Archive for the ‘Male Genetics’ Category

Olaf is almost two months old. (Submitted by Diane Barber)

Contact: Vanessa Beeson

STARKVILLE, Miss.A Mississippi State University partnership with the Fort Worth Zoo has hatched the first of more than 30 metamorphosed toadlets produced through in vitro fertilization.

A Puerto Rican crested toad named Olaf, hatched at the Fort Worth Zoo this year, is what one might call a work of art. ART, or assisted reproductive technologies, developed by scientists in the universitys Mississippi Agricultural and Forestry Experiment Station and the Forest and Wildlife Research Center, helps amphibians like the Puerto Rican crested toad, considered a threatened species by the U.S. Fish and Wildlife Service.

The technologies include hormone therapies, sperm cryopreservation and in vitro fertilization. MSU also is home to the countrys only National Amphibian Genome Bank, a repository of cryopreserved sperm from approximately 10 of the worlds most threatened and endangered amphibian species.

Carrie Vance, assistant research professor in the Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology who co-leads the project, said Olaf is an example of how ART helps increase the genetic diversity and sustainability of populations of threatened amphibians.

Olaf represents the first time we used cryopreserved sperm from a wild Puerto Rican crested toad as a new genetic line to be combined with an egg from a captive female, said Vance, who also is a MAFES scientist at MSU. Whats more is that both of Olafs parents have since died of natural causes so Olaf is truly the last of this particular genetic line.

Vance pointed out that while cryopreserving sperm from wild males, researchers have been able to use hormone therapies to assist breeding. As opposed to other methods, this technique enables researchers to collect sperm without killing the animal, which Vance believes will result in a wider adoption of the practice.

Previously to introduce genetics from wild individuals into a captive population, the animals were brought into captivity, then paired, and often would never breed anyway. If the collection of sperm from testes macerates was needed, it would require the animal be euthanized, Vance said. This new method means we can collect the sperm and release the specimen back into the wild.

Vance said ART is one facet of a larger species survival plan, which includes steps such as habitat restoration, disease control and establishing an assurance colony in captivity.

ART helps when amphibians have difficulty breeding in captivity. Typically, amphibian breeding is cued by environmental factors such as day length, rainfall and temperature, which are things that can be difficult to control in a 10-gallon aquarium. When they dont breed, the genetic lines are lost, and a zoos entire assurance colony can collapse.

Vance said it comes down to overriding the environmental cues and synchronizing the timing of the actual breeding, noting that while it takes males only hours to generate sperm, it can take weeks for females to produce eggs.

The hormone therapy overrides the environmental factors to trigger the production of reproductive hormones, which cause sperm and egg release. Sperm cryopreservation holds the sperm in perpetuity until the eggs are ready for synchronization.

Vance has partnered with Andy Kouba, professor and head of the Department of Wildlife, Fisheries and Aquaculture in MSUs College of Forest Resources and scientist in the Forest and Wildlife Research Center, for more than 20 years developing innovative reproductive technologies for threatened and endangered species.

The researchers also have applied ART to the Mississippi gopher frog, considered one of the most endangered in the U.S. Their pioneering work resulted in thousands of Mississippi gopher frogs being produced by zoos around the country and reintroduced into their native habitat.

Many of the techniques we use on species like the Puerto Rican crested toad were developed using the Mississippi gopher frog, Kouba said. The Mississippi gopher frog was the first endangered species ever produced from frozen sperm. The offspring are still alive and have subsequently produced a second generation of offspring, considered another first of its kind.

Kouba said seeing the applied conservation in action and being able to reintroduce animals back into the wild is what excites him most about the work.

Globally, it is estimated that 30-40 percent of amphibians are threatened with extinction. In the U.S. that number is closer to 50 percent, Kouba said. Our assisted reproductive technologies have led to millions of tadpoles from threatened and endangered species being released into the wild across many species.

Kouba added that amphibians serve as indicator species for the health of their surrounding ecosystems.

They are the canary in the coal mine, Kouba said. Anything happening in the environment soaks through their permeable skin. Also, they have two life stages, an early aquatic stage and a terrestrial stage, which lets scientists know what is happening in two different environments. As an indicator species, it is important to understand why amphibian populations are disappearing and to try and help the populations recover.

In addition to the Puerto Rican crested toad and the Mississippi gopher frog, other species the team focuses on include the Boreal toad, Houston toad, Chiricahua leopard frog and various species of salamanders. Graduate students on the project include doctoral student Allison Julien of Scotts Valley, California; masters student Isabella Burger of Prattville, Alabama; and Kristen Counsell, a spring 2018 masters graduate of Cedar Falls, Iowa. Masters student Amanda Gillis of Fallston, Maryland, and research associate Emmet Guy of Oxford contribute to the labs salamander research.

Support for the Olaf project includes funding from Disneys Conservation Endowment Fund and the Association of Zoos and Aquariums. Longtime funding partner, the Institute of Museums and Library Services, supported the early development of this work and currently sponsors the labs salamander research. Morris Animal Foundation also has provided previous financial support.

For more on the Mississippi Agricultural and Forestry Experiment Station, visit http://www.mafes.msstate.edu. For more on the Forest and Wildlife Research Center, visit http://www.fwrc.msstate.edu.

MSU is Mississippis leading university, available online at http://www.msstate.edu.

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One-of-a-kind toad born through MSU pioneering technology that's saving threatened species - Mississippi State Newsroom

Scientists have discovered a gene linked to male infertility, which they hope could help to account for 50 percent of unexplained cases.

Variants of a gene called SYCP2 could be the reason why some men struggle to conceive, according to the authors of a study published in the American Journal of Human Genetics. So far, they have pinpointed them in four men with fertility problems.

Back in 1991, a 28-year-old man who had been unable to conceive for two years and had a very low sperm count was referred to the authors of the study. The team assessed his chromosomes, and found he had what is known as a chromosomal rearrangement, where parts of the chromosome might be missing, duplicated, or moved around. This appeared to make the SYCP2 gene 20 times more active, they found.

Identifying this abnormality led the researchers to look at how the gene behaved in a lab using cells and yeast. By modeling the rearrangement in yeast, they found it appeared to trigger an issue linked with defective sperm production in mammals, they wrote in the study.

Next, they looked at whether infertile males had variants of this gene, working with scientists at the University of Mnster. Compared with the general population, disruptions to SYCP2 were more common in those with fertility problems, the investigators found.

Co-author Samantha Schilit, an expert in unexplained infertility at Harvard Medical School, commented in a statement that these chromosomal problems in infertile men are rarely followed up beyond reporting a higher risk for an issue, which can lead to recurrent miscarriages.

"This work shows that a chromosomal rearrangement may also disrupt or dysregulated genes important in fertility, and therefore should be considered."

Infertility is one of the most common problems among those aged between 20 to 45 years old, affecting between 10 to 15 percent of couples. Evidence suggests doctors are unable to diagnose the source of the problem in between 40 to 72 percent of men. Of those, between 30 to 50 percent of cases are estimated to be caused by genetics, the authors wrote. "Searching for genes involved in unexplained infertility is a rich endeavor," they said.

Co-author Cynthia Morton, a medical geneticist at the Brigham and Women's Hospital, told Newsweek: "This study focuses our interest in chromosomal rearrangements that underlie infertility beyond what is typically presumedthat is, the infertility results from embryos that are chromosomally unbalanced and may lead to miscarriage."

Morton, whose laboratory has had a longstanding interest in structural rearrangements of human chromosomes that underlie clinical disorders, said: "It brings emphasis to the fact that structural rearrangements in chromosomes may contribute to infertility by dysregulating gene expression of genes with a role in gametogenesis," or the creation of sperm.

More work needs to be done to validate the role of SYCP2 male infertility. This would be strengthened by finding more males with genomic variants in SYCP2, she said. The team envisions SYCP2 one day being included in genetic screenings for males to identify the genetic basis of infertility.

"A diagnosis can be therapeutic in itself -- even if there isn't something that can be done to correct it. It ends the search for the underlying issue and opens the door for enrolling in clinical trials," said Morton. "And I believe there is good reason to be optimistic; we now have better tools for discovery and can begin on the path toward therapy."

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A Gene Linked to Male Infertility Has Been Discovered, and It May Account for 50 Percent of Unexplained Cases - Newsweek

One out of eight couples has trouble conceiving, with nearly a quarter of those cases caused by unexplained male infertility. For the past decade, research has linked that infertility to defective sperm that fail to evict proteins called histones from DNA during development. However, the mechanisms behind that eviction and where this is happening in the sperm DNA has remained both controversial and unclear.

Now, researchers atPenn Medicineshow, using newer genome-wide DNA sequencing tools, the precise genetic locations of those retained histones, as well as a key gene regulating it. The findings were published inDevelopmental Cell.

Taking it a step further, the researchers created a new mouse model with a mutated version of the gene,Gcn5, which allows investigators to closely track the defects in sperm from the early stages of sperm development through fertilization and on. This is an important step forward as it could lead to a better understanding of not only infertility in menand ways to potentially reverse itbut also the suspected epigenetic mutations being passed onto the embryo from males either naturally or through in vitro fertilization.

Epigenetics, the factors influencing an organisms genetics that are not encoded in the DNA, play a strong role in sperm and egg formation.

For men who have unexplained infertility, everything may look normal at the doctors: normal semen counts, normal motility. Yet they can still have problems conceiving, says first authorLacey J. Luense, a research associate in the lab of the study's senior author,Shelley L. Berger, the Daniel S. Och University Professor in the departments of Cell and Developmental Biology in the Perelman School of Medicine and Biology in the School of Arts and Sciences, and director of the Penn Epigenetics Institute. One explanation for persistent problems is histones being in the wrong location, which may affect sperm and then early development. Now, we have a really good model to study what happens when you dont get rid of the histones appropriately in the sperm and what that may look like in the embryo.

Read more at Penn Medicine News.

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Uncovering a defective sperm epigenome that leads to male infertility - Penn: Office of University Communications

Say hello to SoCal's newest mountain lions, P-78 and P-79. (Courtesy, Santa Monica Mountains National Recreation Area/Facebook)

Christmas has come early for Southern California wildlife enthusiasts in the form of two new mountain lions.

On Tuesday, Angela Beatriz Cholo, a ranger with the Santa Monica Mountains National Recreation Area, introduced via Facebook P-78 and P-79 to greater L.A. and to wildlife officials' study of big cats in the region.

Officials found both cats within a day of each other. P-78, a "subadult male," was captured Dec. 11, in the Santa Monica Mountains. P-79, another young male, was spotted and captured in the backyard of a home on Dec. 12.

Both were outfitted with GPS collars and released. P-78 will roam the Santa Monica Mountains, and P-79 will roam the Santa Susana Mountains, according to the Facebook post.

As their designated numbers suggest, P-78 and P-79 are the 78th and 79th mountain lions to join the study overall. Last month, P-77 was captured and tagged in the Santa Monica Mountains. In June, officials captured P-75 in a mobile home park in Pacific Palisades, outfitted her with a tracking collar and released her into the Santa Monica Mountains.

Despite these additions, Southern California's puma population has suffered in recent decades due to habitat loss and inbreeding.

Three big cats were found dead this year. P-53, a 4-year-old female lion, and P-30, a 6-year-old male, had traces of rat poison in their bodies. P-61, a 4-year-old male, died after being hit by a car on the 405 in the Sepulveda Pass area. Researchers think he may have been running from an uncollared puma at the time.

Like her fellow big cats, P-77 has a lot to contend with. There are turf wars with other mountain lions. There's the risk of getting hit by a vehicle while trying to cross freeways, which limit mating opportunities and decrease the genetic diversity of the local mountain lion population. Then there's the risk of mange. Plus, these big cats have to contend with rat poison and other chemicals introduced into the food chain by humans, who are the worst.

Reporter Ryan Fonseca contributed to this story.

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LA Gets Early Christmas Gift In The Form Of 2 New Mountain Lions, P-78 And P-79 - LAist

BOTHELL, Wash. & TOKYO--(BUSINESS WIRE)--Seattle Genetics, Inc. (Nasdaq:SGEN) and Astellas Pharma Inc. (TSE: 4503, President and CEO: Kenji Yasukawa, Ph.D., Astellas) today announced that the U.S. Food and Drug Administration (FDA) granted accelerated approval to PADCEV for the treatment of adult patients with locally advanced or metastatic urothelial cancer who have previously received a PD-1/L1 inhibitor and a platinum-containing chemotherapy before (neoadjuvant) or after (adjuvant) surgery or in a locally advanced or metastatic setting. PADCEV is approved under the FDAs Accelerated Approval Program based on tumor response rate. Continued approval may be contingent upon verification and description of clinical benefit in confirmatory trials. PADCEV is the first FDA approved treatment in the U.S. for these patients. It is a first-in-class antibody-drug conjugate (ADC) that is directed against Nectin-4, a protein located on the surface of cells and highly expressed in bladder cancer.1,3

Metastatic urothelial cancer is an aggressive and devastating disease with limited treatment options, and the approval of PADCEV is a significant advance for these patients who previously had limited options after initial therapies failed, said Jonathan E. Rosenberg, M.D., Medical Oncologist, Chief, Genitourinary Medical Oncology Service, Memorial Sloan Kettering Cancer Center in New York. The PADCEV clinical trial enrolled a range of patients whose cancer was difficult to treat, including those whose disease had spread to the liver.

The FDA approval of PADCEV is welcome news for patients with bladder cancer, said Andrea Maddox-Smith, Chief Executive Officer, Bladder Cancer Advocacy Network. Though new medicines for bladder cancer have been approved in recent years, most people living with advanced stages of this disease face a difficult journey with few treatment options.

This approval underscores our commitment to develop novel medicines that address unmet patient needs, and were grateful to the patients and physicians whose participation led to this outcome, said Andrew Krivoshik, M.D., Ph.D., Senior Vice President and Oncology Therapeutic Area Head, Astellas.

PADCEV is the first antibody-drug conjugate approved for patients facing this aggressive disease, and it is the culmination of years of innovative work on this technology, said Roger Dansey, M.D., Chief Medical Officer, Seattle Genetics.

PADCEV was evaluated in the pivotal trial EV-201, a single-arm phase 2 multi-center trial that enrolled 125 patients with locally advanced or metastatic urothelial cancer who received prior treatment with a PD-1 or PD-L1 inhibitor and a platinum-based chemotherapy.1 In the study, the primary endpoint of confirmed objective response rate (ORR) was 44 percent per blinded independent central review (55/125; 95% Confidence Interval [CI]: 35.1, 53.2). Among patients treated with the single agent PADCEV, 12 percent (15/125) experienced a complete response, meaning no cancer could be detected at the time of assessment, and 32 percent (40/125) experienced a partial response, meaning a decrease in tumor size or extent of cancer in the body. The median duration of response (DoR), a secondary endpoint, was 7.6 months (95% CI: 6.3, not estimable [NE]). The most common serious adverse reactions (3%) were urinary tract infection (6%), cellulitis (5%), febrile neutropenia (4%), diarrhea (4%), sepsis (3%), acute kidney injury (3%), dyspnea (3%), and rash (3%). The most common adverse reaction leading to discontinuation was peripheral neuropathy (6%). The most common adverse reactions (20%) were fatigue (56%), peripheral neuropathy (56%), decreased appetite (52%), rash (52%), alopecia (50%), nausea (45%), dysgeusia (42%), diarrhea (42%), dry eye (40%), pruritus (26%) and dry skin (26%). The most common Grade 3 adverse reactions (5%) were rash (13%), diarrhea (6%) and fatigue (6%).

The FDA's Accelerated Approval Program allows approval of a medicine based on a surrogate endpoint if the medicine fills an unmet medical need for a serious condition. A global, randomized phase 3 confirmatory clinical trial (EV-301) is underway and is also intended to support global registrations.

About PADCEV

PADCEV is a first-in-class antibody-drug conjugate (ADC) that is directed against Nectin-4, a protein located on the surface of cells and highly expressed in bladder cancer.1,2 Nonclinical data suggest the anticancer activity of PADCEV is due to its binding to Nectin-4 expressing cells followed by the internalization and release of the anti-tumor agent monomethyl auristatin E (MMAE) into the cell, which result in the cell not reproducing (cell cycle arrest) and in programmed cell death (apoptosis). PADCEV is co-developed by Astellas and Seattle Genetics.

PADCEV Support Solutions offers access and reimbursement support to help patients access PADCEV. For more information, go to PADCEV Support Solutions at PADCEVSupportSolutions.com.

About Bladder and Urothelial Cancer

Approximately 80,000 people in the U.S. will be diagnosed with bladder cancer this year.4 Urothelial cancer accounts for 90 percent of all bladder cancers and can also be found in the renal pelvis, ureter and urethra.5

Important Safety Information

Warnings and Precautions

Adverse Reactions

Serious adverse reactions occurred in 46% of patients treated with PADCEV. The most common serious adverse reactions (3%) were urinary tract infection (6%), cellulitis (5%), febrile neutropenia (4%), diarrhea (4%), sepsis (3%), acute kidney injury (3%), dyspnea (3%), and rash (3%). Fatal adverse reactions occurred in 3.2% of patients, including acute respiratory failure, aspiration pneumonia, cardiac disorder, and sepsis (each 0.8%).

Adverse reactions leading to discontinuation occurred in 16% of patients; the most common adverse reaction leading to discontinuation was peripheral neuropathy (6%). Adverse reactions leading to dose interruption occurred in 64% of patients; the most common adverse reactions leading to dose interruption were peripheral neuropathy (18%), rash (9%) and fatigue (6%). Adverse reactions leading to dose reduction occurred in 34% of patients; the most common adverse reactions leading to dose reduction were peripheral neuropathy (12%), rash (6%) and fatigue (4%).

The most common adverse reactions (20%) were fatigue (56%), peripheral neuropathy (56%), decreased appetite (52%), rash (52%), alopecia (50%), nausea (45%), dysgeusia (42%), diarrhea (42%), dry eye (40%), pruritus (26%) and dry skin (26%). The most common Grade 3 adverse reactions (5%) were rash (13%), diarrhea (6%) and fatigue (6%).

Lab Abnormalities

In one clinical trial, Grade 3-4 laboratory abnormalities reported in 5% were: lymphocytes decreased, hemoglobin decreased, phosphate decreased, lipase increased, sodium decreased, glucose increased, urate increased, neutrophils decreased.

Drug Interactions

Specific Populations

For more information, please see the full Prescribing Information for PADCEV here.

About Seattle Genetics

Seattle Genetics, Inc. is an emerging multi-product, global biotechnology company that develops and commercializes transformative therapies targeting cancer to make a meaningful difference in peoples lives. The company is headquartered in Bothell, Washington, and has a European office in Switzerland. For more information on our robust pipeline, visit http://www.seattlegenetics.com and follow @SeattleGenetics on Twitter.

About Astellas

Astellas Pharma Inc., based in Tokyo, Japan, is a company dedicated to improving the health of people around the world through the provision of innovative and reliable pharmaceutical products. For more information, please visit our website at https://www.astellas.com/en.

About the Seattle Genetics and Astellas Collaboration

Seattle Genetics and Astellas are co-developing PADCEV (enfortumab vedotin) under a collaboration that was entered into in 2007 and expanded in 2009. Under the collaboration, the companies are sharing costs and profits on a 50:50 basis worldwide.

Seattle Genetics Forward Looking Statements

Certain statements made in this press release are forward looking, such as those, among others, relating to the continued FDA approval of PADCEV (enfortumab vedotin-ejfv) for the treatment of adult patients with locally advanced or metastatic urothelial cancer who have previously received a PD-1/L1 inhibitor, and a platinum-containing chemotherapy in the neoadjuvant/adjuvant, locally advanced or metastatic setting; the conduct of an ongoing randomized phase 3 confirmatory clinical trial (EV-301) intended to verify the clinical benefit of PADCEV and support global registrations; and the therapeutic potential of PADCEV including its efficacy, safety and therapeutic uses. Actual results or developments may differ materially from those projected or implied in these forward-looking statements. Factors that may cause such a difference include the possibility that EV-301 and subsequent clinical trials may fail to establish sufficient efficacy; that adverse events or safety signals may occur; that utilization and adoption of PADCEV by prescribing physicians may be limited by the availability and extent of reimbursement or other factors; and that adverse regulatory actions may occur. More information about the risks and uncertainties faced by Seattle Genetics is contained under the caption Risk Factors included in the companys Quarterly Report on Form 10-Q for the quarter ended September 30, 2019 filed with the Securities and Exchange Commission. Seattle Genetics disclaims any intention or obligation to update or revise any forward-looking statements, whether as a result of new information, future events or otherwise, except as required by law.

