Implication of a sigma-Methane Complex en Route to Elimination of Methane from a Ruthenium Complex: An Experimental and Theoretical Investigation

The five-coordinated 16-electron complex Ru(Me)(dppe)(2)]OTf] (3) undergoes methane elimination at room temperature to afford the ortho-metalated species (dppe){(C6H5)(C6H4)PCH2CH2P(C6H5)(2)}Ru]OTf] (7). Methane elimination, monitored using NMR spectroscopy, revealed no intermediate throughout the reaction. The NOE between Ru-Me protons and ortho phenyl protons and an agostic interaction trans to the methyl group were found in complex 3 by NMR spectroscopy, which form the basis for three plausible pathways for methane elimination and ortho metalation: pathway I (through spatial interaction), pathway II (through oxidative addition and reductive elimination), and pathway III (through agostic interaction). Methane elimination from complex 3 via pathway I was discounted, since it involves interactions through space and not through bonds. Moreover, the calculated energy barrier for the pathway I transition state was quite high (71.3 kcal/mol), which also indicates that this pathway is very unlikely. Furthermore, no spectroscopic evidence for oxidatively added seven-coordinated Ru(IV) species was found and the computed energy barrier of the transition state for pathway II was moderately high (41.1 kcal/mol), which suggests that this cannot be the right pathway for methane elimination and ortho-metalation of complex 3. On the other hand, indirect evidence in the form of chemical reactions point to the most plausible pathway for methane elimination, pathway III, via the intermediacy of a sigma-CH4 complex that could not be found spectroscopically. DFT calculations at several levels on this pathway showed an initial low-barrier rearrangement through TS1 to a square-pyramidal intermediate wherein methyl and agostic C-H are cis to each other. Migration of hydrogen from agostic C-H and elimination of methane proceed through the transition state TS2, which retains a weak metal-H bonding through most parts of the reaction coordinate. Upon comparison of all three pathways, pathway III was found to be the most likely for methane elimination and ortho-metalation of complex 3.

Homology and enzymatic requirements of microhomology-dependent alternative end joining

Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-beta

Actions of transforming growth factor-beta are largely context dependent. For instance, TGF-beta is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-beta also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-beta would change in the context of transformation, we have compared the differential gene regulation by TGF-beta in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-beta in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-beta is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-beta. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-beta in hFhTERT cells and TGF-beta conferred action of these cells was mediated by WNT4. While TGF-beta activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-beta which matched with the response of HT1080 cells to TGF-beta. These data suggest context dependent activation of non-canonical signaling by TGF-beta. (C) 2015 Published by Elsevier Inc.

Atomization Characteristics of Camelina-Based Alternative Aviation Fuels Discharging From Dual-Orifice Injector

The atomization characteristics of blends of bioderived camelina hydrogenated renewable jet (HRJ) alternative fuel with conventional aviation kerosene (Jet A-1) discharging into ambient atmospheric air from a dual-orifice atomizer used in aircraft engines are described. The spray tests are conducted in a spray test facility at six different test flow conditions to compare the atomization of alternative fuels with that of Jet A-1. The fuel sprays are characterized in terms of fuel discharge, spray cone angle, drop size distribution, and spray patternation. The measurements of spray drop size distribution are obtained using laser diffraction based Spraytec equipment. The characteristics of fuel discharge and cone angle of alternative fuel sprays do not show any changes from that of Jet A-1 sprays. The characteristics of spray drop size, evaluated in terms of the variation of mean drop size along the spray axis, for the alternative fuel sprays remain unaffected by the variation in fuel properties between the alternative fuels and Jet A-1. The measurements on spray patternation, obtained using a mechanical patternator at a distance 5.1 cm from the atomizer exit, show an enhanced fuel concentration in the vicinity of spray axis region for the alternative fuel sprays discharging from the dual-orifice atomizer.

Different Modes of Transactivation of Bacteriophage Mu Late Promoters by Transcription Factor C

Transactivator protein C is required for the expression of bacteriophage Mu late genes from lys, I, P and mom promoters during lytic life cycle of the phage. The mechanism of transcription activation of mom gene by C protein is well understood. C activates transcription at Pmom by initial unwinding of the promoter DNA, thereby facilitating RNA polymerase (RNAP) recruitment. Subsequently, C interacts with the (sic) subunit of RNAP to enhance promoter clearance. The mechanism by which C activates other late genes of the phage is not known. We carried out promoter-polymerase interaction studies with all the late gene promoters to determine the individual step of C mediated activation. Unlike at P-mom, at the other three promoters, RNAP recruitment and closed complex formation are not C dependent. Instead, the action of C at P-lys, P-I, and P-P is during the isomerization from closed complex to open complex with no apparent effect at other steps of initiation pathway. The mechanism of transcription activation of mom and other late promoters by their common activator is different. This distinction in the mode of activation (promoter recruitment and escape versus isomerization) by the same activator at different promoters appears to be important for optimized expression of each of the late genes.

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

The annotated whole-genome sequence of Mycobacterium tuberculosis indicated that Rv1388 (Mtihf) likely encodes a putative 20 kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or organization of 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 Arg170, Arg171, and Arg173, which might be involved in DNA binding, and a conserved proline (P150) in the tight turn. The phenotypic sensitivity of Escherichia coli Delta ihfA and Delta ihfB strains to UV and methylmethanesulfonate 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, bind 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 alpha beta. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes compaction of DNA into nucleoid-like or higher-order filamentous structures. We hence 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.

