Tag Archives: Gene

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Simultaneous mutation of two nonessential genes can lead to the death of cancer cells | Instant News


Ludwig cancer research, has revealed a new instance in which simultaneous mutation of two nonessential genes;none of which are vital for the survival of cells can cause cancer cell death.

Headed by member of the Ludwig San Diego Richard Kolodner and published in the current The proceedings of the National Academy of Sciences, the study also shows that it is a deadly combination, or “synthetic lethality” can be reproduced in a drug-like molecule that can be used to treat cancer.

The development and FDA approval of a new generation of drugs called PARP inhibitors, for the treatment of malignant tumors with defects in tumor suppressor genes BRCA1 and BRCA2 that cause breast cancer, ovarian and many other cancers, have generated significant interest in using synthetic lethal interactions to develop cancer treatments.

Scientists, including group Kolodner, are on the hunt for other synthetic lethal interactions in cancers. “PARP inhibitors are a major step forward, but they are not perfect. Patients can become resistant to them, so there’s always a need for new and better treatments.”

Building from research done on yeast cells, Kolodner and his colleagues found that disabling or removing FEN1 gene of mammals, which is essential for DNA replication and repair, is fatal to cancer cells, mutated forms of the genes BRCA1 and 2.

We have provided information that should make people think FEN1 as a potential interesting therapeutic target and showed how yeast can be used to predict a number of synthetic lethal interactions, which can then be tested in a bona FIDE cancer cell lines with genetic instruments”.

Richard Kolodner, Professor, Professor, Department of cellular and molecular medicine, University of California, San Diego

In previous work with yeast Saccharomyces as a model to identify and study genes that maintain the integrity of the genome, Kolodner and his colleagues found that the RAD27 gene, and of synthetic lethal interactions with the 59 other nonessential genes of yeast.

Two such genes, it should be noted RAD51 and RAD52 play a role in recombination of DNA.

FEN1 is a close analogue or homologue, RAD27 in mammals. Based on their studies of yeast, Kolodner and his colleagues predicted that FEN1 synthetic lethal interactions with BRCA1 and BRCA2, which function in the same biochemical reactions in mammals, as RAD51 and RAD52 to do in yeast.

To test this hypothesis, they synthesized four FEN1-blocking molecules and used the best of them, S8, to suppress the activity of FEN1 in tumor cell lines with or without BRCA mutations. C8 proved to be an effective killer of BRCA-mutant cells.

Then they demonstrated that genetic disorders FEN1 expression had the same effect that S8 did for the breast cancer gene-mutant cells, confirming that the S8 worked, causing synthetic lethality.

Finally, the researchers instilled in C8-C8 sensitive and-resistant tumors in mice and showed that C8 significantly inhibited the growth of C8-sensitive tumors, but not in C8-resistant tumors.

Interestingly, not all cancer cell lines and tumors that responded to treatment C8 was deficient BRCA, K, indicating that FEN1 and synthetic lethal interactions with other genes as well.

These results reveal FEN1 as a novel target for drugs for the treatment of various malignant tumors by induction of synthetic lethality.

They also demonstrate that yeast-based screens provide a powerful tool to accelerate the discovery of synthetic lethal interactions for potential therapeutic value;it is an ongoing project in the laboratory Kolodner.

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Neurologists on the map touch the gatekeeper of the brain in unprecedented detail | Instant News


Considered Emily Henderson, Bachelor.22 Jul 2020

Many people with autism experience sensory hypersensitivity, attention deficit, sleep disorders. One area of the brain that were involved in these symptoms of the thalamic reticular nucleus (ryat), which is believed to acts as a gatekeeper for sensory information entering the cortex.

A group of researchers from mit and the broad Institute of MIT and Harvard, is now visible in unprecedented detail TIN, showing that the region contains two different subnets of neurons with different functions. The obtained results have more to offer researchers specific targets for drug development that could facilitate some of the senses, sleep and attention symptoms of autism, says Guoping Feng, one of the leaders of the research team.

The idea is that you can very specifically one group of neurons without affecting the whole brain, and other cognitive functions”.

