EVATAR- Building the female reproductive tract in a dish

EVATAR – a box of reproductive tissues

Researchers have made a hand-sized device with all the tissues of the female reproductive system (1). They call it EVATAR (Eve + Avatar – a digital human).

EVATAR can be used to test if a new medicine is likely to cause hormonal problems or affect fertility in women.

Up until now, such tests could only be performed on human beings.

The device itself is like a box with many compartments. Each compartment contains tissues from the female reproductive organs. These include the ovaries, fallopian tube, uterus, and cervix.

The compartments are connected – allowing them to share nutrients and talk to each other using chemical signals (1).

Special electromagnetic valves control the flow of nutrients between compartments and a computer program controls the amount of nutrient in each compartment (1,2).

Photo credit : Northwestern University, Woodruff Lab

Why do we need this device?

Animals are not the best models for human disease or for studying how people will respond to a new medicine (3,4).

So what do we do when a study/ trial cannot ethically be performed in a human?

Traditionally, a scientist would take out some bits of a tissue from the human body and keep it alive in the lab (5).

They would then use these for further tests and studies.

Many human tissues, however, die or loose their function outside the body.

In the EVATAR device, researchers have succeeded in keeping all the tissues from the female reproductive system alive and functional for the 28-day menstrual cycle (1,6).

 

Testing for the behavior of tissues

During the menstrual cycle, the ovary in a woman’s body produces a mature egg. This mature egg is either fertilized or removed from the body(6)

The reproductive system, brain, and pituitary gland work together to make this happen(6).

The pituitary gland secretes gonadotrophins (namely Follicle Stimulating Hormone and Luteinizing Hormone).

The ovaries in the device responded to an external supply of these hormones by making their own chemical signals/ hormones – oestradiol, progesterone, Inhibin A and B.

The ovaries also produced a mature egg within the device.

Other tissues of the reproductive tract responded to the ovarian hormones.

The lining of the uterus – the endometrium, made more receptors to the hormones progesterone and estrogen at end of the 28-day cycle. The cells of the endometrium also multiplied, as expected during the menstrual cycle.

The ectocervix is the bit of the cervix that is externally exposed. It maintained it’s skin-like appearance and structure through the cycle.

This tissue also made receptors for the hormone progesterone only on day 0 and not on day 14 of the cycle, possibly controlled by the secretions of the ovary.

In the fallopian tube, the hair-like projections or cilia, that nudge the egg from the ovary into the uterus, were beating and functional even after 21 days in the device (7).

Researchers can now use this device to study infections and hormonal problems, as well as the reproductive system itself.

Inclusion of liver tissue in the EVATAR device

Tissue from the liver was included in the EVATAR model in one of the compartments.

The liver is not directly a part of the female reproductive system or cycle. However,  it breaks down a new medicine in the body( 8).

After 28 days, the liver tissue looked normal and made a healthy amount of the protein, albumin, within the EVATAR device.

Researchers can now test a new medicine on the reproductive system and the liver simultaneously.

 

Want to know more about EVATAR and things mentioned in this article? Here are some links:

1. The study describing the construction of the EVATAR device – “A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle” (https://www.nature.com/articles/ncomms14584)

2. Draper Labs on the technology behind EVATAR – http://www.draper.com/news/fighting-cancer-boosting-fertility-promise-first-female-reproductive-system-chip

3. “Man or mouse? Why drug research has taken the wrong turning”, October 2016, NewScientist. (https://www.newscientist.com/article/mg23230973-700-man-or-mouse/)

4. Why do we need animal models? https://speakingofresearch.com/facts/the-animal-model/

5. Tissue culture – http://www.biotechnologynotes.com/animals/animal-cell-culture-history-types-and-applications/671

6. Dr. Woodruff explains the menstrual cycle https://www.coursera.org/learn/reproductive-health (Lecture 2.2)

7. Video of the cilia of the fallopian tube beating – https://www.nature.com/article-assets/npg/ncomms/2017/170328/ncomms14584/extref/ncomms14584-s2.mov

8. How does the liver work? (https://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0072577/)

Thank you, Kelly McKinnon, Hunter Rogers (Dr. Woodruff’s lab) and Bernadette Sztojka for your feedback.

 

Why are obese mice so easy to chase?

Dysfunctional signaling in the brain makes obese mice less active

Summary

Obesity is accompanied by a lack of motivation/desire to exercise. This has lead to the idea that lack of exercise leads to obesity. A new study challenges this by showing that both “lazy” and “active” mice gain weight on a fatty diet [1]. All mice on high fat diet become obese and then move around less than mice fed on standard chow. The researchers go on to show that the lack of motivation to exercise that accompanies obesity may well be brought about by neuronal changes in the regions of the mouse brain that respond to movement.

Introduction

The physical inactivity that accompanies obesity is frustrating for those wanting manage their own weight as well as those who want support a near and dear one who does [2,3]. A better understanding of where this seeming ‘lack of motivation’ to exercise comes from may help design better intervention strategies. Previous studies suggest that obese animals and humans may have defects in dopamine signaling in a region of the brain that controls movement behaviours with the result that they may find physical activity less rewarding [4,5]. However, does lack of exercise cause weight gain?

What did they do and find?

Mice were fed standard chow (lean) or high fat diet (obese) for 18 weeks. Mice become both obese and less active when fed a high fat diet. The researchers of this study wanted to understand if the mice became fat because they were less active. To their surprise they found that low activity and weight gain occurred hand in hand but were not cause and effect. The weight gain however was correlated to the high fat diet.

So what was causing the lower activity in obese mice? There is a bit of the brain called the striatum which is responsible for movement and is disrupted in disorders such as Parkinson’s. There are neurons in this region of the brain that are sensitive to the neurotransmitter dopamine and fire (get activated) during movement. The authors of this study reasoned that perhaps it is this region of the brain that is responsible for inactivity in obese mice.

First they looked for components of dopamine signaling, the levels of dopamine itself and the dopamine receptors which when present on neurons allows them to respond to dopamine. They found that the striatum of obese mice had Dopamine Receptors of a specific kind (D2R receptors) which showed decreased binding while the levels dopamine itself and the other receptor for dopamine was the same in lean and obese mice. This reduction in dopamine 2 Receptor binding did not correlate with weight gain but was correlated with movement loss.

So would lean mice also move less if they had lower binding D2Rs?

Indeed, in genetically modified mice that lacked D2R receptors in the striatal region – lower activity levels were observed even in lean mice. This showed that neuronal changes underlie lower activity levels in obese mice.

To probe this further the researchers measured the activity of neurons in the striatum by inserting an electrode in the brain of live obese and lean mice. These recordings showed that during movement there was less overall firing in the brain of obese mice.

In order to test if these brain regions and neurons were indeed responsible for the lower activity observed in obese mice, the researchers used a special set of mice. These mice are specially modified to express a molecule that is usually produced by active Dopamine signaling via D2R binding (Gi) coupled to an opiod receptor only in the neurons of the striata that naturally express D2R. This allows Gi to be uniquely switched on by use of a synthetic chemical (Salvinorin B). When Gi is artificially produced by the D2R expressing neurons of the striatum both lean mice and obese mice become more active.

Reducing the D2R levels artificially in the neurons of the striatum results in mice with lower activity levels however these mice were not more susceptible to weight gain. Nor are mice with low D2R binding in the beginning of the diet predisposed to weight gain.

Take homes from the study

Experiments on animal behaviour are difficult and sometimes hard to extend beyond specific cases because genetic and environmental effects play a large role in shaping observed behaviour and this study is no different. These data convincingly argue that in mice, obesity is accompanied by and not caused by lack of activity. It also gives us a perspective on how integrated an animal’s body and mind are. At the very least it makes us think that in combating obesity, a role for the mind cannot be ignored.

References

1. Basal Ganglia Dysfunction Contributes to Physical Inactivity in Obesity. Danielle M. Friend, Kavya Devarakonda, Timothy J. O’Neal, Miguel Skirzewski, Ioannis Papazoglou, Alanna R. Kaplan, Jeih-San Liow, Juen Guo, Sushil G. Rane, Marcelo Rubinstein, Veronica A. Alvarez, Kevin D. Hall, Alexxai V. Kravitz, Cell Metab. 2017 Feb 7

2. The mysterious case of the public health guideline that is (almost) entirely ignored: call for a research agenda on the causes of the extreme avoidance of physical activity in obesity. Ekkekakis P, Vazou S, Bixby WR, Georgiadis E, Obes Rev. 2016 Apr;17(4):313-29

3. Exercise does not feel the same when you are overweight: the impact of self-selected and imposed intensity on affect and exertion, P Ekkekakis and E Lind, International Journal of Obesity (2006) 30, 652–660

4. Reward mechanisms in obesity: new insights and future directions. Kenny PJ. Neuron. 2011 Feb 24;69(4):664-79.

5. Obesity and addiction: neurobiological overlaps (Is food addictive). Volkow ND, Wang GJ, Tomasi D, Baler RD. Obes Rev. 2013 Jan;14(1):2-18.

6. Do Dopaminergic Impairments Underlie Physical Inactivity in People with Obesity? Kravitz AV, O’Neal TJ, Friend DM, Front Hum Neurosci. 2016 Oct 14;10:514. eCollection 2016.

7. Increases in Physical Activity Result in Diminishing Increments in Daily Energy Expenditure in Mice. Timothy J. O’Neal,, Danielle M. Friend, Juen Guo, Kevin D. Hall, Alexxai V. Kravitz Curr Biol. 2017 Feb 6;27(3):423-430.

An Interview with Dr. Alexxai V. Kravitz

1. What is causing the change in dopamine signaling in the neurons responsive to movement in obese mice? Do you have more insights into this from your study of Parkinson’s?

This is a great question, but unfortunately one that we don’t know the answer to. Parkinson’s disease is caused by the death of neurons that make dopamine, and we looked at dopamine neurons in obese mice and learned that they were not dying. So in that way, the mechanism underlying the changes in dopamine signaling in obese mice is very different than with Parkinson’s disease. This is a good thing, as it would frankly be scary if a diet high in fat were causing the death of dopamine neurons! Instead, we observed dysfunction in a specific dopamine receptor (a protein that detects dopamine) in obese mice. We’re looking into what exactly is causing the dysfunction of this receptor, but unfortunately we do not currently know.

2. You data does show that mice become both obese and move less on high fat diet, but which bit convinces you that the “laziness” is because of the obesity? Can they not be two parallel outcomes of a high fat diet? If yes, then would a high fructose or high calorie diet lead to a similar outcome?

Let me clarify here – I don’t think the *weight* of the mice is causing the laziness, I believe dysfunction in their dopamine receptors is causing their laziness [More on this in Ref. 6]. And both this dysfunction and weight gain can be caused by the high fat diet. So in that say, yes, they can be two parallel outcomes of the high fat diet. To answer your second question, I’m not sure if other high calorie diets can cause the same dysfunction. This would be a great follow up experiment!

3. In your paper, you describe the limitations of human studies that have measured Dopamine signalling and its links to obesity. Can you tell us a bit more about what the challenges are?

