Tag Archives: DNA

Microbiology

Vaginal sex can shape the composition of urethral microbiome in healthy men

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Reviewed by Emily Henderson, B.Sc.Mar 21 2023

Contrary to common beliefs, your urine is not germ free. In fact, a new study shows that the urethra of healthy men is teeming with microbial life and that a specific activity-;vaginal sex-;can shape its composition. The research, published March 24 in the journal Cell Reports Medicine, provides a healthy baseline for clinicians and scientists to contrast between healthy and diseased states of the urethra, an entrance to the urinary and reproductive systems.

We know where bugs in the gut come from; they primarily come from our surroundings through fecal-oral transfer. But where does genital microbiology come from?”

David Nelson, co-senior author, microbiologist at Indiana University

To flush out the answer, the team of microbiologists, statisticians, and physicians sequenced the penile urethra swabs of 110 healthy adult men. These participants had no urethral symptoms or sexually transmitted infections (STIs) and no inflammation of the urethra. DNA sequencing results revealed that two types of bacterial communities call the penile urethra home-;one native to the organ, the other from a foreign source.

“It is important to set this baseline,” says co-senior author Qunfeng Dong, a bioinformatician at Loyola University Chicago. “Only by understanding what health is can we define what diseases are.”

The researchers found that most of the healthy men had a simple, sparse community of oxygen-loving bacteria in the urethra. In addition, these bacteria probably live close to the urethral opening at the tip of the penis, where there is ample oxygen. The consistent findings of these bacteria suggest that they are the core community that supports penile urethra health.

But some of the men also had a more complex secondary group of bacteria that are often found in the vagina and can disturb the healthy bacterial ecosystem of the vagina. The team speculates that these bacteria reside deeper in the penile urethra because they thrive in oxygen-scarce settings. Only men who reported having vaginal sex carry these bacteria, hinting at the microbes’ origins.

Delving into the participant’s sexual history, the team found a close link between this second bacterial community and vaginal sex but not other sexual behaviors, such as oral sex and anal sex. They also found evidence that vaginal sex has lasting effects. Vagina-associated bacteria remained detectable in the participants for at least two months after vaginal sex, indicating that sexual exposure to the vagina can reshape the male urinary-tract microbiome.

“In our study, one behavior explains 10% of the overall bacterial variation,” says Nelson, when discussing the influence of vaginal sex. “The fact that a specific behavior is such a strong determinant is just profound.”

Although current findings from the study show that vaginal bacteria can spread to the penile urethra, the team’s next plan is to test whether the reverse is true. Using the newly established baseline, the researchers also hope to offer new insights into bacteria’s role in urinary- and reproductive-tract diseases, including unexplained urethral inflammation and STIs.

“STIs really impact people who are socioeconomically disadvantaged; they disproportionately impact women and minorities,” says Nelson. “It’s a part of health care that’s overlooked because of stigma. I think our study has a potential to dramatically change how we handle STI diagnosis and management in a positive way.”

This work was supported by the National Institute of Allergy and Infectious Diseases.

Source:

Cell Press

Journal reference:

Toh, E., et al. (2023). Sexual behavior shapes male genitourinary microbiome composition. Cell Reports Medicine. doi.org/10.1016/j.xcrm.2023.100981

Microbiology

Avanced genome editing technology could be used as a one-time treatment for CD3 delta SCID

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Reviewed by Emily Henderson, B.Sc.Mar 21 2023

A new UCLA-led study suggests that advanced genome editing technology could be used as a one-time treatment for the rare and deadly genetic disease CD3 delta severe combined immunodeficiency.

The condition, also known as CD3 delta SCID, is caused by a mutation in the CD3D gene, which prevents the production of the CD3 delta protein that is needed for the normal development of T cells from blood stem cells.

Without T cells, babies born with CD3 delta SCID are unable to fight off infections and, if untreated, often die within the first two years of life. Currently, bone marrow transplant is the only available treatment, but the procedure carries significant risks.

In a study published in Cell, the researchers showed that a new genome editing technique called base editing can correct the mutation that causes CD3 delta SCID in blood stem cells and restore their ability to produce T cells.

The potential therapy is the result of a collaboration between the laboratories of Dr. Donald Kohn and Dr. Gay Crooks, both members of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA and senior authors of the study.

Kohn’s lab has previously developed successful gene therapies for several immune system deficiencies, including other forms of SCID. He and his colleagues turned their attention to CD3 delta SCID at the request of Dr. Nicola Wright, a pediatric hematologist and immunologist at the Alberta Children’s Hospital Research Institute in Canada, who reached out in search of a better treatment option for her patients.

CD3 delta SCID is prevalent in the Mennonite community that migrates between Canada and Mexico.

Because newborns are not screened for SCID in Mexico, I often see babies who have been diagnosed late and are returning to Canada quite sick.”

Dr. Nicola Wright, pediatric hematologist and immunologist at the Alberta Children’s Hospital Research Institute

When Kohn presented Wright’s request to his lab, Grace McAuley, then a research associate who joined the lab at the end of her senior year at UCLA, stepped up with a daring idea.

