Biology subjects

Nalyvayko, A.

Publications and source records attributed to Nalyvayko, A..

4 recordsLinked to original sources

bioRxiv2024

spotPCR: A Rapid and Efficient Approach for Indexing Individual Template Molecules using Unique Molecular Identifiers

Low-frequency mutations provide valuable insights in various fields, including drug resistance identification, cancer and infectious disease research. One promising strategy to enhance the sensitivity and specificity of mutation detection is the incorporation of unique molecular identifiers (UMIs) during polymerase chain reaction (PCR) amplification and before deep sequencing. However, conventional methods for UMI incorporation oRen necessitate multiple labor-intensive steps. spotPCR (Specific Primer Limited Unique Molecular Identifier Tagging PCR) overcomes these challenges, streamlining the UMI tagging process.

molecular biology↗

bioRxiv2024

How Mycobacterium tuberculosis builds a home: Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation of anti-inflammatory macrophages in infected mouse lungs.

Mycobacterium tuberculosis (MTB) infects and replicates in lung mononuclear phagocytes (MNPs) with astounding ability to evade elimination. ESX-1, a type VII secretion system, acts as a virulence determinant that contributes to MTBs ability to survive within MNPs, but its effect on MNP recruitment and/or differentiation remains unknown. Here, using single-cell RNA sequencing, we studied the role of ESX-1 in MNP heterogeneity and response in mice and murine bone marrow-derived macrophages (BMDM). We found that ESX-1 is required for MTB to recruit diverse MNP subsets with high MTB burden. Further, MTB induces an anti-inflammatory signature in MNPs and BMDM in an ESX-1 dependent manner. Similarly, spatial transcriptomics revealed an upregulation of anti-inflammatory signals in MTB lesions, where monocyte-derived macrophages concentrate near MTB-infected cells. Together, our findings suggest that MTB ESX-1 mediates the recruitment and differentiation of anti-inflammatory MNPs, which MTB can infect and manipulate for survival.

microbiology↗

bioRxiv2022

Circularization of rv0678 for genotypic bedaquiline resistance testing of Mycobacterium tuberculosis

Circular DNA offers benefits over linear DNA in diagnostic and field assays, but currently, circular DNA generation is lengthy, inefficient, highly dependent on the length and sequence of DNA, and can result in unwanted chimeras. We present streamlined methods for generating PCR-targeted circular DNA from a 700bp amplicon of rv0678, the high GC content (65%) gene implicated in Mycobacterium tuberculosis bedaquiline resistance, and demonstrate that these methods work as desired. We employ self-circularization with and without splints, a Gibson cloning-based approach, and novel two novel methods for generating pseudocircular DNA. The circular DNA can be used as a template for rolling circle PCR followed by long-read sequencing, allowing for the error correction of sequence data, and improving the confidence in the drug resistance determination and strain identification; and ultimately improving patient treatment.

molecular biology↗

bioRxiv2017

The Rice XA21 Ectodomain Fused to the Arabidopsis EFR Cytoplasmic Domain Confers Resistance to Xanthomonas oryzae pv. oryzae

O_LIRice (Oryza sativa) plants expressing the XA21 cell surface receptor kinase are resistant to Xanthomonas oryzae pv.oryzae (Xoo) infection. We previously demonstrated that expressing a chimeric protein containing the EFR (ELONGATION FACTOR Tu RECEPTOR) ectodomain and the XA21 endodomain (EFR:XA21) in rice does not confer robust resistance to Xoo.\nC_LIO_LITo test if the XA21 ectodomain is required for Xoo resistance, we produced transgenic rice lines expressing a chimeric protein consisting of the XA21 ectodomain and EFR endodomain (XA21:EFR) and inoculated these lines with Xoo. We also tested if the XA21:EFR rice plants respond to a synthetic sulfated 21 amino acid derivative (RaxX21-sY) derived from the activator of XA21-mediated immunity, RaxX.\nC_LIO_LIWe found that five independently transformed XA21:EFR rice lines displayed resistance to Xoo as measured by lesion length analysis, and showed that five lines express markers of the XA21 defense response (generation of reactive oxygen species and defense response gene expression) after treatment with RaxX21-sY.\nC_LIO_LIOur results indicate that expression of the XA21:EFR chimeric receptor in rice confers resistance to Xoo. These results suggest that the endodomain of the EFR and XA21 immune receptors are interchangeable and the XA21 ectodomain is the key determinant conferring robust resistance to Xoo.\nC_LI

plant biology↗