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December 17, 2020
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https://www.sciencedaily.com/releases/2020/12/201217135244.htm
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Optogenetic method can reveal how gut microbes affect longevity
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Research has shown that gut microbes can influence several aspects of the host's life, including aging. Given the complexity and heterogeneity of the human gut environment, elucidating how a specific microbial species contributes to longevity has been challenging. To explore the influence of bacterial products on the aging process, researchers at Baylor College of Medicine and Rice University developed a method that uses light to directly control gene expression and metabolite production from bacteria residing in the gut of the laboratory worm Caenorhabditis elegans.
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They report in the journal "We used optogenetics, a method that combines light and genetically engineered light-sensitive proteins to regulate molecular events in a targeted manner in living cells or organisms," said co-corresponding author Dr. Meng Wang, Robert C. Fyfe Endowed Chair on Aging and professor of molecular and human genetics and the Huffington Center on Aging at Baylor.In the current work, the team engineered E. coli to produce the pro-longevity compound colanic acid in response to green light and switch off its production in red light. They discovered that shining the green light on the transparent worms carrying the modified E. coli induced the bacteria to produce colanic acid, which protected the worm's gut cells against stress-induced mitochondrial fragmentation. Mitochondria have been increasingly recognized as important players in the aging process."When exposed to green light, worms carrying this E. coli strain also lived longer. The stronger the light, the longer the lifespan," said Wang, an investigator at Howard Hughes Medical Institute and member of Baylor's Dan L Duncan Comprehensive Cancer Center. "Optogenetics offers a direct way to manipulate gut bacterial metabolism in a temporally, quantitatively and spatially controlled manner and enhance host fitness.""For instance, this work suggests that we could engineer gut bacteria to secrete more colanic acid to combat age-related health issues," said co-corresponding author Dr. Jeffrey Tabor, associate professor of bioengineering and biosciences at Rice University. "Researchers also can use this optogenetic method to unravel other mechanisms by which microbial metabolism drives host physiological changes and influences health and disease."
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Biotechnology
| 2,020 |
December 17, 2020
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https://www.sciencedaily.com/releases/2020/12/201217135401.htm
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Scientists simulate a large-scale virus, M13
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Atomistic simulations are a powerful tool to study the movement and interactions of atoms and molecules. In many biological processes, large-scale effects, for example, assembly of large viruses to nanoparticles are important. The assembly processes of these large viruses are of fundamental importance to the design of many devices and viral protein-targeted therapeutics. However, the time and length scale of these assembly processes are usually too large for simulations at molecular resolution.
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Moreover, even though an increase in computing power allows for more complex and longer simulations, virus structures, such as M13, are still beyond reach. That is why a research group from the Singapore University of Technology and Design (SUTD) and the Massachusetts Institute of Technology (MIT) has developed a procedure that links large-scale assembly processes to molecular simulations. Assistant Prof Desmond Loke from SUTD's Science, Mathematics and Technology cluster said, "For the simulation of M13, we started with different sets of force fields. Suitable force fields were chosen and they were used as the inputs for a molecule dynamics simulations with the coarse-grain model designed to capture key pattern of the assembly process.""While we know that M13-based manufacturing can be fundamentally driven by nanoparticle-peptide interactions, which may also be a key principle behind M13-type bioengineering, we have little knowledge of how repeated patterns of short-end-peptides on a M13 surface are actually involved in these interactions. To study this, we ideally have to include a full structure of the viral coat-protein, which is a difficult task for current state-of-the-art molecular dynamics simulations," adds Dr Lunna Li, first author of the article.The procedure allows users to add different types of nanoparticles to a solution, at a realistic level. Inspired by this procedure, Assistant Professor Loke and his colleagues were able to simulate a large-scale virus with nanoparticles and inside a solution for fifty nanoseconds.Dr Li said, "The virus structure and solution contain about 700,000 atoms overall." Considering the shape and size of the features, the complexity of this simulation can be larger than any simulation performed previously."A simulation performed in microseconds would have been possible if a smaller M13 model was used, but it can be worthwhile to reduce the time to actually observe how the full structure may influence the assembly between the M13 and nanoparticles," explained Assistant Prof Loke.MIT Biological Engineering Professor Angela Belcher was also part of the research team that was simulating M13. This research was published in journal
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Biotechnology
| 2,020 |
December 16, 2020
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https://www.sciencedaily.com/releases/2020/12/201216113257.htm
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Oh so simple: Eight genes enough to convert mouse stem cells into oocyte-like cells
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In a new study published in the journal
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On top of providing new insights into the mechanisms of egg cell development, the research may lead to a simple route for generating highly specialized substances unique to oocytes for use in reproductive biology and medicine.Stored in the body until they mature into egg cells ready for fertilization, oocytes represent the very first step in the creation a new human life.Oocytes are extremely unique because of their ability to bring forth the over two hundred kinds of highly differentiated cells needed to create an individual person, and one key to this ability is the complex mixture of substances within the fluid-like cytoplasm filling the cells.So extraordinary are oocytes and their cytoplasm that replacing an oocyte's DNA-containing nucleus with that of a body cell -- a process called somatic cell nuclear transfer -- can produce a new life, as famously demonstrated with Dolly the sheep.Thus, a fundamental understanding of oocytes and their development is important for both advancing reproductive medicine and better grasping how life propagates, but knowledge of the many genes that orchestrate oocyte development is still far from complete.Analyzing the development of oocytes from mice, researchers led by Katsuhiko Hayashi, professor at Kyushu University's Faculty of Medical Sciences, have now identified eight genes for gene-triggering proteins known as transcription factors that not only are necessary for oocyte growth but also can directly convert mouse stem cells into oocyte-like cells."I was initially in complete disbelief to see mouse stem cells so quickly and easily take the form of oocytes based on introducing just a handful of factors, but repeated experiments proved it was true," says Nobuhiko Hamazaki, first author on the study reporting the results and assistant professor at Kyushu University at the time of the research. "To find that eight transcription factors could lead to such big changes was quite astonishing."Working in collaboration with researchers at RIKEN, Hayashi's group found that both mouse embryonic stem cells and induced pluripotent stem (iPS) cells -- which can be created from adult body cells -- consistently converted into oocyte-like cells when forced to produce the set of eight transcription factors, with only four factors being sufficient in some cases though with worse reproducibility."That stem cells can be directly converted into oocyte-like cells without following the same sequence of steps that happen naturally is remarkable," says Hayashi.When grown in the presence of other cells usually found around oocytes, the oocyte-like cells developed structures similar to mature egg cells but with an abnormal chromosome structure. Despite this, the mature oocyte-like cells could be fertilized in vitro and exhibited early development, with some even progressing to an eight-cell stage.Though the modified nuclei of the oocyte-like cells may not be useable in the long run, this is no problem for applications needing mainly the oocyte cytoplasm, such as for studies of reproductive biology and for treatments like mitochondrial replacement therapy, in which parts of oocytes are replaced to prevent mothers from passing to their children diseases related to the mitochondria."Cytoplasm from oocytes is an invaluable resource in reproductive biology and medicine, and this method could provide a novel tool for producing large amounts of it without any invasive procedures," comments Hayashi. "While the processes could still be much more complex for humans, these initial results in mice are very promising."
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Biotechnology
| 2,020 |
December 16, 2020
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https://www.sciencedaily.com/releases/2020/12/201216104642.htm
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Ensuring a proper body plan
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The body plan of an organism, crafted over millennia of evolutionary trial and error, is so exquisitely fine-tuned that even a subtle deviation can be detrimental to individual survival and reproductive success. Now, researchers at the University of Tsukuba have elucidated the workings of an enzyme, lysine demethylase 7a (kdm7a), that facilitates appropriate development of the mouse embryo from tip to tail, 'according to plan' by modulating expression of Hox genes. As these genes, master regulators of embryonal morphogenesis, have been highly conserved over evolution, the findings apply in varying degree to lower species and to all vertebrates, us included.
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It is an astounding fact that the unicellular zygote formed at fertilization contains all the information needed for development into a multicellular organism of immense complexity organized in well-ordered symmetry. How these data are encrypted and decoded is an escalating mystery as emerging answers only unearth further questions. Hox genes allocate regions along the head-tail axis of the developing embryo for development of appropriate structures; in vertebrates they specify the numbers and sequential shapes of the spinal bones.Some histone-modifying enzymes have been implicated in normal morphogenesis as well as in disease. Using CRISPR-Cas9 gene editing technology, the research team first developed knockout mice (Kdm7a?/?) by introducing frameshift mutation. As a result, they obtained mice carrying the mutations for truncated Kdm7a proteins lacking demethylase activity.The researchers analyzed postnatal skeletal preparations of both wild-type and Kdm7a-/-mice. Dr Yasuharu Kanki, senior author, describes the findings. "As expected, all wild-type mice showed a normal axial skeleton. Interestingly, all Kdm7a?/? mice and some heterozygous mutants exhibited vertebral transformation; some vertebrae assumed the characteristics and appendages of their anterior neighbors."The researchers next used RNA sequencing to examine the expression of Hox genes during embryogenesis. Their findings support a functional role of Kdm7a-mediated transcriptional control, especially of the posteriorly situated Hox genes, and suggest that regulation of the repressive histone mark H3K9me2 might be involved."Our data help explain morphogenesis along the anterior/posterior axis in the mouse embryo and, by extension, in all vertebrates including humans," says Dr Kanki. "Deciphering the interplay of various genetic and epigenetic determinants of embryonal morphogenesis as well as the underlying molecular mechanisms increases our knowledge of evolutionary developmental biology and may help in the understanding of disease."
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Biotechnology
| 2,020 |
December 16, 2020
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https://www.sciencedaily.com/releases/2020/12/201216085039.htm
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The DNA regions in our brain that contribute to make us human
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With only 1% difference, the human and chimpanzee protein-coding genomes are remarkably similar. Understanding the biological features that make us human is part of a fascinating and intensely debated line of research. Researchers at the SIB Swiss Institute of Bioinformatics and the University of Lausanne have developed a new approach to pinpoint, for the first time, adaptive human-specific changes in the way genes are regulated in the brain. These results open new perspectives in the study of human evolution, developmental biology and neurosciences. The paper is published in
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To explain what sets human apart from their ape relatives, researchers have long hypothesized that it is not so much the DNA sequence, but rather the regulation of the genes (i.e. when, where and how strongly the gene is expressed), that plays the key role. However, precisely pinpointing the regulatory elements which act as 'gene dimmers' and are positively selected is a challenging task that has thus far defeated researchers (see box).Marc Robinson-Rechavi, Group Leader at SIB and study co-author says: "To be able to answer such tantalizing questions, one has to be able identify the parts in the genome that have been under so called 'positive' selection [see box]. The answer is of great interest in addressing evolutionary questions, but also, ultimately, could help biomedical research as it offers a mechanistic view of how genes function."Researchers at SIB and the University of Lausanne have developed a new method which has enabled them to identify a large set of gene regulatory regions in the brain, selected throughout human evolution. Jialin Liu, Postdoctoral researcher and lead author of the study explains: "We show for the first time that the human brain has experienced a particularly high level of positive selection, as compared to the stomach or heart for instance. This is exciting, because we now have a way to identify genomic regions that might have contributed to the evolution of our cognitive abilities!"To reach their conclusions, the two researchers combined machine learning models with experimental data on how strongly proteins involved in gene regulation bind to their regulatory sequences in different tissues, and then performed evolutionary comparisons between human, chimpanzee and gorilla. "We now know which are the positively selected regions controlling gene expression in the human brain. And the more we learn about the genes they are controlling, the more complete our understanding of cognition and evolution, and the more scope there will be to act on that understanding," concludes Marc Robinson-Rechavi.Most random genetic mutations neither benefit nor harm an organism: they accumulate at a steady rate that reflects the amount of time that has passed since two living species had a common ancestor. In contrast, an acceleration in that rate in a particular part of the genome can reflect a positive selection for a mutation that helps an organism to survive and reproduce, which makes the mutation more likely to be passed on to future generations. Gene regulatory elements are often only a few nucleotides long, which makes estimating their acceleration rate particularly difficult from a statistical point of view.
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Biotechnology
| 2,020 |
December 15, 2020
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https://www.sciencedaily.com/releases/2020/12/201214123538.htm
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One-step method to generate mice for vaccine research
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To develop vaccines and investigate human immune responses, scientists rely on a variety of animal models, including mice that can produce human antibodies through genetically engineered B cell receptors, which are specialized antibodies bound to the B cell membrane. These mice, however, often take several years to develop, requiring a complicated process of genetic modification and careful breeding.
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"The time it takes to generate these specialized mice has been a major factor in delaying vaccine development," says Facundo Batista, PhD, associate director of the Ragon Institute of MGH, MIT and Harvard. "With the recent advances in gene editing technology like CRISPR/Cas9, we knew there had to be a way to speed up this process significantly."Batista's group has developed a new method for generating mouse lines for pre-clinical vaccine evaluation that dramatically shortens this timeline. In a study published recently in the journal To test this technology, the researchers engineered mice to have human B cell receptors that are precursors to what are called broadly neutralizing HIV antibodies. These antibodies are known to be effective in combating HIV, but they are difficult to stimulate through vaccination. The precursors responded to an antigen currently being used in clinical HIV trials by generating broadly neutralizing antibody-like mutations. The ability to quickly evaluate the ability of different antigens to active these precursors has the potential to significantly accelerate vaccine development.The engineered B cells were not just capable of making high-quality antibodies; some became a specialized form of B cell known as memory B cells, which are used to maintain long-lasting immunity once antibodies are produced against a pathogen. This means the mice can likely be used to quickly validate good candidate vaccines for HIV and other pathogens."This new technique may allow scientists studying vaccines and antibody evolution to tremendously speed up their research," says Ragon research fellow Xuesong Wang, PhD, co-first author on the paper.Rashmi Ray, PhD, also co-first author and a Ragon research fellow, agrees: "It will allow researchers to respond much more quickly and flexibly to new developments in the field."
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Biotechnology
| 2,020 |
December 14, 2020
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https://www.sciencedaily.com/releases/2020/12/201214164322.htm
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Compound derived from thunder god vine could help pancreatic cancer patients
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The results of a pre-clinical study led by researchers at the Translational Genomics Research Institute (TGen), an affiliate of City of Hope, suggest how a compound derived from the thunder god vine -- an herb used in China for centuries to treat joint pain, swelling and fever -- is able to kill cancer cells and potentially improve clinical outcomes for patients with pancreatic cancer.
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The medicinal plant's key ingredient, triptolide, is the basis of a water-soluble prodrug called Minnelide, which appears to attack pancreatic cancer cells and the cocoon of stroma surrounding the tumor that shields it from the body's immune system. Investigators recently published the study results in the journal The study found that the compound's mechanism of action is the ability of triptolide (Minnelide) to disrupt what are known as super-enhancers, strings of DNA needed to maintain the genetic stability of pancreatic cancer cells and the cancer-associated-fibroblasts that help make up the stroma surrounding the cancer."The cancer cells rely on super-enhancers for their growth and survival," said Dr. Haiyong Han, a Professor in TGen's Molecular Medicine Division and one of the study's senior authors."We found that by disrupting these super-enhancers triptolide not only attacks the cancer cells, but also the stroma, which helps accelerate cancer cell death."While triptolide has been known to be a general transcriptional inhibitor and a potent antitumor agent, we are the first to report its role in modulating super-enhancers to regulate the expression of genes, especially cancer-causing genes," said Dr. Han, who also is head of the basic research unit in TGen's Pancreatic Cancer Program.Pancreatic cancer is the third leading cause of cancer-related death in the U.S., annually killing more than 47,000 Americans."There is an urgent need to identify and develop treatment strategies that not only target the tumor cells, but can also modulate the stromal cells," said Dr. Daniel Von Hoff, TGen Distinguished Professor and another senior author of the study."Based on our findings, using modulating compounds such as triptolide to reprogram super-enhancers may provide means for effective treatment options for pancreas cancer patients," said Dr. Von Hoff, considered one of the nation's leading authorities on pancreatic cancer.Thunder god vine (Tripterygium wilfordii), also known as léi g?ng téng, is native to China, Japan and Korea. Traditional Chinese medicine has used the vine for more than 2,000 years as a treatment for everything from fever to inflammation and autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. The chemical compound triptolide is among the more than 100 bioactive ingredients derived from the thunder god vine.
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Biotechnology
| 2,020 |
December 11, 2020
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https://www.sciencedaily.com/releases/2020/12/201211115522.htm
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New tool for watching and controlling neural activity
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A new molecular probe from Stanford University could help reveal how our brains think and remember. This tool, called Fast Light and Calcium-Regulated Expression or FLiCRE (pronounced "flicker"), can be sent inside any cell to perform a variety of research tasks, including tagging, recording and controlling cellular functions.
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"This work gets at a central goal of neuroscience: How do you find the system of neurons that underlie a thought or cognitive process? Neuroscientists have been wanting this type of tool for a long time," said Alice Ting, professor of genetics in the Stanford School of Medicine and of biology in the School of Humanities and sciences, whose team co-led this work with the lab of Stanford psychiatrist and bioengineer, Karl Deisseroth.In proof-of-concept experiments, detailed in a paper published Dec. 11 in Cell, the researchers used FLiCRE to take a snapshot of neural activity associated with avoidance behavior in mice. By coupling the FLiCRE snapshot with RNA sequencing, they discovered that these activated neurons primarily belonged to a single cell type, which was inaccessible using genetic tools alone. They then used FLiCRE in combination with an opsin -- a protein for controlling neural activity with light developed by Deisseroth -- to reactivate those same neurons a day later, which led the mice to avoid entering a certain room. The brain region the researchers studied, called the nucleus accumbens, is thought to play an important role in human psychiatric diseases, including depression.FLiCRE is made up of two chains of molecular components that respond to the presence of blue light and calcium. This light sensitivity allows the researchers to precisely control the timing of their experiments, and calcium is an almost-universal indicator of cell activity. To get FLiCRE inside a cell, the researchers package it, in two parts, within a harmless virus. One part of FLiCRE attaches to the cell membrane and contains a protein that can enter the cell's nucleus and drive expression of whatever gene the researchers have selected. The other part of FLiCRE is responsible for freeing the protein under certain specific conditions, namely if the concentration of calcium is high and the cell is bathed in blue light.Whereas existing tagging techniques require hours to activate, the FLiCRE tagging process takes just minutes. The researchers also designed FLiCRE so that they can use standard genetic sequencing to find the cells in which FLiCRE activated. This allows them to study tens of thousands of cells at once, while other techniques tend to require the analysis of multiple microscopic images that each contain hundreds of cells.In one series of experiments, the researchers injected FLiCRE into cells in the nucleus accumbens and used an opsin to activate a neural pathway associated with avoidance behavior in the mice. Once the calcium in FLiCRE-containing cells spiked -- the cellular indication that the mouse is avoiding something -- the cells glowed a permanent red that was visible through a microscope. The researchers also sequenced the RNA of the cells to see which ones contained the fluorescent protein, producing a cell-by-cell record of neural activity."One goal was to map how brain regions are connected to each other in living animals, which is a really hard problem," said Christina Kim, a postdoctoral scholar in genetics at Stanford and co-lead author of the paper. "The beauty of FLiCRE is that we can pulse and activate neurons in one region and then record all of the connected downstream neurons. It is a really cool way to look at long-range brain activity connections."In the next experiments, the researchers used the cellular activity map from the first experiments. They also adjusted FLiCRE so that the protein expressed the opsin protein, which can be controlled by orange light to alter neuronal activity. After activating FLiCRE in the cells, the researchers sent orange light through the fiber optic implant whenever the mice would enter a certain room. In response, the mice steered clear of that room, indicating that FLiCRE had indeed located cells in the brain that drive avoidant behavior.The development and testing of FLiCRE combined chemistry, genetics, biology and neuroscience, and many specialties within those disciplines. As a result, the tool has a wide range of possible applications, including in cells outside the brain, the researchers say."I moved to Stanford in 2016 with the hope of being able to carry out extremely interdisciplinary and collaborative projects such as this," said Ting. "This project has been one of the most rewarding aspects of my move to Stanford -- seeing something this challenging and ambitious actually work out."The researchers are now working on additional versions of FLiCRE, with a goal of streamlining the process. They are hoping to simplify its structure and also make it capable of working with other biochemical events, such as protein interactions or neurotransmitter release.
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Biotechnology
| 2,020 |
December 11, 2020
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https://www.sciencedaily.com/releases/2020/12/201211115511.htm
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The secret behind male ornaments
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The tail feathers of the peacock, the enormous horn of male rhinoceros beetles, the protruding antlers of some deer: In nature, there are countless examples of features which at first sight may only have disadvantages for their owners. After all, it is more difficult to hide from a predator when one is wearing a colourful plumage, and large antlers do not make escaping in the forest any easier. As a rule, it is the male that has such characteristics.
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The evolution of male ornaments has therefore been fascinating to biologists since ever. Already Charles Darwin wondered of how such exaggerated, energy-consuming and in principle harmful structures could have been created by natural selection. Using the example of the swordtail fish (Xiphophorus hellerii), he explained his theory of sexual selection. Darwin's basic idea: If females prefer to mate with the carriers of striking ornamental traits, such traits might become established in the course of evolution even though they are likely to be harmful for their owners.Scientists from Würzburg, Constance and the USA have now been successful in finding the genetic bases of this evolutionary model in Xiphophorus, also well known to aquarists as one of their favourite pets. Among all eligible genes, the researchers identified some that are responsible for the development of the corresponding ornamental trait in this species of fish. Their findings also suggest that in the swordtail a gene that is actually important for neuronal processes in the brain has taken on an additional new function during evolution.The scientists published their findings in the journal "In several species of the genus Xiphophorus, the males carry a so-called 'sword', a striking extension of the lower edge of the tail fin, which is yellow, orange or red in colour and surrounded by a dark black margin," explains Manfred Schartl. The sword develops during puberty and can be as long as the fish itself in some species. This should actually be a disadvantage, because the conspicuous body ornament attracts predators on the one hand and on the other hand makes escaping more difficult as it reduces swimming performance. However, the females of Xiphophorus hellerii and several related species prefer to mate with males that carry a long sword -- males with shorter swords literally lose out in this competition.The genetic bases of this extension of the caudal fin in Xiphophorus have previously been unknown. However, knowledge of this phenomenon is necessary to test hypotheses about the role of sexual selection at the molecular genetic level.The scientists took a gradual approach to pinpointing the responsible genes. They started by looking for all genes that are specifically active in the sword developing part of the tail fin, but not in fin regions that do not form a sword. "This process resulted in a set of 329 differentially expressed genes in all sword transcriptomes," said Schartl, describing the result. The term transcriptome refers to the entirety of genes that are transcribed in a cell or tissue at a certain point in time, i.e. are active.The consideration that genes responsible for sword formation are only expressed in males led to a significant reduction in the number of suspects in the next step. The scientists created transcriptomes of cells from specific areas of the caudal fins in both male and female specimens. If the females showed comparable activities to males, it was clear that these genes are not among the sought-after candidates. After this process, 255 of the original 329 genes remained."Interestingly, this comparison revealed that a spatial pattern of five transcription factors -- Zic1, Hoxb13a, Six2a, Tbx3a and Pax9 -- is responsible for organising the preconditions in the caudal fin for the development of a sword, and that this pattern is also present in females," said Schartl.Genetic mapping came next to further reduce the still high number of 255 candidate genes. For this purpose, swordtail males were crossed with females of a related species whose males had lost their sword in the course of evolution. The male descendants from this mating have swords of different lengths due to the mixing of the parental genomes depending on their random genetic make-up. Sequencing those genomes using special high-throughput techniques then made it possible to correlate certain chromosome segments with sword formation, and those with the list of candidate genes. Finally, three genes were left over.The gene with the scientific name kcnh8 proved to be crucial for the development of the male characteristic. "This gene codes for a potassium channel -- a group of channels that play an important role in particular in the transmission and processing of stimuli in the nervous system," said Schartl. The new findings point to a gene with a primary function in neural cells that was recruited during evolution for developing the male sword about three to five million years ago, i.e. early during the diversification of swordtail fishes. The new function is not due to structural changes within the gene and its product, but to changes in gene regulation.Indeed, experiments show that kcnh8 in the sword during normal development and after treatment with male hormones is highly upregulated in the region where the sword is organised. In all other fin areas of the males and in female caudal fins it is only weakly expressed. In addition, further studies show a direct correlation between the level of gene expression of kcnh8 and the length of swords.Schartl and Meyer received support for their research on potassium channels from an expected source: botanists from the University of Würzburg. Rainer Hedrich, who heads the Department of Molecular Plant Physiology and Biophysics, and his colleague, Professor Dietmar Geiger, have been studying potassium channels for a long time. The techniques they used -- special patch-clamping methods -- could be easily transferred from plants to the fish.Potassium channels transport electrically charged particles and thus cause changes in the membrane potential in cells and tissues. According to the scientists, such channels create tissue-wide bioelectric gradients which affect the overall structure of the cellular microenvironment. Similar phenomena have been observed in the proliferation of cancer cells and have led to hypotheses about the importance of ion gradients for growth control. The role of Kcnh8 in the development of the ventral caudal outgrowth in male swordtails is in good agreement with these models.
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Biotechnology
| 2,020 |
December 11, 2020
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https://www.sciencedaily.com/releases/2020/12/201211100629.htm
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Why 'lab-made' proteins have unusually high temperature stability
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Bioengineers have found why proteins that are designed from scratch tend to be more tolerant to high temperatures than proteins found in nature.
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Natural proteins with high 'thermostability' are prized for their wide range of applications, from baking and paper-making to chemical production. Efforts to enhance protein thermostability -- and to discover the principles behind this -- is one of the hottest topics in biotech.The latest discoveries, described in theResearchers in the relatively young field of protein design have attempted to come up with new types of proteins for myriad medical, pharmaceutical and industrial applications. Until recently, protein engineers have focused on manipulating existing natural proteins. However, these natural proteins are difficult to alter without also distorting the general functioning of the protein -- much like adding a fifth wheel to a car.To avoid this, some protein engineers have begun to build novel proteins entirely from scratch, or what is called de novo protein design.However, this quest has its own set of issues. For example, building proteins from scratch is much harder computationally, and requires a complete understanding of the principles of protein folding -- the multiple levels of how a protein literally folds itself into a particular structure.In biology, structure determines function, much like how a key fits into a keyhole or a cog into a sprocket. The shape of a biological entity is what allows it to do its job within an organism. And upon their production by cells, proteins just fall into their shape, simply as a result of physical laws.But the principles that govern the interaction of these physical laws during the folding process are frustratingly complex -- hence the computational difficulty. They are also still largely unknown. This is why a great deal of effort in protein engineering in recent years has focused on attempting to discover these protein design principles that emerge from physical laws.And one of the mysteries facing protein designers has been the high thermostability of these 'lab-made' proteins."For some reason, de novo proteins have repeatedly shown increased tolerance in the face of quite high temperatures compared to natural proteins," said Nobuyasu Koga, associate professor at Institute for Molecular Science, and an author of the study. "Where others would 'denature', the lab-made proteins are still working just fine well above 100 ºC."The design principles that have been discovered so far emphasize the importance of the backbone structure of proteins -- the chain of nitrogen, carbon, oxygen and hydrogen atoms.On the other hand, these principles have also held that the tight packing of the fatty, hydrophobic (water-resistant) core of naturally occurring proteins -- or rather the molecular interactions that allow them to sit together as snugly as pieces of a jigsaw puzzle -- is the dominant force that drives protein folding. Just as how oil and water don't mix, the fattier part of the protein when surrounded by water will naturally pull itself together without any need for an external 'push'."Indeed, according to our design principles, protein cores were engineered specifically to be as tightly packed and as fatty as possible," Nobuyasu Koga said. "So the question was: Which is more important for high thermostability, backbone structure or the fat and tight core packing?"So the researchers took the de novo proteins they had designed that had shown the highest thermal stability, and began to tweak them with ten amino acids involved with the hydrophobic core packing. As they did this, they saw still folding ability and little reduction in overall thermal stability, suggesting that it is instead the backbone structure, not the hydrophobic core packing, that contributes the most to high thermostability. "It is surprising that the protein can fold with high thermal stability, even the loose core packing," said Naohiro Kobayashi, coauthor and a senior research fellow at RIKEN."Hydrophobic tight core packing may not even be very important for designed proteins," added Rie Koga, coauthor and a researcher at Exploratory Research Center on Life and Living Systems (ExCELLS). "We can create an exceptionally stable protein even if the core packing is not so optimized."The next step for the researchers is to further develop rational principles for protein design, especially with respect to what extent that substructures of the backbone, especially loops within it, can be altered without endangering its folding ability and high thermostability.
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Biotechnology
| 2,020 |
December 10, 2020
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https://www.sciencedaily.com/releases/2020/12/201210145808.htm
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Tasmanian devils may survive their own pandemic
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Amid the global COVID-19 crisis, there is some good news about a wildlife pandemic -- which may also help scientists better understand how other emerging diseases evolve.
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Researchers have found strong evidence that a transmissible cancer that has decimated Tasmanian devil populations likely won't spell their doom.For the first time, a research team led by Washington State University biologist Andrew Storfer employed genomic tools of phylodynamics, typically used to track viruses, such as influenza and SARS-CoV-2 , to trace the Tasmanian devil facial tumor disease. The approach they pioneered has opened the door for application to other genetically complex pathogens.The study, published in the journal "It is cautiously optimistic good news," said Storfer. "I think we're going to see continued survival of devils at lower numbers and densities than original population sizes, but extinction seems really unlikely even though it was predicted a decade ago."Since it was first identified in 1996, Tasmanian devil facial tumor disease has reduced populations of the iconic marsupial by 80%. The devils spread the infection when they fight and bite each other on the face. The disease is still largely fatal to Tasmanian devils who contract it, but it appears to be reaching an equilibrium, according to this study which confirms evidence from previous field studies. The authors say this new evidence means managers should re-consider the practice of releasing captive-bred devils into the wild."Active management may not be necessary and could actually be harmful," Storfer said. "It looks like the devil populations are naturally evolving to tolerate and possibly even resist the cancer. By introducing a whole bunch of genetically naïve individuals, they could breed with the wild individuals, basically mix up the gene pool and make it less well-adapted."The disease-naïve, captive-bred individuals could also increase transmission of the disease among different groups of devils.Researchers have relied on field studies and modeling to try to understand the spread of the Tasmanian Devil facial tumor disease, but this is the first time that phylodynamics has been used successfully to trace the transmissible cancer.Phylodynamics employs genetic sequencing to investigate evolutionary relationships among pathogen lineages to understand and predict how a disease spreads across a population. This method has been used to trace the spread of viruses, including influenza and SARS-CoV-2, which accumulate mutations in their genomes at a relatively fast rate.The Tasmanian devil facial tumor disease is much more genetically complex than a virus, however. Since the disease is a type of cancer, derived from the animals' own cells, the genes that need to be traced are essentially the Tasmanian devils' genes, of which there are thousands more than those of a typical viral pathogen.In this study, the researchers screened more than 11,000 genes from tumor samples to find genes that changed in a "clock-like" manner, showing mutations that were accumulating rapidly. They then identified 28 genes representing more than 430,000 base pairs, the fundamental units of DNA.In comparison, the genome of SARS-CoV-2, the virus that causes COVID-19, has 29,000 base pairs.The breakthrough in using this method took months of pain-staking computational work, which Storfer credits to his doctoral student, Austin Patton, a recent WSU Ph.D. graduate who is now a post-doctoral fellow at University of California, Berkeley."One of the most exciting advances this study presents is the opportunity to apply these types of approaches to virtually any pathogen," said Patton. "It opens the door to using the kind of methods that have been shown to be so important in the study of viruses to a whole new suite of pathogens that impact humans as well as wildlife."
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Biotechnology
| 2,020 |
December 10, 2020
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https://www.sciencedaily.com/releases/2020/12/201210145755.htm
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Using CRISPR, new technique makes it easy to map genetic networks
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CRISPR-Cas9 makes it easy to knock out or tweak a single gene to determine its effect on an organism or cell, or even another gene. But what if you could perform several thousand experiments at once, using CRISPR to tweak every gene in the genome individually and quickly see the impact of each?
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A team of University of California, Berkeley, scientists has developed an easy way to do just that, allowing anyone to profile a cell, including human cells, and rapidly determine all the DNA sequences in the genome that regulate the expression of a specific gene.While the technique will mostly benefit basic researchers who are interested in tracking the cascade of genetic activity -- the genetic network -- that impacts a gene they're interested in, it will also help researchers quickly find the regulatory sequences that control disease genes and possibly find new targets for drugs."A disease where you might want to use this approach is cancer, where we know certain genes that those cancer cells express, and need to express, in order to survive and grow," said Nicholas Ingolia, UC Berkeley associate professor of molecular and cell biology. "What this tool would let you do is ask the question: What are the upstream genes, what are the regulators that are controlling those genes that we know about?"Those controllers may be easier to target therapeutically in order to shut down the cancer cells.The new technique simplifies something that has been difficult to do until now: backtrack along genetic pathways in a cell to find these ultimate controllers."We have a lot of good ways of working forward," he said. "This is a nice way of working backward, figuring out what is upstream of something. I think it has a lot of potential uses in disease research.""I sometimes use the analogy that when we walk into a dark room and flip a light switch, we can see what light gets switched on. That light is like a gene, and we can tell, when we flip the switch, what genes it turns on. We are already very good at that," he added. "What this lets us do is work backward. If we have a light we care about, we want to find out what are the switches that control it. This gives us a way to do that."Ryan Muller, a graduate student in the Ingolia lab, and colleagues Lucas Ferguson and Zuriah Meacham, along with Ingolia, will publish the details of their technique online on Dec. 10 in the journal Since the advent of CRISPR-Cas9 gene-editing eight years ago, researchers who want to determine the function of a specific gene have been able to precisely target it with the Cas9 protein and knock it out. Guided by a piece of guide RNA complementary to the DNA in the gene, the Cas9 protein binds to the gene and cuts or, as with CRISPR interference (CRISPRi), inhibits it.In the crudest type of assay, the cell or organism either lives or dies. However, it's possible to look for more subtle effects of the knockout, such as whether a specific gene is turned on or off, or how much it's turned up or down.Today, that requires adding a reporter gene -- often one that codes for a green fluorescent protein -- attached to an identical copy of the promoter that initiates expression of the gene you're interested in. Since each gene's unique promoter determines when that gene is expressed, if the Cas9 knockout affects expression of your gene of interest, it will also affect expression of the reporter, making the culture glow green under fluorescent light.Nevertheless, with 6,000 total genes in yeast -- and 20,000 total genes in humans -- it's a big undertaking to tweak each gene and discover the effect on a fluorescent reporter."CRISPR makes it easy to comprehensively survey all the genes in the genome and perturb them, but then the big question is, How do you read out the effects of each of those perturbations?" he said.This new technique, which Ingolia calls CRISPR interference with barcoded expression reporter sequencing, or CiBER-seq, solves that problem, allowing these experiments to be done simultaneously by pooling tens of thousands of CRISPR experiments. The technique does away with the fluorescence and employs deep sequencing to directly measure the increased or decreased activity of genes in the pool. Deep sequencing uses high-throughput, long-read next generation sequencing technology to sequence and essentially count all the genes expressed in the pooled samples."In one pooled CiBER-seq experiment, in one day, we can find all the upstream regulators for several different target genes, whereas, if you were to use a fluorescence-based technique, each of those targets would take you multiple days of measurement time," Ingolia said.CRISPRing each gene in an organism in parallel is straightforward, thanks to companies that sell ready-made, single guide RNAs to use with the Cas9 protein. Researchers can order sgRNAs for every gene in the genome, and for each gene, a dozen different sgRNAs -- most genes are strings of thousands of nucleotides, while sgRNAs are about 20 nucleotides long.The team's key innovation was to link each sgRNA with a unique, random nucleotide sequence -- essentially, a barcode -- connected to a promoter that will only transcribe the barcode if the gene of interest is also switched on. Each barcode reports on the effect of one sgRNA, individually targeting one gene out of a complex pool of thousands of sgRNAs. Deep sequencing tells you the relative abundances of every barcode in the sample -- for yeast, some 60,000 -- allowing you to quickly assess which of the 6,000 genes in yeast has an effect on the promoter and, thus, expression of the gene of interest. For human cells, a researcher might insert more than 200,000 different guide RNAs, targeting each gene multiple times."This is really the heart of what we were able to do differently: the idea that you have a big library of different guide RNAs, each of which is going to perturb a different gene, but it has the same query promoter on it -- the response you are studying. That query promoter transcribes the random barcode that we link to each guide," he said. "If there is a response you care about, you poke each different gene in the genome and see how the response changes."If you get one barcode that is 10 times more abundant than any of the others, for example, that tells you that that query promoter is switched on 10 times more strongly in that cell. In practice, Ingolia attached about four different barcodes to each guide RNA, as a quadruple check on the results."By looking more directly at a gene expression response, we can pick up on a lot of subtlety to the physiology itself, what is going on inside the cell," he said.In the newly reported experiments, the team queried five separate genes in yeast, including genes involved in metabolism, cell division and the cell's response to stress. While it may be possible to study up to 100 genes simultaneously when CRISPRing the entire genome, he suspects that, for convenience, researchers would limit themselves to four or five at once.The work was funded by the National Institutes of Health (DP2 CA195768, R01 GM130996).
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Biotechnology
| 2,020 |
December 10, 2020
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https://www.sciencedaily.com/releases/2020/12/201210145751.htm
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Blocking protein restores strength, endurance in old mice
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Blocking the activity of a single protein in old mice for one month restores mass and strength to the animals' withered muscles and helps them run longer on a treadmill, according to a study by researchers at the Stanford University School of Medicine. Conversely, increasing the expression of the protein in young mice causes their muscles to atrophy and weaken.
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"The improvement is really quite dramatic" said Helen Blau, PhD, professor of microbiology and immunology. "The old mice are about 15% to 20% stronger after one month of treatment, and their muscle fibers look like young muscle. Considering that humans lose about 10% of muscle strength per decade after about age 50, this is quite remarkable."The protein hasn't previously been implicated in aging. The researchers show that the amount of the protein, called 15-PGDH, is elevated in old muscle and is widely expressed in other old tissues. Experiments they conducted in human tissue raise hopes for a future treatment for the muscle weakness that occurs as people age.Blau, the Donald E. and Delia B. Baxter Foundation Professor and director of the Baxter Laboratory for Stem Cell Biology, is the senior author of the study, which will be published online Dec. 10 in Muscle loss during aging is known as sarcopenia, and it accounts for billions of dollars of health care expenditures in the United States each year as people lose the ability to care for themselves, experience more falls and become increasingly less mobile. It is due to changes in muscle structure and function: The muscle fibers shrink and the number and function of the cellular powerhouses known as mitochondria dwindle.Blau and her colleagues have long been interested in understanding muscle function after muscle injury and in diseases like Duchenne muscular dystrophy. Previously, they found that a molecule called prostaglandin E2 can activate muscle stem cells that spring into action to repair damaged muscle fibers."We wondered whether this same pathway might also be important in aging," Blau said. "We were surprised to find that PGE2 not only augments the function of stem cells in regeneration, but also acts on mature muscle fibers. It has a potent dual role."Prostaglandin E2 levels are regulated by 15-PGDH, which breaks down prostaglandin E2. The researchers used a highly sensitive version of mass spectrometry, a method for differentiating closely related molecules, to determine that compared with young mice, the 15-PGDH levels are elevated in the muscles of older animals, and the levels of prostaglandin E2 are lower.They found a similar pattern of 15-PGDH expression in human muscle tissues, as those from people in their 70s and early 80s expressed higher levels than those from people in their mid-20s."We knew from our previous work that prostaglandin E2 was beneficial for regeneration of young muscles," Palla said. "But its short half-life makes it difficult to translate into a therapy. When we inhibited 15-PGDH, we observed a systemic elevation of prostaglandin E2 levels leading to a bodywide muscle improvement in aged mice."The researchers administered a small molecule that blocks the activity of 15-PGDH to the mice daily for one month and assessed the effect of the treatment on the old and young animals."We found that, in old mice, even just partially inhibiting 15-PGDH restored prostaglandin E2 to physiological levels found in younger mice," Blau said. "The muscle fibers in these mice grew larger, and were stronger, than before the treatment. The mitochondria were more numerous, and looked and functioned like mitochondria in young muscle."Treated animals were also able to run longer on a treadmill than untreated animals.When Palla and her colleagues performed the reverse experiment -- overexpressing 15-PGDH in young mice -- the opposite occurred. The animals lost muscle tone and strength, and their muscle fibers shrank and became weaker, like those of old animals.Finally, the researchers observed the effect of prostaglandin E2 on human myotubes -- immature muscle fibers -- growing in a lab dish. They found that treating the myotubes with prostaglandin E2 caused them to increase in diameter, and protein synthesis in the myotubes was increased -- evidence that prostaglandin E2 worked directly on the muscle cells, not on other cells in the tissue microenvironment."It's clear that this one regulator, 15-PGDH, has a profound effect on muscle function," Blau said. "We're hopeful that these findings may lead to new ways to improve human health and impact the quality of life for many people. That's one of my main goals."Blau and Palla are studying more about what controls the levels and activity of 15-PGDH during normal aging, and how it might affect the function of other tissues in the body."The mice perform better on a treadmill, but that requires more than just an increase in muscle strength," Blau said. "Other organ systems are involved -- the heart and lungs, for example. It suggests an overall improvement in the function of the whole animal."
