Archive for September, 2019

Images of child sex abuse have reached a crisis point on the internet, spreading at unprecedented rates in part because tech platforms and law enforcement agencies have failed to keep pace with the problem. But less is understood about the issue underlying it all: What drives people to sexually abuse children?

Science in recent years has begun to provide some answers. One thing most pedophiles have in common: They discover, usually as teenagers, that their sexual preferences have not matured like everyone elses. Most get stuck on the same-age boys or girls who first attracted them at the start of puberty, though some retain interest in far younger children.

People dont choose what arouses them they discover it, said Dr. Fred Berlin, director of the Johns Hopkins Sex and Gender Clinic. No one grows up wanting to be a pedophile.

[Read The New York Timess investigation into the spread of online child sex abuse.]

Over the past generation, psychologists, forensic specialists and others have studied pedophilia, a disorder characterized by recurrent, intense arousing fantasies, urges or behaviors involving sexual activity with a prepubescent child, according to psychiatrys diagnostic manual. These experts have interviewed patients in depth, piecing together life histories and performing a variety of psychological and anatomical measures.

While no study offers a complete picture, a portrait is emerging one that helps elucidate the mental dynamic behind the surge in abuse images and the deepening depravity they depict. These findings also defy common stereotypes about what pedophilia is, and what the risks are for engaging in physical abuse.

A majority of convicted offenders are men who prey on children ages 6 to 17. But women also commit hands-on offenses; rough estimates put the rate of pedophilic attraction at 1 to 4 percent in both men and women. Studies suggest that a small subset of male and female pedophiles have an interest in toddlers, or even infants.

As scientists seek to understand how the disorder develops, there is growing consensus that the origin is largely biological. This view is based in part on studies pointing to subtle physical traits that have a higher incidence among pedophiles.

The biological clues attached to pedophilia demonstrate that its roots are prenatal, said James Cantor, director of the Toronto Sexuality Center. These are not genetic; they can be traced to specific periods of development in the womb.

Psychological and environmental factors may also contribute, though it is not yet clear what those are or how they interact with developmental conditions.

By contrast, the common presumption that pedophiles were themselves abused as children now has less support. Child victims are at far greater risk of future substance abuse, depression, persistent traumatic stress or criminal aggression than of becoming molesters. The vast majority of offenders deny any sex abuse in their childhood, even though they could garner sympathy in court by doing so, experts say. A chaotic childhood increases the likelihood of a chaotic adulthood, of any kind, Dr. Cantor said.

The relationship between viewing or collecting images and committing hands-on abuse is a matter of continuing debate among some experts, and one that is critical to evaluating the risk an offender poses. Until recently, the prevailing view was that only a minority of people caught viewing such images, between 5 and 20 percent, also committed physical abuse.

That perception began to change in 2007, when a pair of psychologists at the Federal Bureau of Prisons reported that 85 percent of convicted online offenders acknowledged in therapy that they had raped or otherwise sexually abused children.

That finding circulated widely before the study was formally published, creating an uproar among therapists, researchers and law enforcement specialists. The prisons bureau balked at publishing it at all, and withdrew it from a peer-reviewed journal close to its release date.

Many cited concerns that the study sample was biased: It was based on the confessions of 155 convicts who had sought out therapy in prison, not on a representative sample of pedophiles, a much broader group with diverse habits.

It was what we call a convenience sample that was a legitimate criticism, said Michael L. Bourke, a co-author of the study with Andres E. Hernandez, in a telephone interview. Dr. Bourke is now chief of the behavioral analysis unit of the United States Marshals.

Since then, several other studies have supported the prison finding, if not precisely the 85 percent number. In one, inspectors from an array of government agencies interviewed 127 online offenders shortly after their arrests. Less than 5 percent admitted to previously molesting at least one child.

When agents followed up with more in-depth, polygraph-assisted methods, another 53 percent admitted to hands-on offenses, for a total of nearly 60 percent.

This was not a convenience sample; these were offenders, some of whom had downloaded just a single image, with no known history, from all over the country, interviewed by people from different agencies, Dr. Bourke said. They had zero incentive to admit to a previous offense very much the opposite.

The high rate of previous, hands-on offending undermines another common assumption about pedophiles. We shouldnt assume that viewing online images leads to abuse of a child victim in person, said Joe Sullivan, a specialist in sex crimes against children in Ireland and Britain. In my clinical experience, its the other way around. Most of these men have already committed hands-on offenses.

From this point of view, downloading abuse images and especially connecting with groups of like-minded pedophiles online does not awaken latent desires. The desires are very much awake and, in many cases, have already been acted on. But the images and online communities can help erode inhibitions further, drawing pedophiles into more frequent or more aggressive acts, Dr. Bourke said.

What you see, in their search histories, he said, is that they learn how to evade law enforcement, they become more confident and they begin to use cognitive distortions to overcome their moral inhibitions.

Some therapists and researchers say these findings from law enforcement threaten to unfairly tar people who never act on their desires. This group certainly exists theyre sometimes called virtuous pedophiles but in an era of increasing alarm over the proliferation of online abuse, they are going only further underground.

That is a shame, a tragedy, Dr. Cantor said. That is the group we need to learn about. Thats the kind of person wed like our clients to become, a person whos aware of the urges and learns to effectively manage them.

Learning to manage a drive as visceral, and often consuming, as sexual desire is possible, therapists say, but it cannot be shut off; nor can it be replaced, the way heroin can be swapped for methadone. Treatment can require drugs that reduce circulating testosterone and software that limits online browsing habits.

Often, therapy addresses substance abuse as well. Studies suggest that at least 40 percent of sex offenders were using drugs or alcohol when they committed their crimes.

The important thing, I think, is that people know that treatment is possible, Dr. Berlin said. Theres a subgroup out there, they refer themselves here, and they are quite convinced that they do not want real-life sex with children.

Original post:
Preying on Children: The Emerging Psychology of Pedophiles - The New York Times

Like the deserts of the Antarctic, or the deepest parts of the sea, Mono Lake in California is an inhospitable place for most life forms. Apart from bacteria and algae, it appears only brine shrimp and diving flies can put up with its super-salty waters.

But there's more to this body of water than meets the eye. Researchers at the California Institute of Technology have recently discovered eight more species of microscopic worm thriving in and around the lake, and one of them is a brand new kind of weird.

One of the newly-discovered species of nematode - for now called Auanema sp. - has not one, not two, but three different sexes, the team reports, and it can survive a dose of arsenic 500 times what is humanly possible.

When it comes to differentiation of the sexes, nematode species usually keep it simple, dividing into hermaphrodites and males. But Auanema sp. also has worms of the female sex. Furthermore, they have other interesting sex characteristics, as the researchers note "the arrangement of genital papillae in Auanema sp. males is unique in the genus."

As if that's not radical enough, the team says this microscopic worm also gives birth to live offspring, a unique approach in the typically egg-laying nematode world.

It's an extreme creature in an extreme place, and that's probably not a coincidence. The team thinks this worm's strange characteristics are part of what keeps it alive in the hyper-salty, alkaline waters of Mono Lake.

"Extremophiles can teach us so much about innovative strategies for dealing with stress," says Pei-Yin Shih.

"Our study shows we still have much to learn about how these 1,000-celled animals have mastered survival in extreme environments."

Comparing the strange new species of nematode to others in the same genus, the researchers found a similarly high arsenic resistance among two sister species.

And yet the curious thing was, none of these creatures actually lived in environments with high arsenic levels. There had to be another reason for this astonishing tolerance.

"Previous Auanema species were isolated from rich soils and dung, which can contain high concentrations of phosphate," the authors suggest.

"Since arsenic uptake occurs adventitiously via phosphate transporters, it is conceivable that adaptation to high levels of phosphate in the environment could lead to increased arsenic resistance as well."

In other words, nematodes might be pre-adapted to life as an extremophile. They could have a genetic resiliency and flexibility that makes it easier for them to live in harsh places like Mono Lake.

Before this study, only two other species had been found in this lake - which is three times as salty as the ocean and has an alkaline pH greater than baking soda. Yet even still, the discovery of eight more species wasn't all that surprising to researchers.

Nematodes are the most abundant type of animal on the planet, so even in the harsh environment of Mono Lake, there's a good chance you'll find them.

Over the course of two years, researchers from Caltech isolated nematodes from across the lake and found multiple niches in which these nematodes were thriving. And their numbers included microbe grazers, parasites and predators.

"Thus, nematodes are the dominant animals of Mono Lake in species richness," the authors conclude.

"Phylogenetic analysis suggests that the nematodes originated from multiple colonisation events, which is striking, given the young history of extreme conditions at Mono Lake."

To say these creatures are opportunistic is an understatement. For every human on Earth, there are roughly 57 billion nematodes, and in barely no time at all, these creatures can set up shop in some of the most extreme places on Earth.

"It's just so cool finding another place where there are lots of nematodes and not much else," geoscientist Tullis Onstott who was not involved in the study told The Scientist.

And who knows, maybe when there's not much else, there will still be nematodes.

The findings were published in Current Biology.

The rest is here:
A Worm With Three Sexes Has Been Discovered Thriving in a Nearly Lifeless Lake - ScienceAlert

Fans greeted the news that Sam Neill, Laura Dern and Jeff Goldblum will join the cast of Jurassic World 3 with a roar of approval. Is this a shot in the arm the franchise needs after the relative disappointment of Fallen Kingdom, or is this Universal shamelessly pandering to nostalgia?

Maybe its both. The return of the beloved principals of the first Jurassic Park is certainly welcome, but how effective that return is depends on how well they are used.

The first Jurassic World in 2015 was a huge success, making $652 million here and $1.7 billion worldwide, making it the franchise leader before you adjust for inflation. Although some critics knocked it for being sexist and too derivative of Jurassic Park, it was generally well reviewed, with 72% on Rotten Tomatoes.

Fallen Kingdom came out three yearslater, and something was missing enthusiasm. The sequel made $417 million here, a disappointment somewhat salvaged by making $1.3 billion worldwide, but the reviews hit a franchise low of 48%. Fans and critics alike noted the dour tone, with a structure that seemed too close to The Lost World. Both the 1997 and 2018 contrived reasons to return to dinosaur islands failed to capture the wonder of their predecessors. Both movies also unleashed the dinosaurs on the mainland.

One aspect of the sequel that burned some fans was that the trailers highlighted an appearance by Goldblums Ian Malcolm, but he turned out only to have a cameo, with almost his entire performance contained in the trailer. One hopes Universal isnt making the same mistake again with Dern and Neill, although it sounds like all three will be more central to the story this time.

Jurassic World 3, due out in June 2021, will deal with the aftermath of the dinosaurs reaching the mainland. The Lost Worlds third act dealt with this too, but it cleaned up the mess relatively quickly. That movie had one dinosaur stomping around, while this one will have a whole bunch.

Colin Trevorrow, returning to the directors chair after only co-writing Fallen Kingdom, told EW the storywill be focused storytelling with dinosaurs all over the world. We really wanted this technology, this genetic power, to go open-source at the end of the film. What were suggesting is not just that these specific animals that we care about that were in captivity were freed, but also that the ability to create these animals has gone a little bit wider than our friend Dr. Wu. The open-sourcing of any technology, like nuclear power, thats the scary side for me.

In other words, it sounds like we get not only your traditional T-Rexs and velociraptors, but your mutant hybrid dinosaurs that caused so much trouble in the previous movies. Those greedy humans just never learn, do they? Chris Pratt and Bryce Dallas Howard return as well.

Many fans were delighted to see Neill, Dern and Goldblum back in the mix, with one person on Instagram saying, BEST DAY EVER! YOU GUYS KNOW HOW TO MAKE HAPPY THE FANDOM! A more cynical commenter on EW said, The original title of this story was: Universal resorts to Disney tactics by playing on Gen X nostalgia. (and Millennials disdain for watching ancient 90s movies.)

It almost goes without saying that Jurassic World 3 will make a ton of money. One hopes, however, that Universal isnt just handing out the rose-colored glasses of nostalgia. Sam Neill and Laura Dern came back for Jurassic Park III, and that movie in some corners is like the forgotten stepchild, so theres no guarantee their return will get the franchise back on course.

To be fair, dinosaurs roaming the mainland isnt something the franchise has played with for an entire movie, and if it fulfills the promise of the short film that came out, then Jurassic World 3 will make a good capper for the franchise provided they stop there.

See the original post here:
'Jurassic World 3': The Real Reason the Movie Needs Original Characters to Return - Showbiz Cheat Sheet

Global Male Hypogonadism Market: Snapshot

Hypogonadism in males refers to a condition in the male body where the testes show a significantly reduced level of functioning than normal. The overall result of male hypogonadism is a reduction in the rate of biosynthesis of male sex hormones. This state is more commonly known as interrupted stage 1 puberty. Hypoandrogenism, or the low androgen or testosterone level in a male can vary in severity from person to person. It is often the cause of partial or complete infertility. There are multiple forms of male hypogonadism and even more ways to classify them. Most endocrinologists commonly classify male hypogonadism on the basis of the level of defectiveness of the male reproductive system.

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In many cases, doctors also measure the level of gonadotropins to classify a patient between primary and secondary male hypogonadism. Primary male hypogonadism refers to the cause of the condition being due to defective gonads. There are different types of primary male hypogonadism, including Turner syndrome and Klinefelter syndrome. Secondary male hypogonadism is caused by defects in pituitary or hypothalamic glands. They include Kallmann syndrome and hypopituitarism.

Global Male Hypogonadism Market: Overview

Male Hypogonadism refers to a clinical condition, wherein the testes fail to produce enough testosterone leading to delayed puberty or incomplete development. The condition is related to impaired development of muscle mass, development of breast tissues, impaired body hair growth, and lack of deepening of the voice.

The male Hypogonadism market can be segmented by therapy, type, drug delivery, and geography.

The report presents an in-depth analysis of the global male hypogonadism market with current trends and future estimates to explain the imminent investment pockets. The quantitative analysis of the market for the forecast period from 2017 to 2025 will enable stakeholders to capitalize on the prevailing growth opportunities.

Global Male Hypogonadism Market: Trends and Opportunities

The top driver of the male hypogonadism market includes rising prevalence of testosterone deficiency among men, increasing infertility rates, and increasing awareness among individuals about hypogonadism treatment due to awareness drives organized by several governments across the world. Moreover, high risk of hypogonadism among the geriatric population with obesity and diabetes, and increasing prevalence of chronic disorders among the geriatrics are further expected to boost the markets growth.

However, factors such as high side effects of testosterone products are challenging the growth of testosterone replacement therapy market. Top players in the market are focused on research and development to introduce newer products with fewer or negligible side effects and improved results. For example, LPCN 1111, a product which is under development from Lipocine Inc., is a newer testosterone prodrug that utilizes Lipral technology for enhanced systemic absorption and for enhanced solubility of testosterone. Nevertheless, technological advancements are anticipated to extend new opportunities to the markets growth.

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Global Male Hypogonadism Market: Regional Overview

The global male Hypogonadism market can be analyzed with respect to the regional segments of North America, Asia Pacific, Europe, Latin America, and the Middle East and Africa. North America held the majority share of the global market in the recent past and is expected to retain its dominant position in the near future. This is mainly due to the rise in the number of individuals suffering from primary and secondary conditions of hypogonadism, and rising awareness among individuals about treatment options for the condition. Moreover, the presence of ultra-modern healthcare infrastructure and increasing popularity of technologically advanced products are expected to offer new opportunities for top players in this market. The region is closely followed by Europe.

Asia Pacific is expected to offer lucrative opportunities to this market due to the modernization of the healthcare infrastructure in the emerging economies of India and China and the increasing awareness about the treatment for the condition. In Asia Pacific, the increasing prevalence of hypogonadism and infertility rates along with the rising geriatric population base with diabetes and obesity are propelling the growth of this market. China, Taiwan, and Malaysia are some of the countries that display the highest rate of male hypogonadism.

Major Companies Mentioned in Report

Some of the key players in the male Hypogonadism market include AbbVie Inc., Astrazeneca plc, Eli Lilly and Company Ltd., Merck & Co. Inc., SA, Finox Biotech, Laboratories Genevrier, Teva Pharmaceutical Industries Ltd., Allergan plc, Bayer AG, Endo International plc, IBSA Institut Biochimque, and Ferring.

Key players are focused on product approval for growth considerations and to cater to the changing demand of the industry. The introduction of innovative and technologically advanced products is also the focus of key players to increase their market share and for serving patients in a better manner.

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Male Hypogonadism Market Growth Opportunities and Dynamic Business by 2025 - NewsStoner

Heart attacks can cause immediate death. But in survivors, the blockage of blood flow can kill so many heart muscle cells that heart failure can follow months or years afterwards. Heart disease is the leading cause of hospital admission and death in the United States.

A heart attack causes a loss of muscle and leaves the heart with a scar that does not contract and so impairs the hearts pumping function, says Tim Kamp, a professor of medicine who is co-leader of a new grant designed to attack two roadblocks that have stymied efforts to restore heart muscle with muscle cells grown from stem cells.

Kamp, who directs the Stem Cell and Regenerative Medicine Center at the University of WisconsinMadison, says, Everybody involved in treating these patients knows that this scarring often leads to a continual decline in heart function with heart failure and even death.

The UWMadison researchers used approved surgical devices to locate the damaged heart muscle, and then injected the supportive matrix and committed cardiac muscle cells. The circle outlines target zone established before surgery; black dots show the sites that were injected in this mouse study. Amish Raval, work performed at UWMadison in collaboration with Biologics Delivery Systems.

Sixteen percent of men, and 22 percent of women, develop heart failure after myocardial infarction heart attack. Coronary artery disease the category that includes stoppage of blood flow causes one in seven deaths in the United States.

