| 1. |
- Gupta, Ankit, et al.
(författare)
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Reconstruction of Bacterial and Viral Genomes from Multiple Metagenomes.
- 2016
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 7:April
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Tidskriftsartikel (refereegranskat)abstract
- Several metagenomic projects have been accomplished or are in progress. However, in most cases, it is not feasible to generate complete genomic assemblies of species from the metagenomic sequencing of a complex environment. Only a few studies have reported the reconstruction of bacterial genomes from complex metagenomes. In this work, Binning-Assembly approach has been proposed and demonstrated for the reconstruction of bacterial and viral genomes from 72 human gut metagenomic datasets. A total 1156 bacterial genomes belonging to 219 bacterial families and, 279 viral genomes belonging to 84 viral families could be identified. More than 80% complete draft genome sequences could be reconstructed for a total of 126 bacterial and 11 viral genomes. Selected draft assembled genomes could be validated with 99.8% accuracy using their ORFs. The study provides useful information on the assembly expected for a species given its number of reads and abundance. This approach along with spiking was also demonstrated to be useful in improving the draft assembly of a bacterial genome. The Binning-Assembly approach can be successfully used to reconstruct bacterial and viral genomes from multiple metagenomic datasets obtained from similar environments.
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| 2. |
- Herold, Nikolas, et al.
(författare)
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Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies
- 2017
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 23:2, s. 256-263
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Tidskriftsartikel (refereegranskat)abstract
- The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults'. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP)(2-5), which causes DNA damage through perturbation of DNA synthesis(6). Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment(7-9). Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.
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| 3. |
- Kaur, S., et al.
(författare)
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Biochemical and proteomic analysis reveals oxidative stress tolerance strategies of Scenedesmus abundans against allelochemicals released by Microcystis aeruginosa
- 2019
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Ingår i: Algal Research. - : Elsevier B.V.. - 2211-9264. ; 41
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Tidskriftsartikel (refereegranskat)abstract
- We studied the possible survival strategies of a green alga, Scenedesmus abundans, against allelochemicals secreted by Microcystis aeruginosa. We exposed the monoculture of S. abundans to a cell free-filtrate (allelochemicals)of M. aeruginosa at the start of our experiment and measured the growth behaviour, morphological changes and oxidative stress markers. The results suggest that exposure to allelochemicals induced oxidative stress in S. abundans, which had significantly reduced the growth of green alga with certain morphological changes. However, after seven days, S. abundans found ways to reduce oxidative stress by recovering its morphology and growth close to that of control. To understand possible survival strategies of test alga, we measured biochemical as well as protein level changes in S. abundans. Biochemical response of the green alga clearly showed that as a response to allelochemicals, enzymatic and non-enzymatic antioxidants were induced. Proteomic analysis showed that exposure to allelochemicals induced accumulation of 13 proteins on the 2-DE gel of S. abundans, which falls in three functional categories, i.e., (i)energy metabolism (photosynthesis, carbon fixation and respiration), (ii)ROS scavenging enzymes and molecular chaperones, and (iii)amino acid and protein biosynthesis. After chronic oxidative stress, these proteins presumably retained glycolysis, pentose phosphate pathway and turnover rate of the Calvin-Benson cycle. Moreover, these proteins assisted in the adequate detoxification of ROS and played an important role in the damage removal and repair of oxidized proteins, lipids and nucleic acids. Therefore, our study anticipates that S. abundans embraces biochemical and proteomic reprogramming to thrives against allelochemicals released by M. aeruginosa.
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| 4. |
- Kumar, Sanjiv, et al.
(författare)
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Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution of treponema pallidum subsp. Pallidum, the syphilis spirochete : QC 20181121
- 2018
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Ingår i: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.
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| 5. |
- Mittal, P., et al.
(författare)
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Metagenome of a polluted river reveals a reservoir of metabolic and antibiotic resistance genes
- 2019
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Ingår i: Environmental Microbiomes. - : BioMed Central. - 2524-6372. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Yamuna, a major tributary of Ganga, which flows through the national capital region of Delhi, is among the major polluted rivers in India. The accumulation of various effluents, toxic chemicals, heavy metals, and increased organic load in the Yamuna directly affects the organisms that thrive inside or around this river. It also makes it an ideal site for studying the impact of pollution on the river microflora, which are sentinels of the water quality. Results: In this study, the microbial community structure and functional diversity of the Yamuna river water was assessed from the New Delhi region. The community structure of Yamuna during pre-monsoon (June) was found to be significantly different from the post-monsoon (November) time, with Acinetobacter being the most abundant genus during June, and Aeromonas during November. The functional characterization revealed the higher abundance of Methyl-accepting chemotaxis protein in the river water, which could be important for the microbial chemosensory adaptation in the environment. A higher abundance of genes related to nitrogen and sulfur metabolism, metal tolerance, and xenobiotic degradation, and complete degradation pathways of aromatic compounds such as toluene, xylene, benzene and phenol were identified. Further, the results showed the presence of a pool of antibiotic resistance genes in the bacterial microbiome in the Yamuna alongside a large number of broad-spectrum antibiotics, such as carbapenemases and metallo-β-lactamases. Efflux mechanism of resistance was found to dominate among these microbes conferring multi-drug resistance. The Principal Coordinate Analysis of the taxonomic composition of the Yamuna River water with publicly available freshwater and sewage datasets revealed significant differences in the two Yamuna samples and a greater resemblance of pre-monsoon Yamuna sample to sewage sample owing to the higher pollution levels in Yamuna in the pre-monsoon time. Conclusion: The metagenomic study of the Yamuna river provides the first insights on the bacterial microbiome composition of this large polluted river, and also helps to understand the dynamics in the community structure and functions due to seasonal variations. The presence of antibiotic resistance genes and functional insights on the metabolic potential of a polluted river microbiome are likely to have several applications in health, biotechnology and bioremediation.
