Digging For Clues About Climate Change

Guest post by Rebecca McDonald, science writer

Photo Credit: LeRoy N. Sanchez

While many scientists who study climate change look up to the sky for clues about the Earth’s future, one researcher has spent her career looking down—at the abundance of life in the soil below. Innumerable microorganisms such as bacteria and fungi live in harmony with plant roots, decomposing fallen leaves and dead animals. In addition to acting as the ultimate recyclers, they also stabilize the soil and help to retain water. 

Cheryl Kuske, a microbiologist at Los Alamos National Laboratory, has focused the last two and a half decades on studying this microbial environment. “By decomposing organic matter,” she explains, “microorganisms help cycle carbon and nitrogen through the ecosystem.” Some of the carbon and nitrogen released from the organic matter goes into the soil and is assimilated into roots to help new plants grow—the carbon is incorporated into sugars, and the nitrogen atoms are used to build proteins. But some of these molecules are also released as CO2 and N2 gases into the atmosphere.

The soil ecosystem functions in a delicate balance. Although some organisms release gases into the air, others—including certain bacteria and leafy plants—remove harmful CO2 from the atmosphere for food production.

Kuske and her colleagues at Los Alamos National Laboratory have been investigating the roles of these microbes in carbon and nitrogen cycling to help make better predictions about terrestrial ecosystem responses to climate change. Using a technique called metagenomics to sequence the DNA of all the microbes at once, the team can study the organisms’ genes and the enzymes they produce.

These microrganisms’ lifecycles are so intertwined that their single genomes cannot be isolated for sequencing. However, analyzed jointly, they yield important clues about their collective functions in the environment. Scientists can identify things such as which bacteria or fungi are responsible for fixing nitrogen or carbon, the ratio of bacteria to fungi in the soil, and which microbes are closely associated with root health or plant growth. The researchers can even figure out which enzymes are currently being used through a technique called meta-transcriptomics; this approach sequences only the transcripts of genomic data that are actively being made and used for protein synthesis.

Photo courtesy of Cheryl Kuske

By sampling microbes from various soil environments over long periods of time, Kuske’s team and collaborators are able to understand what happens under the surface when things change aboveground. For instance, in a recent long-term study in Utah, the scientists discovered that slight changes in the summer precipitation pattern, combined with a 2°C rise in soil temperature, resulted in significant changes in the population of microbes below: the types of organisms completely changed, thus altering their overall role in the environment. For example, cyanobacteria—bacteria that create energy through photosynthesis—were no longer present. As a consequence, the new population of microbes no longer had the ability to pull carbon out of the air and had a decreased capacity for fixing nitrogen for protein synthesis.

Increased nitrogen from industrial runoff or fertilizer from agriculture can also have significant effects on the composition of organisms in the soil, as nitrogen is an essential molecule for the growth of both plants and bacteria. A comparison of 15 recent field experiments where nitrogen deposition was measured showed that in an arid environment, an increase in nitrogen had a positive effect on soil health at low concentrations, but too much was toxic to the soil community [1]. In a field experiment in Nevada, higher nitrogen concentrations changed the species composition of bacteria—but not fungi—leading to a fungi-dominated community [2,3].

Although the ramifications of these changes to the microbial world are not yet completely understood, Kuske’s team is continuing their studies, both in the laboratory, under controlled conditions, as well as at various field sites in the American Southwest. What they do know is that the feedback loop is strong. Changes in the aboveground environment—such as rising temperatures, altered precipitation, and increased nitrogen runoff—lead to changes below ground that can have far-reaching consequences.

“The studies being conducted at Los Alamos provide an understanding of the interactive biological processes that are inherent in all types of terrestrial ecosystems and that tightly control carbon and nitrogen fluxes to the atmosphere,” says Kuske. Climate warming and altered weather patterns will disrupt this balance. When the diversity of soil microbes change, the feedback loops that ensue could have lasting effects on the amounts of carbon and nitrogen in the soil and the atmosphere.

Rebecca McDonald is a science writer at Los Alamos National Laboratory specializing in the communication of bioscience research. She has also worked as a freelance writer, and volunteers her time as a communications consultant for a science education non-profit.

Disclaimer: Elena E. Giorgi is a computational biologist in the Theoretical Division of the Los Alamos National Laboratory. She does not represent her employer’s views. LA-UR-16-22406.


[1] Steven B, Kuske CR, Gallegos-Graves LV, Reed SC, & Belnap J (2015). Climate change and physical disturbance manipulations result in distinct biological soil crust communities. Applied and environmental microbiology, 81 (21), 7448-59 PMID: 26276111

[2] Sinsabaugh RL, Belnap J, Rudgers J, Kuske CR, Martinez N, & Sandquist D (2015). Soil microbial responses to nitrogen addition in arid ecosystems. Frontiers in microbiology, 6 PMID: 26322030

[3] Mueller RC, Belnap J, & Kuske CR (2015). Soil bacterial and fungal community responses to nitrogen addition across soil depth and microhabitat in an arid shrubland. Frontiers in microbiology, 6 PMID: 26388845

The Antibacterial Resistance Threat: Are We Heading Toward a Post-Antibiotic Era?

Source: PEW Charitable Trusts

The above graphic, from the Antibiotic Resistance Project by the PEW charitable trusts, summarizes how alarming the emergence of drug resistant bacterial strains has gotten over the past few decades. According to data from the Center for Disease Control (CDC), every year 2 million Americans acquire drug-resistant infections [1], in other words infections that do not respond to treatment with ordinary antibiotics. Not only do drug-resistant infections require much stronger drugs, but, when not deadly, they often leave patients with long-lasting complications.

One of the scariest threats is carbapenem-resistant Enterobacteriaceae (CRE), bacteria that are resistant to several kinds of antibiotics. In 2001, only North Carolina, out of all 50 states had reported one CRE infection. Last year, in 2015, 48 states reported CRE infections to the CDC. And while drug-resistant strains emerge rapidly, the discovery of antimicrobial substances has stalled: in the last decade, only 9 new antibiotics were approved, compared to 29 discovered in the 1980s and 23 in the 1990s. We are fighting a new war, and we are running out of weapons.

How does drug resistance emerge?

