Category Archives: Gene Medicine

Why can some people eat as much as they want, and still stay thin?

In a study published today in the journal Cell, Life Sciences Institute Director Dr. Josef Penninger and a team of international colleagues report their discovery that a gene called ALK (Anaplastic Lymphoma Kinase) plays a role in resisting weight gain.

We all know these people, who can eat whatever they want, they dont exercise, but they just dont gain weight. They make up around one per cent of the population, says senior author Penninger, professor in the Faculty of Medicines department of medical genetics and a Canada 150 research chair.

Dr. Josef Penninger

We wanted to understand why, adds Penninger. Most researchers study obesity and the genetics of obesity. We just turned it around and studied thinness, thereby starting a new field of research.

Using biobank data from Estonia, Penningers team, including researchers from Switzerland, Austria, and Australia, compared the genetic makeup and clinical profiles of 47,102 healthy thin, and normal-weight individuals aged 20-44. Among the genetic variations the team discovered in the thin group was a mutation in the ALK gene.

ALKs role in human physiology has been largely unclear. The gene is known to mutate frequently in several types of cancer, and has been identified as a driver of tumour development.

Our work reveals that ALK acts in the brain, where it regulates metabolism by integrating and controlling energy expenditure, says Michael Orthofer, the studys lead author and a postdoctoral fellow at the Institute of Molecular Biology in Vienna.

When Penningers team deleted the ALK gene in flies and mice, both were resistant to diet-induced obesity. Despite consuming the same diet and having the same activity level, mice without ALK weighed less and had less body fat.

As ALK is highly expressed in the brain, its potential role in weight gain resistance make it an attractive mark for scientists developing therapeutics for obesity.

The team will next focus on understanding how neurons that express ALK regulate the brain at a molecular level, and determining how ALK balances metabolism to promote thinness. Validating the results in additional, more diverse human population studies will also be important.

Its possible that we could reduce ALK function to see if we did stay skinny, says Penninger. ALK inhibitors are used in cancer treatments already, so we know that ALK can be targeted therapeutically.

The study was supported by the Estonian Research Council, the European Union Horizon 2020 fund, and European Regional Development Fund, the von Zastrow Foundation, and the Canada 150 Research Chairs Program.

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UBC scientist identifies a gene that controls thinness - UBC Faculty of Medicine - UBC Faculty of Medicine

INDIANAPOLIS A dime-size nanochip developed by a world-renowned researcher who recently relocated to Indianapolis could help transform the practice of medicine. It could also turn Indianapolis into a manufacturing and research hub for radically new disease and trauma treatment techniques.

It all began in August 2018, when Chandan Sen, one of the worlds leading experts in the nascent field of regenerative medicine, moved his lab from Ohio State University to the Indiana University School of Medicine. He brought along a team of about 30 researchers and $10 million in research grants, and now serves, among a myriad of other positions, as director of the newly formed Indiana Center for Regenerative Medicine and Engineering, to which IU pledged $20 million over its first five years.

IU recruited Sen away from Ohio State in part because of its desire not just to promote academic research in his field but also to help develop practical, commercial products and uses for his breakthroughs.

A scientist prefers to be in the lab and keep on making more discoveries, said Sen, 53.

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But I thought that, unless we participate in the workforce development process and the commercialization process, I dont think that the business people would be ready to do it all by themselves. Because its such a nascent field.

Its definitely new and its potential sounds like the stuff of science fiction.

Regenerative medicine, as its name hints, seeks to develop methods for replacing or reinvigorating damaged human organs, cells and tissues.

For instance, instead of giving a diabetic a lifetimes worth of insulin injections, some of his skin cells could be altered to produce insulin, curing him. Such techniques might also be used for everything from creating lab-grown replacement organs to, someday, regenerating severed limbs.

Regenerative medicine offers a form of medicine that is neither a pill nor a device, Sen said.

It is a completely new platform, where you dont necessarily depend on any given drug, but are instead modifying bodily functions.

A big, tiny breakthrough

Sen and his teams signal contribution to the field is a technique theyve dubbed tissue nanotransfection, or TNT. Put simply, it uses a nanotechnology-based chip infused with a special biological cargo that, when applied to the skin and given a brief electrical charge, can convert run-of-the-mill skin cells into other cell types. Potentially, the technique could be used for everything from regrowing blood vessels in burn-damaged tissue to creating insulin-secreting cells that could cure diabetics.

Obviously, such applications are still down the road a ways. But the technology is far enough along that some products are already making it to marketand investors, entrepreneurs and established companies are sniffing around for opportunities. According to the Alliance for Regenerative Medicine, more than 1,000 clinical trials worldwide are using regenerative medicine technologies.

Thousands of patients are already benefiting from early commercial products, and we expect that number will grow exponentially over the next few years, said Janet Lambert, the alliances CEO.

Lambert predicts that the number of approved gene therapies will double in the next one to two years. Last year, the U.S. Food and Drug Administration predicted it would be approving 10 to 20 cell and gene therapies each year by 2025.

These new techniques could do more than just revolutionize medicine. They could also upend the medical industry as we know it. And the IU School of Medicineand Indianapoliscould lead the way.

There are really only two or three places in the country that did the kind of comprehensive work that Dr. Sens group was doing, said Anantha Shekhar, executive associate dean for research at IU School of Medicine. And they were doing it from the lab all the way to the clinic, where they were already applying those technologies in patients.

So it was very attractive to think of starting with a bang bringing a comprehensive group here and creating a new center.

Ambitious goals

Instead of merely treating chronic conditions, regenerative medicine could end them, once and for all.

For instance, consider a car with an oil leak. The traditional medical approach might be to live with the chronic condition by pouring in a fresh quart of oil every few days. The regenerative medicine approach would fix the leak. Its good for the car, good for the cars owner but not necessarily good for the guy who was selling all those quarts of oil.

Which is why these new techniques, if they catch on, could cause turmoil in the medical industry.

Because regenerative medicine has the potential to durably treat the underlying cause of disease, rather than merely ameliorating the symptoms, this technology has the potential of being extremely disruptive to the current practice of medicine, Lambert said.

This has the potential to be hugely disruptive, Sen added, because so much of medicine today relies on huge industrial infrastructures to manage, not cure, chronic diseases and disabilities.

If such disruption comes to pass, the leaders of 16 Tech, a 50-acre innovation district northwest of downtown that aspires to house dozens of medical-related startups and established firms, would love to be its epicenter.

The Center for Regenerative Medicine will be one of the tenants of 16 Techs first building, a $30 million, 120,000-square-foot research and office building scheduled to open in June.

Regenerative medicine is probably one of the next major waves of medical innovation in the world, 16 Tech CEO Bob Coy said. To have him here doing this work gives Indianapolis and Indiana an opportunity to develop an industrial cluster in regenerative medicine.

Coy believes the most momentous early step on that road was the recent establishment by Sen of masters and doctoral programs in regenerative medicine at the IU School of Medicine. Its the first degree of its type in the country, earning IU and Indianapolis the enviable status of first mover.

I think, for example, of [Pittsburghs] Carnegie Mellon University, which, back in the late 1960s, created the first college of computer science in the country, Coy said. And now you know Carnegie Mellons reputation in computer science.

What isnt in place yet is a state or city program to promote development of a regenerative medicine hub.

We need to start doing that, Coy said. That means putting a lot of the infrastructure in place to support startups that are based on this technology, as well as recruiting companies that want to collaborate with Dr. Sen.

In spite of the lack of a coherent recruitment program, Coys phone has started to ring, thanks largely to Sens presence.

There have been a few meetings Ive had with people who already have relationships with him, who, when they come to town, have reached out to meet and talk about what were doing at 16 Tech, he said.

Fueling entrepreneurship

One of the first 16 Tech startups with designs on the regenerative medicine niche is Sexton Biotechnologies.

The company was groomed by Cook Regentec, a division of Bloomington-based Cook Group charged with incubating and accelerating technologies for regenerative medicine and the related field of cell gene therapy.

Any products that show promise are either folded into the company, turned into their own divisions or, as in Sextons case, spun off as an independent entity with Cook retaining a financial stake.

Its a measure of the newness of this field that Sextons 17 employees arent working on new medicines, but rather marketing basic tools needed to conduct research. The companys offerings include a vial for storing cell and gene products in liquid nitrogen, and a cell culture growth medium.

Theres a ready market for such tailor-made gear, because, for years, researchers in the regenerative medicine field had to make do with jury-rigged equipment.

What most of those companies did was repurpose things like tools from the blood banking industry, or tools from bio pharma, said Sean Werner, Sextons president.

So thats why a lot of newer companies are starting to build tools explicitly for the industry, as opposed to everybody just having to cobble together stuff that was already out there.

Werner said investors recognize the momentous opportunity in regenerative medicine and are flocking to the field.

Its not something you have to explain, he said. Companies and VC groups are trying to get a piece of it.

What has investors and medical researchers charged up is the almost unlimited range of potential applications, from healing burns to, perhaps someday, regenerating limbs.

I think it would be a huge revolution if were able to, for example, regenerate insulin-secreting cells in children who have become juvenile diabetics or have for whatever reason lost their pancreas, Shekhar said. Those are the kinds of things that will start to change the way we see certain diseases.

Lambert predicted that, as the science advances, so will the business case.

While early programs focused primarily on rare genetic diseases and blood cancers, were already seeing the field expand into more common age-related neurological disorders, such as Parkinsons and Alzheimers, she said.

I expect this trend to continue in the coming years, greatly increasing the number of patients poised to benefit from these therapies.

Werner said regenerative medicine also is seeking advancements in manufacturing technologies that will lower the cost of product development.

It all adds up to a huge opportunity the state is well-positioned to seize, Werner believes.

Indiana is a perfect place for this kind of thing to really ramp up, he said. Theres no reason we cant lead the field.

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IU team pursuing breathtaking advancements in regenerative medicine - The Republic

The complement system is part of the bodys immune system to fight pathogens and remove cell debris. Its role in two autoimmune diseases and a mental disorder is a surprise.

The complement system is part of the bodys immune system to fight pathogens and remove cell debris. Its role in two autoimmune diseases and a mental disorder is a surprise.Variants in a gene of the human immune system cause men and women to have different vulnerabilities to the autoimmune diseases lupus and Sjgrens syndrome, according to findings published today in the journal Nature. This extends recent work that showed the gene variants could increase risk for schizophrenia.

The gene variants are a member of the complement system, a cascade of proteins that help antibodies and phagocytic cells remove damaged cells of a persons own body, as well as an infection defense that promotes inflammation and attacks pathogens. Normally the complement system keeps a person healthy in the face of pathogens; it also helps cart away the debris of damaged human cells before the body can mount an autoimmune attack. Now complement gene variants apparently play a contributing role in the diseases systemic lupus erythematosus, Sjgrens syndrome and schizophrenia.

