GN/ CROPSR: A new tool to accelerate genetic discoveries
Genetics biweekly vol.22, 9th February — 23d February
TL;DR
- Scientists have developed CROPSR, the first open-source software tool for genome-wide design and evaluation of guide RNA (gRNA) sequences for CRISPR/Cas9 experiments. This tool significantly shortens the time required to design a CRISPR experiment and reduces the challenge of working with complex crop genomes. It should accelerate bioenergy crop development as well as broader crop improvements and other gene-editing research.
- Researchers have engineered bacteria with internal nutrient reserves that can be accessed when needed to survive extreme environmental conditions. The findings pave the way for more robust biotechnologies based on engineered microbes.
- Researchers have used sound waves to turn stem cells into bone cells, in a tissue engineering advance that could one day help patients regrow bone lost to cancer or degenerative disease.
- In the long-term battle between a herpesvirus and its human host, a virologist and her team of students have identified some human RNA able to resist the viral takeover — and the mechanism by which that occurs.
- A pathway critical for regulating a form of cell death known as necroptosis has been identified. The team’s preclinical findings suggest that an inhibitor targeting this PPP13RG protein complex can help prevent or reduce deaths and severe tissue damage from heart attacks and other inflammation-associated diseases.
- Chemists discovered the structure of a protein that can pump toxic molecules out of bacterial cells. Knowledge of this structure may make it possible to design drugs that could block transport proteins and help resensitize drug-resistant bacteria to existing antibiotics.
- Developing therapies for genetic forms of blindness is extremely challenging, in part because they vary so widely, but scientists have now highlighted a target with great promise for treating a range of these conditions. The scientists have highlighted that a specific gene (SARM1) is a key driver in the damage that ultimately leads to impaired vision (and sometimes blindness), and — in a disease model — showed that deleting this gene protects vision after a chemical kick-starts the chain of dysfunction that mimics a host of ocular conditions.
- Scientists think they may have uncovered a whole new approach to fighting antibiotic-resistant bacteria, which, if successful, would help address a health crisis responsible for more deaths every year than either AIDS or malaria. A team of researchers found a new way to impair antibiotic resistance in bacteria that cause human disease. The team made the bacteria vulnerable again to antibiotics by inhibiting a particular protein that drives the formation of resistance capabilities within the bacteria, called DsbA.
- Tendons connect muscles with bones. When injured, they are really difficult to repair, and the existing therapeutic strategies often have complications. Researchers constructed artificial tendons that were mechanically and biologically similar to normal tendons using human induced pluripotent stem cells. The tendons were successfully implanted in a mouse model of tendon rupture. These findings offer a novel strategy for tendon repair and regeneration.
- Scientists studied the differences in activity of immune genes between male and female Kentish Plovers and found that the immune genes of males were more active. This is evidence that males live longer than females due to differences in their immune systems.
- 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
CROPSR: an automated platform for complex genome-wide CRISPR gRNA design and validation
by Hans Müller Paul, Dave D. Istanto, Jacob Heldenbrand, Matthew E. Hudson in BMC Bioinformatics
Commercially viable biofuel crops are vital to reducing greenhouse gas emissions, and a new tool developed by the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) should accelerate their development — as well as genetic editing advances overall.
The genomes of crops are tailored by generations of breeding to optimize specific traits, and until recently breeders were limited to selection on naturally occurring diversity. CRISPR/Cas9 gene-editing technology can change this, but the software tools necessary for designing and evaluating CRISPR experiments have so far been based on the needs of editing in mammalian genomes, which don’t share the same characteristics as complex crop genomes.
Enter CROPSR, the first open-source software tool for genome-wide design and evaluation of guide RNA (gRNA) sequences for CRISPR experiments, created by scientists at CABBI, a Department of Energy-funded Bioenergy Research Center (BRC). The genome-wide approach significantly shortens the time required to design a CRISPR experiment, reducing the challenge of working with crops and accelerating gRNA sequence design, evaluation, and validation, according to the study.
“CROPSR provides the scientific community with new methods and a new workflow for performing CRISPR/Cas9 knockout experiments,” said CROPSR developer Hans Müller Paul, a molecular biologist and Ph.D. student with co-author Matthew Hudson, Professor of Crop Sciences at the University of Illinois Urbana-Champaign. “We hope that the new software will accelerate discovery and reduce the number of failed experiments.”