“Grace proposed we try base editing, a very new technology my lab had never attempted before,” said Kohn, a distinguished professor of microbiology, immunology and molecular genetics, and of pediatrics.

Base editing is an ultraprecise form of genome editing that enables scientists to correct single-letter mutations in DNA. DNA is made up of four chemical bases that are referred to as A, T, C and G; those bases pair together to form the “rungs” in DNA’s double-helix ladder structure.

While other gene editing platforms, like CRISPR-Cas9, cut both strands of the chromosome to make changes to DNA, base editing chemically changes one DNA base letter into another -; an A to a G, for example -; leaving the chromosome intact.

“I had a very steep learning curve in the beginning, when base editing just wasn’t working,” said McAuley, who is now pursuing an M.D.-Ph.D. at UC San Diego and is the study’s co-first author. “But I kept pushing forward. My goal was help get this therapy to the clinic as fast as was safely possible.”

McAuley reached out to the Broad Institute’s David Liu, the inventor of base editing, for advice on how to evaluate the technique’s safety for this particular use. Eventually, McAuley identified a base editor that was highly efficient at correcting the disease-causing genetic mutation.

Because the disease is extremely rare, obtaining patient stem cells for the UCLA study was a significant challenge. The project got a boost when Wright provided the researchers with blood stem cells donated by a CD3 delta SCID patient who was undergoing a bone marrow transplant.

The base editor corrected an average of almost 71% of the patient’s stem cells across three laboratory experiments.

Next, McAuley worked with Dr. Gloria Yiu, a UCLA clinical instructor in rheumatology, to test whether the corrected cells could give rise to T cells. Yiu used artificial thymic organoids, which are stem cell-derived tissue models developed by Crooks’ lab that mimic the environment of the human thymus -; the organ where blood stem cells become T cells.

When the corrected blood stem cells were introduced into the artificial thymic organoids, they produced fully functional and mature T cells.

“Because the artificial thymic organoid supports the development of mature T cells so efficiently, it was the ideal system to show that base editing of patients’ stem cells could fix the defect seen in this disease,” said Yiu, who is also a co-first author of the study.

As a final step, McAuley studied the longevity of the corrected stem cells by transplanting them into a mouse. The corrected cells remained four months after transplant, indicating that base editing had corrected the mutation in true, self-renewing blood stem cells. The findings suggest that corrected blood stem cells could persist long-term and produce the T cells patients would need to live healthy lives.

“This project was a beautiful picture of team science, with clinical need and scientific expertise aligned,” said Crooks, a professor of pathology and laboratory medicine. “Every team member played a vital role in making this work successful.”

The research team is now working with Wright on how to bring the new approach to a clinical trial for infants with CD3 delta SCID from Canada, Mexico and the U.S.

This research was funded by the Jeffrey Modell Foundation, the National Institutes of Health, the Bill and Melinda Gates Foundation, the Howard Hughes Medical Institute, the V Foundation and the A.P. Giannini Foundation.

The therapeutic approach described in this article has been used in preclinical tests only and has not been tested in humans or approved by the Food and Drug Administration as safe and effective for use in humans. The technique is covered by a patent application filed by the UCLA Technology Development Group on behalf of the Regents of the University of California, with Kohn and McAuley listed as co-inventors.

Source:

University of California – Los Angeles Health Sciences

Journal reference:

McAuley, G.E., et al. (2023) Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing. Cell. doi.org/10.1016/j.cell.2023.02.027.

Microbiology

The ‘Rapunzel’ virus: an evolutionary oddity

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A recent study in the Journal of Biological Chemistry has revealed the secret behind an evolutionary marvel: a bacteriophage with an extremely long tail. This extraordinary tail is part of a bacteriophage that lives in inhospitable hot springs and preys on some of the toughest bacteria on the planet.

Bacteriophages are a group of viruses that infect and replicate in bacteria and are the most common and diverse things on Earth.

“Bacteriophages, or phages for short, are everywhere that bacteria are, including the dirt and water around you and in your own body’s microbial ecosystem as well,” said Emily Agnello, a graduate student at the University of Massachusetts Chan Medical School and the lead author on the study.

Unlike many of the viruses that infect humans and animals that contain only one compartment, phages consist of a tail attached to a spiky, prismlike protein shell that contains their DNA.

Phage tails, like hairstyles, vary in length and style; some are long and bouncy while others are short and stiff. While most phages have short, microscopic tails, the “Rapunzel bacteriophage” P74-26 has a tail 10 times longer than most and is nearly 1 micrometer long, about the width of some spider’s silk. The “Rapunzel” moniker is derived from the fairy tale in which a girl with extremely long hair was locked in a tower by an evil witch.

Brian Kelch, an associate professor of biochemistry and molecular biotechnology at UMass Chan who supervised the work, described P74-26 as having a “monster of a tail.”

Phage tails are important for puncturing bacteria, which are coated in a dense, viscous substance. P74-26’s long tail allows it to invade and infect the toughest bacteria. Not only does P74-26 have an extremely long tail, but it is also the most stable phage, allowing it to exist in and infect bacteria that live in hot springs that can reach over 170° F. Researchers have been studying P74-26 to find out why and how it can exist in such extreme environments.

