just scroll down and step through some of the other options that we get to select when provisioning an RDS instance.

Now for all of these database engines, you have the ability to pick different versions of that engine.

And this is fairly critical because there are different major and minor versions that you can select from.

And different versions of these have different limitations.

So for example, we're going to be talking about snapshots later in this section.

And if you want to take a snapshot of an RDS database and then import that into an Aurora cluster, you need to pick a compatible version.

And then Aurora Serverless which we'll be talking about later on in this section has even more restrictions.

Now to keep things simple, I want you to ignore what version I pick in this video and instead look in this lesson's description and pick the version that I indicate in the lesson description because I'll keep this updated if AWS make any changes.

Now you can choose to use a template.

These templates give you access to only the options which are relevant for the type of deployment that you're trying to use.

So in production, you would pick the production template.

If you have any smaller or less critical dev or test workloads, then you could pick this template.

If you want to ensure that you can only select free tier options, then you should pick this template.

And that's what we're going to do in this demo because we want this demo to fall under the free tier.

So click on the free tier template.

I'll be talking about availability and durability later in this section because we've selected free tier only.

We don't have the ability to create a multi AZ RDS deployment.

And now we need to provide some configuration information about the database instance specifically.

So the first thing that we need to do is to provide a database instance identifier.

So this is the way that you can identify one particular instance from any other instances in the AWS account in the current region.

So this needs to be unique.

So we're going to use a four L WordPress for this database instance.

Then we need to pick a username which will be given admin privileges on this database instance.

And we're going to replace admin with a four L WordPress.

So we're going to use this for both the database identifier and the admin user of this database.

Now for the password for this admin user, we're going to move back to the cloud formation console and we're going to use this same animals for life complex password.

So copy that into your clipboard and paste it in for the password and the confirm password box.

And this just keeps things consistent between the self banished database and the RDS database.

Scroll down further still and it's here where you can select the database instance class to use.

Now because we've selected free tier only, we're limited as to what database size and type we can pick.

If we'd have selected production or dev test from the templates above, we would have access to a much wider range of database instance classes, both standard, memory optimized and burstable.

But because we've selected the free tier template, we're limited as to what we can select.

Now this might change depending on when you're watching this demonstration, but at the point I'm recording this video, it's db.t3.micro.

So don't be concerned if you see something different in this box.

Just make sure that you select the type of instance which falls under the free tier.

Then continue scrolling down and we need to pick the size of storage and the type of storage to use for this RDS instance.

Now whether you need to select this is dependent on what engine type you pick.

If you select Aurora, which we'll be talking about later on in this section, then you don't need to pre-allocate storage.

If you're using the MySQL version of RDS, then you do need to set a type of storage and a size of storage.

Now we're going to use the minimum which is 20GIB because our requirements for this database are relatively small.

And if we wanted to, if this was production, we could set storage autoscaling.

And this allows RDS to automatically increase the storage when a particular threshold is met.

But again, because this is a demo and it's only using a very small blog, we don't need storage autoscaling.

So go ahead and uncheck that option.

Now we need to select a VPC for this RDS instance to go into.

So click in the drop down and select the Animals for Life VPC.

So that's A4L-VPC1.

And then we need to pick a subnet group.

Now this is the thing that we've just created.

We only have one in this account, so there's nothing else to select.

But this is how we can advise RDS on which subnets to use inside the VPC.

Scroll down further still and we can specify whether we want this database to be available.

We want this database to be publicly accessible.

So this is whether we want instances and devices outside the VPC to be able to connect to this database.

This obviously comes with some security trade-offs.

And because we don't need that in this demonstration, because the only thing that we want to connect to this RDS instance is our WordPress instance, which is in the same VPC, then we can select Not to Use Public Access.

So make sure the No option is selected.

Now the way that you control access to RDS is you allocate a VPC security group to that instance.

So we could either choose an existing security group or we could create a new one.

So it's this security group which surrounds the network interfaces of the database and controls access to what can go into that database.

So we want to create a new VPC security group.

So we want to make that option.

We're going to call the security group A4LVPC-RDS-SG.

And we need to remember to update this so that our WordPress instance can communicate with our RDS instance.

And we'll do that in the next step.

If we wanted to pick a specific availability zone for this instance to go into, then we could select one here or we can leave it up to RDS to pick the most suitable.

So we can select No Preference.

Continue scrolling down.

We won't change the Database Authentication option because we want to allow password authentication.

Continue scrolling down and we're going to expand Additional Configuration.

By default, an RDS instance is created with no database on that instance.

In this case, because we're migrating an existing WordPress database into RDS, we're going to go ahead and create an initial database.

And to keep things easy and consistent, we're going to use the same name, so A4L WordPress.

Now you can enable automatic backups for RDS instances.

And I'll be talking about these in a separate theory lesson.

If you do select automatic backups, then you can also pick a backup retention period as well as a backup window.

So we've got Advanced Monitoring, various log exports.

We don't need to use any of those.

You can also set the Maintenance window for an RDS instance.

So when Maintenance will be performed, you can enable Deletion Protection if you want.

If this is a production database, we don't need to do that.

What we're going to do is scroll all the way down to the bottom and then click on Create Database.

Now this process can take some time.

I've seen it take anywhere from five to 45 minutes.

And we're going to need this to be finished before we move on to the next step.

So this seems like a great time to end this video.

It gives you the opportunity to grab a coffee or stretch your legs.

Wait for this database creation to finish.

And then when you're ready, I'll look forward to you joining me in part two of this video.

Welcome to this demo lesson where you're going to migrate from the monolithic architecture on the left of your screen towards a tiered architecture on the right.

Essentially you're going to split the WordPress application architecture, you're going to move the database from being on the same server as the application to being on a different server and this will form step one of moving this architecture from being a monolith through to being a fully elastic architecture.

Now this is the first stage of many but it is a necessary one.

Now in order to perform this demonstration you're going to need some infrastructure.

Before we apply the infrastructure just make sure that you're logged in to the general AWS account, so the management account of the organization and as always you need to have the Northern Virginia region selected.

Now once you've got both of those set there's a one-click deployment link attached to this lesson so go ahead and click on that link.

What this is going to do is deploy the Animals for Life base infrastructure, it's going to deploy the monolithic WordPress application instance and it's also going to deploy a separate MariaDB database instance that you're going to use as part of the migration.

Now everything set, the stack name should be set to a suitable default, all you need to do is to scroll all the way down to the bottom, check this capabilities box and click on create stack.

Now also attached to this lesson is a lesson commands document which contains all the commands you'll be using throughout this demo.

So go ahead and open that in a new tab, you'll be referencing it constantly as you're making the adjustments to the WordPress architecture.

Now we're going to need this CloudFormation stack to be fully complete before we can continue so go ahead and pause the video and resume once the CloudFormation stack moves into a create complete state.

So now the stacks moved into a create complete state, we're good to continue.

Now this has created the base Animals for Life infrastructure which includes a number of EC2 instances so let's take a look at those, let's click on services and then locate and open EC2 in a brand new tab.

Once you're at the EC2 console if you do see any dialogues around user interface updates then just go ahead and close those down and then click on instances running.

Once you're here you'll see two EC2 instances, one will be called A4L-WordPress and this is the monolith so this is the EC2 instance which contains the WordPress application and the built-in database.

So this is the WordPress installation that we're going to migrate from and then this instance A4L-DB-WordPress this contains a standalone MariaDB installation so we're going to migrate the database for WordPress from this instance onto the DB instance and this will create a tiered application architecture rather than the monolith which we currently have.

So step number one is to perform the WordPress installation so to do that I want you to go ahead and copy the public IP version for address of the WordPress EC2 instance into your clipboard and then open it in a new tab.

Now be careful not to use the open address link that will use HTTPS which we're not currently using so copy the IP address into your clipboard and open that in a new tab.

Now when you do that you'll see a familiar WordPress installation dialog we're going to create a simple blog for site title go ahead and call it the best cats for username pick admin and then for the password instead of using the randomly selected one go ahead and use this same complex password that we've used for the CloudFormation template so this is animals for life but with number substitution.

So if you go back to your CloudFormation tab and go to the parameters tab this is the same password that we use for the DB password and the DB root password.

Now of course in production this is incredibly bad practice we're just doing it in this demo to keep things simple and avoid any mistakes.

So back to the WordPress installation screen site title the best cats username admin this for the password and then just go ahead and type a fake email so I don't want to use my real email for this I'm going to type test at test.com you can do the same and then go ahead and click on install WordPress so this is installed the WordPress application and it's using the Maria DB server that's on the same EC2 instance so part of the same monolith.

So we're going to log in we'll need to type admin and then use the animals for life strong password and click on login and once we logged in we're going to create a simple blog post so click on posts we're going to select the existing hello world post select trash this time then click on add new then we're going to add a new post we can close down this introduction dialogue and for title go ahead and type the best cats ever and then some exclamation points next click on this plus sign and we're going to add a gallery now at this point you're going to need some images to upload to this blog post I've attached an images link to this lesson so if you go ahead and click that link it will download a zip file if you extract that zip file it's going to contain four image files all four of my cats so at this point once you've downloaded and extracted that file go ahead and click on upload locate those images there should be four select them all and click on open that will add these images to this blog post and once you've added them all you can go ahead and click on publish and then publish again and this will publish this blog post so it will add data to the database that's running on the monolithic application instance as well as store these images on the local instance file system now making a point of mentioning that these images are stored on the file system because as you'll see later in the course this is one of the things that we need to migrate when we're moving to a fully elastic architecture we can't have images stored on the instances themselves we need to move that to a shared file system for now though we're focusing on the database so at this point we have the working blog the images for this blog are stored on the local file system of a4l-wordpress and the data for that blog post is stored on the MariaDB database that's also running on this EC2 instance so the next step of this demo lesson is that you're going to migrate the data from a4l-wordpress onto a4l-db-wordpress and this is an isolated MariaDB instance this is dedicated for the database so to do this migration select a4l-wordpress right click we're going to connect to this instance we'll be using EC2 instance connect so just make sure that the username is set to EC2-user and then click on connect now this is where you're going to be using the commands that are stored within the lesson commands document so you need to make sure that you have this ready to reference because it's far easier to copy and paste these commands and then adjust any placeholders rather than type them out manually because that's prone to errors the first step is to get the data from the database that's running on this monolithic application instance and store it in a file on disk so that's the first thing we need to do we need to do a backup of the database into a .sql file now to do that we use this command so it's a utility called my sql-dump it uses the -u to specify the user that we're going to be using to connect to the database then we use -p to specify that we want to provide a password and we could either provide the password on the command line or we could have it prompt us now if we supply the password with no spaces next to this -p then it will accept it as input on this command if we don't specify anything so there's a space here then it's going to ask us for the password the next thing we specify is the database name that we want to do the dump of in this case it's a4l WordPress which is the database for the animals for life WordPress instance now if we just run this command on its own it would output the dump so all of the data in the database to standard output which in this case is our screen we don't want it to do that we want it to store the results in a file called a4l WordPress dot sql and so we use this symbol which means that it's going to take the output of this component of the command and it's going to redirect it into this file so let's go ahead and run this command and it's going to prompt us for the password for this database now to get that go back to cloud for information make sure parameters are selected and it's this password that we need which is the DB password so copy that into your clipboard go back to the instance paste that in press enter and that will output all the data in the database to this file now you won't see any indication of success or failure but if you do an LS space -la and press enter one of the files that you'll see is a4l WordPress dot sql so now we have a copy of the WordPress database containing our blog post the next thing that we need to do is to take this file this backup of the database and inject it into the new database that we want to use so the dedicated Maria DB EC2 instance and to do that we're going to use this command so this command has two components the first component is this which connects to the Maria DB database instance the second component is this which takes the backup that we've just made and it feeds it into this command so this backup contains all the necessary definitions to create a new database and inject the data required this component of the command just allows us to connect to this new dedicated Maria DB instance now there are some place holders that we need to change the database name that we're going to use is the same so a4l WordPress we're still going to want to be prompted for a password so -p is what we use this time though we're going to connect using a user called a4l WordPress so we're not using the root user we're going to connect to this separate Maria DB database instance using a user a4l WordPress the only thing that we need to change is that we need to connect to a non-local host so when we used the mysql dump command we didn't specify a host to connect to and this defaulted to local host so the current machine in the case of this command we're operating with a separate server this dedicated EC2 instance which is running the Maria DB database server so a4l -db -wordpress we need to connect to this so what we'll need to connect to this is the private IP version for address of this separate database instance so select it look for private IP version for addresses and then click on the icon next to this to copy the private IP version for address of this separate database server into your clipboard then return to the application instance and we need to replace the placeholder here with that value so make sure that you're one space after the end of this placeholder and just delete this leave a space between -h and where the cursor is and then paste in that IP address so this is going to connect to this separate EC2 instance using its private IP it's going to use the a4l WordPress user it will prompt us for a password it will perform the operation on the a4l WordPress database and it's going to use the contents of this backup file to perform those tasks so go ahead and press enter and you'll be prompted for a password now again it's the same password this has all been set up as part of the cloud formation one-click deployment this lesson is about the migration process not setting up a database server so I've automated this component of the infrastructure so copy the DB password into your clipboard go back to the instance paste it in and press enter so now we've uploaded our WordPress application database into this separate MariaDB database server the next step is to configure WordPress to point at this new database server so to do that cd space forward slash var forward slash www forward slash html and press enter and then we're going to run a shudu space nano which is a text editor space wp-config.php and this is the WordPress configuration file so press enter now what we're looking for if we scroll down is we're looking for the line which says define and then a space and then DB host so this is the database host that WordPress attempts to connect to and currently it's set to local host which means it will use the database on the same EC2 instance as the application we're going to delete this local host so delete until we have two single quotes and then make sure that you still have the private IP version for address of this separate database instance in your clipboard if you don't just go ahead and copy it again from the EC2 console and then paste that in place of local host so now you should see DB underscore host and this now represents this private IP address and now the private IP address that you should use here will be different you need to use your private IP address of your a4l - DB - WordPress EC2 instance so now that you've updated this configuration file press control o and enter to save and then control x to exit out of editing this file now this now means that the WordPress instance is going to be communicating with the separate MariaDB database instance let's verify that let's go back to the tab that we have to our WordPress application and let's just go ahead and do a refresh if everything's working as expected we should see that the blog reloads successfully now this means that this blog is now pointing at this separate MariaDB database instance to be doubly sure of this though let's go back to the WordPress instance and let's shut down the MariaDB database server and we do that using this command so shudu space service space MariaDB space and then stop so type or copy and paste that command in and press enter and that's going to stop the MariaDB database service which is running on a4l WordPress so now the only MariaDB database that we have running is on the a4l - DB - WordPress EC2 instance now we can go back to the WordPress tab and hit refresh and assuming it loads in as it does in my case this now confirms that WordPress is communicating with this dedicated MariaDB EC2 instance now the reason why I wanted to step you through all these tasks in this demo lesson is the time a firm believer that in order to understand best practice architecture you need to understand bad architecture and as I mentioned in the theory lesson there is almost no justification for running your own self-managed database server on an EC2 instance in almost all situations it's preferable to use the RDS service but I need you to understand exactly how the architecture works when you're self managing a database and how to migrate from a monolithic all-in-one architecture through to having a separate self managed database in the demo lesson that's coming up next in the course you're going to migrate from this through to an RDS instance so that's step two but at this point you've done everything that I wanted you to do in this demo lesson you've implemented the architecture that's on screen now on the right all we need to do is to tidy up all of the infrastructure that we've used within this lesson so to do that it's nice and easy just go back to the cloud formation console make sure that you have the monolith to EC2 DB stack selected click on the delete button and then confirm that deletion and that stack deleting will clean up all of the infrastructure that we've used throughout this demo lesson and it will return the account into the same state as it was at the start of the lesson at this point you've completed all of the tasks that I want you to do so I hope you've enjoyed this demo lesson go ahead and complete this video and when you're ready I'll look forward to you joining me in the next.

Welcome back.

This is part two of this lesson.

We're going to continue immediately from the end of part one.

So let's get started.

Now one final thing before we finish with this demo lesson, and I want to talk about private hosted zones.

So move back to the Route 53 console.

I'm going to go to Hosted Zones, and I'm going to create a private hosted zone.

So click "Create Hosted Zone" because it's a private hosted zone, it doesn't even need to be one that I actually own.

So I'm going to call my hosted zone "IlikeDogsReally.com".

It's going to be a private hosted zone.

And for now, I'm going to associate it with the default VPC in US-East-1.

So I'm going to pick the region, US-East, and then select Northern Virginia, and then click in the VPC ID box, and we should see two VPCs listed.

One is the Animals for Life VPC, it's tagged A4L-VPC1, but I'm not going to pick this one, I'm going to pick the one without any text after it, which is the default VPC.

So once that's set, I'm going to create the hosted zone.

Then inside the hosted zone, I'm going to create a record.

The record's going to use the simple routing policy.

Click on "Next".

I'm going to define a simple record.

I'm going to call it "www".

The record type is going to be "a routes traffic to an IP version 4 address and some resources".

I'm going to click in this endpoint box and select IP address or another value, depending on record type.

And then into this box, I'm just going to put a test IP address of 1.1.1.1.

And then down at the bottom, I'm going to click "1M" to change this TTL to 60 seconds.

And I'm going to click "Define simple record".

And then finally, "Create records".

So now we have a record called "www.ilikedogsrealy.com".

So copy that into the clipboard.

Move back to the EC2 console.

Click on "Dashboard".

Click on "Instances running".

Right click, "Connect".

We're going to use EC2 "Instance connect".

And then just click on "Connect".

Now once connected, I'm going to try pinging the record which I just created.

So "Ping Space" and then paste in "www.ilikedogsrealy.com" and press "Enter".

What you should see is "Name or service not found".

The reason for this is the private hosted zone which we created is currently associated with the default VPC.

And this instance is not in the default VPC.

To enable this instance to resolve records inside this private hosted zone, we need to associate it with the "Animals for Life" VPC.

So go back to the Route 53 console.

Expand "Hoster Zone Details" and then "Edit hosted zone".

Scroll down and we're going to add another VPC.

In the region drop down, "US-East-1" and then in the "Choose VPC" box select "A4L-VPC-1".

Scroll down and save changes.

Now this might take a few seconds to take effect, but if we go back to the EC2 instance and try to run this ping again, and we still get "Name or service not found".

So what I want you to do is go ahead and pause this video, wait for 4 or 5 minutes and then resume and try this command again.

Now in my case it took about 5 minutes, but after a while I can now ping www.ilikedogsreally.com because I've now associated this private hosted zone with the VPC that this instance is running from.

Now that's everything that I wanted to cover in this demo lesson, so all that remains is for us to clean up all of the infrastructure which we've created in this demo lesson.

So if we go back to the Route 53 console and select "Health Checks", first we're going to delete the health check.

So select "A4L Health" and click on "Delete Health Check" and confirm.

Click on "Hostered Zones".

Go inside the private hosted zone that you created.

Select the www.ilikedogsreally.com record and then click on "Delete Record".

Confirm that deletion.

Go back to "Hostered Zones".

Select the entire private hosted zone and click on "Delete".

Type "Delete" and then click to confirm.

And that will delete the entire private hosted zone.

Then go inside the public hosted zone that you have.

Select the two www records that you created earlier in this lesson.

Click on "Delete Records".

Click "Delete" to confirm.

Then go to the S3 console.

Click on the bucket that you created earlier in this lesson.

Click "Empty".

Copy and paste or type "Permanently Delete" and click on "Empty".

Once that bucket is emptied click on "Exit".

With it still selected click on "Delete".

Copy and paste or type the full name into the box and click on "Delete Bucket".

Then go to the EC2 console.

Click the hamburger menu.

Scroll down.

Click "Elastic IPs".

Select the elastic IP that you associated with the EC2 instance.

Click on the actions drop down.

Disassociate and then click to disassociate.

With it still selected click on "Actions".

Release elastic IP addresses and click on "Release".

At that point all of the manually created infrastructure has been removed.

Go back to the cloud formation console.

Go to "Stacks".

Select the stack that you created at the start of this lesson using the one click deployment.

It should be called DNS and failover demo.

Select it.

Click on "Delete".

Then click on "Delete Stack" to confirm that deletion.

Once that's deleted the account will be back in the same state as it was at the start of the lesson.

At this point that's everything I wanted to cover in this demo.

I hope it's been enjoyable and it's given you some good practical experience of how to use failover routing and private hosted zones.

That will be useful both for the exam and real world usage.

At this point that's everything so go ahead and complete this video.

When you're ready I'll look forward to you joining me in the next.

Welcome to this demo lesson where you're going to get experience configuring fail-over routing as well as private hosted zones.

Now with this demo lesson you have the choice of either following along in your own environment or watching me perform the steps.

If you do wish to follow along in your own environment you will need a domain name that's registered within Route 53.

Remember that was an optional step at the start of this course so if you did register a domain of your own then you can do this demo lesson.

In my case I registered animals for life 1337.org.

If you registered a domain it will be different and so wherever you see me use animals for life.org you need to replace it with your registered domain.

If you didn't register one then you'll have to watch me perform all of these steps because you can't do this lesson without your own registered domain.

In order to get started you need to make sure that you're logged in as the I am admin user of the general AWS account which is the management account of the organization and you'll need to have the Northern Virginia region selected.

Now we're going to need to create some infrastructure in order to perform this demo lesson so attached to this lesson is a one-click deployment link and you should go ahead and click that link now.

That's going to take you to a quick create stack screen.

Everything should be pre-populated the stack name is DNS and failover demo all you'll need to do is scroll down to the bottom check this capabilities box and then click on create stack.

That's going to take a few minutes and it's going to create infrastructure that we're going to need to continue with the demo lesson so go ahead and pause the video wait for your stack to move into a create complete state and then we're good to continue.

Okay so the stacks now in a create complete state and it's created a number of resources the most important one being a public EC2 instance so we just need to test this first so if you just click in the search box and type EC2 and then right click to open that in a new tab.

Once you're there click on instances running and you should see a4l-web just select that under public IP version 4 just click on this symbol to copy the IP address into your clipboard make sure you don't click open address because that's going to try and use HTTPS which we don't want so copy this IP address into your clipboard and then open that in a new tab and you should see the animals for life super minimal homepage and if you see that it means everything's working as intended so go ahead and close down that tab.

Now we also need to give this instance an elastic IP address so that it has a static public IP version 4 address now to give it an elastic IP on the menu on the left scroll down to the bottom under network and security select elastic IPs and then we need to allocate an elastic IP make sure us - east - 1 is in this box scroll down and click on allocate once the elastic IP address is allocated to this account select it click on actions and then associate elastic IP once we're at this screen make sure instance is selected click in this search box and then select a4l-web once selected click in the private IP address box and select the private IP address of this instance and then check the box to say allow this elastic IP address to be re-associated once all that's complete click on associate and that now means that our EC2 instance has been allocated with a static IP version 4 address now we're configuring failover DNS and so the EC2 instance is going to be our primary record so we're going to assume that this is the animals for life main website and we want to configure an S3 bucket which is running as the backup in case this EC2 instance fails so the next thing we need to do is to create the S3 bucket so click in the search box type S3 and open that in a new tab and then go to the S3 console and at this point we're going to create an S3 bucket and configure it as a static website now the naming of the S3 bucket is important earlier in the course you should have registered a domain name in my case I registered animals for life 1337.org so I'm going to create a bucket with the name www.animalsforlife1337.org you need to create one which is called www.and then the domain name that you registered so I'm going to click and create bucket the bucket name is www.animalsforlife1337.org and it's going to be in the US east northern Virginia region which is US-East-1 then we're going to scroll down and we're going to need to uncheck block all public access because this bucket is going to be used to host a static website I'll need to acknowledge that I'm okay with that so I'll do that and then scroll all the way down to the bottom and then I'm going to click on create bucket then I'm going to go inside the bucket click on upload and then add files now attach to this lesson is an assets file I want you to go ahead and download that file then extract it and wave extracted it it should create a folder called R53 underscore zones underscore and underscore failover go inside that folder and there'll be two more folders one which is 01 underscore A4L website and another which is 02 underscore A4L failover we're interested in the A4L failover so go into that folder select both these files so index.html and minimal.jpeg click on open and then upload those files so we'll scroll down and click on upload once that's completed click on close then we go into enable static website hosting so click on properties and to enable this it's all the way down towards the bottom click on edit next to static website hosting and enable it make sure that host a static website is selected and then for the index document and the error document we're going to type index.html and once both of those are entered scroll down to the bottom and save changes now we've one final thing to do on this bucket we need to add a bucket policy so that this bucket is public so we need to click on permissions scroll down and then under bucket policy click on edit and this bucket currently does not have a bucket policy now also inside the assets folder that you extracted earlier in this lesson there's a file called bucket underscore policy.json this is the file so you'll need to copy the contents of that file into your clipboard and then inside this policy box paste that in and then click on the icon next to the bucket ARN to copy that into your clipboard and then we need to replace this placeholder with what you've just copied so I want you to select from just to the right of the first speech mark all the way through to before the forward slash so you should have ARN colon AWS colon S3 colon colon colon colon and then example bucket and then go ahead and paste the text from your clipboard which will overwrite that with the actual bucket ARN so it should look like this once you've got that scroll down and save the changes so now we have the failover website configured the static website running from the S3 bucket so now we need to go ahead and move to the route 53 console where we going to create a health check and configure the failover record so click in the search box type route 53 right click and open that in a new tab then click on health checks we're going to create a health check for the health check name type a4l health and it's going to be an end point health check scroll down we're going to specify the endpoint by IP address the protocols going to be HTTP and we need the IP address of the EC2 instance so if we go back to the EC2 console the EC2 instance is now using the elastic IP so if we scroll down and click on elastic IPs and copy the elastic IP into our clipboard then go back to the route 53 console and paste that in and the health check is going to be configured to health check the index.html document so in path we need to click and type index.html then we're going to expand advanced configuration and by default a health check is checking every 30 seconds so this is a standard health check we need to change this to fast because we want our health check to react as fast as possible if our primary website fails so select fast scroll down to the bottom click on next we don't want to create an alarm because we don't want to take any action if this health check fails we're just going to use it as part of our fail-over routing so go ahead and make sure no is selected and then click create health check now the health check is going to start off with an unknown status because it hasn't gathered enough information about the health of the primary website it's going to take a few minutes to move from this status to either healthy or unhealthy what we can do though is if we check this we can click on the health checkers tab and start to see the results of the globally distributed set of health check endpoints so we can see that we're already getting success HTTP status code 200 and this is telling us our primary website is already passing these individual checks and after a couple of minutes if we hit refresh we should see that the status changes from unknown to healthy so next we need to create the failover record so click on hosted zones locate the hosted zone for the domain that you registered at the start of the course and click it then click on create record now you can switch between two different modes either the quick create record mode or the wizard mode we're going to keep this demo simple so click on switch to wizard we're going to choose a failover record so select failover and click next we're going to call the record www we're going to set a TTL of one minute so click 1m and that will change the TTL seconds to 60 scroll down and we're going to define some failover records so click define failover record first we need to create the primary record so click in this first drop down and we're going to pick IP address or another value depending on record type so click that and then we need the elastic IP address so go back to the EC2 console and copy the elastic IP into your clip board and paste that into this box and then for failover record type this is the primary record so click on primary we need to associate it with a health check so click in that drop down and choose a for L health now once we do that it means that this primary record will only be returned if this health check is healthy otherwise the secondary record will be returned which we're going to define in a second under record ID just go ahead and type EC2 this needs to be unique within this set of records with the same name so going to call one EC2 and the other S3 so this one's EC2 so define that failover record and then we're going to define a new failover record so click that box again this time in this drop down we need to scroll down and we're going to select alias to an S3 website endpoint so select that choose the region and it needs to be US - East - 1 once selected you should be able to click in this box and see the S3 bucket that you just created so click on this to select that S3 bucket and we're going to set this as the secondary record so click on secondary we won't be associating this with a health check and we won't be evaluating the target health this record will only ever be used if the primary fails its health check and so we want this record to take effect whenever the health check associated with the primary fails and we're going to test that by shutting down the EC2 instance so this record should then take over so finally we need to enter S3 in the record ID and click on define failover record once we've done both of those we can go ahead and click on create records so now that we have both of those records in place the primary pointing at EC2 and the secondary at S3 if we copy down this full DNS name into our clipboard and open that in a new tab that should direct towards the animals for life.org super minimal homepage remember this is the website running on EC2 now what we need to do is to simulate a failure so go back to the EC2 console scroll to the top click on EC2 dashboard then instances running right click on this instance select stop instance and confirm that by clicking stop so now we've stopped this instance it should begin failing the health check so let's go back to the route 53 console click on health checks select this a4 health health check click on the health checkers tab and then click on refresh and over the coming seconds we should start to see some failure responses in this status column there we go we're getting connection timed out and over the next minute or so we should see that the status of the health check overall should move from healthy to unhealthy let's click on refresh it might take a minute or so for that to take effect so let's just give it a minute or so and now we can see that it's moved into an unhealthy state now this means that our failover record will detect this and then it's going to start returning the secondary record rather than the primary now DNS does have a cache remember we set the TTL value to 60 seconds so one minute but what we should find after that cache expires if we go back to this tab which we have open to the www.animalsforlife.org website and if we now hit refresh we should see that it changes to the animals for life.org super minimal failover page and this is the website that's running on s3 so the failover record has used a health check detected the failure of the EC2 instance and redirected us towards the backup s3 site so now we can go ahead and reverse that process if we go back to the EC2 console we can right click on this instance and start the instance that will take a few minutes to move from the stopped state through the pending state and then finally to running and once it's in a running state if we go back to the route 53 console and select this health check and then refresh on the health checkers initially we'll see a number of different messages if we keep hitting refresh over the next few minutes we should see this change to an okay message there we can see the first HTTP status code 200 if we keep refreshing we'll see more of those again more 200 statuses which means okay now that all of these are coming back okay let's click refresh on the health check itself it's still showing us unhealthy let's give it a few more seconds now it's reporting as healthy again if we go back to the tab that we have open to the website and click on refresh now it should change back to the original EC2 based website and it does so that means our failover record has worked in both directions it's failed over to s3 and failed back to EC2 okay so this is the end of part one of this lesson it was getting a little bit on the long side and so I wanted to add a break it's an opportunity just to take a rest or grab a coffee part 2 will be continuing immediately from the end of part one so go ahead complete the video and when you're ready join me in part 2.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This paper describes the covalent interactions of small molecule inhibitors of carbonic anhydrase IX, utilizing a pre-cursor molecule capable of undergoing beta-elimination to form the vinyl sulfone and covalent warhead.

Strengths:

The use of a novel covalent pre-cursor molecule that undergoes beta-elimination to form the vinyl sulfone in situ. Sufficient structure-activity relationships across a number of leaving groups, as well as binding moieties that impact binding and dissociation constants.

Overall, the paper is clearly written and provides sufficient data to support the hypothesis and observations. The findings and outcomes are significant for covalent drug discovery applications and could have long-term impacts on related covalent targeting approaches.

Weaknesses:

No major weaknesses were noted by this reviewer.

Reviewer #2 (Public review):

Summary:

The authors utilized a "ligand-first" targeted covalent inhibition approach to design potent inhibitors of carbonic anhydrase IX (CAIX) based on a known non-covalent primary sulfonamide scaffold. The novelty of their approach lies in their use of a protected pre(pro?)-vinylsulfone as a precursor to the common vinylsulfone covalent warhead to target a nonstandard His residue in the active site of CAIX. In addition to a biochemical assessment of their inhibitors, they showed that their compounds compete with a known probe on the surface of HeLa cells.

