Bioremediation of motor oil-contaminated soil and water by a novel indigenous Pseudomonas otitidis strain DU13 and characterization of its biosurfactant.

Production of biosurfactant by a novel indigenous isolate Pseudomonas otitidis strain DU13 and its role in bioremediation of petroleum hydrocarbon is reported. The identity of the isolate was confirmed by 16S rDNA gene sequencing analysis (Genbank accession: MK177190). The biosurfactant produced by the isolate could reduce the surface tension of petroleum supplemented medium by 46% just after 7 days of treatment. The emulsification index (E 24 ) of the surfactant was found 37, 35, and 20%, respectively, against used motor oil, diesel, and kerosene. The FTIR spectrum of the crude biosurfactant showed the presence of υC-H stretch, υCH2, υ-C=C stretch and υC-H bonding. The isolated strain could degrade 26% of TPH content of used motor oil in liquid culture. Whereas, ex situ pilot-scale field trial demonstrated very high bioremediation potential of the isolate in terms of germination rate of Vigna radiata and Cicer arietinum seeds and plant growth just after 20 days of treatment.

Black tea bioactives as inhibitors of multiple targets of SARS-CoV-2 (3CLpro, PLpro and RdRp): a virtual screening and molecular dynamic simulation study.

The global pandemic due to the novel Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has taken more than a million lives. Lack of definitive vaccine/drugs against this highly contagious virus has accelerated exploratory research on novel natural and synthetic inhibitors. Tea is a rich source of bioactives and known to have antiviral properties. In this study, an in silico strategy involving ADMET property screening, receptor-ligand docking and molecular dynamic (MD) simulation was employed to screen potential tea bio-active inhibitors against three selected targets (RdRp, 3CLpro and PLpro) of SARS-CoV-2. Among the 70 tea bioactives screened, theaflavin 3,3'-di-gallate (TF3), Procyanidin B2 and Theaflavin 3-gallate (TF2a) exhibited highest binding affinities towards RdRp, 3CLpro/Mpro and PLpro targets of SARS-CoV-2 with low docking scores of -14.92, -11.68 and -10.90 kcal/mol, respectively. All of them showed a substantial number of hydrogen bonds along with other interactions in and around the active sites. Interestingly, the top bioactives in our study showed higher binding affinities compared with known antiviral drugs. Further, the top protein-ligand complexes showed less conformational changes during binding when subjected to MD simulation for 100 nanoseconds. The MMPBSA results revealed that RdRp-TF3, 3CLpro-Procyanidin B2 and PLpro-TF2a complexes were stable with binding free energies of -93.59 ± 43.97, -139.78 ± 16.51 and -96.88 ± 25.39 kJ/mol, respectively. Our results suggest that theaflavin 3,3'-digallate, Theaflavin 3-gallate and Procyanidin B2 found in black tea have the potential to act as inhibitors for selected targets of SARS-CoV-2 and can be considered as drug candidates in future studies against COVID-19.

Genome-wide unique insertion sequences among five Brucella species and demonstration of differential identification of Brucella by multiplex PCR assay.

Brucellosis is a neglected zoonotic disease caused by alpha proteobacterial genus Brucella comprising of facultative intracellular pathogenic species that can infect both animals and humans. In this study, we aimed to identify genome-wide unique insertion sequence (IS) elements among Brucella abortus, B. melitensis, B. ovis, B. suis and B. canis for use in species differentiation by conducting an intensive in silico-based comparative genomic analysis. As a result, 25, 27, 37, 86 and 3 unique ISs were identified respectively and they had a striking pattern of distribution among them. To explain, a particular IS would be present in four species with 100% identity whereas completely absent in the fifth species. However, flanking regions of that IS element would be highly identical and conserved in all five species. Species-specific primers designed on these flanking conserved regions resulted in two different amplicons grouping the species into two: one that possesses IS and the other that lacks it. Seeking for species-specific amplicon size for particular species was sufficient to identify it irrespective of biovar. A multiplex PCR developed using these primers resulted in successful differentiation of the five species irrespective of biovars with significant specificity and sensitivity when examined on clinical samples.

TeaMiD: a comprehensive database of simple sequence repeat markers of tea.

