Tag: RNA

Meta-analysis uncovers stress-responsive genes in Arabidopsis

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Plants can be temperamental. Even weeds along the side of highways or pushing their way up in the cracks of concrete sidewalks can get stressed out by dehydration, cold, excess salt and more. Researchers at Hiroshima University have identified 14 genes that thale cress -; a plant commonly used in genetic investigations since its genome is well documented -; express more when responding to five specific stressors, as well as eight genes that the plant suppresses.

They published their results on March 22 in Frontiers in Plant Science.

“Abiotic stresses -; as opposed to biotic stresses like pests or disease -; such as drought, salinity and cold negatively affect plant growth and crop productivity. Understanding the molecular mechanisms underlying plant responses to these stressors is essential for stress tolerance in crops,” said corresponding author Hidemasa Bono, professor in the Laboratory of Genome Informatics at Hiroshima University’s Graduate School of Integrated Sciences for Life. Bono is also affiliated with the Laboratory of Bio-DX in the university’s Genome Editing Innovation Center.

“The plant hormone abscisic acid (ABA) is significantly increased upon abiotic stressors, inducing physiological responses to adapt to stress and regulate gene expression. Although many studies have examined the components of established stress signaling pathways, few have explored other unknown elements.”

To better understand the molecular pathways that allow ABA to increase, the research team analyzed public RNA sequencing data on thale cress, or Arabidopsis thaliana. RNA sequencing is a technique that enables scientists to identify and quantify specific sequences of genetic instructions programmed in an organism’s RNA. This data can reveal how different variables may increase or decrease the expression of certain genes.

Bono and his team specifically focused on five ABA-related stress conditions: ABA, when the hormone is applied directly to the plant; salt, which changes how the plant can use water; dehydration, or how much water the plant has; osmotic, when plant cells swell or shrink inappropriately; and cold.

The data-driven studies have the advantage of analyzing large and independent datasets, which can lead to the identification of novel targets, distinct from the extensively studied established factors and accelerate the development of stress-tolerant crops.”

Hidemasa Bono, Professor, Laboratory of Genome Informatics at Hiroshima University’s Graduate School of Integrated Sciences for Life

The researchers performed a meta-analysis of 216 paired datasets, combining those research results and reanalyzing them to identify where data might overlap or reveal previously unknown connections.

The meta-analysis revealed that 14 genes were commonly up-regulated and eight genes were commonly down-regulated across all five ABA-related stress responses investigated. Bono noted that some genes regulated by salt, dehydration and osmotic treatments were not regulated by ABA or cold stress, suggesting that they may be involved in the plant response through a different signaling pathway.

“Our meta-analysis revealed a list of candidate genes with unknown molecular mechanisms in ABA-dependent and ABA-independent stress responses,” Bono said.

“These genes could be valuable resources for selecting genome editing targets and potentially contribute to the discovery of novel stress tolerance mechanisms and pathways in plants. We will continue to develop methods and utilize data from public databases and conduct comparative analysis from various angles to unravel the unknown mechanisms of stress response in plants.”

Co-authors of the study are Mitsuo Shintani with the Graduate School of Integrated Sciences for Life at Hiroshima University and Keita Tamura with the university’s Genome Editing Innovation Center.

The Center of Innovation for Bio-Digital Transformation (BioDX) and the Japan Science and Technology Agency supported this research.

Source:

Journal reference:

Shintani, M., et al. (2024). Meta-analysis of public RNA sequencing data of abscisic acid-related abiotic stresses in Arabidopsis thaliana. Frontiers in Plant Science. doi.org/10.3389/fpls.2024.1343787.

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April 12, 2024
Unveiling the key role of RNA modification in HIV-1 survival and replication

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A chemical modification in the HIV-1 RNA genome whose function has been a matter of scientific debate is now confirmed to be key to the virus’s ability to survive and thrive after infecting host cells, a new study has found.

This change to HIV-1 RNA, a tiny chemical modification on the adenosine building block of RNA known as m6A, is a common RNA editing process in all life forms that involves altering gene expression and protein production. The functional effect often represents a cellular solution but, in some cases, leads to disease.

By developing technological advances to observe a full length of HIV-1 RNA, researchers at The Ohio State University discovered the m6A modification occurs nearly exclusively at three specific locations on the HIV-1 RNA genome – out of the total 242 potential sites that can harbor an m6A – and these three m6As are crucial in viral replication. The finding suggested that redundancy was built into the system, and further analyses suggested that is, indeed, the case with HIV-1.

“These sites are very important for producing virus proteins and for producing viral genomic RNA,” said senior study author Sanggu Kim, associate professor of veterinary biosciences and an investigator in the Center for Retrovirus Research at The Ohio State University.

“An intriguing question is, why does HIV maintain multiple m6As? Our conclusion is that m6A is so important that HIV wants to have multiples to have redundancy. If it loses one or two, it’s OK. If it loses all three, it’s a problem.”

Though any drug development associated with this work is years away, Kim said the finding suggests targeting the site-specific m6A modifications could be the basis of designing an important new treatment for HIV infection.

The study is published today (April 11, 2024) in the journal Nature Microbiology.

HIV-1, the most common type of the human immunodeficiency virus, attacks immune cells and uses them to make copies of itself. An estimated 1.2 million people in the United States have HIV, according to the 2023 report from the Centers for Disease Control and Prevention.

