A P value below 0.01 means less than a 1% chance that the null hypothesis is true. Typically, a P of 0.05 or below suffices to conclude a significant correlation between two variables.

This probability is derived through a Student's T-test. This test requires the correlation coefficient (r) and the number of samples (n) as inputs.

COVID-19 spread fast. To show this, the authors compared the outbreak data for COVID-19 with those for H1N1, another contagious disease, in China.

To investigate the impact of the Wuhan travel ban on the spread of COVID-19, the authors first evaluated the effectiveness of this ban on stopping the movement of people.

The evaluation was realized by comparing the travel data from the year 2020 with those from the previous two years.

Note that the start and end of the time period of records are fixed with reference to the date of Spring Festival, which, however, varies from year to year.

For the quantitative analysis—linear regression in this work— the input data include official reports of the health commission, mobile phone data, and travel information recorded in online databases.

For details regarding the time span of the data sets and the means of data acquisition, please refer to the first two sections of the Materials and Methods described in the Supplementary Materials.

The authors focused on the first 50 days since the detection of the first COVID-19 case in Wuhan. They summarized the timeline for the implementation of key control measures over this period in Fig. 1.

Total ozone column data can be obtained by combining balloon data with data measured using satellites.

Satellites are able to obtain data at higher altitudes than balloons, which are limited to around 30 kilometers.

The earliest October ozone hole measurements are used as a reference for the trends that follow.

Nevertheless, September and November data can still be used to track the impact human-made chemicals, climate variations, and volcanic eruptions are having on the ozone layer over time.

This approach removes the contributions of climate variation and volcanic eruptions to ozone depletion.

Thus, only the effects of variations in chemistry would remain.

Since aerosols released from volcanic eruptions can also contribute to ozone depletion, the researchers must account for this contribution as accurately as possible.

To calculate the accuracy of the model, the modeled data is compared to measurement data during the years that volcanic eruptions occurred.

M100 is the specific amino acid residue within the protein sequence that has been identified to be critical for the protein’s function. it is very important to determine the ideal amino acid residue for this position. Site saturation mutagenesis is employed.<br> Site-saturation mutagenesis is a form of random mutagenesis, allowing the substitution of a specific amino acid site with one of 20 possible amino acids in a single experiment. In this study, this technique is employed to generate a series of enzymes with enhanced activity and enantiospecificity.

Heme proteins that were readily available were screened to identify the one that gave the highest enantioselectivity. This served as a starting point for directed evolution. Purified heme protein, silane, diazo ester, thiosulfate, methyl cyanide and M9-N buffer as the medium for microbial growth were stirred at room temperature in anaerobic conditions. Reactions were performed in triplicate. Unreacted starting material was obtained in all cases and no further purification was carried out.

Analysis of the sex fate gene expression showed that male markers were significantly decreased, whereas female markers were significantly increased in Kdm6b-RNAi treated gonads compared to controls.

Kdm6b-depleted gonads showed marker expression similar to the female control.

A robust ratio of the Kdm6b-depleted embryos demonstrated a shift from male to female development, despite incubation temperature. This structural data suggests that KDM6B plays an important role in sex determination.

We need to take r values with caution, though. Calculating r values is still possible even for plots where two variables are not linearly associated. It is always wise to look at the scatter plot an r corresponds to.

In the present study, the scatter plot in Figure 2C indeed shows a clear linear association.

The present work addressed this question by studying how the spread of COVID-19 was impacted by the travel bans and the closure of entertainment venues. The travel bans encompass the one for Wuhan city and the suspension of inter- and intra-city public transport.

Given the results demonstrated by Smith and colleagues, the authors decided to look at the size distributions and damage profiled of the RNA recovered from the lung tissue. They found fragments of much greater lengths than 30 nucleotides with fragments ranging from 217-233 nucleotides. They also found no evidence of RNA damage indicating formalin-fixed specimens could serve as a source of well preserved RNA molecules.

These authors took the collection of overlapping DNA fragments produced through sequencing and lined them up to a genome of measles that had already been constructed.

Rma cyt c V75T M100D M103E shows excellent enantioselectivity and turnover over a wide range of substrates. Silicon substrates with weakly electron-donating or -activating methyl substituents (4), strongly electron donating -OMe (5), weakly deactivating -Cl (6), strongly deactivating -CF3 (7), and moderately deactivating COOMe and CONMe (9 and 10 respectively) show moderate to excellent turnover and high selectivity. No direct relationship exists between turnover number and substituent effects from this study. Enantioselectivity is excellent in all substrates. All products were identified using GC-MS, and no traditional organic chemistry techniques were used.

Additional diazo compounds that were successful were R3 = -CH3, -CH2CH3, -Ph.