Adult stem cell injections seemed a logical way to form new heart muscle cells and repair the damaged muscle. But in dozens of experiments, the cells either washed out of the heart or failed to develop into the specialized muscle cells the cardiomyocytes that power cardiac contractions. The benefits were mixed, modest at best, says Kamp.

After years of preliminary investigations, however, Kamp and Amish Raval, a professor of cardiology, researcher and entrepreneur, hope that a combination of two cutting-edge approaches would use a fabric-like material to prevent wash-out and successfully implant cardiomyocytes to damaged hearts.

Aided by a Regenerative Medicine Innovation Project grant from the National Heart, Lung, and Blood Institute, part of the National Institutes of Health, the two will lead a group to test that idea in pigs over two years.

Having committed cells could be a major advance, Raval says. The first stem-cells therapies started with cells that I call the model T. Now, we are moving to the Buick. The cells originate as induced pluripotent stem cells (iPSCs) a relative of embryonic stem cells that is based on reprogramming adult cells.

Two Madison-based businesses, and sources at the University of WisconsinMadison, also helped to fund the research. Fujifilm Cellular Dynamics Inc., one of the largest commercial sources of stem cell products, produces the committed cardiac progenitor cells that will be tested. These committed cells are ready to transform themselves into cardiomyocytes.

Fujifilm bought CDI, a company whose founders included Kamp and UWMadison stem cell pioneer James Thomson, but the operations remain in Madison. Kamp has no ownership position but is a consultant for the company.

Raval is a founder and board chair of the second commercial supporter, Cellular Logistics, Inc., which makes a freeze-dried matrix from the same proteins that naturally holds cardiomyocytes in place in the heart. The material is called extracellular matrix (ECM) because it scaffolds cells from the outside.

When the heart pumps, internal pressures often eject would-be replacement cells through lymph channels and blood vessels. Ravals group has already shown in mice that injecting extracellular matrix proteins along with new cells creates mechanical restraints that avoid the wash-out problem.

The extra-cellular matrix to be used in the NIH grant at UWMadison helped retain stem cells (yellow dots) in a pig heart. When similar cells (blue) were injected without the matrix, the cells spilled out of the heart muscle through the needle track and lymph channels.Eric Schmuck and Amish Raval, work performed at UWMadison. Eric Schmuck and Amish Raval, work performed at UWMadison

The injected scaffold may have another advantage for regenerating muscle after heart attack, Kamp notes. The ECM replenishes the scarred area to become more hospitable to the replacement cardiomyocytes. The effect may be based on chemical and mechanical signaling between the ECM and the regenerating cells.

Pigs hearts are quite close to human hearts in size and structure. The grant will cover tests on four groups of 12 pigs each following myocardial infarction:

If the combination is effective, Raval adds, We plan to proceed toward a Food and Drug Administration application for an investigational new drug, which would allow us to begin human trials.

With the passion and concern of a working cardiac surgeon, Raval says those trials would focus on patients who have not been helped by the best medical management we know today and they are not candidates for heart transplant or mechanical assist devices. The only other option is palliative or hospice care.

As Raval notes, More people are surviving heart attacks, and thats great. But many are left with a scar in the heart muscle a dead zone. That scar can enlarge, and the damage can spread. So we are seeing an increasing number of patients with heart failure. Thats why we are moving forward with this project.

This research is being funded by NIH grant 1U01HL148690-01.

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Moving beyond hype: Could one-two treatment restore damaged heart muscle? - University of Wisconsin-Madison

Instead of testing drugs on patients directly,a cutting-edge precision medicine process is creating miniature beating hearts as primary test subjects.

Be sure to check outPart I,Part II, and Part IIIof our interview series on precision medicine!

Imagine a doctor recommends a strong medicine to a patient, a medicine that often causes cardiac problems in patients.

However, instead of testing the drug on the patient, the doctor gets a lab-grown, mini-sized replica of the patients heart.

The drugs are administered on the mini heart until the right drug is found. Only then is it administered to the patient.

Imagine a situation where you take cells from a particular patient, make these little mini hearts for that patient, and test potential therapies in the in vitro system before you subject the patient to those therapies

Before you start thinking Chromosome 6, (a reference to a Robin Cook book) see what Kevin Costa, Co-Founder and Chief Scientific Officer at Novoheart told The Sociable.

Their MyHeart platforms miniature hearts made from human tissue could be bringing in a revolution in precision medicine as well as drug discovery.

Precision medicine isnt just human-based, its individual based, and you can get increasingly precise, he says.

With a mini heart pumping away like any regular one, one can start to specialize a little bit more. For example, finding out the specifics of a disease in different ethnic backgrounds, like the Jewish population, African-American, Caucasian, or Asian.

So you can go from just having a human heart to an Asian, Caucasian, African, or whatever you want. You can also look at differences between male and female. So thats starting to get a little more precise, he explains.

So we can really get to the level of precision of an individual.

You can even make these tissues from an individual patient. Literally, weve got hearts in our laboratory that were made from a particular patients skin cells, he gushes.

Novoheart focuses on stem cell and tissue engineering for next-generation drug development and discovery. They are primarily a service company providing screening services based on their human tissue engineering technology, which they call the MyHeart platform.

Read more: Deep tech, big data, and their impact on precision medicine

The MyHeart platform consists of several different cardiac assays of human cardiomyocytes, human heart cells that are derived from human induced pluripotent stem (IPS) cells, which means human stem cells that can be differentiated into any cell type of the body.

The IPS cells are then mixed with the cardiomyocytes to make a 3D Structure and then cast into different types of tissues or layers of cells that Novoheart uses to measure electrophysiology or strips of tissue to measure contractility.

Rather than subjecting the patient to testing various cocktails of drugs, if we could get some information early on about whether a particular patient is more susceptible to a therapy, we can treat at a very granular, precise level for each patient

So its kind of like the electrical and mechanical side of how the heart works. We make these little mini hearts that pump like human hearts and give us measurements that clinicians are interested in, for example, cardiac output stroke volume he says.

Everything that Novoheart does is based on human cells. Tissue engineering has evolved to use human cells instead of rodent, and this was the basis for the original idea for Novoheart.

The company combined Costas expertise in tissue engineering in cardiac mechanics, Co-founder and CEO Ronald Lis expertise in human stem cells and cardiac electrophysiology, and Co-founder and Scientific Advisory Board member, Michelle Khines expertise in microfluidic platforms.

Drug discovery currently involves a process that starts with investigating a few thousand compounds in the laboratory, from which a couple of hundred that look promising can be impressed in an animal model.

Then you have to sort of take a leap of faith in moving from testing on animals to human patients. Thats the next step in the clinical trial process, Costa explains.

Costa says for every drug that enters a clinical trial process, 90% of them fail. Maybe, one out of ten that goes back out of several hundred is actually a go, after which clinicians consider candidates and try to get FDA approval.

Its a very inefficient and time-consuming process involving a couple of billion US dollars. Typically, to go from initial concept to approval, it can take over a decade, he says.

The Novoheart team thinks that part of the inefficiency lies in that leap of faith in going from animals to patients.

The way to help improve that process would be if we could get information in a human based heart system before actually testing on patients.

Novoheart has found a less risky way in terms of safety concerns for trying things on patients for the first time.

Also, if a compound doesnt work, you can reiterate in the laboratory and improve its safety and efficacy before moving on to clinical trials. This could ensure an increase in the success rate of clinical trials from 10% to who knows 50% or more.

According to Costa, one of the top reasons that drugs fail in the regulatory approval process is because of cardiac side effects, which is a major roadblock. That is a part of the reason Novoheart focuses on cardiac miniatures.

We focus on cardiac because thats our expertise. But the drugs that we are testing can be for any body part or disease because they all have to go through at least a cardiac safety assessment, he says.

They make two classes of heart tissue, a healthy heart tissue as well as diseased ones.

These organoids are designed thinking ahead towards that day when we will be able to have a little heart organoid, a liver organoid and a little brain organoid, all communicating with one another, kind of like a little humanoid

If you want to find a drug thats going to cure diabetes, you want to ensure it isnt going to give you heart disease in the process. So you can try it on the healthy heart tissue and see if its safe. If it causes arrhythmias or hypertrophy, it would be a problem for the patient, he says.

The other kind of tissue they make is diseased tissue.

If youre trying to develop a drug to cure heart disease, you need to have a model of that disease. So Novoheart is actively involved in that as well, he says.

Will they branch out then to other organoids? How about the liver?

Read more: Machine learning will be able to predict diseases years before onset of symptoms

Costa says Novoheart is thinking about combining different types of organoids together with the technology theyve developed. Costa paints a little picture of the future,

These organoids are designed thinking ahead towards that day when we will be able to have a little heart organoid, a liver organoid and a little brain organoid, all communicating with one another, kind of like a little humanoid.

Currently, its not particularly cost-effective to be able to do this for every single patient. However, Costa says, as the process becomes more streamlined and economical, the future is hopeful.

Imagine a situation where you take cells from a particular patient, make these little mini hearts for that patient, and test potential therapies in the in vitro system before you subject the patient to those therapies, he says.

Precision medicine isnt just human-based, its individual based, and you can get increasingly precise

This will have a major impact on medicine because, often, a cardiologist has to consider multiple therapies for a patient. In the current way of doing things, they try out and see what works on the patient. If not, they go to a second trial, second drug, and see what works best. If this testing process could instead be done on little organoids, it would be helpful.

Not just cardiac drugs, many chemotherapies have cardiac side effects. So rather than subjecting the patient to testing various cocktails of drugs, if we could get some information early on about whether a particular patient is more susceptible to a therapy, we can treat at a very granular, precise level for each patient, he says.

Originally posted here:
How growing mini human hearts is advancing precision medicine, drug discovery - The Sociable

Improving on Current Immunotherapies

Researchers from the Wellcome Sanger Institute, GlaxoSmithKline and Biogen, working under the Open Targets initiative, have demonstrated that thousands of DNA differences that are associated with immune diseases are also connected to the specific switching on of a subtype of immune cells. Previous research has shown that there are thousands of genetic variants common in individuals with immune diseases. The new study was published in the journal Nature Genetics.

Our study is the first in-depth analysis of immune cells and cytokine signals in the context of genetic differences linked to immune diseases, said Blagoje Soskic, lead author of the paper from the Wellcome Sanger Institute and Open Targets. We found links between the disease variants and early activation of memory T-cells, suggesting that problems with regulating this early T-cell activation could lead to immune diseases.

The research teams evaluated parts of the genome active in three types of immune cells accumulated from healthy volunteers, then compared their positions against all the genetic variants associated to different immune diseases. They also added different cytokines to their pool, which then consisted of a total of 55 different cell states that mimic immune disease inflammation. One particular cell type and cell state, early activation of memory T-cells, had the most active DNA as the same regions as the genetic variants associated with immune diseases. Cytokines, however, did not have as significant a role as suspected.

There are thousands of different cell types and states in the body, and finding the cause of autoimmune diseases is like finding a needle in a haystack, said Gosia Trynka, senior author from Wellcome Sanger Institute and Open Targets. We have identified early activation of memory T-cells as being particularly relevant to immune diseases, and will now be able to dive deeper into studying how this is regulated, to discover genes and pathways that could be used as drug targets.

Treating Heart Attacks with an Injectable Hydrogen

Researchers at the University of California San Diego, showed that use of an injectable hydrogel was able to repair damage and restore heart function in patients after a heart attack. This was a Phase I clinical trial sponsored by Ventrix, a UCSD spin-off. The gel, named VentriGel, is manufactured from cardiac connective tissue from pigs. The researchers take the tissue, strip out heart muscle cells, then freeze-dry and grind it into powder, then liquefy into a fluid that allows it to be injected into the heart muscle without surgery. At room temperate the liquid become a semi-solid, porous gel.

The Chromosome Connections Between Humans and Archaebacteria

Archaebacteria are some of the oldest-living organisms on the planet, one of the three biological domains: bacteria, eukaryotes, and archaea. Researchers at the University of Indiana found that the way DNA is organized in archaeal chromosomes has more similarities to human DNA than it does to bacteria. They believe it may help scientists study human DNA and diseases more effectively because the archaea are similar but less complex than human DNA.

How the 2 Strands of DNA are Held Together

DNA is made up of two strands of sugar and phosphate molecules, twisted into a helix. It has been generally accepted that the two strands were held together by hydrogen bondswhich now appears to be incorrect. Researchers at Chalmers University of Technology, Sweden, showed that that molecules have a hydrophobic interior and exist in an environment mostly of watermeaning that the DNA molecules nitrogen basis are hydrophobic, pushing the surrounding water away. The hydrogen bonds appear be more involved in sorting the base pairs rather than connecting the two strands together.

Unexpected Amyotrophic Lateral Sclerosis Findings

Accumulation of a protein, TDP-43, in the brain has been linked to amyotrophic lateral sclerosis (ALS). Researchers used a technique called deep mutagenesis to study all possible mutations in the TDP-43 protein with unexpected results. They developed more than 50,000 mutations of TDP-43 and tracked their toxicity to yeast cells. However, instead of finding the mutant forms to be more toxic, they were less toxic, forming unusual liquid species in the cells. Although unclear, the researchers believe its possible that aggregation of TDP-43 is actually protective, rather than damaging.

Antimicrobial Resistance is Growing Dramatically Around the World

Researchers developed a map of the world showing incidences of antimicrobial activity. The overall picture is of a dramatic increase in antimicrobial resistance, with the highest rates in animals in northeast China, northeast India, southern Brazil, Iran and Turkey. Some of this is related to improved economies, leading to increased meat consumption, linked to dramatic increases in the use of antibiotics in farm animals, but in countries with lower rates of monitoring for antibiotic resistance.

Possible New Weapon for Use in Immunotherapy for Cancer: iNKT Cells

Invariant natural killer T-cells (iNKT) are not as common as other types of immune cells, but they are generally viewed as more powerful. Researchers at UCLA, working in mice, were able to harness iNKT cells to attack cancer cells. They genetically engineered hematopoietic stem cells to develop into iNKT cells, which they then tested on mice with both human bone marrow and human cancers and multiple myeloma and melanoma models. The stem cells differentiated normally into iNKT cells and continued to produce them for the rest of the animals lives. The stem cell-derived iNKT cells also effectively suppressed tumor growth.

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Research Roundup: Improving Current Immunotherapies and More - BioSpace

Researchers Build Microscopic Biohybrid Robots Propelled by Muscles and NervesArnold Lander posted on September 27, 2019 |

An artistic rendering of a new generation of biobotssoft robotic devices powered by skeletal muscle tissue that is stimulated by onboard motor neurons. (Image courtesy of Michael Vincent.)

Researchers at the University of Illinois have developed a biohybrid robot powered by neuromuscular tissue that responds to light. Biohybrid robots are the result of integrating synthetic material and living tissue such as muscle, nerves or bone to produce a device that is capable of independent motion. The addition of neuronal action to control muscle tissue represents a significant step forward in the quest for autonomous biobots.

In 2014 researchers developed the first self-propelled biobots powered by cardiac muscle tissue taken from rats. These early designs, modeled after sperm cells, had a single tail and could swim but could not sense their environment or make decisions.

In this new study, computational models were used to optimize the skeleton design. The previous single-tailed structure was replaced with a new two-tailed model, and the length of the tails was also adjusted. These design improvements resulted in an order of magnitude increase in swimming speed from the previous single-tailed version.

The robot was completed by applying an optogenetic cell culture derived from mouse stem cells adjacent to the muscle tissue. In this process, the neurons advanced toward the muscle and formed neural muscular junctions, with the robot assembling entirely on its own.

The biobot team: (from left) Professor Tahir Saif, graduate student Omar Aydin, graduate student Xiastian Zhang, Professor Mattia Gazzola, graduate student Gelson J. Pagan-Diaz, and Dean of Granger College of Engineering, Rashid Bashir.

The success of this study helps set the stage for the future development of engineered, multicellular living systems with the ability to respond intelligently to environmental cues. These living machines could potentially find applications in the fields of bioengineering, medicine and material science.

The paper Neuromuscular actuation of biohybrid motile bots is availablehere.

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Researchers Build Microscopic Biohybrid Robots Propelled by Muscles and Nerves - ENGINEERING.com

Abstract

The control of stem and progenitor cell fate is emerging as a compelling urgency for regenerative medicine. Here, we propose a innovative strategy to gain optical control of endothelial colony-forming cell fate, which represents the only known truly endothelial precursor showing robust in vitro proliferation and overwhelming vessel formation in vivo. We combine conjugated polymers, used as photo-actuators, with the advantages offered by optical stimulation over current electromechanical and chemical stimulation approaches. Light modulation provides unprecedented spatial and temporal resolution, permitting at the same time lower invasiveness and higher selectivity. We demonstrate that polymer-mediated optical excitation induces a robust enhancement of proliferation and lumen formation in vitro. We identify the underlying biophysical pathway as due to light-induced activation of TRPV1 channel. Altogether, our results represent an effective way to induce angiogenesis in vitro, which represents the proof of principle to improve the outcome of autologous cell-based therapy in vivo.

In recent years, organic semiconductors have emerged as highly promising materials in biotechnology, thanks to several key-enabling features. Differently from silicon-based electronics, they support both electronic and ionic charge transport (1); they can be easily functionalized with specific excitation and sensing capabilities (24); and they are solution processable, soft, and conformable (5). They are highly biocompatible, being suitable for in vivo implantation and long-term operation, as recently reported for many different applications, including electrocorticography, precise delivery of neurotransmitters, electrocardiography, deep brain stimulation, and spinal cord injury (69). An important, distinctive feature of organic semiconductors is their sensitivity to the visible and near-infrared light. Recently, our and other groups have exploited it for optical modulation of cell electrophysiological activity, by using conjugated polymers and organic molecules as exogenous light-sensitive actuators (1012). Interesting applications have been reported in the field of artificial visual prosthesis (5), photothermal excitation or inhibition of cellular activity (13, 14), and modulation of animal behavior (15).

