GN/ Simplifying RNA editing for treating genetic diseases
Genetics biweekly vol.21, 2d February — 16th February
TL;DR
- New research could make it much simpler to repair disease-causing mutations in RNA without compromising precision or efficiency. The new RNA editing technology holds promise as a gene therapy for treating genetic diseases. In a proof of concept, researchers showed that the technology can treat a mouse model of Hurler syndrome, a rare genetic disease, by correcting its disease-causing mutation in RNA.
- Researchers have shown, in mice, that a new class of compounds they developed can improve several aspects of metabolic syndrome. Such conditions often lead to cardiovascular disease, the leading cause of death worldwide.
- Researchers have identified a previously unrecognized key player in cancer evolution: clusters of mutations occurring at certain regions of the genome. These mutation clusters contribute to the progression of about 10 percent of human cancers and can be used to predict patient survival.
- Bioengineers have found a way to radically increase the efficiency of single-cell RNA-sequencing, a powerful tool that can ‘read’ the genetic profile of an individual cell.
- A new study demonstrates ‘species agnostic’ screening of lipid nanoparticles, which could significantly accelerate the development of cutting edge mRNA targeted therapies.
- A new study found in a living embryo that the back ends of moving cell groups push the group forward, with implications for how organs form and cancer spreads.
- Nerves in the intestines help regulate the gut’s acidity, new research shows. That helps keep their bacterial communities in balance.
- Cellular proteins that hold cells and tissues together also perform critical functions when they experience increased tension. A new study observed that when tugged upon in a controlled manner, these proteins — called cadherins — communicate with growth factors to influence in vitro tumor growth in human carcinoma cells.
- Researchers have created a functional catalog of proteins that activate gene expression, with implications for tailored therapy for cancer and other diseases that occur when wrong genes are switched on.
- The microbiome is home to an estimated 100 trillion bacteria, existing as a dense colony of many different strains and species. Similar to all organisms, bacteria must also compete with one another for space and resources, engaging in “warfare” by releasing toxins to kill competitors. One of the many weapons bacteria use in this inevitable fight is the type VI secretion system (T6SS), which delivers toxic effectors into their enemies.
- And more!
Overview
Genetic technology is defined as the term which includes a range of activities concerned with the understanding of gene expression, advantages of natural genetic variation, modifying genes and transferring genes to new hosts. Genes are found in all living organisms and are transferred from one generation to the next. Gene technology encompasses several techniques including marker-assisted breeding, RNAi and genetic modification. Only some gene technologies produce genetically modified organisms.
Modern genetic technologies like genome editing would not be possible without all the previous generations of genetic technologies that have enabled scientists to discover what genes are, what they do and how DNA can be modified to add, remove or replace genes. You can find major genetic technologies development milestones via the link.
Gene Technology Market
- The valuation of the genetic engineering market is projected to escalate to USD 6.90 MN by the end of 2027.
- Global Genetic Engineering Market is projected to grow at 12.48% CAGR during the assessment period (2017–2027).
- North America holds the largest share in the global genetic engineering market, followed by Europe and the Asia Pacific, respectively.
- Another research provider, MarketsandMarkets, forecasts the genome editing, genome engineering market to grow from USD 3.19 billion in 2017 to USD 6.28 billion by 2022, at a compounded annual growth rate (CAGR) of 14.5% during the forecast period. The key factors propelling market growth are rising government funding and growth in the number of genomics projects, high prevalence of infectious diseases (like COVID-19) and cancer, technological advancements, increasing production of genetically modified (GM) crops, and growing application areas of genomics.
Latest News & Research
Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs
by Dhruva Katrekar, James Yen, Yichen Xiang, Anushka Saha, Dario Meluzzi, Yiannis Savva, Prashant Mali in Nature Biotechnology
New research led by bioengineers at the University of California San Diego could make it much simpler to repair disease-causing mutations in RNA without compromising precision or efficiency.
The new RNA editing technology holds promise as a gene therapy for treating genetic diseases. In a proof of concept, UC San Diego researchers showed that the technology can treat a mouse model of Hurler syndrome, a rare genetic disease, by correcting its disease-causing mutation in RNA.
What’s special about the technology is that it makes efficient use of RNA editing enzymes that naturally occur in the body’s cells. These enzymes are called adenosine deaminases acting on RNA (ADARs). They bind to RNA and convert some of the adenosine (A) bases to inosine (I), which is read by the cell’s translation machinery as guanosine (G).
Researchers have been exploring RNA editing approaches with ADARs to correct the G-to-A mutation behind genetic disorders such as cystic fibrosis, Rett syndrome and Hurler syndrome. A big advantage of RNA editing — over DNA editing, for example — is that changes to RNA are only temporary, since RNA has a short lifespan. So even if off-target edits occur, they wouldn’t be there to stay.
