Pemphigus and Fogo Selvagem

Some crop pathogens use a clever trick to multiply and spread infection: they hijack the plant's cellular plumbing. In a new study, researchers unveil a class of bacterial proteins that fold into a straw-like shape and insert themselves into the plant cell membrane, allowing the inside of the leaf to become waterlogged. The researchers also figured out a possible way to block the water channel proteins and prevent infection. Many of the bacteria that ravage crops and threaten our food supply use a common strategy to cause disease: they inject a cocktail of harmful proteins directly into the plant's cells. For 25 years, biologist Sheng-Yang He and his senior research associate Kinya Nomura have been puzzling over this set of molecules that plant pathogens use to cause diseases in hundreds of crops worldwide ranging from rice to apple trees. Now, thanks to a team effort between three collaborating research groups, they may finally have an answer to how these molecules make plants sick -- and a way to disarm them. The findings appear Sept. 13 in the journal Nature. Researchers in the He lab study key ingredients in this deadly cocktail, a family of injected proteins called AvrE/DspE, that cause diseases ranging from brown spot in beans and bacterial speck in tomatoes to fire blight in fruit trees. Ever since their discovery in the early 1990s, this family of proteins has been of great interest to those who study plant disease. They are key weapons in the bacterial arsenal; knocking them out in a lab renders otherwise-dangerous bacteria harmless. But, despite decades of effort, many questions about how they work remain unanswered. Researchers had identified a number of proteins in the AvrE/DspE family that suppressed the plant's immune system, or that caused dark water-soaked spots on a plant's leaves -- the first telltale signs of infection. They even knew the underlying sequence of amino acids that linked to form the proteins, like beads on a string. But they didn't know how this string of amino acids folded into a 3D shape, so they couldn't easily explain how they worked. Part of the problem is that the proteins in this family are huge. Whereas an average bacterial protein might be 300 amino acids long; AvrE/DspE-family proteins are 2000. Researchers have looked for other proteins with similar sequences for clues, but none with any known functions showed up. "They're weird proteins," He said. So they turned to a computer program released in 2021 called AlphaFold2, which uses artificial intelligence to predict what 3D shape a given string of amino acids will take. The researchers knew that some members of this family help the bacteria evade the plant's immune system. But their first glimpse of the proteins' 3D structure suggested an additional role. "When we first saw the model, it was nothing like what we had thought," said study co-author Pei Zhou, a professor of biochemistry at Duke whose lab contributed to the findings. The researchers looked at AI predictions for bacterial proteins that infect crops including pears, apples, tomatoes and corn, and they all pointed to a similar 3D structure. They appeared to fold into a tiny mushroom with a cylindrical stem, like a straw. The predicted shape matched up well with images of a bacterial protein that causes fire blight disease in fruit trees that was captured using a cryo-electron microscope. From the top down, this protein looked very much like a hollow tube. Which got the researchers thinking: Perhaps bacteria use these proteins to punch a hole in the plant cell membrane, to "force the host for a drink" during infection, He said. Once bacteria enter the leaves, one of the first areas they come across is a space between cells called the apoplast. Normally, plants keep this area dry to enable gas exchange for photosynthesis. But when bacteria invade, the inside of the leaf becomes waterlogged, creating a moist cozy haven for them to feed and multiply. Further examination of the predicted 3D model for the fire blight protein revealed that, while the outside of the straw-like structure is water-resistant, its hollow inner core has a special affinity for water. To test the water channel hypothesis, the team joined forces with Duke biology professor Ke Dong and co-first-author Felipe Andreazza, a postdoctoral associate in her lab. They added the gene readouts for the bacterial proteins AvrE and DspE to frog eggs, using the eggs as cellular factories for making the proteins. The eggs, placed in a dilute saline solution, quickly swelled and burst with too much water. The researchers also tried to see if they could disarm these bacterial proteins by blocking their channels. Nomura focused on a class of tiny spherical nanoparticles called PAMAM dendrimers. Used for more than two decades in drug delivery, these dendrimers can be made with precise diameters in a lab. "We were tinkering with the hypothesis that if we found the right diameter chemical, maybe we could block the pore," He said. After testing different sized particles, they identified one they thought might be just the right size for jamming the water channel protein produced by the fire blight pathogen, Erwinia amylovora. They took frog eggs engineered to synthesize this protein and doused them with the PAMAM nanoparticles, and water stopped flowing into the eggs. They didn't swell. They also treated Arabidopsis plants infected with the pathogen Pseudomonas syringae, which causes bacterial speck. The channel-blocking nanoparticles prevented the bacteria from taking hold, reducing pathogen concentrations in the plants' leaves by 100-fold. The compounds were effective against other bacterial infections too. The researchers did the same thing with pear fruits exposed to the bacteria that cause fire blight disease, and the fruits never developed symptoms -- the bacteria didn't make them sick. "It was a long shot, but it worked," He said. "We're excited about this." The findings could offer a new line of attack against many plant diseases, the researchers said. Plants produce 80% of the food we eat. And yet more than 10% of global food production -- crops such as wheat, rice, maize, potato and soybean -- are lost to plant pathogens and pests each year, costing the global economy a whopping $220 billion. The team has filed a provisional patent on the approach. The next step, said Zhou and co-first-author Jie Cheng, a Ph.D. student in Zhou's lab, is to figure out how this protection works, by getting a more detailed look at how the channel-blocking nanoparticles and the channel proteins interact. "If we can image those structures we can have a better understanding and come up with better designs for crop protection," Zhou said. This work was supported by the National Institute of Allergy and Infectious Diseases (1R01AI155441) and the National Institute of General Medical Sciences (GM145026), both at the National Institutes of Health, Duke Science and Technology, and the Howard Hughes Medical Institute.

