Publications by authors named "Konstantin G Skryabin"

63 Publications

A partial genome assembly of the miniature parasitoid wasp, Megaphragma amalphitanum.

PLoS One 2019 23;14(12):e0226485. Epub 2019 Dec 23.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.

Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals conquer new ecological niches. However, this process is poorly understood at the molecular level. Here, we report genomic and transcriptomic features of arguably the smallest known insect-the parasitoid wasp, Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the genome and transcriptome sizes of this parasitoid wasp are comparable to other members of the Chalcidoidea superfamily. Moreover, compared to other chalcid wasps the gene content of M. amalphitanum is remarkably conserved. Intriguingly, we observed significant changes in M. amalphitanum transposable element dynamics over time, in which an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Overall, while the M. amalphitanum genomic data reveal certain features that may be linked to the unusual biological properties of this organism, miniaturization is not associated with a large decrease in genome complexity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226485PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927652PMC
April 2020

Challenges of the Human Proteome Project: 10-Year Experience of the Russian Consortium.

J Proteome Res 2019 12 28;18(12):4206-4214. Epub 2019 Oct 28.

Institute of Biomedical Chemistry , Moscow 119435 , Russia.

This manuscript collects all the efforts of the Russian Consortium, bottlenecks revealed in the course of the C-HPP realization, and ways of their overcoming. One of the main bottlenecks in the C-HPP is the insufficient sensitivity of proteomic technologies, hampering the detection of low- and ultralow-copy number proteins forming the "dark part" of the human proteome. In the frame of MP-Challenge, to increase proteome coverage we suggest an experimental workflow based on a combination of shotgun technology and selected reaction monitoring with two-dimensional alkaline fractionation. Further, to detect proteins that cannot be identified by such technologies, nanotechnologies such as combined atomic force microscopy with molecular fishing and/or nanowire detection may be useful. These technologies provide a powerful tool for single molecule analysis, by analogy with nanopore sequencing during genome analysis. To systematically analyze the functional features of some proteins (CP50 Challenge), we created a mathematical model that predicts the number of proteins differing in amino acid sequence: proteoforms. According to our data, we should expect about 100 000 different proteoforms in the liver tissue and a little more in the HepG2 cell line. The variety of proteins forming the whole human proteome significantly exceeds these results due to post-translational modifications (PTMs). As PTMs determine the functional specificity of the protein, we propose using a combination of gene-centric transcriptome-proteomic analysis with preliminary fractionation by two-dimensional electrophoresis to identify chemically modified proteoforms. Despite the complexity of the proposed solutions, such integrative approaches could be fruitful for MP50 and CP50 Challenges in the framework of the C-HPP.
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http://dx.doi.org/10.1021/acs.jproteome.9b00358DOI Listing
December 2019

Plastid Genomes of Carnivorous Plants and × Reveal Evolutionary Patterns Resembling Those Observed in Parasitic Plants.

Int J Mol Sci 2019 Aug 22;20(17). Epub 2019 Aug 22.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.

Carnivorous plants have the ability to capture and digest small animals as a source of additional nutrients, which allows them to grow in nutrient-poor habitats. Here we report the complete sequences of the plastid genomes of two carnivorous plants of the order Caryophyllales, and × . The plastome of is repeat-rich and highly rearranged. It lacks NAD(P)H dehydrogenase genes, as well as and genes, and three essential tRNA genes. Intron losses are observed in some protein-coding and tRNA genes along with a pronounced reduction of RNA editing sites. Only six editing sites were identified by RNA-seq in plastid genome and at most conserved editing sites the conserved amino acids are already encoded at the DNA level. In contrast, the × plastome has a typical structure and gene content, except for pseudogenization of the gene. × and could represent different stages of evolution of the plastid genomes of carnivorous plants, resembling events observed in parasitic plants in the course of the switch from autotrophy to a heterotrophic lifestyle.
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http://dx.doi.org/10.3390/ijms20174107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747624PMC
August 2019

High-level expression of biologically active human follicle stimulating hormone in the Chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors.

PLoS One 2019 5;14(7):e0219434. Epub 2019 Jul 5.

Laboratory of Mammalian Cell Bioengineering, Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.