Astellas Cautionary Notes

In this press release, statements made with respect to current plans, estimates, strategies and beliefs and other statements that are not historical facts are forward-looking statements about the future performance of Astellas. These statements are based on managements current assumptions and beliefs in light of the information currently available to it and involve known and unknown risks and uncertainties. A number of factors could cause actual results to differ materially from those discussed in the forward-looking statements. Such factors include, but are not limited to: (i) changes in general economic conditions and in laws and regulations, relating to pharmaceutical markets, (ii) currency exchange rate fluctuations, (iii) delays in new product launches, (iv) the inability of Astellas to market existing and new products effectively, (v) the inability of Astellas to continue to effectively research and develop products accepted by customers in highly competitive markets, and (vi) infringements of Astellas intellectual property rights by third parties.

Information about pharmaceutical products (including products currently in development), which is included in this press release is not intended to constitute an advertisement or medical advice.

1 PADCEV [package insert]. Northbrook, IL: Astellas, Inc.2 Rosenberg JE, ODonnell PH, Balar AV, et al. Pivotal Trial of Enfortumab Vedotin in Urothelial Carcinoma After Platinum and Anti-Programmed Death 1/Programmed Death Ligand 1 Therapy. J Clin Oncol 2019;37(29):2592-600.3 Challita-Eid P, Satpayev D, Yang P, et al. Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models. Cancer Res 2016;76(10):3003-13.4 American Society of Clinical Oncology. Bladder cancer: introduction (10-2017). https://www.cancer.net/cancer-types/bladdercancer/introduction. Accessed 05-09-2019.5 National Cancer Institute. Surveillance, Epidemiology, and End Results Program. Cancer stat facts: bladder cancer. https://seer.cancer.gov/statfacts/html/urinb.html. Accessed 05-01-2019.

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FDA Grants Accelerated Approval to Astellas' and Seattle Genetics' PADCEV (enfortumab vedotin-ejfv) for People with Locally Advanced or Metastatic...

As a new mother, she didnt know to look for blue-tinged lips. She could just tell her babys color was off. On a chest X-ray, the clean, white-against-dark curves of his ribs were obscured, clouded by fluid. Pneumonia. That tipped Ray Ballards physicians off: He had a form of severe combined immunodeficiency SCID, for short a genetic mutation that hampered the growth of crucial immune cells, leaving him utterly vulnerable to infection.

The best fix was a transplant of his mothers bone marrow. The attitude was that in three to six months, you should be able to go back to normal life, recalled his mom, Barb Ballard.

That was true at least sort of. He got two more booster transplants before he hit 10. An antibiotic left him with hearing loss, and a virus with digestive tract damage. His lack of B cells meant he needed regular injections of other peoples antibodies, and his T cell counts were never ideal. But he was healthy enough to go to public school, to move through the hallways high-fiving half the guys, to slowly inhale and take aim during rifle team practice.

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His T cells had to be working well enough that he wasnt coming down with everything that walked into the classroom, Ballard said.

Then, when Ray was around 18, his immunity began to wane. For him, it came in the form of a norovirus he couldnt shake. For others with the same rare disease, it appears as pneumonia or gastrointestinal trouble or an unexpected T cell decline. Over the last 10 years, the trend has become increasingly clear: The bone marrow transplants that kept certain babies with SCID alive sometimes stop working after years or decades of providing fairly reliable immune defenses.

Now, to patient advocates, this has become an urgent lesson in the language people use to talk about treatment and not just for SCID. They see their communitys experience as a cautionary tale for anyone developing or receiving a therapy thats marketed as potentially curative.

Theres an expectation and a hope: When they hear about bone marrow transplants, it sounds like a lifetime deal, a forever fix, said John Boyle, president and CEO of the Immune Deficiency Foundation. Weve discovered, as a result of this issue, that bone marrow transplant ended up not being the forever fix we thought it was.

Experts have known for years that some of these transplants wouldnt provide full immune protection over the course of a SCID patients entire life. They say clinicians should have avoided the word cure. But even scientific papers that hinted at such complications called the treatment curative. Just this year, an Immune Deficiency Foundation employee was given the unenviable task of sifting through the organizations thousands of pages of online material, scrubbing out every cure that popped up. It was only there a handful of times sometimes in quotes from clinicians, Boyle said but it was there and it needed to be removed.

The language patients hear can sometimes even change their outcomes. Weve heard of cases where, years later, they realized their immune system isnt as healthy as they thought, but nobody was tracking that because they hadnt maintained a relationship with the physician, or the physician didnt maintain a relationship with them, explained Ballard. The word cure, it gives them a false sense of security.

At a time when seemingly every biotech is promoting the idea of one-and-done therapies and setting prices accordingly these advocates hope companies, too, will be more wary. One of the things Im trying to make them very aware of is the need for lifelong follow-up, said Heather Smith, who runs the SCID Angels for Life foundation. For her, its personal: This summer, her son took part in a clinical trial for a gene therapy in the hope that it would provide the immune protection that his decades-old bone marrow transplant no longer could. My son will be followed for 15 years, she said. But what about after that?

Part of the issue with bone marrow transplants from one person to another is the natural genetic variation between us, particularly in the proteins that help our bodies distinguish its own cells from foreign ones. Receiving cells from someone whose proteins dont match yours could cause a civil war within you. Thats why bone marrow transplants began back in the 1950s with identical twins: Sharing those genes meant increasing the likelihood of harmony between the body and the graft.

But the vast majority of people dont have a protein-matched sibling, let alone an identical twin. So researchers set about figuring out how to transplant bone marrow from a parent to a child in spite of only sharing half of their genes and from a matched unrelated donor to a stranger. Like cooks intent on refining recipes to their taste, the doctors who adapted the technique for SCID often did so slightly differently from one another. Over the past 35 years, those idiosyncrasies have hardened into habits. Right now, everybody transplants their patients their way, said Dr. Sung-Yun Pai, an immune deficiency researcher and co-director of the gene therapy program at Boston Childrens Hospital.

Perhaps the most vociferous controversy has been about whether to use chemotherapy to wipe out the existing stem cells within a recipients bone marrow to make room for the donors. The doctors who do use chemo before a transplant might prescribe different doses; others forego it entirely.

The arguments were sound on both sides. On the one hand, the toxic drugs could clean out the niches within our bone and increase the chances that the donors cells take root. On the other, these chemicals could hamper growth, brain development, and fertility, could make an infant who was already sick even sicker, and could increase the likelihood of certain cancers later in life. Its like being exposed to a bunch of X-rays and sunlight, or other DNA-damaging agents, Pai explained.

Because SCID is so rare the most common subtype is thought to occur in 1 out of every 50,000 to 100,000 newborns and because every hospital was doing transplants slightly differently, it was hard for physicians to systematically study what was working best. But even early on, they could tell that some of the infants whod gotten no chemo were developing incomplete immune systems. They didnt produce their own B cells, for instance, and so needed regular injections of antibodies collected from other peoples blood.

In healthy infants, stem cells migrate from the crevices of the skeleton to an organ in the chest called the thymus, where theyre trained to become T cells. In these infants, the T cell counts grew after transplant but it wasnt necessarily because the sludge was securely taking hold in the niches of their bones. Rather, immunologists say, the donors progenitor cells were only transient. Some were able to head toward the thymus for schooling. Some graduated and started fighting off infections. But as those populations were depleted with age, there werent robust reserves of stem cells in the bone marrow that could arrive to produce more. To Pai, its like trying to fill a kindergarten class in a neighborhood where no ones having babies.

You and I continue to have a slow trickle of new T cells coming out, said Dr. Harry Malech, a senior investigator at the National Institutes of Health, who sits on the board of a gene therapy company, Orchard Therapeutics (ORTX), but does not receive any financial compensation. Instead of a torrent becoming slower, in these patients it goes from a trickle to practically nothing.

Thats why immunity starts to wane in kids like Ray Ballard. To many immunologists, it isnt a surprise, though they still arent sure why chemo-less transplants last longer for some of these kids than others. They can also understand how some families and clinicians might have viewed this treatment as a lifetime fix.

As Malech put it, If I said to you, Your child, instead of dying in infancy, will likely get to adulthood, go to school, have a normal life, you might think the word cure in your mind.

Even for parents who knew the protection might not last forever, the failure of a long-ago bone marrow transplant puts them in a bind. If they do nothing, their child will once again be vulnerable to any passing infection, which could prove fatal. They can try another round of the same procedure, though booster transplants sometimes come with added complications. Or they can try getting their child into a research trial for gene therapy, which comes with the risks of any experimental treatment.

Some feel an irrational guilt when the bone marrow they donated to their child stops functioning. Its your cells, and if it doesnt work, you failed them, said Ballard, who lives in Clifton, Va., about a 40-minute drive from Washington, D.C. Her son Ray had already had three transplants as a child. When his immune system started to fail again in early adulthood, gene therapy at the NIH seemed like the only reasonable choice.

That would involve researchers removing cells from his bone marrow, using an engineered virus as a kind of molecular syringe to slip in a healthy copy of the gene in which he had a defect, and then threading these corrected cells back into his veins a bone marrow transplant to himself. But preparing a virus can be tricky, and there were delays.

Meanwhile, Rays condition was getting worse. His norovirus was preventing him from absorbing much nutrition, and as Ballard put it, his bone structure was just crumbling at that point. His doctors told her he had the skeleton of an 85-year-old.

He died this past February, at 25 years old. One friend got his birth and death dates tattooed onto her shoulder. Another painted a portrait of him for Ballard, in which his arms are crossed, his lips pressed together in a wry smile.

At Boston Childrens, Pai is now helping to lead a randomized trial to better understand what dose of chemo works best for SCID patients receiving transplants. Over the last decade or so, she, Malech, and many other clinicians have also teamed up to track the long-term results of immune deficient patients whove received someone elses bone marrow.

Pai is hopeful that knowing about the phenomenon of waning immunity will give gene therapies a better shot at becoming a durable fix. They probably have a better chance of achieving a one-time, lifelong cure, but its never wrong to be humble, she said. Only after decades more and hundreds or thousands of patients will we know for sure.

Patient advocates point out that even then, these patients will still have the capacity of passing on their SCID-causing gene to future generations, and so the word cure is overly optimistic. Thats why I like the word remission, said Smith. That still gives you the hope. If you were given a cancer diagnosis, you wouldnt go through treatment and then just forget about it for the rest of your life.

As Boyle put it, Weve seen the promise and then weve seen the reality. Everyone who is looking at a transformational therapy should be optimistic, but also realistic, and not assume that this is truly one and done. (Boyles foundation has received financial support from Orchard Therapeutics, which is developing a gene therapy for a form of SCID.)

To Amy Saada, of South Windsor, Conn., that isnt theoretical. Her son Adam is now 12, and the immunity from the bone marrow transplant he got as a baby is wearing off. He isnt yet sick, but his parents know they need to decide between gene therapy or another transplant soon. She has a very clear memory of how long and uncertain the recovery from treatment felt. In some ways, she wishes she didnt know quite as much as she does; that way, she would feel less trepidation about what lies ahead.

Your heart kind of sinks, she said. Youve already been through it once, and it was hell. Its harder the second time.

Go here to see the original:
Waning treatment is a warning for all 'one-and-done' therapies - STAT

The last few decades have seen revolutionary developments in the field of Assisted Reproduction. IVF is over 40 years old. It took hundreds of experiments on animal and human eggs and sperm before Louise Brown was born in Oldham, the UK in 1978. Scientific advancement calls for a healthy interaction of colleagues, scientific bodies and the media.

After much discourse, IVF received scientific and societal acceptance ultimately resulting in the Nobel Prize for Bob Edwards in Medicine or Physiology in 2010 for work leading to the birth of Louise Brown. However, scientific ignorance can thwart major discoveries. Dr Subash Mukhopadhyay created Indias first and the worlds second test-tube baby. Durga was born just 67 days after Louise Brown. The scientific climate did not allow Dr Mukhopadhyay his claim to fame. This resulted in a crushing halt to the spread of ART in India for almost a decade.

The earlier decades saw a plateau in success rates with IVF techniques. Several factors have made IVF a household name making it the standard of care for many infertile conditions.

The introduction of the hormones like LH (Luteinizing Hormone) & FSH (Follicle-stimulating Hormone) to bring about the formation of multiple eggs was a major step to improving success, as there were more eggs and embryos to choose from. Besides, efforts were made to better understand the microenvironment of the embryo. This resulted in nutrient medium satisfying the need for embryos at various stages of development thereby enhancing pregnancy rates.

A major breakthrough occurred for males with very low or no sperm counts, with the introduction of Intracytoplasmic Sperm Injection in 1992 by Dr Palermo & Dr Andre van Steirtegheim in Brussels. This caused a major paradigm shift in treating male infertility. In 1994, our team created Luv Singh the first ICSI baby in South-East Asia. The technique gave a major boost to the success of Assisted Reproduction in India.

The early 90s saw an interest in looking at genetic disorders in couples who were otherwise fertile. Handyside & his group described pregnancies after biopsy of human preimplantation embryos in cases of sex-linked diseases. Techniques were refined over the years so that instead of only 5 chromosomes being checked, today we have the ability not only to check for all 46 chromosomes but to also detect minor variations in the structure and placement of chromosomes by powerful platforms like Next Generation Sequencing.

The technique of Pre Implantation Genetic Testing (PGT) also applies to couples with inheritable mutations for genetic diseases like Thalassemia, Sickle Cell Disease, Duchennes Muscular Dystrophy, and Huntingtons Chorea. This technology has been a boon for couples who would otherwise be at risk of having a genetically compromised baby.

There has been a constant endeavour to enhance pregnancy rates for couples facing fertility issues. The endometrium i.e. the inner lining of the uterus may sometimes be ineffective in helping the process of implantation. We devised a co-culture technique called Cumulus Aided Transfer (CAT) for the first time in the world, in which cells surrounding the oocyte (Cumulus Cells), are used as a feeder layer on which the embryos can grow. This has resulted in better pregnancy chances.

Immunological competence and its vagaries are now better understood. Modulating the womans immune system can prevent the block from implantation.

Today we are witnessing diminishing fertility potential globally due to the presence of different types of pollution affecting the ovaries and testes, thus decreasing egg and sperm counts prematurely. Our team has successfully carried out Ovarian Rejuvenation by instilling Platelet Rich Plasma in the ovaries of such women.

DISCLAIMER: The views expressed are solely of the author and ETHealthworld.com does not necessarily subscribe to it. ETHealthworld.com shall not be responsible for any damage caused to any person/organisation directly or indirectly.

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Better Success Rates of IVF Treatments - Health Files by Dr Firuza R. Parikh - ETHealthworld.com

It remains one of Northern Irelands most notorious cold-cases, still unsolved after 31 years.

It was in April, 1988 when a pretty young blonde schoolgirl from Munich named Inga Maria Hauser, a budding singer with intrepid spirit to match, decided to go backbacking during the Easter break, and full of the anticipation of adventure, primed to see something of other countries and cultures, she set out to visit England, Scotland and Ireland.

Her friend Walter Schreiner, who first met Inga in their local youth club in the mid-1980s, said of the budding young adult: Every person who knew her loved her. She was always smiling, she was always shining and was very intelligent.

A girl with charm and wit and all before her then, setting off to see new sights and meet new people in a different culture to her own; a brave soul, unfazed by the prospect of travelling alone.

Poignantly, Inga had already noted the friendliness of the local people.

During her travels one of the 18-year-olds postcards home from England said: The people here are so helpful and lovely that I cant imagine anything bad could happen to me.

A note in her diary added: The day after tomorrow I am going on to Ireland. Im looking forward to that the best.

She got the ferry from Stranraer, arriving in the port town of Larne on April 6. But she would never make it to her next destinations of Belfast and then Dublin.

Two weeks after she was last seen alive, her dumped mutilated body, having, acorrding to Police, been subjected to a vicious and ruthless sexual and physical assault, was found by a sheep farmer in a remote part of Ballypatrick Forest near Ballycastle.

Here was the life of a beautiful girl cut senselessly short, her dreams and hopes for the future callously denied and a family back in Germany plunged into unimaginable grief.

In the 31 years since the horrific murder both of Ingas distraught parents have died without seeing justice served.

Her mother Almut passed away in October of this year. Her father, Josef, died in 2006 from cancer. Both were hearbroken that the case was never solved; that they were denied answers, a perpetrator or perpetrators brought to justice.

Ingas sister Frederika was particularly badly affected by her sisters death, her son Viktor told a BBC programme last year:

Siblings always have a little tension around them. But they were really close, even though they were quite different. There is a German saying - ein Herz und eine Seele - one heart, one soul.

Of the pursuit of justice for Inga, Viktor said: It would be especially important for my mother. I hope if the murderer gets caught, my mother can finally leave this behind and we can be free of this curse.

SDLP MLA for East Londonderry John Dallat has been involved in the campaign to find Inga Maria Hausers killer for many years; for him the quest to find those responsible for Ingas death has become a life-long priority and passion project.

He was instrumental in getting the case reopened by the PSNI last year after the files were closed due to lack of progress.

John said: I remember when Inga was murdered and my heart went out to her family. I was a young father then to a young girl who I hoped would grow up also and travel the world - and she did. Every parent expects their child to be able to travel without being murdered in the brutal way that Inga Maria was. After ten years of campaigning the parents said they werent coming back and so I assumed that role fighting for justice for Inga. And it has been a long struggle.

This is a personal and emotional struggle for me and it has been a great privilege getting to know Ingas only sister Frederike Leibel. She has one son, Victor, who visited last year, and stayed with us.

In all the years since Inga was murdered they only received a series of letters in English and werent even aware for a long time that they were entitled to legal representation. The support they received was not good.

But we want to focus on getting whoever did this heinous crime to be brought to justice.

Since a memorial stone to Inga was erected in Ballypatrick Forest Park on the 30th year of her death, well-wishers have left tokens at the site, flowers, soft toys, cards, religious objects.

This week John Dallats granddaughter Caitlin laid a wreath at the headstone to mark 31 years. The hope is to send the Hauser family a festive message of support - that their beloved sister and aunt has not been forgotten; the difficult quest for answers continues.

The also plan to erect a German flag to acknowledge Ingas home country and to signify that she was the only international student to die here in Ulster during the Troubles.

Christmas means nothing to the Hauser family since they lost their beloved Inga in one of the most brutal murders ever recorded and all the more reason why we need to show that we really care, continues John.

The family know that Inga hasnt been forgotten and they have recovered hope that justice may still be done.

In May last year, a 59-year-old man was arrested on suspicion of the murder by police investigating the case and later released on bail pending further inquiries.

Detectives said they believe several people may have been involved directly in the murder, or in the subsequent cover-up, and said they only need fractional piece of evidence to bring the chief suspects to justice.

Police also found a male genetic profile at the murder scene, although have yet to find a match.

The Public Prosecution Service is now in receipt of a lengthy file of evidence against the chief suspect.

Dallat continued: The fact that a file has gone to the Public Prosecution Service after 31 years is welcome news and offers some hope to the Hauser Family.

It represents the first chink of light in a long struggle for justice for their daughter Inga who died a most brutal death at the hands of a killer who was intent on raping a young girl over here during a school break to find out about a country that her parents loved.

Perhaps the saddest thing about the murder of Inga is the silence of those who know who did it, but that silence cannot be sustained for much longer.