To work with a phage that thrives in such high temperatures, Agnello had to adjust the conditions of her experiments to coax the phage tail to assemble itself in a test tube. Kelch said Agnello created a system with which she could induce rapid tail self-assembly.

“Each phage tail is made up of many small building blocks that come together to form a long tube. Our research finds that these building blocks can change shape, or conformation, as they come together,” Agnello said. “This shape-changing behavior is important in allowing the building blocks to fit together and form the correct structure of the tail tube.”

The researchers used high-power imaging techniques as well as computer simulations and found that the building blocks of the tail lean on each other to stabilize themselves.

“We used a technique called cryo-electron microscopy, which is a huge microscope that allows us to take thousands of images and short movies at a very high magnification,” Agnello explained. “By taking lots of pictures of the phage’s tail tubes and stacking them together, we were able to figure out exactly how the building blocks fit together.”

They found P74-26 uses a “ball and socket” mechanism to sturdy itself. In addition, the tail is formed from vertically stacking rings of molecules that make a hollow canal.

“I like to think about these phage building blocks as kind of like Legos,” Kelch said. “The Lego has studs on one side and the holes or sockets on the other.”

He added: “Imagine a Lego where the sockets start off closed. But as you start to build with the Legos, the sockets begin to open up to allow the studs on other Legos to build a larger assembly. This movement is an important way that these phage building blocks self-regulate their assembly.”

Kelch pointed out that, compared with most phages, P74-26 uses half the number of building blocks to form stacking rings that make up the tail.

“We think what has happened is that some ancient virus fused its building blocks into one protein. Imagine two small Lego bricks are fused into one large brick with no seams. This long tail is built with larger, sturdier building blocks,” Kelch explained. “We think that could be stabilizing the tail at high temperatures.”

The researchers now plan to use genetic manipulation to alter the length of the phage tail and see how that changes its behavior.

Phages occupy almost every corner of the globe and are important to a variety of industries like healthcare, environmental conservation and food safety. In fact, long-tailed phages like P74-26 have been used in preliminary clinical trials to treat certain bacterial infections.

“Bacteriophages are gaining ever-growing interest as an alternative to antibiotics for treating bacterial infections,” Agnello said. “By studying phage assembly, we can better understand how these viruses interact with bacteria, which could lead to the development of more effective phage-based therapies. … I believe that studying unique, interesting things can lead to findings and applications that we can’t even yet imagine.”

  • Emily Agnello, Joshua Pajak, Xingchen Liu, Brian A. Kelch. Conformational dynamics control assembly of an extremely long bacteriophage tail tube. Journal of Biological Chemistry, 2023; 103021 DOI: 10.1016/j.jbc.2023.103021

    • American Society for Biochemistry and Molecular Biology
    Microbiology

    Researchers Decipher the Inner Workings of a Bacterium

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    A group led by Professor Ralf Rabus, a microbiologist at the University of Oldenburg, and his Ph.D. student Patrick Becker has made significant advancements in comprehending the cellular processes of a widespread environmental bacterium. The team conducted an extensive analysis of the entire metabolic network of the bacterial strain Aromatoleum aromaticum EbN1T and utilized the findings to construct a metabolic model that allows them to forecast the growth of these microbes in various environmental conditions.

    According to their report in the journal mSystems, the researchers uncovered surprising mechanisms that enable the bacteria to adjust to fluctuating environmental conditions. These results are crucial for the study of ecosystems, where the Aromatoleum strain, as a representative of a significant group of environmental bacteria, can act as a model organism. The findings could also have implications for the cleanup of contaminated sites and biotechnological applications.

    The studied bacterial strain specializes in the utilization of organic substances that are difficult to break down and is generally found in soil and in aquatic sediments. The microbes thrive in a variety of conditions including oxygen, low-oxygen, and oxygen-free layers, and are also extremely versatile in terms of nutrient intake. They metabolize more than 40 different organic compounds including highly stable, naturally occurring substances such as components of lignin, the main structural material found in wood, and long-lived pollutants and components of petroleum.

    In particular, substances with a benzene ring composed of six carbon atoms, known as aromatic compounds, can be biodegraded by these microbes – with or without the aid of oxygen. Due to these abilities, Aromatoleum plays an important environmental role in the complete degradation of organic compounds in soil and sediments to carbon dioxide – a process which is also useful in biological soil remediation.

    The aim of the current study was to gain a holistic understanding of the functioning of this unicellular organism. To this end, the researchers cultivated the microbes under both oxic and anoxic conditions – i.e. with and without oxygen – using five different nutrient substrates. For each of these ten different growth conditions, they grew 25 cultures and then examined the various samples using molecular biology methods (technical term: multi-omics) which enable simultaneous analysis of all the transcribed genes in a cell, all the proteins produced, and all its metabolic products.

