Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.     

Description of two new species of ectoparasitic mites of Podapolipus and Podapolipoides (Acari: Podapolipidae) on Oxya sp. from West Bengal,India

Two new species, Podapolipus husbandi and Podapolipoides channabasavannai, collected on Oxya sp. from West Bengal, India are described and illustrated.     

In vitro-generated antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells control the severity of herpes simplex virus-induced ocular immunoinflammatory lesions

Generating and using regulatory T cells (Tregs) to modulate inflammatory disease represents a valuable approach to therapy but has not yet been applied as a means to control virus-induced immunopathological reactions. In this report, we developed a simplified technique that used unfractionated splenocytes as a precursor population and showed that stimulation under optimized conditions for 5 days with solid-phase anti-CD3 monoclonal antibody in the presence of transforming growth factor beta (TGF-beta) and interleukin-2 could induce up to 90% of CD4(+) T cells to become Foxp3(+) and able to mediate suppression in vitro. CD11c(+) dendritic cells were intricately involved in the conversion process and, once modified in the presence of TGF-beta, could convert Foxp3(-) CD4(+) cells into Foxp3(+) CD4(+)cells by producing TGF-beta. The converted cells had undergone cell division, and the majority of them expressed activation markers along with surface molecules that would facilitate their migration into tissue sites. The primary reason for our study was to determine if such in vitro-converted Tregs could be used in vivo to influence the outcome of a virus-induced immunoinflammatory lesion in the eye caused by herpes simplex virus infection. We could show in three separate models of herpetic stromal keratitis that adoptive transfers of in vitro-converted Tregs effectively diminished lesion severity, especially when given in the initial phases of infection. The suppression effect in vivo appeared to be polyspecific. The protocol we have developed could provide a useful additional approach to control virus-induced inflammatory disease.     

Comparison of in vitro Cr(VI) reduction by CFEs of chromate resistant bacteria isolated from chromate contaminated soil

Chromate resistant and reducing strains were isolated from chromium contaminated soil and identified as Bacillus sp. (KCH2 and KCH3), Leucobacter sp. (KCH4) and Exiguobacterium sp. (KCH5). KCH3 and KCH4 showed higher Cr(VI) tolerance (2 mM) and Cr(VI) reduction (1.5 mM) than KCH5 (1.5 mM and 0.75 mM, respectively). Cr(VI) reduction by CFEs of KCH3 and KCH4 showed NAD(P)H dependence, optimum activity at pH 5.5, low K(m) (45-55 microM) and substrate inhibition by Cr(VI) (>75 microM), whereas that of KCH5 showed NADH dependence, pH optimum at 6.0, high K(m) (200 microM) and no inhibition by Cr(VI). Cr(VI) reduction was optimum at 35 degrees C for CFEs of KCH3 and KCH5 and 30 degrees C for that of KCH3. Cr(VI) reduction by CFEs of all the strains were inhibited by Hg(2+) and enhanced by Cu(2+). Activity enhancement by Cu(2+) was more predominant (290%) for KCH4. The characterization of Cr(VI) reduction by CFEs of chromate resistant isolates of different genera is useful for development of Cr(VI) bioremediation.     

All2: A tool for selecting mosaic mutations from comprehensive multi-cell comparisons

Accurate discovery of somatic mutations in a cell is a challenge that partially lays in immaturity of dedicated analytical approaches. Approaches comparing a cell’s genome to a control bulk sample miss common mutations, while approaches to find such mutations from bulk suffer from low sensitivity. We developed a tool, All², which enables accurate filtering of mutations in a cell without the need for data from bulk(s). It is based on pair-wise comparisons of all cells to each other where every call for base pair substitution and indel is classified as either a germline variant, mosaic mutation, or false positive. As All² allows for considering dropped-out regions, it is applicable to whole genome and exome analysis of cloned and amplified cells. By applying the approach to a variety of available data, we showed that its application reduces false positives, enables sensitive discovery of high frequency mutations, and is indispensable for conducting high resolution cell lineage tracing.     