Recombinant human follicle stimulating hormone (FSH), produced in Chinese hamster ovary (CHO) cells, is widely used for treatment of fertility disorders and is subject to biosimilars development. Cell lines with high specific productivities may simplify the FSH production process. Here, we used our previously established expression system based on vector p1.1 to create new cell lines secreting heterodimeric FSH protein. To this end, we linked open reading frames of both FSH subunits by the wild-type internal ribosome entry site from the encephalomyocarditis virus (EMCV IRES). Intact and double-negative for the dihydrofolate reductase CHO cells were stably transfected by the FSH-coding plasmids. Stably transfected intact cells showed higher level of the FSH secretion and were utilized for subsequent methotrexate-driven transgene amplification, which doubled their productivity. The excess of the free α-subunit was corrected by transfecting the cells by the additional p1.1-based plasmid encoding the β-subunit of the FSH. Clonal cell lines obtained secreted mostly the heterodimeric FSH and possessed specific productivities up to 12.3±1.7 pg/cell/day. Candidate clonal cell line C-P1.3-FSH-G4 maintained a constant specific productivity for at least 2 months of culturing without the section pressure. The resulting FSH protein conformed to the international pharmaceutical quality criteria as evidenced by the receptor binding kinetics, distribution pattern of hormone isoforms and biological activity. In conclusion, our expression system offers a simple and cost-effective approach to production of FSH.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219434PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611665PMC
March 2020

Sex specific associations in genome wide association analysis of renal cell carcinoma.

Eur J Hum Genet 2019 10 23;27(10):1589-1598. Epub 2019 Jun 23.

Russian N.N. Blokhin Cancer Research Centre, Moscow, Russian Federation.

Renal cell carcinoma (RCC) has an undisputed genetic component and a stable 2:1 male to female sex ratio in its incidence across populations, suggesting possible sexual dimorphism in its genetic susceptibility. We conducted the first sex-specific genome-wide association analysis of RCC for men (3227 cases, 4916 controls) and women (1992 cases, 3095 controls) of European ancestry from two RCC genome-wide scans and replicated the top findings using an additional series of men (2261 cases, 5852 controls) and women (1399 cases, 1575 controls) from two independent cohorts of European origin. Our study confirmed sex-specific associations for two known RCC risk loci at 14q24.2 (DPF3) and 2p21(EPAS1). We also identified two additional suggestive male-specific loci at 6q24.3 (SAMD5, male odds ratio (OR) = 0.83 [95% CI = 0.78-0.89], P = 1.71 × 10 compared with female odds ratio (OR) = 0.98 [95% CI = 0.90-1.07], P = 0.68) and 12q23.3 (intergenic, OR = 0.75 [95% CI = 0.68-0.83], P = 1.59 × 10 compared with OR = 0.93 [95% CI = 0.82-1.06], P = 0.21) that attained genome-wide significance in the joint meta-analysis. Herein, we provide evidence of sex-specific associations in RCC genetic susceptibility and advocate the necessity of larger genetic and genomic studies to unravel the endogenous causes of sex bias in sexually dimorphic traits and diseases like RCC.
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http://dx.doi.org/10.1038/s41431-019-0455-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777615PMC
October 2019

New insights from Opisthorchis felineus genome: update on genomics of the epidemiologically important liver flukes.

BMC Genomics 2019 May 22;20(1):399. Epub 2019 May 22.

Russian Federal Research Center for Biotechnology, 33/2 Leninsky prospect, Moscow, 119071, Russia.

Background: The three epidemiologically important Opisthorchiidae liver flukes Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, are believed to harbour similar potencies to provoke hepatobiliary diseases in their definitive hosts, although their populations have substantially different ecogeographical aspects including habitat, preferred hosts, population structure. Lack of O. felineus genomic data is an obstacle to the development of comparative molecular biological approaches necessary to obtain new knowledge about the biology of Opisthorchiidae trematodes, to identify essential pathways linked to parasite-host interaction, to predict genes that contribute to liver fluke pathogenesis and for the effective prevention and control of the disease.

Results: Here we present the first draft genome assembly of O. felineus and its gene repertoire accompanied by a comparative analysis with that of O. viverrini and Clonorchis sinensis. We observed both noticeably high heterozygosity of the sequenced individual and substantial genetic diversity in a pooled sample. This indicates that potency of O. felineus population for rapid adaptive response to control and preventive measures of opisthorchiasis is higher than in O. viverrini and C. sinensis. We also have found that all three species are characterized by more intensive involvement of trans-splicing in RNA processing compared to other trematodes.

Conclusion: All revealed peculiarities of structural organization of genomes are of extreme importance for a proper description of genes and their products in these parasitic species. This should be taken into account both in academic and applied research of epidemiologically important liver flukes. Further comparative genomics studies of liver flukes and non-carcinogenic flatworms allow for generation of well-grounded hypotheses on the mechanisms underlying development of cholangiocarcinoma associated with opisthorchiasis and clonorchiasis as well as species-specific mechanisms of these diseases.
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http://dx.doi.org/10.1186/s12864-019-5752-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530080PMC
May 2019

Magnetospirillum kuznetsovii sp. nov., a novel magnetotactic bacterium isolated from a lake in the Moscow region.

Int J Syst Evol Microbiol 2019 Jul 30;69(7):1953-1959. Epub 2019 Apr 30.

1​Institute of Bioengineering, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Leninsky Ave 33, bld. 2, Moscow, Russia.