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Biotechnology
| 2,020 |
December 10, 2020
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https://www.sciencedaily.com/releases/2020/12/201210112118.htm
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Inhaled vaccine induces fast, strong immune response in mice and non-human primates
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Researchers demonstrate in a proof-of-concept study that a phage-based inhalation delivery system for vaccines generates potent antibody responses in mice and non-human primates, without causing lung damage. The findings suggest that a safe and effective lung delivery system could one day be used for vaccines and therapeutics against respiratory diseases. The results appear December 10 in the journal
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"This translational strategy potentially enables more effective delivery of therapeutics or vaccines while reducing the chance of toxic side effects," says co-senior study author Wadih Arap of Rutgers Cancer Institute of New Jersey. "In ongoing research, we hope that this work will play a crucial role in the development of targeted vaccines and treatments to block the spread of respiratory infectious diseases, possibly for the current COVID-19 pandemic, especially in the setting of underserved populations."Pulmonary delivery has many advantages over other routes of administration, particularly for the development of vaccines or therapeutics against respiratory infections, because the vaccines arrive directly at the site of the infection. Inhalation-based vaccination is needle free and minimally invasive, which is especially attractive for administrating multiple doses. It improves therapeutic bioavailability while reducing potential side effects by achieving a more rapid onset of action than needle-based vaccination."The very extensive and accessible layer of cell surfaces in the lungs is highly vascularized, which allows rapid absorption of molecules throughout circulation in much higher concentrations by avoiding the drug-metabolizing enzymes of the gastrointestinal tract and liver," explains co-senior study author Renata Pasqualini of Rutgers Cancer Institute of New Jersey. "Because the lungs are constantly being exposed to pathogens from the air, they likely have a high level of immune defense activity, and therefore represent an efficient site for immune protection against airborne pathogens."Lung delivery could protect against airborne pathogens that cause diseases such as tuberculosis, influenza, Ebola, measles, and COVID-19. But this approach has not been adopted widely, partly because the underlying physiological mechanisms remain largely unknown. Answering this question is critical for designing a general lung delivery system for widespread use.In the new study, Arap and Pasqualini devised and validated a safe, effective lung delivery system that could be used for a broad range of translational applications, and showed how it works. The approach involves the use of phages -- viruses that can infect and replicate within bacterial cells. In certain types of vaccines, phage particles that carry peptides are used to trigger protective immune responses.First, the researchers screened for and identified a peptide -- CAKSMGDIVC -- that could efficiently deliver phage particles across the pulmonary barrier and into the bloodstream. Specifically, phage particles that display CAKSMGDIVC on their surface are absorbed into the body when the peptide binds to and is internalized through its receptor, ?3?1 integrin, on the surface of cells lining the lung airways. Inhaled delivery of CAKSMGDIVC-displaying phage particles elicited a robust antibody response against the phage particles in mice and non-human primates, without damaging the lungs.According to the authors, the new lung delivery system is safe and effective, and has unique advantages for the development of vaccines and therapeutics against airborne pathogens. Phage particles induce very strong and sustained immune responses, without producing toxic side effects. Because they do not replicate inside eukaryotic cells, their use is generally considered safe when compared to other classic viral-based vaccination strategies. In fact, phage particles have been used as antibiotics against multidrug-resistant bacteria and as vaccine carriers for decades.In terms of practical implementation, phage particles are highly stable under harsh environmental conditions, and their large-scale production is extremely cost-effective compared to traditional methods used for vaccine production. Moreover, unlike conventional peptide-based vaccines that often become inactivated, the new lung delivery system has no cumbersome, stringent, or expensive cold-chain requirements for field applications in the developing world. "In addition, phage particles are versatile and can be genetically engineered by standard molecular biology technology," Arap says.Moving forward, the researchers plan to examine the kinetics of pulmonary transport after multiple doses and investigate cell-based immune responses. "It is important to note that all this work was in preclinical models, so we look forward to the translation of our approach to clinical applications such as lung-targeted drug delivery or pulmonary-based vaccination," Pasqualini says.
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Biotechnology
| 2,020 |
December 10, 2020
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https://www.sciencedaily.com/releases/2020/12/201210074712.htm
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Cataloging nature's hidden arsenal: Viruses that infect bacteria
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Scientists are continually searching for new and improved ways to deal with bacteria, be it to eliminate disease-causing strains or to modify potentially beneficial strains. And despite the numerous clever drugs and genetic engineering tools humans have invented for these tasks, those approaches can seem clumsy when compared to the finely tuned attacks waged by phages -- the viruses that infect bacteria.
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Phages, like other parasites, are continually evolving ways to target and exploit their specific host bacterial strain, and in turn, the bacteria are continually evolving means to evade the phages. These perpetual battles for survival yield incredibly diverse molecular arsenals that researchers are itching to study, yet doing so can be tedious and labor-intensive.To gain insight into these defensive strategies, a team led by Berkeley Lab scientists has just developed an efficient and inexpensive new method. As reported in "Despite nearly a century of molecular work, the underlying mechanisms of phage-host interactions are only known for a few pairs, where the host is a well-studied model organism that can be cultured in a lab," said corresponding author Vivek Mutalik, a research scientist in Berkeley Lab's Environmental Genomics and Systems Biology (EGSB) Division. "However, phages represent the most abundant biological entities on Earth, and due to their impact on bacteria, they are key drivers of environmental nutrient cycles, agricultural output, and human and animal health. It has become imperative to gain more foundational knowledge of these interactions in order to better understand the planet's microbiomes and to develop new medicines, such as bacteria-based vaccines or phage cocktails to treat antibiotic-resistant infections."The team's three-pronged approach, called barcoded loss-of-function and gain-of-function libraries, uses the established technique of creating gene deletions and also increasing gene expression to identify which genes the bacteria use to evade the phages. This information also tells the scientists which receptors the phages are targeting without having to analyze the phages' genomes. (However, the scientists do plan to adapt the technique for use on viruses in the future, to learn even more about their function.)Mutalik and his colleagues tested their method on two strains of E. coli that are known to be targeted by 14 genetically diverse phages. Their results confirmed that the method works smoothly by speedily revealing the same suite of phage receptors that had been previously identified through decades of research, and also provided new hits that were missed in earlier studies.According to Mutalik, the approach can also be scaled-up to simultaneously evaluate phage relationships for hundreds of bacteria sampled from diverse environments. This will make it much easier for scientists to study the planet's biological "dark matter," which refers to the unculturable and therefore poorly understood microorganisms that abound in many environments. In fact, it is estimated that 99% of all living microorganisms can't be cultured in a lab.The team's approach also represents an opportunity to standardize genetic resources used in phage research, which has always been an ad-hoc and highly variable process, and create sharable reagents and datasets."The role of phages is a huge 'known-unknown,' as we know there are phages everywhere, but hardly know anything more. For example, we understand less than 10% of the genes encoded in previously sequenced phage genomes," said Mutalik. "Now that we finally have a streamlined tool to look at phages, there are many exciting questions we can start to answer and an opportunity to make a difference in the world."
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Biotechnology
| 2,020 |
December 9, 2020
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https://www.sciencedaily.com/releases/2020/12/201209170642.htm
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Researchers suggest stool transplants can battle serious infections
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Could number two be number one when it comes to combating recurrent
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Using genetic material analysis and machine learning, UBC researchers have pinpointed several key factors to ensure successful fecal microbiota transplants (FMT), which have proven successful in treating bacterial infections in the gut including illnesses like "This therapy is still in its infancy, but studies like ours are helping identify key contributors to its overall success," says Kazemian, a graduate student at UBC Okanagan's School of Engineering.Kazemian and her supervisor, Assistant Professor Sepideh Pakpour, are investigating the internal dynamics of both donors and recipients to set out a formula for the effectiveness of the therapy.Kazemian explains that severely damaged gut ecosystems, like someone who has had "In our study, we showed that the success of gut ecological recovery through FMT is dependent on several factors, including the donor gut microbiome -- the presence of specific bacteria -- as well as the recipient's pre-FMT gut community structures and the absence of specific bacteria and fungi."Some previous studies have pointed to the possibility of "super" donors, but these new findings indicate the relationship between donors and recipients is much more complex. Pakpour says the notion of the super-donor is oversimplified due to the observed short-term fluctuations. A recipient's microbiota may be just as important to consider when predicting treatment outcomes, especially in unbalanced conditions such as ulcerative colitis."Take, for example, blood transplants where we have a strong understanding of the four main blood groups or types, and how they interact with one another," says Pakpour. "With fecal transplants the research up to this point has not been as clear in what constitutes a good match or compatibility."Working with data from the University of Alberta Hospital, Kazemian and Pakpour analyzed the gut composition and DNA from samples extracted before and after FMT.According to Kazemian, their findings indicate that there isn't a "one stool fits all" approach to ensure transplant success."The data illustrates that the unique microorganisms in everyone's bodies respond differently over time, and this has profound implications on whether these transplants work well or not."The researchers suggest that preparing donors and patients' gut ecosystems prior to transplant, maybe by using metabolites, would potentially sync their microbiota leading the way to a higher probability of transplant success.The new research is published in Nature Research's
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Biotechnology
| 2,020 |
December 9, 2020
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https://www.sciencedaily.com/releases/2020/12/201209124921.htm
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Magnesium contact ions stabilize the macromolecular structure of transfer RNA
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In cells transfer RNA (tRNA) translates genetic information from the encoding messenger RNA (mRNA) for protein synthesis. New results from ultrafast spectroscopy and in-depth theoretical calculations demonstrate that the complex folded structure of tRNA is stabilized by magnesium ions in direct contact with phosphate groups at the RNA surface.
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RNA structures consist of long sequences of nucleotides which are composed of a nucleobase, e.g., adenine, uracil, cytosine or guanine, a negatively charged phosphate group, and a sugar unit. The phosphate groups together with the sugars form the backbone of the macromolecule which exists as a folded structure in the cellular environment, the so-called tertiary structure. The tertiary structure of tRNA from yeast has been determined by x-ray diffraction. For maintaining this structure, a basic prerequisite for its cellular function, the repulsive electric force between the negatively charged phosphate groups needs to be compensated by positively charged ions and by water molecules of the environment. How this works at the molecular level has remained unclear so far, there are conflicting pictures of ion and water arrangements and interactions in the scientific literature.Scientists from the Max-Born-Institute in Berlin have now identified contact pairs of positively charged magnesium ions and negatively charged phosphate groups as a decisive structural element for minimizing the electrostatic energy of tRNA and, thus, stabilizing its tertiary structure. Their study which has been published in The Molecular vibrations of the phosphate groups serve as noninvasive probes of the coupling between tRNA and its aqueous environment. The frequency and infrared absorption strength of such vibrations directly reflects the interactions with ions and water molecules. Vibrational spectroscopy of tRNA samples of different magnesium content together with two-dimensional infrared spectroscopy in the femtosecond time domain allow for discerning specific local geometries in which phosphate groups couple to ions and the water shell. The presence of a magnesium ion in the immediate neighborhood of a phosphate group shifts the asymmetric phosphate stretching vibration to a higher frequency and generates a characteristic infrared absorption band used for detection of the molecular species.Experiments at different concentrations of magnesium ions show that a single tRNA structure forms up to six contact ion pairs, preferentially at locations where the distance between neighboring phosphate groups is small and the corresponding negative charge density high. The contact ion pairs make the decisive contribution to lowering the electrostatic energy and, consequently, stabilizing the tertiary tRNA structure. This picture is confirmed in a quantitative way by an in-depth theoretical analysis. The ion pairs impose an electrical force on water molecules nearby and orient them in space, again reducing the electrostatic energy. In contrast, mobile ions in the first five to six water layers around tRNA make a smaller contribution to stabilizing tRNA structure.The new results give detailed quantitative insight in the electric properties of a key biomolecule. They underscore the high relevance of molecular probes for elucidating the relevant molecular interactions and the need for theoretical descriptions at the molecular level.
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Biotechnology
| 2,020 |
December 9, 2020
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https://www.sciencedaily.com/releases/2020/12/201209115209.htm
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A simple rule drives the evolution of useless complexity
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A new study at the University of Chicago has shown that elaborate protein structures accumulate over deep time even when they serve no purpose, because a universal biochemical property and the genetic code force natural selection to preserve them. The work was published on Dec. 9, 2020 in
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Most proteins in our cells form specific complexes with other proteins, a process called multimerization. Like other kinds of complexity in biology, multimers are usually thought to persist over evolutionary time because they confer some functional benefit that is favored by natural selection."How complexity evolves is one of the great questions of evolutionary biology," said senior author Joseph Thornton, PhD, professor of human genetics and ecology and evolution at the University of Chicago. "The classic explanation is that elaborate structures must exist because they confer some functional benefit on the organism, so natural selection drives ever-increasing states of complexity. Clearly in some cases complexity is adaptive, like the evolution of the eye: complex eyes see better than simple ones. But at the molecular level, we found that there are other simple mechanisms that drive the build-up of complexity."The research team, led by Thornton and University of Chicago postdoctoral fellow Georg Hochberg, PhD, set out to study the evolution of multimerization in a family of proteins called steroid hormone receptors, which assemble into pairs (called dimers).They used a technique called ancestral protein reconstruction, a kind of molecular "time travel," Thornton said, that allowed them to recreate ancient proteins in the lab and experimentally examine how they were affected by mutations that happened hundreds of millions of years ago.To their surprise, they found that the ancient proteins functioned no differently when assembled into a dimer than if they had never evolved to dimerize at all. There was nothing useful or beneficial about forming the complex.The explanation for why the dimeric form of the receptor has persisted for 450 million years turned out to be surprisingly simple. "These proteins gradually became addicted to their interaction, even though there is nothing useful about it," explained Hochberg, who is now a group leader at the Max Planck Institute in Marburg, Germany. "The parts of the protein that form the interface where the partners bind each other accumulated mutations that were tolerable after the dimer evolved, but would have been deleterious in the solo state. This made the protein totally dependent on the dimeric form, and it could no longer go back. Useless complexity became entrenched, essentially forever."The researchers showed that simple biochemical, genetic and evolutionary principles make entrenchment of molecular complexes inevitable. The genes that code for every protein are subject to a constant hail of mutations over generations, many of which would disrupt the protein's ability to fold up and function properly. A form of natural selection called purifying selection removes these deleterious mutations from the population.Once a protein evolves to multimerize, the parts that form the interface can accumulate mutations that would be deleterious if the protein were in the solo state, so long as they can be tolerated in the multimer. Purifying selection then entrenches the complex form, preventing a return to the solo state.The researchers showed that a simple and universal rule of biochemistry underlies entrenchment. Proteins are made up of amino acids, which may be water soluble, or hydrophobic, meaning they dissolve easily in oil but not water. Usually, proteins fold so the water-soluble amino acids are on the outside and the hydrophobic amino acids are on the inside. Mutations that make a protein's surface more oil soluble impair its folding, so purifying selection removes them if they occur in solo proteins.If the protein evolves to multimerize, however, those hydrophobic amino acids on the interface surface are hidden from water, and become invisible to purifying selection. The multimer is then entrenched, because returning to the solo state would expose the now-oil-soluble and deleterious interface.This "hydrophobic ratchet" appears to be universal. The researchers analyzed a massive database of protein structures, including hundreds of dimers and related solo proteins, and found that the vast majority of interfaces have become so hydrophobic that the dimeric form is deeply entrenched.This mechanism, operating on thousands of proteins over hundreds of millions of years, could drive the gradual accumulation of many useless complexes inside cells."Some complexes surely have important functions, but even those will be entrenched by the hydrophobic ratchet, making them harder to lose than they would otherwise be," Hochberg said. "With the ratchet constantly operating in the background, our cells have probably built up a massive stock of entrenched complexes, many of which never performed a useful function, or long ago ceased to do so."Future directions include investigating whether or not interactions other than multimerization may be the result of entrenchment. "This was a story about proteins dimerizing with other copies of themselves, which is a super common process," said Thornton. "But there are lots of other interactions in cells, and we think it's possible that some of those may have accumulated during evolution because of a similar kind of acquired dependence on molecular complexity."
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Biotechnology
| 2,020 |
December 9, 2020
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https://www.sciencedaily.com/releases/2020/12/201209094241.htm
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Evolution may be to blame for high risk of advanced cancers in humans
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Compared to chimpanzees, our closest evolutionary cousins, humans are particularly prone to developing advanced carcinomas -- the type of tumors that include prostate, breast, lung and colorectal cancers -- even in the absence of known risk factors, such as genetic predisposition or tobacco use.
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A recent study led by researchers at University of California San Diego School of Medicine and Moores Cancer Center helps explain why. The study, published December 9, 2020 in "At some point during human evolution, the SIGLEC12 gene -- and more specifically, the Siglec-12 protein it produces as part of the immune system -- suffered a mutation that eliminated its ability to distinguish between 'self' and invading microbes, so the body needed to get rid of it," said senior author Ajit Varki, MD, Distinguished Professor at UC San Diego School of Medicine and Moores Cancer Center. "But it's not completely gone from the population -- it appears that this dysfunctional form of the Siglec-12 protein went rogue and has now become a liability for the minority of people who still produce it."Ajit Varki, who is also co-director of both the Glycobiology Research and Training Center and Center for Academic Research and Training in Anthropogeny, led the study with Nissi Varki, MD, professor of pathology at UC San Diego School of Medicine.In a study of normal and cancerous tissue samples, the researchers discovered that the approximately 30 percent of people who still produce Siglec-12 proteins are at more than twice the risk of developing an advanced cancer during their lifetimes, compared to people who cannot produce Siglec-12.Normally, genes that encode such dysfunctional proteins are eliminated by the body over time, and approximately two-thirds of the global human population has stopped producing the Siglec-12 protein. Where the gene still hangs around in humans, it was long thought be of no functional relevance, and there have been very few follow-up studies over the two decades since it was discovered. Meanwhile, chimpanzees still produce functioning Siglec-12.When Nissi Varki's team set out to detect the Siglec-12 in non-cancerous tissue samples using an antibody against the protein, approximately 30 percent of the samples were positive, as expected from the genetic information. In contrast, the majority of advanced cancer samples from the same populations were positive for the Siglec-12 protein.Looking at a different population of patients with advanced stage colorectal cancer, the researchers found that more than 80 percent had the functional form of the SIGLEC-12 gene, and those patients had a worse outcome than the minority of patients without it."These results suggest that the minority of individuals who can still make the protein are at much greater risk of having an advanced cancer," Nissi Varki said.The researchers also validated their findings in mice by introducing tumor cells engineered to produce Siglec-12. The resulting cancers grew much faster, and turned on many biological pathways known to be involved in advanced cancers, compared to control tumor cells without functioning Siglec-12.According to Ajit Varki, this information is important because it could be leveraged for future diagnostics and treatments. The team got a jump start by developing a simple urine test that could be used to detect the presence of the dysfunctional protein, and "we might also be able to use antibodies against Siglec-12 to selectively deliver chemotherapies to tumor cells that carry the dysfunctional protein, without harming non-cancerous cells," he said.Additional co-authors of the study include: Shoib S. Siddiqui, Michael Vaill, Raymond Do, Naazneen Khan, Andrea L. Verhagen, Gen-Sheng Feng, UC San Diego; Wu Zhang, Heinz-Josef Lenz, University of Southern California; Teresa L. Johnson-Pais, Robin J. Leach, University of Texas Health Science Center; and Gary Fraser, Charles Wang, Loma Linda University.Funding for this research came, in part, from the National Institutes of Health (grants R01GM32373, 5U01CA086402, T32GM008666 and DK007202).
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Biotechnology
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December 9, 2020
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https://www.sciencedaily.com/releases/2020/12/201209094219.htm
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When strains of E.coli play rock-paper-scissors, it's not the strongest that survives
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Bacteria is all around us -- not just in bathrooms or kitchen counters, but also inside our bodies, including in tumors, where microbiota often flourish. These "small ecologies" can hold the key to cancer drug therapies and learning more about them can help development new life-saving treatments.
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What happens when different strains of bacteria are present in the same system? Do they co-exist? Do the strongest survive? In a microbial game of rock-paper-scissors, researchers at the University of California San Diego's BioCircuits Institute uncovered a surprising answer. Their findings, titled "Survival of the weakest in non-transitive asymmetric interactions among strains of E. coli," appeared in a recent edition of The research team consisted of Professor of Bioengineering and Molecular Biology Jeff Hasty; Michael Liao and Arianna Miano, both bioengineering graduate students; and Chloe Nguyen, a bioengineering undergraduate. They engineered three strains of E. coli (Escherichia coli) so that each strain produced a toxin that could kill one other strain, just like a game of rock-paper-scissors.When asked how the experiment came about, Hasty commented, "In synthetic biology, complex gene circuits are typically characterized in bacteria that are growing in well-mixed liquid cultures. However, many applications involve cells that are restricted to grow on a surface. We wanted to understand the behavior of small engineered ecologies when the interacting species are growing in an environment that is closer to how bacteria are likely to colonize the human body."The researchers mixed the three populations together and let them grow on a dish for several weeks. When they checked back they noticed that, across multiple experiments, the same population would take over the entire surface -- and it wasn't the strongest (the strain with the most potent toxin). Curious about the possible reasons for this outcome, they devised an experiment to unveil the hidden dynamics at play.There were two hypotheses: either the medium population (called "the enemy of the strongest" as the strain that the strongest would attack) would win or the weakest population would win. Their experiment showed that, surprisingly, the second hypothesis was true: the weakest population consistently took over the plate.Going back to the rock-paper-scissor analogy, if we assume the "rock" strain of E.coli has the strongest toxin, it will quickly kill the "scissor" strain. Since the scissor strain was the only one able to kill the "paper" strain, the paper strain now has no enemies. It's free to eat away at the rock strain slowly over a period of time, while the rock strain is unable to defend itself.To make sense of the mechanism behind this phenomenon, the researchers also developed a mathematical model that could simulate fights between the three populations by starting from a wide variety of patterns and densities. The model was able to show how the bacteria behaved in multiple scenarios with common spatial patterns such as stripes, isolated clusters and concentric circles. Only when the strains were initially distributed in the pattern of concentric rings with the strongest in the middle, was it possible for the strongest strain to take over the plate.It is estimated microbes outnumber human cells 10 to 1 in the human body and several diseases have been attributed to imbalances within various microbiomes. Imbalances within the gut microbiome have been linked to several metabolic and inflammatory disorders, cancer and even depression. The ability to engineer balanced ecosystems that can coexist for long periods of time may enable exciting new possibilities for synthetic biologists and new healthcare treatments. The research that Hasty's group is conducting may help lay the foundation to one day engineer healthy synthetic microbiomes that can be used to deliver active compounds to treat various metabolic disorders or diseases and tumors.Vice Chancellor for Research Sandra Brown said, "Bringing together molecular biology and bionengineering has allowed discovery with the potential to improve the health of people around the world. This is a discovery that may never have occurred if they weren't working collaboratively. This is another testament to the power of UC San Diego's multidisciplinary research."This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (grant R01-GM069811). Michael Liao is supported by the National Science Foundation Graduate Research Fellowship (grant DGE-1650112).Video:
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Biotechnology
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December 8, 2020
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https://www.sciencedaily.com/releases/2020/12/201208163007.htm
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Understanding COVID-19 infection and possible mutations
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The binding of a SARS-CoV-2 virus surface protein spike -- a projection from the spherical virus particle -- to the human cell surface protein ACE2 is the first step to infection that may lead to COVID-19 disease. Penn State researchers computationally assessed how changes to the virus spike makeup can affect binding with ACE2 and compared results to those of the original SARS-CoV virus (SARS).
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The researchers' original manuscript preprint, made available online in March, was among the first to computationally investigate SARS-CoV-2's high affinity, or tendency to bind, with human ACE2. The paper was published online on Sept. 18 in the "We were interested in answering two important questions," said Veda Sheersh Boorla, doctoral student in chemical engineering and co-author on the paper. "We wanted to first discern key structural changes that give COVID-19 a higher affinity towards human ACE2 proteins when compared with SARS, and then assess its potential affinity to livestock or other animal ACE2 proteins."The researchers computationally modeled the attachment of SARS-CoV-2 protein spike to ACE2, which is located in the upper respiratory tract and serves as the entry point for other coronaviruses, including SARS. The team used a molecular modeling approach to compute the binding strength and interactions of the viral protein's attachment to ACE2.The team found that the SARS-CoV-2 spike protein is highly optimized to bind with human ACE2. Simulations of viral attachment to homologous ACE2 proteins of bats, cattle, chickens, horses, felines and canines showed the highest affinity for bats and human ACE2, with lower values of affinity for cats, horses, dogs, cattle and chickens, according to Chowdhury."Beyond explaining the molecular mechanism of binding with ACE2, we also explored changes in the virus spike that could change its affinity with human ACE2," said Chowdhury, who earned his doctorate in chemical engineering at Penn State in fall 2019.Understanding the binding behavior of the virus spike with ACE2 and the virus tolerance of these structural spike changes could inform future research on vaccine durability and the potential for the virus to spread to other species."The computational workflow that we have established should be able to handle other receptor binding-mediated entry mechanisms for other viruses that may arise in the future," Chowdhury said.The Department of Agriculture, the Department of Energy and the National Science Foundation supported this work.
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Biotechnology
| 2,020 |
December 8, 2020
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https://www.sciencedaily.com/releases/2020/12/201208111453.htm
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Research team invents novel light-controlled contamination-free fluidic processor for advanced medical and industrial applications
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A mechanical engineering research team at the University of Hong Kong (HKU) has invented a novel light-controlled, contamination-free fluidic processor, which can serve as a useful tool to greatly reduce the risk of infection of front-line medical workers in testing virus or bacteria in big pandemics like the current COVID-19 pandemic, and to minimise the risk of contamination during the process.
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The new technology has been published in Precision manipulation of various liquids is essential in many fields. The team innovatively uses light as a stimulating force, allowing contactless manipulations in moving, merging, dispensing and splitting liquids, on a specifically designed photo-responsive platform. The platform is non-toxic and non-sticky to all fluids, making it an ideal contamination-free fluidic processor.Professor Wang said the first applications of the new technology can be in biomedical testing and diagnosis, with the aim of lowering the risk of contamination and infection in the process."Testing infectious viruses and bacteria is highly risky, sometimes even fatal. A blood droplet from an Ebola patient can infect medical workers through the skin. For diagnosis, medial workers have to crash, filter and purify a patient's blood sample to obtain the virus's genetic materials. This series of operations, very often in a fluidic medium, is highly infectious. Moreover, fluids stick to surfaces, which will contaminate containers and handling tools, causing potential dangers if the medical wastes are not properly managed." He said.According to WHO reports, healthcare workers are 21 to 32 times more likely to be infected with Ebola and nearly 14% of COVID-19 reported cases are among healthcare workers. Moreover, it is estimated that disposable plastics worth US$20 billion are consumed in testing annually. The used plastics are left with potentially infectious or toxic residues and hazardous wastes that cost another US$10 billion to handle."We hope the newly-invented technique can reduce and even replace the usage of disposable plastics in the biomedical and pharmaceutical industries. The light-control device outperforms its electrical counterpart in the market in terms of operational precision and convenience, whereas the cost is only one-hundredth of it." Professor Wang said.The key technology of the light-controlled fluidic processor is a two-layer photo-responsive platform. With a thickness of only 2mm, it is portable and easy to handle. Its superomniphobic surface interfaces fluids in a frictionless manner, like dew drops rolling on a lotus leaf; and a photothermal pyroelectric layer, which senses the light stimuli and converts it into a force that move, split and dispense fluids.It has great potential in advanced research and applications in DNA analysis, proteomics, cell assay and clinical diagnosis, chemical synthesis and drug discovery. It can handle a wide spectrum of liquids such as water, alcohol, alkanes, and particularly silicone oil, which is particularly challenging because of its ultra-low surface tension. Its maneuverable fluid volume can be from 1000 ?l to tiny droplets at 0.001 ?l, i.e. about 0.02% of the volume of blood in a mosquito bite, which is 100 times smaller than that manipulated by its electrical counterpart."The device functions as a "magic" wetting-proof hand to navigate, fuse, pinch, and cleave fluids on demand, enabling cargo carriers with droplet wheels and upgrading the limit of maximum concentration of deliverable protein by 4000-fold." Professor Wang said.The team will seek to integrate the platform with artificial intelligence (AI) system to work out a fully automatic system for liquid processing. In future, gene editing can be done with the click of a button, instead of repeated pipetting.
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Biotechnology
| 2,020 |
December 8, 2020
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https://www.sciencedaily.com/releases/2020/12/201208090015.htm
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'SCOUT' helps researchers find, quantify significant differences among organoids
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The ability to culture cerebral organoids or "minibrains" using stem cells derived from people has given scientists experimentally manipulable models of human neurological development and disease, but not without confounding challenges. No two organoids are alike and none of them resemble actual brains. This "snowflake" problem has held back the science by making scientifically meaningful quantitative comparisons difficult to achieve. To help researchers overcome those limitations, MIT neuroscientists and engineers have developed a new pipeline for clearing, labeling, 3D imaging and rigorously analyzing organoids.
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Called "SCOUT" for "Single-Cell and Cytoarchitecture analysis of Organoids using Unbiased Techniques," the process can extract comparable features among whole organoids despite their uniqueness -- a capability the researchers demonstrate via three case studies in their new paper in "When you are dealing with natural tissues you can always subdivide them using a standard tissue atlas, so it is easy to compare apples to apples," said study co-lead author Alexandre Albanese, a research scientist in the lab of the paper's senior author, Associate Professor Kwanghun Chung. "But when every organoid is a snowflake and has its own unique combination of features, how do you know when the variability you observe is because of model itself rather than the biological question you are trying to answer? We were interested in cutting through the noise of the system to make quantitative comparisons."Albanese co-led the research with former MIT chemical engineering graduate student Justin Swaney. The team has taken the added step of sharing their software and protocols on GitHub so that it can be freely adopted. Chung said that by sharing many of his lab's tissue processing, labeling and analysis innovations, he hopes to speed up biomedical progress."We are developing all these technologies to enable more holistic understanding of complex biological systems, which is essential to accelerate the pace of discovery and the development of therapeutic strategies," said Chung, an investigator in The Picower Institute for Learning and Memory and the Institute for Medical Engineering and Science as well as a faculty member in Chemical Engineering and Brain and Cognitive Sciences. "Disseminating these technologies is as important as developing them to make a real-world impact."Several of the Chung lab's technologies are components of the SCOUT pipeline. The process starts by making organoids optically transparent so they can be imaged with their 3D structure intact -- a key capability, Chung said, for studying whole organoids as developing systems. The next SCOUT step is to infuse the cleared organoids with antibody labels targeting specific proteins to highlight cellular identity and activity. With organoids cleared and labeled, Chung's team images them with a light-sheet microscope to gather a full picture of the whole organoid at single-cell resolution. In total, each organoid produces about 150 GB of data for automated analysis by SCOUT's software, principally coded by Swaney.The high-throughput process allows for many organoids to be processed, ensuring that research teams can include many specimens in their experiments.The team chose its antibody labels strategically, Albanese said. With a goal of discerning cell patterns arising during organoid development, the team decided to label proteins specific to early neurons (TBR1) and radial glial progenitor cells (SOX2) because their organization impacts downstream development of the cortex. The team imbued SCOUT with algorithms to accurately identify every distinct cell within each organoid.From there, SCOUT could start to recognize common architectural patterns such as identifying locations where similar cells cluster or areas of greater diversity, as well as how close or far different cell populations were from ventricles, or hollow spaces. In developing brains and organoids alike, cells organize around ventricles and then migrate out radially. With the aid of artificial intelligence-based methods, SCOUT was able to track patterns of different cell populations outward from each ventricle. Working with the system, the team therefore could identify similarities and differences in the cell configurations, or cytoarchitectures, across each organoid.Ultimately the researchers were able to build a set of nearly 300 features on which organoids could be compared, ranging from the single-cell to whole-tissue level. Chung said that with further analysis and different molecular label choices even more features could be developed. Notably, the features extracted by SCOUT are unbiased, because they are products of the software's analysis, rather than pre-ordained hypotheses about what is "supposed to be" meaningful.With the analytical pipeline set, the team put it to the test. In one case study they used it to discern trends of organoid development by comparing specimens of different ages. SCOUT highlighted dozens of significant differences not only in overall growth, but also changes in the proportions of cell types, differences in layering, and other changes in tissue architecture consistent with maturation.In another case study they compared different methods of culturing organoids. Harvard University co-authors Paola Arlotta and Silvia Velasco have developed a method that, according to single-cell RNA sequencing analysis, produces more consistent organoids than other protocols. The team used SCOUT to compare them with conventionally produced organoids to assess their consistency at the tissue scale. They found that the "Velasco" organoids show improved consistency in their architectures, but still show some variance.The third case study involving Zika not only proved the utility of SCOUT in detecting major changes, but also led to the discovery of rare events. Chung's group collaborated with virus expert Lee Gehrke, Hermann L.F. von Helmholtz Professor in IMES, to determine how Zika infection changed organoid development. SCOUT spotted 22 major differences between infected and uninfected organoids, including some that had not been documented before."Overall this analysis provided a first-of-its kind comprehensive quantification of Zika-mediated pathology including loss of cells, reduction of ventricles and overall tissue reorganization," the authors wrote. "We were able to characterize the spatial context of rare cells and distinguish group-specific differences in cytoarchitectures. Infection phenotype reduced organoid size, ventricle growth and the expansion of SOX2 and TBR1 cells. Given our observation that SOX2 cell counts correlate with multiscale tissue features, it is expected that Zika-related loss of neural progenitors decreased in the complexity of tissue topography and cell patterning."Chung said his lab is also collaborating with colleagues studying autism-like disorders to learn more about how development may differ.The research was supported by funding sources including the JPB Foundation, the NCSOFT Cultural Foundation, and the National Institutes of Health.
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Biotechnology
| 2,020 |
December 7, 2020
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https://www.sciencedaily.com/releases/2020/12/201207195135.htm
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Why some people may become seriously ill from meningococcal bacteria
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Researchers at Karolinska Institutet in Sweden have come one step closer toward understanding why some people become seriously ill or die from a common bacterium that leaves most people unharmed. In a study published in
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The researchers have also designed and validated a PCR test that can detect these mutations."We found that non-coding RNA mutations within the bacterium The research work began in 2017 after a strain of the This finding prompted the researchers to embark on a quest to collect and investigate more than 7,000 RNAT configurations of These variants shared a common trait in that they produced more and bigger capsules that insulated the bacterium and thus helped it evade the body's immune system."This is the first time we have been able to associate an RNAT's effect on meningitis disease progression," says the paper's first author Jens Karlsson, PhD student at the same department. "This supports further research into this and other non-coding RNAs' potential involvement in the development of bacterial diseases."As part of the study, the researchers also developed a quick PCR test that is capable of distinguishing these RNAT mutations."In the future, this PCR test may be coupled with a simple nose swab at a clinic, and in doing so, facilitate a speedy identification of these mutations, and subsequent treatment," Edmund Loh concludes.The study was funded by the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation and the Swedish Research Council.Facts about RNAs:
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Biotechnology
| 2,020 |
December 7, 2020
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https://www.sciencedaily.com/releases/2020/12/201207153934.htm
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Hormonal cues in plants and animals
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Just like other organisms, plants must respond dynamically to a variety of cues over their lifetime. Going through different developmental stages, or altering their form in response to a drought or drastic temperature change requires altering which of their genes are expressed into proteins and when those processes occur.
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In a new paper in "We saw changes in the level of mRNA NAD+ capping occurring in different plant tissues and in different developmental stages," says Gregory, senior author on the paper and an associate professor in the School of Arts & Sciences' Department of Biology. "This appears to be a potentially quick on/off switch that plants can use to regulate their RNA levels.""Researchers working on mammalian cells had identified an enzyme that appears to perform an analogous action, removing these NAD+ caps," says Yu, a postdoctoral researcher in Greogry's lab and the paper's first author. "Ours is the first study to show this process in a whole, living organism."This work has its origins in preliminary findings that Gregory's lab generated close to a decade ago. While teaching a class on RNA, Gregory had shared with his students a paper about a yeast version of the plant protein DX01, an enzyme now known to be responsible for removing NAD+ from mRNA."I became really intrigued about what it was doing in eurkaryotes," he says. At that time, his lab grew plants with a DX01 mutation and found that their growth was stunted, their leaves were pale green, their development was delayed, and they had defects in fertility."I thought, 'This is cool, we need to work on this,'" Gregory recalls.Pursuing it, they found that the mutants had an abundance of small RNAs, molecules often associated with silencing the expression of other RNA molecules. But ultimately they couldn't piece together a sensible story of how the mutation was causing small RNAs to accumulate, and the work stalled.It stalled that is, until a few years ago, when other scientists who work on mammalian RNA regulation began publishing work showing that mammalian cells possess DX01 as well, and that it could recognize and remove NAD+ caps.With this new understanding of DX01's role, Gregory, Yu, and colleagues decided to pick their own work back up. By studying plants, the group could take the findings in mammals a step further, looking in vivo, at how the enzyme was acting in a live, growing organism.The researchers first confirmed that DX01 acted similarly in plants as in mammals, removing the NAD+ from mRNA transcripts. Plants lacking DX01 developed the problems Gregory had seen years earlier: stunted growth and development. They also used a technique to isolate and sequence only the NAD+-capped mRNAs and found that mRNA transcripts with NAD+ caps occurred frequently for those encoding proteins related to stress response, as well as those involved in processing NAD+ itself. Further analysis confirmed that the NAD+ cap made mRNAs more likely to be broken down.To follow up on the clues pointing to an involvement in stress response, the team applied varying levels of a plant stress hormone, abscisic acid, to plants with or without a functioning DX01. Plants with a mutant DX01 did not appear to be affected by the changing hormone concentration, while those with a functional DX01 were, pointing to a role for NAD+ capping in responding to this hormone.And indeed, they found that the level of NAD+ capping of RNA in response to abscisic acid dynamically changed."It does look like NAD+ capping is tissue-specific and responds to at least one specific physiological cue," says Gregory, "at least in plants. That's pretty neat becaue it looks like it's a strong regulator of RNA stability, so the plant can destabilize different sets of mRNA transcripts, depending on where this process is acting and what cue is being given."The group's findings even tied back to the unusual discovery they had made much earlier, of a build-up of small RNA molecules. In their DX01 mutant plants, they observed that the NAD+ capped mRNA transcripts were processed into small RNAs, which are also unstable. Gregory, Yu, and colleagues believe this may be a secondary mechanism to remove NAD+ and rid themselves of these noncanonically capped transcripts, even in the absence of DX01."What's going on is they're using another pathway, making small RNAs, perhaps to get back the NAD+ so they can use it for other processes," Yu says.Indeed, NAD+ is a critical component in metabolism, so it makes sense that plants would have multiple strategies for ensuring they have enough available to them, the researchers say.In future work, the Gregory lab hopes to continue exploring the NAD+ mark, including working out how it is added and not just removed."Once we learn how to add, recognize, and remove it, it gives us the power to use this process as a tool for regulating various responses in plants," Gregory says, a power that could possibly be used in agriculture.But human health could benefit from these insights as well. The Penn researchers say that the work deserves follow-up in mammalian systems. "I'd be curious to see what types of mRNA transcripts in mammals respond to different hormones," says Gregory.Addition and removal of the NAD+ cap may even be involved in cancer biology, Gregory and Yu say. The abnormal cell metabolism seen in cancer cells often owes to mishaps in the type of regulation that mRNA transcripts undergo, and there's a "real probability," Gregory says, that NAD+ capping and decapping could play a role.For his part, Gregory is pleased to have been able to move forward with an area of research that eluded him years ago, one that is opening up a new area of study for his lab."This is definitely one of those stories that reminds me that science is not a sprint; it's a marathon," Gregory says.
