Traditionally practiced medicinal plant extracts inhibit the ergosterol biosynthesis of clinically isolated dermatophytic pathogens.

OBJECTIVE: Dermatophytes are resistant to some available antibiotics. Development of new plant drugs to control drug resistant microbes is urgently needed. This study evaluates the antidermatophytic potential of 18 selected medicinal plants used by traditional healers in Theni and Virudhunagar Districts of Tamil Nadu, India. MATERIALS AND METHODS: Selected plant parts were collected, shade dried and powdered. Plant powders were extracted with ethanol and their antifungal potency was investigated against and clinical dermatophytes. The antioxidant effect of the extracts was screened using DPPH assay. Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) were estimated for the extracts. Ten plant extracts showed maximum MFC and they were selected to study their efficacy in interfering with ergosterol biosynthesis. Fluconazole-35µg/mL known fungicide was used as control. The most active extracts were taken for biocompatibility studies using 3T3-L1 fibroblast cell lines. RESULTS: The ethanol extract of Phyllanthus reticulates leaves showed good antifungal activity compared to other plant extracts. The MIC and MFC for Phyllanthus reticulatus were 62.5 and 250µg/mL against M. pachydermatitis and T. rubrum respectively. The ethanol extracts of Phyllanthus reticulatus, Coldenia procumbens, Thespesia populnea and Senna alata significantly lowered the release of ergosterol by 16.37, 19.53, 24.79, and 21.44%, respectively. The ethanol extract of Phyllanthus reticulatus leaves was more biocompatible to host cells than other active extracts. CONCLUSION: Our study indicated that the ethanol extract of Phyllanthus reticulates leaves showed promising activity against dermatophytes. It could be a potential material for future development of antidermatophytic agents.

Field evaluation of Bacillus cereus VCRC B540 for mosquitocidal activity - A new report.

A major challenge to control the vector mosquitoes and their diseases. The discovery of bacteria like Bacillus sphaericus Neide (Bs) and B. thuringiensis serovar israelensis de Barjac (Bti), which are highly toxic to dipterans larvae, have opened up the possibility of their use as potential bio-larvicides in mosquito eradication programmes the world over. These bacteria have some important advantages over conventional insecticides in mosquito control operations, besides being safe for non-target organisms including human beings. But, the recent researchers have been reported mosquito resistant against these biological agents. Bacillus cereus VCRC B540 is one of the most potential bio-pesticides which were isolated from the gut contents of the marine fish (Lutjanuas sanguineus) collected in east coastal zone of the Bay of Bengal (Union Territory of Pondicherry, India) to control Culex, Anopheles and followed by Aedes species. The isolated strain was confirmed as Bacillus cereus based on the biochemical characteristics and 16S rDNA gene sequence. The larvicidal activity of B. cereus VCRC B540 was further characterized.

Swertiamarin, a natural steroid, prevent bone erosion by modulating RANKL/RANK/OPG signaling.

Bone erosion is a central feature of rheumatoid arthritis (RA) that is characterized by the infiltration of the synovial lining by osteoclasts and lymphocytes. In the present study, swertiamarin a major secoiridoid glycoside was evaluated for anti-osteoclastogenic property to prevent bone erosion in Freund's complete adjuvant (FCA) induced in-vivo model, in-vitro osteoblast and osteoclasts as well as in co-culture system and in-silico molecular docking analysis. The swertiamarin treatment decreased the expression of TRAP, RANKL, and RANK levels and increased the levels of OPG levels significantly in both in vitro and in vivo models. In in vitro, the compound treatment significantly increased the cell proliferation and ALP levels in osteoblast cells; the high proliferation (153.8600±5.23%) and ALP release (165.6033±4.13%) were observed at 50µg/ml concentration of swertiamarin treatment. At the same time the treatment decreased the TRAP positive cells in osteoclast cells; the high reductions of TRAP positive cells (39.32±3.19%) were observed at 50µg/ml of swertiamarin treatment. The treatment modulated the levels of pro-inflammatory cytokines, MMPs and NF-κB levels in osteoblast and osteoclast co-culture system. In in silico analysis swertiamarin had affinity towards the proteins RANK, RANKL and OPG residues with low binding energy -4.5, -3.92 and -5.77kcal/mol respectively. Thus, the results of this study revealed the anti-osteoclastogenic activity of swertiamarin on the prevention of bone destruction.

An investigation on the diversity of mosquitocidal bacteria and its relationship with incidence of vector borne diseases.