To date there have been a handful of studies that have compared D2 receptor levels in people with obesity vs. normal weight, and a minority have reported dysfunction in D2Rs in people with obesity. It is not clear why some studies have reported lower levels of D2 receptors, while most have not. However, measuring dopamine receptor levels in humans is difficult. The only technique for measuring receptor levels in humans is PET scanning, a technique where a radioactive tracer is injected and the brain is scanned for the location where the tracer binds. If more tracer binds, it is assumed there are more “available” receptors in that brain area. However, this technique can be affected by many factors, including what other transmitters are bound to that receptor. If internal levels of dopamine are higher during the scan, for instance, the amount of a radio-tracer that binds to a dopamine D2 receptor will be lower. The complexity increases when we consider how many things can alter dopamine levels throughout the day, which include caffeine use, food intake, and sleep. These are some of the challenges that face clinical research. Animal studies are less likely to incur these sources of variance, and have more consistently reported decreases in D2 receptors in association with obesity.

4. Are the changes in the striatum reversible, by forced exercise for example or are there natural molecules that could restore Gi signaling?

There are no known ways to reverse these changes, but there is also very little research on this. There is a small amount of evidence in rats that forced exercise increases D2 receptor levels, but this is very preliminary and has not been replicated, nor studied in humans. This idea of how to alter D2 receptor levels is an extremely important concept for future research!

5. Are there common themes about obesity and lower activity levels that have emerged from animal studies and how would you extend them, if at al, to humans? For instance, you say mice and rats are different, then would you expect people to be more similar to mice than rats? Why?

It is very difficult to extend results from mice to humans, so I will be cautious on this one. However, there are some concepts from animal work that are relevant to humans. Many researchers have noted that animals voluntarily over-eat high fat diets, and that this leads to weight gain and obesity. While the specific macronutrient (fat vs. carbs vs. protein) content of human diets is the subject of a lot of debate when it comes to human obesity, it is fair to say that diets that induce over-eating will lead to obesity. Typically, foods that induce over-eating are highly palatable, such as junk foods that pack large numbers of calories into small volumes. While people are all different from one another, understanding the foods that a specific person overeats will inform what is likely to cause that person to gain weight.

As another concept that I believe is relevant to human s, in our study we reported that physical inactivity did not correlate with weight gain in mice. That is, we examined inactive mice that lacked D2 receptors, and found that they gained weight at the same rate as normal active mice. We also examined the natural variation of activity levels of normal mice and did not note any relationship here either. This seems to counter the conventional wisdom that inactivity should cause weight gain. However, this conventional wisdom is based largely on correlations between obesity and inactivity, rather than causal tests of this hypothesis. We all know that correlation does not imply causation, but it is very easy to get caught in this trap. In fact, in causal tests, the contribution of exercise alone (without changes in diet) to weight loss in humans is fairly small, generally resulting in 3-5 pounds of weight loss over the first year. This is consistent with our conclusions in mice. Studies in mice can help us understand at a mechanistic level why changes in activity (both increases and decreases) don’t translate into large changes in body weight [More on this in Ref.7].

6. In their natural habitats animals such as mice and rat consume high fat diets. Do you think your results would hold in wild rodents instead of lab reared ones, especially if they were allowed to interact freely with each other and the environment?

Wow, what a great question! We use lab mice, which have been bred in captivity for many decades. This is somewhat similar to studying domesticated dogs vs. wild dogs. And in many ways, our laboratory mice are quite different from wild mice. However, I believe that even wild mice would become inactive on a high fat diet. The association between obesity and inactivity has been seen in many species including humans adults, children, non-human primates, domesticated cats and dogs, rats, and mice. When an association occurs across so many species of animals, I think it is likely that it would extend to wild mice as well as laboratory mice. This would be a great student project to find some wild mice and test!

A sick mouse’s guide to feasting and fasting

When should you feed a starving mouse and when should you just let it be?

 

Summary

Sick mice, especially those infected with bacteria and viruses often display an anorexic response and eat very little. More than 40 years ago it was recognized that mice sick with a bacterial infection die if you force feed them (1). Is this true for all infections? What about viruses? Should we starve a sick pet or colleague?

In a new series of experiments which explores the scientific basis for the old adage starve a fever, feed a cold, researchers have found that food makes things worse for mice with bacterial infection (such as Listeria monocytogenes) but is required for recovery from viral infections (such as influenza) (2).

Introduction

When a mouse or any host is infected with a pathogen the events that follow can be resolved around 3 types of harm caused by the

i) pathogen itself – related to the number of pathogens, toxins produced by the pathogen etc.
ii) response of the body – collateral damage from the inflammatory response, immune reaction to pathogen, etc., which can often times be non-specific
iii) inability of the body or tissue to repair or take care of the damage

The authors find that it was the third kind – i.e. the ability to cope with tissue damage that ensues when mice sick with bacterial infections are fed and also when mice sick with viral infections are starved. This suggests that in the onslaught by the pathogen, there is a bystander effect upon non-immune tissues caused by host defenses that is a,critical determinant of bouncing back to health.

What did they do and find?

Mice infected with Listeria monocytogenes died when they were force-fed. The pathogen load (bacterial numbers) and defensive/ response molecules secreted by the mouse were not different between the force-fed (test) mice and mice that were not force-fed (control). The authors of the study then used a model for bacterial infection to look at why the mice are dying. In this model, the mice were challenged with a component of the outer membrane of bacteria – this is known to result in a strong inflammatory reaction – and then looked at the effect on mice upon injection of glucose, casein and olive oil. Glucose was found to be the cause of death.

This however is only one part of the story. The researchers then looked at another infection model, of influenza-infected mice, which also display an anorexic response. Here they observed the opposite – that is, if the mice were stopped from using the glucose, they died. In fact, feeding mice made them better. Viruses invoke response pathways, which are distinct from bacteria, so maybe the immune reaction was different between the fed and not-fed mice? Once again the authors ruled both pathogen numbers (viral load) as well as difference in immune responses in both groups. To understand what was causing death in these mice, the authors dissected mice that had been infected with the virus and then were given either normal saline or a molecule that made glucose unavailable to the body. Mice which were starved of glucose had lower heart rate, slightly lower respiratory rate as well as lower body temperatures about a week post infection. This was the first clue that control centres in the brain, which are responsible for these functions, may be affected. The authors extended this finding to a mouse model which cannot mount the normal immune response to viruses and challenged it with a molecular mimic of virus infection (poly I:C). In this mode, they found that when fed a molecule that made glucose unavailable, the mice died.

So why were starving mice dying in viral infections and fed mice dying in bacterial infection models? This work sheds some light on the differences. When the researchers studied glucose uptake in the brain in both models they found that there was glucose uptake in different parts of the brain during viral and bacterial infections. Viruses enter the host cells and use the sub-cellular compartments and cellular machinery to make copies of themselves. One such compartment- known as the endoplasmic reticulum – is needed both by the host cell and the virus to function normally. Infection results in a stress response in this compartment which usually signals to the cell that it should now shut-down (a particular kind of cellular suicide termed apoptosis). In this model of viral infection, glucose helps keep this compartment stress-free and therefore prevents cell death. This is particularly important for cells in the brain. What about bacterial infections then? In the brains of the mice with simulated bacterial infections and glucose injections, the authors find evidence for the accumulation of reactive oxygen species (ROS) in the brain. These molecules are also potent inducers of the cellular suicide pathways. However, the authors note that in this case, it may not be death of brain cells, but their dysfunction that may be the cause of death. This still does not explain the difference between viral and bacterial infections. To get to this, the authors analysed the starvation response. During starvation, the utilization of fats and proteins results in accumulation of ketone bodies, an important alternative fuel source during fasted states, via ketogenesis. Excessive and prolonged accumulation of ketone bodies is known to be toxic for the body. In the case of bacterial infection, this study suggests that the availability of ketone bodies may be helping cells to detoxify ROS.

Take home from this study

This study gives us a new way of thinking about infections, host response to infection (immunity) and the rest of the organs and tissues in the body, particularly the brain which must keep working normally through the pathogen-host cross-fire. There are clearly many unanswered question that this opens up, and while it demonstrates that glucose plays different roles in viral and bacterial infection of mice, the underlying mechanisms still remain to be understood in detail. It is interesting that the main difference of glucose utilization seems to be in the brain. The processes that connect what we eat, to what our body makes of it to how we feel or behave form a fascinating network with new links emerging all the time. It is not too soon to have convictions on what is good for us, our colleagues, our pets or our mice, but it is too early to really know or accept information without doubt.

References

1. Anorexia of infection as a mechanism of host defense.” M J Murray and A B Murray , Am J Clin Nutr. 1979

2. Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation,Andrew Wang et al., Cell. 2016

An interview with L.Harding, S.Huen and A.Wang

Q. The idea that the there is tissue tolerance to injury caused by a pathogen-host battle seems reasonable, can you tell us more about the evolution of this idea and its implications for how people now view disease? Are there biomarkers of tissue tolerance?

The idea evolved from the recognition that oftentimes in sepsis, the immune response is more detrimental to the host than the damage incurred by the pathogen. The robustness of a tissue’s ability to tolerate inflammatory challenge can be measured by the ability of tissues to perform their function during inflammatory challenge. Clinically, physicians use plasma biomarkers of tissue dysfunction—for example, troponins for cardiac dysfunction, creatinine for kidney function, transaminases for liver function—as surrogates for tissue function.

Q. How easy or hard is it to distinguish between bacterial and viral infections in a clinical setting – in humans? Are there good diagnostic tests for this?

It is currently very difficult to distinguish the type of infection at the time of admission. This is an area of active research. Currently, clinicians rely on biomarkers such as procalcitonin, which have poor specificity for infection type, and/or detection of the pathogen itself, which often takes many hours if not days to verify, if at all.

Q. What about mixed infections? How do mice respond to a mixed Listeria and Influenza infections? Your group has explored this co-infection model previously, do you understand it better now?

Historically, it has been observed that mixed infections are worse for the host than either of the infections separately. The most famous example is influenza infection followed by a staphylococcus aureus infection. We have previously looked at influenza followed by listeria monocytogenes, and then at influenza followed by legionella pneumophilia. Generally, it appears that viral “priming” potentiates severe disease from otherwise sublethal challenges with bacteria. The mechanisms operating in these different infection pairs was different, but we are trying to understand if there are more general principles that could make this specific sequence of virus then bacteria more lethal.

Q. Do you plan to study this in humans? If yes, then how would you control for cultural variables, the availability of food and the process of habitual eating that many human beings now live by?

We do plan on studying this in humans. The setting where much of this can be best controlled is the intensive care unit (ICU). In patients admitted to the ICU, many are unconscious for one reason or another. Currently, these patients are fed by tube feeding very shortly after they are admitted. The goal of our initial studies will be to see if restricting glucose in feeds delivered to individuals with documented infections would be better for their outcome compared to standard formula feeds.

