Publications by authors named "Elena N Ilina"

35 Publications

Drift of the Subgingival Periodontal Microbiome during Chronic Periodontitis in Type 2 Diabetes Mellitus Patients.

Pathogens 2021 Apr 22;10(5). Epub 2021 Apr 22.

Ministry of Healthcare of the Russian Federation, A.I. Evdokimov Moscow State University of Medicine and Dentistry, 127473 Moscow, Russia.

Since periodontitis and type 2 diabetes mellitus are complex diseases, a thorough understanding of their pathogenesis requires knowing the relationship of these pathologies with other disorders and environmental factors. In this study, the representability of the subgingival periodontal microbiome of 46 subjects was studied by 16S rRNA gene sequencing and shotgun sequencing of pooled samples. We examined 15 patients with chronic periodontitis (CP), 15 patients with chronic periodontitis associated with type 2 diabetes mellitus (CPT2DM), and 16 healthy subjects (Control). The severity of generalized chronic periodontitis in both periodontitis groups of patients (CP and CPT2DM) was moderate (stage II). The male to female ratios were approximately equal in each group (22 males and 24 females); the average age of the subjects was 53.9 ± 7.3 and 54.3 ± 7.2 years, respectively. The presence of overweight patients (Body Mass Index (BMI) 30-34.9 kg/m) and patients with class 1-2 obesity (BMI 35-45.9 kg/m) was significantly higher in the CPT2DM group than in patients having only chronic periodontitis or in the Control group. However, there was no statistically significant difference in all clinical indices between the CP and CPT2DM groups. An analysis of the metagenomic data revealed that the alpha diversity in the CPT2DM group was increased compared to that in the CP and Control groups. The microbiome biomarkers associated with experimental groups were evaluated. In both groups of patients with periodontitis, the relative abundance of was increased compared to that in the Control group. The CPT2DM group was characterized by a lower relative abundance of / and a higher abundance of compared to those in the CP and Control groups. Furthermore, the CP and CPT2DM groups differed in terms of the relative abundance of (which was decreased in the CPT2DM group compared to CP) and (which was increased in the CPT2DM group compared to CP). In addition, differences in bacterial content were identified by a combination of shotgun sequencing of pooled samples and genome-resolved metagenomics. The results indicate that there are subgingival microbiome-specific features in patients with chronic periodontitis associated with type 2 diabetes mellitus.
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http://dx.doi.org/10.3390/pathogens10050504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8145315PMC
April 2021

Gene Networks Underlying the Resistance of to Inflammatory Factors.

Front Immunol 2020 16;11:595877. Epub 2020 Nov 16.

Department of Molecular Biology and Genetics, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia.

As permanent residents of the normal gut microbiota, bifidobacteria have evolved to adapt to the host's immune response whose priority is to eliminate pathogenic agents. The mechanisms that ensure the survival of commensals during inflammation and maintain the stability of the core component of the normal gut microbiota in such conditions remain poorly understood. We propose a new approach to study the mechanisms of resistance to immune response factors based on high-throughput sequencing followed by transcriptome analysis. This approach allowed us to detect differentially expressed genes associated with inflammation. In this study, we demonstrated that the presence of the pro-inflammatory cytokines IL-6 and TNFα to the growth medium of the GT15 strain changes the latter's growth rate insignificantly while affecting the expression of certain genes. We identified these genes and performed a COG and a KEGG pathway enrichment analysis. Using phylogenetic profiling we predicted the operons of genes whose expression was triggered by the cytokines TNFα and IL-6 . By mapping the transcription start points, we experimentally validated the predicted operons. Thus, in this study, we predicted the genes involved in a putative signaling pathway underlying the mechanisms of resistance to inflammatory factors in bifidobacteria. Since bifidobacteria are a major component of the human intestinal microbiota exhibiting pronounced anti-inflammatory properties, this study is of great practical and scientific relevance.
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http://dx.doi.org/10.3389/fimmu.2020.595877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7701253PMC
July 2021

Identification of Clinically Significant Prostate Cancer by Combined and mRNA Detection in Urine Samples.

Res Rep Urol 2020 17;12:403-413. Epub 2020 Sep 17.

Department of Molecular Biology and Genetics, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia.

Purpose: Preclinical evaluation of and transcript simultaneous detection in urine to diagnose clinical significant prostate cancer (prostate cancer with Gleason score ≥7) in a Russian cohort.

Patients And Methods: We analyzed urine samples of patients with a total serum PSA ≥2 ng/mL: 31 men with prostate cancer scheduled for radical prostatectomy, 128 men scheduled for first diagnostic biopsy (prebiopsy cohort). , , and transcripts were detected by multiplex reverse transcription quantitative polymerase chain reaction, and the results were used for scores for calculation and statistical analysis.

Results: There was no significant difference between clinically significant and nonsignificant prostate cancer PCA3 scores. However, there was a significant difference in the AMACR score (patients scheduled for radical prostatectomy =0.0088, prebiopsy cohort =0.029). We estimated AUCs, optimal cutoffs, sensitivities and specificities for PCa and csPCa detection in the prebiopsy cohort by tPSA, PCA3 score, PCPT Risk Calculator and classification models based on tPSA, PCA3 score and AMACR score. In the clinically significant prostate cancer ROC analysis, the PCA3 score AUC was 0.632 (95%CI: 0.511-0.752), the AMACR score AUC was 0.711 (95%CI: 0.617-0.806) and AUC of classification model based on the score, the AMACR score and total PSA was 0.72 (95%CI: 0.58-0.83). In addition, the correlation of the AMACR score with the ratio of total RNA and RNA of prostate cells in urine was shown (tau=0.347, =6.542e-09). Significant amounts of nonprostate RNA in urine may be a limitation for the AMACR score use.