Hopefully the file which has gone to the Public Prosecution Service will be a historical development which will bring closure to a family who have suffered in silence for far too long, Mr Dallat added.

It is our responsibility and that of the police and courts to redouble our efforts to ensure that those involved in Ingas murder are brought to justice.

I am so sorry that my efforts and that of others havent achieved their purpose so far.

But I remain confident that sooner, rather than later, that justice will be done and that the retribution Almut and Josef were denied will be delivered.

Dallat is clear about what he would like to say to those responsible for, or who know something about, Ingas murder:

I urge them to admit their guilt. Its not easy to give evidence against people who were perhaps once friends. But we believe there are witnesses out there who must do the right thing and demonstrate some level of decency.

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Inga was a beautiful girl with her whole life in front of her, her death was a national shame - Belfast Newsletter

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Have you finished your holiday shopping yet? Sometimes, the hardest people to buy for are your significant other, or those closest to you. Theres always that one big gift you know they want, the one youre sure to givebut what comes next? Sure, you could load up on stocking stuffers, or settle for gift cards. But heres a better idea: Give the gift of health, knowledge, and insight. Give them a 23andMe Health + Ancestry Kit.

The 23andMe Health + Ancestry Kit ($129; was $199) is full of fascinating information on ancestry and family lineage. But it also provides a wealth of biological and genetic insight to help your loved one live a happier, healthier life.

DNA home test kits are all the rage, and with good reason. Its fun to trace your lineage, to track where your ancestors came from, and how you got where you are today. Thats why these kits are so popularespecially this time of year.

Holiday gatherings can be a slog. But sharing ancestral info with family and loved ones is a fantastic conversation piece. Showing up at the family get-together with a family tree, with names, dates, and places of those who came before, is definitely fascinating and fun. But for the seniors in your family, it can be far more than that.

If youve ever watched your grandmother or grandfather reminisce about the old days, you know what we mean. Imagine the faces of the seniors in your family lighting up when you remind them of places and people in the past, of memories theyd forgotten and people theyve missed.

Reminiscing about old times, reliving memories of ancestors passed on, of homes and towns they once adoredor hated!is sure to bring a smile to everyone gathered around the holiday table. Its sure to be a heart-rending, emotional moment. And it will make this holiday season one you, and they, will cherish for the rest of your lives.

Thats why the 23andMe Health + Ancestry Kit makes an unbeatable gift. And right now, you can save $70! Regularly $199, through December 26 you can pick one up for just $129. Thats only $30 more than the Ancestry + Traits kit alone. Thats a 23andMe December deal you cant pass up!

23andMes Health + Ancestry Kit goes deeper than just lineage and genetic traits, though. It provides insight and useful information about health and biological makeup. Your loved one will get more than 150 personalized reports that break down genetic data, the science, and potential next steps to living a healthier life.

Theyll learn how their DNA could affect their chances of developing certain health conditions like Type 2 diabetes*. Theyll find out how their DNA relates to their lifestyle, like muscle composition, diet, and sleep. Particularly valuable is the Carrier Status test, which will let them know if their DNA indicates they may be a carrier for genetic variants linked to certain inherited health conditions.

Of course, the 23andMe Health + Ancestry Kit has the Relative Finder, the Family Tree, and the Ancestry Reports that are so fun and fascinating to share at the holidays. But the valuable information from the more than 150 Trait, Health Predisposition, and Carrier Status reports goes far beyond fun and fascination. Its the kind of knowledge that can help your loved one live a fuller, healthier life. Thats the kind of holiday gift no stocking-stuffer or gift card can possibly beat.

During this 23andMe December deal, youll save $70!

*Customers have the option to choose whether to access their health reports. Visit [https://www.23andme.com/test-info/] for more important test information.

For access to exclusive gear videos, celebrity interviews, and more, subscribe on YouTube!

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The Unbeatable Holiday Gift: Save $70 on a 23andMe Health + Ancestry Kit Today - Men's Journal

PHILADELPHIA -- One out of eight couples has trouble conceiving, with nearly a quarter of those cases caused by unexplained male infertility. For the past decade, research has linked that infertility to defective sperm that fail to "evict" proteins called histones from DNA during development. However, the mechanisms behind that eviction and where this is happening in the sperm DNA has remained both controversial and unclear.

Now, researchers at Penn Medicine show, using newer genome-wide DNA sequencing tools, the precise genetic locations of those retained histones, as well as a key gene regulating it. The findings were published in Developmental Cell.

Taking it a step further, the researchers created a new mouse model with a mutated version of the gene, Gcn5, which allows investigators to closely track the defects in sperm from the early stages of sperm development through fertilization and on. This is an important step forward as it could lead to a better understanding of not only infertility in men -- and ways to potentially reverse it -- but also the suspected epigenetic mutations being passed onto the embryo from males either naturally or through in vitro fertilization.

Epigenetics, the factors influencing an organism's genetics that are not encoded in the DNA, play a strong role in sperm and egg formation.

"For men who have unexplained infertility, everything may look normal at the doctors: normal semen counts, normal motility. Yet they can still have problems conceiving," said first author Lacey J. Luense, PhD, a research associate in the lab of study senior author, Shelley L. Berger, PhD, the Daniel S. Och University Professor in the departments of Cell and Developmental Biology and Biology, and director of the Penn Epigenetics Institute. "One explanation for persistent problems is histones being in the wrong location, which may affect sperm and then early development. Now, we have a really good model to study what happens when you don't get rid of the histones appropriately in the sperm and what that may look like in the embryo."

Healthy sperm lose 90 to 95 percent of histones, the main proteins in chromatin that package DNA and turn genes on and off, and replace them with protamines, which are smaller proteins able to properly pack the DNA into tiny sperm. Given the role of retained histones in infertility and embryonic development, there is great interest in determining the genomic locations so they could potentially be utilized for further study and ultimately treatment.

Past studies have produced conflicting results on the whereabouts of histones. A technology known as MNase-sequencing that uses an enzymatic reaction to pinpoint location has placed the retained histones on important gene promotors. Other studies with the same approach found histones at DNA repeats and placed in so-called "gene deserts," where they play less of a role in regulation.

"There has been controversy in the field trying to understand these discrepant data," Luense said. "In this new study, we found that both of these previously described models are correct. We find histones on genes that appear to be important for embryo development, but we also find them at repetitive elements, places that do need to be turned off and to prevent expression of these regions in the embryo."

The researchers applied a technology known as ATAC-sequencing, a more precise and faster approach, to track waves of histones at unique sites across the genome during the early and late stages of sperm development in mice. ATAC-seq can identify parts of the genome open and closed -- in this case, regions that retain the sperm histones -- and then make a cut and tag the DNA, which can then be sequenced.

In the mouse models created with the mutated Gcn5 gene, the researchers found these mice to have very low fertility. The researchers also showed that retained histones in normal mice sperm correlated with histone positions in very early embryos, supporting the hypothesis that paternal histones transfer epigenetic information to the next generation.

Having this type of mutant model gives scientists a tool to closely study the mechanisms underlying the mutated sperm's trajectory and understand what effect it may have on the embryo and in development. It also opens an opportunity to study potential therapeutic targets.

"Right now, the burden of IVF and other assisted-reproductive technologies fall on women. Even it's the male factor, it's still women who have to go through hormone injections and procedures," Berger said. "Now imagine being able to apply epigenetic therapeutics to change the levels of histones and protamines in males before embryogenesis? That's one of the questions we want to explore and this model will allow us to move toward that direction."

There are numerous available epigenetic drugs used to treat cancer and other diseases. Given their mechanisms, treating sperm with drugs to increase histone eviction is one potential route to explore.

Limitations with human embryos in science have led to a lack of overall research on infertility and the role of the father's epigenome on embryo development, which underscores the importance of studies such as this, the researchers said.

"There are a lot different factors that can alter the sperm epigenome: diet, drugs, alcohol, for example," Luense said. "We are just now starting to understand how that can affect the child and affect development. These initial, basic studies that we are doing are critical, so we can better understand what's driving these epigenetic mutations."

Read the rest here:
Penn researchers uncover defective sperm epigenome that leads to male infertility - Science Codex

As a 30-year-old, Caroline Henaghan was busy. She was working for the UKs Home Office while training to be a barrister, and wondered if the frequent stress and anxiety she was experiencing were just products of overwork and getting older. It felt like getting on a hamster wheel and not being able to get off, she recalls.

Eventually work got to be too much and she took an uncharacteristic short leave of absence. But Henaghans mood didnt improve. She woke up each morning with enormous anxiety, leading to social withdrawal. I would essentially do a disappearing act so I wouldnt have to be around people, she says. She was never suicidal, she stresses. But she did fantasise about leaving things behind. It would be a case of if I could go to sleep and never wake up. Thats how dramatic it was for me.

Though the psychiatric symptoms were the strongest, there were odd physical patterns as well. Henaghan would get bloated and fatigued, sleep excessively, and as a keen gardener shop erratically, for instance buying plants that were out of season. Her family noticed her increasingly strange behaviour as well. She thought it might be bipolar disorder, given the cyclical nature of her ups and downs. For instance, she might spend two weeks each month putting right the damage from the previous week: the fights with loved ones, the untidy home, the slippages at work. Eventually, after what she calls a mini-breakdown, she realised that the recurrence of all these symptoms was linked to her menstrual cycle.

Her doctors dismissed her concerns. She visited five GPs, all male, after the first one commented: Oh, its just PMS. My wife gets that.

But it wasnt just premenstrual syndrome (PMS). Henaghan had to do what many women overlooked by the medical establishment do: her own research. Through her online study, she learned about a condition called premenstrual dysphoric disorder (PMDD).

You might also like:

How the menstrual cycle changes womens brains The effect of childbirth no-one talks about The sexist myths that wont die

PMDD is much more intense than its better-known relative, PMS, with physical symptoms including fatigue and migraines, while the psychological symptoms can include the severe mood swings and anxiety that plagued Henaghan. The disorder can be so debilitating that 15% of those with PMDD have attempted suicide, and some young women affected are opting for hysterectomies.

Read the rest here:
The overlooked condition that can trigger extreme behaviour - BBC News

A memorial has been held for a German teenager who was murdered in Northern Ireland more than thirty years ago.

The body of Munich teenager Inga Maria Hauser was found dumped in a remote part of Ballypatrick Forest, outside Ballycastle, Co Antrim, 14 days after she was last seen alive on a ferry from Scotland.

The 18-year-olds death in April 1988 remains one of the regions most high-profile unsolved murders.

A tribute was paid to Ms Hauser on Saturday at the spot where her body was found.

Mourners laid a Christmas wreath and German flag at the memorial plaque dedicated to her.

Phoenix Law solicitor Claire McKeegan, who attended the memorial, tweeted: Our thoughts are with the Hausers who are pressing the @thePPSNI for a decision on the suspect file. #justiceforINGA.

Last year, on the 30th anniversary of the crime, detectives said they believed a number of people may have been involved either directly in the murder or in the subsequent cover-up, and said they only need fractional pieces of evidence to bring the chief suspects to justice.

Detectives investigating the killing have passed an evidence file on a 59-year-old suspect to the Public Prosecution Service (PPS). The PPS have yet to decide whether there are grounds to prosecute the man.

He was originally arrested in connection with the murder last May and later released on bail pending further inquiries.

Police have a male genetic profile found at the murder scene.

A number of years ago, in one of the largest DNA screenings undertaken in the UK, 2,000 samples failed to produce a definitive match.

Ms Hauser had travelled through England and Scotland and, according to diary entries, intended to travel south to Dublin after her ferry docked at Larne, Co Antrim.

For reasons as yet unexplained, she ended up going in the opposite direction and was found dead in remote woodland two weeks later.

It is understood the IRA carried out its own investigation into the killing 30 years ago.

It is believed republican paramilitaries had considered passing information about the alleged murderer to the RUC at the height of the Troubles, but did not follow through.

Keep up-to-date with all the very latest news, what's on, sport and everything else in Belfast and beyond with the Belfast Live app.

Only select news that interests you by picking the topics you want to display on the app's homepage. Plus, our enhanced user experience includes live blogs, video, interactive maps and slick picture galleries. Download it now and get involved.

Click here to get it from the App Store or here for Google Play .

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Inga Maria Hauser memorial held as those involved in death continue to avoid justice - Belfast Live

Dr Leigh estimates two-thirds of the Wollemi-Hawkesbury koala population has been lost, along with the decimation of several other koala populations around the state.

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Volunteer rescuers included experienced State Emergency Service workers and a specialised climbing team that was called in from Victoria. In total, three males and five females, along with four joeys, were saved.

"We put in some very long hours and we're delighted we've got a good ratio of males and females, and also some joeys," Dr Leigh said.

Koalas are a threatened species and vulnerable to extinction across most of their range inAustralia .

Dr Leigh said that although they were a once-thriving community of 3 to 4 million animals, koala numbers are now as low as 300,000 - the World Wildlife Fund have said that koalas could be extinct in NSW by 2050.

"Saving each and every koala population is vital to the species' survival, she said.

In total, three males and five females, along with four joeys, were rescued.Credit:Taronga Zoo

"We just thought it was really critical even though we got a relatively small number. We just needed to do something and we've done the best we can."

The koalas are being cared for behind-the-scenes by the Taronga Wildlife Hospitals veterinary team and Taronga koala keepers until it is safe to release them back to the wild.

"We are committed to caring for these important koalas to ensure some of this vital genetic diversityfrom the Blue Mountains can be preserved and that the future of this iconic species is secured, saidNick Boyle, Taronga Conservation Societys director of welfare, conservation and science.

"This weeks forecast has multiple days above 40 degrees in western Sydney with gusty andchangeable winds, so it was important that we rescued these animals from impending danger."

Matt Bungard is a journalist at The Sydney Morning Herald.

Original post:
Handful of Blue Mountains koalas successfully relocated to Taronga Zoo - Sydney Morning Herald

Written by Divya Goyal | Ludhiana | Updated: December 17, 2019 11:14:13 am Union Minister for animal husbandry, dairying and fisheries Giriraj Singh said that the plan is to provide dairy farmers with sexed semen for cattle for as cheap as Rs 100 per straw by 2020.

During the recently held 14th Progressive Dairy Farmers Association (PDFA) International Dairy and Agri Expo 2019 at Jagraon, Union Minister for animal husbandry, dairying and fisheries Giriraj Singh had said that the plan is to provide dairy farmers with sexed semen for cattle for as cheap as Rs 100 per straw by 2020. The Indian Express explains the technology behind sexed semen pioneered by the United States, how and when it entered Punjab and if it can help in solving the stray cattle problem:

What is sexed semen, and what is the technology behind it?

Sexed semen is specially processed semen of bulls from which Y chromosomes in sperm cells which lead to the birth of a male calf are either removed through a sorting process or killed. Semen which has only X chromosomes can ensure that a female calf is born.

Dr Prakash Singh Brar, dean, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), said, The reproduction system of cattle is similar to humans. Cows carry XX chromosomes while bull semen carries both X and Y. If the egg fertilises with an X chromosome, a female calf is born and if with Y, a male is born. There are two techniques to produce sexed semen: One is the sorting process in which X and Y chromosomes are separated. X are retained and Y discarded. The other is in which Y chromosomes are altogether killed. Both technologies are pioneered by the United States-based companies and use an instrument called Flow Cytometer. Cows are impregnated using sexed semen through the artificial insemination process with consumption of one straw per cow.

Why is this method being used?

Considered a financial burden, male calves are either killed or abandoned on the roads by farmers as they do not give milk. This had led to an increasing number of cattle roaming the streets, which has caused fatal road accidents as well. Cows are also abandoned when they stop giving milk. If a commercial farmer owns a hundred cows and even fifty of them give birth to male calves, he cannot afford to raise them. They become a burden, said an expert.

Who pioneered the semen sorting technology and its large scale application on cattle?

George E. Seidel, a reproductive physiologist at Colorado State University (CSU) in the US is credited for his pioneering research in the sorting process. According to one of his interviews, some original research had taken place in the 1980s at the Lawrence Livermore National Laboratory in California and their studies of sperm led to discoveries regarding differences between sperm cells carrying X or Y chromosomes. However, for cattle, it was in the late 1990s that Seidel and his team at CSU developed a process for creating sex-sorted cattle semen for freezing and use in artificial insemination and also got patent rights. Through the CSU Research Foundation, they formed a company, XY Inc., which was later sold to Sexing Technologies (ST), based in Texas.

The sperms are pumped through a Flow Cytometer (cell sorter) in tubing past a detector that measures the brightness of individual sperm exposed to laser light. That information is processed by the computer and used to sort sperm, one at a time, at a rate of about 25,000 sperm/second, reads Seidels research paper titled Sex-selected semen.

Currently, ST has rights over semen sorting technology and it installs the entire set-up after taking the royalty amount, said Dr Inderjeet Singh, director, animal husbandry, Punjab and additional CEO, Punjab Livestock Development Board.

After ST, another US-based company ABS Global (formerly American Breeding Services) came up with a cutting edge technique that kills Y chromosome, he added.

Does sexed semen have a 100 per cent success rate?

No, the guarantee of a female calf being born is never 100 per cent. It can be up to 90 per cent. In 10 per cent cases, a male calf might be born despite using sexed semen because even after sorting/killing, some Y chromosomes may pass, said Dr Brar.

The pregnancy rate with sexed semen also reduces as sperm count goes down after sorting or killing. While one straw of conventional semen contains 10-20 million sperms, the count is down to 2-4 million only in sexed semen straws (0.25 ml each). So, while conventional semen will impregnate 60-70 animals out of 100, the rate is 45 to 50 with sexed semen, said Dr Narinder Singh, assistant animal physiologist, GADVASU.

How did sexed semen enter Punjab for farmers? When did the government start procuring and giving it on subsidy?

It was in 2008 that the Progressive Dairy Farmers Association (PDFA), the largest association in Punjab with more than 7,000 registered commercial dairy farm owners, had first imported sexed semen from the US. They have been doing it every year since then. Daljit Singh Sadarpura, president, PDFA, said, We first imported 25,000 sexed semen straws in 2008 from the US. The success rate is better on heifers than adult cows. Since then we are regularly importing it from the US and this year we gave it for Rs 1,500 to 1,700 per straw including transportation cost. Currently, very few rich farmers use it, he said.

In December 2011, the Punjab government imported 5,000 straws followed by 6,280 straws in 2014 and 25,000 straws in 2016- all from the US. Till August last year we were giving straws to farmers on 50 per cent subsidy, so a straw costing Rs 1,200 ($16-17 each) was being given for Rs 600. However, later the subsidy was reduced and it was being given for Rs 1,000. Now we have again proposed more than 75% subsidy and it might be given for Rs 300 only. We have floated tenders to procure sexed semen this year too, says Dr Inderjeet Singh.

How costly is sexed semen compared to conventional ones?

High quality conventional semen straws are available for just around Rs 20-40 per straw only whereas sexed semen costs at least Rs 1,200 per straw without subsidy. It is a costly affair till manufacturing does not start in Punjab itself and currently it is at least 60 times costlier than conventional semen, said Dr Narinder Singh.

Can sexed semen be used for indigenous cattle and buffaloes?

Dr Prakash Singh Brar from GADVASU said, In the 1960s, we bred our indigenous cows with foreign breeds to increase milk production but now governments focus is on promoting pure indigenous breeds. Cross-breeding can be done but it is not highly recommended. Sexed semen units are currently more focused on foreign breeds like Holstein Friesians (HF) and cross-bred bulls and sexed semen is mostly used on HF or cross-bred cows only. It can be produced for indigenous cow breeds like Sahiwal, Gir and also buffaloes like Murrah but in Punjab, majority farmers own foreign cross-bred cattle and problem of abandoning or killing male calf lies with cross-bred not indigenous cattle.

How can sexed semen solve the stray cattle problem in Punjab?

According to the latest Livestock Census 2019 data, there are 25.32 lakh cattle in Punjab of which only 4.26 lakh are indigenous breeds.