The virus is a good example of why research on RNA modification has been getting a lot of attention in recent years, Kim said. Once thought of as the “middle guy” between DNA’s genes and life-sustaining proteins, RNA is now known to contain not just genetic information, but also to possess functional significance – in part because of the chemical modifications that accompany its messenger task.

Especially because HIV is an RNA virus with a very compact RNA genome, it has to encode all of the survival information within its RNA genome – it’s using not only nucleotide sequences, but all of the chemical and structural features of RNA as codes to execute its infection of host cells. We know every aspect of RNA function is very important, but we don’t really know how exactly these chemical and structural modifications of RNAs regulate virus infection.”

Sanggu Kim, associate professor of veterinary biosciences and investigator in the Center for Retrovirus Research at The Ohio State University

Though the m6A (short for N6-methyladenosine) modification was known to exist in HIV-1, previous studies had produced conflicting results about whether it helped or harmed the virus, primarily because its location was unknown and efforts to understand its effect were based on knocking out host cell genes rather than mutating the virus genome itself.

Kim and colleagues used – and refined – a technique called nanopore direct RNA sequencing to view a full length of HIV-1’s RNA genome, which is tricky to observe because RNA is a notoriously unstable and complex molecule.

The team first discovered the three m6A modifications and their specific locations. From there, the researchers analyzed individual RNA molecules with distinct ensembles of m6A modifications, including those with multiple m6As and those with just one of the three m6As. They found that any ensemble of m6A modifications, regardless of the number or the position of m6As, produced similar functional changes. Removal of all three, however, caused devastating effects to viruses – a dead giveaway that these m6As are redundant.

“Until now we didn’t know which exact nucleotides are modified and how they function, and how it’s important for viruses or how it’s important for cells. Our paper addresses the keys to these important questions,” Kim said.

“Why would HIV need all three modifications if they’re functioning in the same way?” he said. “Our study is the first to show that HIV-1 utilizes this unique, important mechanism at the RNA level for its evolutionary benefit.”

Almost all existing HIV drugs block virus replication, but no medications inhibit viral RNA and protein production. There is more to learn about the RNA modification in HIV-1, but Kim said the work hints at the potential to develop therapies that could target these later steps.

This research was funded by the National Institutes of Health, U.S. Department of Defense, U.S. Department of Energy and the C. Glenn Barber Fund Trust.

Co-authors include Alice Baek, Ga-Eun Lee, Sarah Golconda, Anastasios Manganaris, Shuliang Chen, Nagaraja Tirumuru, Hannah Yu, Shihyoung Kim, Christopher Kimmel, Olivier Zablocki and Matthew Sullivan of Ohio State, Asif Rayhan and Balasubrahmanyam Addepalli of the University of Cincinnati, and Li Wu of the University of Iowa.

Source:

The Ohio State University

Journal reference:

Baek, A., et al. (2024). Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As. Nature Microbiology. doi.org/10.1038/s41564-024-01638-5.

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April 12, 2024
Researchers elucidate how gene mutation mechanism causes autism

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Researchers show how mutations of gene transcription and chromatin regulation-related genes cause autism.

The loss-of-function mutation of KMT2C, a gene involved in histone modification, leads to the development of autism and other neurodevelopmental deficits. However, the precise mechanism of the disease progression is still unknown. Now, researchers from Japan have developed an animal model and elucidated the mechanism by which mutation in genes involved in chromatin modification causes autism. They have also discovered a drug that can be used in the treatment of autism.

Autism spectrum disorder (ASD) encompasses neurodevelopmental conditions where patients display repetitive behavior and impaired sociality. Genetic factors have been shown to influence the development of ASD. Additionally, recent studies have shown that the genes involved in chromatin modification and gene transcription are involved in the pathogenesis of ASD. Among the many genes implicated in this process, the gene KMT2C (lysine methyltransferase 2c), which codes for a catalytic unit of H3K4 (histone H3 lysine 4) methyltransferase complex, has been identified to be associated with the development of autism and other neurodevelopmental disorders. Previous studies have shown that haploinsufficiency (a condition where, of the two copies of the gene, only one remains functional) of KMT2C is a risk factor for ASD and other neurodevelopmental disorders. However, the molecular mechanism through which the loss-of-function mutation in KMT2C leads to these conditions remains unclear.

To address this knowledge gap, researchers from Juntendo University, RIKEN, and the University of Tokyo in Japan aimed to provide answers to these questions in a benchmark study published in the journal Molecular Psychiatry on 26 March 2024. The research team included Professor Tadafumi Kato from the Department of Psychiatry and Behavioral Science at Juntendo University Graduate School of Medicine, Dr. Takumi Nakamura and Dr. Atsushi Takata from the RIKEN Center for Brain Science, and Professor Takashi Tsuboi from Graduate School of Arts and Sciences, The University of Tokyo.

To get to the bottom of KMT2C‘s role in ASD pathogenesis, the team developed and analyzed genetically engineered strain mice (Kmt2c^+/fs) having a frameshift mutation that models the KMT2C haploinsufficiency. They then performed various behavioral analyses, in which they observed that the mutant mice exhibited lower sociality, inflexibility, auditory hypersensitivity, and cognitive impairments, which are all ASD-related symptoms.

