Parasites of viruses drive superbug evolution

 Researchers have discovered a previously unknown mechanism by which bacteria share their genetic material through virus parasites. The insights could help scientists to better understand how bacteria rapidly adapt and evolve, and how they become more virulent and resistant to antibiotics.

n a study published today in Cell, one of the most prominent peer-reviewed scientific journals in the field of Biochemistry & Molecular Biology, scientists from the National University of Singapore (NUS) and Imperial College London have discovered a new way by which bacteria transmit their genes, enabling them to evolve much faster than previously understood. Led by Assistant Professor John Chen from the Department of Microbiology and Immunology and the Infectious Diseases Translational Research Programme at the NUS Yong Loo Lin School of Medicine (NUS Medicine), the insights could help scientists to better understand how pathogenic bacteria evolve and become increasingly virulent and resistant to antibiotics.

The ability to share genetic material is the major driver of microbial evolution because it can transform a benign bacterium into a deadly pathogen in an instant. Phages, the viruses of bacteria, can act as conduits that allow genes to transfer from one bacterium to another by a process known as genetic transduction. Currently, there are three known mechanisms of transduction: generalised, specialised, and lateral. Lateral transduction was also discovered by the same groups of researchers in 2018, and it is at least one thousand times more efficient than the next most powerful mechanism, generalised transduction.

The new process is termed lateral cotransduction, and the architects behind this new frequency and speed in bacterial evolution are the Staphylococcus aureus pathogenicity islands (SaPIs), which are selfish DNA elements that exploit and parasitise phages and are commonly found integrated into the chromosomes of S. aureus isolates. S. aureus is a type of bacteria that can cause Staph infections in humans and animals. While it primarily manifests as skin infections, it can become life-threatening if it spreads to the bloodstream and infects organs, bones, or joints.

Professor José R. Penadés from the Department of Infectious Diseases, and Director for the Centre for Bacterial Resistance Biology at Imperial College London, said, "This breakthrough sheds light on a novel pathway through which bacteria evolve. Given the alarming surge of antibiotic-resistant superbugs, comprehending the mechanisms driving bacterial evolution becomes increasingly critical."

This newly discovered process, lateral cotransduction, rivals lateral transduction in terms of efficiency but surpasses the latter in versatility and complexity. While lateral transduction is only known to occur when dormant phages within bacterial genomes become reactivated and initiate reproduction in the lytic cycle, lateral cotransduction can occur during the reactivation process and the infection of new bacterial cells.

Additionally, unlike phages that sacrifice their genes to transmit bacterial host DNA, SaPIs can transfer themselves completely intact with bacterial DNA through lateral cotransduction. This remarkable capability enables them to perpetually repeat the process, making them significantly more potent and efficient in transmitting bacterial genes.

Asst Prof Chen said, "Through the study, we have demonstrated that bacteria can evolve much faster than we understood. While genetic transduction has always been the exclusive domain of phages, in an unexpected twist of irony, our research has shown that parasites of the most prolific parasites on the planet (the phages) are probably the most powerful and efficient transducing agents currently known."

The rise of superbugs has called for new ways to treat antibiotic-resistant strains. One such method that has gained traction in recent years is phage therapy, which involves the use of phages to eliminate harmful bacteria in infections and diseases. However, instead of just fighting bacteria, some therapeutic phages could turn out to be the unwitting accomplices of SaPIs or other related elements capable of lateral cotransduction.

According to Prof Penadés, "This process likely occurs in various other bacterial species as well. This groundbreaking finding marks a paradigm shift in our understanding of bacterial evolution and will immensely influence the ways we combat antibiotic resistance."

"They (phages) could be used to destroy bacteria in the short term but end up spreading harmful genes to other cells in the long term, which could prove to be disastrous. With this new way of understanding the evolutionary mechanisms of disease-causing organisms, it is important for therapeutic phages to be carefully vetted before they are used for therapy," said Asst Prof Chen.