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Biotechnology
| 2,020 |
December 7, 2020
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https://www.sciencedaily.com/releases/2020/12/201207131303.htm
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Synthetic llama antibodies rescue doomed proteins inside cells
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Columbia researchers have created a new technology using synthetic llama antibodies to prevent specific proteins from being destroyed inside cells. The approach could be used to treat dozens of diseases, including cystic fibrosis, that arise from the destruction of imperfect but still perfectly functional proteins.
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In many genetic diseases, including cystic fibrosis, mutated proteins are capable of performing their jobs but are tagged for destruction by the cell's quality control mechanisms."The situation is analogous to ugly fruit," says Henry Colecraft, PhD, the John C. Dalton Professor of Physiology & Cellular Biophysics, who led the research. "Shoppers reject fruit that doesn't look perfect, even though ugly fruit is just as nutritious. If mutated proteins in cystic fibrosis can escape the cell's quality control mechanisms, they work pretty well."In the cell, proteins destined for destruction are marked with a small peptide called ubiquitin. Deubiquitinase enzymes (DUBs) can remove these tags, but simply increasing DUB activity would indiscriminately rescue all proteins in a cell marked for destruction, which would be harmful."A lot of proteins are destroyed by the cell for good reason," Colecraft says, "so a therapy needs to be selective."That's when Colecraft and his graduate student, Scott Kanner, realized they could develop a solution that takes advantage of nanobodies -- small antibodies produced naturally by llamas, camels, and alpacas that were discovered nearly 30 years ago. These small nanobodies bind their targets with exquisite specificity and retain this property inside cells, unlike regular antibodies.The new technology -- called engineered deubiquitinases or enDUBs for short -- combines a synthetic nanobody that recognizes a specific protein with an enzyme that can rescue proteins tagged for destruction.In a new paper in To build each enDUB, the researchers first had to find a nanobody that only recognizes and binds the target protein. Until recently, researchers had to inject their target proteins into llamas, camels, or alpacas and wait for the animal to generate such nanobodies. The Columbia researchers instead fished out binders from a synthetic yeast nanobody display library containing millions of unique nanobodies.Once created, each enDUB was tested in cells that produced the mutated proteins.In both cases, enDUBs prevented the destruction of the proteins, and the proteins migrated to their normal locations in the cell membrane where they performed their normal functions."In the case of one of the cystic fibrosis proteins we tested, we get a remarkable rescue, restoring protein levels in the cell membrane to about 50% of normal," Colecraft says. "If that happened in a patient, it would be transformative."Though both diseases investigated in the study are caused by mutations in ion channel proteins, "the approach can be applied to any protein in the cell, not just membrane proteins or proteins altered by genetic mutations," Colecraft says."It could be applicable to any disease where protein degradation is a factor, including cancer and epilepsy."
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Biotechnology
| 2,020 |
December 7, 2020
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https://www.sciencedaily.com/releases/2020/12/201207112248.htm
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Synthetic biology and machine learning speed the creation of lab-grown livers
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Researchers at the University of Pittsburgh School of Medicine have combined synthetic biology with a machine-learning algorithm to create human liver organoids with blood- and bile-handling systems. When implanted into mice with failing livers, the lab-grown replacement livers extended life.
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The study, published today in "Pregnancy is nine months -- it takes that long and even months after birth for new organs to mature -- but if a person needs a liver, they may not be able to wait that long," said study author Mo Ebrahimkhani, M.D., associate professor of pathology and bioengineering, and member of the Pittsburgh Liver Research Center and the McGowan Institute for Regenerative Medicine. "We showed it's possible to get human liver tissue with four main cell types and vasculature in 17 days. We can mature tissue almost to the third trimester in only three months."Other groups have attempted to coax organoid maturation in a dish using growth factors, but it's expensive, inconsistent and prone to human error, Ebrahimkhani said. Often, there are unwanted tissue or cell types -- such as intestine or brain cells growing in the middle of what should be solid liver.Using genetic engineering is cleaner but also more complex to orchestrate. So, Ebrahimkhani partnered with Patrick Cahan, Ph.D., at Johns Hopkins University to use a machine-learning system that can reverse engineer the genes necessary for human liver maturation.Then, Ebrahimkhani together with his collaborator at Pitt, Samira Kiani, M.D., applied genetic engineering techniques, including CRISPR, to turn a mass of immature liver tissue -- originally derived from human stem cells -- into what the team calls "designer liver organoids."The more mature the organoids got, the more capillaries and rudimentary bile duct cells snaked their way through the thin sheet of tissue, and the more closely the function of the tiny organ rivaled its full-size natural human model. Energy storage, fat accumulation, chemical transport, enzyme activity and protein production were all closer to adult human liver function, though still not a perfect match.Ebrahimkhani imagines designer organoids having three main uses: drug discovery, disease modeling and organ transplant. Since the stem cells can come from the patient's own body, lab-grown organs could be personalized, so there would be no threat of immune rejection.When transplanted into mice with damaged livers, Ebrahimkhani's designer liver organoids successfully integrated into the animals' bodies and continued to work -- producing human proteins that showed up in the animals' blood and prolonging the animals' lives.This is a proof-of-principle to show that it's possible, Ebrahimkhani said. The technique could potentially go much further."Our reference was a nature-designed human liver, but you can go after any design you like. For instance, you can make a genetic switch that protects the tissue from a virus, target the DNA of the virus and destroy it," Ebrahimkhani said. "That sets this method apart."Additional authors on the study include co-first authors Jeremy Velazquez and Ryan LeGraw, as well as Farzaneh Moghadam, Ph.D., Joseph Maggiore, Joshua Hislop and Silvia Liu, Ph.D., of Pitt; Yuqi Tan of Johns Hopkins; Jacquelyn Kilbourne and Christopher Plaisier, Ph.D., of Arizona State University; and Davy Cats and Susana Chuva de Sousa Lopes, Ph.D., of Leiden University Medical Center.Several of the authors have submitted a patent (WO2019237124) for the methods described in this paper.This study was supported by the National Institute of Biomedical Imaging and Bioengineering (R01 EB028532 and R01 EB024562), the National Heart, Lung, and Blood Institute (R01 HL141805), Arizona Biomedical Research Centre (ADHS16-162402), National Institute of Diabetes and Digestive and Kidney Diseases (P30 DK120531) and the National Institute of General Medical Sciences (T32GM008208 and R35GM124725).
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Biotechnology
| 2,020 |
December 7, 2020
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https://www.sciencedaily.com/releases/2020/12/201207112244.htm
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Quick and sensitive identification of multidrug-resistant germs
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Researchers from the University of Basel have developed a sensitive testing system that allows the rapid and reliable detection of resistance in bacteria. The system is based on tiny, functionalized cantilevers that bend due to binding of sample material. In the analyses, the system was able to detect resistance in a sample quantity equivalent to 1-10 bacteria.
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Bacteria that are no longer susceptible to various antibiotics pose a significant threat to our health. In the event of a bacterial infection, physicians require rapid information about potential resistance so that they can respond quickly and correctly.Cantilever systems as an alternative Traditional methods for detecting resistance are based on cultivating bacteria and testing their sensitivity to a spectrum of antibiotics. These methods take 48 to 72 hours to deliver results, and some strains of bacteria are difficult to cultivate. Molecular biological tests are a great deal faster and work by amplifying resistance genes or specific short sequences of genetic material by polymerase chain reaction (PCR), but even this method doesn't deliver satisfactory results for every bacterium.An alternative comes in the form of methods using tiny cantilevers, which bend when RNA molecules bind to their surface, for example -- and this bending can then be detected. RNA molecules are "transcripts" of genes and can be used as instructions for building proteins. In addition, RNA molecules can be used to detect resistance genes in the genetic material of bacteria.Writing in the journal The researchers began by attaching sequences of three genes associated with vancomycin resistance to the cantilevers and then exposed these prepared cantilevers to a flow of RNA extracted from bacteria. If RNA molecules from the resistance genes were present, the matching RNA fragments would bind to the cantilevers, causing them to undergo nanoscale deflection that could be detected using a laser.A clear signal even with point mutations This method allowed the detection of not only resistance genes, but also individual point mutations associated with them. To study this, the researchers used point mutations coupled to genes responsible for resistance to ampicillin and other betalactam antibiotics."The big advantage of the method we've developed is its speed and sensitivity," says Dr. François Huber, first author of the paper. "We succeeded in detecting tiny quantities of specific RNA fragments within five minutes." In the case of single mutations, the detected RNA quantities corresponded to about 10 bacteria. When it came to detecting entire resistance genes, the researchers obtained a clear signal even with an amount of RNA that corresponded to a single bacterium."If we can detect specific genes or mutations in the genome of bacteria, then we know what antibiotic resistance the bacteria will exhibit," explains Professor Adrian Egli from University Hospital Basel, whose team played an essential role in the study. "Our work in the hospital would benefit from this kind of reliable and sensitive information about the resistance of pathogens."
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Biotechnology
| 2,020 |
December 7, 2020
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https://www.sciencedaily.com/releases/2020/12/201207112240.htm
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Useful 'fake' peptides
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Some useful drugs consist of peptides acting on their protein targets. To make them more efficient and stable, scientists have found a way to replace crucial segments of the peptides with ureido units. These oligoureas, which are composed of urea-based units, fold into a structure similar to that of peptides. Oligourea-based "fake" peptides enhance the options for rational drug design, concludes the study published in the journal
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Several drugs are peptides that inhibit or activate the actions of certain proteins. To enhance their efficiency, scientists are investigating peptide mimics. Peptide mimics contain strands of small organic units that resemble amino acids -- the building blocks of peptides -- but are not identical to them. The rationale is that proteolytic enzymes will less likely attack such fake peptide strands, so the drugs would be more effective.However, the synthetic strands -- called oligomers -- must fold into the structure of the original peptide to bind to its target protein properly. Gilles Guichard and his team from CNRS, University of Bordeaux, and colleagues from the University of Strasbourg and Ureka Pharma, Mulhouse, France, have explored oligomers made of ureido units, which are derivatives of urea. These oligoureas fold into a helix, one of the hallmark structures of peptides. However, there are slight differences. "Oligourea helices have fewer residues per turn, a smaller rise per turn, and a larger diameter than the original peptide alpha-helix," says Guichard.To determine whether oligoureas could mimic real peptide structures, the researchers prepared peptide-oligourea hybrids and investigated their binding to target proteins. One of the targets, MDM2, is a natural regulator of the tumor suppressor protein p53. The other one, VDR, is a protein required in the regulation of cell growth, immunity, and other biological functions.For the MDM2-binding peptide mimic, the researchers prepared hybrids by replacing three terminal key amino acids with ureido units. For the VDR-binding peptide mimic, they replaced the middle amino acid segment with ureido units. After some rounds of optimization, the authors found hybrid structures with high binding affinities.The binding affinities were similar to those of the original peptides. Moreover, X-ray analysis revealed that the hybrid structures adopted a regular helical structure. However, the helices were still a bit wider and had larger spaces between the side chains along the oligourea backbone than those of natural peptides.Peptide-oligourea hybrids are expected to resist proteolytic degradation, an important goal in medicinal chemistry. Another advantage is that they allow more modifications. "Alpha amino acids can be substituted at two positions, but ureido units have one site more," says Guichard. This means that hybrid peptide-oligourea drugs offer more options for optimization.
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Biotechnology
| 2,020 |
December 4, 2020
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https://www.sciencedaily.com/releases/2020/12/201204155410.htm
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New CRISPR-based test for COVID-19 uses a smartphone camera
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Imagine swabbing your nostrils, putting the swab in a device, and getting a read-out on your phone in 15 to 30 minutes that tells you if you are infected with the COVID-19 virus. This has been the vision for a team of scientists at Gladstone Institutes, University of California, Berkeley (UC Berkeley), and University of California, San Francisco (UCSF). And now, they report a scientific breakthrough that brings them closer to making this vision a reality.
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One of the major hurdles to combating the COVID-19 pandemic and fully reopening communities across the country is the availability of mass rapid testing. Knowing who is infected would provide valuable insights about the potential spread and threat of the virus for policymakers and citizens alike.Yet, people must often wait several days for their results, or even longer when there is a backlog in processing lab tests. And, the situation is worsened by the fact that most infected people have mild or no symptoms, yet still carry and spread the virus.In a new study published in the scientific journal "It has been an urgent task for the scientific community to not only increase testing, but also to provide new testing options," says Melanie Ott, MD, PhD, director of the Gladstone Institute of Virology and one of the leaders of the study. "The assay we developed could provide rapid, low-cost testing to help control the spread of COVID-19."The technique was designed in collaboration with UC Berkeley bioengineer Daniel Fletcher, PhD, as well as Jennifer Doudna, PhD, who is a senior investigator at Gladstone, a professor at UC Berkeley, president of the Innovative Genomics Institute, and an investigator of the Howard Hughes Medical Institute. Doudna recently won the 2020 Nobel Prize in Chemistry for co-discovering CRISPR-Cas genome editing, the technology that underlies this work.Not only can their new diagnostic test generate a positive or negative result, it also measures the viral load (or the concentration of SARS-CoV-2, the virus that causes COVID-19) in a given sample."When coupled with repeated testing, measuring viral load could help determine whether an infection is increasing or decreasing," says Fletcher, who is also a Chan Zuckerberg Biohub Investigator. "Monitoring the course of a patient's infection could help health care professionals estimate the stage of infection and predict, in real time, how long is likely needed for recovery."Current COVID-19 tests use a method called quantitative PCR -- the gold standard of testing. However, one of the issues with using this technique to test for SARS-CoV-2 is that it requires DNA. Coronavirus is an RNA virus, which means that to use the PCR approach, the viral RNA must first be converted to DNA. In addition, this technique relies on a two-step chemical reaction, including an amplification step to provide enough of the DNA to make it detectable. So, current tests typically need trained users, specialized reagents, and cumbersome lab equipment, which severely limits where testing can occur and causes delays in receiving results.As an alternative to PCR, scientists are developing testing strategies based on the gene-editing technology CRISPR, which excels at specifically identifying genetic material.All CRISPR diagnostics to date have required that the viral RNA be converted to DNA and amplified before it can be detected, adding time and complexity. In contrast, the novel approach described in this recent study skips all the conversion and amplification steps, using CRISPR to directly detect the viral RNA."One reason we're excited about CRISPR-based diagnostics is the potential for quick, accurate results at the point of need," says Doudna. "This is especially helpful in places with limited access to testing, or when frequent, rapid testing is needed. It could eliminate a lot of the bottlenecks we've seen with COVID-19."Parinaz Fozouni, a UCSF graduate student working in Ott's lab at Gladstone, had been working on an RNA detection system for HIV for the past few years. But in January 2020, when it became clear that the coronavirus was becoming a bigger issue globally and that testing was a potential pitfall, she and her colleagues decided to shift their focus to COVID-19."We knew the assay we were developing would be a logical fit to help the crisis by allowing rapid testing with minimal resources," says Fozouni, who is co-first author of the paper, along with Sungmin Son and María Díaz de León Derby from Fletcher's team at UC Berkeley. "Instead of the well-known CRISPR protein called Cas9, which recognizes and cleaves DNA, we used Cas13, which cleaves RNA."In the new test, the Cas13 protein is combined with a reporter molecule that becomes fluorescent when cut, and then mixed with a patient sample from a nasal swab. The sample is placed in a device that attaches to a smartphone. If the sample contains RNA from SARS-CoV-2, Cas13 will be activated and will cut the reporter molecule, causing the emission of a fluorescent signal. Then, the smartphone camera, essentially converted into a microscope, can detect the fluorescence and report that a swab tested positive for the virus."What really makes this test unique is that it uses a one-step reaction to directly test the viral RNA, as opposed to the two-step process in traditional PCR tests," says Ott, who is also a professor in the Department of Medicine at UCSF. "The simpler chemistry, paired with the smartphone camera, cuts down detection time and doesn't require complex lab equipment. It also allows the test to yield quantitative measurements rather than simply a positive or negative result."The researchers also say that their assay could be adapted to a variety of mobile phones, making the technology easily accessible."We chose to use mobile phones as the basis for our detection device since they have intuitive user interfaces and highly sensitive cameras that we can use to detect fluorescence," explains Fletcher. "Mobile phones are also mass-produced and cost-effective, demonstrating that specialized lab instruments aren't necessary for this assay."When the scientists tested their device using patient samples, they confirmed that it could provide a very fast turnaround time of results for samples with clinically relevant viral loads. In fact, the device accurately detected a set of positive samples in under 5 minutes. For samples with a low viral load, the device required up to 30 minutes to distinguish it from a negative test."Recent models of SARS-CoV-2 suggest that frequent testing with a fast turnaround time is what we need to overcome the current pandemic," says Ott. "We hope that with increased testing, we can avoid lockdowns and protect the most vulnerable populations."Not only does the new CRISPR-based test offer a promising option for rapid testing, but by using a smartphone and avoiding the need for bulky lab equipment, it has the potential to become portable and eventually be made available for point-of-care or even at-home use. And, it could also be expanded to diagnose other respiratory viruses beyond SARS-CoV-2.In addition, the high sensitivity of smartphone cameras, together with their connectivity, GPS, and data-processing capabilities, have made them attractive tools for diagnosing disease in low-resource regions."We hope to develop our test into a device that could instantly upload results into cloud-based systems while maintaining patient privacy, which would be important for contact tracing and epidemiologic studies," Ott says. "This type of smartphone-based diagnostic test could play a crucial role in controlling the current and future pandemics."The study entitled "Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy," was published online by Other authors of the study include Gavin J. Knott, Michael V. D'Ambrosio, Abdul Bhuiya, Max Armstrong, and Andrew Harris from UC Berkeley; Carley N. Gray, G. Renuka Kumar, Stephanie I. Stephens, Daniela Boehm, Chia-Lin Tsou, Jeffrey Shu, Jeannette M. Osterloh, Anke Meyer-Franke, and Katherine S. Pollard from Gladstone Institutes; Chunyu Zhao, Emily D. Crawford, Andreas S. Puschnick, Maira Phelps, and Amy Kistler from the Chan Zuckerberg Biohub; Neil A. Switz from San Jose State University; and Charles Langelier and Joseph L. DeRisi from UCSF.The research was supported by the National Institutes of Health (NIAID grant 5R61AI140465-03 and NIDA grant 1R61DA048444-01); the NIH Rapid Acceleration of Diagnostics (RADx) program; the National Heart, Lung, and Blood Institute; the National Institute of Biomedical Imaging and Bioengineering; the Department of Health and Human Services (Grant No. 3U54HL143541-02S1); as well as through philanthropic support from Fast Grants, the James B. Pendleton Charitable Trust, The Roddenberry Foundation, and multiple individual donors. This work was also made possible by a generous gift from an anonymous private donor in support of the ANCeR diagnostics consortium.
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Biotechnology
| 2,020 |
December 4, 2020
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https://www.sciencedaily.com/releases/2020/12/201204110248.htm
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Findings about cilia on cells of the vessel wall may be relevant for diabetes treatment
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A new study from Karolinska Institutet and the Helmholtz Diabetes Research Center shows that primary cilia, hair-like protrusions on endothelial cells inside vessels, play an important role in the blood supply and delivery of glucose to the insulin-producing beta cells in the pancreatic islets. The findings are published in
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When blood glucose levels rise, beta cells in pancreatic islets release insulin into the blood stream. Insulin triggers glucose uptake in a variety of tissues including fat and muscle. Glucose and other nutrients must cross the vascular barrier to reach beta cells inside pancreatic islets. Similarly, newly released insulin must cross the blood vessels into the blood stream to reach its target tissues.Endothelial cells can be found on the inside of blood vessels. Vessels in the pancreatic islets form a dense network with many small pores in the endothelial cell membrane, facilitating the exchange of molecules across the vessel wall.Now, researchers have investigated how pancreatic islet vessel formation and function are affected by primary cilia, small hair-like structures found on beta cells and endothelial cells. Professor Per-Olof Berggren's research group at The Rolf Luft Research Center for Diabetes and Endocrinology, the Department of Molecular Medicine and Surgery, Karolinska Institutet in Sweden and Dr. Jantje Gerdes' research group at the Helmholtz Diabetes Research Center in Munich, Germany, have previously shown that insulin secretion is modulated by cilia on beta cells.In the new study, the researchers examined a mouse model of Bardet-Biedl Syndrome, a disease caused by cilia dysfunction. They were able to show that when endothelial cilia are dysfunctional, the blood supply to the pancreatic islets is less efficient. Newly formed vessels have larger diameters and fewer pores that allow nutrients to pass through the vessel wall."Consequently, the smallest blood vessels, the capillaries, become less efficient at delivering glucose to the beta cells," says Yan Xiong, assistant professor at the Department of Molecular Medicine and Surgery, Karolinska Institutet and first author of the study.Signalling via the growth factor VEGF-A was identified as a key player in this process. Endothelial cells that lack functional cilia are less sensitive to VEGF-A compared to normal endothelial cells, resulting in impaired signalling via the VEGFR2 receptor."In summary, we have demonstrated that primary cilia, specifically those on endothelial cells, regulate pancreatic islet vascularisation and vascular barrier function via the VEGF-A/VEGFR2 signalling pathway," says Dr Gerdes, one of the senior authors of the study.The formation of functional blood vessels is an important factor in transplantation therapies. Beta cell replacement therapy could potentially treat and cure type 1 diabetes, and the formation of a functional interface between beta cells and blood vessels is an important step towards longer graft survival and diabetes remission."This study improves the understanding of how primary cilia facilitate efficient blood vessel formation, and potentially offers novel therapeutic avenues to enable effective pancreatic islet transplantation in diabetes and possibly transplantation of other organs as well," says Dr Berggren, the other senior author of the study.
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Biotechnology
| 2,020 |
December 4, 2020
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https://www.sciencedaily.com/releases/2020/12/201204110212.htm
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'Off switch' during error-prone cell cycle phase may fix CRISPR's unwanted changes problem
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A group of researchers developed a promising fix to CRISPR-Cas9's problem with unwanted genetic changes using a method that allows them to turn off gene-editing until it reaches key cell cycle phases where more accurate repairs are likely to happen.
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Researchers from Hiroshima University and Tokyo Medical and Dental University published on Although previous methods were developed that reported fewer off-target effects associated with the CRISPR technology, the researchers said these often exhibited lower editing efficiency."We aimed to develop the method to avoid the side effect called off-target effect which is one of the most challenging problems in the genome-editing field," said Wataru Nomura, one of the study's authors and a professor at HU's Graduate School of Biomedical and Health Sciences."Our method is like hitting two birds with one stone. We can improve the preciseness of genome editing and suppression of off-target effects at the same time."CRISPR-Cas9 has ushered in a new frontier in gene editing as a simpler and less expensive tool. Acting like scissors, it can snip genetic material you want to alter. The process, however, can also create off-target effects that limit its use in the field of therapeutics.The newest method developed to eliminate off-target effects works by using the anti-CRISPR protein AcrIIA4 which works like an "off switch" that stops the genome editing activity of SpyCas9. The researchers fused AcrIIA4 with the N terminal region of human Cdt1 -- a gene that helps ensure DNA replication happens only once per cell division -- intending to deactivate gene editing until S and GHDR is one of the two DNA repair processes used by organisms along with non-homologous end joining (NHEJ). Of the two, however, HDR is the preferred method as the repair relies on the existence of two chromosome copies in each cell. HDR's use of the duplicate chromosome as a template for repair makes gene editing more precise as opposed to NHEJ which just tends to connect the broken ends of the DNA. HDR occurs during the S and GThe researchers found that the amount of ArIIA4-Cdt1 fusion is dependent on the cell cycle. It increases during the G"The efficiency of HDR using AcrIIA4-Cdt1 was increased approximately by 4.0-fold compared to that using SpyCas9 alone. At target or off-target site 1 (HCN1 gene), the mutation ratio was decreased by 86.5%. Moreover, the mutation ratio at off-target site 2 (MFAP1 gene) was decreased from 8.5% to 0.6% using AcrIIA4-Cdt1," the researchers said in the study."Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppresses off-target effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing."Nomura said they want to further improve the preciseness of the system so it could be used safely in the therapeutic field."We envision to apply our system to other CRISPR/anti-CRISPR combinations as well as other CRISPR based gene editor such as base editors and targeted transcription mediators," he said."Our ultimate goal is to develop a genome editing system which can be used safely in the medical therapeutic field."
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Biotechnology
| 2,020 |
December 4, 2020
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https://www.sciencedaily.com/releases/2020/12/201204110206.htm
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New insights into the mechanisms of neuroplasticity
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Reactive oxygen molecules, also known as "free radicals," are generally considered harmful. However as it now turns out, they control cellular processes, which are important for the brain's ability to adapt -- at least in mice. Researchers from the German Center for Neurodegenerative Diseases (DZNE) and the Center for Regenerative Therapies Dresden (CRTD) at TU Dresden published the findings in the journal
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The researchers focused on the "hippocampus," a brain area that is regarded as the control center for learning and memory. New nerve cells are created lifelong, even in adulthood. "This so-called adult neurogenesis helps the brain to adapt and change throughout life. It happens not only in mice, but also in humans," explains Prof. Gerd Kempermann, speaker of the DZNE's Dresden site and research group leader at the CRTD.New nerve cells emerge from stem cells. "These precursor cells are an important basis for neuroplasticity, which is how we call the brain's ability to adapt," says the Dresden scientist. Together with colleagues he has now gained new insights into the processes underlying the formation of new nerve cells. The team was able to show in mice that neural stem cells, in comparison to adult nerve cells, contain a high degree of free radicals. "This is especially true when the stem cells are in a dormant state, which means that they do not divide and do not develop into nerve cells," says Prof. Kempermann. Current study shows that an increase in the concentration of the radicals makes the stem cells ready to divide. "The oxygen molecules act like a switch that sets neurogenesis in motion."Free radicals are waste products of normal metabolism. Cellular mechanisms are usually in place to make sure they do not pile up. This is because the reactive oxygen molecules cause oxidative stress. "Too much of oxidative stress is known to be unfavorable. It can cause nerve damage and trigger aging processes," explains Prof. Kempermann. "But obviously this is only one aspect and there is also a good side to free radicals. There are indications of this in other contexts. However, what is new and surprising is the fact that the stem cells in our brains not only tolerate such extremely high levels of radicals, but also use them for their function."Radical scavengers, also known as "antioxidants," counteract oxidative stress. Such substances are therefore considered important components of a healthy diet. They can be found in fruits and vegetables. "The positive effect of antioxidants has been proven and is not questioned by our study. We should also be careful with drawing conclusions for humans based on purely laboratory studies," emphasizes Kempermann. "And yet our results at least suggest that free radicals are not fundamentally bad for the brain. In fact, they are most likely important for the brain to remain adaptable throughout life and to age in a healthy way."
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Biotechnology
| 2,020 |
December 3, 2020
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https://www.sciencedaily.com/releases/2020/12/201203200557.htm
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New DNA modification 'signature' discovered in zebrafish
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Researchers at the Garvan Institute of Medical Research have uncovered a new form of DNA modification in the genome of zebrafish, a vertebrate animal that shares an evolutionary ancestor with humans ~400 million years ago.
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Dr Ozren Bogdanovic and his team discovered that unusually high levels of DNA repeats of the sequence 'TGCT' in the zebrafish genome undergo a modification called methylation, which may change the shape or activity of the surrounding DNA. The study, published in "We've revealed a new form of DNA methylation in zebrafish at TGCT repeats, and crucially, the enzyme that makes the modification," says Dr Bogdanovic, who heads the Developmental Epigenomics Lab at Garvan and Senior Research Fellow at the School of Biotechnology and Biomolecular Sciences, UNSW Sydney. "These findings open the field to new possibilities in studying the epigenome -- the additional layer of instructions on DNA that change how genes are read -- and understand how it may be clinically relevant."All species that have DNA -- from plants to humans -- also modify it by attaching molecules called methyl groups."DNA methylation is vital to cellular function, as it controls which genes are turned on and off," explains first author of the paper, PhD student Sam Ross. "This is why the cells in our body can carry out vastly different functions, despite having almost identical DNA."There are four 'base' letters that make up DNA -- C, G, T and A. In vertebrates, methylation occurs mostly where the letter G follows a C ('CG'), but there are some exceptions. One example is methylation at non-CG sites in human brain cells, aberrations of which have been linked to Rett Syndrome, a genetic disorder that impairs growth, movement and speech in children.To investigate non-CG methylation further, the researchers conducted a comprehensive profiling of the zebrafish genome, a vertebrate organism that is a distant evolutionary relative of humans and shares 70% of our genes, which makes it a useful model for studying the effects of human genes.The team discovered that methylation occurred where the sequence 'TGCT' appeared multiple times, close together."We were fascinated to see that methylation levels at TGCT repeats were higher than any non-CG methylation previously observed in the majority of adult vertebrate tissues," says Dr Bogdanovic. "Further, this methylation was present at high levels in the sperm and egg, absent in the fertilised egg, and then appeared again in the growing embryo, reaching its highest levels in adult tissues such as the brain and gonads. While we are yet to reveal how this modification changes gene expression, we believe TGCT methylation to be linked to the 'awakening' of the embryonic genome in zebrafish."The researchers further revealed that the enzyme Dnmt3ba was responsible for methylating the TGCT repeats in the zebrafish genome."While it's unclear if a similar modification occurs in animals more broadly, our discovery in zebrafish is significant, because it means we can start to selectively manipulate this atypical form of methylation in a model organism. It means we can change the levels of Dnmt3ba to see what happens when we remove just one form of methylation, but not another," says Dr Bogdanovic."This could greatly facilitate our understanding of how changes in atypical methylation patterns affect specific tissues such as the brain, to gain further insights into the molecular mechanisms of neurodevelopmental disorders," says Dr Bogdanovic."We hope that our findings will help us develop new experimental models that can be used to study epigenetics in a way that has not been possible thus far."This research was supported by the Australian Research Council (DP190103852).
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Biotechnology
| 2,020 |
December 3, 2020
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https://www.sciencedaily.com/releases/2020/12/201203200555.htm
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Advancing gene editing with new CRISPR/Cas9 variant
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Using a new variant to repair DNA will improve both safety and effectiveness of the much-touted CRISPR-Cas9 tool in genetic research, Michigan Medicine researchers say.
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Those two key problems -- safety and efficacy -- are what continue to hold CRISPR-Cas9 gene targeting back from its full clinical potential, explains co-senior author Y. Eugene Chen, M.D., Ph.D., a professor of internal medicine, cardiac surgery, physiology, pharmacology and medicinal chemistry, from the Michigan Medicine Frankel Cardiovascular Center.The new CRISPR-Cas9 variant improves efficiency when inserting a gene or DNA fragment to a precise location in the genome, known as knocking in. It also reduces the rate of unintended insertions or deletions, known as indels, of base pairs that often happen while gene editing."We name it meticulous integration Cas9, or miCas9, to reflect its extraordinary capacity to enable maximum integration, yet with minimal indels, as well as to recognize its development at the University of Michigan," write senior authors Chen, Jifeng Zhang and Jie Xu for The team previously reported Cas9 genome editing in 2014, and reported beneficial effects of a RAD51 agonist, RS-1, in gene editing in 2016.
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Biotechnology
| 2,020 |
December 3, 2020
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https://www.sciencedaily.com/releases/2020/12/201203122259.htm
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New compact model for gene regulation in higher organisms
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Although the DNA and its double-helix are one of the most familiar molecules of our time, our knowledge of how cells control what genes they want to express still is rather limited. In order to create, for example, an enzyme, the information that's inscribed in our DNA about this enzyme needs to be transcribed and translated. To start this highly complex process special regulatory proteins called transcription factors (TFs) bind to specific DNA regions. That way, they can turn the expression of a gene on and off. The big question is: How can transcription factors find the right place on the DNA to properly regulate gene expression?
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For prokaryotes -- simple cellular organisms without a nucleus, like bacteria -- biophysical models already manage to predict gene expression based on the interaction between TFs and DNA regulatory regions. In prokaryotes, the TF binding sites on DNA are rather long and specific, making it easier for the TFs to find their target. In higher organisms called eukaryotes whose cells have a nucleus, mathematically describing the process of gene regulation proved to be much more difficult. Now, a team of researchers at the Institute of Science and Technology Austria (IST Austria) found a way to describe how the interaction between the different regulatory molecules in eukaryotes could look like.In a new study published in Based on existing data, the researchers argued that individual TFs are limited in their ability to differentiate between specific and random sites on the DNA. Therefore, although each type of TF preferentially binds certain regulatory DNA sequences, TFs bind other non-cognate targets, too. "The main motivation was to find a model to describe how the regulatory elements on the DNA don't get activated by non-cognate transcription factors," says Benjamin Zoller. Their findings suggest that high specificity of gene expression must be a collective effect of the regulatory molecules operating in the "proofreading regime."Furthermore, if a gene is active, the number of proteins it produces fluctuates, creating what scientists call gene expression noise. "What surprised me was the tradeoff between noise and specificity. It seems like if you want to have high specificity, it tends to lead to more noise, which is intriguing," says Benjamin Zoller. High noise is often thought to be detrimental for cells, yet genes in eukaryotes are quite noisy. "So far, we don't really know why this whole transcription machinery has evolved that way. Perhaps an explanation is that high noise is unavoidable if you want high specificity. Within our model, there seems to be no way around it. High specificity will always mean high noise, and it is possible cells have evolved mechanisms to lower the noise later on in the gene expression process," adds Rok Grah. The next step in the collaboration is the experimental test of the new model. Its simplicity makes it perfectly suited for confrontation with precise real-time gene expression measurements, for example, on perturbed DNA regulatory sequences.
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Biotechnology
| 2,020 |
December 3, 2020
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https://www.sciencedaily.com/releases/2020/12/201203113236.htm
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The helix of life: New study shows how 'our' RNA stably binds to artificial nucleic acids
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As medical research progresses, traditional treatment protocols are being rapidly exhausted. New approaches to treat diseases that do not respond to conventional drugs are the need of the hour. In search for these approaches, science has turned to a wide range of potential answers, including artificial nucleic acids. Artificial or xeno nucleic acids are similar to naturally occurring nucleic acids (think DNA and RNA) -- but are produced entirely in the laboratory.
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Xeno nucleic acids are essential for the development of nucleic acid-based drugs. To be effective, they need to be able to stably bind to natural RNA (a cellular single-stranded version of the DNA, which is essential for all body processes). However, it is unclear how, if at all, RNA hybridizes with these xeno nucleic acids. A new study by researchers from Japan sheds light on this mechanism, opening doors to the development of potentially revolutionary nucleic acid-based drugs.In their experimental study published in Natural nucleic acids like DNA and RNA have a sugar-phosphate "backbone" and nitrogen-based components; while the nitrogen-based components in SNA and L-They found that intra-molecular (within-molecule) interactions are important for keeping the helical (twisted) double-stranded structures formed of acyclic nucleic acids and RNA stable. While helical structures of natural nucleic acids are A-type, meaning that they twist towards the right, these synthetic duplex structures seemed to align in a perpendicular pattern, resulting in larger areas between each turn of the helix. In addition, they obtained triple-stranded structures consisting of L-These findings question a lot of things we have so far believed to be fundamental in biology. Ribose, the sugar in the backbone of natural nucleic acids, doesn't seem to be necessary for forming a stable duplex, contrary to the currently accepted knowledge. Then why did nature select ribose? "This is perhaps better answered through future studies looking at the helical structure," says Dr. Kamiya.For now, her team is happy that their findings open up more drug development avenues. "The structural understanding of these duplexes can help us come up with novel designs of nucleic acid-based drugs. We hope that through these findings, the development of nucleic acid drugs will accelerate," she says.These insights, of course, go beyond medical applications. Nucleic acids are the blueprints of the "construction" of all living organisms, but we realize that many of their secrets are still uncovered. These findings shed light on a small but significant chapter of nucleic acids.
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Biotechnology
| 2,020 |
December 3, 2020
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https://www.sciencedaily.com/releases/2020/12/201203094528.htm
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Medicine-carriers made from human cells can cure lung infections
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Scientists used human white blood cell membranes to carry two drugs, an antibiotic and an anti-inflammatory, directly to infected lungs in mice.
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The nano-sized drug delivery method developed at Washington State University successfully treated both the bacterial growth and inflammation in the mice's lungs. The study, recently published in "If a doctor simply gives two drugs to a patient, they don't go directly to the lungs. They circulate in the whole body, so potentially there's a lot of toxicity," said Zhenjia Wang, the study's corresponding author and an associate professor in WSU's College of Pharmacy and Pharmaceutical Sciences. "Instead, we can load the two types of drugs into these vesicles that specifically target the lung inflammation."Wang and his research team have developed a method to essentially peel the membrane from neutrophils, the most common type of white blood cells that lead the body's immune system response. Once emptied, these membranes can be used as nanovesicles, tiny empty sacks only 100 to 200 nanometers wide, which scientists can then fill with medicine.These nanovesicles retain some of the properties of the original white blood cells, so when they are injected into a patient, they travel directly to the inflamed area just as the cells would normally, but these nanovesicles carry the medicines that the scientists implanted to attack the infection.In this study, first author Jin Gao, a WSU research associate, loaded the nanovesicles with an antibiotic and resolvinD1, an anti-inflammatory derived from Omega 3 fatty acids, to treat lungs infected by P. aeruginosa, a common potentially fatal pathogen patients can catch in hospital settings. The researchers used two drugs because lung infections often create two problems, the infection itself and inflammation created by a strong immune system response.Toxicity studies and clinical trials would have to be conducted before this method could be used in human patients, but this study provides evidence that the innovation works for lung inflammation. If the method is ultimately proven safe and effective for humans, Wang said the nanovesicles could be loaded with any type of drug to treat a range of infectious diseases, including COVID-19."I think it's possible to translate this technology to help treat COVID-19," said Wang. "COVID-19 is a virus, not a bacterial pathogen, but it also causes an inflammation response in the lung, so we could load an antiviral drug like remdesivir into the nanovesicle, and it would target that inflammation."
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Biotechnology
| 2,020 |
December 2, 2020
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https://www.sciencedaily.com/releases/2020/12/201202114533.htm
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Visualization reveals how a protein 'hunkers down' to conserve energy
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A visualisation made from nearly 100,000 electron microscope images has revealed the ingenious way a protein involved in muscle activity shuts itself down to conserve energy.
