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1.
Dokl Biochem Biophys ; 496(1): 48-51, 2021 May.
Article in English | MEDLINE | ID: mdl-33689075

ABSTRACT

Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.


Subject(s)
Animals, Genetically Modified/genetics , CRISPR-Cas Systems , Cattle/genetics , Cloning, Organism/methods , Embryo, Mammalian/cytology , Fibroblasts/cytology , Gene Editing/methods , Animals , Animals, Genetically Modified/embryology , Cattle/embryology , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Gene Knockout Techniques/methods , Nuclear Transfer Techniques
3.
Sensors (Basel) ; 19(9)2019 Apr 26.
Article in English | MEDLINE | ID: mdl-31027349

ABSTRACT

The corrosion of steel reinforcement in concrete often leads to huge unbudgeted expenses for maintaining, monitoring and renovating an infrastructure. This is mainly due to the presence of salts or chemical chlorides that pose a danger to the concrete structures. The determination of the existence of these corrosive salts is vital for defining the service life of concrete. This research looked at developing an electromagnetic induction (EMI) sensor for the detection of corrosive salts. The first design adopted a single-loop coil (SLC) concept, and the second was based on a multiple-loop coil (MLC) one using copper wire. Tests were conducted on these two techniques, and with the results obtained, the latter seemed more promising; thus, a prototype sensor was developed using the MLC concept. As this new prototype sensor was able to detect the manifestation of chemical contents in a concrete structure, it could be used as a non-destructive evaluation (NDE) technique for the detection of corrosive chemicals in concrete and has the further possibility of detecting corrosion in concrete.

4.
Dokl Biochem Biophys ; 484(1): 29-32, 2019 May.
Article in English | MEDLINE | ID: mdl-31012007

ABSTRACT

Fifteen chitinases of classes I-V were identified in the transcriptomes of pitchers and adult leaves of the carnivorous plant Nepenthes sp. Ten of these chitinases were identified for the first time, including the chitinases of classes II and V. The expression levels of all found chitinase genes in leaves and at three stages of pitcher development were determined. The maximum level of transcriptional activity in an open pitcher was observed for the genes encoding chitinase NChi4 (class II) and its isoforms. The expression levels of these genes significantly increased as the pitcher developed. In addition, for the first time, transcription of the genes encoding chitinases of all five classes was detected in the leaves of this plant.


Subject(s)
Caryophyllales , Chitinases , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Plant/physiology , Genes, Plant , Plant Proteins , Caryophyllales/enzymology , Caryophyllales/genetics , Chitinases/biosynthesis , Chitinases/genetics , Isoenzymes/biosynthesis , Isoenzymes/genetics , Plant Proteins/biosynthesis , Plant Proteins/genetics
5.
Sci Rep ; 9(1): 1701, 2019 02 08.
Article in English | MEDLINE | ID: mdl-30737432

ABSTRACT

Telomeres are special DNA-protein structures that are located at the ends of linear eukaryotic chromosomes. The telomere length determines the proliferation potential of cells. Telomerase is a key component of the telomere length maintenance system. While telomerase is inactive in the majority of somatic cells, its activity determines the clonogenic potential of stem cells as a resource for tissue and organism regeneration. Reactivation of telomerase occurs during the process of immortalization in the majority of cancer cells. Telomerase is a ribonucleoprotein that contains telomerase reverse transcriptase and telomerase RNA components. The RNA processing mechanism of telomerase involves exosome trimming or degradation of the primary precursor. Recent data provide evidence that the competition between the processing and decay of telomerase RNA may regulate the amount of RNA at the physiological level. We show that termination of human telomerase RNA transcription is dependent on its promoter, which engages with the multisubunit complex Integrator to interact with RNA polymerase II and terminate transcription of the human telomerase RNA gene followed by further processing.


Subject(s)
Promoter Regions, Genetic , RNA/genetics , RNA/metabolism , Telomerase/genetics , Telomerase/metabolism , Feedback, Physiological , HEK293 Cells , Humans , RNA Polymerase II/metabolism , Transcription, Genetic
6.
Ann Surg Oncol ; 25(12): 3556-3562, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30167906