Journal Reference:

1. Melissa Su Juan Chee, Ester Serrano, Yin Ning Chiang, Joshua Harling-Lee, Rebecca Man, Rodrigo Bacigalupe, J. Ross Fitzgerald, José R. Penadés, John Chen. Dual pathogenicity island transfer by piggybacking lateral transduction. Cell, 2023; 186 (16): 3414 DOI: 10.1016/j.cell.2023.07.001

https://www.sciencedaily.com/releases/2023/08/230804123751.htm

Gene-editing technology that eliminates EV-A71 RNA viruses

 A team of scientists from A*STAR's Genome Institute of Singapore (GIS) and the Yong Loo Lin School of Medicine, National University of Singapore (NUS Medicine) has made an important breakthrough in the fight against RNA viruses that cause human diseases and pandemics.

Their research shows that the CRISPR-Cas13 editor delivered by adeno-associated virus (AAV) can directly target and eliminate RNA viruses in laboratory models. AAV are delivery vehicles derived from small viruses that naturally infect humans.They are clinically approved for use in gene therapy drugs which are used to treat diseases such as spinal muscular atrophy, Duchenne muscular dystrophy, and haemophilia.

The EV-A71 virus is the cause of the hand, foot, and mouth disease, and in severe cases, can lead to nervous system disease and death. To treat the viral infection, the team turned to CRISPR-Cas13, an RNA-editing technology that alters RNA in a cell.

CRISPR-Cas13 edits RNA and opens therapeutic avenues to a wide range of diseases that are untreatable by the Nobel Prize-winning CRISPR-Cas9, which edits DNA. CRISPR-Cas13 is programmed by guide RNAs (gRNAs) to target specific RNA sequences. Upon binding to these RNA sequences, the CRISPR-Cas13 cuts the RNA target into pieces, inactivating the RNA. CRISPR-Cas13 could also be utilised for RNA-editing, where a specific RNA sequence is changed to another sequence within the cell.

In this recent work, the team of scientists first developed the Cas13gRNAtor computational programme to design CRISPR gRNAs that cut viral RNA across different viral strains. They show that CRISPR-Cas13 treatment potently reduces viral burden, with less than 0.1% of the viruses remaining in previously infected cells.

Importantly, the research findings show that the AAV-CRISPR-Cas13 therapy clears the EV-A71 infection and prevents organ damage and mortality.

"This is a stunning demonstration that one dose of CRISPR-Cas13 can mean a difference between life and death. We are building on this research to develop further life-changing nucleic acid therapeutics." said Dr Chew Wei Leong, Associate Director and Principal Scientist at A*STAR's GIS.

Associate Professor Justin Chu from NUS Medicine's Department of Microbiology and Immunology and Infectious Diseases Translational Research Programme added, "This amazing study has helped to unlock the new frontiers in antiviral strategies by using AAV-CRISPR-Cas13 to combat human enteroviruses, paving the way for potential therapeutics against viral diseases."

Professor Liu Jian Jun, Acting Executive Director of A*STAR's GIS said, "The CRISPR technology allows the rewriting of the genetic code in almost any organism. This joint research with NUS is an extremely important development which can potentially treat many diseases caused by RNA viruses, and open many avenues for further therapeutic solutions."

These findings demonstrate a therapeutic development pipeline for antiviral AAV-CRISPR-Cas13 against potentially deadly RNA virus infections. Further therapeutic development could bring this technology towards treating human RNA viruses in the clinic. This research was published on 28 June 2023, in eBioMedicine, part of The Lancet Discovery Science.

