Tag Archives: cells

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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Seaweed extract outperforms remdesivir in inhibiting SARS-cov-2 in studies of cells | Instant News


In the test of antiviral efficacy against the virus that causes COVID-19, an extract from edible seaweed, has exceeded remdesivir, the current standard antiviral used to combat the disease. Heparin, a common blood thinner, heparin and variant deprived of its anticoagulant properties, is performed at the same level with remdesivir in inhibiting SARS-cov-2 infection in mammalian cells.

Published online today in Opening Cellsthe study is the latest example of the lure of the strategy, researchers from the center for biotechnology and interdisciplinary studies (CBIS) at Rensselear Polytechnic Institute are developing against viruses such as the coronavirus that triggered the current global health crisis.

The spike protein on the surface of SARS-cov-2 clips on the ACE-2 receptor, a molecule on the surface of human cells. After installation, the virus inserts its genetic material into the cell by hijacking the cell machinery for the production of replica viruses. But the virus can just as easily be convince to lock the bait molecule that offers such devices. Neutralized the virus will be trapped and eventually degraded naturally.

Previous studies have shown that this technique works the decoy in the breeding of wild other viruses, including Dengue, zika and influenza A. to listen to the researchers discuss their findings by watching this video.

We learn to block viral infection, and this knowledge we’ll need if we want to confront the pandemic. The reality is that we have no good antivirals. To protect yourself against future pandemics, we need an Arsenal of approaches that we can quickly adapt to emerging viruses.”

Jonathan Dordick, the lead researcher and Professor of chemical and biological engineering rensselaer Polytechnic Institute

In Opening Cells paper tests of antiviral activity in three variants with heparin (heparin, trisulfated heparin, and without anticoagulant low molecular weight heparin) and two fucoidan (EPI-27 and RPI-28) derived from marine algae. All five compounds are long chains of sugar molecules, known as sulfated polysaccharides, structural conformation that the results of the binding study published earlier this month in anti-virus research is proposed as an effective bait.

Scientists have conducted studies of dose-response is known as the EC50 is the abbreviation for effective concentration of compound that inhibits 50% of viral infectivity — with each of the five compounds on mammalian cells. According to the results the EC50 is given in molar concentration, the lower the value of the signal more powerful composition.

IRTS-27 gave an EC50 value of approximately 83 molar, while similar to the previously published and independent in a test tube remdesivir on the same mammalian cells gave EC50 770 molar. Heparin gave EC50 of 2.1 microns, or about one-third as active as remdesivir, and the anticoagulant heparin gave similar EC50 5.0 µm, about one-fifth as active as remdesivir.

A separate test detected a toxic effect on the cells in any of the compounds, even at the highest concentration.

“What we want is a new way to get an infection,” said Robert Linhardt, Professor Rensselaer chemistry and chemical biology, which cooperates with the Dordick to develop a bait strategy. “Current thinking is that COVID-19 infection starts in the nose, and any of these substances could be the basis for a nasal spray. If you could just treat the infection, or early treatment even before you have the infection, you would have a way to block it before it enters the body”.

Added Dordick that compounds from algae “can serve as the basis for the oral approach, and delivery to address potential gastrointestinal infections.”

In the study of SARS-cov-2 sequencing data, Dordick and Linhardt identified several motifs on the structure of the spike protein, which is promised to Fit compatible with heparin, as a result, it was confirmed during the mandatory study. The spike protein is heavily encrusted in glycans, it is a fixture that protects it from human enzymes that can degrade it, and prepares it for binding with the specific receptor on the cell surface.

“This is a very complex mechanism that we, frankly, don’t know all the details, but we get more information,” said Dordick. “The only thing that was clear from this study is that the larger the molecule, the better suited you. More successful connections of large sulfated polysaccharides that offer a larger number of sites on the molecules to trap the virus.”

Molecular simulation-based binding study showed the sites on the spike protein where heparin can interact, increasing the prospects for such sulfated polysaccharides.

“This is a fascinating study of Professor Dordick and Linhardt among several ongoing studies in CBIS, as well as elsewhere in Rensselaer, for solving problems COVID-19 pandemic using new therapeutic approaches and repurpose existing drugs,” said CBIS Director Deepak Vashishth.

“Sulfated polysaccharides can effectively inhibit the SARS-cov-2 in vitro” was published in Opening Cells supported by the National research Foundation of Korea. In Rensselaer, Dordick and Linhardt were United in the study of Paul S. Kwon, Seok-Joon Kwon, Jin goes, fuming Zhang, and Kate Fraser and researchers at the Korea research Institute of Bioscience and biotechnology in Cheongju, Republic of Korea, and Zhejiang University of technology in Hangzhou, China.

Source:

Journal reference:

Kwon, S. P., et al. (2020) sulfated polysaccharides can effectively inhibit the SARS-cov-2 in vitro. The Opening Of The Cell. doi.org/10.1038/s41421-020-00192-8.

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


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.”

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


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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