Incidence is how much of something first happens within a given amount of time. There is another term called prevalence that is similar, but has an important difference. In diseases, for example, incidence is 'how much new disease there is over a certain time-period,' while prevalence is how much disease there is at a given point in time. This distinction is especially important for diseases that may last a long time. For example, if 100 people each year get HIV, the incidence is 100 new cases of HIV per year. However, because HIV does not go away, the prevalence, or number of people with HIV in a population at a certain time, will be much higher because it will include not just the 100 people who just got HIV, but also all of the people who still have HIV due to infections in previous years.

For example, you can see a graph of measles incidence in the US here. You will notice that it is plotted by when on the X-axis, and how many on the Y-axis.

The incidence of measles significantly drops after vaccination because this provides host resistance to the disease

Host resistance describes how well an individual (the host for the disease) can resist getting a disease (resistance).

For example, if you get the chickenpox vaccine, it is extremely unlikely you will ever get the chickenpox disease. This means that you have a lot of host resistance against chickenpox (because you got the vaccine). It would also be true if you got the chickenpox disease once, you would be resistant to getting it again. Both are examples of host resistance.

In this context, they are describing that the opposite is true. If you get the measles disease there is a prolonged effect decreasing your host resistance to other diseases for 2-3 years.

Keep reading to learn about how this could be the case, and why it is so important around the world.

Opportunistic infections describe pathogens (things that make you sick like bacteria, viruses and fungi) that take advantage of when people's immune systems are weak. Under normal conditions these opportunistic pathogens DO NOT make you sick, but if you have some problem that reduces your immune system's ability to protect you, like a recent sickness or malnutrition, then opportunistic pathogens can make you sick.

Opportunistic pathogens/infections are important in medicine because they can make treating sick people very complicated. For example, AIDS patients don't actually die because the HIV virus made them sick. Victims of AIDS most often die because their immune system is so damaged they cannot fight off opportunistic pathogens. Learn more about why these opportunistic infections are so devastating at AIDS.gov.

This and the subsequent reference discuss the concept of and the dangers associated with gene drives.

Gene drives are techniques that alter genes in such a way that biases their inheritance, making them spread more rapidly than normal through a population.

Mutagenic chain reaction is an example of a powerful gene drive method. They list several checkpoints that they recommend be completed before the use of gene drive in endogenous populations, such as designing and testing techniques for reversal of the gene drive and long term studies to fully understand the consequences of a gene drive strategy.

Because their strategy is so efficient and powerful, many people are raising concerns about whether or not such a tool should be used. It is a hotly debated issue as to whether or not this strategy should be used for research on human tissues in the future.

Additionally, can you imagine what the consequences would be if a research animal carrying an MCR mutation were to accidentally be released into the environment? Essentially 100% of the offspring of that animal would have that mutation, and it would propagate rapidly through the native population.

The authors used a technique called PCR to determine the genotype of F1 flies. They used primers (small DNA fragments that are the same sequence as DNA region of interest) to amplify the either the wildtype y+ locus or the y- locus (with the MCR insertion).

The presence of a DNA band after the wildtype reaction indicates that the fly has wildtype DNA. The presence of a band in either HA (homology arm) reaction indicates that the fly has DNA containing the MCR insertion.

A lab technique that uses short DNA sequences to amplify a specific region of the genome. These short DNA sequences are called primers. They anneal to genomic DNA and serve as a starting point for DNA polymerase to replicate the DNA at that location.

PCR applies multiple cycles of heating and cooling temperatures, creating the ideal conditions for many rounds of replication. This technique is often used to determine the genotype of an animal at a specific gene.

This YouTube video goes over the technique in more detail.

https://www.youtube.com/watch?v=iQsu3Kz9NYo

Although the authors found a strikingly high frequency of entirely y- flies (indicating that the MCR had been successful), they saw a few rare cases in which the flies were only partially y-. This suggests that the CRISPR-Cas9 MCR strategy doesn't work 100% of the time. Sometimes, the CRISPR gRNA doesn't successfully direct the Cas9 protein to the target region of the DNA.

Another potential explanation for the wild-type appearance of some flies is that the non-MCR allele was mutated in a way that made it impossible for the MCR allele to direct it's mutation.

Based on Mendelian inheritance rules, you would predict that a cross between a y- male and a wildtype female (as diagrammed in Figure 2A) would generate all brown progeny; females would inherit one recessive allele from their father and one wildtype allele from their mother, while males would inherit one widltype allele from their mother. But the authors found that nearly all of the female progeny were yellow!

For this to happen, the mutated allele the females inherited from their father subsequently mutated the wildtype allele they obtained from their mother, giving them two copies of a mutated allele and a yellow color. This is further support that the mutagenic chain reaction was successful.

After making their mutagenic chain reaction construct, the authors needed a way to test its efficiency at introducing homozygous mutations. So, they decided to design guide RNAs that would cause mutations in a gene called yellow because they can easily see if this gene was mutated by the color of the flies.

A fly must have 2 copies of the recessive yellow allele to have a yellow color. All other flies are brown. The wildtype allele is X-linked, so males must inherit the allele from their mother, while females inherit the allele from both parents.

This previous study gave Gantz and Bier confidence in choosing the fly yellow locus as the target of their MCR experiment.

The study by Port et al. demonstrated that this locus could efficiently be targeted by CRISPR/Cas9 and that mutations created by CRISPR/Cas9 could easily be detected by change in the color of the flies from brown to yellow.

CRISPR stands for "Clustered Regularly Interspaced Short Palindromic Repeats" and Cas9 refers to the protein that is associated with CRISPR. CRISPRs are DNA sequences that are transcribed into RNA.

Cas9 protein is guided to a specific DNA sequence by the CRISPR RNA. Because Cas9 is a nuclease, meaning that is cuts DNA, it cleaves the DNA at the sequence specified by the CRISPR RNA.

For more about CRISPR, see the following resources:

http://www.nature.com/news/crispr-the-disruptor-1.17673

http://www.nature.com/news/crispr-1.17547

http://www.sciencemag.org/content/347/6228/1300?intcmp=collection-crispr

http://www.sciencemag.org/site/extra/crispr/?intcmp=HP-COLLECTION-PROMO-crispr

http://www.technologyreview.com/news/543721/with-this-genetic-engineering-technology-theres-no-turning-back/

http://www.nytimes.com/2015/11/15/magazine/the-crispr-quandary.html?_r=0

http://www.nytimes.com/2014/03/04/health/a-powerful-new-way-to-edit-dna.html

External validity refers to the ability to say that we could expect these same results in other contexts.

