HDAC inhibitor valproic acid enhances tumor cell kill in adenovirus-HSVtk mediated suicide gene therapy in HNSCC xenograft mouse model

Safety, efficacy and enhanced transgene expression are the primary concerns while using any vector for gene therapy. One of the widely used vectors in clinical. trials is adenovirus which provides a safe way to deliver the therapeutic gene. However, adenovirus has poor transduction efficiency in vivo since most tumor cells express low coxsackie and adenovirus receptors. Similarly transgene expression remains low, possibly because of the chromatization of adenoviral genome upon infection in eukaryotic cells, an effect mediated by histone deacetylases (HDACs). Using a recombinant adenovirus (Ad-HSVtk) carrying the herpes simplex thymidine kinase (HSVtk) and GFP genes we demonstrate that HDAC inhibitor valproic acid can bring about an increase in CAR expression on host cells and thereby enhanced Ad-HSVtk infectivity. It also resulted in an increase in transgene (HSVtk and GFP) expression. This, in turn, resulted in increased cell kill of HNSCC cells, following ganciclovir treatment in vitro as well as in vivo in a xenograft nude mouse model.

Distress call-induced gene expression in the brain of the Indian short-nosed fruit bat, Cynopterus sphinx

Individuals in distress emit audible vocalizations to either warn or inform conspecifics. The Indian short-nosed fruit bat, Cynopterus sphinx, emits distress calls soon after becoming entangled in mist nets, which appear to attract conspecifics. Phase I of these distress calls is longer and louder, and includes a secondary peak, compared to phase II. Activity-dependent expression of egr-1 was examined in free-ranging C. sphinx following the emissions and responses to a distress call. We found that the level of expression of egr-1 was higher in bats that emitted a distress call, in adults that responded, and in pups than in silent bats. Up-regulated cDNA was amplified to identify the target gene (TOE1) of the protein Egr-1. The observed expression pattern Toe1 was similar to that of egr-1. These findings suggest that the neuronal activity related to recognition of a distress call and an auditory feedback mechanism induces the expression of Egr-1. Co-expression of egr-1 with Toe1 may play a role in initial triggering of the genetic mechanism that could be involved in the consolidation or stabilization of distress call memories.

Vertex-Fused Metallaborane Clusters: Synthesis, Characterization and Electronic Structure of [(eta(5)-C5Me5Mo)(3)MoB9H18]

The reaction of the [(eta(5)-C5Me5)MoCl4] complex with [LiBH4 - TH F] in toluene at - 70 degrees C, followed by pyrolysis at 110 degrees C, afforded dark brown [(eta(5)-C5Me5Mo)(3)MoB9H18], 2, in parallel with the known [(eta(5)-C5Me5Mo)(2)B5H9], 1. Compound 2 has been characterized in solution by H-1, B-11, and C-13 NMR spectroscopy and elemental analysis, and the structural types were unequivocally established by crystallographic studies. The title compound represents a novel class of vertex-fused clusters in which a Mo atom has been fused in a perpendicular fashion between two molybdaborane clusters. Electronic structure calculations employing density functional theory yield geometries in agreement with the structure determinations, and on grounds of density functional theory calculations, we have analyzed the bonding patterns in the structure,

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light.

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA-treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had no detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These data indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA- treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had not detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These date indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.

Downregulation of microRNA-498 in colorectal cancers and its cellular effects

miR-498 is a non-coding RNA located intergenically in 19q13.41. Due to its predicted targeting of several genes involved in control of cellular growth, we examined the expression of miR-498 in colon cancer cell lines and a large cohort of patients with colorectal adenocarcinoma. Two colon cancer cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The expression of miR-498 was tested in these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Tissues from 80 patients with surgical resection of colorectum (60 adenocarcinomas and 20 non-neoplastic tissues) were tested for miR-498 expression by qRT-PCR. In addition, an exogenous miR-498 (mimic) was used to detect the miRNA׳s effects on cell proliferation and cell cycle events in SW480 using MTT calorimetric assay and flow cytometry respectively. The colon cancer cell lines showed reduced expression of miR-498 compared to a normal colonic epithelial cell line. Mimic driven over expression of miR-498 in the SW480 cell line resulted in reduced cell proliferation and increased proportions of G2-M phase cells. In tissues, miR-498 expression was too low to be detected in all colorectal adenocarcinoma compared to non-neoplastic tissues. This suggests that the down regulation of miR-498 in colorectal cancer tissues and the direct suppressive cellular effect noted in cancer cell lines implies that miR-498 has some direct or indirect role in the pathogenesis of colorectal adenocarcinomas.

Cost Sharing Mechanisms for Business Clusters with Strategic Firms

A business cluster is a co-located group of micro, small, medium scale enterprises. Such firms can benefit significantly from their co-location through shared infrastructure and shared services. Cost sharing becomes an important issue in such sharing arrangements especially when the firms exhibit strategic behavior. There are many cost sharing methods and mechanisms proposed in the literature based on game theoretic foundations. These mechanisms satisfy a variety of efficiency and fairness properties such as allocative efficiency, budget balance, individual rationality, consumer sovereignty, strategyproofness, and group strategyproofness. In this paper, we motivate the problem of cost sharing in a business cluster with strategic firms and illustrate different cost sharing mechanisms through the example of a cluster of firms sharing a logistics service. Next we look into the problem of a business cluster sharing ICT (information and communication technologies) infrastructure and explore the use of cost sharing mechanisms.

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4-N,N′-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.

Characterization of LRRTM and NGR gene families: Expression and functions

Some leucine-rich repeat (LRR) -containing membrane proteins are known regulators of neuronal growth and synapse formation. In this work I characterize two gene families encoding neuronal LRR membrane proteins, namely the LRRTM (leucine-rich repeat, transmembrane neuronal) and NGR (Nogo-66 receptor) families. I studied LRRTM and NGR family member's mRNA tissue distribution by RT-PCR and by in situ hybridization. Subcellular localization of LRRTM1 protein was studied in neurons and in non-neuronal cells. I discovered that LRRTM and NGR family mRNAs are predominantly expressed in the nervous system, and that each gene possesses a specific expression pattern. I also established that LRRTM and NGR family mRNAs are expressed by neurons, and not by glial cells. Within neurons, LRRTM1 protein is not transported to the plasma membrane; rather it localizes to endoplasmic reticulum. Nogo-A (RTN4), MAG, and OMgp are myelin-associated proteins that bind to NgR1 to limit axonal regeneration after central nervous system injury. To better understand the functions of NgR2 and NgR3, and to explore the possible redundancy in the signaling of myelin inhibitors of neurite growth, I mapped the interactions between NgR family and the known and candidate NgR1 ligands. I identified high-affinity interactions between RTN2-66, RTN3-66 and NgR1. I also demonstrate that Rtn3 mRNA is expressed in the same glial cell population of mouse spinal cord white matter as Nogo-A mRNA, and thus it could have a role in myelin inhibition of axonal growth. To understand how NgR1 interacts with multiple structurally divergent ligands, I aimed first to map in more detail the nature of Nogo-A:NgR1 interactions, and then to systematically map the binding sites of multiple myelin ligands in NgR1 by using a library of NgR1 expression constructs encoding proteins with one or multiple surface residues mutated to alanine. My analysis of the Nogo-A:NgR1 -interactions revealed a novel interaction site between the proteins, suggesting a trivalent Nogo-A:NgR1-interaction. Our analysis also defined a central binding region on the concave side of NgR1's LRR domain that is required for the binding of all known ligands, and a surrounding region critical for binding MAG and OMgp. To better understand the biological role of LRRTMs, I generated Lrrtm1 and Lrrtm3 knock out mice. I show here that reporter genes expressed from the targeted loci can be used for maping the neuronal connections of Lrrtm1 and Lrrtm3 expressing neurons in finer detail. With regard to LRRTM1's role in humans, we found a strong association between a 70 kb-spanning haplotype in the proposed promoter region of LRRTM1 gene and two possibly related phenotypes: left-handedness and schizophrenia. Interestingly, the responsible haplotype was linked to phenotypic variability only when paternally inherited. In summary, I identified two families of neuronal receptor-like proteins, and mapped their expression and certain protein-protein interactions. The identification of a central binding region in NgR1 shared by multiple ligands may facilitate the design and development of small molecule therapeutics blocking binding of all NgR1 ligands. Additionally, the genetic association data suggests that allelic variation upstream of LRRTM1 may play a role in the development of left-right brain asymmetry in humans. Lrrtm1 and Lrrtm3 knock out mice developed as a part of this study will likely be useful for schizophrenia and Alzheimer s disease research.

A Haplotype at the UCP1 Gene Locus Contributes to Genetic Risk for Type 2 Diabetes in Asian Indians (CURES-72)

Background: The gene encoding for uncoupling protein-1 (UCP1) is considered to be a candidate gene for type 2 diabetes because of its role in thermogenesis and energy expenditure. The objective of the study was to examine whether genetic variations in the UCP1 gene are associated with type 2 diabetes and its related traits in Asian Indians. Methods: The study subjects, 810 type 2 diabetic subjects and 990 normal glucose tolerant (NGT) subjects, were chosen from the Chennai Urban Rural Epidemiological Study (CURES), an ongoing population-based study in southern India. The polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. Results: The three polymorphisms, namely -3826A -> G, an A -> C transition in the 5'-untranslated region (UTR) and Met229Leu, were not associated with type 2 diabetes. However, the frequency of the A-C-Met (-3826A -> G-5'UTR A -> C-Met229Leu) haplotype was significantly higher among the type 2 diabetic subjects (2.67%) compared with the NGT subjects (1.45%, P < 0.01). The odds ratio for type 2 diabetes for the individuals carrying the haplotype A-C-Met was 1.82 (95% confidence interval, 1.29-2.78, P = 0.009). Conclusions: The haplotype, A-C-Met, in the UCP1 gene is significantly associated with the increased genetic risk for developing type 2 diabetes in Asian Indians.

Influence of magnetic clusters on electrical and magnetic properties of In1-xMnxSb/GaAs dilute magnetic semiconductor grown by liquid phase epitaxy

In1-xMnxSb films have been grown with different Mn doping concentrations (x = 0.0085, 0.018, 0.029 and 0.04) beyond the equilibrium 14 solubility limit by liquid phase epitaxy. We have studied temperature dependent resistivity, the Hall effect, magnetoresistance and magnetization for all compositions. Saturation in magnetization observed even at room temperature suggests the existence of ferromagnetic clusters in the film which has been verified by scanning electron microscopy studies. The anomalous Hall coefficient is found to be negative. Remnant field present on the surface of the clusters seems to affect the anomalous Hall effect at very low fields (below 350 Gauss). In the zero field resistivity, a variable-range hopping conduction mechanism dominates below 3.5 K for all samples above which activated behavior is predominant. The temperature dependence of the magnetization measurement shows a magnetic ordering below 10 K which is consistent with electrical measurements. (c) 2007 Elsevier Ltd. All rights reserved.