Next, they performed transcriptomic and epigenetic profiling to understand the basis of the molecular changes observed in the mutant mice. What they discovered was remarkable: the genes associated with increased ASD risk showed higher expression in these mutant mice.

This was somewhat unexpected. KMT2C mediates H3K4 methylation, which is thought to activate gene expression, and thereby KMT2C haploinsufficiency was expected to cause reduced expression of target genes.”

Dr. Atsushi Takata, RIKEN Center for Brain Science

To gain mechanistic insights into their finding, the researchers carried out chromatin immunoprecipitation, a technique to determine the location on the DNA where the protein interacts with it. They found an overlap between KMT2C and the differentially expressed genes exhibiting reduced expression, suggesting that KMT2C haploinsufficiency leads to ASD-related transcriptomic changes through an indirect effect on gene expression.

Further, to identify the cell types that contribute more to the pathological changes seen in the mutant mice, the researchers performed single-cell RNA sequencing of newborn mice brains. They observed that the altered genes associated with ASD risk were predominant in undifferentiated radial glial cells. However, a gross change in the cell composition was not observed, implying that the transcriptomic dysregulation does not severely impact cell fate.

Finally, the researchers tested the effects of vafidemstat, a brain penetrant inhibitor of LSD1 (lysine-specific histone demethylase 1A), that could ameliorate histone methylation abnormalities. They found that vafidemstat improved the social deficits in the mutant mice and had an exceptional rescuing effect by changing the expression levels of the differentially expressed genes to their normal expression level. This finding showed that vafidemstat is a valid drug for mutant mice and can potentially help restore the normal transcriptomic state.

What sets this discovery apart is that it challenges the commonly held belief that ASD disability may not be cured and demonstrates the efficacy of vafidemstat in improving ASD-like phenotypes. The results open doors to future research to strengthen the foundation for the pharmacologic treatment of ASD and other neurodevelopmental disorders. Prof. Kato concludes, “Our research shows that drugs similar to vafidemstat may be generalizable to multiple categories of psychiatric disorders.”

Source:

Journal reference:

Nakamura, T., et al. (2024). Transcriptomic dysregulation and autistic-like behaviors in Kmt2c haploinsufficient mice rescued by an LSD1 inhibitor. Molecular Psychiatry. doi.org/10.1038/s41380-024-02479-8.

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April 9, 2024
Fluent BioSciences awarded NIH SBIR grant to commercialize low-cost million-cell transcriptome profiling kits

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Fluent BioSciences, a cutting-edge life sciences company leading the charge in revolutionizing single-cell analysis through simple, cost-effective, and highly scalable single-cell RNA sequencing solutions is thrilled to announce the award of an NIH Small Business Innovation Research (SBIR) Phase II grant, funded by the National Institute of General Medical Sciences (NIGMS, 2 R44 GM137648). This funding will support the commercialization of million-cell analysis kits enabled by low-cost, high-capacity sequencing by Ultima Genomics.

Prototype million-cell PIPseq kits developed by Fluent BioSciences that enable the profiling of an unprecedented number of cells within a single reaction were recently tested in the laboratory of Samantha Morris at Washington University, St. Louis (WUSTL). The WUSTL team employs single-cell lineage tracing to interrogate how to recreate physiologically accurate cell types with cellular reprogramming. Ultima Genomics sequencing of the prototype million-cell PIPseq data revealed clonal lineage trees with restriction of specific cell reprogramming fates. Leveraging the unmatched scalability of PIPseq with the affordable high-capacity sequencing of Ultima Genomics, WUSTL demonstrated that cellular reprogramming studies can be easily and affordably conducted at scale.

Sample preparation was simple and the scale of the million-cell kit provided impressive power to our single-cell lineage tracing observations.”

Samantha Morris, Associate Professor, WUSTL

“The collaboration between Fluent BioSciences and Ultima represents a remarkable synergy in pushing the boundaries of single-cell analysis,” said Brian McKernan, CEO of Fluent BioSciences. “This collaboration harnesses the strengths and expertise of both companies to address a critical challenge faced by researchers – the ability to perform single-cell sequencing on larger cell inputs for improved scalability and reduced cost. By eliminating the need for microfluidics devices or chips, the PIPseq technology offers unparalleled flexibility in the scale of beads per sample.”

“We are excited to work with Fluent BioSciences to demonstrate compatibility of PIPseq on Ultima sequencers, optimize library construction for Ultima-specific sequencing, and demonstrate high-capacity single cell analysis across several applications developed by Fluent, including high cell input neuroscience, multi-sample hashing, and complex CRISPR editing applications. “The single-cell market is undergoing another major wave of growth with larger-scale experiments enabled by low-cost sequencing and advancement of single-cell technology”, said Doron Lipson, CSO of Ultima Genomics.

Through this partnership, Fluent BioSciences and Ultima aim to revolutionize the field of single-cell analysis by enabling researchers to unlock deeper insights into cellular mechanisms and disease pathways with unparalleled throughput and accuracy.

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April 9, 2024
New ovarian atlas paves the way for extended fertility and hormone restoration

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A new “atlas” of the human ovary provides insights that could lead to treatments restoring ovarian hormone production and the ability to have biologically related children, according to University of Michigan engineers.

This deeper understanding of the ovary means researchers could potentially create artificial ovaries in the lab using tissues that were stored and frozen before exposure to toxic medical treatments such as chemotherapy and radiation. Currently, surgeons can implant previously frozen ovarian tissue to temporarily restore hormone and egg production. However, this does not work for long because so few follicles-;the structures that produce hormones and carry eggs-;survive through reimplantation, the researchers say.

