Aplicando

Escribir un punto al final de la siguiente ecuación.

opzoeken -> snap het niet helemaal.

signaleren (= afnemen toets en maken leerlingrapport) -> analyseren (= meer gegevens verzamen over niveau en leerbehoeften leerling) -> plannen (= bepalen welke leerdoelen en op welke wijze) -> handelen (= handelingsplan uitvoeren) -> evalueren -> opnieuw.

for - Youtube - Tukdam talk - An Overview Of Center for Healthy Minds at University of Wisconsin-Madison (CHM)’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson - wellbeing - clear light meditation, meditation at time of death - Tukdam

summary - Professor Davidson speaks on the subject of Tukdam, the Tibetan practice of meditation at the time of death practiced by Tantric practitioners - He contextualizes it in the framework that all sentient beings are sacred, and have the capacity for unfolding the intrinsic sacred that each of us is born with - Davidson's team explores the impact of meditation and mindfulness practices on human health and wellbeing and have formulated a wellbeing framework with four pillalrs - Deep Humanity - impacts of meditation - meditation at time of death

to - Youtube - documentary movie trailer - Tukdam: Between Worlds - https://hyp.is/FJg9XL4PEe-M9OfpvdsFQQ/www.youtube.com/watch?v=dDBEl9bSGMQ

for - trivia - measuring tukdam after death - 24 hour period immediately following death is important but to date, no data captured - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - clear light meditation - Tukdam at time of death - results so far - studied 20 cases - in all cases body doesn't decompose like a normal person's body does at death - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - meditation - Tukdam clear light meditation at time of death - research motivation from HH Dalai Lama - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

Summary - His Holiness Dalai Lama requested the research so that science could validate what Tibetan practitioners have known for a long time, that there is still an awareness present in the advanced meditator even after death has occurred - this is the Tukdam "clear light" meditation practice.

for - wellbeing app - The Healthy Minds program - Richard J. Davidson - mindfulness, meditation and wellbeing

to - Healthy Minds program app - https://hyp.is/bGfwCL4LEe-9cc9rnRiXig/www.portal.hminnovations.org/launch

for - mindfulness meditation research - 4 pillars of wellbeing - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

summary - four pillars of wellbeing - 1 awareness - 2 connection - 3 insight (of the nature of self) - 4 purpose (intention)

for - fourth of four pillars of wellbeing - purpose - finding it in our everyday life here and now - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson - comparison - intention vs attention

comment - Davidson does not provide much rich commentary on purpose, although it is quite an important idea to consider. - Intention is synonymous with purpose - The reason we consider the word intention instead is that we can compare to attention - intention - purpose or focus direction of future work (fourth pillar) - attention - focus awareness (first pillar) - Both of these acts are acts of constraining from the infinite field of our reality to a very narrow one - intention - among the infinite things I CAN do, I choose to do THIS specific one - attention - among all the infinite things I can sense, I choose to sense THIS specific one

for - illusion of self narrative / construction - third pillar - insight - key insight on insight! - not about CHANGING NARRATIVES - but about PENETRATING THE NARRATIVE to understand its essence - - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - third of four pillars of wellbeing - insight - a curiosity driven knowledge of the self - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

comment - this insight is specifically about the nature of self as a narrative construction imposed upon a constellation of changing thoughts and emotions - when we gain the insight that the solid-appearing self is constructed on emptiness, research shows that this insight sets the stage for wellbeing to emerge

for - adjacency - compassion / kindness - early model - HH Dalai Lama - Deep Humanity - mOTHER - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - comparison / key insight - compassion is like language (and also like genetics) - every infant has the biological capacity for these - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

comparison / key insight - compassion is like language (and also like genetics) - compassion, like language and genetics is intrinsic to our human nature. Every newborn comes into the world with the biological capacity for kindness/compassion, language and for genetic expression. However, - how we actually turn out as adults depends on what variables exist in our environment - If we have a compassionate mOTHER, our Most significant OTHER, she will teach us compassion - just like a child raised in a community of other language speakers in the environment will enable the child to cultivate the language capacity and - without a community of language speakers, a feral infant will grow up not understanding language at all - a healthy environment triggers beneficial epigenetic processes - Again, the chinese saying is salient: (hu)man on earth, good at birth. The same nature, varies on nurture

to - feral children - Youtube - https://hyp.is/go?url=http%3A%2F%2Fdocdrop.org%2Fvideo%2FTKaS1RdAfrg%2F&group=world - Chinese saying: (hu)man on earth, good at birth. The same nature, varies on nurture - https://hyp.is/TWOEYrlUEe-Mxx_LHYIpMg/medium.com/postgrowth/rediscovering-harmony-how-chinese-philosophy-offers-pathways-to-a-regenerative-future-07a097b237a0

for - everyone is sacred - everyone has Buddha Nature - different ways of saying - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson - poverty mentality - Chinese saying: (hu)man on earth, good at birth. The same nature, varies on nurture

everyone is sacred - different ways of saying it - We are all born with Buddha nature - We are all born with innate goodness - Chinese saying: (hu)man on earth, good at birth. The same nature, varies on nurture - Not seeing this, we fall into poverty mentality, and all the associated forms of suffering it brings

to - Chinese saying: (hu)man on earth, good at birth. The same nature, varies on nurture - https://hyp.is/TWOEYrlUEe-Mxx_LHYIpMg/medium.com/postgrowth/rediscovering-harmony-how-chinese-philosophy-offers-pathways-to-a-regenerative-future-07a097b237a0

for - innate connection - innate care for others - study of infants with puppets show 100% preference for compassionate play over selfish play - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - second of four pillars of wellbeing - connection - capacity to socially engage with others - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - mindfulness and happiness - research conclusion - wandering mind is an unhappy mind - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - first of four pillars of wellbeing - awareness - capacity to regulate our attention - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - two drivers of plasticity - neuroplasticity and epigenetics - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - explanation - epigenetics and health / wellbeing - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

explanation - epigenetics and health - Richard J. Davidson gives a simple and clear explanation of the connection between epigenetics and health / wellbeing - We are born with DNA that won't change much over the course of a lifetime - However, many of those genes are not active but can be rapidly activated by environmental cues such as emotions, chemical signals, etc

for - meditation - training the mind - scientific measurable effects on wellbeing - brain and body functions - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - wellbeing - is best regarded as a skill - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - quote - everyone is sacred - HH Dalai Lama - via Richard J. Davidson - His Holiness says every human being is the same - We're all built in the same way and every human being has the capacity to flourish - We would even go a little further and we would say that - every human being has the right to flourish and also - has all of the necessary constituents - the necessary components - the underlying mechanisms that enable uh a person to flourish or to have well-being

for - quote - comparison - wellbeing vs happiness - Richard J. Davidson - The idea isn't really to be happy all the time. - If a sad event or something tragic occurred, it would not be appropriate to be happy in that moment - At that moment, it's possible to be sad and have very high levels of wellbeing. That's why we prefer the term wellbeing. - Another term that we also use is "flourishing"

Welcome back and in this lesson I want to cover the serverless architecture.

Serverless is a type of architecture which is relatively commonplace within AWS, mainly because AWS includes many products and services which support its use.

The key thing to understand about the serverless architecture, aside from the fact that there are really servers running behind the scenes, is that it's not one single thing.

Serverless is an architecture, but it's more a software architecture than a hardware architecture.

The aim with the serverless architecture and where its name comes from is that as a developer or an architect or an administrator, you're aiming to manage few, if any, servers.

Servers are things which carry overhead, so cost, administration and risk, and the serverless architecture aims to remove as much of that as possible.

In many ways, serverless takes the best bits from a few different architectures, mostly microservices and event-driven architectures.

Within serverless you break an application down into as many tiny pieces as possible, even beyond microservices, collections of small and specialized functions.

These functions start up, do one thing really, really well, and then they stop.

In AWS, logically, because of this, Lambda is used.

But there are other platforms such as Microsoft Azure, which has their own equivalent, namely Azure Functions.

From an architecture perspective, the actual technology which is used is less relevant.

These functions which make up your application, they run in stateless and ephemeral environments.

Why this matters is because if the application is architected to assume a clean and empty environment, then these functions can run anywhere.

Every time they run, they obtain the data that they need, they do something, and then optionally, they store the result persistently somehow, or they deliver that output to something else.

The reason why Lambda is cheap is because it's scalable.

Each environment is easy to provision, and each environment is the same.

So the serverless architecture uses this to its advantage.

Each function that runs does so in an ephemeral and stateless environment.

And another key concept within serverless is that generally everything is event-driven.

This means that nothing is running until it's required.

Any function code that your application uses is only running on hardware when it's processing a system or customer interaction, an event.

Serverless environments should use fast products such as Lambda for any general processing needs.

Lambda as a service is built based on execution duration, and functions only run when some form of execution is happening.

Because serverless is event-driven, it means that while not being used, a serverless architecture should be very close to zero cost until something in that environment generates an event.

So serverless environments generally have no persistent usage of compute within that system.

Now, where you need other systems beyond normal compute, a serverless environment should use where possible managed services.

It shouldn't reinvent the wheel.

Examples are using S3 for any persistent object storage, or DynamoDB, which we haven't covered yet for any persistent data storage, and third-party identity providers such as Google, Twitter, Facebook, or even corporate identities such as Active Directory instead of building your own.

Other services that AWS provides, such as Elastic Transcode, can be used to convert media files or manipulate these files in other ways.

With the serverless architecture, your aim should be to consume as a service whatever you can, code as little as possible, and use function as a service for any general-purpose compute needs, and then use all of those building blocks together to create your application.

Now, let's look at this visually, because I think an architecture diagram might make it easier to understand exactly what a serverless architecture looks like.

So let's step through a simple serverless architecture, and we're going to do so visually.

And I want your default position to be that unless we state otherwise, you're not using any self-managed compute, so no servers and no EC2 instances, unless we discuss otherwise.

So that should be your starting position.

And at each step throughout this architecture, I'll highlight exactly why the parts are serverless and why it matters.

Now, we're going to use a slightly more inclusive example.

This time, we're going to use PetTube.

There was an uproar about PetTube only being for cats, and so it's rebranded to be a little bit more inclusive.

So to start with, we've got Julie using her laptop, and she wants to upload some woofy holiday videos.

And so to do that, she browsers to an S3 bucket that's running as a static website for the PetTube application.

She downloads some HTML, and that HTML has some JavaScript included within it.

Now, one crucial part of the serverless architecture is that modern web browsers are capable of running client-side JavaScript inside the browser.

And this is what actually provides the front end for the PetTube application, JavaScript that's running in the browser of the user that's downloaded from a static website S3 bucket.

So at this point, the application has no self-managed compute that's being used.

We've simply downloaded HTML from an S3 bucket with some included JavaScript that's now running in Julie's web browser.

Now PetTube uses third-party identity providers for its authentication.

Like all good serverless applications, it doesn't use its own store of identity, its own store of users.

It's lower admin overhead, and also remember there's a limit on the number of IAM users that can exist inside one AWS account.

That's 5,000 IAM users per account.

And so if we used IAM users for authentication, then PetTube would be limited to 5,000 users, and each user of the application would need one additional account.

So one additional username and one additional password.

So instead of doing that, we use a third-party identity provider and one that our users are already likely to have an account inside.

So that reduces the number of accounts that our users are required to maintain.

So the JavaScript that's running in Julie's browser communicates with the third-party identity provider, and we're going to assume that we're using Google.

And you'll have seen the screen that's generated if you've ever logged into Gmail or anything that uses Gmail logins, but this could just as easily be Twitter, Facebook, or any other third-party identity provider.

The key thing to understand is that Julie logs into this identity provider.

It's this identity provider that validates that the user claiming to be Julie is in fact Julie, so it checks her username and password.

And if it's happy with the process or if it's happy with the username and password combination that Julie's provided, then it returns to Julie an identity token.

And this token proves that she's authenticated with the Google identity provider.

Now, AWS can't directly use third-party identities, and so the JavaScript that's running in Julie's browser communicates with an AWS service called Cognito.

And Cognito swaps this Google identity token for temporary AWS credentials, and these can be used to access AWS resources.

So the JavaScript in Julie's browser now has available some temporary AWS credentials that it can use to interact with AWS.

And so it uses these temporary credentials to upload a video of Woofy to an S3 bucket.

This is the original bucket of our application, the bucket where the master videos go that our customers upload.

Notice that so far in this process, no self-managed compute, no servers have been used to provision this service.

We've performed all of these activities without using any compute servers or compute instances that we need to manage or design as solutions architects.

It's all delivered by using managed services, so S3, Cognito, and the Google identity provider.

Now, when the Woofy video arrives inside the original's bucket, that bucket is configured to generate an event.

That event contains the details of the object which was uploaded, and it's set to send that event to and invoke a Lambda function to process that video.

That Lambda function takes in the event and it creates jobs within the elastic transcoder service, which is a managed service offered by AWS which can take in media and manipulate that media.

One of the things that it can do is to transcode the media, so generate media of different sizes from one master video file.

Multiple jobs get created, one for each size of video that's required.

The elastic transcoder gets the location of the original video as part of the initiation of the job and it loads in that video at the start of each job processing cycle.

So each job outputs an object to a transcoder bucket, so one object for each different size of the original video.

In addition, details on each of the new videos are added to a database, in this case DynamoDB.

Now again at this stage, notice that we still have no self-managed servers.

The only resources that are consumed are storage space in S3, DynamoDB, and any processing time used for the Lambda function and any elastic transcoder jobs.

With this architecture so far, we've allowed a customer to upload a master video, we've transcoded it into different video sizes, and at no point have we consumed any self-managed compute, no EC2 instances or no other long-running compute services.

It's all managed services or compute that's used in Julie's browser.

Now the last part of the architecture is where Julie, by clicking another part of the client site that's running inside her browser, can interact with another Lambda function, and we'll call this My Media, and this Lambda function will load data from the database, identify which objects in the transcode bucket are Julie's, and return URLs for Julie to access.

And this is how Julie can load up a web page which show all of the videos that she's uploaded to the PetTube application.

Now this is a simplified diagram, in reality it's a little bit more complex.

For example, API Gateway would generally be used between any client-side processing and the Lambda functions, but conceptually this is actually how it works.

We've got no self-managed servers, we've got no self-managed database servers, we've got little, if any, costs that are incurred for base usage.

It's a fully consumption-based model.

It consumes compute only when it's being used, so when events are generated, either from a system-side or a client-side, and it uses third-party services as much as possible.

Now there are many third-party services to choose from, and you can never expect to know them all end-to-end.

The key thing to understand about serverless is the way to do things, and I've covered that in this lesson.

Later in the section you'll experience how to implement a serverless application within the demo lesson called PetCuddleatron.

And this will show you how to implement a serverless application just like the one that's on screen.

It's slightly less complex, but it's one that uses many of the same architectural fundamentals, and it should start to really cement the theory that you're learning right now.

Now before we move on to this demo, there are a few more services that I need to cover, which the PetCuddleatron demo lesson will utilize.

So for now, that's it for this lesson.

Thanks for watching.

Go ahead and complete this video, and then when you're ready, I'll look forward to you joining me in the next.

Welcome back.

In this lesson, I want to cover CloudWatch events.

We've covered CloudWatch earlier in the course, which focused on metrics and monitoring.

We've also covered CloudWatch logs, which focused on the ingestion and management of logging data.

CloudWatch events delivers a near real-time stream of system events.

These events describe changes in AWS products and services.

When an instance is terminated, started or stopped, these generate an event.

When any AWS products and services which are supported by CloudWatch events perform actions, they generate events that the product has visibility of.

Events Bridge is the service which is replacing CloudWatch events.

It can perform all of the same bits of functionality that CloudWatch events can produce.

It's got a superset of its functionality.

In addition, Events Bridge can also handle events from third parties as well as custom applications.

They do both share the same basic underlying architecture, but AWS are now starting to encourage a migration from CloudWatch events over to Events Bridge.

We've got a lot of architecture to cover, so let's jump in and get started.

Both Events Bridge and CloudWatch events perform at a high level the same basic task.

They allow you to implement an architecture which can observe if X happens or if something happens at a certain time, so Y, then do Z.

X is a supported service which generates an event, so it's a producer of an event.

Y can be a certain time or time period, and this is specified using the Unix Cron format, which is a flexible format letting you specify one or more times when something should occur, and Z is a supported target service to deliver the event to.

Events Bridge is basically CloudWatch events version two.

It uses the same underlying APIs, and it has the same basic architecture, but AWS recommend that for any new deployments, you should use Events Bridge because it has a superset of the features offered by CloudWatch events.

Things created in one are visible in the other for now, but this could change in the future.

So as a general best practice, you should start using Events Bridge by default for any of the functions that you can use CloudWatch events for.

Now, both of these services actually operate using a default entity, which is known as an event bus, and both of them actually have a default event bus for a single AWS account.

A bus in this context is a stream of events which occur from any supported service inside that AWS account.

Now, in CloudWatch events, there is only one event bus available, so it's implicit.

It's not really exposed to the UI.

It just exists.

You interact with it, but because there's only one of them, it's not actually exposed as a visible thing.

You just look for events and then send these events to targets when you want something to occur.

So in CloudWatch events, there is only one event bus, and it's not exposed inside the UI.

In Event Bridge, you can create additional buses, either for your applications or third-party products and services, and you can interact with these buses in the same way as the account default event bus.

Now, with CloudWatch events and Event Bridge, you create rules, and these rules pattern match events which occur on the buses, and when they see an event which matches, they deliver that event to a target.

Alternatively, you also have schedule-based rules which are essentially pattern-matching rules but which match a certain date and time or ranges of dates and times.

So if you're familiar with the Unix Cron system, this is similar.

For a schedule rule, you define a Cron expression, and the rule executes whenever this matches and delivers this to a particular target.

So the rule matches an event, and it routes that event to one or more targets which you define on that rule.

And an example of one target is to invoke a specific Lambda function.

Now, architecturally, at the heart of Event Bridge is the default account event bus, which is a stream of events which are generated by supported services within the AWS account.

Now, EC2 is an example of a supported service, and let's say in this case, we've got Bob changing the state of an EC2 instance, and he's changing the state from stopped to running.

When the instance changes state, an event gets generated which runs through the event bus.

Event Bridge, which sits over the top of any event buses that it has exposure to, monitors all of the events which pass through this event bus.

Now, within Event Bridge or CloudWatch events, which I'm going to start calling just Event Bridge from now on because it makes it easier, but within Event Bridge, we have rules.

Now, rules are created, and these are linked to a specific event bus, and the default is the account default event bus.

The two types of rules are pattern matching rules, and these match particular patterns of the events themselves as they pass through the event bus.

We've also got scheduled rules which match particular cron-formatted times or ranges of times, and when this cron-formatted expression matches a particular time, the rule is executed, and in both of these cases, when a rule is executed, the rule delivers the particular event that it's matched through to one or more targets.

And of course, as I just mentioned, examples of these targets could be to invoke a lambda function.

Now, events themselves are just JSON structures, and the data in the event structure can be used by the targets.

So in the example of a state change of an EC2 instance, the lambda function will receive the event JSON data, which includes which instance has changed state, what state it's changed into, as well as other things like the date and time when the change occurred.

So that's a theory of both CloudWatch events and the event bridge, and both of these products are used as a central point for managing events generated inside an AWS account and controlling what to do with those events.

So at this point, that is everything that I wanted to cover.

Go ahead and complete this lesson, and then when you're ready, I look forward to you joining me in the next.

• [ ] 常用的开发插件 #share

Majority electoral system - A candidate does not need more than 50% to be declared the winner - Requires pure majority(50% + 1)

Preferential voting - Voters assign the number 1 to their first choice, 2 to their second choice and so forth - If a voter's first choice fails to be elected, officials may re-examine their vote and their other preferences will have consideration - Delivers a satisfactory result to most voters

First-past-the-post - the voter gets to cast a single vote for the candidate of their choice - The person with the most ballots wins

Welcome back and in part three of this series, I want to finish off and talk about some advanced elements of Lambda.

Now we've got a lot to cover, so let's jump in and get started.

First, I want to talk about the ways a Lambda function can be invoked.

We've got three different methods for invoking a Lambda function.

We've got synchronous invocation, asynchronous invocation, and invocation using event source mappings.

And I want to step through each of them visually so that you can understand in detail how they work because this is essential for the exam.

So let's start off with synchronous invocation of Lambda.

With this model, you might start off with a command line or API directly invoking a Lambda function.

The Lambda function is provided with some data and it executes that data.

Now all this time, the command line or API is waiting for a response because it's synchronous.

It needs to wait here until the Lambda function completes its execution.

So the Lambda function finishes and it returns that data, whether it's a success or a failure.

Now synchronous invocation also happens if Lambda is used indirectly via the API gateway, which is the use case for many serverless architectures.

So we might have some clients using a web application via API gateway and this proxies through to one or more Lambda functions.

Again, the Lambda function performs some processing all the while the client is waiting for a response within their web application.

And then when the Lambda function responds, this goes back via the API gateway and back through to the client.

The common factors with both of these approaches is that the client sends a request which invokes Lambda and the result be it a success or failure is returned during that initial request.

The client is waiting for any data to be returned.

Another implication of a synchronous invocation is that any errors or retries have to be handled within the client.

The Lambda function runs once, it returns something and then it stops.

If there's a problem or data isn't processed correctly, then the client needs to rerun that request.

And this happens at the client side.

So synchronous invocation is generally used when it's a human directly or indirectly invoking a Lambda function.

Next, let's look at asynchronous invocation.

And this is typically used when AWS services invoke Lambda functions on your behalf.

Let's use an example, an S3 bucket with S3 events enabled.

So we upload a new image of whiskers to this S3 bucket.

This causes an event to be generated and sent through to Lambda.

And this is an asynchronous invocation.

So S3 isn't waiting around for any kind of response.

It basically just forgets about it at this point.

Once it sent that event through to Lambda, it doesn't continue waiting.

It doesn't worry about this event at all.

Now maybe as part of processing this image, it's generating a thumbnail or maybe performing some kind of analysis and storing that data into DynamoDB.

But again, S3 isn't waiting around for any of this.

It's asynchronous.

Lambda is responsible for any reprocessing in the event that there's a failure.

And this reprocessing value is configurable between zero and two times.

Now a key requirement for this is that the function code needs to be idempotent.

And this is important.

If you've never heard this term before, let me explain.