When experiments are conducted in social settings, external validity is not something we can expect to acquire because different social, economic and cultural contexts would have different effects on the outcomes.

This study makes it clear that summer jobs do not reduce violent activity because jobs keep youth occupied.

If this was true, then rates of violence would be expected to return to normal rates soon after the program ends.

However, violent activity continues to drop during the 13 months after the summer jobs program ends.

So if social-emotional learning is capable of reducing violence when it's not part of a summer jobs program, how can we understand why it did not provide even more of an influence in this study?

The author suggests that even without the formality of social-emotional learning curriculum, these skills were still taught on the job, perhaps through the job mentors.

Another idea the author presents is that the act of working helps youth develop better skills for handing conflict.

An important part of interpreting results from data is to consider what other possible explanations might exist. This study concludes that summer jobs have a significant effect on reducing violent crime arrests and that this effect could be attributable to the preventative nature of the program.

Other explanations for why this summer jobs program might reduce violent activity include consideration of the mentorship provided in this program. Mentorship is unique to this program and has not been part of previous jobs programs.

Another possible explanation might be how crime was measured. This study considered crimes by type. Previous studies have only measured crime as a generic category. Because of that kind of measurement, those studies might have been constrained in their ability to detect an effect.

The contribution of this study is to show how a relatively short jobs program can have a substantial and significant effect on youth violent crime behavior.

Previous studies have focused on more long-term jobs programs that are very costly to implement. These previous studies have also been implemented in a remediating manner.

This study, by contrast, operates in a preventative manner. It shows how short and preventative oriented jobs programs can have a significant effect on reducing rates of violent crime among young adults.

One of the hypotheses about why summer jobs can reduce violent activity (the "incapacitation effect) is youth lack the time or opportunity to engage in violence.

If this was the case we would expect to see that arrest rates for violent crime would occur during and most immediately after the summer jobs program. The researcher tests this hypothesis and finds that the 13 months after the summer program has concluded have an effect on violence.

This suggests that the incapacitation effect is not at work in this study.

This study is testing whether or not replacing some work hours with social-emotional learning has a different effect on violent behavior than the effect of only participating in a jobs program.

The findings show that there is no statistically significant difference between the groups who only participated in the summer jobs program and those who participated in both the summer jobs program and the social-emotional learning.

Because of the significant finding that arrest rates drop for those involved in the summer jobs program compared to those who are not involved, this study is also prepared to ask why this might be the case.

Recall that the those youth randomly assigned to the treatment group were also randomly assigned to either take part in social-emotional learning or to not take part in social-emotional learning (SEL).

This random assignment makes it possible to also assess whether social-emotional learning plays any role in reducing violent crime activity.

The researcher is using previous studies on crime and social interventions to make the case for how she is proceeding with analysis.

Previous studies find that crimes originate in different contexts. Violent crimes tend to be conflict oriented. Nonviolent crimes tend to originate from economic circumstances.

She is therefore going to look at how the summer jobs program (One Summer Plus, or OSP) influences rates of crimes according to their violent or non-violent (property, drug, or other crimes) nature.

Proximal refers to something that is near to, or in close proximity to, a point of reference. Proximal is a term commonly used in research because research is always striving to explain cause and effect relationships.

Proximal causes are those causes that are thought to be part of the relationship, but are perhaps not the ultimate source of the relationship.

In this case, hostile attribution bias, or the problem of assuming other people's intent is hostile, even when it isn't, is thought to be the proximal cause.

Assignment refers to the process by which the study population is put into either the treatment or the control group.

Random assignment in this study refers to a technique in which the researcher placed participants into two groups (those who have the jobs program only and those who have the jobs program and the social-emotional learning) in a manner that ensures there are no pre-existing differences between the groups.

Any difference that can be found in the groups at the end of the study can therefore be attributed to the study, and not to pre-existing differences.

A randomized controlled trial is one of many ways to conduct a scientific experiment.

Many people are selected for the experiment, but only some people are randomly selected to receive the intervention that is being tested for.

These kinds of studies are often referred to as the gold standard. When pharmaceuticals are still in the development stage, randomized controlled trials are used to test the drug's effectiveness.

Violence can be considered a public health crisis not only because it can lead to mortality, but because those involved often end up needing health care services.

The Centers for Disease Control has identified youth violence as a public health crisis. They report that in 2008 more than 656,000 young adults between the ages of 10 and 24 were treated in emergency rooms for injuries related to violence.

In terms of mortality, homicide kills more young black males than the 9 leading causes of death combined.

Sewer infrastructure maintenance is a costly business:

https://ichemeblog.org/2014/08/31/combating-sewer-corrosion-day-96/

Sack Sulfates to Preserve Sewers:

http://www.scientificamerican.com/podcast/episode/sack-sulfates-to-preserve-sewers/

Figure 3 is composed of two y-axes:

1) The left one shows the cost as $ per m3 of water treated and is to be used for the blue-colored data.

2) The right one shows the % increase in cost and is to be used for the red-colored data.

Because the point of reference for increase in cost is "no alum dosing", % increase in cost starts from zero even though it in fact costs a certain amount of money.

In other words, the authors assume the increase in cost for "no alum dosing" as zero and calculate the rest according to:

$${(X-Y)}/{Y}*100$$

where; X=cost with alum dosing, Y=cost of no alum

The purpose of this test is to find out how much ferric chloride is going to be necessary to maintain the dissolved sulfide concentration below 0.5 mg S/L.

The test conditions were provided (shown in the Figure 3) as 0, 5, 10, and 15 mg S/L.

The results of this test showed that as sulfate addition levels increase, the amount of ferric chloride necessary to drop the sulfide concentration below 0.5 mg/L will increase. As a result, more ferric chloride use leads to more money spent on corrosion prevention.

A statistical term. Summation of all the elements divided by their number.

Researchers investigated the effect of sulfate-based coagulants in different scenarios:

1-changing source water sulfate concentration,

2-changing hydraulic retention time,

3-changing rising main (pipe that supplies water from ground to upper levels of a building) fraction,

4-changing sewage temperature.