Many sections of the non-coding region of the human genome play a key role in regulating gene activity. But the relationship between non-coding mutations and cancer risk has been a mystery. New research sheds light on that mystery, providing clues that may link mutations to epigenetic changes, and may point to potential drug targets to reduce risk for people born with certain genetic mutations. For many years, the human genome was viewed as a book of life in which sections of great eloquence and economy of expression were interspersed with vast stretches of gibberish. The legible sections contained the code for making cell proteins; the other regions, representing about 90% of the entire genome, were dismissed as "junk DNA," having no discernable purpose. Research has taught scientists otherwise. Far from being useless filler, many non-coding sections have been shown to play a key role in regulating gene activity -- increasing or decreasing it as needed. For cancer scientists, this has raised questions of its own: if mutations in coding regions cause cells to make flawed proteins, what do mutations in non-coding regions do? How does a mutation in the hinterlands of the genome -- in areas devoid of genes -- contribute to cancer? Given that non-coding regions are involved in gene regulation, researchers have hypothesized, naturally, that mutations in these zones play havoc with gene activity in ways conducive cancer. Study after study, however, has found this generally not to be the case, leaving the biological impact of non-coding mutations something of a mystery. Thinking locally In a new paper in the journal Nature Genetics, Dana-Farber investigators provided an answer. They did so by the scientific equivalent of thinking locally -- narrowing the scope of their investigation to the specific DNA in which non-coding mutations occur. They found that in the overwhelming number of cases examined, such mutations have an epigenetic effect -- that is, they change how tightly the DNA at those locations is wrapped. That, in turn, affects how open those locations are to binding to other sections of DNA or certain proteins, all of which can influence the activity of genes involved in cancer. The discovery reveals, for the first time, a pervasive biological mechanism by which non-coding mutations can influence cancer risk. It also opens the way to therapies that, by disrupting that mechanism, can reduce at-risk people's likelihood of developing certain cancers. "Studies have identified an enormous number of mutations across the genome that are potentially involved in cancer," says Alexander Gusev, PhD, of Dana-Farber, the Eli and Edythe L. Broad Institute and Brigham and Women's Hospital, who co-authored the paper with Dana-Farber's Dennis Grishin, PhD. "The challenge has been understanding the biology by which these variations increase cancer risk. Our study has uncovered an important part of that biology." Does mutation change expression? To identify inherited, or germline, mutations that increase a person's risk of developing cancer, investigators conduct what are known as genome-wide association studies, or GWASs. In these, researchers collect blood samples from tens or hundreds of thousands of people and scan their genomes for mutations or other variations that are more common in people with cancer than in those without the disease. Such tests have yielded thousands of such mutations, but only a small percentage of them are in coding portions of the genome that are relatively easy to link to cancer. Breast cancer is one example. "More than 300 mutations have been identified that are associated with an increased risk of the disease," Gusev states. "Less than 10% of them are actually within genes. The rest are in 'desert' regions, and it hasn't been clear how they influence disease risk." To try to make that connection, researchers gather two sets of data: one, GWAS data showing mutations in a specific type of cancer; and two, data on another genomic feature of that cancer type -- such as an abnormally high or low level of activity in certain genes. By looking for areas of overlap between these data sets, in a process called colocalization, researchers can determine whether the mutations correspond with a rise or fall in the activity of those genes. If such a relationship exists, it would help explain how non-coding mutations can lead to cancer. Despite a massive investment in this type of research, however, colocalization studies have turned up very few such correspondences. "The vast number of mutations identified by GWASs have been found to have no colocalizing gene at all," Gusev remarks. "For the most part, non-coding mutations associated with cancer risk don't overlap with the changes in gene expression [activity] documented in public data sets." Looking closer to home With that route looking increasingly unenlightening, Gusev and Grishin tried another, more fundamental approach. Instead of beginning with the premise that non-coding mutations might influence gene expression, they asked how they alter their home environment -- whether they affect the coiling of DNA in their immediate vicinity. "We hypothesized that if you look at the effect of these mutations on local epigenetics -- specifically, whether they caused nearby DNA to be wound more tightly or loosely -- we'd be able to detect changes that wouldn't be evident in expression-based studies," Gusev relates. Their reasoning: "If a mutation has an effect on disease, that effect will probably be too subtle to capture at the level of gene expression but may not be too subtle to capture at the level of local epigenetics -- what is happening right around the mutation," Gusev says. It's as if previous studies sought to understand how a brush fire in California could affect the weather in Colorado, whereas Gusev and Grishin wanted to see its effect on the hillside where it began. To do that, they performed a different type of overlay study. They took GWAS data on cancer-related mutations and data on epigenetic changes in seven common types of cancer and examined whether -- and where -- they intersected. The results came in stark contrast to those from colocalization studies. "We found that whereas most non-coding mutations don't have an effect on gene expression, most of them do have an impact on local epigenetic regulation," Gusev states. "We now have a basic biological explanation of how the vast majority of cancer-risk mutations are potentially linked to cancer, whereas previously no such mechanism was known." Using this approach, the researchers created a database of mutations that can now be linked to cancer risk by a known biological mechanism. The database can serve as a starting point for research into drugs that, by targeting that mechanism, can lower an individual's risk of developing certain cancers. "If we know, for example, that a certain transcription factor [a protein involved in switching genes on and off] binds to one of these cancer-associated mutations, we may be able to develop drugs targeting that factor, potentially reducing the likelihood that people born with that mutation, will contract cancer," Gusev says.