    “With this systems biology approach, you gain a deep understanding of all the inner workings of an organism,” explains Rabus, who heads the General and Molecular Microbiology research group at the University of Oldenburg’s Institute for Chemistry and Biology of the Marine Environment (ICBM). “You break down the bacterium into its individual components and then you can put them back together – in a model that predicts how fast a culture will grow and how much biomass it will produce.”

    Through their meticulous work, the researchers obtained a comprehensive understanding of the metabolic reactions of this bacterial strain. They found that around 200 genes are involved in the degradation processes and determined which enzymes break down the substances added as nutrients and via which intermediates the various nutrients are decomposed. The scientists incorporated their findings about the metabolic network into a growth model, and demonstrated that the model predictions largely corresponded to the measured data.

    “We can now describe the organism with a level of precision that has so far only been possible with very few other bacteria,” says Rabus. This holistic view of the bacteria’s cellular inner workings forms the basis for a better understanding of the interactions between the analyzed strain (and related bacteria) and their biotic and abiotic environment, he adds, and can also help scientists to better predict the activity of these unicellular organisms in polluted soils and thus, for example, determine the optimal conditions for the remediation of a contaminated site.

    By combining different methods, the team was able to uncover unexpected mechanisms in the metabolism of these bacteria. Much to the researchers’ surprise, it emerged that the microbe produces several enzymes which they cannot use under the given growth conditions – which at first glance would seem to be a superfluous expenditure of energy. “Usually the bacterial cells detect whether oxygen is present in their environment and then, via specific mechanisms, activate only the nutrient-specific metabolic pathway with the corresponding enzymes,” Rabus explains.

    But with some substrates, the microbe produced all the enzymes for aerobic and anaerobic degradation pathways regardless of oxygen levels – even though some of these enzymes were entirely superfluous. Rabus suspects that this apparent waste is in fact a strategy for surviving in an unstable environment: “Even if oxygen levels suddenly fluctuate – which is often the case in natural environments – Aromatoleum remains flexible and can utilize this nutrient and produce energy as required,” the microbiologist explains, adding that so far, no other bacteria are known to use such a mechanism.

    Reference: “Systems Biology of Aromatic Compound Catabolism in Facultative Anaerobic Aromatoleum aromaticum EbN1T” by Patrick Becker, Sarah Kirstein, Daniel Wünsch, Julia Koblitz, Ramona Buschen, Lars Wöhlbrand, Boyke Bunk and Ralf Rabus, 29 November 2022, mSystems.
    DOI: 10.1128/msystems.00685-22

    University of Oldenburg

    Microbiology

    New discoveries made regarding autism onset in mouse models

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    Reviewed by Emily Henderson, B.Sc.Mar 10 2023

    Although autism is a common neurodevelopmental disorder, the multiple factors behind its onset are still not fully understood. Animal models of idiopathic autism, especially mice, are often used to help researchers understand the complicated mechanisms behind the disorder, with BTBR/J being the most commonly used mouse model in the world.

    Now, an international research collaboration including Kobe University’s Professor TAKUMI Toru and Researcher Chia-wen Lin et al. have made new discoveries regarding autism onset in mouse models.

    In their detailed series of experiments and analyses of BTBR/J mice and the other subspecies BTBR/R, they revealed that endogenous retrovirus activation increases a fetus’s susceptibility to autism. They also discovered that BTBR/R exhibits autistic-like behaviors without reduced learning ability, making it a more accurate model of autism than the widely-used BTBR/J model.

    It is hoped that further research will contribute towards better classification of autism types, as well as the creation of new treatment strategies for neurodevelopmental disorders.

    These research results were published in Molecular Psychiatry on March 7, 2023

    Main points

    • The researchers analyzed BTBR/J, a widely used mouse model of autism, and its subspecies BTBR/Rusing MRI. This revealed that the corpus callosum, which connects the left and right hemispheres of the brain, was impaired in BTBR/J mice but not in BTBR/R mice.
    • Genome and transcription analysis showed that BTBR mice have increased levels of endogenous retrovirus genes.
    • Furthermore, single-cell RNA analysis of BTBR/R mice revealed changes in the expression of various genes (including stress response genes) that are indicative of endogenous retrovirus activation.
    • Even though BTBR/J and BTBR/R mice have the same ancestry, the results of various behavioral analysis experiments revealed differences in spatial learning ability and other behaviors between the two types of model mice.

    Research background

    Autism (autism spectrum disorder) is a neurodevelopmental disorder that remains largely unexplored despite the rapidly increasing number of patients. Reasons for this continuing increase in people diagnosed with autism include changes to diagnostic criteria and older fathers becoming more common. Autism is strongly related to genetic factors and can be caused by abnormalities in DNA structure, such as copy number variations. Animal models, especially mice, are often used in research to illuminate the pathology of autism. Among these models, BTBR/J is a mouse model of the natural onset of autism that is commonly used. Studies have reported various abnormalities in BTBR/J mice including impairment of the corpus callosum (which connects the left and right hemispheres of the brain) and excessive immune system signaling. However, it is not fully understood why this particular lineage displays autistic-like behavioral abnormalities.