In this framework, the opportunity to use polymer-based phototransduction mechanisms to regulate the very early stages of living cell development has been very scarcely considered (16, 17). The possibility to selectively and precisely regulate a number of cell processes, such as adhesion, differentiation, proliferation, and migration, would be key to regenerative medicine and drug screening. The presently dominant approaches to reliably regulate stem and progenitor cell fate for regenerative purposes mainly rely on the use of chemical cues. However, irreversibility and lack of spatial selectivity represent important limitations of these methods. Whenever targeting in vivo applications, one must face the major, unsolved problem of diffusion of neurotrophic molecules by the conventional intravenous or oral routes. In addition, the therapeutic outcome of autologous cell-based therapy is often impaired by low engraftment, survival, and poor integration of stem cells within the environment of the targeted tissue. Other stimuli, mainly consisting of mechanical and electrical cues, were recently reported to have some notable effects, and recent advances in nanotechnology and material science enabled versatile, robust, and larger-scale modulation of the cell fate. In particular, carbon-based materials and conjugated polymers led to interesting results (18). However, their distinctive visible light absorption was never exploited in optically driven techniques.

Use of light actuation has been proposed either by viral transfer of light-sensitive proteins, by optogenetics tools, or by absorption of endogenously expressed light-sensitive moieties, based on low lightlevel therapies (1921). In the first case, interesting results were obtained (22); however, this approach bears all the drawbacks related to the need for viral gene transfer. Photobiomodulation led to interesting outputs as well, but overall efficiency is hampered by the limited absorption of light-responsive molecules endogenously expressed in living cells.

In this work, we propose to couple the use of conjugated polymers with visible light excitation to gain optical control of cell fate. We focus our attention on endothelial progenitor cells (EPCs) and, in particular, on endothelial colony-forming cells (ECFCs), which are currently considered the bona fide best surrogate of EPCs (23). ECFCs are mobilized from the bone marrow and vascular stem cell niche to reconstruct the vascular network destroyed by an ischemic insult and to restore local blood perfusion (24). ECFCs may be easily harvested from peripheral blood, display robust clonogenic potential, exhibit tube-forming capacity in vitro, and generate vessel-like structures in vivo (24, 25), thereby representing a promising candidate for autologous cell-based therapy of ischemic disorders (24). Manipulating the signaling pathways that drive ECFC proliferation, migration, differentiation, and tubulogenesis could represent a reliable strategy to improve the regenerative outcome of therapeutic angiogenesis in the harsh microenvironment of an ischemic tissue, such as the infarcted heart (24, 25). Intracellular Ca2+ signals play a crucial role in stimulating ECFC proliferation and tubulogenesis by promoting the nuclear translocation of the Ca2+-sensitive nuclear transcription factor B (NF-B) (2628). It has, therefore, been suggested that intracellular Ca2+ signaling could be targeted to boost the regenerative potential of autologous ECFCs for regenerative purposes (29). For the above-mentioned reasons, ECFCs represent a valuable test bed model for assessing the possibility to exploit the visible light sensitivity of conjugated polymers to gain touchless, optical modulation of cell proliferation and function.

In this framework, we demonstrate that polymer-mediated optical excitation during the first steps of ECFC growth leads to a robust enhancement of both proliferation and tubulogenesis through the optical modulation of the Ca2+-permeable transient receptor potential vanilloid 1 (TRPV1) channel and NF-Bmediated gene expression. Our results represent, to the best of our knowledge, the first report on the use of polymer photoexcitation for the in vitro modulation of ECFC fate and function, thereby representing the proof of principle to obtain direct control of progenitor cell fate.

Figure 1A shows a sketch of the bio/polymer interface developed for obtaining optical control of ECFC proliferation and network formation, together with the polymer chemical structure and the optical absorption spectrum. The material of choice for light absorption and phototransduction is a workhorse organic semiconductor, widely used in photovoltaic and photodetection applications, namely, regioregular poly(3-hexyl-thiophene) (P3HT) (6). It is characterized by a broad optical absorption spectrum, in the blue-green visible region, peaking at 520 nm. P3HT outstanding biocompatibility properties have been reported in a number of different systems, both in vitro and in vivo, including astrocytes (30), primary neurons and brain slices (14), and invertebrate models of Hydra vulgaris (15). Chronical implantation of P3HT-based devices in the rat subretinal space did not show substantial inflammatory reactions up to 6 months in vivo (10). Here, polymer thin films (approximate thickness, 150 nm) have been deposited by spin coating on top of polished glass substrates, as detailed in Materials and Methods. Both polymer-coated and glass substrates have been thermally sterilized (120C, 2 hours), coated with fibronectin, and, lastly, used as light-sensitive and control cell culturing substrates, respectively. ECFCs have been isolated from peripheral blood samples of human volunteers and seeded on top of polymer and glass substrates.

(A) P3HT polymer optical absorption spectrum. Insets show the chemical structure of the conjugated polymer and a sketch of the polymer device used for cell optical activation. ECFCs are cultured on top of P3HT thin films, deposited on glass substrates. (B) ECFC viability at fixed time points after plating (24, 48, and 72 hours). Cell cultures were kept in dark conditions at controlled temperature (37C) and fixed CO2 levels (5%). No statistically significant difference was observed between the glass and polymer substrates at any fixed time point (unpaired Students t test). (C) Experimental setup and optical excitation protocol for evaluation of polymer-mediated cell photoexcitation effects on cell fate. Polymer and control samples are positioned within a sterilized, home-designed petri holder. Light scattering effects are completely screened. The geometry and the photoexcitation protocol have been implemented to minimize overheating effects and to keep the overall extracellular bath temperature fairly unaltered. Thirty-millisecond-long green light pulses are followed by 70 ms in dark condition.

ECFC proliferation on polymer substrates has been primarily assessed in dark conditions at three different time points, namely, 24, 48, and 72 hours after plating (Fig. 1B). Polymer-coated samples, while showing from the very beginning a slightly lower number of cells as compared with control substrates, exhibit a proliferation rate fully similar to cells plated on glass substrates (slope of the linear fitting is 0.034 0.003, R2 = 0.99 and 0.034 0.005, R2 = 0.96 for control and P3HT polymer samples, respectively).

Once assessed that the P3HT polymer surface represents a nicely biocompatible substrate for ECFC seeding and proliferation in the dark, we moved to investigate the effect of polymer photoexcitation. In more detail, to evaluate the effect of optical stimulation on cell proliferation and network formation, we continuously shined light for the whole temporal window required for cell growth, and we realized an ad hoc system suitable for operation within the cell incubator. The experimental configuration and the excitation protocol are schematically represented in Fig. 1C. Optical excitation is provided by a light-emitting diode (LED) source, with maximum emission wavelength at 525 nm, incident from the substrate side. The choice of the protocol, continuously administered to the cell cultures during early seeding and proliferation stages, has been mainly dictated by the need to avoid overheating effects, with possible negative outcomes on the overall cell culture viability. On the basis of these considerations, we opted for a protocol based on 30-ms excitation pulses, followed by a 70-ms dark condition, at a photoexcitation density of 40 mW/cm2. The whole protocol is continuously repeated for a minimum of 4 up to 36 hours, depending on the type of functional assay, at controlled temperature (37C) and CO2 levels (5%).

The temporally precise and spatially localized measurement of the temperature variation upon polymer photoexcitation at the polymer/cell interface (i.e., within the cell cleft) is not straightforward because it requires the use of localized, submicrometer probes with a fast response time. However, according to the heat diffusion equation, we expect that dissipation occurs within a few milliseconds, following exponential decrease dynamics (14). Moreover, we used the well-known method of the calibrated pipette (31) to characterize the temperature variation dynamics within the extracellular bath volume, defined by the cylinder with the base area corresponding to the light spot size and the height of about 1 m. This choice is a good approximation of the overall volume occupied by a single ECFC cell; thus, it provides a realistic estimation of the average heating experienced by the cell (fig. S1A). We observe that temperature variation closely follows short optical pulse dynamics, reaching a maximum temperature at the end of the 30-ms illumination period, quickly followed by an almost complete thermal relaxation to the basal temperature during the 70-ms-long dark period. We conclude that our polymer-based system provides a highly spatially and temporally resolved method for optical excitation, making it possible, in perspective, to selectively target single cells and even cell subcompartments. Upon prolonged illumination (hours), one should also consider possible overheating effects of the whole extracellular medium volume. The average temperature of the bath for the entire duration of the long-term experiment was measured by a thermocouple immersed in the medium. Data show that an equilibrium situation is established after 5 hours and that the absolute temperature of the bath is increased by about 1.5 (fig. S1B). The adopted prolonged excitation protocol does not negatively affect overall cell culture viability (see below).

Figure 2 reports specific effects mediated by P3HT substrates and visible light stimulation on ECFC proliferation. ECFCs were plated in the presence of EGM-2 medium to facilitate the adhesion to the substrate. After 12 hours, the medium was switched to EBM-2 supplemented with 2% fetal bovine serum, and the cells were subjected to the long-term lighting protocol for 36 hours at controlled temperature (37C) and CO2 levels (5%). Under these conditions, ECFCs seeded on P3HT and subjected to light stimulation undergo a significant increase in proliferation rate, as compared with the control condition, i.e., to cells also seeded on P3HT polymer substrates but kept in dark conditions for the whole duration of the experiment (+158% versus P3HT dark; P < 0.05). No statistically significant difference in proliferation was observed among cells seeded on glass, whether they were subjected to optical excitation or not (Fig. 2A).

(A) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT thin films, together with corresponding control samples kept in dark conditions. Cell proliferation was measured after 36 hours of culture in the presence of EBM-2 supplemented with 2% fetal calf serum. (B) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence (CTRL) and presence of 10 M capsazepine (CPZ), 10 M ruthenium red (RR), 20 M RN-1734 (RN-1734), and 30 M BAPTA-AM (BAPTA). The results are represented as the means standard error of the mean (SEM) of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way analysis of variance (ANOVA) coupled with Tukey (A) or Dunnetts (B) post hoc test. *P < 0.05.

Recent evidence demonstrated an interesting correlation between processes key to ECFC vascular regeneration, including proliferation and network formation, and activation of TRPV1 channels, which are expected to be endogenously expressed in ECFCs (32). In addition, we recently reported that polymer photoexcitation leads to selective TRPV1 activation in transfected human embryonic kidney (HEK) cell models (33). Therefore, we were prompted to evaluate whether the increase in cell proliferation is distinctively determined by a polymer-mediated photoactivation of the TRPV1 channel. To this goal, we preliminarily checked the actual expression of the TRPV1 channel in the ECFC models by carrying out electrophysiology experiments in patch-clamp configuration. Methods and results are extensively discussed in the Supplementary Materials (fig. S2 and related description). Briefly, the expression of the TRPV1 channel was confirmed, as well as the capability to selectively excite its activity through localized polymer excitation at high optical power density. To establish whether the TRPV1 channel also has a role in the observed increase in cell proliferation upon polymer excitation, we performed the experiments under light illumination upon administration of a highly specific TRPV1 antagonist [capsazepine (CPZ), 10 M], an aspecific TRPV channel inhibitor [ruthenium red (RR), 10 M], and a selective antagonist of a different temperature-sensitive channel, TRPV4, which is also endogenously expressed in ECFCs (RN-1734, 20 M) (34) (Fig. 2B). TRPV1 inactivation by CPZ and RR results in a relative, strong reduction in cell proliferation by 51 and 30%, respectively, as compared with untreated cells. Conversely, in the case of RN-1734 treatment, the proliferation increase due to polymer photoexcitation is completely unaltered.

As mentioned earlier, intracellular Ca2+ signaling has been reported to drive ECFC proliferation (26, 28). To further investigate whether TRPV1-mediated extracellular Ca2+ entry mediates the proangiogenic response to light illumination, we pretreated ECFCs with [1,2-Bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester) BAPTA-AM] (30 M), a membrane-permeable buffer of intracellular Ca2+ levels (26, 28). BAPTA-AM is widely used to prevent the increase in intracellular Ca2+ concentration ([Ca2+]i) induced by extracellular stimuli and inhibits the downstream Ca2+-dependent processes. For instance, BAPTA-AM represents the most suitable tool to prevent the activation of Ca2+-sensitive decoders residing within tens of nanometers from the inner pore of plasmalemmal Ca2+ channels (35). It was recently reported that, in the absence of Ca2+-mobilizing growth factors, it does not impair the low rate of ECFC growth (27). Here, however, BAPTA-AM clearly reduced the light-driven proliferation increase, thus confirming that TRPV1 stimulates ECFCs through an increase in [Ca2+]i (Fig. 2B).

We further examined the physiological outcome of chronic light stimulation by carrying out a tube formation assay within an extracellular matrix protein-based scaffold, which is a surrogate of the basement membrane extracellular matrix. This assay recapitulates many steps of the angiogenic process, including adhesion, migration, protease activity, and tubule formation (27, 28). ECFCs were plated in the presence of EBM-2 medium supplemented with 2% fetal calf serum and subjected to the long-term lighting protocol for 8 hours at controlled levels of temperature and CO2. Control experiments carried out in dark conditions, either onto glass (see Fig. 3A for a representative optical image) or onto polymer substrates (Fig. 3C), as well as control experiments carried out upon photoexcitation of cells seeded on glass substrates (Fig. 3B), do not show remarkable differences. Conversely, ECFC cultures subjected to polymer-mediated optical excitation clearly tend to assemble into an extended bidimensional capillary-like network (Fig. 3D). Cell cultures were monitored up to 24 hours after illumination onset, but results were comparable to observations reported here, after 8 hours of illumination. This qualitative observation is fully confirmed by quantitative morphological analysis (27). As depicted in the sketch of Fig. 3E, we quantitatively evaluated the main features typical of the capillary-like network formation and, in particular, the number of master segments (Fig. 3F), master junctions (Fig. 3G), and meshes (Fig. 3H). In all cases, a notable, statistically relevant difference is observed between cells subjected to polymer-mediated optical excitation and controls. Within the same considered temporal window, the combined use of polymer substrates and visible light stimuli does not lead to sizable toxicity effects or delays in cell proliferation. Conversely, it leads to enhanced cell proliferation (Fig. 2) and allows the achievement of the formation of a more extended and mature tubular network (Fig. 3).

(A to D) Representative images of in vitro tubular networks of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT, as well as on corresponding control samples in dark conditions. Cultures were observed up to 24 hours, but their appearance did not substantially change after pictures were taken after 8-hour culture. Scale bars, 250 m. (E) Sketch representing the main features typical of the capillary-like network that were considered for the topologic analysis. Number of master segments (F), master junctions (G), and meshes (H) analyzed in the different conditions. The results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Tukey post hoc test. **P < 0.01 and ***P < 0.001.

As evidenced for the proliferation rate, the TRPV1 channel activation emerges to play also a fundamental role in tubulogenesis (Fig. 4). The TRPV1 pharmacological blockade with the specific inhibitor CPZ deterministically leads to a marked reduction in network formation (Fig. 4A). Upon CPZ administration, a statistically significant decrease in the relative variation of the number of master segments (Fig. 4E), master junctions (Fig. 4F), and meshes (Fig. 4G) is observed. In line with the results shown in Figs. 2 and 3, RR administration resulted in a less marked but still sizable reduction in the tubular network (Fig. 4, B and E to G), probably due to the minor specificity toward TRPV1, while the protubular effect of light remained fully unaltered in the presence of the TRPV4 inhibitor RN-1734 (Fig. 4, C and E to G). Notably, the treatment with BAPTA-AM (30 M), which affected ECFC proliferation, was able to prevent also in vitro tubulogenesis, thus corroborating the key role of intracellular Ca2+ signaling in the proangiogenic response to light illumination (Fig. 4, D and E to G). Control measurements carried out in dark conditions on polymer substrates upon the considered pharmacological treatments do not show any relevant effect (fig. S4, A to C). Overall, this evidence supports the notion that TRPV1 stimulates ECFC proliferation and network formation and demonstrates that optical excitation, properly mediated by biocompatible polymer substrates, positively affects ECFC fate by spatially and temporally selective activation of the TRPV1 channel.

(A to D) Representative optical images of in vitro tubular network of ECFCs subjected to long-term optical excitation seeded either on bare glass or on P3HT thin films and treated respectively with CPZ (A), RR (B), RN-1734 (C), and BAPTA-AM (D). Scale bars, 250 m. (E to G) Relative variation of number of master segments (E), master junctions (F), and meshes (G) of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence [control (CTRL)] and presence of 10 M CPZ, 10 M RR, 20 M RN-1734 (RN-1734), and 30 M BAPTA-AM (BAPTA). The results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Dunnetts post hoc test. *P < 0.05 and **P < 0.01.

We now turn our attention to elucidating the possible mechanisms leading to optically enhanced tubulogenesis, through TRPV1 channel activation, upon prolonged polymer excitation.

Reliable optical modulation of the cell activity mediated by polymer photoexcitation has been reported in several, previous reports, both in vitro, at the level of single cells, and in vivo, at the level of the whole animal, as evidenced by behavioral studies on both invertebrate and vertebrate models. Three different photostimulation mechanisms, active at the polymer/cell interface, have been proposed so far. These include (i) the creation of an interface capacitance, i.e., of a localized electric field, possibly affecting the cell membrane potential (11); (ii) photothermal processes, establishing a localized temperature increase upon polymer photoexcitation (13, 36); and (iii) photoelectrochemical reactions, mainly oxygen reduction processes, leading to a local variation of extracellular and/or intracellular pH (33) and sizable production of reactive oxygen species (ROS), at a nontoxic concentration, and intracellular calcium modulation (37).

In electrophysiological experiments, carried out at a photoexcitation density higher than the one used in chronic stimulation by about two orders of magnitude, we clearly observe TRPV1 excitation, corresponding however to a small variation of the cell membrane potential, in the order of a few millivolts (Supplementary Materials). Thus, upon much lower light intensity, the effects of either direct photothermal channel activation and of photocapacitive charging are expected to be negligible. To further corroborate this hypothesis, we carry out control experiments aimed at disentangling photoelectrical from photothermal transduction processes.

