The middle cerebral artery is one of the major blood vessels that supplies blood to the brain, occlusion of this would create loss of blood to the brain and thus mimic stroke. This is a method to induce stroke experimentally.

Injury when blood supply to tissues being restricted leads to shortage of oxygen in the tissue, such as during a stroke, where blood supply to brain is restricted.

The authors examine other tissues to show that wherever they observe neutrophil-mediated inflammation it is regulated by PSGL-1. Also, functions of neutrophils (such as formation of extracellular traps that can bind pathogen) are also regulated by PSGL-1.

These experiments provided evidence that interactions of neutrophils with platelets occurs via PSGL-1 and in its absence, recruitment of neutrophils to tissue decreases, and thus inflammation induced because of neutrophils decreases.

Using CT, the authors observed that in mice deficient for PSGL-1, the accumulation of fluid in the lungs (pulmonary edema) during ALI was reduced.

The authors extended the observation of neutrophil-platelet interaction via PSGL-1 during ALI. By inhibiting PSGL-1, they observed that mice were protected from ALI-induced death as the interaction between neutrophils and platelets was inhibited.

Lung blood flow.

Inflammation of the blood vessels.

The authors used another model of pathogenic inflammation. They injected a higher dose of LPS into the blood stream to induce endotoxemia (toxins in the blood). This can lead to sepsis because of drastic activation of the immune system.

The authors confirmed the results obtained earlier with TNF-a–induced inflammation was consistent even during pathogenic inflammation, such as ALI.

Acute Lung injury includes several different symptoms that ultimately lead to excess inflammation in the lungs followed by loss of the ability of the lungs to exchange gases. Functional capacity of the lungs diminishes. Sometimes, during blood transfusions, the presence of donor antibodies may attack recipient leukocytes and this can lead to a cascade of immune activation leading to neutrophil influx into the lungs and release of toxic agents. This causes fluid accumulation in lungs and manifests in several symptoms of ALI.

http://www.transfusionmedicine.ca/articles/transfusion-related-acute-lung-injury-trali

Inflammatory response to a pathogen or an insult. In this paper's context, it also refers to injury-induced inflammation.

The authors reach this conclusion because the original localization of the chemotactic receptor (CXCR2) and adhesive receptor (MAC-1) is disturbed in PSGL-1–deficient mice.

Previously, the authors described that neutrophils from Mac-1–deficient mice and PSGL-1–deficient mice show reduced crawling.

However, inhibiting PSGL-1 in Mac-1–deficient mice (these neutrophils are now deficient in both) did not result in an additive reduction in crawling. This might be possible if signaling through these receptors leads to the same pathway downstream.

The original distribution of these receptors was disturbed in the PSGL-1–deficient mice.

CXCR-2 is a receptor that binds to interleukin 8 (called KC in mice; secreted signaling proteins molecule). This activates neutrophils. Chemokines are secreted signaling molecules that attract cells to an area because of its gradient (mimicking chemoattraction).

Without intact signaling through PSGL-1, neutrophils were unable to migrate into the blood vessels. The loss of signaling through PSGL-1, however, did not affect the polarization of neutrophils.

Cytoplasmic domain is the part of the receptor that is present inside the surface of cells. On stimulation at the surface, the transduction of signal occurs through the cytoplasmic domain/tail that further instructs the cell.

Because of lack of Cdc-42, neutrophils were not able to interact with platelets and their crawling ability was affected.

Guanine nucleotide exchange factor belonging to the family with Rac GTPases that functions in a similar manner to Rac GTPases, described earlier.

Mx-1Cre-Transgenic mice are used to create inducible Cdc42 knockout mice. These mice have hematopoietic cells that are deficient in Cdc42.

Read more on the transgenic mice used:

http://www.informatics.jax.org/allele/MGI:2176073

Read more on inducible gene targeting:

http://www.sciencemag.org/content/269/5229/1427.long

Ref: A Guide to Methods in the Biomedical Sciences. By Ronald B. Corley. Page 113: Description on bone marrow chimeras

Besides using a gene knockout, the authors also used a model where depleting platelets was in their control. This was done by treating the mice with antiplatelet serum.

It is important to support data from gene knockout experiments with blocking experiments to be able to translate one's findings to humans (we will not find gene knockouts in humans).

Just prior to imaging the mice, the authors injected through IV an antibody that blocks PSGL-1.

It has been shown before that without Mac-1 neutrophils are unable to crawl on the vasculature.

The behavior of the neutrophils in the vasculature. In this study, this includes the mobility of the neutrophils.

The authors were able to conclude that the interaction between neutrophils and platelets occurred because of recognition of PSGL-1 on neutrophils and P-selectin on platelets.

In order to visualize some of the molecules, the authors inject dyes/antibodies with a fluorescent color tagged. These will specifically target the molecule of interest. (In vivo means inside the animal.)

The PSGL-1–bearing end of the neutrophils were in a way poking through the blood vessel wall into the inside of the vessel (luminal space). This positioned the cells in an ideal way to interact with cells in circulations (flowing through the blood vessel).

Spinning disk microscopy rapidly captures multiple images in real time, up to 10,000 frames per second. The authors utilize this technique of IVM to capture multiple images to construct a 3D image of cells.

http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html

Inside space of a blood vessel.

Rac GTPases are known to be involved in cell motility and cytoskeletal dynamics in a cells. Thus, regions where they observe Dock-2 fluorescence would suggest "active structural dynamics."

In this case, the authors tagged Dock-2 protein with GFP that allows them to view the molecule through the fluorescence emitted.

They observed Dock-2 fluorescence on sites where PSGL-1 formed clusters.

Rac GTPases are a class of G proteins that respond to a stimulus and transmit a signal from outside of a cell to its inside. They are usually in an "off" or inactive state when bound to a molecule called GDP (two phosphate groups).

Dock-2 helps Rac GTPasq2es in the exchange of GDP to GTP (three phosphate groups) so that the GTPase is now in the "on" or active state to transmit signal.

