To determine the cycle life of the actuator, contractions were repeated until failure. 158,060 mechanical contractions were successfully completed before the actuator ripped, showing that the actuator is able to operate efficiently under large loads for an extended period of time.

Stacking actuators allows for generating larger movement in comparison to one actuator. Since these actuators self heal from electrical damage, the actuation stroke can be increased without permanent damage to the system.

Even with a decrease in flexural strength, the arms of the smart pill still have sufficient rigidity to move forward with the experiment. If the rigidity was affected more drastically, it could lead to the arms breaking inside the stomach. The arms are meant to keep the smart pill large enough so that it is too big to pass through the pylorus, therefore arm breakage could lead to early digestion of the pill.

The smart pill is able to fold into itself to fit inside a capsule for ingestion.

The authors were able to control the porosity of the 3D printed filaments by controlling the salt concentration and pH of the support bath.

Fig. 2A shows the concentration of the drug from a Levora tablet in a pig over the course of 2 days. The concentration reaches it's peak of 199 ± 56 pg/ml (n = 5) at around 6 hours and by 48 hours dropped to 5 ± 4 pg/ml (n = 5).

This method helps determine the load that can be repeatedly applied to the joint area between the central elastomer and the arms while still keeping its structural integrity.

This arm design was initially used because of the higher surface area it allowed in comparison to the other designs.

Using the capsule with three arms, they found concentrations of 45 ± 2 pg/ml on day 3 and 54 ± 29 pg/ml on day 11. These results illustrate that the capsule is steadily releasing the hormone over a 20-day period. One problem with this formulation is that the drug concentration fluctuates significantly over time which may reduce its effectiveness for pregnancy protection.

Using the capsule with half of the drug loaded in poly(sebacic anhydride) and the other half in PDMS, the authors illustrated birth control drug delivery for an extended period. They observed a peak concentration of the drug on day 17. This shows that, unlike the daily tablet, their formulation slowly and steadily releases the drug and provides protection over a longer period.

After a period of 2 weeks in the simulated stomach acid, the strength of the caged arms decreased. The integrity of the polymeric arms are affected by exposure to the acid. The decrease in strength is ~25% after 2 weeks.

After ingestion, the smart pill is released from the capsule and expands to a size larger than the stomach's opening, which prevents it from passing into small intestine. This will allow for the extended release of the birth control drug within the stomach.

Tests are performed in order to understand how this specific polymer reacts when introduced to different pH levels. These are done to ensure the polymer is stable in the low pH of the stomach.

Using the capsule with six loaded arms of PDMS, a maximum concentration was reached on day 2. Note how the maximum concentration is 126 ± 24 pg/ml, as opposed to the latter configuration with a maximum concentration of 55 ± 18 pg/ml.

The simulated gastric fluid is a manmade fluid to mimic the acidic condition in human stomach. This fluid is used for testing the stability of the polymer that the solid arms are made of, for prolonged periods.

PDMS-based material was used in this experiment because there are known uses for this material in other sustained- release products.

A heart valve was printed with the collagen gel and was strengthened using standardized methods.

Heart valves are structures with flaps of tissue (leaflets) that open and close. This allows blood to flow out to the rest of the body, and prevents it from flowing back into the heart (regurgitation). The valve printed here was 28 mm which is within human range, and was tri-leaflet meaning it contained 3 flaps.

This is an important experiment because heart valves experience extreme forces in the body. If the overall goal for the authors is to 3D print full organs, they need to verify that their collagen can withstand these forces.

The authors 3D printed a left ventricle, the bottom left chamber of the heart that pumps blood out to the body. They printed collagen gel for structure, and also used a cellular ink containing human embryonic stem cell-derived cardiomyocytes (hESC-CMs) which are the contracting cells in the heart.

The ventricle was designed as an ellipsoidal shell with an outer and inner wall printed from collagen gel. The hESC-CMs were printed in the space between the two walls along with 2% cardiac fibroblasts, cells that produce connective tissue.

Two types of collagen disks were created: i) solid disks from a mold, and ii) porous disks printed by FRESH v2.0.

These disks were implanted under the skin of live rats to observe cell movement into the porous matrix as a first step towards formation of a network of blood vessels.

After 3 and 7 days, the disks were removed and tested for this result.

In the second version of FRESH, the authors wanted to improve the gelatin microparticles used in the support bath. To do this, they used coacervation which is a chemical method of producing polymer droplets.

The particles produced with this method were smaller, more spherical, and had less size variation. Additionally, the method allowed the elasticity and strength of the particles to be varied.

As stated previously, the FRESH method uses a supporting gel that contains tiny particles to support the bio-inks (such as collagen-containing gel) as they are printed. The supporting gel is thermoreversible which means its properties can be changed with heat.

The authors 3D printed a model of a small coronary artery with type 1 collagen (1.4 mm inner diameter, ~300-micron thick wall). C2Cl2 cells in a collagen gel were then printed around the tube to test its ability to support tissue.

Over 5 days, one tube was left static (no perfusion) and the other underwent active perfusion in which the cells were fed.

The caged arms fracture force was significantly higher than the V-shaped arms. The three-point bend test shows that it takes a higher force to break the caged arms than it takes to break the V- shaped arms.

The arms were made out of a thermoplastic polymer which is a substance that can be heated to become pliable, and upon cooling, it hardens.

Different forces were applied to the material. The physical reactions to those forces were noted in order to determine the mechanical properties of this material.

Examples of Mechanical Properties: strength, toughness, brittleness, etc..

The polymer making up the outer sleeve is for structural support of the dosage form.

Picogram is a unit of measurement of weight and it is equivalent to one-trillionth (10^-12) of a gram. A picogram analysis is done by weighing a sample at the picogram scale and then using other analytical techniques on this sample to get meaningful information.

In order to confirm the sublithospheric features, the authors characterized the structure of these diamonds using a technique called cathodoluminescence imaging. With this technique, light emitted by a sample when irradiated with electron radiation can be measured.

Measurement of isotopic content of primitive basaltic rocks has been a useful method to understand the exact chemical composition of these old rocks. Learn more in this article from Eos explaining the importance of using these techniques. https://eos.org/features/isotope-geochemists-glimpse-earths-impenetrable-interior

The carbon isotope compositions were measured using an instrument called Stable Isotopes mass spectrometer. Watch this video to get an idea about how this instrument works in a science laboratory: https://www.youtube.com/watch?v=SHbzEwMt-1s

The experiment was repeated with a much larger number of tumor cells injected. This is to ensure that the effects seen rely only on the treatment course and not on other factors.

The authors injected groups of 5 mice each with SA1N cells causing fibrocarcinoma.

A third group of mice received anti-CTLA-4 antibodies as treatment.

The authors injected mice with antibodies that bind CTLA-4.

The authors have deposited a few drops of graphene in a colloidal liquid state on a SiO₂ substrate to give it a non-uniform coverage of graphene upon evaporating the solution. Simultaneously, they also deposited a solution containing nanodiamonds to get nanodiamond particles on the SiO₂ surface.

