Researchers have genetically engineered the first mice that get a human-like form of COVID-19, according to a study published online November 1in Nature.

Led by researchers from NYU Grossman School of Medicine, the new work created lab mice with human genetic material for ACE2 -- a protein snagged by the pandemic virus so it can attach to human cells as part of the infection. The mice with this genetic change developed symptoms similar to young humans infected with the virus causing COVID-19, instead of dying upon infection as had occurred with prior mouse models.

"That these mice survive creates the first animal model that mimics the form of COVID-19 seen in most people -- down to the immune system cells activated and comparable symptoms," said senior study author Jef Boeke, the Sol and Judith Bergstein Director of the Institute for Systems Genetics at NYU Langone Health. "This has been a major missing piece in efforts to develop new drugs against this virus."

"Given that mice have been the lead genetic model for decades," added Boeke, "there are thousands of existing mouse lines that can now be crossbred with our humanized ACE2 mice to study how the body reacts differently to the virus in patients with diabetes or obesity, or as people age."

Problem of Large DNA

The new study revolves around a new method to edit DNA, the 3 billion "letters" of the genetic code that serve as instructions for building our cells and bodies.

While famous techniques like CRISPR enable the editing of DNA editing just one or a few letters at a time, some challenges require changes throughout genes that can be up to 2 million letters long. In such cases, it may be more efficient to build DNA from scratch, with far-flung changes made in large swaths of code pre-assembled and then swapped into a cell in place of its natural counterpart. Because human genes are so complex, Boeke's lab first developed its "genome writing" approach in yeast, one-celled fungi that share many features with human cells but that are simpler and easier to study.

More recently, Boeke's team adapted their yeast techniques to the mammalian genetic code, which is made up of not just of genes that encode proteins, but also of many switches that turn on different genes at different levels in different cell types. By studying this poorly understood "dark matter" that regulates genes, the research team was able to design living mice with cells that had more human-like levels of ACE gene activity for the first time. The study authors used yeast cells to assemble DNA sequences of up to 200,000 letters in a single step, and then delivered these "naked" DNAs into mouse embryonic stem cells using their new delivery method, mSwAP-In.

Overcoming the size limits of past methods, mSwAP-In delivered a humanized mouse model of COVID-19 pathology by "overwriting" 72 kilobases (kb) of mouse Ace2 code with 180 kb of the human ACE2 gene and its regulatory DNA.

To accomplish this cross-species swap, the study method cut into a key spot in the DNA code around the natural gene, swapped in a synthetic counterpart in steps, and with each addition, added a quality control mechanism so that only cells with the synthetic gene survived. The research team then worked with Sang Yong Kim at NYU's Rodent Genome Engineering Lab using a stem cell technique called "tetraploid complementation" to create a living mouse whose cells included the overwritten genes.

In addition, the researchers had previously designed a synthetic version of the gene Trp53, the mouse version of the human gene TP53, and swapped it into mouse cells. The protein encoded by this gene coordinates the cell's response to damaged DNA, and can even instruct cells containing it to die to prevent the build-up of cancerous cells. When this "guardian of the genome" itself becomes faulty, it is a major contributor to human cancers.

Whereas the ACE2 experiments had swapped in an unchanged version of a human gene, the synthetic, swapped-in Trp53 gene had been designed to no longer include a combination of molecular code letters -- cytosine (C) next to guanine (G) -- known to be vulnerable to random, cancer-causing changes. The researchers overwrote key CG "hotspots" with code containing a different DNA letter in adenine (A).

"The AG switch left the gene's function intact, but lessened its vulnerability to mutation, with the swap predicted to lead to a 10-to-50 fold lower mutation rate," said first author Weimin Zhang, PhD, a post-doctoral scholar in Boeke's lab. "Our goal is to demonstrate in a living test animal that this swap leads to fewer mutations and fewer resulting tumors, and those experiments are being planned."

The work was funded by National Institutes of Health CEGS grant 1RM1HG009491 and Perlmutter Cancer Center Support Grant P30CA016087. Boeke is a founder of CDI Labs, Inc., a founder of Neochromosome, Inc.; a founder of ReOpen Diagnostics, LLC, and serves or has served on the scientific advisory boards of Logomix Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Sangamo, Inc., Tessera Therapeutics, Inc. and the Wyss Institute. Boeke also receives consulting fees and royalties from OpenTrons, and holds equity in the company. These relationships are managed in accordance with the policies of NYU.

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First mice engineered to survive COVID-19 like young, healthy ... - Science Daily

Researchers have genetically engineered the first mice that get a humanlike form of COVID-19, according to a study published online November 1 in Nature.

Led by researchers from NYU Grossman School of Medicine, the new work created lab mice with human genetic material for ACE2a protein snagged by the pandemic virus so that it can attach to human cells as part of the infection. The mice with this genetic change developed symptoms similar to those of young humans infected with the virus causing COVID-19, instead of dying upon infection, as had occurred with prior mouse models.

That these mice survive creates the first animal model that mimics the form of COVID-19 seen in most peopledown to the immune system cells activated and comparable symptoms, said senior study author Jef D. Boeke, PhD, the Sol and Judith Bergstein Director of the Institute for Systems Genetics at NYU Langone Health. This has been a major missing piece in efforts to develop new drugs against this virus.

Given that mice have been the lead genetic model for decades, added Dr. Boeke, there are thousands of existing mouse lines that can now be crossbred with our humanized ACE2 mice to study how the body reacts differently to the virus in patients with diabetes or obesity, or as people age.

The new study revolves around a new method to edit DNA, the 3 billion letters of the genetic code that serve as instructions for building our cells and bodies.

While famous techniques like CRISPR enable editing DNA just one or a few letters at a time, some challenges require changes throughout genes that can be up to 2 million letters long. In such cases, it may be more efficient to build DNA from scratch, with far-flung changes made in large swaths of code preassembled and then swapped into a cell in place of its natural counterpart. Because human genes are so complex, Dr. Boekes lab first developed its genome writing approach in yeast, one-celled fungi that share many features with human cells but that are simpler and easier to study.

More recently, Dr. Boekes team adapted their yeast techniques to the mammalian genetic code, which is made up not only of genes that encode proteins but also of many switches that turn on different genes at different levels in different cell types. By studying this poorly understood dark matter that regulates genes, the research team was able to design for the first time living mice with cells that had more humanlike levels of ACE gene activity. The study authors used yeast cells to assemble DNA sequences of up to 200,000 letters in a single step, and then delivered these naked DNAs into mouse embryonic stem cells using their new delivery method, mSwAP-In.

Overcoming the size limits of past methods, mSwAP-In delivered a humanized mouse model of COVID-19 pathology by overwriting 72 kilobases (kb) of mouse Ace2 code with 180 kb of the human ACE2 gene and its regulatory DNA. To accomplish this cross-species swap, the study method cut into a key spot in the DNA code around the natural gene, swapped in a synthetic counterpart in steps, and with each addition, added a quality control mechanism so that only cells with the synthetic gene survived. The research team then worked with Sang Y. Kim, PhD, at NYU Langones Rodent Genetic Engineering Laboratory, using a stem cell technique called tetraploid complementation to create a living mouse whose cells included the overwritten genes.

In addition, the researchers had previously designed a synthetic version of the gene Trp53, the mouse version of the human gene TP53, and swapped it into mouse cells. The protein encoded by this gene coordinates the cells response to damaged DNA, and it can even instruct cells containing it to die to prevent the buildup of cancerous cells. When this guardian of the genome itself becomes faulty, it turns into a major contributor to human cancers.

Whereas the ACE2 experiments had swapped in an unchanged version of a human gene, the synthetic, swapped-in Trp53 gene had been designed to no longer include a combination of molecular code letterscytosine (C) next to guanine (G)known to be vulnerable to random cancer-causing changes. The researchers overwrote key CG hot spots with code containing a different DNA letter, adenine (A).

The AG switch left the genes function intact, but lessened its vulnerability to mutation, with the swap predicted to lead to a ten- to fiftyfold lower mutation rate, said first author Weimin Zhang, PhD, a postdoctoral scholar in Dr. Boekes lab. Our goal is to demonstrate in a living test animal that this swap leads to fewer mutations and fewer resulting tumors, and those experiments are being planned.

Along with Dr. Boeke and Dr. Zhang, NYU Langone study authors were Ran Brosh, PhD; Aleksandra Wudzinska, MPhil; Yinan Zhu; Noor Chalhoub; Emily Huang; and Hannah Ashe in the Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology; Ilona Golynker; Luca Carrau, PhD; Payal Damani-Yokota, PhD; Camille Khairallah, PhD; Kamal M. Khanna, PhD; and Benjamin tenOever, PhD; in the Department of Microbiology; and Matthew T. Maurano and Dr. Kim in the Department of Pathology.

The work was funded by National Institutes of Health CEGS grant 1RM1HG009491 and Perlmutter Cancer Center Support Grant P30CA016087. Dr. Boeke is a founder of CDI Labs Inc., a founder of Neochromosome Inc., a founder of ReOpen Diagnostics LLC, and serves or has served on the scientific advisory boards of Logomix Inc., Modern Meadow, Rome Therapeutics, Sample6, Sangamo Therapeutics, Tessera Therapeutics, and the Wyss Institute. Dr. Boeke also receives consulting fees and royalties from Opentrons and holds equity in the company. These relationships are managed in accordance with the policies of NYU Langone Health.

Greg Williams Phone: 212-404-3500 Gregory.Williams@NYULangone.org

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First Mice Engineered to Survive COVID-19 Are Like Young, Healthy ... - NYU Langone Health

A decade ago, scientists outlined the gene editing potential of CRISPR, turning the vestiges of a bacterial immune system into one of the biotechnology industrys most powerful tools.

On Tuesday, a group of advisers to the Food and Drug Administration met to discuss the merits of what could be the first CRISPR medicine approved by the agency: a treatment for sickle cell disease from partners Vertex Pharmaceuticals and CRISPR Therapeutics.

Documents published last week show that FDA scientists are focused on the technical aspects of how CRISPR does its DNA-editing work. They seem relatively convinced the treatment, known as exa-cel, is effective.

BioPharma Dive tracked the daylong meeting and reported on the discussion here. The most recent entries are listed first.

An early wrap

About an hour before the meeting was scheduled to end, OTP Director Verdun thanked public speakers, Vertex and FDA staff as she drew the days discussions to a close.

