Cold beam of isotopically pure Yb atoms by deflection using 1D-optical molasses

We demonstrate the generation of an isotopically pure beam of laser-cooled Yb atoms by deflection using 1D-optical molasses. Atoms in a collimated thermal beam are first slowed using a Zeeman slower. They are then subjected to a pair of molasses beams inclined at 45(a similar to) with respect to the slowed atomic beam. The slowed atoms are deflected and probed at a distance of 160 mm. We demonstrate the selective deflection of the bosonic isotope Yb-174 and the fermionic isotope Yb-171. Using a transient measurement after the molasses beams are turned on, we find a longitudinal temperature of 41 mK.

Mycobacterium tuberculosis DinG Is a Structure-specific Helicase That Unwinds G4 DNA IMPLICATIONS FOR TARGETING G4 DNA AS A NOVEL THERAPEUTIC APPROACH

The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' -> 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' -> 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.

MicroRNA-125a Reduces Proliferation and Invasion of Oral Squamous Cell Carcinoma Cells by Targeting Estrogen-related Receptor alpha IMPLICATIONS FOR CANCER THERAPEUTICS

Estrogen-related receptor (ESRRA) functions as a transcription factor and regulates the expression of several genes, such as WNT11 and OPN. Up-regulation of ESRRA has been reported in several cancers. However, the mechanism underlying its up-regulation is unclear. Furthermore, the reports regarding the role and regulation of ESRRA in oral squamous cell carcinoma (OSCC) are completely lacking. Here, we show that tumor suppressor miR-125a directly binds to the 3UTR of ESRRA and represses its expression. Overexpression of miR-125a in OSCC cells drastically reduced the level of ESRRA, decreased cell proliferation, and increased apoptosis. Conversely, the delivery of an miR-125a inhibitor to these cells drastically increased the level of ESRRA, increased cell proliferation, and decreased apoptosis. miR-125a-mediated down-regulation of ESRRA impaired anchorage-independent colony formation and invasion of OSCC cells. Reduced cell proliferation and increased apoptosis of OSCC cells were dependent on the presence of the 3UTR in ESRRA. The delivery of an miR-125a mimic to OSCC cells resulted in marked regression of xenografts in nude mice, whereas the delivery of an miR-125a inhibitor to OSCC cells resulted in a significant increase of xenografts and abrogated the tumor suppressor function of miR-125a. We observed an inverse correlation between the expression levels of miR-125a and ESRRA in OSCC samples. In summary, up-regulation of ESRRA due to down-regulation of miR-125a is not only a novel mechanism for its up-regulation in OSCC, but decreasing the level of ESRRA by using a synthetic miR-125a mimic may have an important role in therapeutic intervention of OSCC and other cancers.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

Background: mIHF belongs to a subfamily of proteins, distinct from E. coli IHF. Results: Functionally important amino acids of mIHF and the mechanism(s) underlying DNA binding, DNA bending, and site-specific recombination are distinct from that of E. coli IHF. Conclusion: mIHF functions could contribute beyond nucleoid compaction. Significance: Because mIHF is essential for growth, the molecular mechanisms identified here can be exploited in drug screening efforts. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ihfA and ihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHF. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Measuring Glutathione Redox Potential of HIV-1-infected Macrophages

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E-GSH; Grx1-roGFP2) and measured subcellular changes in E-GSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E-GSH (approximately -310 mV), active viral replication induces substantial oxidative stress (E-GSH more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E-GSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about similar to 25 mV in E-GSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E-GSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

Mycobacterium tuberculosis Response Regulators, DevR and NarL, Interact in Vivo and Co-regulate Gene Expression during Aerobic Nitrate Metabolism

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and Delta narL mutant strain during exponential growth in broth cultures with or without nitrate defined an similar to 30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis Delta devR, Delta devS Delta dosT, and Delta narL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR similar to P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

Observation of coherent population trapping in a V-type two-electron system

We observe coherent population trapping (CPT) in a two-electron atom-Yb-174-using the S-1(0), F= 0 -> P-3(1), F `= 1 transition. CPT is not possible for such a transition according to one-electron theory because the magnetic sublevels form a V-type system, but in a two-electron atom like Yb, the interaction of the electrons transforms the level structure into a V-type system, which allows the formation of a dark state and hence the observation of CPT. Since the two levels involved are degenerate, we use a magnetic field to lift the degeneracy. The single fluorescence dip then splits into five dips-the central unshifted one corresponds to coherent population oscillation, while the outer four are due to CPT. The linewidth of the CPT resonance is about 300 kHz and is limited by the natural linewidth of the excited state, which is to be expected because the excited state is involved in the formation of the dark state.

A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as ``sinks'' for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Background: The heterotrimeric M. tuberculosis RecBCD complex, or each of its individual subunits, remains uncharacterized. Results: MtRecD exists as a homodimer in solution, catalyzes ssDNA-dependent ATP hydrolysis, unwinding of DNA replication/recombination intermediates, and interacts with RecA. Conclusion: MtRecD possesses strong 5 3- and weak 3 5-helicase activities. Significance: These findings provide insights into the mechanism underlying DSB repair and homologous recombination in mycobacteria. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5 overhangs relative to the 3 overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having 18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3 overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5 overhangs, it could also catalyze significant unwinding of substrates containing 3 overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5 3 and weak 3 5 unwinding activities. The extent of unwinding of Y-shaped DNA structures was approximate to 3-fold lower compared with duplexes with 5 overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented Delta mimG: Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant Msm Rv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-kappa B, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.