Let's say that you had $10 in your bank account and I wanted to increase this value to $20.

Now there are two ways that I could do this if I operated the bank.

I could simply add $10 to your balance, increasing it from 10 to 20, or I could explicitly set the balance to 20.

Now if I set the balance to 20 and this operation failed at some undetermined point in this process, then I could simply rerun the process, safe in the knowledge that even running it again on your balance would only at worst set the value to $20 again.

This is known as an idempotent operation.

You can run it as many times as you want and the outcome will be the same.

Now if I performed the operation where I added $10 to your account and the operation failed, it could have failed before it added the $10 or after.

If it failed after and I rerun the operation, well now you'd have $30 and this is an example of something which is not idempotent.

When Lambda retries an operation it doesn't really provide any other information.

The function just reruns.

So logically in this example you would need to make sure that your function code isn't additive or subtractive.

It just needs to perform its intended task.

With this example it needs to set your balance to $20.

Generally when designing a Lambda function which is used in this way, the Lambda function needs to finish with a desired state.

It needs to make something true.

If you're using Lambda functions which are designed in a non-idempotent way, you can end up with some questionable results.

Now Lambda can be configured to send any events which it can't process after those automatic retries to a dead letter queue which can be used for diagnostic processing.

And a new feature of Lambda is the ability to create destinations.

So events processed by Lambda functions can be delivered to another destination such as SQS, SNS, another Lambda function and even EventBridge.

And separate destinations can be configured based on successful processing or failures.

So this is asynchronous invocation.

It's generally used by AWS services which are capable of generating events and sending those events to Lambda.

It means that Lambda can automatically reprocess failed events and the original source of the event isn't waiting for processing to complete.

But there is a third type of invocation.

The last type of invocation is known as Event Source Mapping.

And this is typically used on streams or queues which don't generate events.

So things where some kind of polling is required.

Let's look at an example.

Let's say that we have a Kinesis data stream and into this stream, a fleet of producer vans driving around scanning with LIDAR and imaging equipment are all producing data which is being put into a Kinesis stream.

Now Kinesis is a stream based product.

Generally consumers can read from a stream but it doesn't generate events when data is added.

So historically this wouldn't have been an ideal fit for Lambda which is an event driven service.

So what happens is that we have a hidden component called an event source mapping which is polling queues or streams looking for new data and getting back source batches.

So batches of source data from this data source.

Now these source batches are then broken up as required based on a batch size and sent into a Lambda function as event batches.

Now a single Lambda function invocation could in theory receive hundreds of events in a batch.

It depends on how long each event takes to process.

Remember Lambda has a 15 minute timeout so you need to carefully control this event batch size to ensure that the Lambda function doesn't terminate before completing this batch.

Now there's one really important thing that you need to understand about event source mapping.

With a synchronous invocation an event is delivered to Lambda from the source and Lambda doesn't need permissions to the source service unless it actually wants to read more data from that source.

For example, if an object is added to an S3 bucket then S3 generates and delivers an event which contains details of that event.

So which object was uploaded and perhaps some other metadata.

But unless you need to read additional data from S3 maybe to get the actual object well then the Lambda function doesn't need S3 permissions.

With event source mapping invocation the source service isn't delivering an event.

The event source mapping is reading from that source.

And so the event source mapping uses permissions from the Lambda execution role to access the source service.

And this is really important to know because it does come up in the exam.

So even if a Lambda function receives an event batch containing Kinesis data even though the Lambda function doesn't directly read from Kinesis the execution role needs Kinesis permissions because the event source mapping uses them on its behalf to retrieve that data.

Now any batches which consistently fail can be sent to an SQS queue or an SNS topic for further processing or analysis.

Now that's the third type of invocation.

This is event source mapping invocation.

And that's the method used when Lambda functions are processing SQS queues, Kinesis streams, DynamoDB streams and even Amazon managed streaming for Apache Kafka.

And this last one is something that we won't be covering within the course.

But it's important to know all of the different types of products that use event source mapping based invocation.

With that being said that's all of the three types of invocation I wanted to cover.

So let's move on to a different topic.

This time Lambda versions.

With Lambda functions it's possible to define specific versions of Lambda functions.

So you could have different versions of the given function for example, version one, version two and version three.

Now as it relates to Lambda, a version of a function is actually the code plus the configuration of that Lambda function.

So the resources and any environment variables in addition to any other configuration information.

Now when you publish a version, that version is immutable.

It never changes once it's published.

And it even has its own Amazon resource name.

So once you publish a version you can no longer change that version.

There's also the concept of dollar latest and dollar latest points at the latest version of a Lambda function.

Now this can obviously change as you publish later and later versions of the function.

So this is not immutable.

You can also create aliases.

So for example, dev stage and prod.

And these can point at a particular version of a Lambda function.

And these can be changed.

So these aliases are not immutable.

So generally with large scale deployments of Lambda you'd be producing Lambda function versions for all of the major changes.

And using aliases so that different components of your serverless application can point at those specific immutable version numbers.

So that's important to know for the exam.

So the last thing I want to talk about is Lambda startup times.

And to understand that you need to understand how Lambda functions are actually executed.

Lambda code runs inside a runtime environment.

And this is also referred to as an execution context.

Think of this as a small container which is allocated an amount of resource which runs your Lambda code.

When a Lambda function is first invoked, let's say by receiving an S3 event, this execution context needs to be created and configured.

And this takes time.

First the environment itself is created and this requires physical hardware.

Then any run times which are required are downloaded and installed.

Let's say this is for Python 3.8.

Then the deployment package is downloaded and then installed and this takes time.

Now this process is known as a cold start.

And all in this process can take hundreds of milliseconds or more, which can be significant if a Lambda function is performing a task which touches a human who is expecting a response.

Now if this is an S3 event, then maybe this extra time isn't such a big deal.

But you need to be aware that this cold start occurs because an execution context is being created and configured.

Any prerequisites are being downloaded and installed.

The deployment package is being downloaded and installed.

And that's all before the function itself can execute.

Now if the same Lambda function is invoked again without too much of a gap, then it's possible that Lambda will use the same execution context.

And this is known as a warm start.

It doesn't need to set up the environment or download the deployment package because all of that is already contained within the execution context.

This time the context just receives the event and immediately begins processing.

A warm start means the code can be running within milliseconds because there's no lengthy build process.

A Lambda function which invokes again fairly soon after a cold start can reuse an execution context.

But if too long a time period goes between invocations, then the context can be deleted which results in another cold start.

Also one function invocation runs at a time per context.

So if you need 20 invocations of a function at once, then this can result in 20 cold starts.

Now you can make this process more efficient.

You can actually use a feature known as provisioned concurrency where you can inform AWS in advance.

An execution context can be provisioned for you in advance for Lambda invocations.

You might use these when you know that you have periods of high load on a serverless application or if you're preparing for a new production release of a serverless application and want to pre-create all of these execution environments.

Now there are also other things that you can do to improve performance.

You can use the temp space to pre-download things within an execution context.

For example, maybe you're using some animal images as part of your processing.

Well, if another function uses the same execution context, then it too will have access to those same animal images without having to download them a second time.

Now you do need to be careful because your functions need to be able to cope with the environment being new and clean every time they can never assume the presence of anything.

From a code perspective, you can create other things like database connections outside of the Lambda function handler code.

So when you create a Lambda function, generally most things go within the Lambda function handler.

But if you create anything outside of the Lambda function handler, then these will be made available for any future function invocations in the same context.

So anything that you define within a Lambda function handler is limited to that one specific invocation of that Lambda function.

But for anything which you anticipate there being a potential for reuse, you can declare that outside of the Lambda function handler.

And in theory, that will be available for any other invocations of the Lambda function which occur within that same execution context.

But again, you need to make sure that your function doesn't require or expect that.

Every single time a function invokes, it should be absolutely fine with recreating everything.

You should by default assume that execution contexts are stateless and any invocation of a Lambda function is going to be operating in a completely freshly created environment.

But if you want to be efficient, your functions should also be able to reuse common aspects that persist through different function invocations.

Now again, these are all deep dive things that you need to be aware of for the exam.

I've covered a lot of these elements across all three parts of this Lambda deep dive mini series.

But at this point, that's everything I wanted to cover in part three.

And this is the last part of this mini series.

So thanks for watching.

Go ahead and complete this video.

And when you're ready, I look forward to you joining me in the next lesson.

Welcome back to part two of this lesson series going into a little bit more depth on Lambda.

In this part of the series I'm going to be talking about Lambda networking, Lambda permissions and Lambda monitoring.

Now this is a lot to cover in one lesson so let's jump in and get started.

Lambda has two networking modes and you need to be aware of both of them for the exam.

First we have public which is the default and then second we have VPC networking.

Now you need to understand the architecture of both of them so let's step through them in a little bit more detail.

For public networking we start with an AWS environment and inside it a single Lambda function.

Now this is part of a wider application let's say the Categorum Enterprise application running in a VPC which uses Aurora for the database, EC2 for compute and the Elastic file system for shared file storage.

Now this is the default configuration for Lambda where it's running in the public AWS network so Lambda using this configuration can access public space AWS services such as SQS and DynamoDB or internet-based services such as IMDB if the Lambda function wanted to fetch the latest details of cat themed movies and TV shows.

So Lambda running by default using public networking means that it has network connectivity to public space AWS services and the public internet.

It can connect to both of those from a networking perspective and as long as it has the required methods of authentication and authorization then it can access all of those services.

Now public networking offers the best performance for Lambda because no customer specific networking is required.

Lambda functions can run on shared hardware and networking with nothing specific to one particular customer but this does mean that any Lambda functions running with this default have no access to services running within a VPC unless those services are configured with public addressing as well as security rules to allow external access so this is a big limitation that you need to understand for the exam so the architecture on screen now this Lambda function could not access Aurora EC2 or the Elastic File system unless they had public addressing and the security was configured to allow that access.

So in this example without configuration changes the Lambda function could access public services but would have no access to anything running inside the VPC.

Now in most cases in my experience Lambda is used with this public networking model but there are situations where this isn't enough and for those situations Lambda can be configured to run inside a VPC.

Let's look at how.

This time we have the same architecture so a VPC running within AWS but this time the Lambda function is configured to run inside a private subnet at the bottom.

Now this is the same subnet where the Catergram Enterprise infrastructure is running from and for the exam specifically the key thing to understand about Lambda's running inside a VPC is that they obey all of the same rules as anything else running in a VPC because they're actually running within that VPC.

So to start with this means that Lambda functions running inside a VPC can freely access other VPC based resources assuming any network ACLs and security groups allow that access but the flip side of this means they can't access things outside of the VPC unless networking configuration exists within the VPC to allow this external access.

So by default with this architecture the Lambda function couldn't access DynamoDB or any internet based endpoints such as with this example IMDB.

Now if you face any exam questions or you need to design any solutions which involve Lambda functions running within a VPC then just treat them like anything else running in that VPC.

So this means that you could use a VPC endpoint for example a gateway endpoint to provide access to DynamoDB because the Lambda function is running within the VPC it could utilize a gateway endpoint to access DynamoDB or in the case that the Lambda function needed access to AWS public services or the internet you could deploy a NAT gateway in a public subnet and then attach an internet gateway to the VPC.

Remember Lambda running within a VPC behaves like any other VPC based service the same gateways and configurations are needed to allow VPC based Lambda functions to communicate with the AWS public zone and the public internet.

Now you also need to give your Lambda functions EC2 network permissions via the execution role which I'll cover very soon because the Lambda service needs to create network interfaces within your VPC it requires these permissions and this architecture of using network interfaces within a VPC is what I want to quickly cover now.

Now there used to be disadvantages to running Lambda in a VPC significant disadvantages and the reason was the networking architecture that Lambda used.

VPC based Lambda functions don't actually run within your VPC the way they work is similar to Fargate so we have AWS and there's a Lambda service VPC and a customer VPC.

Now let's keep things simple and say that we only have three Lambda functions.

Now the way that this historically worked is that each of these Lambda functions when invoked would create an elastic network interface within the customer VPC and traffic would flow between this service VPC and the customer VPC.

Now the problem is that configuring these elastic network interfaces on a per function per invocation basis would take time and add delay to the execution of the Lambda function code.

In addition this architecture doesn't scale well because parallel function executions or concurrency required additional elastic network interfaces and the more popular a system became the worse the problem became with larger systems you had more and more performance issues and more and more issues with keeping VPC capacity available for larger and larger numbers of ENIs.

Now luckily this is the old architecture this is the way that Lambda used to handle this private networking it's not how it works anymore.

With the new way instead of requiring an elastic network interface per function execution AWS analyze all of the functions running in a region in an account and build up a set of unique combinations of security groups and subnets.

So for every unique one of those one ENI is required in the VPC.

So if all your functions used a collection of subnets but the same security groups then one network interface would be required per subnet if they all used the same subnet and all used the same security group then all of your Lambda functions could use the single elastic network interface.

So a single connection between the Lambda Service VPC and your VPC is created for every unique combination of security groups and subnets used by your Lambda functions.

Now the network interfaces using this architecture are created when you configure the Lambda function and typically this might take 90 seconds but this is done once so when you create the function or when you update the network and configuration this network and configuration is created or updated and that means that it isn't required every single time a Lambda function is invoked so it doesn't delay your function invocations.

Now this means that you can use private networking at scale without increasing the number of elastic network interfaces required.

So where it used to be a bad idea performance-wise to use VPC-based lambdas this is no longer the case.

So that's networking so this is how you configure Lambda functions if you need them to have access to private VPC services and it's important that you understand both the public and VPC networking model especially for the exam because you will face questions on the exam about executing Lambda functions within a VPC.

Again one really important hint that I will provide is just treat Lambda functions running in a VPC like any other VPC-based resource and by now you should know how to architect a VPC so that services running in that VPC have access to everything that they need so just treat Lambda functions in the same way.

Now let's look at the security of Lambda functions.

When it comes to Lambda permissions there are actually two key parts of the permissions model that you need to understand.

One of them is pretty well known and that's covered at the associate level the other not so much.

Now let's start with a typical Lambda environment this is a runtime environment the thing where your Lambda functions execute within so this is running a runtime in this case Python 3.8 it's allocated some resources and the code loads and runs within this environment.

Now for this environment to access any AWS products and services it needs to be provided with an execution role this is a role which is assumed by Lambda and by doing so the code within the environment gains the permissions of that role based on the role's permissions policy so a role is created which has a trust policy which trusts Lambda and the permissions policy that that role has is used to generate the temporary credentials that the Lambda function uses to interact with other resources so in many ways this is just the same as an EC2 instance role so this governs what permissions the function receives which might be something like loading data from DynamoDB and storing output data into S3.

Now this is the most well known aspect of Lambda permissions but there is another part Lambda actually has resource policies now this in many ways is like a bucket policy on S3 it controls who can interact with a specific Lambda function it's this resource policy which can be used to allow external accounts to invoke a Lambda function or certain services to use a Lambda function such as SNS or S3.

The resource policy is something changed when you integrate other services with Lambda and you can manually change it via the CLI or the API unless something's changed between creating this lesson and when you're watching it it currently can't be changed using the console UI so this is only something which can be manipulated using the CLI or the API so that's how security works within a Lambda function now one more thing that I want to cover before finishing up with part two is logging so Lambda uses cloud watch, cloud watch logs and x-ray for various aspects of its logging and monitoring so any logging information generated from Lambda executions that goes into cloud watch logs so the output of Lambda functions any messages that you output to the log any errors details on the duration of the execution that's all stored into cloud watch logs any metrics so details such as invocation successes or failure numbers any retries anything to do with latency that's all stored in cloud watch so cloud watch is the thing that stores metrics and this is important to understand logging goes into cloud watch logs and any details on the number of indications successes or failures anything around metrics goes straight into cloud watch now lambdas can also be integrated into x-ray which I cover elsewhere in the course and this can be used to add distributed tracing capability so if you need to trace the path of a user or the path of a session through a serverless application which uses Lambda then you can use the x-ray service now I don't expect this to feature heavily on the exam but just remember the terms x-ray and distributed tracing because that might come in handy for one or two exam questions if these topics do crop up now one really important thing to remember for the exam is that for Lambda to be able to log into cloud watch logs to generate the output of any of the executions you need to give Lambda permissions via the execution role so there's actually a pre-built policy and role within aws specifically designed to give Lambda functions the basic permissions that they require to log information into cloud watch logs and one really common exam scenario is where you're trying to diagnose why a Lambda function is not working there's nothing in cloud watch logs and one possible answer is that it doesn't have the required permissions via the execution role now that's everything I wanted to cover in part two of this Lambda in-depth mini series so we've covered networking both public and private we've covered security and we've covered logging so go ahead and complete this lesson and when you're ready I look forward to you joining me in part three.

Welcome back and in this multi-part lesson mini series, I want to talk about AWS Lambda.

Lambda is a function as a service or a fast product.

This means that you provide specialized short running and focused code to Lambda and it takes care of running it and billing you only for what you consume.

So a Lambda function is a piece of code which Lambda runs and every Lambda function is using a supported runtime.

So an example of a supported runtime is Python 3.8.

So when you create a Lambda function, you need to define which runtime that piece of code uses.

Now, when you provide your code to Lambda, it's loaded into and executed within a runtime environment.

And this runtime environment is specifically created to run code using a certain runtime, a certain language.

So when you create a Lambda function that uses the Python 3.8 runtime, then the runtime environment that's created is itself specifically designed to run Python 3.8 code.

Now, when you create a Lambda function, you also define the amount of resource that a runtime environment is provided with.

So you directly allocate a certain amount of memory and based on that amount of memory, a certain amount of virtual CPU is allocated, but this is indirect.

You don't get to choose the amount of CPU.

This is based on the amount of memory.

Now, the key thing to understand about Lambda as a service, because it's a function as a service product, because it's designed for short running and focused functions, you only actually build for the duration that a function runs.

So based on the amount of resource allocated to an environment and based on the duration that that function runs for per invocation, that determines how much you'll build for the Lambda product.

So you'll build for the duration of function executions.

Now, Lambda is a key part of serverless architectures running within AWS.

And over this section of the course, you're going to get some experience of how you can use Lambda to create serverless or event-driven architectures.

Architecturally, the way that Lambda works is this.

You define a Lambda function.

Now, you can think of a Lambda function as a unit of configuration.

Yes, you can also use the term Lambda function to describe the actual code.

But when you think of a Lambda function, think of it as the code plus all the associated wrappings and configuration.

Your Lambda function at its most basic is a deployment package which Lambda executes.

So when you create a Lambda function, you define the language which the function is written in.

You provide Lambda with a deployment package and you set some resources.

And whenever the Lambda function is invoked, what actually happens is the deployment package is downloaded and executed within this runtime environment.

Now, Lambda supports lots of different runtimes.

Some of the common ones are various different versions of Python.

We also have Ruby.

We've got Java.

We've also got Go and there's also C# as well as various versions of Node.js.

Now, you can also create custom ones using Lambda layers.

And many of these are created by the community.

For the exam though, one really important point is that if you see or hear the term Docker, consider this to mean not Lambda.

So Docker is an anti-pattern for Lambda.

Now, Lambda does now support using Docker images, but this is distinct from the word Docker.

If you hear the term Docker in the exam, then it generally will be referring to traditional containerized computing.

So that's using a specific Docker image to spin up a container and use it in a containerized compute environment such as ECS.

Now, you can also use container images with Lambda.

Now, that's a different process.

That means that you're using your existing container build processes, the same ones that you use to create Docker images.

But instead, you're creating specific images designed to run inside the Lambda environment.

So don't confuse Docker container images and Docker with images used for Lambda.

They're two different things.

The only thing that they share is that you can use your existing build processes to build Lambda images.

Now, custom runtimes could allow languages such as Rust, which is a very popular community-based language to work within the product.

So if you search using Google or any other popular search engine, you'll be able to find lots of languages which have been added by the community using the Lambda layer functionality.

And I'll be talking about that elsewhere in the course.

Now, you select the runtime to use when creating the function, and this determines the components which are available inside the runtime environment.

So Python code, for instance, requires Python of a certain version to be installed in addition to various Python modules.

Conceptually, think about it like this.

Every time a Lambda function is invoked, which means to execute that function, a new runtime environment is created with all of the components that that Lambda function needs.

Let's say, for example, a Python 3.8-based Lambda function.

So the code loads, it's executed, and then it terminates.

Next time, a new clean environment is created, it does the same thing, and then it terminates.

Lambda functions are stateless, which means no data is left over from a previous invocation.

Every time a function is invoked, it's a brand new invocation, a brand new environment.

Now, I'm going to be talking about this in part 3 of this series, because this isn't always the case, but you have to assume that it is architecturally.

So your code running within Lambda needs to be able to work 100% of the time if it's a new environment.

Lambda runtime environments have no state.

Now, there are some situations where a function might be invoked multiple times within the same environment.

And I'll be talking about that in part 3 of this series.

But as a base level, a default, assume that every time a Lambda function is invoked, it's inside a brand new runtime environment.

Now, you also define the resources that Lambda functions use, and this determines how much resource the runtime environment gets.

Now, you directly define the memory.

And this is anywhere from 128 MB to 10 to 40 MB in one MB steps.

Now, you don't directly control the amount of virtual CPU.

This scales with the memory.

So 1769 MB of memory gives you one VCPU of allocation, and it's linear.

So the less memory means less virtual CPUs, and more memory means additional VCPU capacity.

The runtime environment also has some disk space allocation.

512 MB is mounted as forward slash TMP within the runtime environment.

This is the default amount, but it can scale to 10,240 MB.

Now, you can use this, but keep in mind, you have to assume that it's blank every single time a Lambda function is invoked.

This should only be viewed as temporary space.

Lambda functions can run for up to 900 seconds or 15 minutes.

And this is known as the function timeout.

This is important because for anything beyond 15 minutes, you can't use Lambda directly.

And that's a really important figure to know for the exam.

You know by now I'm not a fan of people memorizing facts and figures, but this is definitely one that you need to remember for the exam.

So 15 minutes is a critical amount of time for a Lambda function.

You can use other things, such as step functions, to create longer running workflows, but one invocation of one function has a maximum of 15 minutes or 900 seconds.

Now, we're going to be covering security in more detail in part two, as well as networking.

But the security for a Lambda function is controlled using execution roles.

And these are IAM roles, assumed by the Lambda function, which provides permissions to interact with other AWS products and services.