Q. Do you suspect that there is a strong genetic component to tissue tolerance, set-points or points of no return?

There is likely a strong genetic component to tissue tolerance. Since the immune response has been subject to great selective pressure, it should follow that tissue response to inflammatory signals generated by the immune response would also be under the same selective pressures, especially because it is ultimately tissue dysfunction that leads to death and thus the inability to transmit genetic material. However, because the field of tissue tolerance is relatively unstudied, no studies that try to identify those genetic components exist.

Q. Is the brain the most vulnerable organ – as opposed to say the kidneys which flush out toxins from the body, in terms of coping with damage from an infection? Did this finding surprise you?

In any injury, there is usually an organ or small set of organs, which, if dysfunctional, becomes limiting for the organism’s survival. The limiting organ in turn depends on the type of insult. In general, if the heart, lungs, or brain fail, it is rapidly lethal for the host in the absence of medical intervention. There is a lot of precedence for central nervous system dysfunction in bacterial sepsis, but we were surprised to find that the brain also appeared to be limiting in our influenza model, which is primarily a lung-injury model.

Q. For some bacterial diseases, tuberculosis is a case in point, we know that malnutrition makes the condition worse. How do you reconcile these observations with your finding?

There is a big difference between acute infection and chronic infection. What we were studying was the response to acute self-limited infections. In chronic infection, the persistence of the inflammatory response, persistence of the pathogen, and the changes that this dynamic imposes on the host is very different than the acute phase response. So, it is likely that the metabolic requirements of chronic infections are very different from the metabolic requirements of acute infections. Also, even in the acute setting, bacteria have co-evolved with their hosts and in the process may have developed mechanisms that interfere with the tissue tolerance mechanisms that we have described here. Therefore, our current work may not be generalizable to the full spectrum of bacterial and viral infections.

Modifying plants for human consumption

– the story of a CRISPR salad

Modifying plants to feed the world

How are we going to feed the 7.45 billion people in the world? Many people including scientists believe that genetically modified plants are one route to food security (1).  However, we as a species and society remain largely afraid of genetically modified organisms, perhaps, because of the seeming “unnaturalness” of it all.

A new technology now allows scientists to modify plants and other organism in a more “nature-identical” way. In fact, plants produced by this method are so indistinguishable(genetically) from naturally occurring varieties that no one an tell the difference. Therefore, the Swedish agricultural board and the United States Department of Agriculture (for mushrooms, fungi not ”technically” plants) have set the precedent by clearing seeds and mushrooms produced by this method for production and consumption.

The method used to produce these is called the CRIPR-Cas9 system (Watch Carl ZImmer’s  video on CRISPR here). It can be used to remove bits of DNA from an organism’s genome in a targeted manner.  Think of it like what the invention of the engine was for transportation. Scientists all over the world are using this metaphorical scissor to snip out pieces of DNA exploring function and consequences, in an effort to understand ourselves, the living world around us, and curing mice of rare mice diseases (a favourite among us lab rats!) (2-5). Why not make varieties of plants that are better suited for human consumption using this method?

Undoubtedly, there are serious and complex socio-economic issues around the use and misuse of plant genetic engineering, but the discussion has often focussed on and suggested that the “science” is not good enough, which in the personal opinion of those of us writing this piece is a problem. We think that this blanket idea of “imprecise science” actually detracts from the actual concerns -mostly social and economic (monopolization, unexpected risks of cultivation of new cultivars, proprietary seeds etc.). How well-founded or rational is our fear of modified crops? What do we know and not know? We thought we would ask the scientist who recently publicly (broadcast on Swedish Public Radio by host Gutaf Klarin) ate a plate of CRISPR generated cabbage and broccoli and has been actively reaching out to people about the use of this technology in agriculture.

Further Reading and References

1. Original Broadcast of the CRISPR dinner from Sverige Radio by Gustaf Klarin (In Swedish, amenable to translation using Google Translate or similar other tools), September 5th, 2016

2. What was on the plate?

References

  1. http://time.com/4521582/2016-election-food/?iid=sr-link1
  2. RNA-guided genome editing in plants using a CRISPR-Cas system. (review)
  3. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy
  4. In vivo gene editing in dystrophic mouse muscle and muscle stem cells.
  5. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.

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An interview with Stefan Jansson

Q. You may be remembered as the first man to eat a CRISPR modified plant, how do you feel about that?

Well, I would of course prefer to be remembered because of my contributions to science, not just because I made some cooking once upon a time. But these two things are closely connected, if I have´t had the scientific credibility that I actually have, I would just have been regarded a ”mad scientist” doing a stunt and the meal would not have got the same positive attention.

Q. As a plant biologist, do you have concerns about genetically modified crops – for example, what if the gene modified leads to increased requirement for water etc.? What measures are in place to assess this?

As with all plant breeding techniques, one can of course create varieties that are better or worse for the environment. I do like those that are likely to have a positive impact but not those that are likely to have a negative impact, regardless of they are made with techniques that fall under the GMO legislation or not

Q. If CRISPR becomes a patented/proprietary technology it may restrict the adoption to richer countries and not actually benefit or ensure food security. What are your thoughts?

It would of course be better for the world with no restriction of access, but I also realize that we cannot change the patent legislation, and as companies are there to make money, we simply have to live with any restrictions that may come. I do hope that they will not restrict the use of those that need it most, but that is beyond yours and my control.

Q. Within the current norms of GMO versus non GMOs, is insertion of genes from the same plant (not-foreign) allowed?

It is considered a GMO, at least under the EU legislation.

Q. Do seed companies make new varieties using random mutagenesis using chemicals and radiation? Is this permitted?

Many breeders are still busy exploiting the variation created by mutagenesis programs conducted many decades ago, but I assume that also run new mutagenesis programs for some species. These are not considered GMO

Q. Plants have been selected by humans since the time we began agriculture. Would comparison of ancient plant genomes to current plant genomes be a good starting point to identify or make an inventory of desirable changes?

Indeed, and I have understood that some breeders do this

Q. Given that most genes in a plant that make protein are useful in some circumstance or other, how many genes do you think can we knock-off and ensure ‘improvement’?

It is of course only few genes that we can knock out and increase the fitness of the plant, if so evolution would probably have already selected those changes. But all genetic changes that we have made so for during the domestication have probably lead to a reduced fitness of the plant, but made them more useful to us in the agricultural system, so there is indeed a huge room for improvement also with new techniques.

Frigate birds keep their eyes wide open during flight – most of the time!

Sleep patterns of flying birds

Snap-Shot of the study

Birds fly enormous distances during migratory flight. It seemed reasonable therefore to think that birds sleep while flying, especially since birds can go to sleep -one brain-half at a time.

But then, how can they function and be attentive to the demands of flying, foraging, avoiding predators, finding their route, often over water, over such long periods of time? Imagine the frigate birds with the wind on their wings as they begin their flight across the ocean, flying continuously and majestically for upto 2 months! In an elegant and well written piece of research, the authors find that frigate birds sleep very little during their long flight (1). By recording brain activity in flying birds, the authors show for the first time that these birds take short and light naps but mostly forgo sleep during migratory flight.

Background to this study

A single late night is enough to send most adult humans into a downward spiral the next day, fruit-flies that don’t sleep have their lives cut short (2-4). Birds however have been shown i) to go on long flights without resting (5) ii) to sleep uni-hemispherically – single eye open – keeping one part of the brain alert to the requirements of the environment while the other part rests (6). iii) are able to function more or less normally even when deprived of sleep (7)

So what happens during flight? Given the high energy/metabolic demands of flying the need for rest and sleep must be high, on the other hand the need for attention is also high during flight, can birds afford to sleep on the wing? One way to find out is to record the patterns of brain activity in migratory birds during flight and look for patterns of sleep and wakefulness.

What did they do?

In this study (1), sleep patterns of 15 female great frigate birds flying over the Pacific Ocean and after returning to their nest on Genovesa Island (Galápagos) were recorded using implanted devices that measured brain activity (EEG – electroencephalogram), movement of the head as well as acceleration. No behavioural differences were observed in the birds with implants during and after removal of the devices. Using these devices, the researchers measured movement of the bird, acceleration, deceleration, flapping of the wings and brain activity near the primary visual area and also collected data on weather conditions. Measuring movement of the head allowed them to separate patterns formed by head movement from actual changes in brain activity.

What did they find?

The overall EEG patterns were similar in the birds on land and during flight, allowing the researchers to look at duration and intensity of sleep. They describe three sleep-awake states roughly – wakefulness, rare episodes of REM sleep (like in humans, this is deep sleep characterized by rapid eye movement) and slow wave sleep – this is the most frequent type of sleep described in birds which can be bi-hemispherical or uni-hemispherical or assymetric (6).

During the day, the birds showed patterns of wakefulness (fast head movements together with high amplitude signals in the EEG), even at night during flapping of wings wakefulness patterns were observed. These were interspersed with slow waves which the authors identify as slow wave sleep. Rarely, between bouts of slow wave sleep, short bursts of deep sleep or REM sleep patterns characterized by dropping of the head, and twitching were also observed. Interestingly, birds ascended in altitude during slow wave sleep and descended during wakefulness. All types of slow wave sleep, including unihemsipherical, asymmetrical (when one hemisphere was more active than the other)  and bihemispherical slow wave sleep were observed during flight. There was an increase in asymmetric sleep in flight than on land but this was not correlated with any one type of movement.

Overall, frigate birds seemed to sleep very little during flight – in shorter bursts and less soundly –  a homeostatic balance was restored when these birds landed. Further, preliminary evidence from this study suggests that these bursts of sleep are enough to sustain the birds during flight.

Why is this interesting?

The very fact that these birds are able to accomplish Himalayan tasks such as follow migration routes, feed themselves, with such low levels of sleep suggests that, at least for frigate birds, sleep may be dispensable during flight. Are they postponing this need? What sort of adaptations allow them to postpone sleep or perform sleeplessly? This study is a step towards understanding adaptations to lack of sleep and perhaps a way to understand the very nature of sleep itself.

1. Evidence that birds sleep in mid-flight. Rattenborg NC, Voirin B, Cruz SM, Tisdale R, Dell’Omo G, Lipp HP, Wikelski M, Vyssotski AL. Nat Commun. 2016 Aug 3;7:12468. doi: 10.1038/ncomms12468. PMID: 27485308

2.Reduced sleep in Drosophila Shaker mutants. Nature. 2005 Apr 28;434(7037):1087-92  Cirelli C, Bushey D, Hill S, Huber R, Kreber R, Ganetzky B, Tononi G.

3. Genetics of sleep and sleep disorders. Cell. 2011 Jul 22;146(2):194-207. doi: 10.1016/j.cell.2011.07.004. Sehgal A, Mignot E.

4. http://www.curiouscascade.com/blogpost/clocking/

5. Frigate birds track atmospheric conditions over months-long transoceanic flights. Science. 2016 Jul 1;353(6294):74-8. doi: 10.1126/science.aaf4374. Weimerskirch H, Bishop C, Jeanniard-du-Dot T, Prudor A, Sachs G.

6. Half-awake to the risk of predation Nature 397, 397-398 (4 February 1999) | doi:10.1038/17037 Niels C. Rattenborg, Steven L. Lima & Charles J. Amlaner

7. Adaptive sleep loss in polygynous pectoral sandpipers. Science. 2012 Sep 28;337(6102):1654-8. Epub 2012 Aug 9 Lesku JA, Rattenborg NC, Valcu M, Vyssotski AL, Kuhn S, Kuemmeth F, Heidrich W, Kempenaers B.