Bacteria constitute an irreplaceable building block of our ecosystem: they are found in soil, water, air, and in every living organism. In humans, it's estimated that they outnumber our cells by 3:1, and numerous studies have shown that not only do they help us digest and produce enzymes that our body wouldn't otherwise be able to break down, but they can also influence gene expression and certain phenotypes (see some of my past posts for more information).

They live in symbiosis with us, yet some bacteria can be highly pathogenic. The overall mortality rate from infectious diseases in the US fell by 75% over the first 15 years following the discovery of antibiotics [3], and researchers estimate that antibiotics have increased our lifespan by 2 to 10 years [4] by enabling us to fight infections that would otherwise be deadly.

However, evolution has taught bacteria to fight back.

Bacteria develop drug resistance through the acquisition of genetic mutations that either modify the bacteria's binding sites (and therefore the drug can no longer enter the membrane), or reduce the accumulation of the drug inside the bacterium. The latter happens through proteins called "efflux pumps", so called because their function is to pump drugs and other potentially harmful chemicals out of the cell. Once these advantageous mutations appear in the population, they spread very quickly, not only because they are selected for, but also thanks to bacteria's ability to transfer genes: the drug-resistant genes form a circular DNA unit called plasmid, and the unit is passed on to nearby bacteria so that they, too, can become drug resistant.

These mechanisms are not new to bacteria, however, what's new is the increasing overuse of antibiotics and antimicrobial chemicals in our modern lifestyle. The antimicrobial agent called triclosan, for example, can be found in all antibacterial soaps, toothpaste, mouthwash, detergents, and even toys and kitchen utensils. Because of its wide use in household and hygiene products, triclosan has been found in water, both natural streams and treated wastewater, as well as human samples of blood, urine, and breast milk. As though that alone wasn't enough to alert consumers, a study published on the Proceedings of the National Academy of Sciences [5] claims that triclosan, which can be absorbed through the skin, can impair the functioning of both skeletal and cardiac muscle. The researchers confirmed these findings both in vitro and in animal models.

Resistance is also spread through the use of antibiotics in industrial farming. In the US alone, the daily consumption of antibiotics amounts to 51 tons, of which around 80% is used in livestock, a little under 20% is for human use, and the rest is split between crops, pets, and aquaculture [3]. A meta-analysis published last year in PNAS [6] found that between 2000 and 2010 the global use of antibiotic drugs increased by 36%, with 76% of the increase coming from developing countries. The researchers projected that worldwide antibiotic consumption would rise by 67% by 2030 due to population growth and the increase in consumer demand.

These frightening statistics prompted CDC director Tom Frieden to issue a warning: “If we are not careful, we will soon be in a post-antibiotic era.” An era when common infections are deadly again.

"We need to be very careful in using antimicrobial agents for everything from hand washing to toothpaste," Harshini Mukundan, microbiologist at Los Alamos National Laboratory, explains. "Increased selection of drug resistant organisms means that future generations will be helpless in fighting even the most common bacterial infections."

Mukundan and her colleagues have been working on biosurveillance and tracking the emergence of drug resistant strains in high disease burden populations where emerging antibiotic resistance is a huge concern. In collaboration with the Los Alamos National Laboratory metagenomics group, and Los Alamos scientists Ben McMahon and Norman Doggett, the team is working on developing new assays for faster diagnosis of drug resistant infections. Another approach to fight drug resistance is trying to understand how bacterial efflux pumps work at excreting the drug out of the bacterium. Gnana Gnanakaran, a computational biologist at Los Alamos National Laboratory, and his team have developed mathematical models to describe the structure of these pumps [7] and find a way to deactivate them.

While this research is highly promising and exciting, we all need to step up and do our part before it's too late: the CDC published a series of recommendations for patients to follow at the doctor's office, and there are smart choices we can make at home, too. In a recent report, the Food and Drug Administration (FDA) claims that there is no evidence that antibacterial soaps do a better job at preventing infections than ordinary soap, and that in fact:

"New data suggest that the risks associated with long-term, daily use of antibacterial soaps may outweigh the benefits."

In its 2011 policy paper, the Infectious Diseases Society of America (IDSA) recommended a substantial reduction in the use of antibiotics for growth promotion and feed efficiency in animal agriculture, and encouraged the FDA to complete and publish risk assessments of antibiotics currently approved for non-therapeutic use.

Just like any other precious resource, antibiotics (and antimicrobial drugs in general) need to be used with parsimony.

Resources:
[1] Antibiotic Resistance Threats in the United States, 2013 (CDC)

[2] PEW Antibiotic Resistance Poject

[3] Armstrong GL, Conn LA, & Pinner RW (1999). Trends in infectious disease mortality in the United States during the 20th century. JAMA, 281 (1), 61-6 PMID: 9892452

[4] Hollis, A., & Ahmed, Z. (2013). Preserving Antibiotics, Rationally New England Journal of Medicine, 369 (26), 2474-2476 DOI: 10.1056/NEJMp1311479

[5] Cherednichenko, G., Zhang, R., Bannister, R., Timofeyev, V., Li, N., Fritsch, E., Feng, W., Barrientos, G., Schebb, N., Hammock, B., Beam, K., Chiamvimonvat, N., & Pessah, I. (2012). Triclosan impairs excitation-contraction coupling and Ca2+ dynamics in striated muscle Proceedings of the National Academy of Sciences, 109 (35), 14158-14163 DOI: 10.1073/pnas.1211314109

[6] Van Boeckel, T., Brower, C., Gilbert, M., Grenfell, B., Levin, S., Robinson, T., Teillant, A., & Laxminarayan, R. (2015). Global trends in antimicrobial use in food animals Proceedings of the National Academy of Sciences, 112 (18), 5649-5654 DOI: 10.1073/pnas.1503141112

[7] Resisting Bacterial Resistance, by Rebecca McDonald, 1663 Magazine.

Allergies: Can Too Much Hygiene Actually Harm Us?

It's that time of the year again. You step out of the house and your eyes itch, your nose starts running and your head feels like an empty balloon. Yes, it's allergy season again. Even the resilient ones, give them enough time and eventually they will develop some form of allergic reaction.

But what are allergies and why do so many people suffer from them?

Allergies are a glitch in our immune system. The immune system is built to recognize and destroy pathogens -- potential threats like viruses and harmful bacteria. Unlike pathogens, allergens are substances that, despite being harmless to the body, still trigger a response from the immune system. As soon as the allergen is detected, the immune system releases a class of antibodies called IgE. These antibodies signal the cells to release histamine, a neurotransmitter that triggers all the pesky symptoms typical of an allergic reaction: wheezing, watery eyes, running nose, coughing, and all the like.