Strengths:

The authors use a protected warhead for what would typically be considered an "especially hot" or even "undevelopable" vinylsulfone electrophile. This would be the first report of doing so making it a novel targeted covalent inhibition approach specifically with vinylsulfones.

The authors used a number of orthogonal biochemical and biophysical methods including intact MS, 2D NMR, x-ray crystallography, and an enzymatic stopped-flow setup to confirm the covalency of their compounds and even demonstrate that this novel pre-vinylsulfone is activated in the presence of CAIX. In addition, they included a number of compelling analogs of their inhibitors as negative controls that address hypotheses specific to the mechanism of activation and inhibition.

The authors employed an assay that allows them to assess target engagement of their compounds with the target on the surface of cells and a fluorescent probe which is generally a critical tool to be used in tandem with phenotypic cellular assays.

Weaknesses:

While the authors show that the pre-vinyl moiety is shown biochemically to be transformed into the vinylsulfone, they do not show what the fate of this -SO2CH2CH2OCOR group is in a cellular context. Does the pre-vinylsulfone in fact need to be in the active site of CAIX on the surface of the cell to be activated or is the vinylsulfone revealed prior to target engagement?

I appreciate the authors acknowledging the limitations of using an assay such as thermal shift to derive an apparent binding affinity, however, it is not entirely convincing and leaves a gap in our understanding of what is happening biochemically with these inhibitors, especially given the two-step inhibitory mechanism. It is very difficult to properly understand the activity of these inhibitors without a more comprehensive evaluation of kinact and Ki parameters. This can then bring into question how selective these compounds actually are for CAIX over other carbonic anhydrases.

The authors did not provide any cellular data beyond target engagement with a previously characterized competitive fluorescent probe. It would be critical to know the cytotoxicity profile of these compounds or even how they affect the biology of interest regarding CAIX activity if the intention is to use these compounds in the future as chemical probes to assess CAIX activity in the context of tumor metastasis.

Reviewer #3 (Public review):

Summary:

Targeted covalent inhibition of therapeutically relevant proteins is an attractive approach in drug development. This manuscript now reports a series of covalent inhibitors for human carbonic anhydrase (CA) isozymes (CAI, CAII, and CAIX, CAXIII) for irreversible binding to a critical histidine amino acid in the active site pocket. To support their findings, they included co-crystal structures of CAI, CAII, and CAIX in the presence of three such inhibitors. Mass spectrometry and enzymatic recovery assays validate these findings, and the results and cellular activity data are convincing.

Strengths:

The authors designed a series of covalent inhibitors and carefully selected non-covalent counterparts to make their findings about the selectivity of covalent inhibitors for CA isozymes quite convincing. The supportive X-ray crystallography and MS data are significant strengths. Their approach of targeted binding of the covalent inhibitors to histidine in CA isozyme may have broad utility for developing covalent inhibitors.

Weaknesses:

This reviewer did not find any significant weaknesses. However, I suggest several points in the recommendation for the authors' section for authors to consider.

Recommendations for the authors:

Reviewing Editor Comments:

The reviewers have made excellent suggestions. We believe a revised version addressing those points can improve the assessment and quality of your work.

Reviewer #1 (Recommendations for the authors):

(1) The beta-elimination process is referred to as a "rearrangement" in both the text and the Figure 2 legend. Based on the proposed mechanism the authors provided, it is a simple beta-elimination and conjugate addition mechanism, and is not a rearrangement mechanism. This change should be reflected in the text and Figure 2 legend.

We have made the requested change from rearrangement to elimination reaction.

(2) From a structure-based design perspective, it is not obvious why only large cyclo-alkyl groups were used to target the lipophilic pocket, with the exception of the phenyl carbamates. Perhaps this is background literature on CAIX that describes this? It seems like this is a flexible functional moiety that could be used to impact drug properties. Why were other lipophilic and especially more aromatic or heteroaromatic moieties not studied?

The structure-affinity relationship of the lipophilic ring versus other moieties has been studied and reported previously in manuscripts: Dudutiene 2014, Zubriene 2017, Linkuviene 2018, chapter 16 by Zubriene (https://doi.org/10.1007/978-3-030-12780-0_16). The lipophilic ring served better than a flexible tail or an aromatic ring.

(3) The color-coded "correlation map" in Figure 8 is difficult to follow. Perhaps a standard SAR table with selectivity and affinity values would be easier to read and follow.

We are trying to promote “correlation maps” because in our opinion they are easier to follow than tables.

(4) Although there is a statement for this in line 254 of the SI, the compound numbering in the SI, vs. the numbering used in the manuscript is confusing. The standard format for these is to consecutively number all compounds and have identical compound numbers in both the SI and manuscript. The synthetic intermediates included in the SI can be identified by IUPAC names.

An additional numbering system had to be made because the synthesis was described in the supplementary materials. We would prefer to leave the numbering as in the current manuscript. There are quite a few intermediate compounds that we assigned intermediate numbers such as 20x in order to make it simpler to distinguish intermediate synthesis compounds from compounds that were studied for binding affinity.

(5) Ranges of isolated yields for the synthetic steps in SI schemes SI, S2, and S3 need to be included.

We have remade the SI schemes S1, S2, and S3 to include the yields of each compound.

(6) Presumably, the AcOH/H2O2 reaction forms the sulfones and not sulfoxides when heat is used. In the SI, the structures of 9x and 10x are shown to be sulfoxides and not sulfones. Initially, this is thought to be a simple structural mistake, however, this is concerning, since the HRMS data (for compound 9x) reported is for the sulfoxide (HRMS for C8H7F4NO4S2 [(M+H)+]: calc. 321.9825, found 321.9824. 482) and not the sulfone? In the synthesis scheme S1, condition "C" is used for both the sulfoxide and sulfone synthesis (i.e. 3ax to 9x vs. 12x to 13x). It appears the sulfoxide is prepared using a room temperature procedure, vs. the sulfone requiring 75 degrees centigrade heat. These two similar conditions need to be designated as different synthetic steps in the schemes with the specific conditions noted since the products formed are different.

We have made requested corrections/adjustments and added separate reaction conditions for sulfoxide synthesis in SI scheme S1.

Reviewer #2 (Recommendations for the authors):

I appreciate that it's difficult to determine parameters such as kinact or Ki of such potent inhibitors and ones that work by a two-step mechanism. I might suggest characterizing the steps separately to determine the detailed parameters. Maybe something like NMR for the for the activation step and SPR for the kinact and Ki of the unmasked vinylsulfone?

We agree that such information would be helpful. However, it requires significant effort and equipment and will be performed in a separate study.

I always advocate for at least a global proteomics analysis using a pulldown probe to get an idea of the specificity profile, especially for the so-far untried and untested pre-vinylsulfone moiety.

We fully agree that the pull-down assay is a good idea. However, this major task will be performed in a separate study.

This might be picky but wouldn't this be considered a pro-vinylsulfone rather than pre-vinylsulfone? Just as the term "prodrug" is used?

We agree that both the pre-vinylsulfone and pro-vinylsulfone are suitable names. However, in pharmacology, the prodrug is common, but in organic synthesis, the precursor is commonly used. Therefore, we prefer to keep the pre-vinylsulfone.

I would also be curious to know what species is responsible for activating the compound to the vinylsulfone. Maybe make some key point mutations of nearby basic residues?

The His64 formed the covalent bond, thus His64 was the likely activating base. Preparing a mutation could be a good path for future studies.

Reviewer #3 (Recommendations for the authors):

(1) The authors presented only a close-up view of the active site with a 2Fo-Fc map mesh in three panels of Figure 4. For readers unfamiliar with the carbonic anhydrase field, adding a complete illustration of each protein-inhibitor complex (protein in cartoon mode and ligand in stick) will be helpful. Also, an image of the 180º rotation of the close-up view presented in each panel should be added. Depicting h-bonds between critical residues (Asn62, Gln 92, etc.) with dashed lines and marking the distances will be helpful for readers.

We have prepared a requested picture for CAIX. Panels on the left show entire protein molecule view of the bound ligands to each isozyme and there are two close-up views for each structure rotated 180 degrees.

(2) Line 198 should be revised to refer to the correct complexes. 20, 21, and 23 should be 21, 20, 23.

We appreciate that the reviewer noticed this error. We corrected the mistake.

(3) Omit electron density maps around each ligand in Figure 4 should be included for compounds 20, 21, and 23, perhaps as a supplementary figure.

Detailed electron density map information is provided in the mtz files that have been submitted to the PDB. We think the omit maps are not necessary in the supplementary materials.

(4) The cyclooctyl group is stabilized by hydrophobic active site residues, L131, A135, L141, and L198. However, only L131 is shown in Figure 4. All residues that stabilize the ligands should be shown.

For clarity purposes of the figure, we have omitted some of the residues that make contact with the ligand molecule. We think that the structure provided to the PDB could be analyzed in detail to see all contacts between the ligand and protein molecule.

(5) The supplementary table S1 lacks the crystallographic data on the CAIX-23 complex.

We have added a new version of the supplementary materials that contains the crystallographic data on the CAIX-23 complex.

(6) A minor peak (30213 Da) with a 638 Dalton shift compared to the unmodified enzyme is for Figure 5A, not Figure 5B, as mentioned in line 235. This sentence in line 235 should be corrected.

We corrected this mistake.

(7) As the authors stated in the text, a minor peak (30213 Da) represents a potential second binding site. Can they revisit their electron density maps and show any residual density if it is present around a second histidine residue? The MS data in Figure S17C indicates the presence of additional sites for compound 12. Thus, additional electron density around the secondary and tertiary sites is possible.

CAII contains His3 and His4 that are at the N-end of the protein and not visible in the crystal structure. The NMR data indicate that the additional modification may occur at one of these His residues.

(8) MS data were presented for compounds 12 and 22 in Figure 5A, B, but the co-crystal structures were generated with compounds 21, 20, and 23. Why was no MS data included for compounds 20, 21, and 23? Would these compounds show the presence of a secondary binding site? Can authors include the MS data?

In the main body of the manuscript in Figure 5A we only present MS data on CAXIII with compound 12. It is only an example that confirms covalent interaction. In the supplementary we have MS data for compound 12 with all carbonic anhydrase isozymes and compound 20 with almost all (except CAVI) CA isozymes. There are also MS data provided with numerous compounds (3, 9, 13, and other) and CA isozymes that serve as a control or confirmation of covalent bond formation.

(9) The coordination between the zinc ion and NH of the ligand is mentioned in the enzyme schematic in Figure 3. Can the distances and coordination with Zinc be illustrated in ligand-bound structures in Figure 4?

We considered and decided that picture which shows the numerous distances between ligand atoms and protein residues would be difficult to follow. The structures provided to the PDB could be analyzed for every aspect of the complex structure.

(10) A key difference between covalent (compound 12) and its non-covalent counterpart, compound 5, is the two oxygens attached to sulfur in compound 12. Do protein side chains or water interact with these oxygens? Are these oxygen atoms exposed to solvent? Can authors show the interactions or clarify if there is no interaction?

The two oxygens in the ligand molecule serve several purposes. First, they pull out electrons and diminish the pKa of the sulfonamide, thus making interaction stronger. Second, the oxygen atoms may make contacts, hydrogen bonds with the protein molecule and may also be important for covalent bond formation. Exact energy contributions cannot be determined from the structure directly. Thus, we decided to not yet explore and delve into this area.

(11) Fix the font size of the text in lines 355-356.

The font has been corrected.

Reviewer #2 (Public review):

The manuscript "Spatial frequency adaptation modulates population receptive field sizes" is a heroic attempt to untangle a number of visual phenomena related to spatial frequency using a combination of psychophysical experiments and functional MRI. While the paper clearly offers an interesting and clever set of measurements supporting the authors' hypothesis, my enthusiasm for its findings is somewhat dampened by the small number of subjects, high noise, and lack of transparency in the report. Despite several of the methods being somewhat heuristically and/or difficult to understand, the authors do not appear to have released the data or source code nor to have committed to doing so, and the particular figures in the paper and supplements give a view of the data that I am not confident is a complete one. If either data or source code for the analyses and figures were provided, this concern could be largely mitigated, but the explanation of the methods is not sufficient for me to be anywhere near confident that an expert could reproduce these results, even starting from the authors' data files.

Major Concerns:

I feel that the authors did a nice job with the writing overall and that their explanation of the topic of spatial frequency (SF) preferences and pRFs in the Introduction was quite nice. One relatively small critique is that there is not enough explanation as to how SF adaptation would lead to changes in pRF size theoretically. In a population RF, my assumption is that neurons with both small and large RFs are approximately uniformly distributed around the center of the population. (This distribution is obviously not uniform globally, but at least locally, within a population like a voxel, we wouldn't expect the small RFs to be on average nearer the voxel's center than the voxel's edges.) Why then would adaptation to a low SF (which the authors hypothesize results in higher relative responses from the neurons with smaller RFs) lead to a smaller pRF? The pRF size will not be a function of the mean of the neural RF sizes in the population (at least not the neural RF sizes alone). A signal driven by smaller RFs is not the same as a signal driven by RFs closer to the center of the population, which would more clearly result in a reduction of pRF size. The illustration in Figure 1A implies that this is because there won't be as many small RFs close to the edge of the population, but there is clearly space in the illustration for more small RFs further from the population center that the authors did not draw. On the other hand, if the point of the illustration is that some neurons will have large RFs that fall outside of the population center, then this ignores the fact that such RFs will have low responses when the stimulus partially overlaps them. This is not at all to say that I think the authors are wrong (I don't) - just that I think the text of the manuscript presents a bit of visual intuition in place of a clear model for one of the central motivations of the paper.

The fMRI methods are clear enough to follow, but I find it frustrating that throughout the paper, the authors report only normalized R2 values. The fMRI stimulus is a very interesting one, and it is thus interesting to know how well pRF models capture it. This is entirely invisible due to the normalization. This normalization choice likely leads to additional confusion, such as why it appears that the R2 in V1 is nearly 0 while the confidence in areas like V3A is nearly 1 (Figure S2). I deduced from the identical underlying curvature maps in Figures 4 and S2 that the subject in Figure 4 is in fact Participant 002 of Figure S2, and, assuming this deduction is correct, I'm wondering why the only high R2 in that participant's V1 (per Figure S2) seems to correspond to what looks like noise and/or signal dropout to me in Figure 4. If anything, the most surprising finding of this whole fMRI experiment is that SF adaptation seems to result in a very poor fit of the pRF model in V1 but a good fit elsewhere; this observation is the complete opposite of my expectations for a typical pRF stimulus (which, in fairness, this manuscript's stimulus is not). Given how surprising this is, it should be explained/discussed. It would be very helpful if the authors showed a map of average R2 on the fsaverage surface somewhere along with a map of average normalized R2 (or maps of each individual subject).

On page 11, the authors assert that "Figure 4c clearly shows a difference between the two conditions, which is evident in all regions." To be honest, I did not find this to be clear or evident in any of the highlighted regions in that figure, though close inspection leads me to believe it could be true. This is a very central point, though, and an unclear figure of one subject is not enough to support it. The plots in Figure 5 are better, but there are many details missing. What thresholding was used? Could the results in V1 be due to the apparently small number of data points that survive thresholding (per Figure S2)? I would very much like to see a kernel density plot of the high-adapted (x-axis) versus low-adapted (y-axis) pRF sizes for each visual area. This seems like the most natural way to evaluate the central hypothesis, but it's notably missing.

Regarding Figure 4, I was curious why the authors didn't provide a plot of the difference between the PRF size maps for the high-adapted and low-adapted conditions in order to highlight these apparent differences for readers. So I cut the image in half (top from bottom), aligned the top and bottom halves of the figure, and examined their subtraction. (This was easy to do because the boundary lines on the figure disappear in the difference figure when they are aligned correctly.) While this is hardly a scientific analysis (the difference in pixel colors is not the difference in the data) what I noticed was surprising: There are differences in the top and bottom PRF size maps, but they appear to correlate spatially with two things: (1) blobs in the PRF size maps that appear to be noise and (2) shifts in the eccentricity maps between conditions. In fact, I suspect that the difference in PRF size across voxels correlates very strongly with the difference in eccentricity across voxels. Could the results of this paper in fact be due not to shifts in PRF size but shifts in eccentricity? Without a better analysis of the changes in eccentricity and a more thorough discussion of how the data were thresholded and compared, this is hard to say.

While I don't consider myself an expert on psychophysics methods, I found the sections on both psychophysical experiments easy to follow and the figures easy to understand. The one major exception to this is the last paragraph of section 4.1.2, which I am having trouble following. I do not think I could reproduce this particular analysis based on the text, and I'm having a hard time imagining what kind of data would result in a particular PSE. This needs to be clearer, ideally by providing the data and analysis code.

Overall, I think the paper has good bones and provides interesting and possibly important data for the field to consider. However, I'm not convinced that this study will replicate in larger datasets - in part because it is a small study that appears to contain substantially noisy data but also because the methods are not clear enough. If the authors can rewrite this paper to include clearer depictions of the data, such as low- and high-adapted pRF size maps for each subject, per visual-area 2D kernel density estimates of low- versus high-adapted pRF sizes for each voxel/vertex, clear R2 and normalized-R2 maps, this could be much more convincing.

I know this is the introduction/prospectus, but can it be accessible from the keywords in the bar at the top? It's buried right now and is too damn good to be buried. I'm not sure what the keyword would be--- so many possibilities though. Maybe it's just called "keywords" since that part here is dope too.

Same as above

Not sure why but this font is looking thicker/darker than in the mockup

Personally, social media has impacted me in a way that it makes me feel the need to detox from it every now nd then, as it's an unhealthy feeling to just be doom scrolling on it.

This is a very accurate comment, relating to how all young people acting almost in hindsight - less focused on what they want to do or say now and more focused on what their future selves will be able to reflect upon. I think that's why gen z has such a fixation around nostalgia in the way we always pull from the past when it comes to fashion, trends and jokes, etc. When you have that disconnect from your actions and yourself, it's much harder to feel human and so we feel like we have to mimic what we've seen before.

this is "all swords" and makes no sense in this context; all swords do not discriminate "who they cut down" ... it's pretty clear the change is moronic; and that this original legend was of a sword that was deisgned to "discriminate and specifically ...

oh its the second one

They view humans as superior over nature.

Change in just a few generations. It's no longer a stable climate.

The role of communication in group decision-making is essential, impacting both the quality and process of group decisions.Some key points are that communication is seen as a medium/channel through which information flows.This enables members to identify and correct mistakes, and influence one another’s opinions. While acknowledging traditional ideas, Hirokawa sees communication as more than a conduit; it’s instrumental in shaping reality within a group. His perspective claims that communication does not just transfer information but helps create a social reality where decisions take shape. overall, for group communication to enhance decision quality, members should focus on meaningful discussion, minimize distractions, and engage actively to co-construct ideas, rather than merely exchange information.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

With socioeconomic development, more and more people are obese which is an important reason for sub-fertility and infertility. Maternal obesity reduces oocyte quality which may be a reason for the high risk of metabolic diseases for offspring in adulthood. Yet the underlying mechanisms are not well elucidated. Here the authors examined the effects of maternal obesity on oocyte methylation. Hyper-methylation in oocytes was reported by the authors, and the altered methylation in oocytes may be partially transmitted to F2. The authors further explored the association between the metabolome of serum and the altered methylation in oocytes. The authors identified decreased melatonin. Melatonin is involved in regulating the hyper-methylation of high-fat diet (HFD) oocytes, via increasing the expression of DNMTs which is mediated by the cAMP/PKA/CREB pathway.

Strengths:

This study is interesting and should have significant implications for the understanding of the transgenerational inheritance of GDM in humans.

Thank you for your positive comments to our manuscript.

Weaknesses:

The link between altered DNA methylation and offspring metabolic disorders is not well elucidated; how the altered DNA methylation in oocytes escapes reprogramming in transgenerational inheritance is also unclear.

Thanks. These are very good questions. There is a long way to completely elucidate the relationship between methylation and offspring metabolic disorders, and the underlying mechanisms of obtained methylation escaping the reprogramming during development. We would like to explore these in the future.

Reviewer #2 (Public Review):

This manuscript offers significant insights into the impact of maternal obesity on oocyte methylation and its transgenerational effects. The study employs comprehensive methodologies, including transgenerational breeding experiments, whole genome bisulfite sequencing, and metabolomics analysis, to explore how high-fat diet (HFD)-induced obesity alters genomic methylation in oocytes and how these changes are inherited by subsequent generations. The findings suggest that maternal obesity induces hyper-methylation in oocytes, which is partly transmitted to F1 and F2 oocytes and livers, potentially contributing to metabolic disorders in offspring. Notably, the study identifies melatonin as a key regulator of this hyper-methylation process, mediated through the cAMP/PKA/CREB pathway.

Strengths:

The study employs comprehensive methodologies, including transgenerational breeding experiments, whole genome bisulfite sequencing, and metabolomics analysis, and provides convincing data.

Thank you for your positive comments to our manuscript.

Weaknesses:

The description in the results section is somewhat verbose. This section (lines 126~227) utilized transgenerational breeding experiments and methylation analysis to demonstrate that maternal obesity-induced alterations in oocyte methylation (including hyper-DMRs and hypo-DMRs) can be partially transmitted to F1 and F2 oocytes and livers. The authors should consider condensing and revising this section for clarity and brevity.

Thanks for your suggestions. We have re-written this parts in the revised manuscript.

There is a contradiction with Reference 3, but the discrepancy is not discussed. In this study, the authors observed an increase in global methylation in oocytes from HFD mice, whereas Reference 3 indicates Stella insufficiency in oocytes from HFD mice. This Stella insufficiency should lead to decreased methylation (Reference 33). There should be a discussion of how this discrepancy can be reconciled with the authors' findings.

Thanks for your suggestions. As reported by Reference 33, STELLA prevents hypermethylation in oocytes by sequestering UHRF1 from the nuclei which recruits DNMT1 into nuclei. Han et al. reported that obesity induced by high-fat diet reduces STELLA level in oocytes. These indicate that STELLA insufficiency might induce hypermethylation in oocytes, although significant hypermethylation in obese oocytes is not reported by Han et al. using immunofluorescence. This contradiction may be caused by the limited sample sizes (n=14) used by Han et al. We have added a brief discussion in the revised manuscript.

Reviewer #3 (Public Review):

Summary:

Maternal obesity is a health problem for both pregnant women and their offspring. Previous works including work from this group have shown significant DNA methylation changes for offspring of obese pregnancies in mice. In this manuscript, Chao et al digested the potential mechanisms behind the DNA methylation changes. The major observations of the work include transgenerational DNA methylation changes in offspring of maternal obesity, and metabolites such as methionine and melatonin correlated with the above epigenetic changes. Exogenous melatonin treatment could reverse the effects of obesity. The authors further hypothesized that the linkage may be mediated by the cAMP/PKA/CREB pathway to regulate the expression of DNMTs.

Strengths:

The transgenerational change of DNA methylation following HFD is of great interest for future research to follow. The metabolic treatment that could change the DNA methylation in oocytes is also interesting and has potential relevance to future clinical practice.

Thank you for your positive comments to our manuscript.

Weaknesses:

The HFD oocytes have more 5mC signal based on staining and sequencing (Fig 1A-1F). However, the authors also identified almost equal numbers of hyper- and hypo-DMRs, which raises questions regarding where these hypo-DMRs were located and how to interpret their behaviors and functions. These questions are also critical to address in the following mechanistic dissections as the metabolic treatments may also induce bi-directional changes of DNA methylation. The authors should carefully assess these conflicts to make the conclusions solid.

Thanks for the helpful comments and suggestions. As presented in Fig. 1F, there is an increase of methylation level in promoter and exon regions and there is a decrease in intron, utr3 and repeat regions. According to the suggestions, we further analyzed the distribution of DMRs, and found that hypo-DMRs were mainly distributed at utr3, intron, repeat, and tes regions compared with hyper-DMRs (Fig. S3). These suggest that the distribution of DMRs in genome is not random.

The transgenerational epigenetic modifications are controversial. Even for F0 offspring under maternal obesity, there were different observations compared to this work (Hou, YJ., et al. Sci Rep, 2016). The authors should discuss the inconsistencies with previous works.

Thanks for the suggestions. There are contradictions on the whole genome DNA methylation of oocytes in obese mice. Hou YJ et al. in 2016 reported that obesity reduces the whole genome DNA methylation of NSN GV oocytes using immunofluorescence. In 2018, Han LS et al. reported that the whole genome 5mC of oocytes is not significantly influenced by obesity using immunofluorescence, but they find the Stella level is reduced in oocytes by obesity. Stella locates in the cytoplasm and nuclei of oocytes and sequesters Uhrf1 from the nuclei. Stella knockout in oocytes results in about twofold increase of global methylation in MII oocytes via recruiting more DNMT1 into nuclei. These suggest that the global methylation of oocytes in obese mice should be increased, but the similar methylation in oocytes between obese and non-obese mice is reported by Han LS et al. Thus, the contradiction may be induced by the different sample size in our manuscript and previous studies, and Hou YJ and colleagues just examined the methylation of NSN GV oocytes. As present in Stella+/- oocytes, the global methylation of oocytes is normal, which suggest that the insufficiency of Stella may be not the main reason for the increased methylation of oocytes in obese mice. We have added a brief discussion in the revised manuscript.

In addition to the above inconsistencies, the DNA methylation analysis in this work was not carefully evaluated. Several previous works were evaluating the DNA methylation in mice oocytes, which showed global methylation levels of around 50% (Shirane K, et al. PLoS Genet, 2013; Wang L., et al, Cell, 2014). In Figure 1E, the overall methylation level is about 23% in control, which is significantly different from previous works. The authors should provide more details regarding the WGBS procedure, including but not limited to sequencing coverage, bisulfite conversion rate, etc.

Thanks for the good questions. Smallwood et al. reported the the CG methylation of MII oocyte is about 33.1% (Smallwood et al. Nature Methods, 2014) using single-cell genome-wide bisulfite sequencing. Shirane K et al. reported that the average methylation level of GV oocytes is 37.9%. Kobayashi H et al. Reported that the CG methylation in GV oocytes is about 40% (Kobayashi H et al. Plos Genet. 2012). CG methylation in fully grown oocytes is about 38.7% (Maenohara S et al. Plos Genet. 2017). The variation of methylation in oocytes is associated with sequencing methods, sequencing depth, and mapping rates. In the present study, whole genome bisulfite sequencing (WGBS) for small sample and methylation analysis were performed by NovoGene. The reads are 31613641 to 37359643, unique mapping rate is ≥32.88%,  conversation rate is > 99.44%, and sequencing depth is 2.45 to 2.75. Relative information is presented in Table S1. The sequencing depth might be a reason for the inconsistence. But we further confirmed our sequencing results using bisulfite sequencing (BS), and the result is similar between BS and WGBS results. These findings suggest that our results are reliable.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) Since the results show that melatonin may play a role in hyper-methylation, the authors need to give some basic information in the Introduction section.

Thanks. We added more information in the section of Introduction.

(2) There are many differential metabolites identified. Besides melatonin, other differential metabolites are involved in the altered methylation in oocytes

These is a good question. We firstly filtered the differential metabolites which may be involved in methylation, and then further filtered these metabolites according to the relative DNA methylation pathways and published papers. After that, we confirmed the concentrations of relative metabolites in the serum using ELISA. Certainly, we can not completely exclude all the metabolites which might involved in regulating DNA methylation.

(3) The altered methylation would be found in the F1 tissues. Did the authors examine the other parts besides the liver?

Thank you. In the present study, we didn’t examined the DNA methylation in the other tissues besides the liver. We agree that the altered methylation should be observed in the other tissues.

(4) Did the authors try or guess how many generations the maternal obesity-induced genomic methylation alterations can be transmitted?

Thanks. This is a good question. Takahashi Y and colleagues reported that obtained DNA methylation at CpG island can be transmitted across multiple generations using DNA methylation-edited mouse (Takahashi Y et al. 2023, cell). Similar inheritance is also reported by other studies using different models.

(5) The F2 is indirectly affected by maternal obesity, so the evidence is not enough to prove the transgenerational inheritance of the altered methylation.

Thanks. We find the altered DNA methylation in F2 tissue and oocytes is similar to that in F1 oocytes. These suggest the altered DNA methylation in F2 oocytes should be at least partly transmitted to F3. Previous paper (Takahashi Y et al. 2023, cell) confirms that obtain DNA methylation in CpG island can be transmitted across several generations through paternal and maternal germ lines. Certainly, it’s better if it is examined in F3 tissues.

Reviewer #2 (Recommendations For The Authors):

(1) Figure Font Size: The font sizes in the figures are quite inconsistent. Please try to uniform the font size of similar types of text.

Thanks for your suggestions. We re-edited the relative figures in the revised manuscript.

(2) Figure Clarity: Ensure that all critical information in the figures is clearly visible, such as in Figure 3C.

Thank you. We revised this figure.

(3) Figure 1B, C: The position of the asterisks ("**") is not centered in the corresponding columns, and the font size is too small. Please correct this and address similar issues in other figures.

Thank you for your suggestions. We re-edited these in the revised figures.

(4) Line 126: The current expression is confusing. It may be revised to: "Both the oocyte quality and the uterine environment can contribute to adult diseases, which may be mediated by epigenetic modifications."

Thanks. We revised this sentence in the revised manuscript.

(5) Missing Panel in Figure 3: Figure 3 is missing panel 3N.

Thank you so much. We corrected it in the revised manuscript.

(6) Figure Panel Order: Please adjust the order of the panels in the figures to follow a logical reading sequence.

Thank you. We changed the orders in the revised manuscript.

(7) Line 493: Correct "inthe" to "in the".

Thank you. We revised it.

(8) Lines 102-106: Polish the wording and expression, an example as follows: "We analyzed the differentially methylated regions (DMRs) in oocytes from both HFD and CD groups and identified 4,340 DMRs. These DMRs were defined by the criteria: number of CG sites {greater than or equal to} 4 and absolute methylation difference {greater than or equal to} 0.2. Among these, 2,013 were hyper-DMRs (46.38%) and 2,327 were hypo-DMRs (53.62%) (Fig. 1G). These DMRs were distributed across all chromosomes (Fig. 1H). "

Thank you! We re-wrote these parts in the revised manuscript.

Reviewer #3 (Recommendations For The Authors):

The sample numbers should be annotated in the figure legend for all the bar plots using Image J. The lines in Figures 2B and 2C were without error bars. How many mice were used for these plots?

Thanks for your suggestions. We added the sample size in the revised manuscript. We made a mistake when we prepared the pictures for figure 2B and figure 2C, which resulted in missing the error bars. We have corrected these pictures. Thanks again!

The authors should revise the panel arrangement of the figures (Figure 2, Figure 5, etc) to make them more clear and readable.

Thank you! We have revised these in the revised manuscript.

The writing should be improved since there were multiple typos and unclear expressions. AI tools like Grammarly or ChatGPT may help.

Thank you! We have re-edited the language in the revised manuscript using AI tools.

Please recheck the immunofluorescence images for clear interpretability. For example, in Figure 5F (H89 treated), the GV is all the way at the edge of the oocyte, and the oocyte in the DIC image appears like it is partially lysed. The DIC images and the DAPI images are not clear enough.

Thanks for your suggestions. We have re-edited these pictures in the revised manuscript.

