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R K Singh

Publications and source records attributed to R K Singh.

At least 19 recordsLinked to original sources
PubMed2026

Sulfated mannan of diatoms selects host-specific microbiota in the sunlit ocean.

BACKGROUND: Diatoms, a keystone phylum in Earth's ecosystems, are responsible for substantial oxygen production and the fixation of carbon dioxide in the form of carbohydrates that fuel global food webs. They host diverse prokaryotes, yet how diatoms preferentially recruit those with complementary metabolic traits remains unknown. RESULTS: We discovered that diatoms exude a C6-sulfated α-1,3-mannan that serves as a selective carbon source for adapted Polaribacter. Its structure was resolved using NMR spectroscopy, chromatography, chemical synthesis, and enzymatic dissection. Biochemical, physiological, and structural analyses demonstrated, that specialized Bacteroidota employ a four-enzyme pathway to metabolize this glycan. Metagenomic and transcriptomic data revealed that sulfated mannan utilization loci are globally abundant and actively expressed in surface ocean bacterioplankton. Because this mannan provides only carbon, oxygen, sulfur, and hydrogen, bacteria must obtain other essential elements elsewhere, reinforcing metabolic interdependence. CONCLUSIONS: Together, these results define a chemically specific interaction between diatoms and specialized bacteria that is mediated by a single sulfated polysaccharide and a dedicated four-enzyme degradation pathway. Presence of this pathway in marine metagenomes and transcriptomes indicates that a sulfated mannan from diatoms exerts selection pressure in the sunlit ocean microbiome. Video Abstract.

Diatoms↗
PubMed2006

Development of an indirect ELISA for the detection of antibodies against Peste-des-petits-ruminants virus in small ruminants.

Peste des petits ruminants (PPR) is an acute, febrile, highly contagious and economically important viral disease of small ruminants. A polyclonal antibody based indirect ELISA was developed for detection of antibodies to PPR virus in the serum samples of goats and sheep using purified PPR viral antigen propagated in Vero cell culture. A threshold (cut-off) value was set as twice the mean of the negative population based on the distribution of known negative serum samples in respect of PPR virus antibodies in the test. A total of 1544 serum samples from goats and sheep were screened by indirect ELISA and competitive ELISA. The indirect ELISA compared very well with competitive ELISA, with a high degree of specificity (95.09%) and sensitivity (90.81%). When compared with virus neutralization test, the present assay had 100% specificity and 80% sensitivity. With serum samples, the assay could clearly differentiate animals from the infected population from uninfected ones. These results suggest that the indirect ELISA may be a good alternative tool to competitive ELISA for seroepidemiological surveys.

Animals↗
PubMed2006

Interplay between human papilloma virus infection and p53 gene alterations in head and neck squamous cell carcinoma of an Indian patient population.

AIM: To investigate the complex interplay between human papilloma virus (HPV) infection and p53 gene alteration in 92 head and neck squamous cell carcinoma (HNSCC) and 28 leukoplakia samples from eastern India. METHODS: DNA isolated from the patient samples was subjected to HPV detection, loss of heterozygosity (LOH) analysis of the chromosome 17p region harbouring p53, genotyping at the p53 codon 72 locus and sequencing of the entire p53 gene to identify somatic mutations. Codon 72 heterozygotes carrying the p53 mutation were further cloned and resequenced to identify the allele harbouring the mutation. RESULTS: HPV positivity in the HNSCC samples was 69%; 21% of the HNSCC were found to harbour p53 mutations in the coding region of the gene. The absence of the p53 mutation in HPV positive tumours was statistically significant compared to the HPV negative tumours (p = 0.01), but the same did not hold true for p53 LOH (p = 1.0). Among the germline p53 codon 72 heterozygotes, the Pro allele was preferentially lost (p = 0.02) while the Arg allele was mutated in the majority of cases. The risk of HPV mediated tumourigenesis increased with the increase in number of Arg alleles at the codon 72 locus. CONCLUSION: It is proposed that genetic and epigenetic alteration of p53 follow distinct pathways during the development of HNSCC from normal epithelium via dysplasia. The p53 mutation and HPV mediated p53 inactivation possibly constitute two independent pathways of tumourigenesis.