Strain LBB-42 was isolated from sediment sampled at Lake Beloe Bordukovskoe, located in the Moscow region (Russia). Phylogenetic analyses based on 16S rRNA gene sequencing results assigned the strain to the genus Magnetospirillum. Major fatty acids were C16 : 1ω7c, C16 : 0 and C18 : 1 ω9/C18 : 1 ω7. Genome sequencing revealed a genome size of 4.40 Mbp and a G+C content of 63.4 mol%. The average nucleotide identity and digital DNA-DNA hybridization values suggested that strain LBB-42 represents a new species, for which we propose the name Magnetospirillum kuznetsovii sp. nov., with the type strain LBB-42 (=VKM B-3270=KCTC 15749).
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http://dx.doi.org/10.1099/ijsem.0.003408DOI Listing
July 2019

The influence of obesity-related factors in the etiology of renal cell carcinoma-A mendelian randomization study.

PLoS Med 2019 01 3;16(1):e1002724. Epub 2019 Jan 3.

National Institute of Public Health, Bucharest, Romania.

Background: Several obesity-related factors have been associated with renal cell carcinoma (RCC), but it is unclear which individual factors directly influence risk. We addressed this question using genetic markers as proxies for putative risk factors and evaluated their relation to RCC risk in a mendelian randomization (MR) framework. This methodology limits bias due to confounding and is not affected by reverse causation.

Methods And Findings: Genetic markers associated with obesity measures, blood pressure, lipids, type 2 diabetes, insulin, and glucose were initially identified as instrumental variables, and their association with RCC risk was subsequently evaluated in a genome-wide association study (GWAS) of 10,784 RCC patients and 20,406 control participants in a 2-sample MR framework. The effect on RCC risk was estimated by calculating odds ratios (ORSD) for a standard deviation (SD) increment in each risk factor. The MR analysis indicated that higher body mass index increases the risk of RCC (ORSD: 1.56, 95% confidence interval [CI] 1.44-1.70), with comparable results for waist-to-hip ratio (ORSD: 1.63, 95% CI 1.40-1.90) and body fat percentage (ORSD: 1.66, 95% CI 1.44-1.90). This analysis further indicated that higher fasting insulin (ORSD: 1.82, 95% CI 1.30-2.55) and diastolic blood pressure (DBP; ORSD: 1.28, 95% CI 1.11-1.47), but not systolic blood pressure (ORSD: 0.98, 95% CI 0.84-1.14), increase the risk for RCC. No association with RCC risk was seen for lipids, overall type 2 diabetes, or fasting glucose.

Conclusions: This study provides novel evidence for an etiological role of insulin in RCC, as well as confirmatory evidence that obesity and DBP influence RCC risk.
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http://dx.doi.org/10.1371/journal.pmed.1002724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317776PMC
January 2019

The complete mitochondrial genome of the carnivorous flowering plant .

Mitochondrial DNA B Resour 2018 Oct 29;3(2):1259-1260. Epub 2018 Oct 29.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.

Carnivorous plants have the ability to capture and digest small animals as a source of additional nutrients, which allows them to grow in nutrient-poor habitats. This study reports the complete mitochondrial genome sequence of a pitcher plant . It was 520,764 bp in size with a GC content of 44.17% and contained 37 protein-coding genes, 2 pseudogenes, 18 tRNA genes and 3 rRNA genes. Four tRNA genes and the gene were probably transferred to mitochondrion form the chloroplast genome. Phylogenetic analysis confirmed that belongs to the order .
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http://dx.doi.org/10.1080/23802359.2018.1532353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7800874PMC
October 2018

The Genes of Leaf and Leaf-Like Organ Polarity in Leafless Plant .

Int J Genomics 2018 24;2018:7203469. Epub 2018 Apr 24.

Federal State Institution "Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences", Moscow 119071, Russia.

is a mycoheterotrophic, nonphotosynthetic plant acquiring nutrients from the roots of autotrophic trees through mycorrhizal symbiosis, and, similar to other extant plants, forming asymmetrical lateral organs during development. The members of the YABBY family of transcription factors are important players in the establishment of leaf and leaf-like organ polarity in plants. This is the first report on the identification of genes in a mycoheterotrophic plant devoid of aboveground vegetative organs. Seven members were identified and classified into four clades. By structural analysis of putative encoded proteins, we confirmed the presence of YABBY-defining conserved domains and identified novel clade-specific motifs. Transcriptomic and qRT-PCR analyses of different tissues revealed transcriptional patterns, which were similar to those of orthologous genes from other angiosperms. These data should contribute to the understanding of the role of the genes in the regulation of developmental and physiological processes in achlorophyllous leafless plants.
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http://dx.doi.org/10.1155/2018/7203469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5941816PMC
April 2018

Genetic Variants Related to Longer Telomere Length are Associated with Increased Risk of Renal Cell Carcinoma.