An important part of our mission is not just evaluating efficacy, but safety, both short- and long-term, and doing what we can to evaluate both known and unknown risk of therapies, Verdun said.

She noted that the FDA will take the committees discussions and recommendations into consideration as it completes its review of exa-cel.

While the FDA doesnt have to follow the advice of its advisory panels, it often does. The agency is set to decide on exa-cels approval by Dec. 8. Gwendolyn Wu

The committee's view

Committee chair Ahsan closed the discussion by summarizing the panelists views. They agreed, she said, that Vertexs analysis of off-target risks was reasonably detailed, but indicated further study could still be useful. So then the question becomes, when have we done enough theoretical analysis to allow us to move forward? she said.

Ahsan added that, to better understand if and how off-target edits emerge, Vertex needs to continue to monitor patients. Yet she acknowledged that approach might have some limitations, however.

It would be nice to see some evaluation of monitoring the edits over real time, looking at clonal expansion, Ahsan said. But it's unsure the technology that would be used to do that, whether whole genome sequencing [or something else], would actually have the detection levels to give us meaningful information there. Jonathan Gardner

Enemy of the good

The committee has ended its cross-examination of Vertex and moved on to the broader discussion. Scot Wolfe, professor of molecular, cell, and cancer biology at the University of Massachusetts medical school, summarized Vertexs methods as pretty detailed, and quibbled only with the depth of the analysis.

We want to be careful to not let the perfect be the enemy of the good, Wolfe said. You want to do as good a job as you possibly can, but at some point, you have to try things in patients. I think in this case, there is a huge unmet need.

That was echoed by Alexis Komor, an assistant professor of chemistry and biochemistry at University of California, San Diego. Do we have the technology to sequence every single patient and do an expansive individualized on-target analysis on each one? Probably, but is that reasonable to expect from them at this point? I dont know, she said. Jonathan Gardner

Cellular sampling

FDA panelists asked a number of questions to agency reviewers and to Vertex. Joseph Wu, one of the panelists and director of the Stanford Cardiovascular Institute, noted how Vertexs cellular off-target analysis used donor cells from only three sickle cell patients, while the company has treated dozens more in the exa-cel trial.

You've had several years with these samples. Why not just do the actual analysis rather than the in silico modeling?" Wu said.

The sickle cell patient samples Vertex used were among 14 donor cell samples that the company has tested, and there's no data to suggest that the result would be different for patients with sickle cell than the other possibilities, Altshuler responded.

Whether we were to do the testing, Altshuler said, we'd end up at the same place, I believe, which is the risk assessment.

Wus line of questioning, as well as the concerns raised by Singh in her analysis, return to a similar theme: how much off-target analysis is enough?

I suspect if there were hard guidelines we wouldn't be having this meeting, committee chair Ahsan said. Gwendolyn Wu

The regulator's view

FDA staff just finished outlining the agencys view of exa-cel. The first presentation, by reviewer Karl Kasamon, gave a clinical overview of the therapy and largely lined up with Vertexs assessment of its efficacy. The second, by bioinformatics expert Komudi Singh, was a detailed review of the days main issue: off-target CRISPR editing.

Singh described how off-target edits, while potentially inconsequential, can be harmful if they occur in regions of the genome that have regulatory functions or code for a protein. There are a number of tools available to assess this likelihood and Vertex took several approaches, including using computer algorithms to identify possible off-target sites and sequencing donor cells that were edited.

Singh raised a number of concerns with Vertexs methods. For example, the database used by the company contains only a small amount of sequencing data from the intended population for exa-cel treatment. It also may not be representative of genetic variants that present higher risk of off-target edits. Vertexs cellular analysis, meanwhile, used a small sample of donor cells.

Singh ended by asking the committee to provide recommendations, setting up the days concluding discussion on whether other studies are needed to gauge exa-cels safety. Ned Pagliarulo

Victoria and Jimi

The open public hearing began with Victoria Gray, a 38-year-old woman who was the first patient to be treated with exa-cel. She spoke of sickle cells pain, which she compared to being simultaneously hit by a truck and lightning, and the diseases effects on her life and family.

She met with physician Haydar Frangoul, who offered her a spot in the exa-cel trial. I said yes without hesitation, knowing that I would be the first person but this was my opportunity to fight, Gray recounted.

She no longer has pain crises after receiving exa-cel, nor does she need blood transfusions. I now work full time and I contribute to my household and my community.

Her experience was similar to that of Jimi Olaghere, who participated in the exa-cel study about three years ago.

Gene therapy has given me the ability to take full control of my life, said Olaghere. In a world where the deck was stacked against me, gene therapy has been a winning hand. While I recognize gene editing wont be the solution for everyone I strongly recommend [sickle cell] warriors to consider this one-time therapy. Ned Pagliarulo

Lunch break

The panel is now on break until 12:35 p.m., when we'll hear from patients and advocates during the open public hearing. Ned Pagliarulo

Like a hammer hitting a wall

The first two patients who entered the exa-cel trial have seen notable improvements since they were treated, said Haydar Frangoul, hematology and oncology medical director at Tristar Centennial Medical Group in Nashville and lead investigator of the exa-cel studies.

One, a 33-year-old woman who couldn't walk or even pull up a spoon to feed herself during pain crises, has been a
ble to take up full-time work and become more active in caring for four children since receiving the therapy. Another, a 13-year-old girl who was hospitalized several times each year because of pain crises, can attend school regularly because she no longer needs as much medical care.

Frangoul also urged exa-cels use early in the disease course because of the cumulative effects of the disease on organs and joints.

Sickle cell disease is like a hammer hitting a wall, he said. I can take away the hammer. But we cannot reverse the damage. We cannot fix the wall. Jonathan Gardner

Safety follow-up

To track exa-cel safety over the long term, Vertex plans to rely on two 15-year studies, including a registry-based study that would begin post-approval and involve sickle cell patients treated with exa-cel. The other is follow-up for an ongoing clinical trial.

The Center for International Blood and Marrow Transplant Research, which has for years collected data on patients receiving cell therapies, will also assess the long-term safety of exa-cel, said Christopher Simard, Vertex's vice president of global patient safety. All planned U.S. exa-cel treatment centers will provide data to the center, he said. Gwendolyn Wu

Visualizing exa-cels benefit

William Hobbs, Vertexs head of hematology clinical development, outlined the clinical trial data showing exa-cels effectiveness. The results are clear: Treatment eliminates, for at least multiple years, the debilitating pain crises that people with sickle cell can experience and keeps them out of the hospital. Ned Pagliarulo

The need for treatment

There are a few drugs currently available to help manage sickle cell symptoms. But they dont fix the condition and dont work for everyone.

A bone marrow transplant of donor stem cells can cure the disease, but is available for less than 20% of people with sickle cell, said Alexis Thompson, division chief of hematology at Childrens Hospital of Philadelphia, who spoke at Tuesdays meeting on behalf of Vertex.

An estimated 100,000 people live with sickle cell in the U.S., and about one-fifth have a severe form of the disease, with recurring symptoms such as severe pain flare-ups, a lung condition known as acute chest syndrome, priapism and splenic sequestration. The resulting organ damage raises the risk of death.

Sickle cell also occurs at "disproportionately high rates" among people of color, particularly people of African ancestry, Thompson said.

People with sickle cell often live in low income areas and communities with high unmet medical need, further adding to substantial healthcare disparities, she said. Gwendolyn Wu

How much testing is enough?

The vastness of the human genome presents a thorny problem for assessing the risk of off-target CRISPR edits. Scientists trying to vet the risk have to focus their analysis, or risk being swamped by a deluge of data that might not actually help suss out potential problems.

Urnov, answering a question from the panelist and National Institutes of Health branch chief John Tisdale, said theres a limit to how much reassurance preclinical editing analyses can provide.

The technology is in fact ready for prime time, said Urnov. We're kind of reaching asymptotic places in terms of how we can de-risk it non-clinically. I don't know what else to do at this point in terms of understanding the benefit-risk.

One advantage to studying blood diseases, Bauer noted, is that samples can be easily obtained, tested and tracked over time.

Taby Ahsan, the committees chair, said Urnovs and Bauers answers set the stage for the rest of the days discussion: When is enough theoretical data sufficient to support a patient specific risk assessment? And where are we in that curve of risk mitigation? Ned Pagliarulo

Risk uncertainty

After deeply technical presentations by Urnov and Daniel Bauer about the risks of off-target edits, committee member Lisa Lee, a bioethics expert from Virginia Tech University, brought it back to the patient level by asking a simple question: If you were talking to a family about this kind of treatment, how would you characterize the consequences of off target edits?

Bauer acknowledged the uncertainty of the risk by noting how most of whats in the human genome doesnt code for any specific function, meaning an off-target edit might have no effect on patients.

The only way to know that is through careful follow up, he said. My guess is it's a relatively small risk in the scheme of the risk-benefit. And that's one of the goals, I would say, of doing this under very careful circumstances is to try to learn what that risk is so that we can continually improve those therapies. Jonathan Gardner

"A whole new world"

Fyodor Urnov is a leading expert on CRISPR gene editing and director of technology and translation at UC Berkeleys Innovative Genomics Institute. His task today: Condense the history of CRISPR gene editing research to a 20-minute presentation.

He starts by noting that, while CRISPR is still relatively new, it builds on decades of research into gene editing by other means.

I need to frame the state of our field of gene editing today by stepping 20 years back, Urnov said. So at the time, the sole method for targeted genetic engineering in human cells was an approach called gene targeting. It was inefficient and toxic, he added, ruling out therapeutic applications.

The discovery of CRISPR has brought about an exponential scale-up in gene editing research and its potential applications a shift Urnov documented with the stylized chart below.

Here we are in 2023, and we are proverbially in a whole new world. There are 27,000 references to the word cas9 in PubMed. Genome editing with cas9 and other tools has been shown to work in every basic and applied research setting where it can be tried, as well as in clinical trials of blood stem cells, T cells, the liver and the eye. Ned Pagliarulo

FDA lays out meeting purpose

OTP director Verdun kicked off the meeting by emphasizing the FDA convened the meeting to specifically evaluate the risk of off-target edits. Her comments again signaled that agency scientists are comfortable exa-cel helps patients and that its safety profile is otherwise acceptable.