It had been known that all three illnesses had common genetic associations with a section of the human chromosome called the major histocompatibility complex, or MHC. This region on chromosome 6 includes many genes that regulate the immune system. However, making an association with a specific gene or with the mutational variants of a specific gene that are called alleles has been difficult, partly because the MHC on human chromosome 6 spans three million base-pairs of DNA.

The Nature paper is a collaboration of 22 authors at 10 institutions in the United States and one in England, along with many members of a schizophrenia working group. Robert Kimberly, M.D., professor of medicine at the University of Alabama at Birmingham and director of the UAB Center for Clinical and Translational Science, is a co-author of the research, which was led by corresponding author Steven McCarroll, Ph.D., assistant professor of genetics at Harvard Medical School.

The identified alleles are complement component 4A and 4B, known as C4A and C4B.

The research showed that different combinations of C4A and C4B copy numbers generate a sevenfold variation in risk for lupus and 16-fold variation in risk for Sjgrens syndrome among people with common C4 genotypes. Paradoxically, the same C4 alleles that previously were shown to increase risk for schizophrenia had a different impact for lupus and Sjgrens syndrome they greatly reduced risk in those diseases. In all three illnesses, the C4 alleles acted more strongly in men than in women.

For the complement proteins that are encoded by the genes for C4 and for complement component 3, or C3, both C4 protein and its effector C3 protein were present at greater levels in men than in women in cerebrospinal fluid and blood plasma among adults ages 20-50. Intriguingly, that is the age range when the three diseases differentially affect men and women for unknown reasons. Lupus and Sjgrens syndrome affect women of childbearing age nine times more than they do men of similar age. In contrast, in schizophrenia, women exhibit less severe symptoms, more frequent remission of symptoms, lower relapse rates and lower overall incidence than men, who are affected more frequently and more severely.

Both men and women have an age-dependent elevation of C4 and C3 protein levels in blood plasma. In men, this occurs early in adulthood, ages 20-30. In women, the elevation is closer to menopause, ages 40-50. Thus, differences in complement protein levels in men and women occur mostly during the reproductive years, ages 20-50.

The researchers say sex differences in complement protein levels may help explain the larger effects of C4 alleles in men, the greater risk of women for lupus and Sjgrens, and the greater vulnerability of men for schizophrenia.

Robert Kimberly, M.D.The ages of pronounced sex differences in complement levels correspond to the ages when men and women differ in disease incidence. In schizophrenia cases, men outnumber women in early adulthood; but that disparity of onset lessens after age 40. In lupus, female cases greatly outnumber male cases during childbearing years; but that difference is much less for disease onset after age 50 or during childhood. In Sjgrens syndrome, women are more vulnerable than are men before age 50.

The researchers say the differing effect of C4 alleles in schizophrenia versus lupus and Sjgrens syndrome will be important to consider in any therapeutic effort to engage the complement system. They also said, Why and how biology has come to create this sexual dimorphism in the complement system in humans presents interesting questions for immune and evolutionary biology.

Co-authors with McCarroll and Kimberly for the paper, Complement genes contribute sex-biased vulnerability in diverse illnesses, are Nolan Kamitaki, Aswin Sekar, Heather de Rivera, Katherine Tooley and Christine Seidman, Harvard Medical School, Massachusetts; Robert Handsaker and Christopher Whelan, Broad Institute of Massachusetts Institute of Technology; David Morris, Philip Tombleson and Timothy Vyse, Kings College London, London, United Kingdom; Kimberly Taylor and Lindsey Criswell, University of California-San Francisco School of Medicine; Loes Olde Loohuis and Roel Ophoff, University of California-Los Angeles; Michael Boehnke, University of Michigan; Kenneth Kaufman and John Harley, Cincinnati Childrens Hospital Medical Center, Ohio; Carl Langefeld, Wake Forest School of Medicine, North Carolina; Michele Pato and Carlos Pato, State University of New York, Downstate Medical Center; and Robert Graham, Genentech Inc., South San Francisco, California.

Support came from National Institutes of Health grants HG006855, MH112491, MH105641 and MH105653; and from the Stanley Center for Psychiatric Research.

At UAB, Kimberly holds the Howard L. Holley Research Chair in Rheumatology.

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Complement genes add to sex-based vulnerability in lupus and schizophrenia - UAB News

In patients with coronavirus disease 2019 (COVID-19), higher sputum cell expression of angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) was observed in certain patients with asthma while lower expression was found in patients who used inhaled corticosteroids (ICS), according to study results published in the American Journal of Respiratory and Critical Care Medicine.

COVID-19, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), may be more severe in patients with chronic lung disease, including patients with asthma, and it appears that demographic or biological factors influence susceptibility to the infection or severity of disease. Because ACE2 and TMPRSS2 mediate viral infection of host cells, researchers reasoned that differences in ACE2 or TMPRSS2 gene expression in sputum cells in patients with asthma may identify subgroups at risk for COVID-19 morbidity.

By analyzing gene expression for ACE2 and TMPRSS2 as well as intercellular adhesion molecule 1 (ICAM-1) in sputum cells from 330 participants and 79 healthy control individuals, researchers found that gene expression of ACE2 was lower than TMPRSS2, and that expression levels of both genes were similar in patients with asthma and healthy individuals. In patients with asthma, however, men, African Americans, and people with diabetes had higher expression of ACE2 and TMPRSS2. In patients with asthma, ICAM-1 expression increased and there were fewer consistent differences related to sex, race, and ICS use. Use of ICS was associated with lower expression of ACE2 and TMPRSS2, while treatment with triamcinolone acetonide did not decrease expression of either gene or ICAM-1.

Higher expression of ACE2 and TMPRSS2 in males, African Americans, and patients with diabetes mellitus provides rationale for monitoring these asthma subgroups for poor COVID-19 outcomes, the study authors wrote. The lower expression of ACE2 and TMPRSS2 with ICS use warrants prospective study of ICS use as a predictor of decreased susceptibility to SARS-CoV-2 infection and decreased COVID-19 morbidity.

Reference

Peters MC, Sajuthi S, Deford P, et al; for the National Heart, Lung, and Blood Institute Severe Asthma Research Program-3 Investigators. COVID-19 related genes in sputum cells in asthma: Relationship to demographic features and corticosteroids [published online April 29, 2020]. Am J Respir Crit Care Med. doi:10.1164/rccm.202003-0821OC

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COVID-19-Related Genes Have Higher Expression in Certain Patients With Asthma - Pulmonology Advisor

May 18, 2020 -Using precision medicine approaches to tailor heart disease therapies could lead to more cost-effective treatments and improved patient outcomes, according to a study led by researchers at the University of Alabama at Birmingham (UAB).

Patients who experience a heart attack have sharply reduced blood flow in coronary arteries, as well as a high risk of heart failure or death. Coronary angioplasty, a procedure to open narrowed or blocked arteries in the heart, and percutaneous coronary intervention (PCI) can restore blood flow to minimize heart damage. These procedures reduce the risk of subsequent major adverse cardiovascular events (MACE), which include heart attacks, strokes, or death.

After these procedures, providers have to make a treatment decision. After PCI, all patients receive two antiplatelet agents for up to one year. The most commonly used antiplatelet combination after PCI is aspirin and clopidogrel. Clopidogrel is converted to its active form by an enzyme called CYP2C19, but patients respond to this treatment differently depending on their genetic makeup.

Over 30 percent of people have loss-of-function variants in the CYP2C19 gene that decreases the effectiveness of clopidogrel. These patients may not get the full benefit of clopidogrel, which would increase their risk of MACE. The FDA recommends that providers consider different treatments for these individuals, such as prasugrel or ticagrelor, to replace clopidogrel.

In 2018, UAB and researchers at nine universities across the US showed that patients with loss-of-function variants who were treated with clopidogrel had elevated risks. The study revealed that there was a twofold risk of MACE in PCI patients, and a threefold risk for MACE among patients with acute coronary syndrome who received PCI, as compared to patients prescribed prasugrel or ticagrelor instead of clopidogrel.

While prasugrel and ticagrelor are not influenced by loss-of-function variants and can substitute for clopidogrel, these drugs are much more costly and can bring a higher risk of bleeding.

Using this real-world data, the research team set out to conduct an economic analysis of the best treatments for heart disease patients. The study compared three main strategies: treating all patients with clopidogrel, treating all patients with ticagrelor, and genotyping all patients and using ticagrelor in those with loss-of-function variants.

The group considered differences in event rates for heart attacks and stent thrombosis in patients receiving clopidogrel versus ticagrelor versus genotype-guided therapy, during the one-year period following PCI. They also considered medical costs from events like admissions, procedures, medications, clinical visits, and genetic testing. The study used an economic measure known as the quality-adjusted life year (QALY).

First, we looked at which strategy provided the highest QALY, Limdi said. The QALY is the gold standard for measuring benefit of an intervention in our case, genotype-guided treatment compared to treatment without genotyping. Universal ticagrelor and genotype-guided antiplatelet therapy had higher QALYs than universal clopidogrel so those are the best for the patient.

Researchers then analyzed whether those interventions that have higher QALYs were also reasonable from a cost perspective, which includes a payers or patients willingness to pay.

In our case, the payor would recognize that ticagrelor is more expensive than clopidogrel $360 per month vs. $10 per month and there is a $100 cost for each genetic test, Limdi said. So, from the payor perspective, the more effective strategy (one with a higher QALY) if more expensive (higher cost) would have to lower the risks of bad outcomes like heart attacks and strokes for the gains in QALY that are at, or below, the willingness-to-pay threshold.

A measure called incremental cost-effectiveness ratios (ICERs) assesses the incremental cost of the benefit, or improvement in QALY. In the US, a treatment is considered cost-effective if its associated ICER is at or below the willingness-to-pay threshold of $100,000 per QALY.

In our assessment, the two strategies with the highest QALY had very different ICERs, Limdi said. The genotype-guided strategy was cost-effective at $42,365 per QALY. Universal ticagrelor was not; it had an ICER of $227,044 per QALY.

The study results demonstrate the effectiveness of genotyping and precision medicine strategies for tailoring treatments and improving patient outcomes.

We showed that tailoring antiplatelet selection based on genotype is a cost-effective strategy, said Nita Limdi, PharmD, PhD. Support is now growing to change the clinical guidelines, which currently do not recommend genotyping in all cases. Evidence like this is needed to advance the field of precision medicine.

