WASHINGTON, October 2, 2020 Weeks after nurse Dawn Wooten came forward with allegations of forced and coerced hysterectomies performed on immigrant women at the Irwin County Detention Center (ICDC), Planned Parenthood Federation of America and leading social justice organizations based in Georgia are reiterating the need for immediate action to end these atrocities. Recognizing the long history of similar abuse in the U.S., Representative Pramila Jayapal introduced a resolution, HR 1153, that condemns unwanted and unnecessary medical procedures without the full, informed consent of individuals, and recognizes that everyone deserves to control their own reproductive choices and make informed choices about their bodies. Additionally, the resolution further states that accountability, oversight and transparency is necessary to protect people in the custody of U.S. Immigration and Customs Enforcement. With 225 co-sponsors in the House of Representatives, todays House passage moves this important resolution one step closer to accountability, oversight, and transparency on this devastating issue.

According to a new report from the New York Times, concerns from women detained at Irwin emerged long before Ms. Wooten came forward. Action cannot wait. SisterSong, Project South, Planned Parenthood Southeast, and other leading reproductive justice and immigrant rights organizations have announced specific demands of state and federal politicians and health care regulators to address the reports, which continued to go unanswered.

The report from Ms. Wooten, who is being represented by lawyers from Project South, is not isolated, but part of a long history of forced sterilization and state violence that targets Black and Latino people. This latest assault is one more example of the horrific abuse, medical neglect, and human rights violations that immigrant detainees have faced under this administration.

Statement from Alexis McGill Johnson, president and CEO, Planned Parenthood Federation of America:

Forced sterilization is a tool of white supremacy used to control and dehumanize Black, Latino, AAPI, and Indigenous people and we know that there is no reproductive freedom without justice for immigrants. These horrifying reports are a reminder that the threats to bodily autonomy, particularly for people of color, have been widespread and too often ignored. This is sadly nothing new. Planned Parenthood is proud to stand with SisterSong, Project South, Dawn Wooten, and the reproductive and immigrant justice leaders on the ground who are leading this movement for change. Were honored to follow the lead of our partners in this fight and work with them in support of the accountability they are seeking from state and federal agencies.

Statement from Monica Simpson, executive director, SisterSong:

Recent reports of forced hysterectomies on ICE detainees at the Irwin Detention Center, privately owned and operated by LaSalle Corrections, have sent shockwaves that have been felt so deeply in Georgia and beyond. As Reproductive Justice leaders and advocates committed to securing our human right to bodily autonomy, we are horrified and angry. This brazen attack on incarcerated Black and Brown bodies is part of a long history of policies and programs inflicting violence and harm on migrants and immigrants seeking refuge, safety and opportunity in this country. Forced gynecological procedures and the calculated sterilization of those incarcerated by ICE are yet another attempt to control who has children and who can make decisions about their reproduction. We know and have always known what we need and are committed to defending our bodies, our families and our human rights.

Statement from Azadeh Shahshahani, Legal & Advocacy Director, Project South:

We have documented conditions at Irwin for many years. The treatment of immigrants at this prison has always been horrid. These new shocking revelations further highlight the extent of the egregious abuses at the facility. The fact that Black and Brown immigrant women are held in an extremely vulnerable position at this prison where they have no control over their bodies and no say about what is done to them is sickening. Irwin should be shut down immediately and people should be freed. The United States Government, as well as the private prison corporation running this prison, should be held accountable.

Statement from Dr. Georgina Amaral, Physician at Planned Parenthood Southeast

As a reproductive health care provider, I was sickened to hear about the inhumane treatment of the detainees at Irwin. It is our job as physicians to respect the dignity and humanity of every patient no matter who they are or where they came from. Bodily autonomy doesnt end at the gates of a detention center. Coercing or forcing patients to undergo medical procedures goes against the hippocratic oath and everything we have committed our lives to as physicians. It is unconscionable to think that a doctor would perform hysterectomies without consent. It is unethical, if not outright criminal, and this physician must be held accountable. We demand the Georgia Medical Board to investigate this doctor and anyone else involved, and revoke their licenses. We also demand that the Department of Homeland Security take action to close down the Irwin County Detention Center and its operator, LaSalle Southwest Corrections. I also urge my fellow medical professionals to stand up and speak out against this atrocity boldly and without hesitation.

You can listen to audio from a September 17, 2020 press call HERE.

Planned Parenthood is the nations leading provider and advocate of high-quality, affordable health care for women, men, and young people, as well as the nations largest provider of sex education. With more than 600 health centers across the country, Planned Parenthood organizations serve all patients with care and compassion, with respect and without judgment. Through health centers, programs in schools and communities, and online resources, Planned Parenthood is a trusted source of reliable health information that allows people to make informed health decisions. We do all this because we care passionately about helping people lead healthier lives.

See more here:
Social Justice Groups Call for Action Following Reports of Gynecological Procedures on ICE Detainees in Georgia - YubaNet

Its one of the most important genes in biology: Sry, the gene that makes males male. Development of the sexes is a crucial step in sexual reproduction and is essential for the survival of almost all animal species.

Today in the journal Science, my international collaborators and I report the surprise discovery of an entirely new part of the Sry gene in mice a part we had no idea existed.

I co-discovered Sry in 1990. It is the gene on the Y (male) chromosome that leads to the development of male characteristics in mice, humans and most other mammals. Since then, Sry has been the subject of intense study worldwide because of its fundamental role in mammalian biology.

We have come to understand, in some detail, how Sry acts to trigger a cascade of gene activity that results in the formation of testes, instead of ovaries, in the embryo. Testes then stimulate the formation of other male characteristics.

But its clear we dont have all the answers just yet. Our results published today take us one step further in the right direction.

For 30 years, we have understood the Sry gene is made up of one exon, a segment of a gene used to code for amino acids, the building blocks of proteins. This can be compared to a computer file consisting of one contiguous block of data, on a hard disk.

Our newest research reveals theres actually a second exon in mouse Sry. This is like finding a whole new separate block of previously hidden data.

The mouse genome, like the human genome, has been extensively characterised due to the availability of advanced DNA sequencing and related technologies. Researchers commonly assume all the genes and all the parts of the genes have already been discovered.

But earlier this year, scientists in Japan uncovered what looked like a new piece of the Sry gene in mice. New sequencing approaches revealed what appeared to be two versions of Sry: a short, single-exon form and a longer, two-exon form. They called this two-exon version Sry-T.

They collaborated with my group at the University of Queensland and removed the new exon using CRISPR, a gene editing tool that lets researchers alter DNA precisely. Together we discovered this prevented Sry from functioning: XY mice (which would normally develop as males) developed as females instead.

Conversely, adding Sry-T to fertilised XX mouse eggs (which would normally develop as females) resulted in males.

Importantly, although human Sry does not have the added exon, our discovery may reveal new functions that might be shared between mouse and human Sry.

The DNA sequence of the new exon in Sry-T may point us towards discovering some of the genes and proteins that interact with Sry, something that has been elusive up till now.

And interactions we find in mice may also occur in humans. Studying what human Sry interacts with may help explain some cases of differences in human sex development, otherwise known as intersex development. This is a common but poorly understood group of mostly genetic conditions that arise in humans.

Currently, we dont know the genetics behind a large proportion of intersex conditions. This is partly because we dont yet know all the genes involved in the human sex development pathway.

Read more: Sex, genes, the Y chromosome and the future of men

Scientifically, this discovery is a bit like discovering a new cell type in the body, or a new asteroid in the Kuiper belt. As with many scientific discoveries, it challenges what we thought we knew and raises many questions.

What is the function of the new exon in Sry-T?

Currently, we only have part of the answer. It turns out the first exon of Sry, the one we already knew about, contains instability sequences at its end. These are sequences that cause proteins to fray and degrade.

An important function of the newly discovered second exon is to mask the instability sequences, seal the end of the Sry protein and prevent it from degrading. In other words, this second exon is crucial to the development of male babies.

Whats more, this protection mechanism represents an unusual and intriguing evolutionary mechanism that has acted to help stop vulnerable Y-chromosome genes from literally falling apart.

But its early days yet. The challenge now is to understand whether there are more functions hidden within the newly discovered exon.

If so, this information may provide some of the missing links that have stood in the way of our full understanding of how Sry works at a molecular level and of how males and females come to be.

Read more: Why education about gender and sexuality does belong in the classroom

Original post:
We discovered a missing gene fragment that's shedding new light on how males develop - The Conversation AU

In the years since Barry Stevens was conceived, much has changed in the world of sperm donation. Back then, in the 1950s, it was a murky affair. As we discover in The World's Biggest Family, a documentary from CBC Docs POV, a woman would show up at her doctor's office and a fresh semen sample would appear from a back room, provided by a man she knew nothing about. Typically, she was told to go home, have sex with her husband and pretend the child was his. Couples were instructed to keep the matter secret. Stevens's parents followed that same advice his mother didn't tell him he was donor-conceived until after his father's death.

In contrast, sperm donation these days happens on an industrial scale. Men are enlisted for a couple of deposits a week, on contract. Their samples are split into multiple portions and sold for about $1,000 each. The men are numbered and catalogued. They provide baby photographs of themselves, voice recordings and catchy philosophical meditations.

But one thing hasn't changed: in many of the world's jurisdictions, sperm donors continue to be anonymous.

That is the case in Canada. Men who donate sperm have the right not to be identified to the parents who used the sample or even to the individuals created with it. Unlike adoptees in most provinces, people conceived through donated sperm have no right to ever know their origins. In fact, their parents are often made to sign contracts promising they'll never even try.

Anonymity in Canada's Assisted Human Reproduction Act

2004Originally, there was a clause in the law stating that "...the identity of the donor or information that can reasonably be expected to be used in the identification of the donor shall not be disclosed without the donor's written consent." Donors had the right to anonymity and donor offspring had no right to information about their donor's identity.

Interestingly, in the original law, two people who were in a sexual relationship, but worried they might be related through a donor, were allowed to write to Health Canada and be informed of their relationship. (This was in the days before widespread genetic testing.) In reality, this was not possible, because Health Canada never collected the information needed to provide this.

2012Both clauses (among many others) were repealed in 2012. Canada's law is now silent on the issue of anonymity, although it continues to tacitly protect it.

"We can't change what happened in the past," says Barbara, one of about 45 half-siblings Stevens has discovered so far. "But now, there is no excuse whatsoever for donors to insist on secrecy."

David, a half-brother, agrees. "Everyone has the right not to be deliberately deceived or deliberately deprived of information about essential aspects of their personal history," he says.

In the early 2000s, Canada was on track to be a trailblazer against anonymity. In a review of an early draft of our law, the Standing Committee on Health recommended an open donation system, citing the right of a child to know its origin. "We feel that, where there is a conflict between the privacy rights of a donor and the rights of a resulting child to know its heritage, the rights of the child should prevail," they wrote. "We want to end the current system of anonymous donation."

But lawmaking is a fickle business. By the time it came to a vote, a new clause protecting anonymity had been inserted, and the onus was on those opposed to get an amendment. The amendment was defeated by one vote. So when the Assisted Human Reproduction Act finally came into force in March 2004, anonymity was enshrined as a right of the donor.

The Act had also initially called for a central health registry. But it was never set up, and eight years after being mandated, the idea was repealed.

In the same period, the U.K. went in a much different direction. It ruled that, thereafter, every person conceived using donated gametes in that country would have the right to find out the identity of their donor. At 16, offspring can learn the country and year of their donor's birth; information about physical features, like eye, hair and skin colour; and personal family and medical history. At 18, they can learn the donor's full name, date and place of birth, and most recent address. No more than 10 families can use the sperm from one donor and it's all tracked.

The U.K. is not alone in opposing anonymity: Germany, Sweden, Norway, Australia and New Zealand, among others, ban it as well. But the world seems divided on the question. Canada, along with the U.S., Russia, Denmark and many others, still allow anonymity to prevail. Some jurisdictions including China, Spain and Israel actually require it.

These days, of course, anonymity is merely theoretical. Cheap, widely-available home genetic testing has made it virtually impossible to stay hidden forever. Yet sperm banks still promise anonymity to donors and peddle it to would-be parents.

Genetic testing has made it possible for families like Stevens's to find each other. But it has also uncovered some uncomfortable truths about the fertility industry.

For starters: the size of sibling groups. Many donors were told they would sire no more than 10 children through the process. But sibling groups of 50 or even 200 have turned up. Stevens reckons his donor family could number 600.

Then there is the fact that information about the donor depends largely on a questionnaire and the honour system. A couple in Port Hope, Ont., thought they'd selected a healthy donor working on his PhD, but when the sperm bank accidentally disclosed his full name, they discovered he had no degrees at all, had been diagnosed with schizophrenia and had a criminal record.

Other cases over the years have underscored that medical screening may be inadequate. One donor passed on a deadly heart condition to nine of the 24 children conceived with his sperm; another seems to have given rise to autism in at least a dozen of his offspring. And because of anonymity, it's difficult to get the word out to all the children, should that need arise as a donor with a heritable but treatable cancer discovered.

But for many donor offspring, even when the news is upsetting, knowing is better than not knowing. This is true even for people and there are now quite a few who discover their biological father was actually their mother's fertility doctor.

Stevens and others believe it's time to end donor anonymity. In The World's Biggest Family, he asks his brother David why the genetic connection matters so much, even though they both loved the dads who raised them. "I haven't the faintest idea," replies David. "But it does."

Watch The World's Biggest Family on CBC Docs POV.

Alison Motluk is a Toronto writer who publishes HeyReprotech, a weekly newsletter on assisted reproduction.

Original post:
When it comes to sperm donor anonymity, Canada is behind the curve - CBC.ca

Women who go private to have their baby are almost three times more likely than public patients to have an elective Caesarean section, according to a study of 75,000 women attending a Dublin maternity hospital.

The increased elective Caesarean rate among private patients is linked in the study of patients at the Coombe Women and Infants Hospital to a desire for continuity of care from an obstetrician from women who are risk-averse.

Women who opt for private care are more likely to have experienced pregnancy loss, infertility treatment and multiple pregnancies, and are on average more than five years older than public patients, the authors found.

This suggests that women are choosing private care, in part, because they are more risk-averse for clinical and sociodemographic reasons. They prefer a model of care where there is continuity of care by a senior obstetrician and where they believe they can optimise a good clinical outcome for their baby and themselves.

Prof Michael Turner, of the UCD Centre for Human Reproduction at the Coombe hospital, pointed out that many of the women would have received IVF, and having invested personally and financially in this technology would be more likely to go privately to an obstetrician for the birth so they got continuity of care and risk minimisation.

The study involved 73,000 women who had a single pregnancy at the Coombe and 2,000 who had multiple births. It found 75 per cent had public care, 11 per cent semi-private and 14 per cent private.

Overall, 71 per cent of births in the study involved vaginal delivery, 14.5 per cent were elective Caesareans and 14.5 per cent were emergency Caesareans.

Elective Caesareans are typically carried out where a doctor thinks a vaginal delivery might be too difficult, where the baby is in the breech position or where complications have arisen.

Private patients were on average 5.3 years older and were more likely to be Irish-born, married and in professional or managerial employment.