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An interview with Alexei Vyssotski

Q. Can you tell us a little more on how you determined that the birds were sleeping? How did you identify the pattern corresponding to sleep when you got all your recordings?

Sleep was identified by visual inspection of 4-sec episodes of raw EEG records. Slow-wave sleep is characterized by large amplitude low-frequency oscillations in EEG. These episodes are easily-detectable. Visual scoring was used because automated methods of sleep staging can’t separate properly large-amplitude EEG events from movement artifacts in all cases. Locomotor artifacts can have similar amplitude to slow-waves during in sleep.

Q. Do you think these birds sleep a lot during an annual cycle? Do migratory birds tend to sleep longer than non-migratory birds on average – there must surely be some compensatory mechanisms?

We have found that frigatebirds sleep on average 9.3 hours per day when on land and only 0.69 hours per day when flying. We did measurements only during breeding period. While the frigatebirds live in equatorial area with relatively small weather seasonal changes, one can suppose that the duration of sleep is linked stronger with the pattern of animal activity than with the time of the year per se. It is known that migratory species can stay on the wing for a long time. Extrapolating our findings to these species one can suppose that they should sleep in the flight smaller amount of time than on land, and might compensate migratory sleep loss on land later like our frigatebirds did. However, the compensatory increase in sleep duration and intensity upon landing after the trip is relatively small comparatively to missed amount of sleep on the wing. One can speak only about partial compensation of sleep loss. The birds have, probably evolutionary formed, an ability to stay without sleep significant amount of time without physiological dysfunctions. Unlike most mammals, the birds do not have so called sleep-deprivation syndrome. If a rat will be forced to stay without a sleep for significant time, it will die, but a homing pigeon can stay month-long awake and still behave properly. Migratory birds definitely reduce amount of sleep during migration, but whether they sleep longer than non-migratory birds in other situations is difficult to say.

Q. Do you think that migratory birds produce neurochemicals that resist the urge to sleep or have special brain structures?

The neurochemistry of avian sleep is investigated in much less extent than mammalian sleep. To the best of my knowledge, no special anatomical structures that are responsible for sleepless in birds have been discovered. It is assumed that the avian sleep control brain system is similar to mammalian. However, additional studies are needed to check how strong this similarity is indeed and what are the differences.

Q. Does the slow sleep wave recordings refer to the activity of only certain regions of the brain?

No. It is assumed that like in mammals, the vigilance states in birds are controlled by deep brain structures that modulate activity of superficial brain regions in a synchronous manner. However, contrary to most of mammals that have only bi-hemispheric sleep, the birds have so- called unilateral sleep, when one hemisphere is sleeping and another is awake. Thus, the avian brain hemispheres are more independent from each other than mammalian. One should note that the phenomenon of “local sleep” has been also discovered in birds. This means that if a particular region of the brain has been used intensively in a wake state, the slow waves of increased amplitude will be observed during the following sleep in this brain area.

Q. Did you have independent recordings of some of the parameters using a high speed camera to set the baseline for each bird?

Do you mean Ca2+ brain cells imaging? No, we did not do this, but of course, if would be nice to monitor how activity of different cells ensembles changes in sleep in birds. The recently developed head-attached microscopes can help to film brain in freely behaving animals.

Q. It is astonishing that you got so much information that you could put together, did you expect that when you captured the 15 birds? What were the unexpected challenges in the study?

That is true, this time we collected more information than in our previous studies. We alreadyhad experience with EEG and GPS logging. This time we compensated luck of GPS precision by the acceleration data to reveal the flight mode of the animal. 3-D acceleration data practically doubled the dataflow, but this was not a problem to log in 1 GB onboard flash memory. We did not predict the particular way of the data analysis in advance, but observing three-modal distribution of sway acceleration leaded us to separate analysis of EEG in three different flight modes (straight flight, circling to the left and to the right). The real challenge was to master the surgery and handling in an animal-friendly way to have the birds back with the equipment. Indeed, the rate of return 14 from 15 birds exceeded our expectations. To be honest, I expected larger losses and was very happy when returns exceeded 50%.

Q. Have you been to the Galapagos? What is it like to do research in that setting? Do you think that nearly 180 years after Darwin’s voyage, the biodiversity of Galapagos still holds new discoveries?

Yes, I have a real luck to spend a week at the Darvin station in Santa Cruz and then a week on Genovesa island working in the bird colony. My colleagues Bryson Voirin and Ryan Tisdale spent two weeks more waiting for birds return. This is the best place for animal study that I have ever seen. Wild birds behave almost like tame animals there and do not run away from humans. Thus, it is easy to handle them. This is, of course, one of the features that attracts biologists. The biodiversity of these islands is definitely not studied completely and will attract scientists for a long time.

Game of Theories: A Policy of Bribery and Punishment

Theoretical study suggests that encouraging complaints from citizens may be the most effective way of reducing incidents of bribery

 

Article

Data analysis is critical for the formation of any robust policy. But reliable data in economics is not always available, since unlike in the natural sciences, controlled experiments at the population level are not possible.  Moreover, present data may not have sufficient predictive power, since population behavior changes over time. Economists use theoretical models to account for variables and parameters to predict end-results in such situations. These models can serve as prototype systems on which to test the possible consequences of a new policy.

In this particular study, the authors use evolutionary game theory to approach bribery, a prevalent problem in may societies.

What is Game Theory?

Economics, broadly defined, is the science of human behavior. And much like in the natural sciences, economists look for “bottom-up” explanations of phenomena, where simple underlying rules give rise to complex behavior. The rules generally take the form of mathematical equations, the solutions to which are expected to capture (and predict) the essential features of social dynamics.

A central theory which has proved useful is game theory. A more accurate name for it is “multi-person decision theory” – because that’s exactly what it is. It deals with situations where multiple players engage with each other, each armed with a repertoire of possible strategies, and the outcome (or “payoff”, as it is called) for each player depends on the strategy adopted by all the players.  The aim of each player is to choose the strategy that maximizes her payoff. As you can see, the theory potentially covers a very broad range of human interactions, and thus the widespread application of game theory in economics is hardly surprising.

But how do we adopt and revise our strategies over time? A set of perfectly rational beings with complete information would quickly reach a unique equilibrium situation (provided such a situation is allowed by the dynamics of the game). But in the real world, people are neither perfect computing machines, nor do they have perfect information. So the strategies we use, and the way we update them with further experience, depends a lot on the context.

What game does this article study?

The study employs different players and their corresponding strategies; an honest officer, a bribe-taking officer, an honest citizen, a bribe-paying citizen who doesn’t report on the crime, and a bribe-paying citizen who reports on the crime. The advantage to reporting, of course, is that you have a chance of getting your bribe refunded. These players (citizens and officers) interact at random and such interactions can potentially involve a corrupt transaction (depending on the strategies of the interacting players) leading to payment of a bribe. Like Kaushik Basu, the authors focus on the problem of harassment bribes. These are bribes paid by citizens to corrupt officers for getting access to a service they are legally entitled to (such as acquiring a passport or getting a driver’s license)

The authors also consider two different ways in which the citizens and officers may update their strategies over time. First is the Replicator model, where an officer or citizen randomly chooses a fellow officer or citizen, and tends to imitate their strategy if they have been more successful. In the second scenario, called the Alternative Strategy Exploration model, they first make their moves and receive their payoffs, then consider whether the possible alternatives may have given them a higher payoff, and if so, update their strategies accordingly. In both the cases, it is found that the population finally reaches a fixed frequency distribution of the various strategies.

What is the question?

The authors consider two major punishment models:

1. Symmetric punishment, where both bribe taker and bribe giver are punished

2. Asymmetric punishment, where only the bribe taker is punished.

In fact, a major motivating point for the study was the claim by economist Kaushik Basu that bribe-giving, as opposed to bribe-taking, should be legalized for harassment bribes, as this would increase the frequency of complaints and help bring down bribery.

The authors numerically simulate the evolution of strategies under both kind of punishment models to find out which might be better in curbing bribery incidents. They further analyze conditions under which a decrease in incidents of harassment bribery might be possible.

What do they find?

Contrary to the claims by Basu, they find that the effect of asymmetric punishment depends on the update strategies used by the players, and cannot be considered a universal solution. Lowering the cost of complaint, however, seems to work under both the asymmetric and symmetric punishment models. However, the extent of bribery reduction depends on other parameters as well.

What are the conclusions?

Bribery is a very complex and dynamic issue. While there can be no single way to get rid of it, the authors suggest that bringing down the cost of complaint to negligible might be important in an overall reduction in incidents of bribery, irrespective of the punishment model. On the other hand, what happens under more complicated updated strategies is still an open question. As the authors say in their article, “It would perhaps be more pragmatic to look at a combination of technological fixes and public policies targeting the myriad underlying causes of bribery in order to effect reduction in bribery and ease the toll it takes on public finances.”

It is interesting to note that even in relatively idealized economic models, a simple and universal solution may prove to be elusive. This should make us more skeptical of quick fixes suggested by policy leaders that seem to make intuitive sense, and look for solutions that are customized to individual scenarios.

References

Bribe and Punishment: An Evolutionary Game-Theoretic Analysis of Bribery,Prateek Verma, Supratim Sengupta, PLoS One. 2015 Jul 23;10(7):e0133441.

An interview with Dr. Supratim Sengupta

Q. Basu (2011) had submitted a report to the Government to inform on policy for bribery punishment. Why did you choose to study this report using game theory and which facets of this report have your study addressed in more depth?

When this report came out, a literature search revealed that there was practically no quantitative analysis of the issues raised and claims made in the report. The principle claim was that incidents of harassment bribery could be substantially reduced if only the bribe-taker but not the bribe-giver was penalized for the crime. It was also evident to us that the problem was ideally suited to be analyzed using evolutionary game theory since it presented a very well-defined scenario of social conflict with mutually exclusive interests of the principle parties (bribe-giver and bribe-taker) involved. Our main objective was to quantitatively examine in great detail the principle claim of Basu’s report (mentioned above) and understand how reduction in incidents of bribery depend on factors like the amount of bribe demanded, the cost of complaining about a bribery incident, the penalty to a bribe-taker if caught. We also examined how the manner in which individuals updates their strategies in response to a bribery incident affect the prevalence of bribery in the population. None of these aspects had been examined in such detail using the framework of evolutionary game theory and our work (carried out with my PhD student Prateek Verma) is the first to do so.

Q. Your models indicate that low cost of complaint may eradicate bribery. In countries with low bribery, are there platforms which make registering complaints easier?

Yes, most western European countries and places like USA, Canada etc., have efficient grievance redressal systems and harassment bribes are practically non-existent for basic services provided by the government. Even in India, a website called ipaidabribe.com started by an NGO in Bangalore has made it easy to report incidents of bribery and bring grievances to the notice of public officials. Such steps have led to positive outcomes which suggest that reducing the cost of reporting bribery incidents and establishing an efficient grievance redressal system by using technology can be quite helpful. If there is one policy proposal that we could advocate to reduce incidents of bribery, it would be this.

Q. Countries with low corruption indices seem to have a higher national income and lower inequality. But these factors do not seem to be explicitly incorporated in your model. Would you like to comment on that?

Bribery is a multi-faceted social problem and has many underlying causes including (but not restricted to) high levels of income inequality, as you have pointed out. However it is quite difficult to quantify the impact of such income inequality on the problem of harassment bribes. We took into consideration the most relevant factors that directly affect harassment bribery and that could be incorporated into a tractable mathematical model that would enable us to obtain insights into the problem and identify steps that can be taken to reduce such incidents of bribery.

Q. Why did you choose the two strategies: Replicator dynamics and alternative strategies dynamics for the study? What other scenarios might have been considered?

We wanted to highlight the difference in outcome when individuals choose two completely different methods to update their strategies over the course of time. We found that in the alternative strategy exploration case, the reduction in incidents of bribery was far less pronounced even when we followed Kaushik Basu’s proposal. However, these two methods are not the only ways individuals can choose to update their strategies and hence the outcome would indeed depend on the method followed. For instance, different individuals in the population could choose different methods to update their strategies and that would affect the outcome.

Q. Since collecting bribery data is difficult due to the secretive nature of the phenomenon, how do we check if some bribery-related policy has worked?

Collecting bribery data may not be as difficult as it seems. The government can ask citizens to fill out a simple questionnaire given by the service provider to report incidents of bribery. For example, the regional passport office can ask citizens applying for a passport to submit their feedback online by filling out an online questionnaire at the end of the process. Statistics based on such documents can be a very useful indicator of the prevailing levels of bribery in different services. It would also provide clues on how to allocate resources to reduce incidents of bribery in different services provided by the government.

Q. Do you expect your study or other related studies to have an impact on government policy?

It is difficult to predict whether any study of this kind will impact public policy. This is a topic of great public interest and Kaushik Basu’s original proposal certainly garnered a lot of attention. But that was in no small measure due to the important position he held at that time (Chief Economic Advisor to the PM) as well as his influence as a well-known economist. However, many (but not all) of the responses to that article in the press at that time were simplistic, knee-jerk reactions. Moreover, they were not grounded on objective analysis. We hope our work along with those of others have contributed towards a critical analysis and will at least rekindle the debate and stimulate new proposals on how to reduce harassment bribery. Despite the controversial nature of the proposal, we believe there are concrete and simple steps that can be taken that may have an impact. As I mentioned in my previous response, using web-based technology to streamline access to services, gather critical feedback and reduce the cost of complaining can definitely have a positive impact. However, we also acknowledge that technological fixes alone cannot and will not get rid of such a complex social problem that is definitely affected by income-inequality, the efficiency of the public justice system etc.

Q. In general, how much do you think game theory models accurately capture real world dynamics?

Evolutionary game theory is employed when the effectiveness of a strategy depends on the presence of other competing strategies in the population. It relies on specifying the net gain or loss (referred to as “payoffs”) to each strategy when it interacts with different strategies. It is very useful in analyzing how the number of individuals employing different strategies change over time and the conditions under which a specific strategy can out-compete all other strategies and take over the population. Unlike conventional game theory used by economists, which is involved only in finding equilibrium solutions (the so-called Nash equilibrium), evolutionary game theory allows us to see how the population gradually progresses towards an equilibrium state by tracking the change in number of individuals employing different strategies over time. The usefulness of evolutionary game theory in accurately capturing real world scenarios depends on the question being asked. There have been some criticisms (valid in my opinion) of using game theory to understand how cooperative behavior can spread or be sustained among groups of individuals. Experiments have revealed that people have an intrinsic tendency to cooperate more than game theoretic analysis predicts, despite cooperation being costly. In such scenarios, it is necessary to be cautious in drawing conclusions based on game theory. However, in the current scenario, such concerns do not apply since the primary interests of the principle players are at cross-purposes. We therefore feel that evolutionary game theory is well suited to accurately address the bribery problem. Nevertheless, some words of caution are in order. Our models employ quantities like the prosecution rate and penalty when prosecuted to understand the effects of punishment and its deterrence on bribery. In reality, these depend on the efficiency and integrity of the public grievance redressal systems and justice systems. These can vary a lot not just across countries but also across jurisdictions within the same country. These factors need to be given serious consideration before framing any public policy dealing with bribery.

Q. What do you think is the role of theory in economics, in general terms? I come from a background in theoretical physics (says Jabali) and now work in biology. And I and others like me sometimes wonder about whether we are making the right kind of assumptions in our models, or whether the phenomena we are describing are really reducible to a mathematical description, given the current state of knowledge. Does a theoretical study in economics contain the same kind of concerns? Or is the picture there very different?

You raise a very pertinent point here that I will not be able to satisfactorily address. I am not an economist by training. Like you, I am a theoretical physicist interested in analyzing complex social and biological systems using quantitative tools inspired by Physics and Mathematics. I am therefore not qualified to comment about the real-world relevance and drawbacks of theoretical models in Economics in general. However, the concerns you raise are surely relevant to studies of this kind and any honest scientist/economist needs to ponder about them. These concerns certainly informed our formulation of the problem and we tried to make the model as realistic as possible. But we also admit that some simplification was inevitable and we could not incorporate every single factor (like income-inequality for example) that may play a role in the prevalence of harassment bribes. Sometimes, even the factors incorporated, like prosecution rate, penalty for taking bribes etc. may not reveal the true complexity of the real world problem where such quantities depend on the efficiency and integrity of the police and justice systems. In some cases, the simplifications (like our assumption of a mixed population) can be addressed by studying the effects of a structured population of citizens and officers, as we are currently doing in an ongoing work (with Prateek Verma and Anjan Nandi) which is being written up. It is therefore important to acknowledge the potential consequences of the underlying assumptions and the associated simplifications while proposing new public policy based on such studies. It is also important for us (the community of social and natural scientists) to continue to strive to develop more realistic models by improving on existing ones whenever possible. Finally, I believe it is necessary for policy makers to pay attention to such studies and start a dialogue with social scientists to critically examine the consequences of such studies on public policy.

A whole new whale

A whole new whale- genetic analysis supports the existence of a new whale species

Snap-shot

In the mysterious dark waters of the deep seas, imaginative explorers have chased mythical creatures – from mermaids to kraken, creating an endless list of adventures. Even in the 21st century, we know very little about the creatures that inhabit these worlds. Now, a shy whale species comes into molecular spotlight as scientists use genetic tools to reveal its existence and decipher its relationship with better known cousins (1).

Background

Public Domain, https://commons.wikimedia.org

The beaked whales are a mysterious and diverse family – characterized by teeth and a prominent beak. Very little is known about this family of whales because they live in remote oceanic habitats, dive deep and seem to be sparse (1, 2). The existence of a black beaked whale is long rumored among Japanese fishermen and was first described as a possible new species by Japanese researchers in 2013 (3). A type of beaked whale known as ‘Baird’s beaked whale’ are seen the north pacific (ranging from Japan to Mexico), these whales are slate-gray in colour. Rare sightings of smaller black whales have also been reported around these regions (only around Japan). Although the black whales are distinguished by their skin tone and smaller size, these criteria alone were not sufficient to to classify them as a new species. One explanation for example could be that the black whales were just the young juveniles of the slate-gray Baird’s beaked whale. A recently published study (1) uses mitochondrial DNA sequencing to show that the black beaked whales are genetically distinct from other beaked whales and are only distantly related to the slate-gray whales with overlapping geographical niches.

What did they do?

The team has analyzed the DNA sequence of a total 178 samples of Baird’s beaked whales. These samples were painstakingly collected  from the rare live animals captured across the entire north pacific range including  two  museum samples. Unsurprisingly, majority of these samples (170) belonged to the slate-gray forms and only 8 belonged to the suspected black whales. They compared the genetic sequence (haplotype https://en.wikipedia.org/wiki/Haplotype) of particular stretches about 450 base pairs (nucleotide lengths) of mitochondrial DNA of these whales. If indeed the 8 black samples belonged to a new species, then their DNA sequence is expected to be quite similar to each other and differ considerably from the slate-gray ones.

What did they find?

Genetic analysis showed that a particular stretch of  mitochondrial DNA from the black and slate-gray whale samples had only 6-7 differences within their own group but differed from each other in 16 positions. This compares with the difference of 12 nucleotides positions that the slate-gray whales have  with a different beaked whale species Arnoux’s beaked whale, a completely  different species found in southern hemisphere (a benchmark for genetic disparity the authors have used to validate the genetic differences-). This analysis in combination with the known physical characteristics of size and colour, positions the rare black whale as a new species. The presence of 6 nucleotide differences in the mitochondrial DNA of the 8 black whales suggests that there may be high diversity within the species as well.

Why is this important?

This work highlights how little we still know about the fascinating world around us. It uses relatively inexpensive, and widely available molecular methods (sequencing short stretches of mitochondrial DNA) to support the claim of a new species of whale in the North Pacific Ocean. This raises new questions about how this species lives and thrives in the remote oceans and how it is affected by human activity.

References

References

1.Morin, Phillip A., C. Scott Baker, Reid S. Brewer, Alexander M. Burdin, Merel L. Dalebout, James P. Dines, Ivan Fedutin, et al. 2016. “Genetic Structure of the Beaked Whale Genus Berardius in the North Pacific, with Genetic Evidence for a New Species.” Marine Mammal Science, July. doi:10.1111/mms.12345.

2. (http://www.beakedwhaleresource.com/aboutbeakedwhales.htm)

3. Kitamura, Shino, Takashi Matsuishi, Tadasu K. Yamada, Yuko Tajima, Hajime Ishikawa, Shinsuke Tanabe, Hajime Nakagawa, Yoshikazu Uni, and Syuiti Abe. 2012. “Two Genetically Distinct Stocks in Baird’s Beaked Whale (Cetacea: Ziphiidae).” Marine Mammal Science, September, doi:10.1111/j.1748-7692.2012.00607.x.

An interview with Dr. Phillip Morin

Q. You have performed analysis on 5 blacks and 8 gray beaked whales, how would you increase sampling? Would you deploy a dedicated team/ facility or co-ordinate more with the fishermen in the area to collect more samples? Does the constraints of sampling limit the interpretation of your finding?

Actually, we have sampled 5 of the new black form, included DNA sequences from the 3 that were previously analyzed in Japan, for a total of 8 of the black form. We included 170 of the gray form in our study, ranging from Japan to Mexico across the North Pacific. We would certainly like to have more samples of the black form, and hope that researchers who have data on additional beached whales that they think may be the black form will contact us, and that researchers, fishermen and others who see live beaked whales can help us to find where they spend their time when they are alive. At the moment we do not have funding to commit to this type of study, but if we are able to get a better idea of where to look for them, that could help us develop a more specific (and less expensive) study. Although the sample size is fairly small, the fact that samples are from both Japan and the Bering Sea strengthens the result, and the fact that we have sampled across the known range of Baird’s beaked whales (the gray ones) also strengthens our interpretation of 2 separate species in the North Pacific.