Conclusion: The AMACR score is a good predictor of clinically significant prostate cancer. Significant amounts of nonprostate RNA in urine may be a limitation for the AMACR score use. Evaluation of the AMACR score and classification models based on it for clinically significant prostate cancer detection with larger samples and a follow-up analysis is promising.
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http://dx.doi.org/10.2147/RRU.S262310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505712PMC
September 2020

Phigaro: high-throughput prophage sequence annotation.

Bioinformatics 2020 06;36(12):3882-3884

Department of Molecular Biology and Genetics, Federal Research and Clinical Centre of Physical-Chemical Medicine, Moscow 119435, Russia.

Summary: Phigaro is a standalone command-line application that is able to detect prophage regions taking raw genome and metagenome assemblies as an input. It also produces dynamic annotated 'prophage genome maps' and marks possible transposon insertion spots inside prophages. It is applicable for mining prophage regions from large metagenomic datasets.

Availability And Implementation: Source code for Phigaro is freely available for download at https://github.com/bobeobibo/phigaro along with test data. The code is written in Python.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa250DOI Listing
June 2020

Deep Functional Profiling Facilitates the Evaluation of the Antibacterial Potential of the Antibiotic Amicoumacin.

Antibiotics (Basel) 2020 Apr 2;9(4). Epub 2020 Apr 2.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow 117997, Russia.

The global spread of antibiotic resistance is forcing the scientific community to find new molecular strategies to counteract it. Deep functional profiling of microbiomes provides an alternative source for the discovery of novel antibiotic producers and probiotics. Recently, we implemented this ultrahigh-throughput screening approach for the isolation of strains efficiently producing the ribosome-targeting antibiotic amicoumacin A (Ami). Proteomics and metabolomics revealed essential insight into the activation of Ami biosynthesis. Here, we applied omics to boost Ami biosynthesis, providing the optimized cultivation conditions for high-scale production of Ami. Ami displayed a pronounced activity against and , including methicillin-resistant (MRSA) strains, which was determined using both classical and massive single-cell microfluidic assays. However, the practical application of Ami is limited by its high cytotoxicity and particularly low stability. The former is associated with its self-lactonization, serving as an improvised intermediate state of Ami hydrolysis. This intramolecular reaction decreases Ami half-life at physiological conditions to less than 2 h, which is unprecedented for a terminal amide. While we speculate that the instability of Ami is essential for ecology, we believe that its stable analogs represent attractive lead compounds both for antibiotic discovery and for anticancer drug development.
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http://dx.doi.org/10.3390/antibiotics9040157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235827PMC
April 2020

Long-term impact of fecal transplantation in healthy volunteers.

BMC Microbiol 2019 12 30;19(1):312. Epub 2019 Dec 30.

R.M.Gorbacheva Memorial Institute of Oncology, Hematology and Transplantation, Pavlov First Saint Petersburg State Medical University, St. Petersburg, Russian Federation.

Background: Fecal microbiota transplantation (FMT) has been recently approved by FDA for the treatment of refractory recurrent clostridial colitis (rCDI). Success of FTM in treatment of rCDI led to a number of studies investigating the effectiveness of its application in the other gastrointestinal diseases. However, in the majority of studies the effects of FMT were evaluated on the patients with initially altered microbiota. The aim of our study was to estimate effects of FMT on the gut microbiota composition in healthy volunteers and to monitor its long-term outcomes.

Results: We have performed a combined analysis of three healthy volunteers before and after capsule FMT by evaluating their general condition, adverse clinical effects, changes of basic laboratory parameters, and several immune markers. Intestinal microbiota samples were evaluated by 16S rRNA gene and shotgun sequencing. The data analysis demonstrated profound shift towards the donor microbiota taxonomic composition in all volunteers. Following FMT, all the volunteers exhibited gut colonization with donor gut bacteria and persistence of this effect for almost ∼1 year of observation. Transient changes of immune parameters were consistent with suppression of T-cell cytotoxicity. FMT was well tolerated with mild gastrointestinal adverse events, however, one volunteer developed a systemic inflammatory response syndrome.

Conclusions: The FMT leads to significant long-term changes of the gut microbiota in healthy volunteers with the shift towards donor microbiota composition and represents a relatively safe procedure to the recipients without long-term adverse events.
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http://dx.doi.org/10.1186/s12866-019-1689-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938016PMC
December 2019

Shifts in the Human Gut Microbiota Structure Caused by Quadruple Eradication Therapy.

Front Microbiol 2019 27;10:1902. Epub 2019 Aug 27.

Federal Research and Clinical Centre of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia.

