Whole-genome sequencing sheds light on adaptation of koala bears to their diet of eucalyptus leaves

The study reported in the Nature Genetics publication was primarily carried out in order to determine the entire koala genome by whole-genome sequencing (WGS). They found that the haploid genome spanned ~3.42 billion nucleotides, i.e. a diploid genome of ~3.42 gigabase pairs (Gbp). An incredible amount of information was gained by these authors in producing a complete and contiguous marsupial reference genome –– including centromeres.

CYP2C enzyme metabolism and substrate specificity of course could be the project for a future grant proposal. However, constructing 31 CYP2C cDNA expression vectors and inserting each of them into either bacteria or yeast in order to determine enzyme substrate specificity –– is no trivial experiment. However, I hope to see those results completed (by someone) within the next few years. 🙂

From: ((Anonymous))
Sent: Thursday, October 04, 2018 4:19 PM


I’m suprised that the journal and the reviewers of this paper did not request proof that one or more of these koala CYP2C enzymes did indeed metabolize camphor or other various eucalyptus oils.
We did something like that with 36 sequence variants of POR (P450-oxidoreductase), tested for CYP1A2 and 2C19 activity. Took about a year, but certainly do-able in a small lab –– if you have a plate reader and a colorimetric assay. For anyone interested, the 2008 Pharmacogenet Genom paper is attached. Also, our 2011 review, on the entire topic, in Mol Cell Endocrinol is attached.

Along these same lines, just this minute I came across this article [abstract pasted below] –– describing a rapid procedure to measure the functional activity of 1,056 discovered single-nucleotide variants, just within the first 192 amino acids of the BRCA1 protein, measuring efficiency of double-strand DNA breaks. Therefore, ’31 CYP2C variants’, or ’36 POR variants’, would now be ‘small potatoes’ if one considers the latest advances in technology. 🙂


Am J Hum Genet. 4 Oct 2018; 103: 498-508
A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function.

Starita LM1, Islam MM2, Banerjee T2, Adamovich AI2, Gullingsrud J3, Fields S4, Shendure J5, Parvin JD6.

Loss-of-function pathogenic variants in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for sequence changes in BRCA1 frequently reveals a missense variant for which the impact on cancer risk and on the molecular function of BRCA1 is unknown. Functional BRCA1 is required for the homology-directed repair (HDR) of double-strand DNA breaks, a critical activity for maintaining genome integrity and tumor suppression. Here, we describe a multiplex HDR reporter assay for concurrently measuring the effects of hundreds of variants of BRCA1 for their role in DNA repair. Using this assay, we characterized the effects of 1,056 amino acid substitutions in the first 192 residues of BRCA1. Benchmarking these results against variants with known effects on DNA repair function or on cancer predisposition, we demonstrate accurate discrimination of loss-of-function versus benign missense variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing.

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Genetic ancestry and population differences in levels of inflammatory cytokines in women

Evolution for the past ~3.8 billion years involves constant (often adverse) environmental signals, being presented as challenges to the cell, to the organism, and the organism’s genome must respond to that challenge in order to survive (i.e., find food, avoid predators, reproduce). Hence, this topic lies within the domain of “gene-environment interactions”. The mammalian immune system provides a primary defense against pathogens external to, as well as within, the body. For the ~300,000 years that modern humans have existed, hominids as they migrated from Africa to the rest of the world encountered vastly different pathogenic environments (e.g. fungus, bacterial, viral), and their survival and reproductive fitness depended on how successful their immune systems fought off infections (before the time of modern medicine).

It has been hypothesized that strong selection pressure due to exposures to life-threatening infectious pathogens endemic to Africa, particularly malaria, shaped a pro-inflammatory immune milieu in populations of African ancestry. This hypothesis is supported by evidence from evolutionary genetic data showing that genomic regions hosting immunity-related genes are under stronger selection pressure than the rest of the human genome. A number of studies have shown that African-ancestry individuals have a higher frequency of DNA variants related to pro-inflammatory cytokines (cell-signalling molecules that help in cell-to-cell communication involving immune responses and stimulate movement of immune cells toward sites of inflammation, infection and trauma), but a lower frequency of variants related to anti-inflammatory cytokines (‘pro-inflammatory’ means to stop infection before it starts; ‘anti-inflammatory’ of course is to fight infection after it has started).

Many variants associated with infectious, autoimmune, and inflammatory diseases discovered from genome-wide association studies (GWAS) display extreme differences in allele frequencies across populations. These ancestral genetic variations –– that were shaped by human evolutionary history –– likely remain influential on the constitutive immune milieu in populations today. The serious smallpox epidemic among Amerindians, following invasion of Europeans into the Americas, represents an extreme example in which the Amerindian genome had never been exposed to smallpox and therefore had virtually no ability to defend against that virus.