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The protein is called myosin and it is known as a molecular motor because of the way it interacts with other proteins and energy molecules to generate force and movement. It is found inside muscle fibres where it forms long myosin filaments made up of hundreds of individual myosin molecules.When muscle activity ceases, the process of forming the myosin filaments goes into reverse: the filaments decouple and return to the individual myosin molecule state.The visualisation -- developed by scientists from the University of Leeds in the UK and East Carolina University in the US -- has revealed how the structure of the molecule changes. It folds up and becomes more compact, meaning it can be moved more easily to where it is next needed in the cell.Their findings -- Structure of the shutdown state of myosin-2 -- are published today (Wednesday, 2 December) in the journal Nature.An individual molecule of myosin is large -- and is made up of a 'head' and a 'tail'. When in an active state, the tails of the molecules come together to form fibrous myosin filaments. The heads within the filament bind with another protein called actin to produce muscle contraction.By combing 96,000 electron microscope images, the scientists were able to see how the molecule adopts an inactive form in unprecedented detail. The tail of each molecule wraps itself around the head and is locked in place by key molecular interactions. That process shuts down its activity and makes it easier for the molecule to be recruited to where it is next needed.Professor Michelle Peckham, from the Astbury Centre for Structural Molecular Biology at Leeds who supervised the research, said: "The analogy here is that the folded myosin is like a Brompton bicycle, kept in a folded state when not needed, and able to be quickly unfolded when it is, by releasing a simple catch."The compact folded myosin is also more easily transported through a crowd to where it's needed."Scientists have been aware of the role of myosin in muscle activity for decades. But until now, they were unclear about how this inactive state was formed or how its formation was so highly controlled.What does the research mean for understanding disease?There are genetic mutations of myosin linked with certain diseases.Professor Peckham explained: "Mutations in muscle myosin cause a wide range of muscle diseases. Our research into the structure of myosin and the way it functions help explain how mutations or defects in the protein may be causing disease. That opens the door to the possibility that scientists can develop therapeutic approaches to ensure myosin functions normally."The Astbury Centre for Structural Molecular Biology at the University of Leeds is an interdisciplinary research group involving biologists, physicists and chemists to investigate the molecular basis of life. One of the major research themes is to understand the process of protein folding.The research was funded by the Medical Research Council and Wellcome Trust.
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Biotechnology
| 2,020 |
December 1, 2020
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https://www.sciencedaily.com/releases/2020/12/201201124223.htm
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Molecular 'barcode' helps decide which sperm will reach an egg
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A protein called CatSper1 may act as a molecular 'barcode' that helps determine which sperm cells will make it to an egg and which are eliminated along the way.
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The findings in mice, published recently in "Male mammals ejaculate millions of sperm cells into the female's reproductive tract, but only a few arrive at the egg," explains senior author Jean-Ju Chung, Assistant Professor of Cellular & Molecular Physiology at Yale School of Medicine, New Haven, Connecticut, US. "This suggests that sperm cells are selected as they travel through the tract and excess cells are eliminated. But most of our knowledge about fertilisation in mammals has come from studying isolated sperm cells and eggs in a petri dish -- an approach that doesn't allow us to see what happens during the sperm selection and elimination processes."To address this challenge, Chung and colleagues, including lead author Lukas Ded, who was a postdoctoral fellow in the Chung laboratory when the study was carried out, devised a new molecular imaging strategy to observe the sperm selection process within the reproductive tract of mice. Using this technique, and combining it with more traditional molecular biology studies, the team revealed that a sperm protein called CatSper1 must be intact for a sperm cell to fertilise an egg.The CatSper1 protein is one of four proteins that create a channel to allow calcium to flow into the membrane surrounding the tail of the sperm. This channel is essential for sperm movement and survival. If this protein is lopped off in the reproductive tract, the sperm never makes it to the egg and dies. "This highlights CatSper1 as a kind of barcode for sperm selection and elimination in the female reproductive tract," says Chung.The findings, and the new imaging platform created by the team, may enable scientists to learn more about the steps in the fertilisation process and what happens afterwards, such as when the egg implants into the mother's uterus."Our study opens up new horizons to visualise and analyse molecular events in single sperm cells during fertilisation and the earliest stages of pregnancy," Chung concludes. "This and further studies could ultimately provide new insights to aid the development of novel infertility treatments."
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Biotechnology
| 2,020 |
December 1, 2020
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https://www.sciencedaily.com/releases/2020/12/201201124212.htm
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CRISPR tagging improves accuracy of model cells grown from stem cells
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A team of biomedical engineers at Duke University has created a new way to turn stem cells into a desired cell type by mastering the language of gene regulatory networks.
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Programming stem cells into other cell types is not a new idea. Several methods already exist, but the results have left something to be desired. Often, programmed stem cells do not mature correctly when cultured in the lab, so researchers seeking adult neuron cells for an experiment might end up with embryonic neurons, which won't be able to model late-onset psychiatric and neurodegenerative conditions."The cells might seem right at first glance," said Josh Black, the Duke Ph.D. student who led the work in Charles Gersbach's lab, "but they are often missing some of the key properties you want in those cells."Using CRISP gene editing, the lab led by Gersbach, The Rooney Family Associate Professor of Biomedical Engineering and the Director of the Center for Advanced Genomic Technologies, created a method to identify which transcription factors -- master controllers of gene activity -- were essential to making a good neuron.Their work, appearing Dec. 1 in CRISPR technology is most often used for editing DNA sequences, known as "genome editing," in which the Cas9 protein is bound to a guide RNA that directs Cas9 to cut the DNA at a specific location, leading to changes in the DNA sequence. "DNA editing has been widely used to alter gene sequences, but that doesn't help in situations where the gene is turned off," Gersbach said.A deactivated Cas9 (dCas9) protein, though, will attach to the DNA without cutting it. In fact, it typically won't do anything without another molecule attached or recruited to it. Gersbach and his colleagues have previously reported a variety of methods to attach different molecular domains to the dCas9 protein can that will tell a cell to turn on a gene and remodel chromatin structure.When Black joined Gersbach's lab, he was interested in using these tools to turn on genes that could convert one cell type into another to create better disease models.In 2016, Black and Gersbach reported an approach to use the CRISPR-based gene activators to turn on gene networks that would convert fibroblasts, an easily accessible cell type that makes up connective tissue, to neuronal cells. This study targeted gene networks that were known to be associated with neuronal specification, but did not generate cells with all of the properties needed to make effective disease models. However, the right gene networks to generate those desired cells were unknown, and there are were thousands of possibilities encoded in the human genome. So Black and Gersbach devised a strategy to test all of the networks in a single experiment.They started with pluripotent stem cells, since this cell type should be able to become any other cell in the human body. To make mature neurons from stem cells, the team engineered stem cells that fluoresced red once they became neuronal. The brighter the fluorescence, the stronger the push towards a neuronal fate. Then they made a pooled library of thousands of guide RNAs targeted to all of the genes that encode transcription factors in the human genome. Transcription factors are the master regulators of gene networks, so to make the desired neurons, they have to get all of the right transcription factors turned on.They introduced the CRISPR gene activator and guide RNA library into the stem cells so that each cell only received a single guide RNA, and therefore turned on its particular corresponding transcription factor gene target. Then they sorted the cells based on how red they became and sequenced the guide RNAs in the most and least red cells, which told them which genes, when turned on, made the cells more or less neuronal.When they profiled the gene expression from the stem cells engineered with the guide RNAs, the results suggested that the corresponding cells generated more specific and more mature types of neurons. They also found genes that worked together when targeted simultaneously. Moreover, the experiment revealed factors that antagonized the neuronal commitment of the stem cells, and when they used CRISPR-based repressors of those genes, they could also enhance the neuronal specification.However, these results were all just measuring markers of neurons. To know if these engineered cells truly recapitulated the function of more mature neurons, they needed to test their ability to transmit electrical signals.For this, they turned to Professor Scott Soderling, the George Barth Geller Distinguished Professor for Research in Molecular Biology and Chair of the Duke Department of Cell Biology. Shataakshi Dube, a grad student in Soderling's lab, used a technique known as patch clamp electrophysiology to measure the electrical signals inside the newly formed neurons. By poking a tiny hole in the cell with a very small pipette, she could look inside the neuron and see if it was transmitting electrical signals known as action potentials. If so, the team knew the neuronal cell had properly matured. In fact, the neurons engineered to activate a particular pair of transcription factor genes were more functionally mature, emitting more action potentials more frequently."I was curious but skeptical on how neuronal these stem cells could become," Dube said, "but it was remarkable to see how much these programmed cells looked just like normal neurons."The process from stem cell to mature neuronal cell took seven days, dramatically shortening the timeframe compared to other methods that take weeks or months. This faster timeline has the potential to significantly accelerate the development and testing of new therapies for neurological disorders.Creating better cells will help researchers in a number of ways. Diseases like Alzheimer's disease, Parkinson's disease, and schizophrenia most often occur in adults and are difficult to study because making the right cells in the lab is challenging. This new method can allow researchers to better model these diseases and others. It can also help with drug screening, as different cells respond to drugs differently.More broadly, the same method for screening transcription factor genes and gene networks could be used to improve methods to make any cell type, which could be transformative for regenerative medicine and cell therapy.For example, Gersbach's group reported a method for using CRISPR-based gene activation to convert human stem cells into muscle progenitor cells that could regenerate damaged skeletal muscle tissue earlier this year."The key to this work is developing methods to use the power and scalability of CRISPR-based DNA targeting to program any function into any cell type," Gersbach said. "By leveraging the gene networks already encoded in our genome, our control over cell biology is dramatically improved."
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Biotechnology
| 2,020 |
December 1, 2020
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https://www.sciencedaily.com/releases/2020/12/201201124157.htm
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Scientists discover role of protein in detecting the common cold virus
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The role of a protein in detecting the common cold virus and kickstarting an immune response to fight infection has been uncovered by a team of scientists from Nanyang Technological University, Singapore (NTU Singapore), the Agency for Science, Technology and Research (A*STAR) and the National University of Singapore.
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In a study published in the journal HRV is a major cause of the common cold and acute respiratory disease in children and adults, which in severe cases, leads to bronchiolitis and pneumonia.The research team said that discovering NLRP1's purpose could lead to new treatments for the symptoms of the common cold, which affects millions of people annually. They plan to work with clinicians to develop drugs that 'turn off' or block NLRP1, to lessen the severity of symptoms for HRV-related diseases. However, the team noted that blocking the protein in human lung cells did not increase the viral load, which refers to the amount of virus in an infected person's blood."Now that we know that NLRP1 is the "on switch" for inflammation after it detects the common cold virus, the next step is to figure out how to block its activation and to minimise the inflammatory response it triggers," said Assistant Professor Franklin Zhong from NTU's Lee Kong Chian School of Medicine and A*STAR's Skin Research Institute of Singapore (SRIS).Asst Prof Zhong is the corresponding author of the study, along with Professor Bruno Reversade from A*STAR's Genome Institute of Singapore and Institute of Molecular and Cellular Biology and first author, Dr Kim S Robinson, Research Fellow at SRIS, A*STAR.Asst Prof Zhong said that their new insights into immune system functions could help scientists to develop more effective treatments for other inflammatory diseases of the human airway."This work represents a significant advance in our understanding of how our immune system uses special proteins to sense and defend against viral pathogens. This knowledge will be useful in the design of treatments for viral diseases including influenza and Covid-19," he said.NLRP1 has been known to scientists for years but its exact purpose was unknown. It is a member of a class called 'Nod-like Receptor' proteins that are sensors in the immune system that trigger the human body's response against invading pathogens.When the team began their study in 2017, they hypothesised that NLRP1 serves as a sensor for viruses, because it is highly abundant in the human skin and lungs -- surfaces that are commonly exposed to viral pathogens.The team screened NLRP1 against several viruses to see if any would trigger the protein. After months of trials, they observed that an enzyme made by HRV called 3Cpro activated NLRP1 in human airway cells.They saw that the 3Cpro enzyme cut into NLRP1 at a specific point, triggering a form of inflammatory 'cell death', which is an important process in rapidly clearing pathogens like HRV during an infection.Prof Reversade, who is also Professor of Genetics at Koç University in Istanbul, Turkey, said that pinpointing NLRP1's purpose marked a key step in understanding how our bodies react to HRV infections."There is immediate value from this finding, as we can better understand why an HRV infection could lead to complications in individuals with weaker immune systems, such as young children, the elderly, and those with asthma," said Prof Reversade.He added that the value from this research could extend to other diseases caused by viruses of the same family."Targeting NLRP1 in patients is likely to provide therapeutic benefits in a number of human diseases. Our findings on the immune response to this class of viruses also bear relevance to Coxsackieviruses which are responsible for hand, foot, and mouth disease (HFMD) in young children."
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Biotechnology
| 2,020 |
December 1, 2020
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https://www.sciencedaily.com/releases/2020/12/201201124115.htm
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Chemical memory in plants affects chances of offspring survival
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Researchers at the University of Warwick have uncovered the mechanism that allows plants to pass on their 'memories' to offspring, which results in growth and developmental defects.
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In order to survive and thrive, plants have the unique capability to sense and remember changes in their environment. This is linked to the chemical modification of DNA and histone proteins, which alters the way in which DNA is packaged within the cell's nucleus and genes are expressed -- a process known as epigenetic regulation.Usually, this epigenetic information is reset during sexual reproduction to erase any inappropriate 'memories' from being passed on to ensure the offspring grows normally. In the paper, 'A new role for histone demethylases in the maintenance of plant genome integrity' published in the journal The researchers identified two proteins in Thale Cress (Arabidopsis), previously known only to control the initiation and timing of flowering, that are also responsible for controlling 'plant memory' through the chemical modification (demethylation) of histone proteins.They showed that plants unable to reset these chemical marks during sexual reproduction, passed on this 'memory' to subsequent generations, resulting in defects in growth and development.Some of these defects were linked to the activation of selfish DNA elements, also known as 'jumping genes' or transposons, thus indicating that the erasure of such 'memory' is also critical for maintaining the integrity of plant genomes by silencing transposons.Prof. Jose Gutierrez-Marcos, a senior author on the paper from the School of Life Sciences at the University of Warwick commented:"Our study into the proteins that regulate plant memory has shown how important it is for chemical marks to be reset during sexual reproduction in order to avoid offspring inheriting inappropriate 'memories' that lead to growth and developmental defects associated with genome instability."The next step is to work out how to manipulate such 'memories' for plant breeding purposes, so that subsequent generations show greater adaptability to allow them to thrive in a changing environment."
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Biotechnology
| 2,020 |
December 1, 2020
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https://www.sciencedaily.com/releases/2020/12/201201124059.htm
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Plants on aspirin
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When pathogens enter a plant, infected cells set off an alarm before they die. They discharge methylsalicylic acid, which is later transformed into salicylic acid, triggering an immune response. Hence, salicylic acid is a stress signal in plants, but it also participates in regulating plant growth and development. In humans, salicylic acid proofed to be useful in a different way: Already in prehistoric times people realized that when they were drinking willow bark tea or taking other willow bark preparations, fever dropped and pain disappeared. Centuries later, scientists developed salicylic acid derivatives such as Aspirin and Ibuprofen. These so called non-steroidal anti-inflammatory drugs (NSAIDs) suppress the inflammatory response of mammalian cells, thereby making us feel better when we have a cold. But how do they affect plants?
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"When I got the idea, I had a really serious toothache and I had some Ibuprofen at hands," explains Shutang Tan, who at that time was a postdoc at the Institute of Science and Technology (IST) Austria working in the group of Professor Ji?í Friml. "I simply used the tablets from the pharmacy and I took the same amount as in my previous experiments with salicylic acid. Then, I observed the effect of the Ibuprofen on Arabidopsis seedlings." The primary roots of the plant were significantly shorter and instead of growing downward, they were curling up, unable to respond to gravity. Furthermore, the plants developed fewer or no lateral roots at all.Together with colleagues at the IST Austria and six other research institutions Shutang Tan looked at the effects of 20 different painkillers on Arabidopsis seedlings. "We found that all of the painkillers we tested, including Aspirin and Ibuprofen, were interfering with the auxin flow," explains Tan. The plant hormone auxin is essential for all developmental processes within a plant. It is especially responsible for a plants ability to stretch its leaves towards the sun and its roots towards the center of the earth. So called PIN proteins regulate the flow of auxin from one cell to the other, depending on which side of the cell they are sitting. If the PIN proteins are not at the right location within the cell, the flow of auxin is disturbed, leading to a faulty development of the plant. Hence, the painkillers seemed to interfere with the localization of the PIN proteins. But it didn't stop there.Looking closely, the scientists discovered that the effect is not limited to PIN proteins, but that the drugs interfere with the whole endomembrane system, suppressing the movement and trafficking of substances within the cells. The painkillers impair the dynamics of the cytoskeleton of the cells, a network of interlinking proteins, which among many other things gives the cell its shape and is involved in the uptake of extracellular material. Together with Markus Geisler's group at the University of Fribourg, Switzerland, the researchers at IST Austria uncovered that one group of painkillers, including the drugs Meclofenamic acid and Flufenamic acid, directly target an immunophilin-like protein, called TWISTED DWARF1, to realize these physiological and cellular activities.Furthermore, the scientists were able to show, that NSAIDs have similar physiological and cell biological effects as auxin transport inhibitors -- important chemical tools in cell biology, which interfere with the transport of auxin. "It would be very interesting to find out, if these auxin transport inhibitors can also be used as painkillers in animals. That is one big question we still need to answer" concludes Tan. Together with IST Professor Ji?í Friml, Shutang Tan, who is now establishing his own laboratory at the University of Science and Technology of China, wants to investigate which additional proteins within the plant are targeted by the painkillers and what pathways they use to do so.
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Biotechnology
| 2,020 |
November 30, 2020
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https://www.sciencedaily.com/releases/2020/11/201130155841.htm
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Microfluidic system with cell-separating powers may unravel how novel pathogens attack
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To develop effective therapeutics against pathogens, scientists need to first uncover how they attack host cells. An efficient way to conduct these investigations on an extensive scale is through high-speed screening tests called assays.
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Researchers at Texas A&M University have invented a high-throughput cell separation method that can be used in conjunction with droplet microfluidics, a technique whereby tiny drops of fluid containing biological or other cargo can be moved precisely and at high speeds. Specifically, the researchers successfully isolated pathogens attached to host cells from those that were unattached within a single fluid droplet using an electric field."Other than cell separation, most biochemical assays have been successfully converted into droplet microfluidic systems that allow high-throughput testing," said Arum Han, professor in the Department of Electrical and Computer Engineering and principal investigator of the project. "We have addressed that gap, and now cell separation can be done in a high-throughput manner within the droplet microfluidic platform. This new system certainly simplifies studying host-pathogen interactions, but it is also very useful for environmental microbiology or drug screening applications."The researchers reported their findings in the August issue of the journal Microfluidic devices consist of networks of micron-sized channels or tubes that allow for controlled movements of fluids. Recently, microfluidics using water-in-oil droplets have gained popularity for a wide range of biotechnological applications. These droplets, which are picoliters (or a million times less than a microliter) in volume, can be used as platforms for carrying out biological reactions or transporting biological materials. Millions of droplets within a single chip facilitate high-throughput experiments, saving not just laboratory space but the cost of chemical reagents and manual labor.Biological assays can involve different cell types within a single droplet, which eventually need to be separated for subsequent analyses. This task is extremely challenging in a droplet microfluidic system, Han said."Getting cell separation within a tiny droplet is extremely difficult because, if you think about it, first, it's a tiny 100-micron diameter droplet, and second, within this extremely tiny droplet, multiple cell types are all mixed together," he said.To develop the technology needed for cell separation, Han and his team chose a host-pathogen model system consisting of the salmonella bacteria and the human macrophage, a type of immune cell. When both these cell types are introduced within a droplet, some of the bacteria adhere to the macrophage cells. The goal of their experiments was to separate the salmonella that attached to the macrophage from the ones that did not.For cell separation, Han and his team constructed two pairs of electrodes that generated an oscillating electric field in close proximity to the droplet containing the two cell types. Since the bacteria and the host cells have different shapes, sizes and electrical properties, they found that the electric field produced a different force on each cell type. This force resulted in the movement of one cell type at a time, separating the cells into two different locations within the droplet. To separate the mother droplet into two daughter droplets containing one type of cells, the researchers also made a downstream Y-shaped splitting junction.Han said although these experiments were carried with a host and pathogen whose interaction is well-established, their new microfluidic system equipped with in-drop separation is most useful when the pathogenicity of bacterial species is unknown. He added that their technology enables quick, high-throughput screening in these situations and for other applications where cell separation is required."Liquid handling robotic hands can conduct millions of assays but are extremely costly. Droplet microfluidics can do the same in millions of droplets, much faster and much cheaper," Han said. "We have now integrated cell separation technology into droplet microfluidic systems, allowing the precise manipulation of cells in droplets in a high-throughput manner, which was not possible before."
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Biotechnology
| 2,020 |
November 30, 2020
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https://www.sciencedaily.com/releases/2020/11/201130131429.htm
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Microbiota linked to dynamics of human immune system
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In recent years, the microbiota -- the community of bacteria and other microorganisms that live on and in the human body -- has captured the attention of scientists and the public, in part because it's become easier to study. It has been linked to many aspects of human health.
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A multidisciplinary team from Memorial Sloan Kettering has shown for the first time that the gut microbiota directly shapes the makeup of the human immune system. Specifically, their research demonstrated that the concentration of different types of immune cells in the blood changed in relation to the presence of different bacterial strains in the gut. The results of their study, which used more than ten years of data collected from more than 2,000 patients, is being published November 25, 2020, in "The scientific community had already accepted the idea that the gut microbiota was important for the health of the human immune system, but the data they used to make that assumption came from animal studies," says Sloan Kettering Institute systems biologist Joao Xavier, co-senior author of the paper together with his former postdoc Jonas Schluter, who is now an assistant professor at NYU Langone Health. "At MSK, we have a remarkable opportunity to follow how the composition of the microbiota changes in people being treated for blood cancers," Dr. Xavier adds.The data that were used in the study came from people receiving allogeneic stem cell and bone marrow transplants (BMTs). After strong chemotherapy or radiation therapy is used to destroy cancerous blood cells, the patient's blood-forming system is replaced with stem cells from a donor. For the first few weeks until the donor's blood cells -- including the white blood cells that make up the immune system -- have established themselves, the patients are extremely vulnerable to infections. To protect them during this time, patients are given antibiotics.But many of these antibiotics have the unwanted side effect of destroying healthy microbiota that live in the gut, allowing dangerous strains to take over. When the patient's immune system has reconstituted, the antibiotics are discontinued, and the gut microbiota slowly starts to grow back."The parallel recoveries of the immune system and the microbiota, both of which are damaged and then restored, gives us a unique opportunity to analyze the associations between these two systems," Dr. Schluter says.For more than ten years, members of MSK's BMT service have regularly collected and analyzed blood and fecal samples from patients throughout the BMT process. The bacterial DNA were processed by the staff at MSK's Lucille Castori Center for Microbes, Inflammation, and Cancer, which played a key role in creating the massive microbiota dataset. "Our study shows that we can learn a lot from stool -- biological samples that literally would be flushed down the toilet," Dr. Xavier notes. "The result of collecting them is that we have a unique dataset with thousands of datapoints that we can use to ask questions about the dynamics of this relationship."This wider effort has been led by Marcel van den Brink, Head of the Division of Hematologic Malignancies, and a team of infectious disease specialists, BMT doctors, and scientists. "For a fair number of patients, we collected daily samples so we could really see what was happening day to day," Dr. van den Brink says. "The changes in the microbiota are rapid and dramatic, and there is almost no other setting in which you would be able to see them."Previous research using samples collected from this work has looked at how the gut microbiota affects patients' health during the BMT process. A study published in February 2020 reported that having a greater diversity of species in the intestinal microbiota is associated with a lower risk of death after a BMT. It also found that having a lower diversity of microbiota before transplant resulted in a higher incidence of graft-versus-host disease, a potentially fatal complication in which the donor immune cells attack healthy tissue.The databank that the MSK team created contains details about the types of microbes that live in the patients' guts at various times. The computational team, including Drs. Schluter and Xavier, then used machine learning algorithms to mine electronic health records for meaningful data. The data from the health records included the types of immune cells present in the blood, information about the medications that patients were given, and the side effects patients experienced. "This research could eventually suggest ways to make BMTs safer by more closely regulating the microbiota," Dr. van den Brink says.Analyzing this much data was a huge undertaking. Dr. Schluter, who at the time was a postdoctoral fellow in Dr. Xavier's lab, developed new statistical techniques for this. "Because experiments with people are often impossible, we are left with what we can observe," Dr. Schluter says. "But because we have so many data collected over a period of time when the immune system of patients as well as the microbiome shift dramatically, we can start to see patterns. This gives us a good start toward understanding the forces that the microbiota exerts on the rebuilding of the immune system.""The purpose of this study was not to say whether certain kinds of microbes are 'good' or 'bad' for the immune system," Dr. Xavier explains, adding that this will be a focus of future research. "It's a complicated relationship. The subtypes of immune cells we would want to increase or decrease vary from day to day, depending on what else is going on in the body. What's important is that now we have a way to study this complex ecosystem."The researchers say they also plan to apply their data to studying the immune system in patients receiving other cancer treatments.
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Biotechnology
| 2,020 |
November 30, 2020
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https://www.sciencedaily.com/releases/2020/11/201130131408.htm
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New method identifies adaptive mutations in complex evolving populations
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A team co-led by a scientist at the University of California, Riverside, has developed a method to study how HIV mutates to escape the immune system in multiple individuals, which could inform HIV vaccine design.
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HIV, which can lead to AIDS, evolves rapidly and attacks the body's immune system. Genetic mutations in the virus can prevent it from being eliminated by the immune system. While there is no effective cure for the virus currently available, it can be controlled with medication."Understanding the genetic drivers of disease is important in the biomedical sciences," said John P. Barton, an assistant professor of physics and astronomy at UCR, who co-led the study with Matthew R. McKay, a professor of electronic and computer engineering and chemical and biological engineering at the Hong Kong University of Science and Technology. "Being able to identify genomic rearrangements is key to understanding how illnesses occur and how to treat them."Barton explained that notable examples of genetic drivers of disease include mutations that allow viruses to escape from immune control, while others confer drug resistance to bacteria."It can be difficult, however, to differentiate between real, adaptive mutations and random genetic variation," he added. "The new method we developed allows us to identify such mutations in complex evolving populations."Evolutionary history, he added, contains information about which mutations affect survival and which simply reflect random variation."However, it is computationally difficult to extract this information from data," he said. "We used methods from statistical physics to overcome this computational challenge. Our method can be applied generally to evolving populations and is not limited to HIV."McKay explained the new method provides a means to efficiently infer selection from observations of complex evolutionary histories."It enables us to sort out which genetic changes provide an evolutionary advantage from those that offer no advantage or have a deleterious effect," he said. "The method is quite general and could be potentially used to study diverse evolutionary processes, such as the evolution of drug resistance of pathogens and the evolution of cancers. The accuracy and high efficiency of our approach enable the analysis of selection in complex evolutionary systems that were beyond the reach of existing methods."Some well-known diseases that have known genetic causes are cystic fibrosis, sickle cell anemia, Duchenne muscular dystrophy, colorblindness, and Huntington's disease."In the case of HIV, an understanding of the genetic mutations that lead to HIV resistance could help researchers determine the most appropriate treatment for patients," Barton said. "Our approach isn't limited to HIV, but there are a few reasons why we focused on HIV as a test system. HIV is highly mutable and genetically diverse. It also mutates within humans to escape from the immune system. Understanding the details of how HIV evolves could therefore help to develop better treatments against the virus."Barton was supported by a grant from the National Institutes of Health. Study results appear in Barton and McKay were joined in the study by Muhammad Saqib Sohail and Raymond H. Y. Louie of Hong Kong University of Science and Technology and the University of New South Wales.
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Biotechnology
| 2,020 |
November 30, 2020
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https://www.sciencedaily.com/releases/2020/11/201130131402.htm
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HIV-like virus edited out of primate genome
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Taking a major step forward in HIV research, scientists at the Lewis Katz School of Medicine at Temple University have successfully edited SIV -- a virus closely related to HIV, the cause of AIDS -- from the genomes of non-human primates. The breakthrough brings Temple researchers and their collaborators closer than ever to developing a cure for human HIV infection.
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"We show for the first time that a single inoculation of our CRISPR gene-editing construct, carried by an adeno-associated virus, can edit out the SIV genome from infected cells in rhesus macaque monkeys," said Kamel Khalili, PhD, Laura H. Carnell Professor and Chair of the Department of Neuroscience, Director of the Center for Neurovirology, and Director of the Comprehensive NeuroAIDS Center at the Lewis Katz School of Medicine at Temple University (LKSOM).Dr. Khalili was a senior co-investigator on the new study, with Tricia H. Burdo, PhD, Associate Professor and Associate Chair of Education in the Department of Neuroscience at LKSOM, who is an expert on the utilization of the SIV (simian immunodeficiency virus)-infected antiretroviral therapy (ART)-treated rhesus macaque model for HIV pathogenesis and cure studies; and with Andrew G. MacLean, PhD, Associate Professor at the Tulane National Primate Research Center and the Department of Microbiology and Immunology at Tulane University School of Medicine, and Binhua Ling, PhD, Associate Professor at the Southwest National Primate Research Center, Texas Biomedical Research Institute. Dr. Ling was previously Associate Professor at the Tulane National Primate Research Center and the Department of Microbiology and Immunology at Tulane University School of Medicine. Pietro Mancuso, PhD, an Assistant Scientist in Dr. Khalili's laboratory in the Department of Neuroscience at LKSOM, was first author on the report, which was published online November 27 in the journal Of particular significance, the new work shows that the gene-editing construct developed by Dr. Khalili's team can reach infected cells and tissues known to be viral reservoirs for SIV and HIV. These reservoirs, which are cells and tissues where the viruses integrate into host DNA and hide away for years, are a major barrier to curing infection. SIV or HIV in these reservoirs lies beyond the reach of ART, which suppresses viral replication and clears the virus from the blood. As soon as ART is stopped, the viruses emerge from their reservoirs and renew replication.In non-human primates, SIV behaves very much like HIV. "The SIV-infected rhesus macaque model studied in Dr. Burdo's lab is an ideal large animal model for recapitulating HIV infection in humans," explained Dr. Khalili.For the new study, the researchers began by designing an SIV-specific CRISPR-Cas9 gene-editing construct. Experiments in cell culture confirmed that the editing tool cleaved integrated SIV DNA at the correct location from host cell DNA, with limited risk of potentially harmful gene editing at off-target sites. The research team then packaged the construct into an adeno-associated virus 9 (AAV9) carrier, which could be injected intravenously into SIV-infected animals.Dr. Burdo, in collaboration with colleagues at Tulane National Primate Research Center, randomly selected three SIV-infected macaques to each receive a single infusion of AAV9-CRISPR-Cas9, with another animal serving as a control. After three weeks, the researchers harvested blood and tissues from the animals. Analyses showed that in AAV9-CRISPR-Cas9-treated macaques, the gene-editing construct had been distributed to a broad range of tissues, including the bone marrow, lymph nodes, and spleen, and had reached CD4+ T cells, which are a significant viral reservoir.Moreover, the Temple researchers demonstrated that the SIV genome was effectively cleaved from infected cells, based on genetic analyses of tissues from treated animals. "The step-by-step excision of SIV DNA occurred with high efficiency from tissues and blood cells," Dr. Mancuso explained. Excision efficiency varied by tissue but reached notably high levels in the lymph nodes.The new study is a continuation of efforts by Dr. Khalili and colleagues to develop a novel gene-editing system using CRISPR-Cas9 technology -- the subject of the 2020 Nobel Prize in Chemistry -- to specifically remove HIV DNA from genomes harboring the virus. The researchers have shown previously that their system can effectively eliminate HIV DNA from cells and tissues in HIV-infected small animal models, including HIV-1 humanized mice.Co-corresponding author Dr. MacLean is encouraged by the findings. "This is an important development in what we hope will be an end to HIV/AIDS," says MacLean. "The next step is to evaluate this treatment over a longer period of time to determine if we can achieve complete elimination of the virus, possibly even taking subjects off of ART."Dr. MacLean is hopeful that this treatment strategy will translate to the human population. The biotech company Excision BioTherapeutics, of which Dr. Khalili is a scientific founder and where Dr. Burdo contributes to preclinical research and development and serves on the Scientific Advisory Board, will assist with funding and infrastructure for larger scale studies and future clinical trials after approval by the Food and Drug Administration."We hope to soon move our work into clinical studies in humans as well," Dr. Khalili added. "People worldwide have been suffering with HIV for 40 years, and we are now very near to clinical research that could lead to a cure for HIV infection."
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Biotechnology
| 2,020 |
November 30, 2020
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https://www.sciencedaily.com/releases/2020/11/201130113603.htm
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The genetic blueprint that results in foot-and-mouth being so infectious
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Scientists have conducted a 'molecular dissection' of a part of the virus that causes foot-and-mouth disease, to try and understand why the pathogen is so infectious.
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Foot-and-mouth disease is a highly contagious infection of cloven-hoofed animals, which impacts on agricultural production and herd fertility. Global economic losses due to the disease have been estimated at between $6.5 billion and $22.5 billion each year, with the world's poorest farmers hit the hardest.A team of scientists from the University of Leeds and University of Ilorin, in Nigeria, has investigated the significance of the unusual way the virus's genome -- or genetic blueprint -- codes for the manufacture of a protein called 3B. The protein is involved in the replication of the virus.Researchers have known for some time that the virus's genetic blueprint contains three separate codes or instructions for the manufacture of 3B. Each code produces a similar but not identical copy of 3B. Up to now, scientists have not been able to explain the significance of having three different forms of the protein.In a paper published in the Dr Oluwapelumi Adeyemi, formerly a researcher at Leeds and now with the University of Ilorin and one of the paper's lead authors, said: "Our experiments have shown that having three forms of 3B gives the virus an advantage and that probably plays a role in why the virus is so successful in infecting its hosts."It is not as straightforward as saying because there are three forms of 3B -- it is going to be three times as competitive. There is a more nuanced interplay going on which needs further investigation."The paper describes how the scientists manipulated the genetic code, creating viral fragments with one form of 3B, two different forms of 3B and all three forms of 3B. Each was then measured to see how well they replicated.They found there was a competitive advantage -- greater replication -- in those samples that had more than one copy of 3B.Dr Joe Ward, post-doctoral researcher at Leeds and second co-lead author of the study, said: "The results of the data analysis were clear in that having multiple copies of the 3B protein gives the virus a competitive advantage. In terms of future research, the focus will be on why is that the case, and how the virus uses these multiple copies to its advantage."If we can begin to answer that question, then there is a real possibility we will identify interventions that could control this virus."The study involved using harmless viral fragments and replicons, fragments of RNA molecules, the chemical that make up the virus's genetic code.The study was funded by the Biotechnology and Biological Sciences Research Council and the Global Challenges Research Fund.
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Biotechnology
| 2,020 |
November 30, 2020
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https://www.sciencedaily.com/releases/2020/11/201130101250.htm
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Small molecules control bacterial resistance to antibiotics
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Antibiotics work in different ways. Some, including penicillin, attack the cell wall of the bacteria by inhibiting their synthesis. But the bacteria are not helpless against this attack. "We have been able to identify a small ribonucleic acid that has a decisive influence on the antibiotic-resistance of the cholera-triggering bacterium Vibrio cholerae," says Kai Papenfort, Professor of General Microbiology at the University of Jena, Germany.
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The protein CrvA is found in the periplasmic space of the bacterium. This lies between the outer cell wall and the basic substance of the cell, the cytoplasm, which is also surrounded by a membrane. There CrvA determines the curvature of the rod-shaped bacterium V. cholerae. "Until now, however, it was not known what controls the activity of this protein. With the small ribonucleic acid VadR we were able to identify a post-transcriptional inhibitor of the synthesis of this protein," says Prof. Papenfort. In contrast to messenger RNA, small ribonucleic acids do not contain any genetic information, but do influence its expression -- usually after the DNA has been transcribed into messenger RNA."Cholera bacteria in which the protein CrvA is not suppressed by VadR show a reduced survival rate on contact with penicillin," says Papenfort. This indicates that the maintenance of the cell form by the small RNA is crucial for antibiotic resistance, the microbiologist adds. The researchers revealed other functions of the small RNA VadR, including the formation of biofilms, which play an important role in the pathogenicity of V. cholerae."VadR is one of many molecules that can intervene in gene expression in V. cholerae. If we understand all these molecules, their functions and their interaction, we can derive new therapeutic approaches. The increasing resistance to antibiotics makes this urgently necessary," says Papenfort, whose research contributes to the Cluster of Excellence "Balance of the Microverse" at the University of Jena.
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Biotechnology
| 2,020 |
November 25, 2020
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https://www.sciencedaily.com/releases/2020/11/201125154808.htm
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Scientists discover a motif that guides assembly of the algal pyrenoid
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The next time you visit a lake or the seashore, take a deep breath. As you exhale, take a moment to be thankful for the little things: specifically, for the microscopic, single-celled algae in the soil and waters all around you that are extracting the carbon dioxide you just exhaled and incorporating it into sugars that will eventually be used by every other organism in the biosphere. About 30% of this activity, globally, is carried out by a specialized structure in algae called the pyrenoid.
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To visualize a pyrenoid, think of a pomegranate. The pyrenoid contains kernels of Rubisco, the enzyme that carries out the molecular work of incorporating carbon dioxide into sugars. These kernels are embedded in a supportive flesh, or matrix, of other proteins, that is itself surrounded by an outer shell made of starch. The fruit is a bit worm-eaten; it is riddled with fingerlike channels -- actually, tubules enclosed by membrane -- that deliver concentrated carbon dioxide to the Rubisco kernels. The tubules are important to pyrenoid function because waterborne algae such as Chlamydomonas reinhardtii would otherwise struggle to get enough carbon dioxide to keep Rubisco operating at peak capacity.The pyrenoid presents several enigmas for scientists. For example, how the proteins that make up the pyrenoid are routed there, and how they organize into such a complex arrangement, has been an enduring mystery. New work from the laboratory of Martin Jonikas, an Assistant Professor in the Department of Molecular Biology at Princeton, and collaborators, has now solved this riddle."The key initial discovery was made by chance," says Jonikas.Research Molecular Biologist Moritz Meyer and colleagues were trying to identify what proteins are present in the pyrenoid besides Rubisco. To do this, they used an antibody: a protein that, like a key, attaches to other proteins that possess a specific, matching lock. Meyer and colleagues planned to crack open algae and then add an antibody that binds a particular matrix protein to the resulting molecular soup. By pulling on the antibody, the scientists could drag that protein out. Any other proteins that bind to the antibody's target protein would come along for the ride, and the scientists could then determine whether any of them were previously unknown pyrenoid components. But the experiment didn't turn out as expected."We noticed that the antibody directly bound to several pyrenoid-localized proteins," says Jonikas. In other words, they'd just discovered that all these proteins possess a lock matching their antibody's key. Closer examination of the proteins revealed the existence of a sequence of amino acids, or motif, that is present in the antibody's original target and also appears in all of the other proteins."We hypothesized that this motif may serve as a signal that targets the proteins to the pyrenoid, and the experiments we did support this hypothesis," explains Jonikas. "Removing the motif from one of the motif-containing proteins caused it to no longer localize to the pyrenoid, while adding it to non-pyrenoid proteins caused them to localize to the pyrenoid."Meyer and colleagues found that the motif binds to Rubisco. This explains how the pyrenoid forms: its component proteins remain loose in the cell until they bump into Rubisco and become trapped."Several of the proteins do not simply localize to the pyrenoid matrix, but rather appear to localize to the interfaces between the matrix and the pyrenoid's two other sub-compartments, the pyrenoid tubules and the starch sheath," notes Jonikas. This may allow the proteins to self-organize into the complex pyrenoid structure."The study represents an exquisite example of investigative science," says Dr. Howard Griffiths, Professor of Plant Science at Cambridge University in the United Kingdom. Dr. Griffiths has collaborated with Jonikas's group on other studies, but he was not involved in this work."They used clever experimental manipulations to prove that a common motif could allow the specific linker to form the Rubisco matrix, and anchor other key elements both internally to the thylakoid tubules, and the starch sheath towards the periphery," says Griffiths. "Overall, the report by Meyer and colleagues has made a significant contribution to our understanding of pyrenoid form and function, with relevance both for understanding aquatic primary productivity, and to underpin approaches seeking to incorporate such a mechanism to 'turbocharge' photosynthesis in terrestrial crop plants."This research was supported by grants to M.C.J. by NSF (IOS-1359682 and MCB-1935444), NIH (DP2-GM-119137), and the Simons Foundation and Howard Hughes Medical Institute (55108535); and to L.C.M.M. by the UK Biotechnology and Biological Sciences Research Council (BB/R001014/1).