Adult↗
PubMed2006

Development and characterization of a stable vero cell line constitutively expressing Peste des petits ruminants virus (PPRV) hemagglutinin protein and its potential use as antigen in enzyme-linked immunosorbent assay for serosurveillance of PPRV.

We developed and characterized a stable Vero cell line constitutively expressing Peste des petits ruminants virus (PPRV) hemagglutinin (H) protein and assessed its potential use as diagnostic antigen in enzyme-linked immunosorbent assay (ELISA). PPRV H gene of the vaccine strain (Sungri-96) was amplified by reverse transcription (RT)-PCR, cloned into a eukaryotic expression vector (pTarget), and subsequently transfected and expressed in Vero cells. A stable Vero cell line was developed after 20 repeated passages by using G418 antibiotic selection pressure (400 to 600 microg/ml). The integration of PPRV H gene in the Vero cell genome and its genomic transcription were confirmed by PCR and RT-PCR assays, respectively, and the 70-kDa PPRV H protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The recombinant protein reacted specifically with PPRV anti-H neutralizing monoclonal and polyclonal antibody in competitive, sandwich, and indirect ELISA, respectively, indicating that the native form of the protein was expressed. Evaluation of the protein in competitive ELISA and indirect ELISA vis a vis whole virus was done using 306 and 146 goat field serum samples, respectively; comparable results were obtained with high degrees of relative diagnostic specificity (93.53% and 100%, respectively) and sensitivity (99.04% and 79.16%, respectively). This study shows that the PPRV H protein could be a sustainable source of safe antigen in countries of nonendemicity without the need to handle infectious virus for serodiagnosis.

Animals↗
PubMed2006

Comparative sequence analysis of major envelope protein gene (B2L) of Indian orf viruses isolated from sheep and goats.

Orf virus (ORFV), the type species of Parapoxvirus, is responsible for contagious ecthyma in sheep and goats. In the present report, sequence analysis of major envelope gene (B2L) of four Indian orf virus isolates originating two each from sheep and goats was carried out. These recent isolates belonged to different outbreaks that occurred in Kumaon hills and adjoining plains during 2004-2005. Preliminary screening of the scab samples was carried out by diagnostic PCR. Full-length B2L gene encoding for immunogenic major envelope protein from all the four ORFV isolates was amplified by PCR and the amplicons (1206 bp) were cloned and sequenced. Comparative sequence analysis revealed an open reading frame of 1137 nucleotides (nt) encoding a polypeptide of 378 amino acids (aa). Indian isolates were highly related amongst themselves with sequence identity of over 97% at the nt and aa level. Further, they showed 97-98% sequence identity with sequences of other ORFV isolates from around the world; while 94-95 and 82.7-83.8% sequence identity was observed, respectively, with pseudocowpox and bovine papular stomatitis viruses--the other members of the genus. Phylogenetic analysis also showed that these Parapoxviruses from sheep and goats are closely related to other orf viruses reported worldwide.

Amino Acid Sequence↗
PubMed2006

Comparative sequence analysis of envelope protein genes of Indian buffalopox virus isolates.

Buffalopox virus (BPXV) is considered to be a close variant of vaccinia virus (VACV), the prototype member of the genus Orthopoxvirus. In the present study, we have analyzed the sequences of H3L, A27L, and D8L gene-homologues of VACV in BPXV to elucidate its genetic relationship to VACV and other orthopoxviruses (OPVs). Products of these genes have been shown to be important in attachment of VACV to host cell surface receptors during viral entry. Additionally, the A27L gene is also responsible for cell fusion during infection, while the H3L gene is required for synthesis of the highly immunogenic major envelope protein p35. Full-length nucleotide sequences of H3L, A27L, and D8L genes of three BPXV isolates were determined by PCR amplification, cloning, and sequencing. The nucleotide (nt) sequence and the deduced amino acid (aa) sequences were compared with published sequences from other members of the genus Orthopoxvirus. Comparative sequence analysis of all the three genes revealed high sequence identity of BPXV isolates with VACV (close to 99% sequence identity) at both the nt and aa level. Phylogenetic analysis based on the deduced aa sequences of the H3L, A27L, and D8L genes also showed that BPXVs are more closely related to VACV than to any of the other OPVs.

Amino Acid Sequence↗
PubMed2006

The current status of sheep pox disease.