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Precision Medicine Informs Cost-Effective Heart Disease Treatments - HealthITAnalytics.com

Breakthrough genome editing companies includingEditas (NASDAQ:EDIT) and Intellia Therapeutics (NASDAQ:NTLA) have been in a tailspin since late 2019, and the latest earnings reports from both of those companies show that their revenue from collaborations and partnerships has started to dry up despite positive revenue growth overall.

Both companies aim to produce gene therapies utilizing CRISPR-based genetic editing in living patients, though their methods of delivering that therapy differ substantially. Neither company has a product on the market, though Editas beat Intellia to clinical trialsin April when it began testing EDIT-101 for Leber congenital amaurosis, a type of congenital blindness. Nonetheless, Editas is many years away from its first therapy being approved for sale, assuming that EDIT-101 proceeds past phase 1.

Investors considering either of these two companies should be aware that both are risky choices with no guarantee of a payoff over any term. There is one significant difference that wise investors will weigh carefully, however: Editas's partnerships and strategic collaborations appear positioned to be far more fruitful for the company than Intellia's.

Image source: Getty Images.

Intellia is a slightly smaller company than Editas, but its pipeline is comparable in breadth. The companies are of similar age, with Editas having been founded in 2013 and Intellia in 2014. However, Intellia's network of collaborations and research partnerships is far less lucrative, and its pipeline projects may soon require new funding to move forward.

Intellia's partners include pharma giantNovartis (NYSE:NVS) and biotechRegeneron (NASDAQ:REGN). Novartis made a substantial equity investment in Intellia as part of that partnership, and Novartis also retained exclusive rights to develop any engineered CAR-T cancer therapies produced by the collaboration. Intellia also agreed to give Regeneron the exclusive right to develop CRISPR-based therapies targeted at any of 10 different genes in the liver.

The terms of these collaborations make Intellia unable to capitalize on major successes beyond extending the depth of integration with its partners. Thus, in the long view, the company's path forward would still require moving its wholly owned therapy candidates to market, even if its approach is proven by a collaborator's success.

Editas's partnerships, on the other hand, are substantially more equitable. Editas's major drug development collaborations include Allergan (now part of AbbVie (NYSE:ABBV) and biopharma giantBristol Myers Squibb (NYSE:BMY). The expectation with these collaborations is that the more mature partner companies will be responsible for clinical-stage development, with Editas providing trial-ready therapy candidates and a technology platform to develop similar therapies according to the partners' needs.

Should these candidates show promise in phase 2 clinical trials investigating preliminary efficacy, the company's collaborators would likely respond by initiating new collaborations to capitalize on Editas's platform before its output is replicated by a competitor like Intellia. But Editas isn't in the same position as Intellia with regard to its major collaborations because it has a chance to capture the upside of collaborators' successes as well.

Editas's collaboration with Allergan specifies that both parties have optionality to co-develop any successful programs, and that Editas will share the revenue and losses of those programs equally with Allergan.And Editas's previous collaborations with companies like Celgene demonstrate that companies collaborating with Editas do so to access its gene-editing platform as customers as much as partners.

Editas also has partnerships with research-stage small preclinical companies such as Sandhill Therapeutics. Sandhill's therapeutic platform could benefit immensely from integrating Editas' genetic editing technologies. A similar research-stage pact with BlueRock Therapeutics initiated in 2019 has already advanced to clinical pipeline collaborations for Editas, proving that working with external peers is one of the company's organizational strengths.

It's important to remember that Editas's collaboration advantage is far from the only ingredient the company needs to survive in the medium term. Reliable revenue remains absent, and collaborations are vulnerable to amendment if the company can't deliver what its collaborators need to move products through the clinical trial process.

Data by YCharts

For the moment, neither Editas nor Intellia warrants a definite buy, and present holders of Intellia may want to consider selling. If Intellia cancels any of its preclinical programs, consider it a strong sign that the company's health is deteriorating. Look at Editas's performance in the second and third quarters to see if they're on the right track for a buy early next year, but understand that waiting until next year to reevaluate the company's situation is probably the wisest path.

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Here's Why Editas Could Beat Intellia to a CRISPR Therapy - Motley Fool

SOUTH SAN FRANCISCO, Calif.--(BUSINESS WIRE)--Genentech, a member of the Roche Group (SIX: RO, ROG; OTCQX: RHHBY), today announced that the U.S. Food and Drug Administration (FDA) has approved Tecentriq (atezolizumab) as a first-line (initial) treatment for adults with metastatic non-small cell lung cancer (NSCLC) whose tumors have high PD-L1 expression (PD-L1 stained 50% of tumor cells [TC 50%] or PD-L1 stained tumor-infiltrating [IC] covering 10% of the tumor area [IC 10%]), as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations.

We are pleased to offer people with certain types of lung cancer a new chemotherapy-free option that can help prolong their lives and be administered on a flexible dosing schedule, including an option for once-a-month Tecentriq infusions, said Levi Garraway, M.D., Ph.D., chief medical officer and head of Global Product Development. Today marks the fifth approval of Tecentriq in lung cancer, as we remain committed to providing an effective and tailored treatment option for every person diagnosed with this disease.

This approval is based on an interim analysis from the Phase III IMpower110 study, which showed Tecentriq monotherapy improved overall survival (OS) by 7.1 months compared with chemotherapy (median OS=20.2 versus 13.1 months; hazard ratio [HR]=0.59, 95% CI: 0.400.89; p=0.0106) in people with high PD-L1 expression (TC3/IC3-wild-type [WT]). Safety for Tecentriq appeared to be consistent with its known safety profile, and no new safety signals were identified. Grade 34 treatment-related adverse events (AEs) were reported in 12.9% of people receiving Tecentriq compared with 44.1% of people receiving chemotherapy.

Tecentriq is the first and only single-agent cancer immunotherapy with three dosing options, allowing administration every two, three or four weeks. The supplemental Biologics License Application for the Tecentriq monotherapy was granted Priority Review, a designation given to medicines the FDA has determined to have the potential to provide significant improvements in the treatment, prevention or diagnosis of a disease.

In the U.S., Tecentriq has received four approvals across NSCLC, including as a single agent or in combination with targeted therapies and/or chemotherapies. It is also approved in combination with carboplatin and etoposide (chemotherapy) for the first-line treatment of adults with extensive-stage small cell lung cancer.

Genentech has an extensive development program for Tecentriq, including multiple ongoing and planned Phase III studies across lung, genitourinary, skin, breast, gastrointestinal, gynecological and head and neck cancers. This includes studies evaluating Tecentriq both alone and in combination with other medicines.

About the IMpower110 study

IMpower110 is a Phase III, randomized, open-label study evaluating the efficacy and safety of Tecentriq monotherapy compared with cisplatin or carboplatin and pemetrexed or gemcitabine (chemotherapy) in PD-L1-selected, chemotherapy-nave participants with stage IV non-squamous or squamous NSCLC. The study enrolled 572 people, of whom 554 were in the intention-to-treat WT population, which excluded people with EGFR or ALK genomic tumor aberrations, and were randomized 1:1 to receive:

The primary efficacy endpoint was OS by PD-L1 subgroup (TC3/IC3-WT; TC2/3/IC2/3-WT; and TC1,2,3/IC1,2,3-WT), as determined by the SP142 assay test. Key secondary endpoints included investigator-assessed progression-free survival (PFS), objective response rate (ORR) and duration of response (DoR).

About lung cancer

According to the American Cancer Society, it is estimated that more than 228,000 Americans will be diagnosed with lung cancer in 2020, and NSCLC accounts for 80-85% of all lung cancers. It is estimated that approximately 85% of lung cancer diagnoses in the United States are made when the disease is in the advanced stages.

About Tecentriq (atezolizumab)

Tecentriq is a monoclonal antibody designed to bind with a protein called PD-L1. Tecentriq is designed to bind to PD-L1 expressed on tumor cells and tumor-infiltrating immune cells, blocking its interactions with both PD-1 and B7.1 receptors. By inhibiting PD-L1, Tecentriq may enable the re-activation of T cells. Tecentriq may also affect normal cells.

Tecentriq U.S. Indications

Tecentriq is a prescription medicine used to treat adults with:

A type of lung cancer called non-small cell lung cancer (NSCLC).

A type of lung cancer called small cell lung cancer (SCLC).

It is not known if Tecentriq is safe and effective in children.

Important Safety Information

What is the most important information about Tecentriq?

Tecentriq can cause the immune system to attack normal organs and tissues and can affect the way they work. These problems can sometimes become serious or life threatening and can lead to death.

Patients should call or see their healthcare provider right away if they get any symptoms of the following problems or these symptoms get worse.

Tecentriq can cause serious side effects, including:

Getting medical treatment right away may help keep these problems from becoming more serious. A healthcare provider may treat patients with corticosteroid or hormone replacement medicines. A healthcare provider may delay or completely stop treatment with Tecentriq if patients have severe side effects.

Before receiving Tecentriq, patients should tell their healthcare provider about all of their medical conditions, including if they:

Patients should tell their healthcare provider about all the medicines they take, including prescription and over-the-counter medicines, vitamins, and herbal supplements.

The most common side effects of Tecentriq when used alone include:

The most common side effects of Tecentriq when used in lung cancer with other anti-cancer medicines include:

Tecentriq may cause fertility problems in females, which may affect the ability to have children. Patients should talk to their healthcare provider if they have concerns about fertility.

These are not all the possible side effects of Tecentriq. Patients should ask their healthcare provider or pharmacist for more information. Patients should call their doctor for medical advice about side effects.

Report side effects to the FDA at 1-800-FDA-1088 or http://www.fda.gov/medwatch.

Report side effects to Genentech at 1-888-835-2555.

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A girl in New Delhi gets a nasal swab to test for the new coronavirus. A rare Kawasaki diseaselike illness linked to the virus is sickening young people.

By Jennifer Couzin-FrankelMay. 21, 2020 , 4:10 PM

Sciences COVID-19 reporting is supported by the Pulitzer Center.

Three children at one London hospital in mid-April, followed the next day by three at anotherfor Elizabeth Whittaker, a pediatric infectious disease doctor at Imperial College London, those first cases raised an alarm. The youngsters had fevers, rashes, stomach pain, and, in some cases, heart problems, along with blood markers that characterize COVID-19 in adults, including one associated with clotting. But in most, nasal swabs failed to reveal any virus.

I dont understandthey look like they have coronavirus, Whittaker recalls thinking. Doctors nonetheless suspected a link. Within days, a survey turned up 19 additional cases across England, and an alert on 27 April asked doctors to be on the lookout for such symptoms in children. Soon after, dozens more cases surfaced in New York along with smaller clusters elsewhere, bolstering a connection to the pandemic. Reports of children on life support and some deaths put parents on edgeand were especially disheartening after earlier signs that COVID-19 largely spares children from serious illness.