Verdun also added a personal note about her experience with sickle-cell patients.

I've had the pleasure of taking care of several sickle cell patients and admire the courageous and resilient patient community, she said. I'm also reminded of the sickle cell disease patient-focused drug development program at FDA in which we heard directly from patients and their caregivers which highlighted the significant unmet need. Jonathan Gardner

Today's schedule

After some housekeeping, advisers will hear first from Nicole Verdun, the FDAs new head of the Office of Therapeutics Products, which oversees gene therapies like exa-cel.

The meeting includes two guest speaker presentations, from gene editing experts Fyodor Urnov, of the University of California, Berkeley, and Daniel Bauer, of Boston Childrens Hospital.

After a short break, Vertex and CRISPR Therapeutics will then walk through their data, followed by an open public hearing. The FDA is up in the afternoon and committee discussion is scheduled from 3:00 p.m. to 4:50 p.m Ned Pagliarulo

Why is this meeting important?

The advisory committee meeting is one of the final steps in
the FDAs review of exa-cel, which the agency expects to complete by Dec. 8. These meetings offer a rare window into the agencys thinking midway through an approval review, as well as a chance to publicly vet companies data.

In this case, the meeting will also serve as a forum for discussing CRISPR gene editing, which has become an important biomedical tool used by a growing number of biotechnology companies. The days agenda shows that advisers will hear from other experts about CRISPRs merits and risks, making Tuesdays meeting a mini-summit on the technology.

A positive review by the panel would up the chances that the regulator grants the therapy a historic approval, as well as boost other gene editing companies like Intellia Therapeutics. Ned Pagliarulo

How does exa-cel work?

Essentially, exa-cel combines CRISPR gene editing with bone marrow transplantation.

To construct the treatment, a patients stem cells are collected from their blood and sent to a manufacturing facility where they are edited using CRISPR/cas9. The DNA snip is made to a specific part of a gene called BCL11A that controls production of a protein known as fetal hemoglobin, or HbF. The edited cells are frozen and shipped back to the treating hospital, where, after a preparatory chemotherapy regimen, they are infused into the patient.

Churning out fetal hemoglobin, the new cells effectively mute the most prominent symptoms of sickle cell, which is caused by damaged adult hemoglobin warping red blood cells into a crescent.

Vertex and CRISPR Therapeutics are seeking approval of exa-cel in people aged 12 years and older who have sickle cell and experience the diseases characteristic pain crises. Ned Pagliarulo

What is the FDA thinking?

Documents published last week showed FDA staff to be laser focused on the risk of off-target edits, or when CRISPR makes unintended cuts to DNA other than what its been programmed to snip. Wayward edits could disrupt cell functions or cause damage that later leads to health problems.

Since unintended edits can disrupt gene expression if present in the coding or regulatory DNA sequences, it is critical that the specificity of [exa-cels targeting component] be thoroughly screened to ensure off-target genome editing is minimized, FDA staff wrote.

Vertex and CRISPR have done several analyzes to document and predict this risk with exa-cel specifically, and claim their work shows no detectable off-target editing.

But the FDA appears concerned that their work might have missed something, and wants its advisers to discuss whether any further study is needed. In particular, they raise questions about how well Vertex and CRISPRs analyses capture the risk of off-target edits in a broad population of people with sickle cell.

On the efficacy side of the equation, however, FDA scientists seemed supportive of exa-cels potential, describing Vertex and CRISPRs data as strongly positive at one point in the documents. Ned Pagliarulo

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A play-by-play of the FDA's meeting on Vertex, CRISPR's sickle cell ... - BioPharma Dive

What is Biotechnology?

Biotechnology is the application of biological systems and materials to create new technologies, products, and services that offer qualitative improvements to human and environmental health. Biotechnology encompasses many disciplines including genetics, molecular biology, bioengineering, pharmaceuticals, agriculture, bioinformatics and more. Among the most widely recognized applications of biotechnology is genomic manipulation, or gene editing. Through gene editing, scientists can make highly targeted alterations to the DNA sequences of a living organisms genome. This enables better understanding of genetic and hereditary diseases and can be used to make DNA more resilient to certain viruses or bacteria. The field also enables the creation of biosynthetics, or novel materials created through organic chemistry, that exhibit superior characteristics and greater environmental sustainability than traditional petroleum-based compounds.

There will be increased risk that biotech advancements make the weaponization of biological and chemical agents more likely. There is no universal standard governing the proliferation of biotech, and export controls fail to keep pace with the rapid development of this sector. The spread of biosynthetic tools will enable more research labs around the world to explore pathogen research and the engineering of novel viruses. Moreover, existing safeguards and governance regimes may not be sufficient to prevent the accidental or nefarious spread of dangerous new compounds, toxins and infectious diseases. The risk of non-state access to various biotechnologies also means that bioterrorism will present as a threat, potentially in the form of intentional sabotage of agricultural systems via release of genetically modified organisms. The multiplying effect of AI-enabled research and development will also contribute to the proliferation of biotechnology inways that have yet to be understood.

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Fact Sheet: Biotechnology - Center for Arms Control and Non-Proliferation

Heather Dewey-Hagborg is on a mission to confront the uncomfortable future, especially when it comes to emerging tech. Stranger Visions features portrait sculptures crafted from analyses of genetic material the transdisciplinary artist, educator and filmmaker literally picked up in public places (one persons discarded cigarette butt is anothers way into a strangers DNA). T3511, a collaboration with cinematographer Toshiaki Ozawa (Laurie Andersons Heart of a Dog), sees an anonymous saliva sample become fodder for the alchemizing of the perfect romantic partner.

Now theres Hybrid: an Interspecies Opera, perhaps Dewey-Hagborgs most ambitious work to date. Opening at NYCs Fridman Gallery on November 1, the multimedia project includes a short documentary/personal narrative set to an original score alongside a set of (robotically-constructed and clay-fired) memorial pig sculptures, which allude to the xenotransplantation topic at hand as well as the question of whether genetically engineering bovine for the sole purpose of harvesting hearts for human transplantation is the ethical easy call Big Tech would like us to make (and believe).

Just prior to the artworks New York debut, Filmmaker reached out to Dewey-Hagborg to learn more about enmeshing the scientific and the personal to shape a career in biopolitical art.

Filmmaker: What initially led you to explore the biomedical realm?

Dewey-Hagborg: This started more than 10 years ago, when I became entranced with emerging possibilities of genomics in my project Stranger Visions. The first community biohacker lab had just opened in Brooklyn (Genspace), and I became a member and learned all about DNA. What I realized at that time was that so much was happening so fast in biotech, but it wasnt getting the same critical, artistic attention as digital technology was. Well, this is still true, and I am committed to changing that.

Filmmaker: How did Hybrid: an Interspecies Opera originate? Do you see it as an extension of that previous work Stranger Visions and (your collaboration with Toshiaki Ozawa) T3511?

Dewey-Hagborg: Yes, but also it is a pretty different approach for me in a number of ways. The musical collaboration with Bethany Barrett was something very new for me, and now working on transforming it into a live opera performance (which will premiere next year at the Exploratorium on March 7 and 8) is a really exciting but also very challenging new direction. The film itself has some similarities to T3511 in that both are unusual forms of documentary and exist as records of my practice, but also hopefully transcend this to stand as emotionally relatable media that draws the viewer into contemplating those topics of DNA privacy and xenotransplantation, respectively, more deeply.

Filmmaker: How did this idea of turning this piece into an opera come about? What was the actual process of developing the score and working with the various musicians?

Dewey-Hagborg: I was invited to work on a new piece about gene editing by the MIT Museum and guest curator William Myers. I had been intrigued by xenotransplantation for quite some time because it was the place where the most simultaneous gene edits had occurredin order to make pigs essentially more human. Usually I like to work hands-on in the lab, but with this piece getting access to the kinds of labs that do this work was really prohibitively difficult, because of the controversy surrounding it and the fragile nature of this very experimental new technique. Additionally, it was during the height of COVID.

So, I started the project with a lot of research. I began interviewing scientists that study pigs and xenotransplantation, as well as archaeologists who study the evolution of the pig. I really wanted to get at this question of whether gene editing was something radically new or a continuation of 10 millennia of domestication and selective breeding (as molecular biologists often posit). I began having these Zoom sessions and recording them, then I started working with the wordstranscribing them, editing themand was struck by the beauty, poetry, humor and drama I was hearing from my interlocutors. I just started pulling sentences and arranging them into small poems, and suddenly I heard them in my head in opera voice. I thought, Maybe that is the form this should take. Maybe music should convey the emotional layers of this emerging technology.

I wrote the libretto and went through several iterations and experiments until finally a friend recommended composer Bethany Barrett, who is based in Berlin. She wrote the music and sent me the names of singers she wanted to work with, and we just continued to pass ideas and recordings back and forth.

Now, in working on the live production, I have a music director, Sam Faustine; an associate director, Becca Wolff; and a local crew of singers in San Francisco. Its really an incredible team. (Also, the staff at the Exploratorium has been wonderful.) We rehearse together because my (speaking) parts are intertwined with the singing. It is such an amazing feeling standing onstage and hearing these powerful voices sing the words I wrote live.

Filmmaker: Why do you bring personal narrative into your art?

Dewey-Hagborg: When I was an undergraduate art student I was taught not to: I was told to keep my work conceptual, impersonal, abstract. And while I love work like that too, ultimately it was not my voice. The personal for me is authentic. I want to put my subjectivity forward. I really enjoy enmeshing the scientific and the personal, the messy and the clean. I call it writing through. I like to write my experience through the scientific and technological critique. It feels real to me and more honest than a standard documentary would. And I hope it brings an emotional layer that people can relate to. But every project is different, and I try to listen to the material and orient my approach in a way to best serve its dimensions.

Filmmaker: Youve spoken in the past about your discomfort with both corporations and governments having such easy access to our genetic material be it through seemingly benign ancestry tracing sites or even COVID testingand you also seem similarly uneasy with xenotransplantation and genetically engineering pigs for human hearts (i.e., for humanitys greater good). So, what sorts of change do you ultimately hope to accomplish through your biopolitical art?

