ALBERT

Description: Trehalose-6-phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti-stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43-fold purification. The protein sequence, determined by N-terminal sequencing and MALDI-TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length gene sequence of TPP of Candida utilis was identified using rapid amplification of cDNA ends - PCR reaction (RACE-PCR). The gene was cloned and expressed in E. coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steady state fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. K m was determined to be ∼0.8 mM. Optimum temperature and pH was found to be 30 °C and 8.5 respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylation mediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations towards understanding the physico-chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalose synthesis and survivability of the organism under thermal stress was established. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: [1]  Structural evolution of monsoon clouds in the core monsoon region of India has been examined using multi-sensor data. Invigoration of warm clouds above 4.5 km (occurring in only 15.4% days of the last 11 monsoon seasons) is associated with a transition from negative to positive normalized rainfall anomaly. Cloud top pressure reduces with an increase in aerosol optical depth at a higher rate of invigoration in drier condition (characterized by large fraction of absorbing aerosols) than wet condition. Cloud effective radius for warm clouds does not show any significant change with an increase in aerosol concentration in presence of high liquid water path, probably due to strong buffering role of meteorology. The structural evolution of monsoon clouds is influenced by both dynamic and microphysical processes that prolong the cloud lifetime, resulting in infrequent rainfall. Our results call for improved representation of aerosol and cloud vertical structures in the climate models to resolve this issue.

Description: A thin layer of fresh water from summer monsoon rain and river runoff in the Bay of Bengal (BoB) has profound influence on air-sea interaction across the south Asian region, but the mechanisms that sustain the low-salinity layer are as yet unknown. Using the first long time series of high-frequency observations from a mooring in the north BoB and satellite salinity data, we show that fresh water from major rivers is transported by large-scale flow and eddies, and shallow salinity stratification persists from summer through winter. The moored observations show frequent 0.2-1.2 psu salinity jumps with time scales of 10 minutes to days, due to O(1-10) km sub-mesoscale salinity fronts moving past the mooring. In winter, satellite sea surface temperature shows 10 km-wide filaments of cool water, in line with moored data. Rapid salinity and temperature changes at the mooring are highly coherent, suggesting slumping of salinity-dominated fronts. Based on these observations, we propose that sub-mesoscale fronts may be one of the important drivers for the persistent fresh layer in the north BoB.

Description: Pointillistic based super-resolution techniques, such as photoactivated localization microscopy (PALM), involve multiple cycles of sequential activation, imaging, and precise localization of single fluorescent molecules. A super-resolution image, having nanoscopic structural information, is then constructed by compiling all the image sequences. Because the final image resolution is determined by the localization precision of detected single molecules and their density, accurate image reconstruction requires imaging of biological structures labeled with fluorescent molecules at high density. In such image datasets, stochastic variations in photon emission and intervening dark states lead to uncertainties in identification of single molecules. This, in turn, prevents the proper utilization of the wealth of information on molecular distribution and quantity. A recent strategy for overcoming this problem is pair-correlation analysis applied to PALM. Using rigorous statistical algorithms to estimate the number of detected proteins, this approach allows the spatial organization of molecules to be quantitatively described.

Description: Oncostatin-M (OSM) is a patho-physiologically important pleiotropic, multifunctional cytokine. OSM mRNA sequence analysis revealed that its 3'UTR contains three highly conserved GC-rich cis -elements (GCREs) whose role in mRNA stability is unidentified. In the present study, the functional role of the proximal GC-rich region of osm 3'-UTR (GCRE-1) in post-transcriptional regulation of osm expression in U937 cells was assessed by transfecting construct containing GCRE-1 at 3' end of a fairly stable reporter gene followed by analysis of the expression of the reporter. GCRE-1 showed mRNA destabilizing activity however, upon PMA treatment the reporter message containing GCRE-1 was stabilized. This stabilization is owing to a time dependent progressive binding of trans -factors (atleast five proteins) to GCRE-1 post PMA treatment. Nucleolin was identified as one of the proteins in the RNP complex of GCRE-1 with PMA treated U937 cytosolic extracts by oligo-dT affinity chromatography of poly-adenylated GCRE-1. Immuno-blot revealed time dependent enhancement of nucleolin in the cytoplasm which in turn directly binds GCRE-1. RNA co-immunoprecipitation confirmed the GCRE-1-nucleolin interaction in-vivo . To elucidate the functional role of nucleolin in stabilization of osm mRNA, nucleolin was overexpressed in U937 cells and found to stabilize the intrinsic osm mRNA, where co-transfection with the reporter containing GCRE-1 confirms the role of GCRE-1 in stabilization of the reporter mRNA. Thus in conclusion, the results asserted that PMA treatment in U937 cells leads to cytoplasmic translocation of nucleolin that directly binds GCRE-1, one of the major GC-rich instability elements, thereby stabilizing the osm mRNA. This article is protected by copyright. All rights reserved