General method for the preparation of phenyl dimethyl silanes: In a 100 mL round bottom flask, chlorodimethylsilane in THF was cooled to zero degrees. A solution of the appropriate Grignard reagent was added dropwise over ~15 minutes. The reaction was allowed to reach room temperature and stirred for ~8 hours. The product mixture was quenched with ammonium chloride solution. The product was extracted into ether and then isolated. Purification was done by column chromatography.

Via site-saturation mutagenesis, V75 and M103 positions along the protein sequence were identified as likely beneficial mutations and were randomized, i.e., the amino acids at these positions are replaced by random ones. A large number of random variants, which together constitute a library, are produced and then screened in an attempt to discover a highly active variant among them. The evolved triple mutant fits the bill.

Recombinant protein is a protein encoded by a gene that has been cloned in a system that supports expression of the gene (in this case, it is M100). Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. In this study, M100D mutation is more highly activating than the wild-type protein.

Turnover frequency for each variant relative to wild-type protein: WT: 1 M100D 2.8 +/- 0.2 V75T M100D 4.6 +/- 0.3 V75T M100D M103E 7.1 +/- 0.4

From this experimental data, it is clear that directed evolution has resulted from changing the enzyme from unselective wild-type into a highly enantioselective variant.

Experiments were conducted with purified heme protein, silane, diazo ester, sodium dithionate, MeCN, and M9-N buffer at room temperature in anaerobic conditions for 1.5 h. Three trials were conducted. Turnover number reported is the average of the three trials. Unreacted reactants were not isolated.

The range of 4.8 to 12.7 million metric tons of waste comes from uncertainty in the total amount of waste generated, the amount of that waste that is plastic, and the amount of mismanaged waste that enters the ocean.

Here the authors restate the goal of their investigation.

The authors visited multiple areas in Hawai'i to study how seeds from plants (originally from the area or recently introduced) were being distributed by invasive birds.

Enantiomeric excess was determined using chiral SFC.

Products were analyzed using gas chromatography.

The authors conducted a new set of experiments to check whether administering anti-CTLA-4 after tumors are detected is as effective as administering it at the same time tumors are introduced. If they are able to successfully treat mice after tumors are already established, then maybe this treatment could work for human patients as well!

The authors injected the mice which were previously treated with unmodified tumor. If they developed an immune memory, they may be able to clear this tumor even though it is not expressing B7 and they have not been given anti-CTLA-4 antibodies.

The mice were split into groups that received different treatment regimens. Two of the groups were untreated or were treated only with anti-CD28 antibodies.

The authors decided to check if there is an effect to changing the tumor dose. They halved the dose to 2x10(^6) and had a group of untreated and a group of anti-CTLA-4 treated mice.

This set of experiments was conducted with a variant of the same murine colon cancer tumor cells, but this time the tumors did not express B7. Thus the tumors are not able to provide the secondary signal to T cells.

The authors had a control group of mice which were injected with tumor cells expressing B7-1 molecules but were not treated with any antibodies.

The authors injected groups of mice with tumor cells expressing B7-1 molecules. The injections were administered within the membrane lining of the abdominal (peritoneal) cavity. These mice were then treated with two different regimens. One group of mice was injected with antibodies targeting CTLA-4 and another with antibodies targeting CD28.

Tips are connected to the shaft which consists of very large molecules composed of many repeating subunits.

The authors made a surgical incision into the abdominal cavity and performed a surgical operation to make an opening in the stomach in order to manually implant the millliposts into a swine stomach. This shows the drug being taken in at a very similar rate as to when the device was ingested.

The weight distribution of the SOMA device out performed devices made solely of PCL showing that weight distribution plays a large role in the ability to self orient.

The authors show that a force of 5 N from the spring allows the milliposts to enter into the tissue of the swine's stomach. They verify that this will be safe by testing a force of 9 N, which is larger than the used 5 N, showing that even a larger force will not cause the tissue to rip or be damaged.

The authors tested in a simulation that the millipost will dissolve within 60 minutes after released, so that it is not staying in the body for too long of a time period after it is released.

The authors show that the device can be stored for an extended period of time in a warm, dry environment, as well as a slightly shorter time in a liquid environment. This demonstrates that the device can be safely stored and it will not affect the device.

When devices with different shapes but the same equivalent mass, volume and density distribution were compared, the SOMA shape was shown to be better at self orienting. This shows that the SOMA shape performs better than the sphere and ellipsoid shapes.

The authors used a technique to determine vibrational modes of molecules to analyze the chemical structure of the API used after having it put under high pressure.

In the case that the millipost did not dissolve, the authors used SOMAs with stainless steel needles to test that problem. They concluded that the SOMAs with needles that did not dissolve did not cause blockages in the GI tract, nor pneumoperitoneum, or the presence of air or gas in the abdomen. This means that the SOMAs do not cause GI blockages, or behavioral changes or other adverse effects in the swine model. This is promising news and could mean that the SOMAs would work in a human model.