The new atlas reveals the factors that enable a follicle to mature, as most follicles wither away without releasing hormones or an egg. Using new tools that can identify what genes are being expressed at a single-cell level within a tissue, the team was able to home in on ovarian follicles that carry the immature precursors of eggs, known as oocytes.

“Now that we know which genes are expressed in the oocytes, we can test whether affecting these genes could result in creating a functional follicle. This can be used to create an artificial ovary that could eventually be transplanted back into the body,” said Ariella Shikanov, U-M associate professor of biomedical engineering and corresponding author of the new study in Science Advances.

The majority of the follicles, called primordial follicles, remain dormant and are located in the outer layer of the ovary, called the cortex. A small portion of these follicles activate periodically and migrate into the ovary, to a region known as the growing pool. Only a few of those growing follicles go on to produce mature eggs that get released into the fallopian tube.

With the ability to guide follicle development and tune ovarian environment, the team believes that engineered ovarian tissue could function for much longer than unmodified implanted tissue. This means that patients would have a longer fertility window as well as a longer period in which their bodies produce hormones that help regulate the menstrual cycle and support muscular, skeletal, sexual and cardiovascular health.

We’re not talking about utilizing a surrogate mother, or artificial insemination. The magic we’re working toward is being able to trigger an immature cell into maturity, but without knowing which molecules drive that process, we’re blind.”

Jun Z. Li, associate chair of U-M’s Department of Computational Medicine and Bioinformatics and co-corresponding author of the study

U-M’s team utilized a relatively new technology, called spatial transcriptomics, to track all of the gene activity-;and where it occurs-;in tissue samples. They do this by reading strands of RNA, which are like notes taken from the DNA strand, revealing which genes are being read. Working with an organ procurement organization, U-M researchers performed RNA sequencing of ovaries from five human donors.

“This was the first time where we could target ovarian follicles and oocytes and perform a transcription analysis, which enables us to see which genes are active,” Shikanov said.

“The majority of ovarian follicles, already present at birth, never enter the growing pool and eventually self-destruct. This new data allows us to start building our understanding of what makes a good egg-;what determines which follicle is going to grow, ovulate, be fertilized and become a baby.”

U-M’s work is part of the Human Cell Atlas project, which seeks to create “maps of all the different cells, their molecular characteristics and where they are located, to understand how the human body works and what goes wrong in disease.”

Shikanov, Li and U-M collaborators such as Sue Hammoud, U-M associate professor of human genetics and urology, are mapping other parts of the female reproductive system, including the uterus, fallopian tubes and ovaries. Other contributors include Andrea Suzanne Kuliahsa Jones, formerly of U-M and now at Duke University, and D. Ford Hannum, a U-M graduate student research assistant in bioinformatics.

The research was partially funded by the Chan Zuckerberg Initiative. Additional financial support was provided by the National Institutes of Health.

Source:

Journal reference:

Jones, A. S. K., et al. (2024) Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing. Science Advances. doi.org/10.1126/sciadv.adm7506.

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April 6, 2024
Neuroscientists identify age-defying RNAs in the brain

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Certain RNA molecules in the nerve cells in the brain last a life time without being renewed. Neuroscientists from Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) have now demonstrated that this is the case together with researchers from Germany, Austria and the USA. RNAs are generally short-lived molecules that are constantly reconstructed to adjust to environmental conditions. With their findings that have now been published in the journal Science, the research group hopes to decipher the complex aging process of the brain and gain a better understanding of related degenerative diseases.

Most cells in the human body are regularly renewed, thereby retaining their vitality. However, there are exceptions: the heart, the pancreas and the brain consist of cells that do not renew throughout the whole lifespan, and yet still have to remain in full working order.

Aging neurons are an important risk factor for neurodegenerative illnesses such as Alzheimer’s. A basic understanding of the aging process and which key components are involved in maintaining cell function is crucial for effective treatment concepts:”

Prof. Dr. Tomohisa Toda, Professor of Neural Epigenomics at FAU and at the Max Planck Center for Physics and Medicine in Erlangen

In a joint study conducted together with neuroscientists from Dresden, La Jolla (USA) and Klosterneuburg (Austria), the working group led by Toda has now identified a key component of brain aging: the researchers were able to demonstrate for the first time that certain types of ribonucleic acid (RNA) that protect genetic material exist just as long as the neurons themselves. “This is surprising, as unlike DNA, which as a rule never changes, most RNA molecules are extremely short-lived and are constantly being exchanged,” Toda explains.

In order to determine the life span of the RNA molecules, the Toda group worked together with the team from Prof. Dr. Martin Hetzer, a cell biologist at the Institute of Science and Technology Austria (ISTA). “We succeeded in marking the RNAs with fluorescent molecules and tracking their lifespan in mice brain cells,” explains Tomohisa Toda, who has unique expertise in epigenetics and neurobiology and who was awarded an ERC Consolidator Grant for his research in 2023. “We were even able to identify the marked long-lived RNAs in two year old animals, and not just in their neurons, but also in somatic adult neural stem cells in the brain.”