So any permissions which a Lambda function needs to be provided with are delivered by creating an execution role and attaching that to a specific Lambda function.

Now, just a few final things before we finish up some common uses of Lambda.

So Lambda forms a core part of the delivery of serverless applications within AWS.

And generally this uses products such as S3, API gateway, and Lambda.

So these three together are often used to deliver serverless applications.

Lambda can also be used for file processing, using S3, S3 events, and Lambda.

So a very common example that's used in training is watermarking images.

So have images uploaded to S3, generate an S3 event, invoke a Lambda function, which applies a watermark, and then terminates.

And you're only billed for the compute resources used during those Lambda function invocations.

You can also use Lambda for database triggers.

So this is using DynamoDB, as well as DynamoDB streams, and then Lambda.

So Lambda can be invoked any time data is inserted, modified, or deleted from a DynamoDB table with streams enabled.

And this is another powerful architecture.

You can also use Lambda to implement a form of serverless cron.

So you can use EventBridge or CloudWatch events to invoke Lambda functions at certain times of day, or certain days of week, to perform certain scripted activities.

And this is something that traditionally you would need to run on something like an EC2 instance, but using Lambda means that you're only billed for the amount of time that these functions are executing.

So this is another really common use case.

And then finally, you can perform real-time stream data processing.

So Lambda's can be configured to invoke whenever data is added to a Kinesis stream.

And this can be useful because Lambda is really scalable.

And so it can scale with the amount of data being streamed into a Kinesis stream.

And again, this is another really common architecture for any businesses that are streaming large quantities of data into AWS, and they require some form of real-time processing.

Now that's everything that I wanted to cover in part one of this series.

Remember, it's a three-part mini-series, part two and part three, are going to introduce some more advanced concepts.

Specifically, though, is that you'll need for the exam.

But at this point, go ahead, complete this lesson, and then 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 the previous architecture can be evolved by using queues.

A queue is a system which accepts messages.

Messages are sent onto a queue and messages can be received or polled off the queue.

In many queues there's ordering.

So in most cases messages are received off the queue in a 5.0 or first in, first out architecture.

Although it's worth noting that this isn't always the case.

Using a queue based decoupled architecture, CatTube would look something like this.

Bob would upload his newest video of whiskers laying on the beach to the upload component.

And once the upload is complete, instead of passing this directly onto the processing tier, it does something slightly different.

It stores the master 4k video inside an S3 bucket.

And it also adds a message to the queue, detailing where the video is located, as well as any other relevant information such as what sizes are required.

This message, because it's the first message in the queue, is architecturally at the front of the queue.

At this point the upload tier, because it's uploaded the master video to S3 and added a message to the queue, it's finished this particular transaction.

It doesn't talk directly to the processing tier and it doesn't know or care if it's actually functioning.

The key thing is that the upload tier doesn't expect an immediate answer from the processing tier.

The queue has decoupled the upload and processing components.

It's moved from a synchronous style of communication where the upload tier expects and needs an immediate answer and it needs to wait for that answer.

Instead, it uses asynchronous or async communications where the upload tier sends the message and it can either wait in the background or just continue doing other things while the processing tier does its job.

Now while this process is going on, the upload component is probably getting additional videos being uploaded and they're added to the queue along with the whiskers video processing job.

Other messages that are added to the queue are behind the whiskers job, because with this queue there is an order.

It's a 5.0 or first in, first out queue.

Now at the other side of the queue we have an auto scaling group which has been configured.

It has a minimum size of 0, a desired size of 0 and a maximum size of 1,337.

So currently it has no instances provisioned.

But it has auto scaling policies which provision or terminate instances based on what's called the queue length.

And the queue length is just the number of items in the queue.

Because there are messages on the queue added by the upload tier, the auto scaling group detects this and so the desired capacity is increased from 0 to 2.

And because of this, instances are provisioned by the auto scaling group.

And these instances start polling the queue and receive messages that are at the front of the queue.

Remember that these messages contain the data for the job, but they also contain the location of the S3 bucket and the location of the object in that bucket.

So once these jobs are received from the queue by these processing instances, they can also retrieve the master video from the S3 bucket.

Now these jobs are processed by the instances and then they're deleted from the queue and this leaves only one job in the queue.

At this point maybe the auto scaling group decides to scale back because of the shorter queue length.

So it reduces the desired capacity from 2 to 1.

And this process terminates one of the processing instances.

The instance that remains polls the queue and receives the one final message.

It completes processing of that message so it performs the transcoding on the videos and it leaves zero messages in the queue.

The auto scaling group realizes this, it scales back the desired capacity from 1 to 0 and that results in the termination of the last processing EC2 instance.

Using a queue architecture, so placing a queue in between two application tiers decouples those tiers.

One tier adds jobs to the queue and doesn't care about the health or the state of the other and another tier can read jobs from that queue and it doesn't care how they got there.

This is unlike the example on the previous screen where application load balancers were used between tiers.

While this did allow for high availability and scaling, the upload tier in the previous example still synchronously communicated with one instance of the processing tier.

This way using the queue architecture no communication happens directly.

The components are decoupled, the components can scale independently and freely and in this case the processing tier which uses a worker fleet architecture.

It can scale anywhere from zero to a near infinite number of instances based only on the length of the queue.

So the number of messages in the queue.

Now this is a really powerful architecture because of the asynchronous communications that it uses.

And it's an architecture that's commonly used in applications such as CatTube where customers upload things for processing and you want to ensure that you've got a worker fleet behind the scenes that can scale to perform that processing.

Now you might be asking why this matters at least in the topic of event driven architectures and I'm getting there, I promise.

If you continue breaking down a monolithic application into smaller and smaller pieces, you'll end up at a microservice architecture which is a collection of as the name suggests microservices.

And microservices do individual things very well.

In this example we have the upload microservice, the processing microservice and the store and manage microservice.

A full application such as CatTube might have hundreds or even thousands of these microservices.

They might be different services or there might just be lots of copies of the same service such as this example which is lucky because it's far easier to diagram.

The upload service is a producer, the processing node is a consumer and the data, store and manage microservice performs both.

Now logically producers produce data or they produce messages.

Consumers as the name suggests consume data or messages and then you've got microservices that can do both things.

Now the things that services produce and consume architecturally are events.

Cues can be used to communicate events as we saw with the previous example but larger microservices architectures can get complex pretty quickly.

With services needing to exchange data between partner microservices, if we do this with a queue architecture then logically we're going to have a lot of queues.

It works but it can be complicated.

Keep in mind a microservice is just a tiny self-sufficient application.

It has its own logic, its own store of data and its own input/output components.

Now if you hear the term event driven architecture I don't want you to be too apprehensive.

Event driven architectures are just a collection of event producers which might be components of your application which directly interact with customers or they might be parts of your infrastructure such as EC2 or they might be systems monitoring components.

They're bits of software which generate or produce events in reaction to something.

If a customer clicks submit that might be an event.

If an error occurs when packing a customer order or an error occurs during the upload of the whiskers holiday video that's an event.

Producers are things which produce events and the inverse of this are consumers.

Pieces of software which are ready and waiting for events to occur.

If they see an event which they care about they will do something with that event.

They will take an action.

It might be displaying something for a customer.

It might be to dispatch a human to resolve an order packing issue or it might be to retry an upload.

Components or services within an application can be both producers and consumers.

Sometimes a component might generate an event for example a failed upload and then consume events to force a retry of that upload.

Now the key thing to understand about event driven architectures is that neither the producers or the consumers are sat around waiting for things to occur.

They're not constantly consuming resources.

They're not running at 100% CPU load waiting for things to happen.

With producers events are generated when something happens when a button is clicked when an upload works or when it doesn't work.

These producers produce events.

Consumers are not waiting around for those events.

They have those events delivered and when they receive an event they take an action and then they stop.

They're not constantly consuming resources.

Now applications would be really complex if every software component or service needed to be aware of every other component.

If every application component required a queue between it and every other component to put events into and access them from it would be a really complex application architecture.

Best practice event driven architectures have what's called an event router.

A highly available central exchange point for events and the event router has what's known as an event bus and you can think of this like a constant flow of information.

When events are generated by producers they're added to this event bus and the router can deliver these to event consumers.

The WordPress system that we've used to date we've been running it on an EC2 instance and an EC2 instance is essentially a consistent allocation of resources.

Whether that WordPress is using low amounts of load or large amounts of load we're still going to be billed for that EC2 instance.

We're still consuming resources.

I want you to imagine a system with lots of small services all waiting for events.

If events are received the system springs into action it allocates resources and it scales components up as required.

It deals with those events and then it returns to the low or no resource usage which is the default state.

Event driven architectures only consume resources as and when required.

So with an event driven architecture there's generally nothing constantly running nothing waiting for things.

We're not constantly polling hoping for things to happen.

We have producers which generate events when something happens.

If you're browsing the Amazon.com website and you click on order that generates an event and actions are taken based on that event.

But the Amazon.com website is not constantly checking your browser each and every second to check if you've clicked submit on that order.

So producers generate events when something happens so when clicks happen when errors occur when criteria are met when uploads complete or any other actions.

So producers they generate event on things occurring and these events are delivered to consumers of those events and that generally happens using an event router.

An event router decides which consumers to deliver events to and when that occurs when these events are delivered to the consumers then actions are taken.

And then once the action is complete the system returns to waiting it goes into a dormant state and doesn't consume resources.

So in summary a mature event driven architecture it only consumes resources while handling events when events are not occurring it doesn't consume resources.

And this is one of the key components of a serverless architecture which I'll be talking about more later in this section.

Now I know that this has been a lot of theory but I promise you as you continue through the course it will really make sense why I introduce this theory in detail at this point in the course.

And it really will help you within the exam.

In the rest of this section we're going to be covering more AWS specific and practical things.

But they'll all rely on your knowledge of this evolution of systems architecture.

So thanks for watching this video.

At this point though you can go ahead finish off this video and when you're ready I'll look forward to you joining me in the next lesson.

Welcome back and in this first technical lesson of this section of the course, we'll be stepping through what an event-driven architecture is and comparing it to other architectures available within AWS.

As a solutions architect, this matters because you're the one who needs to design a solution using a specific architecture around a given set of business requirements.

So you need to have a good base level understanding of all of the different types of architectures available to you within AWS.

You can't build something unless you fully understand the architectures.

So let's jump in and get started because we've got a lot to cover.

Now to help illustrate how an event-driven architecture works, let's consider an example.

And the example that I want to use is a popular online video sharing platform that you've all probably heard of.

Yes, that's right, it's CatTube.

One of the popular ways that CatTube is used is for people to upload holiday videos of their cats.

So Bob uploads a 4k quality video of whiskers on holiday to CatTube.

Now at this point, CatTube begins some processing and it generates lots of different versions of that video at various different quality levels.

For example, 1080p, 720p and 480p.

Now this is only part of the application but it happens to be the most intensive in terms of resource usage.

The website also needs to display videos, manage playlists and channels, and store and retrieve data to and from a database.

Now there are a few ways that we could architect this solution.

Historically, the most popular systems architecture was known as a monolithic architecture.

Now think of this as a single black box with all of the components of the application within it.

So in this example, I'm just showing a subset but we've got the upload component where Bob uploads his collection of videos where whiskers is on holiday, the processing component which does the conversion of videos, and then we have the store and manage component which interacts with the underlying persistent storage.

Now this architecture has a number of considerations, a number of important things to keep in mind.

Because it's all one entity, it fails together as an entity.

If one component fails, it impacts the whole thing end to end.

If uploading fails, it could also affect processing as well as store and manage.

Logically, you know that they're separate things, you know that uploading is different than processing, which is different than store and manage.

But if they're all contained in a single monolithic architecture, one code base, one big monolithic component, then the failure of any part of that monolith can affect everything else.

The other thing to consider when talking about monoliths is they also scale together.

They're highly coupled.

All of the components generally expect to be on the same server directly connected and have the same code base.

You can't scale one without the other.

Generally with monolithic architectures, you need to vertically scale the system because everything expects to be running on the same piece of compute hardware.

And finally, and this is one of the most important aspects of monolithic architectures that you need to be aware of, they generally build together.

All of the components of a monolithic architecture are always running and because of that they always incur charges.

Even if the processing engine is doing nothing, even if no videos are being uploaded, the system capacity has to be enough to run all of them.

And so they always have allocated resources, even if they aren't consuming them.

So using a monolithic architecture tends to be one of the least cost effective ways to architect systems, ranging from small to enterprise scale.

Now we've seen earlier in the course how we can evolve a monolithic design into a tiered one.

With a tiered architecture, the monolith is broken apart.

What we have now is a collection of different tiers and each of these tiers can be on the same server or different servers.

With this architecture, the different components are still completely coupled together because each of the tiers connects to a single endpoint of another tier.

The upload tier needs to be able to send data directly at the processing tier and again this could be on the same server or a different server.

With the WordPress example that you looked at earlier in the course, we separated the database component of the monolithic application onto its own RDS instance and left the EC2 instance running the Apache web server and the WordPress application.

But both of those services still needed to communicate with each other.

They were very tightly coupled.

Now the immediate benefit of a tiered architecture versus a monolith is that these individual tiers can be vertically scaled independently.

Put simply, you can increase the size of the server that's running each of these application tiers.

What this means is that if the processing tier for example requires more CPU capacity, then it can be increased in size to cope with that additional load without having to increase the size of the upload or the store and manage tiers.

But this architecture can be evolved even more.

Instead of each tier directly connecting to each other tier, we can utilize load balances located between each of the tiers.

Remember in the previous section I mentioned internal load balances.

This is an example of when internal load balances are useful.

It means that in this example the upload tier is no longer communicating with a specific instance of the processing tier.

And it means that the store and manage tier is not communicating with a specific instance of the processing tier.

Both of them are going via a load balancer.

And if you remember from the section of the course where I talked about load balances, this means it's abstracted.

It allows for horizontal scaling, meaning additional processing tier instances can be added.

Communication occurs via the load balances, so the upload and store and manage tiers have no exposure to the architecture of the processing tier, whether it's one instance or a hundred.

This means that the processing tier is now able to be scaled horizontally by adding additional instances.

And it's now highly available.

If one instance fails, the load balancer just redistributes the connections across the working instances.

So by abstracting away from individual instance architecture for the individual tiers, using load balances now means we can scale each tier independently, either vertically or horizontally.

Now this architecture isn't perfect for two main reasons.

First, the tiers are still coupled.

The upload tier, for example, expects and requires the processing tier to exist and to respond.

While the load balancer means that we can have multiple instances for the processing tier, for example, the processing tier has to exist.

If it fails completely, then the upload tier itself will fail because the upload tier expects at least one instance of the processing tier to answer it.

If there's a backlog in processing, if the processing tier slows down and it starts to take longer to accept jobs for processing, then that can also impact the upload tier and the customer experience.

The other issue with this architecture is that even if there's no jobs to be processed, the processing tier has to have something running.

Otherwise, there'll be a failure when the upload tier attempts to add an upload job.

So it's not possible to scale the individual tiers of the application back down to zero because the communication is synchronous.

The upload tier expects to perform a synchronous communication with the processing tier.

It expects to ask for a job to be entered and it requires an answer.

So while the tiered architecture improves things, it doesn't solve all of the problems.

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.

high interest in domestic country means lots of capital inflow, wich means high demand for domestic bonds, wich means high demand for domestic currency, wich will lead to an appreciation of the domestic currency

With a fixed exchange rate, there isn't much to speculate about. With floating exchange rates, speculation can lead to destabilising and hindering of the economic growth

open market operation

good example of SFXO. Selling H bonds means supplying the bonds on the market, supply of Domestic currency increases. This depreciates the domestic currency, making home goods relatively cheaper. this increases the demand for home goods and thus the export. Export minus import increases, wich increases the current account. Bop must always be in balance, so this means we are having a negative K, wich is logical because we are buying foreign bonds with SFXO. The demand of these foreign bonds leads to appreciation of the foreign bonds, making them relatively more expensive, wich decreases the demand. Eventually the H currency will appreciate again

for - from - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson - https://hyp.is/FAIHjL4LEe-3CLe2MabNuw/www.youtube.com/watch?v=JObdEbHqqFA

Cette présentation laisse entendre qu'UNE seule étude de 2002 est en cause. C'est inexact. De nombreuses autres études ont mis en évidence ce risque. L'une des dernières en date (https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(19)31709-X/fulltext) montre qu'environ 1 million de femmes ont contracté un cancer du sein dans les pays occidentaux depuis le lancement des THM et que le risque perdure jusqu'à 10 ans après l'arrêt des traitements.

En cliquant sur le lien donné dans l'article on remarque que la phrase citée est assortie d'une réserve importante qui n'est pas citée dans l'article : "Il serait prématuré d’affirmer, à partir de ces résultats, que les associations d’estrogène et de progestérone, ou d’estrogène et de dydrogestérone n’augmentent pas le risque de cancer du sein. D’une part, parce que E3N est la seule étude à avoir évalué le lien entre ces traitements et le risque de cancer du sein. Or, en épidémiologie, les résultats doivent s’inscrire dans un faisceau de preuves avant de pouvoir être admis."

Un examen de la page en question montre en outre qu'elle s'appuie sur des études vieilles de plus de 15 ans ! c'est particulièrement problématique car les dernières données, relayées par l'INCa disent l'exact inverse : l'oestradiol combiné à la progestérone est bien une cause de cancer du sein et de l'endomètre.

cf. https://www.e-cancer.fr/Professionnels-de-sante/Facteurs-de-risque-et-de-protection/Traitements-hormonaux/Les-traitements-hormonaux-de-la-menopause

Affirmation non-étayée et en contradiction avec la dernière revue systématique de Cochrane (2015), cf. supra.

1. Le lien donné dans l'article pointe vers une étude qui ne montre pas la réduction du risque de cancer colorectal chez les femmes THM, mais cherche à évaluer l'effet de la génétique combiné à celui des THM. En outre les scores polygéniques de risque utilisés par les auteurs n'ont aucune valeur probante.

2. La dernière revue systématique publiée sur le sujet remonte à 2021. Sa conclusion est à l'inverse : "La synthèse des données probantes a montré ce qui suit : (1) le THM a montré une hétérogénéité dans les résultats concernant le risque de cancer colorectal avec une légère tendance à un effet neutre ou protecteur ; (2) l'effet du THM était soit neutre soit protecteur sur l'adénome colorectal ; (3) le THM n'avait pas d'impact sur le grade de la tumeur, le sous-site et les types histologiques ; (4) le THM n'était pas associé à la mortalité due au cancer colorectal ; et (5) le THM a montré des effets hétérogènes sur le stade du cancer colorectal et sur son caractère invasif, respectivement. En résumé, malgré certaines données indiquant un effet protecteur de THM sur le cancer colorectal, le THM n'est actuellement pas recommandé par les lignes directrices internationales pour la prévention primaire du cancer colorectal, en raison de plusieurs effets importants et potentiellement nocifs."

cf. https://onlinelibrary.wiley.com/doi/10.1111/cen.14469

Cette formulation suggère que l'étude a été rétractée, ce qui n'est pas le cas. Elle demeure citée dans la littérature.

1. Il aurait été bien de rappeler que l'étude de 2002 du WHI ne montre pas de bénéfices en termes de fractures chez les femmes THM par rapport aux femmes témoins.

2. Pour le risque cardio-vasculaire, le consensus est inverse de ce qui est écrit dans l'article. Conclusion de la dernière review Cochrane (2015) sur le sujet :

"Les résultats de notre revue fournissent des preuves solides que le traitement hormonal des femmes post‐ménopausées dans l'ensemble, que ce soit pour la prévention primaire ou secondaire des événements cardiovasculaires, apporte peu ou pas de bénéfice et provoque une augmentation du risque d'AVC et d'événements thromboemboliques veineux."

cf. https://www.cochranelibrary.com/es/cdsr/doi/10.1002/14651858.CD002229.pub4/full/fr#CD002229-abs-0003

Cette citation est mensongère à deux niveaux. FT veut faire croire que les auteurs de ce papier auraient raté quelque chose dans leur analyse. C'est inexact. Voici la phrase de l'étude en question :

"(among never users, 114 vs 102; HR, 1.06; 95% CI, 0.81-1.38; for women with <5 years of prior use, 32 vs 15; HR, 2.13; 95% CI, 1.15-3.94; for women with 5-10 years of prior use, 11 vs 2; HR, 4.61; 95% CI, 1.01-21.02; and for women with ≥10 years of prior use, 9 vs 5; HR, 1.81; 95% CI, 0.60-5.43; test for trend, z = 2.17)"

On voit donc que pour les femmes "naïves" du traitement à l'inclusion, le risque est augmenté de 6% mais n'est pas retenu faute de puissance statistique. C'est parfaitement identifié comme non statistiquement significatif par les auteurs.

Deuxième mensonge : FT veut faire croire que cette absence de significativité statistique équivaut à une preuve d'absence ce qui est faux et évident quand on voit la progressivité linéaire de l'effet (cf. phrase supra).

Il ne s'agit pas de "peur" mais de précaution, justifiée par la nature des risques.

La titraille ne mentionne que les bénéfices potentiels, et non les risques avérés des THM. La référence à un "tabou" dans leur usage est à mon avis problématique: ce n'est pas un tabou moral qui a conduit à la réduction de leur prescription mais l'existence de risques importants de cancer du sein, en particulier.

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Referee #3

Evidence, reproducibility and clarity

This study by Mordier and colleagues represents an in depth analysis to clarify the evolutionary history and processes of the rapidly evolving Schlafen gene family with a strong focus on primates and rodents.