In a study integrating genetic, molecular and epidemiologic data, authors [see attached article] compared population differences (between 914 African and 855 European ancestry women), examining plasma levels of 14 cytokines involved in innate (defense against infection that can be activated immediately once a pathogen attacks) and adaptive (acquired immunity, i.e. ‘immunological memory’ is established, after an initial response to a specific pathogen, and leads to an enhanced response to subsequent encounters with that pathogen) immunity. Authors found significant differences in seven cytokines. Levels of two pro-inflammatory chemokines, CCL2 and CCL11, were strongly associated with African ancestry. The signal was pinpointed to the Duffy-null allele of rs2814778. These findings confirm strong ancestral footprints in inflammatory chemokine regulation. The Duffy-null allele may indicate a loss of the buffering function for chemokine levels. These substantial immune differences, by ancestry, may have broad implications to health disparities between African and Eurpean populations.

PLoS Genet June 2o18; 14: e1007368

There are more than 3,500 species of mosquitoes. I doubt that wiping out one of them would create a monster vacuum that would then get filled by something even worse. But, eradication of Anopheles gambiae would save hundreds of thousands of human lives every year.

Be bold. All of those dying, incredibly cute kids would like us to do something. Mosquito nets are not doing the job.

Doing something has unprecedented consequences. We would for the first time use our genetic power to wipe out a species.
Doing nothing has consequences, too. We then choose to let hundreds of thousands die every year

What is your choice? The continued death of about 3 million people per decade, or the death of one species of mosquito? I vote people over mosquito.

Agreed, I had previously commented to Steve that “Nature abhors a vacuum” –– meaning that if a species existing in a particular ecological niche is eliminated, it is virtually 100% certain that some other species will move into that niche. It could be an existing organism closely related to the species eliminated, or (via changes in DNA sequence and/or epigenetic events) a new species might evolve.
Concerning gene drive and eradication of pests, this is a really debatable question, Tony James was on the forefront of this for awhile. Genetically- modified organism (GMO) mosquitoes have been introduced in to Florida previously – so the overall approach has already been field-tested. Key question came from Dr Malcome in Jurrassic Park….”Will Mother Nature find a way?”

As a public health question, there is no doubt it should be tried. The expected ecological impacts will likely be minor, but will it ultimately cause extinction of malaria? In my opinion, this is doubtful, nature will find a way to beat the technology, via selection! But generation of GMO mosquitoes may be the best weapon yet!

FYI, we have colleagues here at NC State that are leading an international effort funded by DOD to develop gene drive approaches to eliminate invasive mice on Australian islands. If you think the mosquito is a controversial target….releasing gene-drive-GMO mammals into the environment – brings new meaning to the word “controversy”..!! (See attached links). These are super-cool ideas, and a lot of effort is being invested in saving many species of sea birds from extinction.

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Thalidomide, which caused many birth defects, degrades transcription factor SALL4, necessary for fetal limb and organ development

Thalidomide first came on the U.S. market in the 1950s –– as a nonaddictive, nonbarbiturate sedative drug, having anti-emetic properties. Thus, it became widely used to treat morning sickness in pregnant women. Soon after the arrival of thalidomide on the market, some began to suspect the drug was causing severe birth defects. It wasn’t until 1961 that two independent convincing epidemiological studies confirmed that thalidomide was causative; this became known as the “largest preventable medical disaster in modern history”. Despite this tragedy, thalidomide (and its close derivatives, lenalidomide and pomalidomide), acting as immunomodulatory drugs, are still commonly used to treat several very serious clinical conditions such as multiple myeloma, and 5q-deletion associated myelodysplastic syndrome.

While a potentially successful treatment for certain malignancies, the molecular mechanisms of thalidomide teratogenicity (birth defect-causing), and many of its biological activities, remain elusive. However, recently it was shown that thalidomide and analogs exert their therapeutic effect by binding to the Cullin RING E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN, thereby enhancing degradation of pivotal key efficacy targets –– such as the zinc-finger transcription factors IKAROS, AIOLOS, and ZFP91.

Authors [see attached preprint] showed that immunomodulatory drugs disrupt a broad transcriptional network through the increaed degradation of several C2H2 [(Cysteine)2(Histidine)2; (Cys)2(His)2] zinc-finger transcription factors –– including SALL4 (member of the Spalt-like family of developmental transcription factors). Intriguingly, human genetic studies had shown that heterozygous loss-of-function (LOF) mutations in the SALL4 gene result in a developmental condition that mimics thalidomide-induced birth defects such as absence of thumbs, phocomelia (hands and/or feet attached close to the trunk, the limbs being grossly underdeveloped or absent –– a trait seen in many Vietnamese children exposed to high levels of Agent Orange), defects in ear and eye development, and congenital heart disease. Authors demonstrated that thalidomide induces degradation of SALL4 exclusively in humans, primates, and rabbits –– but not in rodents or fish –– which provides a mechanistic link for the species-specific pattern of pathogenesis that is seen with the thalidomide syndrome.


eLife 2o18; 7: e38430

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Glutamate activates long-distance calcium-based defense system in PLANTS

Just like animals, plants also must respond within minutes to environmental stresses such as wounding, and plants do so –– with both local and system-wide reactions that prime nondamaged regions to mount defenses against the incoming adverse effect. For herbivory (something eating on a plant while it’s still alive!), production of the defense hormone jasmonic acid (JA) and accumulation of toxic, repellent, or digestibility-reducing compounds all aid in deterring future attacks. Hence, this is an example of gene-environment interactions. Reactive oxygen species (ROS), electrical signals, and changes in cytosolic calcium concentration ( [Ca2+]cyt ) are believed to form signaling-networks that support both local and systemic defense responses.