To make a targeted A-to-I (or essentially, an A-to-G) edit on RNA using ADARs, a short accessory strand of RNA — called a guide RNA — is needed to guide ADARs to the target and make the desired change there.
A big challenge with this approach is that traditional guide RNAs are not efficient at using native ADARs in the cell, so they require external ADARs to be brought into the cell to work, explained Prashant Mali, a bioengineering professor at the UC San Diego Jacobs School of Engineering. “But the problem with that,” he added, “is that it makes delivery complicated. And it can result in more off targets.” To overcome these issues, Mali and colleagues engineered a new kind of guide RNA — one that is extremely effective at recruiting the cell’s own ADARs to make edits at a precise target RNA region.
“We can simply deliver just a small piece of RNA inside the cell and repair mutations in vivo. We don’t have to provide any extra enzymes,” said Mali.
The team designed the guide RNAs to target the single G-to-A mutation that causes Hurler syndrome. This mutation prevents the body from producing an enzyme that is necessary for breaking down complex sugars. Buildup of these sugars causes severe tissue damage, skeletal abnormalities, cognitive impairment, and other serious health problems. Systemic injection of the guide RNAs into diseased mice resulted in correction of 7 to 17% of the mutant RNAs after two weeks, as well as a 33% decrease in the buildup of complex sugars.
One aspect that makes the new guide RNAs effective is that they are longer than traditional guide RNAs. “This basically makes them stickier for ADARs already present in the cell to come and bind to them,” said Mali. Other unique design features make them more stable and precise than traditional guide RNAs. They can last for days and stay on the target RNA region for longer periods of time, whereas RNA in general gets quickly destroyed by the cell. That’s because these guide RNAs are built as circular rather than linear molecules; being circular makes them resistant to the cell’s RNA-degrading enzymes. In terms of precision, these guide RNAs only allow changes at the target A and not at any other As nearby. They do this by folding into loop structures at predetermined spots along the target RNA region, which prevents off-target As from getting edited.
The research is still at an early stage, said Mali, “and it remains to be seen how this RNA editing technology will work in primates.” Immediate next steps for the team will focus on improving delivery of the guide RNAs into cells.
“I’m hopeful that this work opens the door even more for RNA editing as another gene therapy tool,” said Mali.
Small molecule SWELL1 complex induction improves glycemic control and nonalcoholic fatty liver disease in murine Type 2 diabetes
by Susheel K. Gunasekar, Litao Xie, Ashutosh Kumar, et al in Nature Communications
A study in mice — led by researchers at Washington University School of Medicine in St. Louis — shows that a new class of compounds the scientists developed can improve multiple aspects of metabolic syndrome. An increasingly common group of conditions that often occur together, metabolic syndrome includes type 2 diabetes, high cholesterol, fat buildup in the liver, and excess body fat, especially around the waist. This syndrome often leads to cardiovascular disease, the leading cause of death worldwide.
Testing one of the compounds referred to as SN-401, the researchers found it treats diabetes by improving the ability of the pancreas to secrete insulin and boosting the ability of other tissues to utilize that insulin to more effectively remove sugar from the bloodstream. In an effort to optimize the treatment, the researchers fine-tuned the compound — creating a class of related compounds — based on their studies of a key protein called SWELL1 (also LRRC8a). The gradual decline of this protein may have a central role in the development of diabetes and other aspects of metabolic syndrome.
“Our goal is to develop better therapies for cardiovascular disease, including diabetes and metabolic syndrome, which are major risk factors for worsening heart and vascular problems,” said senior author Rajan Sah, MD, PhD, an associate professor of medicine. “We have many treatments for diabetes, but even with those therapies, cardiovascular disease remains a leading cause of death among patients with type 2 diabetes. There is a need for new treatments that work differently from the current standard-of-care therapies.”
The protein Sah and his colleagues studied is called SWELL1 because of its role in sensing the size or volume of cells. Their new research reveals that the protein also helps to control insulin secretion from the pancreas and improve insulin sensitivity, including in skeletal muscle and adipose tissue, the body’s fat stores.
Surprisingly, the researchers showed that SWELL1 does both of these seemingly independent tasks because the protein has a previously unknown double life. It acts as a signaling molecule, turning on cellular tasks that govern how well cells use insulin and also facilitates the pancreas’ secretion of insulin into the bloodstream.
“This protein, SWELL1, has a sort of dual personality,” Sah said. “The compound binds to SWELL1 in a manner that stabilizes the protein complex so as to enhance expression and signaling across multiple tissues, including adipose, skeletal muscle, liver, the inner lining of blood vessels, and pancreatic islet cells. This restores both insulin sensitivity across tissue types and insulin secretion in the pancreas.”