Dewey-Hagborg: Some issues are very straightforward, but most are complex and contain layers of tradeoffs. Xenotransplantation is clearly a morally complicated issue. The goal with my work generally is to question the status quo, to advocate for critical attention and debate to topics that are under-discussed. With all the reports in the last year of the remarkable progress in xenotransplantation, there is little to no discussion of the animals whose lives are taken. This is not to say I advocate for a ban on the practice, but I dont think it makes sense to completely skip over discussing the moral dilemma, when we are setting structures into place now that will frame how the future unfolds. When I started the project, I tried to get access to the leading xenotransplantation company in the US to shoot and they told me straight up, We dont want people thinking about pigs. So, my film is for the pigs.

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"My Film is For the Pigs": Heather Dewey-Hagborg on Hybrid: an ... - Filmmaker Magazine

Generation of a glycogene knock out library in HaCaT keratinocytes

HaCaT is a human keratinocyte cell line capable of forming a stratified squamous epithelium, and thus allows evaluating the infection of the skin tropic HSV-1 in both cell and organotypic tissue culture. In order to address the role of specific glycan structures in the HSV-1 infectious cycle, we used precise gene editing to target glycosyltransferases involved in the early steps of core structure synthesis, and in major elongation and branching steps of the main glycosylation pathways, including N-linked glycosylation, mucin type O-linked glycosylation, as well as GSL and GAG synthesis (Fig.1a, Supplementary Table1).

For N-linked glycans we generated MGAT1, MGAT4A, MGAT4B, MGAT5, and MGAT5+4B knock outs (KO). MGAT1 adds the first N-acetylglucosamine to the C-2 of core 3-linked mannose, and lack of this enzyme results in elimination of all N-glycan maturation steps, yielding high-mannose type N-glycans as confirmed by MS-glycoprofiling (Fig.1a, Supplementary Fig.1). MGAT4A, MGAT4B and MGAT5 are responsible for N-glycan branching, where MGAT4A or MGAT4B initiate a 4-linked antenna on the 3-linked mannose, and MGAT5 performs 6-linked branching from the core 6-linked mannose. Lack of MGAT5 results in loss of tetra-antennary N-glycans, and loss of MGAT4 isoforms also strongly diminishes the content of tetra-antennary N-glycans (Supplementary Fig.1). In addition, KO of each of the three branching enzymes resulted in increased relative abundance of hybrid type N-glycans, whereas double KO of MGAT5 and MGAT4B increased the relative abundance of biantennary glycans (Supplementary Fig.1).

For mucin type O-linked glycans, we knocked out core 1 synthase (C1GALT1), its obligate chaperone COSMC (C1GALT1C1), core 2 synthase (GCNT1), as well as the major core 1-capping glycosyltransferase ST3GAL1. Loss of C1GALT1 or COSMC eliminates the 3-linked galactose (core 1 structure), results in truncation of O-linked glycans to the initiating -GalNAc, and prevents assembly on secreted -benzyl GalNAc precursor used in CORA O-glycoprofiling (Fig.1a, Supplementary Fig.2). GCNT1 is the predominant enzyme creating the branched core 2 structure by addition of 6-linked GlcNAc to the GalNAc. Loss of GCNT1 nearly abolished all the disialylated core 2 structures, though some structures matching the composition of monosialylated core 2 could still be detected. Such structures cannot be discriminated from isobaric core 1 structures, and a minor contribution from other GCNTs to core 2 synthesis cannot be excluded either. Finally, loss of ST3GAL1 significantly reduces the 3-linked sialic acid content and results in predominantly non-capped core 1 structures (Supplementary Fig.2). We also targeted the synthesis of GSLs and GAGs. Through KO of B4GALT5 or ST3GAL5, we generated cells with the truncated GSLs, glucosylceramide (GlcCer) and lactosylceramide (LacCer), respectively (Fig.1a). Furthermore, we knocked out B4GALT7, which adds a 4-linked galactose to the initiating xylose in GAG biosynthesis, effectively truncating all classes of GAGs on membrane proteoglycans (Fig.1a). The generated keratinocyte library represents a screening platform for roles of defined cell-surface presented glycan structures in HSV-1 biology in the context of natural infection.

To define the capacity of HSV-1 to complete the infectious cycle in glycoengineered keratinocytes, we infected confluent monolayers of the KO cell lines with HSV-1, and quantified HSV-1 DNA and infectious particles in the growth media at 17h post infection (hpi) by qPCR and plaque titration, respectively. As a measure for viral replication fitness, we calculated the ratio of genome copies/infectious particles for each KO. When infecting cells with truncated O-glycans (C1GALT1C1 KO) a decrease in viral titers was detected (Fig.1b, e). In contrast, the same cells generated close to normal levels of viral DNA (Fig.1c, f), suggesting decreased fitness of virions lacking elongated O-glycans (Fig.1d, g). This feature was unique to complete truncation, and not seen when eliminating branching or sialylation of O-glycans. In cells lacking N-glycan maturation (MGAT1 KO) we also found a lower number of infectious particles (Fig.1b, e) with an apparent decreased fitness as indicated by an increase in the ratio of DNA/infectious particles (Fig.1d, g). This apparent decrease in fitness was not detected in cells with loss of N-glycan branching, and in MGAT4A KO cells we even observed an overall increased viral output (Fig.1b, c). When analysing cells with GSL synthesis defects, we found that lack of LacCer sialylation (ST3GAL5 KO) accelerated virus production (Fig.1b, c, e, f), but without any change in viral fitness (Fig.1d, 1g). Finally, loss of cellular GAGs increased the production of viral particles (Fig.1c). In conclusion, most of the tested glycogene disruptions permitted HSV-1 replication, and only disruption of N- or O-glycan maturation impaired viral fitness. We next evaluated the impact of defined glycan classes to distinct stages of the HSV-1 infectious cycle, including binding and entry, viral assembly and properties of progeny virus, and cell-to-cell spread.

HSV-1 binds and enters human keratinocytes very rapidly, with around 30% of virions bound after 20min on ice, and 80% after 2h28. Most of the bound virions enter keratinocytes within 5min after warming28. Perturbations in each of the investigated glycosylation pathways modulated early virus-host interactions (Fig.2ae). Diminished core 2 O-glycan branching resulted in increased binding also reflected in subsequent entry experiments (Fig.2b, c). Lack of complex N-glycans and reduced 4-antenna branching (MGAT1 KO and MGAT4B KO) showed reduced binding, again also reflected in the entry experiments (Fig.2ac). Interestingly, deletion of MGAT4A, another isoform catalyzing the 4-antenna synthesis on N-glycans, likely on another subset of proteins or sites in proteins29,30, selectively affected viral entry (Fig.2b, c, e). Cells displaying truncated glycolipids showed a reduction in binding to around 50% of that of WT (Fig.2a, b). A similar effect was observed in both B4GALT5 and ST3GAL5 KOs, controlling the consecutive steps in the biosynthesis of the GSL GM3. In addition, an incremental reduction in entry was observed for B4GALT5 KO cells, suggesting involvement of glycolipids in both viral binding and entry to host cells (Fig.2c, e).

HSV-1 binding (20min (a) or 120min (b) on ice) and entry (5min at 37C after 120min on ice (c)) to KO cell lines. Data is shown as WT-normalized mean+SEM of 3 independent experiments for each KO cell line. Two-way ANOVA followed by Dunnetts multiple comparison test was used on raw data to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Proportion of virus bound at 20min compared to 120min (d) or proportion of virus entered at 5min compared to virus bound at 120min (e) is shown as mean+SEM of 3 independent experiments for each KO cell line from a total of 15 experiments. One-way ANOVA followed by Dunnetts multiple comparison test was used to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). f HaCaT WT and B4GALT7 KO cells were probed for HSV-1 gC binding. Enzymatic treatments were included to evaluate contributions of HS and CS GAG chains. Representative of 2 independent experiments. g A simplified overview of GAG synthe sis. h HSV-1 gC binding to a panel of CHO KO cell lines. Data is shown as WT-normalized geometrical means of 3 independent experiments for each KO cell line, and the bar heights indicate mean+SEM. One sample t test was used to evaluate differences from 1. FDR at 5% was controlled by two-stage step-up method of Benjamini, Krieger and Yekutieli (*q<0.05, **q<0.01, ***q<0.001, ****q<0.0001). i Nectin 1 and HVEM surface expression. Data points show background subtracted median fluorescence intensity (MFI) from two independent experiments, and the bar heights indicate the mean. j Percentages of total quantified CS disaccharides in HaCaT WT (Supplementary Table2). k Percentages of total quantified HS disaccharides in HaCaT WT (Supplementary Table3). Source data are provided as a Source Data file for all graphs.

Then, we analysed cells impaired in GAG biosynthesis and found an almost complete loss of binding to cells presenting only the initiating xylose on proteoglycans (B4GALT7 KO). Although we still lack a clear understanding of finer structural requirements of GAGs presented on their core proteins in the context of total cellular glycome, this fits well with the known importance of heparan sulfate (HS) in the initial attachment of HSV-1 (Fig.2a, b)26. To further dissect the importance of GAG binding determinants we investigated the binding of recombinant HSV-1 gC to our HaCaT KO cells. The use of recombinant HSV-1 gC limited the interactions to a single viral protein known to bind to synthetic GAGs in vitro, similarly to intact HSV-127,31,32. As expected, no binding was detected on B4GALT7 KO cells (Fig.2f), and to further confirm the selectivity for HS we treated HaCaT WT cells with heparinases 1, 2, and 3. Loss of HS completely abolished gC binding suggesting minimal interaction with chondroitin sulfate (CS) or dermatan sulfate (DS) presented on the cell surface (Fig.2f). Minimal changes in cell staining for bound HSV-1 gC after chondroitinase ABC treatment further supported this interpretation. Next, we analyzed a library of glycoengineered CHO cells delineating the GAG biosynthesis pathways (Fig.2g) and quantified gC binding by flow cytometry (Fig.2h)33. This library included selective elimination of HS or CS (Extl2+Extl3 KO and Csgalnact1 KO+Csgalnact2 KO+Chsy1 KO, respectively), reduction in chain polymerization of HS or CS (Ext1 KO+Ext2 KO and Chpf KO, respectively), elimination of HS N-sulfation, also effectively diminishing follow-up O-sulfation (Ndst1 KO+Ndst2 KO), as well as elimination of 4-O sulfation of CS and DS units of CS chains (Chst11 KO+Chst12 KO+Chst13 KO+Chst14 KO). In addition, we used B4galt7 KO and B3gat3 KO cells to truncate all GAGs to the initiating xylose and a short linker trisaccharide, respectively (Fig.2g). In agreement with the HaCaT cell staining data, manipulation of HS synthesis and chain length substantially decreased gC binding showing that the interaction was entirely dependent on HS sulfation and not compensated by the presence of CS (Fig.2h). Accordingly, manipulation of CS synthesis only slightly decreased gC binding independent of the predominant 4-O sulfation (Fig.2h). As expected, truncation to the linker also eliminated gC binding (Fig.2h). To our surprise, some binding was retained upon complete GAG truncation, possibly representing unspecific binding due to gross changes in the glycocalyx. In conclusion, by using cell surface presented GAGs, we were able to identify sulfated HS as the major contributor to HSV-1 gC binding and show that CS sulfation is not necessary for interaction with CS, at least in the presence of HS. More generally, the binding and entry assays show that perturbations in the cellular glycome landscape have immediate effects to early virus-cell interactions, which can be further dissected as demonstrated for the interaction between gC and HS.