Women with multiple pregnancies were almost twice as likely to go private than those expecting one child (24.6 per cent versus 14.9 per cent).

The elective Caesarean rate in private patients was 29.4 per cent, compared to 11.1 per cent in public patients, but the emergency Caesarean rate was slightly lower 13.3 per cent versus 14.2 per cent.

The study, published in BMC Pregnancy and Childbirth journal, also looked at women who returned to the hospital for a second pregnancy, and found 90 per cent chose the same package second time around. Women who switched to private for their second pregnancy were more likely to have had an emergency Caesarean or admission to the neonatal unit the first time.

Rising rates of Caesarean section, in Ireland and internationally, have been a source of concern for many years. The study points out that the operation has become much safer for mothers, even while risk factors have increased due to women having children later, rising obesity levels and increased rates of diabetes.

Its a question of balancing the risks and benefits for each individual case. Theres a lot of mythology about the optimum Caesarean rate, when no one knows what it is, said Prof Turner.

There is a big difference in the Caesarean rate between private and public patients, but when you correct for clinical risk it shrinks considerably.

The issue is more complex than it is sometimes portrayed, and people should not apply simplistic, ideological arguments in relation to it.

See more here:
Women who go private to give birth more likely to have Caesarean - The Irish Times

Spoiler Alert: For fans of Amazon Prime, trending now is season one of a show called Utopia, in which John Cusack plays a billionaire madman who wants to solve overpopulation by tricking the world into believing in a global pandemic so he can sell a vaccine that actually sterilizes people.For most of humanity, a medical event ends a persons ability to have a family.

With abortion, the parallels between fact and this fiction are uncomfortably close.

Why women have abortions is a topic much discussed by those who argue that ending a life in the womb is sometimes necessary. The Guttmacher Institute, the abortion industry think tank established by Planned Parenthood, published a survey examining 32 studies in 27 countries and found that [w]orldwide, the most commonly reported reason women cite for having an abortion is to postpone or stop childbearing. What wasnt reported, however, was how one thing might lead to a womans inability to have children at all.

Fully informed consent to any abortion, whether surgical or chemical, should include the long-term implications for womens fertility, as some women will later find theyve lost the ability ever to have a child. The abortion industrys failures to educate women on what might happen to their own bodies following an abortion should be considered malpractice.

Some abortion advocates try to downplay the potential loss. Some argue that telling women about their own bodies is somehow a violation of abortion vendors First Amendment rights, but informed consent is not a violation of our rights but a precondition for fulfilling them.

Abortion ends lives, hurting women and preborn children. Maybe people shouldnt be surprised that a predatory abortion business profiting from such deaths isnt doing enough to make sure womens future fertility survives a visit to an abortionist, fighting common-sense laws for informed consent. Protecting infants is the opposite of their business model, but women deserve to know how choosing abortion might mean they never have a child.

Before any abortion, women must be screened for their blood types. When a woman has an Rh-negative blood type, which affects 15 percent of the population, and her partner is Rh-positive, antibodies can build up in a womans body that may leave a first child alone but will attack future pregnancies, which can result in miscarriage.

There is a solution: a shot of Rh immunoglobulin that will neutralize the antibodies if given in time. If not, those antibodies will remain in the mother, ready to attack the blood of a future child.

The abortion lobby, however, has started to argue that they should not have to screen for Rh-negative status, especially for chemical abortions, because its not a requirement and because so many who are handing out chemical abortion pills havent set up medical practices that stock shots of Rh immunoglobulin to protect women.

Writing in the journal Contraception, a whos who of abortion-industry thought leaders concluded: Early abortion continues to expand outside of traditional clinics, through telemedicine, self-managed medication abortion, or in smaller offices that do not specialize in obstetrical care. Consequently, requiring Rh testing and anti-D immunoglobulin as part of abortion care is becoming a barrier.

The barrier, however, is not for women but for those who want to sell abortion pills without health and safety standards.

Another complication for post-abortive women is the risk of preterm birth, noted by more than 130 studies. In the journal Human Reproduction, an abortion-friendly publication, researchers note the issues for future pregnancies, writing, Although legal induced abortion is considered safe, its potential impact on subsequent fertility, ectopic pregnancy and the length of gestation is a public health concern. Previous studies have identified a significant relationship between history of induced abortion and spontaneous preterm birth.

Damage to a womans body from abortion can come from complications including pelvic infection, fever, tissue retention, bleeding and cervical trauma, which can contribute to early births. One study found that 31.5 percent of preterm births could be traced to abortion.

Preterm birth (occurring before 37 weeks) is a serious event and the leading cause of the deaths of children under five worldwide, according to the World Health Organization. In the United States, the preterm delivery of a baby is the leading cause of infant death. Another study looking at worldwide data noted that the risk of preterm birth increased with the number of abortions.

The World Health Organization also reported that around the world, in countries with reliable data, preterm birth rates are increasing, but they disproportionately affect some people.Consider that although black women make up only 13 percent of the population, in 2016, they had 38 percent of all abortions tracked. That community also experiences a higher pre-term birthrate than women of other races.

Abortions can also increase the chance of a pregnancy implanting in the wrong location, risking the lives of both mother and child.

A study published in the National Library of Medicine notes that abortions can increase a womans chance of an ectopic pregnancy, a condition in which rather than implanting in the womb, a fertilized egg implants in a womans fallopian tube, where it will not be able to survive and a woman can experience life-threatening bleeding. That risk of ectopic pregnancy does not include women who lost a child through miscarriage. The study followed up on one from about 20 years ago that determined there was a 10-fold risk of ectopic pregnancies for those who had an abortion.

Most severe of all is the fact that removing a womans uterus by a hysterectomy sometimes takes place after abortion, ending her ability to carry her own child. The shoddy conditions and gruesome practices of abortion vendors have been well documented. The patients needs and safety are sacrificed in such conditions.

Both chemical and surgical abortion can impose severe short- and long-term complications for women who were told the life-ending event is a quick and easy fix. Some women dont know that agreeing to an abortion means more than just that child will be lost to them forever. One in five cases of abortion can result in a major complication that requires an overnight hospital stay, a blood transfusion or surgery.

Thats just the beginning. Infertility is on the rise in America, as today almost 12 percent of women are having difficulty getting pregnant. To head off that kind of heartbreak, fully informed consent, Rh-negative treatment, and follow-up care after all abortions must be required of the abortion industry so it does not quietly and negligently shut down a familys future.

KristanHawkinsis president of Students for Life of America.

The rest is here:
The Left Won't Tell Women That Abortion Can Cost Their Future Family - The Federalist

Gov. Justice and state officials provide an update for media on COVID-19 preparedness and response plans. For more information about COVID-19 prevention and more, call the States toll-free hotline at 1-800-887-4304 or visit Coronavirus.wv.gov.

Gov. Justice paid his respects to those who passed away from COVID-19 over the weekend. He asked people to be thankful for the years of service these West Virginians gave to their families.

The Rt (reproduction factor) in West Virginia has fallen below one to 0.98. That makes the state 15th best in the country.

The West Virginia Department of Education is reporting outbreaks at 17 schools across the state. The list is available on the WVDE website.

Free testing is ramping up across the state. The goal is to get daily testing up to 10,000. The minimum target number is 7,000. Testing is being done at sites across the state.

Additional free testing is being done in Kanawha County. On Monday, Tuesday and Wednesday there will be a site set up at George Washington High School. It will be held from 3 p.m. to 7 p.m. The Governor stated they will increase testing in Barbour County as well. The announcement of when and where will be coming.

The COVID-19 pandemic has led to modernization of the contact tracing system used by the Department of Health and Human Resources. The change was recently completed at all health departments. The surveillance system allows the DHHR to an additional 150 contact tracers. The DHHR is also working to improve the online dashboard.

The National Guard is working on sanitization missions. They are also working to finish adding WiFi hot spots for the Kids Connection Initiative. They expect to finish those by Wednesday.

Maj. Gen. James Hoyer mentioned recent statistics showing the number of suicides among the U.S. military is up 20-percent since the pandemic began. A crisis hotline has been established for National Guard personnel. Maj. Gen. Hoyer stated West Virginians should be watchful for their neighbors mental wellbeing.

See more here:
West Virginias reproductive factor is below one - WVNS-TV

Utopia spoilers follow.

Amazon's remake of the Channel 4 series Utopia has landed at both the most appropriate and inappropriate time. The series, adapted by Gone Girl's Gillian Flynn, includes the threat of a global flu pandemic and, because it's 2020, it had to come with a disclaimer warning viewers that it was a work of fiction.

But the series is about a lot more than just the threat of a deadly virus. At the centre of its first season is Utopia itself, a graphic novel which holds the secrets of the world's impending doom.

In season one, we discovered that both Utopia and its protagonist Jessica Hyde (Sasha Lane) are real as she teamed up with reluctant helpers Sam (RIP), Ian, Becky, Wilson and Grant to find Utopia and solve the puzzle hidden inside it.

But their search for the book and its answers also led them to Dr Kevin Christie (John Cusack), the CEO of a pharmaceutical company whose plans for the world go far beyond creating a sustainable meat substitute.

Flynn has already confirmed she's working on season two of Utopia, although Amazon is yet to officially announce a second season. But with season one's finale leaving us with more questions than answers, Digital Spy is here to share everything we know about season two and our predictions.

It seems surreal to write that the future of Utopia is up in the air due to a pandemic, but it really is. The first season wrapped filming in October 2019, a year before it aired on Amazon.

Given the scale of the production and the impact of Covid-19 on schedules across the board, we're probably looking at 2022 at the earliest for a second season.

It's safe to assume most of the main cast who survived the finale will return for season two, including the following:

In the season one finale, Arby turned his back on Dr Christie and delivered him (wrapped in duct tape) to Jessica, Wilson, Grant, Ian, Becky and Alice. This looked to be the end for Cusack's character but incredibly, he made it out alive (despite losing his thumb).

Early in the finale, it seemed clear that Christie was Mr Rabbit (he had the tattoo on his side, after all). His plan was to spread a deadly virus while profiting off a vaccine that didn't work. But as the episode went on, his true masterplan was revealed.

The virus that had been spreading through school children is not the actual threat. Instead, it's the vaccine which carries a "world-improving omnivirus" that will not kill but sterilise the population, stopping human reproduction for the next three generations. Christie explained that he believes he is "curing" the world by solving the problem of overpopulation while "saving ourselves from ourselves".

Knowing they must destroy the vaccine before it's delivered en masse across America, Ian Becky, Grant and Alice headed to Christie Labs, while Jessica left to go to Home with Arby, who had discovered his true purpose was to help her. That left Wilson on his own with Christie, with his plan being to record him confessing to all his evils.

But while Wilson had always been the group's biggest conspiracy theorist and the most untrustworthy of the big corporations, he began to come around to his way of thinking when he was left alone with Christie.

As Christie refused to follow Wilson's orders, he appeared to convince him that his plan was actually good for the world. The next we saw them, they were in a car together with Christie now free, having converted a new disciple.

Wilson then picked up Becky, who got into the car and was shocked to see Christie and his son Thomas working alongside Wilson. As they drove off, Christie seemed to have a hunch about where Jessica had disappeared to.

Meanwhile, Grant found himself apprehended by police, after being framed for a mass shooting, while Ian and Alice ended the series very much alive but separated from their group and on the run.

As for Jessica, her season one ending offered another unexpected twist as she followed Arby to Home. Arriving at Christie farms, Jessica went inside her former house, finding her old room with her initials still on the headboard of her bed.

Weakened after being bitten by one of the lab rabbits, Jessica went to lie down, but was woken by Agent Milner. In that moment, it's revealed that he's not a helpful Homeland Agent, after all. Instead, she's intent on capturing Jessica.

And what's more, she had the same symbols as Christie tattooed on her side, leaving us wondering if Mr Rabbit was in fact her all along.

As Jessica tried to escape, she realised she was trapped and threw Utopia out the window. But it was never Utopia that Milner was after, it was Jessica herself, whose back is revealed to be covered in symbols which presumably spell out the key to viruses created by her father.

And in a shocking final twist, Milner headed to the basement, where it was revealed Jessica's father is still alive and looks to be working on a follow-up to Utopia.

Speaking to RadioTimes.com, Flynn described season one's ending as "a very deliberate sort of cliffhanger".

Confirming that work was already under way on season two, Flynn added: "We already are just dipping into season two. And I've told everyone, the world is open, let's see what we want to do."

With so many cliffhangers and swerves in the season one finale, Flynn is certainly left with a lot of directions she can go in.

One of the most pressing questions concerns Milner, who admitted to previously being aligned with Christie.

Speaking to Variety, Flynn said she wanted to leave it to the audience to debate whether Jessica's father was being kept against his will by Milner or for his safety. And with the reveal that Milner also has the tattoo on her side, any certainty about Mr Rabbit's identity has been blown out of the water.

In an interview with IndieWire, Flynn also revealed she wanted to move beyond the pandemic storyline in season two.

"Personally, I like shows that move on and move forward in general," Flynn said. "Aside from whether I want to deal with the current pandemic [in season two], I want the show to move on and go to different objectives."

However, with Christie having made it out alive and finding a new ally in Wilson, it seems unlikely he's going to be walking into the sunset and giving up on his plans to curb the population.

But with the vaccine seemingly destroyed and Stearns leaving Chicago with an egg which holds the last remnants of the virus, he may have to find another way to execute his plan.

Unfortunately, not even Dr Kevin Christie would have the means to bring us a Utopia season two trailer as soon as we'd like it. With the second season mostly likely over a year away, we'll be lucky to get any new footage before the end of 2021.

Utopia season one is available to watch now on Amazon Prime Video.

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Transnational commercial surrogacy and the outsourcing of reproductive labour to women of the global South is arguably the most controversial practice in an expanding market in body parts and reproductive labour. The central role of India in this market represents a particularly challenging example, given the historical symbiosis between reproductive policies and population control in the country. For roughly a decade, India was a hub for commercial surrogacy and biocrossings, facilitated through the global assemblages of a liberalised capitalist economy (Bharadwaj 2008). Within this transnational fertility circuit, bodies of underprivileged Indian women, formerly seen as waste and their reproduction as something to be controlled by the post-independence Indian state and policymakers in the first world, were transformed into sites of profit generation within the reproductive industry of the neo-liberal Indian state (Rao 2010).

While previous research on surrogacy has addressed the stratified reproduction (Colen 1995) of Indian women in terms of class and economic status (Pande 2014; Rudappa 2015; Vora 2015; Deomampo 2016), the question of caste has received little attention (Madge 2015). Responding to the lack of research on assisted reproductive technologies (ARTs) and caste, the aim of this article is to explore the significance of this intersection toARTsin general and commercial surrogacy and egg donation in particular. We draw on in-depth interviews with Dalit feminists whose perspectives onARTsare uncharted.1Our analysis explicates the need to connect these issues with broader questions of social justice that we theorise through the framework ofreproductive justice. This understanding challenges dominant articulations ofARTscentred onreproductive rights.