Sheep are the moving banks of shepherds and their economic contribution in terms of meat, wool and skin/hide is immense. Various infectious diseases jeopardize the optimum productivity; among which sheep pox is more important as the disease restricts the export of sheep and their products besides other economic losses. Although, clinical signs are indicative of the disease but a laboratory confirmation is necessary for unequivocal diagnosis and studying epidemiology. The causative agent, sheep pox virus (SPV), is antigenically and genetically closely related to goat pox virus (GPV) and lumpy skin disease virus (LSDV), the other members of the genus capripox virus. In some countries, SPV and GPV are cross infective to small ruminants posing problem in diagnosis and epidemiology. However, recent studies have showed that the viruses are phylogenetically distinct and can be differentiated by molecular tools. Prophylaxis using attenuated vaccines is the choice of control measure as the immunity is long lasting. Detailed information on isolation, identification, pathology, epidemiology, diagnosis and prophylaxis would not only help in updating the knowledge of scientific fraternity but will be useful to the policy makers in order to formulate appropriate measures for control and eradication of the disease. This synthesis is to present an up-to-date review of the disease and its control to provide the reader with an overview of the problem.

Animals↗
PubMed2006

Doppler effect in fluorine K-Auger line produced in electron-induced core ionization of SF6.

An experimental evidence is reported on the observation of the Doppler effect in fluorine K-Auger line emitted from a core-ionized SF6 molecule under an impact of 16 keV electrons. The emitting source of the Auger line is found to acquire a kinetic energy of 4.7+/-0.3 keV. We propose that such large energy is released from the Coulomb repulsion taking place between F+ and SF5+ fragment ions under influence of an intense focusing field of the incident electrons. In the presence of the Coulomb field of these ions, the Auger line obtains a polarization P = 76%+/-7%.

Journal Article↗
PubMed2006

Ferulic acid esterase production by Streptomyces sp.

Studies were carried out on ferulic acid esterase production using a culture of Streptomyces S10. In optimized condition, enzyme yield was 2.0 mU/ml in MBS medium, containing 1.5% de-starched wheat bran at 30 degrees C and initial pH 6.5 under agitated submerged culture.

Carboxylic Ester Hydrolases↗
PubMed2006

One-step multiplex RT-PCR assay for the detection of peste des petits ruminants virus in clinical samples.

A single-tube one-step multiplex RT-PCR was standardized to amplify both 337 bp and 191 bp fragments of N and M genes of peste des petits ruminants virus (PPRV), respectively, and only a 337 bp fragment of N gene of Rinderpest virus (RPV). The RT-PCR using purified viral RNA was easily adopted for direct detection of PPRV in clinical field samples and its differentiation from RPV. The amplified N and M gene products were confirmed to be PPRV- and RPV-specific by their size in 1.5% agarose gel and restriction analysis. In the assay, the Qiagen one-step RT-PCR kit containing the Ominiscript and Sensiscript reverse transcriptases and Hot star Taq DNA polymerase was utilized. The sensitivity of the assay was found to be 100 fg of PPRV RNA. Compared with a two-step assay, the one-step assay is easier and time-saving as it requires just a single buffer for both reactions, reverse transcription (RT) and PCR. In experimentally infected goats, PPRV was detectable by the one-step RT-PCR in nasal and ocular swabs 7-17 days post infection (p.i.). and in oral swabs 7-15 days p.i. Out of 32 clinical field samples tested, 18 were positive by sandwich ELISA (S-ELISA), while 22 were positive by the one-step RT-PCR.

Animals↗
PubMed2006

Sequence analysis of the haemagglutinin and fusion protein genes of peste-des-petits ruminants vaccine virus of Indian origin.

