BAC-BROWSER: The Tool for Visualization and Analysis of Prokaryotic Genomes

Prokaryotes are actively studied objects in the scope of genomic regulation. Microbiologists need special tools for complex analysis of data to study and identification of regulatory mechanism in bacteria and archaea.We developed a tool BAC-BROWSER, specifically for visualization and analysis of small prokaryotic genomes. BAC-BROWSER provides tools for different types of analysis to study a wide set of regulatory mechanisms of prokaryotes: -transcriptional regulation by transcription factors (TFs), analysis of TFs, their targets, and binding sites.-other regulatory motifs, promoters, terminators and ribosome binding sites-transcriptional regulation by variation of operon structure, alternative starts or ends of transcription.-non-coding RNAs, antisense RNAs-RNA secondary structure, riboswitches-GC content, GC skew, codon usageBAC-browser incorporated free programs accelerating the verification of obtained results: primer design and oligocalculator, vector visualization, the tool for synthetic gene construction. The program is designed for Windows operating system and freely available for download in http://smdb.rcpcm.org/tools/index.html

Reconstruction of Transcription Control Networks in Mollicutes by High-Throughput Identification of Promoters

Bacteria of the class Mollicutes have significantly reduced genomes and gene expression control systems. They are also efficient pathogens that can colonize a broad range of hosts including plants and animals. Despite their simplicity, Mollicutes demonstrate complex transcriptional responses to various conditions, which contradicts their reduction in gene expression regulation mechanisms. We analyzed the conservation and distribution of transcription regulators across the 50 Mollicutes species. The majority of the transcription factors regulate transport and metabolism, and there are four transcription factors that demonstrate significant conservation across the analyzed bacteria. These factors include repressors of chaperone HrcA, cell cycle regulator MraZ and two regulators with unclear function from the WhiA and YebC/PmpR families. We then used three representative species of the major clades of Mollicutes (Acholeplasma laidlawii, Spiroplasma melliferum and Mycoplasma gallisepticum) to perform promoters mapping and activity quantitation. We revealed that Mollicutes evolved towards a promoter architecture simplification that correlates with a diminishing role of transcription regulation and an increase in transcriptional noise. Using the identified operons structure and a comparative genomics approach, we reconstructed the transcription control networks for these three species. The organization of the networks reflects the adaptation of bacteria to specific conditions and hosts

Evaluation of CAR-T Cells’ Cytotoxicity against Modified Solid Tumor Cell Lines

In recent years, adoptive cell therapy has gained a new perspective of application due to the development of technologies and the successful clinical use of CAR-T cells for the treatment of patients with malignant B-cell neoplasms. However, the efficacy of CAR-T therapy against solid tumor remains a major scientific and clinical challenge. In this work, we evaluated the cytotoxicity of 2nd generation CAR-T cells against modified solid tumors cell lines—lung adenocarcinoma cell line H522, prostate carcinoma PC-3M, breast carcinoma MDA-MB-231, and epidermoid carcinoma A431 cell lines transduced with lentiviruses encoding red fluorescent protein Katushka2S and the CD19 antigen. A correlation was demonstrated between an increase in the secretion of proinflammatory cytokines and a decrease in the confluence of tumor cells’ monolayer. The proposed approach can potentially be applied to preliminarily assess CAR-T cell efficacy for the treatment of solid tumors and estimate the risks of developing cytokine release syndrome

Restriction-modification systems of <i>M</i>. <i>gallisepticum</i> S6.

Arrows represent the ORFs; grey, blue, green, and orange colors indicate genes hsdC (controller protein), hsdM (methyltransferase), hsdS (specificity subunit), and hsdR (restriction subunit), respectively. Ori–origin of replication. Faded colors represent disrupted genes.</p

Differences in expression levels of genes with DNA methylations sites between WT and ΔS.MgaS6I strains.

Groups of control genes eno, gadp, tuf, ligA, gyrB, dnaJ_2, and dnaJ_6; genes with the methylation motif in promoters topA, dnaJ_4, tktA_1, scpA, osmC_2, trxA_1, yebC, and dnaB; genes with different numbers of methylation sites or with hemi-methylation status ligA, gyrB, dnaB, hup_2, ung, uvrB, and groEL; genes of methyltransferases hsdM from MgaS6I and mraW have been presented. Genes with significant difference between strains (Student’s t-test, Benjamini–Hochberg correction, P<0.05) are shown using red boxes and asterisks.</p

Genome alignment of Mycoplasma gallisepticum strains obtained using the program Mauve.

RM system MgaS6I is marked in orange, variable lipoproteins vlhA clusters in green, the CRISPR system in blue. M. gallisepticum S6 genome is on the top, below the genomes of the other strains. A–whole genome representation, B–fragment of genomes near MgaS6I genomic context. (PDF)</p

Fig 8 -

A–Difference between the expression levels of paired genes for mutants with EGFP genes under synthetic promoters, with or without methylation sites. For each pair, the difference for EGFP gene in comparison with control genes eno, gadp, tuf, ligA, and gyrB is shown. Genes with significant difference between promoters with or without the methylation site (Student’s t-test, Benjamini–Hochberg correction, PEGFP promoters with or without methylated sites in -10 box, -35 box, between -35 and -10 boxes, and in the TSS. Methylation sites are underlined; methylated adenines in forward and reverse strands are in bold; -10 boxes are in blue and highlighted with blue boxes; -35 boxes are highlighted with green boxes and the sequence with strong consensus to the -35 box is in green; TSSs are in red and marked with red arrows; sequence differences between paired methylated or non-methylated promoters are in pink.</p