The study is of high quality in my opinion, though I do have some minor comments:

1. Fig 2 and Fig 4B present inferred phylogenetic trees of schalfens in primates and rodents - these trees appear to be unrooted or rooted on a single species rather than an outgroup/gene. I suggest that the authors consider whether an outgroup gene could be included or if an outgroup free approach could be used to estimate the position of the root. This is important because the use of an unrooted tree to make inferences on gene family evolution has important implications - for example, there are no clades in an unrooted tree (Wilkinson et al 2007, Trends Ecol Evol).
2. Schlafen proteins beyond mammals are referred to as SLFN11, it is not clear why this is the case because they seem to be co-orthologous to all mammal schalfen groups (except SLFNL1) based on supplementary figure S2. In this context, perhaps this image should form part of the main text?
3. For blast searches parameters should be included - what cutoffs were implied for similarity searches etc. Related to this on line 120-121 homology is described as 'significant'. Homology refers to an evolutionary relationship, sequence similarity may be significant or not based on the search performed but homology is qualitative and simply detectable or not.
4. The first results section describes the results of phylogenetic analyses, however this section relies heavily on what might better be considered interpretation of these analyses, this is great and should be included but I suggest that the branching patterns in the trees and bootstrap values supporting relationships between genes are also reported in the text to link interpretations to actual results.
5. Bustos 2009 included viral genes belonging to the family in their analyses and I think it may be pertinent to do so here also to determine if the results are consistent or not.
6. Was a rate heterogeneity (e.g. gamma rates / +G) parameter considered in phylogenetic analyses or model testing, it is not reported here and very rare for this not to improve model fit and phylogenetic accuracy.
7. The authors state that all data are available in public databases, but this is not the case for the results they generated. Making various file types produced in this study would be good - e.g. alignments, phylogenetic tree files, structures, etc.

Significance

This study is an important step forward in clarifying our understanding of schalfen evolution. I think the manuscript will be of interest to a number of research areas, including gene family evolution because of its focus on an unusually rapidly evolving gene cluster and to those working on the schalfen gene families functional importance in development and immunity. The results may also draw interest from those interested in the confluence of protein structure, function, and evolution. My expertise In the context of this study is in the phylogenetics and evolution of rapidly evolving gene families.

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Referee #2

Evidence, reproducibility and clarity

In the current manuscript, Mordier et al. combine bioinformatic searches, synteny, and phylogenetic analysis to reconstruct the duplicative history of the Schlafen Genes in rodents and primates and then use molecular evolution analyses in combination with structural modeling to make inferences regarding the role of natural selection in the evolution of this gene family. The study represents an update on Bustos et al. (2009), who had already presented evidence that Positive Darwinian selection was likely a factor in the diversification of these genes in mammals. In this context, the contribution of this paper is the identification of sites that are candidates to be evolving under natural selection, and the structural exploration of the location of these sites in the proteins. CODEML strength lies in the detection of signatures of positive selection at the codon level, but it is not that accurate when it comes to pinpointing the actual sites that might be under selection. Hence, without experimental data, these inferences remain speculative. The manuscript is well-written and represents an update on the evolution of this gene family.

Major Issues

The rationale for the choice of species included in the analyses is never presented, and some of it is hard to understand. Why do authors exclude the platypus but include non-mammalian lobe-finned vertebrates is not clear. If they are going to discuss the evolution of these genes outside mammals, the authors need to survey a much wider array of genomes. Even within mammals, there is little discussion on why some species were included and others not. I think that focusing the study on rodents and primates is OK, but I also think that providing a strong justification of the selection of species to include in the study and a tree that justifies splitting the focus on rodents and primates would also be important.

In the trees in Figures 2 and 4, several genes considered as orthologs are not in monophyletic groups. These pattern aligns well with the birth-and-death model of gene family evolution, and has implications for their molecular evolution analyses. The authors need to address this issue explicitly. I would use topology tests to evaluate whether these deviations from the expected topology are significant. In addition, the relevant tests to report here are M8 vs M7 and M8 vs M8a. The M0 vs M1a comparison does not provide evidence for positive Darwinian selection. If the M8 vs M7 and M8 vs M8a tests are not significant, the inferences about sites evolving with dN/dS>1 are not really valid.

CODEML can implements models that are designed to test patterns of gene family evolution, contrasting pre and post duplication branches, which I think would be of value in this family.

Some analyses are described very succinctly, which would make replication challenging.

Minor Issues

Could 2R be responsible for the emergence of SLFN and SLFNL1?

There are several minor issues authors should fix in a revised manuscript. In general, because results are presented before the materials and methods, I think it is easier for readers to have some of the information in the results section.

They need to be consistent in using italics for species names as well as for capitalization.

In the Alignment and maximum-likelihood phylogenies section the authors indicate that they used either Muscle or Mafft for the alignments. What was the rationale for picking one alignment over the other for a given gene? In this section, they also indicate the selected a best-fitting model of substitution using SMS, but then indicate that they used JTT for protein alignments and HKY for nucleotide alignments.

How did the authors ensure that nucleotide alignments remained in frame?

Significance

I think this is a significant contribution to our understanding of the evolution of the Schlafen gene family. There are two key contributions here: the demonstration that gene conversion is a factor obscuring relationships among genes in this gene family, and the mapping of amino acids inferred be evolving under positive selection to structurally important residues of the proteins. These residues should be of interest for functional assays that evaluate the functional role of these proteins.

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Referee #1

Evidence, reproducibility and clarity

Mordier et al. used in-depth phylogenomic methods to analyze the evolution of the mammalian Schlafen gene family. They identified a novel orphan Schlafen-related gene that arose in jawed vertebrates, and they assigned orthology between Schlafen cluster paralogs. This will allow for further accurate selection studies. Throughout the entire manuscript, the authors use nomenclature predating structural and biochemical studies. The nomenclature is purely based on sequence similarities, which are sometimes very weak and not convincing, and not based on the known function of the protein. In my opinion, this causes confusion and does not help scientists in the field. Especially in Figure 3, I wouldn't call it RNAse E (AlbA); instead, tRNA recognition site,endoribonuclease domain, SLFN core domain are the correct domain designations. Since SLFN11 is not a GTPase, why do the authors name the domain GTPase domain? Actually, the SWADL domain comprises a SWAVDL instead of a SWADL sequence motif. Hence, I would name the domain SWAVDL domain instead of SWADL domain, which is, in my opinion, misleading and was wrongly chosen in initial publications.

In e.g. Figure 3 SLFN11 structure it would be better if the authors illustrated the important residues concerning the known RNase active site and ssDNA binding site. Further, a close-up of the SLFN11 interface with labeled amino acids involved in the interaction and highlighting the residues undergoing positive selection would help understand the evolutionary adaptation.

Although, according to Metzner et al., the SLFN11 dimer is built up by two interfaces (I and II), where Interface I is situated in the C-terminal helicase domain and Interface II in the N-terminal SLFN11 core domain. It would be helpful for the reader if the authors stuck to this already introduced and widely accepted nomenclature in the field.

In addition to the antiviral function, SLFN11 expression levels have been reported to show a strong positive correlation with the sensitivity of tumor cells to DNA damaging agents (DDAs). Hence, SLFN11 can serve as a biomarker to predict the response to, e.g., platinum-based drugs. It was revealed that SLFN11 exerts its function by direct recruitment to sites of DNA damage and stalled replication forks in response to replication stress induced by DDAs. Could the authors include this different molecular function of SLFN11 in their discussion of SLFN11s evolution and positive selection?

Even though it seems unclear from the genetic and evolutionary aspect (Figure 4), mouse Slfn8 and Slfn9 complement human cells lacking SLFN11 during the replication stress response and seem to resemble the function of SLFN11 (Alvi et al. 2023). The authors of this study claim that Slfn8/9 genes may share an orthologous function with SLFN11. Could the authors comment on that discrepancy?

Significance

In general, the work is well conducted and provides valuable new insights in an important and growing field of research. However, there are some limitations to the study including the disregard of known protein function (e.g. SLFN11) and the usage of a purely sequence similarity based nomenclature.

烫过的发型

有品位的化妆

eLife Assessment

This study presents a valuable advancement in antiviral research by applying SHAPE-Map to analyze the secondary structure of the Porcine Epidemic Diarrhoea Virus RNA genome in infected cells, identifying promising therapeutic targets within viral genomic RNA. The authors provide convincing evidence of potential antiviral targetable RNA regions through a wide array of data from different methods, supported by well-documented experimental design and data analysis, demonstrating how RNA structural probing can effectively discover RNA targets and enabling further discoveries in the field. The work will be of interest to researchers focused on RNA therapeutics and viral genome studies.

Reviewer #1 (Public review):

Summary:

This study investigates the potential of targeting specific regions within the RNA genome of the Porcine Epidemic Diarrhea Virus (PEDV) for antiviral drug development. The authors used SHAPE-MaP to analyze the structure of the PEDV RNA genome in infected cells. They categorized different regions of the genome based on their structural characteristics, focusing on those that might be good targets for drugs or small interfering RNAs (siRNAs).

They found that dynamic single-stranded regions can be stabilized by compounds (e.g., to form G-quadruplexes), which inhibit viral proliferation. They demonstrated this by targeting a specific G4-forming sequence with a compound called Braco-19. The authors also describe stable (structured) single-stranded regions that they used to design siRNAs showing that they effectively inhibited viral replication.

Strengths:

There are a number of strengths to highlight in this manuscript.

(1) The study uses a sophisticated technique (SHAPE-MaP) to analyze the PEDV RNA genome in situ, providing valuable insights into its structural features.

(2) The authors provide a strong rationale for targeting specific RNA structures for antiviral development.

(3) The study includes a range of experiments, including structural analysis, compound screening, siRNA design, and viral proliferation assays, to support their conclusions.

(4) Finally, the findings have potential implications for the development of new antiviral therapies against PEDV and other RNA viruses.

Overall, this interesting study highlights the importance of considering RNA structure when designing antiviral therapies and provides a compelling strategy for identifying promising RNA targets in viral genomes.

Weaknesses:

I have some concerns about the utility of the 3D analyses, the effects of their synonymous mutants on expression/proliferation, a potentially missed control for studies of mutants, and the therapeutic utility of the compound they tested vs. G-quadruplexes.

Reviewer #2 (Public review):

Summary:

Luo et. al. use SHAPE-MaP to find suitable RNA targets in Porcine Epidemic Diarrhoea Virus. Results show that dynamic and transient structures are good targets for small molecules, and that exposed strand regions are adequate targets for siRNA. This work is important to segment the RNA targeting.

Strengths:

This work is well done and the data supports its findings and conclusions. When possible, more than one technique was used to confirm some of the findings.

Weaknesses:

The study uses a cell line that is not porcine (not the natural target of the virus).

Reviewer #3 (Public review):

Summary:

This manuscript by Luo et al. applied SHAPE-Map to analyze the secondary structure of the Porcine Epidemic Diarrhoea Virus (PEDV) RNA genome in infected cells. By combining SHAPE reactivity and Shannon entropy, the study indicated that the folding of the PEDV genomic RNA was nonuniform, with the 5' and 3' untranslated regions being more compactly structured, which revealed potentially antiviral targetable RNA regions. Interestingly, the study also suggested that compounds bound to well-folded RNA structures in vitro did not necessarily exhibit antiviral activity in cells, because the binding of these compounds did not necessarily alter the functions of the well-folded RNA regions. Later in the manuscript, the authors focus on guanine-rich regions, which may form G-quadruplexes and be potential targets for small interfering RNA (siRNA). The manuscript shows the binding effect of Braco-19 (a G-quadruplex-binding ligand) to a predicted G4 region in vitro, along with the inhibition of PEDV proliferation in cells. This suggests that targeting high SHAPE-high Shannon G4 regions could be a promising approach against RNA viruses. Lastly, the manuscript identifies 73 single-stranded regions with high SHAPE and low Shannon entropy, which demonstrated high success in antiviral siRNA targeting.

Strengths:

The paper presents valuable data for the community. Additionally, the experimental design and data analysis are well documented.

Weakness:

The manuscript presents the effect of Braco-19 on PQS1, a single G4 region with high SHAPE and high Shannon entropy, to suggest that "the compound can selectively target the PQS1 of the high SHAPE-high Shannon region in cells" (lines 625-626). While the effect of Braco-19 on PQS1 is supported by strong evidence in the manuscript, the conclusion regarding the G4 region with high SHAPE and high Shannon entropy is based on a single target, PQS1.

Von der Klima- und Energiestrategie 2030 wird nicht abgerückt. Diese Strategie ist allerdings so unkonkret, dass sie von der neuen Landesregierung nach Belieben umgedeutet werden kann.

Klimaschutz und Klimanpassung kommen im Programm nicht vor. Die Auswirkungen der globalen Erhitzung werden so gut wir nicht genannt, ihre Ursachen – Treibhausgasausstoß und Veränderung der Landnutzung – überhaupt nicht. Wie auch auf anderen Gebieten, vor allem bei der Migration und den geopolitischen Abhängigkeiten der Steiermark – ist das Programm ein Dokument der Verdrängung der Realität. Man suggeriert, es gäbe keine Gletscherschmelze, keine Borkenkäfer und keine klimabedingten Überschwemmungen. Die, wie es mehrfach kitschig heisst, „grüne Mark“ soll durch ökologisch schädliches Wirtschaftswachstum weiter zu den ökologischen Krisen beitragen und zugleich nach außen als eine Idylle im Sinne der 50er oder eher der 30er Jahre erscheinen.

aus: Arbeitsübereinkommen der FPÖ Steiermark und der Steirischen Volkspartei 2024–2029

Dieses Programm hat einen scheinbar „wirtschaftsfreundlichen“, auf Deregulierung ausgerichteten Kern. Bei den für sie wichtigen Themen haben sich die Vertreter kurzfristiger Kapitalinteressen und die ihnen verbundenen Profis in der ÖVP durchgesetzt. Das bedeutet vor allem, dass umwelt- und klimapolitisch begründete Einschränkungen für Investitionen so weit wie möglich abgeschafft werden sollen (was allerdings oft die Kompetenzen einer Landesregierung übersteigt): Weitere Förderung des Verbrenners, weniger Auflagen bei der thermischen Saniierung, Straßenausbau im möglichst großen Stil. Dieser harte Kern des Regierungsprogramms ist in eine braune Packerl-Soße eingetaucht, die die FPÖ anrühren durfte. Heraus kommt ein synthetisches Heimat-Branding à la „Aufsteirern“-

FPÖ Steiermark, & Steirische Volkspartei. (2024, Dezember 17). Starke Steiermark. Sichere Zukunft. Arbeitsübereinkommen der FPÖ Steiermark und der Steirischen Volkspartei 2024–2029.

Die Energie Steiermark kommt im Regierungsprogramm nur zweimal vor: Sie soll den Netzausbau vorantreiben, und sie soll als Stakeholderin in eine „Standort-Partnerschaft“ eingebunden werden. Die wichtigen Fragen, ob das Land einen Teil wieder verkaufen soll und wie das Verhältnis zur Energie Graz gestaltet werden soll, werden nicht angesprochen.

aus: Arbeitsübereinkommen der FPÖ Steiermark und der Steirischen Volkspartei 2024–2029

This talks about how sometimes when reading different scientific papers most of the time you could encounter words that you are not familiar with so it is helpful to know about resources that are available to help aid understanding with scientific papers.

Author response:

The following is the authors’ response to the current reviews.

Reviewer #1 (Public review):

Summary:

In the manuscript "Intergenerational transport of double-stranded RNA limits heritable epigenetic changes," Shugarts and colleagues investigate intergenerational dsRNA transport in the nematode C. elegans. By inducing oxidative damage, they block dsRNA import into cells, which affects heritable gene regulation in the adult germline (Fig. 2). They identify a novel gene, sid-1-dependent gene-1 (sdg-1), upregulated upon SID-1 inhibition (Fig. 3). Both transient and genetic depletion of SID-1 lead to the upregulation of sdg-1 and a second gene, sdg-2 (Fig. 5). Interestingly, while sdg-1 expression suggests a potential role in dsRNA transport, neither its overexpression nor loss-of-function impacts dsRNA-mediated silencing in the germline (Fig. 7).

Strengths:

• The authors employ a robust neuronal stress model to systematically explore SID-1 dependent intergenerational dsRNA transport in C. elegans.

• They discover two novel SID-1-dependent genes, sdg-1 and sdg-2.

• The manuscript is well-written and addresses the compelling topic of dsRNA signaling in C. elegans.

Weaknesses:

• The molecular mechanism downstream of SDG-1 remains unclear. Testing whether sdg-2 functions redundantly with sdg-1could provide further insights.

• SDG-1 dependent genes in other nematodes remain unknown.

We thank the reviewer for highlighting the strengths of the work along with a couple of the interesting future directions inspired by the reported discoveries. The restricted presence of genes encoding SDG-1 and its paralogs within retrotransposons suggests intriguing evolutionary roles for these proteins. Future work could examine whether such fast-evolving or newly evolved proteins with potential roles in RNA regulation are more broadly associated with retrotransposons. Multiple SID-1-dependent proteins (including SDG-1 and SDG-2) could act together to mediate downstream effects. This possibility can be tested using combinatorial knockouts and overexpression strains. Both future directions have the potential to illuminate the evolutionarily selected roles of dsRNA-mediated signaling through SID-1, which remain a mystery.

Reviewer #2 (Public review):

Summary:

RNAs can function across cell borders and animal generations as sources of epigenetic information for development and immunity. The specific mechanistic pathways how RNA travels between cells and progeny remains an open question. Here, Shugarts, et al. use molecular genetics, imaging, and genomics methods to dissect specific RNA transport and regulatory pathways in the C. elegans model system. Larvae ingesting double-stranded RNA is noted to not cause continuous gene silencing throughout adulthood. Damage of neuronal cells expressing double-stranded target RNA is observed to repress target gene expression in the germline. Exogenous short or long double-stranded RNA required different genes for entry into progeny. It was observed that the SID-1 double-stranded RNA transporter showed different expression over animal development. Removal of the sid-1 gene caused upregulation of two genes, the newly described sid-1-dependent gene sdg-1 and sdg-2. Both genes were observed to be negatively regulated by other small RNA regulatory pathways. Strikingly, loss then gain of sid-1 through breeding still caused variability of sdg-1 expression for many, many generations. SDG-2 protein co-localizes with germ granules, intracellular sites for heritable RNA silencing machinery. Collectively, sdg-1 presents a model to study how extracellular RNAs can buffer gene expression in germ cells and other tissues.

Strengths:

(1) Very cleaver molecular genetic methods and genomic analyses, paired with thorough genetics, were employed to discover insights into RNA transport, sdg-1 and sdg-2 as sid-1-dependent genes, and sdg-1's molecular phenotype.

(2) The manuscript is well cited, and figures reasonably designed.

(3) The discovery of the sdg genes being responsive to the extracellular RNA cell import machinery provides a model to study how exogenous somatic RNA is used to regulate gene expression in progeny. The discovery of genes within retrotransposons stimulates tantalizing models how regulatory loops may actually permit the genetic survival of harmful elements.

Weaknesses:

(1) The manuscript is broad, making it challenging to read and consider the data presented. Of note, since the original submission, the authors have improved the clarity of the writing and presentation.

Comments on revised version:

This reviewer thanks the authors for their efforts in revising the manuscript. In their rebuttal, the authors acknowledged the broad scope of their manuscript. I concur. While I still think the manuscript is a challenge to read due to its expansive nature, the current draft is substantially improved when compared to the previous one. This work will contribute to our general knowledge of RNA biology, small RNA regulatory pathways, and RNA inheritance.

We thank the reviewer for highlighting the strengths of the manuscript and for helping us improve the presentation of our results and discussion.

---

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In the manuscript "Intergenerational transport of double-stranded RNA limits heritable epigenetic changes" Shugarts and colleagues investigate intergenerational dsRNA transport in the nematode C. elegans. They induce oxidative damage in worms, blocking dsRNA import into cells (and potentially affecting the worms in other ways). Oxidative stress inhibits dsRNA import and the associated heritable regulation of gene expression in the adult germline (Fig. 2). The authors identify a novel gene, sid-1-dependent gene-1 (sdg-1), which is induced upon inhibition of SID-1 (Fig. 3). Both transient inhibition and genetic depletion of SID-1 lead to the upregulation of sdg-1 and a second gene, sdg-2 (Fig. 5). The expression of SDG-1 is variable, potentially indicating buffering regulation. While the expression of Sdg-1 could be consistent with a role in intergenerational transport of dsRNA, neither its overexpression nor loss-of-function impacts dsRNA-mediated silencing (Fig. 7) in the germline. It would be interesting to test if sdg-2 functions redundantly.

In summary, the authors have identified a novel worm-specific protein (sdg-1) that is induced upon loss of dsRNA import via SID-1, but is not required to mediate SID-1 RNA regulatory effects.

We thank the reviewer for highlighting our findings on SDG-1. We found that oxidative damage in neurons enhanced dsRNA transport into the germline and/or subsequent silencing.

Remaining Questions:

• The authors use an experimental system that induces oxidative damage specifically in neurons to release dsRNAs into the circulation. Would the same effect be observed if oxidative damage were induced in other cell types?

It is possible that oxidative damage of other tissues using miniSOG (as demonstrated in Xu and Chisholm, 2016) could also enhance the release of dsRNA into the circulation from those tissues. However, future experiments would be needed to test this empirically because it is also possible that the release of dsRNA depends on physiological properties (e.g., the molecular machinery promoting specific secretion) that are particularly active in neurons. We chose to use neurons as the source of dsRNA because by expressing dsRNA in a variety of tissues, neurons appeared to be the most efficient at the export of dsRNA as measured using SID-1-dependent silencing in other tissues (Jose et al., PNAS, 2009).

• Besides dsRNA, which other RNAs and cellular products (macromolecules and small signalling molecules) are released into the circulation that could affect the observed changes in germ cells?

We do not yet know all the factors that could be released either in naive animals or upon oxidative damage of neurons that influence the uptake of dsRNA into other tissues. The dependence on SID-1 for the observed enhancement of silencing (Fig. 2) shows that dsRNA is necessary for silencing within the germline. Whether this import of dsRNA occurs in conjunction with other factors (e.g., the uptake of short dsRNA along with yolk into oocytes (Marré et al., PNAS, 2016)) before silencing within the germline will require further study. A possible approach could be the isolation of extracellular fluid (Banse and Hunter, J Vis Exp., 2012) followed by characterization of its contents. However, the limited material available using this approach and the difficulty in avoiding contamination from cellular damage by the needle used for isolating the material make it challenging.

• SID-1 modifies RNA regulation within the germline (Fig. 7) and upregulates sdg-1 and sdg-2 (Fig. 5). However, SID-1's effects do not appear to be mediated via sdg-1. Testing the role of sdg-2 would be intriguing.

We observe the accumulation of sdg-1 and sdg-2 RNA in two different mutants lacking SID-1, which led us to conservatively focus on the analysis of one of these proteins for this initial paper. We expect that more sensitive analyses of the RNA-seq data will likely reveal additional genes regulated by SID-1. With the ability to perform multiplexed genome-editing, we hope in future work to generate strains that have mutations in many SID-1-dependent genes to recapitulate the defects observed in sid-1(-) animals. Indeed, as surmised by the reviewer, we are focusing on sdg-2 as the first such SID-1-dependent gene to analyze using mutant combinations.