Journal Reference:

1. Choong Tat Keng, Thinesshwary Yogarajah, Regina Ching Hua Lee, Irfan Bin Hajis Muhammad, Bing Shao Chia, Suraj Rajan Vasandani, Daryl Shern Lim, Ke Guo, Yi Hao Wong, Chee Keng Mok, Justin Jang Hann Chu, Wei Leong Chew. AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice. eBioMedicine, 2023; 93: 104682 DOI: 10.1016/j.ebiom.2023.104682

https://www.sciencedaily.com/releases/2023/08/230802105830.htm

Derek Lowe: Too Many Bad Clinical Trials

 Looking behind the scenes at clinical trial data can be disturbing, as a paper published in 2020 in the journal Anaesthesia shows, and as this new follow-up article continues to show. The author of the 2020 paper, John Carlisle, is an expert in dealing with such trial data, and several years ago he worked his way through 526 manuscripts that reported doing a real randomized clinical trial (2017-2020). For 153 of these, he was able to get access to the actual data behind these, the (anonymized) “individual participant data” (IPD). Initially, he requested these numbers from the authors of manuscripts where the data looked problematic, then in 2019-2020 he began requesting these automatically from authors submitting from the countries sending in the most manuscripts overall, namely Egypt, China, India, Iran, Japan, South Korea, and Turkey.

He believes that 44% (73) of the 153 papers with IPD available had false data in them, but keep in mind that this ranges from unintentional mistakes (duplicated data entries and the like) up to what seems to be outright fabrication (Carlisle has seen this before!) He was able to pick out false data in 6 of the other 373 manuscripts. As a percentage of total manuscripts, China and Egypt were by far the worst offenders for false data. Carlisle uses the phrase “zombie trials” for ones where the problems were great enough that the trial would have (or should have!) been retracted if its flaws were only noticed after publication. 43 of the manuscripts fell into this category, and 20 of these were from China. Trials can sink to this level either by dishonesty or incompetence, and let’s not rule out both at once, either.

Digging this stuff out is not light work:

There were some zombie trials that I did not detect when I initially inspected their spreadsheets, even though I spent hours editing two of these after they had been provisionally accepted for publication. The spreadsheet for one trial contained repeated sequences of numbers in columns for one group, which I had not initially noticed, and I did not notice until the fourth revision of that paper. The authors explained that an uncredited medical student had made the data up for them. We published a different trial in March 2019, which we subsequently identified as zombie (trial number 198; see online Supporting Information, Appendix S1). Anaesthesia had provisionally accepted it for publication in September 2018 and I had edited it to its published version after five revisions. Anaesthesia had not requested individual patient data, but did so the same month it was published, when analyses of individual patient data became routine. The spreadsheet had 536 rows and 91 columns, which exhibited multiple copied segments that became apparent after I ordered the rows by age, height and weight.

Carlisle notes that “you can gauge whether your threshold for disbelief matched mine” by inspecting the detailed list here. But he believes that his findings are (unfortunately) representative of the broader medical literature; there’s no reason to think that submissions to Anaesthesia are outliers. The new follow-up piece at Nature linked in the first paragraph agrees with that assessment, and shows that there are efforts underway in several other fields to do similar estimates (such as the work of Ben Mol). The results so far are not cheerful - that is, they line up well with Carlisle’s results. Reported trials from Iran, Egypt, Turkey, and China feature prominently in these efforts as well.

What to do? You’ll see many suggestions in the Nature piece. A big one is tightening up criteria for considering a published trial in reviews and meta-analyses, since these things contaminate those papers in turn. There’s also having journals ask (much) more often for anonymized trial data as part of the manuscript review process, although that means a lot more work in the editorial process. And back at the source, there’s trying to get countries with data-trust problems to alter the policies that lead to this stuff in the first place. Egypt, for example, has recently passed a law trying to regulate clinical trials in that country. Universities and other employers who just count up publications or trials as a criterion for promotion are a big part of the problem, as you’d imagine.