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Biotechnology
| 2,020 |
November 25, 2020
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https://www.sciencedaily.com/releases/2020/11/201125154806.htm
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Plants: Scientists solve the mystery behind an enigmatic organelle, the pyrenoid
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Carbon is one of the main building blocks for life on Earth. It's abundant in our planet's atmosphere, where it's found in the form of carbon dioxide. Carbon makes its way into Earthlings' bodies mainly through the process of photosynthesis, which incorporates carbon dioxide into sugars that serve as components for important biomolecules and fuel the global food chain. About a third of this process globally is carried out by single-celled algae that live in the oceans (most of the rest is done by plants).
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The enzyme that performs the first step of the reaction to assimilate carbon dioxide into sugars is a bulky protein called Rubisco assembled from eight identical small subunits and eight identical large subunits arranged together symmetrically. All the parts of this assembly, which is called a holoenzyme, work in concert to perform Rubisco's enzymatic duty. Rubisco's rate of activity -- and by extension, the rate at which plants and algae can grow -- is limited by its access to carbon dioxide. Free carbon dioxide can be scarce in water, so aquatic algae such as "The defining feature of a pyrenoid is the matrix, a giant liquid-like condensate that contains nearly all of the cell's Rubisco," explains Jonikas, an Assistant Professor in the Department of Molecular Biology at Princeton.Rubisco is the main component of the pyrenoid matrix, but not the only one; in 2016, Jonikas's lab discovered another abundant protein in the pyrenoid called EPYC1. In their 2016 paper, Jonikas's group showed that EPYC1 binds to Rubisco and helps concentrate Rubisco in the pyrenoid. The researchers theorized that EPYC1 works like a molecular glue to link together Rubisco holoenzymes. Postdoc Shan He, together with colleagues in Jonikas's lab and collaborators from Germany, Singapore and England, set out to test this theory."In the present work, we demonstrate that this is indeed how it works," says Jonikas, "by showing that EPYC1 has five binding sites for Rubisco, allowing it to 'link' together multiple Rubisco holoenzymes."EPYC1 is a loosely structured, extended protein, and its five Rubisco binding sites are evenly distributed across its length. The researchers also found that Rubisco has eight EPYC1 binding sites distributed evenly across its ball-like surface. Computer modeling showed that the loosely structured and flexible EPYC1 protein can make multiple contacts with a single Rubisco holoenzyme or bridge together neighboring ones. In this way, EPYC1 drives Rubisco to cluster in the pyrenoid matrix.Although this offers a satisfying explanation for how the matrix is assembled, it poses something of a conundrum. Other proteins need to be able to access Rubisco to repair it when it breaks down. If the EPYC1-Rubisco network is rigid, it could block these proteins from accessing Rubisco. However, He and colleagues found that EPYC1's interactions with Rubisco are fairly weak, so although the two proteins may form many contacts with each other, these contacts are exchanging rapidly."This allows EPYC1 and Rubisco to flow past each other while staying in a densely packed condensate, allowing other pyrenoid proteins to also access Rubisco," notes Jonikas. "Our work solves the longstanding mystery of how Rubisco is held together in the pyrenoid matrix."Land plants don't have pyrenoids, and scientists think that engineering a pyrenoid-like structure into crop plants could boost their growth rates. Understanding how the pyrenoid is assembled in algae represents a significant step toward such efforts."He and colleagues provide a very nice molecular study of the protein-protein interactions between the Rubisco small subunit and EPYC1," says Dr. James Moroney, Professor of Biology at the Louisiana State University department of Biological Sciences, whose lab studies photosynthesis in plants and algae."This work is encouraging for researchers trying to introduce pyrenoid-like structures into plants to improve photosynthesis," he adds.In a world beset by hunger and disease, we can use all the boosts we can get.Funding: The work described here was supported by grants to M.C.J. from the National Science Foundation (nos IOS-1359682 and MCB-1935444), National Institutes of Health (no. DP2-GM-119137), and Simons Foundation and Howard Hughes Medical Institute (no. 55108535); to B.D.E. by Deutsche Forschungsgemeinschaft (EN 1194/1-1 as part of FOR2092); to O.M.-C. by the Ministry of Education (MOE Singapore) Tier 2 (no. MOE2018-T2-2-059); to A.J.M. and N.A. by the UK Biotechnology and Biological Sciences Research Council (no. BB/ S015531/1) and Leverhulme Trust (no. RPG-2017-402); to F.M.H by NIH ( R01GM071574); to S.A. P. by Deutsche Forschungsgemeinschaft fellowship (no. PO2195/1-1); and to V.K.C. by a National Institute of General Medical Sciences of the Institutes of Health (no. T32GM007276) training grant.
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Biotechnology
| 2,020 |
November 25, 2020
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https://www.sciencedaily.com/releases/2020/11/201125122321.htm
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Researchers uncover the unique way stem cells protect their chromosome ends
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Telomeres are specialised structures at the end of chromosomes which protect our DNA and ensure healthy division of cells. According to a new study from researchers at the Francis Crick Institute published in
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For the last 20 years, researchers have been working to understand how telomeres protect chromosome ends from being incorrectly repaired and joined together because this has important implications for our understanding of cancer and aging.In healthy cells, this protection is very efficient, but as we age our telomeres get progressively shorter, eventually becoming so short that they lose some of these protective functions. In healthy cells, this contributes to the progressive decline in our health and fitness as we age. Conversely, telomere shortening poses a protective barrier to tumour development, which cancer cells must solve in order to divide indefinitely.In somatic cells, which are all the cells in the adult body except stem cells and gametes, we know that a protein called TRF2 helps to protect the telomere. It does this by binding to and stabilising a loop structure, called a t-loop, which masks the end of the chromosome. When the TRF2 protein is removed, these loops do not form and the chromosome ends fuse together, leading to "spaghetti chromosomes" and killing the cell.However, in this latest study, Crick researchers have found that when the TRF2 protein is removed from mouse embryonic stem cells, t-loops continue to form, chromosome ends remain protected and the cells are largely unaffected.As embryonic stem cells differentiate into somatic cells, this unique mechanism of end protection is lost and both t-loops and chromosome end protection become reliant on TRF2. This suggests that somatic and stem cells protect their chromosome ends in fundamentally different ways."Now we know that TRF2 isn't needed for t-loop formation in stem cells, we infer there must be some other factor that does the same job or a different mechanism to stabilise t-loops in these cells, and we want to know what it is," says Philip Ruis, first author of the paper and PhD student in the DNA Double Strand Breaks Repair Metabolism Laboratory at the Crick."For some reason, stem cells have evolved this distinct mechanism of protecting their chromosomes ends, that differs from somatic cells. Why they have, we have no idea, but it's intriguing. It opens up many questions that will keep us busy for many years to come."The team have also helped to clarify years of uncertainty about whether the t-loops themselves play a part in protecting the chromosome ends. They found that telomeres in stem cells with t-loops but without TRF2 are still protected, suggesting the t-loop structure itself has a protective role."Rather than totally contradicting years of telomere research, our study refines it in a very unique way. Basically, we've shown that stem cells protect their chromosome ends differently to what we previously thought, but this still requires a t-loop," says Simon Boulton, paper author and group leader in the DNA Double Strand Breaks Repair Metabolism Laboratory at the Crick."A better understanding of how telomeres work, and how they protect the ends of chromosomes could offer crucial insights into the underlying processes that lead to premature aging and cancer."The team worked in collaboration with Tony Cesare in Sydney and other researchers across the Crick, including Kathy Niakan, of the Human Embryo and Stem Cell Laboratory, and James Briscoe, of the Developmental Dynamics Laboratory at the Crick. "This is a prime example of what the Crick was set up to promote. We've been able to really benefit from our collaborator's expertise and the access that was made possible by the Crick's unique facilities," says Simon.The researchers will continue this work, aiming to understand in detail the mechanisms of telomere protection in somatic and embryonic cells.
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Biotechnology
| 2,020 |
November 25, 2020
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https://www.sciencedaily.com/releases/2020/11/201125114350.htm
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New insights into how the CRISPR immune system evolved
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With new insights into how the genetic tool CRISPR -- which allows direct editing of our genes -- evolved and adapted, we are now one step closer to understanding the basis of the constant struggle for survival that takes place in nature. The results can be used in future biotechnologies.
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In 2020, the Nobel Prize in Chemistry goes to Emmanuelle Charpentier and Jennifer A. Doudna for their discoveries of the molecular mechanism behind CRISPR-Cas and the use of the technology as a genetic tool. Although CRISPR-Cas has found many uses in biotechnology and medicine, it originates in nature, where it functions as a microbial immune system.Just as our immune system remembers the pathogens we have been exposed to earlier in life, CRISPR-Cas provides microorganisms with an ability to respond quickly to viruses they have previously encountered by storing a small amount of the viral DNA in their own genome.CRISPR-Cas is found naturally in most bacteria as well as the so-called archaea. When examining the origin of life on Earth, archaea are particularly interesting, as they form a kind of "missing link" between bacteria and the cells of higher eukaryotes like our own. Studies of these organisms can therefore provide us with important insights into how the CRISPR-Cas immune system has evolved over hundreds of millions of years.New research results from researchers at the Department of Molecular Biology and Genetics, Aarhus University -- obtained in close collaboration with leading researchers from the University of Copenhagen and Old Dominion University in Virginia, USA, and published in two articles in leading, international journals -- now shed new light on how CRISPR-Cas emerged early during the development of life on Earth, as well as how this immune system is constantly adapting to new challenges.The research group from Aarhus -- led by Associate Professor Ditlev E. Brodersen -- has discovered how a part of CRISPR-Cas that is responsible for incorporating foreign, viral DNA into the microorganism's own genome has originated from another, very common type of genes in bacteria and archaea that surprisingly encode toxins.The new knowledge therefore provides insights into an evolutionary process in which the toxin genes were present early during the development of life, and over time were integrated and adapted as part of the CRISPR-Cas modules that many microorganisms possess to this day. For the first time, we have an answer to a question that has puzzled researchers for a long time, namely why toxin genes exist among the CRISPR-Cas genes."This understanding of how certain proteins are 'recycled' in several different situations, is enormously useful for researchers," explains Ditlev Brodersen, "because when we understand the entire repertoire of functions that certain proteins possess, it opens up for the possibility of using them as specific tools in genetic engineering. For example, it might be possible to get disease-causing bacteria to direct their CRISPR-Cas systems towards themselves and thus avoid infection."In another article, published in the journal, In a boiling mud puddle on Iceland lives a very special organism, an archaea called But even though Sulfolobus has chosen a very unattractive place to live, it still encounters resistance, not the least from small, rod-shaped DNA viruses that constantly poke holes in the cells and shoot their foreign DNA into them, causing Sulfolobus to explode in a wealth of new virus particles. To avoid this fate, Sulfolobus has developed a CRISPR-Cas defence, by which it has stored small parts of the viral DNA in its own genome to be able to withstand these attacks.But in the constantly escalating battle between life and death, the virus has developed a countermeasure: It has managed to cope by producing a small weapon, an anti-CRISPR protein that, like upsetting the applecart, blocks the CRISPR-Cas response in Sulfolobus.The new results from Ditlev E. Brodersen's group at Aarhus University -- generated in close collaboration with Associate Professor Xu Peng from the Department of Biology, University of Copenhagen -- now for the first time show how this fight takes place in the boiling pools.The researchers have been able to visualise how the anti-CRISPR protein binds strongly to the largest protein of the CRISPR-Cas system, thereby directly preventing it from destroying the viral DNA. In this way, the virus bypasses -- at least for some time -- being beated off by CRISPR-Cas. The new results give scientists insights into the arms race that is constantly taking place in nature, and how the evolution of life is in fact a constant struggle for survival.- "We now know the details of how the anti-CRISPR protein can block the CRISPR-Cas immune system, so the question is what will be the next move in this arms race," says Ditlev Brodersen. "Perhaps the microbes will begin to form anti-anti-CRISPR proteins, a third type of protein that can prevent the anti-CRISPR protein from working, but we have yet to find these in Sulfolobus archaea. So right now the ball is back on Sulfolobus' half of the field," says Ditlev Brodersen, "and the cold war is always warm in the boiling pool."
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Biotechnology
| 2,020 |
November 24, 2020
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https://www.sciencedaily.com/releases/2020/11/201124150847.htm
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Commonly used antibiotic shows promise for combating Zika infections
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In 2015, hundreds of children were born with brain deformities resulting from a global outbreak of Zika virus infections. Recently, National Institutes of Health researchers used a variety of advanced drug screening techniques to test out more than 10,000 compounds in search of a cure. To their surprise, they found that the widely used antibiotic methacycline was effective at preventing brain infections and reducing neurological problems associated with the virus in mice. In addition, they found that drugs originally designed to combat Alzheimer's disease and inflammation may also help fight infections.
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"Around the world, the Zika outbreak produced devastating, long-term neurological problems for many children and their families. Although the infections are down, the threat remains," said Avindra Nath, M.D., senior investigator at the NIH's National Institute of Neurological Disorders and Stroke (NINDS) and a senior author of the study published in The study was a collaboration between scientists on Dr. Nath's team and researchers in laboratories led by Anton Simeonov, Ph.D., scientific director at the NIH's National Center for Advancing Translational Sciences (NCATS) and Radhakrishnan Padmanabhan, Ph.D., Professor of Microbiology & Immunology, Georgetown University Medical Center, Washington, D.C.The Zika virus is primarily spread by the Aedes aegypti mosquito. In 2015 and 2016, at least 60 countries reported infections. Some of these countries also reported a high incidence of infected mothers giving birth to babies born with abnormally small heads resulting from a developmental brain disorder called fetal microcephaly. In some adults, infections were the cause of several neurological disorders including Guillain-Barré syndrome, encephalitis, and myelitis. Although many scientists have tried, they have yet to discover an effective treatment or vaccination against the virus.In this study, the researchers looked for drugs that prevent the virus from reproducing by blocking the activity of a protein called NS2B-NS3 Zika virus protease. The Zika virus is a protein capsule that carries long strings of RNA-encoded instructions for manufacturing more viral proteins. During an infection, the virus injects the RNA into a cell, resulting in the production of these proteins, which are strung together, side-by-side, like the parts in a plastic model airplane kit. The NS2B-NS3 protease then snaps off each protein, all of which are critical for assembling new viral particles."Proteases act like scissors. Blocking protease activity is an effective strategy for counteracting many viruses," said Rachel Abrams, Ph.D., an organic chemist in Dr. Nath's lab and the study leader. "We wanted to look as far and wide as possible for drugs that could prevent the protease from snipping the Zika virus polyprotein into its active pieces."To find candidates, Dr. Abrams worked with scientists on Dr. Simeonov's and Dr. Padmanabhan's teams to create assays, or tests, for assessing the ability of drugs to block NS2B-NS3 Zika virus protease activity in plates containing hundreds of tiny test tubes. Each assay was tailored to a different screening, or sifting, technique. They then used these assays to simultaneously try out thousands of candidates stored in three separate libraries.One preliminary screen of 2,000 compounds suggested that commonly used, tetracycline-based antibiotic drugs, like methacycline, may be effective at blocking the protease.Meanwhile, a large-scale screen of more than 10,000 compounds helped identify an investigational anti-inflammatory medicine, called MK-591, and a failed anti-Alzheimer's disease drug, called JNJ-404 as potential candidates. A virtual screen of over 130,000 compounds was also used to help spot candidates. For this, the researchers fed the other screening results into a computer and then used artificial intelligence-based programs to learn what makes a compound good at blocking NS2B-NS3 Zika virus protease activity."These results show that taking advantage of the latest technological advances can help researchers find treatments that can be repurposed to fight other diseases," said Dr. Simeonov.The Zika virus is known to preferentially infect stem cells in the brain. Scientists suspect this is the reason why infections cause more harm to newborn babies than to adults. Experiments on neural stem cells grown in petri dishes indicated that all three drugs identified in this study may counteract these problems. Treating the cells with methacycline, MK-591, or JNJ-404 reduced Zika virus infections.Because tetracyclines are U.S. Food and Drug Administration-approved drugs that are known to cross the placenta of pregnant women, the researchers focused on methacycline and found that it may reduce some neurodevelopmental problems caused by the Zika virus. For instance, Zika-infected newborn mice that were treated with methacycline had better balance and could turn over more easily than ones that were given a placebo. Brain examinations suggested this was because the antibiotic reduced infections and neural damage. Nevertheless, the antibiotics did not completely counteract harm caused by the Zika virus. The weight of mice infected with the virus was lower than control mice regardless of whether the mice were treated with methacycline."These results suggest that tetracycline-based antibiotics may at least be effective at preventing the neurological problems associated with Zika virus infections," said Dr. Abrams. "Given that they are widely used, we hope that we can rapidly test their potential in clinical trials."
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Biotechnology
| 2,020 |
November 24, 2020
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https://www.sciencedaily.com/releases/2020/11/201124150836.htm
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CRISPRi screens reveal sources of metabolic robustness in E. coli
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Metabolic robustness, the ability of a metabolic system to buffer changes in its environment, is not always a welcome feature for microbiologists: it interferes with metabolic engineering or prevents that antibiotics kill bacteria. Therefore it is important to understand the mechanisms that enable metabolic robustness. A massively parallel CRISPRi screen demonstrated that
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In their natural habitat, bacteria like To understand how As Dr. Hannes Link explains: "Our results demonstrated that To answer this question, the team measured the proteome and metabolome of 30 CRISPRi strains. In some strains the proteome responses revealed mechanisms that actively buffered the CRISPRi knockdowns. For example, knockdown of homocysteine transmethylase (MetE) in the methionine pathway caused a compensatory upregulation of all other enzymes in the methionine pathway. In other words, This comprehensive approach creates new possibilities for the development of industrially useful microbes, as Dr. Hannes Link points out: "In the future, we want to use these data to construct metabolic models that are dynamic and predictive. We used a very small dynamic model in the current study, but building larger models remains one of the big challenges. Such models would allow us to engineer
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Biotechnology
| 2,020 |
November 24, 2020
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https://www.sciencedaily.com/releases/2020/11/201124111027.htm
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Enzymatic photocaging for the study of gene regulation through DNA methylation
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The addition and removal of methyl groups on DNA plays an important role in gene regulation. In order to study these mechanisms more precisely, a German team has developed a new method by which specific methylation sites can be blocked and then unblocked at a precise time through irradiation with light (photocaging). As reported in the journal
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Although they look very different and serve completely different functions, all cells in our body have identical DNA. However, they do not use the same genes. Certain genes are turned on and others off, depending on the type of cell and the moment in time. The "switches" are chemical changes in the building blocks of the DNA. These changes are called epigenetic modifications. One significant regulation mechanism is methylation and demethylation, meaning the attachment and removal of a methyl group (-CH(3)). The methylation patterns of cancer cells, for example, differ from healthy cells. During a methylation, enzymes known as methyl transferases (MTases) transfer a methyl group from S-adenosyl-?-methionine (AdoMet) to the target molecule.In order to study the purpose and function of this regulation more closely and determine methylation patterns, it would be useful to have "tools" to specifically inhibit methylation at targeted locations and then lift the inhibition at a defined time. To this end, a team led by Andrea Rentmeister chose to use a method known as photocaging. In this method, a "photocage" is a molecule that falls apart upon irradiation, such as a 2-nitrobenzyl group. The cage first blocks the target location, then targeted irradiation with light acts as a "switch" to remove the blockade.The idea was to equip AdoMet analogues with a photocage that is then transferred to the methylation sites. However, AdoMet analogues decompose in aqueous solutions and cannot enter into cells. Therefore, the team at the University of Münster wanted to produce them in situ. In the body, AdoMet is produced from the amino acid methionine through the action of the enzyme, methionine adenosyl transferase (MAT). Synthesis of the AdoMet analogues requires methionine with an attached nitrobenzyl photocage and a MAT that can use such an altered substrate. Starting with a MAT enzyme from a single-celled organism (Cryptosporidium hominis), the researchers were able to carefully change specific amino acids in the enzyme to increase the size of its hydrophobic binding cavity so that it could contain the nitrobenzyl group. A crystal structure analysis showed that the ADoMet analogue is bound in the cavity of this photocaging MAT (PC-MAT). Based on this information, the team also produced a second PC-MAT based on a thermostable MAT enzyme from the archaeon Methanocaldococcus jannaschii.Both of these PC-MATs are compatible with DNA and RNA MTases and made it possible to attach photocages to all natural methylation sites of a plasmid DNA. Irradiation with light removed the blockade.
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Biotechnology
| 2,020 |
November 24, 2020
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https://www.sciencedaily.com/releases/2020/11/201124101031.htm
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World's first: Drug guides stem cells to desired location, improving their ability to heal
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Scientists at Sanford Burnham Prebys Medical Discovery Institute have created a drug that can lure stem cells to damaged tissue and improve treatment efficacy -- a scientific first and major advance for the field of regenerative medicine. The discovery, published in the
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Toxic cells (green) disappeared when mice with a neurodegenerative condition received both therapeutic stem cells (red) and the drug SDV1a-which corresponded with longer lives and delayed symptom onset. These results suggest that SDV1a can be used to improve the efficacy of stem cell treatments."The ability to instruct a stem cell where to go in the body or to a particular region of a given organ is the Holy Grail for regenerative medicine," says Evan Y. Snyder, M.D. Ph.D., professor and director of the Center for Stem Cells & Regenerative Medicine at Sanford Burnham Prebys and senior author of the study. "Now, for the first time ever, we can direct a stem cell to a desired location and focus its therapeutic impact."Nearly 15 years ago, Snyder and his team discovered that stem cells are drawn to inflammation -- a biological "fire alarm" that signals damage has occurred. However, using inflammation as a therapeutic lure isn't feasible because an inflammatory environment can be harmful to the body. Thus, scientists have been on the hunt for tools to help stem cells migrate -- or "home" -- to desired places in the body. This tool would be helpful for disorders in which initial inflammatory signals fade over time -- such as chronic spinal cord injury or stroke -- and conditions where the role of inflammation is not clearly understood, such as heart disease."Thanks to decades of investment in stem cell science, we are making tremendous progress in our understanding of how these cells work and how they can be harnessed to help reverse injury or disease," says Maria T. Millan, M.D., president and CEO of the California Institute for Regenerative Medicine (CIRM), which partially funded the research. "Dr. Snyder's group has identified a drug that could boost the ability of neural stem cells to home to sites of injury and initiate repair. This candidate could help speed the development of stem cell treatments for conditions such as spinal cord injury and Alzheimer's disease."In the study, the scientists modified CXCL12 -- an inflammatory molecule which Snyder's team previously discovered could guide healing stem cells to sites in need of repair -- to create a drug called SDV1a. The new drug works by enhancing stem cell binding and minimizing inflammatory signaling -- and can be injected anywhere to lure stem cells to a specific location without causing inflammation."Since inflammation can be dangerous, we modified CXCL12 by stripping away the risky bit and maximizing the good bit," says Snyder. "Now we have a drug that draws stem cells to a region of pathology, but without creating or worsening unwanted inflammation."To demonstrate that the new drug is able to improve the efficacy of a stem cell treatment, the researchers implanted SDV1a and human neural stem cells into the brains of mice with a neurodegenerative disease called Sandhoff disease. This experiment showed SDV1a helped the human neural stem cells migrate and perform healing functions, which included extending lifespan, delaying symptom onset, and preserving motor function for much longer than the mice that didn't receive the drug. Importantly, inflammation was not activated, and the stem cells were able to suppress any pre-existing inflammation.The researchers have already begun testing SDV1a's ability to improve stem cell therapy in a mouse model of ALS, also known as Lou Gehrig's disease, which is caused by progressive loss of motor neurons in the brain. Previous studies conducted by Snyder's team indicated that broadening the spread of neural stem cells helps more motor neurons survive -- so the scientists are hopeful that strategic placement of SDV1a will expand the terrain covered by neuroprotective stem cells and help slow the onset and progressive of the disease."We are optimistic that this drug's mechanism of action may potentially benefit a variety of neurodegenerative disorders, as well as non-neurological conditions such as heart disease, arthritis and even brain cancer," says Snyder. "Interestingly, because CXCL12 and its receptor are implicated in the cytokine storm that characterizes severe COVID-19, some of our insights into how to selectively inhibit inflammation without suppressing other normal processes may be useful in that arena as well."
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Biotechnology
| 2,020 |
November 24, 2020
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https://www.sciencedaily.com/releases/2020/11/201118141757.htm
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Super-resolution 'street view' microscopy hits the SPOT
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The ability to "see" the inner workings of structures (organelles) within cells, in real time, offers the promise of advancements in disease diagnosis and treatment. Organelle dynamics drive the self-efficient micro-world of cells, but current super-resolution microscopy techniques used to track these interactions have limitations.
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Now an advanced technique called SPOT (Spectrum and Polarization Optical Tomography) is giving researchers a "street view" of the vital lipid membranes surrounding organelles and by so doing opens up the opporutnies to study the sophisticated world of lipid dynamics.The researchers say it's a significant development, building on earlier work on super-resolution polarization microscopy.The research, published in Lead author Dr Karl Zhangao from UTS-SUSTech Joint Research Centre said that lipid membranes surround most organelles and play a significant role."Their shape, composition and phase synergistically regulate biophysical membrane properties, membrane protein function and lipid-protein interactions.""However it is challenging to observe such a level of complexity due to their similar chemical composition," he said.Simply using one dye that universally stains the lipid membranes, SPOT can simultaneously reveal lipid membrane morphology, polarity, and phase from measuring the intensity, spectrum, and polarization, respectively. Combined with lipophilic probes, the team successfully revealed more than ten types of organelles simultaneously, and their sophisticated lipid dynamics.Using the new imaging platform established at SUStech, researchers observed the multi-organelle interactive activities of cell division, lipid dynamics during plasma membrane separation, tunneling nanotubules formation, and mitochondrial cristae dissociation."This is the first time researchers have been able to quantitatively study the lipid heterogeneity inside subcellular organelles," senior author Professor Dayong Jin says. Professor Jin is Director of UTS-SUStech Joint Research Centre and Director of UTS Institute for Biomedical Materials and Devices."This is a very powerful tool for super-resolution imaging the inner working of each single cells, that will advance our knowledge in understanding how cells function, diagnose when a "factory" or a transportation doesn't work properly within the cell, and monitor the progression of disease," Professor Jin said"With such information it isn't too big a leap to identify pathways for potential drug treatments, as well as examine their efficacy right on the SPOT" he said.Wenhui Liu, a doctoral student in Tsinghua University, and Meiqi Li, a doctoral student in Peking University are also co-first authors of this work.
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Biotechnology
| 2,020 |
November 24, 2020
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https://www.sciencedaily.com/releases/2020/11/201124190532.htm
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Cutting edge technology to bioprint mini-kidneys
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Researchers have used cutting edge technology to bioprint miniature human kidneys in the lab, paving the way for new treatments for kidney failure and possibly lab-grown transplants.
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The study, led by the Murdoch Children's Research Institute (MCRI) and biotech company Organovo and published in The research showed how 3D bioprinting of stem cells can produce large enough sheets of kidney tissue needed for transplants.Like squeezing toothpaste out of a tube, extrusion-based 3D bioprinting uses a 'bioink' made from a stem cell paste, squeezed out through a computer-guided pipette to create artificial living tissue in a dish.MCRI researchers teamed up with San Diego based Organovo Inc to create the mini organs.MCRI Professor Melissa Little, a world leader in modelling the human kidney, first began growing kidney organoids in 2015. But this new bio-printing method is faster, more reliable and allows the whole process to be scaled up. 3D bioprinting could now create about 200 mini kidneys in 10 minutes without compromising quality, the study found.From larger than a grain of rice to the size of a fingernail, bioprinted mini-kidneys fully resemble a regular-sized kidney, including the tiny tubes and blood vessels that form the organ's filtering structures called nephrons.Professor Little said by using mini-organs her team hope to screen drugs to find new treatments for kidney disease or to test if a new drug was likely to injure the kidney."Drug-induced injury to the kidney is a major side effect and difficult to predict using animal studies. Bioprinting human kidneys are a practical approach to testing for toxicity before use," she said.In this study, the toxicity of aminoglycosides, a class of antibiotics that commonly damage the kidney, were tested."We found increased death of particular types of cells in the kidneys treated with aminoglycosides," Professor Little said."By generating stem cells from a patient with a genetic kidney disease, and then growing mini kidneys from them, also paves the way for tailoring treatment plans specific to each patient, which could be extended to a range of kidney diseases."Professor Little said the study showed growing human tissue from stem cells also brought the promise of bioengineered kidney tissue."3D bioprinting can generate larger amounts of kidney tissue but with precise manipulation of biophysical properties, including cell number and conformation, improving the outcome," she said.Currently, 1.5 million Australians are unaware they are living with early signs of kidney disease such as decreased urine output, fluid retention and shortness of breath.Professor Little said prior to this study the possibility of using mini kidneys to generate transplantable tissue was too far away to contemplate."The pathway to renal replacement therapy using stem cell-derived kidney tissue will need a massive increase in the number of nephron structures present in the tissue to be transplanted," she said."By using extrusion bioprinting, we improved the final nephron count, which will ultimately determine whether we can transplant these tissues into people."
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Biotechnology
| 2,020 |
November 24, 2020
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https://www.sciencedaily.com/releases/2020/11/201124101035.htm
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Antimicrobial soap additive worsens fatty liver disease in mice
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University of California San Diego School of Medicine researchers found evidence that triclosan -- an antimicrobial found in many soaps and other household items -- worsens fatty liver disease in mice fed a high-fat diet.
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The study, published November 23, 2020 in "Triclosan's increasingly broad use in consumer products presents a risk of liver toxicity for humans," said Robert H. Tukey, PhD, professor in the Department of Pharmacology at UC San Diego School of Medicine. "Our study shows that common factors that we encounter in every-day life -- the ubiquitous presence of triclosan, together with the prevalence of high consumption of dietary fat -- constitute a good recipe for the development of fatty liver disease in mice."Tukey led the study with Mei-Fei Yueh, PhD, a project scientist in his lab, and Michael Karin, PhD, Distinguished Professor of Pharmacology and Pathology at UC San Diego School of Medicine.In a 2014 mouse study, the team found triclosan exposure promoted liver tumor formation by interfering with a protein responsible for clearing away foreign chemicals in the body.In the latest study, the researchers fed a high-fat diet to mice with type 1 diabetes. As previous studies have shown, the high-fat diet led to non-alcoholic fatty liver disease (NAFLD). In humans, NAFLD is an increasingly common condition that can lead to liver cirrhosis and cancer. Diabetes and obesity are risk factors for NAFLD.Some of the mice were also fed triclosan, resulting in blood concentrations comparable to those found in human studies. Compared to mice only fed a high-fat diet, triclosan accelerated the development of fatty liver and fibrosis.According to the study, here's what's likely happening: Eating a high-fat diet normally tells cells to produce more fibroblast growth factor 21, which helps protects liver cells from damage. Tukey and team discovered that triclosan messes with two molecules, ATF4 and PPARgamma, which cells need to make the protective growth factor. Not only that, the antimicrobial also disrupted a variety of genes involved in metabolism. In addition, the mice exposed to triclosan had less diversity in their gut microbiomes -- fewer types of bacteria living in the intestines, and a makeup similar to that seen in patients with NAFLD. Less gut microbiome diversity is generally associated with poorer health.So far, these findings have only been observed in mice who ingested triclosan. But since these same molecular systems also operate in humans, the new information will help researchers better understand risk factors for NAFLD, and give them a new place to start in designing potential interventions to prevent and mitigate the condition."This underlying mechanism now gives us a basis on which to develop potential therapies for toxicant-associated NAFLD," said Tukey, who is also director of the National Institute of Environmental Health Sciences Superfund Program at UC San Diego.In 2016, the U.S. Food and Drug Administration (FDA) ruled that over-the-counter wash products can no longer contain triclosan, given that it has not been proven to be safe or more effective than washing with plain soap and water. However, the antimicrobial is still found in some household and medical-grade products, as well as aquatic ecosystems, including sources of drinking water.An estimated 100 million adults and children in the U.S. may have NAFLD. The precise cause of NAFLD is unknown, but diet and genetics play substantial roles. Up to 50 percent of people with obesity are believed to have NAFLD. The condition typically isn't detected until it's well advanced. There are no FDA-approved treatments for NAFLD, though several medications are being developed. Eating a healthy diet, exercising and losing weight can help patients with NAFLD improve.Disclosure: Michael Karin is a founder, inventor and an Advisory Board Member of Elgia Therapeutics and has equity in the company.
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Biotechnology
| 2,020 |
November 23, 2020
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https://www.sciencedaily.com/releases/2020/11/201123120726.htm
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Tarantula toxin attacks with molecular stinger
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Oversized, hairy tarantulas may be unsightly and venomous, but surprisingly their hunter toxin may hold answers to better control of chronic pain.
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A bird-catching Chinese tarantula bite contains a stinger-like poison that plunges into a molecular target in the electrical signaling system of their prey's nerve cells.A new high-resolution cryo-electron microscopy study shows how the stinger quickly locks the voltage sensors on sodium channels, the tiny pores on cell membranes that create electrical currents and generate signals to operate nerves and muscles. Trapped in their resting position, the voltage sensors are unable to activate.The findings are published Nov. 23 in "The action of the toxin has to be immediate because the tarantula has to immobilize its prey before it takes off," said William Catterall, professor of pharmacology at the University of Washington School of Medicine. He was the senior researcher, along with pharmacology professor and Howard Hughes Medical Institute investigator, Ning Zheng, on the study of the molecular damage inflicted by tarantula venom.While some might dismiss those tarantulas as ugly, tough and mean, medical scientists are actually interested in their venom's ability to trap the resting state of the voltage sensor on voltage-gated sodium channels and shut them down. Such studies of toxins from these "big, nasty dudes," as Catterall describes them, could point to new approaches to structurally designing drugs that might treat chronic pain by blocking sensory nerve signals.Catterall explained that chronic pain is a difficult-to-treat disorder. Efforts to seek relief can sometimes be a gateway to opiate overdose, addiction, prolonged withdrawal, and even death. The development of safer, more effective, non-addictive drugs for pain management is a vital need.However, because it has been hard to capture the functional form of the tarantula toxin-ion channel chemical complex, reconstructing the toxin's blocking method in a small molecule has so far eluded molecular biologists and pharmacologists seeking new ideas for better pain drug designs.Researchers overcame this obstacle by engineering a chimeric model sodium channel. Like mythical centaurs, chimeras are composed of parts of two or more species. The researchers took the toxin-binding region from a specific type of human sodium channel that is crucial for pain transmission and imported it into their model ancestral sodium channel from a bacterium. They were then able to obtain a clear molecular view of configuration of the potent toxin from tarantula venom as it binds tightly to its receptor site on the sodium channel.This achievement revealed the structural basis for voltage sensor trapping of the resting state of the sodium channel by this toxin."Remarkably, the toxin plunges a 'stinger' lysine residue into a cluster of negative charges in the voltage sensor to lock it in place and prevent its function," Catterall said. "Related toxins from a wide range of spiders and other arthropod species use this molecular mechanism to immobilize and kill their prey."Catterall explained the medical research importance of this discovery. The human sodium channel placed into the chimeric model is called the Nav1.7 channel. It plays an essential role, he noted, in transmission of pain information from the peripheral nervous system to the spinal cord and brain and is therefore a prime target for pain therapeutics."Our structure of this potent tarantula toxin trapping the voltage sensor of Nav1.7 in the resting state," Catterall noted, "provides a molecular template for future structure-based drug design of next-generation pain therapeutics that would block function of Nav1.7 sodium channels."
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Biotechnology
| 2,020 |
November 23, 2020
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https://www.sciencedaily.com/releases/2020/11/201123101021.htm
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Helicates meet Rotaxanes to create promise for future disease treatment
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A new approach to treating cancers and other diseases that uses a mechanically interlocked molecule as a 'magic bullet' has been designed by researchers at the University of Birmingham.
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Called rotaxanes, the molecules are tiny nanoscale structures that resemble a dumbbell with a ring trapped around the central post. Scientists have been experimenting with rotaxanes based on thin, thread-like central posts for a number of years, but this new design uses instead a much larger cylindrical-shaped supramolecular 'helicate' molecule -- around 2nm long and 1nm wide -- which have remarkable ability to bind Y-shaped junctions or forks in DNA and RNA.These forks are created when DNA replicates and, in laboratory tests, the Birmingham researchers have shown that, when they bind to the junctions, the cylinder molecules are able to stop cancer cells, bacteria and viruses from reproducing.To gain control over that binding, the team from the University's Schools of Chemistry and Biosciences, collaborated with researchers in Wuhan, in China, and Marseille, in France, to solve the challenge of identifying a ring structure large enough to fit around this central cylinder molecule. They have now shown that a giant pumpkin-shaped molecule, called a cucurbit, is able to host the cylinder. When the ring is present, the rotaxane molecule is unable to bind.To prevent the cylinder from slipping out of the pumpkin-shaped ring, the researchers added branches to each end of the cylinder. They demonstrated that the cylinder then becomes mechanically locked inside the ring and that they can use this to control the way the supramolecular cylinder interacts with RNA and DNA.The results, published in the Lead researcher, Professor Mike Hannon, explains: "This is a really promising new approach that harnesses robust and proven chemistry in an entirely new way that has potential for targeted treatment of cancers and other diseases."Our approach is very different to leading cancer drugs which commonly affect all cells in the body, not just the cancer cells. The rotaxane molecule holds the promise that, by turning it on and off as required, it can specifically target and inhibit cancer cells with a high degree of accuracy."University of Birmingham Enterprise has a filed patent application covering the structure and design of these novel rotaxanes, and the team has already started work to explore a variety of applications for the approach.
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Biotechnology
| 2,020 |
November 23, 2020
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https://www.sciencedaily.com/releases/2020/11/201123101012.htm
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Proteins in motion
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Proteins are the essential substrate of learning and memory. However, while memories can last a life-time, proteins are relatively short-lived molecules that need to be replenished every couple of days. This poses a huge logistic challenge on over 85 billion neurons in the brain: billions of proteins need to be continuously produced, shipped, addressed and installed at the right location in the cell. Scientists at the Max Planck Institute for Brain Research have now addressed a bottleneck in the protein trafficking system, dendritic branch points. They find that surface diffusion of proteins is more effective at providing proteins to distal dendritic sites than cytoplasmic diffusion.
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"Dendritic arborization of neurons is one of the fascinating features that evolved to increase the complexity of the interactions between neurons. However, a more complex dendritic arbor also increases the difficulty of the logistical task to supply proteins to each part of the neuron," says Tatjana Tchumatchenko, Research Group Leader at the Max Planck Institute for Brain Research who led the study.Neurons distribute thousands of different protein species, necessary for maintaining synaptic function and plasticity across their dendritic arbor. However, the majority of proteins are synthesized hundreds of microns away from distal synapses, in the soma (its cell body). How do proteins reach distal sites? "In this study, we focused on passive protein transport which corresponds to free diffusion. In contrast to active transport via molecular motors, diffusion is energetically cheap. However, there is a downside: passive transport is slow and non-directional," explains Fabio Sartori, graduate student in the Tchumatchenko group and the lead author of the new study.What happens when proteins encounter dendritic branch points? Branch points are like cross roads for traffic, some of the proteins will turn right, others will turn left. Cross roads for cars can be traffic bottlenecks. Similarly, the more branch points proteins meet on their journey, the lower the total protein number downstream. As a result, a neuron needs to produce more proteins to maintain a minimal protein number at distal synapses. "We used experimental data provided by our collaborators and developed a new computational framework to compare two classes of proteins, based on their "transport medium": soluble proteins that diffuse in the cytoplasm and membrane proteins," says Sartori. "Interestingly, we find that surface diffusion is on average 35 percent more effective than cytoplasmic diffusion in providing proteins to downstream locations.Each protein has a typical distance it can cover while diffusing, this is its diffusion length. The higher this value is, the more proteins will reach distal dendrites. If a dendritic branch has a large radius, then it can carry more proteins. The combination of two factors, the width (or "radii") of dendrites and how far proteins can move, determines the number of proteins a neuron needs to produce to supply all synapses. Sartori and colleagues found that by optimizing dendritic radii, a neuron can reduce the total protein count and thereby the protein synthesis cost by several orders of magnitude. "Our results suggest that neuronal dendritic morphologies play a key role in shaping neuronal function and reflect optimization strategies and constraints imposed by protein trafficking," concludes Tchumatchenko.