The human gut microbiome plays an important role both in health and disease. Use of antibiotics can alter gut microbiota composition, which can lead to various deleterious events. Here we report a whole genome sequencing metagenomic/genomic study of the intestinal microbiota changes caused by (HP) eradication therapy. Using approaches for metagenomic data analysis we revealed a statistically significant decrease in alpha-diversity and relative abundance of due to HP eradication therapy, while the relative abundance of increased. We have detected changes in general metagenome resistome profiles as well: after HP eradication therapy, the group, and genes were overrepresented, while and genes were underrepresented. We have confirmed these results with genome-resolved metagenomic approaches. MAG (metagenome-assembled genomes) abundance profiles have changed dramatically after HP eradication therapy. Focusing on gene conferring resistance to macrolides, which were included in the HP eradication therapy scheme, we have shown a connection between antibiotic resistance genes (ARGs) and some overrepresented MAGs. Moreover, some strains isolated from stool samples obtained after HP eradication have manifested greater antibiotic resistance in comparison to other isolates, as well as the higher number of ARGs conferring resistance to macrolides and tetracyclines.
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http://dx.doi.org/10.3389/fmicb.2019.01902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718723PMC
August 2019

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage.

Nat Microbiol 2019 10 8;4(10):1727-1736. Epub 2019 Jul 8.

National Food Institute, Research Group for Genomic Epidemiology, Technical University of Denmark, Kongens Lyngby, Denmark.

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.
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http://dx.doi.org/10.1038/s41564-019-0494-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440971PMC
October 2019

Ultrahigh-throughput functional profiling of microbiota communities.

Proc Natl Acad Sci U S A 2018 09 4;115(38):9551-9556. Epub 2018 Sep 4.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520;

Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate strains demonstrating antimicrobial activity against Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of Proteomics and metabolomics revealed a unique mechanism of self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.
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http://dx.doi.org/10.1073/pnas.1811250115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156654PMC
September 2018

Draft genomes of strains isolated from human feces before and after eradication therapy against .

Data Brief 2018 Feb 27;16:511-514. Epub 2017 Nov 27.

Federal Research and Clinical Centre of Physical-Chemical Medicine, Malaya Pirogovskaya 1a, Moscow 119435, Russia.

The abundance of Enterococci in the human intestinal microbiota environment is usually < 0.1% of the total bacterial fraction. The multiple resistance to antibiotics of the opportunistic spp. is alarming for the world medical community because of their high prevalence among clinically significant strains of microorganisms. Enterococci are able to collect different mobile genetic elements and transmit resistance to antibiotics to wide range of Gram-positive and Gram-negative species of microorganisms, including the transmission of vancomycin resistance to methicillin-resistant strains of . The number of infections caused by antibiotics resistant strains of spp. is increasing. Here we present a draft genomes of strains. These strains were isolated from human feces before and after (1 month) eradication therapy. The samples were subject to whole-genome sequencing using Illumina HiSeq. 2500 platform. The data is available at NCBI https://www.ncbi.nlm.nih.gov/bioproject/PRJNA412824.
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http://dx.doi.org/10.1016/j.dib.2017.11.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734704PMC
February 2018

Links of gut microbiota composition with alcohol dependence syndrome and alcoholic liver disease.

Microbiome 2017 10 17;5(1):141. Epub 2017 Oct 17.

Moscow Institute of Physics and Technology, Institutskiy per. 9, Dolgoprudny, Moscow Region, 141700, Russia.

Background: Alcohol abuse has deleterious effects on human health by disrupting the functions of many organs and systems. Gut microbiota has been implicated in the pathogenesis of alcohol-related liver diseases, with its composition manifesting expressed dysbiosis in patients suffering from alcoholic dependence. Due to its inherent plasticity, gut microbiota is an important target for prevention and treatment of these diseases. Identification of the impact of alcohol abuse with associated psychiatric symptoms on the gut community structure is confounded by the liver dysfunction. In order to differentiate the effects of these two factors, we conducted a comparative "shotgun" metagenomic survey of 99 patients with the alcohol dependence syndrome represented by two cohorts-with and without liver cirrhosis. The taxonomic and functional composition of the gut microbiota was subjected to a multifactor analysis including comparison with the external control group.

Results: Alcoholic dependence and liver cirrhosis were associated with profound shifts in gut community structures and metabolic potential across the patients. The specific effects on species-level community composition were remarkably different between cohorts with and without liver cirrhosis. In both cases, the commensal microbiota was found to be depleted. Alcoholic dependence was inversely associated with the levels of butyrate-producing species from the Clostridiales order, while the cirrhosis-with multiple members of the Bacteroidales order. The opportunist pathogens linked to alcoholic dependence included pro-inflammatory Enterobacteriaceae, while the hallmarks of cirrhosis included an increase of oral microbes in the gut and more frequent occurrence of abnormal community structures. Interestingly, each of the two factors was associated with the expressed enrichment in many Bifidobacterium and Lactobacillus-but the exact set of the species was different between alcoholic dependence and liver cirrhosis. At the level of functional potential, the patients showed different patterns of increase in functions related to alcohol metabolism and virulence factors, as well as pathways related to inflammation.

Conclusions: Multiple shifts in the community structure and metabolic potential suggest strong negative influence of alcohol dependence and associated liver dysfunction on gut microbiota. The identified differences in patterns of impact between these two factors are important for planning of personalized treatment and prevention of these pathologies via microbiota modulation. Particularly, the expansion of Bifidobacterium and Lactobacillus suggests that probiotic interventions for patients with alcohol-related disorders using representatives of the same taxa should be considered with caution. Taxonomic and functional analysis shows an increased propensity of the gut microbiota to synthesis of the toxic acetaldehyde, suggesting higher risk of colorectal cancer and other pathologies in alcoholics.
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http://dx.doi.org/10.1186/s40168-017-0359-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645934PMC
October 2017

Genome analysis of E. coli isolated from Crohn's disease patients.

