Tag Archives: #nucleotide

Researchers Discover Nucleotide Sequence Responsible For Effectively Fighting Pathologies (Biology)

HSE researchers uncover the fundamental mechanisms behind the maturation of microRNA molecules

Researchers from HSE University have discovered nucleotide sequences characteristic of microRNA isoforms (microRNAs with errors). The discovery will help predict errors in microRNA behaviour and create drugs that can detect targets (such as viruses) more effectively. The results of the study have been published in the RNA Biology journal.

MicroRNAs (miRNAs) are very small molecules that regulate all the processes in a cell, including the transformation of inherited information in RNA or proteins (gene expression). Each microRNA has its own unique set of targets–genes whose activity it can suppress. Recent studies show that even slight changes in microRNA nucleotide sequences (so-called microRNA isoforms or isomiRs) can completely rebuild numerous targets. This can drastically alter the biological function of the molecule. However, until recently, researchers did not know why some microRNAs have isoforms, while others do not.

HSE Faculty of Biology and Biotechnology researchers Anton Zhiyanov, Stepan Nersisyan, and Alexander Tonevitsky applied bioinformatics methods to find the answer to this question. The team managed to create an algorithm that characterizes the fundamental differences between microRNAs that have isoforms and those that do not.

Stepan Nersisyan, Junior Research Fellow at the HSE International Laboratory of Microphysiological Systems © Stepan Nersisyan

Their study also has important applications for the creation of artificial molecules similar to microRNAs. Dozens of research teams across the globe are currently working to solve this problem. Researchers artificially synthetize molecules that are similar to microRNAs (so-called short hairpin RNAs or shRNAs) in order to ‘knock down’ the gene they are interested in. In addition to having academic applications, this technology is also used in therapy to suppress ‘bad’ genes that cause diseases.

The authors of the study demonstrated that such artificially synthetized molecules can also have isoforms.

‘Some combinations of nucleotides (AGCU, AGUU) are most often found in microRNAs where no errors occur. Combinations such as CCAG and some of its variations can predict changes and target failure with up to 70% precision. Sequencing short hairpin RNAs from our own experiments revealed that they also have isoforms. This means that it is possible to have a situation where we invent a molecule with a specific list of targets, but in practice, isoforms appear with unintended targets of their own. Our algorithm helps predict such events at the computer analysis stage without having to carry out costly experiments,’ said Stepan Nersisyan, Junior Research Fellow at the HSE International Laboratory of Microphysiological Systems.

Featured image: The pattern discovered by the researchers. Letters that grow upwards represent error-free microRNA processing, while those growing downwards represent a processing pattern with errors. The bigger the letter, the stronger the correlation. © Nersisyan S. et al.


Reference: Anton Zhiyanov, Stepan Nersisyan & Alexander Tonevitsky (2021) Hairpin sequence and structure is associated with features of isomiR biogenesis, RNA Biology, DOI: 10.1080/15476286.2021.1952759


Provided by HSE

Penn Researchers Discover Two Key Events That Turn Normal Cells into Cancer (Medicine)

The findings may inform the development of new therapies that could treat any tumor type.

More than 100 different cancers can arise all over the body, but two universal metabolic pathways may tie them all together, researchers from the Perelman School of Medicine at the University of Pennsylvania report in a new study published today online in Cell Metabolism. Researchers have long believed all cancers are governed by a common set of fundamental processes. Exactly what those were, however, has remained elusive.

The findings may improve the development of new therapies that could treat any tumor type ©Penn state

Having a unifying mechanism could inform new therapeutic approaches to prevent normal cells from transforming into any type of tumor, be it breast, prostate, or colon, for example.

The team discovered how the transformation from a phenotypically normal cell to a cancerous one involves the enhancement of two key elements: antioxidant defense and nucleotide synthesis. Genes associated with cancer, they found, are super charging some cells to fight off oxidative stress and synthesize nucleotides, which cells need to survive and rapidly grow, respectively.

“Since the early 1980s, numerous cancer genes have been identified. However, they often affect multiple cellular processes, which makes it very hard to really summarize what exactly turns cells cancerous,” said senior author Xiaolu Yang, PhD, a professor of Cancer Biology in the Perelman School of Medicine at the University of Pennsylvania. “We took a unique approach and looked at the cellular changes driven by a particular metabolic enzyme, which turned out to be the key here. Strikingly, we found that for a phenotypically normal cell to become a cancer cell, all it needs to do is to be equipped with the extra capacity to endure oxidative stress and produce nucleotides.”

Shut down these metabolic pathways, the study suggests, and the cells don’t become cancerous.

The researchers first overexpressed the gene G6PD, which makes the enzyme glucose-6-phosphate dehydrogenase, in mice and human cells. That enzyme is active in nearly all cells in the body and involved in the normal processing of carbohydrates. They showed that this overexpression alone turned human cells cancerous and led to tumors in the mice.

Next, they analyzed the mechanisms involved in that overexpression to pinpoint what pathways were critical to the transformation. They found that G6PD stimulates production of new NADPH, a crucial co-enzyme for maintaining redox balance (which keeps the cell from being damaged and dying off), as well as more nucleotide precursors to keep them multiplying. Under conditions that elicit oxidative stress, which are often encountered by cancer cells due to their relentless proliferation, often in a wrong place, a normal cell would buckle, but a cancer cell armed with these additions presses on.