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Biotechnology
| 2,020 |
November 21, 2020
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https://www.sciencedaily.com/releases/2020/11/201121104304.htm
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Folding of SARS-CoV2 genome reveals drug targets -- and preparation for 'SARS-CoV3'
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For the first time, an international research alliance has observed the RNA folding structures of the SARS-CoV2 genome with which the virus controls the infection process. Since these structures are very similar among various beta corona viruses, the scientists not only laid the foundation for the targeted development of novel drugs for treating COVID-19, but also for future occurrences of infection with new corona viruses that may develop in the future.
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The genetic code of the SARS-CoV2 virus is exactly 29,902 characters long, strung through a long RNA molecule. It contains the information for the production of 27 proteins. This is not much compared to the possible 40,000 kinds of protein that a human cell can produce. Viruses, however, use the metabolic processes of their host cells to multiply. Crucial to this strategy is that viruses can precisely control the synthesis of their own proteins.SARS-CoV2 uses the spatial folding of its RNA hereditary molecule as control element for the production of proteins: predominantly in areas that do not code for the viral proteins, RNA single strands adopt structures with RNA double strand sections and loops. However, until now the only models of these foldings have been based on computer analyses and indirect experimental evidence.Now, an international team of scientists led by chemists and biochemists at Goethe University and TU Darmstadt have experimentally tested the models for the first time. Researchers from the Israeli Weizmann Institute of Science, the Swedish Karolinska Institute and the Catholic University of Valencia were also involved.The researchers were able to characterise the structure of a total of 15 of these regulatory elements. To do so, they used nuclear magnetic resonance (NMR) spectroscopy in which the atoms of the RNA are exposed to a strong magnetic field, and thereby reveal something about their spatial arrangement. They compared the findings from this method with the findings from a chemical process (dimethyl sulphate footprint) which allows RNA single strand regions to be distinguished from RNA double strand regions.The coordinator of the consortium, Professor Harald Schwalbe from the Center for Biomolecular Magnetic Resonance at Goethe University Frankfurt, explains: "Our findings have laid a broad foundation for future understanding of how exactly SARS-CoV2 controls the infection process. Scientifically, this was a huge, very labour-intensive effort which we were only able to accomplish because of the extraordinary commitment of the teams here in Frankfurt and Darmstadt together with our partners in the COVID-19-NMR consortium. But the work goes on: together with our partners, we are currently investigating which viral proteins and which proteins of the human host cells interact with the folded regulatory regions of the RNA, and whether this may result in therapeutic approaches."Worldwide, over 40 working groups with 200 scientists are conducting research within the COVID-19-NMR consortium, including 45 doctoral and postdoctoral students in Frankfurt working in two shifts per day, seven days of the week since the end of March 2020.Schwalbe is convinced that the potential for discovery goes beyond new therapeutic options for infections with SARS-CoV2: "The control regions of viral RNA whose structure we examined are, for example, almost identical for SARS-CoV and also very similar for other beta-coronaviruses. For this reason, we hope that we can contribute to being better prepared for future 'SARS-CoV3' viruses."The Center for Biomolecular Magnetic Resonance was founded in 2002 as research infrastructure at Goethe University Frankfurt and has since then received substantial funding from the State of Hessen.
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Biotechnology
| 2,020 |
November 20, 2020
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https://www.sciencedaily.com/releases/2020/11/201120132626.htm
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Discovery illuminates how cell growth pathway responds to signals
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A basic science discovery by researchers at the Johns Hopkins Bloomberg School of Public Health reveals a fundamental way cells interpret signals from their environment and may eventually pave the way for potential new therapies.
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The finding involves a signaling pathway in cells, called the Hippo pathway, which normally constrains cell division and regulates the size of organs, and also plays a role in tissue growth and development as well as tumor suppression. The Hippo pathway is so fundamental that it is found in species ranging from humans to flies.The Bloomberg School researchers clarified the working of this signaling pathway by solving a long-standing mystery of how one of its core components, an enzyme called MST2, can be activated by multiple signaling inputs.The discovery is reported in a paper on November 20 in the "We knew that this pathway could be activated by different upstream signals, and here we've revealed the mechanism by which that happens," says study senior author Jennifer Kavran, PhD, assistant professor in the Bloomberg School's Department of Biochemistry and Molecular Biology.The Hippo pathway normally works as a brake on cell division that stops organs from growing larger once they have reached the appropriate size. Mutations or other abnormalities in the pathway that take the brakes off cell division have been found in many cancers, making elements of the Hippo pathway potential targets for future cancer treatments.Due to its fundamental role of tissue and organ growth, the pathway also is of great interest to researchers who are developing techniques to improve wound healing and stimulate the regeneration of damaged tissue.The heart of the Hippo pathway begins with the activation of two highly related enzymes, MST1 and MST2, which are almost identical and perform overlapping functions. A variety of biological events, including cell-to-cell contacts, certain nutrients, stress, and signaling through cell receptors, can cause MST1/2 to become activated -- a process in which the enzyme becomes tagged with sets of phosphorus and oxygen atoms called phosphoryl groups.Once activated by this "autophosphorylation," MST1/2 can send signals downstream to complete the signaling chain and inhibit cell division. Normally, proteins that undergo autophosphorylation are activated by a single molecular "event" -- such as binding a particular molecule or interacting with another copy of the same enzyme. How such a variety of inputs can each trigger MST1/2's activation has been a mystery."In cell biology, we're used to the idea that when an enzyme is transmitting a signal, a single molecular event turned that enzyme on," Kavran says.In the study, she and her colleagues used test tube and cell culture experiments with human MST2 to show that the myriad upstream activators of this enzyme trigger MST2 autophosphorylation the same way -- simply by increasing the local concentration of these enzymes -- thus reducing the distance between the enzymatic sites on individual enzymes and making it easier for them to phosphorylate one another.The researchers believe their discovery is likely to apply not only to MST2 but also its twin MST1 as well as the very similar versions of the enzyme produced in other species.Although this was principally a basic science study, the results should enhance the ability of researchers to manipulate Hippo pathway signaling, both for basic research as well as for potential therapeutic applications for tissue regeneration and anti-cancer therapies."The techniques we used to activate MST2 in cell cultures should be useful to other labs that are studying the Hippo pathway and need a way to turn it on in a controlled manner," Kavran says.She and her lab plan to investigate how other enzymes in the pathway are regulated.The research was supported by the National Institutes of Health (R01GM134000, T32CA009110, R35GM122569).
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Biotechnology
| 2,020 |
November 20, 2020
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https://www.sciencedaily.com/releases/2020/11/201120132620.htm
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Biophysics: Geometry supersedes simulations
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Ludwig-Maximilians-Universitaet (LMU) in Munich physicists have introduced a new method that allows biological pattern-forming systems to be systematically characterized with the aid of mathematical analysis. The trick lies in the use of geometry to characterize the dynamics.
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Many vital processes that take place in biological cells depend on the formation of self-organizing molecular patterns. For example, defined spatial distributions of specific proteins regulate cell division, cell migration and cell growth. These patterns result from the concerted interactions of many individual macromolecules. Like the collective motions of bird flocks, these processes do not need a central coordinator. Hitherto, mathematical modelling of protein pattern formation in cells has been carried out largely by means of elaborate computer-based simulations. Now, LMU physicists led by Professor Erwin Frey report the development of a new method which provides for the systematic mathematical analysis of pattern formation processes, and uncovers the their underlying physical principles. The new approach is described and validated in a paper that appears in the journal The study focuses on what are called 'mass-conserving' systems, in which the interactions affect the states of the particles involved, but do not alter the total number of particles present in the system. This condition is fulfilled in systems in which proteins can switch between different conformational states that allow them to bind to a cell membrane or to form different multicomponent complexes, for example. Owing to the complexity of the nonlinear dynamics in these systems, pattern formation has so far been studied with the aid of time-consuming numerical simulations. "Now we can understand the salient features of pattern formation independently of simulations using simple calculations and geometrical constructions," explains Fridtjof Brauns, lead author of the new paper. "The theory that we present in this report essentially provides a bridge between the mathematical models and the collective behavior of the system's components."The key insight that led to the theory was the recognition that alterations in the local number density of particles will also shift the positions of local chemical equilibria. These shifts in turn generate concentration gradients that drive the diffusive motions of the particles. The authors capture this dynamic interplay with the aid of geometrical structures that characterize the global dynamics in a multidimensional 'phase space'. The collective properties of systems can be directly derived from the topological relationships between these geometric constructs, because these objects have concrete physical meanings -- as representations of the trajectories of shifting chemical equilibria, for instance. "This is the reason why our geometrical description allows us to understand why the patterns we observe in cells arise. In other words, they reveal the physical mechanisms that determine the interplay between the molecular species involved," says Frey. "Furthermore, the fundamental elements of our theory can be generalized to deal with a wide range of systems, which in turn paves the way to a comprehensive theoretical framework for self-organizing systems."
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Biotechnology
| 2,020 |
November 20, 2020
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https://www.sciencedaily.com/releases/2020/11/201120132618.htm
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Ribosome assembly: The final trimming step
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Ribosomes synthesize all the proteins in cells. Studies mainly done on yeast have revealed much about how ribosomes are put together, but an Ludwig-Maximilians-Universitaet (LMU) in Munich team now reports that ribosome assembly in human cells requires factors that have no counterparts in simpler model organisms.
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In every cell, hundreds of thousands of intricate molecular machines called ribosomes fabricate new proteins, extending each growing chain at a rate of a few amino acids per second. Not surprisingly therefore, the construction of these vital protein factories is itself a highly complex operation, in which more than 200 assembly factors are transiently involved. Mature ribosomes are made up of approximately 80 proteins and four ribosomal RNAs. But how these constituents are assembled in the correct order to yield a functional ribosome is still not fully understood. Moreover, most of our knowledge of the process comes from studies carried out on model organisms like bacteria and yeast, and may not necessarily be applicable to the cells of higher organisms. Researchers led by Professor Roland Beckmann (Gene Center, LMU Munich) have now uncovered new details of the crucial steps in the maturation of ribosomes in human cells.Active ribosomes consist of two separately assembled particles, which differ in size and interact with each other only after the first steps in protein synthesis have taken place on the smaller of the two (in human cells, the '40S subunit'). Beckmann's team has used cryo-electron microscopy to determine the structures of several precursors of the 40S subunit isolated from human cells and follow the course of its maturation. "This study follows on from an earlier project, in which we obtained initial insights into the process," says Michael Ameismeier. He is a doctoral student in Beckmann's team and lead author of the new report, which is concerned with the final steps in the assembly of the small subunit.At this late stage in the process, one end of the ribosomal RNA associated with the small particle protrudes from the body of the immature subunit. The last step in the maturation of the 18S subunit consists in the removal of this now superfluous segment. To ensure that this reaction does not occur prematurely, the enzyme responsible -- NOB1 -- is maintained in an inactive state until it is required. The new study shows that the activation of NOB1 is preceded by a conformational change that results in the detachment of a binding partner from the enzyme. This in turn triggers a structural rearrangement in NOB1 itself, which enables the enzyme to snip off the protruding rRNA segment. "The activation of NOB1 is coordinated by another enzyme," Ameismeier explains. Together with a protein we have discovered -- which is not found in yeast -- the latter enzyme inserts like a wedge into the maturing 40S subunit, and this facilitates the decisive conformational change in NOB1."The authors have also shown that yet another protein not found in yeast plays an (as yet) enigmatic role in the maturation of the 40S subunit. "This demonstrates the importance of considering the human system separately from other experimental models," says Beckmann. Use of the evolutionarily simpler yeast system is sufficient for a basic understanding of the process. But certain pathological syndromes have been linked to errors in ribosomal biogenesis in humans, which provides an obvious rationale for the study of ribosomal assembly in human cell systems.
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Biotechnology
| 2,020 |
November 20, 2020
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https://www.sciencedaily.com/releases/2020/11/201120113854.htm
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A biochemical random number
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True random numbers are required in fields as diverse as slot machines and data encryption. These numbers need to be truly random, such that they cannot even be predicted by people with detailed knowledge of the method used to generate them.
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As a rule, they are generated using physical methods. For instance, thanks to the tiniest high-frequency electron movements, the electrical resistance of a wire is not constant but instead fluctuates slightly in an unpredictable way. That means measurements of this background noise can be used to generate true random numbers.Now, for the first time, a research team led by Robert Grass, Professor at the Institute of Chemical and Bioengineering, has described a non-physical method of generating such numbers: one that uses biochemical signals and actually works in practice. In the past, the ideas put forward by other scientists for generating random numbers by chemical means tended to be largely theoretical.For this new approach, the ETH Zurich researchers apply the synthesis of DNA molecules, an established chemical research method frequently employed over many years. It is traditionally used to produce a precisely defined DNA sequence. In this case, however, the research team built DNA molecules with 64 building block positions, in which one of the four DNA bases A, C, G and T was randomly located at each position. The scientists achieved this by using a mixture of the four building blocks, rather than just one, at every step of the synthesis.As a result, a relatively simple synthesis produced a combination of approximately three quadrillion individual molecules. The scientists subsequently used an effective method to determine the DNA sequence of five million of these molecules. This resulted in 12 megabytes of data, which the researchers stored as zeros and ones on a computer.However, an analysis showed that the distribution of the four building blocks A, C, G and T was not completely even. Either the intricacies of nature or the synthesis method deployed led to the bases G and T being integrated more frequently in the molecules than A and C. Nonetheless, the scientists were able to correct this bias with a simple algorithm, thereby generating perfect random numbers.The main aim of ETH Professor Grass and his team was to show that random occurrences in chemical reaction can be exploited to generate perfect random numbers. Translating the finding into a direct application was not a prime concern at first. "Compared with other methods, however, ours has the advantage of being able to generate huge quantities of randomness that can be stored in an extremely small space, a single test tube," Grass says. "We can read out the information and reinterpret it in digital form at a later date. This is impossible with the previous methods."
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Biotechnology
| 2,020 |
November 19, 2020
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https://www.sciencedaily.com/releases/2020/11/201119153938.htm
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Engineered immune cells elicit broad response to HIV in mice, offering hope for vaccine
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Unlike so many other deadly viruses, HIV still lacks a vaccine. The virus -- which continues to infect millions around the world -- has proven especially tricky to prevent with conventional antibodies, in part because it evolves so rapidly in the body. Any solution would require coaxing the body into producing a special type of antibody that can act broadly to defeat multiple strains of the virus at once.
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This week, scientists at Scripps Research moved closer to attaining that holy grail of HIV research with a new vaccine approach that would rely on genetically engineered immune cells from the patient's body.In experiments involving mice, the approach successfully induced broadly neutralizing antibodies -- also called bnabs -- that can prevent HIV infection, says principal investigator James Voss, PhD, of Scripps Research. The study appears in Voss and his team showed in 2019 that it was possible to reprogram the antibody genes of the immune system's B cells using CRISPR so the cells would produce the same broadly neutralizing HIV antibodies that have been found in rare HIV patients.The new study shows that such engineered B cells, after being reintroduced to the body, can multiply in response to a vaccination -- and mature into memory cells and plasma cells that produce high levels of protective antibodies for long periods of time in the body. The team also demonstrated that the engineered genes can be improved to make antibodies that are even more effective against the virus, using a process that normally occurs in B cells that are responding to immunization."This is the first time it has been shown that modified B cells can create a durable engineered antibody response in a relevant animal model," Voss explains.He hopes that his vaccine approach may someday prevent new HIV infections and possibly offer a functional cure to those who already have HIV/AIDS. The virus is still prevalent throughout the world, with an estimated 38 million people with the disease in 2019.Voss notes that in humans, the starting cells to create the vaccine could be obtained easily from a simple blood draw, then engineered in the lab before being reintroduced to the patient. He and his team -- including first author Deli Huang, PhD, Jenny Tran, PhD, Alex Olson, PhD, and graduate student Mary Tenuta -- are now exploring ways to improve the technology so that it would be accessible to the greatest number of people. Because the approach relies on delivering genes to a patient's own immune cells, this could be a significant challenge."People think of cell therapies as being very expensive," Voss says. "We're doing a lot of work towards trying to make the technology affordable as a preventative HIV vaccine or functional cure that would replace daily antiviral therapy."
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Biotechnology
| 2,020 |
November 19, 2020
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https://www.sciencedaily.com/releases/2020/11/201119131028.htm
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Vibrations of coronavirus proteins may play a role in infection
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When someone struggles to open a lock with a key that doesn't quite seem to work, sometimes jiggling the key a bit will help. Now, new research from MIT suggests that coronaviruses, including the one that causes Covid-19, may use a similar method to trick cells into letting the viruses inside. The findings could be useful for determining how dangerous different strains or mutations of coronaviruses may be, and might point to a new approach for developing treatments.
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Studies of how spike proteins, which give coronaviruses their distinct crown-like appearance, interact with human cells typically involve biochemical mechanisms, but for this study the researchers took a different approach. Using atomistic simulations, they looked at the mechanical aspects of how the spike proteins move, change shape, and vibrate. The results indicate that these vibrational motions could account for a strategy that coronaviruses use, which can trick a locking mechanism on the cell's surface into letting the virus through the cell wall so it can hijack the cell's reproductive mechanisms.The team found a strong direct relationship between the rate and intensity of the spikes' vibrations and how readily the virus could penetrate the cell. They also found an opposite relationship with the fatality rate of a given coronavirus. Because this method is based on understanding the detailed molecular structure of these proteins, the researchers say it could be used to screen emerging coronaviruses or new mutations of Covid-19, to quickly assess their potential risk.The findings, by MIT professor of civil and environmental engineering Markus Buehler and graduate student Yiwen Hu, are being published today in the print edition of the journal All the images we see of the SARS-CoV-2 virus are a bit misleading, according to Buehler."The virus doesn't look like that," he says, because in reality all matter down at the nanometer scale of atoms, molecules, and viruses "is continuously moving and vibrating. They don't really look like those images in a chemistry book or a website."Buehler's lab specializes in atom-by-atom simulation of biological molecules and their behavior. As soon as Covid-19 appeared and information about the virus' protein composition became available, Buehler and Hu, a doctoral student in mechanical engineering, swung into action to see if the mechanical properties of the proteins played a role in their interaction with the human body.The tiny nanoscale vibrations and shape changes of these protein molecules are extremely difficult to observe experimentally, so atomistic simulations are useful in understanding what is taking place. The researchers applied this technique to look at a crucial step in infection, when a virus particle with its protein spikes attaches to a human cell receptor called the ACE2 receptor. Once these spikes bind with the receptor, that unlocks a channel that allows the virus to penetrate the cell.That binding mechanism between the proteins and the receptors works something like a lock and key, and that's why the vibrations matter, according to Buehler. "If it's static, it just either fits or it doesn't fit," he says. But the protein spikes are not static; "they're vibrating and continuously changing their shape slightly, and that's important. Keys are static, they don't change shape, but what if you had a key that's continuously changing its shape -- it's vibrating, it's moving, it's morphing slightly? They're going to fit differently depending on how they look at the moment when we put the key in the lock."The more the "key" can change, the researchers reason, the likelier it is to find a fit.Buehler and Hu modeled the vibrational characteristics of these protein molecules and their interactions, using analytical tools such as "normal mode analysis." This method is used to study the way vibrations develop and propagate, by modeling the atoms as point masses connected to each other by springs that represent the various forces acting between them.They found that differences in vibrational characteristics correlate strongly with the different rates of infectivity and lethality of different kinds of coronaviruses, taken from a global database of confirmed case numbers and case fatality rates. The viruses studied included SARS-CoV, MERS-CoV, SATS-CoV-2, and of one known mutation of the SARS-CoV-2 virus that is becoming increasingly prevalent around the world. This makes this method a promising tool for predicting the potential risks from new coronaviruses that emerge, as they likely will, Buehler says.In all the cases they have studied, Hu says, a crucial part of the process is fluctuations in an upward swing of one branch of the protein molecule, which helps make it accessible to bind to the receptor. "That movement is of significant functional importance," she says. Another key indicator has to do with the ratio between two different vibrational motions in the molecule. "We find that these two factors show a direct relationship to the epidemiological data, the virus infectivity and also the virus lethality," she says.The correlations they found mean that when new viruses or new mutations of existing ones appear, "you could screen them from a purely mechanical side," Hu says. "You can just look at the fluctuations of these spike proteins and find out how they may act on the epidemiological side, like how infectious and how serious would the disease be."Potentially, these findings could also provide a new avenue for research on possible treatments for Covid-19 and other coronavirus diseases, Buehler says, speculating that it might be possible to find a molecule that would bind to the spike proteins in a way that would stiffen them and limit their vibrations. Another approach might be to induce opposite vibrations to cancel out the natural ones in the spikes, similarly to the way noise-canceling headphones suppress unwanted sounds.As biologists learn more about the various kinds of mutations taking place in coronaviruses, and identify which areas of the genomes are most subject to change, this methodology could also be used predictively, Buehler says. The most likely kinds of mutations to emerge could all be simulated, and those that have the most dangerous potential could be flagged so that the world could be alerted to watch for any signs of the actual emergence of those particular strains. Buehler adds, "The G614 mutation, for instance, that is currently dominating the Covid-19 spread around the world, is predicted to be slightly more infectious, according to our findings, and slightly less lethal."The research was supported by the MIT-IBM Watson AI Lab, the Office of Naval Research, and the National Institutes of Health.
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Biotechnology
| 2,020 |
November 19, 2020
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https://www.sciencedaily.com/releases/2020/11/201119083925.htm
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Solving a mystery: How the TB bacterium develops rapid resistance to antibiotics
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For a slow-growing microbe that multiplies infrequently,
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Now, TB researchers at San Diego State University have uncovered a crucial clue to the mystery: the answer may lie in the epigenetic domain rather than the genetic domain where most scientists have concentrated their efforts.Their discovery could help advance new diagnostics, therapeutics and vaccine targets.Epigenetics is the study of inheritable changes in gene expression that do not involve a corresponding change to the underlying DNA sequence -- meaning changes to the phenotype but no change in the genotype. This affects only the physical structure of the DNA, through a process called DNA methylation where a chemical 'cap' is added to the DNA molecule, preventing or facilitating the expression of certain genes.The SDSU researchers describe the rapid response phenomenon they discovered as 'intercellular mosaic methylation,' a process by which "We believe this also explains why diagnostic testing in some patients does not predict treatment failure, and why some patients come back months later with the disease reemerging in a far more resistant state," said Faramarz Valafar, a TB expert with SDSU's School of Public Health who studies the genetics and epigenetics of pulmonary diseases. "This is also why CT scans of the lungs of many "cured" patients show lesions with possible bacterial activity."Worldwide, TB is among the top 10 causes of death. It killed 1.4 million people in 2019, and about 10 million people fall ill with it each year, according to the World Health Organization.Valafar's team collected hundreds of samples of drug resistant varieties of the bacteria from patients in India, China, Philippines and South Africa, as well as Europe, through collaborations with TB researchers worldwide.Their study was published in "We've known for decades that bacterial epigenetics can influence the expression of certain genes, which can lead to a variety of phenotypes even when they have identical genotypes," Conkle-Gutierrez said. "We discovered evidence of that phenomenon in the TB bacterium."Antibiotic resistance is typically caused by genomic mutations, but this bacterium is one of several that leverages alternative mechanisms in the epigenetic domain to enable rapid adaptation."We found that some of them had mutations that led to variable DNA methylation and those strains had much more diversity in their epigenome, and thus more potential to be drug resistant," Modlin said.The researchers found there were no set patterns and methylation was fairly random. They used advanced comparative genomic and epigenetic techniques to identify variations across cells within a colony from a single isolate, from a single patient -- including tiny variations that nevertheless impacted gene expression. They were able to do this because, rather than assuming the reference genome has a common structure, they reconstructed each genome from scratch and analyzed its epigenetic signatures.They will now focus on testing and confirming the key genes they identified with methylation signatures. There is more work to be done before their discovery can eventually be used for diagnostics."There is a lot of resistance in TB that escapes current molecular diagnostics and we don't really know why. That's problematic," Valafar said. "This study offers a new domain, new tools, and a new approach to looking for alternative mechanisms. We move away from the classical view of molecular diagnostics and use a novel, comprehensive approach to analyzing bacteria."Current standard of care treatments use two types of antibiotics -- bacteriostatics that prevent bacteria from multiplying but don't kill them, and bactericides that do kill them."We found a new mode of variation and if we can inhibit that diversification mechanism, we can inhibit short-term epigenetic resistance and kill the bacteria before mutations in the genome develop and cause long-term, genetic resistance," Modlin said.This may be how some bacterial populations survive treatment and make the patient ill again with far greater antibiotic resistance or hypervirulence.
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Biotechnology
| 2,020 |
November 18, 2020
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https://www.sciencedaily.com/releases/2020/11/201118141850.htm
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Algae breathe life into 3D engineered tissues
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3D bioprinted algae can be harnessed as a sustainable source of oxygen for human cells in engineered vascularized tissues, researchers report November 18 in the journal
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"The study is the first true example of symbiotic tissue engineering combining plant cells and human cells in a physiologically meaningful way, using 3D bioprinting," says senior study author Y. Shrike Zhang a bioengineer at Harvard Medical School and Brigham and Women's Hospital. "Our study provides a unique example of how we can harness the symbiotic strategy, very often seen in nature, to promote our ability to engineer functional human tissues."There is an increasing demand for artificial tissues to replace those that have been damaged in order to restore organ functions, and over the past decade, 3D bioprinting techniques have been used to fabricate tissue scaffolds for biomedical and tissue engineering applications. This approach typically involves depositing a bioink onto a surface to produce 3D structures with desired architectures and shapes to recapitulate organs and tissues, including the vasculature, which plays a critical role in transporting oxygen and nutrients throughout the body. A bioink is essentially a hydrogel containing living cells, biomaterials, and other growth supplements. It mimics the extracellular matrix of the desired tissue and supports the growth of the embedded cells.Despite advances in the fabrication of 3D tissues, the main limitation has been maintaining sufficient oxygen levels throughout the engineered tissue to promote cell survival, growth, and functioning. Researchers have tried to address this problem by incorporating oxygen-releasing biomaterials, but these typically do not work long enough and are sometimes toxic to cells because they produce molecules such as hydrogen peroxide or other reactive oxygen species. "A method to enable sustained release of oxygen from within the engineered tissues is in urgent demand," Zhang says.To meet this demand, Zhang and his colleagues developed an algae-based 3D bioprinting method to incorporate vascular patterns within engineered tissues and provide a sustainable source of oxygen for human cells in the tissues. Specifically, they used photosynthetic single-celled green algae called Chlamydomonas reinhardtii. This symbiotic strategy also benefits the algae, whose growth is partially supported by carbon dioxide released by the surrounding human cells.The first step involved 3D bioprinting the algae. The researchers encapsulated C. reinhardtii in a bioink composed primarily of cellulose -- the main structural component of plants, algae, and fungi. The bioink was loaded into a syringe fitted with a needle, and extrusion bioprinting was performed using a bioprinter.Next, the researchers embedded both the bioprinted algae and human liver-derived cells in a 3D hydrogel matrix. The bioprinted C. reinhardtii released oxygen in a photosynthetic manner and enhanced the viability and functions of the human cells, which grew to a high density and produced liver-specific proteins. "High cell densities in engineered vascularized human tissues were difficult to obtain before," Zhang says.Finally, the researchers used the enzyme cellulase to degrade the cellulose-based bioink, then filled the hollow microchannels left behind with human vascular cells to create vascular networks in the liver-like tissue. "Development of such a fugitive bioink that allows initial oxygenation and subsequent vessel formation within a single tissue construct has not been reported before," Zhang says. "This a critical step toward successful engineering of viable and functional tissues."In the end, the 3D vascularized, oxygenized engineered tissues hold potential for future implantation to achieve tissue regeneration in humans. These tissues could also be used for drug screening and development, studying disease mechanisms, and possibly personalized medicine if patient-specific cells are used.Another potential application of the 3D bioprinting technology is food engineering. Microalgae represent a rich source of protein, carbohydrates, polyunsaturated fatty acids, carotenoids, vitamins, and essential minerals. These bioactive compounds could be incorporated into innovative, cultured food products to enhance their nutritional value and promote health.But in the meantime, more effort is needed to optimize the method. For example, the culture media could be improved to facilitate the growth of both C. reinhardtii and human cells, and light conditions could be tuned to optimize oxygen supply from the algae. Moreover, detailed studies on biosafety, toxicity, and immuno-compatibility of the algae will be important for clinical translation in the future. "This technology cannot be immediately put to human uses," Zhang says. "It is still proof-of-concept and will require significant follow-up studies to translate."
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Biotechnology
| 2,020 |
November 18, 2020
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https://www.sciencedaily.com/releases/2020/11/201118141820.htm
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Lurking in genomic shadows: How giant viruses fuel the evolution of algae
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Viruses are tiny invaders that cause a wide range of diseases, from rabies to tomato spotted wilt virus and, most recently, COVID-19 in humans. But viruses can do more than elicit sickness -- and not all viruses are tiny.
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Large viruses, especially those in the nucleo-cytoplasmic large DNA virus family, can integrate their genome into that of their host -- dramatically changing the genetic makeup of that organism. This family of DNA viruses, otherwise known as "giant" viruses, has been known within scientific circles for quite some time, but the extent to which they affect eukaryotic organisms has been shrouded in mystery -- until now."Viruses play a central role in the evolution of life on Earth. One way that they shape the evolution of cellular life is through a process called endogenization, where they introduce new genomic material into their hosts. When a giant virus endogenizes into the genome of a host algae, it creates an enormous amount of raw material for evolution to work with," said Frank Aylward, an assistant professor in the Department of Biological Sciences in the Virginia Tech College of Science and an affiliate of the Global Change Center housed in the Fralin Life Sciences Institute.Mohammad 'Monir' Moniruzzaman, a postdoctoral researcher in Aylward's lab, studies endogenous viral elements, which are fragments or whole sequences of raw viral DNA that have been inserted into the infected host's genome.Together, Aylward and Moniruzzaman have recently discovered that endogenous viral elements that originate from giant viruses are much more common in chlorophyte green algae than previously thought.Their findings were recently published in Chlorophytes, a group of green algae, are an important group of photosynthetic organisms that are at the base of the food chain on many ecosystems and produce massive amounts of food and oxygen across the planet. Chlorophytes thrive in our lakes and ponds -- and their dynamics with giant viruses as well as their unique evolutionary history, were central to Aylward and Moniruzzaman's research.Chlorophyte algae are close relatives of land plants, and studying their interactions with giant viruses may shed some light on the roles that the viruses played during the early evolution of plants."We now know that endogenous viral elements are common across chlorophytes, which makes you think that plants might also interact with these giant viruses. There is some data that suggests that some early plants, like moss and ferns, did experience these endogenization events over the evolutionary timeline. But we are not exactly sure about the extent of this phenomenon in other early plants," said Moniruzzaman, the first author on this published paper.To learn more about the prevalence of endogenous viral elements in algae, Moniruzzaman and Aylward performed a bioinformatic analysis on the sequenced genomes of different algae groups.They discovered that 24 of the 65 genomes that were analyzed had some kinds of viral signatures in their genomes, which originated from repeated endogenization of distinct viruses. In one algal organism, Tetrabaena socialis, researchers found that around 10 percent of its genes originated from a virus in the nucleo-cytoplasmic large DNA virus family.Although the endogenization of viruses have been well studied, studies have mostly been limited to small RNA viruses, such as the human immunodeficiency virus (HIV), the retrovirus that is responsible for causing acquired immunodeficiency syndrome (AIDS).Aylward and Moniruzzaman's study is one of the first to put a spotlight on large eukaryotic DNA viruses, which marks a major shift in the field.Electron micrograph image of a AaV, a giant virus that infects and kills a unicellular alga that causes harmful algae blooms. Giant viruses that belong to the same group as AaV can frequently insert their genomes into the genomes of their hosts. Image courtesy of Chuan Xiao and Yuejiao Xian, University of Texas at El Paso; Steven W. Wilhelm and Eric R. Gann, University of Tennessee, Knoxville."These large endogenous viral elements are a lot more common than previously thought. Now that we have a systematic analysis, other researchers are really going to start to pay attention. This study shows that endogenous viral elements are pretty common, and so it might possibly be a common mechanism of genome evolution. I think these results will broaden our view on the role of giant viruses as mere agents of host mortality to significant players in host genome evolution," said Moniruzzaman.Now that Moniruzzaman and Aylward have confirmed that endogenization is happening in larger viruses, they wonder what conditions are causing these viruses to inject EVEs into green algae in the first place -- and why the hosts show no signs of rejecting them."We don't know what the mechanism is or how the DNA is being maintained, but it is possible that the endogenization is a random, almost accidental process. And once the viral DNA is endogenized, it can alter the evolutionary dynamics of the host, and that it could further influence the evolution of that lineage," said Aylward.The idea that there is a potentially beneficial relationship at play between the host and its virus is of particular interest to Moniruzzaman."There might be a reason as to why the host is keeping these viral genomes within them. It's not like these viral genes are causing the hosts to become unsuccessful or unable to survive in the environment. So that's the thing: Are the endogenous viral elements beneficial to the host? And how are they getting in there and staying in there?" asked Moniruzzaman.
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Biotechnology
| 2,020 |
November 18, 2020
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https://www.sciencedaily.com/releases/2020/11/201118141819.htm
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Gut microbiome manipulation could result from virus discovery
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Scientists have discovered how a common virus in the human gut infects and takes over bacterial cells -- a finding that could be used to control the composition of the gut microbiome, which is important for human health.
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The Rutgers co-authored research, which could aid efforts to engineer beneficial bacteria that produce medicines and fuels and clean up pollutants, is published in the journal "CrAssphages are the most abundant viruses infecting bacteria in the human gut. As such, they likely control our intestinal community of microbes (the microbiome)," said co-author Konstantin Severinov, a principal investigator at the Waksman Institute of Microbiology and a professor of molecular biology and biochemistry in the School of Arts and Sciences at Rutgers University-New Brunswick. "Understanding how these tiny viruses infect bacteria may allow scientists to control and manipulate the makeup of the microbiome, either by increasing the proportion of beneficial bacteria in our intestines or decreasing the number of harmful bacteria, thus promoting health and fighting disease."Scientists found that crAssphages use their own enzyme (an RNA polymerase) to make RNA copies of their genes. RNA has the genetic information to make proteins. All cells, ranging from bacterial to human, use such enzymes to make RNA copies of their genes. And these enzymes are very similar in all living matter, implying that they're ancient and related by common ancestry, Severinov said.When the team revealed the atomic structure of a crAssphage enzyme, they were surprised to learn that it is distinct from other RNA polymerases but closely resembles an enzyme in humans and other higher organisms that is involved in RNA interference. Such interference silences the function of some genes and may lead to certain diseases."This is a startling result. It suggests that enzymes of RNA interference, a process that was thought to occur only in cells of higher organisms, were 'borrowed' from an ancestral bacterial virus early in evolution," Severinov said. "The result provides a glimpse of how cells of higher organisms evolved by mixing and matching components of simpler cells and even their viruses.""In addition to deep evolutionary insights, phage (viral) enzymes such as crAssphage RNA polymerase may be used in synthetic biology to generate genetic circuits that do not exist in nature," he said.Synthetic biology involves redesigning organisms so they can, for example, produce a medicine, nutrient or fuel, sense something in the environment or clean up pollutants, according to the National Human Genome Research Institute."We are now trying to match the thousands of different crAssphage viruses in our gut with the bacterial hosts they infect," Severinov said. "By using just the 'right' bacterial virus, we will be able to get rid of bacteria it infects, which will allow us to alter the composition of the gut microbiome in a targeted way."Leonid Minakhin at the Waksman Institute of Microbiology contributed to the study along with scientists at many other institutions.
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Biotechnology
| 2,020 |
November 18, 2020
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https://www.sciencedaily.com/releases/2020/11/201118141801.htm
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Dual brake on transport protein prevents cells from exploding
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A high concentration of salt or sugar in the environment will dehydrate microorganisms and stop them from growing. To counter this, bacteria can increase their internal solute concentration. Scientists from the University of Groningen elucidated the structure of a transport protein OpuA, that imports glycine betaine to counter osmotic stress. The protein belongs to the well-known family of ABC transporters, but it has a unique structure and working mechanism. The results were published in
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Food preservatives are designed to make life difficult for microorganisms. Salt and sugar are well-known preservatives; they increase the electrolyte concentration to above that inside bacteria. The result is that water flows out of these bacteria until concentrations are approximately equal, leaving behind shrivelled cells that can no longer grow.'However, some bacteria have evolved defences against such preservatives,' says Bert Poolman, Professor of Biochemistry at the University of Groningen. Around 20 years ago, a food producer asked him to find ways to defeat those defences. It led to the discovery of OpuA, a transport protein that is triggered by dehydration and responds by importing a substance called glycine betaine. 'This increases the osmolyte concentration inside the cells without compromising the structure of proteins. The result is that the cells absorb more water and start to grow again,' explains Poolman.OpuA belongs to a well-known class of proteins called ABC (ATP-binding cassette) transporters. This protein family is one of the largest known in biology. Humans have around 50 of these transporters, some plants have hundreds of them and bacteria have a number somewhere in between. OpuA is special because it can import glycine betaine in huge amounts, leading to a very high internal osmolyte concentration. That is why Poolman was intrigued to find out how it worked. 'I have worked on this problem, on and off, ever since.'The problem was elucidating the structure of the protein. Until a few years ago, the standard method was to grow crystals from proteins and investigate those using X-ray diffraction. It is very difficult to grow crystals from proteins that are embedded in the cell membrane and for OpuA, it turned out to be impossible. Based on the amino acid sequence and the structure of other ABC transporters, the scientist compiled a model of the structure, but this could not explain the way that OpuA functioned.The breakthrough came with the introduction of cryo-electron microscopy, together with the work of PhD student Hendrik Sikkema and the collaboration with the research group of University of Groningen assistant professor of Cryo-EM Cristina Paulino. A large number of single proteins were scanned in an electron microscope at a very low temperature, after which all the images were combined to provide a direct view of the structure. The results showed not one but five different structures. 'The protein is a dynamic structure, as it changes conformation to suit the function, but the different parts also vibrate on their own,' explains Poolman. 'This means that one protein exists in many variant structures. And you cannot grow crystals amidst such diversity.'The first conclusion from the cryo-EM studies was that most of what they thought they knew about the structure of OpuA was incorrect. 'For example, parts that we believed to be on the inside of the cell membrane sat on the outside.' The real structure was beautiful, according to Poolman. The second conclusion was that OpuA is in part regulated by cyclic di-AMP, a second messenger molecule that was only recently discovered. 'The protein primarily responds to ionic strength, which varies as a function of osmotic stress, but it uses cyclic di-AMP as a second brake to completely stop importing glycine betaine and prevent the cell from exploding under non-stress conditions.'The ionic strength sensor of the OpuA protein carries a positive charge while the membrane has a negative charge. When water is drawn from the cells, the concentration of salts, such as potassium chloride, increases. 'This disrupts the interaction of the ionic strength sensor with the membrane, which activates the pumping mechanism.' Once the glycine betaine concentration is high enough to make the cell swell to its normal proportions, the protein-membrane interaction is normalized. 'However, the pump does not shut down completely, so it continues to import some glycine betaine. This will increase the pressure inside the cell and eventually cause it to pop.' That is why cyclic di-AMP is used to fully shut down the pump.The paper describes the different structures and provides functional data on the transport protein. This combination gives a good insight into the workings of OpuA: a satisfying result for Poolman. 'It is the accumulation of twenty years of research, which has produced seven or eight PhD theses.' The results show how the resistance of bacteria to preservatives, such as salt or sugar, could be overcome. 'Furthermore, we are part of a consortium that is trying to construct a synthetic cell. OpuA is an important part of the design; it is meant to regulate the cell's internal pressure.'