Fluorescently labeled antibodies to Mac-1 and PSGL-1 were injected into the mice so as to trace these molecules by IVM based on the fluorescence they emit.

As a positive control, the authors use mice that lack another cell adhesion molecule, Mac-1. This molecule is required for neutrophils to interact with other cell types at both the leading edge and the uropod.

To confirm whether PSGL-1 on neutrophils is what is mediating these interactions, the authors see what happens in the absence of PSGL-1.

To test the requirement of a molecule, researchers generally question what happens in the absence of the molecule. Thus, they generate knockouts.

http://www.powershow.com/view/24ca0d-ZjkwN/What_is_a_Knockout_Mouse_powerpoint_ppt_presentation

This is done by completely removing the gene that encodes the molecule.

Through IVM the authors observed that the uropod of neutrophils was constantly bumping with the platelets and some of these collisions lasted long enough for them to measure.

CD62L is a type of selectin found on lymphocytes. It is required for lymphocytes to move from circulation through the endothelial cell lining into tissue. CD62L on lymphocytes bind to ligands on endothelial cells, particularly those present in lymph nodes, to facilitate this movement.

Glycosylation is the process of adding a sugar molecule to a protein or lipid.

The uropod is defined as the tail end of a cell.

Motile cells develop a leading front that is made of actin filaments. Actin is a protein that is crucial for cell motility. Lamellipodia behaves like the engine of a motile cell pulling it forward.

Blocking the interaction of neutrophils and platelets that lead to neutrophil migration and inflammation protected mice from excessive injury in the blood vessels (thrombo-inflammatory injury).

Neutrophils are also called white blood cells. These are the first cells that respond to an infection.

They are normally found in the blood vessels, but on chemical induction owing to an infection, they migrate out of the blood vessels and into the site of infection.

Interaction between neutrophils protruding into the blood vessel and platelets inside the blood vessel via PSGL-1 initiated a signaling cascade that allowed neutrophils to redistribute their cell surface receptors, polarize, and eventually migrate.

For mobility of cells, polarization is a process that marks a cell with a front and tail end. The two ends usually differ in the arrangement of proteins (receptors) on the cell surface.

Thus, polarization of cells usually results in the redistribution of receptors.

For more information on Cyclone Klaus, click here.

And for access to the report that is cited here by Gardiner and colleagues, click here.

Hansen and colleagues found that the rate of forest loss in Brazil has slowed considerably from 2000 to 2011 (Figure 3).

This effect is likely the result of Brazilian laws such as the Forest Act, which protects both publicly and privately owned land (e.g., farms) from development (e.g., the trees cannot be removed for slash-and-burn farming).

However, in 2012 measures were passed to change the Forest Act to open up a large portion of this land for development. Find more information here.

Here, Hansen and colleagues show how science (in this case, imaging data and processing) can inform policy, and thereby show just one way that science and society are intertwined.

The United Nations Programme on Reducing Emissions from Deforestation and Forest Degradation was started in 2008 to reduce carbon emissions through forest degradation.

Yet, this program only works with tropical forests and does not regulate temperate or boreal forests.

Find out more here.

An abstract is a short overview of the experiments and results found in a paper. In this case, the abstract outlines Hansen and colleagues' work on mapping forest cover changes across the globe through time.

Science is interdisciplinary. Take a look at the authors who worked together on this project. Some of them are from remote sensing fields at various universities, whereas others are computer scientists and engineers at Google.

Authors:

Matthew C. Hansen (remote sensing scientist at University of Maryland, and associate team member of NASA's MODIS Land Science Team)

Peter V. Potapov (remote sensing scientist at University of Maryland)

R. Moore (Google)

M. Hancher (Google)

S. A. Turudanova (University of Maryland)

A. Tyukavina (University of Maryland)

D. Thau (Google)

S.V. Stehman (Syracuse University)

S. J. Goetz (Woods Hole Research Center)

T. R. Loveland (U.S. Geological Survey)

A. Kommareddy (South Dakota University)

A. Egorov (South Dakota University)

L. Chini (University of Maryland)

C. O. Justice (University of Maryland)

J.R.G. Townshend (University of Maryland)

Also, if you are interested in taking a closer look at the results, the data from this paper are featured here.

Hansen and colleagues verify the changes in forest cover that they saw in the Landsat images by exploring news on natural disturbances.

In this case, they found that drastic and instantaneous reductions in forest cover in southern Sweden in 2005 and southwestern France in 2009 were caused by cyclones.

Brazil has historically had high rates of deforestation (e.g., cutting down the Amazon rainforest for agriculture and logging), however Hansen and colleagues show that this rate is steadily decreasing. Read on to find out why!

On the other side of things, Indonesia is steadily increasing the amount of forest that is cut down each year. Why might this be the case? Check out reference #23 for some potential explanations!

How much is 2.3 million square kilometers of land? For perspective, Alaska is the largest state in the United States and it is 1.7 million square kilometers.

Texas is the second largest state and it is 0.7 million square kilometers. Thus, imagine adding both Alaska and Texas together, and that is roughly how much forest was lost from 2000 to 2012.

Now, how much forest was gained in that same time? If you add the area of Texas to Kentucky (0.1 million square kilometers and the 37th largest state), you roughly have 0.8 million square kilometers.

In reference 23, Thomas Rudel explains how forests change with economies. When an impoverished society's economy starts to grow, the society then cuts down many of the tree in it's surrounding territory.

Once the economy grows to a certain point, however, the deforestation is replaced with reforestation, and the forest is replanted.

At this point the forests may then be managed like a crop in cycles of planting and harvesting timber.

In reference 22, Rodriguez and colleagues investigate land/nature reserves around the globe. When added together, 11.5% of Earth's surface is protected in nature reserves.

They found that although a substantial portion of land is protected, the locations of these reserves do not necessarily cover areas with different types of ecosystems and different types of plant and animal species.

Therefore, the present state of these land reserves is not optimized to protect as many species of plants and animals as possible.