To follow up on our binding and entry data, we aimed to investigate the cellular landscape of HSV-1 entry receptors and other surface molecules that may have an impact on the early virus-cell interactions in the different knock out cells. We first quantified the surface expression levels of Nectin 1 and HVEM in WT HaCaT cells and found very low levels of the latter (Fig.2i, Supplementary Fig.3a, b). MGAT1 KO and B4GALT7 KO cells expressed significantly lower levels of Nectin 1 on the cell surface, whereas MGAT4 KO and GCNT1 KO expressed higher levels (Fig.2i). These results correlate well with the virus binding data, and may help explain the altered proportion of virus bound to cells with alterations in N-glycosylation and O-glycosylation pathways. Importantly, the selective effect on entry to MGAT4 KO was not correlated to availability of the receptor.

For B4GALT7 KO, Nectin 1 presentation decreased by approximately 60%, but this does not explain the complete loss of HSV-1 binding, which is likely a combination of a decrease in GAG and protein receptors. While gC mediates early virus-GAG interactions, facilitating subsequent interactions between gD and its cognate protein entry receptors, 3-O-sulfated HS has also been identified as an independent entry receptor for gD34,35. In order to evaluate the potential contribution of 3-O-sulfated HS to HSV-1 entry in skin cells, we performed disaccharide analysis of HaCaT WT and B4GALT7 KO cells, using our recently developed method, which allows detection of 3-O-sulfated HS36 (Fig.2 j, k, Supplementary Fig.4, Supplementary Table2 and 3). Except for hyaluronan, which is synthesized by a distinct family of enzymes, we did not detect any CS or HS disaccharides in B4GALT7 KO cells (Supplementary Fig.4). HaCaT WT cells expressed high levels of 4-O-sulfated or 6-O-sulfated CS, hyaluronan, as well as N-sulfated, N-/2-O-sulfated, N-/2-O/6-O-sulfated, and non-sulfated HS. We detected very low levels of 3-O-sulfated HS disaccharides, demonstrating that usage of these receptors for HSV-1 entry in human keratinocytes is limited. We therefore suggest that Nectin 1 is the most widely available HSV-1 entry receptor for gD in HaCaT keratinocytes.

The disaccharide expression profiles in skin cells provided additional insight into the gC binding data on the CHO cell library. Namely, N-sulfated GAG motifs required for gC binding to CHO cells were abundantly found on human keratinocytes, and likely play a significant role in vivo. On the contrary, 4-O-sulfated CS, abundantly found on skin cells, is unlikely to be a critical receptor for gC, as seen from CHO data.

We next looked into GSLs expressed in skin cells (Fig.3). We saw comparable levels of Nectin 1 on the surface of WT, B4GALT5 KO and ST3GAL5 KO cells (Fig.2i), and yet HSV-1 binding and entry to these cells was markedly decreased. We thus hypothesized that elongated GSLs may help deliver the viral entry receptors to membrane compartments accessible to incoming virus. We used antibodies and toxins recognizing various (glyco)lipid structures to illuminate their distribution in keratinocytes (Fig.3a). Ceramide and glucosylceramide, representing initial steps of GSL synthesis, were predominantly located intracellularly in WT cells, while some ceramide accumulation could be seen in B4GALT5 KO, devoid of elaborate GSLs (Fig.3b). Interestingly, expression of more complex GSLs was heterogeneous, and different cells appeared committed to a specific GSL subtype. Specifically, we detected Gb3 structures, synthesized from lactosylceramide precursor, in both WT, and ST3GAL5 KO cells with clear surface presentation, but not B4GALT
5 KO (Fig.3b, e). In contrast, GM3, the product of ST3GAL5, was only detected in WT cells (Fig.3b). GM3 partially co-localized with intracellular glucosylceramide-positive structures but were primarily expressed on the cell membrane (Fig.3c). Importantly, GM3 was abundantly found on apical cell surfaces accessible to the extracellular environment (Fig.3d). Gb3 and GM3 were expressed in mostly distinct subsets of cells, and a substantial proportion of skin cells remained unlabeled, presumably expressing more elaborate structures (Fig.3e). In conclusion, we show heterogeneous yet regulated expression of different GSLs in distinct cells and within different cellular compartments, which may be relevant for interaction with extracellular virus.

a The cartoon depicts a simplified human glycosphingolipid biosynthetic pathway. Glycolipid structures highlighted in magenta were probed by antibodies or fluorescently labeled toxins. be Cells grown on cover slips were fixed with 4% PFA and stained for different GSL structures. b Confocal micrographs show distribution of different GSLs in HaCaT WT, B4GALT5 KO and ST3GAL5 KO monolayers. Representative of two independent experiments, scale bars are indicated for each set of micrographs. c z-stack maximal intensity projection of HaCaT WT cells labeled with anti-GlcCer and anti-GM3 antibodies. Representative of 2 independent experiments, scale bar is indicated. d HaCaT WT cells labeled with anti-GM3 antibody. An individual z-slice within a stack is shown, with orthogonal cross sections of the z-volume included, and indicate apical expression of GM3. Nuclei are labeled with DAPI (blue). Representative of 2 independent experiments, scale bar is indicated. e The confocal micrograph shows spatially distinct distribution of Gb3 and GM3 GSLs in HaCaT WT, probed by FITC-labeled Shiga toxin B (StxB-FITC), and anti-GM3 antibody, respectively. Representative of 2 independent experiments, scale bar is indicated.

We next investigated late stages of viral replication in KO cells with changes in protein glycosylation capacity and altered viral propagation dynamics. We probed the expression of gD and gB that promote virion envelopment. In WT most of gD signal was confined to the cell surface, partially overlapping with E-cadherin (Fig.4a), while gB primarily localized to the perinuclear compartment and secondary envelopment sites with some surface presentation, consistent with the literature (Fig.4b)37. In contrast, C1GALT1C1 KO, C1GALT1 KO, and MGAT1 KO cells exhibited a weaker and more dispersed gD immunostaining pattern with partial cytoplasmic accumulation suggesting issues with envelope glycoprotein trafficking (Fig.4a). In addition, gB exhibited poorer surface and perinuclear localization and presented in large clusters within the cells (Fig.4b). ST3GAL1 KO cells, which did not exhibit defects in viral propagation dynamics, displayed similar gB staining as WT (Fig.4b), while exhibiting stronger gD signal (Fig.4a). Overall, the results suggest that lack of core 1 O-glycans or mature N-glycans causes defects in viral particle formation due to suboptimal incorporation of viral proteins, which would fit with the observed diminished titers or loss of fitness. In addition, using an HSV-1 strain with GFP-labeled capsid protein VP26 allowed us to observe differences in the localization of viral capsids. The capsids were found in nuclear assembly compartments, outer nuclear rim, and transitioning through the cytosol in WT and ST3GAL1 KO cells. We observed lower numbers of capsid assembly sites in the nucleus and rare association with the outer nuclear rim in C1GALT1C1 KO, C1GALT1 KO and MGAT1 KO cells, with the most pronounced effect in C1GALT1C1 KO (Fig.4a, b).

a HaCaT cells grown on cover slips were infected with MOI10 of HSV-1 K26-GFP and fixed and permeabilized at 14hpi followed by co-staining for HSV-1 gD (magenta) and E-cadherin (cyan). Histograms on the left indicate intensities of gD and E-cadherin signals across the confocal images (marked with black arrowheads). Pixel overlap from the two channels is shown in white. GFP labeled capsid proteins (VP26) are seen in green. Nuclei were stained with DAPI (blue). Stainings of mock-infected cells are included. Scale bar: 10m. Images are representative of two independent experiments. b HaCaT cells grown on cover slips were infected with MOI10 of HSV-1 K26-GFP and fixed and permeabilized at 14hpi followed by staining for HSV-1 gB (magenta). GFP labeled capsid proteins (VP26) are seen in green. Nuclei were stained with DAPI (blue). Scale bar: 10m. Magnified regions of merged images are indicated with dashed white boxes. Images are representative of 2 independent experiments. c HaCaT WT and C1GALT1C1 KO cells were infected with MOI3 or MOI10 of HSV-1 K26-GFP and viral capsids imaged by live microscopy at 14 and 20hpi. Fluorescent images overlaid with bright field images are also shown. Scale bar: 10m. Images are representative of two independent experiments.

We further explored the viral replication dynamics in WT and C1GALT1C1 KO cells by live imaging of GFP-labeled HSV-1. Features seen in thin optical sections (Fig.4a, b) were also reflected in widefield images (Fig.4c). In WT cells at 14 hpi, multiple capsid assembly sites could be seen in the nucleus and capsids were also associating with the nuclear envelope in most cells irrespective of the viral load (Fig.4c). In C1GALT1C1 KO cells less and smaller assembly sites could be seen, and capsids were less frequently associating with nuclear envelope. This association slightly improved later in infection (20hpi), but the capsid production did not intensify, suggesting HSV-1 infection is generally less robust in C1GALT1C1 KO (Fig.4c).

To evaluate the contribution of viral glycans to fitness of progeny virus for early interactions with wild type host cells, we added equal numbers of infectious particles, produced in propagation experiments, to WT keratinocyte monolayers following the previously outlined strategy. No defects in binding or entry were found with virions lacking different glycan structures (Fig.5ae). In fact, virions lacking O-glycan elongation were capable of accelerated binding, despite low viral titers of HSV-1 produced in C1GALT1C1 KO or C1GALT1 KO (Fig. 5). This suggests the observed propagation defects are related to host and viral factors influencing the formation of infectious virions and not their efficiency in establishing a new infection.