First, we use a different material as a cell-seeding substrate, characterized by optical absorption and heat conductivity similar to the ones typical of P3HT (13) but fully electrically inert (i.e., unable to sustain electronic charge generation upon photoexcitation). The material of choice is a photoresist (MicroPosit S1813). S1813 thin films are realized by spin coating, and deposition parameters are optimized to obtain optical absorbance values similar to the semiconducting polymer samples at the considered excitation wavelength. The capability of photoresist substrates to sustain ECFC proliferation was successfully assessed in a control measurement, obtaining fully comparable results with respect to the P3HT substrates (Fig. 5A). The functional effect eventually driven by photoresist optical excitation on tubulogenesis was then investigated by using the same experimental conditions and analysis technique previously adopted for polymer and glass substrates (Fig. 5B). Data show that long-term photoresist excitation does not lead to sizable enhancement of the cellular network formation, thus pointing out that a purely photothermal effect does not play a major role in boosting the tubulogenesis process at variance with semiconducting polymer substrates. In a complementary experiment, we directly assessed the occurrence of photoelectrochemical reactions at the polymer/extracellular bath interface by measuring ROS production. We previously demonstrated that P3HT polymer thin films exposed to saline electrolytes sustain efficient light-triggered charge generation and charge transfer processes, giving rise to photoelectrochemical reactions (38, 39). Moreover, we also reported that P3HT nanoparticles are efficiently internalized within the cytosol of secondary line cell models (HEK-293) and that their photoexcitation leads to the production of ROS and subsequent intracellular calcium modulation (15, 37). However, the actual capability to sustain photoelectrochemical reactions in the specific experimental conditions used in this work (polymer film deposition conditions, sterilization process, prolonged exposure to specific cellular growth medium in an incubating environment, prolonged exposure to a light excitation protocol, light wavelength, pulses duty cycle, and power density) was never assessed. In particular, direct measurement of intracellular ROS was never carried out in the presence of polymer thin films. To this goal, we realized ECFC cultures on top of polymer and glass control substrates, and we exposed them to the same optical stimulation protocol previously used in the tubulogenesis assay. ROS production was then evaluated by means of a fluorescence experiment based on the use of the well-known ROS probe 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Fig. 5C). Results show that light induces an increase in ROS production both on glass and polymer substrates. Relative percentage variation amounts to +34 and +200%, respectively, thus pointing out that polymer surface photocatalytic activity plays a major role in the phototransduction phenomenon.

(A) An electrically insulating, thermally conducting material (photoresist) is successfully used as an ECFC seeding substrate. (B) Photoresist long-term photoexcitation does not lead to sizable enhancement in tubulogenesis parameters. (C) Evaluation of intracellular ROS production following long-term photoexcitation protocol of ECFC cultures on polymer and glass substrates (glass dark, n = 629; glass light, n = 656; P3HT dark, n = 686; and P3HT light, n = 583). For each panel, the results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with unpaired Students t test (A and B) or one-way ANOVA coupled with Tukey post hoc test (C). ***P < 0.001.

Altogether, data in Fig. 5 indicate that photoelectrochemical reactions induced by light at the interface between the organic semiconducting polymer and the extracellular bath play a key role in triggering the observed enhancement in cell network formation through indirect activation of the TRPV1 channel. The occurrence of faradaic phenomena at the polymer/bath interface may give rise to material degradation effects. The photostability of the polymer substrates was carefully checked by optical absorption, photoluminescence, and Raman spectra measurements. By treating the samples with the same experimental protocol used for cell tubulogenesis assays (photoexcitation density, pulses frequency, overall exposure duration, temperature, and humidity levels), no sign of irreversible polymer degradation was observed, as compared with nonilluminated samples (fig. S5).

The Ca2+-sensitive transcription factor NF-B might provide the missing link between the influx of Ca2+ through TRPV1 and the increase in proliferation and tubulogenesis observed in ECFCs upon photostimulation (26). We therefore monitored the nuclear translocation of the cytoplasmic p65 NF-B subunit via immunofluorescence staining and mRNA levels of a number of genes induced during tubulogenesis in an NF-Bdependent manner (26, 40) (Fig. 6). Our data indicate that ECFCs seeded on polymer and subjected to light stimulation have a significantly enhanced p65 NF-B nuclear translocation compared with the control conditions consisting of cells also seeded on P3HT but kept in dark conditions (+35% versus P3HT dark; P < 0.05; Fig. 6, A and B), and seeded on bare glass (+28% versus glass dark; P < 0.05; Fig. 6B). No differences were observed between samples seeded on glass, whether they were subjected to optical excitation or not (fig. S6).

ECFCs seeded on P3HT samples and glass controls are subjected to long-term photostimulation protocol. Corresponding control samples are kept in dark conditions. After photostimulation, p65 NF-B nuclear translocation (A and B) and mRNA levels of tubulogenic/angiogenic genes that have been shown to be activated downstream of NF-B (C) are evaluated. (A) Representative images of immunofluorescence staining showing p65 NF-B (green) nuclear translocation. Cell nuclei are detected by 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 m. (B) Quantitative evaluation of p65 NF-B nuclear translocation, as evidenced by colocalization experiments. Results are expressed as means SEM of the relative percentage of p65 nucleipositively stained cells to the total number of cells (glass dark, n = 151; glass light, n = 125; P3HT dark, n = 147; and P3HT light, n = 159). Ten fields per condition are analyzed. Data are obtained from two different experiments conducted on cells harvested from two different donors. (C) mRNA levels of intercellular adhesion molecule 1 (ICAM1), selectin E (SELE), and matrix metalloproteinases (MMP1, MMP2, and MMP9) are quantified by real-time polymerase chain reaction (PCR). Data are expressed as means SEM of percentage variation with respect to cells grown in the dark (n = 6). The significance of differences was evaluated with unpaired Students t test (C) or one-way ANOVA coupled with Tukey post hoc test (B). *P < 0.05 and **P < 0.01.

In addition, we have checked the expression of nine genes whose expression is known to be induced in endothelial cells during tubulogenesis/angiogenesis in an NF-Bdependent manner. We considered intercellular adhesion molecule 1 (ICAM1); vascular adhesion molecule 1 (VCAM1); selectin E (SELE), matrix metalloproteinases (MMPs) 1, 2, and 9; vascular endothelial growth factor A (VEGFA); cyclooxygenase 2 (COX2, PTGS2); and cyclin D1 (CCND1) (40). Of these, five are significantly up-regulated by light exposure in cells grown on P3HT substrates, namely, ICAM1 (+90% versus P3HT dark; P < 0.05), SELE (+1119%; P < 0.01), MMP1 (+242%; P < 0.01), MMP2 (+467%; P < 0.05), and MMP9 (+458%; P < 0.05) (Fig. 6C). Conversely, VCAM1, VEGFA, PTGS2, and CCND1 do not show relevant variation upon light stimulation (fig. S7A). Light excitation on cells grown on bare glass substrates does not show any significant effect as compared with control samples in dark conditions (fig. S7B).

Therapeutic angiogenesis via autologous EPC transplantation represents a promising strategy to preserve or, at least, partially restore cardiac function after myocardial infarction (24, 41). Nevertheless, the regenerative outcome of EPC-based therapies in preclinical studies was rather disappointing and did not lead to sufficient neovascularization of the ischemic heart (41). This led to the proposal to boost their angiogenic activity by using emerging technologies, including tissue engineering of vascular niches, pharmacological preconditioning, or genetic and epigenetic reprogramming (42). ECFCs are regarded among the most suitable EPC subtypes to induce therapeutic angiogenesis and cardiac regeneration due to their high clonal proliferative potential and ability to assemble into capillary-like structures (23, 24). In addition, they can be easily isolated and expanded from the peripheral blood of patients and healthy donors. It has recently been suggested that their angiogenic activity could be boosted by targeting the intracellular Ca2+ toolkit (29). Here, we target ECFCs by adopting a fully different approach, i.e., by exploiting visible light as a modulation trigger and by the use of a thiophene-based conjugated polymer as the exogenous, light-responsive actuator. We demonstrate that photoexcitation of the organic material deterministically leads to robustly enhanced proliferation and tubulogenesis. Pharmacological assays, supported by electrophysiology experiments, allow the identification of TRPV1 selective excitation as a key player in the molecular pathway leading to macroscopic outcomes, as observed by quantitative analysis of the angiogenic response.

All data unambiguously show that polymer photoexcitation leads to selective activation of the TRPV1 channel, which has recently been shown to be expressed and drive angiogenesis in human ECFCs (32). TRPV1 is a polymodal Ca2+-permeable channel that integrates multiple chemical and physical cues to sense major changes in the local microenvironment of most mammalian cells (43). TRPV1 is activated by either noxious heat (>42C) and acidic solutions (pH < 6.5), whereas mild acidification (pH 6.3) of the extracellular milieus sensitizes TRPV1 to heat stimulation and results in channel activation at temperature thresholds (30 to 32C) well below the normal one (43). ROS production is also expected to further contribute to TRPV1 activation, as previously reported in mouse coronary endothelial cells (44), in which hydrogen peroxide elicits a depolarizing inward current at negative holding potentials. Likewise, ROS may stimulate TRPV1 to depolarize the membrane potential, thereby triggering trains of action potentials in airway C fibers (45, 46).

On the basis of measurements carried out in cells seeded on the photoresist substrate, as well as on direct evaluation of a limited, local temperature increase upon light stimuli during the long-term photoexcitation protocol, we infer that the excitation of the TRPV1 channel through direct photothermal transduction is not the predominant process leading to enhanced tubulogenesis.

We have previously demonstrated that polymer photoexcitation leads to generation of faradaic current, to electron transfer reactions at the polymer/electrolyte interface, and to sizable intracellular enhancement of ROS (37, 38). Briefly, optical excitation of P3HT polymer thin films leads to photoexcited species (Eq. 1), namely, singlets and charge states, which react with the oxygen dissolved in the cell medium, thus reducing oxygen (Eq. 2)P3HT+hP3HT*(1)P3HT*+O2P3HT++O2(2)

The superoxide further evolves, leading to the generation of different ROS and, lastly, ending up with hydrogen peroxide production. It has been reported that extracellular H2O2 can cross the plasma membrane through aquaporin AQP3, thereby triggering intracellular ROS signaling (47, 48). In line with our previous results, we have demonstrated here that intracellular ROS enhancement does occur in ECFCs upon photoexcitation of polymer thin films, thus contributing to TRPV1 activation.

Altogether, the evidence supports the hypothesis of a transduction mechanism mainly governed by photoelectrochemical reactions. Moreover, these same observations could explain why TRPV4, which is also expressed in ECFCs (34), is not sensitive to optical modulation. Although TRPV4 is activated by moderate heat (24 to 38C), it is supposed to be inhibited by local pH variation, although this is still a matter of debate (49, 50).

On the one hand, the role attributed in the phototransduction mechanism to the capability of the polymer to generate and transport electronic charges, as well as to its photocatalytic activity in an aqueous environment, clearly implies the need for a biocompatible, visible lightresponsive, semiconducting material. This excludes any possible implementation of the reported technique by using a thermally conducting, electrically insulating plastic substrate. Suitable cell-seeding materials have to be selected and developed within the wide arena of organic semiconducting polymers. On the other hand, the key role played by ROS raises additional issues about material photostability, cell viability, and overall safety and reliability of the technique. We extensively verified that the main polymer optoelectronic properties are not substantially altered by the exposure to light and to incubating conditions. From the biological point of view, it is very well known that high ROS levels can induce highly toxic effects and, finally, lead to cell death. We notice, however, that the established photoactivation protocol (illuminator geometry and air flow, light photoexcitation density, duty cycle, and repetition rate) has been implemented to avoid any detrimental effect. Accordingly, no toxicity effects were detected for the overall duration of the experiments, as proven by the robust increase in ECFC proliferation and tubulogenesis exposed to light. This observation is consistent with the emerging notion that appropriate ROS levels can exert a signaling role and control angiogenesis in endothelial cells (51).

The biophysical mechanisms whereby the photoactivation of TRPV1 stimulates in vitro angiogenesis in ECFCs deserve a more detailed discussion as well. Earlier work showed that TRPV1 stimulates proliferation and tube formation in vascular endothelial cells by mediating extracellular Ca2+ entry. The following increase in intracellular Ca2+ concentration ([Ca2+]i) leads to the recruitment of several downstream Ca2+-dependent decoders, such as endothelial nitric oxide synthase and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (52). Recently, TRPV1 was found to induce also proliferation and tube formation in ECFCs by mediating the uptake of the endocannabinoid anandamide (32). This study, however, did not investigate whether TRPV1 activation was per se able to stimulate ECFCs by engaging Ca2+-dependent pathways. Intracellular Ca2+ signaling is a crucial determinant of ECFC fate and behavior (2628). Accordingly, light-induced ECFC proliferation and tube formation were markedly reduced by the pharmacological blockade of TRPV1-mediated Ca2+ entry with CPZ and RR and by preventing the subsequent increase in [Ca2+]i with BAPTA-AM. This finding endorses the view that optical excitation stimulates ECFCs through TRPV1-mediated extracellular Ca2+ entry, and we suggest here that this occurs via downstream activation of transcriptional factor NF-B. NF-B has previously been shown to stimulate cell proliferation and tubulogenesis in endothelial cells (53, 54) and in hepatocytes (55). Our group has shown that NF-B triggers the transcriptional program underlying the angiogenic response to extracellular Ca2+ entry in ECFCs (26). Moreover, NF-B activation in response to extracellular stimulation and Ca2+ entry through TRPV1 has also been demonstrated (56, 57). Under resting conditions, NF-B is retained in the cytoplasm by the complex with the inhibitory protein IB. An increase in [Ca2+]i results in IB degradation by ubiquitination, which is triggered upon the Ca2+-dependent phosphorylation of IB. As a consequence, the p65 NF-B subunit is released from IB inhibition and translocates into the nucleus (58) where it induces the expression of multiple proangiogenic genes (40). Consistently, we found that optical excitation significantly boosted the nuclear translocation of p65 in ECFCs cultured on the conjugated polymer compared with those not exposed to light. Robust up-regulation of several angiogenic genes, such as ICAM, SELE, MMP1, MMP2, and MMP9, which are under NF-Bdependent transcriptional control, was also consequently observed. Intriguingly, NF-B also mediates VEGFA-induced gene expression and angiogenesis in vascular endothelial cells (59, 60) through an increase in [Ca2+]i (61). These observations strongly hint at NF-B as the Ca2+-sensitive decoder that translates optical excitation into an angiogenic response in human ECFCs interfaced with the light-sensitive conjugated polymer.

Overall, our findings represent the proof of principle that optical modulation may be successfully exploited to directly control the fate of a progenitor cell population, i.e., ECFCs, which has been shown to support revascularization of ischemic tissues. The in vitro activation of ECFC angiogenic activity is made possible by the use of a biocompatible, light-sensitive polymer as the phototransduction element.

The combined use of optical excitation and organic polymer technology can open interesting perspectives for several different reasons. First, the use of light modulation allows unprecedented spatial and temporal resolution to be achieved in a fully reversible way. Light temporal and spatial patterns can be specifically designed and adapted to different in vitro cell models, allowing ideally endless combinations of possibilities, to finely tune overall output in cell proliferation and network formation. The demonstrated technology is minimally invasive, allows for massive parallelization of experiments, and can be virtually implemented in any cell therapy model in a straightforward way. In addition, the use of different polymers, with lower energy gap and in the form of nanobeads, may pave the way to the optical enhancement of therapeutic angiogenesis in vivo. Further work is needed to understand whether the pattern and/or intensity of the illumination protocol may be adjusted to further boost the angiogenic response. For instance, the optical excitation protocol consisted of 30-ms-long light pulses that were delivered at 1 Hz for 4 (tubulogenesis) up to 36 (proliferation) hours. This is likely to result in oscillations in [Ca2+]i, which are known to deliver the most instructive signal for ECFCs to undergo angiogenesis by inducing the nuclear translocation of the p65 NF-B subunit (26). As the frequency of intracellular Ca2+ oscillations can be artificially manipulated to regulate NF-Bdependent gene expression in virtually any cell type (62), we envisage an additional layer of specificity and control that could be exploited to further improve the angiogenic response to optical excitation. Future work will also be devoted to assess the outcome of optical modulation on patient-derived ECFCs. One of the main hurdles associated to autologous cell-based therapy is the impairment of the angiogenic activity of EPCs, including ECFCs harvested from cardiovascular patients (29). The therapeutic translation of our findings will require the demonstration that light-induced TRPV1 activation boosts angiogenesis also in ECFCs derived from individuals affected by severe cardiovascular disorders, such as hypertension, atherosclerosis, and heart failure. In this view, the combination of organic semiconductors and genetic manipulation to increase endogenous TRPV1 expression could be sufficient to restore the reparative phenotype of autologous ECFCs from cardiovascular patients.

Regioregular P3HT (99.995% purity; Mn 54,000 to 75,000 molecular weight) was purchased from Sigma-Aldrich and used without any further purification. The samples for cell cultures were prepared by spin coating on a square 18 mm by 18 mm glass (VWR International) substrates carefully rinsed in subsequent ultrasonic baths of ultrapure water, acetone, and isopropanol. P3HT solution was prepared in chlorobenzene at a final P3HT concentration of 20 g/liter and spin coated on the cleaned substrates with a two-step recipe: (i) 3 s at 800 rpm and (ii) 60 s at 1600 rpm. Polymer film thickness is about 150 nm.

Microposit S1813 photoresist was purchased from Shipley and used without any further purification. Photoresist thin films were prepared by spin coating on cleaned substrates with a two-step recipe: (i) 3 min at 300 rpm and (ii) 30 s at 2600 rpm. Parameters were adjusted to obtain homogeneous films and similar optical absorbance to the one of the polymer thin films, at the same excitation wavelength used in the long-term stimulation protocol (see below). All films were thermally treated in an oven at 120C for 2 hours for annealing and sterilization. To promote adhesion, samples were coated with fibronectin (from bovine plasma; Sigma-Aldrich) at a concentration of 2 mg/ml in phosphate-buffered saline (PBS) for at least 30 min at 37C and then rinsed with PBS.

ECFCs were isolated from peripheral blood and expanded as shown elsewhere (26). Blood samples (40 ml) collected in EDTA-containing tubes were obtained from healthy male human volunteers aged from 28 to 38 years. The Institutional Review Board at Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Foundation in Pavia approved all protocols and specifically approved this study. Informed written consent was obtained according to the Declaration of Helsinki of 1975 as revised in 2008. We focused on the so-called ECFCs, a subgroup of EPCs that are found in the CD34+ CD45 fraction of circulating mononuclear cells (MNCs), exhibit robust proliferative potential, and form capillary-like structures in vitro (23). To isolate ECFCs, MNCs were obtained from peripheral blood by density gradient centrifugation on lymphocyte separation medium for 30 min at 400g and washed twice in EBM-2 with 2% fetal calf serum. A median of 36 106 MNCs (range, 18 to 66) was plated on fibronectin-coated culture dishes (BD Biosciences) in the presence of the endothelial cell growth medium EGM-2 MV (Lonza) containing endothelial basal medium (EBM-2), 5% fetal bovine serum (FBS), recombinant human (rh) EGF, rhVEGF, recombinant human Fibroblast Growth Factor-Basic (rhFGF-B), recombinant human Insulin-like Growth Factor-1 (rhIGF-1), ascorbic acid, and heparin and maintained at 37C in 5% CO2 and humidified atmosphere. Nonadherent cells were discarded after 2 days, and thereafter, medium was changed three times a week. The outgrowth of ECFCs from adherent MNCs was characterized by the formation of a cluster of cobblestone-shaped cells. That ECFC-derived colonies belonged to the endothelial lineage was confirmed by staining with anti-CD31, anti-CD105, anti-CD144, anti-CD146, antivon Willebrand factor, anti-CD45, and anti-CD14 monoclonal antibodies and by assessment of capillary-like network formation in the in vitro tube formation assay.

For our experiments, we have mainly used endothelial cells obtained from early-passage ECFCs (P2-4, which roughly encompasses a 15- to 18-day period) with the purpose to avoid, or maximally reduce, any potential bias due to cell differentiation. However, to make sure that the phenotype of the cells did not change throughout the experiments, in the preliminary experiments, we tested the immunophenotype of ECFCs at different passages, and we found no differences. We also tested whether functional differences occurred when early (P2) and late (P6)passage ECFCs were used by testing the in vitro capacity of capillary network formation in a Cultrex assay and found no differences between early- and late-passage ECFC-derived cells (data not shown).

Electrophysiological recordings were performed using a patch-clamp setup (Axopatch 200B; Axon Instruments) coupled to an inverted microscope (Nikon Eclipse Ti). ECFCs were measured in whole-cell configuration with freshly pulled glass pipettes (3 to 6 M), filled with the following intracellular solution: 12 mM KCl, 125 mM K-gluconate, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM EGTA, 10 mM Hepes, and 10 mM ATP (adenosine 5-triphosphate)Na2. The extracellular solution contained the following: 135 mM NaCl, 5.4 mM KCl, 5 mM Hepes, 10 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2. Only single cells were selected for recordings. Acquisition was performed with the pCLAMP 10 software (Axon Instruments). Membrane currents were low pass filtered at 2 kHz and digitized with a sampling rate of 10 kHz (Digidata 1440 A; Molecular Devices). Data were analyzed with Clampfit (Axon Instruments) and Origin 8.0 (OriginLab Corporation).

For optical excitation of the polymer, a homemade petri cell culture illuminator, compatible with the use within the cell incubator, was designed and implemented. Its design included a black spacer made by fused filament fabrication, both to minimize overheating effects in the extracellular bath and to avoid unwanted light scattering/diffusion effects and cross-talk between different specimens. Optical excitation was provided by a green LED system, whose duty cycle, repetition rate, and intensity were set through a custom-made control circuit, comprising a microcontroller, a digital-to-analog converter, and an analog LED driver. The driver was connected to five green LEDs (SMB1N-525V-02; Roithner LaserTechnik GmbH, Vienna, Austria), with maximum emission wavelength at 525 nm, each carrying a collimator lens reducing the emission angle to 22. This way, up to five 3.5-cm petri dishes can be simultaneously treated with a homogeneous photoexcitation density of 40 mW/cm2. The long-term optical excitation protocol adopted for cell fate modulation consists of 30-ms-long pulses, followed by 70-ms-long dark conditions, continuously repeated for a minimum of 4 up to 36 hours in the case of tubulogenesis and proliferation assays, respectively.

Growth dynamics were evaluated by plating a total of 5 103 ECFC-derived cells into 10-mm fibronectin-treated cloning cylinders (5 104/cm2) in the presence of EGM-2 MV medium to facilitate the adhesion. After 12 hours, the medium was switched to EBM-2 supplemented with 2% fetal calf serum. For the pharmacological treatment, one of compounds was added to the medium: BAPTA (30 M), CPZ (10 M), RN-1734 (20 M), or RR (10 M). Cultures were incubated at 37C (in 5% CO2 and humidified atmosphere), and cell growth was assessed after 36 hours since the beginning of the long-term illumination protocol. At this point, cells were recovered by trypsinization from all the dishes, and the cell number was assessed by counting in a hemocytometer. Preliminary experiments showed no unspecific or toxic effect for each agent when used at these concentrations. Each assay was repeated in triplicate.

ECFC-derived cells from early-passage (P2 to P4) cultures were obtained by trypsinization and resuspended in EBM-2 supplemented with 2% FBS. EPC-derived cells (10 103) per well were plated in Cultrex basement membrane extract (Trevigen Inc., Gaithersburg, MD, USA) 10-mm fibronectin-treated cloning cylinders. Plates were then incubated at 37C, 5% CO2, and capillary network formation was assessed starting from 4 to 24 hours later. At least three different sets of cultures were performed every experimental point. Quantification of tubular structures was performed after 8 hours of incubation by measuring the total length of structures per field with the aid of the ImageJ software (National Institutes of Health, USA; http://rsbweb.nih.gov/ij/). To evaluate the role of TRPV1, the same protocol was repeated in the presence of the following drugs: BAPTA (30 M), CPZ (10 M), RN-1734 (20 M), or RR (10 M).

H2DCF-DA (Sigma-Aldrich) was used for the intracellular detection of ROS. ECFCs were seeded onto polymer and control substrates and subjected to the same photoexcitation protocol used for the in vitro tube formation assay. Immediately after the end of the protocol, cell cultures were incubated with the ROS probe for 30 min. After careful washout of the excess probe from the extracellular medium, the fluorescence of the probe was recorded (excitation/emission wavelengths, 490/520 nm; integration time, 70 ms for H2DCF-DA) with an inverted microscope (Nikon Eclipse Ti) equipped with an Analog-WDM Camera (CoolSNAP MYO, Teledyne Photometrics). To minimize the effects of the spectral overlap between the polymer absorption and emission spectra, and the probe emission, samples were turned upside down by using a homemade chamber with a 500-m-thick channel filled with extracellular medium. Variation of fluorescence intensity was evaluated over regions of interest covering single-cell areas, and reported values represent the average over multiple cells. See figure captions for additional details about statistical analysis. Image processing was carried out with ImageJ and subsequently analyzed with Origin 8.0.

Two sets of P3HT thin films (n = 12) were prepared as described above. The optical absorbance, the emission, and the Raman spectrum were measured immediately after fabrication. Then, all samples were exposed to ECFC growth medium (EBM-2 supplemented with 2% FBS) and incubated at 37C, 5% CO2 for 24 hours. The first set was taken in dark conditions (n = 6), and the second one was treated with the same optical excitation protocol used in the tubulogenesis assays (n = 6). After incubation, absorption, emission, and Raman spectrum were measured again in the same conditions as before. Absorption spectra were recorded by using a spectrophotometer (PerkinElmer Lambda 1040) in transmission mode. Photoluminescence spectra were acquired by using a Jobin-Yvon spectrofluorometer; the excitation wavelength was set at the polymer absorption peak wavelength (530 nm). Resonant Raman spectra were recorded by using visible light excitation at 532 nm (HORIBA Jobin-Yvon HR800 micro-Raman spectrometer system). Laser power intensity on the sample was kept at values lower than 0.03 mW to avoid laser-induced sample degradation. Spectra were typically recorded in the region 600 to 2000 cm1 and were calibrated against the 520.5 cm1 line of an internal silicon wafer. The signal-to-noise ratio was enhanced by repeated acquisitions (100). The measurements were conducted at room temperature (RT), and the resulting spectral resolution was 0.4 cm1.

To examine NF-B p65 subunit translocation into the nucleus in the individual ECFCs, the coverslips were fixed with 4% formaldehyde in PBS (20 min at RT) and permeabilized with 0.1% Triton X-100 in PBS (7 min at RT). Primary rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology, catalog no. Sc-372) was applied at a final dilution of 1:100 for 1 hour at 37C in a humidified chamber. After three washes with PBS, secondary chicken anti-rabbit Alexa(488)-conjugated antibody (1:200; Invitrogen, catalog no. A-21441) was applied for 1 hour at RT. After washing (three times in PBS), nuclei were counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI; 1:5000 dilution in PBS; 20 min at RT; Invitrogen, catalog no. D1306). Last, the coverslips with cells were mounted on microscope glass slides using Fluoroshield mount medium (Sigma, catalog no. F6182). Fluorescence images were taken with the same fluorescence microscope used for the electrophysiology experiments, using standard DAPI and fluorescein isothiocyanate filters set for the acquisition of DAPI and Alexa(488) fluorescence emission, respectively.

Cells were lysed in 0.5 ml of TRI Reagent (Sigma, catalog no. T9424), and total RNA was extracted according to the manufacturers protocol. One microgram of total RNA was retrotranscribed using SensiFAST cDNA Synthesis Kit (Bioline, London, UK, catalog no. BIO-65054). Real-time polymerase chain reaction (PCR) was performed using iTaq qPCR master mix according to the manufacturers instructions (Bio-Rad, Segrate, Italy, catalog no. 1725124) on a SFX96 Real-Time System (Bio-Rad). As a control, S18 ribosomal subunit was used, whose expression did not change across the conditions. For each gene, Ct was calculated by using the formula Ct = 2^(Ct(gene) Ct(S18)). The data are expressed as a percentage variation between P3HT light and glass light conditions and P3HT dark and glass dark samples, respectively. Sequences of oligonucleotide primers are listed in table S1.

The significance of differences was evaluated with unpaired Students t test or one-way analysis of variance (ANOVA) coupled with Tukey or Dunnetts post hoc test, as appropriate. Data are represented as means standard error of the mean (SEM). P < 0.05 was considered statistically significant. Statistical analysis was performed using the GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA).

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaav4620/DC1

Fig. S1. Local and global evaluation of the extracellular bath temperature.

Fig. S2. TRPV1 is endogenously expressed in ECFCs, and it is efficiently activated by polymer photostimulation.

Fig. S3. Current clamp measurements in HEK-293 cells.

Fig. S4. Pharmacological study on ECFCs seeded on polymer substrates in the darkEvaluation of effect on tubulogenesis.

Fig. S5. Polymer photostability.

Fig. S6. p65 NF-B nuclear translocation is unaltered in ECFCs seeded on glass subjected to light-induced photostimulation.

Fig. S7. mRNA levels of proangiogenic genes downstream of NF-B signaling.

Table S1. List of oligonucleotide primers used for real-time PCR.

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

Acknowledgments: We gratefully thank I. Abdel Aziz for the characterization of the homemade petri cell culture illuminator used for long-term optical excitation and P. Falvo for the constructive criticism of the manuscript and the helpful scientific discussions. Funding: This work was jointly supported by the European Research Council (ERC) under the European Unions Horizon 2020 research and innovation program LINCE, grant agreement no. 803621 (M.R.A.), the EU Horizon 2020 FETOPEN-2018-2020 Programme LION-HEARTED, grant agreement no. 828984 (F.L., F.M., and M.R.A.), the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (20182022)Department of Biology and Biotechnology L. Spallanzani, University of Pavia (F.M.), and Fondo Ricerca Giovani from the University of Pavia (F.M.). Author contributions: F.L., F.M., and M.R.A. planned the experiments. F.L. carried out the experimental measurements (electrophysiology, short- and long-term photoexcitation, evaluation of effects on proliferation, tubulogenesis, and ROS production). V.R. provided the ECFC models, took care of the cell cultures, and contributed to the tubulogenesis and proliferation experiments. G.T. prepared the polymer samples. A.D. designed, realized, and optimized the experimental setup for the long-term photoexcitation. L.T. and D.L. carried out the immunofluorescence and real-time PCR assays. P.C. contributed to the methodological discussion about gene expression. F.L. and M.R.A. wrote the main manuscript, with help from F.M. All authors contributed to the data interpretation and approved the final manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Conjugated polymers optically regulate the fate of endothelial colony-forming cells - Science Advances

Stem cells are biological cells which have the ability to distinguish into specialized cells, which are capable of cell division through mitosis. Amniotic fluid stem cells are a collective mixture of stem cells obtained from amniotic tissues and fluid. Amniotic fluid is clear, slightly yellowish liquid which surrounds the fetus during pregnancy and is discarded as medical waste during caesarean section deliveries. Amniotic fluid is a source of valuable biological material which includes stem cells which can be potentially used in cell therapy and regenerative therapies. Amniotic fluid stem cells can be developed into a different type of tissues such as cartilage, skin, cardiac nerves, bone, and muscles. Amniotic fluid stem cells are able to find the damaged joint caused by rheumatoid arthritis and differentiate tissues which are damaged. Medical conditions where no drug is able to lessen the symptoms and begin the healing process are the major target for amniotic fluid stem cell therapy. Amniotic fluid stem cells therapy is a solution to those patients who do not want to undergo surgery. Amniotic fluid has a high concentration of stem cells, cytokines, proteins and other important components. Amniotic fluid stem cell therapy is safe and effective treatment which contain growth factor helps to stimulate tissue growth, naturally reduce inflammation. Amniotic fluid also contains hyaluronic acid which acts as a lubricant and promotes cartilage growth.

With increasing technological advancement in the healthcare, amniotic fluid stem cell therapy has more advantage over the other therapy. Amniotic fluid stem cell therapy eliminates the chances of surgery and organs are regenerated, without causing any damage. These are some of the factors driving the growth of amniotic fluid stem cell therapy market over the forecast period. Increasing prevalence of chronic diseases which can be treated with the amniotic fluid stem cell therapy propel the market growth for amniotic fluid stem cell therapy, globally. Increasing funding by the government in research and development of stem cell therapy may drive the amniotic fluid stem cell therapy market growth. But, high procedure cost, difficulties in collecting the amniotic fluid and lack of reimbursement policies hinder the growth of amniotic fluid stem cell therapy market.

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The global amniotic fluid stem cell therapy market is segmented on basis of treatment, application, end user and geography: Segmentation by Treatment Allogeneic Amniotic Fluid stem cell therapy Autologous Amniotic Fluid stem cell therapy Segmentation by Application Regenerative medicines Skin Orthopedics Oncology Fetal tissue reconstruction Kidney regeneration Regeneration of neural tissue Cardiac regeneration Lung epithelial regeneration Others Drug research and development Segmentation by End User Hospital Ambulatory Surgical Centers Specialty Clinics Academic and Research Institutes Segmentation by Geography North America Latin America Europe Asia-Pacific Excluding China China Middle East & Africa

Rapid technological advancement in healthcare, and favorable results of the amniotic fluid stem cells therapy will increase the market for amniotic fluid stem cell therapy over the forecast period. Increasing public-private investment for stem cells in managing disease and improving healthcare infrastructure are expected to propel the growth of the amniotic fluid stem cell therapy market.

However, on the basis of geography, global Amniotic Fluid Stem Cell Therapy Market is segmented into six key regions viz. North America, Latin America, Europe, Asia Pacific Excluding China, China and Middle East & Africa. North America captured the largest shares in global Amniotic Fluid Stem Cell Therapy Market and is projected to continue over the forecast period owing to technological advancement in the healthcare and growing awareness among the population towards the new research and development in the stem cell therapy. Europe is expected to account for the second largest revenue share in the amniotic fluid stem cell therapy market. The Asia Pacific is anticipated to have rapid growth in near future owing to increasing healthcare set up and improving healthcare expenditure. Latin America and the Middle East and Africa account for slow growth in the market of amniotic fluid stem cell therapy due to lack of medical facilities and technical knowledge.

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Some of the key players operating in global amniotic fluid stem cell therapy market are Stem Shot, Provia Laboratories LLC, Thermo Fisher Scientific Inc. Mesoblast Ltd., Roslin Cells, Regeneus Ltd. etc. among others.

The report covers exhaustive analysis on: Amniotic Fluid Stem Cell Therapy Market Segments Amniotic Fluid Stem Cell Therapy Market Dynamics Historical Actual Market Size, 2012 2016 Amniotic Fluid Stem Cell Therapy Market Size & Forecast 2016 to 2024 Amniotic Fluid Stem Cell Therapy Market Current Trends/Issues/Challenges Competition & Companies involved Amniotic Fluid Stem Cell Therapy Market Drivers and Restraints

Regional analysis includes North America Latin America Europe Asia Pacific Excluding China China The Middle East & Africa

Report Highlights: Shifting Industry dynamics In-depth market segmentation Historical, current and projected industry size Recent industry trends Key Competition landscape Strategies of key players and product offerings Potential and niche segments/regions exhibiting promising growth A neutral perspective towards market performance

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Amniotic Fluid Stem Cell Therapy Market to Rear Excessive Growth During 2018 2026 - Herald Space

What if CRISPR could be used not just to edit genes, but also to alter the epigenomethe vast network of chemicals and proteins that orchestrates the actions of genes? A study from biomedical engineers at Duke University describes a new CRISPR technology that could allow scientists to do just that.