The electrical component is dependent on glutamate receptor–like (GLR) proteins, a family of cation-permeable ion channels (if you remember from high school chemistry, ‘cations’ are positive-charged ions) that function in plant processes ranging from pathogen defense to root growth. Authors [see attached article and editorial] asked how GLRs are triggered by wounding and how subsequent Ca2+-related signaling-events operate to mediate systemic defense. Multicellular organisms (both animals and plants) have developed mechanisms to communicate systemically “the occurrence, and location, of a wound” –– to help organisms escape or defend themselves from predators. Because plants are stationary and cannot escape herbivory, they must respond with chemical defenses to deter herbivores (plant-eating animals) and repair damaged tissue.

Authors [see attached] describe long-distance Ca2+-signaling in the model plant, Arabidopsis thaliana (in the mustard family) in response to caterpillar herbivory or mechanical wounding. Authors discovered long-distance calcium signals that require GLR channels for signal propagation. These channels are activated by extracellular glutamate –– which is a well-known neurotransmitter in mammals (including humans) and a more recently uncovered developmental signal in plants. In mammals, glutamate receptors are central to fast excitatory neurotransmission, which is an intriguing parallel to their role as long-distance signals in wounding and defense in plants. Authors found that glutamate is a wound signal in plants. Ion channels of the Arabidopsis thaliana GLUTAMATE RECEPTOR–LIKE family act as sensors that convert this signal into an increase in intracellular Ca2+ ion concentration that subsequently propagates to distant organs (roots, stems or leaves), where defense responses are then induced.


Science 14 Sept 2o18; 361: 1112–1115 [article] & pp 1068–1069 [editorial]

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Meet the Scientists Bringing Extinct Species Back From the Dead

This interesting article was online (Wall Street Journal) the morning of October 9th and out in print in the Oct 10th edition. “Nature” = genes, and “nurture” = environment –– making this a gene-environment interactions topic. 🙂


Meet the Scientists Bringing Extinct Species Back From the Dead
New gene-editing technology could revive everything from the passenger pigeon to the woolly mammoth.

Meet the Scientists Bringing Extinct Species Back From the Dead



By Amy Dockser Marcus

Oct. 9, 2018 10:00 a.m. ET

The pigeons are outwardly unremarkable. Thirteen birds, ages two weeks to three months, occupy a coop at an animal research facility west of Melbourne, Australia. They’re descendants of the common rock pigeon, recognizable denizens of city squares and park benches—with one small but crucial distinction. These are the first pigeons in history with reproductive systems that contain the Cas9 gene, an essential component of the Crispr gene-editing tool. The squabs of this flock will be born with the Cas9 gene in every one of their cells, allowing scientists to edit their offspring with DNA from the extinct passenger pigeon. Those birds, if everything goes to plan, will be the first live animals edited with traits from a species that no longer exists. The flock was created by Ben Novak, an American scientist who has spent the past six years working obsessively on a process known as de-extinction. His goal: to bring back a bird that disappeared from the face of the Earth in 1914.

Over the past six years, new gene-editing technology has given us previously unimaginable control over genetics. The CRISPR-Cas9 system consists of two main parts: an RNA guide, which scientists program to target specific locations on a genome, and the Cas9 protein, which acts as a molecular scissors. The cuts trigger repairs, allowing scientists to edit DNA in the process. Think of CRISPR as a cut-and-paste tool that can add or delete genetic information. CRISPR can also edit the DNA of sperm, eggs and embryos—implementing changes that will be passed down to future generations. Proponents say it offers unprecedented power to direct the evolution of species.

In January 2013 scientists published papers demonstrating that, for the first time, they had successfully edited human and animal cells using CRISPR. The news sparked fears of so-called designer babies edited for traits like intelligence and athleticism, something scientists stay is still far off because of the complexity of those traits. But editing of embryos for research is already under way. In the past 18 months, researchers in the U.S. and China successfully edited disease-causing mutations in viable human embryos not intended for implant or birth.

The technology is widely used in animals. CRISPR has produced disease-resistant chickens and hornless dairy cattle. Scientists around the world routinely edit the genes in mice for research, adding mutations for human diseases such as autism and Alzheimer’s in a search of possible cures. CRISPR-edited pigs contain kidneys that scientists hope to test as transplants in humans.

CRISPR has been discussed as a de-extinction tool since its earliest days. In March 2013 the conservation group Revive & Restore co-organized the first TedXDeExtinction conference in Washington, D.C. Revive & Restore was co-founded by Stewart Brand, the creator of the counterculture Whole Earth Catalog and a vocal advocate for a passenger pigeon revival.