What we shouldn’t do is just agree not to think about it too much, and sadly that’s what people who are trying to fix these problems can encounter. Some authors never respond when asked for more data, some editors don’t seem too worked up about pushing for retractions, some review authors don’t care so much about revising their work when it turns out to include flawed studies, and some institutions don’t seem to think that investigating problematic work is much of a priority, either. But we should take the time, and we should give a damn now and then.

https://www.science.org/content/blog-post/too-many-bad-clinical-trials

Derek Lowe: A New Mode of Cancer Treatment

 

Let’s talk about a really interesting new paper in Cell Chemical Biology, which is also getting some pickup in the wider press. It’s about a molecule designated AOH1996, which seems to have a unique mode of action in tumor cells, one that might make it more more selective for those as compared to normal ones.

The key target here is a protein called PCNA (from its old name of “proliferating cell nuclear antigen”). If you look at that link, you’ll see its very appealing structure in the cell - three PCNA proteins come together and encircle a DNA strand, and the outside of this ring has binding spots for a lot of other proteins involved in replication (such as DNA polymerase epsilon), in DNA repair (ditto), in things like making epigenetic signaling marks on DNA-histone complexes, and in chromatin remodeling in general. That makes it sound like a pretty important protein, and so it is - PCNA is absolutely essential in replication, and it was first noticed as something that got expressed heavily in cell nuclei during DNA synthesis as a runup to cell division.

And this brings up a fundamental conflict in the life of a cell. DNA is constantly being transcribed (read off into RNA for the later translation to proteins as well as other uses), and there are huge numbers of proteins and processes involved in that. Replication for cell division is a whole other process, one that also involves a huge number of proteins descending onto the DNA (every single bit of it, as the entire genome is copied for the daughter cell to come). You can imagine that these two mighty networks of DNA-associated machinery might bang into each other from time to time, and so they do - this review, entitled “Transcription as a Threat to Genomic Integrity”, will take you through a lot of this. From a strict cell-replication standpoint, it would be better if the DNA was locked up in a vault where nothing could mess with it until it came time to make a copy, but that’s impossible: day-to-day (and minute-to-minute) cell biology requires constant messing around with that same DNA. Winding and unwinding around histone proteins, huge transcriptional complexes landing on particular sequences and starting the synthesis of mRNA molecules, transcription factors sitting around on their preferred sequences and blocking/promoting such readouts - the activity around a cell’s DNA makes beehives look pretty laid-back.

Cancer cells, many of whom are constantly replicating, are under particular stress in this regard, and a lot of chemotherapy drugs are specifically interfering with replication and DNA repair pathways. The discovery of a “cancer-associated PCNA” (caPCNA) isoform made that a particularly interesting target. Back about ten years ago there were several reports of small molecules and cell-penetrating peptides as PCNA ligands, and these continue to show up in the literature (although as far as I know, none of them have progressed further). But a peptide in this area has made it through Phase I in the clinic. PCNA continues to be targeted whenever some new mode of action comes along that might make it actionable.

The current molecule is a traditional direct small molecule binder that is selective for caPCNA over the regular type, which is a very attractive advantage to explore. The team behind it has been working on it for several years now to validate that mechanism, and the new paper linked first above is their report of going all the way into animal models. AOH1996 is a very unremarkable-looking molecule - to be honest, it looks like the sort of stuff that you used to see in old combinatorial chemistry libraries in the late 90s and early 2000s, a couple of aryl-rich groups strung together with amide bonds. It’s certainly not going to be the most soluble stuff in the world, but they seem to have been able to formulate it. But I’m definitely not going to make fun of any chemical structure that works!

Mechanistically, it binds to a particular region that’s different in caPCNA (as shown by some very nice structural biology results), and this binding stabilizes the protein’s interaction with the largest subunit (RPB1) of RNA polymerase II - in fact, it stabilizes it so thoroughly that the RPB1 protein gets targeted for degradation by the cellular housekeeping machinery! Binding of AOH1996 also weakens its association with actively-transcribed chromatin regions, and this causes accumulation of double-stranded DNA breaks DSBs), which is just the kind of thing you want to mess up a cancer cell’s replication process (and just the kind of thing you don’t want happening to other cell types). This is an example of deliberately amplifying the transcription/replication conflict - things get so messed up that the transcriptional complex has to be cleared.