Q. What do we know about the behaviour of the black whale – are they solitary or social? Are they hunter carnivores or live on krill? Usually food is abundant in the shallow sea near the coast and in deeper sea basins the food becomes scarce, do we know something about their dietary habit that allows them to survive? Is the restriction of their abundance in particular geographical locations is because of a known nutrient gradient around your sampling sites?

There are 22 known species of beaked whales, and to my knowledge all of them live primarily in deep waters and feed on squid and/or fish at depth, and are naturally not abundant, probably due to this dietary specialization, high energy needed to obtain sufficient food, and patchiness of food sources. All are also typically found in smaller groups, though Baird’s beaked whale is known to travel in larger groups of 30 or 40 at times. Although we don’t know anything about the behavior or habits of the new species, we can infer that, like other beaked whales, it feeds at depth on squid or deep/bottom fish, and probably travels in small groups (a few individuals).

Q. Mitochondrial DNA versus genomic DNA for phenotyping – what are the challenges of using complete genomic sequences – What would the advantages of doing complete genome sequencing be?

Genome sequencing remains relatively expensive and requires quite a lot of high-quality DNA. For the purposes of determining whether there are 2 species, short sequences of mitochondrial DNA are actually very diagnostic (and therefore adequate), very inexpensive, and can be generated even from bone specimens in museums. We will very likely do some additional sequencing from the nuclear genome to get a more precise estimate of the amount and timing of divergence among the 3 species in the genus Berardius, but full genome sequencing is unlikely to contribute much in the absence of more information on the biology, life history, behavior, etc. of the species, so that is were we are better off putting our research time and effort.

Q. When do you call something a new species ?Can you tell us a little bit more about how haplotypes are used to infer distance and diversity? What other lines of evidence would further strengthen the black beaked whale as new species?

That’s a difficult question, as evolution is a continuous process, and taxonomy (the process of describing species and higher taxa) is an artificial hierarchy imposed on evolution to help us understand and describe biodiversity. We are using a species concept that infers species when genetic evidence indicates that they are evolving along independent evolutionary trajectories, e.g., they do not significantly interbreed and have not for long enough that they have evolved different ecosystem niches and functions (e.g., different food sources or feeding behaviors, habitat use, mating systems, acoustics, etc.). The mtDNA data strongly suggests that this is the case, as there are only very small differences between haplotypes of individuals within each form (≤5), and much larger differences between haplotypes of the two forms (16-26). The fact that the 2 adult specimens of the black form from which we have measurements are only about 2/3 the size of adult Baird’s beaked whales (far outside the normal size range for adult Baird’s whales), and that they have darker skin and several other recognizable (to experts) physical differences also indicates that they are different species, not just variation within a species.

Q. Do you see yourself as a sort of molecular explorer? How would you go about naming the new black whale?

This study has very much been like molecular exploration! We’ve used genetics to help us identify specimens that were previously identified as Baird’s beaked whales, and then been able to follow up with additional studies on those specimens. We use genetics that way frequently for marine mammals because it is so difficult to study them in their natural habitats. Official naming of the new species is done by publishing a complete description of the species (as complete as the specimens will allow), including some skull morphology if possible, and identification of a type specimen (the “holotype”) that will mostly likely be a skull in the Smithsonian Museum that was collected in 1948, but previously thought to be from a small Baird’s beaked whale. There is a team of researchers from Japan that are currently working on that publication and will most likely propose a scientific name. They were the first to indicate that this might be a new species in 2013 based on the 3 specimens from Japan, so that is why they have taken the lead in describing and naming it.

Q. What are the implications of your findings for whaling and conservation?

Baird’s beaked whales and several other toothed whales and dolphins are still commercially hunted in Japan, so it will be important for the whaling industry and scientists there to monitor the hunts to make sure that the new species, which appears to be much more rare than Baird’s beaked whales, is not taken. There are also other potential human impacts that could endanger this species, including seismic exploration, navy sonar, increasing ocean noise from other sources, pollution, entanglement in fishing gear, competition with humans for food, and climate change. Now that we know it’s there, we can work to learn more about it and, hopefully, how to protect it from these potential impacts

Not just grease: packing property of cholesterol powers dynein motor proteins

Cholesterol in vesicle membrane helps dynein power cargo trafficking to the lysosome

 

Short Summary

Inside the cell, cargo-containing vesicles move on microtubules (MTs), powered by protein motors called kinesins and dyneins. Motor proteins work uni-directionally: dyneins transport vesicles inwards {into the cell (minus end of MTs)}, while kinesins move vesicles outwards, in the opposite direction.

This paper makes two points about inward vesicular trafficking: (1) that cholesterol-enriched domains (lipid rafts) help drive dynein motor clustering, a mechanism that helps dynein generate sufficient force for a one-way trip of a late phagasome to the lysosome and (2) that Leishmania donovani (causative agent of kala azhar), a successful intracellular pathogen, secretes lipids that disrupts lipid rafts, thus affecting dynein function and stonewalling the transport of the vesicle that contains it.

Phago-lysosomal fusion

Intracellular pathogens are ingested into vesicles called early endosomes that mature, by changing protein and lipid content, into late phagosomes. These finally fuse with the lysosome, where the pathogen can be degraded. Early endosomes have both dyneins and kinesins, which pull in opposite directions, like a tug-of-war, so they move in a sclerotic manner. But as the vesicle matures, kinesins are lost and dyneins are enriched on its surface. This propels the vesicle in one direction.

How do dynein molecules accomplish this from a physical point of view? The force generated by a single dynein molecule is ~1.1pN, while a late phagosome requires high force (~16pN) to move.

The physics angle

One obvious way to generate more force is to add more dyneins, which these vesicles do, two at a time. However, just more is not enough. Given that vesicles are spherical, a homogenous distribution of dynein molecules will not result in sufficient number being available to engage the MT and vesicle. To increase the area of contact between the MT and vesicle, dynein molecules have to be clustered. This paper provides the first evidence that on late phagosomes, dynein is clustered and this clustering is promoted by cholesterol-enriched lipid rafts (Figure 3). This anchoring of dynein molecules is facilitated by Rab7, a membrane-associated protein enriched on late phagosomes.

How is this information useful?

Besides the advance in fundamental knowledge, that lipids can influence motor proteins, and thus vesicular trafficking, this paper goes on to explore how pathogens exploit this mechanism. Leishmania is an intracellular protozoan pathogen: to stall phago-lysosomal fusion, it secretes lipids called lipophosphoglycans (LPG), that disrupts the raft-like clustering of lipids on late phagosomes. This allows it to subvert transport to the lysosome and survive as a pathogen inside the cell.

Experimental technique

This paper uses a microscopy technique called optical trap. As with experimental science, the first two figures in the paper are dedicated to demonstrating that the technique works, and how its data can be utilised. First, a latex bead is fed to a cell. At different time points, the bead, which the cell tries to transport towards the lysosome is recovered. Early on (<10mins), the bead is an early endosome (EE), while >30mins later it is a late phagosome (LP). The beads are trapped optically and the force that it uses to escape the trap is inferred as the force being applied by the dynein molecules. The force measurements are in the order of pN (10-12), allowing precise measurements of very small magnitudes. Measurements from beads are compared to EEs and LPs purified from cells to prove that the in vitro construction reflects what normally happens in a cell. The beads can thus act as a convenient <case study>. From beads, the general observation emerges that LPs have different transport properties than EEs. Fluorescence images reveals that on LPs, but not EEs, dynein cluster and that these clusters are enriched in cholesterol, and proteins that like rafts.

Take Home

Phagosome maturation and fusion with lysosome is critical for clearance of unwanted material from our cells. This process requires dynein-driven phagasome movement to the minus end of the microtubule. The requisite force for unidirectional travel is provided by clustering of dynein, which is underpinned by clustering of lipids by cholesterol. Pathogens try to escape the clearance mechanism by making lipids that disrupt cholesterol-clustering, and thus dynein clustering.

Reference

Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes. Rai A, Pathak D, Thakur S , Singh S, Dubey AK, Mallik R.

An interview with Roop Mallik

Q. The cartoon model of phagasome movement is a ball moving along a stick. However, from a molecular point, is the phagosome tumbling or rolling like a drum i.e., moving as each cholesterol-enriched dynein cluster grasps the MT, or is it walking, the cluster assembling and disassembling rapidly to move along the MT?

Thank you for asking that question. I don’t think that this phagosome is actually rolling along the microtubule. What happens is that once it is in the proper geometrical orientation where a certain group of these motors is close to the microtubule and the motors attach, that will prevent the rolling motion and from our calculations, as you can see in the paper, we think that on this entire surface of the phagosome there are roughly three such clusters, so the average distance between these clusters is likely to be quite large, I mean the angle between them is likely to be quite large. So just by rolling in this manner, going past one of these clusters and finding another cluster, that chance would be fairly low, so I think like most of the time it is on one cluster and then the thing detaches and if it reattaches again to another microtubule it might be on a different cluster. I have no way of proving this but simple physical arguments suggest this kind of a pitch.

Q. Do you think that the mechanism of cholesterol-enriched membrane domains modulating dynein, would be conserved in non-phagocytic cells, like neurons?

Yes, I think so, I don’t have direct evidence for this but the reason is that many of the marker proteins that come on to these phagosomes are pretty much the same whether they are phagocytic cells or non-phagocytic cells. In my view, the only difference is that the cells which are “professional” phagtocytes, their phagocytic rates are much higher and they phagocytose much more and probably the entire process of phagosome maturation is accelerated (is faster) in the professional phagocyte cells. In other generic cells like Cos7 or something like that which are not phagocytic cells, fewer things would be phagocytosed and probably their maturation process would be slower but essentially if you slow down the whole process the sequence of events I don’t think it would be very different but I have not studied this in non-phagocytic cells, so this is just my guess.

Q. Would all cholesterol-enriched domains with Rab7 bind dynein or do you foresee other molecular players that sequester dyenein specifically to Rab7-positive late phagosomes?

There are certainly other molecular players; cholesterol is just probably the scaffold on which things assemble. What we think is that there is a series of proteins via which dynein attaches to these cholesterol-rich domains and I don’t want to take too many names but at least there are proteins which have GPI anchors which specifically bind to cholesterol-rich domains and one of such proteins is a protein called ORP1L, which in turn recruits downstream proteins like Rab7, RILP and then comes dynein and then actin. Interestingly Rab7, which you mention in your question, also has a GPI anchor and we suspect that it plays something like a Picket fence type of role because the Rab7 protein embeds its GPI-anchor probably into the cholesterol-rich domain and then once that happens, the Rab7 also has a property to dimerise. So if you now think of two fences embedded in the membrane and these two poles want to come together, that would stabilize this entire structure right because we embed something and bring them closer that would create stability in the structure. Those kind of models have been discussed in the context of other lipid microdomain-associated proteins, also Rab7, but as you understand these are not things which are easy to visualise most of the biochemical studies which reach these conclusions may have certain artefacts. So that’s where we are right now.