Yet, data like what Hansen and colleagues present in this paper could help improve how and where we select new nature reserves to protect more species.

In reference 21, Brooks and colleagues review the different management strategies that are used to conserve plant and animal species (e.g., biodiversity).

This means that the researchers looked at how often, within the 2013 U.S. Congressional Record, individuals used words taken from the Positive and Negative Affect Schedule, a common scale for measuring mood.

In this study, the researchers were particularly interested in how often the congressmen and congresswomen used "positive affect" words: alert, determined, enthusiastic, excited, proud, etc.

In addition to positive affect, the researchers also looked at individuals' usage of "negative affect" words (e.g., nervous, irritable, upset, scared), "joviality" words (e.g., joyful, delighted, energetic, lively), and "sadness" words (e.g., sad, alone, lonely, downhearted).

For this part of the study, the researchers used official photographs of the members of Congress and had someone trained in the Facial Action Coding System code how intensely both of these muscles were used in the photos.

The key point is that both "sincere" (Duchenne) and "insincere" smiles use the zygomatic major muscle, but only "sincere" smiles tend to also involve the orbicularis oculi muscle.

Defensive self-evaluation is a form of self-evaluation that is affected by external sources, such as the evaluations of other people.

Defensive self-evaluation is less stable than "secure" self-evaluation, which is driven by internal sources.

Like in Study 2, conservatives, compared with liberals, were likely to display less intense activation of the muscles around the eyes (the orbicularis oculi muscle), indicating a less intense/genuine smile.

Conservatives in this study were also more likely than liberals to not use those eye muscles at all (i.e., to display "insincere" or "non-Duchenne" smiles).

In Study 2, if you remember, this difference in likelihood of displaying non-Duchenne smiles was not statistically significant.

The Positive and Negative Affect Schedule scale is a very common way of measuring an individual's mood.

The scale involves the presentation of a list of different emotions (e.g., cheerful, sad, relaxed, distressed), and participants are asked to rate to what extent they currently feel each emotion.

The expanded form of the scale involves emotions that can be split into a number of different dimensions: general positive affect, general negative affect, basic positive emotions, basic negative emotions, and "other" affective states (shyness, fatigue, serenity, and surprise).

In a footnote, the authors explain that "conservatism" is a complex concept, involving many different factors. So in this research, they restrict their research to U.S. samples and use multiple different ways of defining conservatism.

They use participants' self-reported political ideology (Study 1), party affiliation (i.e., Republican versus Democrat) (Study 2), congressional voting records (Study 2), social media use (Study 3), and involvement with liberal and conservative organizations (Study 4).

A "meta-analysis" is a kind of statistical technique for analyzing results across many different studies that all examine the same thing.

So, this meta-analytic review of the topic (done by Onraet, Van Hiel, and Dhont, 2013) looked at data from close to 70,000 participants, across 97 studies, all of which examined the relationship between political ideology and happiness in some way.

"Agency" refers to the extent that people feel like they have personal control over their lives.

The more agentic someone feels, the more he or she feels like they are free to make personal choices.

The "ideological happiness gap" is a way of referring to this self-reported difference in happiness between conservatives and liberals.

That is, there is a "gap" in levels of happiness, based on political ideology.

"Big data" is a relatively new term, and it refers to collecting information from an exceptionally large data set.

Studies that use "big data" do not involve running an experiment in a laboratory; instead, they involve analyzing data that are already out there in the world.

Social media platforms, such as Facebook or Twitter, are common sources of "big data."

"Self-enhancement" is the act of making yourself seem better (or, in this case, happier) than you really are.

It's the idea of putting your best foot forward, and it helps people feel good about themselves and maintain self-esteem.

"Mediation" is a statistical term, which tries to answer the question of 'why' a relationship between two variables occurs.

If X causes Y because of Z, then Z mediates (or explains) the relationship between X and Y.

"Subjective well-being" (SWB) refers to an individual's perception of personal happiness and quality of life.

SWB is often meant to include mood/emotions, general happiness, and overall life satisfaction.

The authors provide a map of their sampling sites in figure S1 (in the supplemental information). It is included here in the annotation tabs for Figure 1. Lakes are shown as open circles (135 different sites) and streams or rivers are shown as closed triangles (73 different sites).

According to the supplemental information, water samples were collected biweekly to monthly during 2011-13.

This video featuring primary author Rose Cory and her coworkers shows some of the sampling sites and overviews the background of this research.

This video explains what the researchers do with the water samples they collect.

In order to make their results from the water samples relevant to actual streams, lakes, and rivers, the authors had to account for how much light is available over the range of depths in each water body.

They measured the amount of incoming sunlight at the field sites and also made field measurements of how much light is available as you go from the water surface to the bottom.

By using the bacterial respiration and photo degradation rates obtained from the water samples and the information about incoming light, the authors are able to approximate the total amount of degradation that takes place within a measured area of surface water each day.

Some of the results of these calculations are shown in Table 1. The results are discussed further in the next paragraph.

The authors used their calculated daily areal degradation rates (Table 1) to sum up the total amount of degradation that would happen in each water body over the course of a year. These calculations were based on daily measurements of sunlight during the ice-free season.

The authors made additional measurements of light availability in the water column at sampling sites beyond those in Figure S1 in order to increase the accuracy of their calculations for the entire river basin.

The results for degradation per year are shown in Table 2.

In recent decades, global average temperatures have been increasing and typical weather patterns have been changing.

Scientists agree that this is caused by human activities, especially the release of excess carbon dioxide into the atmosphere from burning oil, coal, and natural gas.

See this link for more information.

Climate change could increase the importance of sunlight-driven degradation of dissolved organic carbon (DOC)—both by releasing carbon that has been frozen in soils for years and by increasing the amount of time with no ice on the water surface.

If more DOC is processed into carbon dioxide, climate change could become more extreme.

To summarize, the authors found that 55% of organic carbon in arctic waters is broken down into carbon dioxide by either sunlight or microbes, while the other 45% is only partially broken down. (So it is still a form of dissolved organic carbon.)