ABSTRACT

BACKGROUND: Knowledge of a germline pathogenic/likely pathogenic variant (PV) may inform breast cancer management. BRCA1/2 PV often impact surgical decisions, but data for multi-gene panel testing are lacking. Expedited genetic testing reduces turn-around times based on request for treatment-related decision making. This report aims to describe the clinical utility of expedited multi-gene panel testing for patients with newly diagnosed breast cancer. METHODS: Clinical and demographic information were reviewed for patients with newly diagnosed female breast cancer undergoing expedited panel testing between 2013 and 2017. The National Comprehensive Cancer Network guidelines (NCCN, version 1.2018) were evaluated in terms of published management recommendations for the genes in which PVs were identified. RESULTS: The overall PV yield was 9.5% (678/7127) for women undergoing expedited panel testing, with 700 PVs identified among 678 women. PVs were identified in genes other than BRCA1/2 in 55.9% (391/700) of cases. The NCCN guidelines recommend management for the genes in which 96.6% (676/700) of PVs are identified. The NCCN guidelines also recommend risk-reducing mastectomy for 46.0% (322/700) of PVs identified. An additional 45.6% (319/700) of PVs were identified in genes for which NCCN recommends mastectomy based on family history. In addition, 49.9% (349/700) of PVs were in genes with NCCN guidelines recommending prophylactic surgery for tissues other than breast. CONCLUSION: A majority of the patients with newly diagnosed breast cancer were candidates for surgical intervention according to the NCCN guidelines, and half of these patients would have been missed if only BRCA1/2 testing had been ordered. Expedited multi-gene hereditary cancer panel testing should be considered as a first-line approach to provide comprehensive information for breast cancer management.


Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Genetic Predisposition to Disease , Genetic Testing , Germ-Line Mutation , Practice Guidelines as Topic/standards , Biomarkers, Tumor/genetics , Breast Neoplasms/surgery , Disease Management , Female , Humans , Mastectomy , Middle Aged , Prognosis
7.
Acta Naturae ; 10(1): 85-94, 2018.
Article in English | MEDLINE | ID: mdl-29713522

ABSTRACT

The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of HA2 (76-130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2e tandem copies (Flg-4M2e-HA2) provides partial α-helix formation nontypical of native M2e on the virion surface. The C-terminal position of the M2e tandem copies (Flg-HA2-4M2e) largely retained its native M2e conformation. These conformational differences in the structure of the two chimeric proteins were shown to affect their immunogenic properties. Different antibody levels induced by the chimeric proteins were detected. The protein Flg-HA2-4M2e was more immunogenic as compared to Flg-4M2e-HA2, with the former offering full protection to mice against a lethal challenge. We obtained evidence suggesting that the order of linkage of target antigens in a fusion protein may influence the 3D conformation of the chimeric construct, which leads to changes in immunogenicity and protective potency.

8.
Vopr Virusol ; 63(3): 130-135, 2018 Jun 20.
Article in English | MEDLINE | ID: mdl-36494939

ABSTRACT

Virus-like HBc particles formed as a result of the self-assembly of the nuclear antigen of the hepatitis B virus can be used as a highly immunogenic carrier for the presentation of foreign epitopes when creating recombinant vaccines. We use this vehicle to create influenza vaccines based on the conservative antigens of the influenza virus, the extracellular domain of the transmembrane protein M2 (M2e) and the fragment of the second subunit of hemagglutinin (HA2). Presentation on the surface of HBc particles should improve the immunogenicity of these peptides. Using genetic engineering techniques, we obtained a fusion protein in which the HA2 sequence is attached to the N-terminus of the HBc antigen, and the M2e peptide is included in the immunodominant loop region exposed on the surface of HBc particle. The hybrid protein expressed in Escherichia coli and purified under denaturing conditions formed virus-like HBc particles after refolding in vitro. Refolding of this protein in the presence of a previously denatured HBc antigen carrying no inserts resulted in formation of mosaic virus-like particles. The developed method will allow construction of mosaic HBc particles carrying different target epitopes of the influenza virus by combining the corresponding modified HBc proteins, which opens the possibility of creating vaccines with a wider spectrum of protection.