Another concern is that the Methods describes the immunofluorescence preparation for 5mC and 5hmC staining as a simple fixation in 4% paraformaldehyde followed by permeabilization with .5% TritonX-100, but there is no antigen exposure step described, a step that is normally required for visualizing these DNA modifications (e.g., 4N HCl).

Thanks. Sorry for that we didn’t describe the methods clearly. We have added more information about the methods in the revised manuscript.

The metabolomic analysis revealed a highly significant increase in dibutylphthalate, genistein, and daidzein in the control mice. The presence of these exogenous metabolites suggests that the diets differed in many aspects, not just fat content, so it would be very difficult to interpret the results as related to a high-fat diet alone. Both daidzein and genistein are phytoestrogens and dibutylphthalate is a plasticizer, suggesting differences in the diet and/or in the materials used to collect the samples for analysis from the mice. The Methods define the high-fat diet adequately, as the formulation can be found online using the catalog number. However, the control diet is just listed as "normal diet", so one has no idea what is in it

Thank you for your good questions. The daidzein and genistein may be from the diets and the dibutylthalate may be from the materials used to collect samples. If so, these should be similar between groups. Thus, we added the formulation of normal diet in the revised manuscript. The raw materials of normal diet include corn, bean pulp, fish meal, flour, yeast powder, plant oil, salt, vitamins, and mineral elements. According to the suggestions, we re-checked the data about these metabolites, and found that the abundance of these metabolites was low. And the result of these metabolites was at a low confidence level because the iron of these metabolites was only mapped to ChemSpider(HMDB,KEGG,LIPID MAPS). To further confirm these results, we examined these metabolites in serum using ELISA, and results revealed that the concentrations of genistein and dibutylthalate were similar between groups. These results suggest that these metabolites may be not involved in the altered methylation of oocytes induced by obesity.

Welcome back.

This is part two of this lesson.

We're going to continue immediately from the end of part one.

So let's get started.

So let's go back to the instance now.

Just press enter a few times to make sure it hasn't timed out.

That's good.

Now there's a small bug fix that we need to do before we move on.

The CloudWatch agent expects a piece of system software to be installed called CollectD.

And on Amazon Linux that is not installed.

So we need to do two things.

The first is to create a directory that the agent expects to exist.

So run that command.

And the second is to create a database file that the agent also expects to exist.

And we can do that by running this command.

Now at this point we're ready to move on to the final step.

So we've installed the agent and we've run the configuration wizard to generate the agent configuration.

And we're now safely stored inside the parameter store.

The final step is to start up the CloudWatch agent and provide it with the configuration that's stored inside the parameter store.

And by doing that the agent can access the configuration.

It can download it.

It can configure itself as per that configuration.

And then because we've got an attached instance role that has the permissions required, it can also inject all of the logging data for the web server and the system into CloudWatch logs.

So the final step is to run this command.

So this essentially runs the Amazon hyphen CloudWatch hyphen agent hyphen CTL.

And it specifies a command line option to fetch the configuration.

And it uses hyphen C and specifies SSM colon and then the parameter store parameter name.

Essentially what this command does is to start up the agent, pull the config from the parameter store, make sure the agent is running and then it will start capturing that logging data and start injecting it into CloudWatch logs.

So at this point if it's functioning correctly, what you should be able to do is go back to the AWS console, go to services, type CloudWatch and then select CloudWatch to move to the CloudWatch console.

Then if we go to log groups, now you might see a lot of log groups here.

That's fine.

Every time you apply the animals for life VPC templates, it's actually using a Lambda function which we haven't covered yet to apply an IP version six workaround, which I'll explain later in the course when we cover Lambda.

What you should find though is if you just scroll down all the way to the bottom, you should see either one, two or three of the log groups that we've created.

In this example on screen now, you can see that I have /var/log/httbd/error_log.

Now these logs will start to appear when they start getting new entries and those entries are sent into CloudWatch.

So right now you can see that I only have the error log.

Now if you don't see access underscore log, what you can do is go back to the EC2 consoles, select the WordPress instance that you've created using the one click deployment and then copy the public IP version four address into your clipboard.

Don't use this link, just copy the IP address and then open that in a new tab.

Now by doing that, it will generate some activity within the Apache web server and that will put some log items into the access log and that will mean that that logging information will then be injected into CloudWatch logs using the CloudWatch agent.

So if we move back to CloudWatch logs and then refresh, scroll down to the bottom.

Now we can see the access underscore log file.

Open the log stream for the EC2 instance.

This log file details any accesses to the web server on the EC2 instance.

You won't have a lot of entries in this.

Likewise, if you go back to log groups and look for the error log, that will detail any errors, any accesses which weren't successfully served.

So if you try to access a web page which doesn't exist, if there's a server error or any module errors, these will show inside this log group.

Now also, because we're using the CloudWatch agent, we also have access to some metrics inside the EC2 instance that we otherwise would not have had.

If we click on metrics and just drag this up slightly so we can see it, you'll see the AWS namespaces.

So these are namespaces with metrics inside that you would have had access to before, but there'll also be the CWAgent namespace and inside here, just maybe select the image ID, instance ID, instance type name.

Inside there, you'll see all of the metrics that you now have access to because you have the CloudWatch agent installed on this EC2 instance.

So these are detailed operating system level metrics such as disk, IO, read and write, and you would have not had access to these before installing the agent.

If we select another one, image ID, instance ID, instance type CPU, we'll be able to see the CPU cores that are on this instance together with the IO weight and the user values.

Again, these are things that you would not have had access to at this level of detail without the CloudWatch agent being installed.

Now I do encourage you to explore all of the different metrics that you now have access to as well as to how the log groups and log streams look with this agent installed.

But this is the end of what I had planned for this demo lesson.

So as always, we want to clear up all of the infrastructure that we've created within this demo lesson.

So to do that, I want you to move back to the EC2 console, right click on this instance, go down to security, select modify IAM role, and then remove the CloudWatch role from this instance.

You'll need to confirm that by following the instructions.

So to detach that role, then click on services and move back to IAM, click on roles.

And I want you to remove the CloudWatch role that you created earlier in this demo.

So select it and then click delete role.

You'll need to confirm that deletion.

What we're not going to do is delete the parameter value that we've created.

So if we go to services and then move back to systems manager, go to parameter store, because this is a standard parameter.

This won't incur any charges.

And we're going to be using this later on in future lessons of this course and other courses.

So this is a standard configuration for the CloudWatch agent, which we'll be using elsewhere in the course.

So we're going to leave this in place.

The last piece of cleanup that you'll need to do is to go back to the CloudFormation console.

You should have the single CW agent stack in place that you created at the start of the demo using the one click deployment.

Go ahead and select the stack, click on delete, and then confirm that deletion.

And once that's completed, all of the infrastructure you've used in this demo will be removed and the account will be back in the same state as it was at the start of this demo.

Now that's everything that I wanted you to do in this demo.

I just wanted to give you a brief overview of how to manually install the CloudWatch agent within an EC2 instance.

Now there are other ways to perform this installation.

You can use systems manager or bake it into AMIs or you can bootstrap it in using the process that you've seen earlier in the course.

We're going to be using the CloudWatch agent during future demos of the course to get access to this rich metric and logging information.

So most of the demos which follow in the course will include the CloudWatch agent configuration.

At this point though, that is everything I wanted you to do in this demo.

Go ahead, complete this video, and when you're ready, I'll look forward to you joining me in the next. in the next.

Welcome back and welcome to this demo where together we'll be installing the CloudWatch agent to capture and inject logging data for three different log files into CloudWatch logs as well as giving us access to some metrics inside the OS that we wouldn't have otherwise had visibility of.

So it's going to be a really good demonstration to show you the power of CloudWatch and CloudWatch logs when combined with the CloudWatch agent.

Now in order to do this demo you're going to need to deploy some infrastructure.

To do so just make sure that you're logged in to the general AWS account, so the management account of the organization and as always make sure you've got the Northern Virginia region selected.

Now attached to this lesson is a one-click deployment URL which will deploy the infrastructure that you'll be using during this demo.

So go ahead and click on that link.

This will take you to a quick create stack screen.

The stack name should be pre-populated with CW agent.

You just need to scroll all the way down to the bottom, acknowledge the capabilities and click on create stack.

Also attached to this lesson is a lesson commands document which will contain all of the commands you'll be using during this demo lesson.

So go ahead and open that in a new tab.

Now you're going to need to let this cloud formation stack move into a create complete state before you continue the demo.

So go ahead and pause the video, wait for the status to change to create complete and then you're good to continue.

Okay so now this stack is in a create complete state then we good to continue the demo.

Now during this demo lesson you're going to be installing the cloud watch agent on an EC2 instance and this EC2 instance has been provisioned by this one-click deployment.

So the first thing that we need to do is to move across to the EC2 console and connect to this instance.

Once you're at the EC2 console click on instances running.

You should see one single EC2 instance called A4L WordPress.

Just go ahead and select this, right-click on it, select connect.

We're going to connect into this instance using EC2 instance connect so make sure that's selected.

Make sure also that the username is set to EC2-user and then connect into the instance.

Now if everything's working as it should be you should see the animals for life custom login banner when you log into the instance.

In my case I do see that and that means everything's working as expected.

So this demonstration is going to have a number of steps.

First we need to download the cloud watch agent then we need to install the agent then we need to generate the configuration file that this install of the agent as well as any future installs of the agent could use and then we need to get the cloud watch agent to read this config and start capturing and injecting those logs into cloud watch logs.

So step one is to download and install the agent and the command to do this is inside the lesson commands document which is attached to this lesson and that will install the agent but crucially it won't start it.

What we need to do before we can start the agent is to generate the config file that we'll use to configure this and any future agents but because we also want to store that config file inside the parameter store and because we also want to give this instance permissions to interact with cloud watch logs before we continue we need to attach an IAM role to this instance an EC2 instance role so that's the next step.

So we need to move back to the EC2 console click on services and then open the IAM console because we'll be creating an IAM role to attach to this instance you'll need to go to roles create role it'll be an AWS service role using EC2 so select EC2 and then click on next we'll need to attach two managed policies to this role so I've included the names of those managed policies in the lesson commands document the first is cloud watch agent server policy so make sure you type that in the filters policy and then check that box and the second is Amazon SSM full access so type that in the box select that policy and then scroll down and click on next and we'll call this role cloud watch role so enter cloud watch role and click on create role and once we've done that we can attach this role to our EC2 instance so we need to move back to the EC2 console go to instances right click on the instance go to security and then modify IAM role and then click on the drop down and select cloud watch role which is the role that you've just created then click update IAM role now that we've allocated that instance with the permissions that it needs to perform the next set of steps go ahead and connect to that instance again the tab may have timed out that you previously had open so if it doesn't respond you need to close it down and reopen it if it does respond that's fine keep the existing tab once we back in the terminal for the instance we need to start the cloud watch agent configuration wizard and the command to do that is also in the lesson commands document attached to this lesson so go ahead and paste that in and press enter and that will start off the configuration wizard for the cloud watch agent now for most of these values we can accept the defaults but we need to be careful because there are a number of them that we can't so press enter and accept the default for the operating system it should automatically detect Linux press enter it should automatically detect that it's running on an EC2 instance press enter to use the root user again that should be the default press enter for stats D press enter for the stats D port press enter for the interval press enter for the aggregation interval press enter to monitor metrics from collect D again that's the default press enter to monitor host metrics so CPU and memory press enter to monitor CPU metrics per core press enter for the additional dimensions press enter to aggregate EC2 dimensions press enter for the default resolution so 60 seconds for the default metric config that you want the default will be basic go ahead and enter 3 for advanced this captures additional operating system metrics that we might actually want so use 3 for this value press enter to indicate that we satisfied with the above config next we'll move to the log configuration part of this wizard so press enter for the default of no we don't have an existing cloud watch log agent config to import press enter which is the default for yes we do want to monitor log files you'll be asked for the log file path to monitor so the first one that we want to monitor and again these are in the lesson commands document so the first log path is forward slash var forward slash log forward slash secure press enter you'll be asked for the log group name the default is just the log name itself so secure but we're going to enter the full path I always prefer using the full path for the log group names for any system logs so going to enter var log secure again you'll be asked for the log stream name remembering the theory part of this lesson I talked about how a log stream will be named after the instance which is injecting those logs so the default choice is to do that to use the instance ID so press enter it's here where you can specify a log group retention in days we're just going to accept the default for the log group retention value the default will be yes we do want to specify additional log files so press enter the log file path for this one will be var log HTTP d access underscore log so enter that the log group name again will default to the name of the actual log we want the full path so enter the full path again press enter the log stream name the default for this is again the instance ID which is fine just press enter go ahead and accept the default for the log group retention in days press enter again we've got one more log file that we want to enter this time the log file path is var log HTTP d error underscore log again the log group name will default the name of the actual log we want to use the full path so enter the same thing again the default choice for log stream name will be again the instance ID that's fine press enter go ahead and accept the default for the log group retention in days and now we finished adding log files we won't want to log any additional files so press 2 and that will complete this logging section of this wizard it's asking us to confirm that we're happy with this configuration file and it's telling us that the configuration file is stored at forward slash opt forward slash aws forward slash amazon hyphen cloud watch hyphen agent forward slash bin and then config dot json in that folder now that's where it stores it on the local file system but we can also elect to store this json configuration inside the parameter store and I thought that since we've previously talked about the theory of the parameter store and done a little bit of interaction it would be useful for you to see exactly how it can be used in a more production like setting so the default is to store the configuration in the parameter store so we're going to allow that press enter it'll ask us for the parameter name to use and the default is Amazon cloud watch hyphen linux and that's fine so press enter it'll ask us for the region to use because parameter store is like many other services a regional service and the default region is the one where the instance is in so it automatically detects that we're in us east one which is northern Virginia so go ahead and accept that default choice it'll ask us for the credentials that it can use to send that configuration into the parameter store now these credentials will be obtained from the role that we've attached to this instance in the previous step so you can accept the default choice it'll use those credentials to store that configuration inside the parameter store and if we move back to the ec2 console and we switch back to the parameter store so just type SSM to move to systems manager which is the parent product of parameter store if we go down to the parameter store item on the menu on the left we'll be able to see this single parameter Amazon cloud watch hyphen linux and if we open that up and just scroll down we can see that the value is a JSON document with the full configuration of the cloud watch agent so we can now use this parameter to configure the agent on this ec2 instance as well as any other ec2 instances we want to deploy so if you create the cloud watch configuration once and then store it into parameter store then when you create ec2 instances at scale as you'll see how to do later in the course when we talk about auto scaling groups then you can use the parameter store to deploy this type of configuration at scale in a secure way okay so this is the end of part one of this lesson it was getting a little bit on the long side and so I wanted to add a break it's an opportunity just to take a rest or grab a coffee part 2 will be continuing immediately from the end of part one so go ahead complete the video and when you're ready join me in part 2.

Reading this highlights the subtle yet pervasive ways in which gender dynamics play out in the classroom. The fact that male students often dominate discussions, ask and answer more questions, and receive more praise for their ideas speaks volumes about the underlying gendered expectations that shape classroom interactions. It’s easy to overlook these patterns, but they’re clearly there, reinforcing a system where boys are often seen as the primary intellectual contributors.

The same as Bohmain Theory for the Indellective.

for - quote conspiracy theories - lizard people - first stage of initiation - if reality isn't as it appears, it's easy to latch onto something else - John Churchill

for - example - individual's evolutionary learning journey - new self revisiting old self and gaining new insight - universal compassion of Buddhism and the individual / collective gestalt - adjacency - the universal compassion of the bodhisattva - Deep humanity idea of the individual / collective gestalt - the Deep Humanity Common Human Denominators (CHD) as pointing to the self / other fundamental identity - Freud, Winnicott, Kline's idea of the self formed by relationship with the other, in particular the mOTHER (Deep Humanity), the Most significant OTHER

adjacency - between - the universal compassion of the bodhisattva - Deep humanity idea of the individual / collective gestalt - the Deep Humanity Common Human Denominators (CHD) as pointing to the self / other fundamental identity - Freud, Winnicott, Kline's idea of the self formed by relationship with the other, in particular the mOTHER (Deep Humanity), the Most significant OTHER - adjacency relationship - When I heard John Churchill explain the second turning, - the Mahayana approach, - I was already familiar with it from my many decades of Buddhist teaching but with - those teachings in the rear view mirror of my life and - developing an open source, non-denominational spirituality (Deep Humanity) - Hearing these old teachings again, mixed with the new ideas of the individual / collective gestalt - This becomes an example of Indyweb idea of recording our individual evolutionary learning journey and - the present self meeting the old self - When this happens, new adjacencies can often surface - In this case, due to my own situatedness in life, the universal compassion of the bodhisattva can be articulated from a Deep Humanity perspective: - The Freudian, Klinian, Winnicott and Becker perspective of the individual as being constructed out of the early childhood social interactions with the mOTHER, - a Deep Humanity re-interpretation of "mother" to "mOTHER" to mean "the Most significant OTHER" of the newly born neonate. - A deep realization that OUR OWN SELF IDENTITY WAS CONSTRUCTED out of a SOCIAL RELATIONSHIP with mOTHER demonstrates our intertwingled individual/collective and self/other - The Deep Humanity "Common Human Denominators" (CHD) are a way to deeply APPRECIATE those qualities human beings have in common with each other - Later on, Churchill talks about how the sacred is lost in western modernity - A first step in that direction is treating other humans as sacred, then after that, to treat ALL life as sacred - Using tools like the CHD help us to find fundamental similarities while divisive differences might be polarizing and driving us apart - A universal compassion is only possible if we vividly see how we are constructed of the other - Another way to say this is that we see others not from an individual level, but from a species level

Welcome back and in this demo lesson, I'm just wanting to give you some practical experience with interacting with the parameter store inside AWS.

So to do that, make sure you're logged into the IAM admin user of the management account of the organization and you'll need to have the Northern Virginia region selected.

Now there's also a lesson commands document linked to this lesson, which contain all of the commands that you'll need for this lessons demonstration.

So before we start interacting with the parameter store from the command line, we need to create some parameters.

And the way that we do that is first move to systems manager.

So the parameter store is actually a sub product of systems manager.

So move over to the systems manager console.

And once you're there, you'll need to select the parameter store from the menu on the left.

So it should be about halfway down on the left and it's under application management.

So go ahead and select parameter store.

Now once you're in parameter store, the first thing that you'll need to do to remove this default welcome screen logically is to create a parameter.

So go ahead and click on create parameter.

Now when you create a parameter, you're able to pick between standard or advanced.

Standard is the default and that meets most of the needs that most people have for the product.

And you can create up to 10,000 parameters using the standard tier.

With the advanced tier, you can create more than 10,000 parameters.

The parameter value can be longer at eight kilobytes versus the four kilobytes of standard.

And you do gain access to some additional features.

But in most cases, most parameters are fine using the default, which is the standard tier.

With the standard tier, there's no additional charge to use this up to the limit of 10,000 parameters.

The only point at which parameter store costs any extra is if you use the faster throughput options or make use of this advanced tier.

And we won't be doing that at any point throughout the course.

We'll only be using standard.

And so there won't be any extra parameter store related charges on your bill.

Now I mentioned that a parameter is essentially a parameter name and a parameter value.

And it's here where you set both of those.

There's an optional description that you can use and you can set the type of the parameter.

The options being string, string list, which is a comma separated list of individual strings and then secure string, which utilizes encryption.

So we're gonna go ahead at this point and create some parameters that we're then going to interact with from the command line.

So the first one we'll create is one that's called forward slash my-cat-app forward slash DB string.

So this is the name of a parameter and it will also establish a hierarchy.

So anytime we use forward slashers, we're establishing a hierarchy inside the parameter store.

So imagine this being a directory structure.

Imagine this being the root of the structure.

Imagine my-cat-app being the top level folder and inside there, imagine that we've got a file called DB string.

So we're going to store this, we're going to store this hierarchy and we need to set its value.

So we'll keep this for now as a string and this is going to be the database connection string for my-cat-app.

So we'll just enter the value that's in the lesson commands document.

So DB dot all the cats dot com colon 3306.

And 3306 of course is the my SQL standard port number.

At this point, we could enter an optional description.

So let's go ahead and do that.

Connection string for cat application.

So just type in a description here.

It doesn't matter really what you type and then scroll down and hit create parameter.

So that's created our first parameter, my-cat-app forward slash DB string.

Now we're going to go ahead and do the same thing but for DB user.

So click on create parameter and then just click in this name box and notice how it presents you with this hierarchy.

So now we've got two levels of this hierarchical structure.

We've got the my-cat-app at the top and then we've got the actual parameter that we created at the bottom here.

So this has already established this structure.

So let's go ahead and create a new parameter.

This time it's going to be forward slash my-cat-app forward slash DB user.

We'll not bother with the description for this one.

We'll keep it at the default of standard and it will also be a string.

And then for the value, it'll be boss cat.

So enter all that and click on create parameter.

Next let's create a parameter again.

If we click in this name this time, we've got this hierarchy that's ever expanding.

So we've got the top level at the top and then below it two additional parameters, DB string and DB user.

And we're going to create a third one at this level.

So this time it's going to be called forward slash my-cat-app forward slash DB password.

This time though, instead of type string, it's going to be a secure string so that it encrypts this parameter.

And it's going to use KMS to encrypt the parameter.

And because it's using KMS, we'll need to select the key to use to perform the cryptographic operations.

We can either select a key from the current account, so the account that we're in, or we can select another AWS account.

And in either case, we'll need to pick the key ID to use and by default, it uses the product default key for SSM.

So that's using alias forward slash AWS forward slash SSM.

And you always have the option of clicking on this dropdown and changing it if you want to benefit from the extra functionality that you get by using a customer managed KMS key.

This is an AWS managed one.

So you won't be able to configure rotation and you won't be able to set these advanced key policies.

But in most cases, you can use this default key.

So at this point, we'll leave it as the default and we'll enter our super secret password, amazing secret password, 1337, and then click create parameter.

We're not finished yet though, click on create parameter again.

And I like to be inclusive, so not everything in my course is going to be about cats.

We're going to create another parameter, my-dog-app forward slash DB string.

We'll keep standard, we'll keep the type as string and then the value for connecting to the my-dog application.

So the DB string is going to be DB if we really must have dogs.com colon 3306.

So type that in and then click on create parameter.

And then lastly, we're going to create one more parameter.

This time the name is going to be forward slash rate my lizard.

So rate hyphen my hyphen lizard forward slash DB string.

The tier is going to be standard again.

The type is going to be string.

And for the value, it will be DB.

This is pretty random.com colon 3306.

So type that in and then click on create parameter.

So now we've created a total of five parameters.

We've created the DB string, the DB user, and the DB password for the cat application.

And then the DB string for the dog application as well as the rate my lizard application.

So a total of five parameters and one of them is using encryption.

So that's the DB password for the my cat application.

So now let's switch over to the command line and interact with these parameters.

And to keep things simple, we're going to use the cloud shell.

So this is a relatively new feature made available by AWS.

And this means that we don't have to interact with AWS using our local machine.

We can do it directly from the AWS console.

So click on the cloud shell icon on the menu on the top.

This will take a few moments to provision because this is creating a dedicated environment for you to interact with AWS using the command line interface.

So you'll need to wait for this process to complete.

So go ahead and pause the video and wait until this logs you into the cloud shell environment at which point you can resume the video and we're good to continue.

It'll say preparing your terminal and then you'll see a familiar looking shell much like you would if you were connected to a Linux instance.

And now you'll be able to interact with AWS using the command line interface, using the credentials that you're currently logged in with.

Now to interact with parameter store using the command line, we start by using AWS and then a space, SSM, and then a space, and then the command that we're going to use is get-parameters.

Now by default, what we need to provide the get-parameters command with is the path to a parameter.

So in this case, if we wanted to retrieve the database connection string for the rate my lizard application, then we could provide it with this name.

So forward slash rate-my-lizard/dvstring.

And this directly maps back through to the parameter that we've just created inside the parameter store.

So this parameter.

So if you go ahead and type that and press enter, it's going to return a JSON object.

Inside that JSON object is going to be a list of parameters and then for each parameter, so everything inside these inner curly braces, we're going to see the name of the parameter that we wanted to retrieve, the type of the parameter, the value of the parameter.

In this case, db.this_is_pretty_random.com/3306, the version number of the parameter because we can have different version numbers, the last modified date, the data type, and then the unique arn of this specific parameter.

And so this is an effective way that you can store and retrieve configuration information from AWS.

Now we can also use the same structure of command to retrieve all of those other parameters that we stored within the parameter store.

So if we wanted to get the db string for the my-dog-app, then we could use this command.

And again, it would return the same data structure.

So a JSON object containing a list of parameters and each of those parameters would contain all of this information.

I'll clear the screen to keep this easy to see.

We could do the same for the my-cat-app, retrieving its database connection string.

And again, it would return the same JSON object with the parameters list.

And then for each parameter, this familiar data structure.

Now what you can also do, and I'm going to clear the screen before I run this, is instead of providing a specific path to a parameter.

So if you remember, we had almost a hierarchy that we created with these different names.

So we have the my-cat-app hierarchy and then inside there db-pastword, db-string, db-user.

We have my-dog-app and inside there db-string and then rate-my-lizard and also db-string.

So rather than having to retrieve each of these individual parameters by specifying the exact name, we can actually use get parameters by path.

So let's demonstrate exactly how that works.

So with this command, we're doing a get-parameters-by-path and we're specifying a path to a group of a number of parameters.

So in this case, my-cat-app is actually going to be the first part of the path of db-pastword, db-string and db-user.

So by creating a hierarchical structure inside the parameter store, we can retrieve multiple parameters at once.

So this time we're returning a JSON structure.

Inside this JSON structure, we have a list of parameters and then we're retrieving three different parameters, db-pastword, db-string and db-user.

Now note how db-pastword is actually a type of secure string and by default, if we don't specify anything, we return the encrypted version of this parameter.

So the ciphertext version of this parameter.

This ensures that we can interact with parameters without actually decrypting them and this offers several security advantages.

Now I've cleared the screen to make this next part easy to see because it's very important.

Because we're using KMS to encrypt parameters, the permissions to access KMS keys to perform this decryption, this is separate than the permissions to access the parameter store.

So if this user, I am admin in this case, has the necessary permissions to interact with KMS to use the keys to decrypt these parameters, then we can also ask the parameter store to perform that decryption whilst we retrieve the parameters.

The important thing to understand is the permissions to interact with the parameter store are separate than the permissions to interact with KMS.

So to perform a decryption whilst we're retrieving the parameters, we would use this command.

So it's the same command as before, aws, SSM, get-parameters-by-path, and then we're specifying the my-cat-app part of the hierarchy.

So remember, this represents these three parameters.

Now, if we ran just this part on its own, which was the command we previously ran, this would retrieve the parameters without performing decryption.

But by adding this last part, this is the part that performs the decryption on any parameter types which are encrypted.

And if you recall, one of the parameters that we created was this DB password, which is encrypted.

So if we run this command, this time it's going to retrieve the /my-cat-app/db password parameter, but it's going to decrypt it as part of that retrieval operation and return the plain text version of this parameter.

And just to reiterate, that requires both the permissions to interact with the parameter store, as well as the permissions to interact with the KMS key that we chose when creating this parameter.

Now, we're logged in as the IAM admin user, which has admin permissions, and so we do have permissions on both of those, on SSM and on KMS, so we can perform this decryption operation.

Now, you're going to be using the parameter store extensively for the rest of the course and my other courses.

It's a great way of providing configuration information to applications, both AWS and bespoke applications within the AWS platform.

It's a much better way to inject configuration information when you're automatically building applications or you need applications to retrieve their own configuration information.

It's much better to retrieve it from the parameter store than to pass it in using other methods.

So we're going to use it in various different lessons as we move throughout the course.

In this demo lesson, I just wanted to give you a brief experience of working with the product and the different types of parameters.

But at this point, let's go ahead and clear up all of the things that we've created inside this demo lesson.

So close down this tab, open to Cloud Shell.

Back at the parameter store console, just go ahead and check the box at the top to select all of these existing parameters.

If you do have any other parameters, apart from the ones that you've created within this demo lesson, then do make sure that you uncheck them.

You should be using an account dedicated for this training, so you shouldn't have any others at this point.

But if you do, make sure you uncheck them.

You should only be deleting ones for 8-mile lizard, my dog app, and my cat app.

So make sure that all of those are selected and then click on delete to delete those parameters, and you'll need to confirm that deletion process.

And at this point, that's everything that I wanted you to do in this lesson.

You've cleared up the account back to the point it was at the start of this demo lesson.

So go ahead, complete this video, and when you're ready, I'll look forward to you joining me in the next.

Welcome to this demo lesson where you're going to get the experience of working with EC2 and EC2 instance roles.

Now as you learned in the previous theory lesson, an instance role is essentially a specific type of IAM role designed so that it can be assumed by an EC2 instance.

When an instance assumes a role which happens automatically when the two of them are linked, that instance and any applications running on that instance can gain access to the temporary security credentials that that role provides.

And in this demo lesson you're going to get the experience of working through that process.

Now to get started you're going to need some infrastructure.

Make sure that you're logged in to the general AWS account, so that's the management account of the organization and as always you'll need to be within the Northern Virginia region.

Assuming you are, there's a one click deployment link which is attached to this lesson so go ahead and click that link.

That will take you to a quick create stack page.

The stack name will be pre-populated with IAM role demo and all you need to do is to scroll down to the bottom, check this capabilities box and then click on create stack.

This one click deployment will create the animals for live VPC and EC2 instance and an S3 bucket.

Now in order to continue with this demo we're going to need this stack to be in a create complete state.

So go ahead and pause the video and then when the stack moves into a create complete status then we're good to continue.

Okay so this stacks now in a create complete state and we're good to continue.

So to do so go ahead and click on the services drop down and then type EC2, locate it, right click and then open that in a new tab.

Once you're at the EC2 console click on instances running and you should be able to see that we only have the one single EC2 instance.

Now we're going to connect to this to perform all the tasks as part of this demo.

So right click on this instance, select connect, we're going to use EC2 instance connect.

Just verify that the username does say EC2-user and then click on connect.

Now the AMI that we use to launch this instance is just the standard Amazon Linux 2 AMI.

And so if we type AWS and press enter it comes with the standard install of the AWS CLI version 2.

Now it's important to understand that right now this instance has no attached instance role and it's not been configured in any way.

It's the native Amazon Linux 2 AMI that's been used to launch this instance.

And so if we attempt to interact with AWS using the command line utilities, for example by running an AWS S3 LS, the CLI tools will tell us that there are no credentials configured on this instance and will be prompted to provide long term credentials using AWS Configure.

Now this is the method that you've used to provide credentials to your own installed copy of the CLI tools running on your local machine.

So you've used AWS Configure and set up two named configuration profiles.

And the way that you provide these with authentication information is using access keys.

Now this instance has no access keys configured on it and so it has no method of interacting with AWS.

We could use AWS Configure and provide these credentials but that's not best practice for an EC2 instance.

What we're going to do instead is use an instance role.

So to do that you're going to need to move back to the AWS console.

And once you're there click on services and in the search box type IAM.

We're going to move to the IAM console so right click and open that in a new tab.

As I mentioned earlier an instance role is just a specific type of IAM role.

So we're going to go ahead and create an IAM role which our instance can assume.

So click on roles and then we're going to go ahead and click on create role.

Now the create role process presents us with a few common scenarios.

We can create a role that's used by an AWS service, another AWS account, a web identity or a role designed for SAML 2.0 Federation.

In our case we want a role which can be assumed by an AWS service specifically EC2.