To better meet the needs of crop geneticists, the team built software that lifts restrictions imposed by other packages on design and evaluation of gRNA sequences, the guides used to locate targeted genetic material. Team members also developed a new machine learning model that would not avoid guides for repetitive genomic regions often found in plants, a problem with existing tools. The CROPSR scoring model provided much more accurate predictions, even in non-crop genomes, the authors said.
“The goal was to incorporate features to make life easier for the scientist,” Müller Paul said.
Many crops, particularly bioenergy feedstocks, have highly complex polyploid genomes, with multiple sets of chromosomes. And some gene-editing software tools based on diploid genomes (like those from humans) have trouble with the peculiarities of crop genomes.
“It can sometimes take weeks or months to realize that you don’t have the outcome that you expected,” Müller Paul said.
For example, a trait may be regulated by a collection of genes, particularly one involving plant stress where backup systems are useful. A scientist might design an experiment to knock out one gene and be unaware of another that performs the same function. The problem may not be discovered until the plant matures without altering the trait in any way. It’s a particular issue with crops that require specific weather conditions to grow, where missing a season could mean a year-long delay.
Using a genome-wide approach allowed the scientists to tailor CROPSR for plant use by removing built-in biases found in existing software tools. Because they are based on human or mouse genomes, where multiple copies of genes are less common, those tools penalize gRNA sequences that hit the genome in more than one position, to avoid causing mutations in places where they’re not intended. But with crops, the goal is often to mutate more than one position to knock out all copies of a gene. Previously, scientists sometimes had to design four or five mutation experiments to knock out each gene individually, requiring extra time and effort.
CROPSR can generate a database of usable CRISPR guide RNAs for an entire crop genome. That process is computationally intensive and time-consuming — usually requiring several days — but researchers only have to do it once to build a database that can then be used for ongoing experiments.
So, rather than searching for a targeted gene through an online database, then using current tools to design separate guides for five different locations and doing multiple rounds of experiments, scientists could search for the gene in their own database and see all the guides available. CROPSR would indicate other locations to target in the genome as well. Researchers could select a guide that hits all of the genes, making it much easier and quicker to design the experiment.
“You can just hop into the database, fetch all the information you need, ready to go, and start working,” Müller Paul said. “The less time you spend planning for your experiments, the more time you can spend doing your experiments.”
For CABBI scientists, who often work with repetitive plant genomes, having a gRNA tool that allows them to design functioning guides with confidence “should be a step forward,” he said. As the name implies, CROPSR was designed with crop genomes in mind, but it’s applicable to any type of genome.
“CROPSR is also based on human genes, as the data availability for crop genes just isn’t there yet,” Müller Paul said, “but we’re looking into some collaborations with other BRCs to provide a more capable prediction based on biophysics to help mitigate some of the issues caused by the lack of data.”
Going forward, he hopes researchers will record their failed results along with successes to help generate the data to train a crop-specific model. If the collaborations pan out, “we could be looking at some very interesting advancements in training machine learning models for CRISPR applications, and potentially to other models as well.”
Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis
by Klara Szydlo, Zoya Ignatova, Thomas E. Gorochowski in ACS Synthetic Biology
Researchers from the Universities of Bristol and Hamburg have engineered bacteria with internal nutrient reserves that can be accessed when needed to survive extreme environmental conditions. The findings pave the way for more robust biotechnologies based on engineered microbes.
Synthetic Biology allows scientists to redesign organisms, harnessing their capabilities to lead to innovative solutions spanning the sustainable production of biomaterials to advanced sensing of pathogens and disease.
Dr Thomas Gorochowski, joint senior author and a Royal Society University Research Fellow in the School of Biological Sciences at Bristol, said: “Many of the engineered biological systems we have created to date are fragile and break easily when removed from the carefully controlled conditions of the lab. This makes their deployment and scale-up difficult.”
To tackle this problem, the team focused on the idea of building up reserves of protein within cells when times are good, and then breaking these down when conditions are difficult and additional nutrients are needed.