The authors have designed and performed superlubricity experiments by sliding DLC-coated stainless steel balls against a graphene surface. However, after analyzing the initial test results, they needed to modify the design by also incorporating nanodiamonds into the system. They anticipated that the nanodiamonds can act as nano-ball bearings, thereby enhancing the mechanical strength of graphene and contributing to superlubricity.

Molecular dynamics is a computer simulation method that allows for prediction of the time evolution of a system of interacting particles such as atoms and molecules.

The authors hypothesized that because the ZI to PVT projection promotes intake of foods that are pleasurable to eat and also makes mice overcome their aversion to light in order to eat that food, that stimulation of this pathway is pleasurable or rewarding for the mice.

They tested this by placing the animals in a box with two identical compartments. The mice were able to freely move around the box. On one side of the chamber the mice received stimulation of their ZI-PVT neurons, whereas the stimulation was turned off when the mice were on the other side.

A technique that uses light to control the activity of cells, most commonly neurons, in living animals. The cells are genetically modified to express ion channels that are sensitive to light. Shining light on the neurons changes their activity, allowing scientists to understand the role of the neuron in a given behavior or physiological process.

Infection of the neurons with tdTomato-tagged AAV allows the projection of the ZI GABA axons to be visualized.

The authors used this method to determine where in the brain these neurons project to.

To target a neuron population of interest, e.g. those that express GABA, scientists use genetically modified viruses (AAVs) to deliver proteins into the brain (such as optogenetic tools).

This is achieved by using two tools: 1) a mouse line that expressed the enzyme Cre recombinase in a specific population of neurons (e.g. those that express the GABA transporter VGAT) and 2) an AAV that expresses an optogenetic protein only in the presence of Cre. The AAV is injected into the brain region of interest in the Cre mice. This AAV has a tdTomato tag which allows the injection site to be visualized under a fluorescent microscope.

For further information on these tools see how mice optogenetics are used this video.

The ZI in both hemispheres of the brain was injected with the AAV (bilaterally), with the region lying towards the front of the brain (rostral) being targeted. The optogenetic tool used (ChIEF) activates neurons when blue light is shone on the cells.

To study glucose and fructose metabolism in tumors the they traced the breakdown of the molecules.

1. The scientists labeled glucose and fructose with a radioactive atom that can be traced even as the molecule is broken down.
2. They incubated tumor tissues with labeled-glucose, labeled-fructose or a mix of labeled-glucose + fructose, or a mix of labeled-fructose + glucose. These tumor tissues absorb the sugars and metabolize them. Note: Adding a mixture of sugars to the tumor allows the scientists to determine how the metabolic pathways are related.
3. The different components after metabolism are then determined in lab to trace the metabolic pathway of how tumors break down sugars.

In order to investigate tumor metabolism in human tissues scientists used a tissue microarray where tiny samples of human tumors or tissues were cultured and studied. They compared metabolism between 25 different tumors, of varying severity, to normal human intestinal cells.

The authors wanted to test whether tumors near the end (distal) of the intestines or in the colon consume fructose since it can be found in much higher concentrations in the colon than glucose can. Approach: They marked glucose and fructose molecules with C14 (radioactive carbon) which can be traced as the sugars get broken down and metabolized. This way if they find C14 from fructose and glucose in a tumor they can conclude that it metabolizes both sugars.

The scientists repeated experiments from previous work to validate their methods. They fed mice (oral bolus) high fructose corn syrup and then 30 minutes later (to allow for digestion) measured fructose levels in the colon. Similar to previous studies, they found elevated fructose in the colon suggesting the passive transporters in the intestine were saturated and allowed fructose to pass through undigested.

Here they did a two-part experiment: 1) They tested whether high fructose corn syrup itself induced metabolic dysfunction by giving mice a limited about of high fructose corn syrup so that they did not become obese.

2) To test the effects of high fructose corn syrup on colorectal tumor formation and growth the authors compared tumor characteristics between mice fed with different amounts of high fructose corn syrup. First, they treated all mice with tamoxifen to activate tumor formation. Then they broke them down into three groups: HFCS- mice that were treated with a limited amount of high fructose corn syrup to prevent obesity, WB- mice that had high fructose corn syrup mixed in with water so they consume a lot of it, and Con- a control group with no high fructose corn syrup administered. They then compared the formation of tumors and their characteristics to look at the effects of high fructose corn syrup.

The scientists were first interested in looking at how high fructose corn syrup affects an entire mouse, and compare the effects on normal mice and their genetically modified mouse (APC -/-). They did this by mixing high fructose corn syrup into their water and allowing them to drink as much as they wanted (ad libitum). They monitored the mice's weight over time.

The scientists need a mouse model that will develop intestinal tumors so that they can study the effects of sugar-sweetened beverages on the tumor. They manipulated the mouse genes so that after injecting a drug (Tamoxifen) a gene in the intestine is deleted and tumors begin to form. With this genetically engineered mouse the scientists can induce tumor formation of the mouse, then track tumor size and metabolism to look at the effects of high fructose corn syrup in a diet.

The sodium influx caused by veratridine was cancelled out by tetrodotoxin exposure. Tetrodotoxin blocks the influx of sodium ions thereby stopping depolarization. Hence there is no increase in the activity of substance P in the presence of both drugs.

Used to compare average means of two or more samples.

Here, the authors used this test to measure the activity of substance P across the following groups: (1) control, (2) in the presence of tetrodotoxin, (3) in the presence of veratridine, and (4) in the presence of tetrodotoxin and veratridine.

Read more at Khan Academy.

These are explants obtained from nucleus locus ceruleus. These explants are placed in a nutrient medium and no drugs are provided to this group. This group serve as a comparison group to the groups treated with the drugs.

The last step in the dot blot to detect the materials of interest using a radioactive probe. Here, the radioactive probes tagged to proenkephalin were observed for three time points: 0 days, 1 day, and 3 days.

Medullary explants were obtained from adult rats to understand the mechanism behind the different transmitter expression. 

The tyrosine hydroxylase (TH) activity stayed consistent throughout the seven-day period. TH enzyme is responsible for the synthesis of catecholamines. The consistent TH activity indicates that there is no change related to the expression of catecholamines in this experiment. 

Next, the PNMT enzyme is responsible for adrenergic expression. PMNT exhibited a 60% decrease in four hours and was maintained at that level for the remainder time period. 

The [leu]enkephalin (opiate expression) showed a 50-fold increase in four days and continued to increase until seven days. There is a continuous increase in the expression of opiates as opposed to catecholamines.

Technique used to mark the target of interest using an antibody-based test. In this study, tyrosine hydroxylase and dopamine β-hydroxylase are the target molecules of interest. The antibodies against tyrosine hydroxylase—namely dopamine β-hydroxylase—are used to detect the molecule of interest.

Explants were depolarized with veratridine, leading to a decrease in the levels of substance P.

