Timofey Skvortsov

Heterogeneity and degree of TIMP4, GATA4, SOX18, and EGFL7 gene promoter methylation in non–small cell lung cancer and surrounding tissues

We used methylation-sensitive high resolution melting analysis to assess methylation of CpG islands within the promoters of the TIMP4, GATA4, SOX18, and EGFL7 genes in samples of non-small cell lung cancer and surrounding apparently normal tissue and noncancerous lung tissues. We found that the promoter methylation was heterogeneous in both tumor and surrounding normal tissue. This is in contrast to healthy lung tissue, where the promoters were normally either non- or hypomethylated, and the heterogeneity of methylation was low. An increased heterogeneity of methylation in the normal tissues surrounding the tumor may suggest an early start of epigenetic processes preceding genetic and morphologic changes and can be used as a biomarker of early cancerization events. This analysis is an easy and sensitive tool for studying epigenetic heterogeneity and could be used in clinical practice.

Metagenomic Characterisation of the Viral Community of Lough Neagh, the Largest Freshwater Lake in Ireland

Lough Neagh is the largest and the most economically important lake in Ireland. It is also one of the most nutrient rich amongst the world’s major lakes. In this study, 16S rRNA analysis of total metagenomic DNA from the water column of Lough Neagh has revealed a high proportion of Cyanobacteria and low levels of Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes. The planktonic virome of Lough Neagh has been sequenced and 2,298,791 2×300 bp Illumina reads analysed. Comparison with previously characterised lakes demonstrates that the Lough Neagh viral community has the highest level of sequence diversity. Only about 15% of reads had homologs in the RefSeq database and tailed bacteriophages (Caudovirales) were identified as a major grouping. Within the Caudovirales, the Podoviridae and Siphoviridae were the two most dominant families (34.3% and 32.8% of the reads with sequence homology to the RefSeq database), while ssDNA bacteriophages constituted less than 1% of the virome. Putative cyanophages were found to be abundant. 66,450 viral contigs were assembled with the largest one being 58,805 bp; its existence, and that of another 34,467 bp contig, in the water column was confirmed. Analysis of the contigs confirmed the high abundance of cyanophages in the water column.

RNA-Seq Analysis of Mycobacterium avium Non-Coding Transcriptome

Deep sequencing was implemented to study the transcriptional landscape of Mycobacterium avium. High-resolution transcriptome analysis identified the transcription start points for 652 genes. One third of these genes represented leaderless transcripts, whereas the rest of the transcripts had 5′ UTRs with the mean length of 83 nt. In addition, the 5′ UTRs of 6 genes contained SAM-IV and Ykok types of riboswitches. 87 antisense RNAs and 10 intergenic small RNAs were mapped. 6 intergenic small RNAs, including 4.5S RNA and rnpB, were transcribed at extremely high levels. Although several intergenic sRNAs are conserved in M. avium and M. tuberculosis, both of these species have unique intergenic sRNAs. Moreover, we demonstrated that even conserved small RNAs are regulated differently in these species. Different sets of intergenic sRNAs may underlie differences in physiology between conditionally pathogenic M. avium and highly specialized pathogen M. tuberculosis.

A new technique for obtaining whole pathogen transcriptomes from infected host tissues

We propose a novel experimental approach based on coincidence cloning for analyzing sequences of bacterial intracellular pathogens specifically transcribed in affected tissues. Co-denaturation and co-renaturation of excess bacterial genomic DNA with the cDNA prepared on total RNA of the infected tissue allows one to select the bacterial fraction of the cDNA sample. We used this technique for preparing and characterizing the Mycobacterium tuberculosis cDNA pool, representing the transcriptome of infected mouse lungs in the chronic phase of infection. A cDNA pool enriched in fragments of mycobacterial cDNA was analyzed by the high-throughput 454 sequencing procedure. We demonstrated that its composition corresponded to what can be expected in the chronic phase of infection and, after the adaptation of M. tuberculosis to the host immune system, was characterized by an active lipid metabolism and switched from aerobic to anaerobic respiration. The technique is universal and requires no prior knowledge of the pathogen genome sequence. Pools of transcribed sequences obtained by this technique retain the main characteristics of the genome-wide gene transcription pattern within infected tissue, and can be used for in vivo analysis of gene expression of a wide spectrum of infection agents, such as viruses, bacteria, and protista.

Dormant non-culturable Mycobacterium tuberculosis retains stable low-abundant mRNA

BackgroundDormant Mycobacterium tuberculosis bacilli are believed to play an important role in latent tuberculosis infection. Previously, we have demonstrated that cultivation of M. tuberculosis in K+-deficient medium resulted in generation of dormant cells. These bacilli were non-culturable on solid media (a key feature of dormant M. tuberculosis in vivo) and characterized by low metabolism and tolerance to anti-tuberculosis drugs. The dormant bacteria demonstrated a high potential to reactivation after K+ reintroduction even after prolonged persistence under rifampicin. In this work, we studied the transcriptome and stability of transcripts in persisting dormant bacilli under arrest of mRNA de novo synthesis.ResultsRNA-seq-based analysis of the dormant non-culturable population obtained under rifampicin exposure revealed a 30–50-fold decrease of the total mRNA level, indicating global transcriptional repression. However, the analysis of persisting transcripts displayed a cohort of mRNA molecules coding for biosynthetic enzymes, proteins involved in adaptation and repair processes, detoxification, and control of transcription initiation. This ‘dormant transcriptome’ demonstrated considerable stability during M. tuberculosis persistence and mRNA de novo synthesis arrest. On the contrary, several small non-coding RNAs showed increased abundance on dormancy. Interestingly, M. tuberculosis entry into dormancy was accompanied by the cleavage of 23S ribosomal RNA at a specific point located outside the ribosome catalytic center.ConclusionsDormant non-culturable M. tuberculosis bacilli are characterized by a global transcriptional repression. At the same time, the dormant bacilli retain low-abundant mRNAs, which are considerably stable during in vitro persistence, reflecting their readiness for translation upon early resuscitation steps. Increased abundance of non-coding RNAs on dormancy may indicate their role in the entry into and maintenance of M. tuberculosis dormant non-culturable state.

A review of the transcriptome analysis of bacterial pathogens in vivo: Problems and solutions

This review is devoted to the state-of-the-art strategies for the in vivo whole-transcriptome analysis of intracellular pathogens. The methods for the initial enrichment with bacterial RNA are discussed in detail, including the hybridization-based approaches, as well as the methods for cDNA synthesis taking into account the specific features of prokaryotic RNA and the methods for bacterial cDNA analysis, in particular, high-throughput RNA sequencing. The described methods are illustrated with an analysis of Mycobacterium tuberculosis transcriptome in different infection models (cell lines, infected animal tissues and organs, and human lung surgical samples). The advantages and limitations of the discussed methods are discussed.

Novel small RNAs from Mycobacterium avium

Posttranscriptional regulation of gene expression mediated by small RNAs plays an important role in the virulence of pathogenic microorganisms. We have detected the expression of the following Mycobacterium avium genes situated in intergenic loci and coding for small RNAs: MAV_0380-0381 (4.5S RNA), MAV_1034-1035 (trans-encoded small RNA), MAV_1415-1416 (antisense or trans-encoded small RNA), and MAV_1531-1532 (processed 5′-UTR of the 16S r

Catalytic mechanism of Phenylacetone monooxygenases for non-native linear substrates: implications on rational engineering of BVMOs to expand the substrate specificity

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