The new paper shows preclinical toxicity testing in two species (mice and dogs), which is what you need to get to human trials. It seems to pass those very well, with no signs of trouble at 6x the effective dose in either species. And if you were throwing DSBs all over the place in normal tissues, believe me, you’d see tox. It is clean in an Ames test, for example. As for efficacy, in cell assays the concentration needed for 50% growth inhibition across 70 different cancer cell lines averaged around 300nM, while it showed no toxic effects on various non-cancer lines up to 10 micromolar (at least a 30x window). The affected cells show cell-cycle arrest, replication stress, apoptosis, and so on. And application of AOH1996 along with other known chemotherapy agents made the cells much more sensitive to those, presumably because they couldn’t deal with those on top of the problems that AOH1996 was already causing.

It also shows growth arrest in xenograft tumors in mouse models, with a no-effect dose at least six times its effective dose, and combination therapy with a topoisomerase inhibitor showed even more significant effects. The compound has entered a Phase I trial in humans on the basis of the above data, and I very much look forward to seeing it advance to Phase II, where it will doubtless be used in combination with several existing therapies. I hope that human cancers will prove vulnerable to this new mode of attack in the clinic, and that they are not able to mutate around it with new forms of caPCNA too quickly, either. The comparison with the peptide agent mentioned above will be especially interesting, too. There’s only one way to find out - good luck to everyone involved!

https://www.science.org/content/blog-post/new-mode-cancer-treatment

NIH researchers say agency seeks to block unionization effort

 Early-career researchers who work in labs operated by the U.S. National Institutes of Health (NIH) have hit a roadblock on their way to unionizing. In June, a group of postdocs, graduate students, and postbaccalaureate researchers asked the agency that oversees the certification of unions by federal employees, the Federal Labor Relations Authority (FLRA), for permission to hold an election to determine whether early-career researchers at NIH are in favor of forming a union. But on Monday, the organizers say, they were told that NIH had submitted paperwork to FLRA arguing that many of the union’s potential members are not employees and don’t have standing to form a union.

“The NIH treats postdocs like employees when it suits them and then tries to claim we do not have the same rights enjoyed by other workers at the NIH when they think it serves their interests,” says Marjorie Levinstein, a neuropharmacology postdoc at the National Institute on Drug Abuse (NIDA) and an organizer for the proposed union, called NIH Fellows United. “But the fact is, the NIH controls postdocs’ terms of employment, working conditions, intellectual property and more, and we deserve a say in determining our rights at work.”

In an email to Science, an NIH spokesperson declined to confirm the agency’s position. “The NIH is unable to comment on matters that are pending before the Federal Labor Relations Authority. The NIH adheres to all procedures and obligations under the Federal Service Labor-Management Relations Statute.”

Union representatives also declined to send Science the entirety of the document NIH submitted to FLRA. But they did share one excerpt, which argued that most of the potential union’s 4800 likely members cannot be considered employees. “The Agency is of the view that individuals in all categories appointed under the CRTA [Cancer Research Training Award] and IRTA [Intramural Research Training Award] authorities, i.e., all categories other than Clinical Fellows, Research Fellows, and Senior Research Fellows, are not employees under the Statute,” the document states, according to the union.

Another union organizer expressed annoyance with NIH’s apparent maneuver. “I'm not surprised they're taking this avenue,” says Emilya Ventriglia, a neuroscientist who has worked full time at NIDA for the past 3 years as a postbaccalaureate fellow and is starting a Ph.D. at Brown University later this year. “But it is frustrating because at the NIH nothing would get done if it weren't for us. We are the ones with boots on the ground doing the labor—in the bench, on the computer, at conferences. So we are workers and we do have a right to collective bargaining.”

Ventriglia acknowledges that the agency’s intramural training program puts on “workshops and things like that. So, there is an air of supporting growth and … different aspects of training us to be scientists,” she says. “But I do fail to see how that negates us being workers at all.”