The most commonly used gene editing technology, CRISPR-Cas9, uses just one Cas protein to cut DNA. In the CRISPR field, this is known as a class 2 system. Class 1 systems, by contrast, are more complicated because they rely on multiple proteins to bind to DNA and then recruit a Cas3 protein to cut it. That network of proteins is called Cascade (CRISPR-associated complex for antiviral defense).

The Duke team used a class 1 CRISPR system to edit the epigenome in cells. In a study published in Nature Biotechnology, they reported that they were able to attach gene activators to the Cascade complex and regulate levels of gene expression in cells. They also connected a repressor to Cascade to turn genes off altogether.

Synthetic Controls: Best Practices and Regulatory Perspectives

With increasing competition for patients, the ability to effectively and efficiently execute on clinical development plans has increased focus on alternative data sources. In this one-hour session attendees will hear from former FDA leaders and industry experts who will address what Synthetic Control is and how it can be leveraged in clinical development.

"We have found Cascade's structure to be remarkably modular, allowing for a variety of sites to attach activators or repressors, which are great tools for altering gene expression in human cells," said Adrian Oliver, Ph.D., a postdoctoral fellow and lead author of the study, in a statement.

RELATED: A better CRISPR? RNA 'hairpins' could improve gene editing

The potential of CRISPR-Cas3 is already generating some excitement in the biopharma world. In January, Locus Biosciences inked a deal with Johnson & Johnson to develop its platform for using CRISPR-Cas3 to solve the problem of antibiotics resistance. The deal could be worth up to $818 million for Locus, a 2019 Fierce 15 company.

Meanwhile, several academic groups are investigating various ideas for improving CRISPR, including another Duke team thats focused on improving the Cas9 enzyme. In April, it described a technique for adding a short tail to the guide RNA thats used in CRISPR systems to improve the accuracy of gene cuts.

Researchers at Columbia University are hoping to sidestep DNA cutting altogether. Theyre using a transposon, or jumping gene, in a system designed to insert DNA in exact locations in the genome without cutting.

The Duke researchers working onclass 1 CRISPR technology are planning further studies to determine whether it might help solve some of the shortcomings of CRISPR-Cas9 in addressing human disease, including the risk of dangerous immune responses. They are also investigating whether the tool could be used to perform many different genome engineering tasks simultaneously.

"We know CRISPR could have a big impact on human health," said Charles Gersbach, Ph.D., Duke professor of biomedical engineering. "But we're still at the very beginning of understanding how CRISPR is going to be used, what it can do, and what systems are available to us.

See the original post here:
New CRISPR approach could improve gene and cell therapies - FierceBiotech

HIV treatment has come a long way over the years, due in large part to antiretroviral drugs that stop the HIV virus from replicating in the body. This gives the immune system a chance to repair itself and stop further damage. Thanks to these amazing advances, HIV is no longer the death sentence that it was in previous decades.

However, antiretrovirals only keep HIV at bay for as long as theyre taken. Defaulting on the drugs means that the HIV virus comes back. Even worse, it can cause patients to build up resistance to the antiretrovirals so that they do not work so effectively in the future.

In other words, theres still room for improvement when it comes to treatment. Fortunately, researchers from the University of California San Diego School of Medicine are poised to provide help, courtesy of a new genetic-sequencing approach that could possibly provide a kill switch to clear out dormant HIV reservoirs inside cells.

The most exciting part of this discovery has not been seen before, Tariq Rana, professor of pediatrics and genetics at UC San Diego School of Medicine, said in a statement. By genetically modifying a long non-coding RNA, we prevent HIV recurrence in T cells and microglia upon cessation of antiretroviral treatment, suggesting that we have a potential therapeutic target to eradicate HIV and AIDS.

The work is based on the discovery of a recently emerged gene that appears to regulate HIV replication in immune cells, including macrophages, microglia, and T cells. The team refers to this as HIV-1 Enchanced LncRNA (HEAL), and it is elevated in people with HIV. By using CRISPR-Cas9 gene editing, their work suggests that it could stop HIV from recurring in the event that antiretroviral treatment is stopped.

This has the potential for [being a] cure but, [well] have to wait for animal studies, Rana told Digital Trends. As for the next steps, Rana said that future studies will determine if turning this regulator HEAL off can remove viral reservoirs, which are the key source for viral rebound when therapies are discontinued.

A paper describing the work was recently published in the journal mBio.

See the article here:
CRISPR-Cas9 gene editing could one day turn off HIV virus in the body - Digital Trends

[Tweaking an embryos DNA can] help save someone who is already alive.

Take the case of Jessica and Keith, a couple in the Bay Area with a 2 1/2-year-old daughter with Fanconi anemia, a genetic disease that leads to the failure of bone marrow to produce red and white blood cells and carries an increased risk of a number of cancers. The best treatment is a stem cell transplant from a sibling, and Jessica and Keith, who asked that their last name not be used, are now in the process of trying to have another child through IVF who can serve as a donor whats known as a savior sibling.

But making an embryo thats both healthy and a suitable donor match for the older sibling is an exercise in long odds. Its theoretically possible that altering an embryos DNA with the genome-editor CRISPR could improve the process.

He and Jessica understand its too soon to use CRISPR in such cases. The technology is not advanced or precise enough yet and might never be.

But its another example of the ways in which genome-editing could help patients where other reproductive technologies cannot.

Read full, original post: Could editing the DNA of embryos with CRISPR help save people who are already alive?

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CRISPR could save the lives of sick children by tweaking the embryos of their siblings - Genetic Literacy Project

Integration of CRISPR-case9 technology to accelerate the discovery of innovative antibiotics

DEINOVE (Euronext Growth Paris: ALDEI), a French biotechnology company that relies on a radical innovation approach to develop innovative antibiotics and bio-sourced active ingredients for cosmetics and nutrition, announces that it has expanded its technological platform with an advanced genetic tool, the CRISPR-cas9 system, to enhance its ability to optimize various microorganisms.

In the last few years, DEINOVE has set up a high throughput genetic engineering platform specifically dedicated to rare microorganisms and thus demonstrated its ability to adapt genetic tools to poorly described organisms. Thus, the exploitation of Deinococci as microbial plants has allowed the large-scale production of pure high value-added compounds such as carotenoids. It should be recalled that Deinococci are extremophilic microorganisms whose biological and molecular specificities have so far been little studied and therefore unexploited.

After developing a platform dedicated to the identification of novel antibiotic structures produced by rare bacteria (AGIR Program), DEINOVE strengthens its expertise in genetic engineering with the integration of a cutting-edge tool, the CRISPR-cas9 technology, known as molecular scissors, which has revolutionized genetic engineering in recent years.

The objective for DEINOVE is to be able to directly manipulate the strains producing antimicrobial activities or to transfer these activities into phylogenetically close frames. This has been successfully achieved by the Company which has made the Streptomyces chassis an effective producer of a pharmaceutical intermediate initially produced by Microbacterium arobescens (proof of concept DNB101/102).

Genome editing occurs at two levels. First, highlights the cluster of genes at the origin of the antibiotic activity of interest. To optimize the spectrum of activity and eliminate any potential cytotoxicity, the structure of a natural molecule can then be modified by directly, finely and precisely editing the genes responsible for this activity.

This technology opens up many opportunities in the identification and optimized production of new antibiotic structures.

"Our expertise in the genetic engineering of a variety of microorganisms, unusual for some, is unique, and the integration of CRISPR-cas9 extends the possibilities of our platform," says Georges GAUDRIAULT, Scientific Director of DEINOVE. "We continue to structure the various technological bricks of the AGIR platform to be able to drastically accelerate the identification and optimization of new antibiotic structures. This technology is an additional asset in our race against the clock in the face of rising antimicrobial resistance."

ABOUT DEINOVE

DEINOVE is a French biotechnology company, a leader in disruptive innovation, which aims to help meet the challenges of antibiotic resistance and the transition to a sustainable production model for the cosmetics and nutrition industries.

DEINOVE has developed a unique and comprehensive expertise in the field of rare bacteria that it can decipher, culture, and optimize to disclose unsuspected possibilities and induce them to produce biobased molecules with activities of interest on an industrial scale. To do so, DEINOVE has been building and documenting since its creation an unparalleled biodiversity bank that it exploits thanks to a unique technological platform in Europe.

DEINOVE is organized around two areas of expertise:

Within the Euromedecine science park located in Montpellier, DEINOVE employs 60 employees, mainly researchers, engineers, and technicians, and has filed more than 350 patent applications internationally. The Company has been listed on EURONEXT GROWTH since April 2010.

CONTACTS

See the article here:
Integration of CRISPR-case9 technology to accelerate the discovery of innovative antibiotics - GlobeNewswire

BODY LANGUAGE

Cutting-edge medical advancements are changing the way we think about having children. From gene therapy and pre-implantation genetic testing to women carrying children in transplanted uteruses, these medical advancements each come with an array of issues.

Perhaps the most prominent advancement featuring in the debate is that of gene-editing using CRISPR-Cas9 technology.Gene editing has already been used to create tomatoes that can sit in the pantry and ripen slowly for months without rotting.

It has been used to modify mosquitos so that they are unable to transmit malaria, and create ultra-muscular dogs to be used by law enforcement authorities. Gene editing has even allowed us to create cows without horns.

The most controversial application for gene editing which has been considered, is the genetic modification of the human species. Jennifer Doudna, the American biochemist who is credited for developing the CRISPR-Cas9 system, has been quoted as saying: The power to control our species genetic future is awesome and terrifying. Deciding how to handle it may be the biggest challenge we have ever faced.

Beverley Townsend and Donrich Thaldar(Gene editing is risky, but worth it, Mail & Guardian, September 13), suggest that the blanket moratorium called for by the international community blocks scientific progress and, with it, the opportunity to act responsibly.

They offer two specific examples of gene editing of human embryos: that of Chinese scientist He Jiankuis covert gene editing of twin girls in 2018, and the planned gene editing of embryos by Russian molecular biologist Denis Rebrikov.

Townsend and Thaldar provide an explanation of these two examples, but after brief mention of the gene-editing of the twin girls, who were subsequently born alive and are reportedly healthy, they shift their focus to Rebrikovs plans to remove the genetic sequence for inheritable deafness from embryos created using the gametes of a deaf couple.

Rebrikov is not the best example of the considerations in the debate, because the proposed gene editing itself is problematic. The Chinese scientist planned to use gene editing to make the children born resistant to HIV infection. This was clearly a therapeutic aim. But are Rebrikovs plans to remove inheritable deafness from embryos therapeutic? It is arguable whether deafness is a disease or a disability. In fact, a whole section of society does not believe that deafness is a disability, and that such proposed treatment amounts to discrimination against the deaf population.

The implications of what He has done is crucial to the discussion. Rebrikovs expression of his intention provides us with the opportunity to identify and debate issues regarding gene editing, and the control which we are prepared to allow ourselves to exercise over our genetic heritage.

But, the Chinese incident clearly demonstrates exactly why a moratorium on the gene editing of human embryos must be the first words in the conversation.

He Jiankui used CRISPR-Cas9 to delete part the CCR5 gene that allows HIV to enter cells. The world had a mixed reaction to the sudden announcement of the birth of the children, and some hailed it as the medical miracle that signalled the end of HIV. But, there were many who did not reciprocate this sentiment.

For many doctors and scientists, He had acted in haste without any consideration of what was safe or ethical. At the outset, the ethical implications were clear: he had fated these children to be prized cattle in a society already ravaged with the HIV epidemic.

The news that broke in June this year was more devastating, and proves that we must adopt a cautious, conservative approach to gene editing.

According to a study published in Nature Medicine, He may have inadvertently shortened the twins life expectancy. The study showed that people with two disabled copies of the CCR5 gene are 21% more likely to die before the age of 76 than are people with at least one copy of the gene. The reason for the discrepancy is not yet known.

There are useful measures that may help us to determine whether particular uses of gene editing are justified. We can ask whether the proposed gene editing amounts to enhancement of our biological systems, because therapeutic interventions are always ethically easier to justify over enhancements which offer no therapeutic benefit apart from expressing what we want to see in our future children. Consideration of what is in the best interests of the future child is also important.

The best interests of the child principle is firmly entrenched in law and asks us to consider what action would serve a childs interests best. If the proposed gene editing would place a child at a disadvantage, it would not be appropriate to proceed.

In 2008, a deaf British couple requested permission to genetically test their embryos to ensure that the child was deaf. The couple stated that being deaf is not about being disabled, or medically incomplete its about being part of a linguistic minority. Were proud, not of the medical aspect of deafness, but of the language we use and the community we live in.

A public outcry followed because many people viewed the intention to ensure that a child was hearing-impaired was against the best interests of the child.

The Human Fertilisation and Embryology Authority, which oversees the fertility industry in the United Kingdom, refused to permit the selection.

We should also consider whether the proposed gene editing may cause harm to the child which may be born. There is also the bioethical norm of beneficence, which asks us to consider whether we are doing good by our actions.

Townsend and Thaldar correctly state that we are not aware of the risks of gene editing on human embryos, but they conclude that this cannot be a reason to prohibit the treatment.

They correctly identify that there are risks with any therapeutic procedure. But we subject ourselves to therapeutic procedures after at least some of the inherent risks have been identified and measured. We would not do so if there were no studies regarding the efficacy and safety of such procedures.

This is not a risk we would take for ourselves, let alone the earliest and most vulnerable forms of human life.

A fundamental question is: Who gets to decide these issues? The public and legal and scientific communities need to be involved, and this cannot happen overnight.

Moratoria provide us with the opportunity to pause, debate and conduct critical research, which will allow us to safely navigate the course towards clinical treatment. This is responsible scientific progress.

There may be grave consequences of gene editing humans that we do not yet know. When we exercise control over genes, we are determining what people of the future will be like. This cannot be taken lightly, and we cannot allow cowboy scientists to proceed with gene editing of human embryos until the risks are better understood.

The published study comes too late for the two little girls. Perhaps they will live long, healthy lives. But, there is now evidence against that possibility. Gene editing is definitely worth it, but we must proceed with caution.

Sheetal Soni is an academic at the School of Law at the University of Kwa-Zulu-Natal

See original here:
Yes, edit our genes but do it cautiously - Mail and Guardian

We are delighted to be recognized by DTRA for the advanced state of our technology and development efforts. This support will further our mission to harness the power of CRISPR and synthetic biology to develop diagnostic tools that enable effective decision making in any environment at any time, said Rahul K. Dhanda, Sherlocks co-founder, president and CEO. This funding will enable our team to continue to make important progress advancing our platform, while also addressing an urgent need to rapidly identify pathogenic agents and other biothreats.

The multiyear grant will accelerate the companys development of infectious disease diagnostic tests for decentralized settings, such as the battlefield. Further, the support will help the company refine its deep learning and bioinformatics tools for rapid definition and deployment of molecular diagnostic tests.

When we published the call for a new class of rapid diagnostics, we were optimistic that we would identify both a technology and an organization that could deliver a significant leap forward in testing for biothreat and battlefield settings through initiatives driven by the Defense Innovation Unit, said Charles Hong, a science and technology manager for the Detection and Diagnostics Division at DTRA. We are pleased to support Sherlock in its efforts to create novel CRISPR- and synthetic biology-based diagnostic tests that will be sensitive, fast and easily deployable in any setting.

The Defense Innovation Unit identifies commercial industry solutions to support the U.S. military, and through this process, the organization facilitated finding a new class of rapid diagnostic solutions on behalf of DTRA.

This funding provides important validation for the versatility of Sherlocks platform, and our ability to develop simple, impactful diagnostic tools that can be applied in field-based settings, said William Blake, Ph.D., Sherlocks chief technology officer. We are grateful to DTRA for this award and look forward to working with the agency in support of their mission to enable rapid responses to potential threats and crises.

About Sherlock Biosciences

Sherlock Biosciences is dedicated to making molecular diagnostics better, faster and more affordable through Engineering Biology platforms. The company is developing applications of SHERLOCK, a CRISPR-based method to detect and quantify specific genetic sequences, and INSPECTR, a Synthetic Biology-based molecular diagnostics platform that is instrument free. SHERLOCK and INSPECTR can be used in virtually any setting without complex instrumentation, opening up a wide range of potential applications in areas including precision oncology, infection identification, food safety, at-home tests, and disease detection in the field. For more information visit Sherlock.bio.

About DTRA

The Defense Threat Reduction Agency (DTRA), an agency within the United States Department of Defense (DoD), is the official Combat Support Agency for countering weapons of mass destruction (chemical, biological, radiological, nuclear, and high explosives). The Defense Threat Reduction Agency enables DoD, the U.S. government, and international partners to counter and deter weapons of mass destruction and improvised threat networks. Under the auspice of the Chemical and Biological Defense Program, DTRA has the responsibility to manage and integrate the DoD chemical and biological defense science and technology programs. DTRAs continued effort to enhance the combat support mission also advances public health services by developing innovative technologies that protect against biological threats. For more information, visit http://www.dtra.mil.

About Defense Innovation Unit

DIU strengthens our national security by increasing the adoption of commercial technology throughout the military and growing the national security innovation base. Learn more about DIU at http://www.diu.mil.

Continued here:
Sherlock Biosciences Awarded Contract from US Defense Threat Reduction Agency to Support the Development of Ultra-Fast, Ultra-Sensitive Diagnostics -...