It is mostly foreign or cross-bred like HF and Jersey bulls and non-lactating cows which are abandoned by farmers on roads. If a male calf is born, it is also abandoned or killed. Farmers rarely abandon indigenous breeds. If sexed semen is used widely for cross-bred, stray cattle problem might be solved to some extent, says Dr Brar. Over one lakh stray cattle is on roads in Punjab approximately.

If only female calves will be born, can it lead to semen shortage?

No, say experts. We need a controlled male population with high quality genetic potential and they are being bred at semen stations. Semen is frozen and supplied to farmers. Controlled male population is enough to meet semen demand, said Dr Brar.

Sexed semen assures 90 per cent female births not 100 per cent. Also, it wont be possible to cover entire cow population with sexed semen so male births will be there anyway, says Dr Inderjeet Singh.

Is Giriraj Singhs statement to provide farmers with sexed semen Rs 100 per straw viable?

Experts are clueless on the calculation used by the minister here. Even if manufacturing starts in Punjab, initial project installation will cost crores. According to our initial estimates, we might have to pay royalty of $13 per straw (Rs 900 approx) to the operating company. Providing it to farmers for just Rs 100 seems too far-fetched as of now even if 50 per cent subsidy is given, said an official.

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Explained: What is sexed semen and how does the technology work? - The Indian Express

For marginalized people, the tech worlds constant barrage of innovations is getting exhausting. It seems like every week, science and tech pioneers are revealing new projects that pose a clear threat to anyone not white, cisgender, or malewhether its porn deepfakes or algorithms that judge womens boobs.

Enter Harvard Medical School, where researchers are creating a new dating app that matches people based on their DNA. The goal is to create a system that screens out matches that would result in a child with an inherited disease, according to a report aired Sunday night on 60 Minutes.

In other words, its a dating app for eugenicsthe disturbing ideological practice of systematically discriminating against people based on genetic qualities judged to be undesirable or inferior.

The app is being developed by a team of geneticists led by George Church, who, in the same interview, defended accepting money for his lab donated by convicted pedophile Jeffrey Epstein. Churchs lab is most famous for its work on the gene-editing technology CRISPR/Cas9, and its researchers are looking at ways to make humans immune to viruses, reverse the effects of aging, and de-extinct animals. Its 7,000 diseases, its about 5 percent of the population, [and] about $1 trillion a year worldwide in medical expenses, Church told 60 Minutes.

But for anyone not white, cis, able-bodied, or male, its obvious where all this is going.

Eugenics has long been a fascination of Nazis and white supremacists, who dream of creating a white and genetically pure master race. Dystopian sci-fi tales like Gattaca have also warned of the horrifying dangers of organizing society based on the perceived desirability (or undesirability) of peoples genetic code.

For people who exist outside mainstream gender norms, these dangers are very real. Last week, Newsweek reported on a team of researchers at the University of Michigan who are attempting to identify regions of the brain associated with gender dysphoriathe discomfort which occurs when a persons gender does not match the sex they were assigned at birth.

Many, but not all transgender people experience gender dysphoria, and it has been used to establish a system of medical gatekeeping that pathologizes trans people and controls access to treatments like hormone replacement therapy and gender-affirming surgeries. But even if scientists identified some hypothetical trait that causes people to be trans, choosing to edit out those traits would be an attempt to effectively erase trans people from existence.

Meanwhile, research into trans medical treatments remains severely underfunded. The federal government is also trying to make it legal for medical providers to refuse to treat trans patientswhether for gender dysphoria or a broken arm.

In other words, these cis researchers, funders, and policymakers seem more interested in curing or erasing trans people than finding better and cheaper ways of treating themor anyone else labeled as falling outside the norm of biologic desirability. Churchs lab, for example, recently received over $100 million for its work on gene-editing.

Church says he is being careful, and claims his lab has appointed a full-time ethicist on its staff to work toward the goal of genetic equitywhere all people have access to genetic technology, regardless of race or economic status.

But for marginalized people suffering under deeply unequal and discriminatory systems of power, that mission seems dangerously naive. If the people who risk being most harmed by these innovations arent intimately involved in their development, maybe its better toyou knownot make them at all?

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A Genetic Dating App Is a Horrifying Thing That Shouldnt Exist - Free

Chun-Jun Guo

Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA 94305, USA.Jill Roberts Institute for Research in Inflammatory Bowel Disease, Department of Medicine, Weill Cornell Medicine, NY 10021, USA.

Breanna M. Allen

Graduate Program in Biomedical Sciences, Departments of Otolaryngology and Microbiology and Immunology, Helen Diller Family Comprehensive Cancer Center, Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94143, USA.Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.

Kamir J. Hiam

Graduate Program in Biomedical Sciences, Departments of Otolaryngology and Microbiology and Immunology, Helen Diller Family Comprehensive Cancer Center, Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94143, USA.Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.

Dylan Dodd

Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Will Van Treuren

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.

Steven Higginbottom

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.

Kazuki Nagashima

Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA 94305, USA.

Curt R. Fischer

Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA 94305, USA.Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.

Justin L. Sonnenburg

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.

Matthew H. Spitzer

Graduate Program in Biomedical Sciences, Departments of Otolaryngology and Microbiology and Immunology, Helen Diller Family Comprehensive Cancer Center, Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94143, USA.Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.

Michael A. Fischbach

Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA 94305, USA.Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.

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Depletion of microbiome-derived molecules in the host using Clostridium genetics - Science Magazine

Incidence rates for hormone receptor positive (HR+) breast cancers are considerably higher in black men than white men, which is in stark contrast to lower incidence rates of those cancer subtypes in black versus white women to according to a new American Cancer Society appearing in the journalJNCI Cancer Spectrum.

In this study, researchers examined subtype specific breast cancer incidence rates in both black and white men in the U.S. using a contemporary nationwide database.

According to the study results, found that rates for all subtypes were higher among black than white men, with rates for HR+/HER- breast cancers about 41% higher among black men compared to white men; about 65% higher for HR+/HER2+, more than 2.5 times higher for HR-/HER2+, and 2.27 times higher for triple-negative breast cancer. Conversely, among women, rates in blacks were 21% lower for HR+/HER2- and comparable for HR+/HER2+, but 29% and 93% higher for HR-/HER2+ and triple-negative subtypes, respectively.

Reasons for the elevated risk of breast cancer in black men are largely unknown but may involve multitude of risk factors including genetic and non-genetic factors, the authors wrote according to a press release. Racial differences in the prevalence of mammography and menopausal hormone supplements are thought to have contributed to the historically higher incidence rate of HR+ cancers in white women, but these are not factors in breast cancer in men.

Well-known risk factors for male breast cancer include family history of breast and/or ovarian cancers, pathogenic mutations in BRCA2, radiation exposure, and conditions that alter hormonal balance such as Klinefelter syndrome and gynecomastia, and potentially obesity and diabetes. Moreover, previous studies have found higher level of estradiol was found to be associated with increased risk of male breast cancer after controlling for body mass index, suggesting a presence of estrogen-mediated carcinogenesis in male breast cancer. However, whether associations of these risk factors vary by tumor subtypes remains unknown and should be considered in future etiologic studies.

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Breast Cancer Incidence Differs Among Black and White Males - DocWire News

Hemp seed production is vital, as growers get ready to ramp up production. The question is, is there enough feminized hempseed to go around.

Western Farms Seed LLC in Scottsbluff will help in filling the demand, by growing seed for producers at its greenhouse.

The newly created business is owned by cousins, P.J. Hoehn, Mike Hoehn, their uncles Ed and Art Hoehn, and business partner Mark Johnson.

The business kicked off when Mike Hoehn received one of the 10 permits the Nebraska Department of Agriculture allotted to individuals in 2019 to grow hemp. Nebraska did a lottery where they allowed only ten businesses or individuals to grow hemp after the 2018 Farm Bill legalized the plant.

Hoehn grew a one-acre test plot outside of Mitchell, with three varieties of hemp seed.

The current varieties are Wife, Franklin, and Montana, we also have T1s, said P.J. Hoehn, president of the company. Well be crossbreeding them and making new varieties.

The three varieties have been proven to perform well for growers out in the field for the last couple of seasons.

Feminized seeds are bred explicitly in a way that eliminates the male chromosomes, drastically decreasing the chances of producing a male marijuana plant. Male marijuana plants are not desirable to any degree, except for pollination.

The genetics, which we have chosen are specific for industrial hemp, said Johnson, public relations for the company. We feel pretty safe that we wont have an impact from industrial hemps cousin (marijuana).

Western Farms Seed is also working in collaboration with the University of Nebraska-Lincoln through the Panhandle Research and Extension Center on hemp production.

Were producers ourselves, so we want to number one make sure the quality is there and everything else, our germination, our testing will all be there, Hoehn said.

He adds they are also making sure they will be able to advise growers on the equipment, such which as plates to use and vacuum. So, when farmers go to plant, they are ready, and if needed, Western Farms Seed would provide support in the knowledge of equipment, planting, and harvesting.

In terms of growing the crop, Johnson said a hemp crop is similar to corn or dry edible bean crops. Hemp should be planted by May or June and harvested after a 90 to 110 day growing period before frost.

We found hemp to be very resilient after our two hail storms this summer, said Johnson. The crop was able to recover from both hail storms in really good fashion. Ending up producing a nice crop in light of Mother Nature.

The business, with winter, has moved growing operations into the greenhouse. The five interconnected greenhouse buildings have 21,000 sq feet of growing spaces and house the female plants.

The plants will need light at different times, and when they enter the vegetative stage will need light for up to 16 hours a day.

Industrial hemp has two different growth stages, vegetative, which requires more light, and reproductive growth, said Johnson. So people might notice the greenhouse lights being on longer when we go to the next stage of production.

Both Hoehn and Johnson say producers should start small with an acre or so and of course, make sure they have a buyer before they even plant.

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Ag business to grow and market hemp seed - KTIC

While wrestling with the Christmas lights under his tree recently, a wave of sadness washed over Neil Turner. He couldnt help but think of his daughter Colby, who died in 2010 at just two years oldfrom a rare genetic disorder.

Suddenly, the thought of another Christmas without her swept in and replaced my frustration with tears, says Turner, an engineer in Oklahoma and father of two. Not a day goes by that I dont miss her and think about her. But if I focus on just the loss and the heartache, suicidal thoughts come quickly.

Grief isnt linear. It can hit by surprise. It is ongoing and it evolves, says Turner. It is acomplicated emotion for many people, and it can be particularly complex for fathers. Even today, dads might feel pressured to be strong for others and put their own feelings aside after a loss, which can have damaging psychological consequences. And although the expectations regarding so-called masculine behaviorare evolving for the better, many men still feel isolated in their grief and less comfortable opening up about it.

There is a deeply ingrained social conditioning that will take some work to undo and reverse, says David Klow, a licensed marriage and family therapist in the Chicago area and author of You Are Not Crazy: Letters From Your Therapist. A number of men are working to define new models of masculinity, but theres still a lot of work to be done.

Men are generally less willing to talk about their grief, more reticent to express emotion, and less likely to seek support, says Jan Everhart Newman, JD, Ph.D., a psychologist in Charlotte, North Carolina.

Sadly, this pattern can be reinforced when boys and men seek comfort after a loss around more vulnerable emotions such as sadness and are rebuffed and given messages like Dont cry or Stay strong, Newman says. Often, my male clients will report that another family member is more outwardly expressive of intense emotions and that they felt that they couldnt put any more stress on that person [by expressing their own grief].

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Grief from a male perspective has received little research interest, but some of the articles that have been written suggest that mens grief is often diminished or even dismissed. The authors of arecent study of combat veterans noted that grief is a long-overlooked toll of war. In her study of fathers and pregnancy loss, published in 2004, author Bernadette Susan McCreight wrote, the loss can be devastating for fathers yet, very often, the world that surrounds them tends to discount their loss, and emotional support and cultural rituals that are normally available to other bereaved individuals are often absent for this group of men.

Newman agrees. At the funeral of a Special Forces veteran recently, she saw a heartbreaking example of how people dont seem to know how to respond to mens grief. The manwas buried with full military honors, which can be a long affair.Kids clustered in a group poking one another and laughing, Newman says, while adults stood around together, somber and chatting. Then she saw the adult son,who was on his knees at the coffin sobbing entirely alone.

The only person who came to comfort him was his young son, Newman says. There is something about grief that can be frightening and is difficult for others to accept.

Human beings will do anything to avoid discomfort. As it makes them think of their own mortality and lack of control, death is at the top of the list of things that make people uncomfortable, she says. Additionally, traditional gendered expectations might influence how couples deal with grief. Klow says he has counseled women who say they want their male partners to be more in touch with their feelings but dont actually like seeing them cry or express emotions.

Some men might feel isolated in their grief not because they dont know how to feel emotions but because they dont feel its okay to express them.

A web content strategist in the UK, Kevin lost his father last year, shortly before he and his partner found out they were having a baby. He now lives in his fathers house with his family and thinks of his dad often, such as when hes dancing around the kitchen to The Beatles to entertain his son and get him to stop crying. Kevin says he often apologizes for talking about his father even though his partner says she doesnt mind.

It feels wrong that hes not here to enjoy the newborn, Kevin says. It will always feel socially unacceptable for me to express my grief no matter how hard people try to make me feel comfortable.

Cultural background and upbringing have a huge impact on how much men might adhere to stereotypical male tendencies, such as stoicism, that might make them feel less comfortable feeling and expressing grief. And it might be doing men a disservice to expect them to grieve more like women tend to, with outward shows of emotion, according to J. Scott Janssen, MSW, LCSW. Janssen says men who grieve more quietly and keep their emotions in check around others might simply have a more masculine style of grieving that isnt necessarily unhealthy and shouldnt be dismissed.

Of course, caveats exist. You have to be careful with the terms masculine and feminine, which are bound by culture and tradition, and in the age of gender neutrality, this distinction may even be meaningless, Newman says. It comes down to whether a man feels free to express his emotions without judgment and is simply choosing not to versus not expressing emotions because thats not what a man should do.

The latter situation a man feeling like a bad person because theyre experiencing normal, painful emotions is harmful.

There are signs that the walls around male grief are coming down. Recently, comedian Michael Cruz Kayne tweeted on the 10th anniversary of his son Fishers death and received an outpouring of support, as did James Van Der Beekwhen hewrote about the grief he and his wife felt about losing a baby to miscarriage in a heartfelt Instagram post. Comedian Patton Oswalt also has talked openlyabout grieving the death of his first wife, author Michelle McNamara, the mother of his daughter, Alice.

Many men (and women) need time to grieve privately, which is not the same thing as isolating.Although he also talked about his loss with others, Turner says he also needed alone time to process Colbys death.

For quite a few years, two hours into any car ride by myself I would be in tears having that much time alone with my thoughts, Turner says. But if I didnt get that time regularly, my emotions were more likely to come out sideways, in non-preferred ways.

Theres no timeline to it, Klow says. Ten years later, a long solo drive or the dog getting sick can trigger grief all over again. Healthy grieving changes from person to person.It can take a lot of different forms. To help process the loss, it can help to have a social gathering with friends and family to say goodbye and celebrate the life of the person who has died, says Elgin, Illinois, funeral home owner and director, US Army Reserve First Sergeant and father of two Dan Symonds.

Symonds was stationed in Afghanistan when his family told him his father was dying. He lost it for about 15 seconds in front of his Commander, he says, but didnt cry again for a while after his fathers death. He returned home and busied himself arranging military honors for his dad, an example of instrumental grieving that includes task like tending to the estate and cleaning out the house of the person who died. Those tasks shouldnt be dismissed as avoidance they can help people process the loss, Klow says.

Being alone with grief for stretches of time, however, isnt necessarily unhealthy. It can help to put thoughts and feelings into words, Klow says. Humans are social creatures; reaching out to social networks and naming the person theyre grieving and talking about memories and what theyre feeling tends to help.

What helps me is talking about my dad with my children, telling them what he was like and how he would have loved them so much, Symonds says. We keep his memory alive every day.

Klow suggests finding several people to listen to about grief; that can maximize someones avenues of support and alleviate the worry that theyre overburdening one person. That network can include a partner, family members, friends, or a therapist. Klow holds group therapy sessions for men and says many seem relieved to have a safe space in which to express themselves.

Its important not to be alone in grief, Klow says.

Someones partner can be a life-saving source of support, but they might have to work on making the relationship as egalitarian as possible, he adds: You dont have to be perfect, but both partners need to hold space for each other so that there isnt just one person whos the designated feeler of feelings, he says.

It can be difficult to do, but the Turners were able to give each other permission to be in different places in their grief.

We were okay if one of us was sad and the other was not. We werent afraid to give each other space, Turner says. We did see other couples that would get upset with one another with out-of-sync feelings of They need to move on or Why arent they still sad? Im not sure why, but we didnt fall into that trap.

A therapeutic retreat for bereaved parents, if it can fit into the budget, also might be helpful. Turner and his wife went to one after friends suggested it.

I had never been in any therapy session at all, and although it was emotionally and physically exhausting, we found it helpful, he says, but adds, The next year they even invited us back to help lead the retreat as we were the only couple in the group still married. The divorce rate among bereaved parents is really high.

The Turners also found a fulfilling way to process their grief through charity work with the American Heart Association. His daughter, Ella, got involved, too, raising more than $60,000 for the ACS after an event she participated in received media attention.

It gave us the opportunity to talk about Colby and use her story in a positive way, Turner says.

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For Men, Dealing With Grief Is Lonely and Isolating. This Needs to Change - Fatherly

A fathers diet can have a significant effect on the future health of his offspring, affecting everything from blood pressure to heart function and putting them at greater risk of cardiovascular disease, according to research.

The lead author of a British study says the findings show that men who want to start a family should have a healthy, balanced diet from at least three months before conception.

A study from researchers at the University of Nottingham published in the Journal of Physiology shows that poor paternal diet, specifically one that is low in protein, may impact the heart health of the offspring by changing sperm, and the seminal fluid, which bathes sperm.

We have known for a very long time that what a mother eats during pregnancy can influence how her child develops, and whether or not it will develop obesity, type two diabetes and heart disease, explains senior author of the study, and lead researcher, Prof Adam Watkins, assistant professor in reproductive biology at the universitys faculty of medicine and health sciences. However, the importance of the fathers diet on the health of the offspring has been largely ignored or overlooked. We were interested in investigating whether a fathers poor-quality diet at the time of conception might affect the long-term health of its offspring.

Researchers carried out their study on male mice on a poor, low-protein diet, monitoring the cardiovascular health of their offspring. The way mice produce sperm, the way the embryo develops, the way the foetus develops and the way a mouses blood and heart function are all very similar to humans. This means we can use mice to identify important biological processes which we can then look at in human patients.

What his research found, he reports, was both that the way the mices blood vessels worked, and the level of certain important factors in their blood, which regulate heart and blood vessel function, were significantly altered in response to the poor diet of the father: The blood vessels in the offspring did not work as well as they should do. This can ultimately affect blood pressure.

The normal proteins in the blood which would regulate blood vessels and heart function were altered, says Prof Watkins, adding that essentially what this meant was that the young mice were at increased risk of developing cardiovascular ill-health or heart disease.

We know that a poor lifestyle in men does have negative influences on sperm quality and that being overweight or smoking, or consuming excessive alcohol is not good for reproductive health. What we dont know yet is what the long-term implications of a fathers poor diet or lifestyle might be, he says.

We know that the sperm provides genetic information from a father to the egg it fertilises, and we know that poor diet in males can change that. We also know that the seminal fluid in which sperm is carried, interacts with the uterus and initiates a range of responses in the maternal immune system. These responses prime the uterus for the embryo.

We know that the sperm provides genetic information and that the seminal fluid primes the uterus for the embryo, so here are two possible ways that a fathers diet could influence how the offspring might develop.

Essentially, Prof Watkins explains, the Nottingham research shows that the health of mice offspring is influenced by sperm and fluid and that both of them have an equal influence on offspring health.

However, he says, while the research has to date only been carried out on mice, it has significant implications for human fertility in fact the researchers hope to run clinical trials on humans within the next two or three years.