The ganglia was removed from the animal and transferred to a nutrient medium. These explants were maintained in the medium for six months to one year. At several time points, the explants were taken and observed for the activity of substance P.

The neurons are dissected from the animal and grown in a dish. The dish contains supplemental factors and a medium that mimics the composition of the fluid inside the animal.

Neurons are separated from the animal through mechanical or enzymatic disruption. The separated neurons are transferred to a dish or culture plate. The neurons are maintained in the dish.

In order to see if they were accidentally capturing more than one nuclei at a time, the authors mixed nuclei from mouse and human samples prior to running snRNA-seq. If they saw mouse RNA mixed with human RNA, this meant there was a multiplet (or, more than one nuclei was captured).

However, they found that there was very low rates of multiplets, meaning that their experiment is working well.

The authors wanted to see if particular cell types have different gene expression in autistic brains. They also examined two different brain areas to see if there's regional differences.

The authors calculated the total number of genes expressed in the single-nuclei data of controls and ASD patients and found it was about the same. The median gene number is lower than the transcript number because a gene can have multiple transcript forms (called isoforms).

The 10X Genomic system is a platform that isolates single nuclei and isolates "libraries" (a collection of RNA fragments which can be used to identify particular RNAs) from each nuclei.

The authors wanted to make sure that any effects they were seeing were specific to ASD, and not epilepsy, so they included patients with epilepsy alone as an additional control.

This means that the control and ASD subjects didn't differ in age, sex, or RNA quality. This is important because results can be biased by uncontrolled factors (e.g., what if there's more females in one group, and the effect you're seeing is really due to sex?)

Mann-Whitney U test is a statistical test that they used to show that there's no signifiant differences between the controls and ASD subjects.

Veratridine is a drug that causes an increase in the sodium influx. The authors used the drug to cause depolarization.

Fluorescent markers are used to label catecholamine in the neurons and visualized using a fluorescent microscopy.

A technique used to measure the movement or locomotor activity of the animal assessed in the open field box. It is thought that with repeated administration of the drug, the animals can show an increase in locomotor activity, which is a sign of sensitization.

Photobeams are placed on the walls of the box to record the movements of the animal. The mice can explore and get used to the test area of the open field.

The animal is tracked for the distance covered in centimeters using an automated video system. The experiment is repeated on day 1 and on each day following the injection of cocaine (on days 8, 9, 10, 11). The total distance covered by the animal is recorded for each day.

Watch the technique here at: https://www.jove.com/video/53107/assessment-cocaine-induced-behavioral-sensitization-conditioned-place

A three-chamber apparatus is constructed with access to two chambers for the animal for this experiment. There are 3 phases to this experiment: pre-conditioning, conditioning, and testing.

Pre-conditioning: Animal can freely move on any side of the chamber. For each animal, the first preference of the chamber is noted. This was done on days 7 and 8 of the experimental protocol.

Conditioning: Train the animals to saline on its preferred chamber and to cocaine on the least preferred side. This was done on days 9, 10, and 11 of the experimental protocol.

Testing: On day 12 of the experiment, the animals are allowed to have access to both sides of the chamber for 30 minutes. The time spent in the preferred chamber and the less preferred chamber is recorded.

View the video to learn more about the conditioned place preference protocol. The protocol is describing how to measure craving in animals using morphine as the drug of preference. https://www.jove.com/video/58384/a-conditioned-place-preference-protocol-for-measuring-incubation

Slice electrophysiology is a technique that is widely used to study synaptic plasticity. Brain slices containing the nucleus accumbens region was obtained from the mice.

For this technique, stimulating and recording electrodes are needed. Recording electrodes measure the electrical activity of the neurons in the area. Stimulating electrodes are used to stimulate a dendrite (s) in the brain region that can elicit a response which can be recorded via the recording electrode. The stimulus is given at a rate of 1 per minute.

The stimulating electrode was placed in the nucleus accumbens, and the recording electrode was placed near to the stimulating electrodes.

The amplitude or the size of the response can be calculated from each stimulus. Baseline values were obtained. After that, an LTP stimulus was given, and the post-LTP data was collected. The data were normalized to baseline values.

Watch the video here on LTP is done in hippocampus, a brain region involved in memory: https://www.jove.com/video/2330/preparation-acute-hippocampal-slices-from-rats-transgenic-mice-for

The neurons are activated by a high frequency of 100Hz. The protocol used here: 4 trains of 100 Hz tetanus 3 mins apart and are represented below:

Chromatin immunoprecipitation technique is used to measure FosB expression.

Briefly, the brain tissue was fixed with formaldehyde to crosslink the DNA binding proteins. The DNA was sheared into small fragments, some of which contains the DNA binding proteins. Using specific antibodies (H4, H3), the DNA binding protein complex was isolated. The proteins are digested, and the DNA is released. The specific DNA sequences of interest were amplified to see if they precipitated with the antibody.

Watch the video here: https://www.jove.com/science-education/5551/chromatin-immunoprecipitation

mRNA was isolated from the brain tissue using the Trizol reagent. RNA was later reverse transcribed to cDNA or complementary DNA using primers of interest. The fold difference of mRNA over control values is calculated and compared across the groups.

Check the video here on the technique: https://www.youtube.com/watch?v=0MJIbrS4fbQ

Protein was extracted from the tissue. Proteins are separated based on molecular weights in a SDS-Page gel. The gel was transferred to nitrocellulose membrane.

The antibodies to H3 and H4 were applied to the membrane to detect the bands of interest.

Watch the technique here: https://www.jove.com/science-education/5065/the-western-blot

The drug was administered directly to the nucleus accumbens of the mice.

In order to do so, the coordinates of the nucleus accumbens are obtained from the mouse brain atlas. The mouse is placed in a stereotaxic chamber, and the cannula was inserted into the brain to inject the drug every day for 7 days. The cannula was guided to be inserted into the brain using the coordinates

The nuclear fractions are obtained from the mice using a nuclear extraction kit. HDAC activity was measured using the kit.

The mice receive 7 days of nicotine or water, and then the animals are weaned off the drug for 14 days. Cocaine was administered to animals after day 14 of treatment.

What is the duration of nicotine exposure that is needed to obtain the priming response we see in these animals?

Does nicotine need to be given closer to another drug or separated a few days apart?

Next, the authors tested the idea of histone acetylation by using a low dosage of theophylline, an HDAC stimulator. In contrast to SAHA, the theophylline should decrease the response to cocaine.

If nicotine is inhibiting HDAC activity, then by using an HDAC inhibitor, we should be able to mimic the effects of nicotine on LTP and FosB expression. This hypothesis was tested by using SAHA, an HDAC inhibitor.

To confirm that, histone deacetylase activity (HDAC) was measured in the striatum.

The authors next addressed whether the acetylation of residues is due to an increase in activation of acetylases or due to inhibition of deacetylase.

The authors observed the acetylation levels of H3 and H4 after 7 days of nicotine treatment in striatum tissue using chromatin immunoprecipitation and immunoblotting.