Eur Urol 2017 11 7;72(5):747-754. Epub 2017 Aug 7.

Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department Health and Human Services, Bethesda, MS, USA.

Background: Relative telomere length in peripheral blood leukocytes has been evaluated as a potential biomarker for renal cell carcinoma (RCC) risk in several studies, with conflicting findings.

Objective: We performed an analysis of genetic variants associated with leukocyte telomere length to assess the relationship between telomere length and RCC risk using Mendelian randomization, an approach unaffected by biases from temporal variability and reverse causation that might have affected earlier investigations.

Design, Setting, And Participants: Genotypes from nine telomere length-associated variants for 10 784 cases and 20 406 cancer-free controls from six genome-wide association studies (GWAS) of RCC were aggregated into a weighted genetic risk score (GRS) predictive of leukocyte telomere length.

Outcome Measurements And Statistical Analysis: Odds ratios (ORs) relating the GRS and RCC risk were computed in individual GWAS datasets and combined by meta-analysis.

Results And Limitations: Longer genetically inferred telomere length was associated with an increased risk of RCC (OR=2.07 per predicted kilobase increase, 95% confidence interval [CI]:=1.70-2.53, p<0.0001). As a sensitivity analysis, we excluded two telomere length variants in linkage disequilibrium (R>0.5) with GWAS-identified RCC risk variants (rs10936599 and rs9420907) from the telomere length GRS; despite this exclusion, a statistically significant association between the GRS and RCC risk persisted (OR=1.73, 95% CI=1.36-2.21, p<0.0001). Exploratory analyses for individual histologic subtypes suggested comparable associations with the telomere length GRS for clear cell (N=5573, OR=1.93, 95% CI=1.50-2.49, p<0.0001), papillary (N=573, OR=1.96, 95% CI=1.01-3.81, p=0.046), and chromophobe RCC (N=203, OR=2.37, 95% CI=0.78-7.17, p=0.13).

Conclusions: Our investigation adds to the growing body of evidence indicating some aspect of longer telomere length is important for RCC risk.

Patient Summary: Telomeres are segments of DNA at chromosome ends that maintain chromosomal stability. Our study investigated the relationship between genetic variants associated with telomere length and renal cell carcinoma risk. We found evidence suggesting individuals with inherited predisposition to longer telomere length are at increased risk of developing renal cell carcinoma.
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http://dx.doi.org/10.1016/j.eururo.2017.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641242PMC
November 2017

Genome-wide association study identifies multiple risk loci for renal cell carcinoma.

Nat Commun 2017 06 9;8:15724. Epub 2017 Jun 9.

Clinical Center of Serbia (KCS), Clinic of Urology, University of Belgrade-Faculty of Medicine, 11000 Belgrade, Serbia.

Previous genome-wide association studies (GWAS) have identified six risk loci for renal cell carcinoma (RCC). We conducted a meta-analysis of two new scans of 5,198 cases and 7,331 controls together with four existing scans, totalling 10,784 cases and 20,406 controls of European ancestry. Twenty-four loci were tested in an additional 3,182 cases and 6,301 controls. We confirm the six known RCC risk loci and identify seven new loci at 1p32.3 (rs4381241, P=3.1 × 10), 3p22.1 (rs67311347, P=2.5 × 10), 3q26.2 (rs10936602, P=8.8 × 10), 8p21.3 (rs2241261, P=5.8 × 10), 10q24.33-q25.1 (rs11813268, P=3.9 × 10), 11q22.3 (rs74911261, P=2.1 × 10) and 14q24.2 (rs4903064, P=2.2 × 10). Expression quantitative trait analyses suggest plausible candidate genes at these regions that may contribute to RCC susceptibility.
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http://dx.doi.org/10.1038/ncomms15724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472706PMC
June 2017

The loss of photosynthetic pathways in the plastid and nuclear genomes of the non-photosynthetic mycoheterotrophic eudicot Monotropa hypopitys.

BMC Plant Biol 2016 Nov 16;16(Suppl 3):238. Epub 2016 Nov 16.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.

Background: Chloroplasts of most plants are responsible for photosynthesis and contain a conserved set of about 110 genes that encode components of housekeeping gene expression machinery and photosynthesis-related functions. Heterotrophic plants obtaining nutrients from other organisms and their plastid genomes represent model systems in which to study the effects of relaxed selective pressure on photosynthetic function. The most evident is a reduction in the size and gene content of the plastome, which correlates with the loss of genes encoding photosynthetic machinery which become unnecessary. Transition to a non-photosynthetic lifestyle is expected also to relax the selective pressure on photosynthetic machinery in the nuclear genome, however, the corresponding changes are less known.

