Screening of antifungal activity of various plant leaves extracts from Indian plants

The present study was designated to evaluate the antifungal activity and to root out the antifungal plant leaf extracts from this Indian folk-flore. The in vitro antifungal assay was performed by agar diffusion test and minimum inhibitory concentration (MIC) for hexane, ethyl acetate, methanol and distilled water plant leaf extracts. Extraction of 17 different plant leaves was carried out in different solvents such as hexane, ethyl acetate, methanol and distilled water. Among them extractive yield of methanol was maximum than the rest of the three solvents. These extracts were screened for their antifungal activity against nine different fungi. Among these ethyl acetate extracts of Adhatoda vasica, Ocimum sanctum and Holoptelea integrifolia exhibited maximum antifungal activity against Alternaria sp., Aspergillus parasi, Aspergillus nidulans, Trichoderma harzianum and Aspergillus flavus with MIC of 80, 40 and 20 ppm against Aspergillus nidulans and Alternaria sp. Ethyl acetate extracts showed promising antifungal activity against Adhatoda vasica, Ocimum sanctum and Holoptelea integrifolia against Aspergillus nidulans, and Alternaria sp. might be applicable as fungicide against fungal plants disease.     

The evolution of floral nectaries in Disa (Orchidaceae: Disinae): recapitulation or diversifying innovation?

Background and Aims

The Orchidaceae have a history of recurring convergent evolution in floral function as nectar production has evolved repeatedly from an ancestral nectarless state. However, orchids exhibit considerable diversity in nectary type, position and morphology, indicating that this convergence arose from alternative adaptive solutions. Using the genus Disa, this study asks whether repeated evolution of floral nectaries involved recapitulation of the same nectary type or diversifying innovation. Epidermis morphology of closely related nectar-producing and nectarless species is also compared in order to identify histological changes that accompanied the gain or loss of nectar production.

Methods

The micromorphology of nectaries and positionally equivalent tissues in nectarless species was examined with light and scanning electron microscopy. This information was subjected to phylogenetic analyses to reconstruct nectary evolution and compare characteristics of nectar-producing and nectarless species.

Key Results

Two nectary types evolved in Disa. Nectar exudation by modified stomata in floral spurs evolved twice, whereas exudation by a secretory epidermis evolved six times in different perianth segments. The spur epidermis of nectarless species exhibited considerable micromorphological variation, including strongly textured surfaces and non-secreting stomata in some species. Epidermis morphology of nectar-producing species did not differ consistently from that of rewardless species at the magnifications used in this study, suggesting that transitions from rewardlessness to nectar production are not necessarily accompanied by visible morphological changes but only require sub-cellular modification.

Conclusions

Independent nectary evolution in Disa involved both repeated recapitulation of secretory epidermis, which is present in the sister genus Brownleea, and innovation of stomatal nectaries. These contrasting nectary types and positional diversity within types imply weak genetic, developmental or physiological constraints in ancestral, nectarless Disa. Such functional convergence generated by morphologically diverse solutions probably also underlies the extensive diversity of nectary types and positions in the Orchidaceae.     

Intraspecific genetic diversity of Pyrenophora tritici-repentis (Died.) Drechs. (Drechslera tritici-repentis[Died.] Shoem.) detected by random amplified polymorphic DNA assays

Abstract

Random amplified polymorphic DNAs (RAPDs) were used to study the genetic variation of Pyrenophora tritici-repentis isolates causing wheat tan spot. Two independent experiments were conducted in 2002 – 2003. In 2002, 40 isolates collected in Russia (Krasnodar region, Bashkiria), Germany, and the Czech Republic were studied and 35 unique RAPD genotypes were identified. Most of the genetic variation (72%) was observed within populations and 28% between them. In 2003, 69 new isolates from Russia (Dagestan, North Osetia, Bashkiria), Germany, and the Czech Republic were studied and 47 unique RAPD genotypes were identified. As in 2002, most of the genetic variation (75%) was observed within populations and 25% between them. Total gene diversity in each group ranged from 0.67 – 1.00 for 2002 and was 1.00 for 2003. The average gene diversity was estimated between 0.13 and 0.20 in 2002 and between 0.07 and 0.18 in 2003. A dendrogramme based on genetic distances between isolates illustrates that the variation is distributed on a small scale (0.3 – 4.0%). Estimated F_ST values and clustering of isolates on dendrogrammes suggest that groups of isolates from Bashkiria and groups of isolates from Dagestan and North Osetia are separated from others and may be considered as different geographical populations. No clear differentiation between isolates from other sites was revealed.     