Binding (20min (a) or 120min (b) on ice) and entry (5min at 37C after 120min on ice (c)) of HSV-1 produced in different KO cell lines to HaCaT WT. Data is shown as WT-normalized mean+SEM of three independent experiments for eachglycoengineered virus species. Two-way ANOVA followed by Dunnetts multiple comparison test was used on raw data to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Proportion of virus bound at 20min compared to 120min (d) or proportion of virus entered at 5min compared to virus bound at 120min (e) is shown as mean+SEM of three independent experiments for each glycoengineered virus species from a total of 14 experiments. One-way ANOVA followed by Dunnetts multiple comparison test was used to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Source data are provided as a Source Data file for all graphs.

The effect of O-glycosylation on HSV-1 glycoprotein localization, and prior knowledge of O-glycosite modifications compelled us to investigate specific O-glycosites. Eliminating site-specific O-glycosylation may have a more profound effect on protein function than truncation of the O-glycan structure5, 38,39. Therefore, although O-glycan truncation had no deleterious effects on properties of infectious virions, it should not be excluded that individual O-glycosylation sites could play a functional role.

We have previously identified more than 70 O-glycosites on eight out of the 12 HSV-1 surface proteins, including the indispensable fusion machinery comprised of gB, gD, gH, and gL15. Based on available structural data and defined molecular mechanisms, we mutated five out of the identified 21gB O-glycosites and three out of five gD O-glycosites most likely to affect fusion and receptor binding, respectively (Figs.6a, 7a)15. We generated Ser/Thr to Ala substitutions alone or in combination to test cell-cell fusion efficiency using a split luciferase reporter assay as a proxy for viral entry (Supplementary Table4, Supplementary Fig.5). The assay quantifies fusion between two cell types, one (effector) lacking HSV-1 entry receptors and transfected with plasmids encoding the conserved fusion machinery, and the other (target) presenting HSV-1 entry receptors (Fig.6b)40. Each cell type is also transfected with plasmids encoding half of a split luciferase reporter, which upon cell fusion can form a functional enzyme generating luminescence. In addition, we quantified gB and gD surface expression by CELISA40. We used CHO cells, refractory to HSV-1 entry, as effector, and HEK293, an HSV-1 permissive epithelial cell line, as target. We quantified low levels of Nectin 1 and HVEM on HEK293 cells (Supplementary Fig.3c), suggesting other types of receptors and co-receptors may also be involved.

a HSV-1 gB structure (PDB: 2GUM) with select mutated O-glycan acceptor sites indicated within the dashed box. Respective previously identified O-glycans were drawn manually as yellow squares. Domains are numbered in roman numericals according to Heldwein et al., Science 2006. b The cartoon illustrates the principle of split luciferase assay. c Cell surface expression of gB O-glycosite Thr to Ala mutants evaluated by CELISA using mouse anti-gB antibodies. Data is shown as mean absorbance at 450nm+SD of three technical replicates and is representative of three independent experiments. One-way ANOVA followed by Dunnetts multiple comparison test was used to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). d, e Cell-cell fusion activity over 240min using gB O-glycosite Thr to Ala mutants. Data from two independent experiments is shown, where mean normalized luminescence of three technical replicates at each time point is indicated by a dot. Mean values of the two independent experiments are shown as thin lines. Data is normalized to maximum luminescence reading at final time point using WT gB for each experiment. d Data related to gB domain I mutations. e Data related to gB domain V mutations. f Cell-cell fusion activity of gB mutants at t=120min. Data is shown as mean normalized luminescence from two independent experiments. Two-way ANOVA followed by Dunnetts multiple comparison test was used to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). g Average percentages of cell surface expression and fusion efficiency at t=120min from two independent experiments are shown in side-by-side columns. Source data are provided as a Source Data file for all graphs.

a HSV-1 gD structure (PDB: 2C36) with select mutated O-glycan acceptor sites indicated. Positions after removal of signal peptide, often encountered in the literature, are indicated in brackets. Respective previously identified O-glycans were drawn manually as yellow squares. N-terminal region omitted in the crystal structure is drawn as a dashed line. b Cell surface expression of gD O-glycosite mutants evaluated by CELISA. Data is shown as mean absorbance at 450nm +SD of three technical replicates and is representative of three independent experiments. One-way ANOVA followed by Dunnetts multiple comparison test was used to evaluate differences from WT. c Cell-cell fusion activity over 240min using gD O-glycosite mutants. Data from two independent experiments is shown, where mean normalized luminescence of three technical replicates at each time point is indicated by a dot. Mean values of the two independent experiments are shown as thin lines. Data is normalized to maximum luminescence reading at final time point using WT gD for each experiment. d Cell-cell fusion activity of gD mutants at t=120min. Data is shown as mean normalized luminescence from two independent experiments. Two-way ANOVA followed by Dunnetts multiple comparison test was used to evaluate differences from WT. CHO cells stably expressing Nectin 1 (e, f) or HVEM (g, h) were used as target cells to evaluate cell-cell fusion activity using gD O-glycosite mutants. Cell-cell fusion activity over 180min using CHO-Nectin 1 (e) or CHO-HVEM (g) as target. Parental CHO cell line without entry receptors was use for background subtraction. Data is presented as in (c). Cell-cell fusion activity of gD mutants at t=120min using CHO-Nectin 1 (f) or CHO-HVEM (h) as target. Data is shown as mean normalized luminescence from two independent experiments. Two-way ANOVA followed by Dunnetts multiple comparison test was used to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Source data are provided as a Source Data file for all graphs.

For gB single site O-glycan mutants, we focused on the domain directly involved in fusion (I), where we identified three sites on antiparallel beta strands (T169, T267, T268), as well as the arm domain (V) comprised of two alpha helices that undergo structural rearrangements upon fusion, where we found one O-glycosite on each (T690 and T703) (Fig.6a)15, 41,42. All mutations except for T169A and T268A affected gB cell surface expression; T267A and T703A showed moderate reduction, whereas T690A showed increased expression (Fig.6c). T268A exhibited reduced fusion activity, as did T267A. Double or triple mutations in domain I severely decreased surface presentation and fusion activity (Fig.6c, d, f, g). The activity of domain V single mutants did not correlate with changes in surface expression, where T690A exhibited very low fusion activity despite increased surface presentation (Fig.6c, e, f, g). Interestingly, concomitant mutation of T703 (T690A T703A) could partially compensate for the strongly decreased activity of the T690A mutant (Fig.6g).

Though gD does not directly execute fusion, it initiates entry by binding to several different host receptors and compromised interaction with gD would translate to reduced fusion efficiency. For gD, one O-glycan site on the N-terminal tail of the protein (S33 (8)), involved in interaction with both Nectin 1 and HVEM, and two O-glycan sites on an alpha helix undergoing structural changes upon interaction with HVEM (T255 (230) and S260 (235)), were mutated (Fig.7a)15,43,44,45. All mutants maintained close to normal levels of cell surface expression of gD and fusion activity (Fig.7bd). To inspect possible contributions of gD mutations to interactions with distinct HSV-1 entry receptors, we utilized CHO cells overexpressing Nectin 1 or HVEM as target (Fig.7eh, Supplementary Fig.3a, b). Here we saw a modest reduction in Nectin 1-initiated cell-cell fusion, when T255 and S260 were collectively mutated (Fig.7e, f). A more pronounced reduction in cell-cell fusion efficiency was seen in HVEM-mediated entry upon introduction of these mutations (Fig.7g, h).

In conclusion, we identified functionally relevant O-glycan acceptor amino acids on gB, directly executing fusion, but no effects were observed for the initial engager gD in the presence of multiple host entry receptors in HEK293 cells. However, compound mutations in gD affected isolated receptor-mediated entry.

Lastly, we investigated the roles of the specific classes of glycans in direct cell-to-cell spread mediated in part by gE/gI via cell contacts of 2D grown keratinocytes, and unrestricted spread in 3D skin culture, facilitated by tissue destruction and release of free virions (Fig.8a).

a The cartoon illustrates different modes of HSV-1 cell-to-cell spread in 2D glycoengineered HaCaT cell monolayers in the context of a plaque assay, and spread in 3D organotypic skin models. b Plaque diameter on cell monolayers infected with HSV-1 Syn17+ at 48hpi. Data is presented as violin diagrams that include measurements from 3 independent experiments for each KO cell line, with 50 plaques measured for each experiment. Paired WT data includes measurements from 15 independent experiments. The dashed lines within the plots indicate median diameter, whereas the dotted lines indicate the interquartile range. One-way ANOVA followed by Games-Howells multiple comparison test was used to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Source data are provided as a Source Data file. c Cell monolayers grown on cover slips were infected with 200 PFU (MOI<0.0005) of HSV-1 K26-GFP and overlaid with semi-solid media for 48h followed by fixation and staining for HSV-1 gE (magenta). GFP labeled capsid proteins (VP26) are seen in green. Confocal images at two different magnifications were taken to illustrate overviews of plaques (4 combined tiles at 10x, upper panels) as well as gE expression at higher resolution (63x, lower panels). Scale bars for the different magnifications are indicated.

We first performed plaque assays with 2D grown cells, where dissociation of progeny virions is impeded by the dense overlay media, making direct cell-to-cell spread as the predominant mode of spread. Perturbations in core 1 O-glycan biosynthesis resulted in increased plaque size, most notably in C1GALT1 KO and ST3GAL1 KO cells (Fig.8b). Upon plaque immunostaining, WT cells and KO cells exhibiting increased plaque size showed strong gE expression on the cell surface (Fig.8c). In cells lacking N-linked glycan maturation (MGAT1 KO) and those lacking MGAT4B (MGAT4B KO; MGAT5+MGAT4B KO), resulting in reduced 4-antenna branching, we found a markedly reduced cell-to-cell spread (Fig.8b). MGAT1 KO cells showed less pronounced and more punctate gE expression, which may be linked to N-glycosylation sites on gE and help explain the limited spread capacity. Surprisingly, accelerated spread was observed in MGAT4A KO cells, which also contributes to 4-antenna branching, and a similar tendency was observed for MGAT5 KO, devoid in 6-linked antenna branching.