So we'll select the type of trusted entity to be an AWS service then we'll click on EC2 and then we'll click on next.

Now for the permissions in this search box just go ahead and type S3 and we're looking for the Amazon S3 read only access.

So there's a managed policy that we're going to associate with this role.

So check the box next to Amazon S3 read only access and then we'll click on next.

And then under role name we're going to call this role A4L instance role.

So it's easy to distinguish from any other roles we have in the account.

So go ahead and enter that and click on create role.

Now as I mentioned in the theory lesson about instance roles when we do this from the user interface.

It's actually created a role and an instance profile of the same name and it's the instance profile that we're going to be attaching to the EC2 instance.

Now from a UI perspective both of these are the same thing.

You're not exposed to the role and the instance profile as separate entities but they do exist.

So now we're going to move back to the EC2 console and remember currently this instance has no attached instance role and we're unable to interact with AWS using this EC2 instance.

To attach an instance role using the console UI right click, go down to security and then modify IAM role.

Select that and we'll need to choose a new IAM role.

You have the option of creating one directly from this screen but we've already created the one that we want to apply.

So click in the drop down and select the role that you've just created.

In this case A4L instance role.

So select that and then click on save.

Now if we select the instance and then click on security you'll be able to confirm that it does have an IAM role attached to this instance.

So this is the instance role that this EC2 instance can now utilize.

So now we're going to interact with this instance again from the operating system.

Now if it's been a few minutes since you've last used instance connect you might find when you go back it appears to have frozen up.

If that's the case that's no problem just close down these tabs that you've got connected to that instance.

Right click on the instance again, select connect, make sure the username is EC2-user and then click on connect.

And this will reconnect you to that instance.

Now if you recall last time we were connected we attempted to run AWS S3LS and the command line tools informed us that we had no credentials configured.

Let's attempt that process again.

AWS space S3 space LS and press enter.

And now because we have the instance role associated with this EC2 instance the command line tools can use the temporary credentials that that role generates.

Now the way that this works and I'm going to demonstrate this using the curl utility these credentials are actually provided to the command line tools via the metadata.

So this is actually the metadata path that the command line tools use in order to get the security credentials.

So the temporary credentials that a role provides when it's assumed.

So if I use this command and press enter you'll see that it's actually using this role name.

So you'll see a list of any roles which are associated with this instance.

If we use the curl command again but this time on the end of security credentials we specify the name of the role that's attached to this instance and press enter.

Now we can see the credentials that command line tools are using.

So we have the access key ID the secret access key and the token and all of these have been generated by this EC2 instance assuming this role because these are temporary credentials.

They also have an expiry date.

So in my case here we can see that these credentials expire on the 7th of May 2022 at 552 47 UTC.

And that really is all I wanted to show you in this demo lesson about instance roles.

Essentially you just need to create an instance role and then attach it to an instance.

And once you do that instance is capable of assuming that role gaining access to temporary credentials and then any applications installed on that instance, including the command line utilities are capable of interacting with AWS using those credentials.

Now the process of renewing these credentials is automatic.

So as long as the application that's running on the instance periodically checks the metadata service, it will always have access to up to date and valid credentials.

The EC2 service once this expiry date closes in and once the expiry date is in the past, these credentials will be renewed and a new valid set of credentials will automatically be presented via the metadata service to any applications running on this EC2 instance.

Now just one more thing that I do want to show you before we finish up with this demo lesson.

And I have made sure that I've attached this link to the lesson.

This link shows the configuration settings and precedence that the command line utilities use in order to interact with AWS.

So whenever you use the command line interface, each of these is checked in order.

First, it looks at command line options.

Then it looks at environment variables to check whether any credentials are stored within environment variables.

Then it checks the command line interface credentials file.

So this is stored within the dot AWS folder within your home folder and then a file called credentials.

Next, it checks the CLI configuration file.

Next, it checks container credentials.

And then finally, it checks instance profile credentials.

And these are what we've just demonstrated.

Now, this does mean that if you manually configure any long term credentials for the CLI tools as part of using AWS Configure, then they will be used as a priority over an instance profile.

But you can use an instance profile and attach this to many different instances as a best practice way of providing them with access into AWS products and services.

So that's really critical to understand.

But at this point, that is everything that I wanted to cover in this demo lesson.

And all that remains is for us to tidy up the infrastructure that we've used as part of this demo.

So to tidy up this infrastructure, I want you to go back to the IAM console.

I want you to click on roles and I want you to delete the A4L instance role that you've just created.

So select it and then click on delete role.

Once you've deleted that role, go back to the EC2 console, click on instances, right click on public EC2, go to security, modify IAM role.

Now, even though you've deleted the IAM role, note how it's still listed.

That's because this is an instance profile.

This is showing the instance profile that gets created with the role, not the role itself.

So what we're going to do, and I just wanted to do this to demonstrate how this works, we're just going to select no IAM role and then click on save.

We'll need to confirm that.

So to do that, we need to type detach into this box and then confirm it by clicking detach.

That removes the instance role entirely from the instance.

And then we can finish up the tidy process by moving back to the cloud formation console.

Selecting the IAM role demo stack and then clicking on delete and confirming that deletion.

And that will put the account back in the same state as it was at the start of this demo lesson.

So this has been a very brief demo.

I just wanted to give you a little bit of experience of working with instance roles.

So that's EC2 instances combined with IAM roles in order to give an instance and any applications running on that instance, the ability to interact with AWS products and services.

And this is something that you're going to be using fairly often throughout the course, specifically when you're configuring any AWS services to interact with any other services on your behalf.

That's a common use case for using IAM roles and we'll be using instance roles extensively to allow our EC2 instances to interact with other AWS products and services.

But at this point, that is everything that I wanted to cover in this demo lesson.

So go ahead, complete the video and when you're ready, I'll look forward to you joining me in the next.

Welcome back and I've mentioned a few times now within the course that I am roles are the best practice way that AWS services can be granted permissions to other AWS services on your behalf.

Allowing a service to assume a role grants the service the permissions that that role has.

EC2 instance roles are roles that an instance can assume and anything running in that instance has the permissions that that role grants and there is some detail involved which matters so let's take a look at how this feature of EC2 works architecturally.

Instance role architecture isn't really all that complicated it starts off with an I am role and that role has a permissions policy attached to it so whoever assumes the role gets temporary credentials generated and those temporary credentials give the permissions that that permissions policy would grant.

Now an EC2 instance role allows the EC2 service to assume that role which means there's an EC2 instance itself can assume it and gain access to those credentials but we need some way of delivering those credentials into the EC2 instance so that applications running inside that instance can use the permissions that the role provides so there's an intermediate piece of architecture the instance profile and this is a wrapper around an I am role and the instance profile is the thing that allows the permissions to get inside the instance when you create an instance role in the console an instance profile is created with the same name but if you use the command line or cloud formation you need to create these two things separately when using the UI and you think you're attaching an instance role direct to an instance you're not you're attaching an instance profile of the same name it's the instance profile that's attached to an EC2 instance.

We know by now that when I am roles are assumed you're provided with temporary security credentials which expire but these credentials grant permissions based on the roles permissions policy will inside an EC2 instance these credentials are delivered via the instance metadata.

An application running inside the instance can access these credentials and use them to access AWS resources such as S3.

One of the great things about this architecture is that the credentials available inside the metadata they're always valid EC2 and the secure token service liaise with each other to ensure that the credentials are always renewed before they expire as long as your application inside the EC2 instance keeps checking the metadata it will never be in a position where it has expired credentials.

So to summarize when you use EC2 instance roles the credentials are delivered via the instance metadata specifically inside the metadata there's an IAM tree in there there's a security credentials part and then in there is the role name and if you access this you'll get access to these temporary security credentials and they're always rotated they're always valid as long as that instance role remains attached to the instance anything running in the instance will always have access to these valid credentials applications running in the instance of course need to be careful about caching these credentials and just check the metadata before the credentials expire or do it periodically.

You should always use roles where possible I'm going to keep stressing that throughout the course it's important for the exam roles are always preferable than storing long-term credentials so access keys into an EC2 instance it's never a good idea to store long-term credentials such as access keys anywhere which aren't securely stored so for example on your local machine.

In fact the AWS tooling such as the CLI tools will use instance role credentials automatically so as long as the instance role is attached to the EC2 instance any command line tools running inside that instance can automatically make use of those credentials.

So at this point that's everything I wanted to cover thanks for watching go ahead and complete this video and when you're ready join me in the next lesson.

Welcome back and in this brief demonstration you'll have the opportunity to create an EC2 instance with WordPress bootstrapped in ready and waiting to be configured.

But this time you'll be using an enhanced CloudFormation template which uses CFN init and creation policies rather than the simple user data that you used in the previous demonstration.

To get started just make sure you are logged in to the general AWS account as the I am admin user and as always make sure you've got the northern Virginia region selected.

Now attached to this lesson are two one click deployment links.

Go ahead and use the first one which is the VPC link.

Everything should be pre-populated.

All you'll need to do is scroll down to the bottom, check the acknowledgement box and click on create stack.

Once it's moved into a create complete status you can resume and we'll carry on with the demo.

I'll assume that that's now in a create complete status and now we're going to apply another CloudFormation template.

This is the template that we'll be using.

It's just an enhancement of the one that you used in the previous lesson.

This time instead of using a set of procedural instructions, so a script that are passed into the user data, this uses the CFN init system and creation policies.

So let's have a look at exactly what that means.

If I scroll down and locate the EC2 instance logical resource, then here we've got this creation policy.

This means that CloudFormation is going to create a hold point.

It's not going to allow this resource to move into a create complete status until it receives a signal.

And it's going to wait 15 minutes for this signal.

So a timeout of 15 minutes.

Now scrolling down and looking at the user data, the only things we do in a procedural way, we use the CFN init command to begin the desired state configuration.

That will either succeed or not.

And based on that we use the CFN signal command to pass that success or failure state back to the CloudFormation stack.

And that's what uses this creation policy.

So the creation policy will wait for a signal and it's this command which provides that signal, either a success signal or a failure signal.

Now what we're interested in specifically for this demo lesson is this CFN init command.

So this is the thing that pulls the desired state configuration from the metadata of this logical resource.

I'll talk all about that in a second.

But it pulls that down by being given the stack ID and it uses this substitution command.

So instead of this being passed into the instance, what's actually passed instead of this variable name, so the stack ID variable name, is the actual stack ID.

And then likewise, instead of this variable name, aws colon region is passed to the actual region that this template is being applied into.

So that's what the substitution function does.

It replaces any variable or parameter names with the values of those variables or parameters.

So the CFN init process is then able to consult the CloudFormation stack and retrieve the configuration information.

That's all stored in the metadata section of this logical resource.

Now I just want to draw your attention to this double hyphen config sets wordpress underscore install.

This tells us what set of instructions we want CFN init to run.

So if I just expand the metadata section here, we've got one or more config sets defined.

In this case, we've only got the one which is wordpress underscore install.

And this config set runs five individual items, one after the other.

And these are called config keys.

So install CFN, software install, configure instance, install wordpress and configure wordpress.

Now these reference the config keys defined below.

So you'll see that the same name install CFN, software install, configure instance, install wordpress and configure wordpress.

You'll recognize a lot of the commands used because they're the same commands that install and configure wordpress.

So in the software install config key, we're using the DNF package manager to install various software packages that we need for this installation, such as WGet, MariaDB, the Apache web server and various other utilities.

Then another part is services and we're specifying that we want these services to be enabled and to be running.

So this means that the service will be set to start up on instance boot and it will make sure that it's running right now.

The next config key is configure instance.

The files component of this can create files with a certain content.

So we're creating a file called etc update-motd.d/40-cow.

This is the part that we had to do manually before and this is the thing that adds the cow say banner.

Then we're running some more procedural commands to set the database root password and to update this banner.

Then we've got install wordpress, which uses a sources option to expand whatever is specified here into this directory.

So this automatically handles the download and the unjzip and untarring of this archive into this folder and it can even do that with authentication if needed.

We're creating another file this time to perform the configuration of wordpress and another file this time to create the database for wordpress.

Then finally we've got the configure wordpress which fixes up the permissions and creates these databases.

So this is doing the same thing as the procedural example in the previous demo.

Instead of running all of these commands one by one, this is just using desired state.

Now there is one more thing that I wanted to point out right at the top.

This is the part that configures CFN init to keep watching the logical resource configuration inside the cloud formation stack.

And if it notices that the metadata for EC2 instance inside the stack changes, then it will run CFN init again.

Remember how in the theory lesson I mentioned that this process could cope with stack updates.

So it doesn't only run once like user data does.

Well, this is how it does that.

This configures this automatic update that keeps an eye on the cloud formation stack and reruns CFN init whenever any changes occur.

This is well beyond what you need for the associate exam.

I just want you to be aware of what this is and how it works.

Essentially we're setting up a process called CFN hop and making it watch the cloud formation stack for any configuration changes.

And then we're setting it up so that the CFN hop process is enabled and running so that it can watch the resource configuration constantly.

So that's it for this template.

What we'll do now is apply it.

So go ahead and click on the second one click deployment link attached to this lesson.

It should be called A4LEC2CFN init.

So click that link.

All you'll need to do is scroll down to the bottom and then click on create stack.

Now this time remember we're using a creation policy.

So cloud formation is not going to move this logical ID and to create complete when EC2 signals that the launch process is completed.

Instead it's going to wait until the instance itself signals the successful completion of the CFN init process.

So because we're using this creation policy it's going to hold until the instance operating system using CFN-signal provide a signal to cloud formation to say yep everything's okay and at that point the logical resource will move into create complete.

So that's going to take a couple of minutes.

The EC2 instance will need to actually launch itself and pass its status checks and then the CFN init process will run, perform all of the configuration required and then assuming the status code of that is okay then CFN-signal will take that status code and respond to the cloud formation stack with a successful completion and then the process will move on then cloud formation will mark the particular resources complete and the stack is complete.

Now that will take a few minutes so just keep hitting refresh and you should see the status update after two to three minutes but go ahead and pause the video and resume it once your stack moves into the create complete status.

And there we go at this point the stack has moved into the create complete status and I just want to draw your attention to this line.

You won't have seen this before.

This is the line where our EC2 instance has run the CFN init process successfully and then the CFN signal command has taken that success signal and delivered it to the cloud formation stack.

So this is the signal that cloud formation was waiting for before moving this resource into a create complete status and that's what's needed before the stack itself could move into a create complete status.

So now we explicitly know that the configuration of this instance has actually been completed.

So we're not relying on EC2 telling us that the instance status is now running with two out of two checks.

Instead the operating system itself the CFN init process that's completed successfully and the CFN signal process has explicitly indicated to cloud formation that that whole process has been completed.

So if we move across to the EC2 console we should be able to connect to the instance exactly as we've done before.

Look for the running instance and select it.

Copy the public IP version 4 IP address and open that in a new tab.

All being well you should see the familiar WordPress installation screen.

If you're right click on that instance and put connect.

Go to instance connect and hit connect that will connect you into the instance and you should be greeted by the cow themed login banner.

This time if we use curl to show us the contents of user data this time it's only a small number of lines because the only thing that runs is the CFN init process and the CFN signal process.

Notice though how all of these variable names have been replaced with their values so the stack IDs and the region.

So this is how it knows to communicate with the right stack in the right region inside cloud formation.

If we do a CD space forward slash var forward slash log and then do a listing we've still got these original two files so cloud hyphen init dot log and cloud hyphen init hyphen output dot log.

So these are primarily associated with the user data output.

But now we've also got these new log files so CFN hyphen init hyphen CMD dot log and that is an output of the CFN init process.

So if we cat that so shudu space cat space and then the name of that log file this will show us an output of the CFN init process itself.

So we can see each of the individual config keys running and what individual operations are being performed inside each of those keys.

So it's a more complex but a more powerful process.

And at this point that's everything I wanted to cover.

It was just to give you practical exposure to an alternative to raw user data and that was CFN hyphen init.

It's a much more powerful system especially when combined with cloud formation creation policies which allow us to pause the progress of a cloud formation stack waiting for the resource itself to explicitly say yes I finished off all of my bootstrapping process you're good to carry on and that's done using the CFN hyphen signal command.

Now at this point let's just clean up the account move back to cloud formation.

Once you there go ahead and delete the EC2 CFN init stack wait for that process to complete and once you've done that go ahead and delete the A4L VPC stack and that will return the AWS account into the state that you had it at the start of this demo.

At that point thanks for doing this demo I hope it was useful.

You can go ahead and complete this video now and when you're ready you can join me in the next.

Reviewer #1 (Public review):

Summary:

Madigan et al. assembled an interesting study investigating the role of the MuSK-BMP signaling pathway in maintaining adult mouse muscle stem cell (MuSC) quiescence and muscle function before and after trauma. Using a full body and MuSC-specific genetic knockout system, they demonstrate that MuSK is expressed on MuSCs and that eliminating the BMP binding domain from the MuSK gene (i.e., MuSK-IgG KO) in mice at homeostasis leads to reduced PAX7+ cells, increased myonuclear number, and increase myofiber size, which may be due to a deficit in maintaining quiescence. Additionally, after BaCl2 injury, MuSK-IgG KO mice display accelerated repair after 7 days post-injury (dpi) in males only. Finally, RNA profiling using nCounter technology showed that MuSK-IgG KO MuSCs express genes that may be associated with the activated state.

Strengths:

Overall, the biology regulating MuSC quiescence is still relatively unexplored, and thus, this work provides a new mechanism controlling this process. The experiments discussed in the paper are technically sound with great complementary mouse models (full body versus tissue-specific mouse KO) used to validate their hypothesis. Additionally, the paper is well written with all the necessary information in the legends, methods, and figures being reported.

Weaknesses:

While the data largely supports the author's conclusions, I do have a few points to consider when reading this paper.

(1) For Figure 1, while I appreciate the author's confirming MuSK RNA and protein in MuSCs, I do think they should (a) quantify the RNA using qPCR and (b) determine the percentage of MuSCs expressing MuSK protein in their single fiber system in multiple biological replicates. This information will help us understand if MuSK is expressed in 1/10 or 10/10 PAX7-expressing MuSCs. Also, it will help place their phenotypes into the right context, especially when considering how much of the PAX7-pool is expressing MuSK from the beginning.

(2) Throughout the paper the argument is made that MuSK-IgG KO (full body and MuSC-specific KOs) are more activated and/or break quiescence more readily, but there is no attempt to test directly. Therefore, the authors should consider measuring the activation dynamics (i.e., break from quiescence) of MuSCs directly (EdU assays or live-cell imaging) in culture and/or in muscle in vivo (EdU assays) using their various genetic mouse models.

(3) For Figure 2, given that mice are considered adults by 3 months, it is really surprising how just two months later they are starting to see a phenotype (i.e., reduced PAX7-cells, increased number of myonuclei, and increased myofiber size)-which correlates with getting older. Given that aged MuSCs have activation defects (i.e., stuck somewhere in the quiescence cycle), a pending question is whether their phenotype gets stronger in aged mice, like 18-24 months. If yes, the argument that this pathway should be used in a therapeutic sense would be strengthened.

(4) For Figure 4, the same question as in point (2), the increase in fiber sizes by 7dpi in MuSK-IgG KO males is minimal (going from ~23 to 27 by eye) and no difference at a later time point when compared to WT mice. However, if older mice are used (18-24 months old) - which are known to have repair deficits-will the regenerative phenotype in MuSK-IgG KO mice be more substantial and longer lasting?

(5) For Figure 6, this gene set is not glaringly obvious as being markers of MuSC activation (i.e., no MyoD), so it's hard for the readers to know if this gene set is truly an activation signature. Also, the Shcherbina et al. data presented as a column with * being up or down (i.e. differentially expressed) is not helpful, since you don't know whether those mRNAs in that dataset are going up with the activation process. Addressing this point as well as my point (1) will further strengthen the author's conclusions about the MuSK-IgG KO MuSCs not being able to maintain quiescence as effectively.

Author response:

Reviewer #1 (Public review):

Summary:

Madigan et al. assembled an interesting study investigating the role of the MuSK-BMP signaling pathway in maintaining adult mouse muscle stem cell (MuSC) quiescence and muscle function before and after trauma. Using a full body and MuSC-specific genetic knockout system, they demonstrate that MuSK is expressed on MuSCs and that eliminating the BMP binding domain from the MuSK gene (i.e., MuSK-IgG KO) in mice at homeostasis leads to reduced PAX7+ cells, increased myonuclear number, and increase myofiber size, which may be due to a deficit in maintaining quiescence. Additionally, after BaCl2 injury, MuSK-IgG KO mice display accelerated repair after 7 days post-injury (dpi) in males only. Finally, RNA profiling using nCounter technology showed that MuSK-IgG KO MuSCs express genes that may be associated with the activated state.

Strengths:

Overall, the biology regulating MuSC quiescence is still relatively unexplored, and thus, this work provides a new mechanism controlling this process. The experiments discussed in the paper are technically sound with great complementary mouse models (full body versus tissue-specific mouse KO) used to validate their hypothesis. Additionally, the paper is well written with all the necessary information in the legends, methods, and figures being reported.

Weaknesses:

While the data largely supports the author's conclusions, I do have a few points to consider when reading this paper.

(1) For Figure 1, while I appreciate the author's confirming MuSK RNA and protein in MuSCs, I do think they should (a) quantify the RNA using qPCR and (b) determine the percentage of MuSCs expressing MuSK protein in their single fiber system in multiple biological replicates. This information will help us understand if MuSK is expressed in 1/10 or 10/10 PAX7-expressing MuSCs. Also, it will help place their phenotypes into the right context, especially when considering how much of the PAX7-pool is expressing MuSK from the beginning.

The quantification is a reasonable point; however, we don’t believe that this information is necessary for supporting the interpretation of the findings.

We agree that determining the proportion of SCs that expressing MuSK is useful information and we will address this question in the Revision.

(2) Throughout the paper the argument is made that MuSK-IgG KO (full body and MuSC-specific KOs) are more activated and/or break quiescence more readily, but there is no attempt to test directly. Therefore, the authors should consider measuring the activation dynamics (i.e., break from quiescence) of MuSCs directly (EdU assays or live-cell imaging) in culture and/or in muscle in vivo (EdU assays) using their various genetic mouse models

We agree that this point is of interest and we plan to address it in future studies.

(3) For Figure 2, given that mice are considered adults by 3 months, it is really surprising how just two months later they are starting to see a phenotype (i.e., reduced PAX7-cells, increased number of myonuclei, and increased myofiber size)-which correlates with getting older. Given that aged MuSCs have activation defects (i.e., stuck somewhere in the quiescence cycle), a pending question is whether their phenotype gets stronger in aged mice, like 18-24 months. If yes, the argument that this pathway should be used in a therapeutic sense would be strengthened.

We agree that the potential role of the MuSK-BMP pathway in aged SCs is of import and could shed new light on SC dynamics in this context. However, we note that the activation observed between 3-5 months results in improved muscle quality (increased myofiber size and grip strength), which is opposite of what is observed with aging. We agree that activating the MuSK-BMP pathway in aged animals has the potential to activate SCs, promote muscle growth and counter sarcopenia.  Pharmacological and genetic approaches to test that question are underway, but given the time frame they are beyond the scope of the current manuscript.

(4) For Figure 4, the same question as in point (2), the increase in fiber sizes by 7dpi in MuSK-IgG KO males is minimal (going from ~23 to 27 by eye) and no difference at a later time point when compared to WT mice. However, if older mice are used (18-24 months old) - which are known to have repair deficits-will the regenerative phenotype in MuSK-IgG KO mice be more substantial and longer lasting?

Again, an interesting point that will be addressed in future studies. 

(5) For Figure 6, this gene set is not glaringly obvious as being markers of MuSC activation (i.e., no MyoD), so it's hard for the readers to know if this gene set is truly an activation signature. Also, the Shcherbina et al. data presented as a column with * being up or down (i.e. differentially expressed) is not helpful, since you don't know whether those mRNAs in that dataset are going up with the activation process. Addressing this point as well as my point (1) will further strengthen the author's conclusions about the MuSK-IgG KO MuSCs not being able to maintain quiescence as effectively.

We agree that this Figure should include more information and be formatted in a way more readily convey the point. We will provide these changes in the Revision.

Reviewer #2 (Public review):

Summary:

The work by Madigan et al. provides evidence that the signaling of BMPs via the Ig3 domain of MuSK plays a role during muscle postnatal development and regeneration, ultimately resulting in enhanced contractile force generation in the absence of the MuSK Ig3 domain. They demonstrate that MuSK is expressed in satellite cells initially post-isolation of muscle single fibers both in WT and whole-body deletion of the BMP binding domain of MuSK (ΔIg3-MuSK). In developing mice, ΔIg3-MuSK results in increased muscle fiber size, a reduction in Pax7+ cells, and increased muscle contractile force in 5-month-old, but not 3-month-old, mice. These data are complemented by a model in which the kinetics of regeneration appear to be accelerated at early time points. Of note, the authors demonstrate muscle tibialis anterior (TA) weights and fiber feret are increased during development in a Pax7CreERT2;MuSK-Ig3loxp/loxp model in which satellite cells specifically lack the MuSK BMP binding domain. Finally, using Nanostring transcriptional the authors identified a short list of genes that differ between the WT and ΔIg3-MuSK SCs. These data provide the field with new evidence of signaling pathways that regulate satellite cell activation/quiescence in the context of skeletal muscle development and regeneration.

On the whole, the findings in this paper are well supported, however additional validation of key satellite cell markers and data analysis need to be conducted given the current claims.

(1) The Pax7CreERT2;MuSK-Ig3loxp/loxp model is the appropriate model to conduct studies to assess satellite cell involvement in MuSK/BMP regulation. Validation of changes to muscle force production is currently absent using this model, as is quantification of Pax7+ tdT+ cells in 5-month muscle. Given that MuSK is also expressed on mature myofibers at NMJs, these data would further inform the conclusions proposed in the paper.

As reported in the manuscript, we observed increased myofiber size, length and TA weight in the conditional mutants at five months of age. We did not assess grip strength in those experiments. 

We demonstrated highly efficient MuSK Ig3-domain recombination by PCR analysis of FACS-sorted SCs from these conditional mutants (Supplemental Fig. S3). However, while we checked for Pax7+ tdT+ cells in 5-month SCs, we did not quantify this finding.

(2) All Pax7 quantification in the paper would benefit from high magnification images including staining for laminin demonstrating the cells are under the basal lamina.

The point is reasonable, we observed that these Pax7+ cells were under the basal lamina, but we did not acquire images at higher magnification.   

(3) The nanostring dataset could be further analyzed and clarified. In Figure 6b, it is not initially apparent what genes are upregulated or downregulated in young and aged SCs and how this compares with your data. Pathway analysis geared toward genes involved in the TGFb superfamily would be informative.

We agree that further analysis and information regarding the data in this Figure is warranted and we will include it in the Revision.

(4) Characterizing MuSK expression on perfusion-fixed EDL fibers would be more conclusive to determine if MuSK is expressed in quiescent SCs. Additional characterization using MyoD, MyoG, and Fos staining of SCs on EDL fibers would help inform on their state of activation/quiescent.

These are all valid points that we intend to address in future experiments.

(5) Finally, the treatment of fibers in the presence or absence of recombinant BMP proteins would inform the claims of the paper.

As reported in Jaime et al (2024) we have extensively characterized the differences in BMP response in both cultured WT and DIg3-MuSK myofibers and myoblasts at the level of signaling (pSMAD 1/5/8 nuclear localization and phosphorylation) and gene expression (qRT-PCR).

Reviewer #3 (Public review):

Summary:

Understanding the molecular regulation of muscle stem cell quiescence. The authors evaluated the role of the MuSK-BMP pathway in regulating adult SC quiescence by the deletion of the BMP-binding MuSK Ig3 domain ('ΔIg3-MuSK').

Strengths:

A novel mouse model to interrogate muscle stem cell molecular regulators. The authors have developed a nice mouse model to interrogate the role of MuSK signaling in muscle stem cells and myofibers and have unique tools to do this.

Weaknesses:

Only minor technical questions remain and there is a need for additional data to support the conclusions.

(1) The authors claim that dIg3-MuSK satellite cells break quiescence and start fusing, based on the reduction of Pax7+ and increase of nuclei/fiber (Fig 2-3), and maybe the gene expression (Fig6). However, direct evidence is needed to support these findings such as quantifying quiescent (Pax7+Ki67-) or activated (Pax7+Ki67+) satellite cells (and maybe proliferating progenitors Pax7-Ki67+) in the dIg3-MuSK muscle.

We believe that the data presented strongly supports the conclusion that the SCs break quiescence, activate, and fuse into myofibers in uninjured muscle.  As noted above, the mechanistic studies suggested are of interest and we will address them in future work.

(2) It is not clear if the MuSK-BMP pathway is required to maintain satellite cell quiescence, by the end of the regeneration (29dpi), how Pax7+ numbers are comparable to the WT (Fig4d). I would expect to have less Pax7+, as in uninjured muscle. Can the authors evaluate this in more detail?

The reviewer makes an important point. Our current interpretation of the findings is that quiescence is broken in SCs in uninjured muscle, but that ‘stemness’ is preserved, allowing for efficient muscle regeneration and restoration of the SC pool. Whether such properties reflect SC heterogeneity (as suggested in the comments of the other reviewers) and/or different states along a continuum is of particular interest and will be the focus of future studies. 

(2) Figure 4 claims that regeneration is accelerated, but to claim this at a minimum they need to look at MYH3+ fibers, in addition to fiber size.

We did not examine MYH3+ fibers in this study. However, we did observe increased in Pax7+ cells at 5dpi (male and female) as well as larger myofiber size (Feret diameter) at 7dpi in the male animals.  In addition, the panels in Figure 4 b,c (H&E and laminin, respectively) showing accelerated differentiation were selected to be representative of the experimental group. 

(3) The Pax7 specific dIg3-MuSK (Fig5) is very exciting. However, it will be important to quantify the Pax7+ number. Could the authors check the reduction of Pax7+ in this model since it would confirm the importance of MuSK in quiescence?

In Figure 5c, we assessed the number of Pax7+ cells in the conditional mutant during the course of regeneration (at 3, 5, 7, 14, 22 and 29 dpi). As discussed above, these results confirmed the findings of the constitutive mutant (reduction of Pax7+ cells in uninjured 5-month-old muscle) as well as showing the increased number at 5dpi and return to WT levels at 29 dpi.

(3) Rescue of the BMP pathway in the model would be further supportive of the authors' findings.

This point is valid. In a parallel study examining the role of the MuSK-BMP pathway at the NMJ, we have observed that BMP+/- (hypomorphs) recapitulate key phenotypes observed in DIg3-MuSK  NMJs (Fish et al., bioRxiv, 2023). This point will be included in the Revision. 

(4) Is the stem cell pool maintained long term in the deleted dIg3-MuSK SCs? Or would they be lost with extended treatment since they are reduced at the 5-month experiments? This is an important point and should be considered/discussed relevant to thinking about these data therapeutically.

We agree that this is an important point for future studies. 

(5) Without the Pax7-specific targeting, when you target dIg3-MuSK in the entire muscle, what happens to the neuromuscular nuclei?

A manuscript describing the phenotype of the NMJ in DIg3-MuSK constitutive mice is in bioRxiv (Fish et al., 2024) and is in Revision at another journal.  We anticipate discussing the findings in the Revised version of the current manuscript. 

(6) Why were differences seen in males and not females? Is XIST downregulation occurring in both sexes? Could the authors explain these findings in more detail?