It is another surprise from a virus that hasproffered many, and projects worldwide are gearing up to study it. They are combing the blood and sequencing the genomes of patientsand the virus, if it can be isolated from themto search for clues to what makes some children susceptible and how to head off the worst symptoms. Theres hope that whats learned from young patients might help the many adults in whom COVID-19 also triggers a grievous overreaction of the immune system.

In some respects, Its absolutely not shocking to see this, says Rae Yeung, a rheumatologist and immunologist at the Hospital for Sick Children in Toronto, whose center treated 20 children over the past 3 weeks with similar symptoms.Many pathogens occasionally trigger a similar hyperactive immune response in children, known as Kawasaki disease. Its symptoms vary but include rash, fever, and inflammation in medium-size blood vessels. Children can suffer heart problems. In rare cases, blood pressure plummets and shock sets in.

Doctors disagree on whether the variant linked to COVID-19 is Kawasaki disease or something new, with some experts calling it multisystem inflammatory syndrome in children. But as with Kawasaki disease, most recover with treatment, including steroids and immunoglobulins, which calm the immune system.

In linking the inflammatory syndrome to COVID-19,Were going on more than just a hunch, says Jesse Papenburg, a pediatric infectious disease specialist at Montreal Childrens Hospital, in a city thats seen about 25 children with the condition. Kawasaki disease is rare, ordinarily affecting just one to three in every 10,000 children in Western countries, though its more common in children with Asian ancestry. The spikes recorded so far, in COVID-19 hot spots like northern Italy and New York City, track the novel coronavirus march around the world. And although a minority of these children test positive for SARS-CoV-2, a studypublished inThe Lancetby a team in Bergamo, Italy, reported that eight of 10 children with the Kawasaki-like illness had antibodies to the virus, indicating they had been infected. Positive antibody tests have been reported in sick children elsewhere, too.

It was obvious that there was a link, says Lorenzo DAntiga, a pediatrician at the Papa Giovanni XXIII Hospital who led the study. The new coronavirus can elicit a powerful immune response, which he thinks may explain why shock and a massive immune reaction called a cytokine storm are more common in the COVID-19linked cases than in textbook Kawasaki disease. And a time lag between infection and the Kawasaki-like illness could explain why many of the affected children show no evidence of the virus. The immune systems overreaction may unfold over weeks, though virus could also be hiding somewhere in the body.

Theres clearly some underlying genetic component that puts a small number of children at risk, says Tom Maniatis, founding director of Columbia Universitys Precision Medicine Initiative. New York state is investigating 157 cases, and Maniatis is also CEO of the New York Genome Center, which is pursuing whole-genome sequencing of affected children and their parents, as well as sequencing the virus found in children, with family consent. Finding genes that heighten risk of the illness or of developing a severe case could point to better treatments or help identify children who may take a sudden turn for the worse.

Genetics may also help explain a puzzle: why the illness hasnt been reported in Asian countries, even though Kawasaki disease is far more common in children with Asian ancestry. The virus own genetics may be important; an analysis last month indicatedthe predominant viral variant in New York was brought by travelers from Europe. Its also possible that the Kawasaki-like illness is so rare that it only shows up in COVID-19 hotbeds. The areas that have been hardest hit by coronavirus are the areas reporting this syndrome now, says Alan Schroeder, a critical care physician at Lucile Packard Childrens Hospital at Stanford University, which has seen one potentially affected child, a6-month-old baby, who healed quickly.

Yeung is pursuing ways to flag children with COVID-19 who are at risk of this complication. She co-leads an international consortium thats banking blood from affected children both before and after treatment and screening for various markers, including the cytokine molecules that indicate a revved-up immune system. They are also searching for gene variants known to predict poor outcomes in Kawasaki disease. Theres also core COVID stuff that needs to be measured, Yeung says, such as markers of heart function and levels of D-dimer, a protein fragment in the blood that indicates a tendency toward clotting and that surges in many sick adults.

Another project, called DIAMONDSand originally designed to improve diagnostics of pathogens based on patterns of immune response in children with fevers,is recruiting children across Europe with the Kawasaki-like complication, along with those who have run of the mill COVID-19 symptoms. Scientists will study blood for pathogensnot just SARS-CoV-2and the behavior of immune cells such as T cells and B cells.

We have to do a deep dive into the immunology of those patients, says Elie Haddad, a pediatric immunologist and scientist at the St. Justine University Hospital Center in Montreal who,with Yeung and Susanne Benseler at Alberta Childrens Hospital, is leading Canadian research efforts on the new syndrome. These deep dives may also clarify the immune system chaos seen in many sick adults. Children are cleaner, Haddad points outtheyre less likely to have other health burdens, such as diabetes or high blood pressure, that can make it harder to tease out the virus impact on the immune system.

Its possible, too, that the illness affects adults as well but is harder to tease out from their other symptoms. A global effort studying COVID-19 in adults, called the International Severe Acute Respiratory and Emerging Infection Consortium, will look at adults clinical data and blood samples,Whittaker says, to see, is this a uniquely pediatric problem?

Eager as they are to understand this new face of the pandemic, doctors want to avoid overstating the hazards. We need to identify early and we need to intervene early in treating these children, Yeung says. But she also urges calm. The kids were seeing so far, she stresses, they respond to the treatments were giving.

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Doctors race to understand rare inflammatory condition associated with coronavirus in young people - Science Magazine

Abstract

In mammals, the timing of meiosis entry is regulated by signals from the gonadal environment. All-trans retinoic acid (ATRA) signaling is considered the key pathway that promotes Stra8 (stimulated by retinoic acid 8) expression and, in turn, meiosis entry. This model, however, is debated because it is based on analyzing the effects of exogenous ATRA on ex vivo gonadal cultures, which not accurately reflects the role of endogenous ATRA. Aldh1a1 and Aldh1a2, two retinaldehyde dehydrogenases synthesizing ATRA, are expressed in the mouse ovaries when meiosis initiates. Contrary to the present view, here, we demonstrate that ATRA-responsive cells are scarce in the ovary. Using three distinct gene deletion models for Aldh1a1;Aldh1a2;Aldh1a3, we show that Stra8 expression is independent of ATRA production by ALDH1A proteins and that germ cells progress through meiosis. Together, these data demonstrate that ATRA signaling is dispensable for instructing meiosis initiation in female germ cells.

Germ cells exhibit the unique capacity to generate haploid gametes, eventually giving rise to an embryo after fertilization. In mice, primordial germ cells (PGCs) are specified in the epiblast around 6.25 days post-coitum (dpc) and colonize the gonads at around 10.5 dpc (1). At this stage, the gonads start differentiating as testes in XY embryos, or as ovaries in XX embryos (2). In parallel, germ cells loose pluripotency, becoming either prospermatogonia in testes or oogonia in ovaries, both of which further progress into meiotic divisions (3). However, the timing of the initiation of meiosis is sexually dimorphic (4), starting around 8 days postpartum in the testis versus 13.5 dpc in the ovary.

The nature of the signal(s) instructing oogonia to transition from mitosis to meiosis is still debated. Notably, Stra8 (stimulated by retinoic acid 8) is the only gatekeeper currently described that engages the meiotic program. This is evidenced by the failure of premeiotic DNA replication in female Stra8-deficient gonads (5). As Stra8 was originally identified as an all-trans retinoic acid (ATRA)responsive gene in P19 embryonic carcinoma cells (6), ATRA signaling has been proposed as a primary meiosis-instructing factor. This concept is supported by the up-regulation of meiotic markers including Stra8 in embryonic ovaries cultured ex vivo in the presence of ATRA and ATRA receptor (RAR) agonists, or the down-regulation of Stra8 using pan-RAR antagonists (7, 8). Nevertheless, these findings have been brought into question by experiments that demonstrated Stra8 expression and meiosis initiation in embryonic ovaries lacking two of the three ATRA-synthesizing enzymes (ALDH1A2 and ALDH1A3, encoded by the Aldh1a2 and Aldh1a3 genes) (9). Along the same lines, genetic ablation of Aldh1a1 alone does not impair meiosis, although it reduces Stra8 expression and delays meiosis initiation (10). In both mouse models, the remaining Aldh1a isotype(s) that is (are) remaining may, however, be sufficient to produce ATRA and, as a result, induce meiosis.

To clarify the contribution of endogenous ATRA in vivo, we have generated mice deficient for all three Aldh1a isotypes either in the somatic cells of the embryonic ovary or ubiquitously. Using this approach, we have robustly decreased ATRA signaling during PGC colonization of the developing gonad. Detailed analysis of the mutant phenotypes revealed that ALDH1A1, ALDH1A2, and ALDH1A3 are dispensable for meiotic initiation in oogonia.

It has been shown that two potential sources of ATRA coexist in the female urogenital ridges. First, the mesonephros, a transient organ adjacent to the gonad, exhibits both Aldh1a2 mRNA expression at 10.5 and 12.5 dpc and a strong ATRA responsiveness according to the Tg(RARE-Hspa1b/lacZ)12Jrt transgenic reporter (7, 11). Second, ALDH1A1 and ALDH1A2 expression has been detected in the somatic cells within the developing ovary at 12.5 and 13.5 dpc (10, 12). These findings suggest the existence of both external and endogenous sources of ATRA in the female gonad at the onset of meiosis initiation.

Single-cell RNA sequencing and immune-localization analyses (Fig. 1, A and B) revealed that Aldh1a1 expression became readily detectable in the supporting cells of the ovary at the time of meiosis entry (~13.5 dpc), as previously reported (10, 12). Aldh1a2 exhibited robust expression in the somatic progenitor cells of the gonad and in the mesonephros from 10.5 to 13.5 dpc. At 13.5 dpc, Aldh1a2 was also highly expressed in the supporting cells. Aldh1a3 mRNA was nearly absent in the ovary as evidenced by transcriptomic analysis (Fig. 1A). All these observations indicate that the somatic cells of the ovary, but not the germ cells, are able to synthesize endogenous ATRA as early as 10.5 dpc, i.e., 3 days before meiosis entry.

(A) Violin plots showing the expression of Aldh1a1, Aldh1a2, and Aldh1a3 in the progenitor and supporting cells between 10.5 to 16.5 dpc and postnatal day 6 (E10.5 to E16.5 and P6) determined by single-cell RNA sequencing analysis of Sf1-positive somatic cells from female gonads. Expression values are log-transformed reads per kilobase of transcript per million mapped reads (RPKM); small points represent expression in individual cells, and the white point is the median of expression. Statistical analyses were performed using the Wilcoxon-Mann-Whitney test, and P value was adjusted for false discovery rate. (B) Immunodetection of ALDH1A1 or ALDH1A2 (green) and POU5F1 or TRA98 (germ cells, red) in 10.5, 11.5, and 13.5 dpc ovaries. DAPI (blue), nuclei. Scale bars (white), 50 m. Arrowheads highlight examples of ALDH1A-positive cells. **P <0.01 and ***P <0.001 (adjusted p-values, Wilcoxon-Mann-Whitney test).