The amino acid composition of the two surface proteins of peste-des-petits ruminants vaccine virus belonging to lineage four from India were deduced from the nucleotide sequence. The fusion (F) protein gene of PPRV Sungri/96 is 2405 nucleotides long and in relation to the length, it is 80 nucleotides longer than that of PPRV Nigeria/75/1 which are found to be present at the 5'UTR of this virus. The complete F gene alignment with other morbillivirus reveals a homology of 89% with PPRV/Nigeria/75/1 and 48-51% with other morbilliviruses. The F protein of PPRV Sungri/96 exhibited characteristics similarity to those of other morbillivirus F proteins. The overall amino acid similarity with its counterpart PPRV Nigeria/75/1 was 96%; with other morbilliviruses it is 65-74%. The PPRV Sungri/96 haemagglutinin (H) protein gene is 1954 nucleotides long and showed a sequence homology of 90.7% with PPRV/Nigeria/75/1 and with other morbilliviruses it ranged from 33% to 45%. At amino acids level, PPRV Sungri/96 showed a homology of 92.3% with PPRV/Nigeria/75/1 and 34-49% with other morbilliviruses. The phylogenetic tree constructed for F and H gene reveals four separate groups which is very similar to that found in other genes. To the best of our knowledge this is the first report describing the F and H genes of an Indian isolate.

Amino Acid Sequence↗
PubMed2006

Visceral leishmaniasis (kala-azar): challenges ahead.

Indian visceral leishmaniasis (VL) is a parasitic disease caused by a haemoflagellete Leishmania donovani and transmitted by the bite of sand fly Phlebotomus argentipes. It affects various age groups. In India about 1,00,000 cases of VL are estimated to occur annually; of these, the State of Bihar accounts for over than 90 per cent of the cases. Diagnosis of VL typically relies on microscopic examination of tissue smears but serology and molecular methods are better alternatives currently. Notwithstanding the growing incidence of resistance, pentavalent antimony complex has been the mainstay for the treatment of VL during the last several decades. The second line drugs such as amphotericin B, lipid formulations of amphotericin B, paromomycin and recently developed miltefosine are the other alternatives. In spite of significant development in various areas of Leishmania research, there is a pressing need for the technological advancement in the understanding of immune response, drug resistance and the pathogenesis of leishmaniasis that could be translated into field applicable and affordable methods for diagnosis, treatment, and control of the disease.

Aminoquinolines↗
PubMed2005

Serological diagnosis of Indian visceral leishmaniasis: direct agglutination test versus rK39 strip test.

We evaluated the direct agglutination test (DAT), using freeze-dried (FD) and aqueous (AQ) antigen, and the rK39 immunochromatographic strip test in the diagnosis of Indian visceral leishmaniasis (VL). Sera from 508 subjects (150 parasitologically confirmed patients with VL, 100 and 153 healthy controls drawn from non-endemic and endemic regions, respectively, and 105 patients with other diseases presenting with fever and/or splenomegaly) were tested. The sensitivity of the tests were as follows: DAT (FD), 96% (95% CI 91-98); DAT (AQ), 97% (95% CI 93-99); rK39 strip test, 99% (95% CI 95-100). The specificity of DAT (FD), DAT (AQ) and rK39 strip tests were 85% (95% CI 81-88), 87% (95% CI 83-91) and 89% (95% CI 86-92), respectively. A significant correlation (high degree of agreement) was observed between all tests (kappa>0.80). We conclude that the sensitivity of FD antigen is comparable to that of AQ antigen. Similarly, the rK39 strip test is as sensitive as the DAT, but the strip test's greater convenience of use makes it a better tool for diagnosis of VL in peripheral areas of endemic regions.

Agglutination Tests↗
PubMed2005

Analysis of the matrix protein gene sequence of the Asian lineage of peste-des-petits ruminants vaccine virus.

The M gene nucleotide sequence of an Indian peste-des-petits ruminants (PPRV) vaccine virus ("PPRV Sungri/96") belonging to Asian lineage was determined. The gene is 1476 nucleotides long with a single open reading frame (ORF). The nucleotide and predicted amino acid sequence was compared with the homologous region of the African Lineage Vaccine virus "PPRV/Nigeria/75/1". The nucleotide sequence of the "PPRV Sungri/96" was 86% identical to that of "PPRV/Nigeria/75/1", while a homology of 93% and 95% could be observed in the ORF and amino acids level, respectively. The M gene encodes a protein of 335 amino acids, with a predicted molecular weight (MW) of 37.8 kDa. The ORF is flanked by a 3' untranslated region of 436 nucleotides and a high level of sequence divergence (approximately 30%) could be observed in this region between the vaccine viruses of Asian and African lineages. A high degree of conservation of several amino acids of this protein observed previously was also confirmed in this study.

Amino Acid Sequence↗