The SOMAs initially only worked when the stomach was empty, or the fasted state. When the SOMAs were dropped into the stomach where there was food, it would try to inject the insulin, but it would not pierce the stomach wall. When there was water, it pierced the stomach wall, but there was no insulin in the bloodstream.

The authors tested how much pressure could be put on the milliposts, which gave the stiffness of the material to be 730+/- 30 MPa. The maximum pressure the material can withstand before breaking is 20 +/- 0.7 MPa. These numbers are used to make sure a pressure higher than that is not used, ensuring the milliposts will stay intact.

The authors tested the device being ingested, but without the milliposts being inserted into the stomach tissue. No insulin was released, demonstrating that insulin will not leak out of the device without it being implanted in the tissue.

The authors tested how the milliposts would perform with an increased amount of API loaded in it. The device showed that even with more API in it, the drug would be delivered at a constant rate and would continue to be administered for 30 hours.

The release of the drug was shown to be near constant, independent of the concentration that was being released.

The authors test the device in a swine stomach. The device successfully found its way to the wall of the stomach and was able to stand itself up in the correct direction and insert the post into the tissue, without causing any damage.

The authors show that the spring has a high precision, so that the time that the spring will actually be released can be very accurately estimated.

The authors test the relationship between how much force is applied to the milliposts and how deep the post goes into the tissue of the swine.

Oral administration of the device in single doses and repetitive doses was not seen to be hazardous to rats.

A mixture of low and high density materials were used to make the lower portion of the SOMA device.

Experiments confirmed that the device oriented the quickest between the angles of 30° and 135°. The device oriented the slowest between the angles of 45° and 100° or an exact angle of 90°.

When dropped from different angles the simulation predicted that the SOMA device had the fastest average orientation time.

To create blue or red dyes, researchers mixed chemical compounds, methylene blue or amaranth, with water. They also used watercolor paints to create other colors. These solutions helped to camouflage their device.

To change the temperature of the liquids, the researchers would heat or cool them inside an oven or refrigerator before pumping them into the device. This allowed researchers to adjust how the robot is viewed in the infrared spectrum (IR). The IR spectrum reflects the temperature of the emitting body.

The researchers pumped fluids through the microchannels, manually with syringes or with the use of syringe pumps.

Syringe pumps gradually administer, or withdraw, liquid solutions: https://www.chemyx.com/support/knowledge-base/technical-support/chemyx-syringe-pump-work/

By pumping fluids through the microchannels, researchers could change the color and display of the device.

The researchers created color layers with different sized microchannels to change the appearance of the soft robot. Wider microchannels allowed for larger blocks of color to be observed. Whereas smaller microchannels allow for many sharp edges and transitions to be created.

They want readers to realize that many different color-layered microchannel networks can be created to alter how the device may be perceived.

The total volume of the microchannels used in the color layers equates to 0.75 ml, which is similar to a large drop of water. Therefore, they do not require much water to be filled and are lightweight. Once the robot is filled with liquid, via syringe pumps, no further input is needed.

Here, researchers filled only certain microfluidic channels up with a certain color, in order to mimic the background the robot was resting upon. This form of camouflage is called background mimicking. Researchers wanted to see if this soft robot was capable of background mimicking.

Researchers mixed different colored dyes together in order to create different colored solutions. Then, they would place these colored solutions into a pump to be administered into the color layer microchannels of the soft robot.

Here, researchers are filling the color layers up with liquid using a pump. The rate at which liquid is being pumped into the microchannels is 2.25 milliliters (ml) per 1 minute (min). The researchers want to demonstrate that their device can change colors rather quickly and, therefore, show that their device is capable of being used for camouflage.

Although 2.25ml/min may seem like a slow pumping rate, the total volume of the microchannels used in the color layers equates to 0.75 ml, which is similar to a large drop of water. Therefore, it is very reasonable for the microchannels to be filled in 30 seconds (s) with a pumping rate of 2.25ml/min.

The authors excluded the time it took to fill the “tether” when performing this experiment. The tether is simply the tubing that connects the pump to the microchannel. Liquid travels from the pump, through the tether, and into the microfluidic channel.

Because researchers only wanted to know the time it took for the microchannels to change colors within the color layers, they excluded the time needed for the tether to fill with liquid.

The authors tested the mechanical and chemical properties of the milliposts used to deliver the insulin. The tests concluded that the milliposts would be able to successfully delivery the insulin while keeping it stable.

When considering the preferred orientation of the device the SOMA shape was able to self-orient the quickest between the angles of 0° and 45° and the angles of 100° and 180°. This orientation occurs within 100 milliseconds from 85% of all initial positions.

