Clinico-pathological factors affecting lymph node yield in Indian patients with locally advanced squamous cell carcinoma of mandibular Gingivo-Buccal sulcus.

AIM: Lymph node yield (LNY) is a valid marker of prognosis in oral cancer. Precise estimation of LNY in Indian patients with T3/T4 gingivobuccal sulcus squamous cell carcinoma (GBS-SCC) has not been well documented. Hence, the primary objective of the study was to determine the LNY in patients with T3/T4 SCC of mandibular GBS, and the secondary objective was to study the association of LNY with clinicopathological factors such as tumor thickness, histological differentiation, number of positive nodes, and extracapsular spread (ECS). MATERIALS AND METHODS: Study patients comprised biopsy proven T3/T4 SCC of mandibular GBS that underwent unilateral surgery (composite or bite composite resection with level I to level V-neck dissection and pectoralis major flap reconstruction) at our center between January 2012 and October 2014. Grossing of surgical specimens was done as per the guidelines established by the Royal College of Pathologists (December 2009). The data were analyzed using SPSS software (22nd version) and Chi-square test. RESULTS: The surgical specimens of 106 patients yielded 2329 lymph nodes with the mean LNY of 21.97 ± 5.57. Higher mean LNY of over 21 was significantly associated with ECS, number of positive nodes, delay in surgery over 15 days, skin involvement by the tumor, and presence of oral potentially malignant disorders. CONCLUSION: With the single surgeon, pathologist and same surgical procedure, the mean LNY in Indian patients with T3/T4 SCC of mandibular GBS is 21.97 ± 5.57. Although clinicopathological factors affect the estimation of LNY, further studies are needed to validate the findings of this study.

Actinomycetes diversity among rRNA gene clones and cellular isolates from Sambhar salt lake, India.

The vertical stratification of actinomycetes diversity in Sambhar salt lake (India's largest salt lake) was investigated by using cultivable and uncultivable approaches. The isolates from cultured approaches were clustered on the basis of cultural, morphological, biochemical, and cell wall characteristics, and results were further strengthened by 16S rDNA-RFLP into five major groups. 16S rDNA sequencing of the representative isolates from each clusters was identified as belonging to Streptomyces, Actinopolyspora, Microbispora, Saccharopolyspora, and Actinoplanes genera, while culture independent group was established as Streptomyces (130 clones, 20 OTUs), Micromonospora (96 clones, 7 OTUs), Streptosporangium (79 clones, 9 OTUs), Thermomonospora (46 clones, 8 OTUs), and Dactylosporangium (58 clones, 8 OTUs). The diversity assessment using Shannon and Wiener index was found to be 1.55, 1.52, 1.55, and 1.49 from surface lake water, at depth of 1.5 m, shallow layer of water with algal population, and finally at depth of 2.5 m, respectively. We observed diversity in terms of the species richness as Streptomyces is dominant genus in both culture dependent and culture independent techniques followed by Microbispora (culture dependent methods) and Micromonospora (culture independent method) genera, respectively.

Analysis of opportunities and challenges in patenting of Bacillus thuringiensis insecticidal crystal protein genes.

Bacillus thuringiensis (Bt) is the most widely used microbial control agent. The broad spectrum of susceptible hosts, production on artificial media and ease of application has caused the widespread use of this bacterium against several pests in agriculture, forest and vectors of human diseases. B.thuringiensis toxins are highly species specific which provide economic, environmental benefits, potential for future control and spread of the technology worldwide. This makes the B. thuringiensis crystal proteins an interesting tool for the implementation in integrated pest management programs. It has gained importance over the last 100 years for its biocontrol properties which is used in this review as a case study and analysis of the patents granted on B. thuringiensis was carried out. This study categorizes a number of patents related to B.thuringiensis insecticidal crystal proteins, application of B.thuringiensis insecticidal crystal proteins and the development of patentable technologies. The analyses were done using various criteria like patenting trends over the years, assignees playing a major role, comparison of the technology used in different patents and the patenting activity across the insect orders. Patent documents related to bacterium B.thuringiensis contain a trove of technical and commercial information and thus, patent analysis is considered as a useful tool for R management and techno economical development. Patent analysis also helps identifying and evaluating new and alternate technologies, keeping abreast with latest technologies for business interests, finding solutions to technical problems and ideas for new innovative trends.