T cell responses against microsatellite instability-induced frameshift peptides and influence of regulatory T cells in colorectal cancer

High-level microsatellite-unstable (MSI-H) colorectal carcinomas (CRC) represent a distinct subtype of tumors commonly characterized by dense infiltration with cytotoxic T cells, most likely due to expression of MSI-H-related frameshift peptides (FSP). The contribution of FSP and classical antigens like MUC1 and CEA to the cellular immune response against MSI-H CRC had not been analyzed so far. We analyzed tumor-infiltrating and peripheral T cells from MSI-H (n = 4 and n = 14, respectively) and microsatellite-stable (MSS) tumor patients (n = 26 and n = 17) using interferon gamma ELISpot assays. Responses against 4 FSP antigens and peptides derived from MUC1 to CEA were compared with and without depletion of regulatory T cells, and the results were related to the presence of the respective antigens in tumor tissue. Preexisting FSP-specific T cell responses were detected in all (4 out of 4) tumor-infiltrating and in the majority (10 out of 14) of peripheral T cell samples from MSI-H CRC patients, but rarely observed in MSS CRC patients. Preexisting T cell responses in MSI-H CRC patients were significantly more frequently directed against FSP tested in the present study than against peptides derived from classical antigens MUC1 or CEA (p = 0.049). Depletion of regulatory T cells increased the frequency of effector T cell responses specific for MUC1/CEA-derived peptides and, to a lesser extent, T cell responses specific for FSP. Our data suggest that the analyzed FSP may represent an immunologically relevant pool of antigens capable of eliciting antitumoral effector T cell responses.     

Inhibition of both BRAF and MEK in BRAFV600E mutant melanoma restores compromised dendritic cell (DC) function while having differential direct effects on DC properties

Purpose

Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAF^V600 mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAF^V600 mutant melanoma.

Methods

We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib.

Results

Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAF^V600E mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations.

Conclusions

BRAF^V600E mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.     

Patterns of morphological and genetic diversity of Valeriana jatamansi Jones in different habitats and altitudinal range of West Himalaya,India

Importance to know and understand diversity of Himalayan plants is increasingly recognized considering the fact that various natural and anthropogenic pressures might bring about serious influences to morphological and genetic diversity of the vegetation in the region. In this context, Valeriana jatamansi was investigated in detail, taking into account its importance in various Ayurvedic and modern medicines. Randomly selected mature plants from twenty five different populations (located between 1215 m to 2775 m asl) of V. jatamansi were analysed for their morphological attributes. Further, ISSR markers were used to detect genetic variation among 151 plants of selected 25 populations. Use of 20 primers yielded 125 reproducible polymorphic loci which were used to estimate different parameters of genetic diversity. These parameters were in turn applied to develop relationships with habitat types and altitude range. Significant variation (p < 0.05) in above ground dry weight (AGDW) and below ground dry weight (BGDW) across the populations was observed. Nei's genetic diversity index (He) ranged from 0.25 to 0.37 across the populations, with a mean of 0.31. Genetic diversity exhibited a decreasing trend with increasing altitude, and maximum diversity (He = 0.325) was observed in the range of 1201–1500 m asl. Among the different habitat conditions, highest genetic diversity (He = 0.334; Pp = 84.38) was observed in grassland habitats while minimum in mixed forest habitats (He = 0.285; Pp = 72.433). The genetic diversity (He) had significant negative relationships with AGDW, BGDW and rhizome diameter (Pearson r = −0.359, −0.424 and −0.317, respectively; p < 0.05). The genetic characterization of V. jatamansi from the western Himalaya by this study suggests influences of habitat types and the altitudinal range upon genetic diversity, and based on these proposals for conservation strategies in favour of the species are made.     

Association of peroxisome proliferator activated receptor-γ gene with non-alcoholic fatty liver disease in Asian Indians residing in north India

Background

Genetics of non-alcoholic fatty liver (NAFLD) in Asian Indians has been inadequately studied. We investigated the association of polymorphisms C161T and Pro12Ala of peroxisome proliferator-activated receptor gamma (PPARγ) with clinical and biochemical parameters in Asian Indians with NAFLD.

Methods

In this case–control study, 162 NAFLD cases and 173 controls were recruited. Abdominal ultrasound, clinical and biochemical profiles, fasting insulin levels and value of homeostasis model assessment of insulin resistance were determined. Polymerase chain reaction–restriction fragment length polymorphisms of two polymorphisms were performed. The association of these polymorphisms with clinical and biochemical parameters was analysed.