The authors used a scope and looked at the G.I. tracts of the swine that were fed the SOMAs. There were no visible signs of damage from the SOMAs and there was no evidence that these devices were hurting the swine based off of their behavior, eating habits, and the regularity of the excretion of their feces.

The authors designed a membrane that would stop food and fluid from getting into the millipost chamber, keeping the insulin safe, but would allow the millipost to be pushed through the membrane when the spring was activated.https://imgur.com/ncl6OiG

Here the authors gave 3 swine 3 SOMAs each and after the swine pooped them out, they examined them for visible damage. The authors said eight of the nine SOMAs were recovered and the ninth SOMA was accidentally disposed by accident during a cage cleaning. This showed that the SOMAs do not cause G.I. blockages, which means that they are safe to ingest.

To look at the specific finches that experienced the 2004–2005 drought, the researchers genotyped a HMGA2-specific SNP in both survivors and victims (71 total birds).

Lamichhaney and colleagues took a closer look at another 133 birds. Specifically, they investigated an SNP at a certain position in the genome.

Within the 17 SNPs, researchers knew that the large finches were homozygous (LL in Figure 2D) for one haplotype group and small finches were homozygous (SS) for another haplotype group. However, what was going on with the medium ground/tree finches?

This particular SNP was shown to be associated with only beak and body size within these medium finches.

HMGA2 had been identified as a candidate gene, and the SNPs within the 525-kb region within HMGA2 had been located. Therefore, the researchers attempted to see which trait this gene was specifically related to.

Researchers performed pairwise, genome-wide fixation index (see definition in Glossary section) scans across the whole genome. They did this with 15kb windows that were non-overlapping regions.

The following three comparisons were made: 1) large ground/tree versus medium ground/tree; 2) large ground/tree versus small ground/tree; and 3) medium ground/tree versus small ground/tree.

A compilation of SNP calls yielded 44,767,199 variable sites within or between populations. The fixation index score ranged from zero (complete sharing of genetic material) to one (no sharing of genetic material).

Each index value was then transformed into a Z-score, which is simply a measure of how many standard deviations below or above the population mean a raw score is. Further analysis was done if the Z-score of the fixation index was greater than five. See the Supplementary Materials for more information.

The researchers again calculated the fixation index. However, this time it was only for the variable region (~525-kb in size) that contained the HMGA2 gene.

Remember, the fixation index score ranged from zero (complete sharing of genetic material) to one (no sharing of genetic material).

For a good explanation of a SNP, see this video.

DNA extraction and whole genome sequencing was performed using samples from 60 birds. In addition, 120 bird samples from a previous study (Lamichhaney 2015) were used.

The sequence reads were analyzed and trimmed using FASTQC software and FASTX software, respectively. To see more specific software used, see the Supplementary Materials

The researchers used the genome assembly of a medium ground finch as a reference genome. The reads from the samples were aligned to this reference.

Once the silenced genes were introduced to the planaria via diet, amputations were conducted.

Each of these proteins were silenced individually using RNAi and then incorporated into a food source for planarians. The experimental group was divided into three and each was fed one type of the RNAi food for 8 days. Amputations were then completed on all experimental animals on Day 9.

To identify anatomical characteristics that follow an anterior/posterior axis position (head vs tail), the researchers looked at two organs - the primitive brain and the primitive spinal cord of the planaria . This is important to determine, because it allows researchers to identify which molecules direct cells to become a certain cell type as well the molecule that directs the cells to their respective locations in the body.

In this initial part of the experiment, the researchers identified the components of the Wnt signaling pathway found in S. mediterranea and made copies of the genes. Then, using RNAi, each component was silenced individually, and then was silenced in selected groupings to determine the role of each chemical and whether there was overlap in their functions. RNAi was fed to the planaria to introduce the silenced protein.

In a controlled experiment, there are two groups: 1) one is controlled, which means that all conditions are held constant or the same and 2) experimental group, in which a defined variable is changed. In this case, the experimental group was treated with RNAi, while the control group was not. This allows scientists to compare the two groups to determine if what they changed in the experimental group was due to that variable.

The goal for this series of experiments was to better understand the Wnt signaling system because it is found many animal species ranging from invertebrates to vertebrates. The Wnt signaling system is a pathway found in most animal species. It is one of the most important cell signaling systems because of its control over how and when cells divide, change into specific cell types, and move to the area of the body that they belong during embryonic development. This includes directly how stem cells allow for renewal of damaged tissues.

You can find more information on this pathway here: https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/wnt-signaling-pathway

Motor behavior was assessed using amphetamine-induced rotations, head position bias, and locomotion.

Rotations were performed by injecting amphetamine 30 minutes prior to trial and placing the animal in an opaque cyclinder. Ipsilateral (same side as) rotations to the 6-OHDA lesion (clockwise) were added and contralateral (opposite side as) rotations were subtracted.

