Insilico Structural 3D Modelling of Novel Cry1Ib9 and Cry3A Toxins from Local Isolates of Bacillus thuringiensis.

Three-dimensional (3D) models for the 79.2 kDa activated Cry1Ib9 and 77.4 kDa activated Cry3A δ-endotoxins from Bacillus thuringiensis (Bt) native isolates that are specifically toxic to Coleopteran insect pests were constructed by utilizing homology modeling online tool. Evidences presented here, based on the identification of structural equivalent residues of Cry1Ib9 and Cry3A toxin through homology modelling indicate that, they share a common Bt toxin tridimensional structure. The main differences observed in Cry1I9 domain I at positions α2b (S56-I60), α4 (F78-l93) and additionally ß0 (Q10-L12), α8a (T280-V282) were observed, in domain II at positions α9b (P333-L339), ß6(T390-Q393), ß7(V398-W404), ß8 (V418-W425), ß9 (E453-N454), ß10 (S470-I479) where as in domain III the changes were observed at positions ß19 (R601-F607), ß20 (609-L613), ß21 (S618-F627) and α11a (K655-F664), α13, α14 components present at downstream sites, where as in Cry3A main differences observed in domain I is at the position of α4 (P105-I152), α5 (Q163-A185), ß1A(E190-L192), α6 (F193-Y217), Domain II is not consevered and main variations were observed at ß2 (E292-L295), ß3(V299-L308), ß4(I340-F347), ß5(D356-P368), ß6(I375-T377), ß7(V389-F394), ß8(K398-N405), ß9(Y416-Y427), ß10 (T436-Y439), ß12(G476-H495), ß12A (M503-I504) where as in domain III main variations observed at positions of ß18 (P583-I593), ß19(F604-S610), ß20(P611-L615), ß21(N619-G626). Cry1Ib9 and Cry3A contain the most variable regions in the loops of domain II, which determine the specificity of these toxins. These are the first models of Coleopteran-active protein from native isolates of Bt and its importance can be perceived since members of this group of toxins are potentially important candidates for coleoptera insect pest control programs.

Molecular characterization and genetic diversity of insecticidal crystal protein genes in native Bacillus thuringiensis isolates.

The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.

Bacillus thuringiensis isolates from Great Nicobar Islands.

Bacillus thuringiensis (Bt) strains were isolated from soil samples of Great Nicobar Islands, one of the "hottest biodiversity hotspots," where no collection has been characterized previously. The 36 new Bt isolates were obtained from 153 samples analyzed by crystal protein production with light/phase-contrast microscopy, determination of cry gene profile by SDS-PAGE, evaluation of toxicity against Coleopteran, and Lepidopteran insect pests, finally cloning and sequencing. Majority of the isolates showed the presence of 66-35 kDa protein bands on SDS-PAGE while the rest showed >130, 130, 73, and 18 kDa bands. The variations in crystal morphology and mass of crystal protein(s) purified from the isolates of Bt revealed genetic and molecular diversity. Based on the toxicity test, 50 % of isolates were toxic to Ash weevils, 16 % isolates were toxic to cotton bollworm, 38 % isolates were toxic both to ash weevil as well as cotton bollworm, while 11 % of the isolates did not exhibit any toxicity. PCR analysis unveiled prepotency of cry1B- and cry8b-like genes in these isolates. This study appoints the first isolation and characterization of local B. thuringiensis isolates in Great Nicobar Islands. Some of these isolates display toxic potential and, therefore, could be adopted for future applications to control some agriculturally important insect pests in the area of integrated pest management for sustainable agriculture.

Expression of cry3A gene and its toxicity against Asian Gray Weevil Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae).

Coleopterans are the most damaging pests of many agricultural and forestry crops; there is an urgent need to develop effective biopesticides against these insects. Enhancers of Bt toxicity typify an opportunity to improve currently available commercial products into more effective control agents against diverse pests. A 1.9 kb DNA fragment, PCR amplified from native isolates of Bt using cry3A gene specific primers was cloned in expression vector pQE-80L and then used for transformation of Escherichia coli M15 cells. The sequence of the cloned crystal protein gene showed almost complete homology with a Coleopteran active Cry3A toxin gene with 117 mutations scattered in different domain regions encoding a protein of 645 amino acid residues in length, with a predicted molecular mass of 77.4 kDa. Phylogenetic analysis could be compulsive for new/novel Bacillus thuringiensis strains, allowing them to be grouped with related Cry proteins. The toxicity of Bt protein was determined against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) LC50 152 ng cm(-2). Genes coding for Coleopteran active Cry3A proteins have been isolated and their efficient expression will provide the tools necessary to increase the efficacy of Cry-based biopesticide against economically important beetles.

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.

Factors Influencing Antibiotic Prescribing Behavior and Understanding of Antimicrobial Resistance Among Veterinarians in Assam, India.