They showed that important savings can be achieved simply by decreasing the use of sulfate-based coagulants (aluminum sulfate in this case) in all different scenarios except source water sulfate concentration.

The amount of sulfate-based coagulants did not really effect the cost of corrosion prevention when the source water has high levels of sulfate (above 10 mg S/L).

Figure 2 is a bar graph. It shows sulfate concentration on the y-axis and two different scenarios on the x-axis: 1) Without aluminum sulfate dosing during drinking water treatment,

2)With aluminum sulfate dosing during drinking water treatment.

The figure emphasizes that there is a clear difference between the source water and drinking water when the plants use aluminum sulfate during drinking water treatment.

The inset figure is drawn by taking the averages of source and drinking water values in two different scenarios.

Figure 1 is composed of a bar chart and an inset pie chart. It shows two different representation/information from the same data collection process when they monitored the sulfate concentrations in source water, drinking water, and sewage.

On the y-axis, sulfate concentration is shown. The x-axis contains the three data sources: source water, drinking water, and sewage.

Figure 1 draws the ideal water cycle by assuming a fixed sulfate concentration (no loss in the process) during the water cycle. It shows the increase in the sulfate concentration as water is collected from the source until its disposal as sewage.

If the total sulfate concentration in sewage (approximately 17.5 mgS/L as seen from the Figure) is 100%, the contribution of coagulant was calculated as 52% as in the inset pie chart.

Normally, the last step during drinking water purification is a disinfection step.

Chlorine is the most commonly used chemical.

Although effective in killing harmful pathogens, chlorine can react with natural organic matter and form harmful byproducts (e.g., trihalomethanes [THM], bromate, chlorite, etc.).

For additional information

Comes from the natural processes in the environment such as decomposition of organic matter.

Examples include proteins, amino acids, polysaccharides, and humic and fulvic acids, etc. (Reference)

The article connects to NGSS 8 practices for science and engineering Practice 8 and Science practices for AP Biology Practice 7 by explaining why the coagulation process is important in water treatment and relating it to the knowledge of the disinfection process.

The article connects to English Language Arts Standards 11-12.2 by summarizing how sulfide causes sewer corrosion.

Describes how C4.4, a human GPI-anchored protein, was found.

Identifies hAG-2 and hAG-3 (nAG homologs) as ligands for C4.4, a GPI-anchored protein in humans.

hAG-2 (an nAG homolog in humans) is expressed alongside an estrogen receptor in breast-cancer cell lines. The authors propose that hAG may play some role in cancer.

Acetycholine, a neurotransmitter for skeletal muscle, was once thought to be a molecule crucial in limb regeneration. However, this paper shows that when its release is inhibited, the limb still regenerates.

Experiments showing the effects of Prod1 overexpression in regeneration are shown. Overexpression causes distal blastema cells to take on a proximal identity and disrupt proper limb patterning.

This paper shows how Prod1 was discovered in salamanders and provides a basis for this paper, as nAG is ligand.

A research article that shows that vitamin A causes issues with the PD axis during regeneration of salamanders. This paper provides the basis for using vitamin A and other retinoids to disrupt proper limb regeneration.

That is, evolutionarily conserved or kept the same throughout the evolution of salamanders and humans.

A protein that contains a high number of cysteine amino acids.

The 3D tertiary structure of these proteins has not been solved.

Doing this would allow for one to compare how related their structures are at the tertiary level. This is as opposed to looking just at the amino acid sequence (primary structure), which may mislead one into thinking that they are very different.

However, it could very well be the case that the primary structures are very different, but the tertiary structures very similar.

Of note, the 3D structure of Prod1 has now been solved. See, Garza-Garcia et al. (2009) PLOS ONE e7123, and the interaction between mammalian AG2 and C4.4 appears important in models of pancreatic cancer (Arumugam et al., Molecular Cancer Therapeutics 14, 941-51 [2015]).

Human AG2 protein, which is similar to nAG, also likely binds to a GPI-anchored protein (C4.4).

This is very similar to nAG, which binds to Prod1, a GPI-anchored protein.

More than expected or normally found.

A sixth digit on a human hand would be supernumerary.

When cells of different proximal and distal (PD) origin are grafted together on the salamander the blastema still grows. Though, the tissues may not become what is supposed to (i.e., it is not specified correctly).

The experiment referenced had a distal wrist blastema grafted to a proximal shoulder stump. This results in formation of a normal limb regenerate, where the stump now forms the tissues between the shoulder and wrist, and the wrist blastema forms the hand

However, if the limb is denervated this does not occur.

The authors use this reference to suggest that nAG from the nerve is necessary for growth of the blastema even in situations where the PD gradient is abnormal.

Standard deviation. A statistical measure that helps demonstrate how far values are from the mean.

In this case, the mean is 8.3 and most values are expected to be within the range of five to 11.6 (+/- 3.3 from the mean).

How many times over the baseline

For instance, if Jenny has five flowers and Howard has 50, Howard has 10-fold more flowers than Jenny.

For that example, Jenny sets the baseline and Howard has some number-fold greater flowers than her.

A molecule that can be incorporated into DNA like thymidine (T). This is a nonnaturally occurring molecule, so it is useful for determining how often new nucleotides are being incorporated into DNA.

For an in-depth discussion of bromodeoxyuridine (BrdU) pulse labeling, see the annotation for figure 6.

A picture of BrdU versus dT (thymidine) can be seen here

This can be broken into three parts.

Mean: Average

Stimulation index: The ratio of cells in s-phase to the total number of cells counted.

S-phase: The portion of the cell cycle where DNA strands are duplicated or copied.

In other words, the average ratio of number of cells where DNA is being duplicated compared to the total number of cells.

Cos 7 cells transfected with nAG plasmid are expected to release nAG protein into the medium. This is an easy way for the authors to make nAG so that they can add it to limb blastema cells.

To do this, the authors allow the Cos 7 cells some time to secrete the nAG protein into the medium they are contained in and then simply transfer those media to their limb blastemal cells.

The control plasmid will not produce nAG protein and instead acts as control to show what happens when Cos 7 media is transferred to limb blastemal cells.

About 70% of the cells extracted from the limb blastema had Prod1 expressed on the cell surface. This is an interesting finding showing exactly how many cells would be under the influence of nAG in the living organism.