BMC Genomics 2017 07 19;18(1):544. Epub 2017 Jul 19.

Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia.

Background: Escherichia coli (E. coli) has been increasingly implicated in the pathogenesis of Crohn's disease (CD). The phylogeny of E. coli isolated from Crohn's disease patients (CDEC) was controversial, and while genotyping results suggested heterogeneity, the sequenced strains of E. coli from CD patients were closely related.

Results: We performed the shotgun genome sequencing of 28 E. coli isolates from ten CD patients and compared genomes from these isolates with already published genomes of CD strains and other pathogenic and non-pathogenic strains. CDEC was shown to belong to A, B1, B2 and D phylogenetic groups. The plasmid and several operons from the reference CD-associated E. coli strain LF82 were demonstrated to be more often present in CDEC genomes belonging to different phylogenetic groups than in genomes of commensal strains. The operons include carbon-source induced invasion GimA island, prophage I, iron uptake operons I and II, capsular assembly pathogenetic island IV and propanediol and galactitol utilization operons.

Conclusions: Our findings suggest that CDEC are phylogenetically diverse. However, some strains isolated from independent sources possess highly similar chromosome or plasmids. Though no CD-specific genes or functional domains were present in all CD-associated strains, some genes and operons are more often found in the genomes of CDEC than in commensal E. coli. They are principally linked to gut colonization and utilization of propanediol and other sugar alcohols.
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http://dx.doi.org/10.1186/s12864-017-3917-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517970PMC
July 2017

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity.

Proc Natl Acad Sci U S A 2017 03 15;114(10):2550-2555. Epub 2017 Feb 15.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, , and predicted which genera were associated with inhibitory activity.
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http://dx.doi.org/10.1073/pnas.1621226114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347554PMC
March 2017

Data on gut metagenomes of the patients with alcoholic dependence syndrome and alcoholic liver cirrhosis.

Data Brief 2017 Apr 17;11:98-102. Epub 2017 Jan 17.

Moscow Institute of Physics and Technology, Institutskiy per. 9, Dolgoprudny, Moscow Region 141700, Russia.

Alcoholism is associated with significant changes in gut microbiota composition. Metagenomic sequencing allows to assess the altered abundance levels of bacterial taxa and genes in a culture-independent way. We collected 99 stool samples from the patients with alcoholic dependence syndrome (=72) and alcoholic liver cirrhosis (=27). Each of the samples was surveyed using "shotgun" (whole-genome) sequencing on SOLiD platform. The reads are deposited in the ENA (project ID: PRJEB18041).
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http://dx.doi.org/10.1016/j.dib.2017.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5257029PMC
April 2017

Data on gut metagenomes of the patients with infection before and after the antibiotic therapy.

Data Brief 2017 Apr 17;11:68-71. Epub 2017 Jan 17.

Federal Research and Clinical Centre of Physical-Chemical Medicine, Malaya Pirogovskaya 1a, Moscow 119435, Russia; Moscow Institute of Physics and Technology, Institutskiy per. 9, Dolgoprudny, Moscow Region 141700, Russia.

Antibiotic therapy can lead to the disruption of gut microbiota community with possible negative outcomes for human health. One of the diseases for which the treatment scheme commonly included antibiotic intake is infection. The changes in taxonomic and functional composition of microbiota in patients can be assessed using "shotgun" metagenomic sequencing. Ten stool samples were collected from 4 patients with infection before and directly after the eradication course. Additionally, for two of the subjects, the samples were collected 1 month after the end of the treatment. The samples were subject to "shotgun" (whole-genome) metagenomic sequencing using Illumina HiSeq platform. The reads are deposited in the ENA (project ID: PRJEB18265).
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http://dx.doi.org/10.1016/j.dib.2017.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256668PMC
April 2017

State of the Art of Chromosome 18-Centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells.

J Proteome Res 2016 11 29;15(11):4030-4038. Epub 2016 Aug 29.

Institute of Biomedical Chemistry , Pogodinskaya Street, 10, Moscow 119121, Russia.

A gene-centric approach was applied for a large-scale study of expression products of a single chromosome. Transcriptome profiling of liver tissue and HepG2 cell line was independently performed using two RNA-Seq platforms (SOLiD and Illumina) and also by Droplet Digital PCR (ddPCR) and quantitative RT-PCR. Proteome profiling was performed using shotgun LC-MS/MS as well as selected reaction monitoring with stable isotope-labeled standards (SRM/SIS) for liver tissue and HepG2 cells. On the basis of SRM/SIS measurements, protein copy numbers were estimated for the Chromosome 18 (Chr 18) encoded proteins in the selected types of biological material. These values were compared with expression levels of corresponding mRNA. As a result, we obtained information about 158 and 142 transcripts for HepG2 cell line and liver tissue, respectively. SRM/SIS measurements and shotgun LC-MS/MS allowed us to detect 91 Chr 18-encoded proteins in total, while an intersection between the HepG2 cell line and liver tissue proteomes was ∼66%. In total, there were 16 proteins specifically observed in HepG2 cell line, while 15 proteins were found solely in the liver tissue. Comparison between proteome and transcriptome revealed a poor correlation (R ≈ 0.1) between corresponding mRNA and protein expression levels. The SRM and shotgun data sets (obtained during 2015-2016) are available in PASSEL (PASS00697) and ProteomeExchange/PRIDE (PXD004407). All measurements were also uploaded into the in-house Chr 18 Knowledgebase at http://kb18.ru/protein/matrix/416126 .
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http://dx.doi.org/10.1021/acs.jproteome.6b00380DOI Listing
November 2016

Correction for Sotnikova et al., Complete Genome Sequence of Mycobacterium bovis Strain BCG-1 (Russia).

