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).

To figure out how specifically the Cas13 orthologs would target RNA, they created a "library" (collection) of sequences with one or two mutations in the gRNA target site for the Gluc gene. The idea was to see how different a sequence could be and still be recognized by the nuclease.

Remember that the goal is to find a highly specific nuclease. This means that ideally the nuclease would not recognize sequences with mutations.

The authors ran the assay without msfGFP to determine if any of the orthologs do not require stabilization domains. This is important because orthologs that do not require these domains could be used when an experimental construct needs to be small.

Cas13a prefers to recognize target sequences when a spacer (target sequence) is surrounded by specific PFS motifs.

To determine which PFS sequences Cas13b orthologs prefer, the researchers used an ampicillin interference assay. They transformed bacterial cells with two plasmids: One contained a Cas13b ortholog while the second plasmid included an ampicillin resistance gene and a gRNA against this gene with randomly generated 5' and 3' PFS around the target sequence. If the PFS led to the Cas13b ortholog targeting the correct gene, bacterial cells would die because they would lose ampicillin resistance when the gene was cleaved.

The authors collected and analyzed cells that survived to determine which PFS sequences were irrelevant for targeting. They then subtracted these PFSs from the starting pool to determine which PFSs are preferred by Cas13b orthologs.

To monitor the effect of Cas13 nuclease activity, the researchers constructed a vector that contained genes for two different luciferases. These luciferases use different substrates (source materials) to generate light.

One luciferase was used to measure Cas13 interference, and the other was used as a control. In cells where Cas13 did not interfere, both luciferases would emit light. In cells with interference, however, activity from one of the luciferases would decrease.

The neurons are visualized and imaged with a fluorescent microscopy.

Analyzing multiple whole genomes at the same time is so complex that scientists needed to simplify the data to make it easier to see patterns and differences between the genomes. The mathematical tool that they used was PCA analysis, which reduces the complexity of the data and retains its variance.

PCA analysis works by setting principal components (e.g. PC1, PC2, PC3), which are essentially directions in which the data has the largest spread.

In this case, PC1 and PC2 are studied.

The d-loop sequences of mitochondrial DNA are considered to be mutational hotspots (places where mutations appear to happen more frequently than others), and therefore looking at this region can provide important information about how evolution occurred.

Modern dog sequences and seven of the 59 ancient d-loop sequences were already available on a public database. The rest of the sequences were generated from ancient DNA samples by polymerase chain reaction (PCR). The sequences were then compared to each other using DomeTree, a program that creates haplogroup phylogenetic trees based on mitochondrial DNA.

The team of scientists used radiocarbon dating techniques to calculate the dog as 4700-4900 years old.

Since they knew how old the Newgrange dog was, they were able to estimate the time at which the Newgrange dog and the Portuguese village dogs last had a common ancestor, and figure out when they diverged from each other.

Based on the assumption that a new generation of dogs were born every 3 years, the scientists were able to calculate the mutation rate.

The information represented in Figure 1A was used to define the two core groups. The dogs needed to have very high bootstrap values (>90)—high-quality genetic data—to support their placement in the groups.

The Western Eurasian core group consisted of all modern breeds and Portugal village dogs. The East Asian core group consisted of Sharpei, Village dogs from China, Tibet, and Vietnam, and Tibetan Mastiffs.

Three tools for mining the data for ancestry information.

Principal components analysis (PCA) simplifies the data. The number of variables is reduced but the trends and patterns are retained.

D-statistics detects admixture events; it is used to detect gene flow between closely-related species.

TreeMix is a genetics computer program used to estimate ancestral relationships. It can detect population splits and migration events.

This technique allows scientists to estimate the age of a plant or animal based on the amount of carbon-14 that is present at the time of measurement. Carbon-14, also known as radiocarbon, is a weakly radioactive type of carbon molecule that decays over time. The age of samples up to 60,000 years old can be estimated.

To learn more about radiocarbon dating, check out this video from Scientific American.

A type of genetic test that covers 170,000 single-position variations in the genome of a dog. It is essentially a chip, upon which the genetic material of the dog of interest is placed. It contains thousands (170,000 in this case) of probes—short DNA sequences that can stick to the complementary sequence in the sample, if that matching DNA variant is present. Each interaction can be recorded to easily measure the presence of a large number of genes at the same time.

The scientists isolated DNA from a portion of the temporal bone in the dog's skull.