Tea is a highly cross-pollinated, woody, perennial tree. High heterozygosity combined with a long gestational period makes conventional breeding a cumbersome process. Therefore, marker-assisted breeding is a better alternative approach when compared with conventional breeding. Considering the large genome size of tea (~3 Gb), information about simple sequence repeat (SSR) is scanty. Thus, we have taken advantage of the recently published tea genomes to identify large numbers of SSR markers in the tea. Besides the genomic sequences, we identified SSRs from the other publicly available sequences such as RNA-seq, GSS, ESTs and organelle genomes (chloroplasts and mitochondrial) and also searched published literature to catalog validated set of tea SSR markers. The complete exercise yielded a total of 935 547 SSRs. Out of the total, 82 SSRs were selected for validation among a diverse set of tea genotypes. Six primers (each with four to six alleles, an average of five alleles per locus) out of the total 27 polymorphic primers were used for a diversity analysis in 36 tea genotypes with mean polymorphic information content of 0.61-0.76. Finally, using all the information generated in this study, we have developed a user-friendly database (TeaMiD; http://indianteagenome.in:8080/teamid/) that hosts SSR from all the six resources including three nuclear genomes of tea and transcriptome sequences of 17 Camellia wild species. Database URL: http://indianteagenome.in:8080/teamid/.

Evaluation of fimC and bdha based duplex PCR for specific identification and differentiation of Burkholderia pseudomallei from near-neighbor Burkholderia species.

Assays for the rapid detection and accurate differentiation of Burkholderia pseudomallei from near-neighbor species are urgently needed in melioidosis endemic regions due to the high associated mortality and biowarfare importance of the pathogen. PCR-based methods have revolutionized this field due to the accuracy, sensitivity, and specificity that are achievable in a rapid way. In this study, a compound molecular detection system, consisting of a duplex PCR assay, was developed for the specific identification of Burkholderia pseudomallei and differentiation from other Burkholderia species. For accurate identification of B. pseudomallei, we deciphered and adopted a novel gene termed putative fimbrial chaperone (fimC). d-beta hydroxybutyrate dehydrogenase (bdha), reported previously by our group for sequence-based differentiation of B. pseudomallei from other Burkholderia species, was employed as a genus-specific target. Enforcement of an internal amplification control in the PCR format ruled out possible false negative results in the assay. Thus, the developed PCR assay was highly specific (100%) in its detection features, and a clear detection sensitivity of 10 pg/µl for purified gDNA and 3 × 103 CFU/ml for B. pseudomallei spiked urine was recorded. Successful identification of B. pseudomallei from an experimental mouse model at both the genus and species level revealed the accurate diagnostic efficiency of the duplex PCR method.

Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India.

Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively). In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha) was studied both in silico and in vitro for accurate detection of Burkholderia genus. The optimized bdha based PCR assay when evaluated on the Burkholderia isolates of this study, it was found to be highly specific (100%) in its detection feature and a clear detection sensitivity of 10 pg/µl of purified gDNA was recorded. Nucleotide sequence variations of bdha among interspecies, as per in silico analysis, ranged from 8 to 29% within the target stretch of 730 bp highlighting the potential utility of bdha sequencing method in specific detection of Burkholderia species. Further, sequencing of the 730 bp bdha PCR amplicon of each Burkholderia strain isolated could differentiate the species and the data was comparable with recA sequence data of the strains. All sequencing results obtained were submitted to NCBI database. Bayesian phylogenetic analysis of bdha in comparison with recA and 16S rDNA showed that the bdha gene provided comparable identification of Burkholderia species.

Computational identification and analysis of MADS box genes in Camellia sinensis.

MADS (Minichromosome Maintenance1 Agamous Deficiens Serum response factor) box genes encode transcription factors and they play a key role in growth and development of flowering plants. There are two types of MADS box genes- Type I (serum response factor (SRF)-like) and Type II (myocyte enhancer factor 2 (MEF2)-like). Type II MADS box genes have a conserved MIKC domain (MADS DNA-binding domain, intervening domain, keratin-like domain, and c-terminal domain) and these were extensively studied in plants. Compared to other plants very little is known about MADS box genes in Camellia sinensis. The present study aims at identifying and analyzing the MADS-box genes present in Camellia sinensis. A comparative bioinformatics and phylogenetic analysis of the Camellia sinensis sequences along with Arabidopsis thaliana MADS box sequences available in the public domain databases led to the identification of 16 genes which were orthologous to Type II MADS box gene family members. The protein sequences were classified into distinct clades which are associated with the conserved function of flower and seed development. The identified genes may be used for gene expression and gene manipulation studies to elucidate their role in the development and flowering of tea which may pave the way to improve the crop productivity.