Results: Here we report the complete sequence of the plastid genome of Monotropa hypopitys, an achlorophyllous obligately mycoheterotrophic plant belonging to the family Ericaceae. The plastome of M. hypopitys is greatly reduced in size (35,336 bp) and lacks the typical quadripartite structure with two single-copy regions and an inverted repeat. Only 45 genes remained presumably intact- those encoding ribosomal proteins, ribosomal and transfer RNA and housekeeping genes infA, matK, accD and clpP. The clpP and accD genes probably remain functional, although their sequences are highly diverged. The sets of genes for ribosomal protein and transfer RNA are incomplete relative to chloroplasts of a photosynthetic plant. Comparison of the plastid genomes of two subspecies-level isolates of M. hypopitys revealed major structural rearrangements associated with repeat-driven recombination and the presence of isolate-specific tRNA genes. Analysis of the M. hypopitys transcriptome by RNA-Seq showed the absence of expression of nuclear-encoded components of photosystem I and II reaction center proteins, components of cytochrome b6f complex, ATP synthase, ribulose bisphosphate carboxylase components, as well as chlorophyll from protoporphyrin IX biosynthesis pathway.

Conclusions: With the complete loss of genes related to photosynthesis, NADH dehydrogenase, plastid-encoded RNA polymerase and ATP synthase, the M. hypopitys plastid genome is among the most functionally reduced ones characteristic of obligate non-photosynthetic parasitic species. Analysis of the M. hypopitys transcriptome revealed coordinated evolution of the nuclear and plastome genomes and the loss of photosynthesis-related functions in both genomes.
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http://dx.doi.org/10.1186/s12870-016-0929-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123295PMC
November 2016

transcriptome assembly of the mycoheterotrophic plant .

Genom Data 2017 Mar 30;11:60-61. Epub 2016 Nov 30.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.

(pinesap) is a non-photosynthetic obligately mycoheterotrophic plant of the family . It obtains the carbon and other nutrients from the roots of surrounding autotrophic trees through the associated mycorrhizal fungi. In order to understand the evolutionary changes in the plant genome associated with transition to a heterotrophic lifestyle, we performed transcriptomic analysis of using next-generation sequencing. We obtained the RNA-Seq data from flowers, flower bracts and roots with haustoria using Illumina HiSeq2500 platform. The raw data obtained in this study can be available in NCBI SRA database with accession number of SRP069226. A total of 10.3 GB raw sequence data were obtained, corresponding to 103,357,809 raw reads. A total of 103,025,683 reads were filtered after removing low-quality reads and trimming the adapter sequences. The Trinity program was used to assemble 98,349 unigens with an N50 of 1342 bp. Using the TransDecoder program, we predicted 43,505 putative proteins. 38,416 unigenes were annotated in the Swiss-Prot protein sequence database using BLASTX. The obtained transcriptomic data will be useful for further studies of the evolution of plant genomes upon transition to a non-photosynthetic lifestyle and the loss of photosynthesis-related functions.
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http://dx.doi.org/10.1016/j.gdata.2016.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154972PMC
March 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

Draft Genome Sequences of Two Magnetotactic Bacteria, Magnetospirillum moscoviense BB-1 and Magnetospirillum marisnigri SP-1.

Genome Announc 2016 Aug 11;4(4). Epub 2016 Aug 11.

Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation

We report here the draft genome sequences of two recently isolated magnetotactic species, Magnetospirillum moscoviense BB-1 and Magnetospirillum marisnigri SP-1. The genome of M. moscoviense BB-1 has 4,164,497 bp, 65.2% G+C content, and comprises 207 contigs. The genome of M. marisnigri SP-1 consists of 131 contigs and has a length of 4,619,819 bp and 64.7% G+C content.
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http://dx.doi.org/10.1128/genomeA.00814-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982287PMC
August 2016

Deep-sequence profiling of miRNAs and their target prediction in Monotropa hypopitys.

Plant Mol Biol 2016 Jul 20;91(4-5):441-58. Epub 2016 Apr 20.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, Moscow, Russia, 119071.

Myco-heterotroph Monotropa hypopitys is a widely spread perennial herb used to study symbiotic interactions and physiological mechanisms underlying the development of non-photosynthetic plant. Here, we performed, for the first time, transcriptome-wide characterization of M. hypopitys miRNA profile using high throughput Illumina sequencing. As a result of small RNA library sequencing and bioinformatic analysis, we identified 55 members belonging to 40 families of known miRNAs and 17 putative novel miRNAs unique for M. hypopitys. Computational screening revealed 206 potential mRNA targets for known miRNAs and 31 potential mRNA targets for novel miRNAs. The predicted target genes were described in Gene Ontology terms and were found to be involved in a broad range of metabolic and regulatory pathways. The identification of novel M. hypopitys-specific miRNAs, some with few target genes and low abundances, suggests their recent evolutionary origin and participation in highly specialized regulatory mechanisms fundamental for non-photosynthetic biology of M. hypopitys. This global analysis of miRNAs and their potential targets in M. hypopitys provides a framework for further investigation of miRNA role in the evolution and establishment of non-photosynthetic myco-heterotrophs.
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http://dx.doi.org/10.1007/s11103-016-0478-3DOI Listing
July 2016

The complete chloroplast genome of parasitic flowering plant : extensive gene losses and size reduction.