Genome Announc 2016 Jun 2;4(3). Epub 2016 Jun 2.

Microgen, Federal State Unitary Scientific-Industrial Company for Immunobiological Medicines of the Ministry of Health of the Russian Federation, Moscow, Russian Federation.

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http://dx.doi.org/10.1128/genomeA.00535-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891655PMC
June 2016

Complete Genome Sequence of Mycobacterium bovis Strain BCG-1 (Russia).

Genome Announc 2016 Mar 31;4(2). Epub 2016 Mar 31.

Microgen, Federal State Unitary Scientific-Industrial Company for Immunobiological Medicines of the Ministry of Health of the Russian Federation, Moscow, Russian Federation.

Mycobacterium bovisBCG (Bacille Calmette-Guérin) is a vaccine strain used for protection against tuberculosis. Here, we announce the complete genome sequence ofM. bovisstrain BCG-1 (Russia). Extensive use of this strain necessitates the study of its genome stability by comparative analysis.
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http://dx.doi.org/10.1128/genomeA.00182-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816620PMC
March 2016

RNA-Seq gene expression profiling of HepG2 cells: the influence of experimental factors and comparison with liver tissue.

BMC Genomics 2014 Dec 15;15:1108. Epub 2014 Dec 15.

Research Institute of Physico-Chemical Medicine, Malaya Pirogovskaya 1a, Moscow 119435, Russia.

Background: Human hepatoma HepG2 cells are used as an in vitro model of the human liver. High-throughput transcriptomic sequencing is an advanced approach for assessing the functional state of a tissue or cell type. However, the influence of experimental factors, such as the sample preparation method and inter-laboratory variation, on the transcriptomic profile has not been evaluated.

Results: The whole-transcriptome sequencing of HepG2 cells was performed using the SOLiD platform and validated using droplet digital PCR. The gene expression profile was compared to the results obtained with the same sequencing method in another laboratory and using another sample preparation method. We also compared the transcriptomic profile HepG2 cells with that of liver tissue. Comparison of the gene expression profiles between the HepG2 cell line and liver tissue revealed the highest variation, followed by HepG2 cells submitted to two different sample preparation protocols. The lowest variation was observed between HepG2 cells prepared by two different laboratories using the same protocol. The enrichment analysis of the genes that were differentially expressed between HepG2 cells and liver tissue mainly revealed the cancer-associated gene signature of HepG2 cells and the activation of the response to chemical stimuli in the liver tissue. The HepG2 transcriptome obtained with the SOLiD platform was highly correlated with the published transcriptome obtained with the Illumina and Helicos platforms, with moderate correspondence to microarrays.

Conclusions: In the present study, we assessed the influence of experimental factors on the HepG2 transcriptome and identified differences in gene expression between the HepG2 cell line and liver cells. These findings will facilitate robust experimental design in the fields of pharmacology and toxicology. Our results were supported by a comparative analysis with previous HepG2 gene expression studies.
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http://dx.doi.org/10.1186/1471-2164-15-1108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378340PMC
December 2014

Emergence of carbapenemase-producing Gram-negative bacteria in Saint Petersburg, Russia.

Int J Antimicrob Agents 2014 Aug 12;44(2):152-5. Epub 2014 Jun 12.

Research Institute of Children's Infections, Professor Popov Str. 9, Saint Petersburg 197022, Russia; North-West State Medical University, Kirochnaya Str. 41, Saint Petersburg 191015, Russia. Electronic address:

The emergence and spread of carbapenemase-producing Gram-negative bacteria represents a serious public health concern. Here we show that of 477 Gram-negative isolates collected from 18 hospitals between November 2011 and February 2013 in Saint Petersburg (Russia), minimum inhibitory concentrations (MICs) were greater than the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off value of at least one carbapenem antibiotic in 101 isolates (21.2%). The bla(NDM-1) gene was detected by PCR in 17 Klebsiella pneumoniae and 1 Acinetobacter nosocomialis isolate. Multilocus sequence typing (MLST) revealed that all NDM-1-producing K. pneumoniae isolates belonged to sequence type 340 (ST340) and harboured genes encoding additional β-lactamases; presence of the bla(CTX-M-1-like) gene correlated with aztreonam resistance, whilst its absence correlated with susceptibility. The epidemiological situation in Saint Petersburg can be assessed as regional spread of NDM-1-producers. The bla(KPC-2) gene was detected in two K. pneumoniae isolates (ST258 and ST273) and one Enterobacter cloacae isolate. Two E. cloacae isolates harboured the bla(VIM-4) gene, and one K. pneumoniae (ST395) isolate harboured the bla(OXA-48) gene. In NDM-1-producers, MICs of biapenem were the lowest compared with those of other carbapenems. Most isolates were susceptible to tigecycline and polymyxin, except for one K. pneumoniae isolate that was found to be polymyxin-resistant and one E. cloacae isolate that was tigecycline-resistant. Only one patient with a urinary tract infection caused by KPC-2-producing K. pneumoniae had a history of travel abroad (Southeast Asia). Thus, there is an actual threat of the emergence of an alarming endemic situation with NDM-1-producers in Saint Petersburg.
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http://dx.doi.org/10.1016/j.ijantimicag.2014.05.004DOI Listing
August 2014

Long-term dissemination of CTX-M-5-producing hypermutable Salmonella enterica serovar typhimurium sequence type 328 strains in Russia, Belarus, and Kazakhstan.