CAMBRIDGE, Mass., Sept. 26, 2019 (GLOBE NEWSWIRE) Intellia Therapeutics, Inc. (NASDAQ: NTLA) announced today that an arbitration panel issued an Interim Award confirming that certain structural and chemical guide RNA modification technologies were exclusively licensed to Intellia by Caribou Biosciences under the parties July 2014 agreement. This Interim Award is subject to additional negotiations between the parties and potentially further arbitration proceedings before it becomes final.

After concluding that the chemical modification technology was within the scope of Intellias exclusive license from Caribou, the arbitration panel noted that its decision could delay or otherwise adversely impact the continued development of these modified guide RNAs as human therapeutics. It also noted that Intellia currently is not using these modified guide RNAs in any of its active programs. For this reason, the panel stated it will declare that Caribou has leaseback rights, which it described as exclusive, perpetual and worldwide, to the chemically modified guide RNAs. This leaseback only applies to the chemically modified guides and will be subject to terms, including Caribous future payments to Intellia, to be negotiated by the parties or, if unsuccessful, to additional arbitration proceedings.

The leaseback will not include the structural guide modifications intellectual property at issue in the arbitration, any other intellectual property exclusively licensed or sublicensed by Caribou to Intellia under the Caribou license (including but not limited to the foundational CRISPR/Cas9 intellectual property co-owned by University of California, University of Vienna and Dr. Emmanuelle Charpentier), or any other Intellia intellectual property.

Upon, and subject to the terms of, a final award, which will follow negotiations between the parties and potential further legal proceedings, Caribou would be able to use the modified guide RNAs at issue for human therapeutics. Intellia or Caribou may challenge the arbitration panels decisions under limited circumstances.

The Interim Award has no impact on any of Intellias current programs. Additionally, the Interim Award has no effect on any other Intellia rights or Caribou obligations under their agreement.

Background on Intellias License from Caribou BiosciencesIn July 2014, Intellia Therapeutics, Inc. licensed from Caribou Biosciences, Inc. certain intellectual property. On October 17, 2018, the Company initiated an arbitration proceeding with the Judicial Arbitration and Mediation Services (JAMS) against Caribou asserting that Caribouis violating the terms and conditions of theCaribou license, as well as other contractual and legal rights, by using and seeking to license to third parties technology covered by two patent families (described in, for instance, PCT No. PCT/US2016/015145 and PCT No. PCT/US2016/064860, and related patents and applications) relating to specific structural or chemical modifications of guide RNAs.

AboutIntellia TherapeuticsIntellia Therapeuticsis a leading genome editing company focused on developing curative therapeutics using the CRISPR/Cas9 system. Intellia believes the CRISPR/Cas9 technology has the potential to transform medicine by permanently editing disease-associated genes in the human body with a single treatment course, and through improved cell therapies that can treat cancer and immunological diseases, or can replace patients diseased cells. The combination of deep scientific, technical and clinical development experience, along with its leading intellectual property portfolio, puts Intellia in a unique position to unlock broad therapeutic applications of the CRISPR/Cas9 technology and create a new class of therapeutic products. Learn more aboutIntellia Therapeuticsand CRISPR/Cas9 atintelliatx.comand follow us on Twitter @intelliatweets.

Forward-Looking StatementsThis press release contains forward-looking statements ofIntellia Therapeutics, Inc.(Intellia or the Company) within the meaning of the Private Securities Litigation Reform Act of 1995. These forward-looking statements include, but are not limited to, express or implied statements regarding Intellias beliefs and expectations regarding: its ability to advance and expand its CRISPR/Cas9 technology to develop human therapeutic products, as well as maintain, protect and expand its related intellectual property portfolio; its plans to negotiate, and ability to agree to, a leaseback with Caribou, including the scope of such arrangement and the timing and amount of payment under any such arrangement; the potential to initiate additional arbitration or legal proceedings if negotiations are not successful; the potential implications and impact the Interim Award may have on Intellias current programs or on any other intellectual property rights and changes in any of the foregoing in connection with the issuance of a final award; its ability to develop otherin vivoorex vivocell therapeutics of all types; and the impact of any of the foregoing on its collaborations and licensing arrangements.

Any forward-looking statements in this press release are based on managements current expectations and beliefs of future events, and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to: risks related to Intellias ability to protect and maintain our intellectual property position, including as a result of the Interim Award or the finalization of any award; risks related to Intellias relationship with third parties, including our licensors; or risks related to our ability, or the ability of our licensors, to protect and maintain their intellectual property position. For a discussion of these and other risks and uncertainties, and other important factors, any of which could cause Intellias actual results to differ from those contained in the forward-looking statements, see the section entitled Risk Factors in Intellias most recent annual report on Form 10-K as well as discussions of potential risks, uncertainties, and other important factors in Intellias other filings with theSecurities and Exchange Commission. All information in this press release is as of the date of the release, andIntellia undertakes no duty to update this information unless required by law.

Intellia Contacts:Investors:Glenn GoddardExecutive Vice President, Chief Financial Officer+1 857-706-1056glenn.goddard@intelliatx.com

Media:Jennifer Mound SmoterSenior Vice President, External Affairs & Communications+1 857-706-1071jenn.smoter@intelliatx.com

Read more:
Arbitration Decision Affirms Intellia Therapeutics Interpretation of Licensing Agreement with Caribou Biosciences on the CRISPR/Cas9 Technology -...

A Recent Research By MarketResearch.Biz On CRISPR and Cas Genes Market Provides A Professional, In-depth, Comprehensive, and Well-designed Insights that can help you to make better business decisions

The globalCRISPR and Cas Genes Marketis studied and analyzed with the help of a complete primary and secondary market research. The report provides broad industry data on the CRISPR and Cas Genes market, such as size, forecasted growth, profitability, key players, market share and market trends will give you a high-level overview of CRISPR and Cas Genes market opportunities. This report studies the global market size of CRISPR and Cas Genes, especially focuses on the key regions like North America, Europe and Asia-Pacific, South America, Middle East, and Africa

The market size section gives the CRISPR and Cas Genes market revenues, covering historic growth of the market as well as forecasting the future. Moreover, the report covers a host of company profiles, who are making a mark in the industry or have the potential to do so. The profiling of the players includes their market size, key product launches, information regarding the strategies they employ, and others.

The Leading key players covered in this study:Addgene Inc, AstraZeneca Plc., Bio-Rad Laboratories Inc, Caribou Biosciences Inc, Cellectis S.A., Cibus Global Ltd, CRISPR Therapeutics AG, Editas Medicine Inc, eGenesis Bio, GE Healthcare, GenScript Corporation

To Obtain All-Inclusive Information On Forecast Analysis Of GlobalCRISPR and Cas GenesMarket, Request A Pdf Report Herehttps://marketresearch.biz/report/crispr-and-cas-genes-market/request-sample

WorldwideCRISPR and Cas Genes Market: Market Segmentation

Segmentation on the basis of product:

Vector-based CasDNA-free CasSegmentation on the basis of application:

Genome EngineeringDisease ModelsFunctional GenomicsKnockdown/ActivationSegmentation on the basis of end user:

Biotechnology & Pharmaceutical CompaniesAcademic & Government Research InstitutesContract Research Organizations

In the end, The report also gives complete information regarding the research methodology of the CRISPR and Cas Genes market.

High level Business Questions Answered and Covered in this Report: Heres a checklist

What is the market size of the CRISPR and Cas Genes market at the global level?

Which is the preferred age group for targeting CRISPR and Cas Genes for manufacturers?

What are the key factors driving, growth of the market?

What is the impact of the regulations on the growth of the CRISPR and Cas Genes market?

Which is the leading country and region for the growth of the market?

What is the anticipated growth rate of the major regions during the forecast period?

How are the emerging markets for CRISPR and Cas Genes expected to perform in the coming years?

Who are the major players operating in the global CRISPR and Cas Genes market? What is the current market position of the players? Who are the emerging players?

Who are the major distributors, traders, and dealers operating in the CRISPR and Cas Genes market?

For More Actionable Insights Into The Competitive Landscape Of Global Market, Get A Customized Report Herehttps://marketresearch.biz/report/crispr-and-cas-genes-market/#inquiry

Research Methodology:

The report has been consolidated using three research methodologies. The first step centers around exhaustive primary and secondary researches, which includes an extensive collection of information on the Global CRISPR and Cas Genes Market and the parent and peer market.

The next step involves validating the market size, estimations, findings, and assumptions with further accurate information from industry experts. The report obtains a complete estimation of the market size with the help of bottom-up and top-down approaches. Finally, the report obtains the market estimation of all the segments and sub-segments using data triangulation and market breakup procedures.

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Global CRISPR and Cas Genes Market 2019 | Complete Research Study on the Current State of the Global Market with a Focus on the Regional Market - Biz...

The human body is a wonderfully and mind-numbingly complex machine, with any new therapy or medication kickstarting thousands of chain reactions barely perceptible to patients. The relatively-few effects that do matter can be deadly. Prescription drug-related deaths cost the healthcare system and grieving families more than $100 billion annually. Fortunately, cutting-edge consumer genetic kits can sound the alarm on potential problems that could arise if a patient takes a certain drug or drug of a certain class. But the Food and Drug Administration (FDA) never lets a good regulatory opportunity go to waste. The agency is determined to keep key prescription information out of the hands of patients and block some genetic tests altogether pending an onerous paperwork process. The FDAs war on genetic testing will harm patients and make it harder for millions of Americans to get the lifesaving care they need.

The next generation of medications holds significant promise for patients suffering from illnesses ranging from depression to malaria to bladder cancer. But, with this potential, patients must deal with the endless frustration of their bodies not responding properly to newly-available treatments. This is a particularly pressing problem in the world of anti-depressants where patients are asked to go through an agonizing trial-and-error process with different medications. Genetic testing companies such as Color Genomics and 23andMe have a solution for this and received FDA authorization in 2018 to add a depression component to their gene test results (available online to participating consumers).

For the first time, consumers can find out cheaply and quickly whether they have the gene variants typically responsive to anti-depressants. Patients can in turn easily pass this information along to their medical providers, who can prescribe accordingly. This genetic information revolution is trumpeted by scientific bodies such as the Association for Molecular Pathology, which recommends that providers be made aware of any information regarding the tests interpretation[including] A generalized statement to alert healthcare providers when alternate dosage or drug treatment may be considered based on the results.

But just as the FDA giveth, the agency threatens to taketh away. The first troubling sign came in October 2018, when the agency warned consumers and physicians against using these tests to determine whether certain medications should be embraced/avoided. After muddying the waters, the agency began to move against smaller testing companies guilty of the egregious sin of not consulting with the FDA. In April 2019, the agency sent a strictly-worded warning letter to Inova Genomics for, illegally marketing certain genetic tests that have not been reviewed by the FDA for safety and effectiveness.

In the perfect world of monolithic Washington bureaucrats, all companies offering innovative scientific services would submit thousands of oft-redundant pages of research for FDA review at a cost of hundreds of thousands of dollars for each small addition. In reality, smaller companies simply cannot keep pace with these regulatory requirements.

And if consumer genetic testing becomes the sole domain of a few large companies, the quality of testing and incentive to improve and update results will suffer. The FDAs logic only works assuming that the agency is better at gauging efficacy than prescribers and scientific bodies. But if the FDAs sorry track record with vaping products and nutritional foods is any indicator, the agency has had more than its fair share of misfires.

Rather than going down the same, tired road of restricting lifesaving innovations, the FDA should greenlight genetic testing and allow the market to develop. Consumers deserve access to inexpensive tests that could help them and their providers navigate through the tricky world of difficult-to-pronounce medications with scary side effects. The FDA could save thousands of lives and billions in healthcare costs by taking a step back and enabling patients to take charge of their own medical care.

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FDA Must Allow, Not Thwart, Genetic Testing Revolution | Ross Marchand - The Beacon

Israeli artificial intelligence (AI) solutions company RSIP Vision has partnered with BioView, a cell imaging and analysis solutions provider, to use artificial intelligence (AI) for classifying chromosomes for accurate genetic testing.

Chromosome classification for genetic testing, known as karyotype classification, involves pairing of chromosomes to identify any irregularities.

The procedure is often performed manually by cytologists. However, the manual approach is expensive and time-consuming.

The AI solution is expected to reduce time, as well as the expense associated with genetic testing, providing a significant improvement in results.

RSIP Vision CEO Ron Soferman said: Machine learning provides the unique capability to both segment and straighten out chromosomes so that the results that the patient receives are as accurate as possible.

When patients are undergoing genetic testing, it is often a sensitive time for them they are testing for various conditions that could potentially affect their lives forever. Reducing the time for patients to receive the results of genetic testing is a step in making this process easier and smoother, providing patients with the correct answers, faster.

During karyotype classification, cytologists analyse chromosome size, shape and number, as well as abnormal locations and pieces. The information indicates abnormal growth development or body functioning.

Machine learning boosts the genetic testing process through the capture of several data sets for the training phase.

RSIP Vision and BioView leveraged a deep learning technique trained on extensive datasets to facilitate precise data correlations for fast identification of any abnormalities.

BioView deployed the AI solution in Duet Image and Analysis systems at hospitals across the US and Europe. The solution led to increased testing throughput, as well as more accurate results when compared to other solutions, according to the companys customers.

In May, RSIP Vision launched an AI solution to match patients undergoing knee replacement surgery with the best possible implants.

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RSIP Vision and BioView to use AI for accurate genetic testing - Medical Device Network

(Fargo, ND) -- Gene testing is benefiting more women than ever by allowing them to dive deeper into their families history with breast and ovarian cancer.

"Do I wish 10 year ago I would've done this testing? Absolutely."

This is what two time cancer survivor and Sanford Roger Maris Cancer Center patient, Sandy Dunn, had to say about the genetic testing.

"I was diagnosed with breast cancer in 2008. I was 44 years old and then I had a recurrence in 2010," says Sandy.

During this time, her oldest daughter was diagnosed with breast cancer, just two weeks after her 34th birthday.

"They knocked her down pretty hard. She's pretty weak. It's very hard to see,um, she still has that beautiful smile though. I'm sorry. This has been tough. It's the hardest thing I've ever gone through in my life, but we're going to survive this, too."

After her daughter's diagnoses, Sandy took her and her youngest daughter to get genetic testing for breast cancer.

This test is a simple blood draw, but doctors are able to see if there is a gene that is increasing a risk for cancer, like the BRCA Gene.

"They're the ones that we know are associated with breast and ovarian cancer and then also prostate cancer in men," says Genetic Counselor Meghann Reardon.

This type of gene testing also shows doctors if these types of cancers are hereditary.

Reardon explains, "so, if a person test positive, we know that all their children - boys and girls - are at a 50% risk to have inherited that as well."

Knowing that information, Sandy wishes she would've taken the test sooner.

"As we started digging into this, I learned that there was other women in our history that had breast cancer and some of them had not survived. This was not information that we were discussing in our family. So this test brought up these topics of conversation," says Sandy.

While, Sandy said it was scary to have her BRCA test come out positive, she says it was also empowering. Moving forward, she can make her own decisions. Like other surgeries that can help lower her risk of getting breast or ovarian cancer, again.

"I keep saying that I've been driving in this car that's been weaving all over the road and I don't know what it's going to smash into and my driver has been cancer. This BRCA testing puts me in the drivers seat," says Sandy.

Meaning now, she's kicking cancer to the curb.

At this time, BRCA gene testing is only for patients with a concerning personal condition or family history of cancer.

If you'd like to know more, it's advised to speak with your doctor.

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BRCA Gene Testing allowing patients to dive deeper into their families history of breast cancer - Valley News Live

Its hard to regard Ellie as a menace.

When Greg Manteufel is frustrated or feeling down, she sits by him. At night, she sleeps under his covers. At dinner, shes there next to him, knowing hell throw something her way. She belies the stereotype of the vicious pit bull.

We love her like shes our daughter, he said of the dog.

And yet, Ellie may be the reason Manteufel, 49, nearly died.

Gravely ill, he lost parts of his arms and legs, as well as the skin of his nose and part of his upper lip. The cause was capnocytophaga, a germ from Ellies mouth or from another dog he encountered.

READ MORE: Dog owners limbs amputated after contracting rare infection from lick

Capnocytophaga is commonly found in the saliva of cats and dogs and almost never leads to people getting sick, unless the person has a compromised immune system. But Manteufel was perfectly healthy. In fact, he doesnt think hed ever used his health insurance before he fell ill.

The case is extremely rare and doctors at his hospital, Froedtert & the Medical College of Wisconsin, had no explanation for why he got so sick. But over the last 10 years there have been at least five other healthy people who have had severe reactions to the germ. A team of researchers connected with Harvard Medical School has developed a theory on why a gene change in all the victims.

And their finding means doctors cant rule out the capnocytophaga bacteria could strike Manteufel and other victims again.

Greg Manteufel thought he was getting the flu in June of 2018. He had a fever, vomiting and diarrhea. But when he started getting confused, his family took him to the hospital.

Doctors did blood cultures and found capnocytophaga, which caused sepsis, a severe blood infection that led to his blood pressure dropping and many of his organs shutting down.

Do what you have to, to keep me alive, he told the doctors.

In this Aug. 2, 2018 file photo provided by Dawn Manteufel, Greg Manteufel lays in his hospital bed at Froedtert Hospital in Milwaukee. He lost parts of his arms and legs, as well as the skin of his nose and part of his upper lip from capnocytophaga.

He had so much to live for foremost, his wife of 16 years, Dawn, and 26-year-old son, Mike. He was just starting to get really good at his day job, painting houses. He cherished his Harley Davidson Electric Glide. He was in the middle of fixing up his 66 El Camino. And of course there was Ellie, the pup.

And so he persisted, through more than 20 surgeries, including amputations of his left and right arms just below the elbow, and legs through the middle of the knee.