The authors found that dissolved organic carbon (DOC) degrades differently depending on the type of water it is in.

The authors offer this possible explanation: When carbon dissolves out of the soil, it is most likely to enter a small stream first, then a larger river, then a lake.

DOC is degraded by light most easily when it is "new" and has not been exposed to light before (e.g., if it was previously buried in the soil).

DOC in small streams is "newest," so photodegradation is more important in small streams than in lakes or rivers.

Variability in daily weather and the decrease in sunlight over the course of the summer had a large effect on the rate of sunlight-driven dissolved organic carbon degradation.

Kd,λ values in this paper are based on measurements made in the field whereas aCDOM,λ values are based on measurements on water samples taken back to the lab. The authors were able to find a mathematical correlation between the two parameters and use aCDOM,λ to calculate Kd,λ, which they used in their calculations in the information in Table 2.

The details are provided in supplemental information.

This is what aCDOM,λ and Kd,λ measure—see the next sentence/annotation.

pH is a measurement of how acidic a liquid is.

Low pH means very acidic.

The amount of organic carbon broken down or partially broken down by sunlight was between three and 19 times larger than the amount broken down by bacteria when not exposed to sunlight.

Based on this observation, the authors conclude that sunlight controls the break down of organic carbon in streams, lakes, and rivers.

This is different from what scientists previously thought.

This sentence points out a gap that the researchers who wrote this paper want to fill—there are no previous studies that measure both sunlight and microbe-driven breakdown of dissolved organic carbon and account for interactions between the two processes.

In other words, before this study, scientists thought that sunlight played a smaller role than microbes in the break down of dissolved organic carbon to carbon dioxide.

Two of the authors of this paper were also involved in research showing that sunlight can stimulate carbon dioxide release from permafrost soil (reference 11).

This is more than 2 billion tons (!!) of carbon transferred from lakes, streams, and rivers to the atmosphere.

Previous research has measured how much carbon dioxide is released from streams, lakes, and rivers in the Arctic.

George Kling, a professor at the University of Michigan, Ann Arbor, and an author of this paper, contributed to this research and is an author of two of the papers cited here.

Organic carbon is found in all soils and water on Earth and is produced by the decay of living things (plants, animals, etc.).

Organic carbon compounds are made of carbon atoms attached to at least one hydrogen atom.

Organic molecules can be large and contain hundreds of carbon atoms, have only one or two carbon atoms, or anything in between.

Permafrost is a layer of soil that stays frozen all year—even in the summer ("permanently frosted" soil).

Global warming is causing some permafrost in the arctic to thaw.

ERRATUM: actually no difference between the sexes. See Volume 335 page 401

[https://www.youtube.com/watch?v=WzE0liAzr-8]

[http://www.sciencemag.org/content/334/6061/1427.full#ref-1]

[https://www.youtube.com/watch?v=9QdAH6qz240]

In the condition to the far right with a real rat trapped in the restrainer, the free rat is seen to poke its head close to the restrainer and contact the trapped rat several times.

The black dot on the rat's head was used to track the movement depicted in Figure 1B.

The author's wanted to determine whether the rats opened the door because they felt empathy for their cagemate or because of some inherent boldness.

Taking this measurement allowed the authors to determine whether helping is preferentially done by bold rats.

The tendency to feel and express emotions similar to and influenced by those of others.

Voluntary behavior designed to help others.

Of the same species.

An animal that shares the same cage as another.

Experiments in which the variables are controlled so that the effects of varying one factor at a time may be observed.

These rats had regular access to food before the experiment

Both these reviews cite physical restraint of rats in cylindrical tubes as a source of stress.

Previous studies have attempted to differentiate between altruistic and egoistic motivation driving helping behavior in rats.

The studies concluded that the helping actions were driven by sensitization and conditioning, rather than empathy or altruism.

Communication of feelings, thoughts, or attitudes between individuals.

The authors demonstrate that they can indeed detect nAG secreted into the medium by Cos 7 cells.

They use reducing and non-reducing conditions as well. In reducing conditions disulfide bridges in the protein are broken, but in non-reducing conditions disulfide bridges are intact.

For a picture showing disulfide bridges and other bonds found in proteins see here

In other words, the axons in some animals are able to regrow even after being cut. However, nerves that do this are thought have no effect on regeneration.

For these experiments the authors performed immunohistochemistry on sections of newt limbs to determine the location of nAG. This method is further described in the annotation for figure 2.

Why would the authors use 2 different antibodies for nAG and what is the significance of getting the same results from both?

Consider the "scientific process" when answering this question.

This animal had both limbs amputated, but only the right limb was denervated. In addition, the right limb was electroporated with nAG. This causes some number of those cells to express nAG when they normally would not.

This group of animals acts as the experimental group.

Using focal point electroporation (previously described), the authors were able to introduce exogenous DNA into the amputated limb.

The authors chose to use a plasmid (circular DNA) to introduce their gene into the limb.

The authors use an empty vector as the negative control for this experiment. The vector is essentially the exact same as nAG plasmid, but lacks the DNA sequence coding for nAG.

stopped

Cutting in the transverse plane, or across.

Ecology is the scientific study of interactions between organisms and their environment. It considers the relationships between organisms of the same species, organisms of different species, and the nonliving, chemical and physical components of their environment.

Through the use of a drug, or a molecule that has a biological effect on the cells, tissues, or organs of an organism.

A genus of flowering plants whose seeds (coffee beans) are used to make coffee.

A genus of flowering plants including oranges, lemons and limes.

A naturally occurring carbohydrate commonly known as table sugar.

To increase the power or effect of something. Often used in reference to the effect of a drug.

A pair of structures in the brain of insects known to play a role in learning and developing memories of smells.

Picture: Drosophila Mushroom Bodies

A drug that acts as a stimulant of the Central Nervous System found in common beverages like coffee, tea, and some sodas.

Picture: 3D Structure of Caffeine_3D_ball.png)

Relating to the sense of smell.