Klara Szydlo, first author and a PhD student at the University of Hamburg, elaborated: “Cells require building blocks like amino acids to function and survive. We modified bacteria to have a protected reserve of these that could then be broken down and released when nutrients became scarce in the wider environment. This allowed the cells to continue functioning when times were tough and made them more robust to any unexpected challenges they faced.”
To create such a system, the team engineered bacteria to produce proteins that could not be directly used by the cell, but which were recognized by molecular machines called proteases. When nutrients fluctuated in the environment, these proteases could then be called on to release the amino acids making up the protein reserve. The released amino acids allowed the cells to continue growing, even though the environment lacked the nutrients required. The system acted similar to a biological battery that the cell could tap into when the mains power was cut.
Dr Gorochowski added: “Developing such a system like this is difficult because there are many different aspects of the design to consider. How big should the protein reserve be? How quickly does this need to be broken down? What sorts of environmental fluctuation would this approach work for? We had lots of questions and no easy way to assess the different options.”
To get around this problem, the team built a mathematical model that allowed them to simulate lots of different scenarios and better understand where the system worked well and where it broke. It turned out that a careful balance was required between the size of the protein reserve, the speed of its breakdown when required, and the length of time nutrients were scarce. Importantly though, the model also showed that if the right combination of these factors was present, the cell could be completely shielded from changes in the environment.
Professor Zoya Ignatova, joint senior author from the Institute of Biochemistry and Molecular Biology at the University of Hamburg, concluded: “We’ve been able to demonstrate how carefully managing reserves of key cellular resources is a valuable approach to engineering bacteria that need to operate in challenging environments. This capability will become increasingly important as we deploy our systems into complex real-world settings and our work helps pave the way for more robust engineered cells that can operate in a safe and predictable manner.”
Short‐Duration High Frequency MegaHertz‐Order Nanomechanostimulation Drives Early and Persistent Osteogenic Differentiation in Mesenchymal Stem Cells
by Lizebona August Ambattu, Amy Gelmi, Leslie Y. Yeo in Small
Researchers have used sound waves to turn stem cells into bone cells, in a tissue engineering advance that could one day help patients regrow bone lost to cancer or degenerative disease.
The innovative stem cell treatment from researchers at RMIT University in Melbourne, Australia, offers a smart way forward for overcoming some of the field’s biggest challenges, through the precision power of high-frequency sound waves.
Tissue engineering is an emerging field that aims to rebuild bone and muscle by harnessing the human body’s natural ability to heal itself. A key challenge in regrowing bone is the need for large amounts of bone cells that will thrive and flourish once implanted in the target area. To date, experimental processes to change adult stem cells into bone cells have used complicated and expensive equipment and have struggled with mass production, making widespread clinical application unrealistic. Additionally, the few clinical trials attempting to regrow bone have largely used stem cells extracted from a patient’s bone marrow — a highly painful procedure.
In a new study, the RMIT research team showed stem cells treated with high-frequency sound waves turned into bone cells quickly and efficiently. Importantly, the treatment was effective on multiple types of cells including fat-derived stem cells, which are far less painful to extract from a patient.
Co-lead researcher Dr Amy Gelmi said the new approach was faster and simpler than other methods.
“The sound waves cut the treatment time usually required to get stem cells to begin to turn into bone cells by several days,” said Gelmi, a Vice-Chancellor’s Research Fellow at RMIT.
“This method also doesn’t require any special ‘bone-inducing’ drugs and it’s very easy to apply to the stem cells. “Our study found this new approach has strong potential to be used for treating the stem cells, before we either coat them onto an implant or inject them directly into the body for tissue engineering.”
The high-frequency sound waves used in the stem cell treatment were generated on a low-cost microchip device developed by RMIT.
Co-lead researcher Distinguished Professor Leslie Yeo and his team have spent over a decade researching the interaction of sound waves at frequencies above 10 MHz with different materials.
The sound wave-generating device they developed can be used to precisely manipulate cells, fluids or materials.
“We can use the sound waves to apply just the right amount of pressure in the right places to the stem cells, to trigger the change process,” Yeo said. “Our device is cheap and simple to use, so could easily be upscaled for treating large numbers of cells simultaneously — vital for effective tissue engineering.”
The next stage in the research is investigating methods to upscale the platform, working towards the development of practical bioreactors to drive efficient stem cell differentiation.