We know that it can take about 75 days to make a sperm, and that seminal fluid is reproduced every 24-48 hours, says Prof Watkins.

If a man goes on a crash diet a week before getting his partner pregnant, he explains, the sperm will continue to reflect the old, poor quality diet,while the seminal fluid will reflect the newer, better-quality diet.

Therefore there may be a situation where the sperm and the fluid are not compatible to each other, so we are saying that if the sperm and the fluid are different, we see the biggest effect on offspring health.

The potential message is this, he warns: If men and women are thinking about changing their lifestyle and becoming parents, we would say that ideally they begin the changes three months before trying to start a family. That is an ideal time frame to change over from a poor diet and lifestyle to a healthier one in terms of its implications for the mans reproductive health.

The Nottingham research findings have interesting implications for what we know about the role of seminal fluid and sperm DNA fragmentation (a term used for the presence of abnormal genetic material within the sperm, which may lead to male subfertility, in-vitro fertilisation failure and miscarriage) believes Dr Bart Kuczera, consultant gynaecologist and fertility expert at Beacon Care Fertility:

What we know is that men with a poor lifestyle in terms of diet, smoking and drinking can have a condition called sperm DNA fragmentation.

Men are advised to live a healthy lifestyle in order to keep their sperm in the best condition, because, he explains: Sperm DNA fragmentation can be affected by poor diet, stress and overeating, for example. This study would make the case for a good diet and lifestyle for men; that is, a normal balanced protein diet.

Sperm quality of men in the western world, he warned, has been shown to have deteriorated in the last 40 years: We believe this is very linked to lifestyle and the environment, to the sedentary lifestyle and a poor diet which reaches the recommended carbohydrate level but would not include a diversity of food.

In the greater picture you could potentially have a population of children who would be affected in terms of physical health problems and weight gain as a result of the paternal diet at conception. It is important to spread the responsibility between the man and the woman at the time of conception, he says, adding that this study suggests that the father may have an equally significant impact on his offsprings health problems.

This study has implications for our knowledge about diet and lifestyle in terms of fertility and men should be made aware of it, believes Dr Hans Arce, fertility consultant and medical director of ReproMed, a leading Irish fertility and IVF clinic network. The majority of our knowledge in relation to diet and lifestyle in terms of fertility comes because we studied women. Women were the ones who got pregnant and they were the focus. We saw, for example, that women with obesity had children with a higher risk of obesity and diabetes.

However this study showed the offspring of male mice with poor diets ended up having the expression of inflammation, and more of a tendency to high blood pressure, for example.

Men should be made aware of this. Its something the schools, the public health service and the GP should be telling men about that our diets can affect their future childrens health. Studies like these have implication for human beings, he says, adding that the results point in the direction of the fact that the health of a man may have implications for the health of his offspring.

What this study says, he observes, is that a mans diet will not just affect his own health, but potentially has implications for the health of his offspring: We dont have proper human studies yet this is mice but it is pointing in that direction!

Lifestyle is the single biggest issue when it comes to fertility, believes consultant nutritionist Gaye Godkin.

Godkin believes the University of Nottingham study is a further endorsement of what she says, is the role of epigenetics in health outcomes from pre-conception health across the life course.

There is a growing body of evidence showing just how much the fathers diet impacts on the pre-conception phase, in terms of its impact on sperm and seminal fluid and from there on to the long-term health of his offspring.

Epigenetics, she explains, is the environment in which the sperm lives prior to penetrating the egg. Sperm is produced around every 75 days or so but new seminal fluid is produced every 24 to 48 hours.

If the man has a long-term poor diet, it will affect his sperm, she says, adding however, that a man can have healthy sperm, while at the same time his seminal fluid could be of much lower quality because of a poor diet just before conception.

Normal sperm carries DNA. A poor diet has a negative effect on the DNA and the DNA enzymes which in turn are crucial to the formation of a healthy foetus.

In fertility clinics, they measure the level of a condition called DNA fragmentation in the male sperm. This test shows the quality of the sperm. For years I have worked with men who have high levels of DNA fragmentation in their sperm. I believe that it is strongly linked to diet, as well as to lifestyle factors such as smoking, alcohol consumption, excess weight and the effect of pesticides.

While the Nottingham study was based on a mouse model, she says, its findings were moving in the right direction in terms of our understanding of the volatility of sperm quality and what affects it, as well as its relationship with the internal environment of the male body.

While there is no medical treatment available for DNA fragmentation, says Godkin, she has found that 90 days on a good-quality diet which also features a reversal of poor lifestyle factors can lead to fragmentation levels being significantly reduced to the extent that a couple are in a position to use their own sperm to achieve conception.

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Diets of fathers can affect future health of offspring, study finds - The Irish Times

This image shows Channelrhodopsin-eYFP neurons (green) expressed in the central amygdala (CeA) neurotensin (NTS) containing neurons. The magenta is antibody staining for the neuropeptide NTS. Credit: McElligott Lab

The UNC School of Medicine lab of Zoe McElligott, PhD, found that alcohol consumption is regulated by the activity of a particular set of neurons in a specific brain region, a discovery that could lead to a better understanding of why some casual drinkers develop an alcohol use disorder.

Scientists have known that a region of the brain called the central nucleus of the amygdala (CeA) plays a role in behaviors related to alcohol use and consumption in general. Its been less known which precise populations of brain cells and their projections to other brain regions mediate these behaviors. Now, UNC School of Medicine scientists discovered that specific neurons in the CeA contribute to reward-like behaviors, alcohol consumption in particular.

Published in the Journal of Neuroscience, this research pinpoints a specific neural circuit that when altered caused animal models to drink less alcohol.

Zoe McElligott, Ph.D. Credit: University of North Carolina Health Care

The fact that these neurons promote reward-like behavior, that extremely low levels of alcohol consumption activate these cells, and that activation of these neurons drive alcohol drinking in animals without extensive prior drinking experience suggests that they may be important for early alcohol use and reward, said senior author Zoe McElligott, Ph.D., assistant professor of psychiatry and pharmacology. Its our hope that by understanding the function of this circuit, we can better predict what happens in the brains of people who transition from casual alcohol use to subsequent abuse of alcohol, and the development of alcohol use disorders.

McElligott, who is also a member of the UNC Bowles Center for Alcohol Studies, set out to investigate if a population of neurons that express a specific neuropeptide (neurotensin or NTS) contributes to reward-like behaviors and alcohol drinking. She was especially interested in these neurons in the context of inexperienced alcohol use, such as when a person first begins to drink alcohol. Also, NTS neurons are a subpopulation of other neurons in this CeA brain region that have been implicated in anxiety and fear known as the somatostatin and corticotropin releasing factor neurons.

Using modern genetic and viral technologies in male mice, McElligott and colleagues found that selectively lesioning or ablating the NTS neurons in the CeA, while maintaining other types of CeA neurons, would cause the animals to drink less alcohol. This manipulation did not alter anxiety-like behavior. It also did not affect the consumption of other palatable liquids such as sucrose, saccharin, and bitter quinine solutions.

We found that these NTS neurons in the CeA send a strong projection to the hindbrain, where they inhibit the parabrachial nucleus, near the brainstem, McElligott said.

Mara Luisa Torruella Suarez. Credit: University of North Carolina Health Care

Using optogenetics a technique where light activates these neurons the researchers stimulated the terminal projections of the CeA-NTS neurons in the parabrachial and found that this stimulation inhibited the neurons in the parabrachial. When the scientists stimulated this projection with a laser in one half of the animals box, animals would spend more time where the stimulation would occur.

Animals also learned to perform a task to get the laser stimulation to turn on, and they would do this repeatedly, suggesting that they found this stimulation to be rewarding.

Furthermore, when we stimulated this projection, animals would drink more alcohol as compared to when they had an opportunity to drink alcohol without laser stimulation, McElligott said. In contrast to our study where we ablated the NTS neurons, laser stimulation of this parabrachial pathway also caused the animals to consume caloric and non-caloric sweetened beverages. When the animals were presented with regular food and a sweet food, however, laser stimulation did not enhance the consumption regardless of the mouses hunger state. This suggests that different circuits may regulate the consumption of rewarding fluids and solids.

McElligott and her graduate student Mara Luisa Torruella Suarez, the first author of this study, hope to explore how alcohol experience may change these neurons over time.

Would these cells respond differently after animals have been drinking high quantities of alcohol over time? McElligott said. We also want to discover which populations of neurons in the parabrachial are receiving inputs from these neurons. Fully understanding this circuit could be the key to developing therapeutics to help people with alcohol use disorders.###

Reference: Manipulations of central amygdala neurotensin neurons alter the consumption of ethanol and sweet fluids in mice by Mara Luisa Torruella-Surez, Jessica R. Vandenberg, Elizabeth S. Cogan, Gregory J. Tipton, Adonay Teklezghi, Kedar Dange, Gunjan K. Patel, Jenna A. McHenry, J. Andrew Hardaway, Pranish A. Kantak, Nicole A. Crowley, Jeffrey F. DiBerto, Sara P. Faccidomo, Clyde W. Hodge, Garret D. Stuber and Zo A. McElligott, 19 November 2019, Journal of Neuroscience.DOI: 10.1523/JNEUROSCI.1466-19.2019

The National Institutes of Health, The North Carolina Translational Clinical Science (NC TraCS) Institute, the Alcohol Beverage Medical Research Foundation, and The UNC Bowles Center for Alcohol Studies funded this research.

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Alcohol Consumption Is Regulated by Particular Set of Neurons in Specific Brain Region - SciTechDaily

There are some new kids on the block at the Old Man on His Back (OMB) Prairie and Heritage Conservation Area that will help to bring vitality and diversity to the bison herd.

The Nature Conservancy of Canada (NCC) is adding five young bison bulls to the herd as part of a revised management plan to grow the herd.

The bulls have already arrived at OMB and will be integrated with the herd during the annual roundup, when the herd is moved to their winter pasture.

It will definitely shake things up a little bit, because the existing bulls in the herd are all quite a bit older than these ones, NCC Program Director for Southwest Saskatchewan Michael Burak said. We don't anticipate that they'll consider them to be too much of a threat necessarily. There definitely will be a couple of squabbles here and there. It will really depend largely on the attitude of the five that we just brought in. If they're really trying to push boundaries and trying to test some of those older animals, they'll be put in their place pretty quickly.

The introduction of the young bulls during the annual roundup is done on purpose to make their arrival less noticeable, because there is a lot of movement and change during the herds shift from the summer to winter pasture.

Everybody is a little bit confused and there was a really big kind of change and a shift that happened, he said. They all go through a handling system. So they're all a little bit stressed out. It's kind of the perfect time to just push everybody out into their winter pasture at the same time and hopefully they will not notice that there's all of a sudden these extra individuals that weren't there previously.

The five bulls arrived about a month ago. They were released into a separate paddock to give them time to adjust to their new surroundings and their health can also be monitored before their introduction to the herd.

The date for the roundup will depend on when the herd decides to come into the corral, which usually only happens when there is already a good snow cover and they can be attracted with bales of hay.

All five bulls were born in 2017 and they are around two-and-a-half years old. They were born and raised in Canada, but their lineage can be traced back to the northern United States, including the bison herd at Theodore Roosevelt National Park in North Dakota.

They came from three different producers. Three are from different areas of Saskatchewan. Two are from a producer in the Prince Albert area and another one was obtained from a producer near Shaunavon. Two came from a producer in central Alberta.

The OMB herd is genetically pure plains bison and the new arrivals were evaluated to confirm they are also the same. Genetic samples were submitted to the University of California, Davis, where tests were done to determine the presence of any cattle genes in those animals. The Alberta producer has done regular genetic tests on his herd, and he picked bulls that he knew are genetically pure.

These bulls will have a lot more space to roam when they join the herd on the vast plains of the OMB conservation area.

I think it's definitely going to be a bit of a change for them, Burak said. They may not be used to having quite as much open space, but I don't think it would be too big a transition for them. Normally with bison, they tend to just want to stick socially close to each other.

The OMB conservation area covers 13,088 acres (5,297 hectares) and is located in the R.M. of Frontier, 15 kilometres west of Claydon and 210 kilometres southwest of Swift Current. NCC manages OMB as a working ranch, and the bison will graze on about 9,000 acres of land. The remaining 4,000 acres are used for annual grazing by about 200 cattle from around 11 neighbouring ranchers from early June to mid October.

We're really pleased with how the whole bison introduction and the bison program have been going, he said. We're definitely achieving the goals that we set for ourselves as far as keeping the property healthy and making sure the grass is nice and productive, and that it is being grazed in a sustainable way. We're really proud of the way that we are able to showcase the differences or the similarities as well between cattle and bison grazing.

The original introduction of a group of 50 two-year-old plains bison to the OMB conservation area took place in the winter of 2003. They came from Elk Island National Park in central Alberta. There were 25 males and 25 females, but over time the NCC staff realized this 50 per cent split between the two sexes did not work. There were too many male animals, and over time the composition of the herd was changed.

There are currently seven adult bulls (excluding the five new arrivals), 68 adult cow, and nine calves (six yearling heifers and three yearling bulls) that were kept back from the previous calving. The introduction of the five young bulls will help to diversify the genetics of the herd and the goal is to increase the size of the herd to about 100 breeding individuals.

We've been in the process of rewriting a management plan for that herd for probably the better part of a year now to shift towards managing them in a more natural kind of way, Burak said. That would involve the herd being larger for one, as well as we'll have more breadth within our age classes. Right now, the majority of our herd is about 10 years old and older, whereas in a natural herd you would probably have your largest age class from about four to eight years old and then you would have a continuum of younger animals and then some older.

The easiest and least expensive way to grow the herd will be to just keep their own calves back every year, but that will have a potentially negative effect on genetic diversity.

If we're keeping calves with only our current herd bulls left in there, then we have a pretty good chance that they'll actually end up bred back to their relatives or even their fathers, he explained. That's why we brought five more in so that we have a bit more variation and we can spread that out a bit more and dilute the effect of any inbreeding that we might see.

NCC staff still need to do more work to evaluate the carrying capacity of the conservation area as part of the implementation of the revised management plan. They will look at the kind of grass production on the property and that information will be compared with data on the forage needs of bison. They also have to take into account the needs of other foragers, and they will calculate the natural grazing rate of other wild ungulates such as deer.

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Five bulls joining bison herd at Old Man on His Back conservation area - Prairie Post

Interferon Insight

Uncontrolled type I IFN activity has been linked to several human pathologies, but evidence implicating this cytokine response directly in disease has been limited. Here, Duncan et al. identified a homozygous missense mutation in STAT2 in siblings with severe early-onset autoinflammatory disease and elevated IFN activity. STAT2 is a transcription factor that functions downstream of IFN, and this STAT2R148W variant was associated with elevated responses to IFN/ and prolonged JAK-STAT signaling. Unlike wild-type STAT2, the STAT2R148W variant could not interact with ubiquitin-specific protease 18, which prevented STAT2-dependent negative regulation of IFN/ signaling. These findings provide insight into the role of STAT2 in regulating IFN/ signaling in humans.

Excessive type I interferon (IFN/) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2, a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2R148W in homozygosity (but not heterozygosity) were hypersensitive to IFN/, which manifest as prolonged Janus kinasesignal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFN/ signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFN/ signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFN/ activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.

Type I interferons (including IFN/) are antiviral cytokines with pleiotropic functions in the regulation of cellular proliferation, death, and activation. Reflecting their medical importance, type I IFNs have been shown to be essential to antiviral immunity in humans (1), whereas their potent immunomodulatory effects have been exploited to treat both cancer and multiple sclerosis (2, 3).

IFN/ also demonstrates considerable potential for toxicity, which became apparent in initial studies in rodents (4) and subsequent clinical experience in patients (5, 6). Thus, the production of and response to type I IFNs must be tightly controlled (7). Transcriptional biomarker studies increasingly implicate dysregulated IFN/ activity in a diverse spectrum of pathologies ranging from autoimmune to neurological, infectious and vascular diseases (811).

The immunopathogenic potential of IFN/ is exemplified by a group of monogenic inborn errors of immunity termed type 1 interferonopathies, wherein enhanced IFN/ production is hypothesized to be directly causal (12). Neurological disease is typical of these disorders, which manifest as defects of neurodevelopment in association with intracranial calcification and white matter changes on neuroimaging, suggesting that the brain is particularly vulnerable to the effects of excessive type I IFN activity (9). A spectrum of clinical severity is recognized, from prenatal-onset neuroinflammatory disease that mimics in utero viral infectionAicardi-Goutires syndrome (13)to a clinically silent elevation of IFN activity (14).

However, the central tenet of the type I interferonopathy hypothesis, namely, the critical pathogenic role of type I IFNs (12), has yet to be formally established (15). Evidence for an IFN-independent component to disease includes (i) recognition that other proinflammatory cytokines are also induced by nucleic acid sensing, which might contribute to pathogenesis (16); (ii) imperfect correlations between IFN biomarker status and disease penetrance (14); (iii) the absence of neuropathology in mouse models of Aicardi-Goutires syndrome despite signatures of increased IFN activity (17); and (iv) the observation that crossing to a type I IFN receptor deficient background does not rescue the phenotype in certain genotypes (e.g., STING and ADAR1) (18, 19), although it does in others (e.g., TREX1 or USP18) (20, 21). Here, we provide concrete evidence of the pathogenicity of type I IFNs in humans, shedding new light on the critical importance of signal transducer and activator of transcription 2 (STAT2) in the negative regulation of this pathway.

We evaluated two male siblings, born in the United Kingdom to second cousin Pakistani parents. Briefly, patient II:3, born at 34 weeks + 6 days with transient neonatal thrombocytopenia, was investigated for neurodevelopmental delay at 6 months (which was attributed to compensated hypothyroidism). Aged 8 months, he presented with the first of three episodes of marked neuroinflammatory disease, associated with progressive intracranial calcification, white matter disease, and, by 18 months, intracranial hemorrhage (Fig. 1A). These episodes were associated with systemic inflammation and multiorgan dysfunction, including recurrent fever, hepatosplenomegaly, cytopenia with marked thrombocytopenia, raised ferritin, and elevated liver enzymes. Latterly, acute kidney injury with hypertension and nephrotic range proteinuria developed (see Table 1, Supplementary case summary, and table S1).

(A) Neuroimaging demonstrating calcifications [brainstem/hypothalamus (proband II:3, top), cerebral white matter/basal ganglia/midbrain/optic tract (sibling II:4, top and middle)], hemorrhages [occipital/subdural/subarachnoid (proband II:3, middle)], and cerebral white matter and cerebellar signal abnormality with parenchymal volume loss (both, bottom), accompanied by focal cystic change and cerebellar atrophy (sibling II:4). (B) Whole blood RNA-seq ISG profiles: controls (n = 5); proband II:3 (n = 4); and patients with mutations in: TREX1 (n = 6), RNASEH2A (n = 3), RNASEH2B (n = 7), RNASEH2C (n = 5), SAMHD1 (n = 5), ADAR1 (n = 4), IFIH1 (n = 2), ACP5 (n = 3), TMEM173 (n = 3), and DNASE2 (n = 3). (C) IFN scores (RT-PCR) of patients, parents, and n = 29 healthy controls. ****P < 0.001, ANOVA with Dunnetts posttest. (D) Renal histopathology in proband (400 magnification) showing TMA with extensive double contouring of capillary walls (silver stain, top left); endothelial swelling, mesangiolysis, and red cell fragmentation (top right); arteriolar fibrinoid necrosis (bottom left); and myxoid intimal thickening of an interlobular artery (bottom right, all hematoxylin and eosin). (E) Transcriptional response to JAK inhibitor (JAKi) ruxolitinib in both patients (RT-PCR).

HLH, hemophagocytic lymphohistiocytosis; EEG, electroencephalogram.