The findings also lend further evidence shown in clinical trials and other studies that antioxidants in fact support tumor growth, not decrease it. For a tumor to form, it needs a robust antioxidant defense; giving it more antioxidants provides an ideal environment for it to do that. The findings also provide an explanation for the observation that compounds interfering with nucleotide biosynthesis are among the most successful chemotherapeutic drugs for cancer.

Importantly, the study reveals a molecular framework to better understand the process of oncogenesis and a potential road map for new approaches to treat cancer, the authors said.

“Now we can say that the oncogenic transformation comes from two fundamental steps,” Yang said. “Our study also suggests that combining therapeutics that affect both events, some which are already in clinics, would be more effective at preventing normal cells from becoming cancerous.”

The first author of this study, Yang Zhang, is a postdoctoral researcher in Yang’s lab. Other co-authors include Yi Xu, Lili Guo, and Ian A. Blair, also from Penn, as well as Wenyun Lu, Jonathan M. Ghergurovich, and Joshua D. Rabinowitz from Princeton University.

The study was supported by the National Institutes of Health (R01CA184867, R01CA235760, R01CA243520 and R50CA211437).

References: Yang Zhang, Yi Xu, Wenyun Lu, Jonathan M. Ghergurovich, Lili Guo, Ian A. Blair, Joshua D. Rabinowitz, Xiaolu Yang, “Upregulation of Antioxidant Capacity and Nucleotide Precursor Availability Suffices for Oncogenic Transformation”, Cell Metabolism, 2020, ISSN 1550-4131,
https://doi.org/10.1016/j.cmet.2020.10.002.
(http://www.sciencedirect.com/science/article/pii/S1550413120305350)

Provided by Penn State University

A Simple, Cost-effective Molecular Assay May Help Manage Growing Spread Of Drug-resistant Gonorrhea (Medicine)

A multiplex assay based on high-resolution melting technology accurately identified antimicrobial resistant determinants in Neisseria gonorrhoeae, researchers report in the Journal of Molecular Diagnostics.

A dual therapy treatment regimen of expanded-spectrum cephalosporins (ESCs) plus azithromycin (AZM) is the recommended standard of care for gonorrhea. A strain of the Neisseria (N.) gonorrhoeae that is resistant to the ESC and AZM combination has emerged around the world with the potential to make gonorrhea untreatable. The currently used screening methods for antimicrobial resistant (AMR) determinants are slow, expensive, and not widely available. In an article in The Journal of Molecular Diagnostics, published by Elsevier, researchers report a rapid and cheap method that can provide real-time surveillance to help control the spread of AMR strains of N. gonorrhoeae.

Take effective measures against antimicrobial resistant strains of Neisseria gonorrhoeae. ©Junping Peng.

“N. gonorrhoeae has developed resistance to almost all classes of antibiotics that were previously recommended for treatment,” explained lead investigator Junping Peng, MD, NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Key Laboratory of Respiratory Disease Pathogenomics, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. “Timely determination and monitoring of AMR profiles are crucial for appropriately personalized treatment and maintenance of treatment effectiveness.”

Current surveillance systems primarily depend on culture-based methods, which have high sensitivity but long turnaround times. Several nucleic acid amplification testing methods have been developed, but they are expensive and not widely available. Dr. Peng and his team have spent the past several years assessing the status of gonococcal AMR and the development of AMR screening technologies. They designed and developed a multiplex assay based on high-resolution melting (HRM) technology. Compared with other molecular methods that detect a single genetic mutation, HRM technology can detect the most frequent mutations associated with ESCs and AZM resistance in a single test. It uses a real time PCR system, equipment commonly found in most microbiological laboratories and clinical settings.

Forty-eight well-characterized N.gonorrhoeae clinical specimens and 15 non-gonococcal strains were selected for the initial assay establishment. The multiplex HRM assays were able to accurately identify different nucleotide variations of the AMR determinants related to ceftriaxone and azithromycin resistance. Then, results from 556 multiplex HRM tests of clinical isolates and samples were compared with results from whole gene sequencing and PCR sequencing of the same samples. Compared with whole genome sequencing, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex HRM assays for detection of AMR determinants were 98.6 percent, 99.2 percent, 98.6 percent, and 99.2 precent, respectively. The results were available within 90 minutes at a cost of less than $1.00 per sample.

This sequencing-free HRM assay may be applied to large-scale epidemiological programs for increasing surveillance of ESCs and AZM resistance and supporting identification and investigation of antimicrobial-resistant N. gonorrhoeae outbreaks in real-time. By application of this assay, gonococcal AMR surveillance could be enhanced significantly, resulting in improved management programs aimed at controlling the further spread of antimicrobial-resistant N. gonorrhoeae strains and pathogen eradication.

“Our team has been committed to the assessment of the current status of gonococcal AMR and the development of AMR screening technologies to provide scientific and technological support for the effective prevention and treatment of gonorrhea,” observed Dr. Peng. “This fast, simple, and cheap method could have significant implications in resource-limited countries with a high burden of disease,” said Dr. Peng.

References: http://dx.doi.org/10.1016/j.jmoldx.2020.08.003

Provided by Elsevier