The m6A reader YTHDC2 is essential for escape from KSHV SOX-induced RNA decay
by Daniel Macveigh-Fierro, Angelina Cicerchia, Ashley Cadorette, Vasudha Sharma, Mandy Muller in Proceedings of the National Academy of Sciences
In the long-term battle between a herpesvirus and its human host, a University of Massachusetts virologist and her team of students have identified some human RNA able to resist the viral takeover — and the mechanism by which that occurs. This discovery represents an important step in the effort to develop anti-viral drugs to fight off infections.
“This paper is about trying to understand the mechanism that makes these RNA escape degradation,” says senior author Mandy Muller, assistant professor of microbiology. “The next step is to figure out if we can manipulate this to our advantage.”
In the Muller Lab, student researchers work with Muller studying how Kaposi sarcoma-associated herpesvirus (KSHV) hides for years inside the human body before seeking to gain control over human gene expression to complete the viral infection. At that point, people with a weakened immune system may develop Kaposi sarcoma cancer lesions in the mouth, skin or other organs. The researchers use genome-wide sequencing, post-transcriptional sequencing and molecular biology to examine how the human cell or the virus knows how to prevent degradation.
“Viruses are very smart, that’s what I love to say,” Muller says. “They have lots of strategies to stick around, and they don’t do a lot of damage for a very long time, because that’s one way to hide from the immune system.
“But then, at some point — many, many years later — they reactivate. The way they do this is by triggering a massive RNA degradation event where the virus will wipe out the mRNA from the cell. That means the human system can no longer express the proteins that it needs to express, and that means also that a lot of resources are suddenly available for the virus.”
How and why some RNA are able to escape the viral degradation are questions Muller’s team — including lead author and graduate student Daniel Macveigh-Fierro and co-authors and undergraduates Angelina Cicerchia, Ashley Cadorette and Vasudha Sharma — has been investigating.
“We show that RNA that escape have a chemical tag on them — a post-transcriptional modification — that makes them different from the others,” Muller explains. “By having this tag, M6A, they can recruit proteins that protect them from degradation.”
Muller has been studying KSHV since she was an undergraduate in her native France, and her mission continues.
“We know you need this protein to protect the RNA from degradation, but we still don’t know how that physically stops the degradation, so that’s what we’re going to look at now,” she says.
Ultimately, understanding the mechanisms and pathways involved in KSHV infection may lead to the development of RNA therapeutics to treat viral diseases.
“By identifying the determinants of what makes an mRNA either resistant or susceptible to viral-induced decay, we could use those findings to our advantage to better design anti-viral drugs and reshape the outcome of infection,” Muller says.
RIPK1 dephosphorylation and kinase activation by PPP1R3G/PP1γ promote apoptosis and necroptosis
by Jingchun Du, Yougui Xiang, Hua Liu, Shuzhen Liu, Ashwani Kumar, Chao Xing, Zhigao Wang in Nature Communications
Cell death plays an important role in normal human development and health but requires tightly orchestrated balance to avert disease. Too much can trigger a massive inflammatory immune response that damages tissues and organs. Not enough can interfere with the body’s ability to fight infection or lead to cancer.
Zhigao Wang, PhD, associate professor of cardiovascular sciences at the University of South Florida Health (USF Health) Morsani College of Medicine, studies the complex molecular processes underlying necroptosis, which combines characteristics of apoptosis (regulated or programmed cell death) and necrosis (unregulated cell death).
During necroptosis dying cells rupture and release their contents. This sends out alarm signals to the immune system, triggering immune cells to fight infection or limit injury. Excessive necroptosis can be a problem in some diseases like stroke or heart attack, when cells die from inadequate blood supply, or in severe COVID-19, when an extreme response to infection causes organ damage or even death.
A new preclinical study by Dr. Wang and colleagues at the University of Texas Southwestern Medical Center identifies a protein complex critical for regulating apoptosis and necroptosis — known as protein phosphatase 1 regulatory subunit 3G/protein phosphatase 1 gamma (PPP1R3G/PP1γ, or PPP1R3G complex). The researchers’ findings suggest that an inhibitor targeting this protein complex may help reduce or prevent excessive necroptosis.