Sah and his colleagues showed that the SN-401 compound improved multiple aspects of metabolic syndrome in two groups of mice that each developed diabetes from different causes, one because of a genetic predisposition and the other due to a high-fat diet. In addition to improving insulin sensitivity and secretion, treatment with the compound also improved blood sugar levels and reduced fat buildup in the liver. Most of these studies were conducted with an injected form of the compound, but the researchers showed evidence that it also could be effective if taken by mouth.
The researchers further showed that the compound does not have a big impact on blood sugar in healthy mice, which is important for its potential as a future possible therapy. Current medications for diabetes can result in blood sugar levels that are too low. The evidence suggests that this compound does not lower blood sugar in situations when it doesn’t need to.
Mapping clustered mutations in cancer reveals APOBEC3 mutagenesis of ecDNA
by Erik N. Bergstrom, Jens Luebeck, Mia Petljak, Azhar Khandekar, Mark Barnes, Tongwu Zhang, Christopher D. Steele, Nischalan Pillay, Maria Teresa Landi, Vineet Bafna, Paul S. Mischel, Reuben S. Harris, Ludmil B. Alexandrov in Nature
Researchers led by bioengineers at the University of California San Diego have identified and characterized a previously unrecognized key player in cancer evolution: clusters of mutations occurring at certain regions of the genome. The researchers found that these mutation clusters contribute to the progression of about 10% of human cancers and can be used to predict patient survival.
The work sheds light on a class of mutations called clustered somatic mutations — clustered meaning they group together at specific areas in a cell’s genome, and somatic meaning they are not inherited, but caused by internal and external factors such as aging or exposure to UV radiation, for example.
Clustered somatic mutations have so far been an understudied area in cancer development. But researchers in the lab of Ludmil Alexandrov, a professor of bioengineering and cellular and molecular medicine at UC San Diego, saw something highly unusual about these mutations that warranted further study.
“We typically see somatic mutations occurring randomly across the genome. But when we looked closer at some of these mutations, we saw that they were occurring in these hotspots. It’s like throwing balls on the floor and then suddenly seeing them cluster in a single space,” said Alexandrov. “So we couldn’t help but wonder: What is happening here? Why are there hotspots? Are they clinically relevant? Do they tell us something about how cancer has developed?”
“Clustered mutations have largely been ignored because they only make up a very small percentage of all mutations,” said Erik Bergstrom, a bioengineering PhD student in Alexandrov’s lab and the first author of the study. “But by diving deeper, we found that they play an important role in the etiology of human cancer.”
The team’s discoveries were enabled by creating the most comprehensive and detailed map of known clustered somatic mutations. They started by mapping all the mutations (clustered and non-clustered) across the genomes of more than 2500 cancer patients — an effort that in total encompassed 30 different cancer types. The researchers created their map using next-generation artificial intelligence approaches developed in the Alexandrov lab. The team used these algorithms to detect clustered mutations within individual patients and elucidate the underlying mutational processes that give rise to such events. This led to their finding that clustered somatic mutations contribute to cancer evolution in approximately 10% of human cancers.
Taking it a step further, the researchers also found that some of the cancer-driving clusters — specifically those found in known cancer driver genes — can be used to predict the overall survival of a patient. For example, the presence of clustered mutations in the BRAF gene — the most widely observed driver gene in melanoma — results in better overall patient survival compared to individuals with non-clustered mutations. Meanwhile, the presence of clustered mutations in the EGFR gene — the most widely observed driver gene in lung cancer — results in decreased patient survival.
“What’s interesting is that we see differential survival in terms of just having clustered mutations detected within these genes, and this is detectable with existing platforms that are commonly used in the clinic. So this acts as a very simple and precise biomarker for patient survival,” said Bergstrom.
“This elegant work emphasizes the importance of developing AI approaches to elucidate tumor biology, and for biomarker discovery and rapid development using standard platforms with direct line of sight translation to the clinic,” said Scott Lippman, director of Moores Cancer Center and associate vice chancellor for cancer research and care at UC San Diego. “This highlights UC San Diego’s strength in combining engineering approaches in artificial intelligence for solving current problems in cancer medicine.”
In this study, the researchers also identified various factors that cause clustered somatic mutations. These factors include UV radiation, alcohol consumption, tobacco smoking, and most notably, the activity of a set of antiviral enzymes called APOBEC3.
APOBEC3 enzymes are typically found inside cells as part of their internal immune response. Their main job is to chop up any viruses that enter the cell. But in cancer cells, the researchers think that the APOBEC3 enzymes may be doing more harm than good.