They made a library of single-stranded DNA sequences; smaller pieces of DNA that together represent the entire genome of the dog. The DNA library is sequenced on a machine that can read the order of the bases (As, Ts, Gs, and Cs) that make up the genome of the particular dog being studied.

Check out this video from TED-Ed on how to sequence the human genome (it also applies to the dog genome).

They sequenced the d-loop of mitochondrial DNA, an area where mutations happen more often than other parts of mitochondrial DNA.

Seven of the d-loop sequences were already available on a public database, and the others were generated from DNA in bone samples. A very small amount of bone was ground to a fine powder. The scientists were very careful to make sure contamination of the samples did not occur. Once the cells in the bone sample were broken open, and the DNA was isolated from other parts of the cell, the DNA could be sequenced by polymerase chain reaction (PCR).

Check out this video from Khan Academy to learn how DNA is sequenced by PCR.

In this case, the DNA specimens are ~14,000 to 3000 years old.

This is one of the limitations that will be placed on researchers that plan on conducting research to contribute to this database. They restrict a certain proposed sampling to the non-insect and non-nematode ecdysozoans.

They propose a certain focus for researchers that wish to dedicate time to contributing to the database by stating that they should focus more on neglected groups of arthropods, specifically crustaceans.

GIGA aims to set standards for data acquisition and the processing of that data as well as the input of that data into their system. These standards will be progressively revised as better methods are discovered. These standards are to be used for a large variety of invertebrates. This standardization will allow for a more easily accessible and usable bank of information.

The project coordinators will also join with other networks to work together on future projects of common interest. They also understand that different countries have limits on the use of biological samples and thus their governments will be consulted with so that the international program is protected from any government agencies in the countries where the samples come from.

Once the data has been submitted by researchers, it will go through a quality control process to make sure that it reaches the quality and accuracy which the project requires. As well as to avoid duplicate data being published.

The currently available genome sequences at the time were reviewed and the missing phyla were taken note of. These missing areas in the so called "tree of life" were added to the list of species to be researched so that the data could be used to further other research.

The information gathered from experimentation will be unified by being submitted to the GIGA website, thus allowing researchers from all over the world to have access to the information at a moments notice. The website will also be used to host cooperative research projects that scientists from around the world could participate in and contribute data towards.

Selecting which organisms genetic information is to be gathered and recorded first will be determined at a meeting among researchers in the field. The priority of these organisms will also be determined by their accessibility to researchers as well as their ease of retrieving genetic information.

The purpose of the experiment is to create a genetic database of 7000 invertebrate species.

The main goal of the project is to build an international community whose goal is to perform invertebrate research. This community will be used to avoid duplicate research and wasting time on studies that have already been conducted. They will also set in place standards by which the data must be gathered and presented so that is can be universally understood by everyone around the world and so that the data is accurate enough.

Referencing Box 1, this study was reviewed by the author classified this study as looking at population/species and as qualitative or quantitative estimate of flower or fruit production. The big conclusion of this study is that a rare species is seed-limited while dispersal limitation may play a secondary role in determining its abundance.

Referencing Box 1; The author of this paper looks at how plant phenology changes across succession in a community. Peres is studying keystone resources over many different seasons and how phenology changes over time. This aids in answering this papers research question of how phenology can be used in conservation to help keystone species.

Referencing Box 1; Schmidt and his colleagues focused on sustainability in harvesting non-timber forest products. They did this by analyzing flowering and fruiting time, as well as, the abundance and size of the fruits produced. This conservation effort was done using a large scale population control effort.

Referencing Box 1; Rossi and his colleagues focused their studies and conservations efforts on estimating carbon stocks and developing growth models in order to track and plan for future phenological changes and their effects on different tree species. Their models were developed by looking at tree growth rings within the trunks of various trees in order to look at the relative amounts of carbon dioxide intake and atmospheric carbon stocks at the time of the ring growth.

Referencing Box 1; Ali and his colleagues established a calendar for the collection of seeds and other plant resources in order to properly manage the conservation of genetic resources in natural plant populations. This was done throughout population and species scale conservation. They made direct ground observations of plant phenophases (leafing, flowering, fruiting) and the environmental factors that effected these actions. Advancements in this conservation can be seen in efforts such as seed banks.

Research question:

How can phenology be used to further our knowledge and success in conservation biology? What are the influences of climate change on phenology and what are the implications of those influences within a tropical environment?

These are the parameters made to make sure that the experiment itself is not biased and there are different variables being tested.