Trends and Transitions in Surrogacy in India

A world-leading destination for medical tourism (Pande 2011; Deomampo 2016), surrogacy was legalised in India in 2002 and benefited from the active promotion by the Indian government (Rudrappa 2015; Deompampo 2016). As Amrita Pande (2014: 13) notes, [c]linics in India (...), not only operate without state interference but often benefit from explicit state support for clinics catering to medical and reproductive travelers. In 2012, a study conducted by the Confederation of Indian Industry (CII) estimated the fertility industry to $2 billion, with 600 clinics registered with the government, and another 400 under the official radar (Bhatia 2012). Nearly 10,000 foreign clients, of which 30% were single parents or identified as queer, travelled to India for reproductive procedures during that year (Rudrappa 2015: 39). Low costs, the availability of highly qualified English-speaking medical doctors, women willing to work as surrogates, and the lack of legal regulation surrounding surrogacy arrangements are factors that contributed to Indias flourishing fertility industry.

In 2005, the National Guidelines for the Accreditation, Supervision and Regulation ofARTClinics in India,developed by the Indian Council of Medical Research (ICMR) and the Ministry of Health and Family Welfare (MoHFW), were published (ICMR2005). However, through cases such asBaby Manji Yamada v Union of India(2008) and the Jan Balaz case in 2008, insufficient guidelines regarding citizenship of children born through surrogacy or parentage were brought to public attention (Saravanan 2018). Responding to the growing pressure on the Indian government from stakeholders within theARTindustry to provide a legal framework, theICMRand theMoHFWoutlined the Draft Assisted Reproductive Technologies (Regulation) Bill and Rules in 2008, which was revised in 2010 and 2013. Both the 2008 and 2010 versions of the draft were criticised for harbouring a bias towards the private sector and for promoting the interests of the industry, while failing to address the vulnerability of surrogatemothers (Sama 2012). The 2013 draft restricted the issuing of surrogacy visas to married couples, thus excluding single and gay parents. As a consequence, parts of the business moved to Nepal. However, since Nepal banned its female citizens from being hired as surrogates, but permitted foreign women, Indian and Bangladeshi women were taken to Nepal. A number of highly mediatised cases contributed to the present Indian regulation of surrogacy. One pertains to the 2015 earthquake in Nepal, when the Israeli government arranged to bring back Israeli gay couples and their babies from Kathmandu while the surrogate mothers were left to fend for themselves. Thailand, another hub for transnational surrogacy arrangements, banned commercial surrogacy for foreigners in 2015, in the aftermath of the Baby Gammy case and the Mitsutoki Shigeta case in 2014.

In 2016, the Indian Surrogacy (Regulation) Bill was approved by the union cabinet,2which banned all commercial surrogacy, and prohibited foreigners from accessing surrogacy in the country, while permitting altruistic surrogacy for married couples with documented infertility, provided they use a close relative for the procedure. In August 2019, a revised version of the 2016 bill was passed by the Lok Sabha. Arguably, the intent of this bill was to prevent the oppression embedded in the idea of rent a womb, while simultaneously strengthening cultural nationalism. This was evident in the statements made by the external affairs minister, the late Sushma Swaraj, who claimed that the bill hadan Indian ethos, aligned with our [Hindu] values (Hindu2016). Nonetheless, within the ambit of economic globalisation and Prime Minister Narendra Modis nationalist ideology, Indian womens responsibilities remain bracketed as reproducersshifting marginally from (re)producers for the global bioeconomies (within the logics of outsourcing) to procreators for the Indian nation (as mothers of the nation).

Reproductive Rights vs Reproductive Justice

Western feminist discourses on reproductive rights have been centred around values, such as choice and bodily autonomy and have primarily concerned the right to access birth control. This discourse has been criticised by feminists of colour for not addressing the ways in which socio-economic contexts and geopolitical locations shape womens reproductive realities (Twine 2015). They draw attention to how childbearing by privileged women is encouraged and bolstered through the use of advanced technological interventions, while poor women of colour are subject to public policy measures that include invasive and abusive medical procedures, or forced child removal that prohibitmotherhood (Roberts 1996: 944).

Challenging Western liberal notions of reproductive rights, the concept of reproductive justice was coined in the early 1990s. Merging reproductive rights with social justice, this concept addresses how race- and class-based histories of population control, sterilisation abuse, high-risk contraception, poverty, and the effects of environmental pollution on fertility and maternal health shaped the reproductive lives of the third world (as well as women of colour in the first world) (Bailey 2011: 727; Ross and Solinger 2017; Mohapatra 2012). It decentres abortion and contraception to emphasise how issues, such as incarceration, immigration, racism, housing, and adoption policies affect biological and social reproduction. The political dimension of reproduction is visible in current geopolitical conflicts, exemplified by the implementation of the United States (US) zero-tolerance policy at the Mexican border in 2018, and the coerced abortion and sterilisation of Muslim Uyghurss, Kazakhs and other minority groups of women by the Chinese state in 2020 (Briggs 2012).

A reproductive justice perspective is particularly relevant in the Indian setting given the symbiosis between reproductive politics, eugenics and neo-Malthusian ideologies, which have shaped the ideas of over-population during the early 20th century. Neo-Malthusian concerns were transformed into upper-caste anxieties about the lower castes. Upper-class neo-Malthusian agenda interweaved with the upper-caste agenda of Brahminical Hinduism to reduce women to merely reproductive bodies requiring male control, in a reimbrication of patriarchy (Anandhi 1998). In the initial debates on birth control, the seamless welding of Hindu with upper castes, and the conflation of upper-caste practices and norms as Hindu was achieved (Rao 2004: 3602). Central arguments concerned the reproductive excesses of the lower castes and religious minorities, in particular Muslims. Anandhi points out that several political groups articulated the opposition between desexualised reproductive bodies as the ideal norm of respectable female sexuality and sexual bodies as representing immoral and disreputable sexuality (Anandhi 1998: 145). From an international perspective, growing populations in China and India were increasingly seen as geopolitical threats and third world womens sexual behaviour was specifically targeted (Wilson 2018: 92; Briggs 2002: 117).

The post-independence Indian state and policymakers in World WarIhave seen the bodies of poor women in India as waste and their reproduction as something to be controlled (Rao 2010; Wilson 2018). Negative eugenics has been aggressively practised in India and targeted towards vulnerable communities. The widespread use and abuse of sterilisation is a case in point, as exemplified by the Chhattisgarh sterilisation scandal in 2014 (Ghose 2018). With the exception of a short period of forced mass vasectomies between 1975 and 1977, female sterilisation has been the main instrument of Indias population policies and has been the most common form of contraception available since the late 1970s(Deomampo 2016: 40). We wish to draw attention to the way in which what was formerly considered as waste is transformed into sites of profit generation within the reproductive industry of the neo-liberal Indian state.

Colonial Legacies of the Indian Reproductive Industry

In 2011, Alison Bailey argued for a reproductive justice approach to Indian surrogacy, as a response to what she described as an ethnographic turn, replacing earlier studies focused on the normative and ethical dimensions of surrogacy arrangements: either claiming moral legitimacy by using a liberal discourse, which emphasises womens right to decide over their own bodies, or a Marxist understanding, which perceives surrogacy as exploitation and the ultimate human commodification. By focusing on womens agency and lived experiences, it avoids the Eurocentric fallacy of previous scholarshipthe taken for granted of a particular set of moral concernsthat runs the risk of distorting the realities of women in the global South:

The single-pointed focus on choice occidentalises Indian surrogacy work: it makes it difficult to raise questions about the kind of life a woman has to lead to make this work count as a good choice. It obscures the injustice behind these choices: the reality that, for many women, contract pregnancy is one of the few routes to attaining basic social goods such as housing, food, clean water, education and medical care. (Bailey 2011: 722)

Bailey sees Amrita Pandes work as emblematic of this ethnographic turn, as it evades the discursive colonialism of early writings on surrogacy by giving priority to the surrogate mothers own narratives (Mohanty 2003). Pande observes that these women explicitly reject the category of choice, speaking instead ofmajboori(a compulsion) and loyalty with their families. Her ethnographic accounts add a complexity that makes it impossible to see surrogacy as either a win-win situation or one that transforms surrogate mothers into passive victims of exploitation.

However, Bailey is critical of what she perceives as a lack of a normative perspective; a weak moral absenteeism, noting that [i]nterviews are oddly de-politicised, as if documenting surrogacy workers agency and then properly contextualising their choices is sufficient (Bailey 2011: 72526). Departing from the narratives of the surrogate mothers enables the scholar to address the problematics articulated by the women engaging in these practices (Bailey 2011). Reproductive justice, she argues, provides a theoretical framework capable of encompassing both the surrogates local moral worlds and reflections on the morality of a practice that builds on the labour of women living under abject conditions. Such a perspective is attentive to the acute intersectionality at work in practices ofARTs.

Jyotsna Agnihotri Gupta (2006) responds to a similar understanding of intersectionality and inequality when she argues for a transnational feminist response toARTs. As their impact varies significantly between different groups of women, Gupta perceivesARTsas a testing ground for transnational feminism. Her proposal is a framework based on human dignity: a moral framework that values individuals as ends in themselves and not as tools [and] which encompass(es) individual rights claims but go(es) beyond the narrow focus of individualism and autonomy for the protection of womens self-respect and human dignity (Gupta 2006: 35).

Guptas proposal is critically assessed by Michal Nahman (2008), who agrees with the need for a common feminist stance onARTs, but rejects turning to a universal notion of dignity and the human rights discourse that this notion is based on. Modelled on Western conceptions of the human, dignity was used as a tool during colonialism and capitalism to dehumanise and instrumentalise colonised people. Instead, Nahman foregrounds the logic of the marketplace by recognising how one may attempt to gain a sense of dignity within global capitalism by doing precisely what will perpetuate the system, buying and selling (Nahman 2008: 76). Nahmans approach includes a critical recognition of neo-liberal capitalisms capacity to assimilate and live off the very attempts aimed at resisting it. She advocates a shift of focus to the neo-liberal global forces that position women in situations where they feel a need to commodify their bodies at all. This includes being attentive to how certain bodies are perceived as potential biological material: [W]ho is positioned asmore appropriateto sell a bit of their body (Nahman 2008: 77).

Kalindi Voras (2008, 2009, 2012) work offers the kind of account that Nahman argues for. Whilst Black feminists (Roberts 1996; Twine 2015; Weinbaum 2019) have drawn attention to how the current market in reproductive labour is prefigured by theUSslave economy, Vora draws parallels between surrogacy and Indian indentured labour that replaced slave labour after 1807, when the trade in slaves was abolished within the British Empire (Vora 2009; Lowe 2015). She argues that the colonial past offers the conditions of possibility for the present international division of reproductive labour, that is, why some bodies and not others are seen as the possible sources of commodification.

Others note connections between surrogacy and colonialism tangentially, through Indias history of reproductive politics, leading scholars like Bailey (2011) to argue for a reproductive justice approach. Pande (2014) and Sharmila Rudrappa (2015) address Indias history of population control and coercive reproductive policies targeted against marginalised communities. While Pande acknowledges the paradox of an aggressively anti-natalist state becoming a global hub forARTprocedures, Rudrappa explicitly rejects the relevance of a reproductive rights approach to address the distinct stratifications of Indian society. Instead, she argues for a reproductive justice framework that accounts for the endemic social, political and economic inequalities among different communities which shape individuals abilities to access a good life (Rudrappa 2015: 170).

Intersectionality of Dalit Feminism

The assertions by Dalit feminists in the 1990shave been part of a discourse of dissent to both mainstream womens movements and male-dominated Dalit movement. Sharmila Rege (2018) argues that middle class, upper-caste womens experience, or alternatively Dalit male experience became universalised, resulting in a masculinisation of dalithood and a savarnisation of womanhood (Rege 1998, 2018: 12). Akin to these articulations, one of our research participants, an activist from Bengaluru, stated:

One of the things that I have been doing a lot is critiquing Indian feminists: there is a lack of connect [ion] with real life issues of marginalised women. But the fact is that they are the ones who set the agenda and basically define the issues which feminists talk about in India.

Dalit feminism implies an interrogation of privilege and discrimination embedded within the Ambedkarian notion of Brahminical patriarchy, a specific modality of patriarchy governed by a set of discriminatory levels constituting a hierarchical organisation of society based on caste, which is quite unique to the Indian subcontinent (Arya and Rathore 2020: 8). This graded inequality determines the location of all individuals according to caste and gender, with upper-caste men and lower-caste women at the beginning and end of the spectrum. The Ambedkarian understanding of caste positions endogamy as its grounding principle, which makes the control of womens sexuality central to caste ideology (Rege 1998: 165; Velayudhan 2018).

While both Dalit women and caste Hindu women are disempowered by patriarchal practices, Dalit and lower caste women are more prone to violence as they face oppression at three levels (i) caste, (ii) class, and (iii) gender (Dutt 2019; Moon 2000; Malik 1999), that is, the triple burden of economic marginalisation (low wage labourers working for upper-caste landowners as most of the land is owned by upper caste or upwardly mobile castes), caste discrimination, and gender subordination. Our research participants would commonly refer to this intersectionality of oppression. Thus, engaging with a Dalit feminist perspective demonstrates the importance of intersectionality for grasping gender inequality in India. However, prominent feminists, such as Nivedita Menon oppose its relevance to Indian feminism, which Dalit feminists have perceived as a reluctance on the part of mainstream feminists to acknowledge and address their own caste privilege (Menon 2020).

Towards a Dalit Feminist Standpoint Theory

A Dalit feminist standpoint, as elaborated by Rege (2018) and Kanchana Mahadevan (2020), has significant parallels to other feminist standpoint theories (Collins 2009; Harding 2004; Haraway 1988). Standpoint theory designates the epistemological shift that occurs when marginalised communities gain public voice, and foregrounds the concept of experience. The location of the subject affects the experience and thus the knowledge that it generates. From a Dalit feminist perspective, experience is the origin of knowledge, and like standpoint theories, power is seen as integral to epistemology. The failure of dominant groups to critically interrogate their advantaged situation makes their social position a disadvantaged one for generating knowledge. As stated by one of our research participants, a Dalit journalist: we need to articulate womens experience and theory from the perspective of the marginalised sections, which mainstream feminists clearly are not doing. Indian feminists lack an insight or an experience (...) the entire perspective that we would bring to the table. In the same vein, a social activist among our research participants describes the importance of having worked in the slums: my feminist theory sprung from there you know, and my understanding of caste, class, gender came from the slums that I work [in]. What is emphasised in these narratives is the importance of lived experience, a kind of knowledge that has been omitted from traditional epistemologies, which spans over a register that includes feelings and more elusive elements, what Linda Martn Alcoff (1996, 2008: 294) describes as textures.

Grounding knowledge in experience is democratic and provides an alternative to normative understandings. However, this does not imply a belief in unmediated authentic experience. Rather than a subject merely registering the imprint of reality which then qualifies as knowledge, experience is regarded as a dialectical process of collective articulation by persons belonging to conflicting social locations. As Alcoff elaborates

the oppressed do not have an epistemic privilege over understanding oppression generally; they are not more likely, for example, to know the causes of their oppression. However they are more likely to know the lived reality of the oppression, its emotional costs, its subtler manifestations, what it is like to live it. (Alcoff 2008: 294)

Claiming ones experience as the foundation of knowledge and theory is particularly audacious in the Indian context, where a divide has been instituted between theory (theoretical Brahmins) and experience (empirical shudras) (Patil 2020: 219; Guru 2020). As Cynthia Stephens (2009) suggests, Dalit feminist theoretical claim is a conscious effort to break the existing stereotype of Dalit women as mainly activists (doers) who have little to contribute (as thinkers) to ideological discourses in society, politics, governance, ethics, economics, and development.