To assess viral spread in tissue, we infected fully developed 3D epidermises built with the glycoengineered cells (Fig.9a). Different spread characteristics were observed, when viral spread was not limited to cell-to-cell contacts mediated by gE/gI complex. In wild type HaCaT skin equivalents trans epidermal lesions were observed at 36 hpi (Fig.9b, c). To avoid selection bias, we generated ten subsequent tissue sections separated by 30microns and scanned whole sections, which allowed to visualize and compare the extent of the viral lesions (Fig.9a, b, Supplementary Fig.6). We identified lesions spanning several sections and measured the cross-section areas corresponding to the central regions of those lesions (Fig.9d). Large lesions were seen in MGAT1 KO tissues, contrasting the small plaques observed in 2D (Fig.9c, d). Most N-glycan branching KO tissues, especially MGAT4A KO and MGAT5+4B KO, permitted only limited spread in the top layers of the epidermis. MGAT4B KO allowed formation of bigger lesions, but the tissue penetrance was limited, which was also the case for tissues with reduced core 1 sialylation (ST3GAL1 KO) (Fig.9c, d). No significant spread defects were noted for tissues with disruptions in GSL and GAG synthesis.

ad Fully differentiated 3D skin models built with glycoengineered cells were infected with HSV-1 Syn17+ for 36h followed by fixation in formalin and embedding in paraffin. a The cartoon illustrates the procedure for evaluating HSV-1 spread in organotypic skin tissues. FFPE tissues were sectioned every 30m for 10 consecutive slices containing two sections each, spanning from the center of the tissue outwards in two directions. b Consecutive sections were stained with a polyclonal FITC-labeled anti-HSV-1 antibody to visualize virus lesions and whole sections imaged with a microscope slide scanner. An example section series is shown with HSV-1 lesions outlined in purple. c Representative lesions were selected from the scanned section series for each KO tissue. Nuclei were labeled with DAPI. d Three lesions spanning several sections were identified for each KO tissue and lesion area measured at the centermost section. Data is shown as mean+SEM with individual measurement values indicated as black dots. One-way ANOVA followed by Dunnetts multiple comparison test was used to evaluate differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Source data are provided as a Source Data file.

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Glycoengineered keratinocyte library reveals essential functions of ... - Nature.com

China has blown the whistle on the potential dangers of what it calls "genetic weapons" that could prove to be an incredibly potent weapon of mass destruction, the Global Times reports. On Monday, October 20, 2023, the Chinese Ministry of State Security released a statement on WeChat warning that a "certain" foreign non-governmental organization (NGO) had recruited Chinese "volunteers" to collect biodiversity distribution data under the guise of biological species research to steal China's species data.

The ministry did not name the countries in question, nor did it offer evidence to support the claim. While not a new claim, the existence of such weapons has long been dismissed by the mainstream scientific community as a "conspiracy theory." In a February 2020 report, the South China Morning Post (SCMP) reports, the Council on Strategic Risks stated that bioweapons as a deterrent were "irrelevant" because no country was safe from the effects of a pandemic.

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China warns a 'certain country' is making ethnic bioweapons - Interesting Engineering

In the rapidly evolving landscape of technology, innovation continues to shape the world we live in. Frontier tech, also known as cutting-edge or emerging technology, is at the forefront of this transformation. These emerging technologies are poised to redefine industries, disrupt traditional business models, and push the boundaries of what's possible. In this article, we will explore the future of frontier tech, examining key trends and their potential impacts on society and the economy, writes Colin Stevens.

Artificial Intelligence (AI)

Artificial Intelligence, often referred to as AI, has already made significant strides, but its future holds even greater promise. The integration of AI into various aspects of our lives, from autonomous vehicles and healthcare diagnostics to customer service chatbots, is set to become more profound. AI will continue to evolve, with more advanced algorithms, natural language understanding, and improved machine learning models. This progress will lead to better decision-making, increased efficiency, and the emergence of new applications we haven't even imagined yet. Quantum Computing

Quantum computing is a game-changer in the world of computing. Unlike classical computers, quantum computers harness the power of quantum bits (qubits) to perform complex calculations exponentially faster. As quantum technology matures, it will revolutionize fields like cryptography, materials science, drug discovery, and optimization problems. The ongoing research in quantum algorithms and hardware will pave the way for practical quantum computers that can tackle some of the world's most challenging problems.

5G and Beyond

The rollout of 5G networks is just the beginning of a new era in wireless communication. Beyond 5G, we will witness the development of 6G technology, which promises even higher data speeds, lower latency, and greater connectivity. These advancements will fuel the growth of the Internet of Things (IoT), enabling more interconnected devices and immersive experiences. 6G may also enable entirely new applications, such as holographic communication and remote surgery.

Blockchain and Cryptocurrency

Blockchain technology, known for its role in enabling cryptocurrencies like Bitcoin, has applications that extend beyond digital currency. Decentralized finance (DeFi), non-fungible tokens (NFTs), and smart contracts are just a few examples of how blockchain is disrupting traditional finance, art, and legal processes. In the future, we can expect to see more widespread adoption of blockchain in various sectors, including supply chain management, voting systems, and identity verification. Biotechnology and Genetic Engineering

Advances in biotechnology and genetic engineering are poised to revolutionize healthcare, agriculture, and even our understanding of life itself. Gene editing techniques like CRISPR-Cas9 offer the potential to cure genetic diseases, create more resilient crops, and address environmental challenges. As our understanding of the human genome deepens, we may also see breakthroughs in personalized medicine and enhanced human capabilities.

Augmented and Virtual Reality

Augmented Reality (AR) and Virtual Reality (VR) technologies are making their way into various industries, including gaming, healthcare, education, and manufacturing. In the future, AR glasses and VR headsets could become more compact, affordable, and versatile, enabling immersive experiences for everyday tasks. The blending of physical and digital worlds through AR will lead to a wide range of applications, from interactive navigation to enhanced training and remote collaboration.

Space Exploration and Commercialization

Space exploration is no longer the exclusive domain of governments. Private companies like SpaceX, Blue Origin, and Virgin Galactic are rapidly advancing the possibilities of commercial space travel and colonization. These developments have the potential to unlock new economic opportunities in space mining, satellite services, and interplanetary tourism.

Challenges and Considerations

While frontier tech holds immense promise, it also raises important ethical, regulatory, and security concerns. As technology continues to evolve, society must grapple with issues like data privacy, cybersecurity, AI bias, and the ethical implications of genetic engineering. Striking a balance between innovation and responsibility will be a key challenge in the future.

The future of frontier tech is a journey into uncharted territory, where the boundaries of what's possible are constantly expanding. As AI, quantum computing, 5G, blockchain, biotechnology, AR/VR, and space exploration continue to advance, they will create new opportunities and challenges for society. Staying informed and engaged with these emerging technologies will be crucial in shaping a future that harnesses their potential for the benefit of all. The path ahead is full of possibilities, and it's up to us to navigate the horizon of frontier tech responsibly and wisely.

About the author: Colin Stevens founded EU Reporter in 2008. He has more than 30 years of experience as a TV producer, journalist and news editor. He is a past president of the Press Club Brussels (2020-2022) and was awarded an Honorary Doctor of Letters at Zerah Business School (Malta and Luxembourg) for leadership in European journalism.

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Navigating the horizon: The future of frontier tech - EU Reporter

As with the best science fiction, Lems novel The Invincible has as much to teach us about our present situations as any futures we may face.

In the grand tradition of H. G. Wells and Jules Verne, Stanisaw Lems The Invincible tells the story of a space cruiser sent to an obscure planet to determine the fate of a sister spaceship whose communication with Earth has abruptly ceased. Landing on the planet Regis III, navigator Rohan and his crew discover a form of life that has apparently evolved from autonomous, self-replicating machines perhaps the survivors of a robot war. Rohan and his men are forced to confront the classic quandary: What course of action can humanity take once it has reached the limits of its knowledge? In The Invincible, Lem has his characters confront the inexplicable and the bizarre: the problem that lies just beyond analytical reach.

The following is literary critic and theorist N. Katherine Hayles foreword to the 2020 edition of Lems classic novel, originally published in Polish in 1964.

Science fiction has famously predicted many of the important technologies of the 20th century: space travel, satellites, the atomic bomb, television, the internet, and virtual reality, to name a few. In The Invincible, Stanisaw Lem predicts another: artificial life. Although speculations about self-reproducing artificial systems date from the 1940s, the scientific field received its name from Christopher Langton only in 1986, more than two decades after the original publication of The Invincible (1964). One of the central controversies in artificial life is whether evolutionary programs and devices are actually alive (the strong version), or whether they merely simulate life (the weak version). Researchers who follow the strong version argue that the processes embedded in software programs such as genetic algorithms are as natural as life itself; what is artificial is the medium in which these processes take place.

The issue prompted Robert Rosen, among others, to speculate about the essential characteristics of life itself, not only as it evolved on Earth in carbon-based life forms but also about the possibility of life-as-it-could-be in exoplanetary systems, arguing that silicon-based artificial life forms may provide insight into these theoretical speculations.

The Invincible presents a fascinating hybridization of these different views. Dr. Laudas hypothesis proposes that a space ship from the Lycran system landed on Regis III millions of years ago; while the biological visitors perished, the automata did not. There then followed an evolutionary struggle between the automata and the planets indigenous life forms, on the one hand, and between the different kinds of automata, on the other. Such a scenario requires that the survive and reproduce mandate that governs life on Earth could also operate on this planet. Lem minimally fulfills the requirement by postulating that the automata could manufacture themselves with modifications dictated by evolutionary processes. Clearly his interest is not in filling out how this might take place (John von Neumann, encountering a similar problem, imagined metal parts floating on a lake that could self-assemble). Rather, Lems focus is on envisioning an artificial life form that won the evolutionary competition on Regis III for profoundly different reasons than did Homo sapiens on Earth.

The effect is achieved by introducing a significant factor that has a monumental impact on evolutionary trajectories: rather than fulfilling their energy needs through ingesting food, the automata on Regis III evolve to use solar power. The smaller the artificial organism, the less energy it needs. Hence the evolutionary driver is toward smaller forms, which overcome not through superior intellect but through swarm intelligence. Lem added to this the ability of the swarm of flies to generate immensely powerful electromagnetic fields, which meant that the tiny automata are not only the evolutionary winners on their planet but a powerful force against the invading humans. Their tiny size notwithstanding, their awesome potential illuminates the profound ambiguity of the works title, which can be taken to refer either to the spaceships proud name or to the swarms of alien automata that threaten it.