Control of mosquitoes is the most important aspect of public health, as mosquitoes transmit many human diseases, including the fatal infection, Japanese encephalitis. This paper addresses the isolation of new mosquitocidal bacteria from soil samples in the Union Territory of Pondicherry, India, where, no clinical cases of vector borne infections have been reported. Bacterial isolates from soil samples were screened for potential mosquitocidal strains and bioassays against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) were carried out. Genomic DNA of potential mosquitocidal isolates was amplified and species identification was carried out using BLASTn program (NCBI). Phylogenetic analysis of 16S rRNA sequences of mosquitocidal bacteria revealed seven potential isolates. SDS-PAGE results have shown that there was considerable difference in the protein profiles. Numerical analysis revealed 4 distinct groups at similarity level 25%. The relationship between VBDs and prevalence of soil mosquitocidal bacteria in the study sites has elicited considerable interest in the diversity of mosquitocidal bacteria and their application for mosquito borne diseases control.

Sequencing approaches in cancer treatment.

Use of sequencing approaches is an important aspect in the field of cancer genomics, where next-generation sequencing has already been utilized for targeting oncogenes or tumour-suppressor genes, that can be sequenced in a short time period. Alterations such as point mutations, insertions/deletions, copy number alterations, chromosomal rearrangements and epigenetic changes are encountered in cancer cell genomes, and application of various NGS technologies in cancer research will encounter such modifications. Rapid advancement in technology has led to exponential growth in the field of genomic analysis. The $1000 Genome Project (in which the goal is to sequence an entire human genome for $1000), and deep sequencing techniques (which have greater accuracy and provide a more complete analysis of the genome), are examples of rapid advancements in the field of cancer genomics. In this mini review, we explore sequencing techniques, correlating their importance in cancer therapy and treatment.

Isolation of mosquitocidal bacteria (Bacillus thuringiensis, B.sphaericus and B. cereus) from excreta of arid birds.

Mosquitocidal bacteria are environmentally friendly alternatives to chemical insecticides for controlling mosquitoes and therefore, there have been tremendous world-wide efforts to identify novel mosquitocidal bacteria from natural environment. In the present study, excreta from arid-birds were analyzed for identifying mosquitocidal bacteria. The selection of sample for bacterial screening is significant, because, arid-birds are the unique living species and gathering the foods from variety of sources from environment. Out of 1000 samples examined, twelve bacterial strains were identified as mosquitocidal and the 16S rRNA gene sequence alignment depicted that these isolates belonged to Bacillus species (Bacillus thuringiensis, B.sphaericus and B. cereus). Toxicity assay against mosquito vectors have shown that these isolates are potential. The B. sphaericus VCRC-B547 (NCBI: JN377789) has shown a higher toxicity against Cx. quinquefasciatus, An. stephensi, and Aed. aegypti. Result from SDS-PAGE has shown that there was considerable difference in the protein profiles among the new bacterial isolates. Phylogenetic tree with branch length 0.05 revealed three distinct groups with homology among the closely related Bacillus strains. This study therefore throws considerable interest on the diversity of microbial organisms from arid birds and its application in mosquito control.

Purification and characterization of keratinase from feather degrading bacterium useful for mosquito control--a new report.

Every day, food processing industries release wastes, which are environmental menance. Chicken feathers have been discarded in bulk as waste from poultry industries, globally. Degrading these wastes, as unused disposals, without acquiring any additional benefits has led to an idea to develop a new technology. We have reported earlier that Bacillus thuringiensis serovar israelensis (Bti) can be used for biodegradation of feather waste for biopesticide production. In the present study, purification and characterization of keratinase from feather degrading bacterium (Bti) is reported. Protein precipitate obtained at Ammonium sulphate saturation at 60% level and Sephacryl S-200 column chromatography resulted in 2.3 and 11.68 fold purification of the enzyme respectively. The purity was revealed in SDS-PAGE by a single band of molecular weight of 40 kDa and it was characterized. The optimum pH of the enzyme shifted to a more neutral range (6.0-8.0) with the highest activity (7.0). The optimum temperature of the reaction was determined to be 30ºC. The keratinase enzyme retained 51% residual activity (303 U/mg protein) at 70ºC (60 min) and the half-lives of the enzyme were 130 minutes at 40ºC, 90 min at 50ºC and of 60 min at 70ºC, respectively. Keratinase activity was enhanced by calcium and magnesium ions while EDTA, PMSF, ß- mercaptoethanol and manganese inhibited the activity. This is the first report investigating the keratinase from Bti degraded chicken feathers for the bio-synthesis of mosquitocidal toxins.

In vivo and in vitro immunomodulatory potential of swertiamarin isolated from Enicostema axillare (Lam.) A. Raynal that acts as an anti-inflammatory agent.

Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used as a depurative in the Indian system of traditional medicine. The present study evaluated the immunomodulatory activity of isolated swertiamarin. In vivo immunomodulatory activity of swertiamarin (2, 5, and 10 mg/kg b.w.) was evaluated in a model of sheep red blood cells (SRBC) by assessing its effect on organ weight, hemagglutinating antibody titer (HA), plaque-forming cells (PFC), quantitative hemolysis of SRBC, and delayed type hypersensitivity (DTH). In vitro immunomodulatory potential was studied on isolated splenocytes, neutrophils, and peritoneal macrophages. In silico immunomodulatory effects were evaluated by docking of swertiamarin on proinflammatory cytokines to confirm its potential. In in vivo studies, the animals treated with swertiamarin showed a significant (P ≤ 0.05) increase in antibody titer, plaque-forming cells, and also in weight of the thymus and spleen. A decreased response to DTH reaction was recorded with the treatment of swertiamarin. In in vitro studies, treatment with swertiamarin modulated the messenger RNA (mRNA) and protein expression of IFN-γ, IL-10, and IL-4 significantly (P ≤ 0.05) and also favored Th2-mediated response on concanavalin A (Con A)-induced splenocytes. The compound inhibited the release of free radicals significantly (P ≤ 0.05) in phytohemagglutinin (PHA)-induced neutrophils and also ameliorated the mRNA and protein expression of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) in lipopolysaccharide (LPS)-induced macrophages. In in silico, the best docked pose of swertiamarin with the target proteins (TNF-α, IL-1ß, and IL-6) was confirmed that swertiamarin acted as an anti-inflammatory mediator.

Swertiamarin attenuates inflammation mediators via modulating NF-κB/I κB and JAK2/STAT3 transcription factors in adjuvant induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease that leads to pannus formation followed by severe joint destruction, characterized by synovial hyperplasia, inflammation and angiogenesis. Swertiamarin is a secoiridoid glycoside that is used as an anti-inflammatory compound, mainly found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in Indian system of traditional medicine. In the present study, the effect of swertiamarin was evlauated in experimental adjuvant arthritis animal model by the estimation of biochemical (paw thickness, lysosomal enzymes, and urinary degradative products) parameters, proinflammatory cytokines and enzymes along with histopathological and radiographic observations. The proteins of phosphorylated NF-κB/IκB and JAK2/STAT3 transcription factors were also quantified from experimental animals as well as LPS induced RAW 264.7 macrophage cells. In in silico analysis, swertiamarin was docked with proinflammatory enzymes to confirm its potential. The administration of swertiamarin (2, 5, 10mg/kg bw) significantly (P⩽0.05) inhibited the levels of paw thickness, lysosomal enzymes and increased the body weight of experimental animals in a dose dependent manner. In molecular analysis, the treatment decreased the release of proinflammatory cytokines (IL1, TNF, IL-6) and proangiogenic enzymes (MMPs, iNOS, PGE2, PPARγ and COX-2); and also significantly (P⩽0.05) increased the levels of antiinflammatory proteins (IL-10, IL-4) when compared to the disease groups. The swertiamarin treatment significantly (P⩽0.05) inhibited the release of NF-κB p65, p-IκBα, p-JAK2 and p-STAT3 signaling proteins levels on both experimental animals and LPS induced cells. Histopathological and radiological analysis evidenced the curative effect of swertiamarin on bone destruction. The docking studies of swertiamarin on proinflammatory enzymes supported the results from the in vivo experiments. Thus the swertiamarin inhibited the development of arthritis by modulating NF-κB/IκB and JAK2/STAT3 signaling. These findings suggested that swertiamarin acted as an anti-rheumatic agent.

Swertiamarin ameliorates inflammation and osteoclastogenesis intermediates in IL-1ß induced rat fibroblast-like synoviocytes.

OBJECTIVE AND DESIGN: Rheumatoid arthritis is a chronic inflammatory and autoimmune disease that leads to aggressive joint cartilage and bone destruction. Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in the Indian system of traditional medicine. In the present study, the potential of swertiamarin was evaluated in IL-1ß induced fibroblast-like synoviocytes (FLS). METHODS: The FLS were isolated from Freund's Complete Adjuvant induced arthritic (AA) rats and cultured with IL-1ß. The normal FLS and AA-FLS were cultured and used for subsequent experiment in fibroblastic morphology form. The efficacy of swertiamarin (10-50 µg/ml) was evaluated on mRNA and protein expression levels of inflammatory and osteoclastogenesis mediators. The efficacy was also evaluated on p38 MAPKα levels with time course studies (2, 4, 6, 8 and 12 h). RESULTS: IL-1ß induced cell proliferation (149.46 ± 13.73 %) and NO production (162.03 ± 11.03%) in AA-FLS; treatment with swertiamarin controlled proliferation (82.77 ± 4.22%) and NO production (82.06 ± 3.91% at 50 µg/ml) in a dose-dependent manner. It also significantly (P < 0.05) modulated the expression of apoptotic marker (caspase 3), proinflammation mediators (TNFα, IL-6, PGE2, COX-2, iNOS, MMPs) and osteoclastogenic mediator (RANKL) at both the mRNA and protein levels. Treatment with swertiamarin inhibited the levels of p38 MAPKα in a dose-dependent manner and also significantly (P < 0.05) attenuated the release of the same in time dependent mode. CONCLUSION: These findings suggest that treatment with swertiamarin attenuated IL-1ß induced FLS, and it revealed anti-inflammatory potential by attenuating aggressive FLS.

