BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly

We have identified a potent antibacterial agent N-(4-sec-butylphenyl)-2-(thiophen-2-yl)-1H-benzod]imidazole-4-carboxa mide (BT-benzo-29) from a library of benzimidazole derivatives that stalled bacterial division by inhibiting FtsZ assembly. A short (5 min) exposure of BT-benzo-29 disassembled the cytokinetic Z-ring in Bacillus subtilis cells without affecting the cell length and nucleoids. BT-benzo-29 also perturbed the localization of early and late division proteins such as FtsA, ZapA and SepF at the mid-cell. Further, BT-benzo-29 bound to FtsZ with a dissociation constant of 24 +/- 3 m and inhibited the assembly and GTPase activity of purified FtsZ. A docking analysis suggested that BT-benzo-29 may bind to FtsZ at the C-terminal domain near the T7 loop. BT-benzo-29 displayed significantly weaker inhibitory effects on the assembly and GTPase activity of two mutants (L272A and V275A) of FtsZ supporting the prediction of the docking analysis. Further, BT-benzo-29 did not appear to inhibit DNA duplication and nucleoid segregation and it did not perturb the membrane potential of B. subtilis cells. The results suggested that BT-benzo-29 exerts its potent antibacterial activity by inhibiting FtsZ assembly. Interestingly, BT-benzo-29 did not affect the membrane integrity of mammalian red blood cells. BT-benzo-29 bound to tubulin with a much weaker affinity than FtsZ and exerted significantly weaker effects on mammalian cells than on the bacterial cells indicating that the compound may have a strong antibacterial potential.

In-plane elastic responses of circular cell honeycombs and bundled circular tubes in a diamond array structure

Assemblages of circular tubes and circular honeycombs in close packed arrangement are presently both competing and complementing regular honeycomb structures (HCS). The intrinsic isotropy of bundled tubes/rings in hexagonal arrays restricts their use to applications with isotopic need. With the aim of extending the utility of tubes/rings assemblages to anisotropic needs, this paper explores the prospects of bundled tubes and circular honeycombs in a general diamond array structure (DAS) to cater these needs. To this end, effective transverse Young's moduli and Poisson's ratio for thick/thin DAS are obtained theoretically. Analysis frameworks including thin ring theory (TRT), curved beam theory (CBT) and elasticity formulations are tested and corroborated by FEA employing contact elements. Results indicate that TRT and CBT are reasonable for thin tubes and honeycombs. Nevertheless, TRT yields compact formulae to study the anisotropy ratio, moduli spectrum and sensitivity of the assemblage as a function of thicknesses and array structure. These formulae supplement designers as a guide to tailor the structures. On the other hand, elasticity formulation can estimate over a larger range including very thick tubes/rings. In addition, this formulation offers to estimate refined transverse strengths of assemblages. (C) 2015 Elsevier Ltd. All rights reserved.

Experimental Analysis of Water Vapor Diffusion Through Porous Membranes in a Proton Exchange Membrane Fuel Cell

We report the diffusion characteristics of water vapor through two different porous media, viz., membrane electrode assembly (MEA) and gas diffusion layer (GDL) in a nonoperational fuel cell. Tunable diode laser absorption spectroscopy (TDLAS) was employed for measuring water vapor concentration in the test channel. Effects of the membrane pore size and the inlet humidity on the water vapor transport are quantified through mass flux and diffusion coefficient. Water vapor transport rate is found to be higher for GDL than for MEA. The flexibility and wide range of application of TDLAS in a fuel cell setup is demonstrated through experiments with a stagnant flow field on the dry side.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1 beta in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p < 0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-Fc gamma RIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1 beta (IL1 beta) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1 beta neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1 beta which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1 beta as a potential candidate for developing targeted therapy. (C) 2015 Elsevier B.V. All rights reserved.

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.