A substance that interferes with the activity of another. In this case, it blocks the activation of adenosine receptors.

The amount of a substance at which the taste buds are able to detect its bitter taste.

That is, the plants use caffeinated nectar to make sure that pollinators keep returning back to them instead of going to other plants. This makes it more likely that their pollen and ovules will be used to produce new plants, and thus that their genes will be passed on to the next generation.

See here to learn more about the types of chemical defenses produced by plants to protect themselves against herbivores: Plant Defenses Against Herbivory

It's estimated that the amount of caffeine necessary to be lethal is around 5 grams, or 40 gallons of coffee. Read here to learn more.

The ability to acquire and then use knowledge.

Though both are commonly associated with honey bees, the nectar and pollen are two very different things.

Nectar is a sugar-rich liquid produced by flowering plants in order to attract pollinators like honey bees. The more pollinators visit a flower, the more nectar it produces. Nectar is most commonly known as the sugar source for the honey produced by bees.

Pollen is the reason why flowering plants produce nectar in the first place. Pollen is a powder that carries the male sperm cells of a seed plant to the stigma of another flower. There, it can make its way to the female reproductive organ of the flower, where fertilization will occur. This is the process by which flowering plants reproduce.

However, pollen can't move on its own; that's where pollinators like honey bees come in. When a pollinator comes to a flower to drink its nectar, the flower's pollen gets stuck on the pollinator, which then carries it to another flower.

When scientists use the word "significant," they don't necessarily mean to say that something is particularly important or noteworthy.

"Significant" used this way is a specific statistical term that indicates that something is unlikely to have arisen simply by chance.

Since in this context the authors found that the concentration was NOT significantly different, that means that the concentrations were the same between genera, and any differences seen were most likely due to chance rather than due to some systematic process.

That is, if caffeine makes these plants more "fit," or more likely to survive and reproduce, thereby successfully passing on their genes to the next generation of plants.

In chemistry, a measure of the amount of dissolved substance per unit of volume.

Millimolar, or millimoles per liter - a unit of concentration.

In statistics, a regression analysis is a method for estimating relationships among variables.

It helps you understand how one variable (the dependent variable) changes when another variable (the independent variable) is changed, while all other variables remain constant.

This type of analysis is often used for prediction or forecasting.

Logistic regression is a regression analysis where the dependent variable is categorical. This means that it does not have a numerical value, for example a study participant's eye color or blood type.

See this video for more information on how logistic regression works.

A Z-Score is a statistical measurement of the number of standard deviations an observation is above the mean. (To learn more about standard deviations, see here)

Thus, Z-score of 0 means the observation is the same as the mean, a positive Z-score tells you the observation fell that many standard deviations above the mean, and a negative score tells it fell that many standard deviations below the mean.

So in this case, a Z score of -1.09 tells us that the observation fell 1.09 standard deviations below the mean.

See here to learn more about how Z-scores are calculated.

When you perform a statistical test, a p-value helps you determine whether or not your results are significant. A p-value can be calculated by several different statistical tests.

A very small p-value (usually less than or equal to 0.05, though this varies depending on the field of study) indicates that there is strong evidence that the results were not due to chance. Therefore, the results are significant.

A large p-value (usually larger than 0.05) indicates that there is weak evidence that the results were not due to chance, and therefore the results are not considered significant.

In this case, the p-value is 0.272. As this is much greater than 0.05, the median caffeine concentrations in both genera are not considered significantly different.

Adenosine receptors are proteins that, when they come into contact with the molecule adenosine, play an important role in cellular signaling.

Adenosine receptors in the brain regulate the release of neurotransmitters, chemicals that send messages from neurons to other cells.

Caffeine blocks adenosine receptors, thus enhancing the activity of the neurons involved in learning smells and remembering them.

Caffeine is also found in the nectar of some plants.

Therefore, the authors of the study predict that caffeine in the nectar will in some way affect the learning of pollinating insects. They are going to test this hypothesis using behavior tests on bees in the presence of different amounts of caffeine.

Chemicals like caffeine have traditionally been recognized as defense mechanisms for plants; if a predator eats too much of that plant, they will overdose on the chemical and get ill or die. Thus, the chemical acts as a deterrent against predation.

However, the authors of this study have now identified another role for caffeine, and maybe for other chemicals like it. These chemicals can change the behavior of their insect pollinators to the plants' advantage.

In this case, caffeine helped the insects remember that plant, thus causing them to return to it and increase the chances that its pollen will be brought to female reproductive structures. This increases the plant's fitness by increasing the chances that its genes will be passed on to the next generation.

That is, by helping pollinators remember that specific plant, it makes it more likely that the pollinator will return to it and transport its pollen to female reproductive structures. This helps the plant reproduce more often.

Sensilla are little hair-like sensory organs that help insects detect a number of different chemical and mechanical stimuli, like smells or touch.

See figure 1 for these data.

Concurrently; existing or occurring at the same time.

As shown in Figure 2. Bees were more likely to remember an odor presented to them while they fed on sugar and caffeine than if they were fed sucrose alone.

i.e., while looking for food.

The idea here is actually the opposite of what the authors hypothesized, but is a reasonable concern and thus important to consider.

The authors hypothesized that caffeine could enhance a honeybee's memory of a plant, causing them to return to that plant. This is because of the memory-enhancing effect of the chemical's interaction with the bee's mushroom body neurons.

However, the chemical is also very bitter. A bee is as unlikely to want to drink something that is very bitter as a human is. Therefore, if the honeybee can taste the bitter caffeine, it is unlikely to want to return to that plant.

So how do we figure out which of these two opposite effects caffeine actually has on the bees?

The authors of the study offered honeybees sugar solutions containing increasing amounts of caffeine, and observed at what concentration the bees found the solution to be repellent.

If the concentration of caffeine in the nectar of these plants is at that repellent level or higher, it is unlikely that caffeine is attracting bees to those plants.

However, if the amount of caffeine in the nectar is below that concentration, it is unlikely that the bees would detect its bitterness, and so would not be repelled by it.