9.
Vopr Virusol ; 63(3): 138-143, 2018 Jun 20.
Article in English | MEDLINE | ID: mdl-36494941

ABSTRACT

The aim of the study was to investigate immunogenic properties of mosaic recombinant proteins constructed on the data of hepatitis C virus NS4A and NS4B antigens. Four mosaic recombinant proteins, containing the T and B epitopes of the NS4A and NS4B antigens, were created by genetic engineering methods in the E. coli system. To enhance the immune response they were linked in different variations to the nucleotide sequences of murine interleukin-2 (IL-2), the Neisseria meningiditis lipopeptide, and the T helper epitope of the core protein of hepatitis C virus. The immunogenic properties of these recombinant proteins were analyzed by immunoblotting, ELISA and ELISpot using sera from immunized mice and patients infected with hepatitis C virus. Recombinant proteins specifically reacted with the sera of immunized mice and infected patients in immunoblotting. According to the ELISA data, the predominant formation of antibodies to NS4B was observed when mice were immunized with the recombinant proteins containing both antigens. Analysis of gamma-interferon production by T-lymphocytes upon contact with activated dendritic cells showed in ELISpot that the maximum production of this cytokine was detected when adjuvant components were located at the N- and C-ends of the recombinant protein. The highest level of gamma-interferon production during stimulation with this drug was detected in lymphocytes from the bone marrow and lymph nodes. The recombinant protein containing the T and B epitopes of NS4A and NS4B, murine IL-2 and the lipopeptide Neisseria meningiditis had the greatest immunostimulate effect among the four constructions. This recombinant protein formed nanoparticles of 100-120 nm in size.

10.
Mol Biol (Mosk) ; 51(5): 780-786, 2017.
Article in Russian | MEDLINE | ID: mdl-29116064

ABSTRACT

The precise spatial-temporal coordination of cell division and differentiation is necessary for the correct formation of tissues, organs, and the organism as a whole. This coordination has been implemented by the intercellular communication mediated by signaling molecules and receptors that selectively recognize them. Membrane receptor kinases of ERECTA family regulate inflorescence and flower structure, the formation of root epidermis and adaptation responses. The characterization of the ERECTA genes of flowering plant pinesap Monotropa hypopitys with unique development features can enrich the knowledge about the kinase ERECTA functions and conserved development processes with their participation. Transcriptomic and genomic search with the subsequent structural-phylogenetic analysis identified the mRNA of a gene of serine-threonine kinase receptor with leucine-rich repeats of MhyERL1, which is the only ortholog of the ERECTA family kinases of pinesap. A quantitative analysis of the MhyERL1 gene transcripts has revealed its expression in all analyzed pinesap tissues with maximum levels in the flowers. MhyERL1 is probably involved in defining the inflorescence and flower architecture, and the formation of the pinesap root epidermis. The cascades involving ERL1 are apparently conserved. The exception are pathways associated with the development of above-ground vegetative structures, and the immune response to fungal pathogens probably lost in the process of the pinesap adaptation to unfavorable environmental conditions.


Subject(s)
Ericaceae , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Plant/physiology , Plant Proteins , Protein Serine-Threonine Kinases , Ericaceae/enzymology , Ericaceae/genetics , Plant Proteins/biosynthesis , Plant Proteins/genetics , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics
11.
Dokl Biochem Biophys ; 474(1): 204-208, 2017 May.
Article in English | MEDLINE | ID: mdl-28726096

ABSTRACT

The gene encoding the transcription factor LEAFY was identified in the genome of the mycoheterotrophic plant, pinesap Monotropa hypopitys. In the transcriptomes of roots, bracts, and flowers of flowering pinesaps, the MhyLFY gene expression was absent. These data suggest the conservativeness of the LFY-dependent mechanism of flower meristem identity and flower formation in heterotrophic species with some differences associated to the specificity of development and the structure of such plants. The pinesap flowering under the control of the transcription factor MhyLFY may be initiated either in an embryonic inflorescence during spring dormancy release of adventitious root buds or in an inflorescence of a growing reproductive stem after photoperiodic induction.


Subject(s)
Ericaceae/genetics , Flowers/genetics , Meristem/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Evolution, Molecular , Phylogeny , Plant Proteins/chemistry , Transcription Factors/chemistry
12.
Dokl Biochem Biophys ; 473(1): 111-113, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28510136

ABSTRACT

Genes encoding six chitinases, five of which belong to classes I (MhCHI3 and MhCHI4), IV (MhCHI1), V (MhCHI5), and VII (MhCHI2), were identified in the transcriptome of the parasitic mixoheterotrophic plant Monotropa hypopitys. The transcription level of MhCHI5 and MhCHI1 was low; however, in the leaves (bracts) and roots it was higher than in flowers. MhCHI4 transcripts were detected primarily in the flowers and were almost absent in the roots, whereas the expression level of MhCHI3 was relatively high in all organs but maximum in the leaves (bracts).