At the conference, George Church, a CRISPR pioneer and geneticist at Harvard Medical School, laid out a scientific roadmap for reviving a species. Church focused not on the passenger pigeon but on his own pet project, the woolly mammoth. Scientists, Church explained, had partially sequenced the mammoth’s genome using DNA extracted from ancient bones and other remains. Armed with that information, they could use CRISPR to edit DNA from the Asian elephant, the mammoth’s closest living relative. Through genetic cutting and pasting, physical and behavioral traits of the mammoth—its namesake coat and ability to withstand subzero temperatures—could be added to living elephant cells.

The idea that woolly mammoths might once again roam the Earth made headlines around the world. But in his talk, titled “Hybridizing With Extinct Species,” Church said that the intended result of his de-extinction experiment was not a genetic facsimile of the mammoth. With enough mammoth DNA, Church explained, a CRISPR-edited Asian elephant would become something else entirely: a modern hybrid that looked and behaved like a mammoth but shared DNA with a living species.

For many in the audience that day, an idea straight out of science fiction suddenly seemed plausible. “CRISPR put de-extinction on the plate,” says Novak, who spoke at the TedXDeExtinction conference and directs the passenger pigeon project for Revive & Restore.

The passenger pigeon has a cult-like following—a global network of “pigeoners” that includes scientists, conservationists, ornithologists, pigeon breeders, poultry geneticists and avid birders eager to see the species revived. Even among these obsessives, Novak’s passion stands out. Of the 1,500 stuffed passenger pigeons in museums and private collections, he has personally viewed 497.

He understands that his obsession is difficult for most people to understand. He has a hard time explaining it himself. Novak grew up in a town of 200 people in North Dakota. Long before he could read, he was fascinated by the idea of extinction, digging unsuccessfully for fossils in his backyard. “I was an odd child,” he says.

There is no plan to bring back the pterodactyl. De-extinction does not mean ‘Jurassic Park.’

There is no plan to bring back the pterodactyl. De-extinction does not mean ‘Jurassic Park.’

In eighth grade, Novak was working on a science-fair project on the dodo bird when he discovered that the species was essentially “a giant extinct pigeon.” Nothing prepared him for the rush he felt when, at age 14, he came across photos of a passenger pigeon while flipping through a National Audubon Society book. “I thought it was a gorgeous bird,” Novak says.

Male passenger pigeons were particularly colorful, with red breasts, feet and legs and iridescent pink patches that glistened on the sides of their throats. The birds traveled in flocks that could number three billion, and were known for their grace and speed, flying at up to 60 miles per hour. Novak read histories that described passenger pigeon flocks so large, they darkened the skies for days as they passed overhead. These massive flocks played an important ecological role, breaking branches to allow sunlight to rejuvenate forests and enriching the soil with their excrement. The birds were prized for their meat; hunters could see the flocks approaching from miles away. The population went into steep decline in the late 1800s and never recovered.

The last known passenger pigeon—a bird named Martha—died in captivity at a Cincinnati zoo in 1914. Her demise sparked the passing of modern conservation laws to protect other endangered species in the U.S. Shortly after her death, Martha was frozen and shipped to the Smithsonian Institution in Washington, D.C., to be stuffed. She’s no longer on display, but Novak has, of course, seen her. “Martha is in bad shape,” he says. Written history and degraded taxidermy intensified Novak’s desire to revive the species. “No one can tell me what a passenger pigeon was like in real life,” he says. “I feel robbed of history.”

This Gene-Edited Calf Could Transform Brazil’s Beef Industry

This Gene-Edited Calf Could Transform Brazil’s Beef Industry

Gene-edited beef could be coming to dinner plates around the globe. In this episode of Moving Upstream, WSJ’s Jason Bellini travels to Brazil to meet the world’s first cow that’s been engineered for warmer climates.

The first step was to sequence the passenger pigeon genome. The project was led by Beth Shapiro, a professor of ecology and evolutionary biology at the University of California, Santa Cruz and the author of the book “How to Clone a Mammoth.” Shapiro’s lab studies the DNA of extinct animals, extracting fragments from bones and other remains, some dating back hundreds of thousands of years. Novak joined the lab in 2013 to work on the passenger pigeon project; Revive & Restore funded his work.

Sequencing an extinct species’ genome is no easy task. When an organism dies, the DNA in its cells begins to degrade, leaving scientists with what Shapiro describes as “a soup of trillions of tiny fragments” that require reassembly. For the passenger pigeon project, Shapiro and her team took tissue samples from the toe pads of stuffed birds in museum collections. DNA in the dead tissue left them with tantalizing clues but an incomplete picture. To fill in the gaps, they sequenced the genome of the band-tailed pigeon, the passenger pigeon’s closest living relative.

By comparing the genomes of the two birds, researchers began to understand which traits distinguished the passenger pigeon. In a paper published last year in “Science,” they reported finding 32 genes that made the species unique. Some of these allowed the birds to withstand stress and disease, essential traits for a species that lived in large flocks. They found no genes that might have led to extinction. “Passenger pigeons went extinct because people hunted them to death,” Shapiro says.