There is a risk of subsuming Dalit feminism within dominant feminist discourse through a mere acknowledgement of difference, and making room for different voices from non-hegemonic locations within mainstream feminism (Harding 2008: 158). If that was sufficient, caste discrimination would be a concern only for Dalit and other lower-caste women, just as feminists of colour reject seeing race as something only they should attend to. Instead, seriously engaging with Dalit feminist perspectives entails challenging the dominant paradigm of thought. The goal is not difference in itself, but the relations of power that it legitimates. In Reges words, the aim is to address the social relations that convert difference into oppression (Rege 1998: 157). Here, there are significant parallels to Black feminist standpoint theory which challenges what counts as knowledge. Constructing new knowledge is crucial for empowerment because it provides alternatives to the way things are supposed to be (Collins 2009: 286). Reges idea of oppositional Dalit feminist pedagogies resonates with Black feminist thought when she describes that the importance of Dalit womens narratives lies in the potential to destabilise received truths and locate debates in the complexities and contradictions of historical life (Rege 1998: 133).

Importantly, standpoint theorys rethinking of experience is not an excluding gesture. The we designated by Dalit feminism is an acquired community. Although it may not be possible to speak as or for Dalit women, it is possible to reinvent oneself as a Dalit feminist, which entails rejecting the Brahminical, middle-class outlook that structures mainstream Indian feminism, and become sensitive to the specific disempowerment created by the intersection of caste, class and gender. Furthermore, as Gopal Guru (2020) points out, the subject of a Dalit feminist standpoint is not homogenous but multiple, heterogeneous, and sometimes even contradictory.

Caste, Sexuality and Reproductive Labour

Gender and caste are inseparable in both their material and cultural dimensions. Our analysis demonstrates how these intersections and interactions shape the choices Dalit women make in terms of labour and sexuality, not the least in the area of sexual labour. A characteristic way that our research participants approached commercial surrogacy was through the lens of sex work. Prabha Kotiswaran (2011) speaks of two international agendas having shaped perceptions on sex work in India during the last decades: the abolitionist movement and the antiHIV/AIDSprevention efforts. The abolitionist movement has gained force from the United Nations Global Initiative to Fight Human Trafficking (UNGIFT) launched in 2007. Through media representations, such as the Oscar-winning documentaryBorn into Brothelsdirected by Zana Briski and shot in 2004, set in Kolkatas red-light districts, third world enslaved sex workers have become pre-eminent examples of human trafficking (Kotiswaran 2011: 4). The Immoral Traffic (Prevention) Act (ITPA) conflates trafficking and sex work, which defines the discursive space for conversations on sex work in India.

Sex work is commonly referred to as forced labour or sexual slavery by our research participants; coercion and not choice is emphasised, and there are those among our participants that adhered to the abolitionist view, as the journalist who spoke of all commodification as problematic per se:anything that commercialises () the body () has to be seriously questioned. However, rather than radical feminist understandings of sex work, most of our participants highlighted the socio-economic conditions that make women turn to sex work as a livelihood. Entering the surrogacy industry is described as shaped by the same forces. As one of the Dalit academics and social activists with the experience of working with women in prostitution explained:

[R]ural women likeX, sex work, forced by the husband, by the family and also absence of work at the agricultural sector. All this made them ... it is not theirchoice. I feel no women will get into this kind of selling sex, also selling their wombs for surrogacy. And the ... the whole economic conditions forces them to get into this. And I blame the society which has the purityimpurity concept in everyones mind which is also the sexual purity. Where they dont bother about the purityimpurity and they have the sexuality aspect. The most exploited women are the Dalit women []. Surrogacy is another thing that we cannot accept []. One woman in our area she received`30,000 only.

The same academic/social activist said she knew of rural women forced into sex work as a result of cuts in educational budgets, which makes unaffordable private schooling the only remaining option. Lack of jobs and collapsing farm prices trap families who seek ways out through self-exploitation. In combating commercial surrogacy and sex workmodern kinds of slaverythe rejuvenation of the agricultural sector is key, she argues.

The traditional hierarchical political economy of labour on which caste reproduces itself and Dalit demands for dignity of life and labour have to some extent been addressed by constitutional provisions. The government has tried to aid the empowerment of marginalised communities, especially women, Scheduled Castes (SCs) and Scheduled Tribes (STs) through large anti-poverty alleviation programmes, such as the one under the Mahatma Gandhi National Rural Employment Guarantee Act (MGNREGA). But, the same social activist suggests that the government measures are not always sufficient:

they are getting very poor wages, now this uh ... hundred days work ...MGNREGAwork. It also comes once in a while, and then one job for one family, not all the family members are ... and uh how do you expect Dalit women to depend on this.

More specifically addressing reproductive issues, she reflects on the increasing medicalisation of childbirth and the rapid growth inARTclinics in her city. She also brings up the ways in which exposure to pollution and other environmental issues disproportionately affect marginalised communities, not the least in their reproductive lives: The implication of chemical fertilisers on reproductive health systems of women, they keep on talking about. So I feel somewhere that is also an issue. In bringing unequal access to essential resources, such as clean water to the conversation onARTs, she demonstrates an understanding of reproductive justice as intimately connected to social justice. Ultimately, she argues that sex work and surrogacy is a question of the economic realities that Dalit women are confronted with: We are questioning the failure of (the) economic system. So you know like, what is happening to agriculture? What is happening to the economics?

Surrogacy and sex work are seen as parallel social phenomena not only in relation to how social locations shape womens choice of becoming surrogate mothers, but also the conditions in which they find themselves once within the industry. As one of the Dalit academics/social activists stated:

[F]or instance this`30,000 paid to a Dalit woman may not be the case for the non-Dalit woman who may bargain better. With her colour, with her sort of you know social capital. She may bargain, she may have a choice of going into surrogacy and bargaining for better allowance, better kind of conditions of gestation, etc. That may not exist for the Dalit.

The narratives suggest that economic and other exploitative conditions of Dalit surrogates within the reproductive industry parallels the vulnerability of Dalits working in the sex industry. Furthermore, many of our participants referred to the historical exploitation of Dalit womens sexuality, and the practices of devadasis andjoginisas contributing to the coercive forces surrounding womens labour. As stated by one of the journalists:

We have experienced sexual slavery for ... for simply centuries, whereas mainstream feminists havent had this experience. So they should hardly be the ones, you know, wanting to talk about it. I feel we as a society have failed if women have to sell their vaginas or rent their wombs to survive. You see the question is not of reproductive freedom or choice.Where is the choice when they have no choice?.[our emphasis]

One of the academics specified, Dalit devadasis have nothing to do with the art of dancing at all, they never had temple inheritance () Dalit devadasis were performing a kind of temple prostitution. Following this historical pattern, lower caste women constitute the majority of sex workers in contemporary India. This academic drew attention to the embeddedness of prostitution in caste and class, which, as she points out, was referred to as slavery by B R Ambedkar in 1936.

These intersecting levels of gender, caste and class reveal inherent contradictions in the practices of untouchability, as Aloysius et al (2020: 177) notice,

no more apparent than in dominant castes physical or sexual violence against Dalit women, where an undisputed claim is assumed [on] their bodies. Moreover, the touch of the womens agricultural labour in the dominant castes fields and their domestic work in the dominant castes houses are interpreted as essential services that preclude the strict practice of untouchability.

Detailing the vulnerability of Dalit women working in upper-caste families, one of our academic research participants spoke about the endemic problem of sexual harassment:[T]here is a caste link there where the man of the house and the women of the house believe in some way that this woman is sexually available. As Suruchi Thapar-Bjrkert (2006: 782) argues, the ideological construction of purity/pollution are conveniently forgotten when pure upper caste men are engaged in sexual encounters with impure lower caste women. This is not to deny patriarchal inequalities in Dalit households. In fact, as one of our academic participants said, a lot of Dalit women in my own circles are treated very objectively, like objects in their own relationships. For example, in their families, the man is only educated because of the poverty. So all those things disable the women to talk about the next level of you know rights. So the first thing is to survive. To get food on her table, the second thing is to educate herself if possible, the third thing is to enter a healthy relationship where she is not you know insecure or unsafe.

What Dalit women are capable of pursuing as labour depends on these limiting circumstances of income-generation. One of the journalist participants explains that a framework of choice is uncommon among sex workers from Dalit and lower-caste communities: this is stigmatised, this is despised and yet they do it, exposed to constant violence. One of the academic participants elaborates on the notions of respect and dignity introduced by the journalist:

They dont have the choice. Precisely the kind of structural condition which Dalit women are embedded, the question is not to ask about the agency of Dalit women or to talk about the rhetoric of sex work being dignified, to convert it into a dignified work does not arise given the fact that its an extremely embedded kind of condition in which Dalit women go into sex work () So its not a question of you know sex work being a taboo, here the question [is] of who is performing the work? And this is where Dalit women are saying that inevitably all said and done despite all this kind of talk about giving dignity and self-respect, eventually it is our women who are performing it.

The research participants emphasis on the particular vulnerability of Dalit women and the exposure to violence that a life in prostitution often entails, articulates with the Dalit movements understanding of prostitution as caste exploitation: caste privilege sexually exploits women of lower castes and destroys their self-respect thereby preserving the unequal power relations of a caste-based society (Tambe 2008). While economic desperation and social marginalisation is a recurrent reason for entering into sex work and surrogacy, our research participants simultaneously emphasised how this counteracts the equally important struggle for dignity, as both sex work and surrogacy are stigmatised occupations in the Indian context. As reiterated by one of the social activists: money is not a matter, recognition in the society is the matter. Dignity! ... they are not recognised even by the family. The acute need for survival and the equally pressing struggle for respect become irreconcilable realities; seizing economic opportunity simultaneously enhances social vulnerability.

Dismissing the framework of choice and insisting on the importance of structural conditions resonate with debates on sexuality and labour in which Dalit feminists have become a divergent voice. The legislative ban on bar dancing, critiqued by mainstream feminists as moral policing and an attack on womens right to exercise agency, and stances on sex work, are cases in point (Makhija 2010). Partly as a de-exceptionalising and destigmatising move, and with reference to the first national survey of sex work in India,3sex work has been conceptualised by dominant feminists through the liberal framework of bodily autonomy, rights and choice (Menon 2020), whereas reports by the National Federation of Dalit Women (NFDW) and National Campaign on Dalit Human Rights (NCDHR)emphasising that a majority of sex workers come from lower caste backgroundspoint to the caste-ordained linkage between sexuality and labour. Thus, the social position of the women choosing sex work needs to be acknowledged, in addition to their economic status (Rege 1998: 44).

Brahminisation of Surrogacy

Female reproductive biology is a main generative site in the growing global biomarkets, including emerging stem cell industries that are dependent on high volumes of human embryos, oocytes, fetal tissue and umbilical cord blood (Cooper and Waldby 2008, 2014). The gaining ascendancy of oocyte economies in the aftermath of the new regulatory framework on surrogacy was an important revelation during the course of our interviews. The commercialisation of ova, in particular, surfaced when our research participants addressedLGBTQIA+ (lesbian, gay, bisexual, transgender, queer, intersexed, and agender) communities acute lack of livelihood choices. Several of our research participants spoke of an expanding bio-economy in which lesbians and transgender persons sell their eggs and sperms as a mode of survival. As one of our research participants detailed, transmen and lesbians have very difficult time continuing their education because of family, they run away from home or because they have tough lives. So they are outside, they need money. They are running you know, running scared.

Our research participants give us reason to believe that these practices are inflected by gender, class and caste dynamics, as is surrogacy. One of the journalists shared the following account:

[T]he doctors I have discussed with at that time have told me [] they want fair looking babies, they want intelligent babies so they openly ask for Brahmin eggs. Some doctors have told me that people openly ask for some Brahmin eggs, or Brahmin sperm. So for uterus they are hiring lower caste people ... women They wont opt [for] their eggs () but for eggs and sperm they only seek upper caste [donors].

Brahmin eggs and Brahmin sperm are juxtaposed with the womb of the lower-caste woman, whose defiled and impure sexuality makes their own eggs undesirable. As earlier studies suggest, intended parents would ideally prefer a higher caste or Brahmin surrogate, with the expectation that they would produce healthy and good-looking babies (Dhar 2012). Nonetheless, both these cases reinstate the ideology of purity/pollution, whereby the institutionalised inequalities of the Brahminical system are fortified. We suggest that these reproductive practices assist in building caste capital, which confers benefits comparable to those accrued from social capital. As embodied in relations among persons (Coleman 1988: 118), social capital is productive and enables the achievement of certain ends while conferring power and profit to its holders (Skeggs 1998)in this case upper-caste women and menwhose caste capital protect them from economic and social vulnerability. We conceptualise this assemblage of practices as the Brahminisation of Surrogacy; creating new roles for lower caste women while confining them within the frame of non-valuable breeders for the embryos of valuable women (Corea 1985: 276). Significantly, such practices articulate with earlier Indian eugenic discourses and more recent Hindu nationalist arguments on caste supremacy, which construct oppressed castes as unfit to reproduce.

In conclusion, we reinstate the central purpose of our article which was to analyse caste as a significant parameter for understandingARTs, in particular surrogacy and egg donation. By incorporating Dalit feminist perspectives, that have been marginalised in mainstream debates, we demonstrate the relevance of bringing caste and social justice centre stage, and how this is crucial for forging new conversations.

Notes

1 Semi-structured qualitative in-depth qualitative interviews were conducted by the third author with 11 research participants, all of whom identified as women, who work in areas of reproductive healthas activists, journalists and academicsand who are from the Dalit caste. The respondents are based in Bengaluru (Karnataka) and Chennai (Tamil Nadu).Karnataka and Tamil Nadu share a trajectory of strong Dalit movements, and more importantly, Dalit womens movements in which our research participants are involved. Not all the participants identified as primarily Dalits. Some of them, especially the academics, emphasised that they were feminists who were also Dalits. The interviews were conducted in locations suggested by the participants: in their offices, homes and in cafes.

2 The Surrogacy (Regulation) Bill, 2016. The Ministry of Health and Welfare, Government of India, New Delhi, Bill No 257, https://dhr.gov.in/document/acts-circulars/surrogacy-regulation-bill-2016.

3 The survey was published in 2012, and indicates that approximately 71% of female sex-workers have chosen sex-work ahead of other occupations (www.plri.org).

References

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Bailey, Alison (2011): Reconceiving Surrogacy: Toward a Reproductive Justice Account of IndianSurrogacy,Hypatia, Vol 26, No 4, pp 71541.

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Bhatia, Shekhar (2012): Revealed: How More and More Britons Are Paying Indian Women toBecome Surrogate Mothers,Telegraph, 26 May,http://www.telegraph.co.uk/news/health/news/9292343/Revealed-how-more-and-more-Britons-are-paying-Indian-women-to-become-surrogate-mothers.html.