Screening, diversity and partial sequence comparison of vegetative insecticidal protein (vip3A) genes in the local isolates of Bacillus thuringiensis Berliner.

Characterization, direct sequencing of the PCR amplicon and phylogenetic relationship was done to discover a novel Vip protein genes of the Bt isolates, to improve the prospects for insect control, more Vip proteins should be sought out and researched to predict their insecticidal activity. Characterization was based on direct sequencing of PCR amplicon using primers specific to vip3A gene was presented here. 12 out of 18 isolates screened were positive for vip gene-specific primers. Homology search for the partial sequences using BLAST showed that 11 isolates had high similarity to vip3Aa gene and only one fragment with vip3Ae gene (25-100% at nucleotide and amino acid level). Phylogenetic analysis showed that the gene sequences were responsible for geographic separation for divergence within vip genes, consistent with the evaluation of distinct bacterial population. Despite the geographical distances, strains harbouring vip genes have originated from common ancestors may significantly contribute to control resistant insect pests. Some strains have evolved to be quite distinct and others remain as members of closely related groups. The reported method is a powerful tool to find novel Vip3A proteins from large-scale Bt strains which is effective in terms of time and cost. Further the Vip proteins produced by different strains of B. thuringiensis are unique in terms of the sequence divergence and hence may also differ in their insecticidal activities.

Cloning, characterization and diversity of insecticidal crystal protein genes of bacillus thuringiensis native isolates from soils of Andaman and Nicobar Islands.

Bt strains were isolated from soils of Andaman and Nicobar Islands and characterized by microscopic and molecular methods. Diversity was observed both in protein and cry gene profiles, where majority of the isolates showed presence of 65 kDa protein band on SDS-PAGE while rest of them showed 130, 72, 44, and 29 kDa bands. PCR analysis revealed predominance of cry1I and cry7, 8 genes in these isolates. The PCR screening strategy presented here led us to identify putative novel cry genes which could be active against Coleoptera insects. Variation in the nucleotide sequences of cry genes from the isolates suggests that the genetic diversity of Bt isolates results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats in the soils of Andaman and Nicobar islands. The implications of our studies are important from the point of view of identifying novel cry genes that could be toxic to insects other than lepidoptera.

Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of Bacillus species.

Restriction fragment length analysis of PCR amplified 16S rDNA with AluI revealed the presence of a 265 bp fragment in all species of Bacillus with the exception of B. cereus and B. thuringiensis, which contains two restriction sites within this fragment which results in three smaller fragments totalling to 265 bp. Some distant species of Bacillus with no evidence of this fragment could be delineated into other genera based on phenotypic and genotypic parameters. BLAST search for homologous sequences of individual species revealed that it is a highly conserved region. Multiple alignment of the fragment suggests that a region between 160 and 265 bp of the 265 bp fragment was a hypervariable region and were highly species-specific. A set of primers was designed for amplification of this hypervariable region. Partial sequencing of the hypervariable region within the 265 bp fragment seems an index for identification of Bacillus species.

Biochemical and molecular characterization of thermo-alkali tolerant xylanase producing bacteria from thermal springs of Manikaran.

One hundred ten alkalo-tolerant thermophilic bacteria were isolated from 17 samples (water and sediment) collected from Manikaran. Of 110 isolates, 70 showed the production of xylanases and were further screened for growth and production of xylanases at different temperature ranging from 40 to 75°C. Eleven isolates that showed growth and xylanase production at temperatures ≥50°C were selected for quantitative estimation in modified Reese mineral liquid medium containing wheat bran. Maximum xylanase activity was produced by isolate H-7 followed by H-9 and R-9 and was statistically superior to other isolates. The microscopic observation showed that the isolates possessed the typical rod with endospore, characteristic of genus Bacillus. The isolates were found to be oxidase and catalase positive. Using BIOLOG Microlog 3 software, the isolates H7, H9 and R9 were identified as Paenibacillus ehemensis, Bacillus cereus/B. thuringiensis and B. subtilis respectively, based on utilization of 95 carbon sources. PCR-RFLP analysis of 16S rDNA indicated that the isolates were genetically different from each other. DNA sequencing of the three isolates and phylogenetic analysis revealed that all the isolates obtained from Manikaran thermal springs showed 97 to 100% similarity with the sequences within the GenBank. The closest phylogenetic neighbours according to the 16S rRNA gene sequence data for the three isolates H-7, H-9, and R-9 were Paenibacillus ehemensis, Bacillus cereus and Bacillus subtilis, respectively.