In a Harvard lab, Asian elephant cells are being edited with DNA from the extinct woolly mammoth.

In a Harvard lab, Asian elephant cells are being edited with DNA from the extinct woolly mammoth.

In 2014, Shapiro taught a graduate class on de-extinction and asked each student to make a case for bringing one animal back from the dead. Extinct flightless birds—the moa of New Zealand and the dodo—were favorites, along with the Yangtze River dolphin. Some students cited an animal’s ecological importance or value to tourism. Others mentioned the role humans played in the extinction of a species—a cornerstone of Stewart Brand’s argument for reviving the passenger pigeon.

According to Shapiro, none of these arguments justifies de-extinction. “What’s the point of bringing something back if we don’t know why it went extinct?” she asks. “Or if we do know why it went extinct but haven’t fixed the problem?”

The dodo, she says, exemplifies the latter issue. The flightless bird, native to the Indian Ocean island of Mauritius, nested on the ground and laid only one egg at a time. Settlers who arrived in 1638 brought cats, rats and pigs that devoured dodo eggs. “There is no point in bringing the dodo back,” Shapiro says. “Their eggs will be eaten the same way that made them go extinct the first time.”

Revived passenger pigeons could also face re-extinction. The species thrived in the years before European settlement of North America, when vast forests supported billions of birds. Those forests have since been replaced by cities and farmland. “The habitat the passenger pigeons need to survive is also extinct,” Shapiro says.

Her interest in the bird was rooted in conservation rather than de-extinction. Understanding the exact cause of species’ extinction can help scientists protect living animals and ecosystems. Shapiro argues that passenger pigeon genes related to immunity could help today’s endangered birds survive. “I wanted to study the passenger pigeon,” Shapiro says. “Ben wanted to bring the passenger pigeon back to life.”

But what does it mean to bring an extinct species back? Andre E.R. Soares, a scientist who helped sequence the passenger pigeon genome, says most people will accept a look-a-like as proof of de-extinction. “If it looks like a passenger pigeon and flies like a passenger pigeon, if it has the same shape and color, they will consider it a passenger pigeon,” Soares says.

Shapiro says that’s not enough. Eventually, she says, gene-editing tools may be able to create a genetic copy of an extinct species, “but that doesn’t mean you are going to end up with an animal that behaves like a passenger pigeon or a woolly mammoth.” We can understand the nature of an extinct species through its genome, but nurture is another matter. With no living woolly mammoths or passenger pigeons to model social behavior, who will teach these genetic replicas how to behave like their kind?

“We are going to need a new biology and new names for all this,” Soares says.

Church concedes that there are obstacles to de-extinction, not the least of which is public apprehension. But the history of science, he says, is filled with ideas that start out sounding far-fetched, raise complex ethical issues and over time move toward social acceptance. “The more unknowns there are, the more intense the disagreement,” he says. He points to in vitro fertilization, now a routine reproductive technology that has led to the birth of millions of children. When IVF was first proposed, people worried about the ethics, repercussions and possible risks. “As soon as Louise Brown was born in 1978 and completely normal, the disagreement disappeared,” Church says.

In almost every country, the process of de-extinction requires approval from governments, academic committees and the public along the way. To inject the Cas9 gene into his birds, Novak needed permission from the Office of the Gene Technology Regulator in Australia as well as ethics and animal welfare committees. He’ll need another round of approvals to breed and edit the next generation of his pigeons.

In the meantime, Novak is steadily building the flock. In May he injected 19 eggs with the Cas9 gene, but only two pigeons survived hatching. In August, 11 squabs survived from 46 eggs. Novak and a small team of scientists plan to repeat the process until they have 22 pairs of birds for breeding. They’re considering which passenger pigeon traits to add first, combing through the sequencing data for the genes associated with the extinct bird’s distinctive coloring and preference for life in large flocks. After he determines how passenger pigeon DNA manifests in the rock pigeons, Novak hopes to edit the band-tailed pigeon, the passenger pigeon’s closest living relative, with as many of the extinct bird’s defining traits as possible. Eventually, he says, he’ll have a hybrid creature that looks and acts like a passenger pigeon (albeit with no parental training) but still contains band-tailed pigeon DNA. These new-old birds will need a name, which their human creator has already chosen: Patagioenas neoectopistes, which is Latin for “new wandering pigeon of America.”

Write to Amy Dockser Marcus

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The Dose-Response Revolution: How Hormesis Became Significant: An Historical and Personal Reflection

“Hormesis” is defined in toxicology as the “biphasic dose response to any environmental agent — characterized by a low-dose stimulation, or beneficial effect, and then a high-dose inhibitory, or toxic, effect.” In the research areas of biology and medicine, “hormesis” is defined as an “adaptive response of cells and organisms to a moderate (usually intermittent) stress.” Hence, this topic falls within gene-environment interactions and has been previously discussed many times in these GEITP pages.