Q. The Leishmania data suggests that pathogens may actively exclude cholesterol from late phagasomes to prevent being trafficked to the lysosome: low cholesterol means slower trafficking. Therefore, would people who have naturally low levels of cholesterol be more susceptible to infections?

That is a interesting question. There is no data for this but all I can tell you is that the Leishmania parasite actively down regulates cholesterol metabolism when it infects you by targeting a specific microRNA so it is a possibility, but this is something which would be interesting to find out whether such co-relative studies have been done. But if you think about it mostly Leishmania is endemic to rural regions where affluent people do not live. So certainly those people are not going to be eating the kind of diet which gives you too much cholesterol. I guess that’s the best I can do right now.

Q. What was the motivation behind studying Leishmania, over other intracellular pathogens like Salmonella or Mycobacterium?

Right so that is was partly motivation and it was partly whatever you can get your hands on. So we had this reagent, lipophosphoglycan which some people were kind to give to us and we did not have any equivalent eeagent for lets say, mycobacteria or Salmonella. Now that is one part of the reason, the other reason why we chose Leishmania is because there are already published papers from the group of Michel Desjardins which showed that a specific protein, flotilin binds to cholesterol-rich domains; they could show that this clustering is disrupted in Leishmania. So we suspected that Leishmania somehow probably by the use of this lipophosphoglycan maybe disrupting this cholesterol cluster. What we were bringing in new into this picture was the presence of dynein in these cholesterol clusters so the possibility that Leishmania could disrupt these cholesterol clusters is not directly our finding, it is somebody else’s but implication in terms of transport is something which we believe we have contributed

Q. You trained as a physicist, but moved into biology. What inspired this? What would you advice those students who are contemplating such a switch?

I will answer that in two parts. What inspired me, one is that I got to know my present wife who was a biologist around the time I transitioned into this and secondly I would say that I really enjoyed what I did for my PhD, but I was looking for newer things at that time and I am not a person who can plan for months and years in advance; this was something that looked interesting at the point of time and I just took to it and things just worked out. It was not part of a great vision or a plan. I will just be honest with you. And then your second part is that what advice would I give to physicists who would want to come into biology. So in my experience and this is my personal opinion when I talk to physicists who want to do Biology, their idea of doing Biology is going and measuring something and putting some number onto something. That is good to begin with but I think that’s not very useful in the long run. To understand the context of the problem and to kind of realise what is going to be important to Biology, to the body and kind of contextualizing that is more important than putting numbers onto things and as a physicist when you study a biological problem and putting numbers to something is not the greatest thing that you can do. Try to give it your own idea of how things evolve in space and time that is something which I think physicists can bring to the plate, while many biologists in my opinion often miss that point. They more interested in the molecular interactions you know, this interacts with that, which is all very useful but try to think of it, bring in the element of space and time and sequence of things is something that at least I try to do as a physicist, I don’t know what I am a physicist or a biologist, whatever, but that has helped me in thinking about my science. That’s my view.

Can genetic variations define Ayurvedic Prakritis?

-A genome- wide study finds allelic differences between individuals that correlate with Ayurvedic body-types (Prakritis)

Snap-Shot of the study

Do the ayurvedic body types have genetic underpinnings? In a first step to answer this question, the authors evaluated differences between individuals whose body-type had been assigned by both Ayurvedic practitioners and a software. They found 52 variations across the genomes of 262 individuals which allowed them to be classified into ayurvedic Prakritis – Pitta, Kapha and Vata.

 Introduction to Ayurvedic prakritis

In Ayurveda, according to the ancient text Charaka Samhita –the body and mind must be brought together to lead a harmonious existence. People can be classified into Prakritis or types on the basis of relative contribution of the three constituents Pitta, Kapha and Vata (roughly translating to – arising from movement, digestion and accumulation – of toxic metabolites for instance) to their body. According to Ayurvedic philosophy- an understanding of this body type and the ability to maintain a diet and lifestyle suited to that body type translate to balance and health. Prakriti or Ayurvedic body-types which define a person’s intrinsic physical abilities, mental states and also have implications for their susceptibility to disease and response to drugs (1,2).

Background for this study

In a recent study (3), researchers have identified genetic variations associated with the traditional classification of people into Ayurvedic Prakritis – specifically if small differences (SNPs, Single nucleotide polymorphisms) throughout the human genome correlate with the Prakriti classification. All humans are genetically very similar to each other, differences between us (populations, races, ethnic groups etc.) are captured in variations of nucleotides which make up the DNA – these are called single nucleotide polymorphisms. Many studies have shown particular SNP or group of SNPs to be correlated with risk of disease, whether a person will develop resistance to therapy, etc. forming the basis for personalized medicine  (4,6).

 What did they do?

3416 individuals were classified for their prakritis by Ayurvedic practitioners as well as a software. Of these, DNA isolated from blood samples of 262 individuals (male, healthy, between 20-30 years), who were reliably classified as having a clear dominance of one of the three constituents (Vata, Pitta or Kapha) representing the “extreme” Prakritis were used for the genome-wide study. A microarray consisting of 1 million positions / SNPs was used to identify the genotype of these individuals.

 What did they find?

This study found 52 out of 1 million SNPs is sufficient to assign the Prakriti of individuals, irrespective of their ethnic background. One of the challenges in the study was that there was no control group – therefore each prakriti was compared to the other two. Subsequently, these 52 SNPs were able to cluster individuals into distinct groups by Principal Component Analysis. Interestingly, one of the SNPs in a PGM1 gene (Phosphoglucomutase 1), which codes for an important enzyme in sugar (glucose) metabolism, is significantly associated with Pitta dominant group that is known for efficient metabolism.

 Things to keep in mind about the study:

It is to be noted that the three categories compared and defined here represent extremes –and according to Ayurvedic principles most people are a composite of all three- Kapha, Vata and Pitta, with the dominant element defining their type. There are some previous studies, which have suggested that the Ayurvedic Prakriti classification may have a genetic or metabolic basis (2,5). They were conducted on fewer subjects and looked at fewer genes/ phenotypes compared to this study, which rigorously recruited a large number of subjects, used multiple methods of classifications (software and Ayurveda practitioners) and a genome wide approach. There is much work to be done in understanding the scientific basis of the Ayurvedic classification system and whether we can independently and reliably assign people (independent of race, gender and ancestry) to a type.

 What does this mean?

The 52 SNPs defined in this study can now be used independently in other populations and also provide a way of identifying new associations with metabolism and other phenotypes. For more than fifteen years now, we have been able to read our genome i.e – information in our genes but not been able to fully understand how it defines us as individuals. So on the one hand, this study by correlating phenotypes with genetic variations, helps us understand a little bit more about how genes make us who we are. On the other hand, by using modern genetic tools in the context of traditional knowledge, this study provides a rigorous way of assessing the framework of ayurvedic medicine.

Citations

1. Understanding personality from Ayurvedic perspective for psychological assessment: A caseS Shilpa and C. G. Venkatesha Murthy. Ayu. 2011 Jan-Mar; 32(1): 12–19.

2. Classification of human population based on HLA gene polymorphism and the conceptof Prakriti in Ayurveda. Bhushan P 1 , Kalpana J, Arvind C. J Altern Complement Med. 2005 Apr;11(2):349-53.

3. Genome-wide analysis correlates Ayurveda Prakriti. Govindaraj P, Nizamuddin S, Sharath A, Jyothi V, Rotti H, Raval R, Nayak J, Bhat BK, Prasanna BV, Shintre P, Sule M, Joshi KS, Dedge AP, Bharadwaj R, Gangadharan GG, Nair S, Gopinath PM, Patwardhan B, Kondaiah P, Satyamoorthy K, Valiathan MV, Thangaraj K. Sci Rep. 2015 Oct 29;5:15786. d

4. A database of humans SNPs and their recorded associations can be found here: http://www.snpedia.com/index.

5. Whole genome expression and biochemical correlates of extreme constitutional types defined in Ayurveda . Prasher B, Negi S, Aggarwal S, Mandal AK, Sethi TP, Deshmukh SR, Purohit SG, Sengupta S, Khanna S, Mohammad F, Garg G, Brahmachari SK; Indian Genome Variation Consortium, Mukerji M. J Transl Med. 2008 Sep 9;6:48.

6. http://www.scientificamerican.com/article/a-very-personal-problem/

Interview with Dr. K. Thangaraj

Q. What was the overlap between the prediction by the software, by different Ayurvedic practitioners? Have you tried to estimate if this classification is robust enough to suggest underlying genetic differences?

The first assessment was by Ayurvedic physicians and classified using their knowledge. To double check what they have done, we have used a computer software. The software is also based on various parameters specified by the Ayurveda physicians.There were many questions which the individual had to answer to be assigned a Prakriti. Across the three centres – Bangalore, Pune and Udupi, on average, 75% of the individuals were in concordance.

Q. Why not perform an enzyme profile or measure transcriptional differences for metabolic enzymes? Is there an advantage in taking an SNP approach?

The advantage is that the SNP does not changes, it is there from birth till the individual dies. Whereas transcription profiles may change at different times, depending on time of day, tissue type to tissue type etc. We have looked into that also. This is the very first step. We can extend this across countries, across ethnic groups and cluster them.

Q. Ayurvedic medicine believes in holistic changes including those of lifestyle, dietary along with medicines and does not rely overly on mechanistic explanations beyond the classification into types. There is less emphasis, if you will on dissection of cause-effect and more on restoring the overall balance. Do you agree with this? If yes, then what are the challenges with respect to the study design when you tried to apply the modern framework of science- which relies on a reductionist approach, finding a cause and targeting only that specifically, to the ayurvedic system?

I agree that there is a holistic approach, but the basic approach is to classify the individual. Based on the prakriti, they will make the changes to the diet, prescribe treatment etc., so this is a very important stage. The challenge is the following- I am a geneticist looking at diseases,looking at case-control studies is very easy – for every marker we can ask if the mutation is present or if the prevalence is higher in the patients versus the control. In this case all the individuals are normal and within the same age-group. The only difference is the prakiti. So we wanted to see how to differentiate these individuals- as there are three groups, not two. Then we decided to compare one prakriti against the other two types, and try to see if there is genetic variation between the groups. Then there was a lot of statistical analysis. We used 1 million markers, this has many advantages, the disadvantage is the robustness and having to come up with statistical analysis ourselves. (MT: So, by using 1 million regions, you may increase the chances of false associations, is that the worry?) Not, really false associations. For example we may not have information about a particular SNP in an individual. We need to use markers that are consistent between all the individuals. So, when data is not available, we need ways of retrieving the data. In that process we need to use genetic panel of markers which are Indian specific. We developed our own panel of markers – Dravidian, Indo-European and used as a reference and to extract what is the possible marker in a given position.

Q. Have you tried to validate your classification using the 52 SNP panel with an independent population? For instance, if you knew what a person’s SNP state is, how reliably can you assign their Prakriti? Would it be useful to perform a blind study in which both the SNP panel ayurvedic practitioners and the software performed the classification, with the aim of determining how often they match?