Using an ATRA-reporter mouse model carrying the Tg(RARE-Hspa1b/lacZ)12Jrt transgene (11), the mesonephros exhibits a strong response to endogenous ATRA, whereas the ATRA responsiveness is relatively weak in gonads (7, 10), suggesting that only very few cells are responsive to endogenous ATRA in the ovaries. To investigate the nature and fate of these ATRA-responsive cells, we used a novel transgenic reporter mouse line called Tg(RARE-Hspa1b-cre/ERT2), in which RA response elements (RAREs) coupled to the Hspa1b minimal promoter drive expression of the tamoxifen (TAM)inducible CreERT2 recombinase, thus allowing cell lineage tracing of ATRA-responsive cells. In this model, the expression of the membrane-tagged green fluorescent protein (GFP) from the Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo reporter (13) is activated in cells that are ATRA responsive at the time of TAM administration. These cells then become permanently labeled by GFP (fig. S1A). As expected from previous studies (11, 14), the periocular mesenchyme and the developing heart, two tissues that are sensitive to endogenous ATRA, harbored GFP-positive cells ~24 hours after TAM administration (fig. S1B). We then asked whether germ cells in vivo were sensitive to endogenous ATRA. To this aim, TAM was administered to the pregnant females and the gonads from the embryos were collected ~24 hours later for GFP labeling analyses (Fig. 2A). Although a small number of somatic (POU5F1-negative) cells were responsive to endogenous ATRA at 9.5 dpc (evidenced as GFP-positive cells at 10.5 dpc), only a few germ cells were ATRA responsive between 12.5 and 13.5 dpc (because they were GFP positive at 13.5 dpc) (Fig. 2A). Confocal microscopy on whole-mount organs further revealed that the vast majority of germ cells were not ATRA responsive between 13.5 and 14.5 dpc because they were GFP negative at 14.5 dpc (Fig. 2B), although there were a few exceptions (arrowheads in Fig. 2B). Thus, less than 5% of germ cells were ATRA responsive as previously reported (7). We next dissected ovaries at 13.5 dpc and cultured them for 24 hours in the presence of 4-hydroxy-TAM (4-OH-TAM) at either a physiological dose of ATRA (1 nM) (9) or a pharmacological dose of ATRA (100 nM). Dimethyl sulfoxide (DMSO) was used as a negative control (Fig. 2C). Immunostaining analysis using anti-GFP and anti-TRA98 (a germ cell marker) antibodies demonstrated that germ cells from the ex vivo transgenic ovaries lacked GFP-positive germ cells in DMSO-treated samples but were able to respond to a very low concentration of exogenous ATRA (many GFP-positive germ cells in 1 nM ATRAtreated samples). As expected, cells of the mesonephros also responded to ATRA treatment (7). Together, these results indicate that the vast majority of germ cells failed to activate ATRA-responsive genes in vivo, ultimately questioning the requirement of ATRA for meiosis entry.

(A) Immunodetection of ATRA-responsive cells (GFP positive, green) and germ cells (POU5F1- or TRA98-positive cells, red) in 10.5, 11.5, and 13.5 dpc Tg(RARE-Hspa1b-cre/ERT2) ovarian sections after TAM induction at 9.5, 10.5, and 12.5 dpc, respectively. DAPI (blue), nuclei. Scale bars (white), 50 m. (B) Immunodetection of ATRA-responsive cells (GFP, green) and TRA98 (germ cells, red) in 14.5 dpc Tg(RARE-Hspa1b-cre/ERT2) whole ovaries after TAM induction at 13.5 dpc. Scale bars (white), 50 m. White arrowheads, GFP-positive germ cells. (C) Immunodetection of TRA98 (germ cells) (red) and GFP (ATRA-responsive cells) (green) in the presence of either DMSO (control) or 1 or 100 nM ATRA in cultured Tg(RARE-Hspa1b-cre/ERT2) ovaries from 13.5 to 14.5 dpc. Asterisk, GFP-positive cells within the mesonephros.

To functionally test the contribution of ATRA signaling to meiosis entry, we performed conditional deletion of all three ATRA-producing enzymes (Aldh1a1;Aldh1a2;Aldh1a3, hereafter referred to as Aldh1a1-3) using three distinct Cre driver lines (fig. S2, A and B). First, we used the Tg(Nr5a1-cre)2Klp transgenic line (hereafter named Sf1-cre) to induce deletion in SF1-positive somatic cells in the gonads from 11.5 dpc (15). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis in Sf1-Cre;Aldh1a1-3flox/flox embryos at 13.5 dpc demonstrated the efficient loss of mRNA expression for all three Aldh1a1-3 genes (Fig. 3A). Second, to induce an earlier deletion than the Sf1-Cre, we used the Wt1tm2(cre/ERT2)Wtp strain (16) (hereafter referred to Wt1-CreERT2) that expresses a TAM-inducible CreERT2 recombinase in most, if not all, somatic cells of the gonads at 10.5 dpc (17). TAM administration at 10.5 and 11.5 dpc triggered a strong reduction in Aldh1a1, Aldh1a2, and Aldh1a3 mRNA levels at 13.5 dpc (Fig. 3B). The highly efficient ablation of ALDH1A1 and ALDH1A2 was further confirmed by immunodetection at 13.5 dpc (Fig. 3D). Next, we used the Tg(CAG-cre/ERT2)#Rlb line (18) (hereafter referred to as CAGG-CreER), which ubiquitously expresses a TAM-inducible CreERT2 to delete Aldh1a1-3 in all cell types. In TAM-treated CAGG-CreER;Aldh1a1-3flox/flox ovaries, Aldh1a1, Aldh1a2, and Aldh1a3 mRNA levels were significantly reduced in the gonads, as previously reported (19). This was accompanied by a significant decrease of ALDH1A1 and ALDH1A2 protein levels at 13.5 dpc, resulting in severe heart malformations (Fig. 3E and fig. S2C). To summarize, all three mouse models demonstrated efficient elimination of Aldh1a1, Aldh1a2, and Aldh1a3 expression in the embryonic ovaries.

(A) RT-qPCR analysis of Aldh1a1, Aldh1a2, and Aldh1a3 expression in 13.5 dpc control (orange) and Sf1-Cre;Aldh1a1-3flox/flox (blue) gonads. Students t test, unpaired. Bars represent mean + SEM; n = 10 individual gonads. ***P < 0.001. (B) Top: Protocol of induction of Aldh1a1-3 deletion (10.5 dpc onward). Bottom: RT-qPCR analysis of Aldh1a1, Aldh1a2, and Aldh1a3 expression in 13.5 dpc control (orange) and Wt1-CreERT2;Aldh1a1-3flox/flox (blue) gonads. Students t test, unpaired. Bars represent mean + SEM; n = 10 individual gonads. ***P < 0.001. (C) Top: Protocol of induction of Aldh1a1-3 deletion (10.5 dpc onward). Bottom: RT-qPCR analysis of Aldh1a1, Aldh1a2, and Aldh1a3 expression in 13.5 dpc control (orange) and CAGG-CreER;Aldh1a1-3 flox/flox (blue) gonads. However, the deletion was less efficient than using the Wt1-CreERT2 or Sf1-Cre transgenes. Students t test, unpaired. Bars represent mean + SEM; n = 15 individual gonads. ***P < 0.001. (D) Immunodetection of ALDH1A1 or ALDH1A2 (green) and POU5F1 (germ cells, red) in 13.5 dpc control and Wt1-CreERT2;Aldh1a1-3flox/flox ovaries. DAPI (blue), nuclei. Scale bars (white), 50 m. (E) Immunodetection of ALDH1A1 or ALDH1A2 (green) and CDH1 (germ cells, red) in 13.5 dpc control and CGAG-CreERT2;Aldh1a1-3 flox/flox ovaries. DAPI (blue), nuclei. Scale bars (white), 50 m.

To assess the impact of Aldh1a1-3 deletion on ATRA synthesis, we performed metabolomic investigations and evaluated endogenous production of ATRA in mesonephroi, control testes, control ovaries, or Wt1-CreERT2;Aldh1a1-3flox/flox ovaries from 13.5 dpc embryos (fig. S3A). RA can be isomerized in ATRA, the most abundant form, in 9-cis-RA (9cRA), which is almost undetectable in vivo, and in 13-cis-RA (13cRA), which is mostly present in the serum (20). RA isomers have different affinities for nuclear receptors and therefore exert different biological activities: ATRA has the highest affinity for RA receptors (RAR) and 9cRA can bind RAR/RXR. In contrast, 13cRA has a 100-fold lower affinity to RARs than the two others (21). Using single-ion mass spectrometry, we were able to detect all three RA isomers in vivo, even 9cRA isomer, which shows the high sensitivity of this method, and to quantify the relative abundance of ATRA isomer (fig. S3A). The ATRA level was abundant in mesonephroi as previously described (7) but was strongly decreased in Wt1-CreERT2;Aldh1a1-3flox/flox ovaries compared to control ovaries. In addition, this level was similar to the level detected in testes (fig. S3, B and C).

To determine whether this level of ATRA was sufficient to trigger a biological activity in the mutant ovaries, we designed an experimental ATRA-reporter assay by transfecting Chinese hamster ovary (CHO) cells with a plasmid containing the ATRA-responsive hsp68 mouse promoter (11) controlling the expression of the acGFP1 gene encoding the Aequorea coerulescens GFP. We cultured the transfected CHO cells in the absence or presence of increasing ATRA concentrations, or in the presence of cellular suspensions from either mesonephroi, control, or Wt1-CreERT2;Aldh1a1-3flox/flox ovaries dissected from 13.5 dpc embryos (Fig. 4A). Although GFP expression (quantified by Western blot) was stimulated by commercial ATRA from 1 nM onward, mesonephroi, or control ovaries, the GFP level of expression induced by mutant ovaries barely reached the residual level of GFP measured in cultures with medium depleted in RA, confirming that the genetic deletion of Aldh1a1-3 was efficient enough to inhibit ATRA synthesis (Fig. 4B).