Hexamerin a novel protein associated with Bacillus sphaericus resistance in Culex quinquefasciatus.

Bacterial insecticides like, Bacillus sphaericus and Bacillus thuringiensis serovar israelensis, have been used for the control of nuisance and vector mosquitoes for more than two decades. For many years, it was assumed that the use of microbial larvicides based on B. sphaericus would not lead to resistance in mosquitoes. However, recent reports have shown that B. sphaericus toxins are not free from this problem. Therefore, the resistance of mosquito populations to be will seriously threaten the sustainability of current mosquito control programme using these microbial insecticides. In the present study, we have characterised a novel protein responsible for resistance development in the filariasis vector of Culex quinquefasciatus. Laboratory selection experiments with B. sphaericus against the larvae were carried out up to 17 generations, and the occurrence of resistance was reported (resistance ratio (RR) at lethal concentration (LC)50 and LC90 = 1,987 and 2,051 folds, respectively). The protein profiles of B. sphaericus-resistant and susceptible population have confirmed with the expression of a new polypeptide (80 kDa) in the resistant strain only. Sequence result revealed that the newly expressed protein was 'hexamerin', and this factor might conceivably be responsible for the inheritance of resistance. This study is therefore valuable for comprehending the underlining factor and management of B. sphaericus resistance problem in mosquito population.

Identification and characterization of a novel marine Bacillus cereus for mosquito control.

Entomopathogenic bacteria to control mosquitoes are a promising environmentally friendly alternative to synthetic pesticides. In the present study, a novel mosquitocidal bacterium was isolated from marine soil collected from east coastal areas at Pondicherry (India). 16S rRNA gene sequence alignment depicted that this isolate belonged to Bacillus cereus VCRC-B520 (NCBI: KC-119192). Biochemical studies on bacterial growth, biomass, and toxin production have revealed that this strain could possibly be helpful in the production of a biopesticide in mosquito control. Toxicity assay with B. cereus against mosquito larvae has shown that the filariasis vector, Culex quinquefasciatus, is more susceptible than the other two species (Anopheles stephensi and Aedes aegypti). The LC50 and LC90 values for C. quinquefasciatus were 0.30 and 2.21 mg/L, respectively. No effect of B. cereus was found on nontargeted organisms. SDS-PAGE analysis and protein purification result from the cell mass of B. cereus have shown that a well-perceptible polypeptide was the dependable factor (85 kDa) for mosquitocidal action. Protein characterization (M/S MALDI-TOF) has shown that it is an endotoxin-specific insecticidal protein, namely "Cry4Aa". Phylogenetic analyses of 16S rDNA gene sequence from this marine isolate have revealed the presence of homology among closely related Bacillus strains. Therefore, considerable interest has been shown on the identification of a potential mosquitocidal bacterium from marine environment (B. cereus), which was not reported earlier in view of the current scenario of the rapid development of resistance to Bacillus sphaericus in mosquito vector control program.

Genotypic diversity of mosquitocidal bacteria (Bacillus sphaericus, B. thuringiensis, and B. cereus) newly isolated from natural sources.

In the present study, to comprehend the genetic diversity of mosquitocidal bacteria, the genotypic analysis of 30 strains of Bacillus species isolated newly from diverse environmental sources has been conducted. Randomly amplified polymorphic DNA polymerase chain reaction was conducted to characterize the genotype diversity between the bacterial strains. The matrix of scores from each bacterial DNA was analyzed, and the correlation between the co-efficients and the similarity matrix data was computed. Clusters from dendrogram showing diversity among isolates could be distinguished genetically based on their origin of isolates. The first major cluster consists of 43 strains grouped under various subclusters (91.489 %). A second cluster contains only four strains (8.511 %). An average similarity value of 0.36 revealed the dendrogram split into 28 distinct "groups" or "clusters," allowing almost a complete separation of strains within the Bacillus group isolated from various sources and thus facilitating assessment of genetic diversity of species and subspecies level. The conclusion from the result was that there was broad diversity among the mosquitocidal strains, and cluster analysis revealed the associations among the isolates based on their origin. A high level of polymorphism with distinct genetic lineages consequent to the source of origin of bacterial strains is the significant impact of this study.