RAPD cluster analysis and chlorate sensitivity of some Indian isolates of Macrophomina phaseolina from sorghum and their relationships with pathogenicity.

Charcoal rot caused by Macrophomina phaseolina is an economically important disease in sorghum grown during the post rainy season in India. Variations in random amplified polymorphic DNA (RAPD) polymorphisms, chlorate sensitivity and pathogenicity were studied among sorghum isolates of M. phaseolina collected from different parts of India. RAPD data based on 14 random primers of Kit A and C (OPA and OPC) on 20 isolates showed a high degree of polymorphism (98.1%) in different isolates. UPGMA dendrogram on RAPD data produced 7 clusters at the level of 37% similarity. Isolates from the same locations showed a tendency to group closer, substantiating closer genetic relatedness. Sorghum infecting Macrophomina isolates showed a mixed response for sensitivity to potassium chlorate (120 mM). Chlorate-resistant isolates were predominant (>65% of the isolates) over sensitive isolates. Chlorate-sensitive isolates were found to be genetically closer among them than the resistant ones. For the first time it was shown that chlorate sensitivity in Macrophomina had some relations with charcoal rot severity in sorghum.

Intestinal glucose sensing and regulation of intestinal glucose absorption.

SGLT1 (Na(+)/glucose co-transporter 1) transports the dietary sugars, D-glucose and D-galactose, from the lumen of the intestine into enterocytes. SGLT1 regulation has important consequences for the provision of glucose to the respiring tissues and is therefore essential for maintaining glucose homoeostasis. SGLT1 expression is directly regulated in response to changes in the sugar content of the diet. To monitor these variations, there is a requirement for a glucose-sensing system located on the luminal membrane of gut cells. This short review focuses on recent findings on intestinal sugar sensing and the downstream mechanisms responsible for enhancement in SGLT1 expression.

Pseudomonas aeruginosa inducing rice resistance against Rhizoctonia solani: production of salicylic acid and peroxidases.

Three isolates of Pseudomonas aeruginosa were used for seed treatment of rice; all showed plant growth promoting activity and induced systemic resistance in rice against Rhizoctonia solani G5 and increased seed yield. Production of salicylic acid (Sal) by P. aeruginosa both in vitro and in vivo was quantified with high performance liquid chromatography. All three isolates produced more Sal in King's B broth than in induced roots. Using a split root system, more Sal accumulated in root tissues of bacterized site than in distant roots on the opposite site of the root system after 1 d, but this difference decreased after 3 d. Sal concentration 0-200 g/L showed no inhibition of mycelial growth of R. solani in vitro, while at > or =300 g/L it inhibited it. P. aeruginosa-pretreated rice plants challenged inoculation with R. solani (as pathogen), an additional increase in the accumulation of peroxidase was observed. Three pathogenesis-related peroxidases in induced rice plants were detected; molar mass of these purified peroxidases was 28, 36 and 47 kDa. Purified peroxidase showed antifungal activity against phytopathogenic fungi R. solani, Pyricularia oryzae and Helminthosporium oryzae.

Motility and chemotactic response of Pseudomonas fluorescens toward chemoattractants present in the exudate of Macrophomina phaseolina.

Pseudomonas fluorescens strains (LAM1-hydrophilic) and (LAM2-hydrophobic) showed positive chemotaxis towards attractants (sugars, amino acids, polyols and organic acids) present in the exudate of Macrophomina phaseolina (a soil-borne plant pathogenic fungus). The varied response of motility traits such as speed, rate of change in direction (RCDI) and net to gross displacement ratio (NGDR) was observed for different chemoattractants. Swimming speed of the strains was highest in 10-fold diluted exudate or 100-1000 microM strength of different attractants, but further dilutions significantly decreased the swimming speed (P = 0.05). Chemotactic response of P fluorescens was positively correlated with swimming speed (P = 0.05; r = 0.76). Relative to control, the RCDI values decreased 1.5-fold in amino acids or sugars, and 1.2-fold in polyols or organic acids. With increase in swimming speed, the NGDR of both strains also increased, but the RCDI decreased. Both hydrophilic and hydrophobic strains did not show significant differences in their motility traits. The results demonstrate that M. phaseolina exudate contains chemical attractants that serve as signal for flagellar motility of P. fluorescens. Motile P fluorescens strains thus may consume fungal exudate as nutrients, and thus spores could offer a niche for these bacteria in soil.