That is very interesting. What we did was, we has more than 300 samples analyzed for these 1 million genetic markers, from our initial studies on population genetics. We used some of those samples, as these are all populations specific – a very endogamous population. We tried to project some of those individuals into these three clusters, we did find that although the individuals have come from the same ethnic background (more homogeneous), they were falling in 2 or 3 different prakritis. The same ethinic background can be placed into different prakriti. This we tried to do with our own data, this is not as detailed as you suggest. Independent blind studies need to be done.

Q. You have excluded women from your study and restricted yourself to the Indian population, does this limit the applicability of your results?

Yes, of course. At this point we selected only males because we did not want any confounding effects, in the females there are many hormonal changes and so on.

Q. You have started a way of examining traditional medicine in the framework of modern science. What are the challenges and the future of this approach?

(The future of this approach) This has paved the way to do many more things. For example, the discovery of PGM1 has given the clue that you can take the phenotype of the particular prakriti and correlate it with the gene, this gene is involved in metabolism and individuals with the pitta prakirti have high metabolism. We can now use the characteristic feature of the prakriti and look into those genes in a detailed way. These are some of the futuristic aspects, one can take the study further with. We did try to look at the network of all the metabolic pathways genes, the problem is this will need transcription or metabolic profiles from tissues of these normal individuals.

 

Scouting for the forager ant

– Identifying the basis of labour division in carpenter ants

 

Short summary

What determines the way animals behave? Is this almost unalterably in their genes or in their environment or a complex interaction between what is within and without? A recent study explores the division of roles within the worker caste of the carpenter ant and shows for the first time that these roles can be reversed using mind-altering drugs (1).

 

“Minor”carpenter ants are the foragers

Anyone who has observed the incessant activity of a beehive or a column of ants between their nest and a food source can appreciate how wonderfully co-ordinated and orderly it all looks. This coordination is brought about by a fascinating division of labour – well separated roles of who must do what for the colony. How do insects develop these identities? This becomes especially intriguing in insect colonies in which all inhabitants are children of the same parents and hence genetically related to each other. In carpenter ants, which are the subject of this study, there are two castes within the female workers – minor and major. The minors are smaller do most of the foraging whereas the majors rarely forage. This was established by setting up an arena for foraging around an ant nest and measuring to which caste they belonged and how many individuals came out to forage. While foraging activity for both minor and majors increased with age of the ants, the minors still performed most of the foraging. Additionally, the lead foragers – called scouts were also mostly minors and older scouts were much better/faster foragers than young ones -even when they were foraging in unfamiliar arenas. Hence, the foraging activity was established as a minor worker specific behaviour in these ants.

 

What makes “minor” ants better at foraging?

So what determines the foraging behaviour of minors? Genetic differences were undermined by the relatedness of minors and majors (they are sisters (2)) – suggesting that the differences is unlikely to be due to differences in genes. Also, multiple studies suggest that such behaviours are likely to be controlled by epigenetic mechanisms. Epigenetic modifications are modifications of the genetic material, without changes to the genetic material/ DNA itself (check out a beautiful introduction to the world of epigentics from MinuteEarth in the video below). These modifications which are chemical groups added on proteins that bind DNA, determine the context in which genes are expressed i.e form a basis for the conversion of genotype to phenotype. In this study they focused on the presence of a chemical group (an acetyl group) on histones. Previous studies have suggested that these marks may determine caste specific behaviour in other eusocial insects.

Consistent with this idea, a drastic increase in foraging activity of both majors and minors was brought about by feeding them drugs that inhibit the enzymes that remove the acetyl marks from the histone – or histone deacetylase inhibitors (Valproic acid – used to treat mood disorders in humans and Trichostatin A).  However, the minors continued to forage more and performed almost all the scouting.

Molecules and mechanisms of caste-identity

The authors then determined the molecular mechanisms of how the acetyl marks were placed on the histones in the first place what genes were responsive to these changes. When they inhibited the enzyme responsible for this behaviour (the histone acetyl transferase domain of the Creb binding protein (CBP)) they saw a drastic decrease in scouting. This established the scouts as a distinct behavioural caste within the minors and suggested that acetylation of histones by CBP is the molecular mechanisms that generate this behaviour.

They then looked at what makes major and minor workers different from the perspective of gene expression and came up with a different idea. What if there was a basal behaviour – “to forage” in ants and this was actively suppressed by all these molecular mechanisms? This suggested that injecting drugs at an early stage may prevent this suppression of behaviour. Voilà! in ants injected with drugs (Trichostatin A) the majors started to forage actively! In this case, it seems like timing was everything (look above for what happens when the treatment is started later!). Surprisingly, even when tested as a whole colony (with minors), the majors upon treatment participated more often in foraging. To dissect this further, the authors directly inhibited a single enzyme HDAC1 (Histone deacetylase 1 also called Rpd3) and found the same increase in foraging activity in the majors. This suggests a central role for HDAC1 in repressing foraging behaviour in majors.

Learning from ants

Behaviours are baffling and possibly emerge from complex interactions between genes, how these genes get expressed and what triggers them. Such triggers can come from what we eat, what we smell, how we interact with our environment and one another. Animal behaviour – especially in the context of colonies or societies, is likely to involve intricate rules for function and order. Unraveling these rules is an exciting area of ongoing research. In a surprising but retrospectively sensible turn of events, the authors of this study have found that the division of labour among worker ants lies in the mind, is set up very early and can be reversed.

Acknowledgement

Thank you!  Riley J Graham for helping out with this post

More about the cool process of epigenetic inheritance from the wonderful Minute Earth

youtu.be/AvB0q3mg4sQ

An interview with Riley J. Graham

 

Q. You note in your study, that a carpenter ant colony in nature, maintains a 2:1 ratio of minors to major worker ant. What do you think are the mechanisms for maintaining that ratio? Is it likely to be the same mechanism (HDAC and HAT dependent) as you describe?

It’s difficult to say how this could occur, and there is likely a degree of variation in caste ratio in wild colonies. One of our ongoing questions is whether caste fate can be influenced during development by epigenetic drugs. To address this, we are developing methods to deliver controlled treatment doses to during larval development to determine if this influences caste fate. Such a result would strongly suggest that HDAC and HAT activity is important for regulating the generation of caste specific morphological traits, which could account for how this ratio is maintained in our experimental colonies.

Q. Will the drug reversed majors show increased foraging even when there is an abundance of resources? 

Yes, in fact this is precisely what we saw. All of our colonies were fed ad libitum, for 10 days after injection, and majors treated with HDACi foraged significantly more than controls. However, because minor workers can feed their major sisters after foraging, a mixed caste setting may keep majors full of food even when they never forage. To control for this type of between-caste effect we did a different test in which we separated major and minor workers and withheld sugar water. This ensured all of our test subjects had a similar motivation to exit the nest in search of sugar, and prevented intrinsic behaviors of one caste from biasing behavior of the other.

Q. Have you or others seen such role reversals/ caste reversals in a natural setting? For example – colonies that are stressed for food.

Camponotus floridanus and its relatives in the subfamily Formicinae are interesting because of their discrete morphological caste systems, (e.g. minor, major) but all eusocial insect species rely on some form of caste-based division of labor to survive. Brian Herb and colleagues reported differences in genome-wide patterns of DNA methylation between nurse and forager honeybees. These two groups are behavioral subcastes that arise as younger nurse workers age and progressively become active foragers later in life. Experimental reversion of foragers back to nurses caused a coordinated reversal of DNA methylation to reflect this behavioral change, suggesting epigenetic regulation of behavior is a common trait among social insects, and that behavioral castes are sensitive to environmental changes.

Q. What major contribution do you think will come out of studying eusocial insects like ants or honeybees when compared to solitary insects like fruit flies? 

Fruit flies do not exhibit the vast range of behaviors seen in social insects. Over evolutionary time, some eusocial insect species have acquired sophisticated division of labor strategies, enabling colonies to undertake complex collective tasks including nest architecture, cooperative brood care, and even horticulture, as in the leaf-cutter ants Atta and Acromyrmex. Given that single queens can give rise to millions of individuals in thier lifetime, epigenetic regulation, rather than genetic differences between individuals are expected to have an important role in the expression of caste specific traits. We have not found allelic predictors of caste identity in C. floridanus, suggesting that the exceptional phenotypic differences between major and minor workers are likely attributable to epigenetic mechanisms. Eusocial insects are therefore excellent models for the study of how epigenetic changes can contribute to morphological and behavioral variation.

Q. Earlier studies, for example those by Sokolwaski et. al. have shown single locus polymorphisms controlling foraging behaviour in fruit flies. In the light of this evidence, one might think that epigenetic control of foraging behaviour in ants could be an adaptation to their social lives. What do you think?

I believe you are referring to Marla Sokolowski’s work showing that mutations in the gene foraging (for) can lead to differences in foraging behavior in flies. Such polymorphisms might cause variation in foraging behavior in flies, but in ants, this SNP would contribute to increased foraging in all castes, perhaps even the queen. Given that queen foraging would typically be highly damaging to a colony’s survival, this SNP would be evolutionarily suppressed in queens, but could become positively selected for in minor workers. This variation in the fitness landscape between castes is one reason to think that epigenetic regulators could be important when different castes need to express different genetic profiles from a common genome. Molecular heterochrony allows different genes to be expressed at different times in an animal’s life, and while a very young queen might benefit from a SNP causing increased foraging, a mature queen would not. The genome’s ability to activate or suppress genes depending on caste and age is an important aspect of social insect biology that likely relies on epigenetic mechanisms.

Q. Carpenter ant workers in the study are genetically related, which led you to investigate the possible epigenetic mechanisms determining the cast specific behaviours. Would you expect genetic bases for caste identity in species where genetic relatedness among the workers is not as high as the carpenter ants?

A number of studies describing genetic aspects of caste fate also suggest that the interaction of each genotype with the environment influences caste fate. In this light, it seems that genetic variation primarily alters an organism’s likelihood of becoming a particular caste, rather than rigidly determining caste fate. Allelic predictors of caste identity were not found in our ant species, suggesting behavioral and morphological phenotypes in social insects are likely the product of a gene by environment interaction that is facilitated by the epigenome.

Q. Insect colonies are fascinating systems to study genetic links to behaviour. Your study added valuable insights in mechanisms of determination of a caste specific behaviour. How easy (or hard) would it be to study more complex behaviours in other social animals (not necessarily insects)? 

Our work is among the first to look for indications that social insect behavior can be altered by the epigenome without any change in DNA sequence. Ants are a fascinating middle ground between the moderate behavioral variability seen in solitary insects, and the overwhelming complexity of higher order social behaviors, such as the relationships between kin grooming and reproductive hierarchies in primates. As scientists begin to consider more complex social features, they must also consider the vast array of behaviors that can be performed by each individual. In the case of kin grooming, researchers might be compelled to annotate a complex and fluid social network of kin grooming interactions, which may require a model that considers the behavior of each animal, as well as the behaviors of their social partners. This can get complicated quickly. This is not an insurmountable goal, but it is certainly harder to conceptualize and design experiments around. However, any molecular variation in the population that robustly contributes to behavior can hypothetically be measured, so it is not impossible to study organisms with greater behavioral complexity.