His wife and son stayed optimistic, because he was.

Greg said he didnt come this far to lay down and let this beat him, Dawn Manteufel said.

He was out of the in-patient rehab unit in about two weeks, learning to move from his wheelchair to the bed, toilet and car. The usual stay is three to four weeks, said Dr. David Del Toro, medical director for the inpatient rehab unit at Froedtert.

Manteufel made similar quick advances using his arm prosthetics and leg prosthetics.

He does not seem like any other patient Ive met before, Del Toro said. Hes just, you know, full speed ahead.

In this Aug. 19, 2019 photo, Greg Manteufel tries out a new prosthetic arm during occupational therapy at Froedtert & the Medical College of Wisconsin, in Milwaukee, as his wife Dawn Manteufel reads paperwork in the background.

Meanwhile, researchers at Brigham and Womens Hospital in Boston, connected to Harvard Medical School, as well as Dana-Farber Cancer Institute and Beth Israel Deaconess Medical Center had been investigating cases like his.

The team has done genetic testing on five otherwise healthy people who suffered capnocytophaga infections to see if they could find anything in common. They discovered all had a gene connected to the immune system that was working differently a genetic variant.

It was a really thrilling moment, said Elizabeth Fieg, a genetic counselor at Brigham and Womens Hospital. The stakes are so high with these cases and the patients have gone through so much.

READ MORE: Owning a dog is good for your heart study says what we all knew

They believe it makes those people more susceptible to developing severe medical problems from capnocytophaga. But they are also trying to determine if there are other risk factors.

Of the five in the study, three survived with amputations and two did not. Fieg hopes their research can determine why some did not survive.

She also hopes if their theory is confirmed, it will help diagnose cases faster, and perhaps save lives and limbs.

Thats why Greg Manteufel jumped at the chance to take part when he was approached in August.

Researchers need to gather more evidence, but hope to publish their study in the next year to 18 months.

Manteufels life now includes frequent occupational therapy appointments to perfect his use of arm prosthetics the kind with metal moveable hooks at the end. Hes using a fork regularly and hes now working on picking up the TV remote, opening doorknobs, cutting vegetables and doing the dishes.

Hes using shortened leg prosthetics, called stubbies, to get his body conditioned to eventually use to full-sized ones. Those are expected to arrive any day.

Plastic surgeons plan another surgery to perfect his nose. Theyve already moved skin from his forehead there. It looks oversized now, but it will eventually fit in with the rest of his face.

He plans to get his car revamped so he can drive with prosthetics. He wants to get a special pole so he can go fishing again. He is even considering going back to work painting.

Hes also become less quiet and a lot more outgoing. Now everybody I see wants to hear something or talk to me. I tell them a 15-minute story about what happened. They probably want me to leave, you know, he said, chuckling.

Ellies often by his side.

She loves kids. She loves puppies. Other dogs, Manteufel said.

As harmless as she seems, she may have capnocytophaga germ.

In this Aug. 16, 2019 photo, Greg Manteufel takes his dog Ellie from his wife Dawn Manteufel at their home in West Bend, Wis.

The results of Manteufels genetic tests are expected in three to four months. Fieg said people with the gene variant are at increased risk for recurrent capnocytophaga or other infections in the future.

While Manteufel doesnt like the sound of that, he said Ellies accidentally scratched him since hes been home and even licked his mouth. Hes been fine.

And even if he does have the gene variant, he said, it changes nothing.

We didnt even bother testing her, said Manteufel. We werent going to get rid of her if it was her that caused it anyway.

We just love her to death.

Read the original here:
Genetics might explain why this mans limbs had to be amputated after his dog licked him - Global News

A 28-year old woman arrived to the ER wheezing and short of breath. She had asthma and came in requesting a refill of her inhaler.

But when I examined her, I found out that something else much more serious was going on. The patients blood pressure was very high. Her legs and arms were swollen. Her lungs were filled with fluid. She told me that she had given birth a month before. During her pregnancy, she started having wheezing and trouble breathing, which she attributed to her asthma. Actually, she had untreated high blood pressure that progressed and worsened to life-threatening heart failure.

This scenario was tragically typical. As an emergency medicine physician working in ERs in Boston and Washington, D.C., I saw devastating cases like these far too often.

About 700 women in the U.S. die every year from complications of pregnancy, according to the CDC. And more than 50,000 women face severe health consequences in pregnancy. All of this is to say that women giving birth in the U.S. today are actually more likely than their own mothers to die in childbirth. In fact, more people in the U.S. are dying from pregnancy-related complications than in any other developed country, according to an investigation by NPR and ProPublica in 2017.

The public health crisis of maternal mortality is particularly acute for black women, who have three-to-four times the likelihood of dying in pregnancy and childbirth than white women. To some extent, this can be attributed to inequities in access to care, yet we know that these racial disparities persist even when controlling for all other factors. Structural racism in health care is a key contributor, as the research shows that health care providers are less likely to take the pain of black patients seriously. A recent study from the CDC found that three-in-five pregnancy-related deaths can be prevented, compounding the tragedy.

As a mother of a two-year-old, expecting my second child in March, I know that the period spanning pregnancy to parenting an infant is one of the most rewarding yet vulnerable times in a persons life. In medicine, we refer to pregnancy as a physiological stress test. A pregnant womans blood supply increases by nearly 50 percent; hormone levels fluctuate; and because of the growing uterus and increasing demands of the body, lung volumes reduce by five percent, while oxygen consumption increases. The levels of clotting factors in the blood increase in preparation for active labor, which is in itself a marathon that often lasts many hours.

The postpartum period is another time of incredible vulnerability. During this period, new mothers face a wave of new challenges that can accumulate and often compound mental health conditions like postpartum depression. After giving birth, they become full-time caregivers to a newborn, while learning breastfeeding, barely sleeping, and often navigating the return to work. In a country that lacks federal paid parental leave, many new mothers are back to work within weeks of giving birth, which can result in health consequences like maternal stress and anxiety. Medicaid coverage for new mothers, many of whom are people of color, ends at 60 days after delivery, and many people lose health insurance during this time of medical need. Through these challenges, parents often prioritize the care of their infants and push their own health aside; it is estimated that as many as 40 percent of women do not attend a postpartum visit.

In recent months, there has been increased attention to maternal deaths during childbirth. These are important calls to action, but we must also pay attention to the research that shows that the majority of maternal deaths occur outside of the labor and delivery period itself. Approximately two-thirds of maternal deaths occur before or even months after delivery, according to the CDC. These deaths are largely attributed to undertreated chronic illnesses such as heart disease, high blood pressure, and mental illness.

In order to improve maternal health, we have to focus on improving all womens health and access to carenot just during labor and delivery, but before and after pregnancy, and throughout our lives.

It is imperative that we address the barriers to access and the systemic racism that we know is contributing to our astronomically high maternal mortality rates. The onus must be on the health care system to make necessary changes. But we cant wait for systemic change to occur if we need medical care now. Here are a few crucial steps you can take to advocate for yourself and obtain the best care you can:

You can stay on top of your wellness visits. Regardless of whether or not you have given birth or plan to in the future, you can do your best to take care of your health now. Women are often caregivers for our loved ones, but we must care for ourselves to care for others. The best time to keep yourself healthy is before you get sick.

So instead of waiting for something to be up, go ahead and schedule a well-woman visit with your doctor. Your doctor can screen you for common conditions like high blood pressure and diabetes, which are associated with complications during pregnancy including heart disease, preeclampsia and eclampsia (pregnancy-related disorders of high blood pressure), placental abruption (when the placenta separates from the wall of the uterus), and gestational diabetes.

You should also get tested regularly for STIs. If left untreated, STIs can lead to pelvic inflammatory disease and ectopic pregnancy (a pregnancy that grows outside of the uterus), the most common cause of death among women during the first trimester.

Make sure that you are up-to-date with preventive testing such as Pap tests and breast screenings. Even if youre not pregnant, diagnosing and treating medical conditions now can prevent complications during a future pregnancy.

You can choose contraception if youre not looking to get pregnant. Birth control can help you plan and time your pregnancy, which can help some women get pregnant at a time that is best for them and their bodies. Experts estimate that without access to contraceptives, many more mothers would die globally from pregnancy-related complications. Another study found that increasing contraceptive use in developing countries has cut the number of maternal deaths by more than 40 percent over the past two decades.

Birth control can also be beneficial for many patients in ways that dont directly relate with becoming pregnant. It can help with symptoms of other conditions, like polycystic ovarian syndrome (PCOS) and endometriosis.

You can ask for help. This is true for all women, regardless of whether or not theyre pregnant or plan to be pregnant. But pregnancy and the postpartum period can be particularly vulnerable times for mothers, especially those experiencing a mental health condition or substance abuse or addiction.

It can be really hard to know where to turn when you need help, but one place to start would be your primary care doctor or a local health clinic. Remember that your mental health is just as important as your physical health. Tell your doctor if you have feelings of depression, anxiety, and trouble coping. If you find yourself relying on substances such as alcohol or opioids to deal with the stresses of life, you may have a medical condition that treatment can help.

If youre pregnant or have recently given birth and youre experiencing any worrying symptoms, do not hesitate to seek medical attention. For urgent concerns, go to the ER to get care.

The patient whom I mentioned earlier was in the Intensive Care Unit for a week. Though she will now always have a heart condition, she is able to live her life and care for her young daughter. Her outcome could have been vastly different had she not received care in timeand it could still have been different if her high blood pressure was treated much earlier.

Ending maternal mortality wont be easy, but as mothers, future mothers, spouses, and friends, lets start with what we can do right now. After all, to have healthy women, children, and families in the future, we must start with healthy women today.

Leana S. Wen, M.D. M.Sc. FAAEM is an emergency physician and Visiting Professor at the George Washington University Milken School of Public Health. Follow on Twitter: @DrLeanaWen.

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As a Doctor and Mother, Heres What I Wish People Knew About the Maternal Mortality Crisis - Self

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According to the U.S. Department of Health & Human Services Office On Womens Health, hormonal birth control can be broken down into two subtypes: long-acting reversible contraceptives and short-acting hormonal methods.

Suzanna Arul

According to the U.S. Department of Health & Human Services Office On Womens Health, hormonal birth control can be broken down into two subtypes: long-acting reversible contraceptives and short-acting hormonal methods.

Suzanna Arul

Suzanna Arul

According to the U.S. Department of Health & Human Services Office On Womens Health, hormonal birth control can be broken down into two subtypes: long-acting reversible contraceptives and short-acting hormonal methods.

I started taking birth control after my first ever trip to the gynecologist. I went in looking for answers to the typical questions: Why does penetration hurt? Why does my period feel like the scene in Prometheus when Noomi Rapace births an alien? It felt like I only received one question back: Why arent you on birth control?

Although I came in feeling like a strong woman taking charge of her health, I immediately felt like a teenage girl being warned not to get pregnant. My doctor took me through a birth control tour unprompted, showing off an IUD like a brand new Mercedes. I said I would think about it.

When an ultrasound technician found a cyst on my ovary, I received a phone call that amounted to: You have a cyst, were putting you on birth control. I was given no information on the type of ovarian cyst I had (there are many), no other treatment options, and no time to discuss which method was best for me.

According to the U.S. Department of Health & Human Services Office On Womens Health, hormonal birth control can be broken down into two subtypes: long-acting reversible contraceptives and short-acting hormonal methods.

Short-acting hormonal methods include the pill, shot, patch and vaginal ring. These methods work by releasing hormones into the body that thicken the cervical mucus to block sperm and can prevent ovulation altogether, according to Planned Parenthood. Long-acting reversible contraceptives such as hormonal intrauterine devices (IUD) or hormonal implants work the same way, but dont need to be used on a schedule.

I started on the combination pill, which contains estrogen and progestin. Three weeks later, during the week of my placebo pills, I got the flu. I ran a fever, had bouts of shivering so bad I couldnt walk straight, and couldnt even keep Gatorade down.

To give a better sense of just how sick I was, I alerted my roommate to check on me every few hours in case I needed to be carried to the emergency room. I paid $60 for a last-minute Tamiflu prescription and totally recovered within a week.

For the next three months, I got the flu on a consistent schedule of every four weeks. Recognizing that something was up, I made another doctors appointment (I would rack up about $350 in copayments by the end of this story).

My doctor called my case unusual, but did little more than a basic blood test before sending me home with a new prescription for a slightly lowered dosage of the same pill. Between January and April, I tried two new pills that would surely fix my mystery illness, yet I continued to get sick every month like clockwork.

I missed parties that my friends still reminisce on and full weeks of class. I showed up at one male professors office the day a paper was due, mumbling something about hormone levels and blood tests because I knew one mention of my period meant hed stop taking my problem seriously.

If youve ever suffered from a recurring health issue, you know that after a while people start to blame you for not getting better already. I felt like I was constantly over-explaining, trying to prove that I wasnt just having bad periods; I was suffering every month.

When the semester ended, I braced myself for a serious conversation with my doctor. I was leaving for a two-and-a-half-month internship in South Africa, and I couldnt miss work or a travel opportunity because I was sick.

She started me on the Nuvaring, which is a self-inserted vaginal ring containing hormones. She said that the localized hormone dispersal should make my symptoms less severe.

I got on the plane with a box full of Nuvarings and awkwardly told my new roommates that yes, there were contraceptives in the fridge. I had such high hopes for this method that when I still got sick, I called home and cried.

I stayed home instead of going to see the Southernmost tip of Africa because of my birth control. To make matters worse, I felt like my peers didnt even believe I was sick because I couldnt explain what I was sick with.

Research has shown that women are more likely to have their pain dismissed by medical professionals and less likely to receive diagnosis or treatment, according to The New York Times. I couldnt stop thinking about how maybe if birth control were a mens health issue, there would be a name and an explanation for what was happening to me.

I decided then that I would quit hormonal contraceptives cold turkey as soon as I got back to the U.S.

When I told people I was done with birth control, a lot of them responded with, What are you going to do now? in a disapproving tone, as if I couldnt be trusted to make decisions about my own body or keep myself from getting pregnant.

Increasing awareness about sexual health is a good thing, but sometimes hormonal contraceptives are painted as the only responsible option. My story with birth control has shown me that the most responsible way to approach reproductive health is to know your body and trust yourself to decide what it needs.

If you decide to use hormonal contraceptives, it should be because you want to, and it should be prescribed by a physician who will listen to, believe and treat you.

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My story with birth control - The Cluster

The former Oasis frontman Liam Gallagher has spoken about how Hashimoto's thyroiditis might shorten his career but a specialist tells Medscape News UK the condition is very treatable.

Hashimoto's thyroiditis is typically more common in middle-aged women and can run in families.

Liam Gallagher first talked about his diagnosis and hoarse voice in 2017, including in an interview with the Guardian .

Now, ahead of a 47th birthday homecoming concert in Manchesterin an interview with Apple's Beats 1 he's talked about how the condition might end his career: "Im aint going to get any bigger. If anything, its only going to get worse. You are in that zone now, youre looking about and youre going. All right. I kind of see whats going on but, its all about the journey man.

However, he was reflective about his future career: "If it lasts 10 years, it lasts 10 years. If it lasts 5 years, it lasts 5 years. If it ends tomorrow, Ive still had a f-ing blinder do you know what I mean?"

He continued saying Hashimotos disease "makes your voice a lot hoarser. Thats some of the symptoms.

Dr Paul Jenkins is a consultant physician and endocrinologist at The London Endocrine Centre, and a board member of The Thyroid Trust. He spoke to Medscape News UK.

How can Hashimoto's affect the vocal cords?

If it's untreated, it does cause the cords to be swollen, and to result in a hoarse voice. That's one of the classic signs of an underactive thyroid.

What about the singing style?

With a change in their vocal range, the tenor of their voice, in their singing.

Effective treatment should be able to restore this but not always.

And will it, as Liam Gallagher fears, worsen over time?

No, it should not worsen over time. But most of us don't use our vocal cords so strenuously day-to-day. If you're really straining them I guess it's conceivable, but it shouldn't really worsen.

Is a 'rock-and-roll' lifestyle unhelpful for Hashimoto's?

No, I don't think you could say that. Hashimoto's disease is a very common autoimmune condition of the thyroid gland, where the body's immune system starts attacking it.

Liam Gallagher spoke of it possibly ending his career. Would a more positive attitude be helpful?

That's his thoughts on it. I can't say whether it's positive or negative, it depends on one's overall context.

He's been out of the public eye for some time. So he hasn't been singing as much as he used to.

What kind of treatment regime might he be following?

It is thyroid hormone replacement. It's taking exactly the same hormone as the body is not making butin a tiny pill form.

It is for life, because once the thyroid gland has been destroyed, it does not ever heal itself in this condition.

Are some cases more complex?

Some patients need additional treatment, not just with the thyroxin, but they can continue to have symptoms of underactive thyroid, even if they're taking apparently adequate doses of thyroxin. And we increasingly recognise that they require T3, which is the other active thyroid hormone, in addition to thyroxin.

Is Hashimoto's hard to diagnose in primary care?

No, it should not be hard to diagnose overt cases. There is, as always the grey cases, those that you're not absolutely certain about or don't tick all of the boxes. And that's when a specialist can be prepared to even just try some thyroxin to see if it helps. Certainly, specialists are needed to diagnose those individuals who don't convert the thyroxin to the T3, and who need additional T3.

That's a more controversial area but undoubtedly in my experience, very, very much does exist. And that needs specialist input.

Is it helpful for a major celebrity to come forward and talk about a condition like this?

I think it can be helpful for any celebrity to talk about their conditions, if it raises public awareness of the condition and the treatments to restore it.

So the more debate and discussion, and knowledge generally is a good thing, as long as it's responsible knowledge, and people are given the true facts about their condition.

I'm very happy for him to raise awareness of it and to discuss it.

See the article here:
Will Hashimoto's End Liam Gallagher's Singing Career? - Medscape