“Cell death is very complicated process, which requires layers upon layers of brakes to prevent too many cells from dying,” said study principal investigator Dr. Wang, a member of the USF Health Heart Institute. “If you want to protect cells from excessive death, then the protein complex we identified in this study is one of many steps you must control.”
Dr. Wang and colleagues conducted experiments using human cells and a mouse model mimicking the cytokine storm seen in some patients with severe COVID-19 infection. They applied CRISPR genome-wide screening to analyze how cell function, in particular cell death, changes when one gene is knocked out (inactivated).
Receptor-interacting protein kinase (RIPK1) plays a critical role in regulating inflammation and cell death. Many sites on this protein are modified when a phosphate is added (a process known as phosphorylation) to suppress RIPK1’s cell death-promoting enzyme activity. How the phosphate is removed from RIPK1 sites (dephosphorylation) to restore cell death is poorly understood. Dr. Wang and colleagues discovered that PPP1R3G recruits phosphatase 1 gamma (PP1γ) to directly remove the inhibitory RIPK1 phosphorylations blocking RIPK1’s enzyme activity and cell death, thereby promoting apoptosis and necroptosis.
Dr. Wang uses the analogy of a car brake help explain what’s happening with the balance of cell survival and death in this study: RIPK1 is the engine that drives the cell death machine (the car). Phosphorylation applies the brake (stops the car) to prevent cells from dying. The car (cell death machinery) can only move forward if RIPK1 dephosphorylation is turned on by the PPP1R3G protein complex, which releases the brake.
“In this case, phosphorylation inhibits the cell death function of protein RIPK1, so more cells survive,” he said. “Dephosphorylation takes away the inhibition, allowing RIPK1 to activate its cell death function.”
The researchers showed that a specific protein-protein interaction — that is, PPP1R3G binding to PP1γ — activates RIPK1 and cell death. Furthermore, using a mouse model for “cytokine storm” in humans, they discovered knockout mice deficient in Ppp1r3g were protected against tumor necrosis factor-induced systemic inflammatory response syndrome. These knockout mice had significantly less tissue damage and a much better survival rate than wildtype mice with the same TNF-induced inflammatory syndrome and all their genes intact.
Overall, the study suggests that inhibitors blocking the PPP1R3G/PP1γ pathway can help prevent or reduce deaths and severe damage from inflammation-associated diseases, including heart disease, autoimmune disorders and COVID-19, Dr. Wang said. His laboratory is working with Jianfeng Cai, PhD, a professor in the USF Department of Chemistry, to screen and identify peptide compounds that most efficiently inhibit the PPP1R3G protein complex. They hope to pinpoint promising drug candidates that may stop the massive destruction of cardiac muscle cells caused by heart attacks.
High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport
by Alexander A. Shcherbakov, Peyton J. Spreacker, Aurelio J. Dregni, Katherine A. Henzler-Wildman, Mei Hong in Nature Communications
MIT chemists have discovered the structure of a protein that can pump toxic molecules out of bacterial cells. Proteins similar to this one, which is found in E. coli, are believed to help bacteria become resistant to multiple antibiotics.
Using nuclear magnetic resonance (NMR) spectroscopy, the researchers were able to determine how the structure of this protein changes as a drug-like molecule moves through it. Knowledge of this detailed structure may make it possible to design drugs that could block these transport proteins and help resensitize drug-resistant bacteria to existing antibiotics, says Mei Hong, an MIT professor of chemistry.
“Knowing the structure of the drug-binding pocket of this protein, one might try to design competitors to these substrates, so that you could block the binding site and prevent the protein from removing antibiotics from the cell,” says Hong, who is the senior author of the paper.
MIT graduate student Alexander Shcherbakov is the lead author of the study. The research team also includes MIT graduate student Aurelio Dregni and two researchers from the University of Wisconsin at Madison: graduate student Peyton Spreacker and professor of biochemistry Katherine Henzler-Wildman.
Pumping drugs out through their cell membranes is one of many strategies that bacteria can use to evade antibiotics. For several years, Henzler-Wildman’s group at the University of Wisconsin has been studying a membrane-bound protein called EmrE, which can transport many different toxic molecules, including herbicides and antimicrobial compounds. EmrE belongs to a family of proteins called the small multidrug resistance (SMR) transporters. Although EmrE is not directly involved in resistance to antibiotics, other members of the family have been found in drug-resistant forms of Mycobacterium tuberculosis and Acinetobacter baumanii.