Subject(s)
Chitinases/genetics , Ericaceae/enzymology , Ericaceae/genetics , Gene Expression Regulation, Plant , Ericaceae/physiology , Inflorescence/genetics , Rhizome/genetics
13.
Genom Data ; 11: 87-88, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28066711

ABSTRACT

The vast majority of multicellular organisms coexist with bacterial symbionts that may play various roles during their life cycle. Parasitoid wasp Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) belongs to the smallest known insects whose size is comparable with some bacteria. Using 16S rRNA gene sequencing and Whole Genome Sequencing (WGS), we described microbiota diversity for this arthropod and its potential impact on their lifecycle. Metagenomic sequences were deposited to SRA database which is available at NCBI with accession number SRX2363723 and SRX2363724. We found that small body size and limited lifespan do not lead to a significant reduction of bacterial symbionts diversity. At the same time, we show here a specific feature of microbiota composition in M. amalphitanum - the absence of the Rickettsiaceae family representatives that are known to cause sex-ratio distortion in arthropods and well represented in other populations of parasitoid wasps.

15.
Biochemistry (Mosc) ; 81(5): 521-9, 2016 May.
Article in English | MEDLINE | ID: mdl-27297902

ABSTRACT

Besides regular cap-dependent translation of mRNA, eukaryotes exploit internal initiation of translation driven by internal ribosome entry sites (IRESs). It is supposed that internal initiation provides translation of cellular mRNAs under stress conditions where the cap-dependent initiation is reduced. A number of IRESs have been characterized in mammalian mRNAs, but only a few examples are known in lower eukaryotes, particularly in yeasts. Here we identified two IRESs in the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1. These sites are located in 5'-untranslated regions of genes HPODL_02249 and HPODL_04025 encoding a hypothetical membrane protein and actin-binding protein, respectively. In Saccharomyces cerevisiae cells, both IRESs drive expression of a second gene of a bicistronic mRNA, as well as translation of hairpin-containing monocistronic mRNA. The possibility of spurious splicing or presence of a cryptic promoter in the IRES sequences was ruled out, indicating that expression of a second gene of a bicistronic mRNA was IRES-dependent. We evaluated IRES activity of both elements and found that under normal physiological conditions its contribution to the overall translation of the respective mRNAs in yeast cells is about 0.3-0.4%. Therefore, these results suggest that the IRES-dependent translation initiation mechanism exists in Hansenula polymorpha.


Subject(s)
Internal Ribosome Entry Sites/genetics , RNA, Messenger/metabolism , Saccharomycetales/genetics , 5' Untranslated Regions , Base Sequence , Databases, Genetic , Genes, Reporter , Nucleic Acid Conformation , Promoter Regions, Genetic , Protein Biosynthesis/genetics , RNA, Fungal/isolation & purification , RNA, Fungal/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Saccharomyces cerevisiae/metabolism
16.
Genetika ; 52(11): 1249-55, 2016 Nov.
Article in Russian | MEDLINE | ID: mdl-29372787

ABSTRACT

The nucleotide sequence of cryptic plasmid (designated as pBL90) detected in the cells of Brevibacterium lactofermentum DSM 1412 was determined. The length of plasmid DNA is 67826 bp. Comparison of the nucleotide sequence of pBL90 with known plasmid sequences showed no long regions of significant homology. Computer analysis of the plasmid DNA revealed 29 open reading frames (ORFs). The amino acid sequences of 15 ORFs (approximately 25% of plasmid length) have a high (>70%) level of identity to proteins from different plasmids of Corynebacterium representatives, including replicative proteins. Unusual in pBL90 is the presence of replicative genes from two different families and types of replication.


Subject(s)
Bacterial Proteins/genetics , Brevibacterium/genetics , Open Reading Frames , Plasmids/genetics
17.
Mikrobiologiia ; 85(4): 421-435, 2016 Jul.
Article in English | MEDLINE | ID: mdl-28853774