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Biotechnology
| 2,020 |
November 18, 2020
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https://www.sciencedaily.com/releases/2020/11/201118141753.htm
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Brain protein could be starting point for new treatments for pancreatic cancer
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Researchers have discovered that a protein thought to only be involved in the development of neurons in the brain also plays a major role in the development and growth of pancreatic cancer. Their findings demonstrate for the first time how the protein, called Netrin-G1, helps pancreatic cancer cells survive by protecting them from the immune system and supplying them with nutrients.
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Pancreatic cancer is difficult to treat because tumours are often fibrotic, which means they develop extra connective tissue throughout the pancreas. This connective tissue aids the growth of pancreatic cancer and provides a physical and biochemical protective barrier against drugs and the immune system. This protective barrier is made up of a type of cell called a cancer-associated fibroblast (CAF), which interact with cancer cells to help them grow and survive.Dr Edna Cukierman, Associate Professor at Fox Chase Cancer Center in Philadelphia and Co-Director of the Marvin and Concetta Greenberg Pancreatic Cancer Institute, who led the study, said: "Using a system we developed to study CAFs in a fibrous-like environment that mimics how they would behave inside the pancreas, we identified Netrin-G1 as being highly expressed in CAFs, and found that it supports the survival of pancreatic cancer cells. We uncovered that Netrin-G1 allows CAFs to provide cancer cells with nutrition as well as secreting factors that inhibit anti-tumour immune cell function."The study, published in Dr Cukierman, said: "We saw that many patients express Netrin-G1 in CAFs and that these patients tend to survive for a shorter time so in the future detection of Netrin-G1 could be used to help diagnose patients. We believe that limiting Netrin-G1 function provides the starting point for the design of new treatments in a type of cancer that is in dire need for effective therapies."We next plan to continue investigating the biology behind Netrin-G1 expression in CAFs, figure out a practical way to detect it in patients, and partner with industry to design Netrin-G1 blocking drugs. This way we hope that targeting Netrin-G1 could serve, one day, to treat pancreatic cancer patients."In the UK, over 10,000 people are diagnosed with pancreatic cancer each year and over 9,000 people lose their life to the disease. Pancreatic cancer has one of the worst survival rates of any cancer with only around 1 in 20 people surviving for 10 years or more after their diagnosis. Only 1 in 4 people diagnosed with pancreatic cancer in the UK will survive beyond one year.Dr Helen Rippon, Chief Executive of Worldwide Cancer Research, said: "This is a fascinating new finding in cancer research which shows for the first time how a molecule thought to be involved in the brain is also able to help tumours grow in organs elsewhere in the body. We're delighted to see such great progress from Dr Cukierman's project which offers a starting point for the future development of treatments against a particularly deadly type of cancer. These positive findings come at a dark time for all of us and are a stark reminder of how dedicated our researchers are -- working tirelessly towards new cancer cures even amid a global pandemic. I'm sure this news will be welcomed by all of us who have had to experience the loss of a loved one to cancer."
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Biotechnology
| 2,020 |
November 18, 2020
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https://www.sciencedaily.com/releases/2020/11/201118141746.htm
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Coinfection: More than the sum of its parts
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Organ and stem cell transplants are proven and frequently used methods in everyday modern clinical practice. However, even when performed regularly in specialized centers, some patients still experience a number of serious complications afterwards. Among other things, infections with fungi and viruses can jeopardize therapeutic success. For example, coinfection with cytomegalovirus, which belongs to the family of Herpes viruses, and the fungus Aspergillus fumigatus can be critical. This combination of pathogens poses a serious medical threat in organ and stem cell transplantation.
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A team of scientists from several German research institutions and clinics has now developed a new method to examine these two pathogens, their interaction with each other and with the human cells infected by them. The central result: coinfection with the two pathogens is more "than the sum of its parts." Viruses and fungi interact synergistically in the human organism, where they trigger certain genes that only become active when infected with both pathogens simultaneously.The study involved scientists from the Julius Maximilian University of Würzburg (JMU), the Würzburg University Hospital (UKW), the Leibniz Institute for Natural Product Research and Infection Biology in Jena and the Helmholtz Institute for RNA-based Infection Research (HIRI) in Würzburg, a site of the Braunschweig Helmholtz Centre for Infection Research (HZI). The results have now been published in the journal "For our study, we have developed a method called Triple RNA-seq," explains Alexander Westermann. He is junior professor at the Chair of Molecular Infection Biology I at JMU, as well as group leader at the HIRI. Together with Jürgen Löffler from UKW and Sascha Schäuble he is one of the senior authors of the study. The scientists have advanced an established method that has been an integral part of infection research for years: dual RNA-seq.The term "RNA-seq" is short for RNA-sequencing: This technique enables the simultaneous and precise determination of the activities of thousands of genes at the RNA level in a high-throughput process, thus enabling the identification and better understanding of the changes occurring in the course of diseases. The development of dual RNA sequencing has made it possible to document not only the gene activity of a pathogen, but also the reaction of the host cell affected by it. This has enabled scientists to trace complex causal chains over the course of an infection.Now, Triple RNA sequencing dissects the gene expression of three players and their interplay in infection processes. "Up to now, science has in many cases not known why an infection with a certain pathogen can make the affected person more susceptible to an infection with a second pathogen," explains Jürgen Löffler, molecular biologist at the Medical Clinic II of the UKW. In such cases, dual RNA-seq was insufficient to provide the desired answers.In their study, the researchers used the triple RNA-seq method they developed to investigate what happens when certain cells of the immune system (known as monocyte-derived dendritic cells) are infected with both Aspergillus fumigatus and the human cytomegalovirus.They were able to prove that the two pathogens influence each other, whilst also simultaneously affecting the immune cell in a different way than one pathogen alone otherwise would. For example, the cytomegalovirus weakened the fungal-mediated activation of pro-inflammatory signals, while Aspergillus affects viral clearance -- the time it takes for the virus to become undetectable in tests.At the same time, the team has identified specific genes in immune cells whose expression profiles differ significantly during an infection with both pathogens, compared to a single infection. These genes could thus serve as biomarkers for the timely identification of a co-infection after transplantation.The scientists now hope that the triple RNA-seq technology will also help to better understand other cases of common infections, such as viruses and bacteria, and to prevent their potentially serious consequences. "Promising models for understanding how an infection makes the host more susceptible to another pathogen include certain strains of Salmonella and the human immunodeficiency virus (HIV), streptococci and influenza virus, or Chlamydia and human herpes virus," says Westermann. As a next step, Westermann plans to use the triple RNA-seq technique to investigate infections in which two different types of bacteria jointly influence the course of the disease.
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Biotechnology
| 2,020 |
November 18, 2020
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https://www.sciencedaily.com/releases/2020/11/201118080804.htm
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Are high-protein total diet replacements the key to maintaining healthy weight?
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According to the World Health Organization, obesity has nearly tripled worldwide since 1975. In 2016, for example, more than 1.9 billion adults were categorized as overweight. Of these, more than 650 million had obesity. Because obesity is associated with a higher incidence of diabetes, cardiovascular disease and some cancers, the rise in its incidence has led to a global public health emergency.
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Total diet replacements, nutritionally complete formula foods designed to replace the whole diet for a set period of time, have become increasingly popular strategies to combat obesity. Another popular weight management strategy are high-protein diets, which have been shown to promote weight loss and weight maintenance by increasing our sense of fullness, energy expenditure, and ability to maintain or increase fat-free mass. Taken together, the combination of a total diet replacement with a high-protein diet may be a promising strategy for weight management. In fact, several high-protein total diet replacement products are widely available to consumers. The question is do they work?That's the core question addressed by the authors of "A High-Protein Total Diet Replacement Increases Energy Expenditure and Leads to Negative Fat Balance in Healthy, Normal-Weight Adults," published in In order to conduct their experiment, the authors recruited a group of healthy, normal-weight adults between the ages of 18 and 35 via advertisements placed on notice boards at the University of Alberta, Canada. Subjects were then randomly assigned into one of two groups: one group was fed the high-protein total diet replacement, which consisted of 35% carbohydrate, 40% protein, and 25% fat. The second group, the control group, was fed a diet with the same number of calories, but consisting of 55% carbohydrate, 15% protein, and 30% fat, a typical North American dietary pattern. Participants received the prescribed diets for a 32-hour period while inside a metabolic chamber.Compared to the standard North American dietary pattern, the findings of this inpatient metabolic balance study revealed that the high-protein total diet replacement led to "higher energy expenditure, increased fat oxidation, and negative fat balance." In particular, the results of the study provide further evidence that a calorie is not just a calorie. That is, a diet with a higher proportion of protein might lead to an increase in energy expenditure and fat oxidation compared to a diet consisting of the same number of calories, but with a lower proportion of protein as well as a higher proportion of carbohydrate or fat.Dr. Carla Prado, Professor, University of Alberta and the study's principal investigator, commented, "although these results are restricted to a specific population of healthy, normal-weight adults, they can help nutrition scientists and healthcare providers better understand the real physiological effects of a high-protein total diet replacement in humans. In our opinion, it is imperative to first understand the physiological impact of a high-protein total diet replacement in a healthy population group so that the effects are better translated in individuals with obesity and its related comorbidities."In summary, the results of this study suggest that high-protein total diet replacements may be a promising nutritional strategy to combat rising rates of obesity. Lead author Camila Oliveira added, "future studies are needed to better understand the long-term effects of this dietary intervention on the physiology of both healthy and diseased population groups."
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Biotechnology
| 2,020 |
November 18, 2020
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https://www.sciencedaily.com/releases/2020/11/201118090801.htm
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Certain CBD oils no better than pure CBD at inhibiting certain cancer cell lines
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Cannabidiol (CBD) oils are equally or less effective at inhibiting the growth of certain cancer cells compared to pure CBD, according to Penn State College of Medicine researchers. The results of their recent study indicate that future research into the clinical applications of cannabinoids should include an analysis of whether the pure cannabinoid compound or intact plant material is more effective at achieving the therapeutic effect.
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The researchers evaluated whether CBD oils were better than pure CBD at inhibiting the growth of different cancer cell lines. They studied brain, skin and colorectal cancers -- using two cell lines for each cancer type -- and found that pure CBD was able to reduce cell viability in three of the six cell lines tested and that the effect was cell line specific and not specific to select cancers. None of the CBD oils tested were able to reduce viability to a greater extent than pure CBD.Prior research found that CBD or tetrahydrocannabinol (THC) can reduce cancer cell viability in some cancer cell models. Proponents of medical marijuana argue that there is an additive effect between the various compounds in the plant material that increases its therapeutic efficacy compared to individual, pure cannabinoid compounds. Kent Vrana, professor and chair of the Department of Pharmacology, said the study did not support this concept, known as the "entourage" effect."Based on our results, we recommend that specific investigations on the entourage effect be carried out when determining the therapeutic uses of medical marijuana and other cannabinoid products," Vrana said.Wesley Raup-Konsavage, co-author of the study published in the journal After evaluating the viability of the treated cell lines, researchers determined that the CBD had an effect on one of each of the colorectal cancer, melanoma and glioblastoma cell lines tested. The viability of the other cell lines tested was not significantly reduced.Because a previous study evaluating the use of THC for treating breast cancer cells suggested that there is an entourage effect in that context, Vrana cautioned that careful testing of cannabinoids should be done for each proposed therapeutic context."Pure CBD had the ability to reduce certain cancer cell types' viability in this study," Vrana said. "It would be reckless for a consumer to assume that a CBD oil product off the shelf could have the same effects for them, which is why careful studies around the entourage effect are needed for each intended therapeutic application."Vrana said that even if there were cases where the entourage effect were proven for therapeutic uses, cannabinoid products are unregulated and consumers would not be able to know in many cases whether an off-the-shelf or off-the-street product had the right components to result in the desired therapeutic outcome."The variability in composition and activities of botanical extracts highlights difficulties in assessing their therapeutic potential compared to pure chemical compounds," Vrana said. Raup-Konsavage and Vrana plan to continue investigating the "entourage" effect of cannabinoids in other therapeutic applications.
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Biotechnology
| 2,020 |
November 17, 2020
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https://www.sciencedaily.com/releases/2020/11/201117113044.htm
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Researchers improve neuronal reprogramming by manipulating mitochondria
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The replacement of lost neurons is a holy grail for neuroscience. A new promising approach is the conversion of glial cells into new neurons. Improving the efficiency of this conversion or reprogramming after brain injury is an important step towards developing reliable regenerative medicine therapies. Researchers at Helmholtz Zentrum München and Ludwig Maximilians University Munich (LMU) have identified a hurdle towards an efficient conversion: the cell metabolism. By expressing neuron-enriched mitochondrial proteins at an early stage of the direct reprogramming process, the researchers achieved a four times higher conversion rate and simultaneously increased the speed of reprogramming.
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Neurons (nerve cells) have very important functions in the brain such as information processing. Many brain diseases, injuries and neurodegenerative processes, are characterized by the loss of neurons that are not replaced. Approaches in regenerative medicine therefore aim to reconstitute the neurons by transplantation, stem cell differentiation or direct conversion of endogenous non-neuronal cell types into functional neurons.Researchers at Helmholtz Zentrum München and LMU are pioneering the field of direct conversion of glial cells into neurons which they have originally discovered. Glia are the most abundant cell type in the brain and can proliferate upon injury. Currently, researchers are able to convert glia cells into neurons -- but during the process many cells die. This means that only few glial cells convert into functional nerve cells, making the process inefficient.Magdalena Götz and her team investigated potential hurdles in the conversion process and took a new route: While most studies have focused on the genetic aspects of direct neuronal reprogramming, they decided to study the role of mitochondria and cell metabolism in this process. This was inspired by their previous work in collaboration with Marcus Conrad's group at Helmholtz Zentrum München showing that cells die due to excessive reactive oxygen species in the conversion process."We hypothesized that if we were able to help reprogramming the metabolism of glia cells towards the metabolism of a neuron, this could improve the conversion efficiency," explains Gianluca Russo, first-author of the study. Given their previous data, the researchers focused on mitochondria, the cell's powerhouse. The group extracted mitochondria from neurons and astrocytes (a specific type of glia cell) of mice and compared them by studying their proteins in collaboration with Stefanie Hauck's group of proteomic experts at Helmholtz Zentrum München. Surprisingly, they found that mitochondria of neurons and astrocytes differ in 20 percent of their proteome. This means that between astrocytes and neurons every fifth mitochondrial protein is different."Knowing how different the mitochondrial proteome of neurons is from astrocytes, we needed to see if and when neurons converting from astrocytes actually acquire the mitochondrial proteome of a neuron or not," says Giacomo Masserdotti, co-last author of the study. In a standard reprogramming process, glia cells like astrocytes convert to neurons within a few days and develop into functional neurons within two weeks. "It was striking that cells showed mitochondrial proteins, which are typical for neurons, relatively late in the reprogramming process, only after one week. Since most cells die before this time, this could be a hurdle. In addition, cells that failed to be reprogrammed, still expressed astrocyte-enriched mitochondrial proteins." With this new insight, the researchers hypothesized that the failure of turning on neuronal mitochondrial proteins may be blocking the conversion process.To overcome this hurdle, the group employed CRISPR/Cas9 technology in close cooperation with Stefan Stricker's and Wolfgang Wurst's groups at Helmholtz Zentrum München. With new gene activation tools developed by this group, neuron-enriched mitochondrial proteins could be activated at an early stage of the reprogramming process of astrocytes to neurons. By manipulating one to two mitochondrial proteins only, the researchers gained four times more reprogrammed neurons. On top of that, the neurons appeared and matured faster, as revealed by continuous live imaging."I was amazed that changing the expression of few mitochondrial proteins actually drives the speed of reprogramming," says Magdalena Götz, the lead author of the study. "This shows how important the cell-type-specific differences of mitochondrial proteins are. And indeed, together with our proteome experts at Helmholtz Munich, we are discovering further organellar differences between cell types that reach up to 70 percent. This will pave the way to further improve the reprogrammed neurons to resemble as much as possible endogenous neurons also after brain injury in vivo."
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Biotechnology
| 2,020 |
November 16, 2020
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https://www.sciencedaily.com/releases/2020/11/201116112912.htm
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Cellular powerplant recycles waste gases
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Waste gases of many branches of industry contain mainly carbon monoxide and carbon dioxide. Nowadays, these gases are simply blown into our atmosphere, but this may soon change. The idea is to use the power of bacteria to turn toxic waste gases into valuable compounds such as acetate or ethanol. These can be used afterwards as biofuels or basic compounds for synthetic materials. The first real-size test plants are already under evaluation, using this conversion at an industrial scale, and the stars of these process are bacteria that devour carbon monoxide, carbon dioxide and dihydrogen, among which Clostridium autoethanogenum is by far the favorite.
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"In this microbe, the main lines of the metabolism used to operate the gas conversion have been characterized," says Tristan Wagner, leader of the group Microbial Metabolism at the Max Planck Institute for Marine Microbiology. "But there are still a lot of question marks at the molecular level." The one in focus of the scientists from Bremen: How is the toxic carbon monoxide processed by enzymes at such stunning efficiency?The molecular-level knowledge of the carbon monoxide conversion is derived from studies performed in the species Moorella thermoacetica. This is a convenient and well-studied marine model organism but exhibits a poor ability to detoxify waste gases, unlike Clostridium autoethanogenum. Both bacteria use the same enzyme to convert carbon monoxide: the CO-dehydrogenase/Acetyl-CoA synthase, shortened as CODH/ACS. It is a very common enzyme which existed already in primeval times of the earth. "Since both species use the same enzyme to convert carbon monoxide, we were expecting to see exactly the same structure with eventually minor differences," says Wagner.For their research, Wagner and his colleague Olivier N. Lemaire are studying the bacterium Clostridium autoethanogenum to understand how it can thrive at the thermodynamics of Life, using a metabolism similar to that of the first living forms. Olivier N. Lemaire grew the bacteria and purified its CODH/ACS in absence of oxygen, which is detrimental to the enzyme. The two scientists used the crystallization method to obtain crystals of the enzyme CODH/ACS and determine the protein 3D-structure by X-ray crystallography. "When we saw the results, we couldn't believe our eyes," says Wagner. "The CODH-ACS interface from Clostridium autoethanogenum drastically differs from the model of Moorella thermoacetica, even though it was the same enzyme and similar bacteria."Afterwards, the two researchers carried out further experiments to prove that the first structure was not an artifact but the biological reality. Following experiments confirmed the initial model. Thus, the discovery clearly proves wrong the previous assumption that the enzyme CODH/ACS always has the same overall structure. "The enzyme of Moorella thermoacetica has a linear shape," explains Olivier N. Lemaire, first author of the study, which was recently published in the scientific journal But Clostridium autoethanogenum absorbs carbon monoxide directly. "In Clostridium autoethanogenum the enzyme CODH/ACS has not only one opening, but several. In this way it can collect as much carbon monoxide as possible and conduct it into a whole system of tunnels, operating in both directions," says Lemaire. "These results show a reshuffling of internal gas-tunnels during evolution of these bacteria, putatively leading to a bidirectional complex that ensures a high flux of carbon monoxide conversion toward energy conservation and assimilation of carbon monoxide, acting as the main cellular powerplant." At the end of the process also acetate and ethanol are generated, which can be used to produce fuels."We now have a picture of what this very efficient and robust enzyme looks like," says Tristan Wagner. "But our discovery is only one step further. Among other things, it is still an open question how the bacterium can survive and use carbon monoxide to feed their whole cellular energy needs. We have some hypotheses, but we are still at the beginning. To understand the whole chemical process of converting carbon monoxide to acetate and ethanol, further proteins need to be studied."
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Biotechnology
| 2,020 |
November 16, 2020
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https://www.sciencedaily.com/releases/2020/11/201116092231.htm
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Newly discovered enzyme helps make valuable bioactive saponins
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Many plants, including legumes, make naturally occurring chemicals called saponins. For example, the medicinal plant licorice produces the saponin glycyrrhizin, a potent natural sweetener that also has antiviral and other pharmacological activity. Soyasaponins, found in soybeans, have anticarcinogenic and antioxidant properties.
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But exactly how plants make these useful products was unclear. Now a research team at Osaka University, in collaboration with National Agriculture and Food Research Organization (NARO), RIKEN, and Chiba University, has uncovered a vital link in the complex biochemical pathway for saponin synthesis. Their discovery paves the way for improving the commercial production of these high-value products. The team recently published the study in Osaka University researchers Soo Yeon Chung and Hikaru Seki, with collaborators in NARO (Masao Ishimoto et al.), RIKEN, and Chiba University, studied co-expression gene network of saponin synthesis using technologies including gene cloning and sequence comparisons, coupled with biochemical analyses in mutants and genetically modified plants of a model legume species. They discovered a new enzyme in the CSyGT family that are similar in structure to the enzymes producing cellulose in plant cell walls. Unexpectedly, they showed that the new member of the family was responsible for a key step in saponin synthesis, where a sugar molecule is attached to the triterpenoid backbone. This discovery challenged the generally accepted view that a different class of enzyme was probably involved in this step.They went on to insert the gene for the newly discovered CSyGT enzyme, along with genes for other steps in the biochemical pathway, into yeast cells. The engineered cells successfully produced glycyrrhizin from simple sugars, indicating a potential route for industrial manufacture of valuable saponins by growing yeast cells on a large scale."Our multi-disciplinary team showed, for the first time, that this type of enzyme is important in saponin synthesis," says corresponding author Toshiya Muranaka. "Our results fill a gap in previous knowledge and also challenge the accepted view of how this pathway for biosynthesis operates.""We showed that yeast cells can make glycyrrhizin when we insert the necessary plant genes," explains Chung. "This offers the prospect of new ways to produce these valuable substances commercially, and to generate completely novel types of saponin that might have further beneficial applications in medicine or the food industry." Seki adds, "producing the chemicals in cell cultures would also reduce the need to deplete natural plant resources and so help to meet the vitally important Sustainable Development Goals."
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Biotechnology
| 2,020 |
November 13, 2020
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https://www.sciencedaily.com/releases/2020/11/201113141814.htm
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Light shed on the atomic resolution structure of phage DNA tube
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Given that phages are able to destroy bacteria, they are of particular interest to science. Basic researchers from the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) in Berlin are especially interested in the tube used by phages to implant their DNA into bacteria. In collaboration with colleagues from Forschungszentrum Jülich and Jena University Hospital, they have now revealed the 3D structure of this crucial phage component in atomic resolution. The key to success was combining two methods -- solid-state NMR and cryo-electron microscopy. The study has just been published in the journal
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With growing antibiotic resistance, phages have increasingly become the focus of research. Phages are naturally occurring viruses with a very useful property: they implant their DNA into bacteria and proliferate there until the bacterial cell is ultimately destroyed. This is why they are also referred to as bacteriophages (bacteria eaters).This approach has already been shown to fight multidrug-resistant bacteria. Last year, the case of a girl from England hit the headlines, when she was cured from a serious antibiotic-resistant infection using engineered phages.However, the widespread use of phage therapy is still a long way off. Many of the underlying principles that are key to advancing this therapy are not yet understood. For example, little was previously known about the appearance of the exact architecture of the tube used by phages to implant their DNA into bacteria. Now scientists from the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) in Berlin, together with colleagues from Forschungszentrum Jülich and Jena University Hospital, have managed to reveal the 3D structure of this crucial phage component in atomic resolution."The structure and flexibility of the DNA tube attached to the icosahedron-shaped capsid is somewhat reminiscent of a spinal column," stated FMP's Professor Adam Lange, describing one of the new findings. "It seems to be perfectly designed for transporting DNA."The researchers were able to gain fascinating insights into the structure and function of this sophisticated DNA transport pathway -- in this case, from a variant of phage SPP1 -- by innovatively combining solid-state NMR with cryo-electron microscopy (cryo-EM). Lange's research group further developed nuclear magnetic resonance spectroscopy (NMR) especially for this task under an ERC Grant; cryo-EM expert Professor Gunnar Schröder from Forschungszentrum Jülich performed the electron-microscopic investigations. In addition, new modeling algorithms were required for the computer-based combination of the two data sets for structure determination. These algorithms were developed by Professor Michael Habeck from Jena University Hospital. "The key to success was combining the two methods, representing a methodological milestone," commented Professor Lange.While solid-state NMR is ideal for visualizing flexible structures and tiny details, cryo-EM provides insight into the overall architecture. The resulting image shows that six gp17.1 proteins organize into stacked rings, forming a hollow tube. The rings are connected by flexible linkers, making the tube very bendable. "We are now able to understand how negatively charged DNA is repelled from the likewise negatively charged interior wall of the flexible tube, passing through it smoothly," explained FMP's Maximilian Zinke, lead author of the study now published in Nature Communications. "The bacteria are ultimately destroyed via this pathway."According to group leader Adam Lange, besides representing a quantum leap forward in phage research, the work will also advance "integrated structural biology," the term for the combination of these two complementary methods.Thanks to the recent installation of a new high-resolution Titan Krios electron microscope, the infrastructure required to achieve this is now available on Campus Berlin-Buch. Moreover, a 1.2 gigahertz device will soon be added to the existing NMR spectrometers. "Equipped with cryo-EM and the most sensitive NMR spectrometer in the world, we will be very present in integrative structural biology in the future," enthused Adam Lange. "This offers bright prospects for the campus and for the research location of Berlin."
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Biotechnology
| 2,020 |
November 13, 2020
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https://www.sciencedaily.com/releases/2020/11/201113124055.htm
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Circular RNA regulates neuronal differentiation by scaffolding an inhibitory transcription complex
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In a screening for a functional impact to the neuronal differentiation process, Danish researchers identified a specific circular RNA, circZNF827, which surprisingly "taps the brake" on neurogenesis. The results provide an interesting example of co-evolution of a circRNA, and its host-encoded protein product, that regulate each other's function, to directly impact the fundamental process of neurogenesis.
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Correct timing and delicate control of neuronal differentiation is essential for development of a functional nervous system. These events establish a fine-tuned balance between the ability of stem cells to grow/divide and the neuronal progenitors to eventually exit the cell cycle and emerge as mature neurons. A variety of genes become up- or downregulated upon differentiation, giving rise to both neuron-specific proteins and ribonucleic acids (RNAs), including circular RNAs (circRNAs). This class of circRNAs has until recently escaped conventional detection, although these molecules are highly expressed in the mammalian brain. However, the functional roles of brain-expressed circRNAs remain virtually unknown.In a study, spearheaded by postdoc Anne Kruse Hollensen and led by Associate Professor Christian Kroun Damgaard, Molecular Biology and Genetics, Aarhus University, thousands of circRNAs were identified when stem cells become differentiated into mature neurons. In a screening for a functional impact to the differentiation process, the authors identified a specific circRNA, circZNF827, which surprisingly "taps the brake" on neurogenesis.Various biochemical and cell biological assays, revealed that circZNF827 mechanistically functions as a scaffold for a complex of RNA-binding proteins, including its own host-gene-encoded protein, ZNF827, and two known transcriptional regulators, hnRNP K and L. Despite being localized mostly to the cell cytoplasm, circZNF827 apparently "moonlights" in the nucleus, where it nucleates these transcription factors to specific neuronal genes (e.g. NGFR), and hence, repress their expression.The results contribute to the molecular understanding of neurogenesis and in particular how abundant brain-specific circRNAs tap into this fundamental process.
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Biotechnology
| 2,020 |
November 12, 2020
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https://www.sciencedaily.com/releases/2020/11/201112165830.htm
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Once-discounted binding mechanism may be key to targeting viruses
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"Position 4" didn't seem important until researchers took a long look at a particular peptide.
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That part of the peptide drawn from a SARS-CoV virus turned out to have an unexpected but significant influence on how it stably binds with a receptor central to the immune system's ability to attack diseased cells.In a study published by the They say better understanding of the entire mechanism could lead to advancements in immunotherapy that boosts the body's ability to fight disease.Rice computer scientist Lydia Kavraki, alumnus Jayvee Abella and postdoctoral researcher Dinler Antunes, led the study."Finding good targets to trigger a protective immune response is very challenging, especially in cancer research," Antunes said. "The fact that this particular peptide was predicted not to bind to HLAs (human leukocyte antigens) by sequence-based methods highlights a blind spot in our current prediction capacity."By incorporating structural analysis, we can detect the contribution of these secondary interactions to peptide binding and stability, hopefully enabling us to find better targets for antiviral vaccine development and T-cell-based cancer immunotherapy," he said.The researchers used their simulations to illuminate details of how the intracellular SARS peptide, QFKDNVILL, binds to an MHC receptor protein known as HLA-A?24:02, primarily at dominant anchors on both ends of the peptide (at positions 2 and 9) and presents them for inspection to the immune system's T cells.Stable binding of a peptide and MHC is a prerequisite to the activation of T cells, which look for peptides not normally found in healthy cells. If the peptide and protein don't bind, the T cell is not prompted to attack."That much was known from previous studies of the bound and unbound states of many such complexes," Kavraki said. "What they didn't capture was the intermediate states and the transitions that lead from one state to another, especially the unbinding."I think this is the only analysis that shows the unbinding of peptides from the MHC with atomic resolution," Kavraki said. "Other peptides have similar characteristics and we think they would have similar behaviors."All of these interactions were revealed in great detail through Markov state models that analyze how systems change over time. In this case, the models revealed the importance of secondary sites that support the peptide's primary anchors. That's where position 4 stood out."There are the main, canonical anchors that people know, but there are these secondary interactions that contribute to the binding and the stability," Antunes said. "These are harder to capture, but in this study, it seems that position 4 plays a very important role. When you mutate it, it affects the behavior of the peptide as it unbinds from the molecule."The researchers modeled mutations of the MHC to see how they would influence binding and found they supported the importance of position 4 to the stability of the complex."Our computational approach was able to make predictions on the effect of mutations that are then experimentally verified," said co-author Cecilia Clementi, a former Rice professor who recently became Einstein Professor of Physics at the Free University of Berlin.The researchers developed a two-stage process to simplify the computational complexity of atom-scale analysis of large molecules. The first stage used a technique called umbrella sampling to accelerate the initial exploration of the molecules. The second, exploratory stage used adaptive sampling, in which simulations are driven to accelerate the construction of the Markov model."The challenge is that these MHCs are pretty large systems for computational chemists to simulate," said Abella, whose research on the topic formed much of his doctoral thesis. "We had to make some approximations and leverage advances in these classes of methods to move forward."We're not the first one to study unbinding, but what characterizes our work over others is that we keep full atomic resolution in our simulations," he said. "Other works use a technique known as a Markov chain Monte Carlo, whereas we use molecular dynamics, which lets us incorporate time into our computation to capture the kinetics."Their methods can be applied to other peptide-MHC complexes with existing 3D models. "This was, in some sense, a feasibility study to show we can use molecular dynamics and build a Markov state model of a system this size," Abella said.The researchers also noted the study's relevance to the current fight against COVID-19, as the SARS peptide they viewed, QFKDNVILL, is highly similar to the NFKDQVILL peptide in SARS-CoV-2, with the same binding pockets in positions 2, 4 and 9."These results suggest that both peptides can bind to HLA-A*2402 and provide targets for anti-viral T-cell responses, which are of great interest in light of the current pandemic," said co-author Gregory Lizée, a professor in the Department of Melanoma Medical Oncology at MD Anderson. "But these results also shed light on many other potential immune targets, including those of other viruses and even human cancers."Kavraki noted that experimental work by longterm collaborator Lizée and Kyle Jackson, a graduate research assistant at Lizée's lab who produced the mutant proteins, were critical to validate their simulations. Kavraki's own lab won a National Science Foundation (NSF) Rapid Response Research grant to help identify fragments of SARS-CoV-2 viral proteins as possible targets for vaccine development.Kavraki is the Noah Harding Professor of Computer Science and a professor of bioengineering, mechanical engineering and electrical and computer engineering.The Cancer Prevention and Research Institute of Texas, the Gulf Coast Consortia, the NSF, the Einstein Foundation Berlin and the Welch Foundation supported the research.
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Biotechnology
| 2,020 |
November 12, 2020
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https://www.sciencedaily.com/releases/2020/11/201112165824.htm
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Novel insights on cellular suicide could provide new avenues for cancer therapies
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When it comes to complex life -- that of the multicellular variety -- cell death can be just as important as survival. It allows organisms to clean house and prevent the proliferation of damaged cells that could compromise tissue function.
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Several years ago, biologist Denise Montell, a distinguished professor at UC Santa Barbara, found that sometimes cells survive after what was considered the critical step in cellular suicide. Now, she and her lab have identified two key factors involved in this remarkable recovery.The findings, published in Apoptosis is the most common way cells commit suicide, and this process is critical in maintaining an organism's wellbeing. Living things need a way to terminate cells when they are badly injured or their DNA is damaged. Apoptosis is also part of natural turnover, especially in blood cells, skin cells and the lining of the gut."Before our work, people really thought that apoptosis was an all-or-nothing decision," said Montell, Duggan Professor in the Department of Molecular, Cellular, and Developmental Biology. "You either committed to suicide and went through with it, or you didn't."Scientists considered the activation of an enzyme appropriately called "executioner caspase" to be the point of no return. This enzyme essentially slices and dices many of the cell's proteins. But it turns out apoptosis is more nuanced than previously known, and sometimes cells survive the executioner caspase via another process -- anastasis.This phenomenon first came to Montell's attention around 2010. Generally, scientists studying apoptosis use extreme conditions that cause all the cells in their sample to die. A doctoral student in her lab at the time was curious whether cells could survive the activation of caspase if he removed the substance that induced apoptosis. To everyone's surprise, many of them did.Since then scientists have observed anastasis in cells from many different organisms, including humans, mice and fruit flies. Montell and her team decided to search for genes that would either enhance or inhibit the ability of cells to undergo this process.To this end, the researchers applied a technique they developed in 2016. By breeding transgenic fruit flies that express a specific protein that is cut by the executioner caspase, they initiated a series of events that ultimately makes the cells fluoresce green. That permanently identifies any cell that has survived through this phase of apoptosis.With this tool on hand, the team, led by former postdoctoral fellow Gongping Sun, set out to identify the genes involved in anastasis. Given they couldn't investigate all 13,000 genes in the fruit fly genome, the researchers combed their own data as well as the literature to identify candidate genes, eventually settling on about 200 to investigate further.Sun and her lab mates took hundreds of fruit flies and knocked out the expression of a different gene in half of the cells of each animal. This enabled them to control for other factors that might influence the results.In the paper published in 2016, the team found that some cells undergo anastasis during normal development of the fruit fly. In the new paper, they therefore looked for changes in the percentage of cells that went through this process during development. They also tested the genes for their ability to affect anastasis in response to stresses like radiation and heat.Distinguishing between genes involved in anastasis and those that are simply necessary for basic survival was a challenge. "Because if it's necessary for survival, period, then it's also going to be necessary for recovery from the brink of death," Montell said.So, the team looked not only at how many cells in a sample fluoresced green after the experiment, but the ratio of green cells to non-green cells. If the gene in question was necessary for basic survival, but not involved in anastasis, it affects all cells equally. This would impact the overall number of fluorescent cells, but leave the ratio unchanged.The researchers found two proteins, and the genes that coded for them, were instrumental in anastasis. The first, AKT1, is a well-studied and renowned survival protein that is activated in response to growth factors, essentially telling the cell to grow and divide. Scientists were aware that it can block the activation of executioner caspase, but the team discovered it can also make the difference between survival and death after caspase has been triggered.The other protein, CIZ1, is not as well-studied, and shows up in a number of unrelated papers across the literature. In nearly all these instances it appears that CIZ1 also promotes survival from stress. For instance, a decreased amount of CIZ1 is associated with increased age-dependent neurodegeneration in mice.The involvement of these two proteins in anastasis indicates that it is probably a very ancient process. "Not just the phenomenon of cells recovering from the brink of death, but even the mechanism -- the molecules involved -- are so deeply conserved in evolution that flies and mice are using the same molecules," Montell said.These findings are a huge step forward in understanding apoptosis on a fundamental level. They also suggest possible applications -- especially in efforts to combat cancer.Apoptosis serves an important function in maintaining stable equilibrium within complex organisms. Under normal circumstances -- say UV damage to a skin cell -- the body wants the injured cell to die so that it doesn't develop into a condition like melanoma."However, if you were subjected to extreme stress you might not want every cell to commit apoptosis," Montell said. "That might result in permanent tissue damage from which it would be very hard to recover."In response to severe but temporary trauma, it could be beneficial for some of the cells to be able to bounce back. Montell suspects this is the primary reason that organisms evolved a way to circumvent apoptosis.The temporary nature of the stress seems to be the critical factor both in the role anastasis plays in promoting healing and in the mechanism itself. When a cell is under extreme stress, like radiation or chemical exposure, two things happen simultaneously: The cell activates the apoptosis response -- including executioner caspase -- while also activating pro-survival responses."It's like putting on the accelerator and the brake at the same time," Montell said.The apoptotic factors reinforce themselves, so if the stressful conditions persist, the process crosses a threshold and the cell dies. But if the stress is only transient, the pro-survival pathway is already poised to kick in and help the cell recover. Researchers don't fully understand how the cell turns off the apoptotic pathway, but proteins like AKT1 and CIZ1 are likely involved.There is, however, a dark side to this survival mechanism. "Anastasis could be a good thing if you're trying to repair a damaged tissue, but it could be a bad thing in that it might promote the growth of tumors," Montell pointed out, "especially in response to chemotherapy and radiation treatments, which are extreme temporary stresses."This matches the experience of many physicians, Montell explained. A lot of cancer patients initially respond well to treatments; their tumors shrink and their condition improves. But unfortunately, the tumors often grow back. And scientists aren't certain why this is.Some think the resurgence could be the result of drug-resistant cells that exist in the tumor, which then seed the relapse. This paper provides another hypothesis -- "the idea that the treatment itself could induce the cancer cells to undergo this stress-dependent survival process," Montell said.This notion could fundamentally change the way doctors think about preventing relapse. There isn't much you can do against drug-resistant cells, Montell said, but if the relapse is due to this survival mechanism, these findings could inform new therapies.Drugs that inhibit AKT1 are currently in clinical trials. These could be combined with other therapies to increase their effectiveness, potentially enabling doctors and researchers to inhibit anastasis in cancer cells while promoting it normal cells.What's more, successful cancer cells can actually induce apoptosis in the T cells that the immune system sends to attack them, according to Montell. This presents another target for anastasis therapies."There's this ongoing war between the immune system and cancer," Montell said, "and if you can tip the balance even a little bit, you can start to win."
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Biotechnology
| 2,020 |
November 12, 2020
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https://www.sciencedaily.com/releases/2020/11/201112155840.htm
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Chemists discover the structure of a key coronavirus protein
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MIT chemists have determined the molecular structure of a protein found in the SARS-CoV-2 virus. This protein, called the envelope protein E, forms a cation-selective channel and plays a key role in the virus's ability to replicate itself and stimulate the host cell's inflammation response.
