Purification and differentiation of three avian adenoviruses by restriction endonuclease analysis

A rapid method of ultracentrifugation pelleting of avian adenovirus (AAV) from small volume of chloroform treated infected cell culture fluid or allantoic fluid was adapted for isolation of adenoviral DNA. The viral DNA extracted from semipurified viruses was found to be intact on agarose gel and pure enough (A260/280 = 1.85-1.92) for restriction enzyme analysis. Restriction endonuclease analysis of Indian strain of AAV serotype 1, AAV serotype 4 (group I AAVs) and egg drop syndrome-76 (EDS-76) virus genomes (group III AAV) with Hind III enzyme differentiated these viruses. The AAV serotype 1 and serotype 4 strain exhibited identical Hind III profile to European viral strains belonging to same serotypes however, the EDS-76 virus gave similar but not identical profile. The calculated genomic lengths for AAV serotype 1 and EDS-76 virus were approximately found to be 33.9 and 44.4 Kb, respectively.     

TB Treatment Delays in Odisha,India: Is It Expected Even after These Many Years of RNTCP Implementation?

Background

In India, the Revised National TB Control Programme (RNTCP) envisages initiation of TB treatment within seven days of diagnosis among smear-positive patients. After nearly two decades of RNTCP implementation, treatment delays are usually not expected.

Objectives

To determine the proportion of sputum smear-positive TB patients who were initiated on treatment after seven days and their associated risk factors.

Methods

The study was conducted in Cuttack and Rayagada districts of Odisha. It was a retrospective cohort study that involves review of TB treatment registers and laboratory registers for 2013.

Results

Among 1,800 pulmonary TB (PTB) patients, 1,074 (60%) had been initiated on treatment within seven days of diagnosis, 721 (40%) had been initiated on treatment more than seven days, and 354 (20%) had delays of more than 15 days. The mean duration between TB diagnosis and treatment initiation was 21 days with a range of 8–207 days (median = 14 days). Odds of treatment delay of more than seven days were 4.9 times (95% confidence interval [CI] 3.3-6.6) among those who had been previously treated, 6.2 times (95% CI 1.3-29.7) among those infected with HIV, and 1.8 times (95% CI 1.1-2.9) among those diagnosed outside district DMC.

Conclusion

Delay in initiation of TB treatment occurred in majority of the smear-positive patients. The RNTCP should focus on core areas of providing quality TB services with time-tested strategies. To have real-time monitoring mechanisms for diagnosed smear-positive TB patients is expected to be the way forward.     

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.     

Comparative Genomic Analysis of Buffalo (Bubalus bubalis) NOD1 and NOD2 Receptors and Their Functional Role in In-Vitro Cellular Immune Response

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.     

Control of stem cell fate by engineering their micro and nanoenvironment

Stem cells are capable of long-term self-renewal and differentiation into specialised cell types, making them an ideal candidate for a cell source for regenerative medicine. The control of stem cell fate has become a major area of interest in the field of regenerative medicine and therapeutic intervention. Conventional methods of chemically inducing stem cells into specific lineages is being challenged by the advances in biomaterial technology, with evidence highlighting that material properties are capable of driving stem cell fate. Materials are being designed to mimic the clues stem cells receive in their in vivo stem cell niche including topographical and chemical instructions. Nanotopographical clues that mimic the extracellular matrix(ECM) in vivo have shown to regulate stem cell differentiation. The delivery of ECM components on biomaterials in the form of short peptides sequences has also proved successful in directing stem cell lineage. Growth factors responsible for controlling stem cell fate in vivo have also been delivered via biomaterials to provide clues to determine stem cell differentiation. An alternative approach to guide stem cells fate is to provide genetic clues including delivering DNA plasmids and small interfering RNAs via scaffolds. This review, aims to provide an overview of the topographical, chemical and molecular clues that biomaterials can provide to guide stem cell fate. The promising features and challenges of such approaches will be highlighted, to provide directions for future advancements in this exciting area of stem cell translation for regenerative medicine.     

Phytosphingosine promotes megakaryocytic differentiation of myeloid leukemia cells

We report that phytosphingosine, a sphingolipid found in many organisms and implicated in cellular signaling, promotes megakaryocytic differentiation of myeloid leukemia cells. Specifically, phytosphingosine induced several hallmark changes associated with megakaryopoiesis from K562 and HEL cells including cell cycle arrest, cell size increase and polyploidization. We also confirmed that cell type specific markers of megakaryocytes, CD41a and CD42b are induced by phytosphingosine. Phospholipids with highly similar structures were unable to induce similar changes, indicating that the activity of phytosphingosine is highly specific. Although phytosphingosine is known to activate p38 mitogen-activated protein kinase (MAPK)-mediated apoptosis, the signaling mechanisms involved in megakaryopoiesis appear to be distinct. In sum, we present another model for dissecting molecular details of megakaryocytic differentiation which in large part remains obscure. [BMB Reports 2015; 48(12): 691-695]     

Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells.     

DJ-1 Protects Pancreatic Beta Cells from Cytokine- and Streptozotocin-Mediated Cell Death

A hallmark feature of type 1 and type 2 diabetes mellitus is the progressive dysfunction and loss of insulin-producing pancreatic beta cells, and inflammatory cytokines are known to trigger beta cell death. Here we asked whether the anti-oxidant protein DJ-1 encoded by the Parkinson’s disease gene PARK7 protects islet cells from cytokine- and streptozotocin-mediated cell death. Wild type and DJ-1 knockout mice (KO) were treated with multiple low doses of streptozotocin (MLDS) to induce inflammatory beta cell stress and cell death. Subsequently, glucose tolerance tests were performed, and plasma insulin as well as fasting and random blood glucose concentrations were monitored. Mitochondrial morphology and number of insulin granules were quantified in beta cells. Moreover, islet cell damage was determined in vitro after streptozotocin and cytokine treatment of isolated wild type and DJ-1 KO islets using calcein AM/ethidium homodimer-1 staining and TUNEL staining. Compared to wild type mice, DJ-1 KO mice became diabetic following MLDS treatment. Insulin concentrations were substantially reduced, and fasting blood glucose concentrations were significantly higher in MLDS-treated DJ-1 KO mice compared to equally treated wild type mice. Rates of beta cell apoptosis upon MLDS treatment were twofold higher in DJ-1 KO mice compared to wild type mice, and in vitro inflammatory cytokines led to twice as much beta cell death in pancreatic islets from DJ-1 KO mice versus those of wild type mice. In conclusion, this study identified the anti-oxidant protein DJ-1 as being capable of protecting pancreatic islet cells from cell death induced by an inflammatory and cytotoxic setting.     

Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.