Soil biochemical activity and growth response of rice Oryza sativa in flyash amended soil

Soil amended with different proportions of flyash, a solid waste generated from coal-fired thermal power plants, was evaluated as a soil conditioner and nutrient supplement during a field study on the growth of rice, Oryza sativa. Generally, pH and organic carbon (OC) content did not increase significantly (P > 0.05) in flyash amended soil, but significant increases (P < 0.05) in soil conductivity (32%), available phosphorus (48%) and organic matter (OM, 29%) were observed during harvest at the 20 t ha-1 flyash application rate. Amylase, invertase, dehydrogenase and protease activities, and CO2 evolution increased in flyash amended soil over the control. The pigment (chl-a, chl-b, and carotenoid) content in rice plants did not vary significantly (P > 0.05) between different flyash amendments. Total plants biomass and aboveground biomass increased (P < 0.05) significantly (17% and 25%, respectively) at the 20 t ha-1 flyash application. However, there was a retarded growth of underground biomass. Grain and straw yield increased by 21% and 18%, respectively, at 17.5 t ha-1 flyash amendment when compared to the control. Although, a significant increase (P < 0.05) in plant biomass and grain yield in flyash amended soil is encouraging from the point of waste disposal and management, elucidation of reasons for retarded growth in underground biomass will require additional research based on long-term studies.     

Isolation, partial purification and pharmacodynamics of a slow contractile substance in the venom sac extract of the wasp Vespa cincta Fabr

The venom of V. cincta contains acetylcholine (ACh), histamine and 5-hydroxytryptamine (5-HT). Blockers of these agonists did not block completely the hypotensive and smooth muscle contractile activity of venom. On smooth muscle, there was a residual slow contraction. The active substance which produced this slow contraction was separated by solvent extraction, gel filtration and TLC. The purified material (which has been provisionally designated "Vecikinin") lowered cat, rat and guinea pig blood pressure, increased amplitude of cardiac contraction, and increased capillary permeability. Vecikinin contracted several smooth muscle preparations (rat uterus, rat ascending colon, guinea pig ileum, guinea pig colon and rat ileum), while relaxing rat duodenum. Its contractile activity was not lost on boiling, but acid or alkali-boiling reduced its contractile activity. It was inactivated on incubation with chymotrypsin and carboxypeptidase but not with trypsin, pepsin or leucine aminopeptidase. It is a peptide, appears to be of low molecular weight, and could be distinguished from substance P, angiotensin, bradykinin and hornet or wasp kinin.     

Mutants of Chinese hamster ovary (CHO) cells with altered colcemid-binding affinity.

Clones of CHO cells stably resistant to colcemid have been isolated in the presence of the nonionic detergent Tween 80 after mutagen treatment. Successive single-step selections for increasing resistance were performed resulting in lines after three selection steps about 10 fold more resistant to colcemid than the parental cells. Three observations indicate that these colcemid-resistant (CMR) mutants are different from the colchicine-resistant permeability mutants isolated previously. First, their relative resistance to colcemid was not diminished in the presence of detergent which promoted increased drug permeability. Second, the CMR clones displayed limited cross-resistances only to tubulin-binding compounds. Third, the binding affinity of labeled colcemid by cytoplasmic extracts from CMR clones was reduced, and the reduction was greater in the more resistant clones. No reduction in binding of labeled colcemid was observed in the membrane-altered colchicine-resistant mutants. All these observations are consistent with the CMR clones being tubulin-altered mutants. In further support of this conclusion, we observed that tubulin purified from a CMR mutant still possessed reduced colcemid-binding affinity compared with that from parental cells.     

A facile method for the construction of synthetic genes

A simple protocol for rapid assembly of chemically synthesized deoxyoligonucleotides into double stranded DNA is described. Several parameters of a ligation-free method were investigated to allow efficient assembly of a large number of oligonucleotides into double stranded DNA by polymerase chain reaction. Synthesis of a 701 bp DNA was carried out in a single reaction by assembling 28 oligonucleotides designed with partial overlaps at complementary ends. An estimate of error rate was made by sequencing several independent clones of the synthesized DNA