Prevalence of cataract in an older population in India: the India study of age-related eye disease.

PURPOSE: To describe the prevalence of cataract in older people in 2 areas of north and south India. DESIGN: Population-based, cross-sectional study. PARTICIPANTS: Randomly sampled villages were enumerated to identify people aged ≥ 60 years. Of 7518 enumerated people, 78% participated in a hospital-based ophthalmic examination. METHODS: The examination included visual acuity measurement, dilatation, and anterior and posterior segment examination. Digital images of the lens were taken and graded by type and severity of opacity using the Lens Opacity Classification System III (LOCS III). MAIN OUTCOME MEASURES: Age- and gender-standardized prevalence of cataract and 95% confidence intervals (CIs). We defined type of cataract based on the LOCS III grade in the worse eye of: ≥ 4 for nuclear cataract, ≥ 3 for cortical cataract, and ≥ 2 for posterior subcapsular cataract (PSC). Any unoperated cataract was based on these criteria or ungradable dense opacities. Any cataract was defined as any unoperated or operated cataract. RESULTS: The prevalence of unoperated cataract in people aged ≥ 60 was 58% in north India (95% CI, 56-60) and 53% (95% CI, 51-55) in south India (P = 0.01). Nuclear cataract was the most common type: 48% (95% CI, 46-50) in north India and 38% (95% CI, 37-40) in south India (P<0.0001); corresponding figures for PSC were 21% (95% CI, 20-23) and 17% (95% CI, 16-19; P = 0.003), respectively, and for cortical cataract 7.6% (95% CI, 7-9) and 10.2% (95% CI, 9-11; P<0.004). Bilateral aphakia/pseudophakia was slightly higher in the south (15.5%) than in the north (13.2%; P<0.03). The prevalence of any cataracts was similar in north (73.8%) and south India (71.8%). The prevalence of unoperated cataract increased with age and was higher in women than men (odds ratio [OR], 1.8). Aphakia/pseudophakia was also more common in women, either unilateral (OR, 1.2; P<0.02) or bilateral (OR, 1.3; P<0.002). CONCLUSIONS: We found high rates of unoperated cataract in older people in north and south India. Posterior subcapsular cataract was more common than in western studies. Women had higher rates of cataract, which was not explained by differential access to surgery.

Periosteal fixation in third-nerve palsy.

PURPOSE: We present a new technique of anchoring the eyeball to the nasal periosteum using a nonabsorbable suture in acquired isolated third-nerve paresis. METHODS: This was a case series of 4 consecutive adult subjects with isolated third-nerve paresis. After a 12-mm lateral rectus muscle recession in all 4 subjects, we passed 5-O double-armed polyester (NW683 Ethibond; Ethicon, Division of Johnson and Johnson Ltd., Aurangabad, India) on spatulated needles through the periosteum overlying the anterior lacrimal crest (exposed as in a Dacryocystorhinostomy procedure) at its superior part. The needles were brought anterior to the medial rectus muscle insertion and tightened enough to align the eye in 8-10 prism diopters adducted position. RESULTS: Ocular alignment in the primary gaze was satisfactory at 6-12 months of follow up. CONCLUSIONS: The use of a nonabsorbable polyester suture to anchor the globe to the nasal periosteum is an additional technique that holds promise to align the eyes in the primary gaze.

Isolated "one and a half syndrome" with brainstem tuberculoma.

A seldom reported causal association of two rare entities, an isolated brainstem tuberculoma and an isolated One and a half syndrome in a 12 year old girl is presented. MRI showed an isolated inflammatory granuloma in the brainstem which on empirical treatment with anti tubercular drugs resulted in complete restoration of ocular motility, along with resolution of the lesion on follow up MRI at 6 months. The diagnosis and management are discussed.