This study investigates factors influencing veterinarians' antibiotic prescribing behaviors and their understanding of antimicrobial resistance (AMR). The study used a telephone survey of 50 veterinarians conducted in five districts in Assam state, India. The survey sought information on the most prevalent animal diseases, veterinarians' awareness of potential preventive measures, including factors determining antimicrobial prescribing; the types of antimicrobials used for different health conditions in different species, and possible options to reduce antimicrobial use (AMU). The majority (86%) of respondents worked for the government, 98% reported having no written policy for the use of veterinary health products, and 58% have no on-site diagnostic facilities. Ceftriaxone, Enrofloxacin, and Oxytetracycline were the antibiotics (ABX) most frequently prescribed, by 76, 68, and 54% of veterinarians, respectively. These ABX were prescribed mainly for respiratory health problems and mastitis in cattle, and gastrointestinal infections in buffaloes, sheep, goat, and pigs. Severity of clinical symptoms, economic status of the livestock owner, and withdrawal period for ABX were ranked as very important factors for giving ABX. Less than two thirds (64%) were aware of the government ban for Colistin and only 2% were aware of a national plan for AMR. This study highlighted that ABX prescription is mostly based on tentative diagnosis given the lack of diagnostic facilities in most veterinary clinics. There is a need to enhance veterinary healthcare and to improve communication between policy makers and field veterinarians and, importantly, a need to disseminate clear prescribing guidelines on prudent AMU.

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.

Wound healing and protease inhibition activity of Bacoside-A, isolated from Bacopa monnieri wettest.

Bacopa monnieri (L.) Wettest. (Scrophulariaceae) is a well-known medicinal herb. In the Indian system of medicine it is known as Brahmi (Sanskrit) and Indian water hyssop. Methanolic extract of Bacopa monnieri and its isolated constituent Bacoside-A were screened for wound healing activity. Bacoside-A was screened for wound healing activity by excision, incision and dead space wound on Swiss albino rats. Significant wound healing activity was observed in both extract and the Bacoside-A treated groups. The SDS-PAGE caseinolytic zymogram analysis of inhibition of matrix metalloproteases (MMPs) enzyme from the excision wound by Bacoside-A, an isolated constituent, was done with the concentrations 100 and 200 micromg/ml. In Bacoside-A treated groups, epithelialization of the excision wound was faster with a high rate (18.30 +/- 0.01 days) of wound contraction. The tensile strength of the incision wound was increased (538.47 +/- 0.14 g) in the Bacoside-A treated group. In the dead space wound model, the weight of the granuloma was also increased (89.15 +/- 0.08 g). The histological examination of the granuloma tissue of the Bacoside-A treated group showed increased cross-linking of collagen fibers and absence of monocytes. The wound healing activity of Bacoside-A was more effective in various wound models compared to the standard skin ointment Nitrofurazone.

Wound healing activity of embelin isolated from the ethanol extract of leaves of Embelia ribes Burm.

Ethanol extract of the leaves of Embelia ribes Burm. (Myrsinaceae) and its isolated quinone compound embelin were screened for wound healing activity by excision, incision and dead space wound models on Swiss Albino Rats. Significant wound healing activity was observed in both ethanol crude extract (30 mg/ml) and the constituent treated groups. In embelin treated groups (4 mg/ml of 0.2% sodium alginate gel), epithelialization of the incision wound was faster with a high rate of wound contraction. The tensile strength of the incision wound was significantly increased than the ethanol extract. In dead space wound model also the weight of the granulation was increased indicating increase in collagenation. The histological examination of the granulation tissue of embelin treated group showed increased cross-linking of collagen fibers and absence of monocytes. The wound healing effect was comparatively evaluated with the standard skin ointment Framycetin.

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.

Spectrum of purpura fulminans.

Purpura Fulminans is a severe disorder of acute onset with high morbidity and mortality. It is characterized by DIC with thrombocytopenia, hyofibrinogenemia, hypothrombinemia and anemia. It most often occurs in young with sudden appearance of symmetrical, tender, ecchymotic skin lesions usually involving the lower extremities. An infectious and noninfectious etiology has been proposed. Early recognition and early therapy with appropriate antibiotics and heparin is known to limit both morbidity and mortality. This article reports 5 cases of Purpura Fulminans treated at our centre with review of etiology, pathogenesis, clinical features and treatment.

Wound healing activity and docking of glycogen-synthase-kinase-3-beta-protein with isolated triterpenoid lupeol in rats.

A triterpene compound lupeol isolated from petroleum ether extract of leaves of Celastrus paniculatus was screened for wound healing activity (8 mg/ml of 0.2% sodium alginate gel) by excision, incision and dead space wound models on Swiss Albino rats (175-225 g). In lupeol treated groups wound healing activity was more significant (17.83+/-0.48) than the standard skin ointment nitrofurazone (18.33+/-0.42). Epithelialization of the incision wound was faster with a high rate of wound contraction (571.50+/-5.07) as compared with the control group. In dead space wound model also the weight of the granulation tissue of the lupeol treated animal was increased indicating increase of collagenation and absence of monocytes. The comparative docking of isolated lupeol molecule and standard drug nitrofurazone to glycogen synthase kinase 3-beta protein by Wnt signaling pathway also supported the wound healing property of lupeol. The activation domain of GSK3-beta consisted of Tyr216, with residues Asn64, Gly65, Ser66, Phe67, Gly68, Val70, Lys85, Leu132, Val135, Asp181 in the active pocket docked with lupeol at the torsional degree of freedom 0.5 units with Lamarckian genetic algorithm showed the inhibition constant of 1.38 x 10(-7). The inhibition constant of nitrofurazone was only 1.35 x 10(-4).