Mitochondrial DNA B Resour 2016 Mar 28;1(1):212-213. Epub 2016 Mar 28.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.

Plastid genomes of parasitic plants represent apt systems, in which the effects of relaxed selective pressure on photosynthetic function are studied. The complete chloroplast genome sequence of nonphotosynthetic mycoheterotrophic plant was determined. With only 19 protein-coding, four rRNA and 17 tRNA genes in 34 800 bp long genome, it is one of the most reduced plastid genomes characterized until now. chloroplast genome lacks all genes encoding photosynthetic functions and RNA polymerase subunits but retains most of the ribosomal protein genes and housekeeping genes infA and matK. represents the late stages of chloroplast genome decay following the transition to heterotrophy.
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http://dx.doi.org/10.1080/23802359.2016.1155090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7800667PMC
March 2016

Mitochondrial genome of Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae).

Mitochondrial DNA A DNA Mapp Seq Anal 2016 11 30;27(6):4526-4527. Epub 2015 Nov 30.

a National Research Centre, Kurchatov Institute , Moscow , Russian Federation.

The mitochondrial genome of the parasitic wasp Megaphragma amalphitanum is published in this paper. The mitochondrial DNA (mtDNA) is 15 041 base pairs (bp) in length and contained 13 protein-coding genes, 2 rRNA genes and 22 tRNA genes. The overall base composition of the genome in descending order was 45.67% A, 8.71% C, 39.63% T and 5.99% G, with a significant AT bias of 85.30%.
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http://dx.doi.org/10.3109/19401736.2015.1101546DOI Listing
November 2016

Strain Improvement of Streptomyces xanthochromogenes RIA 1098 for Enhanced Pravastatin Production at High Compactin Concentrations.

Indian J Microbiol 2015 Dec 21;55(4):440-6. Epub 2015 May 21.

Laboratory of Biotechnology of Physiologically Active Compounds, Center for Bioengineering, Russian Academy of Sciences, Prospekt 60-Letiya Oktyabrya, 7/1, Moscow, 117312 Russia.

Pravastatin is one of the most popular cholesterol-lowering drugs. Its industrial production represents a two-stage process including the microbial production of compactin and its further biocatalytic conversion to pravastatin. To increase a conversion rate, a higher compactin content in fermentation medium should be used; however, high compactin concentrations inhibit microbial growth. Therefore, the improvement of the compactin resistance of a producer still remains a relevant problem. A multi-step random UV mutagenesis of a Streptomyces xanthochromogenes strain RIA 1098 and the further selection of high-yield compactin-resistant mutants have resulted in a highly productive compactin-resistant strain S 33-1. After the fermentation medium improvement, the maximum bioconversion rate of this strain has reached 91 % at the daily compactin dose equal to 1 g/L and still remained high (83 %) even at the doubled dose (2 g/L). A 1-year study of the mutant strain stability has proved a stable inheritance of its characteristics that provides this strain to be very promising for the pravastatin-producing industry.
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http://dx.doi.org/10.1007/s12088-015-0537-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627957PMC
December 2015

Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant loop region of hepatitis B core antigen: Insertion of multiple copies of M2e increases immunogenicity and protective efficiency.

Vaccine 2015 Jun 29;33(29):3392-7. Epub 2015 Apr 29.

Centre 'Bioengineering', Russian Academy of Sciences, 117312 Prosp. 60-letya Oktyabrya 7-1, Moscow, Russia.

The extracellular domain of the transmembrane protein M2 (M2e) of influenza A virus is a promising target for the development of "universal" vaccines against influenza. M2e is a poor immunogen by itself; however, when M2e is linked to an appropriate carrier, such as hepatitis B virus core (HBc) particles, it becomes highly immunogenic. Insertions of target peptides into the surface-exposed major immunodominant loop region (MIR) of the HBc antigen are especially immunogenic, but such insertions often affect the protein folding and formation of recombinant virus-like particles. To facilitate an appropriate conformation of the M2e insert, we introduced flexible linkers at the junction points between the insert and flanking HBc sequences. This approach allowed the construction of recombinant HBc particles carrying 1, 2 and 4 copies of M2e in the MIR region. These particles were produced in Escherichia coli and purified to homogeneity. The immune response and protective activity of hybrid HBc particles in mice correlated with the number of inserted M2e peptides: the highest immunogenicity and complete protection of mice against the lethal challenge by influenza virus was observed with particles carrying four copies of M2e. The possibility of the simultaneous presentation of M2e peptides from several important influenza strains on a single HBc particle could also facilitate the development of a broad-specificity vaccine efficient not only against influenza A strains of human origin but also for newly emerging strains of animal origin, such as the avian influenza.
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http://dx.doi.org/10.1016/j.vaccine.2015.04.066DOI Listing
June 2015

Understanding and Engineering Thermostability in DNA Ligase from Thermococcus sp. 1519.