Description: ABSTRACT Transforming growth factor-β signaling exerts divergent effects on normal and cancer cells, although mechanism underlying this differential behavior remains unclear. In this study, expression of ninety-four genes pertaining to the TGF-β signaling pathway was compared between tumor and benign tissue samples from the human prostate gland to identify major discriminators driving prostate carcinogenesis. E2F5 was identified as one of the most deregulated genes in prostate cancer tissues, predominantly in samples with Gleason-score 6. Expression of other deregulated components of TGF-β signaling was examined by qRT-PCR, western blot and immune-staining. Function of E2F5 and p38 in prostate cancer was investigated using siRNA-treatment of PC3 cell-line followed by analyses of associated components and cell cycle. Observations revealed that E2F5 overexpression was accompanied by significantly higher phosphorylation of SMAD3 at Ser-208 in the linker region (pSMAD3L) and p38 in tumor tissue. A striking difference in SMAD3 phosphorylation, marked by preponderance of pSMAD3L and pSMAD3C (Ser-423 and 425) in tumor and benign tissues, respectively was noted. Co-localization of E2F5 with pSMAD3L in the nuclei of tumor and PC3 cells indicated a functional interface between the proteins. Downregulation of E2F5 and p38 in PC3 cells resulted in marked reduction of phosphorylation of SMAD3 and perturbation of cell cycle with an arrest of cells in G 1 . Our findings unearthed that E2F5/p38 axis played a cardinal role in uncontrolled cellular proliferation in prostate cancer through pSMAD3L activation. It also underscores a strong potential for E2F5 to be incorporated as a tool in early detection of prostate cancer. This article is protected by copyright. All rights reserved

Description: The effect of upwelling events, storm water discharge, and local circulation on phytoplankton blooms in the central California and Southern California Bight (SCB) coastal zones was analyzed using 10+ years (1997–2007) of remotely sensed surface chlorophyll concentration (CHL, derived from SeaWiFS ocean color), sea surface temperature, and modeled freshwater discharge. Analysis of variability and factors associated with phytoplankton blooms was conducted using the offshore extension of zones of CHL 〉 5 mg m−3; this method excludes terrestrial interference that complicates the use of ocean color to investigate phytoplankton blooms in coastal waters. In the SCB, blooms were most frequent in spring and associated with the spring transition to an upwelling regime. Along the Central Coast, blooms persisted from spring to autumn during seasonal intensification of upwelling. Offshore CHL extensions showed a significant positive trend during 1997–2007, with maxima in 2000–2001 and 2005–2006 that coincided with higher than normal frequency of upwelling events. Upwelling was found to be a major factor driving phytoplankton blooms, although the standard upwelling index derived from large-scale atmospheric circulation was decoupled from the frequencies of both upwelling events and phytoplankton blooms. Areas of longer residence time associated with natural boundaries between coastal ocean regions had more extensive and persistent blooms. The influence of storm water discharge on offshore CHL extension appeared to be limited to areas in close proximity to major river mouths. These “hot spots” were also co-located with ocean outfalls of Publicly Owned Treatment Works (POTW) discharge and, in some cases, longer residence time of coastal waters.

Description: Abstract Microbial dynamics drive the biotic machinery of early soil evolution. However, integrated knowledge of microbial community establishment, functional associations, and community assembly processes in incipient soil is lacking. This study presents a novel approach of combining microbial phylogenetic profiling, functional predictions, and community assembly processes to analyze drivers of microbial community establishment in an emerging soil system. Rigorous submeter sampling of a basalt‐soil lysimeter after 2 years of irrigation revealed that microbial community colonization patterns and associated soil parameters were depth dependent. Phylogenetic analysis of 16S rRNA gene sequences indicated the presence of diverse bacterial and archaeal phyla, with high relative abundance of Actinomyceles on the surface and a consistently high abundance of Proteobacteria (Alpha, Beta, Gamma, and Delta) at all depths. Despite depth‐dependent variation in community diversity, predicted functional gene analysis suggested that microbial metabolisms did not differ with depth, thereby suggesting redundancy in functional potential throughout the system. Null modeling revealed that microbial community assembly patterns were predominantly governed by variable selection. The relative influence of variable selection decreased with depth, indicating unique and relatively harsh environmental conditions near the surface and more benign conditions with depth. Additionally, community composition near the center of the domain was influenced by high levels of dispersal, suggesting that spatial processes interact with deterministic selection imposed by the environment. These results suggest that for oligotrophic systems, there are major differences in the length scales of variation between vertical and horizontal dimensions with the vertical dimension dominating variation in physical, chemical, and biological features.

Description: Abstract Retarding potential analyzers are frequently flown on small satellites as in situ ion probes, from which can be derived a number of ion plasma parameters from a current‐voltage relationship (I‐V curve). The traditional method of analyzing retarding potential analyzer data produces inaccuracies in derived estimates when there is significant noise present in the instrument measurements. In this study we investigate the dependencies between parameters that produce uncertainties in noisy I‐V curves. It is found that multiple combinations of ion velocity and spacecraft floating potential can produce I‐V curves that lie within the noise envelope, which renders it difficult for a traditional curve fitting technique to objectively and accurately estimate parameters from a noisy I‐V curve. In this paper we propose BATFORD—a bootstrap resampling‐based technique to improve the accuracies of parameter estimates. It is particularly useful when signal‐to‐noise ratios are low. The algorithm is tested against a traditional curve fitting method for a simulated data set comprising I‐V curves for the middle‐ and low‐latitude ionosphere at low Earth orbit altitudes around 450 km, where O+ is the predominant species. BATFORD is found to provide more robust and reliable estimates assuming generalized noise distribution characteristics. As further validation, the algorithm is applied to satellite data from an orbit with deep plasma bubbles and hence low signal levels.