The researchers found that cancer cells — which are often rife with circular rings of extrachromosomal DNA (ecDNA) that harbor known cancer driver genes — have clusters of mutations occurring across individual ecDNA molecules. The researchers attribute these mutations to the activity of APOBEC3 enzymes. They hypothesize that APOBEC3 enzymes are mistaking the circular rings of ecDNA as foreign viruses and attempt to restrict and chop them up. In doing so, the APOBEC3 enzymes cause clusters of mutations to form within individual ecDNA molecules. This in turn plays a key role in accelerating cancer evolution and likely leads to drug resistance. The researchers named these rings of clustered mutations kyklonas, which is the Greek word for cyclones.
“This is a completely novel mode of oncogenesis,” said Alexandrov. Along with the team’s other findings, he explained, “this lays the foundation for new therapeutic approaches, where clinicians can consider restricting the activity of APOBEC3 enzymes and/or targeting extrachromosomal DNA for cancer treatment.”
Deterministic scRNA-seq captures variation in intestinal crypt and organoid composition
by Johannes Bues, Marjan Biočanin, Joern Pezoldt, Riccardo Dainese, Antonius Chrisnandy, Saba Rezakhani, Wouter Saelens, Vincent Gardeux, Revant Gupta, Rita Sarkis, Julie Russeil, Yvan Saeys, Esther Amstad, Manfred Claassen, Matthias P. Lutolf, Bart Deplancke in Nature Methods
Single-cell RNA sequencing, or “scRNA-seq” for short, is a technique that allows scientists to study the expression of genes in an individual cell within a mixed population — which is virtually how all cells exist in the body’s tissues. Part of a larger family of “single-cell sequencing” techniques, scRNA-seq involves capturing the RNA of a single cell and, after multiple molecular conversion reactions, sequencing it. Since RNA is the intermediate step from gene (DNA) to protein, it provides an overview about which genes in that particular cell are active and which are not.
Because scRNA-seq captures the activity of all genes in the cell’s genome — thousands of genes at once — it has become the gold standard for defining cell states and phenotypes. This kind of data can reveal rare cell types within a cell population, even types never seen before. But scRNA-seq isn’t just a tool for basic cell biology; it has been widely adopted in medical and pharmacological research as it is capable of identifying which cells are actively dividing in a tissue, or which are reacting to a particular drug or treatment.
“These single-cell approaches have transformed our ability to resolve cellular properties across systems,” says Professor Bart Deplancke at EPFL’s School of Life Sciences. “The problem is that they are currently tailored toward large cell inputs.”
This isn’t a trivial problem, as scRNA-seq methods require over a thousand cells for a useful measurement. Dr Johannes Bues, a researcher in Deplancke’s group, adds: “This renders them inefficient and costly when processing small, individual samples such as small tissues or patient biopsies, which tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs.”
Bues, with Marjan Bioanin and Joern Pezoldt, also in Deplancke’s group, have now developed a new method that allows scRNA-seq to efficiently process samples with fewer cells. The method is dubbed “DisCo” for “deterministic, mRNA-capture bead and cell co-encapsulation dropleting system.” Unlike usual single-cell methods that rely on passive cell capture, DisCo uses machine-vision to actively detect cells and capture them in droplets of oil and beads. This approach allows for continuous operation, and also renders scaling and serial processing of cell samples highly cost efficient.
As shown in the study, DisCo features precise particle and cell positioning, and controls droplet sorting through combined machine-vision and multilayer microfluidics. All this allows for continuous processing of low-input single cell suspensions at high capture efficiency (over 70%) at speeds that can reach 350 cells per hour.
To further showcase DisCo’s unique capabilities, the researchers tested it on the small chemosensory organs of the Drosophila fruit fly, as well as on individual intestinal crypts and organoids. The latter are tiny tissues grown in culture dishes closely resembling actual organs — a field that EPFL has been spearheading for years. The researchers used DisCo to analyze individual intestinal organoids at different developmental stages. The approach painted a fascinating picture of heterogeneity in the organoids, detecting various distinct organoid subtypes of which some had never been identified before.
“Our work demonstrates the unique ability of DisCo to provide high-resolution snapshots of cellular heterogeneity in small, individual tissues,” says Deplancke.
Species-dependent in vivo mRNA delivery and cellular responses to nanoparticles
by Marine Z. C. Hatit, Melissa P. Lokugamage, Curtis N. Dobrowolski, Kalina Paunovska, Huanzhen Ni, Kun Zhao, Daryll Vanover, Jared Beyersdorf, Hannah E. Peck, David Loughrey, Manaka Sato, Ana Cristian, Philip J. Santangelo, James E. Dahlman in Nature Nanotechnology
James Dahlman and Phil Santangelo are helping to define an evolving era in medicine, one in which messenger ribonucleic acid — mRNA — can be delivered directly to cells to fight against disease. And their latest groundbreaking study could clear the way to faster therapeutic discoveries.