Biochemistry 2015 May;54(19):3076-85

The physical chemical principles underlying enzymatic thermostability are keys to understand the way evolution has shaped proteins to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability is also an important factor for engineering more thermoresistant enzymes to be used in the industrial setting, such as, for instance, DNA ligases, which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. Here, we first address the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519 and identify thermosensitive regions using molecular modeling and simulations. In addition, we predict mutations that can enhance thermostability of the enzyme through bioinformatics analyses. We show that thermosensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and we predict that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I, and A387K) eventually produced a significant and additive increase in the half-life of the enzyme when compared to that of the wild type.
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http://dx.doi.org/10.1021/bi501227bDOI Listing
May 2015

Protection against multiple influenza A virus strains induced by candidate recombinant vaccine based on heterologous M2e peptides linked to flagellin.

PLoS One 2015 23;10(3):e0119520. Epub 2015 Mar 23.

Department of Influenza Vaccines, Research Institute of Influenza, Ministry of Health of the Russian Federation, St. Petersburg, Russia.

Matrix 2 protein ectodomain (M2e) is considered a promising candidate for a broadly protective influenza vaccine. M2e-based vaccines against human influenza A provide only partial protection against avian influenza viruses because of differences in the M2e sequences. In this work, we evaluated the possibility of obtaining equal protection and immune response by using recombinant protein on the basis of flagellin as a carrier of the M2e peptides of human and avian influenza A viruses. Recombinant protein was generated by the fusion of two tandem copies of consensus M2e sequence from human influenza A and two copies of M2e from avian A/H5N1 viruses to flagellin (Flg-2M2eh2M2ek). Intranasal immunisation of Balb/c mice with recombinant protein significantly elicited anti-M2e IgG in serum, IgG and sIgA in BAL. Antibodies induced by the fusion protein Flg-2M2eh2M2ek bound efficiently to synthetic peptides corresponding to the human consensus M2e sequence as well as to the M2e sequence of A/Chicken/Kurgan/05/05 RG (H5N1) and recognised native M2e epitopes exposed on the surface of the MDCK cells infected with A/PR/8/34 (H1N1) and A/Chicken/Kurgan/05/05 RG (H5N1) to an equal degree. Immunisation led to both anti-M2e IgG1 and IgG2a response with IgG1 prevalence. We observed a significant intracellular production of IL-4, but not IFN-γ, by CD4+ T-cells in spleen of mice following immunisation with Flg-2M2eh2M2ek. Immunisation with the Flg-2M2eh2M2ek fusion protein provided similar protection from lethal challenge with human influenza A viruses (H1N1, H3N2) and avian influenza virus (H5N1). Immunised mice experienced significantly less weight loss and decreased lung viral titres compared to control mice. The data obtained show the potential for the development of an M2e-flagellin candidate influenza vaccine with broad spectrum protection against influenza A viruses of various origins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119520PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370815PMC
February 2016

The Geoglobus acetivorans genome: Fe(III) reduction, acetate utilization, autotrophic growth, and degradation of aromatic compounds in a hyperthermophilic archaeon.

Appl Environ Microbiol 2015 Feb 21;81(3):1003-12. Epub 2014 Nov 21.

Bioengineering Centre, Russian Academy of Sciences, Moscow, Russia

Geoglobus acetivorans is a hyperthermophilic anaerobic euryarchaeon of the order Archaeoglobales isolated from deep-sea hydrothermal vents. A unique physiological feature of the members of the genus Geoglobus is their obligate dependence on Fe(III) reduction, which plays an important role in the geochemistry of hydrothermal systems. The features of this organism and its complete 1,860,815-bp genome sequence are described in this report. Genome analysis revealed pathways enabling oxidation of molecular hydrogen, proteinaceous substrates, fatty acids, aromatic compounds, n-alkanes, and organic acids, including acetate, through anaerobic respiration linked to Fe(III) reduction. Consistent with the inability of G. acetivorans to grow on carbohydrates, the modified Embden-Meyerhof pathway encoded by the genome is incomplete. Autotrophic CO2 fixation is enabled by the Wood-Ljungdahl pathway. Reduction of insoluble poorly crystalline Fe(III) oxide depends on the transfer of electrons from the quinone pool to multiheme c-type cytochromes exposed on the cell surface. Direct contact of the cells and Fe(III) oxide particles could be facilitated by pilus-like appendages. Genome analysis indicated the presence of metabolic pathways for anaerobic degradation of aromatic compounds and n-alkanes, although an ability of G. acetivorans to grow on these substrates was not observed in laboratory experiments. Overall, our results suggest that Geoglobus species could play an important role in microbial communities of deep-sea hydrothermal vents as lithoautotrophic producers. An additional role as decomposers would close the biogeochemical cycle of carbon through complete mineralization of various organic compounds via Fe(III) respiration.
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http://dx.doi.org/10.1128/AEM.02705-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4292469PMC
February 2015

Optimized method for preparation of IgG-binding bacterial magnetic nanoparticles.