Author Calabrese [see first attachment; which is Chapter 1 of a book to appear later this month] tells the story of how hormesis emerged from a marginalized dose-response concept — that had been significantly weakened by its historical association with homeopathy — to become widely recognized as a central concept in evolution, biology, toxicology, and medicine. The chapter is set within the framework of personal reflections by the author, concerning key aspects of how this dose-response revolution occurred and the nature of its biological and biomedical significance.

During the 1980s decade, the number of citations (of terms ‘hormesis’ or ‘hormetic’) was ~10 per year. By the year 2000, there were ~400 citations for ‘hormesis’ or ‘hormetic’; and, in 2017, the number had jumped to more than 9,200. The areas of hormetic research are very broad, cutting across a wide range of disciplines and topics. Many topics gain attention for a few years, and then recede, due to the fact that they were discredited or oversold. In the case of hormesis, it has always been underestimated.

A major reason for the strong and sustained growth of hormesis is principally because hormesis is at the core of the adaptive response. The biomedical sciences started to appreciate hormesis about a decade ago and realized that medicine and public health successes can be driven and mediated by the up-regulation of adaptive mechanisms. Protective mechanisms in the cell or organism can be effective in healthy aging and be similarly useful in protecting people from a vast array of life-threatening conditions such as heart attack, stroke, traumatic injury to the brain,, numerous neurodegenerative diseases, diabetes, and other debilitating diseases.

For those interested, the second attachment summarizes the Oct 3 2o18 testimony of Calabrese before the Senate “Environment and Public Works Subcommittee on Superfund, Waste Management and Regulatory Oversight.” The presentation specifically addresses the “Oversight of the Environmental Protection Agency’s (EPA’s) Implementation of Sound and Transparent Science in Regulation” — in order to establish the best, safest, and most rational public policies.


The Science of Hormesis in Health and Longevity, (Suresh I, Rattan S, Kyriazi M, eds.), 1st Edition, published 26 October 2018

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BOUNCER: protein necessary and sufficient for species-specific fertilization

This is an intriguing topic that I’m sure few of us have ever thought about. When one species of fish lays her eggs in some shallow pool of water and there are several other species of male fish around — besides the species of the female laying her eggs — how come the only sperm “allowed” to fertilize those eggs, are those from the correct species? For survival of the species, specific “match-making” of the sperm and egg is of course critical. Sperm must come in contact with the egg, and exactly one sperm needs to fertilize exactly one egg. This is because an unfertilized egg cannot develop, and fertilization with too many sperm (polyspermy) is equally fatal.

Fertilization has to provide a barrier between species by creating egg-sperm incompatibilities. Despite these fascinating biological questions, evolutionary implications, and clear applications for human contraception and infertility inherent in understanding the mechanisms by which egg and sperm meet — the mechanisms remain poorly understood. Authors [see attached article & editorial] investigated fertilization in zebrafish and identified Bouncer, as a protein that is important for sperm-egg interaction and which also creates a species-specific barrier.

Fertilization is a highly orchestrated event: in preparation, both egg and sperm must undergo changes. The sperm increases its mobility and the sperm head is reorganized to form a cap-like structure, (acrosome); this prepares the sperm for penetration of the egg coat (a specialized extracellular matrix that surrounds the egg), attachment, and eventual fusion with the egg membrane. Egg activation leads to changes in the egg coat (called the zona pellucida, or chorion). In mammals, the egg coat not only is a structural component that protects the oocyte but also attracts and binds sperm. However, in fish the micropyle (a specific opening in the egg coat) forms the single entry point for sperm. Upon fertilization, the egg coat then becomes less permeable to prevent polyspermy.

Unexpectedly, authors [see attached] found that Bouncer not only is required for sperm-egg interaction but is also sufficient to allow cross-species fertilization between zebrafish and medaka (two species that diverged from one another more than 200 million years ago)..!! This study thus identifies Bouncer as a key determinant of species-specific fertilization in fish. Bouncer’s closest gene homolog in tetrapods (including human) is SPACA4, a gene expressed only in the male germline — which suggests that these findings in fish have relevance to human biology.


Science 7 Sept 2o18; 361: 1029–1033 [article] & pp 974–975 [editorial]

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Presymptomatic change in microRNAs modulates Tau pathology

As these GEITP pages have often described, an individual’s genotype — determined by DNA sequence differences — contributes to any heritable trait (phenotype) such as type-2 diabetes, schizophrenia, risk of coronary artery disease, or response to a drug or environmental toxicant. On the other hand, epigenetic effects — determined by chromosomal events other than DNA sequence differences — also contribute to many complex traits, but they are not usually inherited but rather acquired during one’s life, due to environmental or other pressures. Epigenetic effects include DNA-methylation, RNA-interference (RNAi; microRNA regulation), histone modifications, and chromatin remodeling. Assays are readily available for the first two, but the latter two are still under intense investigation.