The cells were "maintained," or kept in medium containing 1% serum for the rest of their life span.

In vertebrate cell culture, most cells are grown in a medium containing "serum." Often this is fetal bovine serum.

The blood from a cow fetus is withdrawn and only the serum is kept. The serum contains proteins, nutrients, and other molecules, but does not contain any other cells.

The components of fetal bovine serum can vary greatly, but in general, its addition to cell culture medium is necessary for keeping cells healthy.

There is always a lot of "noise" in biology research. The amount of outside influences that could be affecting your results are boundless. Consequently, it can be hard to determine conclusions from this type of data.

To simplify things and reduce the noise, the authors have opted to use cultured blastema cells to further study nAG.

When deciding on an experimental strategy such as this, it is important to consider what is lost and gained by studying these cells outside of the organism.

Like Cos7 cells, newt blastema cells are able to be kept outside of the living organism in an artificial medium that provides nutrients, or in other words, kept in "culture."

Skeletal muscle, like limb regeneration itself, is dependent on a nerve being present. When the authors rescue the denervated limb with nAG they don't see normal muscle regeneration.

They conclude that nAG alone is not sufficient to regenerate the muscle tissue. So although nAG can regenerate the limb structure, it does not necessarily create it exactly as in an innervated limb.

This shows that nAG likely has no effect on the nerve itself, instead it has its effect by some other means. Not by simply making the nerve regenerate, which would go on to allow the limb regenerate.

This control animal has had both limbs amputated . However, the right limb was electroporated with empty vector.

This group of animals functions as a control in this experiment.

On both sides, i.e., both the left and right limb were amputated.

Often large structures are too large to perform immunostaining or other visualization techniques on.

To get around this the tissue can be "sectioned": cut into very thin layers. Often this tissue will be sliced into micrometer-thick slices (1/1000 millimeter).

The space outside the cell.

This was an important experiment to show. Focal point electroporation is by no means a 100% successful procedure. Some cells in that region will just simply not pick up the exogenous DNA.

Here the authors conclude that 30%–50% of cells in the region appear to take in the plasmid DNA. This information can help "temper" our expectations when looking at future data in their paper that use this technique. This may help explain variable results.

Furthermore, the authors show that expression of red fluorescent protein (RFP) lasts 3 weeks. This is important because this demonstrates that RFP is turned on for a long period of time and does not get shut off within minutes, hours, or days. If this were the case, one would expect much different results.

The authors showed that when the nerve was removed and the limb amputated, there was very little nAG present compared with the amputated limb with intact nerves.

This correlation between the presence of the nerve versus nAG presence suggests that nAG may be a molecule necessary for limb regeneration. Further follow-up studies needed to be performed to help support this idea.

You likely have heard of "differentiation," when a stem cell starts turning into a more specialized cell such as a skin cell or muscle cell.

Dedifferentiation is the exact opposite of that. Now, cells that were originally part of the limb start becoming more like progenitor cells.

These dedifferentiated cells can give rise to the cell type of origin.

The layer of skin below the epidermis (the top layer of skin). This holds many different types of living cells including nerves, vasculature, as well as fibroblasts, which make an important contribution to the blastema after limb transection.

For an example using human skin see here

A general term for any part of the body that secretes a substance (i.e., proteins). Remember nAG is likely a secreted protein, so a gland would be a reasonable place to find it.

In the normal (i.e., not amputated) limb the authors found that there was low staining for the presence of nAG. This was done using immunohistochemistry as described in the second figure's annotation panel.

What does this suggest about its possible functions/role? Why would we expect to see none/little of it considering the background information?

Antibodies can have enzymes linked to them. In this instance, an antibody had a phosphatase added to it. Because of that, wherever this antibody binds you will see phosphatase activity.

To exploit this the authors added a yellow solution made of two compounds, which in the presence a phosphatase has a phosphate group removed. This changes the yellow dye into a blue precipitate.

"Pentosan sulfate" cells, an immortal cell line derived from mouse brain cells.

Although it is definitely more complex than this, antibodies generally have an amino acid sequence that they bind heavily to.

This is likely not surprising because, as you can imagine, an antibody for the flu virus is not going to target herpes and vice versa, a herpes antibody will not target the flu.

Why might it be a good idea to test an immunoblot with two different antibodies?

Remember, nAG is a secreted protein. The myc-tagged nAG they have transfected should be secreted just like the wild-type protein.

Transfection of not cell cultures, but whole organisms is now a possible feat. Often this is done using a virus-based transfection system.

The virus contains the gene you want expressed in the organism and that virus then goes and infects cells. These viruses can be specifically designed to target certain organs like the liver or brain, so that only the organ you want has that DNA and is expressing that protein.

This process is becoming so safe and efficient that it is now being used in extreme circumstances on humans. If a person suffers from a disease caused by a mutation in a gene, a working version of it could theoretically be inserted into their cells using viruses.

Often this concept is lumped together under the broad term "genetic engineering," but is more appropriately termed "gene therapy."

Although this field has been plagued with many past disasters, new advances and better understanding of viral biology is making this a viable treatment for many diseases. The field of gene therapy is still recovering after the death of Jesse Gelsinger, among others.

The authors created transgenic cells using a product called lipofectamine. Transgenic cells contain DNA that is not normally found in that organism.

In this case the authors introduced plasmid DNA encoding myc-nAG. This expresses a nAG protein with an added "myc" epitope. See the earlier explanation of epitope-labeled proteins for further information.

Lipofectamine-based transfection (insertion of foreign DNA) is actually a fairly well understood phenomena. For a nice explanation of the theory behind it, please see the manufacturer's website

Often referred to as "coimmunoprecipitation." Protein complexes are able to be separated by binding an antibody to one of the proteins in that complex.

That antibody is usually bound to something heavy (agarose polysaccharides extracted from seaweed) or magnetic to allow for separation.

The authors demonstrated that Prod1 and nAG do in fact bind to each other. This is a major conclusion of their paper and is an important first study.

For the authors' background and follow-up experiments to make sense, these data needed to be shown first.

Epitope-tagged proteins have small stretches of amino acids added to the normal, wild-type protein. Often these additional amino acids create an epitope, an antibody-binding region.

This allows for the use of standard antibodies. Now, an antibody doesn't have to be created for each protein, only to the epitope tag, which usually can be added easily to many different proteins.

