Methylation Silencing of ULK2, an Autophagy Gene, Is Essential for Astrocyte Transformation and Tumor Growth

Glioblastoma (GBM) is the most aggressive type of brain tumor and shows very poor prognosis. Here, using genome-wide methylation analysis, we show that G-CIMP+ and G-CIMP-subtypes enrich distinct classes of biological processes. One of the hypermethylated genes in GBM, ULK2, an upstream autophagy inducer, was found to be down-regulated in GBM. Promoter hypermethylation of ULK2 was confirmed by bisulfite sequencing. GBM and glioma cell lines had low levels of ULK2 transcripts, which could be reversed upon methylation inhibitor treatment. ULK2 promoter methylation and transcript levels showed significant negative correlation. Ectopic overexpression of ULK2-induced autophagy, which further enhanced upon nutrient starvation or temozolomide chemotherapy. ULK2 also inhibited the growth of glioma cells, which required autophagy induction as kinase mutant of ULK2 failed to induce autophagy and inhibit growth. Furthermore, ULK2 induced autophagy and inhibited growth in Ras-transformed immortalized Baby Mouse Kidney (iBMK) ATG5(+/+) but not in autophagy-deficient ATG5(-/-) cells. Growth inhibition due to ULK2 induced high levels of autophagy under starvation or chemotherapy utilized apoptotic cell death but not at low levels of autophagy. Growth inhibition by ULK2 also appears to involve catalase degradation and reactive oxygen species generation. ULK2 overexpression inhibited anchorage independent growth, inhibited astrocyte transformation in vitro and tumor growth in vivo. Of all autophagy genes, we found ULK2 and its homologue ULK1 were only down-regulated in all grades of glioma. Thus these results altogether suggest that inhibition of autophagy by ULK1/2 down-regulation is essential for glioma development.

The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice

Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of lysine 27 of histone H3. The functions of plant PRC2 have been chiefly described in Arabidopsis, but specific functions in other plant species, especially cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss-of-function mutants in E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 as differentially expressed in the emf2b mutant compared with wild type. OsMADS1 and OsMADS6, known to be required for meristem determinacy in rice, have reduced expression in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by quantitative (q)RT-PCR showed that all three genes are presumptive targets of PRC2 in the meristem. Therefore, in rice, and possibly other cereals, PRC2 appears to play a major role in floral meristem determinacy through modulation of the expression of E-function genes.

Beadex Function in the Motor Neurons Is Essential for Female Reproduction in Drosophila melanogaster

Drosophila melanogaster has served as an excellent model system for understanding the neuronal circuits and molecular mechanisms regulating complex behaviors. The Drosophila female reproductive circuits, in particular, are well studied and can be used as a tool to understand the role of novel genes in neuronal function in general and female reproduction in particular. In the present study, the role of Beadex, a transcription co-activator, in Drosophila female reproduction was assessed by generation of mutant and knock down studies. Null allele of Beadex was generated by transposase induced excision of P-element present within an intron of Beadex gene. The mutant showed highly compromised reproductive abilities as evaluated by reduced fecundity and fertility, abnormal oviposition and more importantly, the failure of sperm release from storage organs. However, no defect was found in the overall ovariole development. Tissue specific, targeted knock down of Beadex indicated that its function in neurons is important for efficient female reproduction, since its neuronal knock down led to compromised female reproductive abilities, similar to Beadex null females. Further, different neuronal class specific knock down studies revealed that Beadex function is required in motor neurons for normal fecundity and fertility of females. Thus, the present study attributes a novel and essential role for Beadex in female reproduction through neurons.

Study the effect of electrical and mechanical shock loading on liberation and milling characteristics of mineral materials

Milling is an energy intensive process and it is considered as one of the most energy inefficient processes. Electrical and mechanical shock loading can be used to develop a pre-treatment methodology to enhance energy efficiency of comminution and liberation of minerals. Coal and Banded Hematite Jasper (BHJ) Iron ores samples were taken for the study to know the effect of shock loading. These samples were exposed to 5 electric shocks of 300 kV using an electric shock loading device. A diaphragmless shock tube was used to produce 3 and 6 compressed air shocks of Mach number 2.12 to treat the coal and Iron ore samples. Microscopic, comminution and liberation studies were carried out to compare the effectiveness of these approaches. It was found that electric shock loading can comminute the coal samples more effectively and increases the yield of carbon by 40% at 1.6 gm/cc density over the untreated coal samples. Mechanical shock loading showed improved milling performance for both the materials and 12.90% and 8.1% reduction in the D-80 of the particles was observed during grinding for treated samples of coal and iron, respectively. Liberation of minerals in BHJ Iron ore was found unaffected due to low intensity of the mechanical shock waves and non conductivity of minerals. Compressed air based shock loading is easier to operate than electrical shock loading and it needs to be explored further to improve the energy efficacy of comminution. (C) 2014 Elsevier Ltd. All rights reserved.

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N-5, N-10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in alpha-lcetoacid dehydrogenase reactions. (C) 2014 Elsevier B.V. All rights reserved.

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation

Background. Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH) 1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Methods. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Results. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Conclusions. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation.

Production and characterization of bioflocculants for mineral processing applications

A comparative study of two bacterial strains namely, Bacillus licheniformis and Bacillus firmus in the production of bioflocculants was made. The highest bioflocculant yield of 16.55 g/L was obtained from B. licheniformis (L) and 10 g/L from B. firmus (F). The bioflocculants obtained from the bacterial species were water soluble and insoluble in organic solvents. FTIR spectral analysis revealed the presence of hydroxyl, carboxyl and sugar derivatives in the bioflocculants. Thermal characterization by differential scanning calorimetry (DSC) showed the crystalline transition and the melting point (T-m) at 90-100 degrees C. Effects of bioflocculant dosage and pH on the flocculation of clay fines were evaluated. Highest bioflocculation efficiency on kaolin clay suspensions was observed at an optimum bioflocculant dosage of 5 g/L. The optimum pH range for the maximum bioflocculation was at pH 7-9. Bioflocculants exhibited high efficiency in dye decolorization. The maximum Cr (VI) removal was found to be 85 % for L (bioflocculant dosage at 2 g/L). This study demonstrates that microbial bioflocculants find potential applications in mineral processing such as selective flocculation of mineral fines, decolorization of dye solutions and in the remediation of toxic metal solutions. (C) 2015 Elsevier B.V. All rights reserved.

Enhancement of Flotation Selectivity of Sphalerite Using Mineral-Stressed Paenibacillus polymyxa and Its Cellular Components

The selective flotation of sphalerite from a sphalerite-galena mineral mixture was achieved using cellular components of Paenibacillus polymyxa after adaptation to the above minerals. The soluble and insoluble fractions of the thermolysed bacterial cells adapted to sphalerite yielded higher flotation recoveries of sphalerite with selectivity indices ranging between 22 and 29. The protein profile for the unadapted and mineral-stressed cells was found to differ distinctly, attesting to variation in the yield and nature of extra-cellular polymeric substances. The changes induced in the bacterial cell wall components after adaptation to sphalerite or galena with respect to the contents of phosphate, uronic acid and acetylated sugars of P. polymyxa were quantified. In keeping with these changes, a marginal morphological transition of P. polymyxa from rods to spheres was observed. The role of the dissolved metal ions from the minerals as well as that of the constituents of extracellular secretions in modulating the surface potential of the mineral-stressed cells were demonstrated. These studies highlighted that, mineral stress led to qualitative and quantitative changes in the cellular components, which facilitated the enhancement of flotation selectivity of sphalerite.