Supplementary figure 3 showed proof that the honeybees have sensilla (sensory organs) that are able to detect caffeine.

This is what is called a "proof-of-concept" experiment; it shows that the hypothesis is at least possible. Whether or not it is actually the way things are remains to be seen.

In this case, it needed to be shown that honeybees are capable of detecting caffeine. If not, it doesn't matter that there is caffeine in the nectar of the plants - if the honeybees can't sense it and don't react to it, then caffeine in the nectar won't affect their behavior regardless of its concentration.

That is, in the seeds, stems, leaves, etc. of the plant.

Remember that in this study, the authors were only assessing the importance of nectar caffeine concentrations in altering honeybee behavior.

In other words, certain concentrations of caffeine help pollinators remember a plant's scent, and so the pollinators return to them. Those plants are therefore more likely to reproduce and generate many offspring with similar concentrations of caffeine in their nectar.

However, other plants with very high or very low levels of caffeine are less likely to have pollinators return to them. High amounts of caffeine are bitter and repellant to bees, whereas low amounts aren't as effective at stimulating scent memory. As a result, those plants will be less successful at reproducing, and won't generate as many offspring with very high or very low concentrations of caffeine in their nectar.

This means that over time, the population as a whole will have concentrations of caffeine in their nectar that are sufficient to have an effect on pollinator behavior, without being so high as to drive the pollinators away.

Thus, the pollinators have "driven selection" toward a specific range of nectar caffeine concentrations by specifically helping plants within that range produce more offspring than those outside it.

When adenosine binds to its receptors in the brain, neural activity slows down. This has a number of effects, including dilating the blood vessels in your brain and making you fall asleep.

When caffeine binds to these same receptors, it takes the place that adenosine would normally fill, but it doesn't slow neural activity. This makes it harder for you to fall asleep.

Any substance that blocks the normal activity of another, like caffeine blocks the activity of adenosine in this case, is called an "antagonist."

The hippocampus is the region of the human brain (also present in many other vertebrates) that is important for spatial navigation and for processing information from short-term to long-term memory.

Fun fact: the name "hippocampus" come from the Greek "hippos" (horse) and "kampos" (sea monster) - the scientists who first named the hippocampus thought its shape very closely resembled a seahorse!

Long-term potentiation is thought to be one of the key cellular processes behind learning and memory.

"Synaptic plasticity" is a term used in neurobiology to refer to the ability of synapses, the structure that allows neurons to send signals to each other, to change their strength. That is to say that they can change how strong or weak the signals being passed between neurons are. Memories are thought to be encoded by these changes in synaptic strength.

Long-term potentiation is the persistent, long-lasting strengthening of these synapses. It's one of the processes underlying synaptic plasticity and has been shown to be required for the formation of memories, though scientists are still not completely clear on how that happens.

See here to learn more about long-term potentiation and synaptic plasticity.

Not much is known about the CA2 region of the hippocampus, but it has been shown to be instrumental for certain forms of learning, memory, and social behavior.

Kenyon cells are a type of Mushroom Body neuron that are responsible for learning and memory. The authors highlight similarities between Kenyon cells and mammalian hippocampal neurons, as both are involved in learning and long- and short-term memory.

During Alzheimer's disease in humans, early symptoms like memory loss and disorientation are due to damage to the hippocampus.

See here to learn more!

Associative learning is the process by which one learns to associate one stimulus with another, or with a stimulus and a behavior.

For example, Pavlov's dogs learning to associate the ringing of a bell with receiving a treat is an example of associative learning.

Watch this video to learn more about learning.

i.e. information acquired using the senses: sight, smell, touch, taste, hearing.

Nicotinic aceylcholine receptors (nAChR) are a type of ligand-gated ion channel - ion channels that open in response to binding its specific signal molecule, or ligand.

nAChR can be activated by two possible ligands — acetylcholine, a neurotransmitter, and nicotine.

The authors used DPCPX to identify whether or not the increase in nicotinic acetylcholine receptor activation they saw in response to caffeine was the result of caffeine interacting with adenosine receptors.

DPCPX binds to adenosine receptors, preventing any other molecule, including caffeine, from binding to and interacting with the receptor. This is similar to the antagonistic action of caffeine on adenosine receptors, which blocks its activation by adenosine.

As DPCPX was applied before caffeine, caffeine was unable to bind to adenosine receptors during this experiment. As a result, the authors could be sure that any effects seen after adding caffeine happened independently of caffeine's ability to bind adenosine receptors.

This tells the researchers something about about how caffeine behaves in the absence of this antagonist, too - if it's able to act even when the adenosine receptors are inhibited, that means that those receptors are not necessary for this effect to occur. Conversely, if they saw that caffeine-mediated nicotinic acetylcholine receptor activation disappeared when they blocked caffeine from binding to the adenosine receptors, they would know that the adenosine receptors play an important role in inducing this effect.

The plural of "genus," a group of organisms ranking above "species" and below "family" in modern taxonomy.

When a cell is not firing, it is "at rest." The resting potential of a cell is the difference in charge between the inside of the cell and the environment outside it. For the average human neuron, the resting membrane potential is approximately -70 millivolts, meaning the inside of the cell has a charge that is 70 millivolts lower than the outside.

While a resting potential occurs when a neuron is at rest, an action potential occurs when a neuron is firing.

Neurons send signals down their axons, long arms that conduct electrical impulses down the neuron (typically away from the neuron's cell body.) An action potential occurs when neurons send these signals down their axons, causing a short, fast increase in electrical activity. Scientists therefore often refer to action potentials as nerve "spikes" or "impulses."

Action potentials are caused by the passage of ions through a membrane; the electrical current that results from this passage is how neurons send signals.

When a neuron receives a stimulus, a sodium channel opens first. As the inside of the cell is negative (remember the -70 mV resting potential!), positively-charged sodium ions waiting outside the cell move into the cell through this open channel.