“The SMR transporters have high sequence conservation across key regions of the protein. EmrE is by far the best-studied member of the family, both in vitro and in vivo, which makes it an ideal model system to investigate the structure that supports SMR activity,” Henzler-Wildman says.
A few years ago, Hong’s lab developed a technique that allows researchers to use NMR to measure the distances between fluorine probes and hydrogen atoms in proteins. This makes it possible to determine the structure of a protein as it binds to a molecule that contains fluorine. After Hong gave a talk about the new technique at a conference, Henzler-Wildman suggested that they team up to study EmrE. Her lab has spent many years studying how EmrE transports a drug-like molecule, or ligand, across the phospholipid membrane. This ligand, known as F4-TPP+, is a tetrahedral molecule with four fluorine atoms attached to it, one at each corner.
Using this ligand with Hong’s new NMR technique, the researchers set out to determine an atomic-resolution structure of EmrE. It was already known that each EmrE molecule contains four transmembrane helices that are roughly parallel. Two EmrE molecules assemble into a dimer, so that eight transmembrane helices form inner walls that interact with the ligand as it moves through the channel. Previous studies have revealed the overall topology of the helices, but not of the protein side chains that extend into the channel interior, which are like arms that grab the ligand and help guide it through the channel.
EmrE transports toxic molecules from the inside of a bacterial cell, which is at neutral pH, to the outside, which is acidic. This change in pH across the membrane affects the structure of EmrE. In a 2021 paper, Hong and Henzler-Wildman discovered the structure of the protein as it binds to F4-TPP+ in an acidic environment. In the new study, they analyzed the structure at a neutral pH, allowing them to determine how the structure of the protein changes as the pH changes.
At neutral pH, the researchers found in this study, the four helices that make up the channel are relatively parallel to one another, creating an opening that the ligand can easily enter. As the pH drops, moving toward the outside of the membrane, the helices begin to tilt so that the channel is more open toward the outside of the cell. This helps to push the ligand out of the channel. At the same time, several rings found in the protein side chains shift their orientation in a way that also helps to guide the ligand out of the channel.
The acidic end of the channel is also more welcoming to protons, which enter the channel and help it to open further, allowing the ligand to exit more easily.
“This paper really completes the story,” Hong says. “One structure is not enough. You need two, to figure out how a transporter can actually open to both sides of the membrane, because it’s supposed to pump the ligand or the antibiotic compound from inside the bacteria out of the bacteria.”
The EmrE channel is believed to transport many different toxic compounds, so Hong and her colleagues now plan to study how other molecules travel through the channel.
SARM1 Ablation Is Protective and Preserves Spatial Vision in an In Vivo Mouse Model of Retinal Ganglion Cell Degeneration
by Laura K. Finnegan, Naomi Chadderton, Paul F. Kenna, Arpad Palfi, Michael Carty, Andrew G. Bowie, Sophia Millington-Ward, G. Jane Farrar in International Journal of Molecular Sciences
Developing therapies for genetic forms of blindness is extremely challenging, in part because they vary so widely, but scientists from Trinity College Dublin have highlighted a target with great promise for treating a range of these conditions.
The scientists have highlighted that a specific gene (SARM1) is a key driver in the damage that ultimately leads to impaired vision (and sometimes blindness), and — in a disease model — showed that deleting this gene protects vision after a chemical kick-starts the chain of dysfunction that mimics a host of ocular conditions. This means that therapies targeting suppression of SARM1 activity may hold the key to effective new options for treating a suite of diseases that can have a devastating impact on quality of life, and for many of which there are no treatment options currently available.
First author on the paper, Laura Finnegan, a PhD Candidate at Trinity, said:
“In response to injury SARM1 contributes to a process that leads to the degeneration of specialised cells and their axons in the eye. When this happens it essentially means that the optic nerve can no longer deliver signals from the eye to the brain.
“Impaired vision and blindness is extremely debilitating for millions of people across the globe, which is one of the main motivations for us to seek to better understand the genetic causes and, potentially, develop life-changing therapies.”
