Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis,phenotypic transformation and migration of human neural progenitor cells within cocultures

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell^® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA^®, Live/Dead^® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3β expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.     

Indole acids as a novel PDE2 inhibitor chemotype that demonstrate pro-cognitive activity in multiple species

An internal HTS effort identified a novel PDE2 inhibitor series that was subsequently optimized for improved PDE2 activity and off-target selectivity. The optimized lead, compound 4, improved cognitive performance in a rodent novel object recognition task as well as a non-human primate object retrieval task. In addition, co-crystallization studies of close analog of 4 in the PDE2 active site revealed unique binding interactions influencing the high PDE isoform selectivity.     

Efficiency of RAPD,ISSR and ITS markers in detecting genetic variability among Salacia species sampled from the Western Ghats of Karnataka

Diversity and phylogenetic relationship between four closely related Salacia species, i.e., Salacia chinensis, Salacia macrosperma, Salacia fruticosa and Salacia oblonga, collected from the Western Ghats of Karnataka, India, was assessed. Ten each of RAPD and ISSR primers generated a total of 76 and 68 loci, generating polymorphisms of 92.21 and 89.71%, respectively. Maximum likelihood analysis of the ITS sequences revealed three clades. Dendrogram analyses of RAPD and ISSR revealed two and four clusters, respectively. Overall polymorphism revealed by RAPD was 41.45?±?10%, ISSR was 33.58?±?6.52%, and ITS was 25.50?±?17.25%. Molecular variance revealed significant variance within and among the Salacia species. Tajima’s D neutrality test and Fu’s Fs were negative for all four species, implying presences of rare alleles and population expansion. Comparative study of RAPD, ISSR and ITS for Salacia species has given an insight into the efficiency of each technique in detecting diversity within and among the population sampled in the Western Ghats of Karnataka.

     

Amino acid deprivation and central carbon metabolism regulate the production of outer membrane vesicles and tubes by Francisella

Francisella tularensis is a highly virulent Gram‐negative bacterial pathogen that causes the zoonotic disease tularemia. F. novicida, a model tularemia strain, produces spherical outer membrane vesicles (OMV), as well as novel tubular vesicles and extensions of the cell surface. These OMV and tubes (OMV/T) are produced in a regulated manner and contain known virulence factors. Mechanisms by which bacterial vesicles are produced and regulated are not well understood. We performed a genetic screen in F. novicida to decipher the molecular basis for regulated OMV/T formation, and identified both hypo‐ and hyper‐vesiculating mutants. Mutations in fumA and tktA, involved in central carbon metabolism, and in FTN_0908 and FTN_1037, of unknown function, resulted in severe defects in OMV/T production. Cysteine deprivation was identified as the signal that triggers OMV/T formation in F. novicida during growth in rich medium. We also found that fully virulent F. tularensis produces OMV/T in a similarly regulated manner. Further analysis revealed that OMV/T production is responsive to deprivation of essential amino acids in addition to cysteine, and that the hypo‐vesiculating mutants are defective in responding to this signal. Thus, amino acid starvation, such as encountered by Francisella during host cell invasion, regulates the production of membrane‐derived structures.     

Effect of female group size on harem male roosting behavior of the Indian short-nosed fruit bat <Emphasis Type="Italic">Cynopterus sphinx</Emphasis>

Mate guarding has been known to incur costs and cause constraints for harem males in many polygynous species. However, the effect of female group size on the harem male’s time budget in bats has received very limited attention. The Indian short-nosed fruit bat, Cynopterus sphinx, exhibits resource defense polygyny, in which tent roosting males construct tents and defend multiple female bats. We studied the effect of female group size on three aspects of harem male behavior: social grooming by reciprocal licking, tent maintenance, and tent guarding in the mast tree Polyalthia longifolia. In the process of reciprocal licking, all the bats in the harem were drenched in saliva before emergence, and this activity was positively and significantly correlated with female group size. Once females departed for foraging, harem males remained in their respective tents at night-time between intermittent foraging bouts and engaged in tent maintenance and tent guarding. Time invested by harem male bats in tent maintenance and tent guarding were positively and significantly correlated with female group size. Harem males extended their presence in tent by utilizing tents as feeding roosts. Female group size also influenced the emergence time of harem male bats, where males with largest group emerged later than did the smallest group. Likewise, harem male with the smallest group had more time available for foraging than the male with the largest group. Findings of this study suggest that having a larger harem may indeed be costly for the males by reducing their foraging time.     

Identification of new HLA class I region genes by sample sequencing

 Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region. We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases. Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes. Cosmids were sonnicated to produce fragments of 0.5 – 1 kilobases, subcloned, and sequenced using an automated sequencer. Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm. A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 – 8 gene family. These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens. Received: 10 December 1996 / Revised: 7 January 1997     

Dexamethasone induced alterations in enzymatic and nonenzymatic antioxidant status in heart and kidney of rats

This study was designed to investigate the alterations in thiobarbituric acid reactants (TBA-reactants) and enzymatic and nonenzymatic antioxidant levels induced by dexamethasone (Dex) in heart and kidney and to find out whether these alterations induced by Dex and its hypertensive effect had any role in the maintenance of hypertension in this model. Administration of dexamethasone induced severe loss of body weight, significant increase in heart and kidney weights and also marked electrocardiographic changes. The protein content in heart and kidney increased significantly during Dex administration and returned to near normalcy after withdrawal. Total activity of lactate dehydrogenase showed a significant increase in heart till day 8 of treatment, whereas in serum, it exhibited a significant decrease. The activity of CK in heart showed an increase till day 8 of treatment and approached normalcy thereafter. In serum, CK exhibited a decrease till day 8, remaining insignificant thereafter. CKMB in heart showed an insignificant increase initially, reaching normal levels on Dex withdrawal, whereas in serum, it showed a significant decrease throughout the experimental period. Mean arterial pressure (MAP) and heart rate increased significantly, while a significant elevation in the ST segment was noticed during administration as well as after withdrawal of dex. The TBA-reactants levels were found to increase in heart and kidney during days 12 and 16 of administration with Dex and even after withdrawal of Dex, the levels were insignificantly elevated. The level of glutathione in heart and kidney increased from day 4 onwards and reached normalcy during the later stages of treatment and after withdrawal of Dex. The total sulfhydryl groups exhibited a significant increase in both heart and kidney throughout the experiment. The antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase exhibited a significant decrease in heart during Dex administration whereas, in kidney, they exhibited a significant increase during treatment and after withdrawal of Dex. Thus, Dex induced rise in mean arterial pressure, significant alterations in electrocardiographic parameters and also marked alterations in enzymatic and nonenzymatic antioxidant levels and in the TBA-reactants level in heart and kidney.