• Are sdg-1 or sdg-2 conserved in other nematodes or potentially in other species?  appears to be encoded or captured by a retro-element in the C. elegans genome and exhibits stochastic expression in different isolates. Is this a recent adaptation in the C. elegans genome, or is it present in other nematodes? Does loss-of-function of sdg-1 or sdg-2 have any observable effect?

Clear homologs of SDG-1 and SDG-2 are not detectable outside of C. elegans. Consistent with the location of the sdg-1 gene within a Cer9 retrotransposon that appears to have integrated only within the C. elegans genome, sequence conservation between the genomes of related species is only observed outside the region of the retrotransposon (see Author response image 1, screenshot from UCSC browser). There were no obvious defects detected in animals lacking sdg-1 (Fig. 7) or in animals lacking sdg-2 (data not shown). It is possible that further exploration of both mutants and mutant combinations lacking additional SID-1-dependent genes would reveal defects. We also plan to examine these mutants in sensitized genetic backgrounds where one or more members of the RNA silencing pathway have been compromised.

Author response image 1.

Clarification for Readability:

To enhance readability and avoid misunderstandings, it is crucial to specify the model organism and its specific dsRNA pathways that are not conserved in vertebrates:

We agree with the reviewer and thank the reviewer for the specific suggestions provided below. To take the spirit of the suggestion to heart we have instead changed the title of our paper to clearly signal that the entire study only uses C. elegans. We have titled the study ‘Intergenerational transport of double-stranded RNA in C. elegans can limit heritable epigenetic changes’

• In the first sentence of the paragraph "Here, we dissect the intergenerational transport of extracellular dsRNA ...", the authors should specify "in the nematode C. elegans". Unlike vertebrates, which recognise dsRNA as a foreign threat, worms and other invertebrates pervasively use dsRNA for signalling. Additionally, worms, unlike vertebrates and insects, encode RNA-dependent RNA polymerases that generate dsRNA from ssRNA substrates, enabling amplification of small RNA production. Especially in dsRNA biology, specifying the model organism is essential to avoid confusion about potential effects in humans.

We agree with most statements made by the reviewer, although whether dsRNA is exclusively recognized as a foreign threat by all vertebrates of all stages remains controversial. Our changed title now eliminates all ambiguity regarding the organism used in the study.

• Similarly, the authors should specify "in C. elegans" in the sentence "Therefore, we propose that the import of extracellular dsRNA into the germline tunes intracellular pathways that cause heritable RNA silencing." This is important because C. elegans small RNA pathways differ significantly from those in other organisms, particularly in the PIWI-interacting RNA (piRNA) pathways, which depend on dsRNA in C. elegans but uses ssRNA in vertebrates. Specification is crucial to prevent misinterpretation by the reader. It is well understood that mechanisms of transgenerational inheritance that operate in nematodes or plants are not conserved in mammals.

The piRNAs of C. elegans are single-stranded but are encoded by numerous independent genes throughout the genome. The molecules used for transgenerational inheritance of epigenetic changes that have been identified thus far are indeed different in different organisms. However, the regulatory principles required for transgenerational inheritance are general (Jose, eLife, 2024). Nevertheless, we have modified the title to clearly state that the entire study is using C. elegans.  

• The first sentence of the discussion, "Our analyses suggest a model for ...", would also benefit from specifying "in C. elegans". The same applies to the figure captions. Clarification of the model organism should be added to the first sentence, especially in Figure 1.

With the clarification of the organism used in the title, we expect that all readers will be able to unambiguously interpret our results and the contexts where they apply. 

Reviewer #2 (Public review):

Summary:

RNAs can function across cell borders and animal generations as sources of epigenetic information for development and immunity. The specific mechanistic pathways how RNA travels between cells and progeny remains an open question. Here, Shugarts, et al. use molecular genetics, imaging, and genomics methods to dissect specific RNA transport and regulatory pathways in the C. elegans model system. Larvae ingesting double stranded RNA is noted to not cause continuous gene silencing throughout adulthood. Damage of neuronal cells expressing double stranded target RNA is observed to repress target gene expression in the germline. Exogenous supply of short or long double stranded RNA required different genes for entry into progeny. It was observed that the SID-1 double-stranded RNA transporter showed different expression over animal development. Removal of the sid-1 gene caused upregulation of two genes, the newly described sid-1-dependent gene sdg-1 and sdg-2. Both genes were observed to also be negatively regulated by other small RNA regulatory pathways. Strikingly, loss then gain of sid-1 through breeding still caused variability of sdg-1 expression for many, many generations. SDG-2 protein co-localizes with a Z-granule marker, an intracellular site for heritable RNA silencing machinery. Collectively, sdg-1 presents a model to study how extracellular RNAs can buffer gene expression in germ cells and other tissues.

We thank the reviewer for highlighting our findings and underscoring the striking nature of the discovery that mutating sid-1 using genome-editing resulted in a transgenerational change that could not be reversed by changing the sid-1 sequence back to wild-type.

Strengths:

(1) Very clever molecular genetic methods and genomic analyses, paired with thorough genetics, were employed to discover insights into RNA transport, sdg-1 and sdg-2 as sid-1-dependent genes, and sdg-1's molecular phenotype.

(2) The manuscript is well cited, and figures reasonably designed.

(3) The discovery of the sdg genes being responsive to the extracellular RNA cell import machinery provides a model to study how exogenous somatic RNA is used to regulate gene expression in progeny. The discovery of genes within retrotransposons stimulates tantalizing models how regulatory loops may actually permit the genetic survival of harmful elements.

We thank the reviewer for the positive comments.

Weaknesses:

(1) As presented, the manuscript is incredibly broad, making it challenging to read and consider the data presented. This concern is exemplified in the model figure, that requires two diagrams to summarize the claims made by the manuscript.

RNA interference (RNAi) by dsRNA is an organismal response where the delivery of dsRNA into the cytosol of some cell precedes the processing and ultimate silencing of the target gene within that cell. These two major steps are often not separately considered when explaining observations. Yet, the interpretation of every RNAi experiment is affected by both steps. To make the details that we have revealed in this work for both steps clearer, we presented the two models separated by scale - organismal vs. intracellular. We agree that this integrative manuscript appears very broad when the many different findings are each considered separately. The overall model revealed here forms the necessary foundation for the deep analysis of individual aspects in the future.

(2) The large scope of the manuscript denies space to further probe some of the ideas proposed. The first part of the manuscript, particularly Figures 1 and 2, presents data that can be caused by multiple mechanisms, some of which the authors describe in the results but do not test further. Thus, portions of the results text come across as claims that are not supported by the data presented.

We agree that one of the consequences of addressing the joint roles of transport and subsequent silencing during RNAi is that the scope of the manuscript appears large. We had suggested multiple interpretations for specific observations in keeping with the need for further work. To avoid any misunderstandings that our listing of possible interpretations be taken as claims by the reader, we have followed the instructions of the reviewer (see below) and moved some of the potential explanations we raised to the discussion section.

(3) The manuscript focuses on the genetics of SDGs but not the proteins themselves. Few descriptions of the SDGs functions are provided nor is it clarified why only SDG-1 was pursued in imaging and genetic experiments. Additionally, the SDG-1 imaging experiments could use additional localization controls.

We agree that more work on the SDG proteins will likely be informative, but are beyond the scope of this already expansive paper.  We began with the analysis of SDG-1 because it had the most support as a regulator of RNA silencing (Fig. 5f). Indeed, in other work (Lalit and Jose, bioRxiv, 2024), we find that AlphaFold 2 predicts the SDG-1 protein to be a regulator of RNA silencing that directly interacts with the dsRNA-editing enzyme ADR-2 and the endonuclease RDE-8. Furthermore, we expect that more sensitive analyses of the RNA-seq data are likely to reveal additional genes regulated by SID-1. Using multiplexed genome editing, we hope to generate mutant combinations lacking multiple sdg genes to reveal their function(s).

We agree that given the recent discovery of many components of germ granules, our imaging data does not have sufficient resolution to discriminate between them. We have modified our statements and our model regarding the colocalization of SDG-1 with Z-granules to indicate that the overlapping enrichment of SDG-1 and ZNFX-1 in the perinuclear region is consistent with interactions with other nearby granule components.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

Major

(1) As presented, the manuscript is almost two manuscripts combined into one. This point is highlighted in Figure 7h, which basically presents two separate models. The key questions addressed in the manuscript starts at Figure 3. Figures 1 and 2 are interesting observations but require more experiments to define further. For example, as the Results text describes for Figure 1, "These differences in the entry of ingested dsRNA into cells and/or subsequent silencing could be driven by a variety of changes during development. These include changes in the uptake of dsRNA into the intestine, distribution of dsRNA to other tissues from the intestine, import of dsRNA into the germline, and availability of RNA silencing factors within the germline." Presenting these (reasonable) mechanistic ideas detracted from the heritable RNA epigenetic mechanism explored in the later portion of the manuscript. There are many ways to address this issue, one being moving Figures 1 and 2 to the Supplement to focus on SID-1 related pathways.

Since this manuscript addresses the interaction between intercellular transport of dsRNA and heritable epigenetic changes, it was necessary to establish the possible route(s) that dsRNA could take to the germline before any inference could be made regarding heritable epigenetic changes. As suggested below (pt. 2), we have now moved the alternatives we enumerated as possible explanations for some experimental results (e.g., for the differences quoted here) to the discussion section.

(2) The manuscript includes detailed potential interpretations in the Results, making them seem like claims. Here is an example:

"Thus, one possibility suggested by these observations is that reduction of sdg-1 RNA via SID-1 alters the amount of SDG-1 protein, which could interact with components of germ granules to mediate RNA regulation within the germline of wild-type animals."

This mechanism is a possibility, but placing these ideas in the citable results makes it seem like an overinterpretation of imaging data. This text and others should be in the Discussion, where speculation is encouraged. Results sections like this example and others should be moved to the discussion.

We have rephrased motivating connections between experiments like the one quoted above and also moved such text to the discussion section wherever possible.

(3) A paragraph describing the SDG proteins will be helpful. Homologs? Conserved protein domains? mRNA and/or protein expression pattern across worm, not just the germline? Conservation across Caenorhabditis sp? These descriptions may help establish context why SDG-1 localizes to Z-granules.

We have now added information about the conservation of the sdg-1 gene in the manuscript. AlphaFold predicts domains with low confidence for the SDG-1 protein, consistent with the lack of conservation of this protein (AlphaFold requires multiple sequence alignments to predict confidently). In the adult animal, the SDG-1 protein was only detectable in the germline. Future work focused on SDG-1, SDG-2 and other SDG proteins will further examine possible expression in other tissues and functional domains if any. Unfortunately, in multiple attempts of single-molecule FISH experiments using probes against the sdg-1 open reading frame, we were unable to detect a specific signal above background (data not shown). Additional experiments are needed for the sensitive detection of sdg-1 expression outside the germline, if any.  

(4) Based on the images shown, SDG-1 could be in other nearby granules, such as P granules or mutator foci. Additional imaging controls to rule out these granules/condensates will greatly strengthen the argument that SDG-1 protein localizes to Z-granules specifically.

We have modified the final model to indicate that the perinuclear colocalization is with germ granules broadly and we agree that we do not have the resolution to claim that the observed overlap of SDG-1::mCherry with GFP::ZNFX-1 that we detect using Airyscan microscopy is specifically with Z granules. Our initial emphasis of Z-granule was based on the prior report of SDG-1 being co-immunoprecipitated with the Z-granule surface protein PID-2/ZSP-1. However, through other work predicting possible direct interactions using AlphaFold (Lalit and Jose, bioRxiv, 2024), we were unable to detect any direct interactions between PID-2 and SDG-1. Indeed, many additional granules have been recently reported (Chen et al., Nat. Commun., 2024; Huang et al., bioRxiv 2024), making it possible that SDG-1 has specific interactions with a component of one of the other granules (P, Z, M, S, E, or D) or adjacent P bodies.

Minor

(1) "This entry into the cytosol is distinct from and can follow the uptake of dsRNA into cells, which can rely on other receptors." Awkard sentence. Please revise.

We have now revised this sentence to read “This entry into the cytosol is distinct from the uptake of dsRNA into cells, which can rely on other receptors”

(2) Presumably, the dsRNA percent of the in vitro transcribed RNA is different than the 50 bp oligos that can be reliably annealed by heating and cooling. Other RNA secondary structure possibilities warrant further discussion.

We agree that in vitro transcribed RNA could include a variety of undefined secondary structures in addition to dsRNAs of mixed length. Such structures could recruit or titrate away RNA-binding proteins in addition to the dsRNA structures engaging the canonical RNAi pathway, resulting in mixed mechanisms of silencing. Future work identifying such structures and exploring their impact on the efficacy of RNAi could be informative. We have now added these considerations to the discussion and thank the reviewer for highlighting these possibilities.

eLife Assessment

In this report, the authors present valuable findings identifying a novel worm-specific protein (sdg-1) that is induced upon loss of dsRNA import via SID-1, but is not required to mediate SID-1 RNA regulatory effects. The genetic and genomic approaches are well-executed and the revision contain generally solid support for the central findings of the work. These findings will be of interest to those working in the germline epigenetic inheritance field.

Reviewer #1 (Public review):

Summary:<br /> In the manuscript "Intergenerational transport of double-stranded RNA limits heritable epigenetic changes," Shugarts and colleagues investigate intergenerational dsRNA transport in the nematode C. elegans. By inducing oxidative damage, they block dsRNA import into cells, which affects heritable gene regulation in the adult germline (Fig. 2). They identify a novel gene, sid-1-dependent gene-1 (sdg-1), upregulated upon SID-1 inhibition (Fig. 3). Both transient and genetic depletion of SID-1 lead to the upregulation of sdg-1 and a second gene, sdg-2 (Fig. 5). Interestingly, while sdg-1 expression suggests a potential role in dsRNA transport, neither its overexpression nor loss-of-function impacts dsRNA-mediated silencing in the germline (Fig. 7).

Strengths:<br /> • The authors employ a robust neuronal stress model to systematically explore SID-1 dependent intergenerational dsRNA transport in C. elegans.<br /> • They discover two novel SID-1-dependent genes, sdg-1 and sdg-2.<br /> • The manuscript is well-written and addresses the compelling topic of dsRNA signaling in C. elegans.

Weaknesses:<br /> • The molecular mechanism downstream of SDG-1 remains unclear. Testing whether sdg-2 functions redundantly with sdg-1could provide further insights.<br /> • SDG-1 dependent genes in other nematodes remain unknown.

Reviewer #2 (Public review):

Summary:

RNAs can function across cell borders and animal generations as sources of epigenetic information for development and immunity. The specific mechanistic pathways how RNA travels between cells and progeny remains an open question. Here, Shugarts, et al. use molecular genetics, imaging, and genomics methods to dissect specific RNA transport and regulatory pathways in the C. elegans model system. Larvae ingesting double-stranded RNA is noted to not cause continuous gene silencing throughout adulthood. Damage of neuronal cells expressing double-stranded target RNA is observed to repress target gene expression in the germline. Exogenous short or long double-stranded RNA required different genes for entry into progeny. It was observed that the SID-1 double-stranded RNA transporter showed different expression over animal development. Removal of the sid-1 gene caused upregulation of two genes, the newly described sid-1-dependent gene sdg-1 and sdg-2. Both genes were observed to be negatively regulated by other small RNA regulatory pathways. Strikingly, loss then gain of sid-1 through breeding still caused variability of sdg-1 expression for many, many generations. SDG-2 protein co-localizes with germ granules, intracellular sites for heritable RNA silencing machinery. Collectively, sdg-1 presents a model to study how extracellular RNAs can buffer gene expression in germ cells and other tissues.

Strengths:

(1) Very cleaver molecular genetic methods and genomic analyses, paired with thorough genetics, were employed to discover insights into RNA transport, sdg-1 and sdg-2 as sid-1-dependent genes, and sdg-1's molecular phenotype.

(2) The manuscript is well cited, and figures reasonably designed.

(3) The discovery of the sdg genes being responsive to the extracellular RNA cell import machinery provides a model to study how exogenous somatic RNA is used to regulate gene expression in progeny. The discovery of genes within retrotransposons stimulates tantalizing models how regulatory loops may actually permit the genetic survival of harmful elements.

Weaknesses:

(1) The manuscript is broad, making it challenging to read and consider the data presented. Of note, since the original submission, the authors have improved the clarity of the writing and presentation.

Comments on revised version:

This reviewer thanks the authors for their efforts in revising the manuscript. In their rebuttal, the authors acknowledged the broad scope of their manuscript. I concur. While I still think the manuscript is a challenge to read due to its expansive nature, the current draft is substantially improved when compared to the previous one. This work will contribute to our general knowledge of RNA biology, small RNA regulatory pathways, and RNA inheritance.

Mensen willen referentiekader (oriëntering). Door breuk met natuur weten ze dat er verschillende mogelijkheden zijn om hun bestaan vorm te geven. Hierdoor zoeken van verbondenheid.

• definition - Tukdam - John Dunne
  • is a word with multiple meanings (polysemy)
  • first - honorific term for samaya - Sanskrit for Tantric vows
    • second - commitment / promise
    • third - chosen deity
    • fourth - practicing any of the above
      • specifically, it could mean accomplishing the goals of Tantric practice, especially at the time of death

for - Tibetan Buddhism - Tukdam - John Dunne - Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

В конце строки.

В моём варианте число занимает целую строку, а запятая болтается в начале следующей строки.

Может, тут можно убрать слово именно? Дальше снова повторяется. И думаю, что тут непревышение надо слитно. Если проверяется, не превышает ли значение число.

У меня в конце строки.

eLife Assessment

This is a valuable study regarding the role of gasdesmin D in experimental psoriasis. The study contains solid evidence for such a role, involving neutrophils, from murine models of skin inflammation, as well as correlative data of elevated gasdermin D expression in human psoriatic skin. The findings will be of interest to researchers trying to unravel pathways of skin inflammation.

Reviewer #1 (Public review):

Summary:

Recommendations for the authors In this study, Liu, Jiang, Diao et.al. investigated the role of GSDMD in psoriasis-like skin inflammation in mice. The authors have used full-body GSDMD knock-out mice and Gsdm floxed mice crossed with the S100A8- Cre. In both mice, the deficiency of GSDMD ameliorated the skin phenotype induced by the imiquimod. The authors also analyzed RNA sequencing data from the psoriatic patients to show an elevated expression of GSDMD in the psoriatic skin.

Strengths:

It has the potential to unravel the new role of neutrophils.

Comments on revisions:

The authors have addressed the majority of comments and concerns and highlighted the potential limitations wherever not possible.

Reviewer #2 (Public review):

Summary:

The authors describe elevated GSDMD expression in psoriatic skin, and knock-out of GSDMD abrogates psoriasis-like inflammation.

Strengths:

The study is well conducted with transgenic mouse models. Using mouse-models with GSDMD knock-out showing abrogating inflammation, as well as GSDMD fl/fl mice without neutrophils having a reduced phenotype.

My major concern would be the involvement of other inflammasome and GSDMD bearing cell types, esp. Keratinocytes (KC), which could be an explanation why the experiments in Fig 4 still show inflammation.

Comments on revisions:

The authors have sufficiently addressed my questions.

Author response:

The following is the authors’ response to the original reviews.

eLife Assessment

This is a potentially interesting study regarding the role of gasdesmin D in experimental psoriasis. The study contains useful data from murine models of skin inflammation, however the main claims (on neutrophil pyroptosis) are incompletely supported in its current form and require additional experimental support to justify the conclusions made.

We sincerely appreciate the positive assessment regarding the significance of our study, as well as the valuable suggestions provided by the reviewers. We have included new data, further discussions and clarifications in the revised manuscript to adequately address all the concerns raised by the reviewers and better support our conclusions.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this study, Liu, Jiang, Diao et.al. investigated the role of GSDMD in psoriasis-like skin inflammation in mice. The authors have used full-body GSDMD knock-out mice and Gsdm floxed mice crossed with the S100A8- Cre. In both mice, the deficiency of GSDMD ameliorated the skin phenotype induced by the imiquimod. The authors also analyzed RNA sequencing data from the psoriatic patients to show an elevated expression of GSDMD in the psoriatic skin.

Overall, this is a potentially interesting study, however, the manuscript in its current format is not completely a novel study.

Strengths:

It has the potential to unravel the new role of neutrophils.

Weaknesses:

The main claims are only partially supported and have scope to improve

We thank the reviewer for the positive evaluation of the interest and potential of our work. In response to reviewers’ suggestions, we have added new content, including additional data and discussions, to further demonstrate the important role of GSDMD-mediated neutrophil pyroptosis in the pathogenesis of psoriasis, thereby enhancing the completeness of our research.

Reviewer #2 (Public review):

Summary:

The authors describe elevated GSDMD expression in psoriatic skin, and knock-out of GSDMD abrogates psoriasis-like inflammation.

Strengths:

The study is well conducted with transgenic mouse models. Using mouse-models with GSDMD knock-out showing abrogating inflammation, as well as GSDMD fl/fl mice without neutrophils having a reduced phenotype.

I fear that some of the conclusions cannot be drawn by the suggested experiments. My major concern would be the involvement of other inflammasome and GSDMD bearing cell types, esp. Keratinocytes (KC), which could be an explanation why the experiments in Fig 4 still show inflammation.

Weaknesses:

The experiments do not entirely support the conclusions towards neutrophils.

We appreciate the reviewers’ positive evaluation regarding the application of our mouse models. We also thank the reviewers for insightful comments and suggestions that can improve the quality of our work. Addressing these issues has significantly strengthened our conclusions. Our responses to the above questions are as follows.

Specific questions/comments:

Fig 1b: mainly in KC and Neutrophils?

In Figure 1b, we observed that GSDMD expression is higher in the psoriasis patient tissues compared to control samples. As the role of GSDMD in keratinocytes during the pathogenesis of psoriasis has already been explored[1], we focused our study on GSDMD in neutrophils. In response to the comments, we have added co-staining results of the neutrophil marker CD66b and GSDMD in the revised manuscript (see new Figure 3b in the revised manuscript). This addition further substantiates the expression of GSDMD in neutrophils within psoriasis tissue.