The authors asked the question: does nicotine increase FosB expression by altering the chromatin structure at FosB promoter?

The animals were given cocaine in drinking water for 7 days. Later, the mice were administered nicotine for 4 days. FosB mRNA levels were measured.

These experiments were performed to determine if the nicotine pretreatment combined with cocaine injection produces similar results to cocaine pretreatment combined with nicotine injection.

Nicotine was added to the drinking water for the mice.

7 days treatment: The mice were fed with the nicotine-containing water for 7 days. For the next 4 days, mice received a cocaine injection intraperitoneally (injection into a body cavity) with continuous exposure to nicotine-containing water. The injections were given once per day.

24 hours of treatment. The mice were exposed to the nicotine-containing water for 24 hours, and the next 4 days; the mice received a cocaine injection (once per day) intraperitoneally with continuous exposure to nicotine-containing water.

The authors predicted that the effect of the values affirmation intervention would be greater for women who more strongly endorse the gender stereotypes.

The authors' main predication was that women who completed values affirmation would have a smaller gender gap than women who did not complete values affirmation.

The authors tested whether using a values affirmation intervention in their college physics course could reduce the performance gap between men and women.

The key variable in this experiment is whether a student experiences the values affirmation intervention.

In the values affirmation intervention, students briefly write about a value they find personally important.

The authors compared the similarities and differences between species' interactions across different locations around the island, taking into account the unique environments of each site.

The authors hypothesize that because stimulation of the ZI to PVT pathway evokes a large increase in food intake in a short amount of time, long-term stimulation should lead to weight gain.

The authors followed an optogenetic protocol by intermittently turning on the stimulation light for 10 seconds followed by 30 seconds of no stimulation. With each 10 seconds of light on, they measured how long it took for the mice to begin eating.

The authors bred two different mouse lines together: one parent expressed Cre in VGAT-positive neurons and the other parent expressed a protein that emits green fluorescence (GFP) in vGlut2-positive neurons.

The researchers than used the offspring of this cross to record from GFP-positive cells in a slice and ask whether VGAT cells in the ZI provide input to these neurons.

The authors recorded the activity of the ChIEF-expressing neurons in brain slices using electrodes. Stimulating the slice with blue light activated the ChIEF-expressing neurons, causing them to fire in the same pattern with which they were stimulated (i.e. high fidelity). Hz (hertz) refers to the number of times the light flashes per second, i.e. 20Hz corresponds to 20 flashes of light per second which caused the neurons to fire 20 times per second.

This virtual lab demonstrates electrophysiological recordings of neurons: Neurophysiology Virtual Lab

The authors identified the neurons that lie upstream and provide input to PVT neurons.

They targeted excitatory PVT neurons using vGluT2-Cre mice and used a modified rabies virus that traffics into neurons that provide input to the starting population of cells.

Mice were placed in a chamber with two compartments—one with no lights and one brightly illuminated. Mice are innately averse to light and so will usually spend more time in the unlit compartment.

The authors gave the mice a small amount of food to eat overnight, which meant that they were hungry during the experiment. Therefore, control mice commenced eating with short latency at at the onset of the stimulation protocol.

The authors assessed the role of the neurons downstream (postsynaptically) of the ZI neurons that project to the PVT. They examined how food intake was affected when PVT excitatory neurons were optogentically stimulated.

Given that GABA is an inhibitory neurotransmitter, the PVT neurons would normally be inhibited when the ZI to PVT projection is active. Thus, when the PVT neurons are stimulated, food intake should increase. Indeed, this is what the authors found.

The authors selectively killed ZI GABA neurons by using an AAV to express a caspase in these neurons. Caspase-3 is an enzyme that induces cell death.

Silencing of neurons in the PVT that receive input from ZI GABAergic neurons should increase food intake given that these neurons are inhibited by ZI GABA neurons, which increase food intake.

The authors used a chemogenetic approach in which a modified (DREADD) receptor is expressed in the neurons using AAVs. The receptor is activated specifically by a synthetic drug (CNO) that has no other biological effect.

The authors used this approach over an optogenetic method to silence the neurons as currently available optogenetic tools for inhibition are not very efficient.

To confirm that the caspase virus was killing cells, a tdTomato reporter, which makes the cells red under a fluorescent microscope, was injected at the same time as the caspase virus.

The authors found that few tdTomato cells were present in mice that also received the caspase, compared to control mice that were injected with the tdTomato only. Thus, the caspase virus efficiently killed the PVT neurons.

In other words, how many bases in a row translate into one amino acid?

Let's do a thought experiment (which is considerably cheaper than a laboratory experiment):

Assume that each amino acid is coded for by two bases in a row. The code would have one of four different bases in the first position of the code (A, G, C, T) and one of four different bases for the second. How many combinations of pairs would be possible?

For example: (1) A A (2) A G (3) A C (4) A T (5) G A (6) G G (7) G C (8) G T …

If you continued to write out every combination, you would come up with 16 possible pairs of bases. However, that's four short of the 20 natural amino acids. This is a good sign that two bases is not enough to code for all possible amino acids (and, in fact, we now know that it takes three bases in a row).

How many combinations would be possible if the code were a grouping of three bases?

This "frame shift" experiment tests whether the bases are read in singlets, pairs, or triplets.

https://ghr.nlm.nih.gov/primer/illustrations/frameshift.jpg

By incorporating acridine into genetic material, Crick and coworkers produced mutations in DNA. These mutations led to either the addition or subtraction of one base pair in the genetic code.

A secondary outcome measure was student scores on the Force and Motion Conceptual Evaluation. Because this test is administered throughout the country, the authors can compare their findings to the normal results for the population.

Students in the control group spent time writing about a value that was not important to their identity. This ensures that any difference between the values affirmation group and the control group is due to the act of self-reflection and affirmation, and not simply a result of general writing.

Level of endorsement of the stereotype that men perform better than women in physics is a moderating variable. Its value influences how much the values affirmation intervention affects course performance.

One of the key features of this study is that it takes places in an authentic college classroom, rather than being an artificial, one-time laboratory experiment.

The investigators initially had to decide that they were going to use samples from six species of finches. Then, they screened the entire genome of these species to look for genetic variants in different individuals. The objective was to see if any variation at any given location was associated with size and/or size-related traits.

The genome-wide fixation index scan found that a region around 525 kb in size showed the most striking differences.

Lamichhaney and colleagues constructed another phylogenetic tree based on the alignment of this region in all of the samples.

The nucleotide alignment of the variable positions from all 180 samples (60 birds plus 120 from previous study) allowed the scientists to generate a phylogeny using software FastTree.

See here to learn more about FastTree and its maximum-likelihood method.

Lamichhaney and colleagues sequenced a total of 60 birds. Sequencing is a technique used to actually read the DNA.

For a history of DNA sequencing and assembly, this resource from HHMI BioInteractive is a great tool.

This video shows specifically how Illumina Sequencing Technology works.