The scientists further studied the relationship between histone proteins and stress levels after acknowledging that a possible cause and effect exists.

By discovering that antibodies connected to the histones were valid, it also proved the oyster to be the best model to test on.

The authors recorded and purified DNA from the gill tissue of the oysters in order to measure DNA methylation.

Later, the DNA sequences were amplified.

By using PCR methods, the samples were digested in order to asses the patterns of methylation and used to identify loci using Gowers Coefficient of Similarity.

The purpose in the experiment was conducted in the extraction of histone proteins. By using classic centrifuging and electrophoresis techniques, the histone proteins were separated in order to retrieve the different types of anti-proteins. The antibodies were then analyzed with the Western Blot using the antibodies.

The collection of the the oysters were at different time intervals in order to monitor and view different relationships between the algae concentrations and oyster infection number.The gills were the main focus of the time intervals to indicate how much of the algae was found in the oysters system.

A 24h simulation using a culture of K. brevis and the eastern oyster specimens.

They were fed less prior so that during the simulation they would actively feed.

There were 4 time points where data was collected: 0hr, 3hr, 5hr, and 24hr.

The oysters were opened and the gills were flash frozen for later examination. This is because the brevetoxins contact the gills first and thus would experience DNA damage first.

Explains how oysters were collected and kept.

Collected: Rookery Bay National Estuarine Research Reserve, Naples FL

Kept: In lab tanks with controlled conditions. Fed twice a day.

In one experiment, one of the authors (Suarez-Ulloa) had previously studied the cause-effect relationship between environmental factors and subsequent epigenetic modifications triggering adaptive responses in marine invertebrates.

In previous experiments conducted on Eastern Oysters, the author has successfully used bivalve mollusks as sentinel organisms.

When doing comparison, it helps to create a table to have a better understanding on the differences in leaf defense.

This was done to determine the different variables that affect insect populations and which ones were more important than others. With this information they set the parameters to create an observable experiment with a limited amount of discrepancies.

The authors have a clear goal in their experiment. Through this observation that is lacking a response they build an experiment to identify natures processes.

A molecular probe is created against a specific DNA sequence, in this case rRNA from either ascomycetes or basidiomycetes. The probe is designed with a recognizable tag on the end. Then a second probe is used to match the first probe, and finally a third probe to match the second one with a fluorescent tag on it. By using multiple probes we can amplify even the smallest signals to our first probe. These probes are then mixed with a biological sample that is fixed in place. If fluorescence is observed the probe has found a target.

In some experiments, as in this example, researchers use multiple probes in a single experiment. This provides information about how close two organisms of interest are in a given sample.

Often researchers only publish a single gene for a newly described taxon, as it is sometimes too expensive to generate sequence information for the entire genome. When research first began working with DNA sequence data to determine evolutionary relationships it was decided that the ribosomal DNA (rDNA), shared by every living thing, was the best single gene to determine evolutionary relationships between taxa. Currently this is hotly debated, however, the use of rDNA is still very common in eukaryotic taxonomy.

Phylogenies are often used to determine the taxonomy of a lineage, that is the evolutionary context of an organism within the greater tree of life. The researchers could recognize that the new symbiont was a basidiomycete from the sequence information, but only by comparing the sequence information of the new organism to the previously known organisms can we determine when and from which ancestor the new species evolved. To account for the millions of years that have passed after all the basidiomycetes diverged from one another, the researchers incorporate as much sequence data as possible. Here, they use 349 loci to determine the placement of the new lineage.

Refers to sequencing of target genes obtained by a procedure called polymerase chain reaction (PCR) using gene-specific primers. Amplicon sequencing provides a snapshot of the microbial populations in an environment.

Researchers attempted to grow and isolate basidiomycete cells independent from their lichen partners, the algae and the ascomycete fungus. Often times it is very difficult to culture new organisms because we do not know enough about the growth requirements. This is frequently the case with organisms that form symbiotic relationships, as they often lose the ability to grow independent of their symbiotic partners.

Primers are small oligonucleotide sequences that are used as probes to determine if something is present in an unknown mixed sample. In this case, the primer is designed to match a piece of ribosomal DNA specifically in basidiomycete genomes found in the previous experiments. If the primer finds a target in a biological sample it is highly likely that a basidiomycete is present in the sample. In these experiments, the biological samples are other wild lichens found next to Bryoria lichens in Montana forests. Primers operate on the molecular level, and so very little biological material is needed to determine if basidiomycetes are present.