ABSTRACT

Drainage waters at the metal mining areas often have low pH and high content of dissolved metals due to oxidation of sulfide minerals. Extreme conditions limit microbial diversity in- such ecosystems. A drainage water microbial community (6.5'C, pH 2.65) in an open pit at the Sherlovaya Gora polymetallic open-cast mine (Transbaikal region, Eastern Siberia, Russia) was studied using metagenomic techniques. Metagenome sequencing provided information for taxonomic and functional characterization of the micro- bial community. The majority of microorganisms belonged to a single uncultured lineage representing a new Betaproteobacteria species of the genus Gallionella. While no.acidophiles are known among the cultured members of the family Gallionellaceae, similar 16S rRNA gene sequences were detected in acid mine drain- ages. Bacteria ofthe genera Thiobacillus, Acidobacterium, Acidisphaera, and Acidithiobacillus,-which are com- mon in acid mine drainage environments, were the minor components of the community. Metagenomic data were -used to determine the almost complete (-3.4 Mb) composite genome of the new bacterial. lineage desig- nated Candidatus Gallionella acididurans ShG14-8. Genome analysis revealed that Fe(II) oxidation probably involved the cytochromes localized on the outer membrane of the cell. The electron transport chain included NADH dehydrogenase, a cytochrome bc1 complex, an alternative complex III, and cytochrome oxidases of the bd, cbb3, and bo3 types. Oxidation of reduced sulfur compounds probably involved the Sox system, sul- fide-quinone oxidoreductase, adenyl sulfate reductase, and sulfate adenyltransferase. The genes required for autotrophic carbon assimilation via the Calvin cycle were present, while no pathway for nitrogen fixation was revealed. High numbers of RND metal transporters and P type ATPases were probably responsible for resis- tance to heavy metals. The new microorganism was an aerobic chemolithoautotroph of the group of psychrotolerant iron- and sulfur-oxidizing acidophiles of the family Gallionellaceae, which are common in acid mine drainages.


Subject(s)
Gallionellaceae/genetics , Genome, Bacterial , Metagenome , Microbial Consortia/genetics , RNA, Ribosomal, 16S/genetics , Wastewater/microbiology , Acidithiobacillus/classification , Acidithiobacillus/genetics , Acidithiobacillus/isolation & purification , Acidithiobacillus/metabolism , Acidobacteria/classification , Acidobacteria/genetics , Acidobacteria/isolation & purification , Acidobacteria/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Electron Transport Chain Complex Proteins/genetics , Electron Transport Chain Complex Proteins/metabolism , Electron Transport Complex III/genetics , Electron Transport Complex III/metabolism , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Gallionellaceae/classification , Gallionellaceae/isolation & purification , Gallionellaceae/metabolism , Gene Expression , Humans , Hydrogen-Ion Concentration , Iron/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Metals/chemistry , Metals/metabolism , Mining , NADH Dehydrogenase/genetics , NADH Dehydrogenase/metabolism , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Photosynthesis/genetics , Phylogeny , Quinone Reductases/genetics , Quinone Reductases/metabolism , Siberia , Sulfate Adenylyltransferase/genetics , Sulfate Adenylyltransferase/metabolism , Thiobacillus/classification , Thiobacillus/genetics , Thiobacillus/isolation & purification , Thiobacillus/metabolism
18.
Prikl Biokhim Mikrobiol ; 51(5): 465-71, 2015.
Article in Russian | MEDLINE | ID: mdl-26596082

ABSTRACT

An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of ß-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and ß-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA.


Subject(s)
Cephalosporins/metabolism , Escherichia coli/enzymology , Ligases/genetics , Cloning, Molecular , Escherichia coli/genetics , Genome, Bacterial , Ligases/isolation & purification , Ligases/metabolism , Penicillin Amidase/genetics , Penicillin Amidase/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolism
19.
Genetika ; 51(5): 519-28, 2015 May.
Article in Russian | MEDLINE | ID: mdl-26137633

ABSTRACT

Uncultured microorganisms represent a significant part of the Earth's biodiversity. Natural ecosystems contain less than 0.1-1% of the microorganisms that can be cultured in the laboratory. Therefore, new methodological approaches are required for the identification and description of uncultured microorganisms, for studies of their genetic diversity and the structure of microbial associations, and for an understanding of their ecological importance in the biosphere. Metagenomics, a method of analyzing the collective genome.of a microbial community without cultivation, makes it possible to unravel fundamental matters of the microbiology and ecology of microorganisms. Another efficient method of analysis of uncultured forms of microorganisms is "single cell genomics," which involves the isolation of single cells from microbial communities and the sequencing of their genomes. Developed in the last decade, the high throughput technologies of next-generation sequencing provide important input into the investigation of genome reconstruction for all of the microorganisms residing and interacting within ecosystems. This review describes the major methodological approaches used in metagenomic analysis of microbial communities, as well as accomplishments in the search for new uncultured microorganism, the unraveling of their genomes, and an elucidation of their role in ecosystems.


Subject(s)
Genome, Bacterial , Metagenome , Metagenomics/methods , Microbial Consortia/genetics
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