If a finch genome is 1 Gbp (one trillion base pairs), sequencing "to ~10x coverage per individual" would mean that you obtain 10 Gbp of sequencing data.

"Using 2 x 125-base pair paired-end reads" refers to the fact that the fragments sequenced were sequenced from both ends and not just one. Refer to the videos above for more information.

Genotyping establishes a genetic code for each individual finch. With birds, this can typically be done using plucked feathers, blood, or eggshell membranes.

The researchers here used blood samples that were collected on FTA paper and then stored at -80°C. FTA paper is treated to bind and protect nucleic acids from degrading.

This type of scanning is used to identify specific gene markers that are highly variable. Researchers wanted to identify a locus that showed beak size variation.

To more rigorously compare the off-target activity of two systems, the authors performed sequencing with higher coverage.

Recall that earlier they used 12.5x coverage. Here, they used 125x coverage.

Why is this important? Cellular genes are expressed at different levels which leads to a different number of individual mRNA molecules. The more abundant a particular molecule is, the easier it is to detect it at a given coverage. When you increase the coverage, you have a chance to catch molecules that are less abundant in the cell.

This is exactly what happened in the experiment with the REPAIRv1. At 125x coverage, the authors detected off-targets in the majority of transcripts (18385 of around 20000 protein-coding genes in our genome). By contrast, the REPAIRv2 system was astonishingly more specific and produced off-targets 1000 times less frequently.

Inspired by other explorations into 3' and 5' motifs, the authors looked at transcripts with off-target effects—specifically at two nucleotides surrounding the edited adenosine.

The authors looked at two characteristics of the modified protein variants.

First, they tested whether the mutant had changed its editing activity. This was calculated by looking at the restoration of the Cluc luciferase signal. While the authors didn't necessarily want increased editing activity, they wanted to avoid a loss of editing activity. 

However, catalytic activity sometimes leads to more off-target effects. Therefore, they authors calculated the ratio between the Cluc signal in targeting and non-targeting conditions, i.e. the specificity score. The higher the score, the more specific a mutant variant was.

To create the reporter, the researchers "broke" the gene for the Cluc luciferase by introducing a mutation in the UGG codon, changing it to UAG (a nonsense mutation, which signals the ribosome to stop translation). Since this codon was positioned in the beginning of the Cluc transcript, no luciferase was synthesized in the cell.

The A (adenosine) in the UAG codon was the target for RNA editing. Both Cas13b-mediated RNA recognition and ADAR-mediated editing were required to remove the stop codon at the beginning of the Cluc transcript, which would restore Cluc expression.

This means that Cluc luminescence would only be seen where editing (both targeting and cleavage) was successful.

The next question was to understand the specificity of Cas13 in the whole cellular transcriptome (the portion of the genome that's transcribed). In the previous experiments, the researchers looked at the expression of only one target (unmodified or modified). Here, they narrowed their focus from the entire genome to just the transcriptome.

To do that, the authors transfected the cells with Cas13, LwaCas13a or PspCas13b, a gRNA to target Gluc, and a plasmid containing the gene for Gluc.

The control cells got an irrelevant gRNA instead of the gRNA targeting Gluc. As an additional control for comparison, the authors used shRNA-mediated knockdown of Gluc in a parallel cell culture.

After 48 hours the researchers collected the cells, extracted mRNA, and determined the sequences and the number of copies for each transcript.

The cells were transfected with a nuclease, a gRNA for the non-mutated target site and a whole library with all possible plasmid variants. After 48 hours the researchers collected the cells, extracted RNA, and determined which mutated sequences from the library were left uncleaved.

The transcript with the unmodified sequence was depleted most efficiently so that its level was the lowest after the cleavage. The levels of all other sequences with substitutions decreased to a lesser extent or did not decrease at all. The better Cas13 cut the sequence, the higher the depletion of this sequence was.

The authors then compared the sequences by their "depletion scores."

Substitutions in the PFS motifs did not affect how well Cas13a and Cas13b found and cut the target sequences. As a result, sequences with such substitutions were depleted as successfully as the control sequence, which was unmodified.

To figure out which parts of the Gluc or Cluc RNA molecules were the best targets for Cas13, the authors generated a series of gRNA guides where each guide was shifted one to several nucleotides relative to the previous one. This is called tiling.

In this way, the guides together could cover the whole sequence or a part of a sequence that a researcher was interested in. See figure 4A and 4C or figure 5A for a visual of how the guides were "tiled."

The authors took six of the best-performing orthologs from the previous part of the study and replaced the msfGFP domain at the C-terminus of each ortholog with different localization sequences. They then tested Gluc knockdown in the same way they previously tested LwaCas13a.

To directly compare the effectiveness of Cas13a, b, and c orthologs, the authors transfected cells with two luciferases, Cas13 and two different gRNAs targeting Gluc luciferase.

They measured Gluc luciferase activity. Reduced Gluc luciferase activity indicated interference from the Cas13 ortholog and successful targeting by the gRNA.

They determined the expression of Cas13 to see whether Gluc knockdown was dependent on the quantity of Cas13 rather than the specific orthology.

In this article, the authors describe how they created a system that can edit RNA molecules. They used a Cas13 protein fused to an adenine deaminase. The Cas13 protein recognized specific sequences on an RNA molecule, and the adenine deaminase edited bases, which can convert A to I (which is functionally read as a G).

The authors improved the targeting specificity (accuracy) and editing rate (precision) by Cas13 and deaminase mutagenesis, and determined the sequences for which this system is most effective. They showed one application of this technology by correcting a series of disease-causing mutations at the cellular level.

Authors have conducted theoretical simulation to investigate the sliding behavior of an ensemble of graphene sheets to elucidate the macroscale scrolling phenomena. To explore the mesoscopic friction behavior, number density (number of particles per unit volume) of the patches as a function of the coefficient of friction and time is calculated by grouping the friction coefficients collected over the ensemble of graphene patches.

To understand the transition of friction from the nanoscale to the macroscopic superlubric condition observed in experiments, authors have simulated a mesoscopic scenario. They have created and analyzed an ensemble (assembly of systems) of graphene patches and nanodiamonds between the DLC and graphene substrate subjected to sliding friction.

To understand the role of defects in superlubricity, the authors performed computer simulations by introducing double vacancies and Stone-Wales defects on graphene sheets. Studies were conducted in both dry and humid environments.

Upon observing experimentally the effect of humidity on friction conditions, authors have extended their studies. They have performed computer simulations to further analyze the interaction between water molecules and graphene in the DLC-nanodiamond-graphene system in a humid environment.

Since this paper was published, the authors refined and optimized the devise and tested it under desert conditions with record high efficiency.

See "Related Content" tab for: Kim, Hyunho, et al. "Adsorption-based atmospheric water harvesting device for arid climates." Nature communications 9.1 (2018): 1191.

To test the material in a laboratory setup, the authors use an enclosed chamber in which conditions such as humidity, temperature, and solar illumination can be regulated. This guarantees control over the experimental conditions and reproducibility.