This clinical phenotype was reminiscent of a particularly severe form of type I interferonopathy. In keeping with this observation, IFN-stimulated gene (ISG) transcripts in whole blood, measured by RNA sequencing (RNA-seq) and reverse transcription polymerase chain reaction (RT-PCR), were substantially elevated over multiple time points at similar magnitudes to recognized type I interferonopathies (Fig. 1, B and C), notably without evidence of concomitant induction of IFN-independent inflammatory pathways (fig. S1). Disease in the proband, which not only met the diagnostic criteria for hemophagocytosis but also included features of a thrombotic microangiopathy (TMA) (Fig. 1D), was partially responsive to dexamethasone and stabilized with the addition of the Janus kinase (JAK) inhibitor ruxolitinib (Fig. 1E and fig. S2). Sadly, however, this child succumbed to overwhelming Gram-negative bacterial sepsis during hematopoietic stem cell transplantation.

Patient II:4, his infant brother, presented with abnormal neurodevelopment and neuroimaging in the neonatal period, characterized by apneic episodes from 3 weeks of age in conjunction with parenchymal calcifications and hemorrhage, abnormal cerebral white matter, and brainstem and cerebellar atrophy (Fig. 1A). Blood tests revealed an elevated ISG score (Fig. 1, B and C), anemia, elevation of D-dimers, and red cell fragmentation on blood film, together with proteinuria and borderline elevations of ferritin and lactate dehydrogenase; renal function was normal, and blood pressure was on the upper limit of the normal range for gestational age. Introduction of ruxolitinib led to prompt suppression of ISG expression in whole blood (Fig. 1E) and an initial reduction in apneic episodes, but neurological damage was irretrievable, and he succumbed to disease at 3 months of age. Mothers pregnancy with patient II:4 had been complicated by influenza B at 23 weeks gestation.

Whole-exome sequencing analysis of genomic DNA from the kindred, confirmed by Sanger sequencing (Fig. 2, A and B), identified an extremely rare variant in STAT2 (c.442C>T), which substituted tryptophan for arginine at position 148 in the coiled-coil domain (CCD) of STAT2 (p.Arg148Trp, Fig. 2C). The Arg148Trp variant was present in the homozygous state in both affected children and was heterozygous in each parent and one healthy sibling, consistent with segregation of an autosomal recessive trait (table S2). This variant was found in the heterozygous state at extremely low frequency in publicly available databases of genomic variation [frequency < 0.00001 in Genome Aggregation Database (22)], and no homozygotes were reported. A basic amino acid, particularly arginine, at position 148 is highly conserved (fig. S3). In silico tools predicted that this missense substitution was probably deleterious to protein function (table S2). STAT2 protein expression in patient cells was unaffected by the Arg148Trp variant, in contrast to the situation for pathogenic loss-of-expression STAT2 variants, which resulted in a distinct phenotype of heightened viral susceptibility (Fig. 2D) (23, 24). Filtering of exome data identified an additional recessive variant in CFH (c.2336A>G and p.Tyr779Cys; fig. S4) present in the homozygous state in II:3 but absent from II:4. We considered the possibility that this contributed to TMA in the proband, but functional studies of this variant showed negligible impact on factor H function (fig. S5).

(A) Pedigree, (B) capillary sequencing verification, (C) protein map, and (D) immunoblot (fibroblasts) showing normal expression of STAT2 protein. DBD, DNA binding domain; LD, linker domain; SH2, Src homology 2 domain; TAD, trans-activation domain.

The transcription factor STAT2 is essential for transcriptional activation downstream of the receptors for the innate IFN-/ (IFNAR) and IFN- and their associated JAK adaptor proteins. In the current paradigm (25), STAT2 is activated by tyrosine phosphorylation, associated with IFN regulatory factor 9 (IRF9) and phosphorylated STAT1 (pSTAT1) to form the IFN-stimulated gene factor 3 (ISGF3) to effect gene transcription by binding to IFN-stimulated response elements in the promoters of ISGs. Although loss-of-function variants in STAT2 increase susceptibility to viral disease (23, 24), evidence here suggested pathological activation. Germline gain-of-function variants have been reported in STAT1 (26, 27) and STAT3 (28, 29) but not hitherto STAT2. Consistent with the apparent gain of IFN activity associated with mutant STAT2R148W, we observed in patient fibroblasts (Fig. 3, A and B) and peripheral blood mononuclear cells (PBMCs; fig. S6) the enhanced expression of ISG protein products across a range of IFN concentrations. However, basal and induced production of IFNB mRNA by fibroblasts was indistinguishable from controls (Fig. 3C); nor was IFN protein substantially elevated in patient samples of cerebrospinal fluid (II:3) or plasma (II:4) as measured by a highly sensitive digital enzyme-linked immunosorbent assay (ELISA) assay (30), albeit samples were acquired during treatment (table S3). Thus, the response to type I IFNs, but not their synthesis, was exaggerated. This heightened IFN sensitivity was accompanied by enhancement of key effector functions, as revealed by assays of IFN-mediated viral protection (Fig. 3D) and cytotoxicity (Fig. 3E). Collectively, these data indicated that STAT2R148W was not constitutively active but rather resulted in an exaggerated response upon IFN exposure. To confirm that the Arg148Trp variant was responsible for this cellular phenotype, we transduced STAT2-null U6A cells (31) and STAT2-deficient primary fibroblasts (23) with lentiviruses encoding either wild type (WT) or STAT2R148W, recapitulating the heightened sensitivity of cells expressing the latter to IFN (Fig. 3, F and G, and fig. S7).

Unless stated, all data are from patient II:3 and control fibroblasts. (A) ISG expression (immunoblot, IFN for 24 hours) and (B) densitometry analysis (n = 3, t test). MX1, MX dynamin like GTPase 1; IFIT1, IFN-induced protein with tetratricopeptide repeats 1; RSAD2, radical S-adenosyl methionine domain containing 2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) IFNB mRNA (RT-PCR) external polyinosinic:polycytidylic acid (poly I:C) treatment (25 g/ml for 4 hours; n = 3, t test). US, unstimulated. (D) Antiviral protection assay (mCherry-PIV5). Twofold dilutions from IFN (16 IU/ml), IFN (160 IU/ml) n = 7 replicates, representative of n = 2 experiments (two-way ANOVA with Sidaks posttest). (E) Cytopathicity assay (IFN for 72 hours; n = 3, t test). (F) As in (A), ISG expression in STAT2/ U6A cells reconstituted with STAT2WT or STAT2R148W (immunoblot, IFN for 24 hours). (G) As in (B), n = 3 to 4, t test. Data are presented as means SEM of repeat experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s., nonsignificant.

To explore the underlying mechanism for heightened type I IFN sensitivity, we first probed STAT2 activation in IFN-stimulated fibroblasts. In control lysates, levels of pSTAT2 had almost returned to baseline between 6 and 24 hours of treatment despite the continued presence of IFN (Fig. 4, A and B). In contrast, pSTAT2 persisted for up to 48 hours in patient cells. This abnormally prolonged pSTAT2 response to IFN was also observed in PBMCs of both patients (fig. S8). Consistent with immunoblot data, immunofluorescence analysis showed persistent ( 6 hours) nuclear localization of STAT2 in patient fibroblasts after IFN treatment, at times when STAT2 staining was predominantly cytoplasmic in control cells (Fig. 4, C and D, and fig. S9). This was accompanied by continued expression of ISG transcripts for 36 hours after the washout of IFN in patient cells as measured by RNA-seq and RT-PCR (Fig. 4, E and F). Thus, the type I IFN hypersensitivity of patient cells was linked to prolonged IFNAR signaling.

All data are from patient II:3 and control fibroblasts. (A) pSTAT2 time course [immunoblot, IFN (1000 IU/ml)] and (B) densitometry analysis (n = 5 experiments, two-way ANOVA with Sidaks posttest). (C) Immunofluorescence analysis [IFN (1000 IU/ml); scale bar, 100 m; representative of n = 3 experiments] with (D) image analysis of STAT2 nuclear translocation (n = 100 cells per condition, ANOVA with Sidaks posttest). A.U., arbitrary units. (E) RNA-seq analysis of IFN-regulated genes (n = 3 controls) with (F) validation by RT-PCR (n = 3, two-way ANOVA with Sidaks posttest). CPM, read counts per million. (G) pSTAT2 decay (immunoblot). IFN (1000 IU/ml; 30 min) followed by extensive washing and treatment with 500 nM staurosporine (STAU). Times relative to STAU treatment. (H) No significant differences by densitometry analysis (n = 3, t test). Data are presented as means SEM of repeat experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

The IFNAR signaling pathway is subject to multiple layers of negative regulation that target STAT phosphorylation directlythrough the action of tyrosine phosphatasesor indirectly by disrupting upstream signal transduction (7). Prolonged tyrosine phosphorylation is reported with gain-of-function mutations in STAT1, in association with impaired sensitivity to phosphatase activity (27). By contrast, we observed no impairment of dephosphorylation of STAT2R148W in pulse-chase assays with the kinase inhibitor staurosporine (Fig. 4, G and H), implying instead a failure of negative feedback upon the proximal signaling events that generate pSTAT2.

To localize this defect, we analyzed by phosflow and immunoblot the successive activation steps downstream of IFNAR ligand binding in Epstein-Barr virus (EBV)transformed B cells from the proband (II:3) and a heterozygous parent (I:2). As was the case for STAT2 phosphorylation, we also observed prolonged phosphorylation of both JAK1 and STAT1 after IFN treatment (Fig. 5, A to D). This points to a defect in regulation of the most proximal IFNAR signaling events, upstream of STAT2 (7). We observed no evidence of this phenotype in cells bearing STAT2R148W in the heterozygous state, consistent with autosomal recessive inheritance and the lack of clinical disease or up-regulation of IFN activity in heterozygous carriers. This genetic architecture provides a notable contrast to gain-of-function mutations affecting other STAT proteins, all of which are manifest in the heterozygous state (2629).

Time course of IFN stimulation (1000 IU/ml) in EBV B cells from patient II:3 [homozygous (hom)], parent I:2 [heterozygous (het)], and n = 3 controls. (A) Immunoblot and (B) densitometry analyses. (C) Representative histograms (flow cytometry) and (D) mean fluorescence intensity (MFI). Data are means SEM of three repeat experiments (*P < 0.05, **P < 0.01, t test).

Known negative regulators of IFNAR signaling are suppressor of cytokine signaling (SOCS) 1 and SOCS3 (32) and the ubiquitin-specific protease 18 (USP18) (33). SOCS1 and SOCS3 participate in regulation of additional JAK-STAT signaling pathways, such as those activated by IFN and interleukin 6 (IL-6) (34, 35), whereas USP18 acts specifically upon IFNAR signaling (33). To better localize the molecular defect in patient cells, we examined the signaling responses to IFN (STAT1 phosphorylation) and IL-6 (STAT3 phosphorylation), based on the prediction that defects of SOCS1 or SOCS3 regulation would manifest under these conditions. These experiments revealed that regulation of STAT1 and STAT3 phosphorylation was normal in patient fibroblasts (fig. S10). Together with the absence of evidence of up-regulation of the IFN and IL-6 pathways in the analysis of whole blood RNA-seq data (fig. S1), these observations effectively ruled out the involvement of SOCS1 and SOCS3 in the clinical phenotype, leading us to suspect a defect of USP18 regulation.

To investigate this possibility, we primed patient and control cells with IFN for 12 hours, washed them extensively, and rested and restimulated them with IFN or IFN after 48 hours. In these experiments, IFN-induced pSTAT2 and pSTAT1 were strongly inhibited by priming in control cells, consistent with desensitization, a well-established phenomenon of type I IFN biology (Fig. 6, A and B) (36). In marked contrast, the response to IFN restimulation in patient cells was minimally suppressed, indicating a failure of desensitization. Desensitization has been shown to be exclusively mediated by USP18, an IFN-induced isopeptidase (37), through its displacement of JAK1 from the receptor subunit IFNAR2 (38, 39)a function that is independent of its isopeptidase activity toward the ubiquitin-like protein ISG15 (33). STAT2 plays a critical role as an adaptor protein by supporting binding of USP18 to IFNAR2 (Fig. 6C) (40). Both the clinical and cellular effects of STAT2R148W resemble homozygous USP18 deficiency, which was recently described as the molecular cause of a severe pseudo-TORCH syndrome associated with elevated type I IFN expression (table S4) (41). Although this STAT2:USP18 interaction has been shown to be essential for negative regulation of type I IFN signaling in vitro (40), its significance in vivo has not previously been examined. Furthermore, the precise residue(s) of STAT2 that bind USP18 were unresolved, although this interaction had been localized to a region including the CCD and/or DNA binding domain(s) of STAT2 (40).

(A) Desensitization assay (immunoblot, fibroblasts) with (B) pSTAT densitometry analysis (pSTAT/tubulin, ratio to unprimed; n = 4, ANOVA with Sidaks posttest). (C) Schematic of USP18 mechanism of action and proposed model of STAT2R148W pathomechanism. (D) Modeling of exposed WT (R148)/mutant (W148) residue, demonstrating charge-change (blue, positive; red, negative) and possible steric restriction. (E) Coimmunoprecipitation of USP18 by STAT2 in U6A cells expressing STAT2WT or STAT2R148W with (F) densitometry analysis (USP18/STAT2, ratio to WT; one-sample t test). Data are means SEM (**P < 0.01, ****P < 0.0001). IB, immunoblot.

Because USP18 was induced normally in patient cells (Fig. 6, A and B) and in vivo (Fig. 1B), our data implied that STAT2R148W impedes the proper interaction of STAT2 with USP18, compromising its regulatory function (Fig. 6C). Molecular modeling of STAT2R148W placed the substituted bulky aromatic tryptophan, and resulting charge change, at an exposed site within the CCD (Fig. 6D). Consistent with our suspicion that this might impair the STAT2:USP18 interaction through electrostatic or steric hindrance, coimmunoprecipitation experiments in U6A cells stably expressing WT or STAT2R148W demonstrated a statistical significance reduction of USP18 pull down STAT2R148W compared with WT (Fig. 6, E and F), providing a molecular mechanism for the USP18 insensitivity of patient cells.

Although disruption to the STAT2R148W:USP18 interaction was the most plausible explanation for the clinical and molecular phenotype, we also considered the contribution of alternative regulatory functions of STAT2. Beyond the role of tyrosine phosphorylated STAT2 in innate IFN signal transduction, the unphosphorylated form of STAT2 (uSTAT2) has additional, recently described functions in the regulation of other cytokine signaling pathways. For example, uSTAT2 negatively regulates the activity of IFN (and other inflammatory cytokines that signal via STAT1 homodimers) by binding to uSTAT1 via its CCD (42). This interaction appears to limit the pool of STAT1 available for incorporation into transcriptionally active (tyrosine phosphorylated) STAT1 homodimers. Conversely, uSTAT2, induced by type I IFN signaling, has been reported to promote the transcriptional induction of IL6 through an interaction with the nuclear factor B subunit p65 (43). To investigate the potential relevance of these regulatory functions of STAT2, we first examined the induction of IL6 by RT-PCR analysis of RNA isolated from whole blood of patients, their heterozygous parents, and healthy controls. We found no evidence of increased expression of IL6 or its target gene SOCS3 (fig. S11, A and B), consistent with our previous pathway analysis of RNA-seq data (fig. S1) and implying that STAT2R148W does not influence IL-6 induction. Next, to explore any impact on STAT2s negative regulatory activity toward STAT1, we examined the transcriptional responses to IFN in patient fibroblasts and in U6A cells expressing STAT2R148W. Although we were able to reproduce the previously reported findings of heightened transcription of the IFN-regulated gene CXCL10 in U6A cells lacking STAT2, alongside a nonsignificant trend for IRF1 (fig. S12, A and B) (42), STAT2R148W did not enhance transcript levels of either CXCL10 or IRF1 above WT, in agreement with the data showing the preserved ability of STAT2R148W to bind STAT1 in a coimmunoprecipitation assay (fig. S12, C and D). Together, these studies effectively exclude a contribution of the USP18-independent regulatory functions of STAT2 to the disease phenotype.

To conclusively demonstrate the impairment of STAT2:USP18-mediated negative regulation in patient cells, we tested the impact of overexpression or knockdown of USP18. First, we probed IFNAR responses in fibroblasts stably expressing USP18. As predicted, USP18 was significantly impaired in its ability to suppress IFN signaling in patient cells, relative to controls, both in terms of STAT phosphorylation (Fig. 7, A and B) and STAT2 nuclear translocation (Fig. 7, C and D), recapitulating our prior observations with IFN priming (Fig. 6A). The reciprocal experiment, in which USP18 expression was stably knocked down using short hairpin RNA (shRNA), revealed significantly prolonged STAT2 phosphorylation in control cells at 24 hours, recapitulating the phenotype of patient cells (Fig. 7, E and F). In contrast, there was no effect of USP18 knockdown in patient cells, demonstrating that they are USP18 insensitive. Incidentally, we noted that the early peak (1 hour) of STAT2 phosphorylation in USP18-knockdown control fibroblasts was marginally reduced (Fig. 7E). This subtle reduction was also apparent in STAT2R148W patient fibroblasts (Fig. 4B), although not in EBV B cells (Fig. 5). We speculate that the cell typespecific induction of other negative regulator(s) of IFNAR signaling at early times after IFN treatment, such as SOCS1, might be responsible for this observation. RT-PCR analysis confirmed the increased expression of SOCS1 mRNA in whole blood of patients (fig. S11C), whereas examination of RNA-seq data from IFN-treated fibroblasts revealed an eightfold enhancement of SOCS1 expression at 6 hours in patient cells as compared with controls (Padj = 0.0001; Fig 4E). Together, these data provide preliminary support for the hypothesis that alternative negative regulator(s) of IFNAR signaling may be up-regulated in patient cells. Nevertheless, such attempts at compensation are clearly insufficient to restrain IFNAR responses in the context of STAT2R148W, reflecting the nonredundant role of STAT2/USP18 in this process (39). Collectively, these data support a model in which the homozygous presence of the Arg148Trp STAT2 variant compromises an essential adaptor function of STAT2 toward USP18, rendering cells USP18 insensitive and culminating in unrestrained, immunopathogenic IFNAR signaling.

All data are from patient II:3 and control fibroblasts. (A) STAT phosphorylation in USP18 and vector expressing fibroblasts (immunoblot) with (B) pSTAT densitometry analysis (pSTAT/tubulin, ratio to unprimed; n = 3, ANOVA with Sidaks posttest). (C) Immunofluorescence analysis of STAT2 nuclear translocation [IFN (1000 IU/ml 30 min); representative of n = 3 experiments] with (D) image analysis (n = 100 cells per condition, ANOVA with Sidaks posttest). (E) Time course of STAT phosphorylation upon IFN stimulation (1000 IU/ml for 0, 1, 6, and 24 hours) of cells transduced with USP18 shRNA or nontargeting (NT) shRNA with (F) densitometry analysis of pSTAT2 (n = 3, t test). Data are means SEM (**P < 0.01, ***P < 0.001, ****P < 0.0001).

We report a type I interferonopathy, caused by a homozygous missense mutation in STAT2, and provide detailed studies to delineate the underlying molecular mechanism. Our data indicate the failure of mutant STAT2R148W to support proper negative regulation of IFNAR signaling by USP18revealing an essential regulatory function of human STAT2. This defect in STAT2 regulation results in (i) an inability to properly restrain the response to type I IFNs and (ii) the genesis of a life-threating early-onset inflammatory disease. This situation presents a marked contrast with monogenic STAT2 deficiency, which results in heightened susceptibility to viral infection due to the loss of the transcription factor complex ISGF3 (23, 24). Thus, just as allelic variants of STAT1 and STAT3 are recognized that either impair or enhance activity of the cytokine signaling pathways in which they participate (44), we can now add to this list STAT2. Our findings also highlight an apparently unique property of human STAT2: That it participates directly in both the positive and negative regulation of its own cellular signaling pathway. Whether this is true of STAT2 in other species remains to be determined. Our findings also localize the interaction with USP18 to the CCD of STAT2, indicating a specific residue critical for this interaction. This structural insight may be relevant to efforts to therapeutically interfere with the STAT2:USP18 interaction to promote the antiviral action of IFNs.