Guoping Feng, the James and Patricia Poitras Professor of neuroscience at MIT and a member of the Institute of mit McGovern Institute for brain research

Feng; SMA-Fu, Deputy Director of neurobiology in the center of the broad Institute of psychiatric research, Stanley; and Joshua Levin, senior group leader at broad Institute, senior author of the study, which appears today in Nature. Leading the report’s authors, former post-doctoral research fellowship at MIT, Yinqing Li, a former postdoc of the broad Institute Violeta Lopez-Huerta, and a wide researcher of the Institute of Xian Adiconis.

Some populations

When you receive sensory information from the eyes, ears and other sensory organs to our brain, it goes first to the thalamus, which then relays it to the cortex for higher level processing. The disadvantages of these thalamo-cortical circuit may lead to attention deficit, hypersensitivity to noise and other stimuli, and sleep problems.

One of the main ways, which controls the flow of information from the thalamus and cortex TRN, which is responsible for blocking distracting stimuli. In 2016, Feng and MIT associate Professor Michael Halassa, who is also the author of the new Nature the paper found that the loss under the Ptchd1 gene significantly affect the function of the RNN. In boys, the loss of this gene, which is carried on the X chromosome, may lead to attention deficit, hyperactivity, aggression, mental retardation and autism spectrum disorders.

In this study, the researchers found that when the Gene Ptchd1 was knocked out in mice, animals showed many of the same behavioral defects seen in humans. When he was knocked out only in TRN, the mice showed only hyperactivity, attention deficit, sleep disturbances, assuming that the BCH is responsible for these symptoms.

In the new study, the researchers wanted to try to learn more about specific types of neurons found in the BCH, in the hope of finding new methods of treating hyperactivity and attention deficit. Currently, these symptoms are most commonly treated with stimulants, such as ritalin, which have a wide impact on the entire brain.

“Our goal was to find a particular part, to modulate functions of the thalamo-cortical output and link it to neurological development,” says Feng. “We decided to try using single-cell technology to analyze what types of cells are there and what genes are expressed. There are certain genes that are amenable to therapy with drugs that are included as a target?”

To explore this possibility, the researchers sequenced the messenger RNA molecules found in the neurons of the RNN, which reveals the genes that are expressed in these cells. This allowed them to identify several hundred genes that can be used to differentiate the cells into two subpopulations, based on how strongly they Express certain genes.

They found that one of these cell populations is at the core of TIN, and the other forms a very thin layer around the nucleus. These two populations also form connections of various parts of the thalamus, the researchers found. On the basis of these compounds, the researchers suggest that cells mainly involved in transmission of sensory information to the cortex when cells in the outer layer appear to help to coordinate information across different senses, such as sight and hearing.

“Targets amenable to therapy with drugs included”

Now scientists plan to study the different roles that these two populations of neurons can have different neurological symptoms, including attention deficit, hypersensitivity, and sleep disturbance. Using genetic and optogenetic methods, they hope to determine the effects of activation or inhibition of different TIN cell types, or genes that are expressed in those cells.

“This may help us in the future to develop specific tasks, amenable to therapy with preparations that have the potential to modulate different functions,” says Feng. “Thalamo-cortical circuits control many different things such as sensory perception, sleep, attention, and cognition, and it may well be that they can be targeted more specifically.”

This approach can also be useful for treating disorders of attention or sensitivity, even when not caused by defects in the function of TIN, say the researchers.

“Trn-target where if you can improve its function, you may be able to fix the problems caused by violations of thalamo-cortical circuits,” says Feng. “Of course, we are far from development of any kind of treatment, but the potential that we can use single-cell technology to not only understand how the brain organizes itself, but also how brain function can be separated, allowing to identify more specific targets that modulate specific functions.”

Source:

Massachusetts Institute of Technology

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Health

McGill researchers discover bacterial organelles involved in gene expression | Instant News


Considered Emily Henderson, Bachelor.20 Jul 2020

Scientists from McGill University have discovered a bacterial organelle involved in gene expression, suggesting that the bacteria may not be as easy as many people think. This discovery may offer new targets for developing new antibiotics.