Heating under reduced pressure lowers the boiling point of liquids. This allows all the solvent and water molecules trapped in the MOF to evaporate easily, emptying all the cavities before starting the experiment.

In order to understand the wear properties of the graphene-nanodiamond compound after the sliding experiments, the authors performed electron microscopy studies which can reveal the structure of the material in the wear debris.

Raman spectroscopy is a chemical analysis technique capable of probing the chemical structure, crystallinity, and molecular interactions of materials.

The authors defined contact area as the area of graphene atoms which are in the range of chemical interactions from the DLC tip atoms. The normalized contact area is defined as the contact area at any time (t) with respect to the initial contact area at time t=0 (when the graphene patches are fully expanded).

In order to elucidate the mechanism of graphene nanoscroll formation and the origin of superlubric state, the authors have conducted computer simulation studies.

To investigate the effect of environmental conditions on nanoscale friction and superlubricity, the authors have conducted experiments in humid air in place of dry nitrogen.

For example, if a temperature profile was taken on Feb. 19th, for each depth in that profile the authors subtracted out the average value for all Februaries over the last 50 years for each depth point. This removes the seasonal temperature changes from the data-set, allowing the authors to focus on the long term variability instead.

By examining the ocean in distinct depth increments of 500m each, the authors aimed to determine where most of the cooling and heating of the North Atlantic is occurring.

The authors calculated how much heat is being stored in the deep sea by looking at the cumulative temperature change from 1955-59 to 1970-74, as well as from 1970-74 to 1988-92.

By integrating, or combining, the temperature data for all depths between 0 and 300m and between 0 and 3000 m, the authors aimed to examine where the heat is going - into the surface ocean or the deep ocean.

The authors combine 5 years of data into one value, typically by averaging all values. A running composite (sometimes known as a moving or running average) is calculated by creating a series of averages of different subsets of the full data set in order to compare to the original data set.

Calculating a running composite is a common technique used with time series data in order to smooth out short-term fluctuations and highlight longer-term trends or cycles.

For more information on how to calculate a moving average: http://www.statisticshowto.com/moving-average/

The authors calculated temperature anomalies by subtracting the seasonal temperature cycle from monthly data values.

Every now and then a shipboard temperature measurement can malfunction or be used incorrectly, recording temperatures far higher or far lower than what is realistic. These values need to be excluded to accurately study the oceans. To make sure these errors do not affect the study, the authors considered a particular range of data points with cutoffs at the higher and lower end of the range.

Unlike their previous work, the authors used a less strict cutoff for when data values were considered good enough to use in their analysis. This is because they found that some large-scale temperature features were mistakenly being flagged as "bad" data under the stricter cutoff, despite those features being real and measurable events in the ocean.

Using data available in the World Ocean Database, Levitus et al. looked at both annual temperature data and average season temperatures within each year (for winter, spring, summer, fall).

However, because temperature changes over the course of a year due to the changing of the seasons (summers are warm, winters are cold), this seasonality must be taken into account when studying the overall change in ocean temperatures. To "objectively analyze" the data, the natural seasonal temperature cycle was subtracted from each data point in order to focus on the trends over time.

For each monthly temperature data point, the average temperature for that month the sample was measured was subtracted. The difference between the data point and the monthly average is called an anomaly.

The authors averaged monthly oceanographic data acquired by ship-of-opportunity and research vessels into annual temperature measurements for every year from 1960 to 1990.

Scientists measure locations on Earth using longitude (180° E ↔ 180° W) and latitude (90° N ↔ 90° S).  Lines of longitude and latitude cross to create a grid across the planet.

For this study, Levitus et al. combined temperature data for every 1° longitude by 1° latitude area of the ocean. Where multiple ships frequented the same location, those multiple data points were averaged into one value for each matching depth.

Refers to a statistical method that searches for multivariate relationships between two data sets.

This method is most often used in genetics and ecological sciences. Learn more about why, how, and when to use it here.

Though the authors had already characterized PFS preferences for Cas13b, they needed to check that the fusion of Cas13b with ADAR did not change its targeting efficiency and specificity. The researchers also wanted to confirm that PFS would work when generating RNA edits. This is important as DNA base editors are limited by the PAM of Cas9 and Cpf1 making RNA more powerful since you can target anywhere in the transcriptome. Therefore, it was important to check PFS constraints again.

The researchers mutated amino acids in ADAR involved with binding to the RNA target or catalytic deamination to test whether they affected deamination.

The authors measured genetic diversity through the inbreeding coefficient and average nucleotide diversity, finding that the pattern of decreasing genetic diversity was as expected for an inbreeding population.

The authors measure allele frequencies to see if they are changing, which is an indication of continuing natural selection.

The authors analyzed body size and several components of bill size to give a more complete comparison of the Big Bird lineage to both of its parental species. Results of these analyses are found in Figure 3.

Based upon previous findings that the cortex and STN are connected, the investigators wanted to know if driving M1 layer V neurons had an effect on STN neuronal firing and subsequent behavioral output. So they placed an optrode over M1 and a recording electrode in the STN.

Thy1 (thymocyte differentiation antigen 1) is expressed in the axonal projections of mature neurons. When its promoter is placed in control over ChR2 expression, the protein would be expressed in the projection neurons as opposed to the somata of local neurons.

Since the subthalamic nucleus is excitatory, meaning the neurons within release the neurotransmitter glutamate, selectively targeting this region can be accomplished via the promoter calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Placing a gene downstream of the CaMKIIα promoter will cause the gene to be selectively expressed only in excitatory neurons.

The authors placed the gene sequence for halorhodopsin under the control of the CaMKIIα promoter and were able to selectively inhibit the firing of excitatory glutamatergic neurons in the subthalamic nucleus.

A quantitative method to detect mRNA levels in the explants. Here, mRNA levels of proenkephalin is measured using this method.

Learn more with this video from Abnova.

A technique used to separate or eliminate a particular nerve supply to specific area(s) in the nervous system.

The authors examined different parameters to optimize the water harvesting properties of the material. The porosity and thickness of the material are assumed to be the parameters having the largest effect because they determine the amount of water that can be adsorbed for a chosen compacted adsorbent layer.

Refers to a computational technique to model the probability of microbial metagenomics data by representing the data as a frequency matrix of the number of times each taxa is observed in a sample.

The researchers wanted to ensure that the phenotypes of interest were not falsely correlated with microbiota genera. To avoid overfitting, they reduced the phenotypic data in different stages. By approaching the problem in a series of distinct stages, the researchers could determine the most important variables that affect the abundance and diversity of gut microbiota.

n the final experiment, the researchers presented participants with a scenario in which an AV would have to sacrifice passengers to minimize overall casualties. They were then asked whether governments should regulate autonomous vehicles, so that they are programmed to minimize casualties; whether they would buy a vehicle that was subject to those regulations; and whether they would buy a vehicle that was not regulated in that way.