(A) CHO cells transfected with the ATRA-reporter construct and incubated with either DMSO (vehicle), increasing concentrations of ATRA from 1 to 100 nM, cellular suspensions of mesonephroi, Aldh1a1-3 flox/flox, or Wt1-CreERT2;Aldh1a1-3flox/flox ovaries dissected from 13.5 dpc embryos (top, brightfield picture; bottom, GFP detection). (B) Immunodetection by Western blot of GFP and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in the same CHO cells transfected (+) or not () with the ATRA-reporter construct (RARE-acGFP1 plasmid) after protein extraction (upper), and corresponding quantification of GFP band intensity after normalization with GAPDH expression (lower). (C) RT-qPCR analysis of RAR, Cyp26a1, Crabp1, and Crabp2 expression in 13.5 dpc control (orange), Wt1-CreERT2;Aldh1a1-3flox/flox (blue) gonads, or whole 10.5 dpc embryos (positive control, gray). Students t test, unpaired. Bars represent mean + SEM; n = 10 individual gonads. ***P < 0.001.

We next analyzed the expression of RAR, Cyp26a1, and Crabp1-2, four ATRA-target genes used to monitor the inhibition of RA signaling (9) in control and Wt1-CreERT2;Aldh1a1-3flox/flox ovaries at 13.5 dpc. Although RAR, Crabp1, and Crabp2 mRNA levels were significantly down-regulated in the Wt1-CreERT2;Aldh1a1-3flox/flox ovaries compared to the controls, we did not detect any Cyp26a1 expression, neither in the control nor in the mutant ovaries (Fig. 4C), demonstrating that RA signaling was impaired in Wt1-CreERT2;Aldh1a1-3flox/flox ovaries. Together, these results indicate that the very low level of ATRA remaining in Wt1-CreERT2;Aldh1a1-3flox/flox ovaries was not sufficient to promote the expression of a RARE reporter and of universal target genes.

Because ATRA signaling has been reported to regulate germ cell proliferation in the developing ovary (22), we next examined the proliferation of POU5F1- or DDX4-positive germ cells at 11.5 and 12.5 dpc in control and Wt1-CreERT2;Aldh1a1-3flox/flox ovaries treated by TAM at 9.5 and 10.5 dpc. A 3-hour pulse of 5-bromo-2-deoxyuridine (BrdU) incorporation permanently labeled the cells transitioning through the S phase of the cell cycle. Quantification of the number of BrdU-positive germ cells indicated that despite the significant loss of Aldh1a1, Aldh1a2, and Aldh1a3 expression upon TAM treatment (Fig. 5A), proliferation was not affected in Wt1-CreERT2;Aldh1a1-3flox/flox gonads (Fig. 5B). Moreover, the pluripotency markers Pou5f1 (also known as Oct3/4), Sox2, and Dazl, driving oogonia differentiation (23), were expressed at similar levels in control and Aldh1a1-3deficient gonads at 11.5 and 12.5 dpc (fig. S4). Together, these results indicate that the genetic ablation of ALDH1A1, ALDH1A2, and ALDH1A3 does not hinder the proliferation or differentiation of PGCs.

(A) Protocol of induction of Aldh1a1-3 deletion (9.5 dpc onward). RT-qPCR analysis of Aldh1a1, Aldh1a2, and Aldh1a3 expression in 11.5 dpc control (orange) and Wt1-CreERT2;Aldh1a1-3flox/flox (blue) ovaries. Students t test, unpaired. Bars represent mean + SEM; n = 10 individual gonads. ***P < 0.001. (B) Immunodetection of BrdU (proliferating cells, green), POU5F1 (PGCs, red), and GATA4 (gonadal somatic cells, cyan) at 11.5 dpc in control (left) and Wt1-CreERT2;Aldh1a1-3flox/flox (right) ovaries. Histograms: Percentage of BrdU-positive (proliferating) versus POU5F1-positive (total) germ cells in control (orange) and Wt1-CreERT2;Aldh1a1-3flox/flox (mutant, blue) ovaries at 11.5 dpc. Students t test, unpaired. Bars represent mean + SEM; n = 10 sections of each genotype (four to seven ovaries per genotype). ns, not significant. Bottom: Immunodetection of BrdU (proliferating cells, green) and DDX4 (germ cells, red) at 12.5 dpc in control (left) and Wt1-CreERT2;Aldh1a1-3flox/flox (right) ovaries. Histograms: Percentage of BrdU-positive (proliferating) versus DDX4-positive (total) germ cells in control (orange) and Wt1-CreERT2;Aldh1a1-3flox/flox (mutant, blue) ovaries at 12.5 dpc. Students t test, unpaired. Bars represent mean + SEM; n = 10 sections of each genotype (four to seven ovaries per genotype). Arrowheads highlight examples of BrdU-positive germ cells.

We next assessed whether germ cells initiated meiosis after genetic deletion of Aldh1a1-3 using the different mouse models described above. In Sf1-Cre;Aldh1a1-3flox/flox embryos, Stra8 expression was reduced by ~20% at 13.5 dpc (fig. S5A). Stra8 expression was also reduced at 13.5 dpc in ovaries of Wt1-CreERT2;Aldh1a1-3flox/flox embryos treated by TAM at 9.5 and 10.5 dpc (fig. S5B). Nevertheless, under these conditions, TAM treatment induced frequent embryonic lethality. Accordingly, TAM was administrated at 10.5 and 11.5 dpc in the following experiments, leading to an almost identical decrease of Aldh1a1 and Aldh1a2 mRNA expression (fig. S6). Upon this TAM treatment, germ cells in Wt1-CreERT2;Aldh1a1-3flox/flox ovaries expressed Stra8 at 13.5 dpc, although its mRNA levels were reduced by ~25% when compared to controls (Fig. 6, A and C). At 14.5 dpc, this deficit was partly compensated in the Wt1-CreERT2;Aldh1a1-3flox/flox ovaries, suggesting a delay in the onset of Stra8 expression in the absence of ALDH1A isotypes (Fig. 6B). Notably, the reduction in Stra8 expression did not impair meiosis entry, as evidenced by the normal levels of Rec8 mRNA, a gene that encodes a component of the cohesin complex accumulating during the meiotic S phase (Fig. 6B), and of Spo11 mRNA, which is expressed during the leptotene stage of meiotic prophase I (Fig. 6A). Together, these results demonstrate that genetic deletion of the three Aldh1a1-3 genes does not impair meiosis entry.

(A) RT-qPCR analysis of Stra8, Rec8, and Spo11 expression in 13.5 dpc control (orange) and Wt1-CreERT2;Aldh1a1-3flox/flox (blue) ovaries. Students t test, unpaired. Bars represent mean + SEM; n = 10 individual ovaries. **P < 0.01. (B) RT-qPCR analysis of Pou5f1, Dazl, Stra8, and Rec8 expression in 14.5 dpc control (orange) and Wt1-CreERT2;Aldh1a1-3flox/flox (blue) ovaries. Students t test, unpaired. Bars represent mean + SEM; n = 10 individual gonads. *P < 0.05. (C) In situ hybridization using Stra8 riboprobe at 13.5 dpc in control (left) and Wt1-CreERT2;Aldh1a1-3flox/flox (right) ovaries. Scale bars (white), 50 m. (D) Immunodetection of DAZL (red) and SYCP3 (green) in 16.5 dpc control and Wt1-CreERT2;Aldh1a1-3flox/flox ovaries. Bottom: Immunodetection of phosphohistone H2AX (red) and DDX4 (green) in 16.5 dpc control and mutant ovaries. DAPI (blue), nuclei. Scale bars (white), 50 m.

We next examined whether meiosis further progressed despite Aldh1a1-3 ablation by looking at specific chromosomal features of meiosis. During meiotic progression, homologous chromosomes pair and the synaptonemal complex promotes chromosome recombination. Immunodetection of the synaptonemal complex protein 3 (SYCP3), which appears in leptotene stage and becomes enriched in the zygotene stage, revealed similar thread-like synaptonemal complex structures in TAM-treated control and Wt1-CreERT2;Aldh1a1-3flox/flox ovaries (Fig. 6D), further demonstrating that germ cells entered meiosis in the absence of all ALDH1A isotypes. Quantification of the germ cells positive for phospho-H2AX, which is associated with DNA double-strand breaks occurring during the leptotene stage, confirmed these observations (Fig. 6D and fig. S7). Hence, these results demonstrate that female germ cells progress to meiotic prophase even when Aldh1a1-3 are deleted from the ovary from 10.5 dpc onward.

In TAM-treated CAGG-CreER;Aldh1a1-3flox/flox ovaries, the mRNA levels of meiotic markers such as Mei1, Dmc1, Rec8, Syce1, and Spo11 were not significantly changed as evidenced by RT-qPCR and in situ hybridization experiments (fig. S8, A and B). Accordingly, phospho-H2AX expression was comparable between TAM-treated control and CAGG-CreER;Aldh1a1-3flox/flox ovaries at 16.5 dpc (fig. S8C). Together, these results indicate that genetic ablation of Aldh1a1-3 does not prevent meiosis entry in the developing mouse ovary.

Evidence that PGCs do not enter meiosis because of an intrinsic autonomous property (i.e., cell division timing) but are rather instructed by a signaling molecule produced by somatic ovarian cells led to a search for a meiosis-inducing substance (MIS) (24). Although ATRA has been suggested to be one such MIS, its role in this biological process has been questioned, giving rise to conflicting reports (79). In all three distinct models of Aldh1a1-3 genetic deletion we have used, we observed only a minor decrease in Stra8 expression, and this reduction was not robust enough to prevent meiosis initiation and germ cells from progressing into meiotic prophase I. Thus, we conclude that ATRA signaling does not initiate but contributes to meiosis by making Stra8 expression on time. Accordingly, ectopic expression of Cyp26a1, which has the most efficient catalytic activity on ATRA degradation, does not affect meiosis entry or Stra8 expression, although Stra8 mRNA levels were reduced by ~30%, i.e., in the same range than the mRNA reduction observed in the present study (25). This indicates that ATRA does regulate Stra8 transcription maintenance rather than Stra8 initiation of transcription. In agreement with our findings, the study from Vernet et al. (this issue) describes a similar delay in expression of Stra8 and a normal progression into meiosis in fetal ovaries of mice lacking the RARs RAR, RAR, and RAR. Because the fetal lethality in our different mice models prevented us to study the postnatal ovary, the fate of the ATRA-responsive female germ cells in vivo requires further investigations.

When identified, Stra8 was not classified in the class of immediate and early, ATRA-responsive gene, as indicated by the kinetics of its mRNA accumulation in P19 pluripotent carcinoma cells treated with ATRA (6). It was shown that ATRA regulates the phosphorylation status of the STRA8 protein in P19 cells (6), indicating that ATRA might also control STRA8 posttranslational modifications. The functional relevance of ATRA-induced phosphorylation in STRA8 activity or stability has not been characterized. Nevertheless, our results indicate that Aldh1a1-3 are not required for Stra8 expression in germ cells, suggesting that either ATRA does not phosphorylate STRA8 in vivo or STRA8 phosphorylation has no impact on its activity in germ cells. Together, our results, those from Vernet et al. and Bellutti et al. (25), indicate that ATRA signaling is dispensable for meiosis entry.