Alternative Formulation of the Discrete Linear Quadratic Regulator (DLQR)

In this paper, an alternative apriori and aposteriori formulation has been derived for the discrete linear quadratic regulator (DLQR) in a manner analogous to that used in the discrete Kalman filter. It has been shown that the formulation seamlessly fits into the available formulation of the DLQR and the equivalent terms in the existing formulation and the proposed formulation have been identified. Thereafter, the significance of this alternative formulation has been interpreted in terms of the sensitivity of the controller performances to any changes in the states or to changes in the control inputs. The implications of this alternative formulation to adaptive controller tuning have also been discussed.

Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7(+) (spslu7(+)) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3' splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3' splice site distance, intron length, and the impact of its A/U content at the 5' end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1(+), a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.

Novel Alternative Splice Variants of Mouse Cdk5rap2

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

Lower-Bound Axisymmetric Formulation for Geomechanics Problems Using Nonlinear Optimization

A lower-bound limit analysis formulation, by using two-dimensional finite elements, the three-dimensional Mohr-Coulomb yield criterion, and nonlinear optimization, has been given to deal with an axisymmetric geomechanics stability problem. The optimization was performed using an interior point method based on the logarithmic barrier function. The yield surface was smoothened (1) by removing the tip singularity at the apex of the pyramid in the meridian plane and (2) by eliminating the stress discontinuities at the corners of the yield hexagon in the pi-plane. The circumferential stress (sigma(theta)) need not be assumed. With the proposed methodology, for a circular footing, the bearing-capacity factors N-c, N-q, and N-gamma for different values of phi have been computed. For phi = 0, the variation of N-c with changes in the factor m, which accounts for a linear increase of cohesion with depth, has been evaluated. Failure patterns for a few cases have also been drawn. The results from the formulation provide a good match with the solutions available from the literature. (C) 2014 American Society of Civil Engineers.

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.

Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-alpha, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKC delta-MAPK pathway to suppress beta-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.

Genomic amplification upregulates estrogen-related receptor alpha and its depletion inhibits oral squamous cell carcinoma tumors in vivo

The ESRRA gene encodes a transcription factor and regulates several genes, such as WNT11 and OPN, involved in tumorigenesis. It is upregulated in several cancers, including OSCC. We have previously shown that the tumor suppressor miR-125a targets ESRRA, and its downregulation causes upregulation of ESRRA in OSCC. Upregulation of ESRRA in the absence of downregulation of miR-125a in a subset of OSCC samples suggests the involvement of an alternative mechanism. Using TaqMan (R) copy number assay, here we report for the first time that the genomic amplification of ESRRA causes its upregulation in a subset of OSCC samples. Ectopic overexpression of ESRRA led to accelerated cell proliferation, anchorage-independent cell growth and invasion, and inhibited apoptosis. Whereas, knockdown of ESRRA expression by siRNA led to reduced cell proliferation, anchorage-independent cell growth and invasion, and accelerated apoptosis. Furthermore, the delivery of a synthetic biostable ESRRA siRNA to OSCC cells resulted in regression of xenografts in nude mice. Thus, the genomic amplification of ESRRA is another novel mechanism for its upregulation in OSCC. Based on our in vitro and in vivo experiments, we suggest that targeting ESRRA by siRNA could be a novel therapeutic strategy for OSCC and other cancers.

An alternative approach to study nonlinear inviscid flow over arbitrary bottom topography

This paper deals with a new approach to study the nonlinear inviscid flow over arbitrary bottom topography. The problem is formulated as a nonlinear boundary value problem which is reduced to a Dirichlet problem using certain transformations. The Dirichlet problem is solved by applying Plemelj-Sokhotski formulae and it is noticed that the solution of the Dirichlet problem depends on the solution of a coupled Fredholm integral equation of the second kind. These integral equations are solved numerically by using a modified method. The free-surface profile which is unknown at the outset is determined. Different kinds of bottom topographies are considered here to study the influence of bottom topography on the free-surface profile. The effects of the Froude number and the arbitrary bottom topography on the free-surface profile are demonstrated in graphical forms for the subcritical flow. Further, the nonlinear results are validated with the results available in the literature and compared with the results obtained by using linear theory. (C) 2015 Elsevier Inc. All rights reserved.

High temperature thermoelectric properties of Zr and Hf based transition metal dichalcogenides: A first principles study

We investigate the electronic and thermal transport properties of bulk MX2 compounds (M = Zr, Hf and X = S, Se) by first-principles calculations and semi-classical Boltzmann transport theory. The band structure shows the confinement of heavy and light bands along the out of plane and in-plane directions, respectively. This results in high electrical conductivity (sigma) and large thermopower leading to a high power factor (S-2 sigma) for moderate n-type doping. The phonon dispersion demonstrates low frequency flat acoustical modes, which results in low group velocities (v(g)). Consequently, lowering the lattice thermal conductivity (kappa(latt)) below 2 W/m K. Low kappa(latt) combined with high power factor results in ZT > 0.8 for all the bulk MX2 compounds at high temperature of 1200 K. In particular, the ZT(max) of HfSe2 exceeds 1 at 1400 K. Our results show that Hf/Zr based dichalcogenides are very promising for high temperature thermoelectric application. (C) 2015 AIP Publishing LLC.