Note that in this paper the authors use both antibodies to the sequence of nAG itself, as well as antibodies to an epitope tag added to nAG. Why are they using both approaches?

An immortal cell line derived from mouse kidney that acts like fibroblasts (the cells that produce collagen in your skin, among other functions).

A common type of tertiary structure found in proteins. It gets it's name from protein thioredoxin, which has this type of fold in it.

A mucus secreting gland found at the head of frog embryos. It helps stick the frog to rocks and other substrates.

Many AG family proteins are found in glands and secretory epithelia.

Using yeast two-hybrid screening the authors determine that there are two secreted proteins that could potentially bind to Prod1.

Because the yeast two-hybrid screens can sometime give false positives, the authors followed up with experiments to confirm this in later panels of figure 1.

What is the significance of finding a secreted protein that binds two Prod1? Remember, where exactly do we find Prod1 in the cell? Why would a cytoplasmic protein probably be a false positive?

Yeast two-hybrid prey libraries are derived by reverse transcription (mRNA to cDNA) of total RNA extracted from the unamputated (normal) limb or the limb blastema.

Which RNAs are present is determined by what genes are expressed. The likely reason the authors chose to use RNA from normal limbs and the limb blastema is because they thought that the expressed genes would be different in each.

As a result of all this, the cDNA library for normal limbs and limb blastema are different (with some overlap). By doing this the authors increase their chance of finding prey for their nAG bait.

For more information on yeast two-hybrid screens, please see the annotation for figure 1.

For an overview of how cDNA libraries are made, please see the following diagram

N (amino) or C (carboxyl) terminals begin and end a protein, respectively. That is, there is a free NH2 at the start of a protein and a free COOH at the end of it.

Often there can be special sequences of amino acids shortly before or after these terminals. These often act as "zip codes" in that they tell the protein what to do and where to go.

For the case of Prod1, the N terminus signals for secretion and the C terminus signals for anchoring and modification with GPI.

The authors in this experiment wisely chose not to include both these sequences in their yeast two-hybrid screen. Yeast are eukaryotic cells and can perform many of the same functions as verterbrate cells.

In turn, leaving these sequences on may very well have caused the Prod1 protein to be secreted. This is not something you want for experiment that relies on your protein being in the nucleus!

Many papers that are limited in word count, such as the ones in Science and Nature, often cut to the chase and state their major conclusions at the end of the introduction or background section. This acts as a "hook" for readers to look at the data supporting these claims.

Remember, the authors had previously found that Prod1 is a plasma membrane–bound protein.

This is really the main question the authors are trying to answer. Which molecule, if any, is producing the effect seen in the previous experiments?

Motor neurons are necessary for controlling skeletal muscle tissue and sensory neurons for sensing the environment (i.e., temperature, pain, etc.).

Nerve impulses from motor neurons travels toward the periphery of the organism to control muscle function.

Nerve impulses from sensory neurons travel toward the central nervous system.

Previous data from JP Brockes have shown that blastemal cells do, in fact, form in the denervated limb. However, the main observation from his paper is that these cells do not appear to grow or proliferate. There appears to be something special about the nerve that makes it necessary for the blastemal cells to function.

Specialized epidermal tissue surrounding the area where the limb has been removed. This tissue is found adjacent to the mesenchymal progenitor cells.

The region and environment where stem cells responsible for forming the new limb are found.

These cells are supported by local signals from other cells and tissues, which ensures that stem cells in this location remain stem cells.

Focal electroporation allows for introduction of exogenous DNA into a small region of tissue. To do this, a pulse of electricity is spread across the region of interest. This generates holes in the plasma membrane of the cells, which allows the DNA to enter into the cell.

In this case Echeverri and Tanaka introduced a Prod1 overexpressing plasmid into distal cells of the larval axolotl blastema. Normally the cells here would not be expressing as much Prod1 as the proximal cells, but by electroporating in this plasmid these cells now start expressing a lot more Prod1 protein.

The end result of this is cells becoming more proximal-like rather than becoming distal.

There is higher expression (i.e., more protein) in the proximal limb and the farther distal you travel the less-and-less Prod1 expression you find.

Lymphocyte antigen 6 (LY-6). A superfamily of proteins found in a variety of organisms that often share the characteristic of being GPI-anchored.

The function of these proteins is extremely diverse. They have a 3D structure that allows them to interact with a large variety of other proteins. In turn, this provides them with a large array of potential functions.

In other words, a determinant of what makes the structure distal or proximal.

Unlike embryonic stem cells (ESCs), mesenchymal stem cells are a type of multipotent stem cell. That is, they cannot become every single type of cell found in the body, but they can become a smaller subset of cell types.

These cells are able to become mesenchymal tissue such as cartilage, muscle, and connective tissue.

Salamander is a broad term for "lizard-bodied," amphibious animals. They include such animals as axolotls and newts and are sometimes referred to as urodeles.

Of note is that salamanders have had a rich history of regeneration research. This is not surprising considering the amazing regenerative abilities of many species within this order.

In fact, this field appears to be in a "renaissance." Because of the massive amount of new molecular and genetic techniques becoming available it is now possible to answer questions that were once unanswerable.

A good overview of limb regeneration, specifically in salamanders. Discusses some molecular mechanisms in regards to limb specification and muscle regeneration.

AP Biology Essential knowledge 3.A.2.a.2: The cell cycle is directed by internal controls or checkpoints. Internal and external signals provide stop-and-go signs at the checkpoints.

AP Biology Essential Knowledge 3.A.2.a.3: Cyclins and cyclin-dependent kinases control the cell cycle.

AP Biology Essential Knowledge 2.B.3: Eukaryotic cells maintain internal membranes that partition the cell into specialized regions.

This article discusses the use of genetic profiling in tumors to determine the ideal course of treatment, something that has gained increasing attention as our sequencing technology improved.

This article provided evidence that p21 might play a role in the G2-M checkpoint in addition to its established role at the G1-S checkpoint.

Additionally, the paper looked at the protein Rb (retinoblastoma) and its role in checkpoint activation. Rb prevents cells from entering S phase when signals from growth factors are absent.

This paper showed that the levels of p21 normally oscillate throughout the cell cycle. However, in cells that did not have functional p53, no p21 was detected, implying that p21 expression is regulated by p53.

This paper discusses research on the process of centrosome duplication.