Jane Farrar, Professor in Trinity’s School of Genetics and Microbiology, senior author on the paper, said:
“Another important finding was that visual function was still preserved when reassessed four months after SARM1 was deleted, indicating that the benefits can remain over time. This raises hopes that a targeted therapy delivered early enough may offer people diagnosed with an ocular neuropathy long-lasting preservation of sight.
“We have a way to go before such a therapy is available but this work represents a significant step, sheds light on the pathway forward and offers hope that a range of diseases involving the optic nerve — from maternally inherited conditions such as Leber Hereditary Optic Neuropathy to the more commonly known glaucoma — will one day be treatable via such therapies.”
Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding
by Nikol Kaderabkova, R Christopher D Furniss, et al in eLife
A team of researchers led by Despoina Mavridou of The University of Texas at Austin found a new way to impair antibiotic resistance in bacteria that cause human disease, including E. coli, K. pneumoniae and P. aeruginosa, which are responsible for the majority of harm caused by resistant infections. The team made the bacteria vulnerable again to antibiotics by inhibiting a particular protein that drives the formation of resistance capabilities within the bacteria.
“It’s a completely new way of thinking about targeting resistance,” said Mavridou, an assistant professor of molecular biosciences.
Bacteria are becoming increasingly resistant to existing antibiotics, and researchers have struggled to identify new bacteria-fighting drugs, leaving the world vulnerable to deadly superbugs.
Antibiotic resistant bacteria have a host of different proteins in their arsenals that neutralize antibiotics. To function properly, these resistance proteins must be folded into the right shapes. The researchers discovered that yet another protein, called DsbA, helps fold resistance proteins into those shapes. For their proof-of-concept study, Mavridou and her fellow scientists inhibited DsbA using chemicals that cannot be used directly in human patients. The team plans now to work on developing inhibitors that can achieve the same outcome and be safely used in humans.
“Other approaches focus on inhibiting resistance proteins, but nobody had thought to try and prevent their formation in the first place,” Mavridou said.
Their goal is to combine a DsbA inhibitor with existing antibiotics to restore the drugs’ ability to kill bacteria. Because it targets the machinery that helps assemble antibiotic-resistance proteins in dangerous bacteria, the approach would render several types of proteins critical for resistance ineffective by preventing their ability to fold or create disulfide bonds.
“Since the discovery of new antibiotics is challenging, it is crucial to develop ways to prolong the lifespan of existing antimicrobials,” said Christopher Furniss, one of the lead authors of this study at Imperial College London. “Our findings show that by targeting disulfide bond formation and protein folding, it is possible to reverse antibiotic resistance across several major pathogens and resistance mechanisms. This means that the development of clinically useful DsbA inhibitors in the future could offer a new way to treat resistant infections using currently available antibiotics.”
DsbA is mostly a house-keeping protein in bacteria that promotes protein stability and folding. Before this study, scientists already knew that DsbA is also involved in a range of functions in pathogens, such as helping build toxins that attack host cells, or assisting with the assembly of needle-like systems that can deliver these toxins into human cells and cause disease. But Mavridou, who studied DsbA for many years, suspected that it might also play an important role in the folding of proteins that help bacteria resist antibiotics. She started investigating this possibility while at Imperial College London, before joining the UT Austin faculty in 2020.
Generation of a tendon-like tissue from human iPS cells
by Hiroki Tsutsumi, Ryota Kurimoto, Ryo Nakamichi, Tomoki Chiba, Takahide Matsushima, Yuta Fujii, Risa Sanada, Tomomi Kato, Kana Shishido, Yuriko Sakamaki, Tsuyoshi Kimura, Akio Kishida, Hiroshi Asahara in Journal of Tissue Engineering
Tendons are tissues that connect muscles to bones and are important for movement and locomotion. Injuries to tendons are quite common, with millions of people — particularly athletes — affected worldwide, and can often take many months to recover from, significantly impacting quality of life. Furthermore, while many options for treatment exist, none of them are perfect cures and many result in pain, immunogenicity, or long-term treatment failure. Therefore, a novel therapeutic strategy for tendon repair is needed.
In a study, researchers from Tokyo Medical and Dental University (TMDU) have successfully induced human stem cells to create artificial tendon-like tissue that mimics tendon properties and offers significantly improved tendon reconstruction in a mouse tendon-rupture model.
Human induced pluripotent stem cells, or hiPSCs, are special stem cells that can be derived from any adult cells and can be differentiated into any specialized cell-type. “Using hiPSCs with Mohawk (Mkx), we could produce artificial tendon tissue.” explains Hiroki Tsutsumi, lead author of the study. Mohawk is a transcription factor that promotes the expression of genes involved in tendon-formation and thus drives differentiation of stem cells into tendon cells. These Mohawk-expressing stem cells were then put in a specialized 3D culture system that exerts mechanical force on the cells while they are growing. This simulates the conditions for tendon development and enhances the cell alignment and organization, allowing them to create tendon-like tissues.
Next, the research team tested the artificial tendon in a mouse model of tendon rupture. The results were exciting. Six weeks after the implantation, the artificial tendon had similar mechanical properties to a normal undamaged mouse tendon. In addition, the implanted tendon-like tissue was able to recruit and mobilize tendon cells from the host that can further participate in the repair process. This confirmed a good integration of the tissue.
“We demonstrated that the bio-tendons derived from human induced pluripotent stem cells have similar mechanical and biological properties to normal tendons and can be fully integrated relatively quickly after a transplant surgery in a mouse model, making them an attractive strategy for clinical application in tendon injuries. The next step towards clinical translation would be to test them in large animal models to assess their capacity as a biomaterial on a larger scale,” concludes Hiroshi Asahara, lead author of the study. These promising results suggest that a novel medical strategy for tendon repair may be clinically available in the future.
Sex differences in immune gene expression in the brain of a small shorebird
by José O. Valdebenito, Kathryn H. Maher, Gergely Zachár, Qin Huang, Zhengwang Zhang, Larry J. Young, Tamás Székely, Pinjia Que, Yang Liu, Araxi O. Urrutia in Immunogenetics
Whilst human males tend to suffer more than females from infectious diseases like Covid19 or flu, for birds it’s the males that appear to have stronger immune systems, suggests a new study led by the University of Bath.
A team of international scientists led by the University of Bath looked at differences between the sexes in the expression, or activity, of immune genes in Kentish Plovers, a common species of shorebird that live on coasts and lakes all over the world. The researchers studied populations living in coastal and high-altitude inland areas in China. Whilst they found no significant differences in immunity between the birds from the two habitats, they found evidence of a difference between the immune systems of male and female birds.
In humans, males have an X and a Y chromosome whereas females have two copies of the X chromosome, however only one of these two X chromosomes is activated, meaning both males and females have just one active X chromosome.
In contrast, in birds it’s the males that have two copies of the sex chromosome Z, with females having two different chromosomes, Z and W. However in males, both copies of the Z chromosome are active. Many of the genes linked with immunity are on the Z chromosome, so the researchers suggest that by having two active copies of these genes, males might have increased activity in their immune systems, resulting in lower mortality compared with females.
Dr José Valdebenito, Research Associate from the Milner Centre for Evolution at the University of Bath, said: “Whilst in humans women tend to live longer than men, the opposite is true of birds, so we wanted to find out why.
“For the first time, we’ve found evidence that there is a difference in the activity of the immune genes in male and female plovers, suggesting that males have stronger immune systems which could explain why they tend to survive longer than females. “In plovers, the higher mortality in females causes an imbalance in the sex ratio, which has a knock-on effect on the mating and parenting behaviours of this species.
“Currently we are working on a project that investigates the relationship between sex differences in immunity and mating system variation, by looking at the immune genes across several shorebird species. “Hopefully this will help us expand our understanding on the drivers behind sex differences in mortality in birds.”
Dr Araxi Urrutia, Senior Lecturer from the Milner Centre for Evolution and senior author on the paper, said: “This was an exciting project, and I was glad to coordinate fieldwork, data analyses and writing up of this joint project between the Milner Centre, Chinese and Hungarian scientists.
“The next step is to expand this approach to other shorebirds. From demographic data we know that in some shorebirds the males live longer than the females whereas in other shorebirds the male lives longer than the females.
“My group is striving to understand the genomic causes of the sex different mortalities — this work is important not only for fundamental science but also biodiversity conservation since these data will help protecting these species in their natural habitats.”
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