Head position bias was determined by the number of head tilts over time, where a greater than 10 degree deviation left or right from midline was measured.

Locomotion was measured using software called Viewer that tracked motion and calculated distance.

This group has led the field of optogenetics by laying the groundwork for how this "tool" can be employed in neuroscience research.

Opsins are transmembrane proteins (passing through the cell membrane) that are ion channels/pumps which allow for various ions like sodium, potassium, hydrogen, chloride, and calcium to move in or out of the cell.

There are three main families of opsins: channelrhodopsins, halorhodopsins, and bacteriorhodopsins. Each opsin is light sensitive due to a chromophore retinal molecule within the transmembrane domain of the channel, and each family is sensitive to a specific wavelength of light.

Second-generation lentiviruses encoding the enhanced halorhodopsin were created using three plasmids.

One plasmid containing the gene of interest (eNpHR) under the control of the promoter (CaMKIIα) is called the transfer vector. The transfer vector usually encodes a fluorescent gene to monitor expression: in this experiment, enhanced yellow fluorescent protein (EYFP) was added after the eNpHR sequence.

A second plasmid containing the envelope gene, usually VSV-G (vesicular stomatitis virus), allows for a broader degree of infectivity in various cells. The third plasmid contains all of the packaging genes necessary to create a functional viral unit.

When all three plasmids are added to HEK293 (human embryonic kidney) cells, viral particles are released from the cells and suspended in the culture media. Learn more about lentiviruses here.

Since inhibiting excitatory neurons in the STN with eNpHR and activating glial cells with ChR2 were both insufficient at correcting motor deficits in hemiparkinsonian rats, the authors attempted to mimic the high-frequency stimulation using ChR2 in the excitatory neurons.

In other words, beneficial effects could arise by activating or targeting axonal projections entering the STN as opposed to direct STN interventions.

The specificity of optogenetic treatments is of particular clinical interest and relevance for neuroscientists. Because individual cells can be targeted in the living organism, optogenetics allows scientists to better understand how different brain cells function and communicate.

An optrode is a combined optical fiber with a metal electrode that can both stimulate via light and record via a tungsten wire. The tungsten wire was tightly attached to the optical fiber with the electrode tip being slightly deeper to ensure proper recording from light-stimulated neurons.

S100β, also known as calcium-binding protein B, is expressed by mature astrocytes. S100β can also be used as a peripheral biomarker of blood-brain barrier permeability.

The main way the authors assessed student performance was to compare scores on multiple-choice exams. These scores are referred to as the main outcome measure.

The authors used a double-blind study design, meaning that neither the students nor the teaching assistants working with the students knew the purpose of the study, or to which group each student was assigned. Double-blind studies are meant to reduce unintentional bias on the part of the participant or the researcher interpreting the data.

With the knowledge from the previous experiment that regulation of β-catenin occurs earlier in the Wnt signaling pathway, the researchers were able to use the anterior/posterior markers to identify the time period during which the stem cells of the blastema differentiated in both the control and experimental animals.

In this set of experiments, Alvarado and colleagues were trying to identify if increased amounts of β-catenin regulates that expression of Smed-APC-1.

In this fourth set of experiments, the researchers wanted to identify the role of β-catenin at different amputation locations for the experiment animals while different genes were silenced and then comparing this to control animals with no genes silence but the same amputations. Amputations included removing the head or tail to see if the head or tail itself would regenerate. In addition, some animals were divided in half longitudinally (lenthwise) and some were divided in half mediolaterally (divided into anterior and posterior halves).

In the fourth series of experiments using the various amputation location, it was noted that in some of the experimental animals that several photoreceptors and brains developed in the wrong location on the same animal. To understand why this happened, unamputated worms had the same genes silenced as the experimental animals and were observed for two weeks.

The use of the anterior and poster markers will allow the researchers to determine if posterior cells are indeed moving to the posterior end and if anterior cells are moving to the anterior end in the RNAi treated animals.

Genes that are expressed only at the anterior or posterior ends were tagged to identify their location in both control and experimental animals while they are regenerating. This allows the researchers to identify the extent to which the stem cells were affected when silencing the proteins. Researchers can compare the control animal's cell migration to the the cell migration in the experimental animals for each of the silenced genes and gene combinations.

In order to determine whether the changes in anterior/posterior identity were temporary changes that influenced the cells to act like a different cell type or whether DNA expression itself was permanently change, Alvarado and colleagues examined the location of the anatomical structures that regenerated. In addition, they identified the active molecular markers, which are chemicals that will help identify the activity of cells in a specific area to determine what type of cell they are. In this research, the markers direct the cells where to go (anterior or posterior). Some are active only for certain time periods of the regeneration process.