Long before the Covid-19 pandemic put a global spotlight on mRNA-based vaccines, these two researchers in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University were combining their distinct skillsets to leverage the clinical potential of mRNA.
“Our work is very compatible,” said Dahlman, associate professor and McCamish Foundation Early Career Professor. “Phil’s lab designs and manufactures really high-quality mRNA, and my lab develops the lipid nanoparticles to deliver it.”
Therapeutics made from mRNA or DNA hold promise in addressing lots of diseases, explained Santangelo, a professor in Coulter BME, “but they’re not much good if they can’t get where they need to go. If you make cargo, which is essentially what we do in my lab, you need delivery, so James and I have a very natural collaboration.” Their partnership, which began when Dahlman arrived at Georgia Tech in 2016, consistently yields results published in high-impact journals and garners generous research grants from federal agencies, including the National Institutes of Health (NIH) and the Defense Advanced Research Projects Agency (DARPA).
“We’re reporting an improved barcoding system that would make animal pre-clinical nanoparticle studies more predictive, speeding up the development of RNA therapies,” Dahlman said.
Several years ago, Dahlman and collaborators developed a technique called “DNA barcoding,” which allows for the rapid, simultaneous screening of many of his custom-made delivery vehicles — what are called lipid nanoparticles, or LNPs. Scientists insert unique snippets of DNA into different LNPs, which are injected into mice. Genetic sequencing is then used to determine which barcodes have reached which specific targets.
“Lipid nanoparticles are usually developed in mice, but when you move them into another species, like a non-human primate — because that’s the natural progression, a primate is more like a human — they frequently don’t work as well,” Santangelo said. “When they don’t, you have to go back and make adjustments.”
But what if you could streamline the process? The genes that affect LNP delivery vary between pre-clinical species and humans, though the extent of those differences is unknown because studies comparing nanoparticle delivery across species have been very difficult to perform. Until now. To speed that process, the researchers developed a new testing system they’re calling Species Agnostic Nanoparticle Delivery Screening, or SANDS.
Using SANDS, the team compared nanoparticle delivery simultaneously in mouse, primate, and living human cells, all within specially engineered mice.
“We can actually put the same group of nanoparticles in all three and compare delivery across species,” Dahlman said. “We found what you might expect: delivery in the primate cells predicted really well how delivery in the human cells would go, whereas the mouse cells were less predictive.”
Unlike the previous barcoding system, which worked well in mouse cells, SANDS needed a different kind of marker for screening, a molecule called reporter mRNA. Santangelo’s lab developed one, “and it basically gets around the limitations of the old system,” he said. “Now we can screen new lipid nanoparticles in mice with primate and human cells.”
SANDS already is facilitating further studies for the research team. Going forward, Dahlman and Santangelo believe that deeper understanding of the different mechanisms driving delivery in mouse cells and other cells will result in a more efficient selection process for LNPs, making pre-clinical nanoparticle studies more predictive and accelerating the development of RNA therapies. That sense of building momentum has been kind of a theme for the Dahlman-Santangelo partnership since it began. Dahlman remembered interviewing at Georgia Tech and Emory and being immediately impressed when he met Santangelo.
“I explained to him my vision for barcoding, and he immediately got it; he explained to me his vision for improving payloads, and I immediately got that,” Dahlman said. “You could have the world’s best nanoparticle, but if you don’t put optimized mRNA in it, that’s not going to be any good.”
They immediately recognized the value and the necessity for collaboration, especially because, as Santangelo put it, “This is a wildly competitive time in mRNA research.”
Rear traction forces drive adherent tissue migration in vivo
by Naoya Yamaguchi, Ziyi Zhang, Teseo Schneider, Biran Wang, Daniele Panozzo, Holger Knaut in Nature Cell Biology
Cells push and pull on surrounding tissue to move in groups as they form organs in an embryo, track down invading bacteria, and as they become cancerous and spread.
A new study found in a living embryo that the back ends of moving cell groups push the group forward. This runs contrary to previous findings, where cell groups grown in dishes of nutrients (cultures) pulled themselves forward with their front edges.