A study published in The proceedings of the National Academy of Sciencesis the first to show that E. coli uses a similar strategy to regulate the transcription of genes as other more complex types of cells.

Just as the human body is composed of organs that perform specialized functions of individual cells contain specialized departments, such as energy-producing organelles called mitochondria. Complex cells contain many different organelles, most of which are surrounded by a membrane that holds them together. Because bacteria do not have membrane-bound organelles, they have been adopted in the absence of them altogether.

Stephanie Weber, assistant Professor, Department of biology, McGill, and her team first showed that bacteria do have such specialized units.

“Our paper provides evidence for the bacterial organelles that are held together by “sticky” proteins, not membrane,” says Weber, who is senior author of the study.

Bacterial organelles described in the study are formed like membraneless compartments for cell phones found in more complex eukaryotes (cells with a nucleus) through a process called phase separation, the same phenomenon that makes oil and vinegar as a dressing for salads.

This is the first direct evidence of phase separation in bacteria, so it can be a universal process in all cell types, and may even have been involved in the Origin of life.”

Stephanie Weber, assistant Professor of biology McGill

Due to the small size of bacterial cells that they studied, the team Weber used a method of imaging – photo-activated localization microscopy – to monitor organelle-shaping proteins.

Weber now trying to understand exactly how proteins assemble into organelles. Because they are involved in the first stages of gene expression – transcription – she believes that they can also become an interesting target for the development of a new generation of antibiotic drugs, which are urgently needed to combat drug resistance.

Source:

Journal reference:

Holiday Home A. et al. (2020) concentrations of bacterial RNA polymerase biomolecular condensate that collects through the liquid–liquid phase separation. Zpz. doi.org/10.1073/pnas.2005019117.

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SARS-cov-2 serological test for 50 cents a sample! | Instant News


Given the continued and alarming increase in both cases and deaths due COVID-19 around the world, scientists are still in search of effective tools for diagnosis of infection. One important way of testing for severe acute respiratory syndrome coronavirus 2 (SARS-cov-2) – serology, but this requires a supply of high-quality recombinant viral antigens.

A recent study published in the preprints server medRxiv* in July 2020 shows that it is indeed possible. The development of this inexpensive, but reliable and sensitive test could change the face of preventive measures for COVID-19 limited resources.

Spike Antigen

SARS-coronavirus-2 spike antigen is one of the most important viral antigens, with researchers focusing on it for the production of vaccines, antivirals and serological tests. Serology is important to track cases, contacts to display, epidemiological surveillance and identification of asymptomatic patients, and identify the mechanism of antiviral immunity.

In Spike protein consists of two subunits, S1 and S2, which mediate receptor binding and membrane fusion, respectively. This allows the virus to penetrate the cell. This protein is a target for neutralizing antibodies, and the most common serological tests for SARS-cov-2 is an enzyme immunosorbent analysis (ELISA) – based protein.

This test is popular for its simplicity and cross-reactivity against the spike proteins of other coronaviruses in the current edition. Moreover, its intensity corresponds to the level of neutralizing antibodies.

Study: characterizing the low-cost IFA

This study was based on the identification of the key features that will allow you to develop an inexpensive test enzyme immunoassay, based on the immunological reaction of antibodies with protein. The goal was to produce a test that will allow large-scale screening for infection in low-income.

Researchers have focused mainly on reducing the cost of manufacturing test by developing optimum means of production of the antigen and making the necessary changes in the method of collecting antigen and sample processing. As a result, they were able to develop a test that will cost less than 50 cents per sample.

To Produce Recombinant Antigen

The researchers cultured SARS-coronavirus-2 protein is in a stable condition prior to the merge in cell culture using a method that allows protein to be expressed stably and as a constitutive feature. It was through the integration of the transgene into the genome of cells in culture.

This adaptation leads to increased scalability, and low cost recombinant proteins. They also used methods of cotransfection gene’s, as well as open source plasmid, which has a selection handle to avoid having to wait for a synthetic gene that needs to be built and sent through the pandemic-disrupted supply chain. Thus, the result is a recombinant cell line expressing high levels of protein were produced within 24 days of transfection and still shows steady expression up to 100 days.