Fig 2a: PASI includes erythema, scaling, thickness and area. Guess area could be trick, esp. in an artificial induced IMQ model (WT) vs. the knock-out mice.

In our model, to accurately assess the disease condition in mice, we standardized the drug treatment area on the dorsal side (2*3 cm). Therefore, the area was not factored into the scoring process, and we have included a detailed description of this in the revised manuscript.

Fig 2d: interesting finding. I thought that CASP-1 is cleaving GSDMD. Why would it be downregulated?

Regarding the downregulation of CASP in GSDMD KO mouse skin tissue, existing studies indicate that GSDMD generates a feed-forward amplification cascade via the mitochondria-STING-Caspase axis [2]. We hypothesize that the absence of GSDMD attenuates STING signaling’s activation of Caspase.

Line 313: as mentioned before (see Fig 1b). KC also show a stron GSDMD staining positivity and are known producers of IL-1b and inflammasome activation. Guess here the relevance of KC in the whole model needs to be evaluated.

Our research primarily focuses on the role of neutrophil pyroptosis in psoriasis, this does not conflict with existing reports indicating that KC cell pyroptosis also contributes to disease progression[1]. Both studies underscore the significant role of GSDMD-mediated pyroptotic signaling in psoriasis, and the consistent involvement of KC cells and neutrophils further emphasizes the potential therapeutic value of targeting GSDMD signaling in psoriasis treatment. We have expanded upon this discussion in the revised manuscript.

Fig 4i - guess here the conclusion would be that neutrophils are important for the pathogenesis in the IMQ model, which is true. This experiment does not support that this is done by pyroptosis.

To address the question, we analyzed the publicly available single-cell transcriptomic data (GSE165021) and found that, compared to the control group, neutrophils infiltrating in IMQ-induced psoriasis-like tissue display a higher expression of pyroptosis-related genes (see new Figure 3e in the revised manuscript). These results strengthen our conclusions about the role of neutrophil pyroptosis in the progression of psoriasis.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Specific Comments:

• Figure 1: Micro abscesses would already be dead, which would likely reflect as non-specific staining. Authors should consider double staining (e.g., GSDMD+Ly6G).

We thank the reviewer for the useful suggestion. We have added co-staining results of the neutrophil marker CD66b and GSDMD in the revised manuscript (see new Figure 3b in the revised manuscript). This addition further substantiates the expression of GSDMD in neutrophils within psoriasis tissue.

• Figures 1 b, c, and d do not have the n number for representative experiments and images.

We apologize for our oversight. We have added the relevant information in the revised manuscript and have reviewed and corrected the entire text.

• What is the difference between psoriasis patients in Figure 1 versus Figure 3 as the staining patterns are different? It is difficult to interpret from Figure 1 that expression is limited to neutrophils. Authors should consider double staining (e.g., GSDMD+Ly6G). How many samples were stained to draw this conclusion?

We thank the reviewer for the suggestion. In Figure 1b, we observed that GSDMD expression is higher in the psoriasis patient tissues compared to control samples. We have added co-staining results of the neutrophil marker CD66b and GSDMD in the revised manuscript (see new Figure 3b in the revised manuscript). For each staining group, we examined samples from 3-5 patients to draw the conclusion.

• Figure 2: GSDMD deficiency mitigates psoriasis-like inflammation in mice has been shown before (PMID#37673869). The paper showed that the GSDMD was mainly expressed in keratinocytes. What is the view of the authors on it and how does this data correlate with the data presented in this manuscript by the authors?

Consistent with previous studies[1], we observed increased expression of pyroptosis-related proteins in psoriatic lesions. However, our research focused specifically on the role of neutrophil pyroptosis in psoriasis, this does not conflict with existing reports indicating that KC cell pyroptosis also contributes to disease progression. Both studies underscore the significant role of GSDMD-mediated pyroptotic signaling in psoriasis, and the consistent involvement of KC cells and neutrophils further emphasizes the potential therapeutic value of targeting GSDMD signaling in psoriasis treatment. We have expanded upon this discussion in the revised manuscript.

• Figure 3d: It is unclear if the IF shows an epidermal or dermal area. As shown by authors in other figures (human psoriatic skin), do authors observe more GSDMD in the micro abscess, which is localized in the epidermis? The authors should also show the staining of GSDM/Ly6G in the whole skin sample.

The region we presented for immunofluorescence staining corresponds to the dermis of the mice, as we did not observe typical neutrophil micro abscesses similar to those in human psoriasis in the epidermis of IMQ-induced classical psoriasis vulgaris (PV) model. Therefore, we have only shown the staining in the dermal area.

• Figure 3e: PI staining also represents necrotic cells and TUNEL staining would not represent just apoptotic cells. It is unclear how the authors conclude an ongoing pyroptosis in neutrophils. A robust dataset is needed to provide evidence supporting neutrophil pyroptosis in the IMQ-challenged mice.

We thank the reviewer for the valuable suggestion. GSDMD is the effector protein of pyroptosis. To further confirm that cells are undergoing pyroptosis, it is necessary to morphologically stain the GSDMD N-terminal protein. Although there is currently no GSDMD N-terminal fluorescent antibody available, we detected the cleaved N-terminus of GSDMD by WB in mouse psoriasis-like skin tissue, and its increased expression suggested increased cell pyroptosis (see new Figure 1d in the revised manuscript). Moreover, we analyzed the publicly available single-cell transcriptomic data (GSE165021) and found that, compared to the control group, neutrophils infiltrating in IMQ-induced psoriasis-like tissue display a higher expression of pyroptosis-related genes (see new Figure 3e in the revised manuscript). These results strengthen our conclusions about the role of neutrophil pyroptosis in the progression of psoriasis.

• Figure 4: The authors did not clarify the reason for choosing D4 over the usual D7 for the imiquimod experiment. S100A8-Cre is also reported in monocytes and granulocytes/monocyte progenitors. And, the authors also show the expression in macrophages and neutrophils, but in the text, only neutrophils are mentioned. The authors should state the results in the text as well to avoid misrepresentation of the data.

We thank the reviewer for the useful suggestion. We have repeated many times of experiments in our previous studies and observed that the IMQ-induced mouse psoriasis model showed the obvious signs of self-resolution after Day 4 even with continuing topical IMQ application, thus we chose 4 days over 7 days for the imiquimod experiment, which are consistent with many other studies[3, 4].

Many studies use S100A8-Cre mice for neutrophil-specific gene knockout[5, 6]. Moreover, we used Ly6G antibody to eliminate neutrophils in GSDMD-cKO mice and control mice. It was found that the difference in lesions between the two groups was abolished after neutrophil depletion, indicating that neutrophil pyroptosis plays an important role in the pathogenesis of imiquimod-induced psoriasis-like lesions in mice. As the database analysis results showed that macrophages have slight expression of S100a8, according to the suggestion of the reviewer, we have added a more precise description in the revised manuscript.

• Figure S2a: Ly6G antibody reduced the ly6G positive, but also negative cells compared to PBS. If this is correct, what is the explanation, and how this observation has been considered for concluding results?

Neutrophils play an important role in regulating inflammatory responses, and their deletion can reduce the overall inflammatory level in the body, which also results in a decrease in other non-neutrophil cells. However, this change does not affect our conclusions. Our results show that after the deletion of neutrophils, there is no difference in the pathological manifestations between the cKO group and the control group. This further that GSDMD in neutrophil plays an important role in the pathogenesis of miquimod-induced psoriasis-like lesions in mice.

• The conclusion in Figure 4i is incorrect as Ly6G administration had an effect on the wt, so it shows neutrophils play a role, but not neutrophil pyroptosis.

- 321 "It was found that the difference in lesions between the

- 321 two groups was abolished after neutrophil depletion (Fig4i, S2a), indicating that

- 322 neutrophil pyroptosis plays an important role in the pathogenesis of

- 323 imiquimod-induced psoriasis-like lesions in mice"

Our results show that after the deletion of neutrophils, there is no difference in the pathological manifestations between the cKO group and the control group. This further indicates that the lower disease scores observed in cKO mice, in the absence of neutrophil deletion, depend on the presence of neutrophils. In the revised manuscript, we have changed the statement to “It was found that the difference in lesions between the two groups was abolished after neutrophil depletion (Fig4i, S2a), indicating that GSDMD in neutrophil plays an important role in the pathogenesis of miquimod-induced psoriasis-like lesions in mice”

• The effect of LyG Ab: reduced PASI in the wt, but the effect on the ko remains the same. What are the other molecular changes observed? What was the level of neutrophils in the wt and the S1A008Cre GsdmDfl/fl mice under steady state and how are they change upon imiquimod challenge? A complete profiling of the immune cells is needed for all the experiments.

As demonstrated by the results, the deletion of neutrophils did not significantly alter the pathological phenotype of cKO mice. We believe that this outcome precisely highlights the crucial role of GSDMD in regulating neutrophil inflammatory responses.

• Figure S2b: The authors conclude that Il-1b in the imiquimod skin is mainly expressed by neutrophils, but the analysis presented in the figure does not support this conclusion. Both neutrophils and macrophages are majorly positive for I1-b, with some expression on Langerhans and fibroblasts. No n numbers are provided for the experiment

As we discussed in the manuscript, we speculate that neutrophil pyroptosis may release cytokines, which in turn activate other cells to secrete cytokines, forming a complex inflammatory network in psoriasis. This may suggest that neutrophil pyroptosis may be involved in the pathogenesis of psoriasis by affecting the secretion of cytokines such as IL-1B and IL-6 by neutrophils, thereby affecting the function of other immune cells such as T cells and macrophages.

We have added the n number in the revised manuscript.

• For clarity and transparency, a list of antibodies with the associate clone and catalogue number should be provided or integrated into the method text.

We thank the reviewer for the useful suggestion. We have added the associate clone and catalogue number of antibodies used in the method text of revised manuscript.

Reviewer #2 (Recommendations for the authors):

Fig 3b: psoriasis and pustular psoriasis have a different pathophysiology (autoimmune vs. autoinflammatory). Neutrophils are centrally important for GPP for the cleavage of IL-36. Guess as not further referred to pustular psoriasis in the paper, that comparison is rather deviating from the story.

In Figure 3b, we stained for GSDMD and CD66b in both plaque psoriasis (PV) and generalized pustular psoriasis (GPP), not to compare the expression differences between the two types of psoriasis, but rather to demonstrate that significant GSDMD expression is present in neutrophils in different types of psoriasis. Unfortunately, due to the lack of a well-established animal model for GPP, we were only able to conduct studies using the established PV animal model. We acknowledge this limitation in our research. In our revised manuscript, we have added the following explanation in the discussion section: “Although we observed significantly increased GSDMD in neutrophils in pustular psoriasis, we were constrained to studying the established PV animal model due to the current absence of a mature GPP animal model. This represents a limitation of our study.”

In summary, we appreciate the Reviewer’s comments and suggestions. We feel that the inclusion of new data addresses the concerns in a comprehensive manner and adds further support to our original conclusions. We hope you will now consider the revised manuscript worthy of publication in eLife.

References:

(1) Lian, N., et al., Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis. Cell Death & Disease, 2023. 14(9): p. 595.

(2) Han, J., et al., GSDMD (gasdermin D) mediates pathological cardiac hypertrophy and generates a feed-forward amplification cascade via mitochondria-STING (stimulator of interferon genes) axis. Hypertension, 2022. 79(11): p. 2505-2518.

(3) Lin, H., et al., Forsythoside A alleviates imiquimod-induced psoriasis-like dermatitis in mice by regulating Th17 cells and IL-17a expression. Journal of Personalized Medicine, 2022. 12(1): p. 62.

(4) Emami, Z., et al., Evaluation of Kynu, Defb2, Camp, and Penk Expression Levels as Psoriasis Marker in the Imiquimod‐Induced Psoriasis Model. Mediators of Inflammation, 2024. 2024(1): p. 5821996.

(5) Stackowicz, J., et al., Neutrophil-specific gain-of-function mutations in Nlrp3 promote development of cryopyrin-associated periodic syndrome. Journal of Experimental Medicine, 2021. 218(10): p. e20201466.

(6) Abram, C.L., et al., Distinct roles for neutrophils and dendritic cells in inflammation and autoimmunity in motheaten mice. Immunity, 2013. 38(3): p. 489-501.

for - language and perception - Alfred Korzybski - paper - The role of language in the perceptual processes - Alfred Korzybski

eLife Assessment

This important work is a versatile new addition to the chemical protein modifications and bioconjugation toolbox in synthetic biology. The technology developed cleverly uses Connectase to irreversibly fuse proteins of interest together so they can be studied in their native context, with convincing data showing the technique works for various protein partners. This work will help multiple fields to explore multi-function constructs in basic synthetic biology. This work will also be of interest to those studying fusion oncoproteins commonly expressed in various human pathologies.

Reviewer #1 (Public review):

Fuchs describes a novel method of enzymatic protein-protein conjugation using the enzyme Connectase. The author is able to make this process irreversible by screening different Connectase recognition sites to find an alternative sequence that is also accepted by the enzyme. They are then able to selectively render the byproduct of the reaction inactive, preventing the reverse reaction, and add the desired conjugate with the alternative recognition sequence to achieve near-complete conversion. I agree with the authors that this novel enzymatic protein fusion method has several applications in the field of bioconjugation, ranging from biophysical assay conduction to therapeutic development. Previously the author has published on the discovery of the Connectase enzymes and has shown its utility in tagging proteins and detecting them by in-gel fluorescence. They now extend their work to include the application of Connectase in creating protein-protein fusions, antibody-protein conjugates, and cyclic/polymerized proteins. As mentioned by the author, enzymatic protein conjugation methods can provide several benefits over other non-specific and click chemistry labeling methods. Connectase specifically can provide some benefits over the more widely used Sortase, depending on the nature of the species that is desired to be conjugated. However, due to a similar lengthy sequence between conjugation partners, the method described in this paper does not provide clear benefits over the existing SpyTag-SpyCatcher conjugation system. Additionally, specific disadvantages of the method described are not thoroughly investigated, such as difficulty in purifying and separating the desired product from the multiple proteins used. Overall, this method provides a novel, reproducible way to enzymatically create protein-protein conjugates.

The manuscript is well-written and will be of interest to those who are specifically working on chemical protein modifications and bioconjugation.

Reviewer #2 (Public review):

Summary:

Unlike previous traditional protein fusion protocols, the author claims their proposed new method is fast, simple, specific, reversible, and results in a complete 1:1 fusion. A multi-disciplinary approach from cloning and purification, biochemical analyses, and proteomic mass spec confirmation revealed fusion products were achieved.

Strengths:

The author provides convincing evidence that an alternative to traditional protein fusion synthesis is more efficient with 100% yields using connectase. The author optimized the protocol's efficiency with assays replacing a single amino acid and identification of a proline aminopeptidase, Bacilius coagulans (BcPAP), as a usable enzyme to use in the fusion reaction. Multiple examples including Ubiquitin, GST, and antibody fusion/conjugations reveal how this method can be applied to a diverse range of biological processes.

Weaknesses:

Though the ~100% ligation efficiency is an advancement, the long recognition linker may be the biggest drawback. For large native proteins that are challenging/cannot be synthesized and require multiple connectase ligation reactions to yield a complete continuous product, the multiple interruptions with long linkers will likely interfere with protein folding, resulting in non-native protein structures. This method will be a good alternative to traditional approaches as the author mentioned but limited to generating epitope/peptide/protein tagged proteins, and not for synthetic protein biology aimed at examining native/endogenous protein function in vitro.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Fuchs describes a novel method of enzymatic protein-protein conjugation using the enzyme Connectase. The author is able to make this process irreversible by screening different Connectase recognition sites to find an alternative sequence that is also accepted by the enzyme. They are then able to selectively render the byproduct of the reaction inactive, preventing the reverse reaction, and add the desired conjugate with the alternative recognition sequence to achieve near-complete conversion. I agree with the authors that this novel enzymatic protein fusion method has several applications in the field of bioconjugation, ranging from biophysical assay conduction to therapeutic development. Previously the author has published on the discovery of the Connectase enzymes and has shown its utility in tagging proteins and detecting them by in-gel fluorescence. They now extend their work to include the application of Connectase in creating protein-protein fusions, antibody-protein conjugates, and cyclic/polymerized proteins. As mentioned by the author, enzymatic protein conjugation methods can provide several benefits over other non-specific and click chemistry labeling methods. Connectase specifically can provide some benefits over the more widely used Sortase, depending on the nature of the species that is desired to be conjugated. However, due to a similar lengthy sequence between conjugation partners, the method described in this paper does not provide clear benefits over the existing SpyTag-SpyCatcher conjugation system.  Additionally, specific disadvantages of the method described are not thoroughly investigated, such as difficulty in purifying and separating the desired product from the multiple proteins used. Overall, this method provides a novel, reproducible way to enzymatically create protein-protein conjugates.

The manuscript is well-written and will be of interest to those who are specifically working on chemical protein modifications and bioconjugation.

Reviewer #2 (Public review):

Summary:

Unlike previous traditional protein fusion protocols, the author claims their proposed new method is fast, simple, specific, reversible, and results in a complete 1:1 fusion. A multi-disciplinary approach from cloning and purification, biochemical analyses, and proteomic mass spec confirmation revealed fusion products were achieved.

Strengths:

The author provides convincing evidence that an alternative to traditional protein fusion synthesis is more efficient with 100% yields using connectase. The author optimized the protocol's efficiency with assays replacing a single amino acid and identification of a proline aminopeptidase, Bacilius coagulans (BcPAP), as a usable enzyme to use in the fusion reaction. Multiple examples including Ubiquitin, GST, and antibody fusion/conjugations reveal how this method can be applied to a diverse range of biological processes.

Weaknesses:

Though the ~100% ligation efficiency is an advancement, the long recognition linker may be the biggest drawback. For large native proteins that are challenging/cannot be synthesized and require multiple connectase ligation reactions to yield a complete continuous product, the multiple interruptions with long linkers will likely interfere with protein folding, resulting in non-native protein structures. This method will be a good alternative to traditional approaches as the author mentioned but limited to generating epitope/peptide/protein tagged proteins, and not for synthetic protein biology aimed at examining native/endogenous protein function in vitro.

I would like to sincerely thank both reviewers for their insightful and constructive feedback on the manuscript. I have addressed reviewer #1’s comments below:

(1) The benefits over the SpyTag-SpyCatcher system. Here, the conjugation partners are fused via the 12.3 kDa SpyCatcher protein, which is considerably larger than the Connectase fusion sequence (20 aa). This is briefly mentioned in the introduction (p. 1 ln 24-25). In a related technology, the SpyTag-SpyCatcher system was split into three components, SpyLigase, SpyTag and KTag  (Fierer et al., PNAS 2014). The resulting method introduces a sequence between the fusion partners (SpyTag (13aa) + KTag (10aa)), which is similar in length to the Connectase fusion sequence. I mention this method in the discussion (p. 8, ln 296 - 297), but preferred not to comment on its efficiency. It appears to require more enzyme and longer incubation times, while yielding less fusion product (Fierer et al., Figure 2).

(2) Purification of the fusion product. The method is actually advantageous in this respect, as described in the discussion (p. 8, ln 257-263). I plan to add a figure showing an example in the revised article.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

This is an interesting manuscript from two groups of experts in Notch signaling biology with complementary expertise in Drosophila genetics (Klein) and in biophysical studies of the Notch pathway (Sprinzak). The paper provides a cutting-edge structure-function dissection of the E3 ubiquitin ligase Neuralized and its mammalian homologs, Neurl1a and Neurl1a. The work is particularly relevant since the functions of mammalian Neurl1a and Neurl1b have been questioned, and more subtle altogether than those of fly Neuralized (as summarized by the authors in Fig. 1C). This is in part due to the dominant effects of the E3 ubiquitin ligase Mindbomb1 (Mib1) in Notch ligand-expressing cells from mammalian systems. The authors use careful structure-function work in fly development (mostly wing imaginal discs) and in mammalian cell culture systems, including a clever approach to study the function of mammalian Neurl1a and Neurl1b and mammalian/fly Notch ligand hybrids in Drosophila to draw new conclusions about the function of Neurl1a/b, showing that they can function as activators of Notch signaling mediated by the Notch ligands Dll1 and Jag1, and not by Dll4 and Jag2, tracing these differential effects to the recognition of a short NXXN consensus sequence in the N-terminal region of the ligand's intracellular domain.

__response: __We thank the reviewer for highlighting the novelty of our findings and experimental approach.

Specific questions: -The current title of the manuscript is not very information-rich and would not allow a reader to gather key information about the findings without reading at least the abstract. Could this be improved? For example, by referring to differential activation of individual Notch ligands, or some other more direct description of the key findings?

__Response: __We appreciate the reviewer's suggestion; however, we believe that the general nature of the title is appropriate in this case.

-The authors design most key experiments documenting agonistic effects of Neurl1a/1b in a Mib1-deficient background, both in flies and in cell culture systems. This is understandable experimentally to isolate Neurl1a/b's effects in these experimental systems. However, this leaves open questions as to the prevailing effects of Neurl1a/b in cells that also express Mib1 (which the authors comment on in the discussion based on past findings, including some suggesting that Neurl1a/1b can function as Notch inhibitors through a ligand ubiquitination mechanism that may differ from their activating function).

Do the authors actually have data that could shed light on this discussion? For example, have they performed cell coculture assays in which Neurl1a or Neurl1b is co-expressed with a Notch ligand, but in the presence of Mib1? This condition seems to be systematically omitted from all the coculture experiments that are presented. It would be interesting to evaluate the net effect of Neurl1a/Neurl1b expression in a Mib1-sufficient system as well.

Response: We have systematically removed MIB1 in our experiments because it activates all ligands, making its removal necessary to show the differential activity of Neurls. The question regarding competition between Mib1 and Neurls, as highlighted by the reviewer, is indeed intriguing. However, systematically investigating this competition would require varying the relative levels of the two proteins in a controlled manner, which is beyond the scope of this manuscript.

That said, we will perform the competition experiments suggested by the reviewers (co-expressing ligands with both Neurl1 and Mib1) and test their activity as controls. While these experiments may provide some insight into the competition, they will not comprehensively address the entire topic.