Led by researchers from NYU Grossman School of Medicine and the NYU Courant Institute of Mathematical Sciences, the study used a new technique to measure the forces applied by a cell group as it moved along a “road-like” tissue membrane and into place in a developing animal. Specifically, the study found for the first time in an animal tissue that proteins called integrins on the surfaces of the cells at the rear attach in greater numbers to the membrane as they move along, and exert more force in one direction, than the cells in the group’s front. The integrin clusters (focal adhesions) observed in the embryo were smaller than those seen in culture studies, and broke down faster. Confirmation of such mechanistic details in living tissue have important implications, say the researchers, as many cancers spread in cell groups, and may use the newfound “rear engine propulsion.”
“Our results clarify how cell groups that will become organs move into place, and reaffirm that cells behave differently when removed from their natural environments,” said senior study author Holger Knaut, PhD, associate professor in the Department of Cell Biology at NYU Langone Health.
The study results are based on mechanisms of cell movement established by past studies. For instance, a protein called actin is known to form the protein “skeleton” of cells, with actin chains able to grow in a certain direction, and apply force that change a cell’s shape. Integrins, proteins built into outer cell membranes, interact both with actin networks, and proteins outside of cells. These and other proteins form a system that a cell uses to briefly attach to and “roll along” a basement membrane, a pliable mesh of proteins and sugars. What was unknown going into the current study was how tissues in living animals apply force in groups to generate this motion.
The new study examined cell group motion in a zebrafish embryo, a major model in the study of development because it shares many cellular mechanisms with human cells, and because zebrafish embryos development externally, such that each stage in development can be directly observed using high-powered microscopes. In this way the team tracked the movement of the primordium — a tissue made up of about 140 cells — as it migrated during development from behind the ear to the tip of the zebrafish tail, where it matures into an organ that senses water flow.
“In the first study of its kind, we combined advanced microscopy with automated, high-throughput computational modeling to measure cellular forces in living organisms,” says co-corresponding author Daniele Panozzo, PhD, an associate professor at the Courant Institute of Mathematical Sciences at New York University.
Using “bleached” dots on the basement membrane to measure shape changes (deformations) on a minute scale, and a new software called embryogram to calculate how far the dots move as the primordium “grips” the membrane, the researchers determined how much the cells pulled and pushed on the membrane, “like a tire on pavement.” The effect is much like the high school physics experiment where students draw two dots on a rubber band, and calculate the force applied as they stretch the band by measuring the change in distance between the dots.
With these tools in hand, the team showed that the primordium cells link the force-generating actin-myosin network at the back end of the moving group through integrin clusters on the side closest to the basement membrane. The team theorizes that cells attached to membrane toward the back push on the cells in front of them to move the entire group. The researches also gained new insights on an established mechanism where cells have surface proteins that let them “sense” and follow a guidance cue called a chemokine, from low concentration to high concentration. The new study found, however, that cells toward the back end of the primordium sense the chemokine gradient more strongly.
Interestingly, the study found that the primordium moved in a “continuous breaststroke” by pushing the basement membrane downward, sideways and backwards, much like the arms of a swimmer. The authors do not know why this is, but speculate that this is the most efficient way to move forward. They note that banana slugs also use the rear edge of the “foot” they apply to the ground, suggesting that evolution favors rear engine propulsions because they are most efficient at different size scales.
The study suggests that group cell movement have the potential to be harnessed to stop cancer spread, perhaps by designing treatments that block the action of integrins, say the authors. Integrin inhibitors have been tested as drugs for cardiovascular and autoimmune disease in clinical trials, but their use against cancer spread has been limited by the need for a better understanding of the mechanisms.
Enteric nervous system modulation of luminal pH modifies the microbial environment to promote intestinal health
by M. Kristina Hamilton, Elena S. Wall, Catherine D. Robinson, Karen Guillemin, Judith S. Eisen in PLOS Pathogens
Nerves in the intestines help regulate the gut’s acidity, new research from the University of Oregon shows. That helps keep their bacterial communities in balance.
“We found an unexpected connection between the nervous system of the intestine and the community of gut microbes,” says UO microbiologist Karen Guillemin. “The nervous system is regulating the microbes.”
Guillemin co-led the new work with Judith Eisen, a neuroscientist at UO. Scientists have known for years that gut bacteria are important for digestive health. And other studies have demonstrated a strong connection between the gut and the brain. The new work links those two mostly distinct areas of research together.
To make the connection, Eisen and Guillemin studied zebrafish with a genetic mutation that leads to missing nerves in the gut. In humans, mutations in this gene are associated with Hirschsprung’s disease, which disrupts gut nervous system development and can cause bouts of severe intestinal inflammation.
Eisen and Guillemin had previously shown that zebrafish missing gut nerves had similar inflammation. But understanding the roots of inflammation can be tricky: So many factors, from diet and exercise to genetics, can impact gut health. And one kind of inflammation often fuels other inflammation, making it tough to figure out where the cycle begins.
