Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

Partial cloning and production of polyclonal antiserum against recombinant capsid protein of Hepatopancreatic Parvovirus (HPV) and its application for diagnostics in penaeid shrimp

Hepatopancreatic Parvovirus (HPV) causes infection in the early stages of shrimp leading to retarded growth, ultimaltely resulting in monetary loss to the shrimp farmers. To over come this situation screening of post-larvae (PL) by immunology-based diagnostics is required. Hence, the specific gene of capsid protein for HPV was cloned into pRSET B expression vector and rHCP overexpressed with 6-histidine tagged fusion protein in Escherichia coli BL21(DE3). Immunology-based methods like Western blot, dot blot and ELISA techniques were employed to detect HPV in infected samples using the antiserum raised in rabbits against recombinant HCP of HPV. The dot blot assay using anti-rHCP was found to be capable of detecting HPV in HPV infected post-larvae as early as at 24 h post infection. The antiserum could detect the HPV in the infected samples at 1 ng of total protein. HPV infection estimated by ELISA using anti-HCP and pure r-HCP as a standard was found to increase gradually during the course of infection from 24 h post infection. The sensitivity of antibody-based diagnostics employed in the present study was compared with that of PCR diagnostic method to screen the post-larvae for the detection of HPV.     

Bispidine-Amino Acid Conjugates Act as a Novel Scaffold for the Design of Antivirals That Block Japanese Encephalitis Virus Replication

Background

Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties.

Methodology/Principal Findings

In this study we have identified 3,7-diazabicyclo[3.3.1]nonane, commonly called bispidine, as a privileged scaffold to synthesize effective antiviral agents. We have synthesized derivatives of bispidine conjugated with amino acids and found that hydrophobic amino acid residues showed antiviral properties against JEV. We identified a tryptophan derivative, Bisp-W, which at 5 µM concentration inhibited JEV infection in neuroblastoma cells by more than 100-fold. Viral inhibition was at a stage post-entry and prior to viral protein translation possibly at viral RNA replication. We show that similar concentration of Bisp-W was capable of inhibiting viral infection of two other encephalitic viruses namely, West Nile virus and Chandipura virus.

Conclusions/Significance

We have demonstrated that the amino-acid conjugates of 3,7-diazabicyclo[3.3.1]nonane can serve as a molecular scaffold for development of potent antivirals against encephalitic viruses. Our findings will provide a novel platform to develop effective inhibitors of JEV and perhaps other RNA viruses causing encephalitis.     

In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells

Solid malignancies contain sphere-forming stem-like cells that are particularly efficient in propagating tumors. Identifying agents that target these cells will advance the development of more effective therapies. Recent converging evidence shows that c-Met expression marks tumor-initiating stem-like cells and that c-Met signaling drives human glioblastoma multiforme (GBM) cell stemness in vitro. However, the degree to which tumor-propagating stem-like cells depend on c-Met signaling in histologically complex cancers remains unknown. We examined the effects of in vivo c-Met pathway inhibitor therapy on tumor-propagating stem-like cells in human GBM xenografts. Animals bearing pre-established tumor xenografts expressing activated c-Met were treated with either neutralizing anti- hepatocyte growth factor (HGF) monoclonal antibody L2G7 or with the c-Met kinase inhibitor PF2341066 (Crizotinib). c-Met pathway inhibition inhibited tumor growth, depleted tumors of sphere-forming cells, and inhibited tumor expression of stem cell markers CD133, Sox2, Nanog, and Musashi. Withdrawing c-Met pathway inhibitor therapy resulted in a substantial rebound in stem cell marker expression concurrent with tumor recurrence. Cells derived from xenografts treated with anti-HGF in vivo were depleted of tumor-propagating potential as determined by in vivo serial dilution tumor-propagating assay. Furthermore, daughter xenografts that did form were 12-fold smaller than controls. These findings show that stem-like tumor-initiating cells are dynamically regulated by c-Met signaling in vivo and that c-Met pathway inhibitors can deplete tumors of their tumor-propagating stem-like cells.     

Solvothermal Preparation of ZnO Nanorods as Anode Material for Improved Cycle Life Zn/AgO Batteries

Nano materials with high surface area increase the kinetics and extent of the redox reactions, thus resulting in high power and energy densities. In this study high surface area zinc oxide nanorods have been synthesized by surfactant free ethylene glycol assisted solvothermal method. The nanorods thus prepared have diameters in the submicron range (300∼500 nm) with high aspect ratio. They have uniform geometry and well aligned direction. These nanorods are characterized by XRD, SEM, Specific Surface Area Analysis, solubility in alkaline medium, EDX analysis and galvanostatic charge/discharge studies in Zn/AgO batteries. The prepared zinc oxide nanorods have low solubility in alkaline medium with higher structural stability, which imparts the improved cycle life stability to Zn/AgO cells.     

Microbiome: A New Lease to Microbiology

Indian Journal of Microbiology -     

Pharmacological inhibition of the MEK5/ERK5 and PI3K/Akt signaling pathways synergistically reduces viability in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.