-The paper suggests important predictions about mammalian functions of Neurl1a/1b, including the neurological effects that have been reported, in double-deficient mice, namely that that there are cells that only express Neurl1a/1b and not Mib1 and do rely on Dll1 and Jag1 for signaling. Could the authors at least comment on this prediction? Are there are any single cell atlases where candidate cells like that can be identified? Or would the authors predict that Neurl1a/1b could actually function as Notch agonist even in cells expressing Mib1? (see also previous comment)

Response: This is an interesting suggestion. We will try to find if we can identify any specific expression patterns of E3 ubiquitin ligases across different tissues.

-Some minor typos: line 305 should likely read "flies homozygous for (...)". Line 408, "for providing" repeated twice.

Response: We thank the reviewer for pointing out this typo.

Reviewer #1 (Significance (Required)):

Thank you for the opportunity to review this lovely collaborative paper. As indicated in my comments to the authors, the findings provide novel structure-function information about an understudied aspect of Notch signaling and clarify conflicting past data about the mammalian homologs of fly Neuralized. The approach is elegant and multidisciplinary, notably in regards to the combination of cell co-culture systems and Drosophila as a platform to study mammalian Neuralized proteins and hybrid Notch ligand molecules. The findings will be interesting to the field and will generate discussion. I would suggest that some additional information would be a plus to substantiate predictions about mammalian functions of Neurl1a/b, and also to clarify its effects in the presence or absence of concomitant Mib1 expression.

We thank the reviewer for their positive evaluation of our work and for suggesting potential future direction regarding the concomitant expression of Mib1 and Neurls proteins.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary

The manuscript describes an analysis of specificity of functional interactions between mammalian Neuralized proteins and different human ligands for Notch. To investigate this, the authors take the approach of constructing hybrid proteins that contain the intracellular domain of the human ligands and the extracellular domain of the Drosophila Delta or Serrate, and investigate their activity in vivo, in the Drosophila wing disc. The latter is a well-established model tissue for assessing Notch ligand activity. As a second assay they express mammalian neutralized constructs in human cells for luciferase-based Notch signal reporter assays. The experiments are well presented and described and make a strong case for the conclusions that both Neurl1 and 2 can activate Notch signalling by Dll1 and Jag1 but not Dll4 and Jag2. Use of different mutant intracellular domains is used to show the importance of the NXXN motif, which in Drosophila is required for Neuralized interaction with Delta and Serrate. The use of missense mutations and in particular the reactivation of the cryptic NXXD site in Dll4 by substitution to N is convincing for establishing the importance of the motif. There is also colocalization data to support the conclusion that there is likely to be NXXN-dependent complex formation between the ligand and Neuralized proteins. This latter conclusion would be made firmer fi there were pull down data to support it, although to be fair it is most unlikely that another explanation, other than complex formation could account for the observation of both colocalization and ligand activation.

__Response: __We appreciate the reviewer's positive assessment of our manuscript and their support for the conclusions drawn from our experiments. We intend to conduct the suggested co-IP experiments with our cell culture assays to further supplement our current data.

__ Major comments__ The main limitation of the work is that it is mostly based on overexpression of constructs to activate ectopic expression rather than gene editing endogenous genes. It would be helpful if the authors could comment on the limitations of the work in discussion.

Two points of data included in the work are important in mitigating this limitation. Firstly, the experiments in the wing disc and cell culture are taking place in a mindbomb mutant background and the activation is observed is therefore a rescue of activity that has been lost.

Secondly, and importantly, the final experiment makes use of a Dl mutant Drosophila line which shows embryo lethality when homozygous, with the characteristic neurogenic phenotype. Rescue of lethality can be brought about by knock-in experiments which restore Dl function and this is also true for the ligand hybrid constructs that introduce mammalian ligand intracellular domains only when they include the NXXN motif This indicates the importance of the motif in normal development- Overall, the data presented in the paper is convincing as regards the conclusions made.

__Response: __We thank the reviewer for their very positive evaluation and his constructive suggestions, which have helped to improve the manuscript. In line with these suggestions, we will include additional data analyzing the bristle SOP selection, a process dependent on Neur. Our Results show that homozygosity of the DlattP-Dl-DLL1 allele, but not the DlattP-Dl-DLL4 allele, leads to correct Notch mediated selection. This finding provides further evidence that Neur requires the NxxN motif in the ICD of a ligand to activate DSL ligands. Notably, we previously showed that this selection relies on the NxxN motif of Dl (Troost et al., 2023). We will further emphasize in the discussion the ability of Dl-DLL4 hybrid ligands, containing a reconstructed NxxN motif, to rescue the neurogenic phenotype of Dl mutants.

Minor points In figure 1 the legend for D says that cryptic sites are substitutions of N for E or Q, but the figure and main text indicate that the substitutions are N to E or D.

Response: We thank the reviewer for pointing this out. We will correct this mistake.

In the remain figures it would be helpful to include in the figure legends and indications of the numbers of wing discs, embryos for which the images shown are representative of.

__Response: __We will quantify the experiments conducted in the wing imaginal discs of Drosophila by measuring the wing field size along the dorsal-ventral axis relative to the anterior-posterior axis. Statistical analysis will be performed to demonstrate statistical significance across n=5 experiments for each sample.

In Fg 3 The activation of Notch, by neural1 and Dl-Jag1 in B'" is stronger in the ventral side of the disc than the dorsal whereas, although activation of the same ligand by Neurl2 in C'" is weaker the majority of the ectopic wingless expression is on the dorsal compartment. Is there any reason for the switch in preference between the two neutralized proteins? Overgrowth of the wing disc seems to be similar on both sides and so am wondering if the picture is representative of the ectopic wingless distribution in this case.

Response: As discussed above we will perform quantification and statistical analysis across multiple experiments to confirm that our images are truly representative.

Reviewer #2 (Significance (Required)):

Significance

Previous work on double genetic knockouts of the two mouse Neuralized genes cast doubt as to whether Neuralized proteins play a role in Notch signal activation in mammals, unlike in Drosophila. There is, however, some genetic indications that spatial memory requires both Notch and neutralized proteins and may represent a specialised function limited to the Neuralized interaction. There are likely to be more subtle contexts waiting to be uncovered. The work is therefore showing important proof of principle for establishing the functionality of the mammalian Neurl proteins and highlights new findings indicting specialisation of the different ligands for interactions with Notch components. Elucidation of such specialisations will help understand why the diversity of different homologues of Notch and ligand have evolved and are maintained in the vertebrate genome compared to the single Notch and two ligands in Drosophila. Since Notch and it misregulation are widely involved in development, health and disease and there is much interest in developing therapeutic interactions that alter Notch activity then the work is likely of broad interest.

We thank the reviewer for the very positive evaluation and his useful suggestions which were helpful in improving the manuscript.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

**Summary**

Notch signalling is one of the major evolutionarily conserved signalling pathways involved in numerous developmental, physiological and pathological processes. Activation of the Notch receptor first requires ubiquitination of its ligands (collectively temed DSL), leading to a 'pulling force" that, upon ligand-receptor engagement, exposes Notch to intramembrane proteolysis leading to the nuclear translocation of the receptor's intracellular domain and activation of target genes with its DNA-binding co-factors.

While both Neuralized (Neurl) and Mind bomb are the E3 ubiquitin ligases for Notch ligands required for Drosphila development, in mammals, the Neur homologues Neur1 (officially Neurl1) and Neur2 (officially Neurl1B) are dispensable for development since double Neur1/2 knock-out mice have no developmental phenotype (but both Neur homologues are involved the the memory-related functions of Notch pathway in adulthood). Rather, just one of the two mammalian Mind bomb homologues, Mib1, functions as the chief E3 ligase for Notch ligands during mammalian development as evidenced by its Notch-related knockout phenotype.

Therefore, it has not been fully established whether and how the NEUR proteins regulate the mammalian Notch ligands. To clarify this issue, the authors assessed the capability of Drosophila Neur and mammalian NEUR1 and 2 proteins to activate the various hybrid Notch ligands (containing extracellularly Drosophila Delta and intracellularly the various Notch ligands' intracellular domains) in Drosophila wing dics and mammalian cell culture. The authors found that NEUR proteins only activate the Notch ligands containing a Neuralized binding motif, with the consensus sequence NxxN, that is present in DLL1 and JAG1, but not in DLL4 and JAG2. The authors also analyse the intracellular domains of mammalian Notch ligands DLL1, DLL4, JAG1 and JAG2 in Drosophila by generating knock-in alleles where endogenous Dl expression had been substituted for those of hybrid Notch ligands. This analysis showed that only in Dl-DLL1 and Dl-JAG1 flies but not in Dl-DLL4 and Dl-JAG" flies is the embryonic lethality rescued, the results being in agreement with the hybrid Dl-DLL experiments on wing dics reported earlier in this work.

The authors conclude that their findings suggest that the activation mechanism of Notch during development differs between Drosophila (where both Neur and Mib1 are required for Notch-related developmental processes ) and mammals and that this could possibly explain the apparently lesser relevance of mammalian NEUR proteins for developmental Notch signalling.

*Evidence and clarity*

The manuscript is quite laconic but clearly written. The evidence presented by the authors, given the heterologous and in vitro nature (i.e using mammalian or hybrid Notch ligands and mammalian E3 ligases thereof in Drosophila and cell cultures) of the study is generally trustworthy but limited in the sense that it probably does not allow definitive conclusions to be drawn as to the differing nature of the action of the E3 ligases of Notch ligands in flies vs mammals in vivo.

__Response: __We thank the reviewer for their positive evaluation of our work and their constructive criticism. We would like to clarify that we do not conclude that the activation mechanism differs between mammals and flies. Our findings demonstrate that the signalling mechanisms of fly Neur and mammalian Neurl's follow the same fundamental rules. Moreover, our study does not aim to provide a definitive answer to how signalling differs between species. Instead, we utilized the 'humanized fly' system to show that Neurl proteins specifically activate Dll1 and Jag1, but not Dll4 and Jag2, which lack a neuralized binding site.

*Reproducibility*

As will be mentioned a number of times, these reviewers would like to enquire as to the reasons for not providing a statistical analysis of variation in the fly wing disc-based experiments (where the readout was either resuce of Wg expression or induction of ectopic Wg expression).

Response: We thank the reviewer for raising this important point. As outlined below, we will quantify the fly experiments and conduct statistical analysis across multiple experimental datasets to further substantiate our claims.

Also, while the constructs used in the study were inserted into the same genomic landing sites to achieve comparable levels of expression of the various proteins, these reviewers would like to see data on the levels of expression of NEUR1 and 2 as well as the hybrid Notch ligands.

**Major comments**

Comment on fly wing disc experiments:

The authors study both the capability of two different mammalian E3 ubiquitin ligases, Neuralized-like 1 and 2 (mouse Neur1 and human NEUR2) to activate four different Notch receptors (DLL1 and 2, JAG1 and 2) in flies and mammalian cell culture system. In flies, they first analyse the capability of Drosophila Neur (as a positive control) and Neur1 and NEUR2 to activate the various Notch ligands (based on wingless activation as a readout) in wild-type wings (where, Mind bomb 1, or Mib1 is the only E3 ligase for Notch ligands present) and Mib1 mutant wing discs (which lack any E3 ligands of Notch receptors). The authors then test four humanised, hybrid Notch ligands (all five N ligands bar Dll3 since the latter does not transactivate the Notch receptor) - where mammalian Notch ligands' intracellular domains, or ICDs, have been attached to fly Dl (Dl-Dll1, Dl-Dll4, Dl-JAG1, Dl-JAG2) - for their capacity to mediate Mib1-dependent activation of Notch (with ectopic Wg expression in wing discs as its readout). They found that all 4 ligands can activate Nocth in wild-type wings (where Mib1 is present), with Dl-JAG2 being less effective than the other 3 hybrid ligands, implying that such hybrid, humanised ligands can be usd in studying Notch pathway activation in Drosophila (thereby constituting a mixed/heterologous experimental system). The reviewers would like to get a comment as to the reason for the weaker activity of Dl-JAG2 in this set-up?.

Response: We do not have a definitive answer as to why the ICDs differ in their activity within MIb1-dependent signalling, since this question was not addressed in the scope of this work. However, it our findings demonstrate that the hybrid ligands are functional in Drosophila and that their differential behavior in Neur-mediated signaling is not attributed to a trivial explanation, e. g. that the hybrid ligands generally display no activity. There are several potential explanations for these differences. One possibility is variations in position, arrangement, or number of targeted lysines among the ICDs. These lysines serve as substrates for ubiquitylation and determine the rate of endocytosis, which in turn impacts the signaling activity of the corresponding ligand/hybrid. Another plausible explanation is differences in affinity of the binding sites of Mib1, which would similarly result in variations in ubiquitylation and endocytosis rates. Regardless, we emphasize that resolving this question does not affect any of the conclusions of the manuscript.

Also, the reviewers would like to get a comment as to why was not a Neur mutant set-up used, only Mib1 mutant dics?

Response: Neur is only expressed at a very late stage in wing development and is restricted to specific single cells (sensory organ precursors). Consequently, even if mutants were present, their impact would be limited to these cells. Moreover, the Neur promoter has a highly complex architecture, which makes it exceedingly difficult to manipulate for experiments involving this mutation. We will address these considerations in the revised manuscript.

The authors then found that only two of these hybrid ligands - Dl-DLL1 and Dl-JAG1 but not Dl-DLL4 or Dl-JAG2 - can be used to activate Notch in the above wing assay when Mib1 was mutant. This is consistent with the fact that the NxxN-based Neuralized Binding motif (NBM) is present in DLL1 and JAG1 only. Using the wing paradigm, the authors also show by either mutating the full NBM (NxxN) in DLL1 or changing the cryptic, "weak" NBM in DLL4 (containing NxxD sequence) into "full/strong" NxxN one that the NBM in the various Notch ligands is required and sufficient for activation of the Notch pathway.

Overall, the fly experiments are convincing in showing diffrential activation of Notch ligands. However, no statistical analysis of the experimental variation in these studies - neither for the number of wing discs analysed per (hybrid) Notch ligand tested nor the extent of a given experimental manipulation's effect is included. We deem that if the images presented in Figures 2 and 3 are truly representative, this needs to be made explicitly clear.

Response: We thank the reviewer for their positive evaluation of our work and for the constructive comments, which we will consider and include into the manuscript. While we have repeated all experiments with multiple flies, we acknowledge the critique regarding the absence of statistical analysis.

To address this, we will quantify the experiments conducted in the wing imaginal discs of Drosophila. We will do that by measuring the wing field size along the dorsal-ventral axis relative to the anterior-posterior axis. We will perform statistical analysis to assess the statistical significance between experiments, using data from n=5 experiments for each sample.

Comment on fly embryonic Delta neurogenic phenotype's rescue experiments by replacing Dl with the hybrid ligands: The authors analysed the capacity of the ICDs of the mammalian ligands to rescue the Dl phenotype in Drosophila, ie. their activation capability at the organismal level. This was achieved by generating knock-in alleles expressing the hybrid ligands in place of Dl. The notion that only NBM-containing hybrid ligands was strengthened by this analysis since it showed that only NBM-containing hybrid ligands - Dl-DLL1 and Dl-JAG1 - but not Dl-DLL4 nor Dl-JAG2 rescued the Dl neurogenic embryonic lethal phenotype. Since this experimental set-up relied on the endogoneous Drosophila E3 ligases for activating the Notch ligands, the capacity of mammalian NEUR1 and 2 proteins to complement the capacity of the hybrid ligands to activate Notch to activate these ligands was not addressed. Please comment as to the reasons for this apparent omission and if such an analyis lies beyond the scope of current work, what would be the expected results of such experiment in the light of other experiments conducted in the course of this work?

Response: Testing whether mammalian Neurl1 and Neurl2 can replace Drosophila Neur in an endogenous setting is an intriguing question; however, it falls outside the focus of this study. Performing such an experiment would be highly challenging due to complex and not well understood architecture of Neur gene in Drosophila. Additionally, we believe it is highly unlikely that the mammalian NEURLs proteins would fully compensate for the loss of function in a Drosophila Neur mutant.

Journal-agnostic peer review: evaluate the paper as it stands independently from potential journal fit.

Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

Generaly yes, put please see the above comments on the absence of statistical analysis of reproducibility/ variation (if any) in fly wing disc experiments.

**Reviewer's additional recommendations:**

To publish in a higher-ranking journal, the co-localisation analyses of Notch ligands and its various E3 ubiquitin ligases studied probably needs to be replaced by a more rigorous, ideally FRET-based approach.

Response: We thank the reviewer for the comment. The co-localization assay is quite a robust and functional approach, as it provides clear evidence that endocytosis into a different compartment has occurred with functional ligands, as opposed to non-functional ligands. This serves as a quantitative and rigorous indicator for functional differences between these ligand types.

Nevertheless, we acknowledge that co-localization is not a direct measure of molecular interactions between Neurl1 and Notch ligands. To address this, as suggested by the reviewer, we will perform co-IP to show the differential interaction between Neurl1 and specific Notch ligands. Additionally, we will attempt a proximity ligation assay (PLA), which we consider to be a more direct and suitable method for detecting interactions between NEURLs and Notch ligands in this context, compared to FRET.

Since previous studies have shown that the Notch ligands are (mostly) poly- or mono-ubiquitylated by the E3 ubiquitin ligases Mib and the NEUR proteins, ideally, this or its follow-up study would benefit from analysis of the ubiquitylation status of the various hybrid Notch ligands.

Response: We thank the reviewer for the suggestion. The ubiquitylation pattern by Neurl1 is beyond the topic of the current manuscript.

Also, it would be useful to show the strength of interaction between the hybrid Notch ligands and NEUR1 and NEUR2 by ising a co-immunoprecipitation based approach.

Response: As suggested by the reviewer, we plan to perform co-IP and/or PLA to show the differential binding of NEURL1 to the different ligands. However, due to the observed toxicity of NEURL2 in our cells, it has been excluded from our assays.

Please request additional experiments only if they are essential for the conclusions. Alternatively, ask the authors to qualify their claims as preliminary or speculative, or to remove them altogether.

These reviewers do not strictly request any further rexperiments. However, since the mammalian NEUR2 could not be studied in cell cultures of U2OS cells due to its toxicity, we would like the auhtors to explain the choice of this cell line. Perhaps a cell line whose viability is not impaired by NEUR2 should be (or should have been) used?

Response: The decision not to use other cell lines was based on several strict experimental requirements. The most stringent requirement was the need to generate a MIB1 knockout cell line, as MIB1 strongly activates all ligands. The availability of having MIB1 KO U2OS cells enabled these experiments.

If you have constructive further reaching suggestions that could significantly improve the study but would open new lines of investigations, please label them as "OPTIONAL".

As mentioned above, the NEUR2's capacity to activate the hybrid ligands in U2OS cells could not be addressed to due to its toxicity. A more optimal cell line will have to be used in follow-up studies.

Also, these findings ultimately warrant in vivo studies using mice to definitively ascertain whether they also hold equally true there.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated time investment for substantial experiments.

The suggested experiments are optional apart from statistical analysis of variation (if any) in the fly wing disc experiments. If there is no (apparently significant) variation in these data, this needs to explicitly stated.

Response: We thank the reviewer for their thoughtful assessment. We will conduct the requested statistical analysis and perform some of the suggested supporting experiments as detailed in the response.

Are the data and the methods presented in such a way that they can be reproduced?

Generally yes, but see above about the lack of statistical data on the variation (if any).

Are the experiments adequately replicated and statistical analysis adequate?

Generally yes, but again, please see above about the lack of statistical data on the variation (if any).

**Minor comments**

Comment#1 (on the abstract and introduction):

In the Abstract, the authors state that there are four Notch ligands in mammals (lines 21 and 22): "Thus, it is unclear how NEURL proteins regulate the four mammalian Notch ligands". In the Introduction, they correctly state that there are five Notch ligands in mammals (lines 38 and 39): „In mammals, there are five ligands, three from the Delta-like (Dll) family (Dll1, Dll3, Dll4), and two from the Jagged (Jag) family (Jag1 and Jag2)." There are five Notch ligands in mammals (Dll1, Dll3, Dll4, Jag1, Jag2), and it is obvious that the authors are very well aware of this (they state in lines 146-147): "We excluded the ICD of DLL3 since it is not a ligand capable of trans-activation of Notch" (the four ligands included were Dll1, Dll4, Jag1 and Jag2)." Therefore, a claricifaction is required in the part of Abstract (i.e lines 21 ansd 22) - did the authors mean the four mammalian Notch ligands they actually studied (i.e Dll1, Dll4, Jag1, Jag2) or is there an oversight and the auhtors actually intended to write "the five Nocth ligands in mammals".? In either case, a correction is required in this reviewer's opinion.

Response: We are fully aware of this point, and will address it by providing clarification in the abstract as suggested.

Specific experimental issues that are easily addressable.

NEUR2 could not be studied in mammalian cell cultures due to its toxicity in the U2OS cell line, the one used by the authors. The use of another cell line would not be probably overly time-consuming; however, if this experiment lies outside the scope of current work, we would like to hear the authors' comment on this matter.

Response: This is addressed above.

Are prior studies referenced appropriately? Generally yes, but four prior studies go unmentioned: the two 2001 mouse Neur1 knock-out studies reporting no Notch-like developmental phenotype (Ruan et al, PNAS; Vollrath et al, Mol Cell Biol), the 2002 study of mouse, rat and human NEUR1 expression, subcellular localisation (Timmusk et al, Mol Cell Neuroscience) and the 2009 cell culture-based study of NEUR2's interaction with DLL1 and DLL4 (Rullinkov et al, BBRC). The non-requirement of NEUR1 and 2 proteins in mammalian developmental Notch signalling could partly be explained by the fact that NEUR1 is not highly expressed during mouse embryonic/foetal development - its expression becomes considerably more pronounced only postnatally (Timmusk et al, 2002).

Response: We will incorporate these references into the introduction and discuss the low expression of Neurls during development as a possible reason for the non-requirement in this context.

Are the text and figures clear and accurate?

Yes. These reviewers find the cartoon-based explanations of the experimental set-up in each figure helpful for enhancing the manuscript's overall clarity.

Response: We thank the reviewers for the positive feedback!

Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Please see above about the lack of statistical data on the variation (if any) in fly wing dic experiments and referencing of the 4 papers that are currently excluded.

Response: These will be corrected in the revised version.