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Link:
Dalit Feminist Voices on Reproductive Rights and Reproductive Justice - Economic and Political Weekly

Newswise Scientists from the University of Portsmouth and Royal Botanic Gardens, Kew, have come up with a formula to help plant breeders and farmers around the world grow crops in a more sustainable way.

The new checklist, which has just been published in theBotanical Journal of the Linnean Society, will guide plant breeders to better understand the species they are trying to improve. It will also help them find ways to increase growth and yield of crops using wild plant species from which they were once domesticated.

There is an urgent and critical need for changes in farming techniques due to the growing challenges of global warming faced by crop producers. Plants that were selected and bred to suit certain climates, now need more help from the humans who are damaging the environment in which they grow.

Study author Dr Rocio Perez-Barrales, Senior Lecturer in the School of Biological Science at the University of Portsmouth said: "When the human race first domesticated crops, the climate and environment were completely different - what we are seeing in the last 50 years is a rapid change in climate. The world is now frequently facing catastrophic climate events like droughts and in the UK we are now seeing some crops being harvested up to a month earlier than they used to be.

"When plants were domesticated, they were artificially selected for a specific desirable trait. Artificial selection and farming have led to quality improvements in foods such as meat, milk, and fruit. However, over hundreds of years, there has been a negative impact to this process - a reduction in plant genetic diversity.

"Scientists believe genetic diversity is important for plants to cope with a change in environment. This leads to a choice of using an artificial process such as the use of pesticides, to protect crops against pests. An alternative for plant breeders is to use wild crop relatives and use the natural genetic variation in those species that protects them against the natural enemies.

"Climate change is altering the way crops behave. Crops have lost so much genetic diversity they are less able to adapt and respond to climate change. Scientists are now looking at wild crop relatives to see what traits can be improved to make crops better adapted to the current environmental challenges."

The researchers re-visited guidelines set out 50 years ago that have since become outdated. They used this classification as a basis to deliver a new method of improving crops, without destroying the very few natural environments left in the world to grow food.

Dr Perez-Barrales explained: "Some crops have just a few closely related species, whilst others might have a hundred or so. For example, linseed has more than 150 related species, and the challenge is how do we select the relevant traits and from what wild relatives? In answering this question, we realised that we needed to learn more from the biology of the species, which can only be done by using modern classification developed using the latest science. The classification developed in the early 1970's needed to be updated, and in effect rebooted, to integrate this modern information."

This new toolset for crop breeders relies on identifying which wild crop relative needs to be explored in order to improve the crops. Dr Perez-Barrales said: "There may be a demand to grow linseed, for example, in countries at different latitudes. Linseed (Linum usitatissimum) was domesticated in the Middle East 10,000 years ago, and we can grow it in England because it naturally captured genes from pale blue flax, Linum bienne, allowing the crop to grow in northern and colder environments. My research looks at the natural variation in flowering of wild Linum species to see if we can use it to improve linseed. That way the right genes can be selected and introduced into the crop, something that plant breeders do regularly. These new guidelines will help plant breeders become more sustainable and efficient. We believe it is the future of farming."

This paper recommends guidelines for plant breeders to select the one right species to improve the crops. The guidelines include:

2. To understand if there is genetic compatibility between species. This includes understanding variation in chromosome numbers. For two plant species to successfully cross, they need to have the same number of chromosomes. As with humans, if the number of chromosomes don't match there will be problems in reproduction. However, plants can have very different chromosome numbers, ranging from 14 to more than 100. It is important to understand chromosome variation so we can understand their compatibility.

3. To gather all the information of the pollination biology, reproduction and the mechanisms to avoid inbreeding. Plants can prevent self-pollination and inbreeding, so the pollen of a flower does not fertilise the ovules from the same flower. Just like in animals, inbreeding can cause genetic diseases. But the mechanisms that avoid selfing can create barriers between the crop and the wild relative, making it difficult to create new hybrids that could be tested to validate the newly improved crop

Dr Juan Viruel from Kew Garden said: "In this study we advise plant breeders to use phylogenetic distance metrics, cytogenetic compatibility data (for example, chromosome number and ploidy) and information about the breeding system to shortlist wild species for plant breeding programmes". With this information we can better select the wild species to improve our crops. It is an invaluable checklist for plant breeders and will help production of crops in a more sustainable way."

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Read more from the original source:
Scientists help reboot 50 years of plant advice to solve one of nature's biggest challenges - Newswise

Staphylococcus aureus is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5-triphosphate (ATP)binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an open conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

See more here:
Structural insight into the Staphylococcus aureus ATP-driven exporter of virulent peptide toxins - Science Advances

INTRODUCTION

-Synuclein (-syn) is a small ubiquitous protein that is especially abundant in the brain. In neurons, -syn is presynaptically enriched and associates with the neurosecretory vesicles as -helical multimers (1), while its C terminus interacts with vesicle-associated membrane protein 2 (VAMP2) (2). Under this conformation, -syn regulates the kinetics of neurotransmitter release in the synaptic cleft (1, 2). Before becoming associated with the membrane of presynaptic vesicles, however, -syn transits in the neuronal cytosol as its free, soluble, monomeric form (1, 3, 4). In this latter context, -syn oscillates between a variety of different conformational states: It is an intrinsically disordered protein (5). Certain conformations that are enriched in beta folds can incidentally get favored and stabilized (6). Their nucleation forms seeds that trigger the templated growth of amyloid structures building up at the expense of neuronal -syn. This process results in the formation of long intraneuronal amyloid fibrils that partly get packed and stored in inclusion bodies called Lewy bodies (LBs) (7) and partly get passed to neighboring neurons where they propagate the amyloid build-up process (8, 9). This self-regenerative mechanism provides an explanation for the progressive intracerebral spread of LBs observed during Parkinsons disease (PD) progression (10) and for the gradual worsening of other synucleinopathies (11).

Notably, during amyloid stacking, -syn can adopt several distinct structural folds, resulting in the emergence of fibril polymorphs (1218). Together with their specific fold, their supramolecular properties also get inherited and propagated as the polymorphs self-replicate by templated growth (1922). It is thus tempting to speculate that the distinctive properties of these different polymorphs are responsible for the different clinical presentation of the neurodegenerative diseases that are underpinned by a synucleinopathy (2325). We noticed, however, that the biological characterization of structurally defined -syn polymorphs had often been limited to the demonstration of their cellular toxicity or pro-aggregative action without consideration of their ability to reproduce their distinctive amyloid fold inside cells. The original aim of our study was, thus, to determine whether structurally characterized -syn polymorphs could demonstrate autonomous self-replication of their structural traits during propagation not only in vitro but also in living neurons. We incidentally found the existence of amyloid polymorphs acutely spreading in neurons. Not only were these amyloids invisible to thioflavin T (ThT), but also they could be preferentially revealed using SYBR Green. On the basis of this, we designed a simple multiplexed assay (which we termed the fibrilloscope) to track their propensity toward self-replication.

We first sought (i) to isolate/characterize different -syn polymorphs self-assembled under simple shaking in saline (see Materials and Methods) and (ii) to determine whether these untouched assemblies could self-replicate and spread in neurons (untouched meaning here without artificially disrupting the assemblies using sonication). A single batch of NMR (nuclear magnetic resonance)grade recombinant -syn (labeled with 13C and 15N) was divided into seven identical aliquots (Fig. 1A). One was left at 4C; the other six were then shaken together. Notably, using the classical amyloid probe ThT (26) to monitor the fibrillization (17, 18, 27), we observed a major variability in the development of the ThT signal from one tube to another (Fig. 1B). Certain aliquots showed a very rapid and strong rise in ThT fluorescence; others had a slower increase but reaching comparable final values, and others had an almost flat ThT curve. As it is widely assumed that the buildup of a ThT signal under these conditions is synonymous with proper -syn amyloid assembly (17, 18) [see current best practice guidelines (27)], we simply sought to compare the bioactivity of iso1, a presumably successfully fibrillized ThT-high sample, with that of iso3, a presumably failed ThT-low sample. To that end, we developed and validated a high-content analysis (HCA) assay based on primary mouse cortical neurons (28, 29), allowing the observation of a seeded synucleinopathic buildup in fully mature primary cultures (30 days in vitro) (fig. S1) [see also (30)]. The neurons were challenged with iso1 and iso3, immediately upon completion of the 40-hour fibrillization period without any other manipulation (untouched). The results (Fig. 1C) were completely opposite to expectation: Iso3, which according to ThT was supposed to be virtually devoid of amyloids (27), induced a massive synucleinopathy, causing a widespread buildup of secondary aggregates, while no synucleinopathy developed in the neurons treated with the ThT-positive iso1. To explain this counterintuitive lack of activity, we reasoned that unspecific clumping could have incidentally occurred in the iso1 sample, impeding neuronal uptake of the seeds. However, using nanoparticle tracking analysis (NTA), no clumping was detectable in iso1 compared to iso3 (Fig. 1D, dotted lines). Nevertheless, we proceeded with sonication that drastically reduced the size of the particles in both samples, producing a distinctive peak of smaller 25- to 50-nm-wide particles in iso1 that was absent in sonicated iso3 (Fig. 1D, solid lines). With particle size reduction, sonicated iso3 exhibited an increased activity in neurons (Fig. 1E). Sonicated iso1 was, however, still unable to trigger a synucleinopathy (Fig. 1E).

(A) Schematic representation of the fibrillization and testing workflow. (B) Development of the ThT signal in the six -syn tubes, sampled after 0, 20, and 40 hours of shaking at 37C or for 40 hours for the unshaken, 4C tube. Three measurement replicates for each sample and time point. (C) HCA of the synucleinopathy induced by 10 nM (equivalent monomeric -syn concentration) of untouched iso1 and iso3 in primary mouse cortical neurons (n = 4, nine measures per replicate, intrareplicate measurement means are shown). Quantitation and representative imaging fields are shown with a full intensity range lookup table (visual saturation of the highest pixel values). Scale bars, 100 m. Close-up of the iso3 condition with an intensity range lookup table focused on the highest pixel values, showing pS129 syn in green and tau in red. The somatic pS129 syn aggregates are perikaryal. (D) NTA shows that untouched iso1 is not more clumped than iso3 and that sonication decreases the size of the assemblies present in both samples. Note that iso1 is more frangible than iso3 with a distinctive size peak at 25 to 50 nm appearing after sonication for iso1. The spectra shown correspond to the average of five consecutive detection runs per sample. (E) The lack of neuronal activity of iso1 in the HCA assay is not enhanced by sonication, while the one of iso3 is increased (n = 3, nine measurement fields per replicate; data points: interreplicate mean of randomly matched fields). Corresponding representative HCA close-ups showing pS129 and phase contrast for sonicated iso1 and iso3. P values of the group differences were calculated using t test. AU, arbitrary units.

We thus proceeded with a further comparative characterization of iso1 and iso3 using the Congo red derivative X-34 (Fig. 2A) (31), density floatation (Fig. 2B), velocity sedimentation (Fig. 2C) (32), and electron microscopy (Fig. 2D). All these approaches indicated that iso3 contained as much -syn amyloid assemblies as iso1, with the iso3 fibrils appearing slightly shorter but straighter and more often bundled at an ultrastructural level (Fig. 2D). Although this became apparent only after sonication using NTA, velocity sedimentation by ultracentrifugation in the presence of sarkosyl revealed that the native amyloid assemblies populating iso1 and iso3 had different size distribution patterns. Iso1 contained smaller assemblies than iso3 (Fig. 2C). However, the similarity of the density floatation patterns observed for iso1 and iso3 (Fig. 2B) indicated that all the amyloid assemblies present in the two preparations shared a common and unique amyloid compactness, irrespective of their size. Overall, these data pointed to the possibility that iso1 and iso3 contained two distinct amyloid -syn polymorphs that had spontaneously emerged from a single condition and endowed with (i) distinct all-or-none ThT visibilities and (ii) distinct all-or-none natural propensities to spread in mouse neurons. This possibility was strengthened by the observation that 23 days after treatment, higher levels of iso3 persisted inside neurons compared to iso1 (Fig. 2E), a result compatible with the notion that distinct -syn polymorphs could be cleared with different efficiencies in neurons (33).

(A) Fluorescence of the amyloid probe X-34 indicates that there are same amounts of amyloids in iso1 and iso3 despite an over 10-fold difference in ThT signal; table with mean values (n = 3) and corresponding graph with iso1 to iso3 signal ratios. P values of the group differences were calculated using t test. (B) Schematic representation of the density floatation protocol: Iso1 and iso3 were solubilized with sarkosyl, loaded in the middle of iodixanol gradients, and fractionated by density upon isopycnic equilibrium ultracentrifugation. The collected fractions (numbered from top to bottom of the gradient) were analyzed for human -syn content (using the anti-human -syn antibody MJFR1) by filter trap (representative pictures of the immunoblots under the graph) by quantification from n = 3 replicates of the absolute chemiluminescence signal (AU, graph). Data presented are the mean curves for each group. (C) Schematic representation of the sedimentation velocity protocol: Iso1 and iso3 were solubilized with sarkosyl and loaded on top of linear iodixanol gradients and fractionated by sedimentation velocity upon ultracentrifugation. The collected fractions were collected and revealed/quantified as in (B). While the density of the iso1 and iso3 amyloid assemblies are strictly equal and monodisperse (B), their size distributions are different (C). (D) Transmission electron micrographs of iso1 and iso3 showing their prototypical fibrillary structure. (E) Neuronal persistence of iso1 and iso3 at DIV 30 (23 days after treatment) imaged and quantified by HCA using MJFR1 (n = 3, nine measurement fields per replicate; data points: interreplicate mean of randomly matched fields). Iso3 is more persistent than iso1. P values of the group differences were calculated using t test. (F) Solid-state NMR hNH experiments of fully protonated iso1 and iso3 showing that they correspond to two distinct amyloid polymorphs at a local level. ppm, parts per million.

We thus performed two-dimensional (2D) solid-state NMR spectroscopy to assess the hNH (Fig. 2F) and hCH (fig. S2A) spectral fingerprints of the fully protonated iso1 and iso3 samples at fast magic angle spinning (100 kHz). Iso1 and iso3 exhibited two clearly distinct structural conformations at the atomic level, as revealed by two different single sets of chemical shift resonances. Both NMR fingerprints exhibited a single set of sharp signals, indicating the presence in each sample of a single nonpolymorphic amyloid structure, highly ordered at the atomic level.

Furthermore, we recorded 3D hCANH experiments on iso1 and iso3 to extract 2D NCA planes and compared them to previous solid-state NMR data of four different polymorphs (1316) spanning the entire structure family spectrum defined by Guerrero-Ferreira and co-workers (18) (fig. S2B).

Notably, iso1 turned out to correspond to the type 2 fibril polymorph species (fig. S2B) (14, 18). As to the stealth polymorph iso3, there was no match with any of the polymorph structure families previously characterized (fig. S2B) (1318). However, despite their structural differences (documented in the panel iso3 versus Gath et al. 2011 tris 5 mM in fig. S2B), iso3 shares its ThT invisibility with the ribbon amyloids assembled under low-salt conditions (13, 19). A feature of ribbons that stands out compared to all other fibril types is the particular beta structure of the N terminus (16): This could suggest the possible existence of a similar trait in iso3. These results indicate that -syn can spontaneously and randomly assemble into distinct amyloids under a single experimental condition and that previously unnoticed ThT-negative polymorphs ( polymorphs) can proliferate, crowd the fibrillar population, and cause exacerbated neuronal spread.