    The aim of the current study was to shed light on the onset mechanism of these autistic-like behavioral abnormalities by conducting comparative analysis on BTBR/J and its subspecies BTBR/R.

    Research findings

    First of all, the researchers conducted MRI scans on BTBR/J and BTBR/R mice to investigate structural differences in each region of the brain. The results revealed that there were differences between BTBR/J and BTBR/R mice in 33 regions including the amygdala. A particularly prominent difference discovered was that even though BTBR/J’s corpus callosum is impaired, BTBR/R’s is normal.

    Next, the research group used the array CGH method to compare BTBR/R’s copy number variations with that of a normal mouse model (B6). They revealed that BTBR/R mice had significantly increased levels of endogenous retroviruses (ERV) in comparison to B6 mice. Furthermore, qRT-PCR tests revealed that these retroviruses were activated in BTBR/R mice. On the other hand, in B6 mice there was no change in the expression of LINE ERV (which is classified in the same repetitive sequence), indicating that this retroviral activation is specific to BTBR.

    Subsequently, the researchers carried out single-cell RNA analysis on the tissue of embryonic BTBR mice (on the AGM and yolk sac). The results provide evidence of ERV activation in BTBR mice, as expression changes were observed in a group of genes downstream of ERV.

    Lastly, the researchers comprehensively investigated the differences between BTBR/J and BTBR/R on a behavioral level. BTBR/R mice were less anxious than BTBR/J and showed qualitative changes in ultrasound vocalizations, which are measured as a way to assess communicative ability in mice. BTBR/R mice also exhibited more self-grooming behaviors and buried more marbles in the marble burying test. These two tests were designed to detect repetitive behavioral abnormalities in autistic individuals. From the results, it was clear that BTBR/R exhibits more repetitive behaviors (i.e. it is more symptomatic) than BTBR/J. The 3-chamber social interaction test, which measures how closely a mouse will approach another mouse, also revealed more pronounced social deficits in BTBR/R than BTBR/J mice (Figure 4i). In addition, a Barnes maze was used to conduct a spatial learning test, in which BTBR/J mice exhibited reduced learning ability compared to B6 (normal mice). BTBR/R mice, on the other hand, exhibited similar ability to B6.

    Overall, the study revealed that retrovirus activation causes the copy number variants in BTBR mice to increase, which leads to the differences in behavior and brain structure seen in BTBR/J and BTBR/R mice (Figure 5).

    Further developments

    BTBR/J mice are widely used by researchers as a mouse model of autism. However, the results of this study highlight the usefulness of the other lineage of BTBR/R mice because they exhibit autistic-like behavior without compromised spatial learning ability. The results also suggest that it may be possible to develop new treatments for autism that suppress ERV activation. Furthermore, it is necessary to classify autism subtypes according to their onset mechanism, which is a vital first step towards opening up new avenues of treatment for autism.

    Source:

    Kobe University

    Journal reference:

    Lin, C-W., et al. (2023) An old model with new insights: endogenous retroviruses drive the evolvement toward ASD susceptibility and hijack transcription machinery during development. Molecular Psychiatry. doi.org/10.1038/s41380-023-01999-z.

    Microbiology

    Antibiotics can destroy many types of bacteria, but increasingly, bacterial pathogens are gaining resistance to many commonly used …

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    Antibiotics can destroy many types of bacteria, but increasingly, bacterial pathogens are gaining resistance to many commonly used types. As the threat of antibiotic resistance looms large, researchers have sought to find new antibiotics and other ways to destroy dangerous bacteria. But new antibiotics can be extremely difficult to identify and test. Bacteriophages, which are viruses that only infect bacterial cells, might offer an alternative. Bacteriophages (phages) were studied many years ago, before the development of antibiotic drugs, and they could help us once again.

    Image credit: Pixabay

    If we are going to use bacteriophages in the clinic to treat humans, we should understand how they work, and how bacteria can also become resistant to them. Microbes are in an arms race with each other, so while phages can infect bacteria, some bacterial cells have found ways to thwart the effects of those phages. New research reported in Nature Microbiology has shown that when certain bacteria carry a specific genetic mutation, phages don’t work against them anymore.

    In this study, the researchers used a new technique so they could actually see a phage attacking bacteria. Mycobacteriophages infect Mycobacterial species, including the pathogens Mycobacterium tuberculosis and Mycobacterium abscessus, as well as the harmless Mycobacterium smegmatis, which was used in this research.

    The scientists determined that Mycobacterial gene called lsr2 is essential for many mycobacteriophages to successfully infect Mycobacteria. Mycobacteria that carry a mutation that renders the Lsr2 protein non-functional are resistant to these phages.

    Normally, Lsr2 aids in DNA replication in bacterial cells. Bacteriophages can harness this protein, however, and use it to reproduce the phage’s DNA. Thus, when Lsr2 stops working, the phage cannot replicate and it cannot manipulate bacterial cells.