MicroRNAs (miRs) constitute an abundant class of noncoding RNA molecules (i.e. they don’t make protein) that are endogenously expressed in multicellular organisms. These miRs are 18–23 nucleotides in length, and they often regulate gene expression by messenger-RNA (mRNA) degradation or translational repression (blocking the process of going from mRNA to the protein). Many miRs show high levels in brain, and are known to contribute to brain development, neural plasticity, and neuroprotection. The levels of many miRs are altered in neurodegenerative diseases (e.g. 35 miRs were shown to be altered in prefrontal cortex of late-onset Alzheimer Disease patients). If one deletes Dicer (a critical enzyme involved in miR synthesis) in adult brain — this results in Tau hyperphosphorylation and neuronal loss, which are the pathological traits associated with tau-opathy (i.e. manifested as deposition of intracellular neurofibrillary tangles). However, it is not known whether or not miR changes are already present at the presymptomatic stages of neurodegenerative diseases, and if such miR changes can directly impact biological pathways that may subsequently lead to neurodegeneration.

Tau-opathies include a wide range of neurodegenerative diseases such as progressive supranuclear palsy, Pick’s disease, parkinsonian tremor linked to chromosome 17, and Alzheimer Disease. Previously, miRs have been identified as modulators of Tau that contribute to Tau pathology. Authors [see attached article] first determined which (mouse brain hippocampal) miRs might be altered at the presymptomatic and symptomatic stages of

tau disease using the “Tau mouse” (rTg4510 mouse), which is a well-characterized lab animal model. By pairing analysis of miR-RNA, using QIAGEN Ingenuity Pathway Analysis (IPA), authors found 401 genes that can be regulated by 71 miRs altered in the Tau mouse hippocampus at the presymptomatic stage. Among several miRs confirmed experimentally, miR142−3p and miR142−5p in the Tau hippocampus was significantly up-regulated by 2 weeks of age and later in life.

Transcriptome studies by RNA-seq and IPA revealed several overlapping biological and disease-associated pathways affected by either Tau or miR142 over-expression — including ‘signal transducer & activator of transcription-3’ (Stat3) and ‘tumor necrosis factor receptor-2’ (Tnfr2) signaling pathways. Similar to what was observed in Tau brains, over-expression of miR142 in cortical neurons of wild-type mice augments mRNA levels of ‘glial fibrillary acidic protein’ (Gfap) and ‘colony-stimulating factor-1’ (Csf1), accompanied by a significant increased numbers of microglia and reactive astrocytes. Taken together, these data suggest that miR alterations by Tau over-expression appear to contribute to the phenotype of neuroinflammation that is seen in the brains of Tau mice. This is an excellent example of an epigenetic effect influencing the individual’s phenotype.

Sci Rep (Nature) 2018; 8: 9251

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Rapid onset” of transgender mentality in the American society ???

Okay, okay: “Gene-environment interactions” is a topic that comes in many flavors — including behavioral traits (phenotypes). It seems clear that “genetic susceptility” (genotype or, more genereally, ‘genetic architecture’) renders some persons more prone than others to be affected by social media (i.e. constant bombardment of environmental signals). Some of us in academia have discussed this remarkable “shift” (seen almost exclusively among Western cultures) in mindset that has been observed in “children of ages 10 to 30” over the past 10-20 years. Clearly, the internet — and subsequently the “instantaneous exchange of ideas and opinions” among young people wh have much more leisure time on their hands — represents an underlying cause of “rapid onset gender dysphoria” (ROGD) that is being seen in high schools, colleges, and young adults. Instead of simply “male” or “female”, some colleges now offer their students up to eight or ten “categories” of “what you feel like calling yourself, this semester.”

ROGD [see attached 2-page editorial] summarizes this trait — a “sudden unease, among teenagers and young adults, with the gender they were assigned at birth”. An incendiary publication about ROGD appeared in Aug 2o18 PLoS ONE [abstract pasted below], written by physician-scientist Lisa Littman of Brown University; her publication has infuriated transgender activists — leading to a debate about “academic freedom”, calling it “a flawed study that reflects an anti-transgender agenda.”


The study remains freely available, but last week, PLoS ONE announced it is conducting a post-publication investigation of its methodology and analysis. “This is not about suppressing academic freedom or scientific research. This is about the scientific content itself — whether there is anything that needs to be looked into or corrected,” PLoS ONE Editor-in-Chief Joerg Heber said. In order to appear “politically correct” (PC), Brown University officials decided to remove the university’s press release, highlighting this publication, from its website. It seems that many universities these days do not wish to offend their students, or the parents, rather than choosing to deal with any issue in an objective manner. And that is unfortunate. 🙁

Science 7 Sept 2o18; 361: 958–959
PLoS ONE 2018 Aug 16;13(8):e0202330.
Rapid-onset gender dysphoria in adolescents and young adults: A study of parental reports.

Littman L1.

Author information


In on-line forums, parents have been reporting that their children are experiencing what is described here as “rapid-onset gender dysphoria,” appearing for the first time during puberty or even after its completion. The onset of gender dysphoria seemed to occur in the context of belonging to a peer group where one, multiple, or even all of the friends have become gender dysphoric and transgender-identified during the same timeframe. Parents also report that their children exhibited an increase in social media/internet use prior to disclosure of a transgender identity. The purpose of this study was to document and explore these observations and describe the resulting presentation of gender dysphoria, which is inconsistent with existing research literature.