This paper established a role for p53 in the regulation of centrosomes, which enable the cell to separate its chromosomes during mitosis. Cells without functional p53 had abnormal numbers of centrosomes, leading to unequal chromosome segregation.

This paper looked at the role of the protein SMAD4 in a pathway called TGF-β signaling. The authors used homologous recombination to disrupt the SMAD4 gene.

This article investigated the G2-M checkpoint in irradiated colorectal cancer cells and found that p53 induced the expression of a protein called 14-3-3, which triggers G2 arrest.

This paper showed that mitotic checkpoints are often defective in cancer cells, leading to the chromosomal instability that may drive carcinogenesis (the development of cancer).

This paper described the protein geminin, which prevents DNA replication from occurring more than once per cell cycle. Geminin accumulates after DNA replication to block repeated replication, and it is degraded in mitosis so that the cell can replicate its DNA in the next S phase.

This article looked into the survival of different cell lines treated with radiation. The authors observed the appearance of "giant cells" that had undergone extra rounds of replication, which in turn generated multiple nuclei.

This paper showed that when cells lacking p21 were treated with DNA-damaging agents, they frequently underwent extra rounds of replication, which led to deformed nuclei and cell death.

This paper established a role for p21 in senescence, an age-dependent decline in the cell's ability to divide.

This paper showed that p21 functioned downstream of p53 in the activation of the G1-S checkpoint.

This review discusses what was known at the time about the regulation of the cell cycle and how we could potentially use this information to design new cancer drugs.

Southern blotting is a technique that uses radioactive probes to detect DNA sequences. The probes will only stick to complementary DNA, enabling researchers to look for very specific sequences.

Here, the authors are using it to confirm that cells have incorporated a foreign DNA sequence.

Lipofectamine is a lipid-based compound that helps cells take up foreign DNA, a process called transfection. Researchers can generate a DNA sequence, combine the DNA with lipofectamine, and add this mix to the cell culture. The foreign DNA will be able to enter the cell and code for a protein of interest.

What happens when a cell has abnormal p53?

This video explains how p53 protects us against cancer by identifying DNA damage:

https://www.youtube.com/watch?v=XfyfcBEQkCk

This paper had important implications for cancer treatment. p53 is required to stop the cell cycle and allow cells to repair DNA damage. Many cancer cells have mutated versions of p53 that are unable to activate this cell cycle arrest. As a result, DNA damage that normally causes cells to arrest and fix the damage may be ignored in cancer cells.

Therefore, if we treat a patient with DNA-damaging agents, their normal cells will stop dividing and repair the damage, but the cancer cells will keep going. DNA replication or cell division in the presence of major DNA damage can be lethal to the cell. So, in theory, the treatment selectively kills only cancer cells.

Unfortunately, though, it is not that simple. In practice, such drugs have many effects on both normal cells and cancer cells, beyond the ones that impact the cell cycle.

Another checkpoint in the cell cycle occurs during mitosis. This checkpoint prevents division until all chromosomes are correctly attached to the mitotic spindle, a network of microtubules that pull sister chromatids to opposite sides of the cell.

When cells are treated with nocodazole, which prevents chromatid attachment, they arrest at the spindle checkpoint. The authors conclude that the two proteins do not play a role at this checkpoint, because cells that lack p53 or p21 still arrest when the attachment is disrupted by nocodazole.

Previous work has clearly shown a role for p53 and p21 in the G1-S checkpoint, but there have been interesting results that suggested a potential role in the G2-M checkpoint.

This paper was able to explain those results by investigating this G2-M role.

The authors have conclusively shown that p53 and p21 are important regulators at the G2-M checkpoint. Previously, these proteins were thought to only play an important regulatory role at the G1-S checkpoint.

The initial separation of two cells that occurs at the end of mitosis.

A structure in animal cells that is involved in mitosis. During cell division, two centrosomes are located at opposite ends of the cell, and they extend fibers called microtubules that pull the chromosomes apart at anaphase. If there are more than two centrosomes, the chromosomes may not be evenly divided among daughter cells, which could lead to cell death or the development of tumors.

Also, centrosomes are duplicated simultaneously with DNA during S phase. So, if there are more than two centrosomes, there is likely also an excess of DNA generated by extra rounds of replication.

The critical point in cell division when the sister chromatids are physically pulled apart to opposite sides of the cell.

These types of experiments can monitor the level or location of a protein over the course of minutes, hours, or even days.

Here, the authors tracked a fluorescently labeled histone protein for 36 hours, taking pictures at specific intervals (see Figure 5). This allowed them to detect defects in mitosis when p53 or p21 was removed.

Here, the authors used a combination of two proteins fused together: (1) a histone protein (H2B), which is found only in the nucleus of a cell and helps organize the DNA, and (2) a green fluorescent protein (GFP), originally found in jellyfish, which glows green in response to a particular wavelength of light.

This combination enables scientists to watch the nucleus of a cell, because only H2B will glow green.

The authors incorporated foreign DNA into cells with the drug resistance gene. The resistance allows cells that contain the foreign DNA to survive treatment with the drug, while other cells will die.

Connective tissue cells that produce the extracellular matrix and collagen, which hold tissues together and help repair tissue damage.

The parental cells are those that did not have disrupted p53 or p21 genes. It was important that the authors used the same cell line as a control because different cell lines have different features that could influence the studied effect.

This result supported the conclusion that p53 and p21 are important in preventing cells from entering mitosis in the presence of DNA damage.

The authors introduced a piece of DNA that is similar in sequence to the p53 gene and the surrounding area. But instead of p53, this DNA encodes a resistance gene that allows a cell to tolerate a toxic drug. The cell recognizes the "p53-like" DNA, triggering a repair response.

Homologous recombination replaces the normal p53 gene with the antibiotic resistance gene. But only a small number of cells will undergo this replacement. After treatment with the drug, though, only cells that have incorporated the resistance gene will survive. This allows scientists to isolate only the cells that incorporated the resistance gene (and are therefore lacking the p53 gene). They now have a line of cells without p53, and they can use this line to study the role of the gene.

Previous research found that p21 triggers cell cycle arrest by inhibiting proteins called cyclins, which regulate progression through the cell cycle. Specifically, cyclin B1 and cdc2 form a complex that initiates entry into M phase (mitosis). p21 prevents mitotic entry by inhibiting this complex.