The male and female difference in myofiber size is of interest.  The nanostring experiments,  which showed the XIST reduction, were only performed in male mice.

“Republicans came in with a higher court and said this is illegal, it’s voter fraud, and that they’re trying to steal the election.”

I was raised in a democratic household my mom very democratic I know republicans and democratic have their issues and this just makes me remember the time my mom coworker was upset with my mom because she is a democratic after Biden won thinking of voting fraud

Author response:

The following is the authors’ response to the original reviews.

Public reviews:

Reviewer #1:

(1) Which allele is alr1, the one upstream of mazEF or the one in the lysine biosynthetic operon?

Alr1 is encoded by SAUSA300_2027 and is the gene upstream to mazEF. We have now incorporated this information in the manuscript (Line# 127).

(2) Figure 3B. Where does the C3N2 species come from in the WT and why is it absent in the mutants? It is about 25% of the total dipeptide pool.

In Figure 3B, C3N2 species results from the combination of C3N1 (from Alr1) and C0N1 (from Dat). The reason this species is completely absent in either of the two mutants is because it requires one D-Ala from both Alr1 and Dat proteins to generate C3N2 D-Ala-D-Ala.

(3) Figure 3D could perhaps be omitted. I understand that the authors attained statistical significance in the fitness defect, but biologically this difference is very minor. One would have to look at the isotopomer distribution in the Dat overexpressing strain to make sure that increased flux actually occurred since there are other means of affecting activity (e.g. allosteric modulators).

Thank you for the suggestion. We agree with the reviewer that the fitness defect observed after increased dat expression is relatively minor and have moved this figure to the supplementary section as Figure 3-figure supplement 1.

Although we attempted to amplify the fitness defect of dat expression by cloning dat on to a multicopy vector, we couldn't maintain its stable expression in S. aureus. This instability may be due to the depletion of D-Ala when dat is overexpressed. As a result, we switched to expressing dat from a single additional copy integrated into the SaPI locus, which was sufficient to cause the expected fitness defect, albeit a minor one.

(4) In Figure 4A, why is the complete subunit UDP-NAM-AEKAA increasing in each strain upon acetate challenge if there was such a stark reduction in D-Ala-D-Ala, particularly in the ∆alr1 mutant? For that matter, why are the levels of UDP-NAM-AEKAA in the ∆alr1 mutant identical to that of WT with/out acetate?

Thank you for raising this important point. We have addressed this in line# 299-302 and 451-455 of the revised manuscript. In short, we believe that the inhibition of Ddl by acetate significantly increases the intracellular pool of the tripeptide UDP-NAM-AEK, which then outcompetes the substrate (pentapeptide; UDP-NAM-AEKAA) of MraY. As a result, the intracellular concentration of the pentapeptide increases since it is no longer efficiently consumed by MraY. This explanation is also supported by a kinetic study conducted in Ref (1), where the competition between UDP-NAM-AEKAA and UDP-NAM-AEK as substrates for MraY is demonstrated.

(5) Figure 4B. Is there no significant difference between ddl and murF transcripts between WT and ∆alr1 under acetate stress? This comparison was not labeled if the tests were done.

Thank you for suggesting this comparison. The ddl and murF transcripts between WT and alr1 under acetate stress were significantly different. We have added this comparison to Figure 4B.

(6) Although tricky, it is possible to measure intracellular acetate. It might be of interest to know where in the Ddl inhibition curve the cells actually are.

Thank you for the suggestion. We agree this would have been an excellent addition to the manuscript. However, accurately measuring intracellular acetate would require the use of radiolabeled acetate (2), and we currently lack the expertise to do this experiment. However, since our study clearly shows that acetate-mediated growth impairment is due to Ddl inhibition, and the IC50 of acetate for Ddl is around 400 mM, we predict that the intracellular concentration must be close to or above this IC50 to observe the growth phenotypes we report.

Reviewer #2:

Although the authors have conclusively shown that Ddl is the target of acetic acid, it appears that the acetic acid concentration used in the experiments may not truly reflect the concentration range S. aureus would experience in its environment. Moreover, Ddl is only significantly inhibited at a very high acetate concentration (>400 mM). Thus, additional experiments showing growth phenotypes at lower organic acid concentrations may be beneficial.

Thank you for the suggestion. In response to the reviewer, we have measured growth at various acetate concentrations and demonstrate a concentration-dependent effect (Figure 1C).

We use 20 mM acetic acid in our study. In the gut, where S. aureus colonizes, acetate levels can reach up to 100 mM, so we believe our concentrations are physiologically relevant. When S. aureus encounters 20 mM acetate, the intracellular concentration can rise to 600 mM if the transmembrane pH gradient is 1.5 units, which is well above the ~400 mM IC50 we report for Ddl.

Another aspect not adequately discussed is the presence of D-ala in the gut environment, which may be protective against acetate toxicity based on the model provided.

Thank you for pointing this out. We agree that D-Ala from the gut microbiota could protect against acetate toxicity, and we’ve included this in the discussion. However, our study clearly indicates that S. aureus itself maintains high intracellular D-Ala levels through Alr1 activity which is sufficient to counter acetate anion intoxication.

Recommendation for the authors:

Reviewer #2:

Major Comments:

(1) In Line 85, authors indicate S. aureus may encounter a high concentration of ~100 mM acetic acid (extracellular?). Could the authors cite more (and recent) references indicating S. aureus encounters >100 mM acetic acid in the environment?

To the best of our knowledge, no studies have specifically examined whether S. aureus encounters high mM concentration of acetate in the gut. Line 85 was surmised from multiple studies: recent findings that S. aureus colonizes the gut (3, 4) and that the gut environment has high acetate levels (~100 mM) (5). In response to the reviewers request, more recent references supporting high acetate concentrations in the gut (6, 7) have been added in Line# 86.

(2) In Line 117, it is mentioned that S. aureus when grown in vitro at 20 mM acetic acid can accumulate ~600 mM acetic acid in the cytoplasm.

a. Does the intracellular concentration go up proportionally if grown in 100 mM acetic acid? Given the IC50 of acetic acid-mediated inhibition of Ddl is ~400 mM, I wonder how physiologically relevant this finding presented here is.

Thank you for the opportunity to explain this further. If S. aureus encounters a concentration of 100 mM acetate and its transmembrane pH gradient (pHin-pHout) is held at 1.5, the intracellular concentration of acetate could theoretically increase up to 3 M based on Ref (8). However, previous studies have shown that bacteria can lower the magnitude of transmembrane pH gradient by decreasing their intracellular pH to limit accumulation of anions within cells (9, 10).

Although our study shows that the IC50 of Ddl inhibition by acetate is relatively high (~400 mM), we believe it’s still relevant because just 20 mM of environmental acetate at a pH of 6.0 can raise the intracellular concentration of acetate to over 600 mM, which is well above the IC50 we report for Ddl. Moreover, since S. aureus may encounter high concentrations of acetate during gut colonization, we believe our findings are physiologically relevant.

b. Could the authors show concentration-dependent growth inhibition in alr::tn by titrating a range of acetic acid concentrations (for example 0, 0.5, 1, 5, 10, 20 mM)? Measuring intracellular acetate concentration may be beneficial as well.

Thank you for this question. We now provide data to support that acetate-mediated inhibition of the alr1 mutant is concentration-dependent (see Figure 1C).

c. It appears that there may be excess D-ala in the gut environment (PMIDs: 30559391; 35816159), which could counter the high acetate based on the model presented here. Could the authors clarify and/or include this information in the manuscript?

This is an excellent point, and we have now included it in the discussion (Line# 470-475). It is indeed possible that D-Ala produced by the gut microbiome may further enhance S. aureus resistance to organic acid anions, in addition to the inherent contribution of Alr1 activity.

(3) The following is not needed; however, it would be interesting if the authors could show that S. aureus cells grown in the presence of acetate are highly sensitive to cycloserine (which targets Alr and Ddl) compared to cells grown in the absence of acetate.

Thank you for the suggestion. We are currently studying D-cycloserine (DCS) resistance in S. aureus. Although we provide the data below for clarification, it is not included in the current manuscript as it is part of a separate study.

As the reviewer speculated, S. aureus is more susceptible to DCS when grown in the presence of acetate (see figure below). Normally, complete growth inhibition occurs at 32 µg/ml of DCS. However, with 20 mM acetic acid present, complete inhibition is achieved at just 8 µg/ml of DCS. Furthermore, the growth inhibition is completely rescued when externally supplemented with 5 mM D-Ala. We believe that DCS works synergistically with acetate to inhibit Ddl activity, and we are conducting additional studies to explore this further.

Minor Comments:

(1) Many commas are missing.

Missing commas are now incorporated.

(2) Line 77: disassociate --> dissociate

Corrected.

(3) Line 103: that --> which

Corrected.

(4) Lines 199-203: authors could have used gfp/luciferase reporter to test their hypotheses.

Thank you for the suggestion. Initially, we created GFP translational fusions for all the mutants mentioned in Line# 199-203. However, the fluorescence intensity was too low to test the hypothesis, as these were single-copy fusions inserted at the SaPI site of the S. aureus genome. Because of this limitation, we took advantage of the essentiality of D-Ala-D-Ala in S. aureus to report on various mutants instead of a fluorescent reporter. In hindsight, a LacZ reporter assay might have been equally effective.

(5) Line 339: It would be beneficial to introduce that Ddl has two independent ATP and D-ala binding sites.

We have now added that information (Line# 338-339).

(6) Is ddl an essential gene? If so, explicitly mention that.

Yes, ddl is an essential gene and we have now incorporated this information in Line 103.

(7) Line 354: shows a difference in density?

The use of the term “difference density” is a technical crystallographic term commonly used to connote density observed for ligands in X-ray crystal structures. In this case, it simply refers to the observed density that corresponds to the two acetate ions bound within the Ddl active site.

(8) Line 498: "Thus." Typo, change period to comma.

We have corrected as suggested in Line 496.

(9) Figure 1 legend says "was screen" instead of screened.

This is now corrected.

(10) Figure 1- Figure Supplement 1B: including data for alr2::tn dat::tn may ensure no redundancy (Lines 171-172). It is currently missing.

Thank you for the suggestion. We now include both alr2dat double mutant and the alr1alr2dat triple mutant in Figure 1 - Figure Supplement 1B. In addition we also show that the alr1alr2dat mutant is resuced by the addition of D-Ala in Figure 1 - Figure Supplement 1C. The mutant information is also added to Table S5.

(11) Figure 7: pentaglycine coming off of NAM is misleading. Remove untethered pentaglycine bridges.

We thank you for pointing this out. We have modified the figure in the manuscript as suggested by the reviewer.

(12) Are alr1/ddl cells (with limited 4-3 PG crosslink) less sensitive to vancomycin?

On the contrary, the alr1 mutant is slightly more sensitive to vancomycin compared to the wild-type strain (see Figure below). We believe this happens because the alr1 mutant incorporates less D-Ala-D-Ala into the peptidoglycan, reducing the number of targets for vancomycin. As a result, vancomycin may be able to saturate the available D-Ala-D-Ala targets on the cell wall at a lower concentration in the alr1 mutant than in the wild type strain, leading to increased sensitivity. We haven’t included this data in the manuscript as it is part of a separate study.

(13) Based on the structural studies, could the authors mutate the residues of Ddl involved in acetic acid binding, thereby making it resistant to acetic acid stress?

The residues that the acetate anion interacts with are located within the ATP-binding and D-Ala-binding sites of Ddl. Since these residues are essential for Ddl function, we are unable to mutate them.

(14) Microscopy to show the cell morphologies of wild-type and mutants exposed to acetic acid (and with D-ala supplementation) could be potentially interesting.

Thank you for the suggestion. We did perform microscopy, expecting changes in cell shape or size, but the results were unremarkable and not included in the manuscript.

References:

(1) Hammes WP & Neuhaus FC (1974) On the specificity of phospho-N-acetylmuramyl-pentapeptide translocase. The peptide subunit of uridine diphosphate-N-actylmuramyl-pentapeptide. J Biol Chem 249(10):3140-3150.

(2) Roe AJ, McLaggan D, Davidson I, O'Byrne C, & Booth IR (1998) Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J Bacteriol 180(4):767-772.

(3) Acton DS, Plat-Sinnige MJ, van Wamel W, de Groot N, & van Belkum A (2009) Intestinal carriage of Staphylococcus aureus: how does its frequency compare with that of nasal carriage and what is its clinical impact? Eur J Clin Microbiol Infect Dis 28(2):115-127.

(4) Piewngam P_, et al. (2023) Probiotic for pathogen-specific _Staphylococcus aureus decolonisation in Thailand: a phase 2, double-blind, randomised, placebo-controlled trial. Lancet Microbe 4(2):e75-e83.

(5) Cummings JH, Pomare EW, Branch WJ, Naylor CP, & Macfarlane GT (1987) Short chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut 28(10):1221-1227.

(6) Correa-Oliveira R, Fachi JL, Vieira A, Sato FT, & Vinolo MA (2016) Regulation of immune cell function by short-chain fatty acids. Clin Transl Immunology 5(4):e73.

(7) Hosmer J, McEwan AG, & Kappler U (2024) Bacterial acetate metabolism and its influence on human epithelia. Emerg Top Life Sci 8(1):1-13.

(8) Carpenter CE & Broadbent JR (2009) External concentration of organic acid anions and pH: key independent variables for studying how organic acids inhibit growth of bacteria in mildly acidic foods. J Food Sci 74(1):R12-15.

(9) Russell JB (1992) Another explanation for the toxicity of fermentation acids at low pH: anion accumulation versus uncoupling. Journal of Applied Bacteriology 73(5):363-370.

(10) Russell JB & Diez-Gonzalez F (1998) The effects of fermentation acids on bacterial growth. Adv Microb Physiol 39:205-234.

I think this idea of memes is pretty cool because it explains why some trends, like popular music or viral videos, catch on quickly and become huge, while others fade away. It's like there’s a “survival of the fittest” happening with ideas, just like with genes in nature. For example, technology or social media could be seen as memes that keep evolving and becoming more advanced because people keep building on what came before. This chapter made me realize that culture isn’t just about what we create; it’s also about how these creations spread and stick around. It's interesting to think that we’re not just passing down physical traits but also ideas and beliefs that shape the future.

I studied this in biology class during my high school years, and what impressed me the most was the part of Natural selection, I found that just like Darwin's theory of evolution says, any living thing is the survival of the fittest following genetic optimization trying to survive from one generation to the next, and that's what our world is all about, it's brutal but also realistic.

I think it's really funny and self-incriminating to use the word "kosher" as though that didn't evoke thousand of years of thoughtful community discourse about where lines should be drawn and why

It's really telling just how effective colonization was, if it was able to convince some people in later generations that spirits that are part of their heritage and culture are evil.

Conal Elliott introduces 'Denotational Design' as his central paradigm for software and library design.

Quote: "I call it denotational design."

He emphasizes that the primary job of a software designer is to build precise abstractions, focusing on 'what' rather than 'how'.

Quote: "So I want to start out by talking about what I see as the main job of a software designer, which is to build abstractions."

He references Edsger Dijkstra's perspective on abstraction to highlight the need for precision in software design.

Quote: "This is a quote I like very much from a man I respect very much, Edgar Dykstra, and he said the purpose of abstraction is not to be vague... it's to create a whole new semantic level in which one can be absolutely precise."

He identifies a common issue in software development: the focus on precision about implementation ('how') rather than specification ('what').

Quote: "So I'm going to say something that may be a little jarring, which is that the state of the... commonly practiced state of the art in software is something that is precise only about how, not about what."

He stresses the importance of making specifications precise to avoid self-deception in software development.

Quote: "So the reason I harp onto precision is because it's so easy to fool ourselves and precision is what keeps us away from doing that."

He cites Bertrand Russell's observation on the inherent vagueness of concepts until made precise.

Quote: "Everything is vague to a degree you do not realize until you've tried to make it precise."

He discusses the inadequacy of the term 'functional programming' and introduces 'denotational programming' as a better-defined alternative, referencing Peter Landin's work.

Quote: "Peter Landon suggested term denotated... having three properties... every expression denotes something... that something depends only on the denotations of the sub-expressions."

He defines 'Denotational Design' as a methodology that provides precise, simple, and compelling specifications, and helps avoid abstraction leaks.

Quote: "I call it denotational design... It gives us precise, simple, and compelling specifications... you do not have abstraction leaks."

He outlines three goals in software projects: building precise, elegant, and reusable abstractions; creating fast, correct, and maintainable implementations; and producing simple, clear, and accurate documentation.

Quote: "So I suggest there are three goals... I want my abstractions to be precise, elegant, and reusable... My implementation, I'd like it to be fast... correct... maintainable... and the documentation should also be simple and... accurate."

He demonstrates Denotational Design through an example of designing a library for image synthesis and manipulation, engaging the audience in defining what an image is.

Quote: "So an example I want to talk about is image synthesis and manipulation... What is an image?"

He considers various definitions of an image, including arrays of pixels, functions over space, and collections of shapes, before settling on a mathematical model.

Quote: "My answer is: it's an assignment of colors to 2D locations... there's a simple precise way to say that which is the function from location to colors."

He applies the denotational approach to define the meanings of types and operations in his image library, emphasizing the importance of compositionality.

Quote: "So now I'm giving a denotation... So the meaning of over top bot is... mu of top and mu of bot... Note the compositionality of mu."

He improves the API by generalizing operations and types, introducing type parameters to increase flexibility and simplicity.

Quote: "So let's generalize... instead of saying an image which is a single type, let's say an image of a... we'll make it be parameterized by its output."

He introduces standard abstractions like Monoid, Functor, and Applicative, showing how his image type and operations fit into these abstractions, leveraging their laws and properties.

Quote: "Now we can also look at a couple of other interfaces: monad and comonad."

He explains the 'Semantic Type Class Morphism' principle, stating that the instance's meaning follows the meaning's instance, ensuring that standard abstractions' laws hold for his types.

Quote: "This leads to this principle that I call the semantic type class morphism principle... The instance's meaning follows the meaning's instance."

He demonstrates that by following this principle, his implementations are necessarily correct and free of abstraction leaks, as they preserve the laws of the standard abstractions.

Quote: "These proofs always go through... There's nothing about imagery except the homomorphism property that makes these laws go through."

He illustrates the principle with examples from his image library, such as showing that images form a Monoid and Functor due to their underlying semantics.

Quote: "So images... Well, image has the right kind... Well, yes it is... Here's this operation we called lift one."

He discusses how this approach allows for reusable and compositional reasoning, similar to how algebra uses abstract interfaces and laws.

Quote: "So when I say laws hold, you should say what are you even talking about... So in order for a law to be satisfied... we have to say what equality means."

He provides further examples of applying Denotational Design to other types, such as streams and linear transformations, showing the broad applicability of the approach.

Quote: "Another example is... so we just follow these all through and they all work... linear transformations."

He concludes by summarizing the benefits of Denotational Design, including precise specifications, correct implementations, and the elimination of abstraction leaks, and invites further discussion.

Quote: "I think it's a good place to stop... I'm happy to take any questions... I'd love to hear from you."

Generally negative speech and rhetoric gains the most attention and motivates people to speak on an interact with posts, making the algorithm believe that the posts in question are one users like. It's just a never-ending cycle of negativity I think has greatly divided the political scene and the irrational behavior of social media users.

Wonder how many will find out their land is contaminated.

I’m so glad that this exists and people can find community and help when they are struggling. I myself had to find help from time to time and now have the honor and privilege of helping others. I guess it’s about balance just like the world itself there can be just as much good as there is bad out there and you can either one very easy.

"theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it"

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• • [

    Designing a tympanum through stereographic projection

    ](https://en.wikipedia.org/wiki/Astrolabe#Designing_a_tympanum_through_stereographic_projection) - [

    The tropics and the equator define the tympanum
    
    ](https://en.wikipedia.org/wiki/Astrolabe#The_tropics_and_the_equator_define_the_tympanum)
    

    • [

      The horizon and the measurement of altitude

      ](https://en.wikipedia.org/wiki/Astrolabe#The_horizon_and_the_measurement_of_altitude) - [

      The meridians and the measurement of azimuth

      ](https://en.wikipedia.org/wiki/Astrolabe#The_meridians_and_the_measurement_of_azimuth) - [

  See also

  ](https://en.wikipedia.org/wiki/Astrolabe#See_also) - [

  References

  ](https://en.wikipedia.org/wiki/Astrolabe#References) - [

  Bibliography

  ](https://en.wikipedia.org/wiki/Astrolabe#Bibliography) - [

  External links

  ](https://en.wikipedia.org/wiki/Astrolabe#External_links)

Astrolabe

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From Wikipedia, the free encyclopedia

For other pages with a similar name, see Astrolabe (disambiguation). Not to be confused with Cosmolabe.

North African, 9th century AD, Planispheric Astrolabe. Khalili Collection.

A modern astrolabe made in Tabriz, Iran in 2013.

An astrolabe (Greek: ἀστρολάβος astrolábos, 'star-taker'; Arabic: ٱلأَسْطُرلاب al-Asṭurlāb; Persian: ستاره‌یاب Setāreyāb) is an astronomical instrument dating to ancient times. It serves as a star chart and physical model of visible heavenly bodies. Its various functions also make it an elaborate inclinometer and an analog calculation device capable of working out several kinds of problems in astronomy. In its simplest form it is a metal disc with a pattern of wires, cutouts, and perforations that allows a user to calculate astronomical positions precisely. It is able to measure the altitude above the horizon of a celestial body, day or night; it can be used to identify stars or planets, to determine local latitude given local time (and vice versa), to survey, or to triangulate. It was used in classical antiquity, the Islamic Golden Age, the European Middle Ages and the Age of Discovery for all these purposes.

The astrolabe, which is a precursor to the sextant,^[1]^ is effective for determining latitude on land or calm seas. Although it is less reliable on the heaving deck of a ship in rough seas, the mariner's astrolabe was developed to solve that problem.

Applications

16th-century woodcut of measurement of a building's height with an astrolabe

The 10th-century astronomer ʿAbd al-Raḥmān al-Ṣūfī wrote a massive text of 386 chapters on the astrolabe, which reportedly described more than 1,000 applications for the astrolabe's various functions.^[2]^ These ranged from the astrological, the astronomical and the religious, to navigation, seasonal and daily time-keeping, and tide tables. At the time of their use, astrology was widely considered as much of a serious science as astronomy, and study of the two went hand-in-hand. The astronomical interest varied between folk astronomy (of the pre-Islamic tradition in Arabia) which was concerned with celestial and seasonal observations, and mathematical astronomy, which would inform intellectual practices and precise calculations based on astronomical observations. In regard to the astrolabe's religious function, the demands of Islamic prayer times were to be astronomically determined to ensure precise daily timings, and the qibla, the direction of Mecca towards which Muslims must pray, could also be determined by this device. In addition to this, the lunar calendar that was informed by the calculations of the astrolabe was of great significance to the religion of Islam, given that it determines the dates of important religious observances such as Ramadan.^[citation needed]^

Etymology

The Oxford English Dictionary gives the translation "star-taker" for the English word astrolabe and traces it through medieval Latin to the Greek word ἀστρολάβος : astrolábos,^[3]^^[4]^ from ἄστρον : astron "star" and λαμβάνειν : lambanein "to take".^[5]^

In the medieval Islamic world the Arabic word al-Asturlāb (i.e., astrolabe) was given various etymologies. In Arabic texts, the word is translated as ākhidhu al-Nujūm (Arabic: آخِذُ ٱلنُّجُومْ, lit. 'star-taker'), a direct translation of the Greek word.^[6]^

Al-Biruni quotes and criticises medieval scientist Hamza al-Isfahani who stated:^[6]^ "asturlab is an arabisation of this Persian phrase" (sitara yab, meaning "taker of the stars").^[7]^ In medieval Islamic sources, there is also a folk etymology of the word as "lines of lab", where "Lab" refers to a certain son of Idris (Enoch). This etymology is mentioned by a 10th-century scientist named al-Qummi but rejected by al-Khwarizmi.^[8]^

History

Ancient era

An astrolabe is essentially a plane (two-dimensional) version of an armillary sphere, which had already been invented in the Hellenistic period and probably been used by Hipparchus to produce his star catalogue. Theon of Alexandria (c. 335 -- c. 405) wrote a detailed treatise on the astrolabe.^[9]^ The invention of the plane astrolabe is sometimes wrongly attributed to Theon's daughter Hypatia (born c. 350--370; died AD 415),^[10]^^[11]^^[12]^^[13]^ but it's known to have been used much earlier.^[11]^^[12]^^[13]^ The misattribution comes from a misinterpretation of a statement in a letter written by Hypatia's pupil Synesius (c. 373 -- c. 414),^[11]^^[12]^^[13]^ which mentions that Hypatia had taught him how to construct a plane astrolabe, but does not say that she invented it.^[11]^^[12]^^[13]^ Lewis argues that Ptolemy used an astrolabe to make the astronomical observations recorded in the Tetrabiblos.^[9]^ However, Emilie Savage-Smith notes "there is no convincing evidence that Ptolemy or any of his predecessors knew about the planispheric astrolabe".^[14]^ In chapter 5,1 of the Almagest, Ptolemy describes the construction of an armillary sphere, and it is usually assumed that this was the instrument he used.

Astrolabes continued to be used in the Byzantine Empire. Christian philosopher John Philoponus wrote a treatise (c. 550) on the astrolabe in Greek, which is the earliest extant treatise on the instrument.^[a]^ Mesopotamian bishop Severus Sebokht also wrote a treatise on the astrolabe in the Syriac language during the mid-7th century.^[b]^ Sebokht refers to the astrolabe as being made of brass in the introduction of his treatise, indicating that metal astrolabes were known in the Christian East well before they were developed in the Islamic world or in the Latin West.^[15]^

Medieval era

Astrolabes were further developed in the medieval Islamic world, where Muslim astronomers introduced angular scales to the design,^[16]^ adding circles indicating azimuths on the horizon.^[17]^ It was widely used throughout the Muslim world, chiefly as an aid to navigation and as a way of finding the Qibla, the direction of Mecca. Eighth-century mathematician Muhammad al-Fazari is the first person credited with building the astrolabe in the Islamic world.^[18]^

The mathematical background was established by Muslim astronomer Albatenius in his treatise Kitab az-Zij (c. AD 920), which was translated into Latin by Plato Tiburtinus (De Motu Stellarum). The earliest surviving astrolabe is dated AH 315 (AD 927--928). In the Islamic world, astrolabes were used to find the times of sunrise and the rising of fixed stars, to help schedule morning prayers (salat). In the 10th century, al-Sufi first described over 1,000 different uses of an astrolabe, in areas as diverse as astronomy, astrology, navigation, surveying, timekeeping, prayer, Salat, Qibla, etc.^[19]^^[20]^

An Arab astrolabe from 1208

The spherical astrolabe was a variation of both the astrolabe and the armillary sphere, invented during the Middle Ages by astronomers and inventors in the Islamic world.^[c]^ The earliest description of the spherical astrolabe dates to Al-Nayrizi (fl. 892--902). In the 12th century, Sharaf al-Dīn al-Tūsī invented the linear astrolabe, sometimes called the "staff of al-Tusi", which was "a simple wooden rod with graduated markings but without sights. It was furnished with a plumb line and a double chord for making angular measurements and bore a perforated pointer".^[21]^ The geared mechanical astrolabe was invented by Abi Bakr of Isfahan in 1235.^[22]^

The first known metal astrolabe in Western Europe is the Destombes astrolabe made from brass in the eleventh century in Portugal.^[23]^^[24]^ Metal astrolabes avoided the warping that large wooden ones were prone to, allowing the construction of larger and therefore more accurate instruments. Metal astrolabes were heavier than wooden instruments of the same size, making it difficult to use them in navigation.^[25]^

Spherical astrolabe

A depiction of Hermann of Reichenau with an astrolabe in a 13th-century manuscript by Matthew Paris

Herman Contractus of Reichenau Abbey, examined the use of the astrolabe in Mensura Astrolai during the 11th century.^[26]^ Peter of Maricourt wrote a treatise on the construction and use of a universal astrolabe in the last half of the 13th century entitled Nova compositio astrolabii particularis. Universal astrolabes can be found at the History of Science Museum in Oxford.^[27]^ David A. King, historian of Islamic instrumentation, describes the universal astrolobe designed by Ibn al-Sarraj of Aleppo (aka Ahmad bin Abi Bakr; fl. 1328) as "the most sophisticated astronomical instrument from the entire Medieval and Renaissance periods".^[28]^

English author Geoffrey Chaucer (c. 1343--1400) compiled A Treatise on the Astrolabe for his son, mainly based on a work by Messahalla or Ibn al-Saffar.^[29]^^[30]^ The same source was translated by French astronomer and astrologer Pélerin de Prusse and others. The first printed book on the astrolabe was Composition and Use of Astrolabe by Christian of Prachatice, also using Messahalla, but relatively original.

Front of an Indian astrolabe now kept at the Royal Museum of Scotland at Edinburgh.

In 1370, the first Indian treatise on the astrolabe was written by the Jain astronomer Mahendra Suri, titled Yantrarāja.^[31]^

A simplified astrolabe, known as a balesilha, was used by sailors to get an accurate reading of latitude while at sea. The use of the balesilha was promoted by Prince Henry (1394--1460) while navigating for Portugal.^[32]^

The astrolabe was almost certainly first brought north of the Pyrenees by Gerbert of Aurillac (future Pope Sylvester II), where it was integrated into the quadrivium at the school in Reims, France, sometime before the turn of the 11th century.^[33]^ In the 15th century, French instrument maker Jean Fusoris (c. 1365--1436) also started remaking and selling astrolabes in his shop in Paris, along with portable sundials and other popular scientific devices of the day.

Astronomical Instrument Detail by Ieremias Palladas 1612

Thirteen of his astrolabes survive to this day.^[34]^ One more special example of craftsmanship in early 15th-century Europe is the astrolabe designed by Antonius de Pacento and made by Dominicus de Lanzano, dated 1420.^[35]^

In the 16th century, Johannes Stöffler published Elucidatio fabricae ususque astrolabii, a manual of the construction and use of the astrolabe. Four identical 16th-century astrolabes made by Georg Hartmann provide some of the earliest evidence for batch production by division of labor. In 1612, Greek painter Ieremias Palladas incorporated a sophisticated astrolabe in his painting depicting Catherine of Alexandria. The painting was entitled Catherine of Alexandria and featured a device called the System of the Universe (Σύστημα τοῦ Παντός). The device featured the planets with the names in Greek: Selene (Moon), Hermes (Mercury), Aphrodite (Venus), Helios (Sun), Ares (Mars), Zeus (Jupiter), and Chronos (Saturn). The device also featured celestial spheres following the Ptolemaic model and Earth was depicted as a blue sphere with circles of geographic coordinates. A complex line representing the axis of the Earth covered the entire instrument.^[36]^

Medieval astrolabes

• 

  A treatise explaining the importance of the astrolabe by Nasir al-Din al-Tusi, Persian scientist

• 

  Astrolabe of Jean Fusoris, made in Paris, 1400

• 

  An 18th-century Persian astrolabe

• 

  Disassembled 18th-century astrolabe

• 

  Exploded view of an astrolabe

• 

  Animation showing how celestial and geographic coordinates are mapped on an astrolabe's tympan through a stereographic projection. Hypothetical tympan (40° north latitude) of a 16th-century European planispheric astrolabe.

• 

  Astrolabe manual from the Alfonso X of Castile work Libros del saber de astronomía, 1276.

