Biology subjects

Makharashvili, N.

Publications and source records attributed to Makharashvili, N..

3 recordsLinked to original sources

bioRxiv2022

Rapid Phenotypic Detection of Carbapenem-Resistant E. coli with Fluorogenic Culture Media

Faster, and simpler methods that determine the susceptibility of bacterial pathogens to antibiotics are needed to enable more effective treatment of bacterial infections and reduce the overuse of antibiotics. Here, we demonstrate a simple method for rapidly detecting bacteria and simultaneously determining their antibiotic susceptibility status. We demonstrate the method with E. coli, a high-impact pathogen, and meropenem, a member of the carbapenem class of antibiotics which is usually reserved for multi-drug resistant infections. Inclusion of a fluorogenic oligonucleotide substrate for endonuclease I, a well-conserved nuclease of the Enterobacteriaceae family, in a tryptic soy agar-based media enabled fluorescence-based detection of E. coli growth within 9 hours. Plating established carbapenem-resistant and -susceptible E. coli strains on this media with and without inclusion of meropenem, followed by fluorescent imaging yielded a clear phenotypic measure of the antibiotic susceptibility status of the strains in 9 hours. In addition to its simplicity and fast turnaround time, advantages of this approach include its ability to provide a measure of the bacterial load of the tested samples, and its easy integration into current microbiology laboratory workflows.

microbiology↗

bioRxiv2022

Reaction Conditions Promoting the Specific Detection of SARS-CoV-2 NendoU Enzymatic Activity

Methods that enable rapid detection of SARS-CoV-2 provide valuable tools for detecting and controlling Covid-19 outbreaks and also facilitate more effective treatment of infected individuals. The predominant approaches developed use PCR to detect viral nucleic acids or immunoassays to detect viral proteins. Each approach has distinct advantages and disadvantages, but alternatives that do not share the same limitations could enable substantial improvements in outbreak detection and management. For instance, methods that have comparable sensitivity to PCR, but that are not prone to the false-positive results that stem from the tendency of PCR to detect molecular degradation products could improve accurate identification of infected individuals. An alternative approach with potential to achieve this entails harnessing the unique enzymatic properties of SARS-CoV-2 enzymes to generate SARS-Cov-2-specific signals that indicate the presence of the virus. This route benefits from the high sensitivity provided by enzymatic signal amplification and also the fact that signal is generated only by intact viral enzymes, not degradation products. Here, we demonstrate enzymatic reaction conditions that enable the preferential detection of NendoU of SARS-CoV-2, versus several of its orthologues, with a fluorogenic oligonucleotide substrate. These compositions provide a possible technical foundation for a novel approach for detecting SARS-CoV-2 that has distinct advantages from current approaches.

microbiology↗

bioRxiv2018

Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5 flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells.

molecular biology↗