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| 6. |
- Pudelko, Linda, et al.
(författare)
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Glioblastoma and glioblastoma stem cells are dependent on functional MTH1
- 2017
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Ingår i: Oncotarget. - : Impact Journals LLC. - 1949-2553. ; 8:49, s. 84671-84684
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Tidskriftsartikel (refereegranskat)abstract
- Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease.
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| 7. |
- Puthenveetil, Robbins, et al.
(författare)
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The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola.
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N-and C-terminal domains, MOSPN and MOSPC. Biophysical analysis of constructs refolded in vitro demonstrated that MOSPC, previously shown to possess porin activity, forms amphiphilic trimers, while MOSPN forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSPC is OM-embedded and surface-exposed, while MOSPN resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the beta-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli.
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| 8. |
- Radolf, Justin D, et al.
(författare)
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The Treponema pallidum Outer Membrane
- 2017
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Ingår i: Spirochete Biology. - Cham : Springer. ; , s. 1-38
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Bokkapitel (refereegranskat)abstract
- The outer membrane (OM) of Treponema pallidum, the uncultivatable agent of venereal syphilis, has long been the subject of misconceptions and controversy. Decades ago, researchers postulated that T. pallidum's poor surface antigenicity is the basis for its ability to cause persistent infection, but they mistakenly attributed this enigmatic property to the presence of a protective outer coat of serum proteins and mucopolysaccharides. Subsequent studies revealed that the OM is the barrier to antibody binding, that it contains a paucity of integral membrane proteins, and that the preponderance of the spirochete's immunogenic lipoproteins is periplasmic. Since the advent of recombinant DNA technology, the fragility of the OM, its low protein content, and the lack of sequence relatedness between T. pallidum and Gram-negative outer membrane proteins (OMPs) have complicated efforts to characterize molecules residing at the host-pathogen interface. We have overcome these hurdles using the genomic sequence in concert with computational tools to identify proteins predicted to form β-barrels, the hallmark conformation of OMPs in double-membrane organisms and evolutionarily related eukaryotic organelles. We also have employed diverse methodologies to confirm that some candidate OMPs do, in fact, form amphiphilic β-barrels and are surface-exposed in T. pallidum. These studies have led to a structural homology model for BamA and established the bipartite topology of the T. pallidum repeat (Tpr) family of proteins. Recent bioinformatics has identified several structural orthologs for well-characterized Gram-negative OMPs, suggesting that the T. pallidum OMP repertoire is more Gram-negative-like than previously supposed. Lipoprotein adhesins and proteases on the spirochete surface also may contribute to disease pathogenesis and protective immunity.
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| 9. |
- Sharma, Ashok K., et al.
(författare)
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Prediction of peptidoglycan hydrolases- a new class of antibacterial proteins.
- 2016
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Ingår i: BMC Genomics. - : BioMed Central. - 1471-2164. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The efficacy of antibiotics against bacterial infections is decreasing due to the development of resistance in bacteria, and thus, there is a need to search for potential alternatives to antibiotics. In this scenario, peptidoglycan hydrolases can be used as alternate antibacterial agents due to their unique property of cleaving peptidoglycan cell wall present in both gram-positive and gram-negative bacteria. Along with a role in maintaining overall peptidoglycan turnover in a cell and in daughter cell separation, peptidoglycan hydrolases also play crucial role in bacterial pathophysiology requiring development of a computational tool for the identification and classification of novel peptidoglycan hydrolases from genomic and metagenomic data.RESULTS: In this study, the known peptidoglycan hydrolases were divided into multiple classes based on their site of action and were used for the development of a computational tool 'HyPe' for identification and classification of novel peptidoglycan hydrolases from genomic and metagenomic data. Various classification models were developed using amino acid and dipeptide composition features by training and optimization of Random Forest and Support Vector Machines. Random Forest multiclass model was selected for the development of HyPe tool as it showed up to 71.12 % sensitivity, 99.98 % specificity, 99.55 % accuracy and 0.80 MCC in four different classes of peptidoglycan hydrolases. The tool was validated on 24 independent genomic datasets and showed up to 100 % sensitivity and 0.94 MCC. The ability of HyPe to identify novel peptidoglycan hydrolases was also demonstrated on 24 metagenomic datasets.CONCLUSIONS: The present tool helps in the identification and classification of novel peptidoglycan hydrolases from complete genomic or metagenomic ORFs. To our knowledge, this is the only tool available for the prediction of peptidoglycan hydrolases from genomic and metagenomic data.AVAILABILITY: http://metagenomics.iiserb.ac.in/hype/ and http://metabiosys.iiserb.ac.in/hype/ .
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| 10. |
- Sharma, Ashok K., et al.
(författare)
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Woods : A fast and accurate functional annotator and classifier of genomic and metagenomic sequences
- 2015
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Ingår i: Genomics. - : Academic Press. - 0888-7543 .- 1089-8646. ; 106:1, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- Functional annotation of the gigantic metagenomic data is one of the major time-consuming and computationally demanding tasks, which is currently a bottleneck for the efficient analysis. The commonly used homology-based methods to functionally annotate and classify proteins are extremely slow. Therefore, to achieve faster and accurate functional annotation, we have developed an orthology-based functional classifier 'Woods' by using a combination of machine learning and similarity-based approaches. Woods displayed a precision of 98.79% on independent genomic dataset, 96.66% on simulated metagenomic dataset and >97% on two real metagenomic datasets. In addition, it performed >87 times faster than BLAST on the two real metagenomic datasets. Woods can be used as a highly efficient and accurate classifier with high-throughput capability which facilitates its usability on large metagenomic datasets.
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