This achievement gives the opportunity to “develop a less expensive, long lasting batch refeed or perfusion technology for cell culture,” according to researchers. They also succeeded in finding an inexpensive nutrient media to support cell growth and high level production of protein. Thus, they have developed a workflow using low-cost methods to achieve the goal of increasing the density of cells in culture with high s secretion of the protein.

Affinity purification chromatography (AC) used resin was more expensive than originally planned ultrafiltration/diafiltration filter (UF/DF), but has become necessary because of failure of the latter to remove smaller contaminants protein. However, they found that it can be used for more than 30 cycles, which reduces the cost of its use.

Then they created an ELISA to detect anti-s antibodies in human serum, plasma, and eluted whole blood samples, called s-ufrj website IFA. They set the amount of highly purified protein (from AC) needed to ensure legible results between negative and positive samples, 150 NG.

Evaluation of sensitivity and specificity

Then the website ufrj Elisa was used to test 210 is negative and positive serum samples, 66 samples from 38 symptomatic COVID-19 patients, 124 samples from the pandemic, and 20 of COVID-19 negative people. They received 122 out of 124 negative samples, the specificity 98%. In addition, the samples 53/66 were positive for IgG, to reduce the sensitivity to 80%.

For comparison, obtained from a commercial IgG rapid diagnostic tests (RDTs), as approved by the Brazilian regulatory health organization, conducted by the Agency only 46% sensitivity.

Then they re-IgG-negative samples from S website ufrj ELISA IgM rapid test. They found that most samples that were negative for IgG and negative for IgM and IgG positive in the first test was IgM positive result of the proximate analysis as well. They, therefore, came to the conclusion that the two false-negative results from symptomatic patients, might have been samples collected early in the disease.

The increase in false positives with increasing duration of symptoms

When the website ufrj ELISA sample results were compared against the duration since symptom onset, they were more likely to be positive, as the duration increased, as a result of seroconversion some individuals who were PCR-positive, negative, scored on IFA for the first time, but positive for anti-s IgG antibodies for the second time. The level of seroconversion to Anti-s IgG antibodies with this test increased from 42% to 100% in direct proportion to the time of onset of symptoms, and from the tenth day, he was consistently above 90%.

An important finding was that the real test detects seroconversion earlier than the rapid test, which had a peak detection rate of 71%, even 20 days from the onset of symptoms.

They also tested the neutralizing ability COVID-19 patients with neutralization of plaque (MFN). Samples with high anti-s IgG antibodies titer was the highest titer of neutralization.

Simplify collection and storage of blood

The researchers also sought to overcome the traditional bottleneck of sample collection and processing in the clinical laboratory under refrigerated storage. They created a simple system for the collection of fingerprick blood in filter paper strips. The use of dried blood spot on filter paper showed comparable results of testing serum.

Dried blood samples (DBS) obtained by a prick of the finger with commercially available devices for piercing. 2.5 cm (W) x 7.5 cm (l) paper filter with three spots of blood from the same volunteers and a commercially available paper hole punching devices have been used DB (arrow), which eluted in the blood for ELISA testing.

The consequences

Thus, low cost of consumables, along with labor, transportation, and equipment costs, all must fit in a half dollar per test, which is approximately 200 times less than the tests currently used in the United States. Another advantage is that spots of blood in sealed plastic bags can be stored for 2 months at least, but still return accurate results serological.

Thus, this study concludes: “on the S website ufrj enzyme immunoassay, including the use of eluates from whole blood pricking her finger as samples, allows a wide serological survey of the population, irrespective of their geographical and socio-economic aspects”. It will be invaluable for the formation of public health strategies to prevent further waves of the pandemic.

*Important Notice

medRxiv publishes preliminary research reports that are not reviewed and therefore should not be considered as a convincing guide to clinical practice/behavior, health-related, or be considered as reliable information.