In addition, the researchers discovered that the long-lived RNAs, that they referred to as LL-RNA for short, tend to be located in the cells’ nuclei, closely connected to chromatin, a complex of DNA and proteins that forms chromosomes. This indicates that LL-RNA play a key role in regulating chromatin. In order to confirm this hypothesis, the team reduced the concentration of LL-RNA in an in-vitro experiment with adult neural stem cell models, with the result that the integrity of the chromatin was strongly impaired.

“We are convinced that LL-RNAs play an important role in the long-term regulation of genome stability and therefore in the life-long conservation of nerve cells,” explains Tomohisa Toda. “Future research projects should give a deeper insight into the biophysical mechanisms behind the long-term conservation of LL-RNAs. We want to find out more about their biological function in chromatin regulation and what effect aging has on all these mechanisms.”

Source:

Friedrich-Alexander-Universität Erlangen-Nürnberg

Journal reference:

Zocher, S., et al. (2024) Lifelong persistence of nuclear RNAs in the mouse brain. Science. doi.org/10.1126/science.adf3481.

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April 5, 2024
Unlocking the secrets of long-lived RNAs in brain cells

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After two decades in the United States, Martin Hetzer returned home to Austria in 2023 to become the 2^nd President of the Institute of Science and Technology Austria (ISTA). A year into his new role, the molecular biologist remains engaged in the realm of aging research.

Hetzer is fascinated by the biological puzzles surrounding the aging processes in organs like the brain, heart, and pancreas. Most cells comprising these organs are not renewed throughout a human’s entire life span. Nerve cells (neurons) in the human brain, for instance, can be as old as the organism, even up to more than a century, and must function for a lifetime. This remarkable age of neurons might be a major risk factor for neurodegenerative disorders such as Alzheimer’s disease. Crucial to comprehending these kinds of ailments is a deeper understanding of how nerve cells function over time and maintain control. This potentially opens doors to therapeutically counteract the aging processes of these specific cells.

The latest collaborative publication by Hetzer, Tomohisa Toda from the Friedrich-Alexander University Erlangen-Nürnberg (FAU), who is also associated with the Max Planck Center for Physics and Medicine, Erlangen, and colleagues, gives new insights into this underexplored field of intricate mechanisms. For the first time in mammals, the study shows that RNA-;an essential group of molecules important for various biological processes inside the cell-;can persist throughout life. The scientists identified specific RNAs with genome-protecting functions in the nuclei of nerve cells of mice that remain stable for two years, covering their entire lives. The findings, published in the journal Science, underpin the importance of long-lived key molecules for maintaining a cell’s function.

Longevity of key molecules

The inside of cells is a very dynamic place. Some components are constantly renewed and updated; others stay the same their whole lives. It is like a city in which the old buildings blend in with the new ones. DNA found in the nucleus-;the city’s heart-;for instance, is as old as the organism. “DNA in our nerve cells is identical to DNA within the developing nerve cells in our mother’s womb,” explains Hetzer.

Unlike stable DNA, which is constantly being repaired, RNA, especially messenger RNA (mRNA), which forms proteins upon DNA’s information, is characterized by its transient nature. The cellular scope, however, extends beyond mRNA to a group of so-called non-coding RNAs. They do not turn into proteins; instead, they have specific duties to contribute to the overall organization and function of the cell. Intriguingly, their lifespan remained a mystery. Until now.

RNAs that last the whole life

Hetzer and Co. set out to decipher that secret. Therefore, RNAs were labeled, i.e. “marked”, in the brains of newborn mice. “For this labeling, we used RNA analogs-;structurally similar molecules-;with little chemical hooks that click fluorescent molecules on the actual RNAs,” explains Hetzer. This assured efficient tracking of the molecules and powerful microscopic snapshots at any given time point in the mice’s lives.

Surprisingly, our initial images revealed the presence of long-lived RNAs, in various cell types within the brain. We had to further dissect the data to identify the ones in the nerve cells. Fruitful collaboration with Toda’s lab enabled us to make sense of that chaos during brain mapping.”

Martin Hetzer, Institute of Science and Technology Austria

Collaboratively, the researchers were able to focus solely on long-lived RNAs in neurons. They quantified the molecules’ concentration throughout a mouse’s life, examined their composition and analyzed their positions.

While humans have an average life expectancy of around 70 years, the typical lifespan of a mouse is 2.5 years. After one year, the concentration of long-lived RNAs was slightly reduced compared to newborns. However, even after two years, they remained detectable indicating a lifelong persistence of these molecules.

RNAs help protect the genome

Additionally, the scientists proved long-lived RNAs’ prominent role in cellular longevity. They found out that long-lived RNAs in neurons consist of mRNAs and non-coding RNAs and accumulate near the heterochromatin-;the densely packed region of the genome, typically homing inactive genes. Next they further investigated the function of these long-lived RNAs.

In molecular biology, the most effective approach to achieve this is by reducing the molecule of interest and observing its subsequent effects. “As their name and our previous experiments suggest, these long-lived RNAs are extremely stable,” says Hetzer. The scientists, therefore, employed an in vitro (outside a living organism) approach, using neuronal progenitor cells-;stem cells with the capacity to give rise to neural cells, including neurons. The model system allowed them to effectively intervene with these long-lived RNAs. A lower amount of long-lived RNAs caused problems in the heterochromatin architecture and stability of genetic material, eventually affecting the cells’ viability. Thus, the important role of long-lived RNAs’ in cellular longevity was clarified.