Universities have made a similar argument—that early-career researchers aren’t employees—in cases adjudicated by the National Labor Relations Board (NLRB), FLRA’s sibling agency that oversees the certification of unions by private sector employees. In 2018, for instance, Columbia University opposed a petition by postdocs to form a union, arguing that “such individuals are research trainees who require additional mentoring and training on how to conduct independent research.” But NLRB dismissed that argument, opening the door for the country’s first postdoc union at a private university. (Other postdoc unions had previously been established at public universities, but their formation was governed by state labor laws.)

NLRB has also weighed in on whether graduate students who do labor for a private university can be considered employees. After a series of conflicting rulings over recent decades, it ruled most recently in 2016 that graduate students at Columbia University could be considered employees with a right to unionize, establishing the current precedent.

FLRA, however, hasn’t yet ruled on the legal standing of unionization efforts by postdocs and graduate students at NIH or other federal agencies. As an independent agency, “it is not bound by National Labor Relations Board precedent or the decisions by analogous state public sector labor relations agencies,” says William Herbert, a collective bargaining scholar at Hunter College, City University of New York. “However, precedent from those agencies can be persuasive authority for the FLRA.”

Union representatives say the ball is in the agency’s court to decide what happens next, but it’s likely that FLRA will opt to hold a hearing to give both sides a chance to voice their arguments. “We’ve been working with a legal team to make it an effective case,” Ventriglia says.

https://www.science.org/content/article/nih-researchers-say-agency-seeks-to-block-unionization-effort

Italy's lower house approves 'right to be forgotten' law for cancer survivors

 An Italian law giving cancer survivors the right not to declare their past condition to banks and insurance agencies, shielding them from discrimination, has been approved by the lower house of parliament.

The so-called "right to be forgotten" (RTBF) bill was passed on Thursday with a unanimous vote, indicating rare cross-party support for a reform that will need to be also voted by the upper Senate house before entering into force.

Recovering cancer patients in Italy have been facing numerous challenges due to their medical history as they are more likely to be refused loans or insurance and even be excluded from adoption procedures.

This is "a law that restores dignity and hope to many people," Marco Furfaro from the opposition Democratic Party, one of the sponsors of the bill, said on X, the social media platform formerly known as Twitter.

In Italy there are one million people who recovered from cancer, Foreign Minister Antonio Tajani also said on X.

"Those who have defeated cancer cannot be branded for life as second-class citizens," he said.

In June, Prime Minister Giorgia Meloni said her right-wing government would offer the necessary support to make sure that the RTBF law gets approved in "the shortest time possible."

The proposals would allow recovering cancer patients not to share information about their previous condition with financial institutions, or adoption authorities, provided that 5-10 years had passed since the successful end of their treatment.

Similar laws are already in place in France, Luxembourg, the Netherlands, Belgium and Portugal, according to the Italian Medical Oncology Association (AIOM).

https://www.yahoo.com/lifestyle/italys-lower-house-approves-forgotten-102913689.html

'Zelenskiy vows more work to fight graft, names no targets'

 Ukrainian President Volodymyr Zelenskiy on Saturday promised a fresh round of institutional "cleaning," a reference to his ongoing campaign to impress Western partners by showing Kyiv has moved on from a history of deep-rooted graft.

"Next week will be a continuation of our work on cleaning state institutions of those who tried to drag from the past all those old habits, old schemes that weakened Ukraine for a very long time, for decades," he said in his nightly address.

Zelenskiy gave no details about who might be a target. He has recently expressed indignation at corruption uncovered during an audit of Ukraine's military recruitment centers, but is anxious to root out graft in general as he presses to join NATO and the European Union.

On Saturday he vowed "no more old formats" in Ukraine, where he said some sought to reap rewards by putting the state and others at their own service. "Whoever a person is, a military commissar, a deputy, or an official, everyone must work only for the interests of the state," he said.

https://uk.news.yahoo.com/zelenskiy-vows-more-fight-graft-225025912.html