Spring is a particularly dreaded time of the year for allergy sufferers because of all the pollen released in the air. Global warming has impacted the duration and spread of pollen allergies: shorter winters and warmer temperatures translate into longer pollen seasons, which in turn increase the duration and severity of symptoms for allergy sufferers. In addition, they also increase the exposure and possible sensitization of people who don't suffer from allergies ... yet [1].

Are allergies on the rise?

In his 2015 review [2], Thomas Platts-Mills, of the University of Virginia School of Medicine, looks at the prevalence over the past five decades of asthma, hay fever, and peanut allergy, and reports a progressive increase in pediatric asthma, as well as a "dramatic" increase in food allergies. Allergies are more prevalent in developed countries, and particularly in urban settings, suggesting that something in the industrialized lifestyle may have triggered the increase. However, given the many drastic changes introduced in these countries over the past century, it's hard to pin-point one specific cause. Several factors have been suggested as possible explanations: changes in hygiene, for example, together with a decrease in outdoor life, smaller families and no more exposure to farm animals, have significantly reduced our exposure to bacteria; the progressive use of antibiotics and antimicrobial products have also reduced such exposure; less outdoor time also means less physical activity, more exposure to indoor allergens, and an increase in body mass.

First proposed in 1989 [3], the "hygiene hypothesis" -- the theory that the rise in allergic reactions is caused by a decrease in childhood exposure to harmless bacteria -- has grown to encompass many other disorders, not just allergies. The theory originally spurred from the observation that children with a higher number of siblings had a lower risk of developing asthma, something that led researchers to think that this was due to a higher exposure to bacteria.

The human microbiome is the set of all bacteria coexisting in our body. They are estimated to outnumber our cells by 3:1 and the vast majority of these organisms are not only harmless, they actually play an important role in our health. For example, by modulating the concentration of chemicals that are precursors of important neurotransmitters, they can affect our mood and mental health [4]. They can also influence our propensity to certain phenotypes such as leanness or obesity by affecting gene expression in our guts [5].

Scientists have used a mouse model to show that by transferring gut micriobiota from allergic mice to resistant mice they could actually transfer the food allergy to the latter [6], proving a correlation between the two. Tolerance to food is acquired during infancy thanks to the interaction between the immune system and the gut microbiota, and therefore, early development of the gut microbiome is believed to play a fundamental role in the predisposition to allergies and other diseases later in life. Indeed, in the industrialized countries that are experiencing an increase in allergies, scientists have observed a delayed gut colonization after birth, less biodiversity in the gut microbiome, and reduced turnover of gut bacterial strains in infants [6].

Three major factors could be responsible for this: (i) natural birth versus C-section (a C-section deprives the newborn of beneficial exposure to commensal bacteria residing in the birth canal); (ii) breast-feeding versus formula; (iii) early exposure to antibiotics. All three practices -- C-section, formula feeding, and the use of antibiotics and antimicrobial products -- have been increasingly used in developed countries, and all three affect the development of the gut microbiome of infants. While studies that have looked at possible associations between any one of them and the risk of allergies so far have not yielded conclusive results, the differences in microbiomes between healthy people and those with asthma and allergies are an indication that early exposure to bacteria may protect against these conditions [7].

Is there such a thing as too much protection?

These observations don't mean that we should all stop washing our hands and start living filthy. They do, however, point to a trend in overuse of antimicrobial household products (soaps, laundry detergents, kitchen cleaners, etc.). These products should be used with care and only when truly needed. In most instances, natural substitutes like vinegar to clean surfaces are a better choice, as they keep your kitchen clean without killing microorganisms that are actually beneficial to our health. As much as we strive to protect our little ones, remember that childhood exposure to pathogens makes your child's immune system grow stronger and well trained to recognize bigger dangers. (On a side note, vaccines equally stimulate the immune system without the hassle of all the symptoms.) Finally, global measures like recycling gray water can benefit both the planet and our own health, as it saves gallons of drinking water from being used in landscaping and farming, while restoring important bacteria into the soil and back into our environment.

References

[1] Ziska, L., Knowlton, K., Rogers, C., Dalan, D., Tierney, N., Elder, M., Filley, W., Shropshire, J., Ford, L., Hedberg, C., Fleetwood, P., Hovanky, K., Kavanaugh, T., Fulford, G., Vrtis, R., Patz, J., Portnoy, J., Coates, F., Bielory, L., & Frenz, D. (2011). Recent warming by latitude associated with increased length of ragweed pollen season in central North America Proceedings of the National Academy of Sciences, 108 (10), 4248-4251 DOI: 10.1073/pnas.1014107108

[2] Platts-Mills, T. (2015). The allergy epidemics: 1870-2010 Journal of Allergy and Clinical Immunology, 136 (1), 3-13 DOI: 10.1016/j.jaci.2015.03.048

[3] Strachan DP (1989). Hay fever, hygiene, and household size. BMJ (Clinical research ed.), 299 (6710), 1259-60 PMID: 2513902

[5] Ridaura VK, Faith JJ, Rey FE, Cheng J, Duncan AE, Kau AL, Griffin NW, Lombard V, Henrissat B, Bain JR, Muehlbauer MJ, Ilkayeva O, Semenkovich CF, Funai K, Hayashi DK, Lyle BJ, Martini MC, Ursell LK, Clemente JC, Van Treuren W, Walters WA, Knight R, Newgard CB, Heath AC, & Gordon JI (2013). Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science (New York, N.Y.), 341 (6150) PMID: 24009397

[4] Li, Q., & Zhou, J. (2016). The microbiota–gut–brain axis and its potential therapeutic role in autism spectrum disorder Neuroscience DOI: 10.1016/j.neuroscience.2016.03.013

[6] Molloy, J., Allen, K., Collier, F., Tang, M., Ward, A., & Vuillermin, P. (2013). The Potential Link between Gut Microbiota and IgE-Mediated Food Allergy in Early Life International Journal of Environmental Research and Public Health, 10 (12), 7235-7256 DOI: 10.3390/ijerph10127235

[7] Riiser, A. (2015). The human microbiome, asthma, and allergy Allergy, Asthma & Clinical Immunology, 11 (1) DOI: 10.1186/s13223-015-0102-0

An open letter to all science lovers who want to defend science ... please don't.