The study highlights that long-lived RNAs may function in the lasting regulation of genome stability. “Lifelong cellular maintenance during aging involves an extended life span of key molecules like the long-lived RNAs, we just identified,” Hetzer adds. The precise mechanism, however, remains unclear. “Together with unidentified proteins, long-lived RNAs likely form a stable structure that somehow interacts with the heterochromatin.” Upcoming research projects in Hetzer’s lab are set on finding these missing links and understanding the biological characteristics of these long-lived RNAs.

Source:

Institute of Science and Technology Austria

Journal reference:

Zocher, S., et al. (2024) Lifelong persistence of nuclear RNAs in the mouse brain. Science. doi.org/10.1126/science.adf3481.

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April 5, 2024
Novel SARS-CoV-2 mutations found in floodwaters near homeless communities
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In a recent study published in the journal Environmental Science & Technology Letters, researchers conducted environmental surveillance to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in two flood control channels in the United States (US), influenced by homeless individuals. They detected SARS-CoV-2 RNA (short for ribonucleic acid) and novel spike gene mutations in the channels during COVID-19 (short for coronavirus disease 2019) outbreaks, emphasizing the efficacy of environmental surveillance for assessing public health in the homeless population.

Study: Environmental Surveillance of Flood Control Infrastructure Impacted by Unsheltered Individuals Leads to the Detection of SARS-CoV-2 and Novel Mutations in the Spike Gene. Image Credit: CROCOTHERY / Shutterstock

Background

During the COVID-19 pandemic, overwhelmed public health laboratories in the US prompted the initiation of the National Wastewater Surveillance System (NWSS) to support traditional surveillance efforts in March 2020. The program could effectively detect SARS-CoV-2 RNA, antimicrobial resistance markers, and emerging variants, offering early detection for public health priorities. Several studies have reported the presence of viruses and human fecal material in flood control channels due to various factors like overflowing sanitary sewers and direct human inputs. In cities where homelessness is common, environmental surveillance of flood control channels can aid in understanding disease transmission among people experiencing homelessness, which is often overlooked in clinical surveillance data.

RNA of SARS-CoV-2 can sustain in water bodies for extended periods, while infected individuals can continue shedding significant amounts of viral RNA in fecal matter for up to seven months. Despite previous research demonstrating the presence of SARS-CoV-2 RNA in surface waters, conducting whole genome sequencing (WGS) from flood control channels for variant identification is less frequent, primarily due to difficulties in collecting and analyzing samples. Researchers in the present study aimed to identify SARS-CoV-2 RNA in environmental water samples from flood control infrastructure impacted by homeless individuals, perform WGS, compare variants with those found in the local community, and potentially reveal any novel mutations.

About the study

In the present study, water sample processing was performed by concentrating primary effluent from wastewater treatment plants (WWTPs) using hollow fiber ultrafiltration, followed by extraction and synthesis of cDNA (short for complementary deoxyribonucleic acid). Environmental water samples from two sources (Flamingo Wash and Tropicana Wash) were processed similarly. A total of 57 samples were collected and analyzed.

SARS-CoV-2 RNA quantification was performed using quantitative polymerase chain reaction (qPCR). Further, library preparation for amplicon-based WGS made use of a SARS-CoV-2 panel and Illumina NextSeq 500. Data analysis included adapter trimming, read alignment, primer masking, variant calling, and determination of variant composition. Low-frequency and novel mutations were identified and validated using various databases.

Results and discussion

SARS-CoV-2 RNA was detected in 15 samples (33% in treated water and 20% in freshwater), with concentrations between 2.8 and 4.8 log10 gc/L. Higher detection frequencies occurred in the first two months of 2022, corresponding to the peak of the first Omicron wave. This aligns with the maximal concentrations observed at the WWTP. PMMoV (short for pepper mild mottle virus), a fecal indicator virus, was detected in almost all samples, with concentrations between 4.0 and 6.3 log10 gc/L, consistent with previous studies. Detection frequencies of PMMoV were slightly higher in this study than in earlier ones, possibly due to the increased sensitivity of sample processing methods or the study of areas with higher densities of unsheltered individuals.

The detected variants were majorly classified as Omicron, Delta, and Alpha, especially in environmental water samples. Notably, Alpha detection in freshwater indicated potential persistent shedding or low circulation levels. Delta variant signals were observed, correlating with shedding timelines, suggesting variable loadings could influence variant composition in environmental samples.

Previously unreported mutations of the SARS-CoV-2 spike protein, including Tyr636Phe, Ser943Thr, and Phe1103Val, were identified in the samples. These mutations, not residing in the receptor-binding domain (RBD), were observed more than once, with Tyr636Phe being the most frequently detected. While the origin and significance of these mutations remain uncertain, their presence suggests potential circulation within the local community rather than being unique to flood control channels or municipal wastewater.

The findings suggest that COVID-19 transmission within unsheltered populations may reflect trends in the general community. However, a direct comparison of variant prevalence could not be made due to limited clinical surveillance data for unsheltered individuals.

Conclusion

In conclusion, the study found that the SARS-CoV-2 variants detected in environmental water samples influenced by human waste from homeless individuals were like those circulating in the broader community, as observed through wastewater and clinical surveillance. The highest concentrations of SARS-CoV-2 RNA coincided with the peak of the initial Omicron surge, followed by a decline correlating with decreased wastewater concentrations and confirmed case counts. The study emphasizes the utility of environmental surveillance for understanding public health conditions and infectious disease transmission, particularly among vulnerable homeless populations.
Journal reference:

Environmental Surveillance of Flood Control Infrastructure Impacted by Unsheltered Individuals Leads to the Detection of SARS-CoV-2 and Novel Mutations in the Spike Gene. Anthony Harrington et al., Environmental Science & Technology Letters (2024), DOI: 10.1021/acs.estlett.3c00938, https://pubs.acs.org/doi/10.1021/acs.estlett.3c00938
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April 5, 2024
Study highlights anti-inflammatory properties of herbal medicine, Erigeron breviscapus to treat osteoarthritis

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In a recent study published in Nutrients, researchers explored using Erigeron breviscapus (EB) as a treatment for osteoarthritis (OA).

Study: Anti-Inflammatory, Analgesic, Functional Improvement, and Chondroprotective Effects of Erigeron breviscapus (Vant.) Hand.-Mazz. Extract in Osteoarthritis: An In Vivo and In Vitro Study. Image Credit: Dragana Gordic/Shutterstock.com

Background

Osteoarthritis, a degenerative bone disorder, causes persistent discomfort, function loss, and joint damage. The worldwide aging trend and a lack of effective medicines are driving up demand for therapy.

The discovery of safe and effective solutions is a public health concern, as existing conservative treatments fail to correct OA’s inflammatory pathology. Erigeron breviscapus is an herbal medication from East Asia with powerful anti-inflammatory qualities that promote disease-fighting benefits across several systems.

However, the existing scientific evidence for EB primarily focuses on cardiovascular diseases and central nervous system disorders, warranting further research.

About the study

In the present study, researchers investigated the therapeutic potential of E. breviscapus for osteoarthritis, specifically the anti-inflammatory-based modulatory effects.

The researchers examined the functional benefits, analgesic effects, and suppression of cartilage breakdown caused by EB among acetic acid-inflicted peripheral-type pain murine animals and monosodium iodoacetate (MIA)-induced osteoarthritis rat models.

They also investigated the inflammation-lowering properties of EB in cartilage tissues and serum in the in vivo settings and lipopolysaccharide (LPS)-induced RAW 264.7 macrophages.

The researchers extracted a powder from dried EB stems and analyzed its components using high-performance-type liquid chromatography (HPLC). They performed the analysis using Sprague-Dawley rats and ICR mice.

The mice received EB extracts (EBE) in 200 mg/kg and 600 mg/kg concentrations, ibuprofen 200 mg/kg, and water as the study control.

After 30 minutes of oral therapy, they administered 0.7% acetic acid in 10 mL/kg concentration intraperitoneally to observe writhing responses 10 minutes later.
The MIA-induced OA rat model included five groups: EB extract 300, indomethacin (INDO 3), sham, and control (CON).

They anesthetized the rats with a combination of oxygen and 2.0% isofluorane and intraarticular MIA injections in 40 mg/mL concentration to induce osteoarthritis in the EBE, indomethacin, and control groups.

They disarticulated and macroscopically scored right-side knee joints to assess articular cartilage deterioration.

The researchers drew blood from the abdominal vein to form a blood clot within thirty minutes. The separated serum was tested for interleukin-1 beta (IL-1β) and IL-6 levels. They treated RAW264.7 macrophages with 500 ng/mL LPS and EBE for 24 hours to determine cell viability and EB cytotoxicity.

They extracted ribonucleic acid (RNA) from RAW264.7 cells for quantitative-type real-time polymerase chain reaction (qRT-PCR) analysis.

They used Western blot analysis to determine the protein expression of interleukin-1 beta, interleukin-6, prostaglandin E receptor 2 (Ptger2), nitric oxide synthase 2 (NOS2), matrix metalloproteinase 1 (MMP1), MMP8, MMP13, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Results

In vitro and in vivo, EB significantly reduced functional impairment, pain, and cartilage deterioration associated with osteoarthritis. It also showed dose-dependent inhibition of pro-inflammatory cytokine molecules such as interleukins-1β, 6, MMP- 13, and NOS2 compared to controls.

HPLC analysis identified 7.5 mg/g of chlorogenic acid as the primary anti-inflammatory component of EB. EBE effectively alleviated acetic acid-induced peripheral discomfort in rats, resulting in fewer writhing responses.

EBE treatment dramatically increased the weight-bearing ability of MIA rats, equivalent to INDO3. EBE reduced MIA injection-induced cartilage erosion and recovered cartilage degeneration at a rate similar to INDO3.

The EBE- and INDO3-treated groups dramatically reduced cartilage degradation caused by CON. EBE lowered NO levels, indicating powerful anti-inflammatory effects.

The EBE group saw a dose-dependent drop in blood concentrations of interleukin-1 beta and interleukin-6 compared to the control group, with downregulation effects on MMP-1, MMP-8, MMP-13, PTGER2, IL-1β, IL-6, and NOS2.

At 300 µg/mL, EB extract exhibited minimal cytotoxic effects in RAW264.7 macrophages. DEX1 and EB extract decreased the expression of tumor necrosis factor-alpha (TNF-α), cyclooxygenase 2 (COX-2), IL-1β, IL-6, MMP-1,13, PTGER2, NOS2 messenger RNA (mRNA).