This monogenic disease of STAT2 regulation provides incontrovertible evidence of the pathogenic effects of failure to properly restrain IFNAR signaling in humans. The conspicuous phenotypic overlap with existing defects of IFN/ overproduction, particularly with regard to the neurological manifestations, provides compelling support for the type I interferonopathy hypothesis, strengthening the clinical rationale for therapeutic blockade of IFNAR signaling (15). JAK1/2 inhibition with ruxolitinib was highly effective in controlling disease in the proband; however, the damage that already accrued at birth in his younger brother was irreparable, emphasizing the importance of timely IFNAR blockade in prevention of neurological sequelae. A notable aspect of the clinical phenotype in patient II:3 was the occurrence of severe TMA. Our studies did not support a pathogenic contribution of the coinherited complement factor H variant in patient II:3. This evidence, together with clinical hematological and biochemical results suggestive of incipient vasculopathy in patient II:4who did not carry the CFH variantsuggests that type I IFN may have directly contributed to the development of TMA. Although it is not classically associated with type I interferonopathies, TMA is an increasingly recognized complication of both genetic (41, 42) and iatrogenic states of IFN excess (43), consistent with the involvement of vasculopathy in the pathomechanism of IFN-mediated disease. The fact that STAT2R148W is silent in the heterozygous state at first sight offers a confusing contrast with gain-of-function mutations of its sister molecules STAT1 and STAT3, both of which produce autosomal dominant disease with high penetrance (2629). However, the net gain of IFNAR signaling activity results from the isolated loss of STAT2s regulatory function, which evidently behaves as a recessive trait. There are other examples of autosomal recessive loss-of-function disorders of negative regulators, including USP18 itself (41, 45); the unique aspect in the case of STAT2R148W is that the affected molecule is itself a key positive mediator within the regulated pathway.

In light of the intimate relationship between STAT2 and USP18 revealed by these and other recent data (40), it is reasonable to conclude that the clinical manifestations of human USP18 deficiency are dominated by the loss of its negative feedback toward IFNAR rather than the STAT2-independent functions of USP18 including its enzymatic activity (40, 46, 47). In mouse, white matter pathology associated with microglia-specific USP18 deficiency is prevented in the absence of IFNAR (21). There are now three human autosomal recessive disorders that directly compromise the proper negative regulation of IFNAR signaling and thus produce a net gain of signaling function: USP18 deficiency, which leads to embryonic or neonatal lethality with severe multisystem inflammation (41); STAT2R148W, which largely phenocopies USP18 deficiency; and ISG15 deficiency, in which there is a much milder phenotype of neurological disease without systemic inflammation (45). ISG15 stabilizes USP18, and human ISG15 deficiency leads to a partial loss of USP18 protein (41). Thus, a correlation is clearly evident between the extent of USP18 dysfunction and the clinical severity of these disorders, with STAT2R148W closer to USP18 deficiency and ISG15 on the milder end of the spectrum (table S4). Those molecular defects that result in a failure of negative regulation of IFNAR signaling (i.e., STAT2R148W and USP18/) lead to more serious and extensive systemic inflammatory disease than do defects of excessive IFN/ production (41), suggesting that the STAT2:USP18 axis acts to limit an immunopathogenic response toward both physiological (48) and pathological (41) levels of IFN/. Thus, variability in the efficiency of this process of negative regulation might be predicted to influence the clinical expressivity of interferonopathies. Determining the cellular source(s) of physiological type I IFNs and the molecular pathways that regulate their production are important areas for future investigation.

Some limitations of our results should be acknowledged. Although strenuous efforts were made, we were only able to identify a single kindred, which probably reflects the rarity of this variant. As more cases are identified, our understanding of the clinical phenotypic spectrum will inevitably expand. Furthermore, for practical and cultural/ethical reasons, limited amounts of cellular material and tissues were available for analysis. As a result, we were unable to formally evaluate the relevance of STAT2 regulation toward type III IFN signaling; however, existing data suggest that USP18 plays a negligible role in this context (38). Together, our findings confirm an essential regulatory role of STAT2, supporting the hypothesis that type I IFNs play a causal role in a diverse spectrum of human disease, with immediate therapeutic implications.

We investigated a kindred with a severe, early-onset, presumed genetic disease, seeking to determine the underlying pathomechanism by ex vivo and in vitro studies. Written informed consent for these studies was provided, and ethical/institutional approval was granted by the NRES Committee North East-Newcastle and North Tyneside 1 (ref: 16/NE/0002), South Central-Hampshire A (ref: 17/SC/0026), and Leeds (East) (ref: 07/Q1206/7).

Dermal fibroblasts from patient II:3 and healthy controls were obtained by standard methods and cultured in Dulbeccos modified Eagles medium supplemented by 10% fetal calf serum and 1% penicillin/streptomycin (DMEM-10), as were human embryonic kidney 293 T cells and the STAT2-deficient human sarcoma cell line U6A (31). PBMCs and EBV-transformed B cells were cultured in RPMI medium supplemented by 10% fetal calf serum and 1% penicillin/streptomycin (RPMI-10). Unless otherwise stated, cytokines/inhibitors were used at the following concentrations: human recombinant IFN-2b (1000 IU/ml; Intron A, Schering-Plough, USA); IFN- (1000 IU/ml; Immunikin, Boehringer Ingelheim, Germany); IL-6 (25 ng/ml; PeproTech, USA); and 500 nM staurosporine (ALX-380-014-C250, Enzo Life Sciences, NY, USA). Diagnostic histopathology, immunology, and virology studies were conducted in accredited regional diagnostic laboratories to standard protocols.

Whole-exome sequencing analysis was performed on DNA isolated from whole blood from patients I:1, I:2, II:3, and II:4. Capture and library preparation was undertaken using the BGI V4 exome kit (BGI, Beijing, China) according to manufacturers instructions, and sequencing was performed on a BGISEQ (BGI). Bioinformatics analysis and variant confirmation by Sanger sequencing are described in the Supplementary Materials.

RNA was extracted by lysing fibroblasts in TRIzol reagent (Thermo Fisher Scientific) or from whole blood samples collected in PAXgene tubes (PreAnalytix), as described previously (49). Further details, including primer/probe information, are summarized in the Supplementary Materials and table S5.

Whole-blood transcriptome expression analysis was performed using nine whole blood samples, from the proband taken before and during treatment, and five controls. In addition, the four patient II:3 samples taken before treatment and samples from six patients with mutations in TREX1, three with mutations in RNASEH2A, seven with mutations in RNASEH2B, five with mutations in RNASEH2C, five with mutations in SAMHD1, four with mutations in ADAR1, two with mutations in IFIH1, three with mutations in ACP5, three with mutations in TMEM173, and three with mutations in DNASE2 were analyzed, as described in the Supplementary Materials. RNA integrity was analyzed with Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA purification and fragmentation, complementary DNA (cDNA) synthesis, and target amplification were performed using the Illumina TruSeq RNA Sample Preparation Kit (Illumina). Pooled cDNA libraries were sequenced using the HiSeq 4000 Illumina platform (Illumina). Fibroblasts grown in six-well plates were mock-treated or treated with IFN for 6 or 12 hours, followed by extensive washing and 36-hour rest, before RNA extraction. The experiment was performed with patient II:3 and control cells (n = 3) in triplicate per time point. RNA was extracted using the ReliaPrep RNA Miniprep kit (Promega) according to manufacturers instructions and processed as described above, before sequencing on an Illumina NextSeq500 platform. Bioinformatic analysis is described in the Supplementary Materials. PMBC and fibroblast STAT2 patient and control data have been deposited in ArrayExpress (E-MTAB-7275) and Gene Expression Omnibus (GSE119709), respectively.

Details of lentiviral constructs, mutagenesis, and preparation are included in the Supplementary Materials. Cells were spinoculated in six-well plates for 1.5 hours at 2000 rpm, with target or null control viral particles, at various dilutions in a total volume of 0.5 ml of DMEM-10 containing hexadimethrine bromide [polybrene (8 g/ml); Sigma-Aldrich]. Cells were rested in virus-containing medium for 8 hours and then incubated in fresh DMEM-10 until 48 hours, when they were subjected to selection with puromycin (2.0 g/ml) or blastocidin (2.5 g/ml) (Sigma-Aldrich). Antibiotic-containing medium was refreshed every 72 hours.

EBV B cells were seeded at a density of 8 105 cells/ml in serum-free X-VIVO 15 medium (Lonza, Basel, Switzerland) and stimulated with IFN (1000 IU/ml) for the indicated times. After staining with Zombie UV (BioLegend, San Diego, CA, USA), cells were fixed using Cytofix buffer (BD Biosciences, Franklin Lakes, NJ, USA). Permeabilization was achieved by adding ice-cold PermIII buffer (BD Biosciences, Franklin Lakes, NJ, USA), and cells were incubated on ice for 20 min. After repeated washing steps with phosphate-buffered saline (PBS)/2% fetal bovine serum (FBS), cells were stained for 60 min at room temperature with directly conjugated antibodies (table S6). Samples were acquired on a Symphony A5 flow cytometer (BD Biosciences) and analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA). The gating strategy is shown in fig. S13.

Immunoblotting was carried out as previously described (1) and analyzed using either a G:BOX Chemi (Syngene, Hyarana, India) charge-coupled device camera with GeneSnap software (Syngene) or a LI-COR Odyssey Fc (LI-COR, NE, USA). Densitometry analysis was undertaken using ImageStudio software (version 5.2.5, Li-COR). For complement studies, sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions was performed on patient/parental serum [diluted 1:125 in nonreducing buffer (PBS)] or affinity-purified factor H (diluted to 200 ng in nonreducing buffer), separated by electrophoresis on a 6% SDS-PAGE gel, and transferred to nitrocellulose membranes for immunoblotting (antibodies in table S6). Blots were developed with Pierce ECL Western blotting substrate (Thermo Fisher Scientific) and imaged on a LI-COR Odyssey Fc (LI-COR).

U6A cells were lysed in immunoprecipitation buffer [25 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate, and 10 mM sodium fluoride, with complete protease inhibitor (Roche, Basel, Switzerland)]. Lysates were centrifuged at 13,000 rpm at 4C for 10 min. Soluble fractions were precleared for 1 hour at 4C with Protein G Sepharose 4 (Fast Flow, GE Healthcare, Chicago, USA) that had been previously blocked with 1% bovine serum albumin (BSA) IP buffer for 1 hour. Precleared cell lysates were immunoprecipitated overnight with blocked beads that were incubated with anti-STAT2 antibody (A-7) for 1 hour and then washed three times in IP buffer before boiling with 4 lithium dodecyl sulfate buffer at 95C for 10 min to elute the absorbed immunocomplexes. Immunoblot was carried out as described above.

Fibroblasts grown on eight-well chamber slides (Ibidi, Martinsried, Germany) were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature before blocking/permeabilization with 3% BSA/0.1% Triton X-100 (Sigma-Aldrich) in PBS. Cells were incubated overnight with anti-STAT2 primary antibody (10 g/ml; C20, Santa Cruz Biotechnology, Dallas, USA) at 4C, and cells were washed three times with PBS. Secondary antibody [goat anti-rabbit Alexa Fluor 488 (1 g/ml), Thermo Fisher Scientific] incubation was performed for 1 hour at room temperature, followed by nuclear staining with 4,6-diamidino-2-phenylindole (DAPI; 0.2 g/ml; Thermo Fisher Scientific). Cells were imaged with an EVOS FL fluorescence microscope with a 10 objective (Thermo Fisher Scientific). The use of STAT2-deficient cells (23) demonstrated the specificity and lack of nonspecific background of the staining approach. Image analysis was performed in ImageJ. The DAPI (nuclear) image was converted to binary, and each nucleus (object) was counted. This mask was overlaid onto the STAT2 image, and the mean fluorescence intensity of STAT2 within each nucleus was calculated (see also fig. S9). About n = 100 cells were analyzed per image.

The structure of human STAT2 has not been experimentally determined. We therefore used comparative modeling to predict the structure. The sequences of both the WT and mutant were aligned to mouse STAT2 (Protein Data Bank code 5OEN, chain B). For each sequence, 20 models were built using MODELLER (50), and the one with the lowest discrete optimized protein energy score was chosen. Protein structures and electrostatic surfaces were visualized with PyMOL (Schrodinger, USA).

Fibroblasts grown on 96-well plates were treated with IFN (1000 or 10,000 IU/ml) or DMEM-10 alone for 72 hours. Cells were fixed in PBS containing 5% formaldehyde for 15 min at room temperature and then incubated with crystal violet stain. Plates were washed extensively then allowed to air dry. The remaining cell membrane-bound stain was solubilized with methanol and absorbance at 595 nm measured on a TECAN Sunrise plate reader (Tecan, Switzerland). Background absorbance was subtracted from all samples, and the results were expressed as a percentage of the absorbance values of untreated cells.

Fibroblasts grown on 96-well plates were pretreated in septuplicate for 18 hours with twofold serial dilutions of IFN and IFN, followed by infection with mCherry-expressing parainfluenza virus 5 (PIV5) in DMEM/2% FBS for 24 hours. Monolayers were fixed with PBS containing 5% formaldehyde, and infection was quantified by measuring mean fluorescence intensity of mCherry (excitation, 580/9; emission, 610/20) using a TECAN Infinite M200 Pro plate reader (Tecan, Switzerland). Background fluorescence was subtracted from all samples, and the results were expressed as a percentage of the fluorescence values of untreated, virus-infected cells.

Unless otherwise stated, all experiments were repeated a minimum of three times. Data were normalized/log10-transformed before parametric tests of significance in view of the limitations of ascertaining distribution in small sample sizes and the high type II error rates of nonparametric tests in this context. Comparison of two groups used t test or one-sample t test if data were normalized to control values. Comparisons of more than one group used one-way analysis of variance (ANOVA) or two-way ANOVA as appropriate, with posttest correction for multiple comparisons. Statistical testing was undertaken in GraphPad Prism (v7.0). All tests were two-tailed with 0.05.

immunology.sciencemag.org/cgi/content/full/4/42/eaav7501/DC1

Materials and Methods

Supplementary case summary

Fig. S1. Ingenuity pathway analysis of whole blood RNA-seq data.

Fig. S2. Longitudinal series of laboratory parameters.

Fig. S3. Multiple sequence alignment of STAT2.

Fig. S4. Factor H genotyping and mutant factor H purification strategy.

Fig. S5. Functional analysis of factor H Tyr779Cys variant.

Fig. S6. Immunoblot analysis of MX1 expression in PBMCs.

Fig. S7. Transduction of STAT2-deficient primary fibroblasts.

Fig. S8. Prolonged STAT2 phosphorylation in PBMCs.

Fig. S9. STAT2 immunofluorescence image analysis.

Fig. S10. STAT phosphorylation is not prolonged in patient cells in response to IFN or IL-6.

Fig. S11. RT-PCR analysis of gene expression in whole blood.

Fig. S12. STAT2R148W does not impair regulation of STAT1 signaling.

Fig. S13. Phosflow gating strategy.

Table S1. Laboratory parameters, patients II:3 and II:4.

Table S2. Rare variants segregating with disease.

Table S3. Digital ELISA detection of IFN protein concentration.

Table S4. Phenotypes of monogenic defects of USP18 expression and/or function.

Table S5. RT-PCR primers and probes.

Table S6. Antibodies.

Data file S1. Raw data (Excel).

References (5159)

Acknowledgments: We are grateful to the patients and our thoughts are with their family. Funding: British Infection Association (to C.J.A.D.), Wellcome Trust [211153/Z/18/Z (to C.J.A.D.), 207556/Z/17/Z (S.H.), and 101788/Z/13/Z (to D.F.Y. and R.E.R.)], Sir Jules Thorn Trust [12/JTA (to S.H.)], UK National Institute of Health Research [TRF-2016-09-002 (to T.A.B.)], NIHR Manchester Biomedical Resource Centre (to T.A.B.), Medical Research Foundation (to T.A.B.), Medical Research Council [MRC, MR/N013840/1 (to B.J.T.)], MRC/Kidney Research UK [MR/R000913/1 (to Vicky Brocklebank)], Deutsche Forschungsgemeinschaft [GO 2955/1-1 (to F.G.)], Agence Nationale de la Recherche [ANR-10-IAHU-01 (to Y.J.C.) and CE17001002 (to Y.J.C. and D.D.)], European Research Council [GA 309449 (Y.J.C.); 786142-E-T1IFNs], Newcastle University (to C.J.A.D.), and ImmunoQure for provision of antibodies (Y.J.C. and D.D.). C.L.H. and R.S. were funded by start-up funding from Newcastle University. D.K. has received funding from the Medical Research Council, Wellcome Trust, Kidney Research UK, Macular Society, NCKRF, AMD Society, and Complement UK; honoraria for consultancy work from Alexion Pharmaceuticals, Apellis Pharmaceuticals, Novartis, and Idorsia; and is a director of and scientific advisor to Gyroscope Therapeutics. Author contributions: Conceptualization: C.J.A.D., S.H., and T.A.B. Data curation: C.F., G.I.R., A.J.S., J.C., A.M., R.H., Ronnie Wright, and L.A.H.Z. Statistical analysis: C.J.A.D., B.J.T., R.C., G.I.R., F.G., D.F.Y., S.C.L., V.G.S., A.J.S., L.A.H.Z., C.L.H., D.K., and T.A.B. Funding acquisition: C.J.A.D., D.D., Y.J.C., R.E.R., D.K., S.H., and T.A.B. Investigation: C.J.A.D., B.J.T., R.C., F.G., G.I.R., D.F.Y., Vicky Brocklebank, V.G.S., B.C., Vincent Bondet, D.D., S.C.L., A.G., M.A., B.A.I., R.S., Ronnie Wright, C.L.H., and T.A.B. Methodology: C.J.A.D., B.J.T., R.C., F.G., D.F.Y., A.J.S., D.D., K.R.E., Y.J.C., R.E.R., C.L.H., and D.K. Project administration: C.J.A.D., K.R.E., S.H., and T.A.B. Resources: S.M.H., Robert Wynn, T.A.B., J.H.L., J.P., E.C., S.B., K.W., and D.K. Software: C.F., A.J.S., M.Z., L.A.H.Z., and Ronnie Wright. Supervision: C.J.A.D., K.R.E., Y.J.C., D.D., C.L.H., R.E.R., D.K., S.H., and T.A.B. Validation: B.J.T., R.C., A.J.S., V.G.S., and C.L.H. Visualization: C.J.A.D., B.J.T., R.C., and S.C.L. Writing (original draft): C.J.A.D., with B.J.T., R.C., S.H., and T.A.B. Writing (review and editing): C.J.A.D., G.I.R., A.J.S., S.C.L., M.Z., S.M.H., K.R.E., R.E.R., D.K., S.H., and T.A.B. Competing interests: The authors declare that they have no competing interests. Data and materials availability: GEO accession: GSE119709. ArrayExpress accession: E MTAB-7275. Materials/reagents are available on request from the corresponding author(s). MBI6 is available from Claire Harris under a material agreement with Newcastle University. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the UK Department of Health.

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Severe type I interferonopathy and unrestrained interferon signaling due to a homozygous germline mutation in STAT2 - Science

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The Cannadabis team is eager to unveil their propriety strains to the domestic and international medical markets. Over the past few years, the founders have started breeding their own cultivars. Currently, the team has focused on a selection of stabilised true breeds (landrace or F5+) for creating original F1 breeds.

Where the F1 generation is created by breeding male and female plants that are distinctly unique from each other; traditional F1s are created by crossing landrace indicas with landrace sativas.

These crosses need to be done with highly stable and uniquely different parents to produce a true F1 progeny that has abundant hybrid vigour. A plant with true hybrid vigour will typically have higher potency, increased pest resistance, and a higher yield than both parent plants; on average yield can be as high as 20% more than either parent.