Last week I had an animated discussion on Facebook over an older post in which I describe some literature I dug out on possible (underline “possible”!) correlations with autism. True, my post is highly incomplete, but it was meant as a discussion starter to point at things that scientists have been looking at in an attempt to unravel what feels like a rise in autism. Is autism the new childhood plague of our modern society or has it always been around and we just became more aware of it? And if the rise is real, what caused it?

To me the most intriguing bit is that if you type 'autism gut microbiota' into the PubMed search field (for those not familiar with PubMed, it's a repository for medical literature), you find an incredible number of studies and reviews: apparently there is an association between autism and disruptions of the gut microbiota, but whether the two are truly correlated or the correlation is spurious is still unclear.

Before I go on analyzing the literature I found on this topic, let me open a parenthesis on the Facebook discussion because it's something I deeply care about. You might think that the animated discussion I got into was with anti-vaxxers who believe that vaccines cause autism. Instead, my post was criticized by pro-vaccine people who, with the same unflinching certainty typical of the anti-vaxxers, believe that the rise in autism is fiction invented by anti-vaxxers, that autism has always been around, and that any difference between gut microbiota of autistic children and non-autistic children has been disproved. "By whom?" I asked. By this one report:

"Children with autism have no unique pattern of abnormal results on endoscopy or other tests for gastrointestinal (GI) disorders, compared to non-autistic children with GI symptoms, reports a study in the Journal of Pediatric Gastroenterology and Nutrition."

Notice that this opening line is a bit misleading because here is the actual paper [1] whose conclusion, quoting from the abstract, are a bit more cautiously stated:

"This study supports the observation that children with autism who have symptoms of gastrointestinal disorders have objective findings similar to children without autism. Neither non-invasive testing nor endoscopic findings identify gastrointestinal pathology specific to autism, but may be of benefit in identifying children with autism who have atypical symptoms."

Notice also the difference from the abstract and the title of the report. You can tell which one was written by a scientist, right? Because when you do a search on PubMed using keywords autism and gut microbiota you find a long list of references and decades of research. So to me what this says is that the question is still open and we need to understand the issues better. It takes way more than one paper to disprove hypothesis-raising questions spurred from decades of research.

Now here's the mother of all problems: the Internet has made everyone (EVERYONE!) an expert. Today you no longer need a medical degree to speak authoritatively about vaccines, disease, and health. This has generated movements like the anti-vaxxers, but, even more unfortunate is the rise of groups that reply to the anti-vaxxers without a scientific mind-set: these people are doing even more damage to the community than the anti-vaxxers themselves. I found myself in a conversation that had the same one-ended arguments used by anti-vaxxers except these were people who are actually in favor of vaccines: for every paper on autism and gut microbiota I brought up they would dismiss it with another one that said the opposite, demonstrating no understanding of the difference between raising hypotheses and making a claim.

As a scientist, I can tell you that this behavior is the very opposite of scientific thinking. All the people who are in favor of science but DO NOT adopt a scientific attitude when counter-arguing non-scientific claims are hurting the scientific community. It's happening for vaccines, for evolution, and for global warming. For example, people who support intelligent design are mistaken about evolution because they don't understand the meaning of the word "theory" and they don't understand how scientific thinking works. We need to educate people on scientific thinking, not give bad examples of undebatable and absolute notions.

So, PLEASE, all science fans, I beg of you: support us by giving us a cheer, by always citing original papers, and by keeping an open mind because that's what a real scientist would do. We are raising hypotheses, not discussing the meaning of Bible verses. And if you know you can't do any of the above, then the best support you can give us is to shut up. Let real science speak for itself.

I'm fully aware that I'm preaching to the choir so I'll stop now and resume my discussion on autism and gut microbiota. As an additional side note, let me emphasize how difficult it is to discuss a topic like autism because of its extreme complexity: it's a relatively new diagnosis (first described in the early twentieth century), and even though no exact etiology has been found of date, the genetic studies conducted so far have implicated as many as 400 genes such that a malfunction in any of these genes could possibly result in autism [2].

Let's start from the facts: our body hosts more microbial cells than human cells, with the vast majority residing in the gut. These organisms, which we collectively call the "human microbiota" (and “gut microbiota” when referring to the ones residing in the gut) interact with our cells in symbiosis and in fact, some experiments have shown that they can affect our health and even gene expression (see this old post for a striking example of how genes expressed by gut bacteria can affect whether we are fat or lean). All this has been known for a long time, but it's only recently that, thanks to the advent of new DNA sequencing techniques that scientists have been able to look deeper into the composition and classification of the human microbiota. Metagenomic studies have found over 3 million distinct microbial genes (collectively called the "microbiome") in human stools, which is astonishing if you think that the human genome, in comparison, contains about 20-30 thousand genes. The gut microbiome is rich in enzymes without which our body would be unable to digest important nutrients. In fact, it's estimated that roughly 10% of our dietary energy intake comes from byproducts of fermentation from the gut bacteria.

That's all fine and dandy, but what does this have to do with behavior and brain health? A lot, actually, to the point that scientists coined the phrase "gut-brain axis" to denote the deep interaction between the nervous system and the gut microbiota. A 2011 PNAS study [3] used a mouse model to demonstrate how the gut microbiota affects mammalian brain development and behavior. This can happen in a number of ways, but one interesting hypothesis is that a healthy gut microbiome can help modulate the concentration of chemicals that are important for brain development as well as important nutrients that are precursors of neurotransmitters like serotonin.

Several studies done on different populations of children affected by autism spectrum disorders (ASD) have reported some form of gastro-intestinal (GI) dysfunction (such as food intolerances, abdominal pain, diarrhea and flatulence), with proportions ranging from 20-60% of the study population [4]. It's true that ASD children are often very picky eaters with drastic dietary habits, which would of course cause the GI issues. However, given the previously mentioned evidence that the gut microbiota shapes brain development since early infancy, the question of which is the cause and which is the effect at this point is legitimate. In other words, what came first, the chicken or the egg?