The author initially looked only at the expressed gene copies (contiguous sections of messenger RNA, or contigs) that corresponded either to known ascomycete or known algal DNA. They limited their search to these two because until then, a lichen was thought to only consist of those two partners. When they could not find any difference that would explain the phenotype differences between the two lichens, they expanded their search to see what other kinds of DNA was found in every single lichen, and perhaps had not been accounted for yet. They found 506 contiguous snippets of DNA that were found in every lichen that appeared to come from a second fungus, a basidiomycete, that wasn’t supposed to occur in lichens.

A transcriptome is a way for researchers to count the number of messenger RNA copies that has been produced from each gene in an organism, which is a measure of how much this gene is expressed. By comparing the level of gene expression between two different species, researchers can get an idea of which genes might be involved in producing different phenotypes in those species.

For a recent study on the history of dogs in the Americas (published by some of the same authors of this paper), check out the 2018 research article in the "Related content" tab.

This sentence describes how the researchers used the conditioning assay to test whether mating is rewarding. The two odors they used came from the chemicals ethyl acetate (EA) and isoamyl alcohol (IAA). The specifics of these two chemicals are not important—any two clearly distinct smelling chemicals could have been used. Male flies were exposed to one of the two odors in the presence of sexually receptive females (who the males presumably mated with), and were exposed to the remaining odor in the absence of females. They then tested to see whether males preferred the females/mating-associated odor over the other neutral odor.

Here, the researchers are aiming to verify their theory about how mating, ethanol, and activity of the NPF circuit are connected (for more on this, check out the previous paragraph) by proving that each of these three things are inherently rewarding.

An example of how they could do this would be to repeatedly expose the flies to alcohol while simultaneously also exposing them to a particular odor cue—let's call it odor A. After repeated exposure to alcohol and odor A in conjunction, the flies will learn that if they smell odor A, alcohol must be near.

Once this association has formed, the researchers could put the flies in a Y maze, with one prong containing odor A, and the other prong containing a random odor—say odor B. Since odor A predicts alcohol, if the flies spend the majority of their time in the prong containing odor A, it would indicate that they are seeking out alcohol, presumably because it is rewarding.

This type of manipulation is exactly what the researchers did here, except that they also investigated mating and manual activation of the NPF circuit (in addition to alcohol) as possible rewarding elements.

Since the initial experiment does not allow us to clearly conclude why the virgin males preferred the alcohol (the list of possible reasons is given in the prior sentence), the researchers designed a series of follow-up experiments, each designed to examine a slightly different possibility.

The group of male flies that were exposed only to females who had already mated and were thus no longer sexually receptive. These male flies experience sexual rejection.

The group of male flies that experienced repeated, lengthy mating sessions with multiple virgin female flies.

Can detect admixture events; it is used to detect gene flow between closely related species.

The fly algorithm is so much more efficient that it can perform more computations for the same cost as the LSH algorithm. This is the because the cost per projection is so different—for each projection in LSH, it requires 2d operations. For each projection in the fly algorithm, it only requires 0.1d operations. To even it out so that the total operational cost is the same means that the fly algorithm is able to generate 20 times the number of projections as the LSH algorithm.

The mean average precision (MAP) is a way of measuring how well an algorithm is doing at a task.

Here, the authors took each algorithm (fly and LSH) and ran it against each data set (SIFT, GLOVE, and MNIST) for 1,000 different query inputs. This produced a list—also called a rank—of all the images or words that were similar to the query. Each item in the rank comes with a certain precision value that represents its accuracy.

Next, the authors added up all the precision values in the ranking (with the top-ranked values carrying the most weight) and divided the sum by the total number of items in the ranking to get the average precision (AP). They then took the average of all 1,000 APs (one for each query) to get the MAP.

Calculating a MAP for each algorithm and data set allowed the authors to compare the effectiveness of the algorithms.

SIFT, GLOVE, and MNIST are three well-known data sets that researchers and computer scientists often use to test similarity search algorithms.

MNIST is the Modified National Standards of Instruments and Technology data set. It consists of handwritten digits, and each row of the matrix corresponds to an image.

SIFT stands for Scale-Invariant Feature Transform. It consists of reference images and can be used to extract specific features of those images (e.g. the corners of a door frame).

GLOVE, or Global Vectors for Word Representation, consists of word vectors. It is useful for measuring the linguistic or semantic similarity between two words.