EBE injection reduced the synthesis of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, NOS2, MMP-1, and MMP-13, as demonstrated by Western blot imaging. EBE had equivalent anti-inflammatory effects as positive controls for all cytokines.

Conclusions

The study found that Erigeron breviscapus extract improves clinical symptoms of osteoarthritis (OA), such as pain, functional decline, and cartilage breakdown.

It has considerable anti-inflammatory effects on pro-inflammatory mediators such as IL-1β, IL-6, MMP13, and NOS2 that contribute to the inflammatory pathophysiology of OA.

EBE is a possible disease-modifying osteoarthritis drug (DMOAD) candidate that requires more investigation to evaluate its effectiveness in altering the complicated inflammatory pathophysiology of OA.

Future research could explore EBE’s multi-component and multi-target effectiveness, using network pharmacology and bioinformatic approaches to determine the detailed mechanism of action and critical signaling pathways.

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April 4, 2024
Atomic-level structural models of enzymes provide disease insights

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When nucleic acids like DNA or RNA build up in a cell’s cytoplasm, it sets off an alarm call for the immune system. Enzymes usually clear these nucleic acids before they cause an issue, but when these enzymes don’t work and the immune system gets called in, it can lead to autoimmune and inflammatory diseases.

In a new study published on March 26, 2024 in the journal Structure, Scripps Research scientists present the previously undescribed structure of two of these nucleic acid-degrading enzymes-;PLD3 and PLD4. Understanding these enzymes’ structures and molecular details is an important step toward designing therapies for the various diseases that arise when they malfunction, which include lupus erythematosus, rheumatoid arthritis and Alzheimer’s disease.

These enzymes are important for cleaning up the cellular environment, and they also set the threshold for what is considered an infection or not. I’m hoping someday we may be able to help patients based on this information.”

David Nemazee, PhD, senior author, professor in the Department of Immunology and Microbiology at Scripps Research

Enzymes are proteins that speed up chemical reactions by binding and reacting to specific molecules called substrates. In the case of PLD3 and PLD4, the substrate is a strand of RNA or DNA, which the enzymes break down nucleotide by nucleotide.

The team used X-ray crystallography to build atomic-scale models of the PLD3 and PLD4 in multiple states or situations, allowing them to examine how their shapes changed over the course of the catalytic reaction. This included when the enzymes were resting, or when they were actively bound to a substrate.

“These models allow us to visualize PLD3 and PLD4 very clearly and with high resolution, so we know exactly how every atom interacts, meaning we can deduce how the enzymes work,” says first author Meng Yuan, a staff scientist in the Department of Integrative Structural and Computational Biology at Scripps Research.

The structural analyses revealed that PLD3 and PLD4 are structurally similar and that they degrade DNA and RNA in a very similar fashion, even though PLD4 is a larger protein. Both enzymes degrade nucleic acids via a two-step process.

“We call this process a two-step catalysis: bite down and release,” says Yuan. “In the first step, the enzyme bites down on the DNA strand and separates a single ‘brick’ or nucleotide from the rest of the strand, and in the second step, it opens its ‘mouth’ and releases the brick to be recycled.”

Because the enzymatic reaction happens so quickly-;within milliseconds-;researchers needed to use an alternative substrate to visualize the enzymes’ structure during catalysis. To do this, they incubated the enzymes together with a molecule that looks very similar to the DNA that the enzyme usually degrades, but that the enzymes degrade much more slowly.

This method uncovered a previously unknown function for one of the enzymes: In addition to biting off nucleotides from single-stranded RNA and DNA, PLD4 also showed phosphatase activity, which means it might also be involved in breaking down DNA’s phosphate backbone.

“I think it’s amazing that the crystal structure told us about this phosphatase activity,” says Nemazee. “To discover new enzymatic activity is unheard of in structural biology. It’s only because Meng was able to solve such an amazingly accurate and detailed structure that he could inform us about this extra enzymatic activity that we had no idea about.”

After they had elucidated PLD3 and PLD4’s usual structure, the researchers examined the structure of variants that are associated with diseases, including Alzheimer’s and spinocerebellar ataxia. These analyses revealed that some of these variants had decreased enzymatic capability, while others-;including a mutation associated with late-onset Alzheimer’s-;appeared to be more active.

“Some of our data suggests that one of these Alzheimer’s-associated enzyme variants might function better, which was a surprise to me, but it also may be less stable and more easily aggregated,” says Nemazee.

The researchers plan to continue investigating the structure and function of these enzymes. Their next steps include exploring possible ways of inhibiting the enzymes in scenarios where they are overactive, and they also plan to investigate the possibility of replacing the enzymes in people who carry non-functional (or non-working) versions.

In addition to Nemazee and Yuan, authors of the study, “Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4,” were Linghang Peng, Deli Huang, Amanda Gavin, Fangkun Luan, Jenny Tran, Ziqi Feng, Xueyong Zhu, Jeanne Matteson, and Ian Wilson, all of Scripps Research.

This study was supported by the National Institutes of Health (grants R01AI142945 and RF1AG070775) and Skaggs Institute for Chemical Biology at Scripps Research.

Source:

Scripps Research Institute

Journal reference:

Yuan, M., et al. (2024). Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4. Structure. doi.org/10.1016/j.str.2024.02.019.

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April 3, 2024