Studies have pointed at alterations of the gut microbiota in ASD children who experience gastro-intestinal issues, and some have reported that ASD children receiving antibiotics seemed to experience behavioral improvements. Drastic changes in diet (for example adopting a gluten-free and/or casein free diet) have shown behavioral improvements in some ASD studies, but not in all (meaning that some studies still didn't observe any improvement). Some papers report a higher risk of ASD in children who have not been breast-fed or who have been weaned after the first month of life. All of these instances would cause the gut microbiota to change, including breast feeding, which plays a fundamental role in establishing a healthy bacterial flora in infants. But why aren't any of these studies conclusive? And why are some conclusions the opposite of others? Such differences in results can be explained by differences in sample sizes (too few patients, for example, would cause a false negative), and also by the fact that many of these children have impaired communication skills, and therefore the symptoms, rather than being self-reported, are gathered from the observations of the parents, which can potentially introduce a bias.

Studies that have compared the microbial composition of stools in children affected by ASD with healthy children have had mixed results: the majority report some differences in the composition of the microbial populations, while a few found no significant differences. And despite many studies have looked into it, no ASD-specific gut disturbance has been found, meaning that whatever gut issues ASD children may experience, they are no different than the ones healthy children may experience as well. At the same time, there is some evidence that probiotics help relieve some of the gastro-intestinal issues ASD children experience and at the same time, improve some of their behavioral issues.

What conclusion can we draw from this? Well, first of all that there's no black and white but a lot of gray and anyone who will tell you it's either black or white does not understand how science works. Look at Lamarck's theory of the evolution of traits, first dismissed by Darwin and now (sort of) coming back in the form of epigenetics. Science is not a means to get a definitive and absolute truth, rather, it is our drive to keep asking questions in the search for working answers. [On a side note, this is exactly why I do not like certain showmen out there who proclaim themselves scientists just because they promote science "truths"; real science educators should be promoting scientific thinking, instead.] More than once in the history of science we've corrected and generalized theories. That doesn't mean that we were wrong, rather, it means that we've expanded our knowledge and acquired better investigative tools.

Unfortunately we don't have historic data on autism, since the term was first used in the early 1900s and the definition of the disorder has changed over time. This questions whether or not case prevalence has been truly rising over time, or, instead, the rise we're seeing is simply the effect of a more comprehensive diagnosis. Regardless of whether this is true or not, the fact that most cases are reported in industrialized countries raises an important speculation: these are countries that have seen the most drastic dietary changes over the past 100 years and also lifestyle changes in terms of hygiene and use of antibacterial products, both in household items, as well as in livestock farming (and the use of antibiotics in livestock farming has indeed been increasing over the past few decades). There is no denying that dietary changes and increased use in antimicrobial products will affect the bacteria coexisting in our environment. Are these changes significant? Can they be play a role in the rise in autism prevalence? Can they play a role in the etiology of other disease whose prevalence appears to be on the rise, such as asthma, food allergies, and autoimmune disorders?

I do believe that these are legitimate questions that call for a deeper understanding of how our body interacts with the environment, both outside and inside. Throughout time, evolution has provided us with ways to adapt, but such adaptations are slow. Instead, over the past 100 years we've introduced drastic changes both in the environment as well as in our lifestyle in ways that are too fast for our genetic make-up to adapt. Anything concerning humans is complex, layered by multiple interactions between genetics, environment, and behavior. That’s why we need to keep looking and, most importantly, that’s why we need to always keep an open mind on things. Anyone who claims to know the absolute truth has misunderstood what science is about. Fighting bogus facts like the ones brought forth by the anti-vaxxers with analogous “absolute truths” will only reinforce the globally spread misunderstanding of what science is and what function it covers in our path toward understanding the world. The day we stop asking questions because we’ve found all the answers is the day we’ve stopped growing.

[1] Kushak RI, Buie TM, Murray KF, Newburg DS, Chen C, Nestoridi E, & Winter HS (2016). Evaluation of Intestinal Function in Children with Autism and Gastrointestinal Symptoms. Journal of pediatric gastroenterology and nutrition PMID: 26913756

[2] Li, Q., & Zhou, J. (2016). The microbiota–gut–brain axis and its potential therapeutic role in autism spectrum disorder Neuroscience DOI: 10.1016/j.neuroscience.2016.03.013

[3] Heijtz, R., Wang, S., Anuar, F., Qian, Y., Bjorkholm, B., Samuelsson, A., Hibberd, M., Forssberg, H., & Pettersson, S. (2011). Normal gut microbiota modulates brain development and behavior Proceedings of the National Academy of Sciences, 108 (7), 3047-3052 DOI: 10.1073/pnas.1010529108

[4] Mulle, J., Sharp, W., & Cubells, J. (2013). The Gut Microbiome: A New Frontier in Autism Research Current Psychiatry Reports, 15 (2) DOI: 10.1007/s11920-012-0337-0

Yes, autism is on the rise. Read this before blaming vaccines.

Waiting for the rain, © EEG

Because I work on HIV vaccine design, lately I've often been involved in debates concerning the safety of vaccines. I have the greatest respect for parents who struggle with disabilities of any kind, especially in children. I'm a parent too and can't even imagine what life is like when your child has a permanent disability. But I'm also a scientist, and I believe in the good cause of my work. My boss has been working day and night for thirty years on a vaccine against HIV because her best friend died of AIDS. We have pictures of AIDS orphans on our desks. We are not monsters, we are not part of a conspiracy, we are not paid by companies to fool people.

In fact, because we do basic research, our salary will be paid whether or not we do succeed in finding a vaccine. It's just our job, and we have no financial gain in this. If you want to point fingers, do it at companies who do make a profit out of health care, or out of selling plastic (and hence bypassing necessary health testing), or out of selling food. As a parent, I am the first to be concerned about the health of our children. I don't accept anything blindly without doing research, be it a vaccine or a drug or a type of food.

I've discussed aluminum in vaccines and why it's a good idea to spread out the shots during the first year of life; I've also discussed why I decided to wait before letting my daughter have the HPV shot. At the same time, parents concerned about autism are right to be alarmed: if you look at the latest numbers published by the CDC, the prevalence of autism in children has doubled. However, this trend has supposedly started in the last two decades whereas vaccines have been around much longer than that [1]. It's true that the US have an aggressive vaccine schedule for infants and I suspect it's tailored to reduce the number of office visits as copays are expensive and insurance companies need to make their profits. So yes, just like other parents, I am bitter at the system. I am bitter at companies profiting out of the health of my own children, not at researchers working hard at finding a cure for deadly diseases. My plea today is to separate the two: the cure, which, just like any other cure, should be used wisely and with good measure and balance, and the people making profits out of the cure.    