An important step in the authors' research was the determination of the body masses of the fossil mammals they unearthed. Body mass was estimated by measuring the length × width (L×W) of the first lower molar. This has been found to be an accurate indicator of body mass through comparisons to molar size and the body mass of living mammals. This data is important to the authors since body mass has been linked with characteristics such as energy expenditure, gestational period, temperature regulation, and niche ecology. When reconstructing life during the time period being studied, body mass distributions in mammalian communities can be used to infer ancient environmental conditions. Therefore, determining the body mass of a fossil mammal is an important step toward understanding its paleoecological role. Brain size (cranial size) usually increases with the size of the animal. However, the relationship is fairly inaccurate, as cranial size does not correlate as closely to body mass as the area of the first molar does.

The fossil record shows that after the K-Pg extinction event, there was an exponential increase in the maximum size of mammals. About 40 million years ago, this size increase leveled off. It is hypothesized that diversification to fill ecological niches was the primary driver of this rapid increase and that environmental temperature and land area have acted to constrain the continued increase.

This mammal was a specialist and was proof that mammals were evolving and becoming larger as they specialized. Increased plant diversity fueled this growth.

These are compact masses of mineral matter, usually spherical or disk-shaped. They are carried along by water and become embedded in host rock that has a different composition. Concretions form in sediments before the sediments become rocks. The authors focused on searching for and cracking open concretions.

Fossil pollen reveals how plant species evolved after the K-Pg extinction. The authors compared pollen from both sides of the K-Pg boundary. The authors discovered that immediately after the impact, there were few plants.

To determine the age of some of the rocks in their study area, the authors used uranium-lead dating. It is most often used to date volcanic and metamorphic rock and very old rocks. This technique involves the radioactive decay of U-238 and U-235 into lead (Pb). The 238 and 235 refer to the sum of the number of protons (92) and neutrons in the nucleus of each of these forms of radioactive uranium. The half-life, which is how long it takes half of a sample of the U-238 to undergo radioactive decomposition and become Pb-206, is 4.47 billion years. The time it takes for half of a sample of U-235 to decay into Pb-207 is 704 million years. Since the two different forms of uranium have different half-lives and decompose into different forms of lead, they are a good check when calculating the age of a rock or fossil. This dating technique is best used on rocks that are from 1 million to 4.5 billion years old.

This technique was used by the authors to date rock strata. Chemical Abrasion-Isotope Dilution-Thermal Ionization Mass Spectrometry (CA-ID-TIMS) is a multistep, high-precision technique used to determine the relative amounts of uranium-238 and lead-206 present in zircon crystals. Zircon crystals, formed from volcanic or metamorphic rock, are extremely durable and resistant to chemical breakdown. They can survive major geologic events. Over time, new zircon layers form on top of the original crystal. The center of the zircon remains unchanged, keeping the chemical characteristics of the rock in which it originally formed. Every radioactive element has a decay rate. The length of time it takes half of the radioactive atoms in a sample to decay (form into a different element) is referred to as its half-life. The half-life of uranium-238 to lead-206 is 4.47 billion years. The authors selected zircon crystals from their study area and dated them using CA-ID-TIMS.

Image of thermal-ionization mass spectrometer: https://www.usgs.gov/media/images/usgs-thermofinnigan-triton-thermal-ionization-mass-spectrometer

This is the study of the magnetism in rocks induced by Earth's magnetic field. The minerals in certain rocks lock in the direction and strength of the magnetic field at the time of their formation. The authors used this method to determine the age of their fossil finds.

To analyze what happened in these basin areas, the authors used sequence stratigraphy. They looked for variations in the successive layers of sedimentary rocks and the composition of the rocks. The order in which the different layers were deposited was carefully recorded. The chronostratigraphy aspect of their fieldwork involved tracking changes in the character of the rocks through geologic time. Fossils found in each layer can then be better placed in terms of when these organisms evolved, and in some cases, became extinct.

Learn more about stratigraphy from HHMI BioInteractive: https://www.biointeractive.org/classroom-resources/stratigraphic-principles

The authors used descriptive statistics, calculating the mean and the standard error of the mean to quantify the variation in the populations. n=42 means that 42 measurements were included in the calculations.

The Grants began studying finches on Daphne Major Island in 1972. Along with team members, they returned there every year until 2012 to observe the finches. Observations included identities of mating pairs, number of offspring (used to construct pedigrees) and mortality of birds (recording deaths of birds).

The techniques used included catching the birds and banding them (installing identification bands around their legs), as well as taking measurements of the birds (including body mass and beak measurements). Blood samples were also collected for later DNA analysis.

ADMIXTURE is a program that can quantify inbreeding by estimating allele frequencies and ancestry proportions. It helps scientists to understand how much of a genome is derived from inbreeding with other populations in the past.