Due to the nature of the F1 progeny, very few breeders release true F1 seeds. If highly stable progenitors are not used, the seedstock will be incredibly variable, which is unfavourable for consumers, who typically want consistency in their seed. However, as commercial cultivators, Cannadabis believes that F1 hybrids are essential for producing at large scale. The breeding and phenotyping can be a long and arduous process, the fruits of labour are not without commercial benefit.

Building upon the tissue culture and breeding practices, Cannadabis is quickly developing polyploidisation methods for creating ultra-premium cultivars. Polyploidisation is another common horticultural practice that Cannadabis expects to apply to their cannabis breeding projects.

Polyploidisation is a naturally occurring mechanism where the chromosomes of the plant cells become doubled within the same nucleus. This mechanism has played a significant role in speciation of crops, occurring frequently in nature, usually due to stress response.

In the 100 years since scientists discovered polyploidy, there has been rapid development of polyploid breeds. It is estimated that up to 80% of all flowering plants have polyploid varieties.2 Common polyploid cultivars includes wheat, coffee, banana, strawberry, potato, etc.

Polyploidy has been researched since the early 1900s. Scientists first used heat and electrical stress to induce those mechanisms. Today polyploidy is more commonly, and consistently, induced with radiation and stressing chemicals. Interestingly, induced polyploidy is explicitly exempt by most organic certification bodies. These types of breeds typically do not fall under genetically modified until foreign, non-similar species, DNA is introduced to the plant cell.

These polyploids are called autopolyploid (same species), and plants made with dissimilar species are called allopolyploids. Cannadabis will also be exploring organic permitted cell fusion; this would allow breeding with two male plants, or two female plants.

In the past, the following horticulture benefits have been derived from polyploidy and cell fusion, which Cannadabis hopes to similarly apply to the cannabis plant:3

The same can apply to cannabis. Strains can be developed that would never seed regardless of direct pollination; massive utility available to outdoor or indoor cultivators with seeding problems.

Cannadabis hopes to release their first polyploid strains in late 2020.

Cannadabis has begun manufacturing premade tissue culture mediums and are currently distributing them to Western Canadian horticulture stores and Amazon Marketplace; the mediums are a standard blend that works on 95%+ of the founders cultivars. The founders tissue culture experience is being provided to the public in both consumer and commercial grade products.

The introductory products show unfamiliar users how to do tissue culture at home, using proven methods that do not require expensive laboratory equipment. Besides what comes in the starter kit, the everyday home grower will usually have all the remaining materials at home. Commercial format mediums are intended for growers that want the best value and space savings.

Cultivators of any background can find information or help on tissue culture through the Cannadabis homepage. They are posting helpful videos and literature on cannabis tissue culture and hope to share the benefits with every grower. All horticulturalists, cannabis or not, can benefit from having their cloning area be 100x more efficient, through stackable containers. Furthermore, their mother plants can easily be maintained with minimal care. 100-1000 mother cultures can be stored within a refrigerator for 4-8 months, no adding nutrient or water. For larger cultivators, Cannadabis provides PGR matrices to more easily troubleshoot difficult cultivars. They also will custom blend and sterilise mediums to customer preference.

Cannadabis has begun developing an automated cell culture process for mass propagation of cultivars. The economies of scale of which are expected to change the supply chain of the entire cannabis industry. Automated cell culturing will provide starting materials to the industry at a fraction of the cost of inhouse cloning. Clones produced through cell culturing will also have the benefit of being totally sterile and free from disease.

Cannadabis has been offered an NRC-IRAP grant for initial developments of the process and are in early negotiations with a Canadian cannabis company to commercialise. The founders are expecting to file patents, mid 2020, and begin construction of a commercial scale process by mid-2021. Cannadabis anticipates that a 5000 sq ft facility will produce 5+ million clones annually, with minimal labour.

The project is looking to possibly incorporate the production of artificial seeds, which would simplify transportation and ease of storage for cultivators. They will also be developing cryogenic preservation methods. Cultivators around the world are encouraged to reach out to Cannadabis if they are looking to simplify their process, access cell culture benefits, and maximise growing space.

Working with Cannadabis cultured clones will be the most affordable, safe, and efficient way of acquiring starting material. Their services would include meristem culturing to remove systemic disease, and long-term storage of genetic inventory. Partners who end up with a pest could rest easy knowing their mother cultures will be perfectly preserved in tissue culture, and fifty thousand clones for the next crop are still on the way.

Cannadabis Medical and Delta 9 Cannabis have teamed up to provide an affordable, turnkey, tissue culture laboratory, complete with operating procedures, equipment, and cannabis medium recipes.

The two companies have co developed this system for their own commercial use and have recently made the system available for other cultivators. Both companies have recognised that the cannabis industry is still reliant on black market methods of propagation, and as a result, there have been countless incidents of crop and genetic loss in the legal industry; many of the stories circulating are understandably refuted by the companies experiencing such loss.

Rather than ignore the inevitable pest problems, the two companies are going toe to toe with mother nature, developing half century old technology and making it specifically for cannabis. Hopefully delivering the same modicum of control to the rest of the industry; cultivators slow to develop tissue culture science may soon find their genetics and crop totally destroyed by a single, often microscopic pest. On a commercial scale, these pests become essentially impossible to remove without the use of tissue culture.

With feet rooted in genuine care, Cannadabis and Delta 9 are prepared and excited to deliver a tissue culturing system to the global cannabis industry. They recognise the value and utility available to growers, and they also recognise that learning tissue culturing can feel out of reach for cultivators with no prior knowledge, or excess funding to hire an inhouse specialist.

Instead of missing out or paying specialists, cultivators can rely on Cannadabis and Delta 9 to deliver a ready to use laboratory, the development of which was based on maximising value for the growers.

The laboratory comes with only bare essentials and extensive, yet simple, operating procedures. Training materials will detail cannabis specific mediums, sanitation protocols, along with troubleshooting methods for finicky cultivars; an inexperienced grower will be comfortably blending and using mediums on the same day of commissioning. The whole system, equipment and all, will be much more affordable than hiring a tissue culture specialist.

Over the next three years, Cannadabis will be working to establish an expanded cultivation with the hope of supplying medical, organic, indoor grown cannabis to domestic and international markets.

They will also pioneer an original cell culture process that expects to be the most affordable source for starting materials in the world; Cannadabis is especially excited to deliver their polyploid cultivars as starting materials to industry members.

Cannadabis would like to offer an open invitation to all scientists, entrepreneurs, and industry professionals for collaboration. We are actively seeking partners who share a similar vision for the cannabis industry. Any professionals who are driven by a sense of genuine care and have a passion for cannabis medicine are encouraged to reach out.

References

1 hempindustrydaily.com/hemp-cultivators-tissue-culture-increase-propagation-preserve-genetics/2 Meyers, L. A., and Levin, D. A. (2006). On the abundance of polyploids in flowering plants. Evolution 60, 11981206. doi: 10.1111/j.0014 3820.2006.tb01198.x3 http://www.slideshare.net/ranganihennayaka/plant-polyploids4 http://www.frontiersin.org/articles/10.3389/fpls.2019.00476/full5 plantbreeding.coe.uga.edu/index.php?title=5._Polyploidy

Alexander CalkinsCEOCANNADABIS Medical INC+1 306 552 4242alexander@cannadabismedical.caTweet @cannadabiscannadabismedical.ca

This article will appear in the first issue ofMedical Cannabis Networkwhich will be out in January.Clickhereto subscribe.

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Cannadabis: tissue culture and the future of cannabis cultivation - Health Europa

Female cyclists are at a lower risk of suffering Sudden Cardiac Death than male athletes, but women should still learn about ways to screen for heart problems before engaging in endurance sports.

Dr. Mehreen Quhreshi is a cardiologist with advanced training in stress testing and cardiac imaging from Columbia University Medical Center in New York. She practices in Harrisburg, Pennsylvania and serves as the director of the Preventative Cardiology Program and the Nuclear Stress Lab at UPMC Pinnacle Heart and Vascular Institute. Dr. Bill Apollo, an amateur bike racer, runner, and duathlete is a Harrisburg, Pennsylvania-based cardiologist, who directs the UPMC Pinnacle Sports and Exercise Cardiology Clinic.

At the Paris Olympics in 1900, endurance sports were exclusively dominated by men; a mere 22 women participated, competing in the five gentrified events of croquet, equestrian, golf, tennis, and sailing. It took until the latter half of the twentieth century for the world to witness women competing in major Olympic endurance sports such as cycling (Los Angeles, 1984) and triathlon (Sydney, 2000).

Wider womens participation in the Olympics roughly coincided with the establishment of Title IX of the United States Educational Amendments of 1972, which mandated equal access for women in any program that received Federal funding including sports in public schools and universities. These two major developments fueled an explosion of female participation in a variety of events at all skill levels. The percentage of women finishers in marathons in the U.S. rose from only 10% in 1980 to a robust 45% by 2015. Women set a new record for Olympic participation at the 2016 Rio Olympics, with nearly equal numbers (5,176 athletes, or 45% of total), and with representation in all events included in the games.

Paradoxically, women have generally been under-represented in medical research studies looking at cardiac health, adaptation to endurance training and its potential consequences. Despite this surge of female athletic participation, we still havent achieved gender equality when it comes to understanding and caring for the female athletes heart. And recent small-scale studies suggest that there are in fact important cardiac differences between the sexes.

Some of the key questions are: to what extent do underlying genetic and hormonal factors impact normal changes in a womans heart related to exercise? How do these influences alter her risk for developing chronic heart problems or sudden cardiac death during competition? Are women better equipped to handle endurance training by design? Some recent research suggests that pregnancy subjects the female body to cardiac stresses similar to those that male athletes experience in even the most competitive events, including events like the Tour de France.

Below we examine the current understanding of cardiac development and risks in women endurance athletes, how and why women may differ from men in this regard, and recommended precautions that should be taken in training and competition by elite female endurance athletes.

Sudden cardiac death (SCD) during athletic competition is fortunately a rare occurrence, and it tends to affect men more commonly than women. In fact, a womans risk of SCD during endurance sports is estimated to be some 10 times lower than for her male colleagues. Professional cycling, during the past 3 seasons, has seen a total of 6 elite men tragically die directly from heart problems during races (5 in road racing, 1 on the track), with the most recent being Robbert de Greef in March 2019. During the same time period, there were zero incidents involving women, and indeed there are no known reports of SCD during elite womens cycling events for the past 20 years. Professional female cyclists are far more likely to die from training accidents (usually involving automobile collisions) than from heart problems.

Interestingly, these observations regarding SCD in cycling seem not to be true for other endurance sports. Marathon running has a huge participant base much larger than the womens pro peloton with nearly a half million participants in 2019 alone. This huge statistical sampling clarifies the measure of SCD risk: 1 incident per 150,000 participants overall, but more commonly occurring in men (1/ 100,000), and much less likely to occur in women (1/243,000).

Despite this fairly low risk of SCD in women, the sheer volume of running participants makes it easier to find reports of SCD. For example, Taylor Ceepo, age 22, died in May 2019 less than 1 mile from the finish line at the Rite-Aid Cleveland Marathon. The medical examiners report indicated that Ceepo experienced sudden cardiac death in association with physical exertion, pseudoephedrine use (a fairly benign over-the-counter decongestant) and cardiomyopathy. Her tragedy should remind us that even in very young and apparently healthy women, undiagnosed heart disease is still a common killer (3rd behind unintentional injuries and cancer in her age group), and her autopsy findings highlight the importance of screening women for underlying heart problems.

The most common causes of SCD are generally driven by age rather than sex. Athletes under age 35 both men and women alike are susceptible to genetically inherited structural heart problems including hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC), as well as potentially lethal heart rhythm problems called channelopathies. Above age 35, coronary artery disease predominates, with women being preferentially protected by their higher estrogen levels, until they reach menopause. Initially, the ten-fold higher incidence of SCD in men was thought to be simply due to the much larger numbers of men participating in endurance sports. But now that participation rates are becoming nearly equal, womens risk of SCD is still not as high as that experienced in the male population.

Several theories exist that might explain why women appear to be more protected from SCD during intense competition. One explanation may lie in the sympathetic nervous system, which is responsible for the bodys fight or flight response. Male physiology is observed to be wound more tightly, meaning that their arteries and blood vessels tend to constrict more during intense activity than women. The increased blood pressure adds resistance to blood the heart is pumping out. When this increased pressure load is coupled with an outpouring of adrenaline during competition, the strains placed on the heart may trigger lethal rhythm problems in susceptible individuals generally those with underlying inherited cardiac problems or acquired fibrosis (scarring) from long-term training. For unclear reasons, even in the context of equal training volumes, men more commonly develop potentially lethal fibrosis substrate, placing them at higher risk of SCD than women.

Another possible explanation relates to obvious hormonal differences between men and women. In some animal models, testosterone has been shown to affect the way the heart conducts impulses making men, at least in theory more susceptible than women to developing electrical instability resulting in malignant heart arrhythmias. Clinically, testosterone promotes thickening of the heart muscle, which may explain why men are more susceptible than women in developing complications from diseases like HCM and ARVC. Estrogens, on the other hand, are protective in this regard, and delay that same process of heart muscle thickening. Despite equal patterns of genetic transmission of HCM and ARVC between both sexes, hormonal differences may explain why these maladies tend to remain latent for a longer period of time in women, presumably translating to a survival advantage and lower risk of SCD.

Sports medicine screening programs are designed to identify potential cardiac risks in individuals who exhibit no outward symptoms of heart problems. Such programs aim to increase participation but to do so with a reasonable level of caution, to ensure the safety of the athlete. Despite the lower risk of SCD in women, screening is still important.

Pre-participation screening typically involves a comprehensive medical history review, focused physical examination, and in some cases an electrocardiogram (EKG). EKG tests are proven to be more sensitive than history and physical examination alone in detecting pathology, especially regarding heart rhythm issues. EKG interpretation should always be completed by a skilled reader able to distinguish the fine line between normal adaptation to exercise and pathology. Guidelines like the International Recommendations for EKG Interpretation in Athletes will increase reading accuracy and reduce the number of false findings, which often lead to expensive and unnecessary longitudinal testing. Men exhibit changes in their EKG patterns more often than women, and these variations in many instances are considered normal purely as the result of physiologic adaptation to training. On the other hand, women are less likely to stray from normal parameters, so most EKG changes are concerning and more likely represent a real problem.

Consistent endurance training induces physiologic remodeling, or normal adaptations to the heart resulting in improved efficiency of an athletes engine. Cyclists are unique because they typically perform the most prolonged exercise pattern more hours per day and more days per year than nearly any other athletes. Cyclists often sustain markedly elevated heart rates for extended periods of time during two distinct types of high cardiac output workouts. First, high intensity aerobic workouts at near peak efficiency, coupled with sustained elevations in heart rate, create a dynamic stress, or a volume load on the heart. And second, long tempo efforts punctuated by intense anaerobic dashes create static stress, exposing the heart to a pressure load because of sustained increases in blood pressure.

Cyclists therefore typically exhibit prominent changes in heart structure due to a combination of dynamic stress (volume overload) and static stress (pressure overload) resulting in generally increased cardiac mass, with mildly enlarged hearts and mildly increased heart wall thickness at least in men. Statistically, women are generally smaller than men with lower lean body mass. Due to their higher estrogen levels, women tend to adapt to exercise in a qualitatively similar manner, but quantitatively different than men showing only minimal heart enlargement and virtually no heart wall thickening. In fact, only about 7% of healthy women show any significant increase in their heart size due to habitual exercise, whereas 47% of men show cardiac enlargement.

Symptoms of heart problems in women are often different to those reported by men. For example, women are less likely to experience classic chest pain due to a heart problem, but may report more subtle symptoms like indigestion, heartburn, fatigue, or poor exercise performance. Misinterpretation of these sometimes confusing symptoms often leads to a delay in diagnosis and poorer long-term outcomes for women. An unexplained decline in athletic performance is obviously concerning to any elite athlete whether male or female because this may be the only clue to a serious underlying heart problem.

However, in young women, such nonspecific symptoms are often incorrectly blamed on things like menstrual problems, eating disorders, iron deficiency anemia, pregnancy, or thyroid disease. In many cases it is the womans primary care provider who must be savvy enough to exclude these other diagnoses, realizing there is a potential heart problem and then making an appropriate referral to a cardiologist.

Estrogen generally protects women from developing CAD at young ages, but the risk rises as they reach menopause. And paradoxically, some young women may actually be at increased risk for CAD because of a syndrome called Relative Energy Deficiency in Sports (RED-S). Sports which favor lean body mass are often associated with heavy training loads and dieting to achieve optimal body weight. In some women this results in the Female Athlete Triad of menstrual dysfunction, unexplained decline in performance (with or without an eating disorder), and decreased bone density, leading to increased probability of fractures.

Prolonged endurance training in young women can lead to menstrual irregularities resulting in the same kind of reduced estrogen levels typically seen in older postmenopausal women. These athletes should be evaluated for the more traditional cardiac risk factors such as high blood pressure, cholesterol problems, and diabetes, with appropriate intervention to modify their risk. Treatment of the Female Athlete Triad is challenging and may require a multidisciplinary approach to improve an athletes overall energy balance. Strategies include decreasing training volume, modifying dietary habits, medically replacing estrogen levels, promoting bone health with dietary supplements, and seeking appropriate professional help to correct eating disorders if present. Due to the focused and highly competitive nature of many endurance athletes, this is often a tall order to fill since they may resist decreasing their training volume.

Regular exercise is the cornerstone of prevention and treatment of many cardiac and non-cardiac diseases. But some researchers suggest that the benefits of exercise are like a drug the benefits of moderate training reach a plateau and exceeding that plateau, or overdosing, may be detrimental to the athletes health. Several studies have reported unexpected abnormalities in endurance athletes primarily in men suggesting either transient or permanent heart damage which puts them at risk for chronic heart issues. Findings have included a five-fold increased risk of atrial fibrillation (AFIB), increased coronary artery calcium deposits (which indicate clinically silent CAD), and scarring of the heart muscle. However, there are several general guidelines that all athletes should be aware of:

The biological adaptation to handle the stress of pregnancy may be a key reason for the apparently better female adaptation to endurance training. Recent research has highlighted that during pregnancy, the body functions at a basal metabolic rate of 2.2 times the normal burning up to 4000 calories a day. Extended over a period of 40 weeks, pregnancy can essentially be considered the ultimate endurance event a true test on the limits of human performance. Under typical circumstances, a body functioning above 2.5 times the normal metabolic rate over a prolonged period will begin to break down. But most women emerge from pregnancy and go on to live healthy lives, having tolerated a level of metabolic strain considered by some to be similar to that experienced by athletes participating in some of the most competitive endurance events.

There are also massive changes in the amount of fluid in a womans body during pregnancy, creating cardiac stresses similar to endurance training. In order to support the developing fetus, she must increase her blood volume by a massive 50%, and her cardiac output by 40-50% constituting the ultimate dynamic stress on the heart. The female body appears to require less adaptation by the heart muscle and chambers to accommodate these changes.

More overlap in research examining the similarities between the effects of endurance training in women and the cardiac demands placed on them during pregnancy may help to explain these gender-based differences in adaptation to exercise and related cardiac risk. Additional research specifically devoted to women is critical to a better understanding of how gender influences normal cardiac adaptation to exercise, as well as to more accurately identify pathologic conditions which sometimes seem to overlap with normal physiology.

Despite the substantially lower risk of SCD in women, cardiac risk screening of female endurance athletes and at-risk pregnant women is still important, and should be carried out by clinicians familiar with the differences in adaptive physiology between men and women. Women often experience challenging and atypical cardiac symptoms, requiring a high index of suspicion on the part of their doctors often at the primary care level to identify these underlying problems. As the current generation of elite female athletes matures into tomorrows Masters champions, we will undoubtedly learn a great deal more about the long-term cardiac implications of endurance training in women.

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The Outer Line: The impact of endurance training on the cardiac health of women - VeloNews