PLoS One 2014 15;9(10):e109914. Epub 2014 Oct 15.

Centre Bioengineering, Russian Academy of Sciences, Moscow, Russia.

In this study, the optimized method for designing IgG-binding magnetosomes based on integration of IgG-binding fusion proteins into magnetosome membrane in vitro is presented. Fusion proteins Mbb and Mistbb consisting of magnetosome membrane protein MamC and membrane associating protein Mistic from Bacillus subtilis as anchors and BB-domains of Staphylococcus aureus protein A as IgG-binding region were used. With Response Surface Methodology (RSM) the highest level of proteins integration into magnetosome membrane was achieved under the following parameters: pH 8.78, without adding NaCl and 55 s of vortexing for Mbb; pH 9.48, 323 mM NaCl and 55 s of vortexing for Mistbb. Modified magnetosomes with Mbb and Mistbb displayed on their surface demonstrated comparable levels of IgG-binding activity, suggesting that both proteins could be efficiently used as anchor molecules. We also demonstrated that such modified magnetosomes are stable in PBS buffer during at least two weeks. IgG-binding magnetosomes obtained by this approach could serve as a multifunctional platform for displaying various types of antibodies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109914PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198182PMC
June 2015

Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

BMC Biotechnol 2014 Jun 14;14:56. Epub 2014 Jun 14.

Laboratory of Mammalian Cell Bioengineering, Centre "Bioengineering", Russian Academy of Sciences, 60-letija Oktyabrya 7, Moscow 117312, Russia.

Background: Establishing highly productive clonal cell lines with constant productivity over 2-3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure.

Results: We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative.

Conclusions: The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.
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http://dx.doi.org/10.1186/1472-6750-14-56DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067061PMC
June 2014

Draft Genome Sequence of Magnetospirillum sp. Strain SO-1, a Freshwater Magnetotactic Bacterium Isolated from the Ol'khovka River, Russia.

Genome Announc 2014 Apr 10;2(2). Epub 2014 Apr 10.

Centre Bioengineering RAS, Moscow, Russia.

Here, we present the draft genome sequence of Magnetospirillum sp. strain SO-1, a freshwater magnetotactic spirillum isolated from the sediments of the Ol'khovka River, Russia.
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http://dx.doi.org/10.1128/genomeA.00235-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983295PMC
April 2014

Complete mitochondrial genome of the cephalosporin-producing fungus Acremonium chrysogenum.

Mitochondrial DNA 2015 10;26(6):943-4. Epub 2014 Jan 10.

a Centre "Bioengineering" RAS , Moscow , Russia.

Complete nucleotide sequence of the 27,266 bp mitochondrial genome of the сephalosporin C producing fungus Acremonium chrysogenum have been determined using whole genome shotgun sequencing approach. The circular mapping molecule encodes a usual set of mitochondrial proteins and RNA genes, including large and small ribosomal RNAs, 19 proteins and 26 tRNA genes and contains 2 introns. All structural genes are located on one strand and are apparently transcribed in one direction. Comparative analysis of this and previously sequenced Pezizomycotina mtDNAs revealed more extensive similarity between A. chrysogenum genome and those of Fusarium clade and specific synthenic patterns characteristic of Hypocrealean mitogenomes. Phylogenetic analysis based on catenated mitochondrial protein sequences confirmed current taxonomic position of A. chrysogenum within Hyprocreales and related taxa.
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http://dx.doi.org/10.3109/19401736.2013.863301DOI Listing
March 2016

Study of Alzheimer family case reveals hemochromotosis-associated HFE mutation.

Hum Genome Var 2014 31;1:14004. Epub 2014 Jul 31.

Center 'Bioengineering', Russian Academy of Sciences , Moscow, Russia.

We report a family case of type II early-onset Alzheimer's disease (AD) inherited over three generations. None of the patients in the family had mutations in the genes believed to be the major risk factors for AD, such as APP, presenilin 1 or 2. Targeted exome sequencing of 249 genes that were previously reported to be associated with AD revealed a rare mutation in hemochromatosis (HFE) gene known to be associated with hemochromotosis. Compared to previous studies, we show that HFE mutation can possess the risk of AD in transferrin-, APOE- and APP-normal patients.
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http://dx.doi.org/10.1038/hgv.2014.4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785525PMC
April 2016