In PGC-like cells (PGCLCs) derived from embryonic stem cells, Stra8 expression is induced without ATRA treatment (26), further highlighting that ATRA is not instrumental for meiosis initiation in this system and that other signals are responsible for initiation of Stra8 expression. In vitro generation of female PGCLCs expressing Stra8, SYCP3, and other meiotic markers requires bone morphogenetic proteins (BMPs), which activate transforming growth factor (TGF)/SMAD signaling. In these experiments, ATRA increased the number of meiotic germ cells, indicating that ATRA signaling served to enhance a preexisting situation (26).

Meiosis initiation is timed by epigenetic factors such as polycomb repressive complex PRC1 that promotes structural modifications of chromatin and consequently times the expression of Stra8 (27). Recent data show that deficiency in vitamin C during gestation induces incomplete DNA demethylation of key germline genes and thus delays meiosis initiation in the embryos (28), indicating that molecules from the maternal nutrition participate in regulating meiotic gene expression in the germ cells of the progeny. In addition, in the absence of Rspo1, an activator of WNT/-catenin signaling in the female fetal gonad, a proportion of PGCs neither expressed Stra8 nor entered meiosis (29), suggesting a control of Stra8 expression by WNT/-catenin. The Msx genes, which encode homeodomain transcription factors, are direct targets of WNT/-catenin signaling in murine embryonic stem cells (30), and in mutant embryos lacking both Msx1 and Msx2, germ cells failed to initiate meiosis (31). In gonadal cultures, BMP4 stimulates the expression of Msx1 and Msx2 that, in turn, directly regulate Stra8 expression (29), suggesting that TGF signaling is also involved in Stra8 regulation. However, in mouse germ cells, the central transducer Smad4 is dispensable for Stra8 expression, although it is required to up-regulate other key genes involved in meiosis (30). Last, the DMRT1 transcription factor also contributes to the switch from mitosis to meiosis by directly regulating Stra8 expression in female germ cells (32). Together, these studies indicate that convergent pathways other than ATRA signaling collaborate to regulate Stra8 expression and the transition from mitosis to meiosis cycle.

Early studies based on the observation that female germ cells cultured together with fetal testes were prevented from initiating meiosis (33, 34) led to the concept of a secreted masculinizing meiosis preventing substance (MPS) in the male gonad (35). The P450 enzyme CYP26B1, expressed in the supporting cells of the fetal testis but not the ovary, has been proposed to be MPS. Analysis of Cyp26b1-null mutant mice demonstrated that CYP26B1 activity prevented germ cells from entering into meiosis in male mice (36). Whereas CYP26B1 is able to degrade ATRA (37), the data from Kumar et al. and Bellutti et al. (25) suggest that CYP26B1 metabolizes a substrate other than ATRA to prevent meiosis initiation (9). So far, knowledge about the metabolic ligands of CYP26B1 is poor, and the nature and the molecular identity of CYP26B1 substrate(s) in the fetal testis remain unknown.

Identifying the molecules controlling the fundamental decision of germ cell to enter meiosis and defining whether they have MPS or MIS functions represents a major challenge for the reproductive medicine community in the upcoming years. The main clinical consequences of defects in germ cell development are infertility and increased susceptibility to germ cell tumors. Therefore, understanding how germ cells change their gene expression profiles in response to somatic signals will provide knowledge on the etiology of human genetic diseases.

The aim of this study was to investigate the role of endogenous ATRA signaling by using an in vivo genetic approach of deletion of Aldh1a1, Aldh1a2, and Aldh1a3 genes (i.e., mice lacking all sources of ATRA in vivo) to challenge the admitted concept of ATRA being the MIS. The number of samples was determined on the basis of experimental approach, availability, and feasibility required to obtain definitive results. No data were excluded from the analyses. The numbers of replicates are specified in Materials and Methods. The researchers were not blinded during data collection or analysis.

The experiments described here were carried out in compliance with the relevant institutional and European animal welfare laws, guidelines, and policies. All the experiments were approved by the French Ministre de lEducation Nationale, de lEnseignement Suprieur et de la Recherche (APAFIS # 3771-2016012110545580v13). All mice were kept on a 129/Sv-C57BL/6J mixed background. Mouse lines were obtained from the Jackson Laboratory. The Wt1tm2(cre/ERT2)Wtp, Tg(Nr5a1-cre)2Klp, Tg(CAG-cre/ERT2)#Rlb, Aldh1a1flox/flox, Aldh1a2flox/flox, Aldh1a3flox/flox, and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo mice were described previously and genotyped as reported (13, 15, 16, 38, 39, 40). Genotyping was performed using DNA extracted from tail tips or ear biopsies of mice. To activate the CreERT recombinase in embryos, TAM (catalog no. T5648, Sigma-Aldrich) was directly diluted in corn oil to a concentration of 40 mg/ml, and two TAM treatments (200 mg/kg body weight) were administrated to pregnant females by force-feeding at 9.5 and 10.5 dpc or 10.5 and 11.5 dpc. This resulted in embryos in which Aldh1a1-3 were deleted upon TAM induction when they were carrying the creERT transgene in contrast with their control littermates. For proliferation assays, BrdU (catalog no. B5002, Sigma-Aldrich) was diluted to a concentration of 10 mg/ml in sterile H2O and administrated to the pregnant females at a final concentration of 10 g/ml by intraperitoneal injection, and pregnant females and their embryos were sacrificed after 3 hours.

Single-cell RNA sequencing was performed as described in (41). Briefly, somatic cells of developing mouse female gonads were purified by fluorescence-activated cell sorting (FACS) using the Tg(Nr5a1-GFP) at six stages of development (10.5, 11.5, 12.5, 13.5, and 16.5 dpc and postnatal day 6). Single-cell isolation, reverse transcription, and complementary DNA (cDNA) amplification were performed using the Fluidigm C1 Auto Prep system, and single-cell sequencing library was prepared with Illumina Nextera XT following Fluidigm protocol. Library was multiplexed and sequenced on an Illumina HiSeq2000 platform with 100base pair (bp) paired-end reads at an average depth of 10 million reads per single cell. Obtained reads were mapped on the mouse reference genome (GRCm38.p3), and gene expression was quantified in RPKMs (reads per kilobase of exon per million reads mapped). Cells were clustered using principal components analysis and hierarchical clustering, and cell types were identified according to the expression level of marker genes and gene ontology enrichment tests. Significance of the difference in expression level between cell types was assessed in R version 3.6.0 using Wilcoxon-Mann-Whitney tests, and P values were adjusted for false discovery rate using Bonferroni.

Individual gonads without mesonephroi were dissected in phosphate-buffered saline (PBS) from 11.5, 12.5, 13.5, and 14.5 dpc embryos. RNA was extracted using the Qiagen RNeasy Kit and reverse-transcribed using the RNA RT-PCR Kit (Stratagene). Primers and probes were designed by the Roche Assay Design Center (https://www.rocheappliedscience.com/sis/rtpcr/upl/adc.jsp). All real-time PCR assays were carried out using the LightCycler FastStart DNA Master Kit (Roche) according to the manufacturers instructions. qPCR was performed on cDNA from one gonad and compared to a standard curve. They were repeated at least twice. Relative expression levels of each sample were quantified in the same run and normalized by measuring Sdha expression (which represents the total gonadal cells). For Aldh1a1, Aldh1a2, and Aldh1a3 real-time PCR, the following specific primers were used: 5-actttcccaccattgagtgc-3 and 5-caccatggatgcttcagaga-3 (Aldh1a1), 5-catggtatcctccgcaatg-3 and 5-gcgcatttaaggcattgtaac-3 (Aldh1a2), and 5-tctgggaatggcagagaact-3 and 5-ttgatggtgacggttttcac-3 (Aldh1a3). For each sample, relative expression levels were quantified and normalized. For each genotype (n = 10), the mean of these 10 absolute expression levels (i.e., normalized) was calculated and then divided by the mean of the 10 absolute expression levels of the control samples considered as the reference (=1 when divided by itself), leading to the fold of change.

For each genotype, the mean of the normalized expression levels was calculated, and graphs show mean fold change + SEM. All the data were analyzed by Students t test using Microsoft Excel. Asterisks highlight the pertinent comparisons and indicate levels of significance: *P < 0.05, **P < 0.01, and ***P < 0.001. Data are shown as mean + SEM.

For metabolomic analysis, 12 mesonephroi or 12 gonads of each genotype (Aldh1a1-3flox/flox ovaries, Aldh1a1-3flox/flox testes, Wt1-CreERT2;Aldh1a1-3flox/flox ovaries) from 13.5 dpc littermate embryos were dissected and subjected to methanol-chloroform extraction. The methanol phase was collected for mass spectrometry, whereas the interphase was used for quantification of protein concentration. Single ion monitoring analysis of RA was performed using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Compounds were loaded onto a Phenomenex Synergi 4 m Hydro-RP 80A 250 2 mm. RA isomers were separated with a 25-min gradient at a flow rate of 0.350 ml/min (mobile phase A water and 0.1% formic acid, mobile phase B acetonitrile and 0.1% formic acid gradient, 30% A for 5 min and 90% B in 15-min return initial condition for 5 min). The column was then thoroughly cleaned with 10 blank runs using a 2-l injection of 30% MeCN, 30% isopropanol, and 0.1% formic acid after each run. The mass spectrometry method was done in positive electrospray ionization mode, which increases the sensitivity for RA isomers. We used a targeted SIM (Selected Ion Monitoring) scan on the M+H: 301.216. This led to an improved signal-to-noise ratio and to lower detection limits. The method consisted of full scans and targeted SIM scan. Full scans were acquired with AGC (Automatic Gain Control) target value of 1 106, resolution of 70,000 full width at half maximum (FWHM) at 200 mass/charge ratio (m/z), and maximum ion injection time of 100 ms. The target was monitored with a 4-min window, AGC target value of 1 105, resolution of 280,000 FWHM at 200 m/z, and maximum ion injection time of 500 ms.

RA levels (all isomers) were quantified by normalizing the peak area given by the mass spectrometer by the protein concentration of each sample. ATRA-specific peak area was measured using ImageJ software and normalized with the protein concentration.