Enter: zebrafish. “Many conditions can cause intestinal inflammation, and it’s very difficult to sort out in people,” Eisen said. “In zebrafish, you can do really controlled manipulations,” including controlling what kind of bacteria are present in their intestines at birth.
Postdoctoral researcher Kristi Hamilton came in with a hunch — she suspected that the diseased zebrafish might have problems with their gut pH. Sure enough, when she fed zebrafish larvae acid-sensing dyes, the ones with missing gut nerves had more acidic guts. That, in turn, led to overgrowth of harmful vibrio bacteria.
Giving the fish a heartburn drug called omeprazole (widely known as Prilosec) calmed down the acidity and restored the bacterial balance. On the other hand, giving zebrafish a drug that increases acidity (acetazolamide, used for altitude sickness, epilepsy, and many other conditions) had the opposite effect, they found. It led to too many vibrio bacteria in zebrafish with previously healthy guts.
The findings suggest that the nerves in the gut do more than control the contractions that keep things moving — they also help regulate gut acidity, ensuring a healthy bacterial population. For these researchers, following their intuition paid off with a new understanding of what it means to have a good gut feeling.
Mechanical disruption of E-cadherin complexes with epidermal growth factor receptor actuates growth factor–dependent signaling
by Brendan Sullivan, Taylor Light, Vinh Vu, Adrian Kapustka, Kalina Hristova, Deborah Leckband in Proceedings of the National Academy of Sciences
The cellular proteins that hold cells and tissues together also perform critical functions when they experience increased tension. A new University of Illinois Urbana-Champaign study observed that when tugged upon in a controlled manner, these proteins — called cadherins — communicate with growth factors to influence in vitro tumor growth in human carcinoma cells.
The study, led by chemical and biomolecular engineering professor Deborah Leckband, found that cadherins that bond with growth factor receptors can sense mechanical force and respond by altering cell communication and growth.
When bound to cadherin molecules in normal tissue, growth factor receptors cannot communicate with growth factor proteins — the substance they need to promote tissue growth. However, the study shows that changes in tensional stress on cadherin bonds disrupt the cadherin-growth factor interaction to switch on growth signals in tissues.
To demonstrate how tension influences tissue growth, the researchers set up an experiment to observe how in vitro human carcinoma cells convert mechanical information into biochemical signals, Leckband said.
The team used a self-built “cell stretcher” in which the carcinoma cells are grown in a thin layer on the surface of a flexible medium. When the cells are stretched, the researchers observed changes that could increase tissue growth and tumorigenesis.
“This study confirms that cadherins use force to switch on biochemical growth signaling,” Leckband said. “By confirming these force-induced disruptions, we may be able to find a way to mutate cadherin molecules in order to prevent certain types of tissue growth, such as metastatic transformation and tumorigenesis.”
The team has observed the cadherin-growth factor receptor complex in human epithelial tissue and plans to expand this concept by working with in vitro human breast tissue.
Structure of a bacterial Rhs effector exported by the type VI secretion system
by Patrick Günther, Dennis Quentin, Shehryar Ahmad, Kartik Sachar, Christos Gatsogiannis, John C. Whitney, Stefan Raunser in PLOS Pathogens
The microbiome is home to an estimated 100 trillion bacteria, existing as a dense colony of many different strains and species. Similar to all organisms, bacteria must also compete with one another for space and resources, engaging in “warfare” by releasing toxins to kill competitors. One of the many weapons bacteria use in this inevitable fight is the type VI secretion system (T6SS), which delivers toxic effectors into their enemies. The groups of Stefan Raunser from the Max Planck Institute of Molecular Physiology in Dortmund and John Whitney from McMaster University in Canada, have now together uncovered the high-resolution 3D structure of such an effector from Pseudomonas protegens by cryo-electron microscopy. The effector protein, called RhsA, has a toxic component that sits unlocked and ready to be fired within a molecular cocoon sealed by a cork-like structure. Their findings will not only help in understanding how the T6SS machinery works, but will also promote the future development of antibacterial treatments and plant protection strategies.
The beneficial bacterium Pseudomonas protegens protects plants from fungi and bacteria. However, behind this seemingly selfless act lies a complex system by which the bacteria try to occupy a biological niche by eliminating their competitors. For this purpose, bacteria have developed a whole arsenal of poisons and a variety of injection systems to prepare them for battle.
One of the most widely-used injection machinery in Gram-negative bacteria is the type VI secretion system. When this machinery is activated, a nanotube is assembled in the cell interior, through which a poison dart with deadly toxic proteins on its tip is shot into a competitor. The 3D structure of one of these toxic proteins, the bacterial RhsA effector, has now been solved by the team of Stefan Raunser along with the team of John Whitney. The scientists found that the RhsA effector consists of three connecting pieces: the toxic weapon itself, a cocoon surrounding it, and a cork-like plug which seals the toxin encapsulating cocoon entirely.