Note that the spontaneous emergence of distinct polymorphs seen here could either reflect a stochastic process taking place during fibrillization or a preexisting process, possibly stochastic as well, having formed several distinct soluble progenitors before sample splitting and subsequent fibrillization. If the latter did hold true, then one would have to also assume that during sample splitting, the progenitors would have been heterogeneously sorted into the different tubes. This could occur only under conditions of extreme dilution with only a few individual molecules of progenitors present in the sample. While this heterogenous sorting of preformed progenitors during sample splitting seems unlikely, it cannot be ruled out. This does not exclude, however, the development of a secondary stochastic process taking place during the fibrillization step as well.

In any case, with regard to the current methodological guidelines (27), these results argue against the exclusive use of ThT to monitor -syn amyloids: This practice can lead to the conclusion that damped or null fluorescence rates of change correspond to inhibited or failed fibrillizations, while they can, in fact, reflect the emergence and growth of polymorphs. This can, for instance, become a substantial concern when studying drug candidates aimed at inhibiting fibrillization. The generalized practice of upstream screening fibrillization products with ThT also resulted in the artificial orientation of structural studies toward a restricted subset of -syn amyloid structures excluding polymorphs [e.g., (17)].

We thus decided to build a structural assay capable of discriminating between and regular ThT-positive polymorphs and to monitor amyloid species generation. The absence of ThT signal in the iso3 fibrils could be due to only subtle changes in the ThT-binding region of the assemblies. ThT is considered to bind to the outer surface of the amyloid assemblies, parallel to the main fibril axis. The binding site is different from the one occupied by Congo red and its derivatives like X-34 (31). With regard to the different amyloid folds adopted by -syn in iso3 and iso1 (fig. S2), differences in the outer exposure of the C and N termini at each amyloid stack level can be hypothesized. This could have an impact on the accessibility of ThT to its binding site, on the affinity of the probe for the site, on the fluorescence of the bound probe due to the electrophilic environment, or a combination of these factors.

Thus, we hypothesized that fluorescent compounds with a slightly different chemical structure, but sharing with ThT its trimethine cyanine skeleton (34, 35), could detect iso3. Notably, SYBR Green, the well-known DNA intercalating dye (Fig. 3A), was capable of sensing the amyloid structure of iso3 but appeared to be much less capable of doing so for iso1, giving an almost mirror image of the ThT readings: Iso1 was ThT-high and SYBR Greenlow, while iso3 was ThT-low and SYBR Greenhigh (Fig. 3B). In contrast, the fluorescent Congo red derivative X-34, which binds to a distinct region in amyloids (31, 36), detected iso1 and iso3 with a similar sensitivity (Fig. 3B): X-34 provided a polymorphism-independent readout of the total amounts of amyloids in each sample. Thus, as with prion strains and luminescent conjugated polymers (LCPs) (37), multiple external probing using these different molecules could discriminate the -syn polymorph identities revealed by solid-state NMR. We therefore combined these probes into a multiplex assay to derive a fingerprint of the emerging -syn assemblies at high throughput and termed it the fibrilloscope (Fig. 3C), a simple assay with four readouts: the three dyes ThT, SYBR Green, and X-34 and the measurement of ultraviolet (UV) light attenuation to exploit Mie scattering and sense the emergence of particulate assemblies in the 30- to 300-nm-diameter range (38, 39).

(A) 2D structures of ThT and SG molecules showing their trimethine cyanine skeleton circled in green. (B) Fluorescence intensities of ThT, SG, and X-34, as well as light attenuation at 280 nm (A280) of iso1 versus iso3 samples (AU, table: mean of n = 3 measures). Ratios of iso1/iso3 signal intensities were calculated and plotted (bar graph) for each sample and probe, showing a specific detection of iso1 by ThT (more than five times higher than for iso3) and a specific detection of iso3 by SG (about two times higher than for iso1), and a higher light attenuation (almost five times), while detection with X-34 was similar for iso1 and iso3 (n = 3 measures). (C) Cartoon model of ThT, SG, and X-34 interaction of iso1 (top) and iso3 (bottom schemes). Putative identical exposed residues are depicted in blue bricks, showing a distinct quaternary structure of iso1 and iso3 assemblies affecting the behavior of the different probes under illumination. The A280 light attenuation difference between iso1 and iso3 is explained by the different relative contributions of Rayleigh (small particles, <30 nm) and Mie (larger particles with diameter from 30 to 300 nm) scattering by the assemblies composing iso1 and iso3.

Using the fibrilloscope, we compared a second series of spontaneous fibrillizations with a series of fibrillizations seeded with 1% of untouched iso1 or iso3 (Fig. 4A). In this second round, we used -syn monomers bearing a single S129A substitution to ascertain that the phosphorylated aggregates appearing during the subsequent assays in neurons would correspond to assemblies newly formed at the expense of the endogenous pool of -syn and not to a phosphorylation of the exogenous seeds (fig. S1) (27, 28). Both iso1 and iso3 were able to prompt the appearance and the buildup of amyloids in vitro in comparison with all the spontaneous fibrillizations, with iso1 being even more potent than iso3 (see the X-34 and UV light attenuation readouts) (Fig. 4B). Moreover, all the amyloids that developed in the presence of 1% of iso1 (iso1.1 to iso1.5) were ThT-high and SYBR Greenlow, while all the amyloids observed in the tubes inoculated with 1% of iso3 (iso3.1 to iso3.5) were ThT-low and SYBR Greenhigh (Fig. 4B), corresponding to an exact reproduction of the parental fibrilloscope fingerprint (Fig. 3B). The iso1 and iso3 samples thus contained distinctive amyloid particles capable of imposing their own core structural arrangement on monomers by conformational templating, driving the assembly of daughter amyloid structures. Notably, the second-generation samples retained the exact biological activity of their parents, i.e., all the samples descending from iso1 were inactive on cortical neurons, and all the iso3 offspring triggered an extensive synucleinopathy (Fig. 4C). This indicated that after inoculation, amyloid particles proliferated and that they retained both the fold and the neuronal spread propensity of the parent assemblies.

(A) Schematic representation of the secondary fibrillizations, with and without inoculation, and corresponding nomenclature. (B) Radar plots showing for each tube the intensity values of ThT, SG, X-34, and A280 at 24, 48, and 72 hours during the fibrillization process. The P value of the difference observed with each probe for the different fibrillization groups (1% iso1; 1% iso3; none; n = 5 for each group) was calculated using two-way analysis of variance (ANOVA) with Tukey correction. Distinctive kinetics indicative of seeded aggregation and speciation can be appreciated. (C) Radar plot of the extent of the synucleinopathic spread sparked by the 72-hour secondary fibrillization products in primary cultures of mouse cortical neurons. The latter HCA neuronal bioactivity tests were all run simultaneously, and each individual value plotted in the radar corresponds to the mean of 27 neuronal field quantifications made in three replicate culture wells. The corresponding 72-hour ThT and SG values already shown in (B) are superimposed for facilitating the appreciation of correlations. (D) Inheritance of specific ThT, SG, and bioactivity traits across seed generations. In this graph, all three parameter values were divided for each single sample by its X-34 value to normalize the parameters with respect to the total amyloid load characterizing each sample. The latter normalization was not shown in the title blocks for the sake of simplicity. The statistical significance of the intergeneration (children versus grandchildren for each group) and intergroup (iso1 versus iso3 for each generation) differences was determined using the Hotellings T2 test with Bonferronis correction for multivariate analysis simultaneously exploiting the three parameters characterizing every sample: iso1 children versus iso1 grandchildren, P = 0.70; iso3 children versus iso3 grandchildren, P = 0.84; iso1 children versus iso3 children, P < 1015; and iso1 grandchildren versus iso3 grandchildren, P = 0.012. ns, not significant.

To explore the sustainability of this particle proliferation, we performed a third round of simultaneous fibrillizations in which we compared a series of conditions inoculated with 1% of the second-generation fibrillization products (fig. S3). This resulted in third-generation amyloids that exhibited the same fibrilloscope fingerprint and the same specific bioactivity as their ancestors (Fig. 4D). These results indicate that the novel polymorph iso3 is capable of replicating its distinctive amyloid fold with the same fidelity and sustainability as iso1, a prototypical ThT-positive type 2 polymorph species member (fig. S2) (18).

We then looked more closely at the neuronal seeding activity of the different amyloid species that emerged in our experiments. First, we tried to determine why the iso1 strain members, already described as belonging to the type 2 polymorph species (18) and which proliferated most efficiently in vitro, were unable to spread in neurons. First, we observed that iso1 was cleared more rapidly than iso3 by neurons (Fig. 2D), suggesting that fewer iso1 seeds would remain available intracellularly to trigger a secondary fibrillization. In addition, previous studies concerning this polymorph (23) were indirectly suggestive of the existence of a monomer concentration threshold for conformational templating. We thus reasoned that these limitations could be overcome by forcing the mouse neurons to overexpress human -syn, a condition that could favor the probability of interaction of more templating-compatible human monomers with the iso1 seeds. Notably, this overexpression was enough to rescue the bioactivity of iso1 to a level comparable to that of iso3 (Fig. 5A). In addition, comparative mass spectrometry (MS) analysis of the neuronal assemblies that formed under these conditions indicated that endogenous mouse -syn was excluded from the neo-aggregates elicited by iso1.1, while those sparked by iso3.1 contained both human and mouse -syn (Fig. 5, B and C). Thus, beyond a monomer concentration threshold and a fast clearance of the exogenous seeds, the inability of the iso1 family members to replicate in mouse neurons was probably also associated with a defective templating of mouse -syn monomers compared to a more natural templating of human -syn monomers, i.e., this was suggestive of a host-species specificity of the templating process for iso1.

(A) Neuronal synucleinopathic spread initiated by iso1 and iso3 in nave and human -syn overexpressing mouse neurons [see also (C)]. P value of the difference in bioactivity observed for the groups (n = 18 for each group) was calculated using a t test. (B) Schematic representation of the extraction analysis of insoluble proteins from neurons treated with iso1 and iso3 as in (A). (C) Pellets from (B) were analyzed by MS. Three peptides specific to mouse -syn, three specific to human -syn, and five common to both species were identified by MS. The species, sequence, and hit counts are represented in the table, as well as their sums, represented in the master protein rows. (D) Left images show typical somatic -syn inclusions that beacon the experimental synucleinopathy triggered by iso3 and 1B in our HCA assay (scale bars, 2.5 m). Right quantifications show that the intranuclear crisscrossed inclusions are distinctive of the synucleinopathy triggered by 1B and its offspring. The counts correspond to the number of somatic aggregates detected in 18 standardized image acquisition fields for each condition. (E) Left images show the extended neuritic synucleinopathy (and its analytical segmentation) caused by 1B compared to iso3 in the neuronal HCA assay. Right quantifications show the 1B/iso3 activity ratio with respect to the induction of neuritic synucleinopathy: This ratio appears to be a constant among all the particle generations produced from 1B and iso3; n = 9 for each group; the individual ratio values correspond to the ratio of random pairs of 1B group and iso3 group measurements. AAV, adeno-associated virus; DAPI, 4,6-diamidino-2-phenylindole.

In addition to iso3, we identified by serendipity a second polymorph, which we named 1B. It was also obtained by spontaneous generation in saline (fig. S3). Similar to iso3, this species was ThT-negative and was able to induce an extensive synucleinopathy in mouse cortical neurons (fig. S4). However, from a qualitative point of view, the synucleinopathy induced by 1B was characterized by many linear -syn aggregates in the lumen of the neuronal processes and by the occasional presence of a single v-shaped or crisscrossed -syn aggregate embedded in the nuclear chromatin of certain neurons (Fig. 5, D and E). In comparison, the synucleinopathy induced by iso3 was characterized by the frequent presence of cytoplasmic round or fuzzy -syn aggregates located in the soma and of few linear structures in the processes, while intranuclear aggregates were never observed in this case (Fig. 5, D and E). The ability to induce these specific patterns was inheritable among seed generations (parents, first generation; children, second generation; and grandchildren, third generation) and constituted distinctive sustainable traits (Fig. 5, D and E). This indicates that as with the type 1 and type 2 polymorph families that have been previously well characterized (1418), polymorphs are also populated by structurally distinct subfamilies, endowed with distinctive and inheritable replication patterns in neurons.

With regard to the florid neuritic synucleinopathy triggered by the polymorph 1B in cultured neurons, we reasoned that this polymorph could be particularly prone to trigger a synucleinopathy spreading over long distances along interconnected brain regions in vivo (40). Ten wild-type (WT) mice were unilaterally injected at the level of the substantia nigra pars compacta, with the polymorph 1B, and 10 sham-injected controls were performed in parallel (fig. S5A). Four months after the injection, secondary -syn aggregates were selectively found in the soma of neurons located in the ipsilateral dorsal striatum of the 1B-injected animals, indicating that the synucleinopathy propagated between interconnected brain regions (fig. S5, A to D). The rostral pattern of spread was generally consistent between animals. In some cases, however, the synucleinopathy also spread to the insular cortex and to the core of nucleus accumbens, suggesting transmission from the ventral tegmental area as well (fig. S5B). In agreement with our observation in cultured neurons, the synucleinopathy triggered by 1B resulted not only in the appearance of numerous somatic and neuritic aggregates (fig. S5, B to D) but also in neuronal nuclear inclusions (fig. S5, E to G).

These results prompted us to determine whether the aggregates that were newly formed in neurons exposed to polymorphs were conformational replicas of the exogenous seeds: in other words, whether they resulted from a bona fide conformational templating. To proceed with a comparative analysis of the polymorph iso3 and of its neuronal propagation products, we first tried to identify distinctive immunological and physicochemical features that could differentiate iso3 from the regular type 2 polymorph iso1 (14, 18) and that could be used to characterize the newly formed assemblies (Fig. 6). In that respect, we observed that the two types of amyloid particles isolated by density floatation exhibited different affinities for the conformation-dependent antibody Syn-F1. The staining of iso1 was much weaker than that of the polymorph iso3 (Fig. 6A). To determine whether this feature was inheritable, we put the daughter amyloid particles iso1.1 and iso3.1 and the grandchild particles iso1.1.1 and iso3.1.1 under scrutiny. After simple trapping on nitrocellulose and aldehyde fixation, we found that the two particle species indeed exhibited inheritance of their level of sensitivity toward Syn-F1 (Fig. 6B and fig. S6). Note that the conformational epitope recognized by Syn-F1 partially maps to the C terminus of -syn. Although the C terminus does not participate in the amyloid stack, it is likely that the topology of the amyloid fold that involves upstream regions of the protein still determines its level of exposure, its orientation, and probably its dynamics at the periphery of the fibrillar axis. Depending on the polymorph, aldehyde fixation could thus freeze the C terminus in different positions at the surface of the fibrils. This could account for the differential binding of Syn-F1 to iso1 and iso3.