This analysis and the inbreeding coefficient analysis helped to confirm the founder's species as G. conirostris.

A statistical calculation that estimates the probability that an individual inherited two identical genes from one ancestor that occurs on both sides of the pedigree. The higher it is, the more inbred the organism is.

It was used to both help assign the founder bird to a species and to quantify the genetic diversity of the Big Bird population as it bred amongst itself.

A computer program was used to create many possible pedigrees based on the genetic data and to analyze the likelihood of each relationship in the pedigree using the genomic data. The most likely pedigree inferred from the data was mostly in agreement with the pedigree based on observations, with only a few changes.

The authors prefer to 3D print collagen in its natural form, but this comes with a challenge. Unmodified collagen transforms into a gel by forming links between individual structures, and this process is difficult to control at physiological pH values of 7.4.

To resolve this issue, the authors prepared collagen bioinks by dissolving collagen in an acidic solution. By dispensing this bioink in a buffered support gel, the pH of the collagen ink is quickly neutralized to 7.4 which promotes the linking process.

This allowed the authors to 3D print unmodified collagen, with higher density and strength, in complex shapes and structures.

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

Previous experiments have only demonstrated small-scale applications such as printing a ventricle and a valve. So, in their last experiment, the authors printed an infant-sized heart to show the larger-scale printing capability of FRESH v2.0 towards printing a fully functional artificial organ.

MRI uses magnetic field and radio-frequency waves to create detailed 3-dimensional pictures of organs within the body. The authors developed a 3D model of the adult human heart was developed by using magnetic resonance imaging (MRI) images.

Due to low resolution of MRI imaging, their 3D heart model included valves and large vessels, but was missing the smaller-scale vessels. The authors later added the small vessels to their model by using a special computer program. The authors then used this 3D model to FRESH-print human heart.

The authors observed the printed ventricle as it contracted to see how the walls thickened during the contractions. This was done because wall thickening is a typical behavior of ventricle contraction.

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

In conducting their experiments, the research team acknowledged the inherent variability in the ³He/⁴He ratio inside each diamond. To minimize these differences, the scientists used diamonds that were free of inclusions thereby eliminating the possibility of inconsistencies in helium ratios originating from these inclusions. Then, they crushed the diamond in a vacuum to verify that 90% of the helium is present in the groundmass of the diamond. This was then followed by step-heating the crushed diamond to measure the variability in the ³He/⁴He ratio in the diamond.

The outer rim may be contaminated by helium-4 (4He) from the surrounding material. Removing the outer rim consisting of helium-4 isotopes from the diamonds narrows the search to helium-3 isotopes located in very ancient chemical reservoirs. This removal is an experimental design to attempt to get a pure analysis of the helium only from the diamond.

The research team sought to identify an untouched, untapped chemical reservoir. To do this, they decided to examine primordial diamonds that contained both helium-4 and helium-3 isotopes.

The helium-4 isotopes are formed from radioactive decay of trace elements and hence take a long time to form. Conversely, the helium-3 isotopes exist inside Earth from the early days of formation and there has been no new generation of helium-3 isotope since then.

The authors carefully inspected 24 diamonds excavated from the Juina area of Brazil. This location was chosen because the diamonds excavated from this area show characteristics similar to the ones that belong to the Earth's transition zone (410 to 660 km depth). Kimberlite pipes are an explosive and geologically "quick" process that brings diamonds up to the Earth's surface with little to no interaction with the surrounding rocks, making them ideal for this study. The sizes of these diamonds ranged between 1.3 mm to 6 mm. Despite the size limitation, the research team could successfully detect the helium gases trapped in these diamonds.

Fig. 2D shows X-ray images of the smart pill containing long-acting formulation–2 over 29 days. Two arms of the pill broke off in pig 3, but the pill was still able to remain in the stomach and provide consistent drug concentrations.

For extended release, the birth control pill (levonorgestrel (LNG)) can be loaded within the structural polymer that makes up the arms.

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.

Single and two-unit planar HASEL actuators are built as explained in Figures S8 and S9. Linear actuators are vertical, with the load applied downward in the same direction of gravity. DC voltage is increased in small quantities, activating the planar actuator. The maximum strain detected is a 79% strain under a 250 g load.

To test the effectiveness of their design, the authors fabricated a dielectric (DE) actuator and a HASEL actuator with same dimensions. They tested the amount of in-plane expansion as a function of voltage. HASEL actuators expanded more (46% compared to 12%) under same conditions.

Two stacks of HASEL actuators are tilted and fixed to form a gripper for other items, as seen in Figure 2 C-G. When a voltage is applied, the grippers are able of grabbing fragile objects including an egg and a raspberry.