    In the video above, by first study author Charles Dulberger, a genetically engineered mutant phage infects Mycobacterium smegmatis. First, one phage particle (red dot at 0.42 seconds) binds to a bacterium. The phage DNA (green fluorescence) is injected into the bacterial cell (2-second mark). The bright green dots at the cells’ ends are not relevant. For a few seconds, the DNA forms a zone of phage replication, and fills the cell. Finally, the cell explodes at 6:25 seconds. (About three hours have been compressed to make this video.)

    The approach used in this study can also be used to investigate other links between bacteriophages and the bacteria they infect.

    “This paper focuses on just one bacterial protein,” noted co-corresponding study author Graham Hatfull, a Professor at the University of Pittsburgh. But there are many more opportunities to use this technique. “There are lots of different phages and lots of other proteins.”

    Sources: University of Pittsburgh, Nature Microbiology


    Carmen Leitch

    Microbiology

    The SARS-CoV-2 virus and the illness it causes, COVID-19, have made an indelible mark on our lives. It …

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    The SARS-CoV-2 virus and the illness it causes, COVID-19, have made an indelible mark on our lives. It seems that is also true in more ways than one; new research has shown that when the virus infects cells, portions of the viral genome integrate into the genome of host cells in a phenomenon known as reverse transcription. While this is a relatively rare even for SARS-CoV-2, so many people have been infected with the virus that integration has probably happened many times. Scientists have now used several techniques to show that SARS-CoV-2 can integrate into a host cell genome, and the findings have been reported in the journal Viruses. This study is confirmation of previous work reported in the Proceedings of the National Academy of Sciences in 2021.

    Colorized scanning electron micrograph of a cell (red) infected with the Omicron strain of SARS-CoV-2 virus particles (blue), isolated from a patient sample. Image captured at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID

    This research may help explain why some people continue to test positive for the virus long after their infection has subsided and they have recovered. In reverse transcription, RNA molecules, in this case from SARS-CoV-2, are transcribed into cDNA, a flip of the typical process in which active genes are transcribed into RNA molecules. Those reverse-transcribed cDNA molecules are then stitched into the host cell genome. If some of those cells are captured during a COVID-19 test, PCR would recognize and amplify the viral DNA in the host cell, causing a positive test result.

    This study has also shown that simply inserting viral RNA into cells is not enough to cause genomic integration, so it seems unlikely based on the evidence we have now that mRNA from the COVID-19 vaccines would cause integration into cells’ DNA.

    “This paper puts our data on a very firm footing. Hopefully, it will clarify some of the issues raised in the discussion that followed the first paper, and provide some reassurance to people who were worried about the implications for the vaccine,” said corresponding study author Rudolf Jaenisch, a founding member of the Whitehead Institute.

    Since the integration of the SARS-CoV-2 genome into cells’ DNA is unusual, the researchers had to use a very sensitive method called digital PCR, which detects very specific genetic sequences, to identify instances in which viral RNA had been integrated into the genome of a cell.

    The digital PCR results found viral RNA that had been reverse-transcribed to cDNA in about 4 to 20 of every 1,000 cells, but this includes all molecules of the sort, whether they ended up being integrated into a genome or not. Thus, the researchers suggested that viral integration is even more rare than that.

    Whole genome sequencing can be used to show when that integration also occurred, because those events are typically accompanied by a reverse transcription complex called LINE1. The LINE1 sequences act as an indicator of integration. However, WGS is usually only used on a handful of cells, so when other investigators looked for those sequences, they could not usually be found.

    “Because the human cell genome coverage by whole genome sequencing is very limited, you would need to run the sequencing experiment many times in order to have a good chance of detecting one viral genome copy,” explained postdoctoral researcher and first study author Liguo Zhang.

    In this study, the researchers created cells that would overexpress LINE1, and make viral integration more common artificially. This time, the digital PCR showed that viral cDNA appeared in fourteen to twenty of every 1,000 cells, and WGS identified instances of integration along with LINE1. Further work with a tool called TagMap confirmed viral integration without overexpressing LINE1.

    “This is unambiguous proof of viral genomic integration,” Zhang said. When this approach was repeated with cells that were treated with SARS-CoV-2 vaccine, there was no evidence of integration.

    “We need to do further testing, but our results are consistent with vaccine RNA not integrating,” Jaenisch said.

    Sources: Whitehead Institute for Biomedical Research, Viruses


    Carmen Leitch

    Microbiology

    Study finds unique epigenetic biosignature in individuals with post-COVID syndrome

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    Reviewed by Emily Henderson, B.Sc.Feb 28 2023

    A reprogramming of which genes are active, and which are not, is visible in post-COVID sufferers. This is shown in a study from Linköping University, Sweden, on a small group of individuals. The researchers can see that genes associated with taste and smell, as well as cell metabolism, are affected in individuals with post-COVID syndrome. These findings may ultimately contribute to the development of new diagnostic tools for this and similar diseases.

    There are many external factors that can affect which of all the genes in a cell are used at a certain point in time. The body’s ability to switch genes on and off contributes to our ability to adapt to various conditions. This gene use regulation is called epigenetics.