Jake Glanville, chief executive officer of Bay Area-based Centivax. (Photo courtesy of Centivax) When COVID-19 shut down the world’s economies, governments across the globe, including the United States, began to pour money into vaccine research and development. Those funds led to the rapid development and deployment of vaccines, but few therapeutics against the virus have achieved the same speed of development. And that may be a problem as the virus shifts from pandemic to endemic. “The virus will not be eradicated through vaccination alone. That means... we need our medicines. That was a failure of 2020 that doesn’t get enough emphasis,” Jake Glanville, chief executive officer of Bay Area-based Centivax, told BioSpace in an interview. Glanville praised the swift response from the industry to develop vaccines that are highly effective against the original strain of the virus, as well as limited efficacy against emerging strains. However, he said more medications are needed, particularly as it appears herd immunity may not be achieved any time soon. Medicines such as antivirals and monoclonal antibodies will be needed to combat the continued spread of COVID-19, he said. And that is where Centivax can play a role, Glanville believes. The company is developing a monoclonal antibody, among other therapeutics, that could play a role in stemming widespread COVID-19 infections. From previous antibody therapies that have been authorized to treat COVID-19, including Eli Lilly’s and AbCellera’s bamlanivimab, Glanville said data shows they can have a significant impact in treating patients. However, the rise of more virulent strains of COVID-19 have called for changes in strategy. Recently Eli Lilly and AbCellera sidelined bamlanivimab as a standalone treatment for COVID-19. Now the antibody must be paired with etesevimab as a more effective therapy. But, this combined monoclonal antibody treatment still requires an infusion, whereas Centivax’s experimental antibody is an ultrahigh concentration, which means can be administered via an intramuscular injection. And that can be beneficial in the long run. Glanville said his company’s experimental broadly neutralizing antibody is easy to administer, especially when many infusion centers are already functioning at peak capacity administering other treatments. “And more importantly it’s 99.5% effective against all strains in the USA including the all the scary new strains,” Glanville said. He added that globally, they’re seeing an efficacy of about 98% against the known strains based on tests in hamsters, animals commonly used to test antivirals. “This is telling us the drug will be useful.” Centivax is working in conjunction with the U.S. Navy on the development of the antibody. If successful, ships could keep a stock of the antibody in order to prevent an onboard outbreak that could destabilize the vessel. The Centivax antibody is expected to enter the clinic by Aug. 1, but Glanville is hoping the timeline can be moved up to July. He anticipates an accelerated clinical program, but not at the same urgent speed Eli Lilly and Regeneron had when developing their antibody treatments. The Phase I trial is expected to last about three months, then could shift into a Phase II/III. It’s possible the company could seek emergency use authorization in early 2022. A single shot should provide 28-42 days of protection, he said. It can also be used as a prophylactic, which will benefit family members who are around someone with the virus. “It’s like a vaccine but it works instantly, just doesn’t last as long,” he said. If the antibody is approved, Glanville said people could receive the drug at their doctor’s office following a COVID-19 diagnosis, or possibly pick it up at the corner drugstore and administer it at home. “As soon as you have a positive test, the drug could be administered,” he said. “The way antivirals work, the earlier you can give the medicine, it’s more effective at preventing that runaway wildfire infection in your body.” Centivax was initially a therapeutics spinout of Distributed Bio, a contract research organization. Earlier this year, Glanville sold Distributed Bio, to Charles River Laboratories for $83 million in upfront money. Last year, the company struck several partnerships to combat COVID-19. The company partnered with SwiftScale Biologics on an antibody. It also struck a deal with Thermo Fisher Scientific, which evaluated several variants of Centivax's antibody candidates for use in research, including flow cytometry, histopathology, in vitro and in vivo neutralization, Luminex and ELISA applications.