HASEL uses a liquid dielectric instead of a solid dielectric. Dielectric breakdown can be considered like a lightening strike. The sky and the ground are the opposite sides of the dielectric. When the power is too high there is a breakdown and the opposite sides connect and conduct electricity, a lightening strike.

The connection is extremely powerful, just like a lightening bolt, and will often burn holes through the dielectric. HASEL actuators, using a liquid dielectric, are protected from these breakdowns. When a hole is burnt through the liquid dielectric, it is filled in with the surrounding liquid, effectively healing it.

The relative size of the electrode determines the mount of force it can generate.

Smaller electrodes can squish less liquid dielectric material between the electrodes. However, they generate a larger force by causing a smaller strain (rise) over a larger area in the donut.

Larger electrodes can squish more liquid dielectric material between the electrodes. However, they generate a smaller force by causing a larger strain (rise) over a smaller area in the donut.

You can control the force generated by the actuator by changing the area of the electrode with respect to the overall area of the actuator.

Think of what the water balloon looks like when you push it down with one finger compared to two. The more fingers you used, the larger the area of the balloon you touch, and the higher the sides of the balloon would rise.

Once the snap-in voltage is passed, the electrodes move further towards each other with increased voltage. The more voltage applied to the electrodes, the more HASEL actuator is squished.

HASEL actuator is initially shaped like a disk, but when voltage (exceeding the snap-in voltage) is applied, it deforms into a donut shape.

As you increase the voltage to the HASEL electrodes the dielectric become more polarized. The electrostatic attraction is fighting against the pressure of the liquid dielectric in HASEL. The electrostatic attraction is equivalent to your finger pushing down on the water balloon and the liquid dielectric pressure is equivalent to the balloon resisting your push.

As voltage is increased, the electrostatic (attractive) force becomes greater than the liquid pressure and the electrodes begin to move towards another. Similar to: when you finally push hard enough, the balloon begins to squish.

The voltage high enough to cause this change is called the "snap-in voltage".

When the dielectric is polarized it, it generates a force which pulls the two electrodes together. Electrostatic Maxwell Stress is a mathematical model that explains the strain caused by these two electrodes being electrostatically attracted to each other. This electrostatic force is like pushing the water balloon in the middle.

Researchers chose HASEL's structure because they wanted stable uniform force to be distributed from the actuator.

When you push in the middle of the water balloon you raise all the sides around your finger equally. If you push off-center, or squish balloon on the side, you have a less equal distribution of pressure. Therefore, the researchers placed the electrodes in the middle of the actuator (on top and bottom surfaces).

A HASEL actuator is like a half-filled water balloon. If you press in the middle of the balloon with your finger, you force the water from the middle of the balloon to the outer edges.

HASEL actuator is filled with a liquid dielectric that does not conduct electricity. Electrical power is supplied to the electrodes placed in the middle of the top and bottom surfaces of a HASEL actuator.

The electrical power polarizes the dielectric, and generates electrostatic force in the middle of the actuator, similar to pressing your finger in the middle of the water balloon. This force help inflate the sides of the actuator, generating a hydraulic force in the vertical direction.

The researchers named their actuator "HASEL". They explain that HASEL is a higher-performance, versatile, muscle mimetic soft transducer, combining the advantages of fluidic and electrostatic actuators.

Higher-performance: compared to other soft-robot actuators HASEL uses less energy to generate larger movement.

Versatile: HASEL can lift weights or used to grab delicate objects (soft gripper).

Muscle Mimetic: HASEL mimics the movement of human muscles based on extension and contraction of muscle fibers.

Soft transducer: HASEL transfers electric energy into mechanical energy.

HASEL actuators are able to sense strain through capacitance, which is a direct relationship: when strain is low, capacitance is low; when strain is high, capacitance is high. In this experiment, two planar HASEL actuators are combined in parallel to simultaneously measure capacitance during various movements of a robotic arm. Capacitance is low when the arm is flexed, while capacitance is high when the arm is extended.

The actuation voltage signal and sensing voltage signal were combined in LabVIEW and output from a data acquisition system to a high voltage amplifier. The amplified voltage signal was then applied to the HASEL actuator. Current and voltage were monitored from the high voltage amplifier and input to the data acquisition system, allowing self-sensing of HASEL actuators.

The force of actuation can be significantly increased by combining multiple planar HASEL actuators. In this experiment, authors combined six planar actuators in parallel to test the work and strain. A gallon of water weighing 8.82 lbs was lifted, and a linear actuation strain of 69% was achieved.

In this experiment, the authors tested the ability of planar HASEL actuators to self-heal using a six-unit actuator. Voltage was steadily applied at 0.5 kV/s until dielectric breakdown occurred. After a minute, voltage was reapplied until dielectric breakdown occurred again. This cycle was repeated 50 times to evaluate the self-healing performance of the actuator.

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.

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.