For example, nobody argues that antibiotics save lives. Unfortunately, today you find antibacterial stuff in soaps, detergent, even toothpaste. Doctors overprescribe antibiotics all the time. And then of course, poultry, beef and pork come loaded with antibiotics. This has led to extremely aggressive, antibiotic resistant superbugs like CRE. Yet nobody dreams of refusing antibiotics when they are really needed. That's because we all know that if you don't take them you might in fact lose your life.

What our society needs is stop pointing fingers, quit all the conspiracy crap, and instead sit at the table and discuss better health practices that don't put profits first but health and good care instead.

How should we address the rise in autism cases? I don't have an answer to this, but I did find a bunch of papers that got me thinking. I list them below.

DISCLAIMER: I'm not discussing these papers to point at a cause of autism. In fact, I believe that we will never find a cause, just like we will never find a cause of cancer. Like I stated in my post last week, we need to think of our lives as a complex orchestra where DNA, RNA, proteins and the environment all play together to create the beautiful symphony of our life. There never is one such thing as a direct cause. Often it's just genetics. Even more often is a genetic predisposition combined with multiple sets of environmental exposures, lifestyle, and diet. If your child has autism, please focus your energy in taking care of that child rather than trying to find a cause.

1) This study [1] looked into the raising numbers of autism cases:

"Diagnosed autism prevalence has risen dramatically in the U.S over the last several decades and continued to trend upward as of birth year 2005. The increase is mainly real and has occurred mostly since the late 1980s. In contrast, children's exposure to most of the top ten toxic compounds has remained flat or decreased over this same time frame. Environmental factors with increasing temporal trends can help suggest hypotheses for drivers of autism that merit further investigation [1]." 

So the threat is real. Yet vaccines have been around much longer than the 1980s.

2) Studies have found a higher incidence of autism in California, in higher educated families. This may be biased by the fact that people with a higher education will be more inclined to have their children tested for autism. But one study in particular [2] found another possible association:

"Our study adds to previous work in California showing a relation between traffic-related air pollution and autism, and adds similar findings in an eastern US state, with results consistent with increased susceptibility in the third-trimester [2]." 

The researchers monitored the air particulate at the birth address of the child starting from preconception through the child's first birthday.

 3) Breast feeding may play a protective role against autism spectrum disorders [3].

4) Inflammation may play a role. Le Belle et al. [4] used a mouse model to test the following hypothesis:

"A period of mild brain overgrowth with an unknown etiology has been identified as one of the most common phenotypes in autism. Here, we test the hypothesis that maternal inflammation during critical periods of embryonic development can cause brain overgrowth and autism-associated behaviors as a result of altered neural stem cell function [4]."

What they found supports the idea that, paired with genetic susceptibility, an infection in the pregnant mother could indeed higher the risk of developing autism in the child.

5) But one of the most fascinating associations I found is between gut microbiome and autism. Newborns are born without any bacteria in their guts and colonization begins right after birth. Vaginal birth vs. cesarean, breast fed vs. formula seem to be factors associated to the gut microbiota found in infants.

"Over the first years of life the gut microbiome is changing and remodeling, ultimately resembling an adult gut microbiome by year 3. This suggests there is a “core microbiome” that is the hallmark of a healthy individual [5]." 

This is particularly important because the microbiota community carries millions of genes whose expression affects our own physiology. The type and number of bacteria in our guts can influence the health and good functioning of our immune system.

Now, here's the worrisome bit:

"Broad-spectrum antibiotics are often prescribed to infants in the Western world in an attempt to protect the developing child from disease. In addition to conferring antibiotic resistance in infancy, antibiotic over usage can significantly disrupt the overall ecology of the gut microbiota, alter the abundances of resident gut bacteria, and potentially bias the child toward certain diseases [6]."

I'm not making a case that antibiotics are bad, just like I will never say that vaccines are bad. I'm just raising a flag that, like in all things, a good measure should be practiced. Antibiotics are a great means to fight infections. But is it safe to use them routinely to prevent infection?

The following study [7] is from 2000, so maybe a bit outdated, and the sample number is awfully low. Still, this is what it had to say:

"In most cases symptoms of autism begin in early infancy. However, a subset of children appears to develop normally until a clear deterioration is observed. Many parents of children with "regressive"-onset autism have noted antecedent antibiotic exposure followed by chronic diarrhea. We speculated that, in a subgroup of children, disruption of indigenous gut flora might promote colonization by one or more neurotoxin-producing bacteria, contributing, at least in part, to their autistic symptomatology [7]."

The study has a huge limit: they tested their hypothesis on 11 children that matched the above criteria (the onset of autism symptoms were observed after administration of antibiotics and subsequent diarrhea), which is an extremely small number. The children were given oral antibiotics and a slight improvement in behavior was noted, not the effects had completely waned by follow-up. Nothing conclusive, but definitely this study makes a case for further investigation.

In a more recent review, Critchfield et al. suggest that:

"Autism spectrum disorders are a diverse group of disorders caused by a complex interplay between genetic and environmental components. There is a range of indications that alterations in the intestinal microbiota in the gut might contribute to the disorder in a substantial number of individuals. Probiotics can be useful to restore the microbial balance in the intestine, to relieve gastrointestinal problems and to attenuate immunological abnormalities. Whether the use of probiotics by children with autism can lead to improvements in behaviors needs to be established in well-controlled trials with sufficient group sizes [8]." 

Please don't take any of this as prescriptions or recommendations. I am NOT a medical doctor. I'm a scientist and I like to pose questions and investigate possible answers. If you have particular concerns about your children, talk to your doctor. The references mentioned above are meant as guidelines. Print them out, read them carefully, and then discuss them with your physician.