Two new species of Gomphonema (Bacillariophyceae) from Doon Valley, Uttarakhand, India

This paper describes two new Gomphonema species from the lesser Himalayas, India, with their valve morphology using light and scanning electron microscopy. The two species, Gomphonema juettnerii sp. nov. and G. doonensis sp. nov., were found in Nalota stream in Doon Valley of Uttarakhand State. Main features of G. juettnerii are valve outline, shape of the areolae and striae pattern and presence of stigma. Main features of G. doonensis are rounded headpole, striae made by doubly punctate striae and features in central area. A detailed discussion on doubly punctate striae in members of gomphonemoid diatoms is presented.

Tuning of dielectric, pyroelectric and ferroelectric properties of 0.715Bi(0.5)Na(0.5)TiO(3)-0.065BaTiO(3)-0.22SrTiO(3) ceramic by internal clamping

This study systematically investigates the phenomenon of internal clamping in ferroelectric materials through the formation of glass-ceramic composites. Lead-free 0.715Bi(0.5)Na(0.5)TiO(3)-0.065BaTiO(3)-0.22SrTiO(3) (BNT-BT-ST) bulk ferroelectric ceramic was selected for the course of investigation. 3BaO - 3TiO(2) - B2O3 (BTBO) glass was then incorporated systematically to create sintered samples containing 0%, 2%, 4% and 6% glass (by weight). Upon glass induction features like remnant polarization, saturation polarization, hysteresis losses and coercive field could be varied as a function of glass content. Such effects were observed to benefit derived applications like enhanced energy storage density similar to 174 k J/m(3) to similar to 203 k J/m(3) and pyroelectric coefficient 5.7x10(-4) Cm-2K-1 to 6.8x10(-4) Cm-2K-1 by incorporation of 4% glass. Additionally, BNT-BT-ST depolarization temperature decreased from 457K to 431K by addition of 4% glass content. Glass incorporation could systematically increases diffuse phase transition and relaxor behavior temperature range from 70 K to 81K and 20K to 34 K, respectively when 6% and 4% glass content is added which indicates addition of glass provides better temperature stability. The most promising feature was observed to be that of dielectric response tuning. It can be also used to control (to an extent) the dielectric behavior of the host ceramic. Dielectric permittivity and losses decreased from 1278 to 705 and 0.109 to 0.107 for 6% glass, at room temperature. However this reduction in dielectric constant and loss increases pyroelectric figures of merit (FOMs) for high voltage responsivity (F-v) high detectivity (F-d) and energy harvesting (F-e) from 0.018 to 0.037 m(2)C(-1), 5.89 to 8.85 mu Pa-1/2 and 28.71 to 61.55 Jm(-3)K(-2), respectively for 4% added ceramic-glass at room temperature. Such findings can have huge implications in the field of tailoring ferroelectric response for application specific requirements. (C) 2015 Author(s).

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Mycobacterium tuberculosis RecG Protein but Not RuvAB or RecA Protein Is Efficient at Remodeling the Stalled Replication Forks IMPLICATIONS FOR MULTIPLE MECHANISMS OF REPLICATION RESTART IN MYCOBACTERIA

Aberrant DNA replication, defects in the protection, and restart of stalled replication forks are major causes of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB, and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen, responds to different types of replication stress and DNA damage are unclear. Here, we show that M. tuberculosis RecG rescues E. coli Delta recG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB(MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without a leading strand single-stranded DNA gap. Interestingly, single-stranded DNA-binding protein suppresses the MtRecG- and MtRuvAB-mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG-catalyzed replication fork remodeling and restart pathways in vivo.

Seismotectonics of the April-May 2015 Nepal earthquakes: An assessment based on the aftershock patterns, surface effects and deformational characteristics

Occurrence of the April 25, 2015 (Mw 7.8) earthquake near Gorkha, central Nepal, and another one that followed on May 12 (Mw 7.3), located similar to 140 km to its east, provides an exceptional opportunity to understand some new facets of Himalayan earthquakes. Here we attempt to assess the seismotectonics of these earthquakes based on the deformational field generated by these events, along with the spatial and temporal characteristics of their aftershocks. When integrated with some of the post-earthquake field observations, including the localization of damage and surface deformation, it became obvious that although the mainshock slip was mostly limited to the Main Himalayan Thrust (MHT), the rupture did not propagate to the Main Frontal Thrust (MFT). Field evidence, supported by the available InSAR imagery of the deformation field, suggests that a component of slip could have emerged through a previously identified out-of-sequence thrust/active thrust in the region that parallels the Main Central Thrust (MCT), known in the literature as a co-linear physiographic transitional zone called PT2. Termination of the first rupture, triggering of the second large earthquake, and distribution of aftershocks are also spatially constrained by the eastern extremity of PT2. Mechanism of the 2015 sequence demonstrates that the out-of-sequence thrusts may accommodate part of the slip, an aspect that needs to be considered in the current understanding of the mechanism of earthquakes originating on the MHT. (c) 2015 Elsevier Ltd. All rights reserved.