Optimization of an improved,efficient and rapid in vitro micropropagation protocol for Petunia hybrida Vilm. Cv. “Bravo”

An efficient protocol for in-vitro propagation of an important ornamental crop, Petunia hybrida Vilm. Cv. “Bravo” was developed. The explants that were used to carry out the experiment were Leaf segments, nodal segments and shoot tips. Nodal segments recorded highest per cent asepsis followed by shoot tips and leaf segments. Asepsis was found to be highest when the explants were sterilized with Fungicide (Carbendazim) 0.02% for the duration of 30 min followed by 0.1% HgCl₂ for duration of 10 min and then ethanol 70% for 10 s. Longer duration of the sterilant treatment showed more necrotic effects on the explants, thus mercuric chloride treatment when given for 5 min proved to be more effective in terms of survival of the explants. Maximum establishment per cent was recorded in Murashige and Skoog (MS) media fortified with BAP (1.5 mg L⁻¹) and IBA (0.5 mg L⁻¹) in shoot tips and nodal segments, i.e. 97.90 and 95.74% respectively. Callus was efficiently induced and developed when PGR amalgamation of BAP (0.1 mg L⁻¹) and 2,4-D (1.5mg L⁻¹) was used. Kinetin at the concentration of 2.0 mg L⁻¹ along with IBA at 0.5mg L⁻¹ recorded highest callus regeneration in both leaf and internodal segment derived callus. Maximum proliferation percent of shoots (97.90%), highest number of shoots (20.50 explant⁻¹) and maximum length of shoot (2.70 cm) was recorded in PGR combination of IBA and BAP both at 0.5 mg L⁻1 concentration level. Rhizogenesis was recorded to be highest in the MS media containing IBA 1.00 mg L⁻¹. Best hardening media which recorded maximum survival per cent 92.50% was noticed on the media formulation comprised of equal ratio of perlite and vermiculite mix, under poly house conditions.     

Antagonistic assessment of Trichoderma spp. by producing volatile and non-volatile compounds against different fungal pathogens

Trichoderma spp. are well-known biological agents that have significant antagonistic activity against several plant pathogenic fungi. In the present study, Trichoderma spp. were tested in vitro for their antagonistic activity against different spp. of Fusarium and Alternaria viz. Alternaria alternata, A. brassicae, A. solani, Fusarium oxysporum and F. solani using dual plate assay and by the production of volatile and non-volatile compounds. The results obtained revealed that Trichoderma harzianum and T. viride effectively inhibited the growth and spore production of different spp. of Fusarium and Alternaria. The highest growth inhibition was found in A. alternata 62.50% and 60.00% by non-volatile compounds of T. harzianum and T. viride, respectively. Similarly, the volatile compounds inhibit the maximum growth of A. alternata 40.00% and 35.00% by T. harzianum and T. viride, respectively. Volatile and non-volatile compounds of Trichoderma spp. were analysed by GC-MS technique and the properties of distinguished compounds showed antifungal, antimicrobial and antibiotic activities. Volatile compounds of T. harzianum and T. viride showed highest percent abundance for glacial acetic acid (45.32%) and propyl-benzene (41.75%), respectively. In case of non-volatile compounds, T. harzianum and T. viride showed D-Glucose, 6-O-α-D-galactopyranosyl- (38.45%) and 17-Octadecynoic acid (36.23%), respectively. The results of present study confirmed that T. harzianum can be used as a promising biological control agent against Alternaria and Fusarium spp. that cause diseases in various vegetables and crops.     

GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes

Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor alpha1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool.     

Osmotin is a homolog of mammalian adiponectin and controls apoptosis in yeast through a homolog of mammalian adiponectin receptor

The antifungal activity of the PR-5 family of plant defense proteins has been suspected to involve specific plasma membrane component(s) of the fungal target. Osmotin is a tobacco PR-5 family protein that induces apoptosis in the yeast Saccharomyces cerevisiae. We show here that the protein encoded by ORE20/PHO36 (YOL002c), a seven transmembrane domain receptor-like polypeptide that regulates lipid and phosphate metabolism, is an osmotin binding plasma membrane protein that is required for full sensitivity to osmotin. PHO36 functions upstream of RAS2 in the osmotin-induced apoptotic pathway. The mammalian homolog of PHO36 is a receptor for the hormone adiponectin and regulates cellular lipid and sugar metabolism. Osmotin and adiponectin, the corresponding "receptor" binding proteins, do not share sequence similarity. However, the beta barrel domain of both proteins can be overlapped, and osmotin, like adiponectin, activates AMP kinase in C2C12 myocytes via adiponectin receptors.     

The beneficial effect of OST-6 (OsteoCare), a herbomineral formulation, in experimental osteoporosis.

OST-6 (OsteoCare), a herbomineral formulation, was evaluated for its inhibitory effect on the progress of bone loss induced by ovariectomy in rats. Ovariectomized (Ovx) rats were administered with OST-6 at 250 and 500 mg/kg b.wt., orally daily for 90 days. On 91st day, ovariectomized rats showed reduced bone mineral content and increased serum alkaline phosphatase levels, excretion of urinary calcium and pyridinium cross-links levels. Histologically, bone sections revealed narrowed and disappearance of trabeculae and widened medullary spaces. The total numbers of Tartrate-resistant acid phosphatase (TRAP) positive cells were significantly increased both in-vivo and in-vitro methods. OST-6, at a dose of 500 mg/kg, significantly improved bone mineral contents, serum alkaline phosphatase levels, reduced the elevated urinary calcium and pyridinium cross-links excretion, number of TRAP positive cells and reversal of the above mentioned histological features. These results indicate the usefulness of OST-6 in the management of osteoporosis in a natural way through herbal resources.     