Recruitment information with a link to a 90-question survey, consisting of multiple-choice, Likert-type and open-ended questions, was placed on three websites where parents had reported rapid onsets of gender dysphoria. Website moderators and potential participants were encouraged to share the recruitment information and link to the survey with any individuals or communities that they thought might include eligible participants to expand the reach of the project through snowball sampling techniques. Data were collected anonymously via SurveyMonkey. Quantitative findings are presented as frequencies, percentages, ranges, means and/or medians. Open-ended responses from two questions were targeted for qualitative analysis of themes.

There were 256 parent-completed surveys that met study criteria. The adolescent and young adult (AYA) children described were predominantly female sex at birth (82.8%) with a mean age of 16.4 years. Forty-one percent of the AYAs had expressed a non-heterosexual sexual orientation before identifying as transgender. Many (62.5%) of the AYAs had been diagnosed with at least one mental health disorder or neurodevelopmental disability prior to the onset of their gender dysphoria (range of the number of pre-existing diagnoses 0-7). In 36.8% of the friendship groups described, the majority of the members became transgender-identified. The most likely outcomes were that AYA mental well-being and parent-child relationships became worse since AYAs “came out”. AYAs expressed a range of behaviors that included: expressing distrust of non-transgender people (22.7%); stopping spending time with non-transgender friends (25.0%); trying to isolate themselves from their families (49.4%), and only trusting information about gender dysphoria from transgender sources (46.6%).

Rapid-onset gender dysphoria (ROGD) describes a phenomenon where the development of gender dysphoria is observed to begin suddenly during or after puberty in an adolescent or young adult who would not have met criteria for gender dysphoria in childhood. ROGD appears to represent an entity that is distinct from the gender dysphoria observed in individuals who have previously been described as transgender. The worsening of mental well-being and parent-child relationships and behaviors that isolate AYAs from their parents, families, non-transgender friends and mainstream sources of information are particularly concerning. More research is needed to better understand this phenomenon, its implications and scope.

PMID: 30114286
Or turning to religion. Or believing in AT LEAST SOMETHING.
Interesting email. Thanks for sending it. If you’re interested in a detailed discussion about the subjects (gender as well as academic freedom) I highly recommend the following book:

Best, Jack

From Wikipedia:

Galileo’s Middle Finger is a 2015 book about the ethics of medical research by Alice Dreger, an American bioethicist and author. Dreger explores the relationship between science and social justice by discussing a number of scientific controversies. These include the debates surrounding intersex genital surgery, autogynephilia, and anthropologist Napoleon Chagnon’s work.

From: Anonymous
Sent: Tuesday, October 08, 2018 10:21 AM

This is a topic that should be discussed while drinking beer. Personally, I think we put way too much emphasis on our plumbing, and the current onset of transgender is just one more rejection of limitations and labels.

In the end, why should we care which pronouns you prefer?

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CRISPR/Cas editing with no detectable genome-wide off-targets — method of validation

These GEITP pages have previously discussed CRISPR/Cas9, the new gene-editing methodology that has revolutionized molecular biology and molecular genetics studies –– especially in laboratory animal and plant model systems. Clinically, this type of gene-editing will be commonplace, as well. Very briefly summarized, CRISP/Cas9 allows the specific removal (cutting out one or more DNA nucleotides) and/or addition (insertion of one or more nucleotides) and/or replacement (substituting one or more different nucleotides) into the genome of an animal or plant. The possible downside of this technique has been that other (undesirable) DNA locations (so-called ‘off-targets’) might also be interfered with, and this error could lead to perturbations in some physiological function or even a disease, including cancer.

Authors [see attached article] describe ‘Verification of In Vivo Off-targets’ (VIVO), a highly sensitive strategy that can robustly identify any genome-wide off-target effects of CRISPR–Cas activity in the intact cell, tissue, or organism. VIVO consists of two steps. First, a superset of potential off-target cleavage sites for the nucleases is identified, using circularization for reporting cleavage effects in cell culture by sequencing (CIRCLE-seq); this sensitive method avoids potential confounding effects associated with cell-based assays and can successfully identify supersets of sites that include bona fide off-targets in cultured mouse or human cells. Second, sites identified by CIRCLE-seq are then examined for insertion-deletion (indel) mutations in target tissues that have been treated with Cas9 (which stands for ‘CRISPR-associated protein-9’, which is an RNA-guided DNA endonuclease enzyme).

Authors [see attached article] used VIVO, comparing it with a “guide RNA” (deliberately designed to be promiscuous) to show that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse liver in the intact animal. Using VIVO, authors then show that appropriately designed guide RNAs can direct efficient editing in liver of the intact mouse with no detectable off-target mutations. Thus, VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms. This technique thereby provides a blueprint to foster the development of therapeutic strategies by which gene-editing in the intact cell or tissue can be used –– clinically, as well as in mouse.


Nature 20 Sept 2o18; 561: 416–419

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