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If researchers could devise ways to block this channel, they may be able to reduce the pathogenicity of the virus and interfere with viral replication, says Mei Hong, an MIT professor of chemistry. In this study, the researchers investigated the binding sites of two drugs that block the channel, but these drugs bind only weakly, so they would not be effective inhibitors of the E protein."Our findings could be useful for medicinal chemists to design alternative small molecules that target this channel with high affinity," says Hong, who is the senior author of the new study.MIT graduate student Venkata Mandala is the lead author of the paper, which appears in Nature Structural and Molecular Biology. Other authors include MIT postdoc Matthew McKay, MIT graduate students Alexander Shcherbakov and Aurelio Dregni, and Antonios Kolocouris, a professor of pharmaceutical chemistry at the University of Athens.Hong's lab specializes in studying the structures of proteins that are embedded in cell membranes, which are often challenging to analyze because of the disorder of the lipid membrane. Using nuclear magnetic resonance (NMR) spectroscopy, she has previously developed several techniques that allow her to obtain accurate atomic-level structural information about these membrane-embedded proteins.When the SARS-CoV-2 outbreak began earlier this year, Hong and her students decided to focus their efforts on one of the novel coronavirus proteins. She narrowed in on the E protein partly because it is similar to an influenza protein called the M2 proton channel, which she has previously studied. Both viral proteins are made of bundles of several helical proteins."We determined the influenza B M2 structure after about 1.5 years of hard work, which taught us how to clone, express, and purify a virus membrane protein from scratch, and what NMR experimental strategies to take to solve the structure of a homo-oligomeric helical bundle," Hong says. "That experience turned out to be the perfect training ground for studying SARS-CoV-2 E."The researchers were able to clone and purify the E protein in two and half months. To determine its structure, the researchers embedded it into a lipid bilayer, similar to a cell membrane, and then analyzed it with NMR, which uses the magnetic properties of atomic nuclei to reveal the structures of the molecules containing those nuclei. They measured the NMR spectra for two months, nonstop, on the highest-field NMR instrument at MIT, a 900-megahertz spectrometer, as well as on 800- and 600-megahertz spectrometers.Hong and her colleagues found that the part of the E protein that is embedded in the lipid bilayer, known as the transmembrane domain, assembles into a bundle of five helices. The helices remain largely immobile within this bundle, creating a tight channel that is much more constricted than the influenza M2 channel.Interestingly, the SARS-CoV-2 E protein looks nothing like the ion channel proteins of influenza and HIV-1 viruses. In flu viruses, the equivalent M2 protein is much more mobile, while in HIV-1, the equivalent Vpu protein has a much shorter transmembrane helix and a wider pore. How these distinct structural features of E affect its functions in the SARS-CoV-2 virus lifecycle is one of the topics that Hong and her colleagues will study in the future.The researchers also identified several amino acids at one end of the channel that may attract positively charged ions such as calcium into the channel. They believe that the structure they report in this paper is the closed state of the channel, and they now hope to determine the structure of the open state, which should shed light on how the channel opens and closes.The researchers also found that two drugs -- amantadine, used to treat influenza, and hexamethylene amiloride, used to treat high blood pressure -- can block the entrance of the E channel. However, these drugs only bind weakly to the E protein. If stronger inhibitors could be developed, they could be potential drug candidates to treat Covid-19, Hong says.The study demonstrates that basic scientific research can make important contributions toward solving medical problems, she adds."Even when the pandemic is over, it is important that our society recognizes and remembers that fundamental scientific research into virus proteins or bacterial proteins must continue vigorously, so we can preempt pandemics," Hong says. "The human cost and economic cost of not doing so are just too high."The research was funded by the National Institutes of Health and the MIT School of Science Sloan Fund.
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Biotechnology
| 2,020 |
November 12, 2020
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https://www.sciencedaily.com/releases/2020/11/201112144032.htm
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Breaking it down: How cells degrade unwanted microRNAs
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UT Southwestern researchers have discovered a mechanism that cells use to degrade microRNAs (miRNAs), genetic molecules that regulate the amounts of proteins in cells.
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The findings, reported online today in Scientists have long known that genes contain the instructions for making every protein in an organism's body. However, various processes regulate whether different proteins are produced and in what amounts. One of these mechanisms involves miRNAs, small pieces of genetic material that break down complementary pieces of messenger RNA (mRNA) in cells, preventing the mRNA sequence from being translated into proteins.Since the discovery of miRNAs in 1993, researchers have amassed a wealth of knowledge about the hundreds of different miRNA molecules and their targets as well as mechanisms that control their production, maturation, and roles in development, physiology, and disease. However, explains Joshua Mendell, M.D., Ph.D., professor and vice chair of the department of molecular biology at UTSW, and postdoctoral fellow Jaeil Han, Ph.D., very little was known about how cells dispose of miRNAs when they're finished using them."As long as miRNA molecules stick around in a cell, they reduce the production of proteins from their target mRNAs," explains Mendell, a Howard Hughes Medical Institute (HHMI) investigator and member of the Harold C. Simmons Comprehensive Cancer Center. "So understanding how cells get rid of miRNAs when they are no longer needed is pivotal for fully appreciating how and when they do their jobs."To answer this question, Mendell, Han, and their colleagues harnessed CRISPR-Cas9, a gene-editing tool that recently won the 2020 Nobel Prize in Chemistry for two scientists who developed it. By serving as "molecular scissors," Mendell says, this system can cut out individual genes, allowing researchers to explore their functions.In a human cancer cell line known as K562, the researchers used CRISPR-Cas9 to target most of the 20,000 protein-coding genes in the human genome, looking for any that caused a normally short-lived miRNA known as miR-7 to linger in cells. Their search turned up at least 10 genes that are needed to degrade this miRNA.The researchers learned that the proteins encoded by these genes come together in cells to form a larger assembly known as a ubiquitin ligase, which functions to tag other proteins for destruction. This particular ubiquitin ligase had never been described before, Mendell says, but like other ubiquitin ligase complexes, it appears to mark proteins destined for degradation. However, rather than tag miR-7 itself, further investigation showed that this complex instead tags a protein called Argonaute, which ferries miRNAs through cells.Once the Argonaute protein attached to miR-7 is targeted for degradation, this miRNA is left naked in the cell -- a state that triggers cells to destroy the miRNA using RNA-degrading enzymes.The research team found that this ubiquitin ligase complex is key for degrading not only miR-7 in K562 cells, but also a variety of other miRNAs in other cell types and species. These results suggest that this mechanism for miRNA decay acts broadly to control the levels of miRNAs during animal development and across tissues. Because other studies have shown that abnormal levels of various miRNAs are associated with a variety of diseases and infections, finding ways to control miRNA degradation -- either to eradicate problematic miRNAs in cells or hold on to beneficial ones -- could represent a new way to treat these conditions."For over a decade, researchers have been searching for mechanisms through which cells degrade miRNAs," says Han. "Now that we've discovered new cellular machinery that can accomplish this, we will be able to apply this discovery to better understand how miRNAs are regulated and, we hope, eventually develop new therapies."Other UTSW researchers who contributed to this study include Collette A. LaVigne, Benjamin T. Jones, He Zhang, and Frank Gillett.This study was funded by grants from the National Institutes of Health (R35CA197311, P30CA142543, and P50CA196516), the Welch Foundation (I-1961-20180324), American Heart Association (19POST34380222), the Cancer Prevention and Research Institute of Texas (RP150596), and the HHMI.
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Biotechnology
| 2,020 |
November 12, 2020
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https://www.sciencedaily.com/releases/2020/11/201112120509.htm
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Researchers create artificial cell organelles for biotechnology
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Cells of higher organisms use cell organelles to separate metabolic processes from each other. This is how cell respiration takes place in the mitochondria, the cell's power plants. They can be compared to sealed laboratory rooms in the large factory of the cell. A research team at Goethe University has now succeeded in creating artificial cell organelles and using them for their own devised biochemical reactions.
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Biotechnologists have been attempting to "reprogram" natural cell organelles for other processes for some time -- with mixed results, since the "laboratory equipment" is specialised on the function of organelles. Dr Joanna Tripp, early career researcher at the Institute for Molecular Biosciences has now developed a new method to produce artificial organelles in living yeast cells.To this end, she used the ramified system of tubes and bubbles in the endoplasmic reticulum (ER) that surrounds the nucleus. Cells continually tie off bubbles, or vesicles, from this membrane system in order to transport substances to the cell membrane. In plants, these vesicles may also be used for the storage of proteins in seeds. These storage proteins are equipped with an "address label" -- the Zera sequence -- which guides them to the ER and which ensures that storage proteins are "packaged" there in the vesicle. Joanna Tripp has now used the "address label" Zera to produce targeted vesicles in yeast cells and introduce several enzymes of a biochemical metabolic pathway.This represents a milestone from a biotechnical perspective. Yeast cells, the "pets" of synthetic biology not only produce numerous useful natural substances, but can also be genetically changed to produce industrially interesting molecules on a grand scale, such as biofuels or anti-malaria medicine.In addition to the desired products, however, undesirable by-products or toxic intermediates often occur as well. Furthermore, the product can be lost due to leaks in the cell, or reactions can be too slow. Synthetic cell organelles offer remedies, with only the desired enzymes (with "address labels") encountering each other, so that they work together more effectively without disrupting the rest of the cell, or being disrupted themselves."We used the Zera sequence to introduce a three-stage, synthetic metabolic pathway into vesicles," Joanna Tripp explains. "We have thus created a reaction space containing exactly what we want. We were able to demonstrate that the metabolic pathway in the vesicles functions in isolation to the rest of the cell."The biotechnologist selected an industrially relevant molecule for this process: muconic acid, which is further processed industrially to adipic acid. This is an intermediate for nylon and other synthetic materials. Muconic acid is currently won from raw oil. A future large-scale production using yeast cells would be significantly more environment-friendly and sustainable. Although a portion of the intermediate protocatechuic acid is lost because the vesicle membrane is porous, Joanna Tripp views this as a solvable problem.Professor Eckhard Boles, Head of the Department of Physiology and Genetics of Lower Eukaryotes observes: "This is a revolutionary new method of synthetic biology. With the novel artificial organelles, we now have the option of generating various processes in the cell anew, or to optimise them." The method is not limited to yeast cells, but can be utilised for eukaryotic cells in general. It can also be applied to other issues, e.g. for reactions that have previously not been able to take place in living cells because they may require enzymes that would disrupt the cell metabolic process.
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Biotechnology
| 2,020 |
November 11, 2020
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https://www.sciencedaily.com/releases/2020/11/201111180651.htm
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Internal clocks drive beta cell regeneration
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Certain parts of our body, such as the skin or liver, can repair themselves after a damage. Known as cell regeneration, this phenomenon describes how cells that are still functional start to proliferate to compensate for the loss. For the past 30 years, scientists have been investigating the regenerative potential of beta cells, pancreatic cells in charge of the production of insulin. Beta-cell population is indeed partially destroyed when diabetes occurs, and regenerating these cells represents an outstanding clinical challenge. By studying diabetic mice, scientists from the University of Geneva (UNIGE) and the University Hospitals of Geneva (HUG, observed that this regeneration mechanism was under the influence of circadian rhythms -- the molecular clocks regulating metabolic functions according to a 24-hour cycle of alternating day-night. In addition, the scientists identified the essential role of the core clock component BMAL1 in this process. These results, to be read in the journal
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Compensatory proliferation, in which cells begin to actively divide to replace those that have been damaged, is a biological mechanism that is both well-known and poorly understood. "And this is particularly true for pancreatic beta cells, whose regenerative mechanism stays largely unexplored despite decades of research," explains Dr Charna Dibner, head of the Circadian Endocrinology Laboratory at UNIGE Faculty of Medicine's the Departments of Medicine and Cell Physiology and Metabolism, as well as at the Diabetes Centre, and at the HUG. "However, deciphering this phenomenon and above all finding out how to promote it could be a game changer for controlling diabetes."To explore the connection between internal biological clocks and beta cell regeneration, Charna Dibner's team first observed two groups of mice with only 20% beta cells remaining after targeted massive ablation. Mice in a first group were arrhythmic, whereas the control group had perfectly functional clocks. "The result was very clear: the mice bearing dysfunctional clocks were unable to regenerate their beta cells, and suffered from severe diabetes, while the control group animals had their beta cells regenerated; in just a few weeks, their diabetes was under control," says Volodymyr Petrenko, a researcher in Dr. Dibner's laboratory and the leading scientist in this study. By measuring the number of dividing beta cells across 24 hours, the scientists also noted that regeneration is significantly greater at night, when the mice are active.The arrhythmic mice were lacking the BMAL1 gene, which codes for the protein of the same name, a transcription factor known for its key action in the functioning of circadian clock. "Our analyses show that the BMAL1 gene is essential for the regeneration of beta cells," adds Volodymyr Petrenko. In addition, large-scale transcriptomic analyses over a 24-hour period, conducted in collaboration with Prof. Bart Vandereycken at the Mathematics Department of the UNIGE, revealed that the genes responsible for regulating cell cycle and proliferation were not only upregulated, but also acquired circadian rhythmicity. "BMAL1 seems to be indeed central for our investigation," stresses Charna Dibner. "However, whether the regeneration requires functional circadian clocks themselves, or only BMAL1, whose range of functions goes beyond clocks remains unclear. That is what we would like to find out at present." The scientists also want to explore the function of alpha cells, which produce glucagon, the hormone that antagonises insulin, in this model. The arrhythmic mice indeed showed very high levels of glucagon in the blood. "A detailed understanding of these mechanisms must now be pursued, in an attempt to explore the possibility of triggering beta cell regeneration in humans in the future" conclude the authors.
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Biotechnology
| 2,020 |
November 11, 2020
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https://www.sciencedaily.com/releases/2020/11/201111144401.htm
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How organ functions were shaped over the course of evolution
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A large-scale study conducted by molecular biologists from Heidelberg University has yielded groundbreaking new insights into the evolution and regulation of gene expression in mammalian organs. The scientists investigated RNA synthesis and subsequent protein synthesis in the organs of humans and other representative mammals, and with the aid of sequencing technologies, they analysed more than 100 billion gene expression fragments from various organs. They were able to demonstrate that the finely tuned interplay of the two synthesis processes during evolution was crucial for shaping organ functions.
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A complex interplay of activity between a large number of genes -- known as gene expression -- underlies organ functions. "Until now, our understanding of these essential genetic programmes in mammals was limited to the first layer of gene expression -- the production of messenger RNAs," explains Prof. Dr Henrik Kaessmann, group leader of the "Functional evolution of mammalian genomes" research team at the Center for Molecular Biology of Heidelberg University (ZMBH). "The next layer -- the actual synthesis of proteins at the ribosome through the translation of the messenger RNAs -- remained largely unknown."It is this second synthesis process that the Heidelberg researchers have now studied more closely. Using so-called next-generation sequencing technologies, they analysed the gene expression of various organs on both layers. They studied the brain, liver and testes from humans and other selected mammals, including rhesus monkeys, mice, opossum and platypus. "On the basis of these data, we could jointly investigate both gene expression layers and compare them across mammalian organs using state-of-the-art bioinformatics approaches," explains Dr Evgeny Leushkin of the ZMBH.In their large-scale study, the ZMBH researchers showed that the finely tuned interplay of the two synthesis processes during evolution was critical for shaping organ functions. For the first time, they were able to show that -- in addition to regulation of messenger RNA production -- other regulatory mechanisms at the layer of translation are crucial for optimising the amount of protein produced in all organs. This is especially true in the testes, where translational regulation is key for sperm development. Another important finding concerns mutational changes in gene expression regulation that arose during evolution. These changes were often balanced between the two layers. Changes that offset one another were primarily maintained to ensure the production of consistent amounts of protein.Researchers from France and Switzerland contributed to the study. Funding was provided by the German Research Foundation and European Research Council. The data are available in a public access database. Their research results were published in
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Biotechnology
| 2,020 |
November 11, 2020
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https://www.sciencedaily.com/releases/2020/11/201111122838.htm
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Researchers light-up mouse brain, revealing previously hidden areas susceptible to opioids
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Winding and twisting like a labyrinth, the brain consists of an elaborate network of passages through which information flows at high speeds, rapidly generating thoughts, emotions, and physical responses. Much of this information is relayed by chemical messengers, or neurotransmitters -- like dopamine and serotonin.
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Although fine-tuned and evolved for complex processing, the brain and its neurotransmitters are vulnerable to hijacking by chemical substances, including opioid drugs such as oxycodone, psychostimulants such as cocaine, and alcohol. Chronic use of any of these substances enhances the activity of a molecule known as the kappa opioid receptor (KOR), which is active in the brain's reward circuitry. KOR activation produces dysphoria and an inability to feel pleasure. Its enhanced activity following chronic drug or alcohol use plays a crucial role in substance abuse.KORs have been known to exist in certain brain regions, particularly those involved in pain processing, reward, and stress responses, but new work at the Lewis Katz School of Medicine at Temple University (LKSOM) shows that these receptors actually are distributed widely throughout the brain. The Temple researchers made this discovery after lighting up the brains of mice using a technique called CLARITY followed by three-dimensional (3D) fluorescent imaging. The study is the first to apply the imaging technique to better understand opioid receptor localization across the whole brain in 3D images."Typically, we look at the brain in sections, thus yielding two-dimensional (2D) images, in which case we are not really able to see get a big picture view of protein distribution," explained Lee-Yuan Liu-Chen, PhD, Professor in the Center for Substance Abuse Research and Department of Pharmacology at LKSOM and senior investigator on the new study. "But with CLARITY we are able to produce 3D images of the entire brain, as a whole organ, and this allowed us to expose the full extent of KOR distribution."The study was published online in the journal The CLARITY technique renders brain tissue transparent, enabling researchers to visualize fluorescent probes linked to a protein of interest, in this case KOR. Fluorescence emitted from the probes is then detected via confocal imaging methods to yield highly detailed 3D images of the specific protein's distribution in the whole brain.Seeking to gain a deeper understanding of KOR localization in the brain, Dr. Liu-Chen and colleagues applied CLARITY to preserved brains from mice that had been engineered to express a fluorescent tag known as tdTomato on KOR proteins. Upon imaging, very specific regions of the KOR-tdTomato mouse brain lit up a bright shade of red, revealing the 3D distribution of KOR throughout the brain. The researchers then examined 2D sections of brain tissue to gain detailed information on the spatial localization of KOR at the cellular level.The 3D analyses and observations from brain sectioning enabled the researchers to map out the specific places of KOR expression. They identified extensive tracts related to pain and reward, building on existing knowledge of KOR's relevance to these pathways, and they discovered many neural tracts not previously known to express KOR."Seeing KOR in 3D space led to the realization that the receptor is expressed in areas of the brain beyond those that had been described before," Dr. Liu-Chen said. "The function of KOR in these additional neural circuits is unknown." A major goal for the team now is to figure out what KOR does in these newly identified circuits.The success of the team's approach in itself is significant and could open doors to the study of other neurotransmitter receptors in the brain. KOR and other opioid receptors are types of G-protein coupled receptors (GPCRs). "No one has done a 3D study of GPCR distribution in the brain before," Dr. Liu-Chen said. "The approach we used is a very useful tool and could be applied to study many different types of GPCRs and other proteins across neural tracts."
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Biotechnology
| 2,020 |
November 10, 2020
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https://www.sciencedaily.com/releases/2020/11/201110151142.htm
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New technique may revolutionize accuracy and detection of biomechanical alterations of cells
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Scientists have developed an optical elastography technique that could revolutionise the accuracy and ease to which health professionals can detect biomechanical alterations of cells and tissues.
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A study derived by an international collaboration between the University of Exeter, Gloucestershire Hospitals NHS Foundation Trust, the University of Perugia (Italy) and the Institute of Materials of the National Research Council of Italy (IOM-CNR) applied an innovative biophotonic approach to highlight how the microscopic processes drive mechanical modification in biological tissues.The team of experts, coordinated by Dr Francesca Palombo from the University of Exeter and Prof. Daniele Fioretto from the University of Perugia, Italy, analysed the great potential of the technique in the investigation of tissue on the microscale.While the mechanical properties of both cells and tissues play a fundamental role in the function of cells and how disease develops, the traditional methods to study these properties can be limited and invasive.Scientists have recently utilised Brillouin microscopy -- a form of imaging that uses light to create an acoustic measurement of the cells and tissue -- as a way of carrying out non-invasive studies of these biomechanical properties.However, a complicating factor in these measurements is the contribution of water both to tissue and cell biomechanics, as well as the Brillouin spectrum itself.Now, for the new study, the team utilised natural biopolymer hydrogels to mimic human tissue and to compare results against measurements taken in human tissue samples.They found that this new technique allows investigations of tissue functional properties (and alterations) to a subcellular scale -- meaning professionals can gain information from analysing a new spatiotemporal region of biological processes.The results of this study demonstrate that, whilst water plays a major role in determining mechanical properties, the effect of the solute including proteins, lipids and other components is apparent especially on viscosity, which is relevant for the transport of metabolites and active molecules.The research was published in Dr Palombo, an Associate Professor in Biomedical Spectroscopy at the University of Exeter, said: "We set out to understand the bases of Brillouin signals in biomedical samples."While taking a step back to analyse the fundamentals of this light scattering process, we made a substantial advancement in that we now understand the distinctive contribution of interfacial dynamics, beyond bulk water, to the viscoelastic response of biological tissues."This has wide-ranging implications in that phase changes, as well as acoustic anisotropy, are ideal scenarios where Brillouin imaging provide unique information. We are still working on establishing the relevance of this technique in medical sciences, however it is undisputable that it offers an invaluable contrast mechanism to detect physiological and disease states."
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Biotechnology
| 2,020 |
November 10, 2020
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https://www.sciencedaily.com/releases/2020/11/201110133200.htm
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Organoids produce embryonic heart
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There was a time when the idea of growing organs in the lab was the stuff of science fiction. Today, stem cell biology and tissue engineering are turning fiction into reality with the advent of organoids: tiny lab-grown tissues and organs that are anatomically correct and physiologically functional.
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The appeal of organoids is obvious. Essentially, they can provide us with an on-demand production of tissues and mini-organs for pharmaceutical and medical research, without constantly having to rely on donors. And although that goal might still be a long way off, we're slowly getting closer.EPFL has been involved in the development of organoids for a while now, with the lab of Matthias Lütolf at the School of Life Sciences leading the charge. This year alone, Lütolf's group has published papers on standardizing organoid growth, 3D-printing organoids, and actually producing a functional organoid-based mini-intestine -- a groundbreaking Nature paper that is leading the way in this field.Now, Lütolf's lab has successfully produced a mouse heart organoid in its early embryonic stages. The project was led by Giuliana Rossi, a post-doctoral researcher from Lütolf's laboratory, and published in the journal The researchers grew their organoids from mouse embryonic stem cells, which, under the right conditions, can self-organize into structures that "mimic aspects of the architecture, cellular composition, and function of tissues found in real organs," as the researchers put it in the paper. Placed in cell-culture under specific conditions, the embryonic stem cells form a three-dimensional aggregate called a "gastruloid," which can follow the developmental stages of the mouse embryo.The idea behind this study was that the mouse gastruloid can be used to mimic the early stages of heart development in the embryo. This is a rather unexplored use of organoids, which are generally grown to mimic adult tissues and organs. But there are three features of mouse gastruloids that make them a suitable template for mimicking embryonic development: they establish a body plan like real embryos, and they show similar gene expression patterns. And when it comes to the heart, which is the first organ to form and function in the embryo, the mouse gastruloid also preserves important tissue-tissue interactions that are necessary to grow one.Armed with this, the researchers exposed mouse embryonic stem cells to a "cocktail" of three factors known to promote heart growth. After 168 hours, the resulting gastruloids showed signs of early heart development: they expressed several genes that regulate cardiovascular development in the embryo, and they even generated what resembled a vascular network.But more importantly, the researchers found that the gastruloids developed what they call an "anterior cardiac crescent-like domain." This structure produced a beating heart tissue, similar to the embryonic heart. And much like the muscle cells of the embryonic heart, the beating compartment was also sensitive to calcium ions.Opening up an entirely new dimension to organoids, the breakthrough work shows they can also be used to mimic embryonic stages of development. "One of the advantages of embryonic organoids is that, through the co-development of multiple tissues, they preserve crucial interactions that are necessary for embryonic organogenesis," says Giuliana Rossi. "The emerging cardiac cells are thus exposed to a context similar to the one that they encounter in the embryo."
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Biotechnology
| 2,020 |
November 10, 2020
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https://www.sciencedaily.com/releases/2020/11/201110081600.htm
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Making 3D nanosuperconductors with DNA
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Three-dimensional (3-D) nanostructured materials -- those with complex shapes at a size scale of billionths of a meter -- that can conduct electricity without resistance could be used in a range of quantum devices. For example, such 3-D superconducting nanostructures could find application in signal amplifiers to enhance the speed and accuracy of quantum computers and ultrasensitive magnetic field sensors for medical imaging and subsurface geology mapping. However, traditional fabrication tools such as lithography have been limited to 1-D and 2-D nanostructures like superconducting wires and thin films.
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Now, scientists from the U.S. Department of Energy's (DOE) Brookhaven National Laboratory, Columbia University, and Bar-Ilan University in Israel have developed a platform for making 3-D superconducting nano-architectures with a prescribed organization. As reported in the Nov. 10 issue of "Because of its structural programmability, DNA can provide an assembly platform for building designed nanostructures," said co-corresponding author Oleg Gang, leader of the Soft and Bio Nanomaterials Group at Brookhaven Lab's Center for Functional Nanomaterials (CFN) and a professor of chemical engineering and of applied physics and materials science at Columbia Engineering. "However, the fragility of DNA makes it seem unsuitable for functional device fabrication and nanomanufacturing that requires inorganic materials. In this study, we showed how DNA can serve as a scaffold for building 3-D nanoscale architectures that can be fully "converted" into inorganic materials like superconductors."To make the scaffold, the Brookhaven and Columbia Engineering scientists first designed octahedral-shaped DNA origami "frames." Aaron Michelson, Gang's graduate student, applied a DNA-programmable strategy so that these frames would assemble into desired lattices. Then, he used a chemistry technique to coat the DNA lattices with silicon dioxide (silica), solidifying the originally soft constructions, which required a liquid environment to preserve their structure. The team tailored the fabrication process so the structures were true to their design, as confirmed by imaging at the CFN Electron Microscopy Facility and small-angle x-ray scattering at the Complex Materials Scattering beamline of Brookhaven's National Synchrotron Light Source II (NSLS-II). These experiments demonstrated that the structural integrity was preserved after they coated the DNA lattices."In its original form, DNA is completely unusable for processing with conventional nanotechnology methods," said Gang. "But once we coat the DNA with silica, we have a mechanically robust 3-D architecture that we can deposit inorganic materials on using these methods. This is analogous to traditional nanomanufacturing, in which valuable materials are deposited onto flat substrates, typically silicon, to add functionality."The team shipped the silica-coated DNA lattices from the CFN to Bar-Ilan's Institute of Superconductivity, which is headed by Yosi Yeshurun. Gang and Yeshurun became acquainted a couple years ago, when Gang delivered a seminar on his DNA assembly research. Yeshurun -- who over the past decade has been studying the properties of superconductivity at the nanoscale -- thought that Gang's DNA-based approach could provide a solution to a problem he was trying to solve: How can we fabricate superconducting nanoscale structures in three dimensions?"Previously, making 3-D nanosuperconductors involved a very elaborate and difficult process using conventional fabrication techniques," said Yeshurun, co-corresponding author. "Here, we found a relatively simple way using Oleg's DNA structures."At the Institute of Superconductivity, Yeshurun's graduate student Lior Shani evaporated a low-temperature superconductor (niobium) onto a silicon chip containing a small sample of the lattices. The evaporation rate and silicon substrate temperature had to be carefully controlled so that niobium coated the sample but did not penetrate all the way through. If that happened, a short could occur between the electrodes used for the electronic transport measurements."We cut a special channel in the substrate to ensure that the current would only go through the sample itself," explained Yeshurun.The measurements revealed a 3-D array of Josephson junctions, or thin nonsuperconducting barriers through which superconducting current tunnels. Arrays of Josephson junctions are key to leveraging quantum phenomena in practical technologies, such as superconducting quantum interference devices for magnetic field sensing. In 3-D, more junctions can be packed into a small volume, increasing device power."DNA origami has been producing beautiful and ornate 3-D nanoscale structures for almost 15 years, but DNA itself is not necessarily a useful functional material," said Evan Runnerstrom, program manager for materials design at the U.S. Army Combat Capabilities Development Command Army Research Laboratory of the U.S. Army Research Office, which funded the work in part. "What Prof. Gang has shown here is that you can leverage DNA origami as a template to create useful 3-D nanostructures of functional materials, like superconducting niobium. This ability to arbitrarily design and fabricate complex 3-D-structured functional materials from the bottom-up will accelerate the Army's modernization efforts in areas like sensing, optics, and quantum computing.""We demonstrated a pathway for how complex DNA organizations can be used to create highly nanostructured 3-D superconducting materials," said Gang. "This material conversion pathway gives us an ability to make a variety of systems with interesting properties -- not only superconductivity but also other electronic, mechanical, optical, and catalytic properties. We can envision it as a "molecular lithography," where the power of DNA programmability is transferred to 3-D inorganic nanofabrication."
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Biotechnology
| 2,020 |
November 9, 2020
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https://www.sciencedaily.com/releases/2020/11/201109124732.htm
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Yin and Yang: Two signaling molecules control growth and behavior in bacteria
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Bacteria are considered to be true experts in survival. Their rapid adaptive response to changing environmental conditions is based, among other things, on two competing signaling molecules. As the "Yin and Yang" of metabolic control they decide on the lifestyle of bacteria, as reported by researchers from the University of Basel. The new findings also play a role in the context of bacterial infections.
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Whether they are pathogens, deep-sea microbes or soil-dwelling organisms, in order to survive, microorganisms must be able to adapt rapidly to diverse changes in their environment, including nutrient depletion. Bacteria owe their extraordinary ability to quickly adjust to adverse living conditions to small signaling molecules.Scientists headed by Professor Urs Jenal and Professor Tilman Schirmer from the Biozentrum, University of Basel, have now discovered that bacteria use two chemically related signaling molecules to adapt their lifestyle to the prevailing living conditions. The researchers present their results in the latest issue of The researchers investigated the antagonistic nature of the two signaling molecules ppGpp and c-di-GMP in the cell using Caulobacter crescentus as a model organism. This bacterium can slip into two different roles: It can be found in a free-swimming form that is unable divide and in a surface-attached, reproductive state.Both the lifestyle and the environmental conditions are reflected in the concentration of the two signaling molecules. This information is detected by a protein that binds both signaling molecules and acts a molecular switch, controlling growth, metabolism and lifestyle of the bacterium.The signaling molecules ppGpp und c-di-GMP compete for binding to the master switch. "In swarming bacteria with high levels of ppGpp, the protein is switched on; it is active," explains Urs Jenal. "In this state, glucose consumption is in full swing. Simultaneously, the resulting harmful oxygen radicals are efficiently neutralized." This ensures, that metabolic reactions adapt to the high energy demand of the motile swimmer cells and cell damage is averted.Under favorable living conditions, providing sufficient nutrients, the level of c-di-GMP increases constantly, forcing the swimmer to develop into a sessile form. "In this case, c-di-GMP displaces ppGpp from the protein's binding pocket, it changes its structure and turns itself off," says Jenal. "This redirects metabolic reactions allowing bacteria to settle, grow and reproduce. The production of building blocks for the cell is boosted along with adhesive substances for surface attachment."With the molecular master switch, the scientists have discovered the link between two large regulatory networks, which until now have been thought to operate independently. Although Caulobacter is a harmless environmental bacterium, the newly uncovered "Yin and Yang" mechanism could also play an important role in pathogens.This may prove to be of key importance: Both ppGpp and c-di-GMP influence bacterial virulence and persistence as well as antibiotic resistance in different ways, thus influencing the course of many infections.
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Biotechnology
| 2,020 |
November 9, 2020
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https://www.sciencedaily.com/releases/2020/11/201109074103.htm
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Cell aging can be slowed by oxidants
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At high concentrations, reactive oxygen species -- known as oxidants -- are harmful to cells in all organisms and have been linked to ageing. But a study from Chalmers University of Technology, Sweden, has now shown that low levels of the oxidant hydrogen peroxide can stimulate an enzyme that helps slow down the ageing of yeast cells.
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One benefit of antioxidants, such as vitamins C and E, is that they neutralise reactive oxygen species -- known as oxidants -- which may otherwise react with important molecules in the body and destroy their biological functions. Larger amounts of oxidants can cause serious damage to DNA, cell membranes and proteins for example. Our cells have therefore developed powerful defence mechanisms to get rid of these oxidants, which are formed in our normal metabolism.It was previously believed that oxidants were only harmful, but recently we have begun to understand that they also have positive functions. Now, the new research from Chalmers University of Technology shows that the well-known oxidant hydrogen peroxide can actually slow down the ageing of yeast cells. Hydrogen peroxide is a chemical used for hair and tooth whitening, among other things. It is also one of the oxidants formed in our metabolism that is harmful at higher concentrations.The Chalmers researchers studied the enzyme Tsa1, which is part of a group of antioxidants called peroxiredoxins."Previous studies of these enzymes have shown that they participate in yeast cells' defences against harmful oxidants," says Mikael Molin, who leads the research group at Chalmers' Department of Biology and Biological Engineering. "But the peroxiredoxins also help extend the life span of cells when they are subjected to calorie restriction. The mechanisms behind these functions have not yet been fully understood."It is already known that reduced calorie intake can significantly extend the life span of a variety of organisms, from yeast to monkeys. Several research groups, including Mikael Molin's, have also shown that stimulation of peroxiredoxin activity in particular is what slows down the ageing of cells, in organisms such as yeast, flies and worms, when they receive fewer calories than normal through their food."Now we have found a new function of Tsa1," says Cecilia Picazo, postdoctoral researcher at the Division of Systems and Synthetic Biology at Chalmers. "Previously, we thought that this enzyme simply neutralises reactive oxygen species. But now we have shown that Tsa1 actually requires a certain amount of hydrogen peroxide to be triggered in order to participate in the process of slowing down the ageing of yeast cells."Surprisingly, the study shows that Tsa1 does not affect the levels of hydrogen peroxide in aged yeast cells. On the contrary, Tsa1 uses small amounts of hydrogen peroxide to reduce the activity of a central signalling pathway when cells are getting fewer calories. The effects of this ultimately lead to a slowdown in cell division and processes linked to the formation of the cells' building blocks. The cells' defences against stress are also stimulated -- which causes them to age more slowly."Signal pathways which are affected by calorie intake may play a central role in ageing by sensing the status of many cellular processes and controlling them," says Mikael Molin. "By studying this, we hope to understand the molecular causes behind why the occurrence of many common diseases such as cancer, Alzheimer's disease, and diabetes shows a sharp increase with age."The fact that researchers have now come a step closer to understanding the mechanisms behind how oxidants can actually slow down the ageing process could lead to new studies, for example looking for peroxiredoxin-stimulating drugs, or testing whether age-related diseases can be slowed by other drugs that enhance the positive effects of oxidants in the body.The Chalmers researchers have shown a mechanism for how the peroxiredoxin enzyme Tsa1 directly controls a central signalling pathway. It slows down ageing by oxidising an amino acid in another enzyme, protein kinase A, which is important for metabolic regulation. The oxidation reduces the activity of protein kinase A by destabilising a portion of the enzyme that binds to other molecules. Thus, nutrient signalling via protein kinase A is reduced, which in turn downregulates the division of cells and stimulates their defence against stress.
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Biotechnology
| 2,020 |
November 9, 2020
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https://www.sciencedaily.com/releases/2020/11/201109132439.htm
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A key player behind parental chromosome matching during meiosis
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A research collaboration based in Kumamoto University (Japan) has clarified how homologous chromosome pairing -- a process necessary for sperm and egg formation where paternally- and maternally-derived chromosomes match and exchange genetic information during meiosis -- attracts factors that play a monitoring role. Since details on the genetic information exchange mechanism during meiosis have not yet been clarified, this research may lead to future advances in reproductive medicine, such as the identification of the causes of infertility.
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Meiosis is a special type of cell division that takes place in the ovaries and testes to produce eggs and sperm. Here, maternal and paternal chromosomes of the same type are aligned in a process called "homologous chromosome pairing." This meiotic recombination results in the partial exchange of genetic information. Homologous chromosome pairing is an essential process for matching paternal and maternal DNA and facilitating the exchange of DNA sequences between them, but if it doesn't work, meiotic recombination doesn't occur normally and eggs and sperm cannot form.Homologous chromosome pairing is known to have a mechanism to monitor whether accurate matching is occurring. However, details about how the mechanism is induced on chromosomes is still unclear. Its dysfunction is an important but poorly understood problem with direct implications for reproductive medicine. Researchers at Kumamoto University's >Institute of Molecular Embryology and Genetics (IMEG), in collaboration with the University of Tokyo's Institute for Quantitative Biosciences, have shown that a protein complex called cohesin calls for a watchdog factor during homologous chromosome pairing, and that it also plays an important role as the backbone of the axial structure that binds chromosomes.In previous studies, a protein called HORMAD1 was shown to emerge along the top of the chromosome during meiosis to monitor the successful matching of homologous chromosomes (Shin et al., 2010, Daniel et al., 2011). Mass spectrometry analysis revealed that HORMAD1 binds to two proteins, SYCP2 and cohesin, both of which are major components of meiotic chromosomes. Furthermore, HORMAD1 appeared normally on chromosomes by binding to cohesin even after the SYCP2 gene was knocked out using genome editing, but it did not function normally when cohesin was knocked out. Thus, it became clear that cohesin acts as a marker to call HORMAD1 to the chromosome prior to homologous chromosome pairing.During meiosis, chromosomes are held together by special "axis" structures that play a pivotal role in the normal matching of paternal and maternal chromosomes and the exchange of genetic information. Importantly, the cohesin molecule acts as the backbone for these axis structures."These results were validated in mice, but cohesin and HORMAD1 proteins work in human germ cells as well," said Associate Professor Keiichiro Ishiguro, who led the research project. "If this mechanism fails, meiotic recombination will not work properly. This leads to a significant decrease in sperm and egg formation resulting in infertility. There are many cases of human infertility where the cause is unknown, but we hope that this discovery will help to clarify the pathogenesis for many of the people affected."
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Biotechnology
| 2,020 |
November 7, 2020
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https://www.sciencedaily.com/releases/2020/11/201107133911.htm
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How cell processes round up and dump damaged proteins
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In a new paper with results that senior author Eric Strieter at the University of Massachusetts Amherst calls "incredibly surprising," he and his chemistry lab group report that they have discovered how an enzyme known as UCH37 regulates a cell's waste management system.
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Strieter says, "It took us eight years to figure it out, and I'm very proud of this work. We had to develop a lot of new methods and tools to understand what this enzyme is doing."As he explains, a very large protease called a proteasome is responsible for degrading the vast majority of proteins in a cell; it may be made up of as many as 40 proteins. It has been known for more than 20 years that UCH37 is one of the regulatory enzymes that associates with the proteasome, he adds, "but no one understood what it was doing."It turns out that the crux of the whole process, he adds, is how complicated modifications in a small protein called ubiquitin can be. "In addition to modifying other proteins, ubiquitin modifies itself resulting in a wide array of chains. Some of these chains can have extensive branching. We found that UCH37 removes branchpoints from chains, allowing degradation to proceed."Writing this week in This advance could eventually lead to a new cancer treatment, Strieter says, because cancer cells need the proteasome to grow and proliferate. "Many cancer cells are essentially addicted to proteasome function," he points out. "Its cells produce proteins at such a fast rate that mistakes are made, and if these are not cleared out, cells can't function. Since UCH37 aids in clearing out proteins, it could be a useful therapeutic target to add to the proteasome inhibitors that have already been successful in the clinic."To begin their years-long process, Strieter says, "we had to come up with a way to generate a wide variety of ubiquitin chains that would represent the potential diversity in a cell. Using that new library of ubiquitin chains allowed us to interrogate the activity of UCH37 in a controlled setting. That series of experiments gave us the first clue that this enzyme was doing something unique."Another new method they developed uses mass spectrometry to characterize the architecture of ubiquitin chains in complex mixtures. "This allowed us to see that the activity we discovered with our library of substrates was also present in a more heterogenous mixture," Strieter says. Finally, the chemists used the CRISPR gene editing tool to remove UCH37 from cells to measure the impact of UCH37 on proteasome-mediated degradation in vitro and in cells.This technique led to one more surprise. "Instead of acting as expected and opposing the degradation process, it turned out that UCH37 was removing branchpoints from ubiquitin chains to help degrade proteins," Strieter says. "You would think that by removing the signal for degradation that degradation would be impaired," he adds, "but it didn't work that way."In future experiments, Strieter and colleagues hope to further explore the degradation process and learn in more detail how UCH37 manages to regulate cellular function.
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Biotechnology
| 2,020 |
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