As the positively-charged ions enter the cell, the charge inside the cell increases slowly at first. If the potential inside the cell doesn't increase to a certain firing threshold (in human neurons, -55 mV), the result is a small, localized increase in potential called a graded potential. The neuron doesn't fire in response, and the signal is not sent down the neuron's axon.

If, however, the firing threshold is achieved, voltage-gated sodium channels open and the resulting rapid influx of sodium ions causes the spike in electrical activity that we call an action potential. The charge inside the cell rapidly approaches 0 mV, then crosses it to reach a maximum potential of about +40 mV. This rapid increase in the cell's potential is called depolarization.

In response to the increasingly positive charge inside the cell, the voltage-gated sodium channels close and potassium channels slowly begin to open. As the cell is now positively charged relative to the environment outside it, positively-charged potassium ions inside the cell exit through the channels.

The action potential then decreases back down to -70 mV, a process called repolarization, and actually passes a little bit below it (hyperpolarization), as the potassium channels stay open slightly longer than necessary.

Ion levels within the cell gradually balance out to resting levels with the help of Sodium-Potassium ion pumps, and the cell returns to its -70 mV resting potential.

All action potentials for neurons of the same size will always have the same magnitude. So in the case of human neurons, the action potential will always reach +40 mV, no more, no less. So how do our nerves convey signals of different intensities?

While the magnitudes of these action potentials don't vary, their frequencies can. The more action potentials occurring in a given time frame, the more intense a signal the neuron is sending.

For a great visual explanation of these concepts, watch the Crash Course video here.

The authors took these recordings using a technique called "whole-cell patch-clamp electrophysiology."

Patch-clamp electrophysiology allows researchers to measure the current flowing through an individual channel in a cell membrane. These currents result from the passage of ions through these channels, and act to send signals from neuron to neuron.

Researchers isolate a tiny patch of the cell membrane using a glass micropipette. The surface of the cell membrane is suctioned into the tiny opening at the end of the pipette and forms a tight seal. This tight seal allows the current across that portion of the membrane to be measured very precisely, without any distortion of the data by surrounding factors.

Whole-cell patch-clamp electrophysiology is a specific kind of patch-clamp in which multiple channels are individually measured at the same time. The same seal is made over a patch of the membrane using a glass pipette, but in the case of whole-cell recording, the suction is strong enough that the membrane is ruptured, allowing access to the interior of the cell.

To see patch-clamp electrophysiology in action, see here, here, here and here.

Classical conditioning is a form of learning in which a subject learns to associate a neutral stimulus (in this case, the floral odor), with a stimulus of biological significance (in this case, receiving sugar), which elicits an innate, often reflexive, response (the extension of the honeybee's proboscis, or mouthpart).

As a result of this association, the first stimulus (the floral odor) is able to elicit the conditioned response (proboscis extension), even though it previously elicited no response at all.

See here for a great review of the history of classical conditioning of honeybees, as well as a detailed description of the methods involved.

In order to train the bees to associate the floral odor with receiving sucrose, the authors exposed a honeybee to the odor they wanted it to learn, then immediately fed it sucrose solution. This process was repeated 6 times per bee, with a 30 second interval between each trial.

In order to test the effect of caffeine on the development of olfactory memory, the bees were split into eight groups. One group was fed either sucrose alone, while the other seven were fed solutions containing seven different concentrations of caffeine.

The authors tested the bees memory of the smell twice - once 10 minutes after the last conditioning trial and once 24 hours later. The bees were tested with both the floral odor they had been taught to associate with sucrose and with another, unrelated odor to see if their memory to the floral scent was specific.

In the field, the authors had observed that bees will spend an average of 3.77 seconds at each flower they visit, and then will spend an average of 20.3 seconds traveling from one flower to the next, about 24 seconds total.

As a result, the authors decided to use a 30 second interval between trials in this study in order to use a time frame between "flowers" (exposure to floral scent) that is similar to that seen in bees feeding in nature.

The Mann-Whitney U Test is a statistical test used to test the null hypothesis that two populations (in this case, the caffeine concentrations from C. canephora and C. arabica) are the same, against an alternative hypothesis (in this case, that the mean caffeine concentration in one genus is greater than that of the other).

Put simply, this test allowed the scientists to determine whether or not the caffeine concentrations were significantly different.

This particular test is necessary when you are comparing two groups (in this case, the mean caffeine concentration of Citrus and Coffea) whose data is not distributed across a bell-curve, or in other words is not normally distributed (see here).

See here for a more detailed explanation of nonparametric statistics and the Mann-Whitney U Test.

An introductory textbook to the subject of "ecological biochemistry." This science is the study of the biochemistry of interactions between animals, plants and the environment, including the effect of plant toxins on animals.

It is generally believe that drug abuse is the result of triggering reward systems in the brain. However, the most commonly used drugs are plant neurotoxins that evolved to deter herbivores from eating the plants, not reward them for it. How do we explain this contradiction?

This paper outlines a few theories to explain this paradox, including the idea that humans may have evolved mechanisms to exploit these plant defenses.

This and the publications listed immediately above and below it are meant to serve as evidence that while caffeine can be a stimulant and have positive effects, it is lethal when ingested at very high doses.

This study showed that high doses of nicotine was lethal and caused convulsions in rats regardless of age. However, the older the rat was, the longer it took for the nicotine to be lethal. The older rats also had reduced neurobiological responses to the nicotine.

The authors hypothesized that these effects reflected the older rats' reduced brain sensitivity to nicotine and decreased ability to metabolize nicotine in the liver.

This study published as a Nature Brief Communication showed that caffeine at high doses is lethal to slugs and snails.

This and the two publications listed below it are meant to serve as evidence that while caffeine can be a stimulant and have positive effects, it is lethal when ingested at very high doses.

This study has an important real-world application - as caffeine is a product that is already classified as "GRAS" (Generally Recognized as Safe) by the U.S. Food and Drug Administration, these results suggest caffeine has potential to be used as a safe alternative to some pesticides used on farm crops.

This paper reviews available data on caffeine dependence, tolerance, reinforcement and withdrawal, including the effects of all of the above on humans and animals.