Antimicrob Agents Chemother 2014 Sep 23;58(9):5202-10. Epub 2014 Jun 23.

Institute of Antimicrobial Chemotherapy, Smolensk State Medical Academy, Smolensk, Russian Federation.

In this paper, we present evidence of long-term circulation of cefotaxime-resistant clonally related Salmonella enterica serovar Typhimurium strains over a broad geographic area. The genetic relatedness of 88 isolates collected from multiple outbreaks and sporadic cases of nosocomial salmonellosis in various parts of Russia, Belarus, and Kazakhstan from 1996 to 2009 was established by multilocus tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). The isolates belong to sequence type 328 (ST328) and produce CTX-M-5 β-lactamase, whose gene is carried by highly related non-self-conjugative but mobilizable plasmids. Resistance to nalidixic acid and low-level resistance to ciprofloxacin is present in 37 (42%) of the isolates and in all cases is determined by various single point mutations in the gyrA gene quinolone resistance-determining region (QRDR). Isolates of the described clonal group exhibit a hypermutable phenotype that probably facilitates independent acquisition of quinolone resistance mutations.
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http://dx.doi.org/10.1128/AAC.02506-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135874PMC
September 2014

Spoligotyping of Mycobacterium tuberculosis complex isolates using hydrogel oligonucleotide microarrays.

Infect Genet Evol 2014 Aug 9;26:41-6. Epub 2014 May 9.

Research Institute of Physical - Chemical Medicine, Moscow, Russian Federation.

Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. This circumstance underscores the relevance of population studies of tuberculosis for transmission dynamics control. In this study, we describe a conversion of the spoligotyping of M.tuberculosis complex isolates on a platform of custom designed hydrogel microarrays (biochips). An algorithm of automated data processing and interpretation of hybridization results using online database was proposed. In total, the 445 samples were tested. Initially, 97 samples representing multiple species of M.tuberculosis complex and nontuberculous mycobacteria were used for protocol optimization and cut-off settings. The developed assay was further evaluated on the out-group of the 348 mycobacterial samples. Results showed high concordance with the conventional membrane-based spoligotyping method. Diagnostic sensitivity and diagnostic specificity of the spoligo-biochip assay were 99.1% and 100%, respectively. The analytical sensitivity was determined to be 500 genomic equivalents of mycobacterial DNA. The high sensitivity and specificity, ease of operation procedures, and the automatic processing of measured data make the developed assay a useful tool for the rapid and accurate genotyping of M. tuberculosis.
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http://dx.doi.org/10.1016/j.meegid.2014.04.024DOI Listing
August 2014

Genome-wide Mycobacterium tuberculosis variation (GMTV) database: a new tool for integrating sequence variations and epidemiology.

BMC Genomics 2014 Apr 25;15:308. Epub 2014 Apr 25.

St, Petersburg State University, Theodosius Dobzhansky Center for Genome Bioinformatics, 41 Sredniy prospect, St, Petersburg, Russia.

Background: Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome.

Description: Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes.

Conclusions: Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains.
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http://dx.doi.org/10.1186/1471-2164-15-308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234438PMC
April 2014

Unusual large-scale chromosomal rearrangements in Mycobacterium tuberculosis Beijing B0/W148 cluster isolates.

PLoS One 2014 8;9(1):e84971. Epub 2014 Jan 8.

Research Institute of Physical - Chemical Medicine, Moscow, Russian Federation.

The Mycobacterium tuberculosis (MTB) Beijing family isolates are geographically widespread, and there are examples of Beijing isolates that are hypervirulent and associated with drug resistance. One-fourth of Beijing genotype isolates found in Russia belong to the B0/W148 group. The aim of the present study was to investigate features of these endemic strains on a genomic level. Four Russian clinical isolates of this group were sequenced, and the data obtained was compared with published sequences of various MTB strain genomes, including genome of strain W-148 of the same B0/W148 group. The comparison of the W-148 and H37Rv genomes revealed two independent inversions of large segments of the chromosome. The same inversions were found in one of the studied strains after deep sequencing using both the fragment and mate-paired libraries. Additionally, inversions were confirmed by RFLP hybridization analysis. The discovered rearrangements were verified by PCR in all four newly sequenced strains in the study and in four additional strains of the same Beijing B0/W148 group. The other 32 MTB strains from different phylogenetic lineages were tested and revealed no inversions. We suggest that the initial largest inversion changed the orientation of the three megabase (Mb) segment of the chromosome, and the second one occurred in the previously inverted region and partly restored the orientation of the 2.1 Mb inner segment of the region. This is another remarkable example of genomic rearrangements in the MTB in addition to the recently published of large-scale duplications. The described cases suggest that large-scale genomic rearrangements in the currently circulating MTB isolates may occur more frequently than previously considered, and we hope that further studies will help to determine the exact mechanism of such events.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084971PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885621PMC
September 2014

Chromosome 18 transcriptoproteome of liver tissue and HepG2 cells and targeted proteome mapping in depleted plasma: update 2013.