In Study 4, participants were given a "budget" of 100 points to assign to different algorithms, which was a way for researchers to look at their priorities.

For example, if there were three algorithms, a participant could choose to allocate 20 points to the first one, 30 points to the second, and 50 to the third. This allowed the authors to directly compare how participants felt about the different algorithms presented in Study 4.

There were three algorithms, and participants had a separate budget for each in which they answered the questions:

1. How moral is this algorithm relative to the others?
2. If self-driving cars were programmed with this particular algorithm over the others, how comfortable would you be with it?
3. How likely would you buy a self-driving car programmed with this algorithm?

Participants read scenarios in which the self-driving vehicle would sacrifice its single passenger to save pedestrians, with the number of pedestrians ranging from one to 100.

The participants were asked which situation (saving the passenger versus some number of pedestrians) would be the most moral choice.

With the last question in each study, the authors were checking if the participant was still paying attention. If the participant got the question wrong, their answers were discarded because the authors assumed that the participant was not actively engaged.

Attention checks can help make sure that the data you are collecting is higher quality. For example, participants could start speeding through the survey without thinking through their answers, which results in data that is not informative or useful.

Amazon Mechanical Turk (MTurk) is a website that lets users earn money for doing small tasks. It has become increasingly popular for researchers, who use it to reach a wide audience online.

With MTurk, a large and diverse set of data can be collected in a short period of time.

The scenarios presented in the study always assumed that someone would be killed by the autonomous vehicle. The authors did not look into situations where the outcome was uncertain, like if the passenger had a greater chance of survival than a pedestrian.

In the final two studies, the authors wanted to discover if people approve of governments legally enforcing utilitarian algorithms in AVs, and whether people would want to buy AVs if these regulations existed.

Regression analyses allow you to understand how different things may or may not be related. For example, how does the number of views for a YouTube video change based on the video's content?

In this study, the authors used regression to investigate how factors like gender, age, and religion might be related to someone's enthusiasm about self-driving cars.

This is important for finding potential covariates in the experiment. Covariates are characteristics of the people in an experiment, like age, gender, and income. These variables cannot be controlled like experimental ones, but they can affect the outcome of an experiment and bias your results in an unexpected way. They can also reveal interesting trends in society.

By determining the covariates that could sway the experiment, scientists can make the model more accurate. Here, the authors determined that the experiments should account for age and gender.

Even though the situations where AVs would sacrifice their passengers are rare, these are emotional events that would have a large influence on the public despite their infrequency.

In Fig. 1C, the car must decide between killing its own passenger or several pedestrians. A utilitarian viewpoint, which calls for the choice resulting in the "most good," would be to sacrifice one (the passenger) to save many.

However, people who prioritize their own safety will not want to buy a car that's programmed this way because the result could be that their car would sacrifice them.

The authors used bill size and body mass (morphological measurements) and genetic data.

The genetic data used was the entire DNA sequence for each bird (whole genome sequencing) for sections to analyze, not just particular sections of DNA, as had been done in the past.

The phylogenetic tree analysis consists of scanning the genomes of all 46 members of the Big Bird lineage and comparing them to the genomes of 180 other finches from Daphne Major for which there are genome data.

A computer program is used to generate many phylogenetic trees, and mathematical parameters are used to ensure that the data are properly interpreted in the program. Then, a statistical test is used to evaluate the reliability of each split in the tree.

This sentence is saying that the higher number of insects you see during the growing season and the lower numbers seen throughout the rest of the year is because of the relationships between insects and temperature, where warmer temperatures result in greater insect populations. This pattern, fewer insects during colder months and more insects during warmer months, is called diapause and means that insects, and also some other animals, go into a dormancy until environmental conditions are more favorable for survival.

Throughout this paper, the researchers have been making the claim that activity of the NPF system signals the fly's "reward state." According to this theory, when the fly experiences something rewarding (such as mating), the amount of NPF in the brain and the activity of associated neurons increases, which in turn leads to a reduced urge to seek additional rewards (such as alcohol). To this end, they have shown that there is an increased amount of NPF in the flies' brains following mating. However, if their theory were true, then the opposite should also be observed. That is, the consumption of alcohol should also increase NPF levels in the brain, since alcohol is also a source of reward (just like mating).

To test this hypothesis, the researchers decided to measure NPF levels after exposure to both air and an alcohol vapor (the former being neutral/not rewarding, and the latter being rewarding). If their theory about NPF generally signalling reward were true, one would expect to see increased levels of NPF in the brain following exposure to alcohol (just like was observed following mating in Figure 2A).

You might be wondering why this step is necessary, especially for alcohol, since the researchers have already shown that flies consume more alcohol-laden food. However, in the alcohol/food consumption paradigm, it is impossible to completely dissociate the contribution of the hunger and the caloric contribution of alcohol from the objective enjoyment of the experience. Here, what is being tested for is the preference for odor (which is otherwise neutral) that has been paired in memory with the experience of alcohol consumption, independent of any confounding factors. Further, the actual test phase occurs 24 hours after the alcohol exposure, when the flies are fully sober. For these reasons, this type of odor-paired conditioning assay is a much better indicator of whether an experience of substance is objectively rewarding for a fly.

Here, the researchers put forth their theory for the mechanism driving all of the effects they have observed so far. This mechanism is based on the concept of homeostasis, which basically states that there is an "ideal state" for one's internal physiological make-up. Any time the body deviates from the ideal state, it will attempt to correct itself and return to this state. The researchers thus theorize that the amount of NPF in the brain is a marker for the "reward state."

If the fly mates repeatedly, it experiences something highly rewarding, leading to an increase in NPF beyond "normal" levels. The fly's body thus wants to return to a normal level of NPF. To do this, it must forgo alternate rewards such as alcohol. This would explain why mated flies do not have a preference for alcohol. Sexually deprived flies, on the other hand, experience a lack of reward, which leads to a decrease in NPF beyond normal levels. To compensate for this, when these flies are exposed to a second possible reward in the form of alcohol, they consume it excessively. This would explain why sexually deprived, virgin flies show an alcohol preference.

Since past work in Drosophila, mice, and C. elegans has shown that altering NPF/NPY can affect how organisms react to alcohol, the researchers in this study hypothesized that sexual deprivation might lead to changes in levels of NPF in the flies' brains, which in turn caused them to consume more alcohol. To test this, they extracted the brains from male flies with three distinct sexual histories (mated, virgin, and sexually rejected), and measured their NPF levels.

In order to further confirm this causal relationship, the authors used the protein dTRPA1 (which activates specific neurons at a high temperature). Specifically, they inserted dTRPA1 into all of the same neurons that also contained NPF. Therefore, at a low temperature there would be no effect, but at a high temperature all of the neurons related to NPF would be activated. If the relationship between NPF and alcohol preference were indeed causal, then artificially increasing the activity of NPF-related neurons should reduce preference for alcohol.