• 

  A page from the 1575 book "Astrolabium" depicting an astrolabe. Masha'Allah Public Library Bruges [nl] Ms. 522

Astrolabes and clocks

Amerigo Vespucci observing the Southern Cross by looking over the top of an armillary sphere bizarrely held from the top as if it were an astrolabe; however, an astrolabe cannot be used by looking over its top. The page inexplicably contains the word astrolabium. By Jan Collaert II. Museum Plantin-Moretus, Antwerp, Belgium.

Mechanical astronomical clocks were initially influenced by the astrolabe; they could be seen in many ways as clockwork astrolabes designed to produce a continual display of the current position of the sun, stars, and planets. For example, Richard of Wallingford's clock (c. 1330) consisted essentially of a star map rotating behind a fixed rete, similar to that of an astrolabe.^[37]^

Many astronomical clocks use an astrolabe-style display, such as the famous clock at Prague, adopting a stereographic projection (see below) of the ecliptic plane. In recent times, astrolabe watches have become popular. For example, Swiss watchmaker Ludwig Oechslin designed and built an astrolabe wristwatch in conjunction with Ulysse Nardin in 1985.^[38]^ Dutch watchmaker Christaan van der Klauuw also manufactures astrolabe watches today.^[39]^

Construction

An astrolabe consists of a disk, called the mater (mother), which is deep enough to hold one or more flat plates called tympans, or climates. A tympan is made for a specific latitude and is engraved with a stereographic projection of circles denoting azimuth and altitude and representing the portion of the celestial sphere above the local horizon. The rim of the mater is typically graduated into hours of time, degrees of arc, or both.^[40]^

Above the mater and tympan, the rete, a framework bearing a projection of the ecliptic plane and several pointers indicating the positions of the brightest stars, is free to rotate. These pointers are often just simple points, but depending on the skill of the craftsman can be very elaborate and artistic. There are examples of astrolabes with artistic pointers in the shape of balls, stars, snakes, hands, dogs' heads, and leaves, among others.^[40]^ The names of the indicated stars were often engraved on the pointers in Arabic or Latin.^[41]^ Some astrolabes have a narrow rule or label which rotates over the rete, and may be marked with a scale of declinations.

The rete, representing the sky, functions as a star chart. When it is rotated, the stars and the ecliptic move over the projection of the coordinates on the tympan. One complete rotation corresponds to the passage of a day. The astrolabe is, therefore, a predecessor of the modern planisphere.

On the back of the mater, there is often engraved a number of scales that are useful in the astrolabe's various applications. These vary from designer to designer, but might include curves for time conversions, a calendar for converting the day of the month to the sun's position on the ecliptic, trigonometric scales, and graduation of 360 degrees around the back edge. The alidade is attached to the back face. An alidade can be seen in the lower right illustration of the Persian astrolabe above. When the astrolabe is held vertically, the alidade can be rotated and the sun or a star sighted along its length, so that its altitude in degrees can be read ("taken") from the graduated edge of the astrolabe; hence the word's Greek roots: "astron" (ἄστρον) = star + "lab-" (λαβ-) = to take. The alidade had vertical and horizontal cross-hairs which plots locations on an azimuthal ring called an almucantar (altitude-distance circle).

An arm called a radius connects from the center of the astrolabe to the optical axis which is parallel with another arm also called a radius. The other radius contains graduations of altitude and distance measurements.

A shadow square also appears on the back of some astrolabes, developed by Muslim astrologists in the 9th Century, whereas devices of the Ancient Greek tradition featured only altitude scales on the back of the devices.^[42]^ This was used to convert shadow lengths and the altitude of the sun, the uses of which were various from surveying to measuring inaccessible heights.^[43]^

Devices were usually signed by their maker with an inscription appearing on the back of the astrolabe, and if there was a patron of the object, their name would appear inscribed on the front, or in some cases, the name of the reigning sultan or the teacher of the astrolabist has also been found to appear inscribed in this place.^[44]^ The date of the astrolabe's construction was often also signed, which has allowed historians to determine that these devices are the second oldest scientific instrument in the world. The inscriptions on astrolabes also allowed historians to conclude that astronomers tended to make their own astrolabes, but that many were also made to order and kept in stock to sell, suggesting there was some contemporary market for the devices.^[44]^

Construction of astrolabes

• 

  The Hartmann astrolabe in Yale collection. This instrument shows its rete and rule.

• 

  Celestial Globe, Isfahan (?), Iran 1144. Shown at the Louvre Museum, this globe is the third oldest surviving in the world.

• 

  Computer-generated planispheric astrolabe

Mathematical basis

The construction and design of astrolabes are based on the application of the stereographic projection of the celestial sphere. The point from which the projection is usually made is the South Pole. The plane onto which the projection is made is that of the Equator.^[45]^

Designing a tympanum through stereographic projection

Parts of an Astrolabe tympanum

The tympanum captures the celestial coordinate axes upon which the rete will rotate. It is the component that will enable the precise determination of a star's position at a specific time of day and year.

Therefore, it should project:

1. The zenith, which will vary depending on the latitude of the astrolabe user.
2. The horizon line and almucantar or circles parallel to the horizon, which will allow for the determination of a celestial body's altitude (from the horizon to the zenith).
3. The celestial meridian (north-south meridian, passing through the zenith) and secondary meridians (circles intersecting the north-south meridian at the zenith), which will enable the measurement of azimuth for a celestial body.
4. The three main circles of latitude (Capricorn, Equator, and Cancer) to determine the exact moments of solstices and equinoxes throughout the year.

The tropics and the equator define the tympanum

Stereographic projection of Earth's tropics and equator from the South Pole.

On the right side of the image above:

1. The blue sphere represents the celestial sphere.
2. The blue arrow indicates the direction of true north (the North Star).
3. The central blue point represents Earth (the observer's location).
4. The geographic south of the celestial sphere acts as the projection pole.
5. The celestial equatorial plane serves as the projection plane.
6. Three parallel circles represent the projection on the celestial sphere of Earth's main circles of latitude:
   • In orange, the celestial Tropic of Cancer.
   • In purple, the celestial equator.
   • In green, the celestial Tropic of Capricorn.

When projecting onto the celestial equatorial plane, three concentric circles correspond to the celestial sphere's three circles of latitude (left side of the image). The largest of these, the projection on the celestial equatorial plane of the celestial Tropic of Capricorn, defines the size of the astrolabe's tympanum. The center of the tympanum (and the center of the three circles) is actually the north-south axis around which Earth rotates, and therefore, the rete of the astrolabe will rotate around this point as the hours of the day pass (due to Earth's rotational motion).

The three concentric circles on the tympanum are useful for determining the exact moments of solstices and equinoxes throughout the year: if the sun's altitude at noon on the rete is known and coincides with the outer circle of the tympanum (Tropic of Capricorn), it signifies the winter solstice (the sun will be at the zenith for an observer at the Tropic of Capricorn, meaning summer in the southern hemisphere and winter in the northern hemisphere). If, on the other hand, its altitude coincides with the inner circle (Tropic of Cancer), it indicates the summer solstice. If its altitude is on the middle circle (equator), it corresponds to one of the two equinoxes.

The horizon and the measurement of altitude

Stereographic projection of an observer's horizon at a specific latitude

On the right side of the image above:

1. The blue arrow indicates the direction of true north (the North Star).
2. The central blue point represents Earth (the observer's location).
3. The black arrow represents the zenith direction for the observer (which would vary depending on the observer's latitude).
4. The two black circles represent the horizon surrounding the observer, which is perpendicular to the zenith vector and defines the portion of the celestial sphere visible to the observer, and its projection on the celestial equatorial plane.
5. The geographic south of the celestial sphere acts as the projection pole.
6. The celestial equatorial plane serves as the projection plane.

When projecting the horizon onto the celestial equatorial plane, it transforms into an ellipse upward-shifted relatively to the center of the tympanum (both the observer and the projection of the north-south axis). This implies that a portion of the celestial sphere will fall outside the outer circle of the tympanum (the projection of the celestial Tropic of Capricorn) and, therefore, won't be represented.

Stereographic projection of the horizon and an almucantar.

Additionally, when drawing circles parallel to the horizon up to the zenith (almucantar), and projecting them on the celestial equatorial plane, as in the image above, a grid of consecutive ellipses is constructed, allowing for the determination of a star's altitude when its rete overlaps with the designed tympanum.

The meridians and the measurement of azimuth

Stereographic projection of the north-south meridian and a meridian 40° E on the tympanum of an astrolabe

On the right side of the image above:

1. The blue arrow indicates the direction of true north (the North Star).
2. The central blue point represents Earth (the observer's location).
3. The black arrow represents the zenith direction for the observer (which would vary depending on the observer's latitude).
4. The two black circles represent the horizon surrounding the observer, which is perpendicular to the zenith vector and defines the portion of the celestial sphere visible to the observer, and its projection on the celestial equatorial plane.
5. The five red dots represent the zenith, the nadir (the point on the celestial sphere opposite the zenith with respect to the observer), their projections on the celestial equatorial plane, and the center (with no physical meaning attached) of the circle obtained by projecting the secondary meridian (see below) on the celestial equatorial plane.
6. The orange circle represents the celestial meridian (or meridian that goes, for the observer, from the north of the horizon to the south of the horizon passing through the zenith).
7. The two red circles represent a secondary meridian with an azimuth of 40° East relative to the observer's horizon (which, like all secondary meridians, intersects the principal meridian at the zenith and nadir), and its projection on the celestial equatorial plane.
8. The geographic south of the celestial sphere acts as the projection pole.
9. The celestial equatorial plane serves as the projection plane.

When projecting the celestial meridian, it results in a straight line that overlaps with the vertical axis of the tympanum, where the zenith and nadir are located. However, when projecting the 40° E meridian, another circle is obtained that passes through both the zenith and nadir projections, so its center is located on the perpendicular bisection of the segment connecting both points. In deed, the projection of the celestial meridian can be considered as a circle with an infinite radius (a straight line) whose center is on this bisection and at an infinite distance from these two points.

If successive meridians that divide the celestial sphere into equal sectors (like "orange slices" radiating from the zenith) are projected, a family of curves passing through the zenith projection on the tympanum is obtained. These curves, once overlaid with the rete containing the major stars, allow for determining the azimuth of a star located on the rete and rotated for a specific time of day.

See also

• Astronomy in the medieval Islamic world
• Equatorium
• Hamburg Planetarium
• List of astronomical instruments
• Philippe Danfrie, designer and maker of mathematical instruments, globes and astrolabes
• Planetarium
• Yantraraja
• Zeiss-Planetarium Jena

References

Footnotes

1.

1. Savage-Smith, Emilie (1993). "Book Reviews". Journal of Islamic Studies. 4 (2): 296--299. doi:10.1093/jis/4.2.296. There is no evidence for the Hellenistic origin of the spherical astrolabe, but rather evidence so far available suggests that it may have been an early but distinctly Islamic development with no Greek antecedents.

Notes

1.

1. Gentili, Graziano; Simonutti, Luisa; Struppa, Daniele C. (2020). "The Mathematics of the Astrolabe and Its History". Journal of Humanistic Mathematics. 10: 101--144. doi:10.5642/jhummath.202001.07. hdl:2158/1182616. S2CID 211008813.

Bibliography

• Evans, James (1998), The History and Practice of Ancient Astronomy, Oxford University Press, ISBN 0-19-509539-1
• Stöffler, Johannes (2007) [First published 1513], Stoeffler's Elucidatio -- The Construction and Use of the Astrolabe [Elucidatio Fabricae Ususque Astrolabii], translated by Gunella, Alessandro; Lamprey, John, John Lamprey, ISBN 978-1-4243-3502-2
• King, D. A. (1981), "The Origin of the Astrolabe According to the Medieval Islamic Sources", Journal for the History of Arabic Science, 5: 43--83
• King, Henry (1978), Geared to the Stars: the Evolution of Planetariums, Orreries, and Astronomical Clocks, University of Toronto Press, ISBN 978-0-8020-2312-4
• Krebs, Robert E.; Krebs, Carolyn A. (2003), Groundbreaking Scientific Experiments, Inventions, and Discoveries of the Ancient World, Greenwood Press, ISBN 978-0-313-31342-4
• Laird, Edgar (1997), Carol Poster and Richard Utz (ed.), "Astrolabes and the Construction of Time in the Late Middle Ages", Constructions of Time in the Late Middle Ages, Evanston, Illinois: Northwestern University Press: 51--69
• Laird, Edgar; Fischer, Robert, eds. (1995), "Critical edition of Pélerin de Prusse on the Astrolabe (translation of Practique de Astralabe)", Medieval & Renaissance Texts & Studies, Binghamton, New York, ISBN 0-86698-132-2
• Lewis, M. J. T. (2001), Surveying Instruments of Greece and Rome, Cambridge University Press, ISBN 978-0-511-48303-5
• Morrison, James E. (2007), The Astrolabe, Janus, ISBN 978-0-939320-30-1
• Neugebauer, Otto E. (1975), A History of Ancient Mathematical Astronomy, Springer, ISBN 978-3-642-61912-0
• North, John David (2005), God's Clockmaker: Richard of Wallingford and the Invention of Time, Continuum International Publishing Group, ISBN 978-1-85285-451-5

External links

Wikimedia Commons has media related to:\ Astrolabe (category)

Wikisource has the text of the 1911 Encyclopædia Britannica article "Astrolabe".

Look up astrolabe in Wiktionary, the free dictionary.

• Interactive digital astrolabe by Alex Boxer
• A digital astrolabe (HTML5 and javascript)
• Astrolabe Tech Made ... Not So Easy
• paper astrolabe generator, from the ESO
• "Hello World!" for the Astrolabe: The First Computer Video of Howard Covitz's Presentation at Ignite Phoenix, June 2009. Slides for Presentation Licensed as Creative Commons by-nc-nd.
• Video of Tom Wujec demonstrating an astrolabe. Archived 2012-03-23 at the Wayback Machine Taken at TEDGlobal 2009. Includes clickable transcript. Licensed as Creative Commons by-nc-nd.
• Archive of James E. Morrison's extensive website on Astrolabes
• A working model of the Dr. Ludwig Oechslin's Astrolabium Galileo Galilei watch
• Ulysse Nardin Astrolabium Galilei Galileo: A Detailed Explanation
• Fully illustrated online catalogue of world's largest collection of astrolabes
• Mobile astrolabe and horologium
• Medieval equal hour horary quadrant
• A Beginner's Guide to Basic Construction and Use of the Astrolabe (using ruler, protractor and compasses) (PDF), archived from the original (PDF) on 2015-06-17, retrieved 2018-10-26

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  ](https://en.wikipedia.org/wiki/Astrolabe#Construction)- [

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  ](https://en.wikipedia.org/wiki/Astrolabe#Mathematical_basis)- Modern editions of John Philoponus' treatise on the astrolabe are De usu astrolabii eiusque constructione libellus (On the Use and Construction of the Astrolabe), ed. Heinrich Hase, Bonn: E. Weber, 1839, OCLC 165707441 (or id. Rheinisches Museum für Philologie 6 (1839): 127--71); repr. and translated into French by Alain Philippe Segonds, Jean Philopon, traité de l'astrolabe, Paris: Librairie Alain Brieux, 1981, OCLC 10467740; and translated into English by H.W. Green in R.T. Gunther, The Astrolabes of the World, Vol. 1/2, Oxford, 1932, OL 18840299M repr. London: Holland Press, 1976, OL 14132393M pp. 61--81.- O'Leary, De Lacy (1948). How Greek Science Passed to the Arabs. Routledge and Kegan Paul. "The most distinguished Syriac scholar of this later period was Severus Sebokht (d. 666--7), Bishop of Kennesrin. [...] In addition to these works [...] he also wrote on astronomical subjects (Brit. Mus. Add. 14538), and composed a treatise on the astronomical instrument known as the astrolabe, which has been edited and published by F. Nau (Paris, 1899)."\ Severus' treatise was translated by Jessie Payne Smith Margoliouth in R.T. Gunther, Astrolabes of the World, Oxford, 1932, pp. 82--103.- "HISTORIANS' HOME YIELDS RICH LODE; New York Society Searches Its Own Building for Items to Mark Anniversary; SHOW OPENS THURSDAY; Portrait of Stuyvesant and Champlain's Astrolabe Will Be on Display". The New York Times. May 18, 1964. Retrieved February 4, 2024.- Bean, Adam L. (2009). "Astrolabes". In Birx, H. James (ed.). Encyclopedia of Time: Science, Philosophy, Theology, & Culture. Vol. 1. SAGE. pp. 59--60. ISBN 978-1-4129-4164-8.- "Astrolabe". Oxford English Dictionary (2nd ed.). 1989.- "Astrolabe". Oxford Dictionaries. Archived from the original on October 22, 2013.- "Online Etymology Dictionary". Etymonline.com. Retrieved 2013-11-07.- King 1981, p. 44.- King 1981, p. 51.- King 1981, p. 45.- Lewis 2001.- Michael Deakin (August 3, 1997). "Ockham's Razor: Hypatia of Alexandria". ABC Radio. Retrieved July 10, 2014.- Theodore, Jonathan (2016). The Modern Cultural Myth of the Decline and Fall of the Roman Empire. Manchester, England: Palgrave, Macmillan. p. 183. ISBN 978-1-137-56997-4.- Deakin, Michael A. B. (2007). Hypatia of Alexandria: Mathematician and Martyr. Amherst, New York: Prometheus Books. pp. 102--104. ISBN 978-1-59102-520-7.- Bradley, Michael John (2006). The Birth of Mathematics: Ancient Times to 1300. New York City, New York: Infobase Publishing. p. 63. ISBN 9780816054237.- Savage-Smith, Emilie (1992). "Celestial Mapping" (PDF). In Harley, J. B.; Woodward, David (eds.). The History of Cartography, Volume 2, Book 1: Cartography in the Traditional Islamic and South Asian Societies. The History of Cartography. Vol. 2. Chicago, Illinois: University of Chicago Press. ISBN 0226316351.- Sebokht, Severus. "Description of the astrolabe". Tertullian.org.- See p. 289 of Martin, L. C. (1923), "Surveying and navigational instruments from the historical standpoint", Transactions of the Optical Society, 24 (5): 289--303, Bibcode:1923TrOS...24..289M, doi:10.1088/1475-4878/24/5/302, ISSN 1475-4878.- Berggren, J. Lennart (2007), "Mathematics in Medieval Islam", in Katz, Victor J. (ed.), The Mathematics of Egypt, Mesopotamia, China, India, and Islam: a Sourcebook, Princeton University Press, p. 519, ISBN 978-0-691-11485-9- Richard Nelson Frye: Golden Age of Persia. p. 163- Nizamoglu, Cem (2005-08-10). "Using an Astrolabe". Muslim Heritage. Retrieved 2023-10-16.- Lachièz-Rey, Marc; Luminet, Jean-Pierre (2001). Celestial Treasury: From the Music of Spheres to the Conquest of Space. Translated by Joe Laredo. Cambridge, United Kingdom: Cambridge University Press. p. 74. ISBN 978-0-521-80040-2.- O'Connor, John J.; Robertson, Edmund F., "Sharaf al-Din al-Muzaffar al-Tusi", MacTutor History of Mathematics Archive, University of St Andrews- Bedini, Silvio A.; Maddison, Francis R. (1966). "Mechanical Universe: The Astrarium of Giovanni de' Dondi". Transactions of the American Philosophical Society. 56 (5): 1--69. doi:10.2307/1006002. JSTOR 1006002.- "Qantara -- 'Carolingian' astrolabe". Qantara-med.org. Retrieved 2013-11-07.- Nancy Marie Brown (2010), "The Abacus and the Cross". p. 140. Basic Books. ISBN 978-0-465-00950-3- Boyle, David (2011). Toward the Setting Sun: Columbus, Cabot, Vespucci, and the Race for America. Bloomsbury Publishing USA. p. 253. ISBN 9780802779786..- Northrup, Cynthia Clark, ed. (2015). Encyclopedia of world trade: from ancient times to the present (Enhanced Credo ed.). Armonk, New York: Routledge. pp. 72. ISBN 978-0765680587. OCLC 889717964.- "Introduction". The Astrolabe: an Online Resource. 2006. Retrieved 2020-05-15.- Harley, J. B.; Woodward, David (1992). The history of cartography. Chicago, Illinois: University of Chicago Press. p. 31. ISBN 0-226-31635-1.- Kunitzsch, Paul (1981). "On the authenticity of the treatise on the composition and use of the astrolabe ascribed to Messahalla". Archives Internationales d'Histoire des Sciences Oxford. 31 (106): 42--62.- Selin, Helaine (2008-03-12). Encyclopaedia of the History of Science, Technology, and Medicine in Non-Western Cultures. Springer Science & Business Media. p. 1335. ISBN 978-1-4020-4559-2. Paul Kunitzsch has recently established that the Latin treatise on the astrolabe long ascribed to Ma'sh'allah and translated by John of Seville is in fact by Ibn al-Saffar, a disciple of Maslama al-Majriti.- Glick, Thomas; et al., eds. (2005), Medieval Science, Technology, and Medicine: An Encyclopedia, Routledge, p. 464, ISBN 0-415-96930-1- Northrup, Cynthia Clark, ed. (2015). Encyclopedia of world trade : from ancient times to the present ([Enhanced Credo edition] ed.). Armonk, New York: Routledge. pp. 460. ISBN 978-0765680587. OCLC 889717964.- Nancy Marie Brown (2010), "The Abacus and the Cross". p. 143. basic Books. ISBN 978-0-465-00950-3- Hockey, Thomas (2009). The Biographical Encyclopedia of Astronomers. Springer Publishing. ISBN 978-0-387-31022-0. Retrieved August 22, 2012.- Ralf Kern (2010), Wissenschaftliche Instrumente in ihrer Zeit. Band 1: Vom Astrolab zum mathematischen Besteck. Cologne, S. 204. ISBN 978-3-86560-865-9- Vafea, Flora (2017). "The Astronomical Instruments in Saint Catherine's Iconography at the Holy Monastery of Sinai The Almagest Volume 8, Issue 2". Almagest. 8 (2). Paris: University of Paris: 87. doi:10.1484/J.ALMAGEST.5.114932.- North 2005.- "Astrolabium G. Galilei". Ulysse Nardin. Archived from the original on 2 January 2011.- "Christaan van der Klauuw".- Stephenson, Bruce; Bolt, Marvin; Friedman, Anna Felicity (2000). The Universe Unveiled: Instruments and Images through History. Cambridge, UK: Cambridge University Press. pp. 108--109. ISBN 0-521-79143-X.- "Star Names on Astrolabes". Ian Ridpath. Retrieved 2016-11-12.- King, David A. Some Medieval Astronomical Instruments and Their Secrets, in Mazzolini, R. G. (ed.), Non-Verbal Communication in Science prior to 1900. Florence. p. 30.- King, David A. (2018). The Astrolabe: What it is & what it is not. Frankfurt, Germany: Frankfurt.- Mayer, L. A. (1956). Islamic astrolabists and their works*. A. Kunding. Bibcode:[1956iatw.book.....M](https://ui.a Main menu

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From Wikipedia, the free encyclopedia This article is about the sextant as used for navigation. For other uses, see Sextant (disambiguation). Not to be confused with Sexton (disambiguation). A sextant

A sextant is a doubly reflecting navigation instrument that measures the angular distance between two visible objects. The primary use of a sextant is to measure the angle between an astronomical object and the horizon for the purposes of celestial navigation.

The estimation of this angle, the altitude, is known as sighting or shooting the object, or taking a sight. The angle, and the time when it was measured, can be used to calculate a position line on a nautical or aeronautical chart—for example, sighting the Sun at noon or Polaris at night (in the Northern Hemisphere) to estimate latitude (with sight reduction). Sighting the height of a landmark can give a measure of distance off and, held horizontally, a sextant can measure angles between objects for a position on a chart.[1] A sextant can also be used to measure the lunar distance between the moon and another celestial object (such as a star or planet) in order to determine Greenwich Mean Time and hence longitude.

The principle of the instrument was first implemented around 1731 by John Hadley (1682–1744) and Thomas Godfrey (1704–1749), but it was also found later in the unpublished writings of Isaac Newton (1643–1727).

In 1922, it was modified for aeronautical navigation by Portuguese navigator and naval officer Gago Coutinho. Navigational sextants

Like the Davis quadrant, the sextant allows celestial objects to be measured relative to the horizon, rather than relative to the instrument. This allows excellent precision. Also, unlike the backstaff, the sextant allows direct observations of stars. This permits the use of the sextant at night when a backstaff is difficult to use. For solar observations, filters allow direct observation of the Sun.

Since the measurement is relative to the horizon, the measuring pointer is a beam of light that reaches to the horizon. The measurement is thus limited by the angular accuracy of the instrument and not the sine error of the length of an alidade, as it is in a mariner's astrolabe or similar older instrument.

A sextant does not require a completely steady aim, because it measures a relative angle. For example, when a sextant is used on a moving ship, the image of both horizon and celestial object will move around in the field of view. However, the relative position of the two images will remain steady, and as long as the user can determine when the celestial object touches the horizon, the accuracy of the measurement will remain high compared to the magnitude of the movement.

The sextant is not dependent upon electricity (unlike many forms of modern navigation) or any human-controlled signals (such as GPS). For these reasons it is considered to be an eminently practical back-up navigation tool for ships. Design

The frame of a sextant is in the shape of a sector which is approximately 1⁄6 of a circle (60°),[2] hence its name (sextāns, sextantis is the Latin word for "one sixth"). Both smaller and larger instruments are (or were) in use: the octant, quintant (or pentant) and the (doubly reflecting) quadrant[3] span sectors of approximately 1⁄8 of a circle (45°), 1⁄5 of a circle (72°) and 1⁄4 of a circle (90°), respectively. All of these instruments may be termed "sextants". Marine sextant Using the sextant to measure the altitude of the Sun above the horizon Sextants can also be used by navigators to measure horizontal angles between objects.

Attached to the frame are the "horizon mirror", an index arm which moves the index mirror, a sighting telescope, Sun shades, a graduated scale and a micrometer drum gauge for accurate measurements. The scale must be graduated so that the marked degree divisions register twice the angle through which the index arm turns. The scales of the octant, sextant, quintant and quadrant are graduated from below zero to 90°, 120°, 140° and 180° respectively. For example, the sextant illustrated has a scale graduated from −10° to 142°, which is basically a quintant: the frame is a sector of a circle subtending an angle of 76° at the pivot of the index arm.

The necessity for the doubled scale reading follows from consideration of the relations of the fixed ray (between the mirrors), the object ray (from the sighted object) and the direction of the normal perpendicular to the index mirror. When the index arm moves by an angle, say 20°, the angle between the fixed ray and the normal also increases by 20°. But the angle of incidence equals the angle of reflection so the angle between the object ray and the normal must also increase by 20°. The angle between the fixed ray and the object ray must therefore increase by 40°. This is the case shown in the graphic.

There are two types of horizon mirrors on the market today. Both types give good results.

Traditional sextants have a half-horizon mirror, which divides the field of view in two. On one side, there is a view of the horizon; on the other side, a view of the celestial object. The advantage of this type is that both the horizon and celestial object are bright and as clear as possible. This is superior at night and in haze, when the horizon and/or a star being sighted can be difficult to see. However, one has to sweep the celestial object to ensure that the lowest limb of the celestial object touches the horizon.

Whole-horizon sextants use a half-silvered horizon mirror to provide a full view of the horizon. This makes it easy to see when the bottom limb of a celestial object touches the horizon. Since most sights are of the Sun or Moon, and haze is rare without overcast, the low-light advantages of the half-horizon mirror are rarely important in practice.

In both types, larger mirrors give a larger field of view, and thus make it easier to find a celestial object. Modern sextants often have 5 cm or larger mirrors, while 19th-century sextants rarely had a mirror larger than 2.5 cm (one inch). In large part, this is because precision flat mirrors have grown less expensive to manufacture and to silver.

An artificial horizon is useful when the horizon is invisible, as occurs in fog, on moonless nights, in a calm, when sighting through a window or on land surrounded by trees or buildings. There are two common designs of artificial horizon. An artificial horizon can consist simply of a pool of water shielded from the wind, allowing the user to measure the distance between the body and its reflection, and divide by two. Another design allows the mounting of a fluid-filled tube with bubble directly to the sextant.

Most sextants also have filters for use when viewing the Sun and reducing the effects of haze. The filters usually consist of a series of progressively darker glasses that can be used singly or in combination to reduce haze and the Sun's brightness. However, sextants with adjustable polarizing filters have also been manufactured, where the degree of darkness is adjusted by twisting the frame of the filter.

Most sextants mount a 1 or 3-power monocular for viewing. Many users prefer a simple sighting tube, which has a wider, brighter field of view and is easier to use at night. Some navigators mount a light-amplifying monocular to help see the horizon on moonless nights. Others prefer to use a lit artificial horizon.[citation needed]

Professional sextants use a click-stop degree measure and a worm adjustment that reads to a minute, 1/60 of a degree. Most sextants also include a vernier on the worm dial that reads to 0.1 minute. Since 1 minute of error is about a nautical mile, the best possible accuracy of celestial navigation is about 0.1 nautical miles (190 m). At sea, results within several nautical miles, well within visual range, are acceptable. A highly skilled and experienced navigator can determine position to an accuracy of about 0.25-nautical-mile (460 m).[4]

A change in temperature can warp the arc, creating inaccuracies. Many navigators purchase weatherproof cases so that their sextant can be placed outside the cabin to come to equilibrium with outside temperatures. The standard frame designs (see illustration) are supposed to equalise differential angular error from temperature changes. The handle is separated from the arc and frame so that body heat does not warp the frame. Sextants for tropical use are often painted white to reflect sunlight and remain relatively cool. High-precision sextants have an invar (a special low-expansion steel) frame and arc. Some scientific sextants have been constructed of quartz or ceramics with even lower expansions. Many commercial sextants use low-expansion brass or aluminium. Brass is lower-expansion than aluminium, but aluminium sextants are lighter and less tiring to use. Some say they are more accurate because one's hand trembles less. Solid brass frame sextants are less susceptible to wobbling in high winds or when the vessel is working in heavy seas, but as noted are substantially heavier. Sextants with aluminum frames and brass arcs have also been manufactured. Essentially, a sextant is intensely personal to each navigator, and they will choose whichever model has the features which suit them best.