    One of the regulation mechanisms entails that a small chemical group, a methyl group, is switched on and removed from the DNA strand. Reduced methylation of a gene may be a sign of it becoming easier for the cell to read and use, whereas high methylation most often means that the gene is not used. The researchers in Maria Lerm’s research group at Linköping University have previously found that exposure to the tuberculosis bacteria is visible in individuals’ DNA by looking at certain epigenetic changes.

    In their new study, published in Clinical Epigenetics, the researchers studied blood samples from ten individuals having had persistent post-COVID symptoms for more than 12 weeks. The most common symptoms were a feeling of not being able to draw in enough air, palpitations, muscle weakness and loss of smell and taste.

    These individuals were compared with two other groups: healthy COVID-19 convalescents, and individuals who had not had COVID-19 when the samples were taken. The researchers measured the methylation pattern on 850,000 sites of the DNA and then used an algorithm that can find data similarities and differences. It turned out that the three groups differed from each other and had distinct methylation profiles. The researchers then identified the genes that differ in methylation patterns between the groups.

    “We have found that, for example, signaling pathways that control taste and smell have been affected. This confirms that the epigenetic differences may in fact be associated with the set of symptoms and be physiologically relevant,” says Maria Lerm, Professor of Medical Microbiology at the Department of Biomedical and Clinical Sciences, BKV, at Linköping University.

    A previous study conducted by the research group concerned individuals who had recently recovered from COVID-19 and who showed a similar epigenetic reprogramming of signaling pathways associated with taste and smell.

    In their new study, the researchers also found epigenetic changes in what is known as the angiotensin-2 system in post-COVID sufferers. This could be biologically relevant as the coronavirus which causes COVID-19, i.e., the SARS-CoV-2 virus, uses the angiotensin-2 system to enter and infect cells.

    One of several conditions similar to post-covid is chronic fatigue syndrome, CFS, which is also known as myalgic encephalomyelitis, ME.

    “One important finding is that we can see that the cells’ energy factories, the mitochondria, are affected in the post-COVID group. Other studies have shown that the cells’ energy factories have also been affected in cases of chronic fatigue,” says Maria Lerm.

    There is currently no test that doctors can use to decide whether a person has post-COVID syndrome. The researchers are hoping that their recent findings can contribute to the development of diagnostic tools for health care providers, tools that might perhaps even make it possible to distinguish post-COVID from similar conditions.

    The study was financed with support from the Swedish Heart Lung Foundation and the Swedish Research Council. The methylation pattern of study participants’ DNA was analyzed at Clinical Genomics, a SciLifeLab platform at Linköping University and Region östergötland.

    Source:

    Linköping University

    Journal reference:

    Defining post-acute COVID-19 syndrome (PACS) by an epigenetic biosignature in peripheral blood mononuclear cells, Frida Nikesjö, Shumaila Sayyab, Lovisa Karlsson, Eirini Apostolou, Anders Rosén, Kristofer Hedman and Maria Lerm, (2022), Clinical Epigenetics 14:172, published online on 14 December 2022 https://doi.org/10.1186/s13148-022-01398-1

    Microbiology

    Using the origami technique to design RNA nanostructures

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    Reviewed by Emily Henderson, B.Sc.Feb 28 2023

    Researchers from Aarhus University and Berkeley Laboratory have designed RNA molecules, that folds into nanoscale rectangles, cylinders, and satellites, and have studied their 3D structure and dynamics with advanced nanotechnological methods. In an article in the journal Nature Nanotechnology, the researchers describe their work and how it has led to the discovery of rules and mechanisms for RNA folding that will make it possible to build more ideal and functional RNA particles for use in RNA-based medicine.

    The RNA molecule is commonly recognized as messenger between DNA and protein, but it can also be folded into intricate molecular machines. An example of a naturally occurring RNA machine is the ribosome, that functions as a protein factory in all cells. Inspired by natural RNA machines, researchers at the Interdisciplinary Nanoscience Center (iNANO) have developed a method called “RNA origami”, which makes it possible to design artificial RNA nanostructures that fold from a single stand of RNA. The method is inspired by the Japanese paper folding art, origami, where a single piece of paper can be folded into a given shape, such as a paper bird.

    Frozen folds provide new insight

    The research paper in Nature Nanotechnology describes how the RNA origami technique was used to design RNA nanostructures, that were characterized by cryo-electron microscopy (cryo-EM) at the Danish National cryo-EM Facility EMBION. Cryo-EM is a method for determining the 3D structure of biomolecules, which works by freezing the sample so quickly that water does not have time to form ice crystals, which means that frozen biomolecules can be observed more clearly with the electron microscope. Images of many thousands of molecules can be converted on the computer into a 3D map, that is used to build an atomic model of the molecule. The cryo-EM investigations provided valuable insight into the detailed structure of the RNA origamis, which allowed optimization of the design process and resulted in more ideal shapes.

    With precise feedback from cryo-EM, we now have the opportunity to fine-tune our molecular designs and construct increasingly intricate nanostructures.”