Since cis-vaccenyl acetate (cVA) is used by females to turn away unwanted mating attempts by males, it is only released by females who have already mated. Virgin females, who may still be looking to mate, do not release this chemical cue. This difference raises the possibility that the increased preference for alcohol amongst the rejected males could be because they smelled this chemical cue (which the mated male flies would not have smelled).

To test this possibility, they exposed the virgin male flies to dead female flies. These virgin males were still exposed to the visual cue of a female fly (especially the rear half of the body where mating occurs). However, they were neither able to mate, nor were they exposed to active rejection by a female.

For the first follow-up experiment, the researchers ensured that both the mated male flies and the virgin male flies were housed in large groups (consisting exclusively of other male flies). This was done to see if social isolation (as the virgin male flies in the initial experiment might have experienced) might have been the cause for the alcohol preference.

To test the possibility that cis-vaccenyl acetate (cVA) was the driving factor behind the alcohol preference amongst rejected males, the researchers conducted a follow up experiment in which one group of males was exposed to mated females, and the other was exposed to dead virgin females. Both of these groups thus experienced sexual rejection. However, the group exposed to the dead virgin females would not be exposed to the cVA cue, whereas the group exposed to mated females would. If there were a difference in alcohol preference between these two cohorts of male flies, then one could conclude that the preference for alcohol was not caused by sexual deprivation specifically, but instead due to exposure to cVA.

To test the effect of sexual experience on alcohol consumption, the researchers first had to create two groups of male flies, each with a different sexual history. They did this by allowing one group to mate freely with multiple different females for four days. This became the "mated-grouped cohort." The second group was only exposed to female flies who had already mated. Female flies who have already mated once will not mate a second time until they have laid their eggs. For this reason, the second group of males experienced repeated sexual rejection by the mated females for four days. This became the "rejected-isolated cohort." These two cohorts, (one mated and the other sexually rejected) could then be tested for the differences between them.

A calculated score, from –1 to 1, that indicates how much the flies preferred the alcohol-spiked food relative to the regular food. A number above zero means the flies ate more food with alcohol in it, and a number below zero means they ate more regular, nonalcoholic food.

Drosophila are typically fed a jellylike substance in the lab consisting largely of sugar. In this experiment, the flies were given a choice between just a regular tube of food or a tube that contained the food mixed with alcohol.

A behavioral model in which an organism is offered a choice between two alternatives. Typically, the organism is allowed to freely choose amongst these alternatives, and its choices are used to define whether or not the organism has a preference for one alternative over another.

Different cell types specialized to performing particular functions require different proteins for their work. Therefore, they have distinct transcriptomes.

Moreover, a cell's transcriptome changes depending on where it is in the cell cycle and the conditions and needs of the surrounding environment.

As a result, off-targets of the REPAIR system may vary from one cell type to another, reflecting changes in the presence and abundance of individual transcripts.

Earlier the authors only counted off-targets and did not look at the effect of adenosine modification on the transcript function. Here, they determined the position of the modification and the possible impact on the amino acid choice if the edit was in the protein coding region.

The recovery of the Cluc signal is an easy way to get the first look at the activity of the mutants. However, a single transcript cannot reflect changes in the whole transcriptome.

It would be too expensive to test all the mutants by RNA sequencing to find whole-transcriptome off-targets. Instead, the authors looked at a single transcript for Cluc luciferase.

The authors assumed that if luciferase activity was restored even non-targeting guide RNAs, it would mean that ADAR2 increased off-target activity.

AAVs can accommodate up to 4.7kb, meaning dCas13b and the extra regulatory sequences are too big to fit in one virus. To solve this problem, the authors truncated (shortened) dCas13b by removing sections of the protein with unnecessary functions.

The dCas13b nuclease is composed of different domains (parts), some of which are used for RNA binding and some which are used for cleavage. The RNA cleavage is mediated by two well-defined HEPN domains which are located close to the N- and C-terminus of the protein. We are still unsure which domains mediate binding.

However, the REPAIRv1 system needs only the RNA binding ability of dCas13b. Therefore, the researchers were able to remove sections of dCas13b containing HEPN while retaining its binding function.

Almost the entire AAV genome can be replaced with a desired construct of max length of 4.7kb. This limits how much genetic material the virus can carry, so some essential viral genes are carried in an additional construct or constructs (the "helper plasmid" in the picture below).

Learn more about AAV at Addgene.

The researchers chose 34 disease causing mutations to test. Each mutation was a G to A substitution that changed the amino acid sequence of the protein and disrupted normal function.

The authors chose three 50-nt spacers to target two genes carrying disease-relevant mutations. As was determined at the previous step, the spacers of 50-nt length showed higher rates of editing but more off-target effects than 30-nt spacers.

After the authors had generated successful REPAIRv1-mediated editing of mRNA for an exogenous gene (i.e., a gene introduced from outside the organism), they chose two endogenous genes (i.e., native to the organism) to further explore the power of REPAIRv1.

They tested Cas13b nuclease activity in the same way as they did with the exogenous gene.

The dCas13b and ADAR2 protein parts are joined together via a stretch of amino acids called a linker. There are more than a thousand linker variants in different multi-domain proteins. The sequence of a linker can influence protein folding and stability, as well as functional properties of individual domains. Therefore, the choice of linker is an important step in protein design.

The authors tested linkers of different length and flexibility and found that shorter and more flexible variants produced the best results.

The ADAR deaminase can introduce changes not only into the target adenosine but also in the surrounding adenosine bases. To check how specific the dCas13-ADAR2 protein was in targeting the correct adenosine base, the researchers sequenced edited Cluc transcripts and determined all positions with A to I substitutions.

Further, they used tiling gRNA molecules in order to determine the influence of the spacer (PFS) length and the mismatch distance on the off-target editing.

The authors directly compared the RNA knockdown efficiency of two technologies, Cas13 cleavage and RNA interference (RNAi). Both technologies require guide RNA (gRNA) molecules for targeted recognition. Cas13 is directed by a gRNA, while RNAi complex uses a molecule termed shRNA.

Different parts of an RNA molecule can be more or less accessible to gRNAs. Therefore, gRNA and shRNA target sequences have to be selected such that they are close to each other, i.e. position-matched, so that a more fair comparison between Cas13 and RNAi can be made.

Why did the authors test several NESs from different proteins?

Over 200 NESs from different proteins have been described. Each NES is around 10 amino acids long and has a unique structure. The variation between NESs means that they have different effects on export efficiency and protein stability in different environments.

The authors hypothesized that even without nuclease activity, Cas13b would still be able to recognize target molecules directed by a gRNA.

Mutations of the key part of the protein responsible for RNA cleavage eliminated Cas13b's catalytic ability. The next step was to test whether the mutated Cas13b was still able to find the target sequences, even if it couldn't cleave them.

The sequences with substitutions in the middle part (between 12 and 26 nucleotides) had the lowest depletion scores. A lower depletion score means that the nuclease was not as successful at cleaving the RNA sequence (i.e. depleting expression).

The neurons are visualized and imaged with a fluorescent microscopy.