A protein that can increase or decrease the expression of a particular gene in response to a signal. In this case, activated p53 increases the expression of p21.

The authors ran a western blot (a technique to separate and identify specific proteins) to analyze the levels of p53 and p21 protein (see Figure 4A).

They authors found that in cells lacking p53 (the last two lanes on the right), there is a small amount of p21 in cells that were subjected to radiation. This tells them that something other than p53 must be turning on expression of p21 in response to DNA damage.

As a control, the authors disrupted a gene that is involved in a different pathway than p53 (the Smad4 gene) to make sure that gene disruption itself did not affect their results.

p53 is a transcription factor that can affect the expression of many target genes. Thus, when researchers look at cells lacking p53, it can be difficult to determine exactly what target genes are responsible for any observed defects.

The authors conclude that the absence of functional p53 allows cells to eventually enter mitosis (M phase) despite DNA damage. Although the authors observed a brief arrest in cells with mutant p53, the arrest was only temporary, and the cells eventually progressed to M phase.

Programmed cell death. In mature organisms, this mechanism eliminates unhealthy or damaged cells. During development, it removes unnecessary cells.

The genetic makeup of a cell at the DNA level as opposed to "phenotypes," which are the observed traits of a cell or an organism.

This gene codes for a protein that provides resistance to a harmful drug, typically an antibiotic. Scientists can transfer this gene to new cells to make the cells resistant to a drug, allowing these cells to be selectively propagated.

A targeting vector is a piece of foreign DNA that can be taken up by a cell. The goal is often to incorporate the sequence in the host cell genome.

Recall that cancer cell lines can carry lots of mutations and other changes in important regulatory genes (see above).

The researchers had to be careful when choosing a cell line to study because there could be additional factors other than p53 status (so-called "confounding variables") that would affect their results. So they started with a cell line that had p53 genes and various other checkpoint genes intact, and then disrupted only the p53 gene by homologous recombination.

A process of exchanging sections of DNA between two similar DNA sequences that cells can use to repair broken DNA.

In molecular biology, scientists can take advantage of this process to replace specific genes (targets) with modified DNA sequences.

Because the p53 mutant cells were progressing into mitosis when they shouldn't be, the researchers concluded that in cells with intact p53 genes, p53 must be responsible for preventing mitotic entry in the presence of DNA damage.

A measure of the amount of cells in a population that are currently undergoing mitosis.

Microtubules are tubular protein structures that are a component of the cell's skeleton (the cytoskeleton). They are also important for cell division, during which they pull sister chromatids apart in anaphase.

A drug that causes cells to arrest in M phase, right before cell division. It is often used in experiments to collect cells in M phase, either to study mitotic pathways or to make sure that all cells in a culture are synchronized in the same phase.

The nonmitotic portion of the cell cycle (G1, S, and G2 phases) during which a cell grows and replicates its DNA.

Growth and replication are followed by division, the mitotic (or M) phase of the cycle.

All of these cancer cell lines were derived from patients. One of the most interesting stories about cancer cell lines concerns a woman named Henrietta Lacks. Her cells (aka "HeLa" cells) were essential in developing the polio vaccine and were used in many other scientific landmark studies.

Read more in Smithsonian Magazine: http://www.smithsonianmag.com/science-nature/henrietta-lacks-immortal-cells-6421299/?no-ist

Here, the authors wanted to compare cells with normal p53 with cells with mutant p53. They treated each of the six lines with radiation to induce DNA damage, and then looked at what happened to the different cells. In this way, they could investigate the role of p53 in the DNA damage response.

Researchers can take a sample of cells from a patient and grow them in a dish. These cells are called a "line," and can be repeatedly grown and studied for years.

Many different lines are commercially available for researchers. Because each line comes from a different person and a different tissue type, they will all have different characteristics. Particularly with cancer cell lines, there will often be mutations in important genes, so it is critical to select a line that is appropriate for what you are studying.

These are proteins that initiate DNA replication.

Replication must occur once and only once during each cell cycle. Therefore, the licensing factors are tightly controlled (spatially and temporally) so that replicated DNA does not replicate a second time before cell division.

In the G2 phase, cells have already duplicated their DNA, resulting in four sets of chromosomes (or 4n). If a 4n cell mistakenly undergoes another DNA replication before it divides, it will have a DNA content of 8n.

This has been shown to occur in some cases when cells were missing p53 or p21.

A stop in the cell cycle before the cell progresses to the M phase where mitosis takes place. This occurs as a result of the activation of a checkpoint at the end of G2.

A cyclin-CDK (cyclin-dependent kinase) complex is what drives a cell through different phases of the cell cycle.

Previous work has shown that the p21 protein inhibits this complex at the G1 checkpoint, thereby preventing DNA replication in the S phase.

A stop in the cell cycle before the cell progresses to the S phase where DNA is replicated. This occurs as a result of the activation of a checkpoint at the end of G1.

Checkpoints are specific points in the cell cycle at which the cell must confirm certain characteristics, such as its size or DNA content, before passing to the next phase. This is to ensure that cells do not replicate DNA or divide when major problems exist, such as DNA damage.

As this paper explains, p53 plays a very important role in checkpoints.

Last phase of the cell cycle during which mitosis (cell division) occurs. The cell divides into two daughter cells that each have the same number of chromosomes.

First gap phase of the cell cycle, between mitosis (M phase) and DNA replication (S phase). Cells typically grow in size during G1.

A checkpoint toward the end of this phase ensures that the cell has no DNA damage to move on to the next phase (the S phase).

"G" is short for "gap."

Phase of the cell cycle during which DNA replication (synthesis) occurs. "S" is short for "synthesis."

Scientists have been trying to understand how cells respond to and repair DNA damage because many cancer treatments work by causing DNA damage to cancer cells.

Cytokinesis occurs during the M phase of the cell cycle and refers to the division of the cytoplasm during cell division.

Cyclin-dependent kinases (CDKs) are important cell cycle regulators. Kinases are enzymes that add a phosphate to other proteins to activate or inhibit their function—a process known as phosphorylation.

CDKs are always present in the cell but become active only when they are bound to other proteins called cyclins. Cyclins were originally identified as proteins whose concentration cycles up and down, depending on the phase of growth. Not all proteins in this class fluctuate in this manner. Regardless, the association of CDK-cyclin complexes regulates the progression through each phase like clockwork.

Some additional regulatory proteins activate CDK-cyclin complexes, stimulating the cell cycle. Others inactivate CDK-cyclins or prevent their activation, which inhibits cell cycle progression. Among the inhibitory proteins are p53 and p21.

p53 is an important protein that is inactivated in many cancers. p53 can sense DNA damage, and accordingly causes the cell cycle to arrest.

The arrest of the cell cycle allows for the DNA to be repaired. Alternatively, p53 can initiate cell death, or apoptosis, in response to DNA damage.

An early review of microglial origin in birds.

This study showed that invasion of embryonic tissue by yolk sac macrophages in zebrafish fails in the absence of macrophage colony-stimulating factor-1.

The authors of this study generated the RUNX1 fate mapping model used in this study and showed that yolk sac cells expressing Runx1 can develop into fetal lymphoid progenitors and adult hematopoietic stem cells (HSCs).

As shown in this paper, microglia are not affected by the autosomal recessive mutation osteopetrosis (op) that leads to a general skeletal sclerosis and decreased numbers of macrophages in various tissues.

A review of the role of the colony-stimulating factor-1, also known as macrophage stimulating factor, in immunity and inflammation.

This study suggested that microglia derive from yolk sac progenitors and actively proliferate in situ.

This study examines the origin and differentiation of dendritic cells.

A review of the developmental origins of hematopoietic stem cells and the molecular mechanisms that regulate lineage-specific differentiation.

In this review of the studies on the origins, phenotype, and function of embryonic phagocytes the authors conclude that yolk sac–derived macrophages constitute a separate lineage.

This study showed that limited recruitment of cells by the recipient in parabiotic models can be attributed to a short half-life of the donor-derived cells in the blood.

In this study the authors identified a unique population of peripheral Ly-6C(hi)CCR2(+) monocytes, which are preferentially recruited into the lesioned brain following irradiation.

Using parabiosis and irradiation in an experimental autoimmune encephalitis mouse model of multiple sclerosis, the authors found that peripheral monocytes infiltrate the brain and contribute to the progression of the disease but do not contribute to the pool of resident microglial cells.

Yet another early study supporting the hypothesis of microglial turnover via division and recruitment of peripheral monocytes into the brain parenchyma in the absence of the overt blood-brain barrier disruption.

One of the earlier studies of microglial origin, supporting their monocytic origin.

This is a comprehensive review of the ontogeny and functions of microglial cells.

The study is also described here:

http://phys.org/news/2010-10-scientists-brain-immune-cells.html

Pial is an adjective derived from"pia mater," the delicate, fibrous, and highly vascular membrane forming the innermost of the three coverings of the brain and spinal cord.

Microglia observed in the adult brain under nonpathological conditions trace their origin to yolk sac macrophages.

In contrast to previous reports that relied on bone marrow irradiation that created experimental artifacts, this study showed that the pool of brain microglia arises from the yolk sac macrophages and is not replenished by circulating blood cells in an adult organism.

The influx of yolk sac macrophages into the brain occurs between embryonic days 8.5 and 9.5.

Na+/Ca2+ exchanger 1

Using a mouse model knock-out for a sodium calcium exchanger 1 gene (Ncx-1), the authors were able to show that established and functional vasculature is necessary for the migration of yolk sac macrophages into the brain.

Colony-stimulating factor-1 receptor.

This indicated that yolk sac progenitors infiltrate the brain via a developed and functional vascular network.

In this experiment the authors again crossed two knock-in mice strains.

The first one had CRE fused to an estrogen receptor and expressed under the Runx1 promoter as described previously.

The second knock-in strain was similar to the strain expressing eYFP downstream of the ROSA26 transcription start site described above, but instead of using eYFP, the authors used another reporter gene called LacZ. LacZ encodes the protein β-galactosidase (β-gal), which can be easily detected by immunohistochemistry.

In the ROSA26 lacZ reporter (ROSA26R) mouse, a transcriptional stop cassette flanked by loxP sites was inserted just downstream of a transcription start site at ROSA26 locus. When intact, this cassette prevents expression of β-gal from the downstream lacZ coding sequence. The stop cassette is excised upon exposure to CRE recombinase, which mediates recombination between the loxP sites, thus permitting expression from the lacZ reporter.

The activity of galactosidase can easily be detected with single-cell resolution by staining tissues with X-Gal. Thereby, CRE activity eventually results in a blue dye precipitate.

This indicates that blood cells such as granulocytes and lymphocytes, as well as others tissue macrophages, do not arise from the same Runx1 yolk sac progenitors as microglia.

The partial labeling would explain about 60% of F4/80-positive cells not expressing YFP (Figure 3, panel A: brain rudiment).

This indicated that the obtained results could not be attributed to the confounding effect of the genetic model used by the authors.

DNA recombination involves the exchange of genetic material either between multiple chromosomes or between different regions of the same chromosome.

The authors used two transgenic mouse strains.

The first strain expressed a CRE recombinase protein fused to two murine estrogen-receptor ligand-binding domains (MERs) downstream of the promoter of the Runx1 gene. That ensured that CRE would only be expressed in Runx1-positive cells and will be functional only following its activation with the estrogen receptor antagonist tamoxifen.

The second strain of mice had enhanced yellow fluorescent protein (eYFP) cDNA knocked-in (inserted) downstream of a transcription start site at the ubiquitously expressed ROSA26 locus. A transcriptional stop cassette flanked by loxP sites was inserted between the transcription start site and the eYFP cDNA. When intact, this cassette prevents expression of eYFP. The stop cassette is excised upon exposure to CRE recombinase activated by tamoxifen, permitting expression of the eYFP reporter gene.

After crossing these two strains of mice, the authors injected pregnant dams with 4-hydroxytamoxifen, which is an active metabolite of tamoxifen and selective estrogen receptor antagonist. It bound to the estrogen receptor in the MER-CRE-MER fusion protein construct, which then entered the nucleus allowing CRE to act on the floxed genes.

This recombination resulted in progeny that expressed eYFP in Runx1-expressing cells.

In genetics, a locus (plural loci) from the Latin word for "place" is the specific location of a gene, DNA sequence, or position on a chromosome.

In genetics, a promoter is a region of DNA that initiates transcription of a particular gene.

Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (toward the 5' region of the sense strand).