CHO cells were cultured in Dulbeccos minimum essential medium (DMEM):F12 medium (1:1) (Gibco) supplemented with recombinant human epidermal growth factor (EGF) (10 ng/ml; catalog no. PHG0314, Gibco), ITS (insulin-transferrin-selenium; 100; catalog no. 41400045, Gibco), and 100 nonessential amino acids (catalog no. 1140068, Gibco) in the absence of serum. Cells (15,000 per well) were seeded in a 24-well plate 24 hours before transfection. Then, CHO cells were transfected with 800 ng of plasmid described hereafter, using Lipofectamine 2000 reagent (catalog no. 11668019, Invitrogen) according to the manufacturers procedure. The ATRA-reporter construct was designed as follows: A DNA fragment encompassing the RA-responsive hsp68 mouse promoter as previously published (10), the rabbit -globin intron, the coding sequence of the acGFP1 gene (Aequorea coerulescens GFP), two STOP codons, and the SV40 late polyadenylation signal was synthesized by Sigma-Aldrich and cloned by the manufacturer in the kanamycin-resistant plasmid pUC57. Two copies in tandem of the 5HS4 insulator were then added by cloning in appropriate restriction sites at the 3 end of the polyadenylation signal. The sequence of the whole construct is available upon request. Twenty-four hours after transfection, CHO cells were incubated with either DMSO (vehicle), commercial ATRA (catalog no. R2625, Sigma-Aldrich) from 1 to 100 nM in DMSO, or cellular suspensions from mesonephroi, Aldh1a1-3flox/flox, or Wt1-CreERT2;Aldh1a1-3flox/flox ovaries from 13.5 dpc littermate embryos freshly dissected (n = 6 mesonephroi or gonads per experiment, experiment performed in two replicates). Twenty-four hours later, GFP endogenous signal was visualized with an Axio Imager Z1 microscope (Zeiss) coupled with an AxioCam MRm camera (Zeiss) and images were processed with AxioVision LE and ImageJ.

Cells were solubilized in 50-ml lysis buffer containing 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 0.2% NP-40, protease inhibitor (cOmplete; catalog no. 4693116001, Sigma-Aldrich), and phosphatase inhibitor cocktails (PhosSTOP; catalog no. 4906845001, Sigma-Aldrich). Proteins (25 mg per lane) were resolved on SDS polyacrylamide gel electrophoresis and transferred onto Immobilon-P membrane (Millipore). The membrane was saturated with PBS supplemented with 5% milk and 0.1% Tween 20 and incubated with the following antibodies: GFP (1:1000; catalog no. ab6673, Abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000; sc32233, Santa Cruz Biotechnology). Western blot chemiluminescence detection was performed using a Fusion FX7 Spectra imager (Vilber). Band intensity was quantified and normalized using ImageJ software.

Samples were dissected and fixed overnight with 4% (w/v) paraformaldehyde and then processed for paraffin embedding. Microtome sections of 7-m thickness were processed for in situ hybridization. Stra8 digoxigenin-labeled riboprobe was synthesized, and in situ hybridization analyses were performed as described in (29). Imaging was performed on an MZ9.5 microscope (Leica) coupled with a DHC490 camera (Leica) and Leica application suite V3.3.0 software and processed with Adobe Photoshop. For each genotype, n = 3 to 5 embryos.

Samples were fixed overnight with 4% (w/v) paraformaldehyde and then processed either for paraffin embedding or directly for whole-mount immunostaining. Microtome sections of 5-m thickness were processed for immunostaining. Immunofluorescence analyses were performed as described (29). The following dilutions of primary antibodies were used: ALDH1A1 (1:50; catalog no. 52492, Abcam), ALDH1A2 (1:500; catalog no. HPA010022, Sigma-Aldrich), CDH1 (1:100; catalog no. 610182, BD Transduction Laboratories), DAZL (1:200; catalog no. GTX89448, GeneTex), DDX4 (1:200; catalog no. 13840, Abcam), GATA4 (1:200; catalog no. 1237, Santa Cruz Biotechnology), GFP (1:750; catalog no. TP401, Torrey Pines Bio Labs), POU5F1 (1:250; catalog no. 611202, BD Transduction Laboratories), phospho-H2AX (1:300; catalog no. 16193, Millipore), SOX2 (1:200; catalog no. 97959, Abcam), SYCP3 (1:200; catalog no. 15093, Abcam), TRA98 (1:150; catalog no. 82527, Abcam), and FUT4 (SSEA1) (1:200; catalog no. 21702, Santa Cruz Biotechnology). Slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) diluted in the mounting medium at 10 g/ml (Vectashield, Vector Laboratories) to detect nuclei. Imaging was performed with a motorized Axio Imager Z1 microscope (Zeiss) coupled with an AxioCam MRm camera (Zeiss), and images were processed with AxioVision LE and ImageJ. ImageJ software was used for quantification of proliferating germ cells versus total germ cells. Whole-organ imaging was performed on an LSM 780 NLO inverted Axio Observer Z1 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a Plan Apo 10 dry NA (numerical aperture) 0.45 objective. In monophoton mode, images were acquired using an argon laser (488 nm) and DPSS (green and yellow diode-pumped solid state) (561 nm). The microscope z-drive was used for z acquisitions, and an automated xy stage was used for multiposition recording acquisitions (Mrzhuser, Wetzlar, Germany). For each genotype, n = 3 to 5 embryos.

Paraffin sections from each genotype were processed for immunohistological experiments with POU5F1, DDX4, or GATA4 antibody. Then, proliferation analysis was performed on the same sections by BrdU labeling, and detection was performed using an appropriate kit (catalog no. 11 296 736 001, Roche). Total germ cells and proliferating cells were quantified on the entire section using ImageJ software. For each picture, the number of BrdU-positive germ cells (proliferating) and the number of either OCT4- or DDX4-positive cells (total) were counted. Then, the percentage of BrdU-positive versus OCT4- or DDX4-positive germ cells was determined. For each genotype (n = 4 to 7; 15 pictures per genotype), the mean and mean + SEM of these percentages were calculated and reported on a graph after statistical analysis (for details, see the Statistical analysis section).

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We thank L. Turchi, F. Massa, S. Lachambre, and S. Rekima for their help. Funding: This work was supported by grants from the Agence Nationale de la Recherche (ANR-13-BSV2-0017-02 ARGONADS, ANR-11-LABX-0028-01, and ANR-18-CE14-0012 SexSpecs). M.L.R. was supported by a fellowship from La Ligue Nationale Contre le Cancer. A.S. was supported by a grant from La Ligue Nationale Contre le Cancer (Equipe Labellise Ligue Nationale Contre le Cancer). The microscopy was done in the Prism facility, Plateforme Prism, and the histological experiments were done at the Experimental Histopathology Platform, IBV-CNRS UMR 7277-INSERM U1091-UNS. Author contributions: A.-A.C., M.-C.C., and N.B.G. designed the study, analyzed the data, and wrote the paper. A.-A.C., M.L.R., I.S., G.J., J.-M.G., and F.D.S. performed the experiments and analyzed the data. E.P., S.N., and A.S. discussed the results and commented on the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Retinoic acid synthesis by ALDH1A proteins is dispensable for meiosis initiation in the mouse fetal ovary - Science Advances

Global Precision Medicine Marketto grow with a substantial CAGR in the forecast period of 2019-2026. Growing prevalence of cancer worldwide and accelerating demand of novel therapies to prevent of cancer related disorders are the key factors for lucrative growth of market

Key Market Players:

Few of the major competitors currently working in the global precision medicine market are Neon Therapeutics, Moderna, Inc, Merck & Co., Inc, Bayer AG, PERSONALIS INC, GENOCEA BIOSCIENCES, INC., F. Hoffmann-La Roche Ltd, CureVac AG, CELLDEX THERAPEUTICS, BIONTECH SE, Advaxis, Inc, GlaxoSmithKline plc, Bioven International Sdn Bhd, Agenus Inc., Immatics Biotechnologies GmbH, Immunovative Therapies, Bristol-Myers Squibb Company, Gritstone Oncology, NantKwest, Inc among others.

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Global Precision Medicine MarketBy Application (Diagnostics, Therapeutics and Others), Technologies (Pharmacogenomics, Point-of-Care Testing, Stem Cell Therapy, Pharmacoproteomics and Others), Indication (Oncology, Central Nervous System (CNS) Disorders, Immunology Disorders, Respiratory Disorders, Others), Drugs (Alectinib, Osimertinib, Mepolizumab,Aripiprazole lauroxil and Others), Route of Administration (Oral,Injectable), End- Users (Hospitals, Homecare, Specialty Clinics, Others), Geography (North America, South America, Europe, Asia-Pacific, Middle East and Africa) Industry Trends and Forecast to 2026

Competitive Analysis:

The precision medicine market is highly fragmented and is based on new product launches and clinical results of products. Hence the major players have used various strategies such as new product launches, clinical trials, market initiatives, high expense on research and development, agreements, joint ventures, partnerships, acquisitions, and others to increase their footprints in this market. The report includes market shares of mass spectrometry market for global, Europe, North America, Asia Pacific and South America.

Market Drivers

Market Restraints

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Market Definition:

Precision medicines is also known as personalized medicines is an innovative approach to the patient care for disease treatment, diagnosis and prevention base on the persons individual genes. It allows doctors or physicians to select treatment option based on the patients genetic understanding of their disease.

According to the data published in PerMedCoalition, it was estimated that the USFDA has approved 25 novels personalized medicines in the year of 2018. These growing approvals annually by the regulatory authorities and rise in oncology and CNS disorders worldwide are the key factors for market growth.

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Key Developments in the Market:

Competitive Analysis:

Global precision medicine market is highly fragmented and the major players have used various strategies such as new product launches, expansions, agreements, joint ventures, partnerships, acquisitions, and others to increase their footprints in this market. The report includes market shares of global precision medicine market for Global, Europe, North America, Asia-Pacific, South America and Middle East & Africa.

Market Segmentation:

By technology:-big data analytics, bioinformatics, gene sequencing, drug discovery, companion diagnostics, and others.

By application:- oncology, hematology, infectious diseases, cardiology, neurology, endocrinology, pulmonary diseases, ophthalmology, metabolic diseases, pharmagenomics, and others.

On the basis of end-users:- pharmaceuticals, biotechnology, diagnostic companies, laboratories, and healthcare it specialist.

On the basis of geography:- North America & South America, Europe, Asia-Pacific, and Middle East & Africa. U.S., Canada, Germany, France, U.K., Netherlands, Switzerland, Turkey, Russia, China, India, South Korea, Japan, Australia, Singapore, Saudi Arabia, South Africa, and Brazil among others.

In 2017, North America is expected to dominate the market.

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COVID-19 UPDATE: Precision Medicine Market 2020: Industry Analysis and Detailed Profiles of Top Industry Players are Neon Therapeutics, Moderna, Inc,...