(A) Density floatations of iso1 and iso3. Fractions filter-trapped and analyzed with Syn-F1 and MJFR1 (n = 3). Mean curves, bold lines; SD, shaded areas. (B) -syn monomers (gray), iso1.1 (dark green), iso1.1.1 (light green), iso3.1 (dark red), and iso3.1.1 (light red) filter-trapped and revealed as in (A). Syn-F1 to MJFR1 ratio plotted in a bar graph with individual n = 4 samples per group. P values were calculated with two-way ANOVA with Tukey correction. (C) Velocity sedimentations of iso1.1 (green) and iso3.1 (red) and of extracts from primary neurons (WT or AAV-hSyninfected) treated with iso1.1 or iso3.1. The fractions were filter-trapped and revealed with MJFR1, Syn-F1, and EP1536Y (top, representative immunoblots). Top: Iso1.1 PFF conditions are overexposed compared to iso3.1 to allow better visualization. Graphs show the quantification from n = 3 replicates per group of the absolute chemiluminescence signal (AU). Data presented are the mean curves (bold lines) with SDs (lighter shaded areas). (D) Filter trap of monomeric and -syn fibrils revealed with Syn-F1, Syn1, and MJFR1. Ratios of Syn-F1 and Syn1 to MJFR1 are plotted in a bar graph with individual n = 4 and n = 8 for monomers and fibrils, respectively. Multiple t tests, Holm-Sidak corrected for multiple comparisons, gave the indicated P values reflecting a specific immunoreactivity of -syn fibrils and monomers toward Syn-F1 and Syn1, respectively. (E) Iso1.1 (green) and iso3.1 (red) were subjected to guanidinium (GdnHCl), urea, and PK treatments [0 to 3.5 M GdnHCl, 1 hour, 25C; 0 to 6 M urea, 6 hours, 25C; and PK (0 to 3.5 g/ml), 30 min, 37C]; reaction aliquots were then filter-trapped and revealed with Syn-F1 (fibrillar, top graphs) or Syn1 (monomers, bottom graphs) (n = 3). Representative immunoblots are shown in fig. S6.

We next focused on iso1.1 and iso3.1 and added two upstream steps (i) harsh treatment with sarkosyl, mimicking the extraction and recovery procedures of insoluble -syn amyloids from neurons, and (ii) velocity sedimentation for size separation. These additional steps completely abolished the Syn-F1 immunoreactivity of iso1.1, while that of iso3.1 was preserved, further widening the immunoreactivity gap between the two species (Fig. 6C).

In the gradients of Fig. 6C, the MJFR1 antibody (human -synspecific) selectively detected the exogenous seeds and permitted their fate to be followed 3 weeks after treatment [see PFF (preformed fibril) and WT neurons rows]. EP1536Y, on the other hand, specifically detected the seeded newly formed amyloid aggregates. By comparing the results obtained with the two stains, it appears that neurons progressively clear the exogenous fibrils (MJFR1 column) and that the amyloids that newly replicate inside neurons (EP1536Y column) span a shorter size range compared to the input fibril seeds. As to the conformation-specific antibody Syn-F1, as one could have expected at this point, the newly formed -syn assemblies that proliferated inside the neurons expressing human -syn also inherited the specific all-or-none Syn-F1 immunoreactivity of the seed species used to inoculate the cultures (Fig. 6C and fig. S6). This indicated that seed speciesspecific templated growth occurred during propagation inside living neurons for both the regular type 2 polymorph iso1 (14, 18) and for the polymorph iso3.

Note that, apart from its solid-state NMR and fibrilloscope fingerprints and distinctive reactivity of its Syn-F1 epitope, the polymorph iso3 was otherwise undistinguishable from iso1 with respect (i) to its sensitivity to proteinase K (PK), (ii) to its resistance to urea or guanidinium chloride (Fig. 6, D and E), and (iii) to its gross fibrillary appearance (Fig. 2D). This indicates that both the fibrilloscope fingerprint and the Syn-F1 epitope exposure identify subtle arrangements of the fibril surface not reflected macroscopically yet encode a size distribution pattern of the amyloid assemblies (Figs. 2C and 6C), a particle proliferation rate (Fig. 4B), and neuronal and cytopathological spread patterns (Fig. 5) that are distinctive of polymorphs.

Our study demonstrates that spontaneous and uncontrolled emergence of polymorphs with exacerbated neuronal spreading abilities can take place during the standard preparation of PFFs (27). This clearly begs the question as to whether the bioactivity characterizing PFF preparations could be partly due to the unsuspected presence of these polymorphs. In that respect, it will be important to distinguish how much the increased pathology triggered by polymorphs in our study is due to its higher stability in mouse neurons versus a higher engagement with mouse -syn per se. Note that despite a minor stability compared to iso3 (Fig. 2E), replication of iso1 is restored when mouse neurons overexpress human -syn (Fig. 5A). This can be taken as an indication that engagement could be a greater determining factor here. This raises the question as to whether polymorphs can emerge in patients and contribute to the formation and spread of the cytopathological inclusions in synucleinopathies.

The ThT invisibility of polymorphs reported here could be taken as an indication that they are not the main components of these inclusions, as the latter consistently appears ThT positive in neuropathological studies. However, it should be kept in mind that our present ThT measurements concern native protein assemblies in saline and are both bulk phase and no wash. This means that when detected, the ThT signals are above the fluorescence background of unbound ThT under conditions where the probed volume is represented at >99% by the solvent phase that properly contains unbound ThT. Under these conditions, polymorphs like iso3 exhibit a very low ThT signal that is ~10-fold smaller than that of iso1 yet is ~4-fold higher than the corresponding monomer condition (Fig. 2A). This fourfold change detected in our bulk phase conditions is likely to be sufficient to grant an apparent ThT-positive status to inclusions containing polymorphs under histopathological detection conditions. In histopathology, ThT staining is performed on aldehyde-fixed sections in which the protein assemblies are cross-linked and often treated with organic solvents and/or detergents. The ThT fluorescence images are then acquired after washing the unbound ThT fraction. In addition, imaging interrogates the sample in a pixelwise fashion, and thus, for the pixels located at the proper level of an inclusion, probably over 50% of the probed volume corresponds to the assembly itself. Thus, inclusions concentrating polymorphs should appear ThT positive over a darker background in histopathology. Note, however, that in neuropathological sections, the ThT difference between the and regular polymorphs might also be blunted because of a possible impact of fixation/organic solvents/detergents on the physicochemical topology of the fibrils.

Recent observations suggest that polymorphs might well emerge under diseased conditions. Reminiscent of the ThT dichotomy characterizing and regular -syn polymorphs, -syn fibrils amplified from the cerebrospinal fluid and postmortem brain extracts of patients with multiple system atrophy (MSA) are respectively ~3 and ~10 times less ThT positive than the ones amplified from comparable samples of patients with PD (25). The ThT dichotomy reported in (25) appears likely to correspond to the detection of distinct -syn polymorphs, as the measurements with the HC-169 amyloid probe excluded a quantitative bias due to the amount of amyloids generated by the amplification procedure (25). In another study, -syn fibrils amplified from brain extracts from different patients with PD and MSA also exhibited variable ThT intensities and stained differently with a set of external fluorescent probes (41).

From a cytopathological point of view, the polymorph 1B triggers a florid Lewy neurite pathology and the frequent formation of -syn inclusions crisscrossing neuronal nuclei. This is intriguing since Lewy neurites are rather reminiscent of the neuropathological signs of PD, while the presence of neuronal nuclear inclusions is, instead, a very specific and early sign of MSA (42, 43).

Collectively, these results justify further characterization of the molecular and supramolecular organization of polymorphs and the identification of their specific neuronal protein interactome compared to ThT-positive -syn fibrils. This should aid in understanding the correlations that exist between the amyloid fold, the outer surface topology of the fibril polymorphs, and their specific interactions with the surrounding intraneuronal environment.

Escherichia coli strain BL21(DE3) plysS was transformed with pET24-syn vector by electroporation and plated onto Luria broth agar plate containing kanamycin (30 g/ml). A preculture in 5 ml of Luria broth medium was inoculated with one clone and incubated at 37C under 200 rpm shaking for 4 hours. The expression on -syn was carried out in M9 minimal medium containing 13C glucose (2 g/liter) and 15NH4Cl (1 g/liter) as carbon and nitrogen sources. Cells from the Luria broth preculture were recovered by centrifugation (1000g, 10 min) and used for inoculating 200 ml of M9 medium. Cells were grown overnight at 37C under 200 rpm shaking and then diluted in 2 liters of culture. Protein expression was induced by adding 1 mM isopropyl--d-thiogalactopyranoside during exponential phase, evaluated at an optical density at 600 nm reaching 0.8. Cells were harvested after 4 to 5 hours of culture at 37C by 6000g centrifugation (JLA 8.1, Beckman Coulter), and pellet was kept at 20C before purification. The site-specific nonphosphorylatable mutant S129A was obtained by site-directed mutagenesis of pET24-syn.

Pellet was thawed in 10 mM tris-HCl (pH 8.0), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and Pierce EDTA-free protease inhibitor tablet (Thermo Fisher Scientific) buffer and sonicated three times for 45 s (Bandelin SONOPULS, VS70T probe) before centrifugation. The supernatant was boiled for 20 min and centrifuged. Streptomycin sulfate was added to the supernatant to a final concentration of 10 mg/ml, and the solution was stirred for 15 min at 4C and then centrifuged. Ammonium sulfate was added to the supernatant to a final concentration of 360 mg/ml, and the mixture was stirred for 15 min at 4C before centrifugation. These four centrifugations were performed at 20,000 rpm for 30 min and at 4C with a Beckman Coulter JA-25.5 rotor. The pellet was resuspended in 25 mM tris-HCl (pH 7.70) and dialyzed against the same buffer to eliminate salts. The dialyzed sample was injected onto a HiTrap Q HP column previously equilibrated with 25 mM tris-HCl (pH 7.70), and -syn was eluted around 250 M NaCl by steps from 0 to 500 mM NaCl with an KTA pure system. Fractions containing the protein were dialyzed against 20 mM tris-HCl (pH 7.40) and 100 mM NaCl buffer before loading onto a 75-pg HiLoad 26/600 Superdex column equilibrated with the same buffer with KTA pure system. Monomeric fractions were collected and concentrated if needed by using Vivaspin 15R 2-kDa cutoff concentrator (Sartorius Stedim). Purification fractions were checked by using polyacrylamide gel electrophoresis (PAGE) tris-tricine 13% dying with ProBlue Safe Stain. Protein concentration was evaluated spectrophotometrically by using absorbance at 280 nm and extinction coefficient of 5960 M1 cm1. Quantification of the preparations with a Pierce chromogenic LAL kit indicated a low endotoxin [lipopolysaccharide (LPS)] residual value: 0.03 to 0.06 EU per g of recombinant protein (44).

Solutions of monomeric -syn at 4 to 5 mg/ml in saline [H2O, 100 mM NaCl, and 20 mM tris-HCl (pH 7.40)] were sterilized by filtration with 0.22-m Millipore single-use filters and stored in sterile 15-ml conical falcon tubes at 4C. Sterilized stock was then distributed into safe-lock Biopur individually sterile-packaged 1.5-ml Eppendorf tubes as 500-l aliquots. The tubes were cap-locked and additionally sealed with parafilm. All previous steps were performed aseptically in a particle-free environment under a microbiological safety laminar flow hood. For comparative fibrillizations, all samples were loaded simultaneously in a ThermoMixer (Eppendorf) in a 24-position 1.5-ml Eppendorf tube holder equipped with a heating lid. Temperature was set to 37C, and shaking was set to 2000 rpm. Sampling for measurements during the fibrillization process was done by temporarily returning the samples under the microbiological safety laminar flow hood. Raising LPS by exogenous addition from the residual value of ~0.018 to 100 g/ml did not favor the appearance of ThT-positive assemblies under these fibrillization conditions (21).

Samples from the fibrillized -syn aliquots or of the control -syn monomers stored at 4C were diluted to 0.1 mg/ml in 100 l of phosphate-buffered saline (PBS) containing either 20 M ThT, 20 M X-34, 0.1% SYBR Green I (SG) commercial stock solution, or nothing else. The diluted samples were distributed either in 96-well plates (samples with probes) or in UVettes (Eppendorf) (samples without probe). After 20 min at room temperature protected from ambient light, the plates were read under orbital shaking in a BMG LABTECH FLUOstar OPTIMA fluorimeter. Excitation/emission wavelength pairs were 380 nm/520 nm for X-34, 450 nm/480 nm for ThT, and 485 nm/520 nm for SG. The UVettes were read at 280 nm using the 1-cm optical path with an Eppendorf biophotometer in absorbance mode to generate the A280 light attenuation readout.

Samples from the fibrillized -syn aliquots or of the control -syn monomers stored at 4C were diluted to 0.1 mg/ml in 100 l in PBS and distributed in cap-locked, sterile 0.5-ml polymerase chain reaction (PCR) tubes (Thermo Fisher Scientific). When relevant, sonication was performed at 25C in a Bioruptor Plus water bath sonicator (Diagenode) equipped with thermostatic control and automated tube carousel rotator. The sonication power was set to high, and 10 cycles of 30-s on followed by 10-s off were applied. The impact of this sonication protocol on the particle contents of the samples was scored using an NanoSight NS300 NTA platform (Malvern) (see Fig. 1D).

Timed pregnant C57BL/6J female mice were received from Charles River Laboratories 2 days before initiation of the primary culture. The cultures were synchronized with the -syn fibrillizations to use untouched amyloids in the bioassay. Cortices were harvested from embryonic day 18 mouse embryos and dissociated enzymatically and mechanically (using neural tissue dissociation kit, C Tubes, and an Octo Dissociator with heaters; Miltenyi Biotech, Germany) to yield a homogenous cell suspension. The cells were then plated at 20,000 per well in 96-well plates (Corning, BioCoat poly-d-lysine imaging plates) in neuronal medium (MACS Neuro Medium, Miltenyi Biotech, Germany) containing 0.5% penicillin-streptomycin, 0.5 mM alanyl-glutamine, and 2% NeuroBrew supplement (Miltenyi Biotech, Germany). The cultures were maintained with 5% CO2 at 37C in humidified atmosphere. The medium was changed by one-third every 3 days, until a maximum of 30 DIV (days in vitro). In such cultures, and under control conditions, neurons represented approximately 85 to 95% of the cell population; thus, in the text and for the sake of simplicity, they are referred to as neurons. After 7 DIV, vehicle and -syn amyloidcontaining samples were added at a final concentration of 10 nM equivalent monomeric concentration. When relevant, neurons were infected at DIV 10 with -syn AAV (adeno-associated virus) particles (multiplicity of infection, 1000). All antibodies used and shown in the study are summarized in table S1. For live-dead analysis, DIV 30 cultures were loaded with 1 M calcein-AM for 30 min at 37C, the plates were washed with Hanks balanced salt solution (pH 7.4), and the de-esterified calcein retained in the living neurons was immediately imaged using Incucyte S3.