MicroRNA Profiling in Intraocular Medulloepitheliomas

Purpose

To study the differential expression of microRNA (miRNA) profiles between intraocular medulloepithelioma (ME) and normal control tissue (CT).

Material and Methods

Total RNA was extracted from formalin fixed paraffin embedded (FFPE) intraocular ME (n=7) and from age matched ciliary body controls (n=8). The clinical history and phenotype was recorded. MiRNA profiles were determined using the Affymetrix GeneChip miRNA Arrays analyzed using expression console 1.3 software. Validation of significantly dysregulated miRNA was confimed by quantitaive real-time PCR. The web-based DNA Intelligent Analysis (DIANA)-miRPath v2.0 was used to perform enrichment analysis of differentially expressed (DE) miRNA gene targets in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

Results

The pathologic evaluation revealed one benign (benign non-teratoid, n=1) and six malignant tumors (malignant teratoid, n=2; malignant non-teratoid, n = 4). A total of 88 miRNAs were upregulated and 43 miRNAs were downregulated significantly (P<0.05) in the tumor specimens. Many of these significantly dysregulated miRNAs were known to play various roles in carcinogenesis and tumor behavior. RT-PCR validated three significantly upregulated miRNAs and three significantly downregulated miRNAs namely miR-217, miR-216a, miR-216b, miR-146a, miR-509-3p and miR-211. Many DE miRNAs that were significant in ME tumors showed dysregulation in retinoblastoma, glioblastoma, and precursor, normal and reactive human cartilage. Enriched pathway analysis suggested a significant association of upregulated miRNAs with 15 pathways involved in prion disease and several types of cancer. The pathways involving significantly downregulated miRNAs included the toll-like receptor (TLR) (p<4.36E-16) and Nuclear Factor kappa B (NF-κB) signaling pathways (p<9.00E-06).

Conclusions

We report significantly dysregulated miRNAs in intraocular ME tumors, which exhibited abnormal profiles in other cancers as well such as retinoblastoma and glioblastoma. Pathway analysis of all dysregulated miRNAs shared commonalities with other cancer pathways.     

A Genome Wide Meta-Analysis Study for Identification of Common Variation Associated with Breast Cancer Prognosis

Objective

Genome wide association studies (GWAs) of breast cancer mortality have identified few potential associations. The concordance between these studies is unclear. In this study, we used a meta-analysis of two prognostic GWAs and a replication cohort to identify the strongest associations and to evaluate the loci suggested in previous studies. We attempt to identify those SNPs which could impact overall survival irrespective of the age of onset.

Methods

To facilitate the meta-analysis and to refine the association signals, SNPs were imputed using data from the 1000 genomes project. Cox-proportional hazard models were used to estimate hazard ratios (HR) in 536 patients from the POSH cohort (Prospective study of Outcomes in Sporadic versus Hereditary breast cancer) and 805 patients from the HEBCS cohort (Helsinki Breast Cancer Study). These hazard ratios were combined using a Mantel-Haenszel fixed effects meta-analysis and a p-value threshold of 5×10⁻⁸ was used to determine significance. Replication was performed in 1523 additional patients from the POSH study.

Results

Although no SNPs achieved genome wide significance, three SNPs have significant association in the replication cohort and combined p-values less than 5.6×10⁻⁶. These SNPs are; rs421379 which is 556 kb upstream of ARRDC3 (HR = 1.49, 95% confidence interval (CI) = 1.27–1.75, P = 1.1×10⁻⁶), rs12358475 which is between ECHDC3 and PROSER2 (HR = 0.75, CI = 0.67–0.85, P = 1.8×10⁻⁶), and rs1728400 which is between LINC00917 and FOXF1.

Conclusions

In a genome wide meta-analysis of two independent cohorts from UK and Finland, we identified potential associations at three distinct loci. Phenotypic heterogeneity and relatively small sample sizes may explain the lack of genome wide significant findings. However, the replication at three SNPs in the validation cohort shows promise for future studies in larger cohorts. We did not find strong evidence for concordance between the few associations highlighted by previous GWAs of breast cancer survival and this study.     

Anti-quorum sensing and antibiofilm potential of Alternaria alternata, a foliar endophyte of Carica papaya, evidenced by QS assays and in-silico analysis

In the present study, secondary metabolites from an endophytic fungus, Alternaria alternata, colonizing Carica papaya, demonstrated antiquorum sensing properties against Pseudomonas aeruginosa. This study reports the antagonistic effects of fungal crude extract of A. alternata against the various quorum sensing (QS) associated virulent factors such as percentage decrease in production of pyocyanin, alginate, chitinase and rhamnolipid; significant decrease in proteases activity such as LasA protease activity, staphylolytic activity, Las B elastase; and a marked decrease in biofilm formation and associated factors such as exopolysaccharide (EPS) production and cell surface hydrophobicity (CSH). Further, motility pattern i.e., swimming and swarming was also found to be inhibited. This down regulation of QS and associated factors are further supported by in-silico analysis of interaction between QS receptor LasR and bioactive molecules viz., sulfurous acid, 2-propyl tridecyl ester and 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester present in fungal crude extract, found based on GCMS analysis, sketches the modulating ability of QS expression. This is the first report on an endophytic fungus of C. papaya having a role in QS inhibition against P. aeruginosa and lays a platform to explore further the endophytes for potent therapeutic agents in QS.