[1] Nevison CD (2014). A comparison of temporal trends in United States autism prevalence to trends in suspected environmental factors. Environmental health : a global access science source, 13 PMID: 25189402

[2] Kalkbrenner AE, Windham GC, Serre ML, Akita Y, Wang X, Hoffman K, Thayer BP, & Daniels JL (2015). Particulate matter exposure, prenatal and postnatal windows of susceptibility, and autism spectrum disorders. Epidemiology (Cambridge, Mass.), 26 (1), 30-42 PMID: 25286049

[3] Al-Farsi YM, Al-Sharbati MM, Waly MI, Al-Farsi OA, Al-Shafaee MA, Al-Khaduri MM, Trivedi MS, & Deth RC (2012). Effect of suboptimal breast-feeding on occurrence of autism: a case-control study. Nutrition (Burbank, Los Angeles County, Calif.), 28 (7-8) PMID: 22541054

[4] Le Belle JE, Sperry J, Ngo A, Ghochani Y, Laks DR, López-Aranda M, Silva AJ, & Kornblum HI (2014). Maternal inflammation contributes to brain overgrowth and autism-associated behaviors through altered redox signaling in stem and progenitor cells. Stem cell reports, 3 (5), 725-34 PMID: 25418720

[5] Mulle, J., Sharp, W., & Cubells, J. (2013). The Gut Microbiome: A New Frontier in Autism Research Current Psychiatry Reports, 15 (2) DOI: 10.1007/s11920-012-0337-0

[6] Arrieta, M., Stiemsma, L., Amenyogbe, N., Brown, E., & Finlay, B. (2014). The Intestinal Microbiome in Early Life: Health and Disease Frontiers in Immunology, 5 DOI: 10.3389/fimmu.2014.00427

[7] Sandler RH, Finegold SM, Bolte ER, Buchanan CP, Maxwell AP, Väisänen ML, Nelson MN, & Wexler HM (2000). Short-term benefit from oral vancomycin treatment of regressive-onset autism. Journal of child neurology, 15 (7), 429-35 PMID: 10921511

[8] Critchfield, J., van Hemert, S., Ash, M., Mulder, L., & Ashwood, P. (2011). The Potential Role of Probiotics in the Management of Childhood Autism Spectrum Disorders Gastroenterology Research and Practice, 2011, 1-8 DOI: 10.1155/2011/161358

Synthetic gene circuits with a memory!

Imagine having a USB port in the body that we could use to insert a "flash drive" and transfer genetic data, therapies, or monitoring devices. The flash drive would have to be some kind of removable biological entity that has no problem getting in and out of the body. If you think about it, bacteria are the perfect candidates to be such devices. So, what if bacteria could be used as storage for genetic memory?

This is not so far-fetched if you think that recent studies have shown for example that genes expressed by bacteria in our guts can affect our propensity to be lean or fat. Bacteria have genes that "record" and "affect" what's going on in our body. The question is: can we control them?

Bacteria have a way of turning "on" or "off" their genes based on stimuli from the environment. Synthetic biology studies ways of using these "switches" to make "gene circuits". Genetic regulatory circuits are the biological analog of electric circuits, where genes, instead of light bulbs, are being turned on or off (by activating other genes).

Genetic circuits have numerous applications in medicine. For example, Auslaender et al. [1] used synthetic biology to create a pH sensor for cells. The researchers then implanted these cells into mice and used it as a device to detect diabetes. Lack of insulin causes an excess of acidity in the blood, and the pH drops below 7.35. Changes in pH induced by diabetes were quickly detected by the pH-sensor cells in the implanted mice. The pH information was processed and triggered a transgene expression response that resulted in the secretion of alkaline phosphatase to counteract the acidity. Basically, what the cells were able to do in the mice is: (1) detect the drop in pH; (2) trigger a response to restore the pH to normal levels.

In an electrical circuit you assemble elements like resistance and capacity. In a genetic circuit you assemble genes and "operators" able to edit the DNA in order to activate or deactivate the genes. One of such "editors" is a class of enzymes called recombinases. Apparently, there aren't many of these enzymes available, which limits the number of gene circuits one can make.

A recent study published in Science [2], however, presented a new class of such enzymes, derived from the bacteriophage Lambda, which is a virus that infects Escherichia coli. The novelty of the method doesn't stop here. You see, the goal is not just to have a working circuit, but to also make it autonomous. In other words, ideally, one wants a system able to detect responses and readjust the output based on the input it receives. The researchers devised genetic regulatory circuits able to "write", "input" and "read" genetic information.

Farzadfard and Lu [2] "converted genomic DNA into a 'tape recorder' for memorizing information in living cell populations." Their circuit, named SCRIBE (Synthetic Cellular Recorders Integrating Biological Events), responds to gene regulatory signals by generating single-stranded (ssDNA). The ssDNA is then coexpressed with a recombinase and introduces specific mutations in targeted positions of the cell DNA. The fraction of cells in the bacterial culture that carry the introduced mutations represents the biological memory at the population level.

For example, when the researchers exposed the cultures to an exposure input for 12 days (the equivalent of 120 generations in the bacterial population), they found that the

"frequency of mutants in these populations was linearly related to the total exposure time. Furthermore, we demonstrate that SCRIBE-induced mutations can be written and erased and can be used to record multiple inputs across the distributed genomic DNA of bacterial populations [2]."

It's a 'collective memory' embedded in the observed frequency of the mutation in the bacterial population. And the applications are almost infinite. I truly can't wait to see where this kind of research will take us in the future.

[1] Ausländer D, Ausländer S, Charpin-El Hamri G, Sedlmayer F, Müller M, Frey O, Hierlemann A, Stelling J, & Fussenegger M (2014). A synthetic multifunctional mammalian pH sensor and CO2 transgene-control device. Molecular cell, 55 (3), 397-408 PMID: 25018017

[2] Farzadfard, F., & Lu, T. (2014). Genomically encoded analog memory with precise in vivo DNA writing in living cell populations Science, 346 (6211), 1256272-1256272 DOI: 10.1126/science.1256272

Viruses and bacteria could be the missing piece in the missing heritability puzzle

© EEG

I've discussed the issue of the missing heritability before: in layman words, same mutations shared across people don't lead to the same phenotype. This is particularly true for diseases. Many whole genome studies have looked at possible associations between DNA mutations and diseases, but, alas, the mutations that have been found generally explain only 10% of the cases. This suggests that there's a lot more to who and what we are than genes alone, and that complex interactions between DNA, RNA and proteins come to play. If you've been following the blog for a while you know that I love to talk about epigenetics (so much so that \begin{plug} I wrote a detective thriller based on epigenetics \end{plug}): I do believe a good portion of the missing heritability puzzle relies on epigenetics, which studies the mechanisms that turn our genes "on" and "off". These mechanisms are not coded in our genes, yet they can be carried on for 2-4 generations.

There are other factors, besides epigenetics and complicated genetic interactions, that could explain the missing heritability. Bacteria and viruses for example could be playing a fundamental role. In a recent post I discussed a study that points at the gut bacteria as responsible for the inheritance of a propensity towards an obese phenotype rather than a lean one. Another example is Crohn Disease: there are some specific mutations that make an individual prone to the disease, yet not everyone with those mutations manifest the symptoms. A 2010 study [1] on a mouse model found that in the presence of the mutation, the disease manifested only after infection with a particular strain of MNV (murine norovirus), the mouse variant of norovirus, a family of viruses that cause viral gastroenteritis. So, rather than the mutation alone, it's an interaction between genetic predisposition and viral infection that seems to cause Crohn Disease.

In a recent study published in PNAS [2], Edwards et al. proposed an yeast model to study the interaction between chromosomal mutations and non-chromosomal elements. In the yeast case, the non-chromosomal elements were:

"... the presence or absence of the yeast killer dsRNA virus and the other was varying mitochondria among two backgrounds with distinct differences in their genome sequence. The two mitochondrial genomes we selected show considerable variation, with about two to three SNPs per kilobase between them and 10 times as many insertions and deletions per kilobase between them as found in the chromosomal genome [2]."

The researchers induced chromosomal changes in different strains of yeast expecting their phenotype to change accordingly: they examined 17 single gene deletions that induced growth defects, expecting to observe much smaller populations. It turns out that this didn't work as a "switch". In other words, the belief "you turn on the green eye gene, you get green eyes" (which, sadly, is a very common misconception that people have about genes) is a myth. Yeast bacteria don't have green eyes, of course, but the researchers saw that despite changing certain genes, they were still getting a broad spectrum of growth phenotypes. Despite having the induced mutations, whether the yeast colonies did or didn't grow depended on the presence or absence of the dsRNA virus and the variation in mitochondrial genes. With their experiment, Edwards et al. showed that

"the heritability of a trait can depend on nonlinear interactions between chromosomal and nonchromosomal information that is transmitted from generation to generation. The nonchromosomal information can interact with the various chromosomal alleles at a locus to modify phenotype significantly. [...] Our results show that the nonchromosomal contribution to heritability can be large and, in some cases, can completely mask the effect of a chromosomal mutation [2]."

[1] Cadwell K, Patel KK, Maloney NS, Liu TC, Ng AC, Storer CE, Head RD, Xavier R, Stappenbeck TS, & Virgin HW (2010). Virus-plus-susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes in intestine. Cell, 141 (7), 1135-45 PMID: 20602997

[2] Edwards, M., Symbor-Nagrabska, A., Dollard, L., Gifford, D., & Fink, G. (2014). Interactions between chromosomal and nonchromosomal elements reveal missing heritability Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1407126111

Forget the "obesity gene": it's the "obesity microbes" that we need to fight.

Antelope Canyo ©EEG

This is going to be gross. So, if you're eating, finish up your snack first.

Ready?

Let's do the following experiment: take two twins, one chubby, the other lean. Why one is chubby and the other is lean is a question we'll leave for another time. For the time being, all we do is take fecal samples from both, extract the microbiota (the bacteria living in feces) and transplant them in mice.

You'd been warned it was kinda gross.

The reason for such an experiment is that large intestine microbiota (the microorganisms that live in our bowels) have been implied in many physiological processes. I've discussed in other posts, for example, how commensal microbiota modulates the immune system. Some studies also suggest that they could be involved in the heritability of some epidemiological markers.

In a 2009 paper, Turnbaugh et al. transplanted human fecal microbiota in germ-free mice (mice that didn't have any pre-existing intestinal micriobiota) and showed that changes in the mice diets could alter the microbiota and the genes they expressed. In particular:

"Humanized mice fed the Western diet have increased adiposity; this trait is transmissible via microbiota transplantation [1]."

The human microbiota not only transplanted successfully in the mice, but it was even transmitted to the offspring, which could possibly validate the question raised in one of the epigenetic papers I discussed last week: are dietary changes that do not affect the DNA "inherited" through the gut microbiota?

Ridaura et al., in a 2013 Science paper [2], continued in this line of experiments, this time using samples from twins that differed in obesity: one was lean, the other obese. After the transplant all mice were fed the same low-fat diet. What did the researchers find?

"The increased adiposity phenotype of each obese twin in a discordant twin pair was transmissible: The change in adipose mass of mice that received an obese co-twin’s fecal microbiota was significantly greater than the change in animals receiving her lean twin’s gut community within a given experiment and was reproducible across experiments (P ≤ 0.001, one-tailed unpaired Student’s t test; n = 103 mice phenotyped) [2]."

As a further control, the researchers took the mice that had received the "obese" microbiota and transplanted them a second time with "lean" microbiota. They noticed that the excess adiposity was shed if the mice were kept at a low-fat diet, but not if the diet was high in fat and low in fiber.

The microbiota composition in the two mouse population is different, and the researchers showed that the "lean" microbiota produce higher quantities of short-chain fatty acids. One of the hypothesis raised, therefore, is that these short-chain fatty acids can protect against accumulation of fat and increase energy expenditure.

While these studies come with the usual caveat that humans are typically far more complicated than mouse models, I find them extremely fascinating, especially in light of the epigenetic papers I discussed last week that pointed at how phenotypic changes induced by diet can affect multiple generations even though they are not encoded in the genome. A possible cause for the transmissibility across generations could be the gut microbiota, paired with a high-fat diet which ends up affecting parents and children alike if they all live under the same roof.

[1] Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, & Gordon JI (2009). The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Science translational medicine, 1 (6) PMID: 20368178

[2] Ridaura VK, Faith JJ, Rey FE, Cheng J, Duncan AE, Kau AL, Griffin NW, Lombard V, Henrissat B, Bain JR, Muehlbauer MJ, Ilkayeva O, Semenkovich CF, Funai K, Hayashi DK, Lyle BJ, Martini MC, Ursell LK, Clemente JC, Van Treuren W, Walters WA, Knight R, Newgard CB, Heath AC, & Gordon JI (2013). Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science (New York, N.Y.), 341 (6150) PMID: 24009397