Reviewer #3 (Significance (Required)):

1. Significance Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested. The following aspects are important: General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? This study uses the amenability of Drosophila to study the mammalian NEUR proteins' (NEUR1 and NEUR2) activity upon Notch ligands using hybrid Notch ligands containing mammalian ICDs (intracellular domains) fused to the extracellular domain of Drosophila Delta (Dl). It confirms and extends prior studies showing that Notch ligands can be (strongly) activated only by the E3 ubiquitin ligases containing the Neuralized Binding Motif (NBM).

Response: We respectfully disagree with the reviewer's assessment on this point. Our study is the first to demonstrate that Neurl proteins differentially activate Dll1 and Jag1, but not Dll4 and Jag2. This findings is further supported by the significance comments of the other reviewers.

However, since this study was based on using hybrid ligands containing mammalian ICDs of Notch ligands fused to the extracellular domain of Drosophila Delta (Dl), it is somewhat artificial. While NEUR1 was also studied in mammalian cell cultures (but not NEUR2 due to its toxicity), only an in vivo study using mice expressing with systematic changes to the Notch ligands' NBM will definitively reveal whether the conclusions reached by the authors hold true in vivo in a non-heterologous system.

Response: We firmly believe that our combined 'humanized fly' model and quantitative cell culture assay represents an innovative and rigorous approach for testing humanized proteins in in-vivo settings, without the need for extensive mouse genetics. The conclusions of our experiments should not be dismissed solely on the grounds of "not being performed in mice," as this would undermine much of current scientific research.

Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...). The study's advances are chiefly mechanistic and functional since they show more definitively that the reason underlying the differing activation of four mammalian Notch ligands by mammalian NEUR1 and NEUR2 is mostly based upon the presence or otherwise of a conserved Neuralized Binding Motif, NBM. Audience: describe the type of audience ("specialised", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?

The audience for this study is the research studying the Notch signalling pathway. Since dysregulation of this pathway is implicated in a number of devastating diseases, any improved understanding of its mechanistic underpinnings could in the long run lead to better therapeutic management of diseases with significant involvement of malfunctioning Notch signalling.

Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Molecular biology, molecular neuroscience, developmental biology, cell-cell signalling, Notch signalling. All parts of the manuscript fall within our expertise.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #3

Evidence, reproducibility and clarity

Summary

Notch signalling is one of the major evolutionarily conserved signalling pathways involved in numerous developmental, physiological and pathological processes. Activation of the Notch receptor first requires ubiquitination of its ligands (collectively temed DSL), leading to a 'pulling force" that, upon ligand-receptor engagement, exposes Notch to intramembrane proteolysis leading to the nuclear translocation of the receptor's intracellular domain and activation of target genes with its DNA-binding co-factors.

While both Neuralized (Neurl) and Mind bomb are the E3 ubiquitin ligases for Notch ligands required for Drosphila development, in mammals, the Neur homologues Neur1 (officially Neurl1) and Neur2 (officially Neurl1B) are dispensable for development since double Neur1/2 knock-out mice have no developmental phenotype (but both Neur homologues are involved the the memory-related functions of Notch pathway in adulthood). Rather, just one of the two mammalian Mind bomb homologues, Mib1, functions as the chief E3 ligase for Notch ligands during mammalian development as evidenced by its Notch-related knockout phenotype.

Therefore, it has not been fully established whether and how the NEUR proteins regulate the mammalian Notch ligands. To clarify this issue, the authors assessed the capability of Drosophila Neur and mammalian NEUR1 and 2 proteins to activate the various hybrid Notch ligands (containing extracellularly Drosophila Delta and intracellularly the various Notch ligands' intracellular domains) in Drosophila wing dics and mammalian cell culture. The authors found that NEUR proteins only activate the Notch ligands containing a Neuralized binding motif, with the consensus sequence NxxN, that is present in DLL1 and JAG1, but not in DLL4 and JAG2. The authors also analyse the intracellular domains of mammalian Notch ligands DLL1, DLL4, JAG1 and JAG2 in Drosophila by generating knock-in alleles where endogenous Dl expression had been substituted for those of hybrid Notch ligands. This analysis showed that only in Dl-DLL1 and Dl-JAG1 flies but not in Dl-DLL4 and Dl-JAG" flies is the embryonic lethality rescued, the results being in agreement with the hybrid Dl-DLL experiments on wing dics reported earlier in this work. The authors conclude that their findings suggest that the activation mechanism of Notch during development differs between Drosophila (where both Neur and Mib1 are required for Notch-related developmental processes ) and mammals and that this could possibly explain the apparently lesser relevance of mammalian NEUR proteins for developmental Notch signalling.

Evidence and clarity

The manuscript is quite laconic but clearly written. The evidence presented by the authors, given the heterologous and in vitro nature (i.e using mammalian or hybrid Notch ligands and mammalian E3 ligases thereof in Drosophila and cell cultures) of the study is generally trustworthy but limited in the sense that it probably does not allow definitive conclusions to be drawn as to the differing nature of the action of the E3 ligases of Notch ligands in flies vs mammals in vivo.

Reproducibility

As will be mentioned a number of times, these reviewers would like to enquire as to the reasons for not providing a statistical analysis of variation in the fly wing disc-based experiments (where the readout was either resuce of Wg expression or induction of ectopic Wg expression). Also, while the constructs used in the study were inserted into the same genomic landing sites to achieve comparable leves of expression of the various proteins, these reviewers would like to see data on the levels of expression of NEUR1 and 2 as well as the hybrid Notch ligands.

Major comments

Comment on fly wing disc experiments:

The authors study both the capability of two different mammalian E3 ubiquitin ligases, Neuralized-like 1 and 2 (mouse Neur1 and human NEUR2) to activate four different Notch receptors (DLL1 and 2, JAG1 and 2) in flies and mammalian cell culture system. In flies, they first analyse the capability of Drosophila Neur (as a positive control) and Neur1 and NEUR2 to activate the various Notch ligands (based on wingless activation as a readout) in wild-type wings (where, Mind bomb 1, or Mib1 is the only E3 ligase for Notch ligands present) and Mib1 mutant wing discs (which lack any E3 ligands of Notch receptors). The authors then test four humanised, hybrid Notch ligands (all five N ligands bar Dll3 since the latter does not transactivate the Notch receptor) - where mammalian Notch ligands' intracellular domains, or ICDs, have been attached to fly Dl (Dl-Dll1, Dl-Dll4, Dl-JAG1, Dl-JAG2) - for their capacity to mediate Mib1-dependent activation of Notch (with ectopic Wg expression in wing discs as its readout). They found that all 4 ligands can activate Nocth in wild-type wings (where Mib1 is present), with Dl-JAG2 being less effective than the other 3 hybrid ligands, implying that such hybrid, humanised ligands can be usd in studying Notch pathway activation in Drosophila (thereby constituting a mixed/heterologous experimental system). The reviewers would like to get a comment as to the reason for the weaker activity of Dl-JAG2 in this set-up?.

Also, the reviewers would like to get a comment as to why was not a Neur mutant set-up used, only Mib1 mutant dics? The authors then found that only two of these hybrid ligands - Dl-DLL1 and Dl-JAG1 but not Dl-DLL4 or Dl-JAG2 - can be used to activate Notch in the above wing assay when Mib1 was mutant. This is consistent with the fact that the NxxN-based Neuralized Binding motif (NBM) is present in DLL1 and JAG1 only. Using the wing paradigm, the authors also show by either mutating the full NBM (NxxN) in DLL1 or changing the cryptic, "weak" NBM in DLL4 (containing NxxD sequence) into "full/strong" NxxN one that the NBM in the various Notch ligands is required and sufficient for activation of the Notch pathway.

Overall, the fly experiments are convincing in showing diffrential activation of Notch ligands. However, no statistical analysis of the experimental variation in these studies - neither for the number of wing discs analysed per (hybrid) Notch ligand tested nor the extent of a given experimental manipulation's effect is included. We deem that if the images presented in Figures 2 and 3 are truly representative, this needs to be made explicitly clear. Comment on fly embryonic Delta neurogenic phenotype's rescue experiments by replacing Dl with the hybrid ligands: The authors analysed the capacity of the ICDs of the mammalian ligands to rescue the Dl phenotype in Drosophila, ie. their activation capability at the organismal level. This was achieved by generating knock-in alleles expressing the hybrid ligands in place of Dl. The notion that only NBM-containing hybrid ligands was strengthened by this analysis since it showed that only NBM-containing hybrid ligands - Dl-DLL1 and Dl-JAG1 - but not Dl-DLL4 nor Dl-JAG2 rescued the Dl neurogenic embryonic lethal phenotype. Since this experimental set-up relied on the endogoneous Drosophila E3 ligases for activating the Notch ligands, the capacity of mammalian NEUR1 and 2 proteins to complement the capacity of the hybrid ligands to activate Notch to activate these ligands was not addressed. Please comment as to the reasons for this apparent omission and if such an analsyis lies beyond the scope of current work, what would be the expected results of such experiment in the light of other experiments conducted in the course of this work? Journal-agnostic peer review: evaluate the paper as it stands independently from potential journal fit.

Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

Generaly yes, put please see the above comments on the absence of statistical analysis of reproducibility/ variation (if any) in fly wing disc experiments.

Reviewer's additional recommendations:

To publish in a higher-ranking journal, the co-localisation analyses of Notch ligands and its various E3 ubiquitin ligases studied probably needs to be replaced by a more rigorous, ideally FRET-based approach. Since previous studies have shown that the Notch ligands are (mostly) poly- or mono-ubiquitylated by the E3 ubiquitin ligases Mib and the NEUR proteins, ideally, this or its follow-up study would benefit from analysis of the ubiquitylation status of the various hybrid Notch ligands. Also, it would be useful to show the strength of interaction between the hybrid Notch ligands and NEUR1 and NEUR2 by ising a co-immunoprecipitation based approach. Please request additional experiments only if they are essential for the conclusions. Alternatively, ask the authors to qualify their claims as preliminary or speculative, or to remove them altogether. These reviewers do not strictly request any further rexperiments. However, since the mammalian NEUR2 could not be studied in cell cultures of U2OS cells due to its toxicity, we would like the auhtors to explain the choice of this cell line. Perhaps a cell line whose viability is not impaired by NEUR2 should be (or should have been) used? If you have constructive further reaching suggestions that could significantly improve the study but would open new lines of investigations, please label them as "OPTIONAL". As mentioned above, the NEUR2's capacity to activate the hybrid ligands in U2OS cells could not be addressed to due to its toxicity. A more optimal cell line will have to be used in follow-up studies. Also, these findings ultimately warrant in vivo studies using mice to definitively ascertain whether they also hold equally true there.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated time investment for substantial experiments.

The suggested experiments are optional apart from statistical analysis of variation (if any) in the fly wing disc experiments. If there is no (apparently significant) variation in these data, this needs to explicitly stated.

Are the data and the methods presented in such a way that they can be reproduced?

Generally yes, but see above about the lack of statistical data on the variation (if any).

Are the experiments adequately replicated and statistical analysis adequate?

Generally yes, but again, please see above about the lack of statistical data on the variation (if any).

Minor comments

Comment#1 (on the abstract and introduction):

In the Abstract, the authors state that there are four Notch ligands in mammals (lines 21 and 22):<br /> "Thus, it is unclear how NEURL proteins regulate the four mammalian Notch ligands". In the Introduction, they correctly state that there are five Notch ligands in mammals (lines 38 and 39): „In mammals, there are five ligands, three from the Delta-like (Dll) family (Dll1, Dll3, Dll4), and two from the Jagged (Jag) family (Jag1 and Jag2)." There are five Notch ligands in mammals (Dll1, Dll3, Dll4, Jag1, Jag2), and it is obvious that the authors are very well aware of this (they state in lines 146-147): "We excluded the ICD of DLL3 since it is not a ligand capable of trans-activation of Notch" (the four ligands included were Dll1, Dll4, Jag1 and Jag2)." Therefore, a claricifaction is required in the part of Abstract (i.e lines 21 ansd 22) - did the authors mean the four mammalian Notch ligands they actually studied (i.e Dll1, Dll4, Jag1, Jag2) or is there an oversight and the auhtors actually intended to write "the five Nocth ligands in mammals".? In either case, a correction is required in this reviewer's opinion.

Specific experimental issues that are easily addressable.

NEUR2 could not be studied in mammalian cell cultures due to its toxicity in the U2OS cell line, the one used by the authors. The use of another cell line would not be probably overly time-consuming; however, if this experiment lies outside the scope of current work, we would like to hear the authors' comment on this matter. Are prior studies referenced appropriately? Generally yes, but four prior studies go unmentioned: the two 2001 mouse Neur1 knock-out studies reporting no Notch-like developmental phenotype (Ruan et al, PNAS; Vollrath et al, Mol Cell Biol), the 2002 study of mouse, rat and human NEUR1 expression, subcellular localisation (Timmusk et al, Mol Cell Neuroscience) and the 2009 cell culture-based study of NEUR2's interaction with DLL1 and DLL4 (Rullinkov et al, BBRC). The non-requirement of NEUR1 and 2 proteins in mammalian developmental Notch signalling could partly be explained by the fact that NEUR1 is not highly expressed during mouse embryonic/foetal development - its expression becomes considerably more pronounced only postnatally (Timmusk et al, 2002).

Are the text and figures clear and accurate?

Yes. These reviewers find the cartoon-based explanations of the experimental set-up in each figure helpful for enhancing the manuscript's overall clarity.

Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Please see above about the lack of statistical data on the variation (if any) in fly wing dic experiments and referencing of the 4 papers that are currently excluded.

Significance

Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested. The following aspects are important:

General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? This study uses the amenability of Drosophila to study the mammalian NEUR proteins' (NEUR1 and NEUR2) activity upon Notch ligands using hybrid Notch ligands containing mammalian ICDs (intracellular domains) fused to the extracellular domain of Drosophila Delta (Dl). It confirms and extends prior studies showing that Notch ligands can be (strongly) activated only by the E3 ubiquitin ligases containing the Neuralized Binding Motif (NBM). However, since this study was based on using hybrid ligands containing mammalian ICDs of Notch ligands fused to the extracellular domain of Drosophila Delta (Dl), it is somewhat artificial. While NEUR1 was also studied in mammalian cell cultures (but not NEUR2 due to its toxicity), only an in vivo study using mice expressing with systematic changes to the Notch ligands' NBM will definitively reveal whether the conclusions reached by the authors hold true in vivo in a non-heterologous system.

Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...). The study's advances are chiefly mechanistic and functional since they show more definitively that the reason underlying the differing activation of four mammalian Notch ligands by mammalian NEUR1 and NEUR2 is mostly based upon the presence or otherwise of a conserved Neuralized Binding Motif, NBM.

Audience: describe the type of audience ("specialised", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field? The audience for this study is the research studying the Notch signalling pathway. Since dysregulation of this pathway is implicated in a number of devastating diseases, any improved understanding of its mechanistic underpinnings could in the long run lead to better therapeutic management of diseases with significant involvement of malfunctioning Notch signalling.

Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Molecular biology, molecular neuroscience, developmental biology, cell-cell signalling, Notch signalling. All parts of the manuscript fall within our expertise.

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Referee #2

Evidence, reproducibility and clarity

Summary

The manuscript describes an analysis of specificity of functional interactions between mammalian Neuralized proteins and different human ligands for Notch. To investigate this, the authors take the approach of constructing hybrid proteins that contain the intracellular domain of the human ligands and the extracellular domain of the Drosophila Delta or Serrate, and investigate their activity in vivo, in the Drosophila wing disc. The latter is a well-established model tissue for assessing Notch ligand activity. As a second assay they express mammalian neutralized constructs in human cells for luciferase-based Notch signal reporter assays. The experiments are well presented and described and make a strong case for the conclusions that both Neurl1 and 2 can activate Notch signalling by Dll1 and Jag1 but not Dll4 and Jag2. Use of different mutant intracellular domains is used to show the importance of the NXXN motif, which in Drosophila is required for Neuralized interaction with Delta and Serrate. The use of missense mutations and in particular the reactivation of the cryptic NXXD site in Dll4 by substitution to N is convincing for establishing the importance of the motif. There is also colocalization data to support the conclusion that there is likely to be NXXN-dependent complex formation between the ligand and Neuralized proteins. This latter conclusion would be made firmer fi there were pull down data to support it, although to be fair it is most unlikely that another explanation, other than complex formation could account for the observation of both colocalization and ligand activation.

Major comments

The main limitation of the work is that it is mostly based on overexpression of constructs to activate ectopic expression rather than gene editing endogenous genes. It would be helpful if the authors could comment on the limitations of the work in discussion. Two points of data included in the work are important in mitigating this limitation. Firstly, the experiments in the wing disc and cell culture are taking place in a mindbomb mutant background and the activation is observed is therefore a rescue of activity that has been lost. Secondly, and importantly, the final experiment makes use of a Dl mutant Drosophila line which shows embryo lethality when homozygous, with the characteristic neurogenic phenotype. Rescue of lethality can be brought about by knock-in experiments which restore Dl function and this is also true for the ligand hybrid constructs that introduce mammalian ligand intracellular domains only when they include the NXXN motif This indicates the importance of the motif in normal development

Overall, the data presented in the paper is convincing as regards the conclusions made.

Minor points

In figure 1 the legend for D says that cryptic sites are substitutions of N for E or Q, but the figure and main text indicate that the substitutions are N to E or D.

In the remain figures it would be helpful to include in the figure legends and indications of the numbers of wing discs, embryos for which the images shown are representative of.

In Fg 3 The activation of Notch, by neural1 and Dl-Jag1 in B'" is stronger in the ventral side of the disc than the dorsal whereas, although activation of the same ligand by Neurl2 in C'" is weaker the majority of the ectopic wingless expression is on the dorsal compartment. Is there any reason for the switch in preference between the two neutralized proteins? Overgrowth of the wing disc seems to be similar on both sides and so am wondering if the picture is representative of the ectopic wingless distribution in this case.

Significance

Previous work on double genetic knockouts of the two mouse Neuralized genes cast doubt as to whether Neuralized proteins play a role in Notch signal activation in mammals, unlike in Drosophila. There is, however, some genetic indications that spatial memory requires both Notch and neutralized proteins and may represent a specialised function limited to the Neuralized interaction. There are likely to be more subtle contexts waiting to be uncovered. The work is therefore showing important proof of principle for establishing the functionality of the mammalian Neurl proteins and highlights new findings indicting specialisation of the different ligands for interactions with Notch components. Elucidation of such specialisations will help understand why the diversity of different homologues of Notch and ligand have evolved and are maintained in the vertebrate genome compared to the single Notch and two ligands in Drosophila. Since Notch and it misregulation are widely involved in development, health and disease and there is much interest in developing therapeutic interactions that alter Notch activity then the work is likely of broad interest.

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Referee #1

Evidence, reproducibility and clarity

This is an interesting manuscript from two groups of experts in Notch signaling biology with complementary expertise in Drosophila genetics (Klein) and in biophysical studies of the Notch pathway (Sprinzak). The paper provides a cutting-edge structure-function dissection of the E3 ubiquitin ligase Neuralized and its mammalian homologs, Neurl1a and Neurl1a. The work is particularly relevant since the functions of mammalian Neurl1a and Neurl1b have been questioned, and more subtle altogether than those of fly Neuralized (as summarized by the authors in Fig. 1C). This is in part due to the dominant effects of the E3 ubiquitin ligase Mindbomb1 (Mib1) in Notch ligand-expressing cells from mammalian systems. The authors use careful structure-function work in fly development (mostly wing imaginal discs) and in mammalian cell culture systems, including a clever approach to study the function of mammalian Neurl1a and Neurl1b and mammalian/fly Notch ligand hybrids in Drosophila to draw new conclusions about the function of Neurl1a/b, showing that they can function as activators of Notch signaling mediated by the Notch ligands Dll1 and Jag1, and not by Dll4 and Jag2, tracing these differential effects to the recognition of a short NXXN consensus sequence in the N-terminal region of the ligand's intracellular domain.

Specific questions:

• The current title of the manuscript is not very information-rich and would not allow a reader to gather key information about the findings without reading at least the abstract. Could this be improved? For example, by referring to differential activation of individual Notch ligands, or some other more direct description of the key findings?
• The authors design most key experiments documenting agonistic effects of Neurl1a/1b in a Mib1-deficient background, both in flies and in cell culture systems. This is understandable experimentally to isolate Neurl1a/b's effects in these experimental systems. However, this leaves open questions as to the prevailing effects of Neurl1a/b in cells that also express Mib1 (which the authors comment on in the discussion based on past findings, including some suggesting that Neurl1a/1b can function as Notch inhibitors through a ligand ubiquitination mechanism that may differ from their activating function). Do the authors actually have data that could shed light on this discussion? For example, have they performed cell coculture assays in which Neurl1a or Neurl1b is co-expressed with a Notch ligand, but in the presence of Mib1? This condition seems to be systematically omitted from all the coculture experiments that are presented. It would be interesting to evaluate the net effect of Neurl1a/Neurl1b expression in a Mib1-sufficient system as well.
• The paper suggests important predictions about mammalian functions of Neurl1a/1b, including the neurological effects that have been reported, in double-deficient mice, namely that that there are cells that only express Neurl1a/1b and not Mib1 and do rely on Dll1 and Jag1 for signaling. Could the authors at least comment on this prediction? Are there are any single cell atlases where candidate cells like that can be identified? Or would the authors predict that Neurl1a/1b could actually function as Notch agonist even in cells expressing Mib1? (see also previous comment)
• Some minor typos: line 305 should likely read "flies homozygous for (...)". Line 408, "for providing" repeated twice.

Significance

Thank you for the opportunity to review this lovely collaborative paper. As indicated in my comments to the authors, the findings provide novel structure-function information about an understudied aspect of Notch signaling and clarify conflicting past data about the mammalian homologs of fly Neuralized. The approach is elegant and multidisciplinary, notably in regards to the combination of cell co-culture systems and Drosophila as a platform to study mammalian Neuralized proteins and hybrid Notch ligand molecules. The findings will be interesting to the field and will generate discussion. I would suggest that some additional information would be a plus to substantiate predictions about mammalian functions of Neurl1a/b, and also to clarify its effects in the presence or absence of concomitant Mib1 expression.

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جدول قابل للتفاعل عند التمرير

يتم استخدام خاصية border-bottom مع العنصر tr (الصفوف) لتطبيق حدود أسفل كل صف.

الفواصل الافقية

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Curator: @dawnn.marie

SciCrunch record: RRID:SCR_006457

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What is this?