Biology subjects

Tkachenko, E.

Publications and source records attributed to Tkachenko, E..

6 recordsLinked to original sources

bioRxiv2026

cloneXplorer: A high-throughput clone discovery platform based on conical microwell arrays

Antigen-specific T cell populations are of great value for studying immune recognition but tedious to generate by limiting dilution or cloning. Here, we develop a streamlined approach to generate antigen-specific T cell clones directly from peripheral blood using the cloneXplorer, a live-cell analysis and clone isolation platform based on conical microwell arrays. This platform continuously monitors cell proliferation, cytokine secretion, and surface markers in up to 100,000 single cell co-cultures, enabling the identification of rare, functionally defined T cells, which can be recovered for clonal expansion or sequence analysis. We benchmark the platform by performing several key demonstrations. First, we show that this platform can efficiently generate monoclonal cell populations from cell lines and human T cells. Next, we demonstrate that antigen-specificity can be identified at single cell resolution using a co-culture of Jurkat cells expressing NFAT-GFP, CD8, and a T cell receptor and K562 antigen presenting cells (APC) expressing a peptide library. Thereafter, we show that immune activation in mouse and human primary samples can be monitored by time lapse analysis of Interferon gamma (IFN-{gamma}) secretion in individual microwell co-cultures using a fluorescent sandwich assay. Finally, we combine these capabilities in a proof-of-concept demonstration, which uses IFN-{gamma} secretion and the presence of CD8 surface markers as hierarchical gates to isolate and expand antigen-specific T cells from human peripheral blood, and we verify their specificity by tetramer staining. Together, these results showcase potential applications of the cloneXplorer platform in cell line development, and in screening and validating immune receptor interactions with specific antigens. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/699323v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1aafc29org.highwire.dtl.DTLVardef@91272dorg.highwire.dtl.DTLVardef@1a306eeorg.highwire.dtl.DTLVardef@1bfd54_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2018

Precision Identification of Diverse Bloodstream Pathogens from the Gut Microbiome

Bloodstream infection is the most common infectious complication in hematopoietic cell transplantation recipients. To evaluate the genomic concordance of bloodstream pathogens and bacterial strains within the intestinal microbiome using whole genome sequencing, we developed StrainSifter, a bioinformatic pipeline to compare nucleotide variation between bacterial isolate strains and stool metagenomes. We applied StrainSifter to bloodstream isolates and stool metagenome samples from hematopoietic stem cell transplant recipients with bloodstream infections. StrainSifter is designed to identify single nucleotide variants between isolate and metagenomic short reads using stringent alignment, coverage, and variant frequency criteria for strain comparison. We identified enteric BSI isolates that were highly concordant with those in the gut microbiota, as well as highly concordant strains of typically non-enteric bacteria. These findings demonstrate the utility of StrainSifter in strain matching and provide a more precise investigation of the intestine as a reservoir of diverse pathogens capable of causing bloodstream infections.

genomics↗

bioRxiv2017

Household triclosan and triclocarban exposure impacts the adult intestinal microbiome but not the infant intestinal microbiome

In 2016, the US Food and Drug Administration banned the use of specific microbicides in some household and personal wash products. This decision was due to concerns that these chemicals might induce antibiotic resistance or disrupt human microbial communities. Triclosan and triclocarban (referred to as TCs) are the most common antimicrobials in household and personal care products, but the extent to which TC exposure perturbs microbial communities in humans, particularly during infant development, was unknown. We conducted a randomized intervention of TC-containing household and personal care products during the first year following birth to characterize whether TC exposure from wash products perturbs microbial communities in mothers and their infants. Longitudinal survey of the intestinal microbiota using 16S ribosomal RNA amplicon sequencing showed that TC exposure from wash products did not induce global reconstruction of either infant or maternal intestinal microbiotas following 10 months of exposure after birth. However, broadly antibiotic-resistant species from the phylum Proteobacteria were enriched in stool samples from mothers in TC households only after the introduction of triclosan-containing toothpaste. Despite the minimal effects of TC exposure from wash products on the gut microbial community of infants and adults, these results suggest detected taxonomic differences are associated with potential harmful effects on host physiology, highlighting the need for consumer safety testing of self-care products not subject to the ban on the human microbiome and health outcomes.

microbiology↗

bioRxiv2017

De novo assembly of microbial genomes from human gut metagenomes using barcoded short read sequences

Although shotgun short-read sequencing has facilitated the study of strain-level architecture within complex microbial communities, existing metagenomic approaches often cannot capture structural differences between closely related co-occurring strains. Recent methods, which employ read cloud sequencing and specialized assembly techniques, provide significantly improved genome drafts and show potential to capture these strain-level differences. Here, we apply this read cloud metagenomic approach to longitudinal stool samples from a patient undergoing hematopoietic cell transplantation. The patients microbiome is profoundly disrupted and is eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allows us to predict that particular mobile element integrations result in increased antibiotic resistance, which we further support using in vitro antibiotic susceptibility testing. Thus, we find read cloud sequencing to be useful in identifying strain-level differences that underlie differential fitness.

bioinformatics↗

bioRxiv2017

Long-term taxonomic and functional divergence from donor bacterial strains following fecal microbiota transplantation in immunocompromised patients

Immunocompromised individuals are at high risk of developing Clostridium difficile-associated disease (CDAD). Fecal microbiota transplantation (FMT) is a highly effective therapy for refractory or recurrent CDAD and, despite safety concerns, has recently been offered to immunocompromised patients. We investigated the genomics of bacterial composition following FMT in immunocompromised patients over a 1-year period. Metagenomic, strain and gene-level bacterial dynamics were characterized in two CDAD-affected hematopoietic stem cell (HCT) recipients following FMT. We found alterations in gene content, including loss of virulence and antibiotic resistance genes. These alterations were accompanied by long-term bacterial divergence at the species and strain levels. Our findings suggest limited durability of the specific bacterial consortium introduced with FMT and indicate that alterations of the functional potential of the microbiome are more complex than can be inferred by taxonomic information alone. Our observation that FMT alone cannot induce long-term donor-like alterations of the microbiota of HCT recipients suggests that FMT cannot indefinitely supersede environmental and/or host factors in shaping bacterial composition.

genomics↗

bioRxiv2016

Blood flow directs arterial-venous remodeling through Notch activation and endothelial cell migration

Arteries and veins are formed independently by different types of endothelial cells (ECs). In vascular remodeling, arteries and veins become connected and some arteries become veins. It is unclear how ECs in transforming vessels change their type and how fates of individual vessels are determined. In embryonic trunk, vascular remodeling transforms arterial intersegmental vessels (ISVs) into a functional network of arteries and veins. We found that, once an ISV is connected to venous circulation, venous blood flow promotes upstream migration of ECs that results in displacement of arterial ECs by venous ECs, completing the transformation of this ISV into a vein without trans-differentiation of ECs. Arterial blood flow initiated in two neighboring ISVs prevents their transformation into veins by activating Notch signaling in ECs. Together, different responses of ECs to arterial and venous blood flow lead to the formation of a balanced network with equal numbers of arteries and veins.

developmental biology↗