From a broader cosmic perspective, the best of human science, engineering, and weaponry may reveal humans to be completely out of our depth, mere kindergarteners bidding for a place in the universes adult civilizations.

Contemporary research in artificial life has validated Lems insight that swarms of artificial beings require only a few simple rules to manifest complex behaviors and hence each member needs to carry only a little cognitive power onboard. Computer simulations that have accurately depicted swarm behaviors in fish, birds, bees, and other biota demonstrate that each individual responds only to the four or five closest to it, with rule sets that take up only a few lines of code. For example, a school of fish swimming to evade a predator is guided by the fish closest to the predator. The direction this most imperiled individual follows determines how the entire school will run as it flashes back and forth, a simple strategy that makes excellent sense, since the fish that has the most to lose will try hardest to escape. Although each fishs behaviors are simple, the collective result nevertheless generates swarm intelligence of considerable complexity.

Decades before these ideas became disseminated within the scientific community, Lem intuited that different environmental constraints might lead to radically different evolutionary results in automata compared to biological life forms. Although on Earth the most intelligent species (i.e., humans) has tended to fare the best, their superior intelligence comes with considerable costs: a long period of maturation; a lot of resources invested in each individual; socialization patterns that emphasize pair bonding and community support; and a premium on individual achievement. But these are not cosmic universals, and different planetary histories might result in the triumph of very different kinds of qualities.

The contrasts between humans and the automata swarm are brought out most poignantly in the scene between Captain Horpach and First Officer Rohan, in which the captain delegates to Rohan the decision whether to put another crew member in grave danger to determine if the missing four men have indeed perished, as seems all but certain, or whether one or more might still be alive. The assumptions that make this gamble even remotely worth taking are revealing: human life is precious; human solidarity depends on the crews belief that everything possible will be done to save them if they are in peril; and every human is unique and therefore uniquely valuable. None of these, of course, holds true for the swarm, whose individual members are virtually identical to one another, with each tiny automaton easily replaced and therefore disposable. Consequently, none is valuable in itself; only the swarm has evolutionary survival value. The contest, then, is not only between different life forms but also between the different values that have resulted from the divergent evolutionary pathways of humans on Earth and the flies on this strange planet. As with Solaris, Lem suggests that assumptions born and bred of Earth may appear hopelessly provincial in light of human encounters with radically different life forms. From a broader cosmic perspective, the best of human science, engineering, and weaponry may reveal humans to be completely out of our de
pth, mere kindergarteners bidding for a place in the universes adult civilizations. The reduction of crew members to infancy when attacked by the flies may be a metaphor for this realization.

Of all the human characters, Rohan has the strongest claim to have encountered the planet on its own terms. He has traversed its terrain with his own feet; he has mixed his sweat with its crevices, valleys, and hills; he has breathed its native atmosphere into his lungs. The insight he gains from his heroic trek therefore commands our respect. When he concludes that not everything everywhere is for us [humans], we are right to hear in this pronouncement Lems own challenge to the anthropocentric assumptions that continue to dominate human ethical frameworks as well as human exploitations of planet Earth. As with the best science fiction, The Invincible has as much to teach us about our present situations as any futures we may face.

N. Katherine Hayles is Distinguished Research Professor of English at the University of California, Los Angeles.

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Stanisaw Lem's Prescient Vision of Artificial Life - The MIT Press Reader

In 2020 the World Health Organization (WHO) publicised a global strategy to accelerate the elimination of cervical cancer as a public health problem. To achieve this goal, the organisation recommends that 90% of girls be fully vaccinated with the human papilloma virus(HPV) vaccine by the age of 15; and that 70% of women be screened with a high-performance test by the age of 35.

For more than four decades, the University of Cape Towns (UCT) Professor Lynette Denny one of the countrys and continents leading cervical cancer researchers has been at the forefront of this fight. During a recent conference, arranged to shine a spotlight on the advances in prostate and cervical cancer research organised by the International Centre for Genetic Engineering and Biotechnology (ICGEB) Professor Denny highlighted the importance of adopting effective screen-and-treat strategies to adequately address the burden of this disease on women.

We started looking at alternative strategies to the pap smear because of our failure to make a dent on [the burden caused by] cervical cancer.

Denny told the audience that her team at the Khayelitsha Cervical Cancer Screening Project (KCCSP), have for years been testing and evaluating alternative, effective methods to the pap smear a well-known procedure used to test for cervical cancer. The KCCSP fast tracks research into cervical cancer prevention and treatment options and provides vital HPV screening and treatment. The clinic is located on the Khayelitsha Community Health Clinic precinct in Site B.

Evaluating alternative strategies

We started looking at alternative strategies to the pap smear because of our failure to make a dent on [the burden caused by] cervical cancer, Denny said.

Cervical cancer is the fourth most common cancer globally, and in 2020 claimed the lives of approximately 350000 women. More than 80% of cases occur in low- to middle-income countries in sub-Saharan Africa, Melanesia, Asia and Southeast Asia. In South Africa, thousands of cases of cervical cancer are diagnosed annually, and the prognosis is seldom good.

Over the past couple of years, Denny and her team have explored several alternative techniques to the pap smear. One, she explained, included evaluating the effectiveness of visual inspection with acetic acid (VIA), both with and without magnification a simple and inexpensive test used to detect cervical pre-cancerous lesions. The next alternative was exploring the use of visual automated evaluation using artificial intelligence and machine learning. This technique shows promise, but, said Denny, further investigation into its effectiveness is still required. The third option was assessing the feasibility of molecular testing using nucleic acid amplification(NAATs). NAATS is a unique HPV DNA test that checks the presence of specific cancer-causing strains, and this technique revealed some pleasantly surprising results.

Using HPV NAATs as the primary screening test prevents cancer and saves more lives than [the use of] VIA cytology as the primary screening test, Denny said. [Therefore] the WHO now encourages the use of HPV NAATs once testing infrastructure is operational and affordable.

Randomised control trial

While on a quest to locate effective, safe and affordable methods to prevent cervical cancer, Denny said the team designed a randomised clinical control trial to investigate the efficacy and feasibility of two specific screen-and-treat strategies. More than 6000 non-pregnant women, who were previously unscreened for HPV, were recruited from clinics in Khayelitsha and participated in the study.

She said a group of women were randomly selected for an HPV [NAATs] screen-and-treat round, in which HPV-positive women received prior therapy; while the second group of participants followed a VIA screen-and-treat approach and HPV-positive women received prior therapy as well.

Performance characteristics of HPV and VIA as the primary screening tests you can see that [with] HPV testing, we [achieved] a 90% sensitivity [to detecting pre-cancerous lesions], 85% specificity, and a 99% negative predictive value. This is very important for national screening programmes.

However, she pointed out that the VIA screen-and-treat results revealed a below 50% sensitivity to detecting pre-cancerous lesions, an 80% specificity and 97% negative predictive value.

If we compare these two screen-and-treat strategies, to reduce the accumulative prevalence of CIN2+ [cervical cancer] by 36 months, we see that we needed 23 patients screened [with HPV NAATS] to prevent one case of CIN2, compared to 50 cases of VIA. This gives you a graphic description, Denny said.

The way forward

However, she added, the right screen-and-treat strategy depends on the environment and the clinics location.

The impact of cervical cancer and mortality must be measured and demonstrated. Without knowing the impact, the process of secondary prevention will fail.

And as scientists and clinicians work towards winning the war on cervical cancer, Denny said there is an urgent need to meticulously evaluate screen-and-treat strategies, while keeping various contexts in the country, the continent and the world top of mind. Further, she said, performing situational analyses to assess these contexts prior to introducing the preferred strategy is essential to ensure that it will benefit the patient and will not break the system.

We need to create a menu of options. What exactly is needed for successful [screen-and-treat] implementation? The ultimate goal is the elimination of cervical cancer as a public health problem, Denny said. The impact of cervical cancer incidence and mortality must be measured and demonstrated. Without knowing the impact, the process of secondary prevention will fail.

Excerpt from:
Screen and treat essential to beat cervical cancer - University of Cape Town News

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The potential of CO2-based production cycles in biotechnology to ... - Nature.com

An anonymous reader shares a report: PFAS stands for "per- and polyfluoroalkyl substances," a family of upwards of 15,000 or more human-made and incredibly durable chemical compounds that have been used in countless industrial and consumer applications for decades. Firefighting foams, waterproof hiking boots, raincoats, nonstick frying pans, dental floss, lipstick, and even the ink used to label packaging -- all can contain PFAS. The compounds are ubiquitous in drinking water and soil, even migrating to Arctic sea ice. PFAS are called forever chemicals because once present in the environment, they do not degrade or break down. They accumulate, are transferred throughout the watershed, and ultimately persist. The quest to reduce the amount of PFAS in the environment is what led me to an industrial park in a southern suburb of Grand Rapids, Michigan. The jar of PFAS concentrate in my hand is part of a demonstration arranged by my hosts, Revive Environmental, during a tour of the company's PFAS destruction site, one of the first in the country to operate commercially and at scale. A few yards in front of me sits the company's PFAS "Annihilator" in a white shipping container.

The Annihilator represents just one of several technologies now vying to break down and destroy PFAS. These span the gamut from established processes like electrochemical oxidation and supercritical water oxidation to emerging techniques relying on ultraviolet light, plasma, ultrasound, or catalyst-driven thermal processes. Some are deployed in field tests. Other companies are actively running pilot programs, many with various divisions of the US Department of Defense and other government agencies. And many other technologies are still undergoing laboratory research. There's good reason for this. Not only are PFAS everywhere around us; they're also in us. Humans can't break down PFAS, and our bodies struggle to clear them from our systems. Studies suggest they're in my blood and yours -- the majority of Americans,' in fact -- and they have been linked to increased risks of kidney and testicular cancer, decreased infant birthweights, and high blood pressure. And that's only what we know about now: researchers continue to grapple with the full impacts of PFAS on human and environmental health.

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The Race To Destroy PFAS, the Forever Chemicals - Slashdot