J Proteome Res 2014 Jan 13;13(1):183-90. Epub 2013 Dec 13.

Orekhovich Institute of Biomedical Chemistry of the Russian Academy of Medical Sciences , 10 Pogodinskaya Street, Moscow 119121, Russia.

We report the results obtained in 2012-2013 by the Russian Consortium for the Chromosome-centric Human Proteome Project (C-HPP). The main scope of this work was the transcriptome profiling of genes on human chromosome 18 (Chr 18), as well as their encoded proteome, from three types of biomaterials: liver tissue, the hepatocellular carcinoma-derived cell line HepG2, and blood plasma. The transcriptome profiling for liver tissue was independently performed using two RNaseq platforms (SOLiD and Illumina) and also by droplet digital PCR (ddPCR) and quantitative RT-PCR. The proteome profiling of Chr 18 was accomplished by quantitatively measuring protein copy numbers in the three types of biomaterial (the lowest protein concentration measured was 10(-13) M) using selected reaction monitoring (SRM). In total, protein copy numbers were estimated for 228 master proteins, including quantitative data on 164 proteins in plasma, 171 in the HepG2 cell line, and 186 in liver tissue. Most proteins were present in plasma at 10(8) copies/μL, while the median abundance was 10(4) and 10(5) protein copies per cell in HepG2 cells and liver tissue, respectively. In summary, for liver tissue and HepG2 cells a "transcriptoproteome" was produced that reflects the relationship between transcript and protein copy numbers of the genes on Chr 18. The quantitative data acquired by RNaseq, PCR, and SRM were uploaded into the "Update_2013" data set of our knowledgebase (www.kb18.ru) and investigated for linear correlations.
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http://dx.doi.org/10.1021/pr400883xDOI Listing
January 2014

Mutation in ribosomal protein S5 leads to spectinomycin resistance in Neisseria gonorrhoeae.

Front Microbiol 2013 10;4:186. Epub 2013 Jul 10.

Research Institute of Physico-Chemical Medicine Moscow, Russia.

Spectinomycin remains a useful reserve option for therapy of gonorrhea. The emergence of multidrug-resistant Neisseria gonorrhoeae strains with decreased susceptibility to cefixime and to ceftriaxone makes it the only medicine still effective for treatment of gonorrhea infection in analogous cases. However, adoption of spectinomycin as a routinely used drug of choice was soon followed by reports of spectinomycin resistance. The main molecular mechanism of spectinomycin resistance in N. gonorrhoeae was C1192T substitution in 16S rRNA genes. Here we reported a Thr-24→Pro mutation in ribosomal protein S5 (RPS5) found in spectinomycin resistant clinical N. gonorrhoeae strain, which carried no changes in 16S rRNA. In a series of experiments, the transfer of rpsE gene allele encoding the mutant RPS5 to the recipient N. gonorrhoeae strains was analyzed. The relatively high rate of transformation [ca. 10(-5) colony-forming units (CFUs)] indicates the possibility of spread of spectinonycin resistance within gonococcal population due to the horizontal gene transfer (HGT).
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http://dx.doi.org/10.3389/fmicb.2013.00186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706878PMC
July 2013

Comparative genomic analysis of Mycobacterium tuberculosis drug resistant strains from Russia.

PLoS One 2013 20;8(2):e56577. Epub 2013 Feb 20.

Research Institute of Physical-Chemical Medicine, Moscow, Russian Federation.

Tuberculosis caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains is a growing problem in many countries. The availability of the complete nucleotide sequences of several MTB genomes allows to use the comparative genomics as a tool to study the relationships of strains and differences in their evolutionary history including acquisition of drug-resistance. In our work, we sequenced three genomes of Russian MTB strains of different phenotypes--drug susceptible, MDR and XDR. Of them, MDR and XDR strains were collected in Tomsk (Siberia, Russia) during the local TB outbreak in 1998-1999 and belonged to rare KQ and KY families in accordance with IS6110 typing, which are considered endemic for Russia. Based on phylogenetic analysis, our isolates belonged to different genetic families, Beijing, Ural and LAM, which made the direct comparison of their genomes impossible. For this reason we performed their comparison in the broader context of all M. tuberculosis genomes available in GenBank. The list of unique individual non-synonymous SNPs for each sequenced isolate was formed by comparison with all SNPs detected within the same phylogenetic group. For further functional analysis, all proteins with unique SNPs were ascribed to 20 different functional classes based on Clusters of Orthologous Groups (COG). We have confirmed drug resistant status of our isolates that harbored almost all known drug-resistance associated mutations. Unique SNPs of an XDR isolate CTRI-4(XDR), belonging to a Beijing family were compared in more detail with SNPs of additional 14 Russian XDR strains of the same family. Only type specific mutations in genes of repair, replication and recombination system (COG category L) were found common within this group. Probably the other unique SNPs discovered in CTRI-4(XDR) may have an important role in adaptation of this microorganism to its surrounding and in escape from antituberculosis drugs treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056577PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3577857PMC
September 2013

Multiple-locus variable number tandem repeat analysis of Neisseria gonorrhoeae isolates in Russia.