Results

Higher frequency of Ala and T alleles of PPARγ was obtained in cases. Ala/Ala genotype of PPARγ (Pro12Ala) was associated with significantly higher serum triglycerides (TG), alkaline phosphatase (ALK) and waist–hip ratio in cases as compared to controls. In C161T polymorphism, TT genotype was significantly increased TG (p = 0.04), total cholesterol (p = 0.01), ALK (p = 0.04) and gamma-glutamyl transpeptidase (p = 0.007) in cases. The linkage disequilibrium for these two single-nucleotide polymorphisms of PPARγ was differed in cases (D1 = 0.1; p = 0.006) and controls (D1 = 0.07; p = 0.1). Using a multivariate analysis after adjusting for age, sex and body mass index, the presence of NAFLD was linked to these two polymorphisms (odds ratio 1.64 (95% CI: 1.09–2.45, p = 0.05)].

Conclusion

Asian Indians in north India carrying the alleles Ala and T of PPARγ (Pro12Ala and C161T) polymorphisms are predisposed to develop NAFLD.     

A prawn transglutaminase: Molecular characterization and biochemical properties

In this study, we report the bioinformatics characterization, gene expression, transglutaminase activity and coagulation assays of transglutaminase (TGase) of freshwater prawn Macrobrachium rosenbergii identified from the constructed cDNA library by GS FLX™ technology. Even though, TGase have sequence similarity, they differ extensively in their substrate specificity and are thought to play an important in variety of functions such as development, tissue differentiation and immune responses etc. Gene expression studies show that MrTGase is widely distributed in the tissues such as heart, muscle, intestine, brain, etc., but higher amounts are found in hemocyte. Results of TGase mRNA relative expression in hemocyte, before and after infected with white spot syndrome baculovirus (WSBV) and Vibrio harveyi show that the gene expression initially increases up to 24 h and then it falls down. Coagulation assay results showed that the endogenous TGase is involved in the rapid assembly of a specific, plasma clotting protein. Structural studies show that MrTGase contains a typical TGc domain between 323 and 424, and two putative integrin-binding motifs at Arg¹⁸⁰–Gly¹⁸¹–Asp¹⁸² and Arg²⁶⁹–Gly²⁷⁰–Asp²⁷¹. The predicted 3D model of MrTGase contains 47.04% coils (366 amino acid residues), 26.74% extended strand (208 residues), 21.72% α-helix (169 residues) and 4.5% beta turns (35 residues). BLASTp analysis of MrTGase exhibited high sequence similarities with other crustacean TGase, with the highest observed in white shrimp (77.1%). Moreover, the phylogenetic analysis also showed that MrTGase clustered with the other members of crustacean TGase. Overall, these results suggested that MrTGase is a major and functional TGase of M. rosenbergii for haemolymph coagulation and also in spread of infection.     

Molecular analysis of the bacterial microbiome in the forestomach fluid from the dromedary camel (Camelus dromedarius)

Rumen microorganisms play an important role in ruminant digestion and absorption of nutrients and have great potential applications in the field of rumen adjusting, food fermentation and biomass utilization etc. In order to investigate the composition of microorganisms in the rumen of camel (Camelus dromedarius), this study delves in the microbial diversity by culture-independent approach. It includes comparison of rumen samples investigated in the present study to other currently available metagenomes to reveal potential differences in rumen microbial systems. Pyrosequencing based metagenomics was applied to analyze phylogenetic and metabolic profiles by MG-RAST, a web based tool. Pyrosequencing of camel rumen sample yielded 8,979,755 nucleotides assembled to 41,905 sequence reads with an average read length of 214 nucleotides. Taxonomic analysis of metagenomic reads indicated Bacteroidetes (55.5 %), Firmicutes (22.7 %) and Proteobacteria (9.2 %) phyla as predominant camel rumen taxa. At a finer phylogenetic resolution, Bacteroides species dominated the camel rumen metagenome. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism were the most abundant SEED subsystem representing 17 and 13 % of camel metagenome, respectively. A high taxonomic and functional similarity of camel rumen was found with the cow metagenome which is not surprising given the fact that both are mammalian herbivores with similar digestive tract structures and functions. Combined pyrosequencing approach and subsystems-based annotations available in the SEED database allowed us access to understand the metabolic potential of these microbiomes. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the rumen microbiota regardless of rumen type. The present study provides a baseline for understanding the complexity of camel rumen microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments.