Hunting For The Signatures of Cancer

Signatures of Mutational Processes Extracted from the Mutational Catalogs of 21 Breast Cancer Genomes. Credit:http://dx.doi.org/10.1016/j.celrep.2012.12.008

Cancer is the second leading cause of death worldwide, with approximately 14 million new cases and 8.2 million cancer related deaths each year (Source: WHO). A family history of cancer typically increases a person's risk of developing the disease, yet most cancer cases have no family history at all. This suggests that a combination of both genetics and environmental exposures contribute to the etiology of cancer. In this context, "genetics" means the genetic make-up we are born with and inherited from our parents. For example, women born with specific mutations in the BRCA1 and BRCA2 genes are known to have a much higher risk of developing breast cancer later in life.

However, besides the genetic make-up we carry from birth, there are many geographical and environmental factors that contribute to the risk of cancer. For example, the incidence of breast cancer is over 4 times higher in North and West Europe compared to Asia and Africa (Source: WHO). Stomach cancer, on the other hand, is much more prevalent in Asia than the US. If you think that this may be linked to the genetic differences across ethnicities, think again. The National Cancer Institute published a summary of several studies that compared the incidence of first and second generation immigrants in the US with the local population. They found that:

"cancer incidence patterns among first-generation immigrants were nearly identical to those of their native country, but through subsequent generations, these patterns evolved to resemble those found in the United States. This was true especially for cancers related to hormones, such as breast, prostate, and ovarian cancer and neoplasms of the uterine corpus and cancers attributable to westernized diets, such as colorectal malignancies."

According to the World Health Organization (WHO),

"around one third of cancer deaths are due to the 5 leading behavioral and dietary risks: high body mass index, low fruit and vegetable intake, lack of physical activity, tobacco use, alcohol use."

Cancer is the result of a series of cellular mechanisms gone awry: every time a cell divides, somatic mutations accumulate in the cell's genome. These are not mutations we are born with, inherited from our parents. Rather, these are changes that accumulate in certain cells as we grow old and are not  the same across all cells in the body. Many environmental exposures contribute to this process and affect the rate at which these mutations accumulate. However, cells have various mechanisms that are normally able to repair harmful mutations or, when the damage is beyond repair, to trigger cell death. The immune system is also "trained" to recognize cancer cells and destroy them.

When all these defense mechanisms fail, cancer cells start dividing uncontrollably.

As a result, all cancer cells carry a number of somatic mutations that set them apart from healthy cells, and some tend to be the same across different cancer patients: for example, specific mutational patterns found in lung cancer have been attributed to tobacco exposure and were indeed reproduced in animal models. Another set of mutations has been attributed to UV exposure and has been found in skin cancers [1, 2].

This prompts the ambitious question: can we find common mutations across individuals with the same cancer? And how many of these mutational patterns that are common across individuals can we attribute to particular exposures and/or biological processes? Distinguished postdoctoral researcher Ludmil Alexandrov, from the Los Alamos National Laboratory, has been working on this problem since his he was a PhD student at the Wellcome Trust Sanger Institute.

"It's like lifting fingerprints," Alexandrov explains. "The mutations are the fingerprints, but now we have to do the investigative work and find the 'perpetrator', i.e., the carcinogens that caused them." During his graduate studies, under the supervision of Mike Stratton of the Wellcome Trust Sanger Institute, Alexandrov developed a mathematical model that, given the cancer genomes from a number of patients, is able to pick the "common signals" across the patients -- i.e. mutation patterns that are common across the patients -- and classify them into "signatures."

"When formulated mathematically," Alexandrov explains, "the question can be expressed as the classic 'cocktail party' problem, where multiple people in a room are speaking simultaneously while several microphones placed at different locations are recording the conversations. Each microphone captures a combination of all sounds and the problem is to identify the individual conversations from all the recordings." Taking from this analogy, each cancer genome is a "recording", and the task of the mathematical model is to reconstruct each conversation, in other words, the mutational patterns. These are sets of somatic mutations that are the observed across the cancer genomes and that characterize certain types of cancers.

In 2013, Alexandrov and colleagues analyzed 4,938,362 mutations from 7,042 patients, spanning 30 different cancers, and extracted more than 20 distinct mutational signatures [2]. "Some patterns were expected, like the known ones caused by tobacco and UV light," Alexandrov says. "Others were completely new."

Of the new signatures found, many are involved in defective DNA repair mechanisms, suggesting that drugs targeting these specific mechanisms may benefit cancers exhibiting these signatures [3]. But the most exciting part of this research will be finding the 'perpetrator' or, as Alexandrov explains, the mutations triggered by carcinogens like tobacco, UV radiation, obesity, and so on. The challenge will be to experimentally associate these mutational patterns to the exposures that caused them. In order to do this, the scientists will have to expose cultured cells and model organisms to known carcinogens and then analyze the genomes of the experimentally induced cancers.

In the meantime, the signatures found so far are only the beginning: Alexandrov and colleagues have teamed up with the Los Alamos High Performance Computing Organization in order to analyze the genomes of almost 30,000 cancer patients.

"The amount of data we will have to handle for this task is enormous, on the order of petabytes," Alexandrov says. "Few places in the world have the capability to handle this many data. Under normal circumstances, it takes months to answer a question on 10 petabytes of data. The supercomputing facility at Los Alamos can provide an answer within a day."

Because of his research, in 2014 Alexandrov was listed by Forbes magazine as one of the “30 brightest stars under the age of 30” in the field of Science and Healthcare. In 2015 he was awarded the AAAS Science & SciLifeLab Prize for Young Scientists in the category Genomics and Proteomics [2] and the Weintraub Award for Graduate Research. He is now the recipient of the prestigious Oppenheimer fellowship at Los Alamos National Laboratory.

References
Siegel, R., Miller, K., & Jemal, A. (2015). Cancer statistics, 2015 CA: A Cancer Journal for Clinicians, 65 (1), 5-29 DOI: 10.3322/caac.21254

[1] Alexandrov LB (2015). Understanding the origins of human cancer. Science (New York, N.Y.), 350 (6265) PMID: 26785464

[2] Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, Bignell GR, Bolli N, Borg A, Børresen-Dale AL, Boyault S, Burkhardt B, Butler AP, Caldas C, Davies HR, Desmedt C, Eils R, Eyfjörd JE, Foekens JA, Greaves M, Hosoda F, Hutter B, Ilicic T, Imbeaud S, Imielinski M, Jäger N, Jones DT, Jones D, Knappskog S, Kool M, Lakhani SR, López-Otín C, Martin S, Munshi NC, Nakamura H, Northcott PA, Pajic M, Papaemmanuil E, Paradiso A, Pearson JV, Puente XS, Raine K, Ramakrishna M, Richardson AL, Richter J, Rosenstiel P, Schlesner M, Schumacher TN, Span PN, Teague JW, Totoki Y, Tutt AN, Valdés-Mas R, van Buuren MM, van 't Veer L, Vincent-Salomon A, Waddell N, Yates LR, Australian Pancreatic Cancer Genome Initiative, ICGC Breast Cancer Consortium, ICGC MMML-Seq Consortium, ICGC PedBrain, Zucman-Rossi J, Futreal PA, McDermott U, Lichter P, Meyerson M, Grimmond SM, Siebert R, Campo E, Shibata T, Pfister SM, Campbell PJ, & Stratton MR (2013). Signatures of mutational processes in human cancer. Nature, 500 (7463), 415-21 PMID: 23945592

[3] Alexandrov LB, Nik-Zainal S, Siu HC, Leung SY, & Stratton MR (2015). A mutational signature in gastric cancer suggests therapeutic strategies. Nature communications, 6 PMID: 26511885

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.

April IWSG roundup

This is a monthly event started by the awesome Alex J. Cavanaugh and organized by the Insecure Writer's Support Group. Click here to find out more about the group and sign up for the next event. You can also sign up for the newsletter.

I know many of you are busy doing the A-Z challenge this month, so I'll keep it short.

As you know, I'm working on two projects at the same time, which is something I never did before. This results in both projects being slower but I fear that if I miss the spontaneity of the moment and put wither one aside, when I'll get back to it later on the voice won't sound half as good. Or at least that's what I tell myself, haha. :-)

I explain this process in a podcast interview with two good friends of mine, and awesome writers, Jason Anspach and Kevin G. Summers. Kevin and Jason started the Literary Outlaws podcast this year and they've already interviewed some pretty cool people. If you have time during your commute to work, I highly recommend you check them out. :-)

That's all folks, hope all is well with your writing, hope you're not sneezing too much this spring but instead enjoying the outdoors and warmer temperatures. And if you are in the southern hemisphere, enjoy the beauty of fall.

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