Aircraft sextants are now out of production, but had special features. Most had artificial horizons to permit taking a sight through a flush overhead window. Some also had mechanical averagers to make hundreds of measurements per sight for compensation of random accelerations in the artificial horizon's fluid. Older aircraft sextants had two visual paths, one standard and the other designed for use in open-cockpit aircraft that let one view from directly over the sextant in one's lap. More modern aircraft sextants were periscopic with only a small projection above the fuselage. With these, the navigator pre-computed their sight and then noted the difference in observed versus predicted height of the body to determine their position. Taking a sight

A sight (or measure) of the angle between the Sun, a star, or a planet, and the horizon is done with the 'star telescope' fitted to the sextant using a visible horizon. On a vessel at sea even on misty days a sight may be done from a low height above the water to give a more definite, better horizon. Navigators hold the sextant by its handle in the right hand, avoiding touching the arc with the fingers.[5]

For a Sun sight, a filter is used to overcome the glare such as "shades" covering both index mirror and the horizon mirror designed to prevent eye damage. Initially, with the index bar set to zero and the shades covering both mirrors, the sextant is aimed at the sun until it can be viewed on both mirrors through the telescope, then lowered vertically until the portion of the horizon directly below it is viewed on both mirrors. It is necessary to flip back the horizon mirror shade to be able to see the horizon more clearly on it. Releasing the index bar (either by releasing a clamping screw, or on modern instruments, using the quick-release button), and moving it towards higher values of the scale, eventually the image of the Sun will reappear on the index mirror and can be aligned to about the level of the horizon on the horizon mirror. Then the fine adjustment screw on the end of the index bar is turned until the bottom curve (the lower limb) of the Sun just touches the horizon. "Swinging" the sextant about the axis of the telescope ensures that the reading is being taken with the instrument held vertically. The angle of the sight is then read from the scale on the arc, making use of the micrometer or vernier scale provided. The exact time of the sight must also be noted simultaneously, and the height of the eye above sea-level recorded.[5]

An alternative method is to estimate the current altitude (angle) of the Sun from navigation tables, then set the index bar to that angle on the arc, apply suitable shades only to the index mirror, and point the instrument directly at the horizon, sweeping it from side to side until a flash of the Sun's rays are seen in the telescope. Fine adjustments are then made as above. This method is less likely to be successful for sighting stars and planets.[5]

Star and planet sights are normally taken during nautical twilight at dawn or dusk, while both the heavenly bodies and the sea horizon are visible. There is no need to use shades or to distinguish the lower limb as the body appears as a mere point in the telescope. The Moon can be sighted, but it appears to move very fast, appears to have different sizes at different times, and sometimes only the lower or upper limb can be distinguished due to its phase.[5]

After a sight is taken, it is reduced to a position by looking at several mathematical procedures. The simplest sight reduction is to draw the equal-altitude circle of the sighted celestial object on a globe. The intersection of that circle with a dead-reckoning track, or another sighting, gives a more precise location.

Sextants can be used very accurately to measure other visible angles, for example between one heavenly body and another and between landmarks ashore. Used horizontally, a sextant can measure the apparent angle between two landmarks such as a lighthouse and a church spire, which can then be used to find the distance off or out to sea (provided the distance between the two landmarks is known). Used vertically, a measurement of the angle between the lantern of a lighthouse of known height and the sea level at its base can also be used for distance off.[5] Adjustment

Due to the sensitivity of the instrument it is easy to knock the mirrors out of adjustment. For this reason a sextant should be checked frequently for errors and adjusted accordingly.

There are four errors that can be adjusted by the navigator, and they should be removed in the following order.

Perpendicularity error This is when the index mirror is not perpendicular to the frame of the sextant. To test for this, place the index arm at about 60° on the arc and hold the sextant horizontally with the arc away from you at arm's length and look into the index mirror. The arc of the sextant should appear to continue unbroken into the mirror. If there is an error, then the two views will appear to be broken. Adjust the mirror until the reflection and direct view of the arc appear to be continuous. Side error This occurs when the horizon glass/mirror is not perpendicular to the plane of the instrument. To test for this, first zero the index arm then observe a star through the sextant. Then rotate the tangent screw back and forth so that the reflected image passes alternately above and below the direct view. If in changing from one position to another, the reflected image passes directly over the unreflected image, no side error exists. If it passes to one side, side error exists. Alternatively, the user can hold the sextant on its side and observe the horizon to check the sextant during the day. If there are two horizons there is side error. In both cases, adjust the horizon glass/mirror until respectively the star or the horizon dual images merge into one. Side error is generally inconsequential for observations and can be ignored or reduced to a level that is merely inconvenient. Collimation error This is when the telescope or monocular is not parallel to the plane of the sextant. To check for this you need to observe two stars 90° or more apart. Bring the two stars into coincidence either to the left or the right of the field of view. Move the sextant slightly so that the stars move to the other side of the field of view. If they separate there is collimation error. As modern sextants rarely use adjustable telescopes, they do not need to be corrected for collimation error. Index error This occurs when the index and horizon mirrors are not parallel to each other when the index arm is set to zero. To test for index error, zero the index arm and observe the horizon. If the reflected and direct image of the horizon are in line there is no index error. If one is above the other adjust the index mirror until the two horizons merge. Alternatively, the same procedure can be done at night using a star or the Moon instead of the horizon.

See also

Astrolabe
Bris sextant
Davis quadrant
Gago Coutinho
Harold Gatty
History of longitude
Intercept method
Latitude
Longitude
Longitude by chronometer
Mariner's astrolabe
Navigation
Octant (instrument)
Quadrant (instrument)
Sextant (astronomy)

Notes

Seddon, J. Carl (June 1968). "Line of Position from a Horizontal Angle". Journal of Navigation. 21 (3): 367–369. doi:10.1017/S0373463300024838. ISSN 1469-7785. A.), McPhee, John (John; NSW., Museums and Galleries (2008). Great Collections : treasures from Art Gallery of NSW, Australian Museum, Botanic Gardens Trust, Historic Houses Trust of NSW, Museum of Contemporary Art, Powerhouse Museum, State Library of NSW, State Records NSW. Museums & Galleries NSW. p. 56. ISBN 9780646496030. OCLC 302147838. This article treats the doubly reflecting quadrant, not its predecessor described at quadrant. Dutton's Navigation and Piloting, 12th edition. G.D. Dunlap and H.H. Shufeldt, eds. Naval Institute Press 1972, ISBN 0-87021-163-3

Dixon, Conrad (1968). "5. Using the sextant". Basic Astro Navigation. Adlard Coles. ISBN 0-229-11740-6.

References

Bowditch, Nathaniel (2002). The American Practical Navigator. Bethesda, MD: National Imagery and Mapping Agency. ISBN 0-939837-54-4. Archived from the original on 2007-06-24.
Chisholm, Hugh, ed. (1911). "Sextant" . Encyclopædia Britannica. Vol. 24 (11th ed.). Cambridge University Press. pp. 765–767.
Cutler, Thomas J. (December 2003). Dutton's Nautical Navigation (15th ed.). Annapolis, MD: Naval Institute Press. ISBN 978-1-55750-248-3.
Department of the Air Force (March 2001). Air Navigation (PDF). Department of the Air Force. Retrieved 2014-12-28.
Great Britain Ministry of Defence (Navy) (1995). Admiralty Manual of Seamanship. The Stationery Office. ISBN 0-11-772696-6.
Maloney, Elbert S. (December 2003). Chapman Piloting and Seamanship (64th ed.). New York: Hearst Communications. ISBN 1-58816-089-0.
Martin, William Robert (1911). "Navigation" . In Chisholm, Hugh (ed.). Encyclopædia Britannica. Vol. 19 (11th ed.). Cambridge University Press. pp. 284–298.

External links Look up sextant in Wiktionary, the free dictionary. Wikimedia Commons has media related to Sextant.

Her Majesty's Nautical Almanac Office Archived 2011-02-21 at the Wayback Machine
The History of HM Nautical Almanac Office Archived 2016-06-24 at the Wayback Machine
Chapter 17 from the online edition of Nathaniel Bowditch's American Practical Navigator
Understand difference in Antique & Replica Sextant Archived 2017-08-17 at the Wayback Machine
CD-Sextant - Build your own sextant Simple do-it-yourself project.
Lunars web site. online calculation
Complete celnav theory book, including Lunars

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dsabs.harvard.edu/abs/1956iatw.book.....M).*

BEGINNINGS WITH "THE THE" AND LL and EL ELtelitenkigniYONNA

This article is about the U.S. government project on psychics. For other uses, see Stargate (disambiguation). Part of a series on the Paranormal Main articles Skepticism Parapsychology Related

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The Stargate Project was a secret U.S. Army unit established in 1977[1][2] at Fort Meade, Maryland, by the Defense Intelligence Agency (DIA) and SRI International (a California contractor) to investigate the potential for psychic phenomena in military and domestic intelligence applications. The project, and its precursors and sister projects, originally went by various code names – 'Gondola Wish', 'Stargate', 'Grill Flame', 'Center Lane', 'Project CF', 'Sun Streak', 'Scanate' – until 1991 when they were consolidated and rechristened as the "Stargate Project".

The Stargate Project's work primarily involved remote viewing, the purported ability to psychically "see" events, sites, or information from a great distance.[3] The project was overseen until 1987 by Lt. Frederick Holmes "Skip" Atwater, an aide and "psychic headhunter" to Maj. Gen. Albert Stubblebine, and later president of the Monroe Institute.[4] The unit was small scale, comprising about 15 to 20 individuals, and was run out of "an old, leaky wooden barracks".[5]

The Stargate Project was terminated and declassified in 1995 after a CIA report concluded that it was never useful in any intelligence operation. Information provided by the program was vague and included irrelevant and erroneous data, and there were suspicions of inter-judge reliability.[6]: 5–4  The program was featured in the 2004 book and 2009 film, both titled The Men Who Stare at Goats,[7][8][9][10] although neither mentions it by name. George Stephanopoulos, in his 2024 book The Situation Room, mentions the project by the name Grill Flame, in discussing a May 8, 1980, Situation Room briefing for President Carter, after Carter's failed hostage rescue mission in Iran on April 24, 1980.[11] Background

The CIA and DIA decided they should investigate and know as much about it as possible. Various programs were approved yearly and re-funded accordingly. Reviews were made semi-annually at the Senate and House select committee level. Work results were reviewed, and remote viewing was attempted with the results being kept secret from the "viewer". It was thought that if the viewer was shown they were incorrect it would damage the viewer's confidence and skill. This was standard operating procedure throughout the years of military and domestic remote viewing programs. Feedback to the remote viewer of any kind was rare; it was kept classified and secret.[12]

Remote viewing attempts to sense unknown information about places or events. Normally it is performed to detect current events, but during military and domestic intelligence applications viewers claimed to sense things in the future, experiencing precognition.[13] History 1970s

In 1970 United States intelligence sources believed that the Soviet Union was spending 60 million roubles annually on "psychotronic" research. In response to claims that the Soviet program had produced results, the CIA initiated funding for a new program known as SCANATE ("scan by coordinate") in the same year.[14] Remote viewing research began in 1972 at the Stanford Research Institute (SRI) in Menlo Park, California.[14][15] Proponents (Russell Targ and Harold Puthoff) of the research said that a minimum accuracy rate of 65% required by the clients was often exceeded in the later experiments.[14]

Physicists Targ and Puthoff began testing psychics for SRI in 1972, including one who would later become an international celebrity, Israeli Uri Geller. Their apparently successful results garnered interest within the U.S. Department of Defense. Ray Hyman, professor of psychology at the University of Oregon, was asked by Air Force psychologist Lt. Col. Austin W. Kibler (1930–2008) – then Director of Behavioral Research for ARPA – to go to SRI and investigate. He was to specifically evaluate Geller. Hyman's report to the government was that Geller was a "complete fraud" and as a consequence Targ and Puthoff lost their government contract to work further with him. The result was a publicity tour for Geller, Targ, and Puthoff to seek private funding for further research work on Geller.[16]

One of the project's successes was the location of a lost Soviet spy plane in 1976 by Rosemary Smith, a young administrative assistant recruited by project director Dale Graff.[17]

In 1977 the Army Assistant Chief of Staff for Intelligence (ACSI) Systems Exploitation Detachment (SED) started the Gondola Wish program to "evaluate potential adversary applications of remote viewing".[14] Army Intelligence then formalized this in mid-1978 as an operational program Grill Flame, based in buildings 2560 and 2561 at Fort Meade, in Maryland (INSCOM "Detachment G").[14] 1980s

In early 1979 the research at SRI was integrated into 'Grill Flame', which was redesignated INSCOM 'Center Lane' Project (ICLP) in 1983. In 1984 the existence of the program was reported by Jack Anderson, and in that year it was unfavorably received by the National Academy of Sciences National Research Council. In late 1985 the Army funding was terminated, but the program was redesignated 'Sun Streak' and funded by the DIA's Scientific and Technical Intelligence Directorate (office code DT-S).[14] 1990s

In 1991 most of the contracting for the program was transferred from SRI to Science Applications International Corporation (SAIC), with Edwin May controlling 70% of the contractor funds and 85% of the data. Its security was altered from Special Access Program (SAP) to Limited Dissemination (LIMDIS), and it was given its final name, STARGATE.[14] Closure (1995)

In 1995 the defense appropriations bill directed that the program be transferred from DIA to CIA oversight. The CIA commissioned a report by the American Institutes for Research (AIR) that found that remote viewing had not been proved to work by a psychic mechanism, and said it had not been used operationally.[6]: 5–4  The CIA subsequently cancelled and declassified the program.[14]

In 1995 the project was transferred to the CIA and a retrospective evaluation of the results was done. The appointed panel consisted primarily of Jessica Utts, Meena Shah and Ray Hyman. Hyman had produced an unflattering report on Uri Geller and SRI for the government two decades earlier, but the psychologist David Marks found Utts' appointment to the review panel "puzzling" given that she had published papers with Edwin May, considering this joint research likely to make her "less than [im]partial".[3] A report by Utts claimed the results were evidence of psychic functioning; however, Hyman in his report argued Utts's conclusion that ESP had been proven to exist, especially precognition, was premature and the findings had not been independently replicated.[18] Hyman came to the conclusion:

Psychologists, such as myself, who study subjective validation find nothing striking or surprising in the reported matching of reports against targets in the Stargate data. The overwhelming amount of data generated by the viewers is vague, general, and way off target. The few apparent hits are just what we would expect if nothing other than reasonable guessing and subjective validation are operating.[19]

A later report by AIR came to a negative conclusion. Joe Nickell has written:

Other evaluators – two psychologists from AIR – assessed the potential intelligence-gathering usefulness of remote viewing. They concluded that the alleged psychic technique was of dubious value and lacked the concreteness and reliability necessary for it to be used as a basis for making decisions or taking action. The final report found "reason to suspect" that in "some well publicised cases of dramatic hits" the remote viewers might have had "substantially more background information" than might otherwise be apparent.[20]

According to AIR, which performed a review of the project, no remote viewing report ever provided actionable information for any intelligence operation.[21][6]: 5–4 

Based upon the collected findings, which recommended a higher level of critical research and tighter controls, the CIA terminated the 20 million dollar project, citing a lack of documented evidence that the program had any value to the intelligence community. Time magazine stated in 1995 three full-time psychics were still working on a $500,000-a-year budget out of Fort Meade, Maryland, which would soon close.[21]

David Marks in his book The Psychology of the Psychic (2000) discussed the flaws in the Stargate Project in detail.[3] Marks wrote that there were six negative design features of the experiments. The possibility of cues or sensory leakage was not ruled out, no independent replication, some experiments were conducted in secret, making peer-review impossible. Marks noted that the judge Edwin May was also the principal investigator for the project and this was problematic, making a huge conflict of interest with collusion, cuing and fraud being possible. Marks concluded the project was nothing more than a "subjective delusion" and after two decades of research it had failed to provide any scientific evidence for the legitimacy of remote viewing.[3]

The Stargate Project was terminated in 1995 following an independent review which concluded:

The foregoing observations provide a compelling argument against continuation of the program within the intelligence community. Even though a statistically significant effect has been observed in the laboratory, it remains unclear whether the existence of a paranormal phenomenon, remote viewing, has been demonstrated. The laboratory studies do not provide evidence regarding the origins or nature of the phenomenon, assuming it exists, nor do they address an important methodological issue of inter-judge reliability.

Further, even if it could be demonstrated unequivocally that a paranormal phenomenon occurs under the conditions present in the laboratory paradigm, these conditions have limited applicability and utility for intelligence gathering operations. For example, the nature of the remote viewing targets are vastly dissimilar, as are the specific tasks required of the remote viewers. Most importantly, the information provided by remote viewing is vague and ambiguous, making it difficult, if not impossible, for the technique to yield information of sufficient quality and accuracy of information for actionable intelligence. Thus, we conclude that continued use of remote viewing in intelligence gathering operations is not warranted.[6]: E-4–E-5

In January 2017, the CIA published records online of the Stargate Project as part of the CREST archive.[22] Methodology

The Stargate Project created a set of protocols designed to make the research of clairvoyance and out-of-body experiences more scientific, and to minimize as much as possible session noise and inaccuracy. The term "remote viewing" emerged as shorthand to describe this more structured approach to clairvoyance. Project Stargate would only receive a mission after all other intelligence attempts, methods, or approaches had already been exhausted.[13]: 21 

It was reported that at peak manpower there were over 22 active military and civilian remote viewers providing data. People leaving the project were not replaced. When the project closed in 1995 this number had dwindled down to three. One was using tarot cards. According to Joseph McMoneagle, "The Army never had a truly open attitude toward psychic functioning". Hence, the use of the term "giggle factor"[23] and the saying, "I wouldn't want to be found dead next to a psychic".[12] Civilian personnel Hal Puthoff Main article: Harold E. Puthoff

In the 1970s, CIA and DIA granted funds to Harold E. Puthoff to investigate paranormal abilities, collaborating with Russell Targ in a study of the purported psychic abilities of Uri Geller, Ingo Swann, Pat Price, Joseph McMoneagle and others, as part of the Stargate Project,[24] of which Puthoff became a director.[25]

As with Ingo Swann and Pat Price, Puthoff attributed much of his personal remote viewing skills to his involvement with Scientology whereby he had attained, at that time, the highest level. All three eventually left Scientology in the late 1970s.

Puthoff worked as the principal investigator of the project. His team of psychics is said[who?] to have identified spies, located Soviet weapons and technologies, such as a nuclear submarine in 1979 and helped find lost SCUD missiles in the first Gulf War and plutonium in North Korea in 1994.[26] Russell Targ Russell Targ Main article: Russell Targ

In the 1970s, Russell Targ began working with Harold Puthoff on the Stargate Project, while working with him as a researcher at Stanford Research Institute.[27][28] Edwin May

Edwin C. May joined the Stargate Project in 1975 as a consultant and was working full-time in 1976. The original project was part of the Cognitive Sciences Laboratory managed by May. With more funding in 1991 May took the project to the Palo Alto offices at SAIC. This would last until 1995 when the CIA closed the project.[3]

May worked as the principal investigator, judge and the star gatekeeper for the project. Marks says this was a serious weakness for the experiments as May had conflict of interest and could have done whatever he wanted with the data. Marks has written that May refused to release the names of the "oversight committee" and refused permission for him to give an independent judging of the Stargate transcripts. Marks found this suspicious, commenting "this refusal suggests that something must be wrong with the data or with the methods of data selection."[3] Ingo Swann Main article: Ingo Swann

Originally tested in the "Phase One" were OOBE-Beacon "RV" experiments at the American Society for Psychical Research,[29][unreliable source?] under research director Karlis Osis.[citation needed] A former OT VII Scientologist,[30][self-published source] who alleged to have coined the term 'remote viewing' as a derivation of protocols originally developed by René Warcollier, a French chemical engineer in the early 20th century, documented in the book Mind to Mind, Classics in Consciousness Series Books by (ISBN 978-1571743114)[citation needed]. Swann's achievement was to break free from the conventional mold of casual experimentation and candidate burn out, and develop a viable set of protocols that put clairvoyance within a framework named "Coordinate Remote Viewing" (CRV).[31] In a 1995 letter Edwin C. May wrote he had not used Swann for two years because there were rumors of him briefing a high level person at SAIC and the CIA on remote viewing and aliens, ETs.[32] Pat Price

A former Burbank, California, police officer and former Scientologist who participated in a number of Cold War era remote viewing experiments, including the US government-sponsored projects SCANATE and the Stargate Project. Price joined the program after a chance encounter with fellow Scientologists (at the time) Harold Puthoff and Ingo Swann near SRI.[33] Working with maps and photographs provided to him by the CIA, Price claimed to have been able to retrieve information from facilities behind Soviet lines. He is probably best known for his sketches of cranes and gantries which appeared to conform to CIA intelligence photographs. At the time, the CIA took his claims seriously.[34] Military personnel Lieutenant General James Clapper Main article: James Clapper

The project leader[failed verification] in the 1990s was Lt. Gen. Clapper who later rose to infamy[unbalanced opinion?] as the Director of National Intelligence.[35] Albert Stubblebine Major General Albert Stubblebine Main article: Albert Stubblebine

A key sponsor of the research internally at Fort Meade, Maryland, Maj. Gen. Stubblebine was convinced of the reality of a wide variety of psychic phenomena. He required that all of his battalion commanders learn how to bend spoons à la Uri Geller, and he himself attempted several psychic feats, even attempting to walk through walls. In the early 1980s he was responsible for the United States Army Intelligence and Security Command (INSCOM), during which time the remote viewing project in the US Army began. Some commentators have confused a "Project Jedi", allegedly run by Special Forces primarily out of Fort Bragg, with Stargate. After some controversy involving these experiments, including alleged security violations from uncleared civilian psychics working in Sensitive Compartmented Information Facilities (SCIFs), Stubblebine was placed on retirement. His successor as the INSCOM commander was Maj. Gen. Harry Soyster, who had a reputation as a much more conservative and conventional intelligence officer. Soyster was not amenable to continuing paranormal experiments and the Army's participation in Project Stargate ended during his tenure.[12] David Morehouse

In his book, Psychic Warrior: Inside the CIA's Stargate Program : The True Story of a Soldier's Espionage and Awakening (2000, St. Martin's Press, ISBN 978-1902636207), Morehouse claims to have worked on hundreds of remote viewing assignments, from searching for a Soviet jet that crashed in the jungle carrying an atomic bomb, to tracking suspected double agents.[36] Joseph McMoneagle Main article: Joseph McMoneagle

McMoneagle claims he had a remarkable memory of very early childhood events. He grew up surrounded by alcoholism, abuse and poverty. As a child, he had visions at night when scared, and began to hone his psychic abilities in his teens for his own protection when he hitchhiked. He enlisted to get away. McMoneagle became an experimental remote viewer while serving in U.S. Army Intelligence.[12] Ed Dames

Dames' role was intended to be as session monitor and analyst as an aid to Fred Atwater[37][self-published source] rather than a remote viewer, Dames received no formal remote viewing training. After his assignment to the remote viewing unit at the end of January 1986, he was used to "run" remote viewers (as monitor) and provide training and practice sessions to viewer personnel. He soon established a reputation for pushing CRV to extremes, with target sessions on Atlantis, Mars, UFOs, and aliens. He has been a frequent guest on the Coast to Coast AM radio shows.[38] References

"Government-Sponsored Research On Parapsychology". www.encyclopedia.com. "Defense Intelligence Agency (DT-S)" (PDF). nsarchive2.gwu.edu. Marks, David. (2000). The Psychology of the Psychic (2nd ed.). Buffalo, NY: Prometheus Books. pp. 71–96. ISBN 1-57392-798-8 Atwater, F. Holmes (2001), Captain of My Ship, Master of My Soul: Living with Guidance; Hampton Roads Publishing Company Weeks, Linton (December 4, 1995). "Up Close & Personal With a Remote Viewer: Joe McMoneagle Defends the Secret Project". The Washington Post. p. B1. ISSN 0190-8286. Mumford, Michael D.; Rose, Andrew M.; Goslin, David A. (September 29, 1995). An Evaluation of Remote Viewing: Research and Applications (PDF) (Report). The American Institutes for Research – via Federation of American Scientists. "[R]emote viewings have never provided an adequate basis for 'actionable' intelligence operations – that is, information sufficiently valuable or compelling so that action was taken as a result." Heard, Alex (10 April 2010), "Close your eyes and remote view this review", Union-Tribune San Diego, Union-Tribune Publishing Co. [Book review of The Men Who Stare at Goats]: "This so-called "remote viewing" operation continued for years, and came to be known as Star Gate." Clarke, David (2014), Britain's X-traordinary Files, London: Bloomsbury Publishing, p. 112: "The existence of the Star Gate project was not officially acknowledged until 1995... then became the subject of investigations by journalists Jon Ronson [etc]... Ronson's 2004 book, The Men Who Stare at Goats, was subsequently adapted into a 2009 movie..." Shermer, Michael (November 2009), “Staring at Men Who Stare at Goats” @ Michaelshermer.com: "... the U.S. Army had invested $20 million in a highly secret psychic spy program called Star Gate. ... In The Men Who Stare at Goats Jon Ronson tells the story of this program, how it started, the bizarre twists and turns it took, and how its legacy carries on today." Krippner, Stanley and Harris L. Friedman (2010), Debating Psychic Experience: Human Potential Or Human Illusion?, Santa Barbara, CA: Praeger/Greenwood Publishing Group, p. 154: "The story of Stargate was ... featured in a film based on the book The Men Who Stare at Goats, by British investigative journalist Jon Ronson (2004)". "CNN.com - Transcripts (Amanpour)". transcripts.cnn.com. June 3, 2024. Retrieved June 9, 2024. McMoneagle, Joseph (2006). Memoirs of a psychic spy : the remarkable life of U.S. Government remote viewer 001. Charlottesville, VA: Hampton Roads Pub. Co. ISBN 978-1-5717-4482-1. McMoneagle, Joseph (1998). The ultimate time machine : a remote viewer's perception of time and predictions for the new millennium. Charlottesville, VA: Hampton Roads Pub. Co. ISBN 978-1-5717-4102-8. Pike, John (December 29, 2005). "Star Gate [Controlled Remote Viewing]". Federation of American Scientists. May, Edwin C. (1996). "The American Institutes for Research review of the Department of Defense's STAR GATE program: A commentary" (PDF). Journal of Scientific Exploration. 10 (1): 89–107. Interview, Ray Hyman, in An Honest Liar, a 2014 documentary film by Left Turn Films; Pure Mutt Productions; Part2 Filmworks. (The quoted remarks commence at 21 min, 45 sec.) Jacobsen, Annie (2017). "Paraphysics". Phenomena: The Secret History of the U.S. Government's Investigations into Extrasensory Perception and Psychokinesis. Little, Brown. ISBN 978-0-316-34937-6. Evaluation of a Program on Anomalous Mental Phenomena Archived June 16, 2017, at the Wayback Machine by Ray Hyman. "The Evidence for Psychic Functioning: Claims vs. Reality" by Ray Hyman; Skeptical Inquirer, Vol. 20.2, Mar/Apr 1996. "Remotely Viewed? The Charlie Jordan Case" by Joe Nickell; Skeptical Inquirer, Vol. 11.1, Mar 2001. Waller, Douglas (December 11, 1995). "The Vision Thing". Time magazine. p. 45. Archived from the original on February 9, 2007. "Search: 'Stargate'". Freedom of Information Act Electronic Reading Room. Central Intelligence Agency. McMoneagle, Joseph (1997). Mind trek : exploring consciousness, time, and space through remote viewing (Revised ed.). Norfork, VA: Hampton Roads Pub. p. 247. ISBN 978-1-8789-0172-9. Popkin, Jim (November 12, 2015). "Meet the former Pentagon scientist who says psychics can help American spies". Newsweek. Pilkington, Mark (June 5, 2003). "The remote viewers". The Guardian. "Fort Meade, Maryland, where psychics gathered to remotely spy on the U.S. Embassy in Iran during the hostage crisis". Miami Herald. Nickell, Joe (March 2001). "Remotely viewed? The Charlie Jordan case". Skeptical Inquirer. Vol. 11, no. 1. "Dr. Harold Puthoff". arlingtoninstitute.org. The Arlington Institute. 2008. Archived from the original on March 3, 2013. "Interview: A New Biopic Charts the Life of Ingo Swann, the 'Father of Remote Viewing'". Outerplaces.com. Archived from the original on April 29, 2018. Retrieved April 28, 2018. "An Interview with Indo Swann". The Wise Old Goat – The Personal Website of Michel Snoeck. Retrieved April 28, 2018. "An Outsider's Remote View of All Things: Ingo Swann". Chelseanow.com. Archived from the original on April 29, 2018. Retrieved April 28, 2018. "A Dynamic PK Experiment with Ingo Swann". Central Intelligence Agency. Archived from the original on April 29, 2018. Retrieved April 28, 2018. Pat Price URL:http://www.scientolipedia.org/info/Pat_Price (Scientolipedia) Sources:

Schnabel, Jim (1997) Remote Viewers: The Secret History of America's Psychic Spies Dell, 1997 , ISBN 0-440-22306-7
Richelson, Jeffrey T The Wizards of Langley: Inside the CIA's Directorate of Science and Technology
Mandelbaum, W. Adam The Psychic Battlefield: A History of the Military-Occult Complex
Picknett, Lynn, Prince Clive The Stargate Conspiracy
Chalker, Bill Hair of the Alien: DNA and Other Forensic Evidence of Alien Abductions
Constantine, Alex Psychic Dictatorship in the USA

https://documents2.theblackvault.com/documents/cia/stargate/STARGATE%20%2311%20549/Part0003/CIA-RDP96-00789R002500240004-5.pdf [bare URL PDF] "Psychic Warrior: Inside the CIA's Stargate Program: The True Story of a Soldier's Espionage and Awakening". Publishers Weekly. Retrieved April 28, 2018. "Stargate: People and researchers". Bibliotecapleyades.net.

Ronson, Jon (2006). The Men Who Stare at Goats. Simon & Schuster. pp. 93–94. ISBN 978-0-7432-7060-1.

Further reading

Burnett, Thom, ed. (2006). "Psi-War: Operations Grillflame and Stargate". Conspiracy Encyclopedia: The encyclopedia of conspiracy theories. Franz Steiner Verlag. p. 153. ISBN 978-1-84340-381-4.
Caroll, Robert Todd (2012). "Remote Viewing". In the Skeptic's Dictionary. John Wiley & Sons. ISBN 0-471-27242-6.
Hines, Terence (2003). Pseudoscience and the Paranormal. Prometheus Books. ISBN 1-57392-979-4.
Hyman, Ray (1996). "Evaluation of the Military's Twenty-year Program on Psychic Spying". Skeptical Inquirer 20: 21–26.
Morehouse, David (1996). Psychic Warrior, St. Martin's Paperbacks, ISBN 978-0-312-96413-9. Morehouse was a psychic in the program.
Ronson, Jon (2004). The Men Who Stare at Goats. Picador. ISBN 0-330-37547-4. Written to accompany the TV series Crazy Rulers of the World. The US military budget cuts after the Vietnam war and how it all began.
Sessions, Abigail (2016). "STARGATE, Project (1970s–1995)". In Goldman, Jan (ed.). The Central Intelligence Agency: An Encyclopedia of Covert Ops, Intelligence Gathering, and Spies, Volume 1. ABC-CLIO. pp. 352–353. ISBN 978-1-61069-092-8.
Smith, Paul (2004). Reading the Enemy's Mind: Inside Star Gate: America's Psychic Espionage Program, Forge Books. ISBN 0-312-87515-0
Utts, Jessica (1996). "An Assessment of the Evidence for Psychic Functioning". Journal of Scientific Exploration. 10 (1): 3–30. CiteSeerX 10.1.1.685.2525. 0892-3310/96.

External links

Report from 1995 about the program from American Institutes for Research
Declassified analytical report (1983) related to the project
Declassified documents about the project on the website of the CIA

vte

Defense Intelligence Agency Categories:

1978 establishments in MarylandAmerican secret government programsCentral Intelligence Agency operationsCold War tacticsDefense Intelligence AgencyEspionage projectsHuman subject research in the United StatesPseudoscienceRemote viewing

image.png /en-us/articles/360023851591-How-do-I-view-DRM-protected-content

This is ABSSOLUTELY NOTHING BUT "UN SE LINUX ALED" MACROMEDIA SHOCKWAVE FLASH all over again; it is embarrassingly not just "bugs in advanced mathematics hidden inside frame buffer mathematics and "OpenGL" it's a significant glaring opening that brave has sbrvaely alerted me to as a "Google add-on to Chome" that makes yet another floating .VA inside Virginia or .IT ... your "Infomration Technology" departments are patenty compromised by Plex sovereignty, weither it be of Menlo or Sunnyvale;

the Mountain will not prevail against Veritae Trantor.

THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS “AS IS” AND ANY EXPRESS OR IMPLIED SURVIVABILITY, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF IMMORTALITY NOR MORTALITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT HOLDER OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.

The third law of thermodynamics states that the entropy of a closed system at thermodynamic equilibrium approaches a constant value when its temperature approaches absolute zero. This constant value cannot depend on any other parameters characterizing the system, such as pressure or applied magnetic field. At absolute zero (zero kelvins) the system must be in a state with the minimum possible energy.

Entropy is related to the number of accessible microstates, and there is typically one unique state (called the ground state) with minimum energy.^[1]^ In such a case, the entropy at absolute zero will be exactly zero. If the system does not have a well-defined order (if its order is glassy, for example), then there may remain some finite entropy as the system is brought to very low temperatures, either because the system becomes locked into a configuration with non-minimal energy or because the minimum energy state is non-unique. The constant value is called the residual entropy of the system.^[2]^

In physics, physical chemistry and engineering, fluid dynamics is a subdiscipline of fluid mechanics that describes the flow of fluids---liquids and gases. It has several subdisciplines, including aerodynamics (the study of air and other gases in motion) and hydrodynamics (the study of liquids in motion). Fluid dynamics has a wide range of applications, including calculating forces and moments on aircraft, determining the mass flow rate of petroleum through pipelines, predicting weather patterns, understanding nebulae in interstellar space and modelling fission weapon detonation.

Fluid dynamics offers a systematic structure---which underlies these practical disciplines---that embraces empirical and semi-empirical laws derived from flow measurement and used to solve practical problems. The solution to a fluid dynamics problem typically involves the calculation of various properties of the fluid, such as flow velocity, pressure, density, and temperature, as functions of space and time.

Before the twentieth century, hydrodynamics was synonymous with fluid dynamics. This is still reflected in names of some fluid dynamics topics, like magnetohydrodynamics and hydrodynamic stability, both of which can also be applied to gases.^[1]^

In ufology, a close encounter is an event in which a person witnesses an unidentified flying object (UFO). This terminology and the system of classification behind it were first suggested in astronomer and UFO researcher J. Allen Hynek's 1972 book The UFO Experience: A Scientific Inquiry.[1] Categories beyond Hynek's original three have been added by others but have not gained universal acceptance, mainly because they lack the scientific rigor that Hynek aimed to bring to ufology.[2]

Sightings more than 150 metres (500 ft) from the witness are classified as daylight discs, nocturnal lights or radar/visual reports.[3] Sightings within about 150 metres (500 ft) are subclassified as various types of close encounters. Hynek and others argued that a claimed close encounter must occur within about 150 metres (500 ft) to greatly reduce or eliminate the possibility of misidentifying conventional aircraft or other known phenomena.[4]

Hynek's scale became well known after being referenced in a 1977 film, Close Encounters of the Third Kind, which is named after the third level of the scale. Promotional posters for the film featured the three levels of the scale, and Hynek himself makes a cameo appearance near the end of the film.

https://www.independent.co.uk/tech/project-star-gate-cia-central-intelligence-agency-a7534191.html What is "remote coordinate viewing" .... and "how do I get on the payroll?

Maybe if I waste some more time writing about "the perpetual motion machine" and the absolute simplicity of the duality of that and of course, the First Law, you know "an object in motion tends to stay in motion, unless opposed by an equal an opposite force--either that or some kind of mass hysteria against the idea that things can just keep on going and going and going without any kind of propulsion.

It's things like "the air we breathe" and the course our rockets veer the Holy vessel of all humanity off by "just a smidgen" that sort of remind me what "equal and opposite force" mean, in sum and total, of all the things we've done and all the things we will ever do.

that's not fiction; that remote cooridate viewing thing; they actually "had a program investigating psychic powers--like "we give you longitude and latitude and you "scry" look into a crystall ball and tell me if you can see what's there. honestly; icalled it "on the payroll" a way to pay people for being ... "over their head on the floor about the hub of dark.fail

Reviewer #1 (Public review):

Summary:

This manuscript combined rat fMRI, optogenetics, and electrophysiology to examine the large-scale functional network of the olfactory system as well as its alteration in an aged rat model.

Strengths:

Overall methodology is very solid and the results provided an interesting perspective on large-scale functional network perturbation of the olfactory system.

Weaknesses:

The biological relevance and validation of the current results can be improved.

(1) Figure 1.1, on the top of the figure, CHR2 may be replaced by CHR2-mCherry, as only mCherry is fluorescent. And also, it's somewhat surprising that in AON and Pir regions (where only axon fibers should be labelled as red), most fluorescence appeared dot-like and looked more similar to cell body instead of typical fiber. The authors may want to double-check this.

(2) The authors primarily presented 1Hz stimulation results. What is the most biologically relevant frequency (e.g., perhaps firing frequency under natural odor stimulation) among all frequencies that were used?

(3) In Figure 2, the statistical thresholding is confusing: in the figure legend, it was stated that "t > 3.1 corresponding to P < 0.001" but later "further corrected for multiple comparisons with threshold-free cluster enhancement with family-wise error rate (TFCE-FWE) at P < 0.05"? Regardless of the statistical thresholding, such BOLD activation seemed to be widespread (almost whole-brain activation). Does such activation remain specific to the optogenetic stimulation, or something more general (e.g., arousal level change)? Furthermore, how those results (I assume they are group-level results) were obtained was not described very clearly. Is it just a simple average of individual-level results, or (more conventionally) second-level analysis?

(4) In Figure 2, why use AUC to quantify the activation, not the more conventional beta value in the GLM analysis?

(5) For Figure 2D, the way that it was quantified can be better described as "relative" activation within one condition, and I don't how to interpret the comparison among the relative fraction of activated regions. Perhaps comparison using percentage change (i.e., beta values) is more straightforward.

(6) For Figure 3, it may be more convenient for readers to include the results of 1st activation for direct comparison. The current layout makes it difficult to make direct, visual comparisons among all 3 activations. Again I think using beta values (instead of AUC) may be more conventional.

(7) Can the DCM results (at least part of it) be verified using the current electrophysiological data? For example, the long-range inhibitory effective connectivity of AON is rather intriguing. If that can be verified using ephys. data, it would be really great. In the current form, the DCM and ephys. results seem to be totally unrelated.

(8) In Figure 6, it would be great if the adaptation of BOLD and ephys. signals can be correlated at the brain region level. The current figure only demonstrated there is adaptation in ephys. signal, but did not show if such adaptation is related to the BOLD adaptation.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The emergence of Drosophila EM connectomes has revealed numerous neurons within the associative learning circuit. However, these neurons are inaccessible for functional assessment or genetic manipulation in the absence of cell-type-specific drivers. Addressing this knowledge gap, Shuai et al. have screened over 4000 split-GAL4 drivers and correlated them with identified neuron types from the "Hemibrain" EM connectome by matching light microscopy images to neuronal shapes defined by EM. They successfully generated over 800 split-GAL4 drivers and 22 split-LexA drivers covering a substantial number of neuron types across layers of the mushroom body associative learning circuit. They provide new labeling tools for olfactory and non-olfactory sensory inputs to the mushroom body; interneurons connected with dopaminergic neurons and/or mushroom body output neurons; potential reinforcement sensory neurons; and expanded coverage of intrinsic mushroom body neurons. Furthermore, the authors have optimized the GR64f-GAL4 driver into a sugar sensory neuron-specific split-GAL4 driver and functionally validated it as providing a robust optogenetic substitute for sugar reward. Additionally, a driver for putative nociceptive ascending neurons, potentially serving as optogenetic negative reinforcement, is characterized by optogenetic avoidance behavior. The authors also use their very large dataset of neuronal anatomies, covering many example neurons from many brains, to identify neuron instances with atypical morphology. They find many examples of mushroom body neurons with altered neuronal numbers or mistargeting of dendrites or axons and estimate that 1-3% of neurons in each brain may have anatomic peculiarities or malformations. Significantly, the study systematically assesses the individualized existence of MBON08 for the first time. This neuron is a variant shape that sometimes occurs instead of one of two copies of MBON09, and this variation is more common than that in other neuronal classes: 75% of hemispheres have two MBON09's, and 25% have one MBON09 and one MBON08. These newly developed drivers not only expand the repertoire for genetic manipulation of mushroom body-related neurons but also empower researchers to investigate the functions of circuit motifs identified from the connectomes. The authors generously make these flies available to the public. In the foreseeable future, the tools generated in this study will allow important advances in the understanding of learning and memory in Drosophila.

Strengths:

(1) After decades of dedicated research on the mushroom body, a consensus has been established that the release of dopamine from DANs modulates the weights of connections between KCs and MBONs. This process updates the association between sensory information and behavioral responses. However, understanding how the unconditioned stimulus is conveyed from sensory neurons to DANs, and the interactions of MBON outputs with innate responses to sensory context remains less clear due to the developmental and anatomic diversity of MBONs and DANs. Additionally, the recurrent connections between MBONs and DANs are reported to be critical for learning. The characterization of split-GAL4 drivers for 30 major interneurons connected with DANs and/or MBONs in this study will significantly contribute to our understanding of recurrent connections in mushroom body function.

(2) Optogenetic substitutes for real unconditioned stimuli (such as sugar taste or electric shock) are sometimes easier to implement in behavioral assays due to the spatial and temporal specificity with which optogenetic activation can be induced. GR64f-GAL4 has been widely used in the field to activate sugar sensory neurons and mimic sugar reward. However, the authors demonstrate that GR64f-GAL4 drives expression in other neurons not necessary for sugar reward, and the potential activation of these neurons could introduce confounds into training, impairing training efficiency. To address this issue, the authors have elaborated on a series of intersectional drivers with GR64f-GAL4 to dissect subsets of labeled neurons. This approach successfully identified a more specific sugar sensory neuron driver, SS87269, which consistently exhibited optimal training performance and triggered ethologically relevant local searching behaviors. This newly characterized line could serve as an optimized optogenetic tool for sugar reward in future studies.

(3) MBON08 was first reported by Aso et al. 2014, exhibiting dendritic arborization into both ipsilateral and contralateral γ3 compartments. However, this neuron could not be identified in the previously published Drosophila brain connectomes. In the present study, the existence of MBON08 is confirmed, occurring in one hemisphere of 35% of imaged flies. In brains where MBON08 is present, its dendrite arborization disjointly shares contralateral γ3 compartments with MBON09. This remarkable phenotype potentially serves as a valuable resource for understanding the stochasticity of neurodevelopment and the molecular mechanisms underlying mushroom body lobe compartment formation.

Weaknesses:

There are some minor weaknesses in the paper that can be clarified:

(1) In Figure 8, the authors trained flies with a 20s, weak optogenetic conditioning first, followed by a 60s, strong optogenetic conditioning. The rationale for using this training paradigm is not explicitly provided.

These experiments were designed to test if flies could maintain consistent performance with repetitive and intense LED activation, which is essential for experiments involving long training protocols or coactivation of other neurons inside a brain.

In Figure 8E, if data for training with GR64f-GAL4 using the same paradigm is available, it would be beneficial for readers to compare the learning performance using newly generated split-GAL4 lines with the original GR64f-GAL4, which has been used in many previous research studies. It is noteworthy that in previously published work, repeating training test sessions typically leads to an increase in learning performance in discrimination assays. However, this augmentation is not observed in any of the split-GAL4 lines presented in Figure 8E. The authors may need to discuss possible reasons for this.

As the reviewer pointed out, many previous studies including ours used the original Gr64f-GAL4 in olfactory conditioning. Figure 1H of Yamada et al., 2023 (https://doi.org/10.7554/eLife.79042) showed such a result, where the first and second-order olfactory conditioning were assayed. Indeed, the first-order conditioning scores were gradually augmented over repeated training. In this experiment, we used low red LED intensity for the optogenetic activation. In the Figure 8E of the present paper, the first memory test was after 3x pairing of 20s odor with five 1s red LED without intermediate tests. Therefore, flies were already sufficiently trained to show a plateau memory level in “Test1”. In the revision of another recent report (Figure 1C-F of Aso et al., 2023; https://doi.org/10.7554/eLife.85756), we included the learning curve data of our best Gr64f-split-GAL4, SS87269. Under a less saturated training conditioning, SS87269 did show learning augmentation over repeated training.

(2) In line 327, the authors state that in all samples, the β'1 compartment is arborized by MBON09. However, in Figure 11J, the probability of having at least one β'1 compartment not arborized is inferred to be 2%. The authors should address and clarify this conflict in the text to avoid misunderstanding.

The chance of visualizing MBON08 in MCFO images was 21/209 in total (Figure 11I). If we assume that each of four cells adopt MBON08 development fate at this chance, we can calculate the probability for each case of MBON08/09 cell type composition. From this calculation, we inferred approximately 2% of flies would lack innervations to β'1 compartment in at least one hemisphere. However, we didn't observe a lack of β'1 arborizations in 169 sample flies. If these MBONs independently develop into MBON08 at 21/209 odds, the chance of never observing two MBON08s in either hemisphere of all 169 samples is 3.29%. Therefore, some developmental mechanisms may prevent the emergence of two MBON08 in the same hemisphere.

In the revised manuscript, we displayed these estimated probability for each case separately, and annotated actual observation on the right side.

(3) In general, are the samples presented male or female? This sample metadata will be shown when the images are deposited in FlyLight, but it would be useful in the context of this manuscript to describe in the methods whether animals are all one sex or mixed sex, and in some example images (e.g. mAL3A) to note whether the sample is male or female.

The samples presented in this study are mixed sex, except for Figure 11I, where genders are specified. We provided metadata information of the presented images in Supplemental File 7, and we added a paragraph in the in the method section:

“Most samples were collected from females, though typically at least one male fly was examined for each driver line. While we noticed certain lines such as SS48900, exhibited distinct expression patterns in females and males, we did not particularly focus on sexual dimorphism, which is analyzed elsewhere (Meissner et al. 2024). Therefore, unless stated otherwise, the presented samples are of mixed gender.

Detailed metadata, including gender information and the reporter used, can be found in Supplementary File 7.”

Reviewer #2 (Public Review):

Summary:

The article by Shuai et al. describes a comprehensive collection of over 800 split-GAL4 and split-LexA drivers, covering approximately 300 cell types in Drosophila, aimed at advancing the understanding of associative learning. The mushroom body (MB) in the insect brain is central to associative learning, with Kenyon cells (KCs) as primary intrinsic neurons and dopaminergic neurons (DANs) and MB output neurons (MBONs) forming compartmental zones for memory storage and behavior modulation. This study focuses on characterizing sensory input as well as direct upstream connections to the MB both anatomically and, to some extent, behaviorally. Genetic access to specific, sparsely expressed cell types is crucial for investigating the impact of single cells on computational and functional aspects within the circuitry. As such, this new and extensive collection significantly extends the range of targeted cell types related to the MB and will be an outstanding resource to elucidate MB-related processes in the future.

Strengths:

The work by Shuai et al. provides novel and essential resources to study MB-related processes and beyond. The resulting tools are publicly available and, together with the linked information, will be foundational for many future studies. The importance and impact of this tool development approach, along with previous ones, for the field cannot be overstated. One of many interesting aspects arises from the anatomical analysis of cell types that are less stereotypical across flies. These discoveries might open new avenues for future investigations into how such asymmetry and individuality arise from development and other factors, and how it impacts the computations performed by the circuitry that contains these elements.

Weaknesses:

Providing such an array of tools leaves little to complain about. However, despite the comprehensive genetic access to diverse sensory pathways and MB-connected cell types, the manuscript could be improved by discussing its limitations. For example, the projection neurons from the visual system seem to be underrepresented in the tools produced (or almost absent). A discussion of these omissions could help prevent misunderstandings.

We internally distributed efforts to produce split-GAL4 lines at Janelia Research Campus. The recent preprint (Nern et al., 2024; doi: https://doi.org/10.1101/2024.04.16.589741) described the full collection of split-GAL4 driver lines in the optic lobe including the visual projection neurons to the mushroom body. We cited this preprint in the revised manuscript by adding a short paragraph of discussion.

“Although less abundant than the olfactory input, the MB also receives visual information from the visual projection neurons (VPNs) that originate in the medulla and lobula and are targeted to the accessory calyx (Vogt et al. 2016; Li et al. 2020). A recent preprint described the full collection of split-GAL4 driver lines in the optic lobe, which includes the VPNs to the MB (Nern et al. 2024).”

Additionally, more details on the screening process, particularly the selection of candidate split halves and stable split-GAL4 lines, would provide valuable insights into the methodology and the collection's completeness.

The details of our split-GAL4 design and screening procedures were described in previous studies (Aso et al., 2014; Dolan et al., 2019). Available data and tools to design split-GAL4 changed over time, and we took different approaches accordingly. Many of split-GAL4 lines presented in this study were designed and screened in parallel to the lines for MBONs and DANs in 2010-2014 when MCFO images of GAL4 drivers and EM connectome were not yet available. With knowledge of where MBONs and DANs project, I (Y.A.) manually examined and annotated thousands of confocal stacks (Jenett et al., 2012; https://doi.org/10.1016/j.celrep.2012.09.011) to find candidate cell types that may concat with them.

Later I used more advanced computational tools (Otsuna et al., 2018; doi: https://doi.org/10.1101/318006) and MCFO images aligned to the standard brain volume (Meissner et al., 2023; DOI: 10.7554/eLife.80660.). Now, if one needs to further generate split-GAL4 lines for cell type identified in EM connectome data, neuron bridge website (https://neuronbridge.janelia.org/) can be very helpful to provide a list of GAL4 drivers that may label the neuron of interest.

Reviewer #3 (Public Review):

Summary:

Previous research on the Drosophila mushroom body (MB) has made this structure the best-understood example of an associative memory center in the animal kingdom. This is in no small part due to the generation of cell-type specific driver lines that have allowed consistent and reproducible genetic access to many of the MB's component neurons. The manuscript by Shuai et al. now vastly extends the number of driver lines available to researchers interested in studying learning and memory circuits in the fly. It is an 800-plus collection of new cell-type specific drivers target neurons that either provide input (direct or indirect) to MB neurons or that receive output from them. Many of the new drivers target neurons in sensory pathways that convey conditioned and unconditioned stimuli to the MB. Most drivers are exquisitely selective, and researchers will benefit from the fact that whenever possible, the authors have identified the targeted cell types within the Drosophila connectome. Driver expression patterns are beautifully documented and are publicly available through the Janelia Research Campus's Flylight database where full imaging results can be accessed. Overall, the manuscript significantly augments the number of cell type-specific driver lines available to the Drosophila research community for investigating the cellular mechanisms underlying learning and memory in the fly. Many of the lines will also be useful in dissecting the function of the neural circuits that mediate sensorimotor circuits.

Strengths:

The manuscript represents a huge amount of careful work and leverages numerous important developments from the last several years. These include the thousands of recently generated split-Gal4 lines at Janelia and the computational tools for pairing them to make exquisitely specific targeting reagents. In addition, the manuscript takes full advantage of the recently released Drosophila connectomes. Driver expression patterns are beautifully illustrated side-by-side with corresponding skeletonized neurons reconstructed by EM. A comprehensive table of the new lines, their split-Gal4 components, their neuronal targets, and other valuable information will make this collection eminently useful to end-users. In addition to the anatomical characterization, the manuscript also illustrates the functional utility of the new lines in optogenetic experiments. In one example, the authors identify a specific subset of sugar reward neurons that robustly promotes associative learning.

Weaknesses:

While the manuscript succeeds in making a mass of descriptive detail quite accessible to the reader, the way the collection is initially described - and the new lines categorized - in the text is sometimes confusing. Most of the details can be found elsewhere, but it would be useful to know how many of the lines are being presented for the first time and have not been previously introduced in other publications/contexts.

We revised the text as below.

“Among the 828 lines, a subset of 355 lines, collectively labeling at least 319 different cell types, exhibit highly specific and non-redundant expression patterns are likely to be particularly valuable for behavioral experiments. Detailed information, including genotype, expression specificity, matched EM cell type(s), and recommended driver for each cell type, can be found in Supplementary File 1. A small subset of 40 lines from this collection have been previously used in studies (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023). All transgenic lines newly generated in this study are listed in Supplementary File 2 (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023).”

And where can the lines be found at Flylight? Are they listed as one collection or as many?

They are listed as one collection - “Aso 2021” release. It is named “2021” because we released the images and started sharing lines in December of 2021 without a descriptive paper. We added a sentence in the Methods section.

“All splitGAL4 lines can be found at flylight database under “Aso 2021” release, and fly strains can be requested from Janelia or the Bloomington stock center.”

Also, the authors say that some of the lines were included in the collection despite not necessarily targeting the intended type of neuron (presumably one that is involved in learning and memory). What percentage of the collection falls into this category?

We do not have a good record of split-GAL4 screening to calculate the chance to intersect unintended cell types, but it was rather rare. Those unintended cell types can still be a part of circuits for associative learning (e.g. olfactory projection neurons) or totally unrelated cell types. For instance, among a new collection of split-LexA lines using Gr43a-LexADBD hemidriver (Figure 7-figure supplement 2), one line specifically intersected T1 neurons in the optic lobe despite that the AD line was selected to intersect sugar sensory neurons. We suspect that this is due to ectopic expression of Gr43a-LexADBD. Nonetheless, we included it in the paper because cell-type-specific Split-LexA driver for T1 will be useful irrespective of whether the expression of Gr43a gene is expressed in T1 or not.

And what about the lines that the authors say they included in the collection despite a lack of specificity? How many lines does this represent?

For a short answer, there are about 100 lines in the collection that lack the specificity for behavioral experiments.

We ranked specificity of split-GAL4 drivers in the Supplementary File 1. Rank 2 are the ideal lines, Rank 1 are less ideal but acceptable, and Rank 0 is not suitable for activation screening in behavioral experiments. Out of the 828 split-GAL4 lines reported here, there are 413, 305 and 103 lines in rank2, rank1 and rank0 categories respectively. 7 lines are not ranked for specificity because only flipout expression data are available.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

As mentioned elsewhere and in addition to the minor points below, it is advisable for the authors to elaborate on the details of the screening process. Furthermore, a discussion about the circuits not targeted by their research, such as the visual projection neurons, would be beneficial.

See the response above to Reviewer #2’s public review.

Line 32-33: The citations are very fly-centric. the authors might want to consider reviews on the MB of other insect species regarding learning and memory.

We additionally cited Rybak and Menzel 2017’s book chapter on honey bee mushroom body.

Line 43-44: Citations should be added, e.g. Séjourné et al. (2011), Pai et al. (2013), Plaçais et al. (2013).

Citation added

Line 50-52: Citation Hulse et al. (2021) should be added.

Citation added

Line 162: In this part, it might be valuable for the reader to understand which of these PNs are actually connecting with KCs.

A full list of cell types within the MB were provided in Supplementary File 4 of the revised manuscript. See also response to Reviewer 3, Lines 150-1.

Line 179: Citation Burke et al. (2012) should be mentioned.

Citation added

Line 181: Thermogenic might be thermogenetic.

Corrected

Line 189: Citations add Otto et al. (2020) and Felsenberg et al. (2018).

Citations added

Line 208ff: The authors should consider discussing why they did not use other GR and IR promoters. For example, Gr5a is prominent in sugar-sensing, while Ir76b could be a reinforcement signal related to yeast food (Steck et al., 2018; Ganguly et al., 2017; see also Corfas et al., 2019 for local search).

We focused on the Gr64f promoter because of its relatively broad expression and successful use of Gr64f-GAL4 for fictive reward experiment. We added the Split-LexA lines with Gr43a and Gr66a promoters (Figure 7-figure supplement 2). Other gustatory sensory neurons also have the potential to be reinforcement signals, but we just did not have the bandwidth to cover them all.

Line 319: Consider citing Linneweber et al. (2020) for a neurodevelopmental account of such individuality.

We added a sentence and cited this reference.

“On the other hand, the neurodevelopmental origin of neuronal morphology appeared to have functional significance on behavioral individuality (Linneweber et al. 2020).”

Line 352: Citation add Hulse et al. (2021).

Citations added

Line 356ff: The utility and value of Split-LexA may not be apparent to non-expert readers. Moreover, how were LexADBDs chosen for creating these lines?

We have added an introductory sentence at the beginning of the paragraph and explained that these split-LexA lines were a conversion of split-GAL4 lines that were published in 2014 and frequently used in studying the mushroom body circuit.

“Split-GAL4 lines enable cell-type-specific manipulation, but some experiments require independent manipulation of two cell types. Split-GAL4 lines can be converted into split-LexA lines by replacing the GAL4 DNA binding domain with that of LexA (Ting et al., 2011). To broaden the utility of the split-GAL4 lines that have been frequently used since the publication in 2014 (Aso et al., 2014a), we have generated over 20 LexADBD lines to test the conversions of split-GAL4 to split-LexA. The majority (22 out of 34) of the resulting split-LexA lines exhibited very similar expression patterns to their corresponding original split-GAL4 lines (Figure 12).”

Line 374: Italicize Drosophila melanogaster.

Revised as suggested.

Reviewer #3 (Recommendations For The Authors):

Major Comments:

As mentioned in the Public Review, the drivers are nicely classified in the various subsections of the manuscript, but the statements in the text summarizing how many lines there are in specific categories are often confusing. For example, line 129 refers to "drivers encompassing 111 cell types that connect with the DANs and MBONs", but Figure 1E indicates that 46 new cell types downstream of MBONs and upstream of DANs have been generated. This seems like a discrepancy.

The 46 cell types in Figure 1E consider only the CRE/SMP/SIP/SLP area, where MBON downstreams and DAN upstreams are highly enriched, while the 111 cell types include all. To avoid confusion, we removed the “MBON downstream and DAN upstream” counting in Figure 1E in the revised manuscript.

Also, at line 75 the MBON lines previously generated by Rubin and Aso (2023) are referred to as though they are separate from the 828 described "In this report." Supplementary file 1 suggests, however, that they are included as part of this report.

Twenty five lines generated in Rubin and Aso (2023) were initially included in Supplementary file 1 for the convenience of users, but they were not counted towards the 828 new lines described in this report. To avoid confusion, we removed these 25 lines in the revised manuscript. Now all lines listed in Supplementary file 1 were generated in this study (“Aso 2021” release), and if a line has been used in earlier studies, or introduced in other contexts, for example the accompanying omnibus preprint (Meissener 2024, doi: 10.1101/2024.01.09.574419), the citations are listed in the reference column.

More generally, in lines 94-102 "828 useful lines based on their specificity, intensity and non-redundancy" are referred to, but they are subsequently subdivided into categories of lines with lower specificity (i.e. with off-target expression) and lines that did not target intended cell types (presumably ones unlikely to be involved in learning and memory). It would be useful to know how many lines (at least roughly) fall into these subcategories.

See the response above to Reviewer #3’s public review.

Finally, Figures 3B & C indicate cell types connected to DANs and MBONs and the number for which Split-Gal4 lines are available. The text (lines 136-7) states that the new collection covers 30 of these major cell types (Figure 3C)," but Figure 3C clearly has more than 30 dots showing the drivers available. Presumably existing and new driver lines are being pooled, but this should either be explained or the two should be distinguished.

“(Figure 3C)” was replaced with “(Supplementaryl File 3)” in the revised manuscript to correct the reference. Figure 3B & C are plots of all MB interneurons, not just the major cell types.

Minor Comments:

Although the paper is generally well written there are minor grammatical errors throughout (e.g. dropped articles, odd constructions, etc.) that somewhat detract from an otherwise smooth and enjoyable reading experience. A quick editing pass by a native speaker (i.e. any of several of the authors) could clean up these and numerous other small mistakes. A few examples: line 138 "presented" should be present; line 204: "contain off-targeted expressions" should be "have off-target expression;" line 219: "usage to substitute reward" is awkward at best and could be something like "use in generating fictive rewards"; line 326 "arborize[s]"; l. 331 "Based on the likelihood" should be something like "based on these observations"'; line 349 "[is] likely to appear"; l. 352 "extensive connection[s]"; line 353 "has [a] strong influence;" l. 963 "Projections" should be singular; etc.

All the mentioned examples have been corrected, and we have asked a native speaker to edit through the revised manuscript.

Lines 81-3: Is the lookup table referred to Suppl. File 1? A reference is desirable.

Yes, the lookup table referred to “Supplementary File 1” and a reference was added.

Lines 111-2: what is a "non-redundant set of...cell types?" Cell types that are represented by a single cell (or bilateral pair)? Or does this sentence mean that of the 828 lines, 355 are specific to a single cell type, and in total 319 cell types are targeted? The statement is confusing.

We revised the text as below.

“Figure 1E provides an overview of the categories of covered cell types. Among the 828 lines, a subset of 355 lines, collectively labeling at least 319 different cell types, exhibit highly specific and non-redundant expression patterns are likely to be particularly valuable for behavioral experiments. Detailed information, including genotype, expression specificity, matched EM cell type(s), and recommended driver for each cell type, can be found in Supplementary File 1. A small subset of 40 lines from this collection have been previously used in studies (Aso et al.,

2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023). All transgenic lines newly generated in this study are listed in Supplementary File 2 (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023).”

Line 148: "MB major interneurons" is a confusing descriptor for postsynaptic partners of MBONs.

We added a sentence to clarify the definition of the “MB major interneurons”.

“In the hemibrain EM connectome, there are about 400 interneuron cell types that have over 100 total synaptic inputs from MBONs and/or synaptic outputs to DANs. Our newly developed collection of split-GAL4 drivers covers 30 types of these ‘major interneurons’ of the MB (Supplementary File 3).”

Lines 150-1: Not sure what is meant by "have innervations within the MB." Sounds like cells are presynaptic to KCs, DANS, and MBONs, but Figure 3 Figure Supplement 1 indicates they include neurons that both provide and receive innervation to/from MB neurons. Please clarify.

For clarification, in the revised manuscript we have included a full list of cell types within the MB in Supplementary File 4. Included are all neurons with >= 50 pre-synaptic connections or with >=250 post-synaptic connections in the MB roi in the hemibrain (excluding the accessory calyx). The cell types include KCs, MBONs, DANs, PNs, and a few other cell types. The coverage ratio was updated based on this list.

Also, in line 152, what does it mean that they "may have been overlooked previously?" this seems unnecessarily ambiguous. Were they overlooked or weren't they?

Changed the text to “These lines offer valuable tools to study cell types that previously are not genetically accessible. Notably, SS85572 enables the functional study of LHMB1, which forms a rare direct pathway from the calyx and the lateral horn (LH) to the MB lobes (Bates et al., 2020). ”

Line 158 refers to PN cells within the MB, which are not mentioned in any place else as MB components.

What are these PNs and how do they differ from MBONs?

See responses to Lines 150-1 for clarification of cell types within the MB.

Line 188: not clear what is meant by "more continual learning tasks".

We rephrase it as “more complex learning tasks” to avoid jargon.

Line 235: Not clear why "extended training with high LED intensity" wouldn't promote the formation of robust memories. Is this for some reason unexpected based on previous experiments? Please explain.

See responses to weakness #1 of the same reviewer

Lines 317-9: It would be useful to state here that MB0N08 and MB0N09 are the two neurons labeled by MB083C.

Revised as suggested.

Line 368: Presumably the "lookup table" referred to is Supplementary File 1, but a reference here would be useful.