Aromatase inhibitors and xenograft studies

Aromatase inhibitors (AIs) have become the front-line choice for treatment of ER+ breast cancer. Nevertheless, although patients are responsive initially, they may acquire resistance and become unresponsive to further treatment. In addition, approximately 25% of breast cancers do not express the estrogen receptor (ERα) and consequently, are innately resistant to endocrine therapy. We have investigated the mechanisms associated with this lack of treatment response using xenograft models. We found that in cells and tumors that acquired resistance to the AI letrozole therapy, expression of the ER was reduced whereas growth factor signally was enhanced, including a marked increase in HER2 expression. Treatment with trastuzumab (HER2 antibody) resulted in a significant down-regulation of HER2 and p-MAPK as well as restoration of ERα expression. Thus, when trastuzumab was added to letrozole treatment at the time of tumor progression, there was significantly prolonged tumor suppression compared to trastuzumab or letrozole alone. This suggests that inhibition of both HER2 and ERα signaling pathways are required for overcoming resistance and restoring treatment sensitivity. ER negative tumors are innately resistant to endocrine therapy. Repression of the ERα has been found to be due to epigenetic modifications such as increased methylation and histone deacetylation. We found that entinostat (ENT), a histone deacetylase inhibitor (HDACi), activated not only expression of ERα but also aromatase in MDA-MB-231 ER-negative breast cancer cells, resulting in their ability to respond to estrogen and letrozole. Treatment with ENT in combination with letrozole significantly reduced tumor growth rate in xenografts compared to control tumors (p < 0.001). ENT plus letrozole treatment also prevented the colonization and growth of MDA-MB-231 cells in the lung with a significant reduction (p < 0.03) in both visible and microscopic foci. These results provide a strong indication for possible use of AIs in combination with HDAC inhibitors for the treatment of ER-negative breast cancer.     

Synthesis and characterization of a novel chitosan based E. coli cytosine deaminase nanocomposite for potential application in prodrug enzyme therapy

Cytosine deaminase is a non-mammalian enzyme of widespread interest for prodrug enzyme therapy due to its ability to convert prodrug 5-fluorocytosine into anticancer drug 5-fluorouracil. Cytosine deaminase enzyme has been purified to homogeneity from E. coli K-12 MTCC 1302 strain. K_m values for cytosine and 5-fluorocytosine were found to be 0.26 mM and 1.82 mM, respectively. We developed a chitosan-entrapped cytosine deaminase nanocomposite. Atomic force microscopy and transmission electron microscopy images showed an elongated sphere shape nanocomposite with an average size of 80 nm diameter. Fourier transform infrared spectroscopy and X-ray diffraction results confirmed gel formation and entrapment of cytosine deaminase within the nanocomposite. Sustained release of cytosine deaminase from the nanocomposite up to one week depicted its potential implication in prodrug inducted enzyme therapy.     

Chitosan and its derivatives for gene delivery

Gene delivery can particularly be used for the treatment of diseases by the insertion of genetic materials (DNA and RNA) into mammalian cells either to express new proteins or to prevent the expression of existing proteins. Chitosan, a natural polymer is nontoxic, biocompatible, and biodegradable and it is used as a support material for gene delivery. However, practical use of chitosan has been mainly limited to its unmodified forms, and thus modified chitosans can be used for the wide range of biomedical applications including the interaction and intracellular delivery of genetic materials. In this context, this review paper provides the recent development on chitosan derivatives available for gene delivery.     

Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I–V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.     

High proportions of deleterious polymorphisms in constrained human genes

Previous studies on human mitochondrial genomes showed that the ratio of intra-specific diversities at nonsynonymous-to-synonymous positions was two to ten times higher than the ratio of interspecific divergences at these positions, suggesting an excess of slightly deleterious nonsynonymous polymorphisms. However, such an overabundance of nonsynonymous single nucleotide polymorphisms (SNPs) was not found in human nuclear genomes. Here, genome-wide estimates using >14,000 human-chimp nuclear genes and 1 million SNPs from four human genomes showed a significant proportion of deleterious nonsynonymous SNPs (～ 15%). Importantly, this study reveals a negative correlation between the magnitude of selection pressure and the proportion of deleterious SNPs on human genes. The proportion of deleterious amino acid replacement polymorphisms is 3.5 times higher in genes under high purifying selection compared with that in less constrained genes (28% vs. 8%). These results are explained by differences in the extent of contribution of mildly deleterious mutations to diversity and substitution.     

An assessment of the DNA barcodes of Indian freshwater fishes

Freshwater fishes in India are poorly known and plagued by many unresolved cryptic species complexes that masks some latent and endemic species. Limitations in traditional taxonomy have resulted in this crypticism. Hence, molecular approaches like DNA barcoding, are needed to diagnose these latent species. We have analyzed 1383 barcode sequences of 175 Indian freshwater fish species available in the databases, of which 172 sequences of 70 species were generated. The congeneric and conspecific genetic divergences were calculated using Kimura's 2 parameter distance model followed by the construction of a Neighbor Joining tree using the MEGA 5.1. DNA barcoding principle at its first hand approach, led to the straightforward identification of 82% of the studied species with 2.9% (S.E = 0.2) divergence between the nearest congeners. However, after validating some cases of synonymy and mislabeled sequences, 5% more species were found to be valid. Sequences submitted to the database under different names were found to represent single species. On the other hand, some sequences of the species like Barilius barna, Barilius bendelisis and Labeo bata were submitted to the database under a single name but were found to represent either some unexplored species or latent species. Overall, 87% of the available Indian freshwater fish barcodes were diagnosed as true species in parity with the existing checklist and can act as reference barcode for the particular taxa. For the remaining 13% (21 species) the correct species name was difficult to assign as they depicted some erroneous identification and cryptic species complex. Thus, these barcodes will need further assay and inclusion of barcodes of more specimens from same and sister species.     

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.     

First parturition of tigers in a semi-arid habitat,western India

Long-term data of large felids is important to understand their reproductive biology and behavior for effective conservation planning. We used camera trap data and direct sightings from 2005 to 2013 to estimate the age of the first parturition of Bengal tigers (Panthera tigris) in a semi-arid habitat in India. We monitored 11 females in the Ranthambhore Tiger Reserve (RTR) from when they were 2–6 months old. The mean age at first reproduction (impregnation leading to cubs) was 51.3?±?(SE) 4.5 months. The tiger population in RTR is an important source population and genetic pool in the western most distribution of tiger. Thus, continuous monitoring of tiger populations is important to develop an understanding of reproductive biology.