“The cocoon protects the bacterium from its self-produced toxin,” Stefan Raunser says. “We already observed a very similar strategy in bacterial Tc toxins.” The scientists have shown that the effector protein itself cleaves the seal and the toxin from the rest of the protein, thereby unlocking the deadly weapon. However, the release of the toxic component is not yet possible since the seal keeps the cocoon secured. “We suspect that when the poison dart penetrates the enemy bacterium, mechanical force is generated to remove the cleaved seal, similar to when a champagne cork pops. This would ensure that the toxin is released in the right place at the right time” Stefan Raunser says.
In a series of earlier collaborative projects, the scientists have already gained a lot of knowledge about how the T6SS injection system works. They were able to reveal how effectors are transported inside the cell, how they are loaded on the poison dart and how the dart is then delivered into the host cell. “Our latest collaborative work now provides molecular insights into the arming process of Rhs effectors and its importance for toxin release. I am quite optimistic that our continued collaboration will uncover even more details of the T6SS machinery. This could one day allow for the engineering of bacteria with improved pathogen suppression capabilities useful for antibacterial and antifungal applications” John Whitney says.
Identification and functional characterization of transcriptional activators in human cells
by Nader Alerasool, He Leng, Zhen-Yuan Lin, Anne-Claude Gingras, Mikko Taipale in Molecular Cell
Also known as transcriptional activators for their ability to induce transcription of genes into RNA messages, these proteins are essential for the cells to function properly. Yet little is known about these proteins, and it wasn’t clear how many activators there might be in human cells — until now.
The research was led by Mikko Taipale, an associate professor of molecular genetics in the Donnelly Centre for Cellular and Biomolecular Research at the Temerty Faculty of Medicine, in collaboration with Anne-Claude Gingras, a senior investigator at the Lunenfeld-Tanenbaum Research Institute, Sinai Health System and professor of molecular genetics at U of T.
In the article, the researchers describe the first unbiased proteome scale study that has expanded the number of known transcriptional activators from a handful to around 250. They have also established how these proteins combine with other cellular machineries to turn genes on, and how protein misregulation can lead to cancer.
“This study was a classic fishing expedition where we did not know what we were going to find,” said Taipale, who holds Canada Research Chair in Functional Proteomics and Protein Homeostasis. “Grant reviewers typically frown upon research that is not hypothesis driven, but that’s the beauty of proteomics. It allows you to cast a net in an unbiased way, and we have found some interesting stuff.
“We now have a better understanding of which proteins are very strong activators. And we can begin to understand the mechanisms by which they activate transcription.”
To find the activators, the researchers tested the majority of 20,000 human proteins for their ability to activate gene expression in human cells. Many activators were transcription factors (TFs), which directly bind DNA and turn on their target genes, whereas others were helper proteins, or co-factors, that bind TFs and activate their targets together. They also found that TFs that are highly similar can talk to different co-factors, explaining why two TFs with essentially identical DNA binding specificities can trigger distinct gene expression programs.
“These activators are not activators in all contexts. It could be that in a gene X they activate, but in gene Y they might actually repress,” Taipale said.
Transcriptional activation occurs through the interaction of the so-called transactivation domains, which are present in the TFs, with the activators. Since the sequences of activation domains are not conserved, they can’t be pinpointed by computational methods. For that reason, the team resorted to chopping up 75 activators into pieces and tested the ability of each piece to activate transcription. They identified around 40 activation domains this way.
They also used AlphaFold, a revolutionary bioinformatic tool developed for the prediction of protein structures, to find the interaction interfaces between the TFs and their activators. Although AlphaFold was not designed to predict protein-protein interactions, this unexpected feature was a highlight for Taipale, who said the software will become the standard tool for these kinds of studies to find functional connections between proteins.
“This has been previously nearly impossible to do computationally,” Taipale said.
While many of the identified proteins are novel, some of them were previously detected in tumours in which a TF and its helper protein are permanently joined in an oncogenic fusion protein which ends up activating the wrong genes. Piecing together the puzzle of how TFs interact with different activators could be a major step towards tailored therapy. One challenge in therapeutics development has been that TFs are not amenable to targeting by small-molecule drugs.
“Transcription factors are really hard to target because they often don’t have druggable pockets, but many of the co-activators are enzymes which means they have pockets that can be targeted,” said Taipale. “For example, when you have a cancer fusion of the transcription factor to the co-activator and you understand the co-activator that the transcription factor interacts with, you may be able to target the co-activator to halt cell proliferation.”
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