Sulfurization of sputtered Ag-In precursors for AgInS2 solar cell absorber layers

Silver indium sulfide (AgInS2) thin films are deposited by sequential sputtering of metallic precursor Ag/In] followed by sulfurization. Effect of substrate temperature (Tsub) during sulfurization process on the film growth is studied by varying the substrate temperature from 350 to 500 degrees C. Films prepared above 350 degrees C showed a mixture of orthorhombic and tetragonal phases of AgInS2 with tetragonal phase being dominant. Better crystalline, nearly stoichiometric and p-type films are obtained at a substrate temperature of 500 degrees C. The characteristic A(1) mode of AgInS2 chalcopyrite structure is observed in the Raman spectra at 274 cm(-1) for the films prepared above 350 degrees C. The grain size of the film increases from 489 to 895 nm with the increase in substrate temperature. The binding energies of the constituent elements are determined using XPS. The band gap of AgInS2 films is in the range of 1.64-1.92 eV and the absorption coefficient is found to be >10(4) cm(-1). Preliminary studies on the AgInS2/ZnS solar cell showed an efficiency of 0.3%. (C) 2015 Elsevier B.V. All rights reserved.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp-CrP14, obtained from stem tissues, and Talaromyces radicus-CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 mu g/ml and 20 mu g/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus-CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus-CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 mu g/l) in modified M2 medium and of vinblastine (70 mu g/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

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.

Differential proteomic and genomic profiling of mouse striatal cell model of Huntington's disease and control; probable implications to the disease biology

Huntington's disease (HD) is an autosomal dominant disorder of central nervous system caused by expansion of CAG repeats in exon1 of the huntingtin gene (Htt). Among various dysfunctions originated from the mutation in Htt gene, transcriptional deregulation has been considered to be one of the most important abnormalities. Large numbers of investigations identified altered expressions of genes in brains of HD patients and many models of HD. In this study we employed 2D SDS-PAGE/MALDI-MS coupled with 2D-DIGE and real-time PCR experiments of an array of genes focused to HD pathway to determine altered protein and gene expressions in STHdh(Q111)/Hdh(Q111) cells, a cell model of HD and compared with STHdh(Q7)/Hdh(Q7) cells, its wild type counterpart. We annotated 76 proteins from these cells and observed differential expressions of 31 proteins (by 2D-DIGE) involved in processes like unfolded protein binding, negative regulation of neuron apoptosis, response to superoxides etc. Our PCR array experiments identified altered expressions of 47 genes. Altogether significant alteration of 77 genes/proteins could be identified in this HD cell line with potential relevance to HD biology. Biological significance: In this study we intended to find out differential proteomic and genomic profiles in HD condition. We used the STHdh cells, a cellular model for HD and control. These are mouse striatal neuronal cell lines harboring 7 and 111 knock -in CAG repeats in their two alleles. The 111Q containing cell line (STHdh(Q111)/Hdh(Q111)) mimics diseased condition, whereas the 7Q containing ones (STHdh(Q7)/Hdh(Q7)), serves as the proper control cell line. Proteomic experiments were performed earlier to obtain differential expressions of proteins in R6/2 mice models, Hdh(Q) knock -in mice and in plasma and CSF from HD patients. However, no earlier report on proteomic alterations in these two HD cell lines and control was available in literature. It was, therefore, an important objective to find out differential expressions of proteins in these two cell lines. In this study, we annotated 76 proteins from STHdh(Q7)/Hdh(Q7) and STHdh(Q111)/Hdh(Q111) cells using 2D-gel/mass spectrometry. Next, by performing 2D-DIGE, we observed differential expressions of 31 proteins (16 upregulated and 15 downregulated) between these two cell lines. We also performed customized qRT-PCR array focused to HD pathway and found differential expressions of 47 genes (8 gene exptessions increased and 39 genes were decreased significantly). A total of 77 genes/proteins (Htt downregulated in both the studies) were found to be significantly altered from both the experimental paradigms. We validated the differential expressions of Vim, Hypk, Ran, Dstn, Hspa5 and Sod2 either by qRT-PCR or Western blot analysis or both. Out of these 77, similar trends in alteration of 19 out of 31 and 38 out of 47 proteins/genes were reported in earlier studies. Thus our study confirmed earlier observations on differential gene/protein expressions in HD and are really useful. Additionally, we observed differential expression of some novel genes/proteins. One of this was Hypk, a Htt-interacting chaperone protein with the ability to solubilize mHtt aggregated structures in cell lines. We propose that downregulation of Hypk in STHdh-Qm (Q111)/Hdh(Q111) has a causal effect towards HD pathogenesis. Thus the novel findings from our study need further research and might be helpful to understand the molecular mechanism behind HD pathogenesis. (C) 2015 Elsevier B.V. All rights reserved.