Recombinant AAV9-CMVie/SynP-wtsyn-WPRE vector containing the sequence of human -syn put under control of the human synapsin promoter was produced by polyethylenimine-mediated triple transfection of low-passage human embryonic kidney293T/17 cells (American Type Culture Collection; catalog number CRL-11268). The AAV expression plasmid pAAV2-CMVie/hSyn-wtsyn-WPRE-pA was cotransfected with the adeno helper pAd Delta F6 plasmid (Penn Vector Core, catalog number PL-F-PVADF6) and AAV Rep Cap pAAV2/9 plasmid (Penn Vector Core, catalog number PL-T-PV008). Cells are harvested 72 hours after transfection, resuspended in lysis buffer [150 mM NaCl and 50 mM tris-HCl (pH 8.5)], and lysed by three freeze-thaw cycles (37C/80C). The cell lysate was treated with Benzonase (150 U/ml; Sigma-Aldrich, St. Louis, MO) for 1 hour at 37C, and the crude lysate was clarified by centrifugation. Vectors are purified by iodixanol step gradient centrifugation and concentrated and buffer-exchanged into lactated Ringers solution (Baxter, Deerfield, IL) using Vivaspin 20 100-kDa cutoff concentrator (Sartorius Stedim, Gttingen, Germany).The genome-containing particle titer was determined by quantitative real-time PCR using the LightCycler 480 SYBR Green Master mix (Roche, catalog number 04887352001) with primers specific for the AAV2 inverted terminal repeats (forward, 5-GGAACCCCTAGTGATGGAGTT-3 and reverse, 5-CGGCCTCAGTGAGCGA-3) on a LightCycler 480 instrument. Purity assessment of vector stocks was estimated by loading 10 l of vector stock on 10% SDS acrylamide gels, and total proteins were visualized using the Krypton Infrared Protein Stain according to the manufacturers instructions (Life Technologies).

Stock preparations of amyloid fibrils [4 to 5 mg/ml in tris-buffered saline (TBS)] were diluted to a final concentration of 0.1 mg/ml in TBS. The equivalent of the quantity of recombinant human -syn fibrils used for the treatment of one petri dish of primary neurons (1.66 g) was diluted in 500 l of final volume with solubilization buffer (SB): 10 mM tris (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, cOmplete EDTA-free protease inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche), with a final concentration of 1% (w/v) N-lauroyl-sarcosine (sarkosyl, Sigma-Aldrich), 2 mM MgCl2, and Benzonase nuclease (~0.5 U/l; Millipore). For treated primary neuron solubilization, 500 l of SB was added per petri dish at room temperature. Cells were scraped with a scraper before transferring the lysate in a 1.5-ml centrifuge tube. Fibrils or primary neuron lysates were then solubilized by incubating at 37C under constant shaking at 600 rpm (ThermoMixer, Eppendorf) for 45 min.

Sedimentation velocity and density floatation gradient fractionations were performed as previously published (32). Briefly, for velocity sedimentations, a volume of 400 l was loaded on top of an 11-ml continuous 10 to 25% iodixanol gradient [OptiPrep, 60% (w/v) iodixanol, Sigma-Aldrich] in SB with 0.5% (w/v) sarkosyl linearized directly in ultracentrifuged 12-ml tubes (Seton) with a Gradient Master (Biocomp). For density floatation gradients, 400 l of solubilized material was diluted in SB with 50% iodixanol and 0.5% (w/v) sarkosyl to make a final concentration of 40% iodixanol. This sample-containing cushion was loaded within an 11.4-ml 10 to 60% discontinuous iodixanol gradient in SB with 0.5% (w/v) sarkosyl. The gradients were centrifuged at 200,000g for 2.5 hours at room temperature (velocity) or at 180,000g for 16 hours at 4C (density) in a swinging-bucket SW-41 Ti rotor using an Optima LE-80K ultracentrifuge (Beckman Coulter). Gradients were then segregated into 16 equal fractions from the top using a piston fractionator (Biocomp) and a fraction collector (Gilson). Fractions were aliquoted for further analysis of their content by dot blot, immunoblot on SDS-PAGE, or native PAGE. Gradient linearity was verified by refractometry.

For filter trap assays, native fractions were spotted onto nitrocellulose 0.2-m membranes (Protran, GE) using a dot blot vacuum device (Whatman). Nitrocellulose membranes were fixed for 30 min in PBS with paraformaldehyde (PFA) at 0.4% (v/v) (Sigma-Aldrich) final concentration. After three washes with PBS, membranes were blocked with 5% (w/v) skimmed powder milk in PBS-Tween 0.5% (v/v) and probed with primary and secondary antibodies in PBS-Tween with 4% (w/v) bovine serum albumin (table S1). Immunoreactivity was visualized by chemiluminescence (Bio-Rad). The amount of the respective protein in each fraction was determined by the Image Studio Lite software after acquisition of chemiluminescent signals with a ChemiDoc digital imager (Bio-Rad). The profiles obtained were normalized and plotted with SD, all with respective Students t test and analysis of variance (ANOVA) using the Prism software.

Fibril-treated primary neuron lysates were prepared from a petri dish as previously described for velocity and density gradients. For extraction of insoluble proteins, 500 l of solubilized lysates were mixed 1:1 with SB 40% (w/v) sucrose, without sarkosyl, MgCl2, and Benzonase, in 1-ml thickwall polycarbonate ultracentrifuge tubes (Beckman Coulter) and centrifuged at 250,000g for 1 hour at room temperature with a TLA 120.2 rotor using an Optima XP benchtop ultracentrifuge (Beckman Coulter). Supernatants were collected by pipetting. Pellets were resuspended in 50 l of PBS, and total protein concentration was determined by bicinchoninic acid assay (Pierce) before equalization and denaturation for 5 min at 100C in Laemmli buffer.

Sample preparation and protein digestion. Ten micrograms of total protein per sample was deposited onto SDS-PAGE gel for concentration and cleaning purpose. Separation was stopped once proteins have entered the resolving gel. After colloidal blue staining, bands were cut out from the SDS-PAGE gel and subsequently cut into 1 mm by 1 mm gel pieces. Gel pieces were destained in 25 mM ammonium bicarbonate/50% acetonitrile (ACN), rinsed twice in ultrapure water, and shrunk in ACN for 10 min. After ACN removal, gel pieces were dried at room temperature, covered with the trypsin solution (10 ng/l in 50 mM NH4HCO3), rehydrated at 4C for 10 min, and lastly incubated overnight at 37C. Spots were then incubated for 15 min in 50 mM NH4HCO3 at room temperature with rotary shaking. The supernatant was collected, and an H2O/ACN/HCOOH (47.5:47.5:5) extraction solution was added onto the gel slices for 15 min. The extraction step was repeated twice. Supernatants were pooled and dried in a vacuum centrifuge. Digests were lastly solubilized in 0.1% HCOOH.

Nanoliquid chromatographytandem MS analysis and label-free quantitative data analysis. Peptide mixture was analyzed on an UltiMate 3000 Nano LC system (Dionex) coupled to an Electrospray Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). Ten microliters of peptide digests was loaded onto a 300-minner diameter 5-mm C18 PepMap trap column (LC Packings) at a flow rate of 10 l/min. The peptides were eluted from the trap column onto an analytical 75-mminner diameter 50-cm C18 PepMap column (LC Packings) with a 4 to 40% linear gradient of solvent B in 105 min (solvent A was 0.1% formic acid, and solvent B was 0.1% formic acid in 80% ACN). The separation flow rate was set to 300 nl/min. The mass spectrometer operated in positive ion mode at a 1.8-kV needle voltage. Data were acquired using Xcalibur 4.1 software in data-dependent mode. MS scans [mass/charge ratio (m/z), 375 to 1500] were recorded at a resolution of R = 120,000 (at m/z of 200) and an AGC target of 4 105 ions collected within 50 ms. Dynamic exclusion was set to 60 s, and top speed fragmentation in HCD mode was performed over a 3-s cycle. Tandem MS scans with a target value of 3 103 ions were collected in the ion trap with a maximum fill time of 300 ms. In addition, only +2 to +7 charged ions were selected for fragmentation. Other settings were as follows: no sheath or auxiliary gas flow; heated capillary temperature, 275C; normalized HCD collision energy of 30%; and an isolation width of 1.6 m/z. Monoisotopic precursor selection was set to peptide, and an intensity threshold was set to 5 103.

Database search and results processing. Data were searched by SEQUEST through Proteome Discoverer 2.3 (Thermo Fisher Scientific) against the Mus musculus Reference Proteome Set (from UniProt 2019-07; 55,121 entries). Spectra from peptides higher than 5000 Da or lower than 350 Da were rejected. The search parameters were as follows: Mass accuracy of the monoisotopic peptide precursor and peptide fragments was set to 10 parts per million and 0.6 Da, respectively. Only b-ions and y-ions were considered for mass calculation. Oxidation of methionines (+16 Da) and protein N-terminal acetylation (+42 Da) were considered as variable modifications, and carbamidomethylation of cysteines (+57 Da) was considered as fixed modification. Two missed trypsin cleavages were allowed. Peptide validation was performed using Percolator algorithm, and only high confidence peptides were retained corresponding to a 1% false-positive rate at peptide level. Peaks were detected and integrated using the Minora algorithm embedded in Proteome Discoverer. Proteins were quantified on the basis of unique peptide intensities. Normalization was performed on the basis of total protein amount. Protein ratios were calculated as the median of all possible pairwise peptide ratios. A t test was calculated on the basis of background population of peptides or proteins. Quantitative data were considered for proteins quantified by a minimum of two peptides, fold changes above two, and a statistical P value lower than 0.05.

Fibril stock preparations (4 to 5 mg/ml in TBS) were diluted to a final concentration of 0.1 mg/ml in TBS. For each guanidine hydrochloride (guanidinium, GdnHCl; Sigma-Aldrich), urea (Sigma-Aldrich), and PK (Sigma-Aldrich) concentration, 10 l (1 g) of PFF was mixed 1:1 with 10 l of stock solutions to make 0, 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 M GdnHCl final concentrations; 0, 0.5, 1, 2, 3, 4, 5, and 6 M urea final concentrations; and 0, 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 g/ml PK final concentrations. Mixtures were gently vortexed before incubation for 1 hour at room temperature for GdnHCl, 6 hours at room temperature for urea, and 30 min at 37C for PK. At the end of the incubation period, treatments were stopped by quickly diluting the samples with 500 l of PBS and directly subjecting them (100 l per immunoblotting antibody) to filter-trap assay as previously described. The different -syn species were quantified by immunolabeling with conformation-specific, monomeric, or pan -syn antibodies (table S1) and expressed as a percentage of related untreated samples, allowing to draw curves of PFF disassembly, denaturation, and proteolysis with GdnHCl, urea, and PK, respectively.

In vivo experimental synucleinopathy. Adult male C57BL/6 mice were bred at the Institute of Lab Animal Science (Chinese Academy of Medical Sciences, Beijing, China). All mice were housed in a temperature-controlled (22C) and light-controlled environment on a 12-hour light/12-hour dark cycle at the Institute of Lab Animal Science animal care facility with access to food and water ad libitum. The study design was approved by the Institute of Lab Animal Science Institutional Animal Care And Use Committee (Chinese Academy of Medical Sciences, Beijing, China), and all experimental procedures were conducted in accordance with the European Communities Council Directive (2010/63/EU) for care of laboratory animals in an AAALAC-accredited facility. The mice (2 months old) unilaterally received 2 l of sonicated polymorph -syn fibrils 1B (2.5 mg/ml) by stereotactic delivery to the region immediately above the right substantia nigra (coordinates from bregma: AP, 2.9; L, 1.3; DV, 4.5) at a flow rate of 0.4 l/min, and the pipette was left in place for 5 min after injection to avoid leakage. Animals were euthanized after 4 months. Ten mice were used in each group, with male and female mixed. The brains were perfused with saline, postfixed for 3 days in 10 ml of 4% PFA at 4C, cryoprotected in gradient 20% sucrose in PBS before being frozen by immersion in a cold isopentane bath (60C) for at least 5 min, and stored immediately at 80C until sectioning. After serial sectioning, the sections were stained using the primary antibodies EP1536Y (Abcam) for detecting phospho S129positive -syn amyloid aggregates [dilution 1:5000 for immunohistochemistry (IHC) and 1:500 for immunofluorescence (IF)]. The slides were acquired using a Pannoramic slide scanner for IHC, and an Incucyte S3 High Content Imager (Sartorius) with a homemade 3D-printed slide holder for IF.

Electron microscopy. Aliquots of fibrillized samples were diluted with water, and one drop was applied to glow-discharged 300-mesh carbon-coated copper grids for 1 min. Grids were washed with one drop of water before being negatively stained with 2% uranyl acetate and dried for 5 min in the dark. Samples were observed using a Philips CM 120 (high-voltage 120 kV, lanthanum hexaboride filament) transmission electron microscope in low dose mode. Images were acquired using a Gatan US1000 (charge-coupled device) camera.

Solid-state NMR. All spectra were recorded on a Bruker NEO 800-MHz (1H Larmor frequency) spectrometer with an ultrafast Bruker 0.7-mm HCND probe. The 0.7-mm rotor was placed into a 1.3-mm rotor with a rubber plug from the bottom. Approximately ~200 l of fibrils resuspended in H2O was pipetted in a funnel of filling tool, and it was spun for ~18 hours at 12C at 29,500 rpm (with an SW32Ti Beckman ultracentrifugation rotor). The detailed NMR experimental parameters are described in tables S2 and S3.

Acknowledgments: We thank L. A. Largitte, L. Basurco, M. L. Thiolat, T. H. Nguyen, N. Biendon, and S. Dovero for technical help; T. Boraud and J.-P. Mazat for critical reading of the manuscript; and A. R. Crossman, Emeritus Professor, University of Manchester, UK, who read the revised the manuscript and made suggestions to improve readability. Funding: The project was conducted using financial support from the Region Nouvelle Aquitaine, the Grand Prix from the Del Duca Foundation, the European Research Council (ERC-2015-StG GA no. 639020), the Swiss National Science Foundation for early postdoc mobility project P2EZP2_184258, the CAMS Innovation Fund for Medical Sciences (CIFMS) grant (2016-I2M-2-006), the SAFEA: Introduction of Overseas Talents in Cultural and Educational Sector (G20190001626), the National Natural Science Foundation of China Grant (31970510), and the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement no. 116060 (IMPRiND). This Joint Undertaking receives support from the European Unions Horizon 2020 research and innovation programme and EFPIA. This work is supported by the Swiss State Secretariat for Education Research and Innovation (SERI) under contract number 17.00038. The opinions expressed and arguments used herein do not necessarily reflect the official views of these funding bodies. We thank the IR-RMN THC FR3050 CNRS and the Biophysical and Structural Chemistry platform (BPCS) at IECB for the access granted to their facilities. Author contributions: Conceptualization: F.D.G. and F.I. Validation and supervision: F.D.G., E.B., and F.I. Investigation and formal analysis: F.D.G., F.L., F.I., F.Z., A.Le., M.B., X.Y., E.M., A.G., E.D., B.H., N.D., J.D., S.C., and A.Lo. Methodology: F.D.G., F.I., F.L., B.H., E.F., C.Q., and A.Lo. Visualization: F.L., F.D.G., and F.I. Writing (original draft preparation): F.I. Writing (review and editing): F.D.G., F.L., E.B., B.H., A.Lo., and F.I. 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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Novel self-replicating -synuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons - Science Advances