It is referred to in this paper to support the idea that low doses of caffeine are mildly rewarding and enhance cognitive performance and memory retention.

This and the two publications listed above it are meant to serve as evidence that while caffeine can be a stimulant and have positive effects, it is lethal when ingested at very high doses.

This paper describes the case of a 21-year-old woman who ingested 10,000 mg of caffeine (and incredibly high dose). The woman went into cardiac arrest, and though she was resuscitated, she died in the hospital three days later.

This paper counters the previously-held belief that the self-pollinating Coffea arabica plant did not benefit from insect pollinators. It shows that honeybees not only help with pollination, but also ultimately boost crop yields by over 50%.

This paper, as its title suggests, outlines an argument for the economic value of tropical forests to coffee production (and so argues against deforestation.) It is cited here to support the claim that plants of the genera Citrus and Coffea produce more fruits and seeds when pollinated by bees.

Found here

This paper describes the experimental procedure behind the classical conditioning of proboscis extension in honeybees. It appears to be the first paper to do so, indicating its authors first established this protocol.

This paper shows that caffeine inhibits adenosine receptors and induces long-term potentiation of neurons in the hippocampus. This long-term potentiation of hippocampal neurons is critical for memory formation.

This review discusses insect mushroom bodies. It is cited here as it discusses the evidence that mushroom bodies in insect brains participate in olfactory memory.

This paper describes the first demonstration of long-term synaptic plasticity in the honeybee brain. It is cited to show the long-term potentiation in insect neurons that is associated with memory formation.

This paper showed that blocking long-term potentiation of hippocampal neurons interferes with learning and memory formation.

This paper found that there were significant amounts of caffeine in the pollen and nectar of citrus flowers. They further found that the presence of caffeine and other alkaloids was linked to the time during which the flowers were in full bloom (i.e., when a honeybee would be interested in them.)

This paper is an overview of the history of research on mushroom bodies. Included in this overview is a discussion of the data showing that insect mushroom bodies are associated with olfactory learning.

This paper is cited here as it shows that caffeine can block A1 receptors (a presynaptic adenosine receptor in the brain).

This paper showed that Kenyon Cells undergo associative plasticity after appetitive odor learning (as performed here with the conditioned scent.)

This paper describes the first time that associative LTP was found in an interneuron of the insect nervous system (the authors stimulated honeybee Kenyon Cells and looked to see what happened.)

Spike-timing-dependent plasticity adjusts the strength of connections between neurons in the brain based on the relative timing of a particular neuron's output and input spikes (or action potentials).

This paper showed that spike-timing-dependent plasticity (STDP), which is responsible for adjustments in the strength of connections between neurons in the brain based on the relative timing of a particular neuron's output and input spikes, plays an important role in associative learning.

A memory is the thing remembered (a scent, a sound, an event, etc.), but the "memory trace" is the structural alteration of brain cells that occurs after learning that, in a way, serves as a physical representation of that memory.

In order to look at the distinctions between the memory of an odor and a memory trace, the authors of this study established a protocol to look specifically at memories after trace conditioning.

This study also describes the formation of memory traces that convey information about the initial odor that was used to stimulate the formation of the memory.

This paper tested the effects of certain defense chemicals produced by plants on honeybees. The alkaloids they tested, a group of chemicals that includes caffeine, were toxic and repellent to honeybees at relatively high concentrations.

The study described in this reference is very similar to the one being performed here with one notable difference: the concentrations of caffeine being used were very high. The study's authors found that honeybees were repelled by very high concentrations of caffeine.

Interestingly, such high concentrations did have have the same positive effect on the development of olfactory memory that low doses of caffeine had on the honeybees tested in this paper.

This study looked to see how honeybees reacted to compounds found naturally occurring in the nectar of some plants. The authors found that (with one exception) naturally occurring levels of these compounds did not have a repellent effect on the bees.

However, concentrations of caffeine higher than those seen naturally occurring in floral nectar were repellent to the honeybees.

This study showed that honeybees can learn to detect toxins in nectar, remember that toxin and the negative effects it previously had, and in the future will avoid flowers whose nectar contains that toxin.

This study did not use caffeine, but it proves the point that if caffeine was present in floral nectar at repellant concentrations, the bees would remember to avoid those flowers, which ultimately would have a negative effect on the plant's reproductive success.

This review sums up current knowledge of caffeine in the field of plant biology. It is cited here to back up the claim that caffeine has been found in the vegetative and seed tissues (as opposed to nectar and pollen) of plants in the genus Coffea at concentrations as high as 24 mg/mL. These values are quite high relative to the concentrations found in the nectar of these same plants.

This paper addresses the question of why flowers produce scent by looking at how olfactory learning of pollinators (like honeybees) influences how plants evolve to produce scents and chemicals in their nectar.

This study examines what conditions influenced foraging bumblebees' decisions to continue foraging from the same species of plant or move to a different species. They found a wide variety of conditions that affect honeybee foraging behavior, but one of the most important determinants was the amount of reward a bee received from a flower (i.e. nectar).

This paper looked at how pollinators, like honeybees, can drive the selection of flower color.

In short, because honeybees prefer some colors to others, plants with flowers that are that color are more frequently visited by pollinators. Therefore, they are able to reproduce more and become more common.

This paper showed that mustard plants with larger flowers (which in this case were more attractive to pollinators) had more pollen transferred to pollinators per visit than plants with smaller flowers.

This paper is cited in the supplementary materials as the source of the protocol for making electrophysiological recordings from neurons located in a sensilla on the tip of the honeybee's proboscis.

This paper is cited in the supplementary materials as the source of the protocol for the bee's sensitivity to bitter compounds.

This paper is cited in the supplementary materials as the source of the protocol for part of their conditioning assay. As in this study, bees were trained by placing them in the conditioning arena and exposing it to a four-second pulse with an odor.

This study was cited in the supplementary materials as the source for the 30 second time interval used between trials during their conditioning experiments.