    Ebbe Sloth Andersen, Associate Professor at iNANO, Aarhus University

    Discovery of a slow folding trap

    Cryo-EM images of an RNA cylinder sample turned out to contain two very different shapes, and by freezing the sample at different times it was evident that a transition between the two shapes was taking place. Using the technique of small-angle X-ray scattering (SAXS), where the samples are not frozen, the researchers were able to observe this transition in real time and found that the folding transition occurred after approx. 10 hours. The researchers had discovered a so-called “folding trap” where the RNA gets trapped during transcription and only later gets released (see video).

    “It was quite a surprise to discover an RNA molecule that refolds this slow since folding typically takes place in less than a second” tells Jan Skov Pedersen, Professor at Department of Chemistry and iNANO, Aarhus University.

    “We hope to be able to exploit similar mechanisms to activate RNA therapeutics at the right time and place in the patient”, explains Ewan McRae, the first author of the study, who is now starting his own research group at the “Centre for RNA Therapeutics” at the Houston Methodist Research Institute in Texas, USA.

    Construction of a nanosatellite from RNA

    To demonstrate the formation of complex shapes, the researchers combined RNA rectangles and cylinders to create a multi-domain “nanosatellite” shape, inspired by the Hubble Space Telescope.

    “I designed the nanosatellite as a symbol of how RNA design allows us to explore folding space (possibility space of folding) and intracellular space, since the nanosatellite can be expressed in cells”, says Cody Geary, assistant professor at iNANO, who originally developed the RNA-origami method.

    However, the satellite proved difficult to characterize by cryo-EM due to its flexible properties, so the sample was sent to a laboratory in the USA, where they specialize in determining the 3D structure of individual particles by electron tomography, the so-called IPET-method.

    “The RNA satellite was a big challenge! But by using our IPET method, we were able to characterize the 3D shape of individual particles and thus determine the positions of the dynamic solar panels on the nanosatellite”, says Gary Ren from the Molecular Foundry at Lawrence Berkeley National Laboratory, California, USA.

    The future of RNA medicine

    The investigation of the RNA origamis contributes to improving the rational design of RNA molecules for use in medicine and synthetic biology. A new interdisciplinary consortium, COFOLD, supported by the Novo Nordisk Foundation, will continue the investigations of RNA folding processes by involving researchers from computer science, chemistry, molecular biology, and microbiology to design, simulate and measure folding at higher time resolution.

    “With the RNA design problem partially solved, the road is now open to creating functional RNA nanostructures that can be used for RNA-based medicine, or act as RNA regulatory elements to reprogram cells”, predicts Ebbe Sloth Andersen.

    Source:

    Aarhus University

    Journal reference:

    McRae, E.K.S., et al. (2023) Structure, folding and flexibility of co-transcriptional RNA origami. Nature Nanotechnology. doi.org/10.1038/s41565-023-01321-6.

    Microbiology

    Scientists have found that a gene that has been previously identified in many animals and their associated microbes …

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    Scientists have found that a gene that has been previously identified in many animals and their associated microbes can enable resistance to antimicrobial drugs. The resistance gene encodes for an enzyme called EstT, which can deactivate antibiotic drugs known as macrolides. The enzyme can disrupt the chemical ring structure of these antibiotics through hydrolysis. When the ring is broken or opened with water, the antibiotic loses both its active shape, and its target affinity, explained study leader Dr. Tony Ruzzini PhD, an assistant professor at the Western College of Veterinary Medicine (WCVM) of the University of Saskatchewan. The findings have been reported in the Proceedings of the National Academy of Sciences.

    Image credit: Pixabay

    This gene can take macrolide antibiotics out of commission, and illnesses can no longer be treated effectively. Macrolides such as tylosin, tilmicosin and tildipirosin are often used to treat cattle with bovine respiratory disease or liver abscesses, and may also be used to treat other diseases in livestock and companion animals.

    In this study, the researchers analyzed genes that were found within microbes that were living in watering bowls at a beef cattle feedlot in western Canada. The investigators isolated the microbes that were in the water, and compared the genes in the microbes to databases of antimicrobial resistance genes.

    A bacterium called Sphingobacterium faecium WB1 was found to carry the EstT gene, which was contained within a cluster of three antibiotic resistance genes (ARGs). It was also near plasmids and retrotransposons, suggesting it can move easily from one microbe to another. EstT is commonly found in microbes in the human microbiome too.

    “This gene, even though we found it in an environmental organism, it is also present in pathogens that are responsible for causing bovine respiratory disease,” noted Ruzzini.

    “Our finding adds to the considerable database of ARGs, which can be crossmatched to a bacteria’s DNA to determine if the bacterium has the potential to be resistant to a particular antimicrobial,” said first study author Dr. Poonam Dhindwal PhD, a postdoctoral fellow at WCVM.

    The researchers are continuing to study EstT to learn more about how it works.

    “As [antimicrobial resistance] surveillance systems rely more on molecular tools for detection, our knowledge of this specific gene and its integration into those systems will help to better inform antimicrobial use,” said Ruzzini.

    Sources: University of Saskatchewan, Proceedings of the National Academy of Sciences (PNAS)


    Carmen Leitch