Influence of growth and environmental conditions on cell surface hydrophobicity of Pseudomonas fluorescens in non-specific adhesion.

The relative cell surface hydrophobicity (CSH) of 18 soil isolates of Pseudomonas fluorescens, determined by phase exclusion, hydrophobic interaction chromatography (HIC), electrostatic interaction chromatography (ESIC), and contact angle, revealed large degrees of variability. Variation in the adhesion efficiency to Macrophomina phaseolina of the hyphae/sclerotia of these isolates was also examined. Two such isolates with maximum (32.8%; isolate 12-94) and minimum (12%; isolate 30-94) CSH were selected for further study. Early- to mid-log exponential cells of these isolates were more hydrophobic than those in stationary phase, and the CSH of these isolates was also influenced by fluctuations in temperatures and pH. Isolate 12-94 exhibited high CSH (32.3%) at 30 degrees C, compared to lower values (28-24%) in the higher temperature range (35-40 degrees C). Increasing concentrations of either Zn2+, Fe3+, K+, and Mg2+ in the growth medium were associated with the increased CSH. Trypsin, pepsin, and proteinase K (75 to 150 micrograms.mL-1) reduced the CSH of isolate 12-94 cells. CSH was reduced, following exposure to DTT, SDS, Triton X-100, or Tween 80. Prolonged exposure of cells to starvation (60 days) also caused a significant decline in CSH. Several protein bands (18, 21, 23, 26 kDa) of the outer cell membrane were absent in 60-day starved cells compared to unstarved cells. In conclusion, our findings demonstrate that CSH of P. fluorescens isolates may contribute to nonspecific attachment/adhesion onto M. phaseolina hyphae/sclerotia, and the efficiency of adhesion is regulated by growth and other environmental conditions.

A method for large-scale multiplication of Curculigo orchioides through bulbil formation from leaf explant in shake flask culture.

An efficient method has been developed for large-scale multiplication of Curculigo orchioides (Hypoxidaceae), an endangered medicinal plant, through direct bulbil formation from leaf explants in shake flask cultures. Leaf-segments (7 x 10 mm) were cultured in B5 liquid medium containing KNO3 (200 mgNL-1), (NH4)2SO4 (50 mgNL-1), benzyl adenosine (2.2 microM), adenine (0.11 mM), indole butyric acid (1.0 microM) and polyvinyl pyrrolidone (250 mgL-1). About 95% of explants produced maximum number of bulbils (546/flask at 6 weeks growth) in the medium. Shake flask cultures yielded 2737 bulbils/L medium whereas static cultures yielded 624 bulbils/L medium. Germination of bulbils was maximum (90.62%) on agar-gelled B5 medium containing benzyl adenosine (2.2 microM) and gibberellic acid (3.5 microM). Plantlets developed in vitro were successfully transferred to soil with a high rate of survivability (90%) and were comparable to natural population in growth and vigour.

Effect of environmental conditions and inoculum density on infection of guava fruits by Colletotrichum glososporioides.

The influence of environmental factors (temperature and humidity), inoculum density on infection by Colletotrichum glososporioides and development of anthracnose lesions were determined on uninjured, sand-injured and punctured fruits. The optical temperature for severe infection was 30 degrees C, whereas the disease incidence was less at 20 and 35 degrees C. Inoculated guavas that received 1-60 h of continuous free moisture developed lesions, but the disease was minimal (0-7%) after 1-6 h free moisture. Infection rates of uninjured, sand-injured and punctured fruits receiving 60 h of free moisture were 34, 70 and 100%, respectively. Disease incidence increased as inoculum density increased from 101 to 106 conidia/ml. In field conditions, the development of anthracnose lesions was greater on punctured guavas than on uninjured or sand-injured ones, in both rainy and winter seasons. In general, the number of lesions was highest in sand-injured fruits, followed by punctured and uninjured fruits. In rainy season the number of lesions on injured and uninjured fruits was greater than similarly treated guavas in winter.