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Single-cell RNA-seq answers key questions in islet cell biology and diabetes research | Instant News


The pancreas is a stomach organ that produces digestive enzymes and hormones that regulate blood sugar levels. This hormone-producing function is localized on the island of Langerhans, which forms a group of various types of endocrine cells.

Among these are beta cells, which produce the hormone insulin which is needed to lower glucose levels (a type of sugar) in our blood, as well as alpha cells, which produce glucagon hormones whose job is to increase glucose levels in the blood.

Type 1 diabetes is a chronic disease where the immune system mistakenly attacks and destroys pancreatic insulin-producing beta cells. Regenerative medicine aims to replenish beta cell mass, and thus support and ultimately replace current insulin replacement therapy.

Changes in island composition, including lack of beta cell function and beta cell dedifference, also contribute to type II diabetes.

Therefore, a deeper understanding of the identity and crosstalk of various islet cell types leads to better characterization of both forms of diabetes and can contribute to the development of new therapeutic concepts.

Single cell transcriptomes are powerful techniques for characterizing cellular identity. Previously, CeMM researchers from the groups Christoph Bock and Stefan Kubicek at CeMM published the first single cell transcriptome from primary human pancreatic islet cells.

Advances in technology have enabled applications for the generation of global single cell atlase human and mouse transcript. Despite this progress, the single cell approach remains technologically challenging given the very small amount of RNA used in the experiment. Therefore, it is important to ensure the quality and purity of the resulting single cell transcriptome.

CeMM researchers in two laboratories who contributed unexpectedly identified high hormone expression in non-endocrine cell types, both in their own dataset and published single cell studies.

They set out to explain whether this would be the result of contamination by RNA molecules, for example from dying cells, and how they could be removed to obtain a more reliable dataset.

Such contamination appears to be present in RNA-seq single cell data from most tissues but is most visible on pancreatic islets. Islet endocrine cells are exclusively devoted to the production of a single hormone, and insulin in beta cells and glucagon in alpha cells is expressed to a higher level than a typical “household” gene.

Thus, the redistribution of these transcripts to other cell types is very clear. Based on these observations, their goal is to develop, validate and apply methods to experimentally determine and computationally eliminate the contamination.

In their investigation, the CeMM researchers used prickly cells of different cell types, both rat and human samples, which they added to their pancreatic islet samples. Importantly, the transcriptomes of these spike cells are fully characterized.

This enables them to internally and accurately control the level of RNA contamination in a single RNA-seq cell, providing that the human transcripts detected in mouse spike-in cells are contaminated RNA.

In this way, they found that the sample had a contamination rate of up to 20%, and was able to determine the contamination in each sample. They then developed a new bioinformatics approach to computationally eliminate contaminated readings from single cell transcriptomes.

Having now obtained the “decontamination” transcriptome, from which false signals have been removed, they proceed to characterize how cellular identities in different cell types respond to treatment with three different drugs.

They found that small molecular blockers of the FOXO1 transcription factor induced dedifferentiation of both alpha and beta cells.

Next, they studied artemeter, which has been found to reduce alpha cell function and can induce insulin production in both in vivo and in vitro studies. Effects of species-specific drug species and cell types.

In alpha cells, a small proportion of cells increase insulin expression and gain aspects of beta cell identity, both in rat and human samples. Importantly, the researchers found that in human beta cells, there was no significant change in insulin expression, whereas in mouse islands, beta cells reduced insulin expression and overall beta cell identity.

This study is the result of interdisciplinary collaboration from the laboratories of Stefan Kubicek and Christoph Bock at CeMM with Patrick Collombat at the Institute of Biology Valrose (France).

This is the first study to apply single cell sequencing to analyze dynamic drug responses in intact isolated tissue, which benefits from the high quantitative accuracy of the decontamination method.

Thus not only provides a new method for single cell decontamination and a very quantitative single cell analysis of drug responses in intact tissue, but also answers current questions that are important in islet cell biology and diabetes research. These findings could open up potential therapeutic avenues for treating type 1 diabetes in the future.

Source:

CeMM Research Center for Molecular Medicine from the Austrian Academy of Sciences

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