Infect Genet Evol 2013 Mar 19;14:8-14. Epub 2012 Dec 19.

Group of Pharmacogenomics, Institute of Chemical Biology and Fundamental Medicine, Lavrentjeva, 8, Novosibirsk 630090, Russia.

In the present study, a multiple-locus variable number tandem repeat analysis (MLVA) was used to assess the molecular epidemiology of Neisseria gonorrhoeae clinical isolates originating from different regions of Russia. MLVA, based on seven loci, was performed on 218 isolates that were previously tested for susceptibility to penicillin, tetracycline and ciprofloxacin and for the presence of certain genetic determinants of drug resistance. In total, 83 MLVA types were identified, indicating that MLVA is a highly discriminatory technique with a Hunter-Gaston discriminatory index of 0.963 (95% CI, 0.950-0.977). MLVA type 16 was shown to be the most prevalent type and is undoubtedly associated with a multidrug resistant phenotype. The spread of MLVA type 16 from Moscow to Irkutsk suggests that this type has a highly successful transmission rate. Hierarchical cluster analysis of the MLVA profiles classified 208 isolates (95%) into six large groups (containing more than 10 isolates). Clusters differed in geographical characteristics and susceptibility profiles. MLVA cluster A comprised in total 34 isolates and was unambiguously associated with multidrug resistance. Most isolates in cluster A carried mutations in penA, ponA, rpsJ, mtrR, gyrA, and parC genes. MLVA cluster D was associated with resistance to penicillin and with mutations in ponA and rpsJ genes and the presence of plasmid-borne bla(TEM-1) gene. MLVA clusters B, C and E were associated with susceptibility to ciprofloxacin and had a lack of mutations in ponA, rpsJ, gyrA, and parC genes. We conclude that MLVA will be a useful tool for N. gonorrhoeae epidemiological studies.
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http://dx.doi.org/10.1016/j.meegid.2012.11.020DOI Listing
March 2013

Chromosome 18 transcriptome profiling and targeted proteome mapping in depleted plasma, liver tissue and HepG2 cells.

J Proteome Res 2013 Jan 20;12(1):123-34. Epub 2012 Dec 20.

Orekhovich Institute of Biomedical Chemistry of the Russian Academy of Medical Sciences, Russia.

The final goal of the Russian part of the Chromosome-centric Human Proteome Project (C-HPP) was established as the analysis of the chromosome 18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells with the sensitivity of 10(-18) M. Using SRM, we have recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2 cells. On the basis of the results of the survey, the SRM assays were drafted for 250 proteins: 41 proteins were found only in the liver tissue, 82 proteins were specifically detected in depleted plasma, and 127 proteins were mapped in both samples. The targeted analysis of HepG2 cells was carried out for 49 proteins; 41 of them were successfully registered using ordinary SRM and 5 additional proteins were registered using a combination of irreversible binding of proteins on CN-Br Sepharose 4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq and RT-PCR has shown a significant correlation (r = 0.78) for 42 gene transcripts. A pilot affinity-based interactome analysis was performed for cytochrome b5 using analytical and preparative optical biosensor fishing followed by MS analysis of the fished proteins. All of the data on the proteome complement of the Chr 18 have been integrated into our gene-centric knowledgebase ( www.kb18.ru ).
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http://dx.doi.org/10.1021/pr300821nDOI Listing
January 2013

Molecular surveillance of clinical Neisseria gonorrhoeae isolates in Russia.

J Clin Microbiol 2010 Oct 21;48(10):3681-9. Epub 2010 Jul 21.

Research Institute of Physicochemical Medicine, Ministry of Public Health of Russian Federation, Moscow, Russia.

The choice of adequate methods for epidemiological purposes remains a challenging problem in Neisseria gonorrhoeae molecular monitoring. In this study, the collection of geographically unrelated gonococci (n = 103) isolated in Russian clinics was comparably tested by (i) a traditional serotyping scheme, (ii) por typing, (iii) Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST), and (iv) multilocus sequence typing (MLST). It is shown that, according to sequencing data, a third of the strains carried new porB1 alleles, as well as tbpB ones, and more than half of the samples had new sequence types (STs) as determined by NG-MAST or MLST. The discriminatory power for each typing method was calculated by using the Hunter-Gaston discriminatory index, D. Commonly, modern nucleic acid-based typing methods (por typing, NG-MAST, and MLST) appeared to be more efficient than the classical serotyping scheme. While the traditional serotyping gave a D value of 0.82, the por typing, NG-MAST, and MLST approaches yielded D values of 0.97, 0.98, and 0.91, respectively. Each typing technique revealed the distribution of gonococci slightly correlated with their geographical sources. However, only the MLST method STs were highly associated with certain phenotypes. Although ST1594, ST1892, and ST6720 were typical for susceptible gonococci, ST1901 and ST6716 were undoubtedly associated with a multidrug-resistant phenotype. We conclude that every tested nucleic acid-based typing method is suitable for N. gonorrhoeae molecular surveillance. However, the MLST method seems to serve large-scale epidemiological purposes, whereas the NG-MAST and por typing approaches are more appropriate for the investigation of local outbreaks.
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http://dx.doi.org/10.1128/JCM.00565-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953076PMC
October 2010
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