Synergistic effect of polymorphism, substrate conductivity and electric field stimulation towards enhancing muscle cell growth in vitro

Poly(vinylidene difluoride), a well-known candidate for artificial muscle patch applications is a semi-crystalline polymer with a host of attributes such as piezo- and pyroelectricity, polymorphism along with low dielectric constant and stiffness. The present work explores the unique interplay among the factors (conductivity, polymorphism and electrical stimulation) towards cell proliferation on poly(vinylidene difluoride) (PVDF)-based composites. In this regard, multi-walled carbon nanotubes (MWNTs) are introduced in the PVDF matrix (limited to 2%) through melt mixing to increase the conductivity of PVDF. The addition of MWNTs also led to an increase in the fraction of piezoelectric beta-phase, tensile strength and modulus. The melting and crystallization behaviour of PVDF-MWNT together with FT-IR confirms that the crystallization is found to be aided by the presence of MWNT. The conducting PVDF-MWNTs are used as substrates for the growth of C2C12 mouse myoblast cells and electrical stimulation with a range of field strengths (0-2 V cm(-1)) is intermittently delivered to the cells in culture. The cell viability results suggest that metabolically active cell numbers can statistically increase with electric stimulation up to 1 V cm(-1), only on the PVDF + 2% MWNT. Summarising, the current study highlights the importance of biophysical cues on cellular function at the cell-substrate interface. This study further opens up new avenues in designing conducting substrates, that can be utilized for enhancing cell viability and proliferation and also reconfirms the lack of toxicity of MWNTs, when added in a tailored manner.

Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing

Cell lines derived from tumor tissues have been used as a valuable system to study gene regulation and cancer development. Comprehensive characterization of the genetic background of cell lines could provide clues on novel genes responsible for carcinogenesis and help in choosing cell lines for particular studies. Here, we have carried out whole exome and RNA sequencing of commonly used glioblastoma (GBM) cell lines (U87, T98G, LN229, U343, U373 and LN18) to unearth single nucleotide variations (SNVs), indels, differential gene expression, gene fusions and RNA editing events. We obtained an average of 41,071 SNVs out of which 1,594 (3.88%) were potentially cancer-specific. The cell lines showed frequent SNVs and indels in some of the genes that are known to be altered in GBM-EGFR, TP53, PTEN, SPTA1 and NF1. Chromatin modifying genes-ATRX, MLL3, MLL4, SETD2 and SRCAP also showed alterations. While no cell line carried IDH1 mutations, five cell lines showed hTERT promoter activating mutations with a concomitant increase in hTERT transcript levels. Five significant gene fusions were found of which NUP93-CYB5B was validated. An average of 18,949 RNA editing events was also obtained. Thus we have generated a comprehensive catalogue of genetic alterations for six GBM cell lines.

Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing

Cell lines derived from tumor tissues have been used as a valuable system to study gene regulation and cancer development. Comprehensive characterization of the genetic background of cell lines could provide clues on novel genes responsible for carcinogenesis and help in choosing cell lines for particular studies. Here, we have carried out whole exome and RNA sequencing of commonly used glioblastoma (GBM) cell lines (U87, T98G, LN229, U343, U373 and LN18) to unearth single nucleotide variations (SNVs), indels, differential gene expression, gene fusions and RNA editing events. We obtained an average of 41,071 SNVs out of which 1,594 (3.88%) were potentially cancer-specific. The cell lines showed frequent SNVs and indels in some of the genes that are known to be altered in GBM-EGFR, TP53, PTEN, SPTA1 and NF1. Chromatin modifying genes-ATRX, MLL3, MLL4, SETD2 and SRCAP also showed alterations. While no cell line carried IDH1 mutations, five cell lines showed hTERT promoter activating mutations with a concomitant increase in hTERT transcript levels. Five significant gene fusions were found of which NUP93-CYB5B was validated. An average of 18,949 RNA editing events was also obtained. Thus we have generated a comprehensive catalogue of genetic alterations for six GBM cell lines.

Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model

Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing.

Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis

Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to -5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy.