CALGB randomized patients with newly diagnosed clear cell

CALGB 90206 randomized patients with newly diagnosed clear cell RCC to IFN or IFN plus bevacizumab (Rini et al., 2008). The primary endpoint was overall survival, and secondary endpoints were progression free survival and safety. The majority (85%) of patients underwent a cytoreductive nephrectomy, and 90% had favorable or intermediate prognosis based on number of MSKCC adverse clinical risk factors. At the interim analysis, the median PFS was 5.2months in the IFN group and 8.5months in the IFN plus bevacizumab group (p<0.0001). However, no statistically significant difference in OS was observed between the two groups. The median OS was 17.4months in the IFN group and 18.3months in the combination arm (Rini et al., 2010). Furthermore, subset analysis failed to identify any clinical variable associated with treatment response. Therefore, no clinical variable other than MSKCC adverse clinical risk factors were included in our final model. An important limitation of prior biomarkers studies is that they included all stages of RCC (Takahashi et al., 2001; Sultmann et al., 2005; Kosari et al., 2005; Jones et al., 2005; Zhao et al., 2006; Cancer Genome Atlas Research N, 2013; Brannon et al., 2010), limiting their applicability to patients with metastatic disease. However, many of these studies used an unbiased, genome-wide approach to discovery of prognostic markers, and provided a wealth of candidate gene filipin markers for us to evaluate. An increased understanding of pathways and mechanisms driving clear cell RCC provided additional candidate markers. In univariate analysis, we found that 21 of the candidate biomarkers were significant predictors of OS using q<0.05 (Table 2 and Supplemental Table 2). These results are validation of prior discovery studies of prognostic markers. Our multivariable analysis identified an 8-gene model of OS. Following VHL inactivation, PBRM1 is the second major gene in ccRCC, with truncating mutations in 41% of cases (Varela et al., 2011). Genes in pathways deregulated following PBRM1 knockdown in RCC cell lines were included as candidate genes in this study. In our final prognostic model, 4 genes (CRYL1, HSD17B10, CEP55 and HGF) were PBRM1 related genes; 3 (CRYL1, HSD17B10 and CEP55) were also differentially expressed when comparing ccRCC to normal kidney (Tun et al., 2010). HGF, filipin which binds the proto-oncogene c-MET, has been linked to invasiveness and VHL inactivation in ccRCC (Horie et al., 1999; Koochekpour et al., 1999; Harshman and Choueiri, 2013). Both TRAF2 (Vasselli et al., 2003) and USP6NL (Zhao et al., 2006) were previously identified as prognostic genes is microarray-based studies of RCC. PCNA was included as a candidate gene because it is a classic marker of proliferation and has been previously associated with RCC prognosis (Nogueira and Kim, 2008). CDK1, a cell cycle regulator, was included as a candidate gene because it was previously reported to predict response to antiangiogenic and epidermal growth factor targeted therapy in RCC (Tsavachidou-Fenner et al., 2010). When generating our prognostic signature, genes were favored that provided independent and non-redundant prognostic information. Therefore, it is not surprising that our 8 genes have been associated with a wide range of functions important to cancer progression such as proliferation (CEP55, PCNA, CDK1), apoptosis (TRAF2), metabolism (CRYL1, HSD17B10) and invasion (HGF) (, 2015).
The genetic heterogeneity of RCC is well documented (Gerlinger et al., 2012, 2014). However, the clonal evolutionary tree has a common “trunk” that links all genomic mutations. In addition, there are common histologic features that pathologists use to classify renal tissue as RCC. Therefore, it is reasonable to expect that there are markers, particularly expression markers that directly reflect the phenotype of RCC. To generate a signature that was less sensitive to sampling artifacts produced by tumor heterogeneity, we performed a separate analysis using untreated primary tumors from metastatic clear cell RCC patients that were sampled in two different areas. Genes with heterogeneous expression within individual patients were excluded from consideration in our multimarker models.

Infections with eleven species of pathogens

Infections with eleven species of pathogens associated with cancers are classified as Group 1 carcinogens, definitely “carcinogenic to humans”, by the IARC. These agents include Helicobacter pylori, hepatitis B virus (HBV), hepatitis C virus (HCV), Opisthorchis viverrini, Clonorchis sinensis, Schistosoma haematobium, human papillomavirus (HPV), Epstein-Barr virus (EBV), human T-cell lymphotropic virus type 1 (HTLV-1), human herpes virus type 8 (HHV-8) and human immunodeficiency virus type 1 (HIV-1) (Bouvard et al., 2009; IARC, 2012; de Martel et al., 2012). Among parasitic diseases, infections with the two fish-borne liver flukes of the family Opisthorchiidae (trematodes), specifically Opisthorchis viverrini and Clonorchis sinensis, can induce cholangiocarcinoma, and infection with the blood fluke Schistosoma haematobium may cause cancer of the urinary Senexin B (Bouvard et al., 2009). Although malaria per se is not considered carcinogenic to humans by the IARC, the geographical association between the occurrence of malaria and that of Burkitt lymphoma provides a clue that malaria plays as a co-carcinogenic factor, together with EBV infection, for the development of Burkitt lymphoma (Molyneux et al., 2012). Other species of the genera Opisthorchis and Schistosoma are thought likely to be carcinogenic (Sripa et al., 2007; Pakharukova and Mordvinov, 2016). Intriguingly, Trypanosoma cruzi, the etiological agents of Chagas disease, displays apparently paradoxical roles in malignancy in exerting carcinogenic and anticancer properties (Krementsov, 2009; Sacerdote de et al., 1980). Potential causative roles of other parasitic infections have been postulated (Machicado and Marcos, 2016).
Here, we summarize current concepts and facts on associations of parasite infections, namely schistosomiasis, opisthorchiasis, clonorchiasis, strongyloidiasis, malaria, and Chagas disease with human cancers and review mechanisms by which parasites may promote, or impede carcinogenesis (Table 1).

Schistosomiasis and Cancer
Schistosomiasis is a neglected disease caused by infection with blood fluke trematodes of the genus Schistosoma. Out of 207 million cases of schistosomiasis currently estimated worldwide, 90% occur in sub-Saharan Africa (Steinmann et al., 2006). Schistosomiasis is considered the most important helminth parasite of humans in terms of morbidity and mortality. The five species of Schistosoma that infect humans are Schistosoma haematobium, S. mansoni, S. japonicum, S. intercalatum, and S. mekongi. Most human infections are due to S. haematobium, S. mansoni, and S. japonicum. Of those, S. haematobium is the most ubiquitous species in Egypt and in sub-Saharan Africa and causes urogenital schistosomiasis (UGS). The prevalence of schistosomiasis is associated with exposure-related factors, in particular with a favourable environment for the imperative intermediate host snails, sub-optimal sanitation infrastructure, and host genetic factors. Adult worms are usually found in human hosts; their interactions with the host and parasite-derived products including their eggs strongly induce carcinogenesis (Brindley et al., 2015). With regard to schistosomiasis at large, clearly UGS i.e. chronic infection with S. haematobium, is carcinogenic and thus classified as a Group 1 carcinogen by the IARC (IARC, 2012). Any carcinogenicity of infection with other schistosomes is far less evident. Liver and colorectal cancers and lymphoid tumors may be associated with chronic schistosomiasis. Nonetheless, infection with S. japonicum is classified by the IARC as Group 2B, i.e. possibly carcinogenic to humans (IARC, 2012; IARC, 1994).
Bladder cancer is a common malignancy of the urinary tract with approximately 400,000 new cases and 150,000 deaths occurring annually (Ferlay et al., 2010). Histological types of bladder cancer include urothelial carcinoma, squamous, adenocarcinoma, micropapillary, small cell and plasmacytoid neoplasms. Urothelial carcinomas account for >90% in the developed world, whereas squamous cell carcinoma is seen predominating in UGS endemic regions (Knowles and Hurst, 2015). Further important risk factors for the induction of bladder cancer are host immune responses and host genetic factors (Fig. 1).

Unsupervised clustering of molecular data such

Unsupervised clustering of molecular data, such as gene TASIN-1 or DNA methylation, provides a method of classifying intrinsic subtypes within cancer populations (Heiser et al., 2012; Hoadley et al., 2014). Identification of cancer subtypes has provided insight into the etiological factors TASIN-1 underlying molecular and clinical heterogeneity in other cancers and has provided clinical biomarkers to predict prognosis and subtype-specific therapeutic response (Heiser et al., 2012; Hoadley et al., 2014; Marisa et al., 2013). Four HNSCC subtypes have been identified by clustering of gene expression data (Chung et al., 2004; Keck et al., 2015; Lawrence et al., 2015; Walter et al., 2013). We have previously reported our identification of five HNSCC subtypes based on clustering of integrated DNA methylation and gene expression data from 310 HNSCCs from The Cancer Genome Atlas (TCGA) study (Gevaert et al., 2015).
DNA methylation, i.e., the covalent addition of methyl groups to CpG dinucleotides to form 5-methylcytosine (5mC), is the best-known epigenetic mechanism of transcriptional regulation, and is widely altered in virtually all cancers, as an early and potentially causative event (Jones and Baylin, 2002; Fernandez et al., 2012). Typical patterns of abnormal methylation in cancer include silencing of tumor suppressor genes by aberrant methylation (hypermethylation) of gene promoters, particularly at promoter CpG islands, as well as general loss of DNA methylation overall (hypomethylation), potentially resulting in genomic instability and reactivation of oncogenes (Jones and Baylin, 2002; Jones, 2012).
DNA methylation patterns are altered by smoking (Shenker et al., 2013; Massion et al., 2008), HPV (Lleras et al., 2013) and age (Xu and Taylor, 2014), and may therefore capture important information about etiological drivers of HNSCC. Moreover, cancer molecular subtypes tend to differ depending on the molecular analyte (such as DNA methylation and gene expression) used for clustering (Heiser et al., 2012). Therefore, we have investigated the clinical, etiological, and molecular attributes of DNA methylation HNSCC subtypes in the complete set of TCGA HNSCC patients (n=528), in order to gain insight into the factors that drive intertumoral heterogeneity.
We have reproduced five DNA methylation subtypes, which differ from the reported gene expression subtypes, and which more clearly segregate with etiological subgroups defined by HPV status and smoking. As most research into molecular heterogeneity has focused on differences between HPV+ and HPV− HNSCC, we have focused primarily on heterogeneity within HPV− HNSCC. Most importantly, we identified two atypical HNSCC subtypes, including a molecularly distinct subtype that is reproducible in additional data sets, providing molecular classification for atypical HNSCC.



Herein, we confirmed our previous finding of five HNSCC MethylMix subtypes (Gevaert et al., 2015), now within the complete TCGA HNSCC data set. These MethylMix subtypes segregated with HPV status and smoking, the best-known risk factors for HNSCC, indicating that they represent biologically meaningful subtypes.
HPV+ HNSCCs clustered into a single, almost ubiquitously HPV+ MethylMix subtype, agreeing with previous studies reporting a clear HPV DNA methylation signature (Lleras et al., 2013; Anayannis et al., 2015). Moreover, our MethylMix subtypes segregated with HPV status more perfectly than gene expression subtypes, consistent with previous reports that HPV+ HNSCCs occur in two gene expression subtypes (Keck et al., 2015), or make up a subset of the AT expression subtype (Chung et al., 2004; Lawrence et al., 2015). This provided proof of principle that our MethylMix subtypes capture key etiological heterogeneity in HNSCC, as HPV+ HNSCC is known to be a clinically and biologically distinct subtype (Sethi et al., 2012; Poling et al., 2014). The original TCGA paper (Lawrence et al., 2015), and other reports (Seiwert et al., 2015); (Lawrence et al., 2015) have focused on molecular differences between HPV+ and HPV− HNSCC, while Lleras et al. described DNA methylation features of HPV+ HNSCC (Lleras et al., 2013). Therefore, we took advantage of the segregation of HPV+ from HPV− HNSCC in our study to investigate less well-studied heterogeneity within the four HPV− subtypes.

Las facultades fiscales del Congreso de la Uni n

Las facultades fiscales del Congreso de la Unión (Cámara de Diputados y de Senadores) fueron minuciosamente reglamentadas. Así, el Congreso tenía la facultad “para imponer las contribuciones necesarias Madecassoside cubrir el presupuesto” (art. 73, fracción VII), bajo esta premisa, básicamente cualquier cosa podía ser sujeto de gravamen federal en detrimento de las facultades fiscales locales; también podía aprobar los empréstitos de la Nación, reconocer y en su caso mandar pagar la deuda nacional (art. 73, fracción VIII); fue facultado también tanto para expedir aranceles al comercio exterior y para, en su caso, impedir que en el comercio de Estado a Estado se establezcan restricciones (art. 73, fracción IX), así como para legislar en materia de comercio interno, minería e instituciones de crédito, lo que incluía también los impuestos derivados de estas actividades (art. 73, fracción X); también podía expedir leyes sobre el uso y aprovechamiento de vías generales de comunicación, sobre postas y correos, aguas de jurisdicción federal (art. 73, fracción XVII) y casas de moneda (art. 73, fracción XVIII); por otro lado, como los ingresos y los egresos habrían de estar contenidos en ordenamientos jurídicos aprobados por el Congreso de la Unión, el primero en una ley y el segundo en un presupuesto, dicho órgano podría examinar la cuenta anual presentada por el Poder Ejecutivo, examinando la conformidad de las partidas gastadas y la justificación de dichos gastos (art. 73, fracción XXX). Finalmente, para salvaguardar aún más las facultades fiscales federales en manos del Poder Legislativo, éste quedó facultado para expedir “todas las leyes que sean necesarias a objeto de hacer efectivas las facultades anteriores” (art. 73, fracción xxXI). Únicamente, y en atención a que era la representante directa de los contribuyentes, la Cámara de Diputados quedó exclusivamente facultada para “aprobar el presupuesto anual de gastos discutiendo primero las contribuciones que a su juicio deben decretarse para cubrir aquel” (art. 74, fracción IV). En resumidas cuentas, todas estas facultades fiscales del Poder Legislativo tendrían como resultado una Ley de Ingresos del Erario Federal, aprobada por el Congreso de la Unión y un Presupuesto de Egresos aprobado por la Cámara de Diputados.
Hasta aquí, es posible observar los numerales que conformaron el fundamento constitucional del sistema tributario desde 1917 hasta 1935. Sin embargo, las contribuciones que se cobraban en la República mexicana estaban contenidas en diversas disposiciones. No obstante que, para la época, al menos en el ámbito federal, para que su cobro fuera legal y legítimo era necesario que estuviesen incluidas en la Ley de Ingresos del Erario Federal, cuya principal característica era su anualidad. Es decir, cada año se incluían las contribuciones que el Poder Ejecutivo incluía en el proyecto de Ley, mismo que era discutido por la Cámara de Diputados para su aprobación. Únicamente se podían cobrar los impuestos en ella contenidos. Debido a que el Código Fiscal de la Federación fue promulgado hasta 1938 no es posible utilizar la clasificación que sobre las contribuciones se incluyó en él, por lo que hemos recurrido a la revisión de las leyes de Ingresos del Erario Federal de 1920 hasta 1935, publicados en el Diario Oficial de la Federación, para terminar de presentar un esquema del sistema tributario vigente desde 1917 hasta 1935 (véase Imagen 1).
Dividido en 11 grandes rubros, cada uno de ellos subdivididos en diversos impuestos, constituían a Hybridoma la gran masa tributaria del momento. Algunos de los más importantes todavía eran: 1) los impuestos a la importación, contenidos en su mayoría en el impuesto general de importación y otros especiales como a la gasolina; 2) el de exportación, que incluía al impuesto general de exportación y al impuesto a la exportación del petróleo; 3) impuesto a la industria, como la de transformación: tabacos, hilados y tejidos, gasolina, industria de alcoholes y bebidas alcohólicas, alcoholes, aguardientes, tequilas, mezcales y zotoles, licores, vinos y demás bebidas alcohólicas de producción nacional, aguamiel y productos de su fermentación, cervezas, impuestos sobre la producción de azúcar, así como el diez por ciento sobre las entradas brutas de los ferrocarriles constituían los impuestos a los transportes; 4) Impuesto sobre la renta; 5) Impuestos sobre capitales: herencias y legados, donaciones, loterías, rifas, y tesoros; 6) Impuesto de la Renta Federal del Timbre: actas, documentos y contratos, así como la contribución federal; 7) Impuesto sobre migración; 8) Impuestos sobre la explotación de recursos naturales: extensión superficial de lotes mineros, producción de metales y compuestos metálicos, producción del petróleo, terrenos petrolíferos y contratos petroleros, uso y aprovechamiento de aguas de jurisdicción federal, pesca, buceo, caza y similares, maderas y bosques, salinas, guano, nitratos y magnesita, otros recursos similares; 9) Diez por ciento adicional sobre los enteros que se hagan por los impuestos y derechos establecidos en los ocho rubros anteriores, cuando así lo determinasen las necesidades del Erario; 10) Derechos por la prestación de servicios públicos: a) consulares: certificación de documentos conforme a las prevenciones de la Ordenanza General de Aduanas, visación de facturas comerciales, legalización de firmas, certificados sobre constitución legal de sociedades extranjeras, certificados y demás actos especificados en otras disposiciones vigentes, expedición, refrendo y “visto bueno” de pasaportes en los Consulados, b) de tráfico marítimo, de navegación y terrestre: de patente de navegación, matrícula y registro, de barra, de tráfico marítimo, de tráfico marítimo interior, de carga y descarga, de arqueo, de tránsito, c) aduanales: de guarda y almacenaje, servicios extraordinarios, d) de comunicación: correo, telégrafo, radio-comunicación, e) de salubridad: certificación de medicinas de patente, especialidades y productos de tocador y belleza, desinfección, inspección, certificación y otros servicios sanitarios, f) de inspección y verificación: pesas y medidas, animales, semillas, frutas, plantas y cereales, instalaciones centrales eléctricas y telefónicas, instituciones bancarias, especiales, g) de registro: patentes de invención y marcas de fábrica, propiedad artística, literaria y dramática, h) diversos: fundición, afinación, ensayo y amonedación, enseñanza secundaria y profesional y expedición de títulos profesionales, visitas a museos, ruinas, monumentos y edificios históricos, d) publicaciones, e) otros servicios; y, 11) Productos y aprovechamientos: multas, recargos y rezagos de impuestos federales y aprovechamientos diversos. A grandes rasgos, estos eran los impuestos que se cobraban en México desde 1917 hasta 1935.

br LC MS MS data acquisition

LC–MS/MS data acquisition

LC–MS/MS data analysis, statistical analysis and visualization
Data were analyzed and filtered on MaxQuant version 1.2.2 [5] (20ppm error tolerance) with a FDR setting of 1% against the SPROT Yeast database and at a cutoff of at least 2 peptides seen to assign quantitation ratio. The exact MaxQuant settings used can be found in attached document. Each experiment was normalized to the ratio of the bait protein, i.e. SSA1 files using SSA1 ratio and HSP82 files normalized using HSP82 ratio. This produced a list of interactors and their respective quantitated changes upon DNA damage. Proteins were removed from the file if they were labeled as “Contaminants”, “Reverse” or “Only identified by site”. Three biological replicates were performed, with each biological replicate split into technical replicates (18O forward (FWD) labeling and 18O reverse (REV) labeling). A protein was considered identified if detected in at least three of the six replicates.
Statistical analysis was performed using the R statistical package ( Proteins with three out of six observations within each group (SSA1 and HSP82) were retained. Missing values were imputed using row mean imputation. Z-score normalization was performed on the log of all protein ratios. An ANOVA test was then performed to identify proteins that indicate significant variability (P-value<0.05) between biological replicates within each group. These were removed from consideration. The full data obtained was uploaded to the PRIDE repository and can now be found under reference number PXD001284.
Data, experimental design, materials and methods
A protein report summarizing the analysis of protein chemokine receptor was generated chemokine receptor in the body and headfoot of infected B. siamensis goniomphalos (Supplementary file 1 and 2 respectively). This file contains the experiment design, model description, statistical model and data summary as well as a detailed summary of each protein including peptide relative expression estimates in addition to protein level estimates.
Furthermore, a detailed spreadsheet containing the raw data from the iQuantitator analysis including ID, mean, standard deviation, credible interval, log2 and description for every protein identified in the body and headfoot samples (Supplementary file 3 and 4 respectively) was generated for each timepoint studied.

Ethics statement
The protocols used for animal experimentation were approved by the Animal Ethics Committee of Khon Kaen University, as described in [1].

This work was supported by Project (613669) and program (1037304) grants from the National Health and Medical Research Council of Australia (NHMRC) and a Tropical Medicine Research Collaboration (TMRC) grant from the National Institute of Allergy and Infectious Disease, National Institutes of Health, USA (P50AI098639). AL is supported by a NHMRC principal research fellowship. Funding from Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant no. PHD/0027/2551) to SP and ST is also gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Value of the data

Data, experimental design, materials and methods

Specifications table

Value of the data

Data, experimental design, materials and methods

2-Dimensional gel electrophoresis (2-DE)
2-DE was performed as described previously [2]. Gel images were analyzed using the Melanie software (GeneBio, Geneva, Switzerland) as described previously [3]. Spot intensities were subjected to statistical analysis to identify differentially abundant proteins. Only those spots that showed statistical significant differences in roots or leaves of the two tested genotypes and exhibited more than 2-fold change in abundance were accepted as candidate drought-responsive proteins (DRPs). The details of number of reproducibly detected spots and the number of spots showed significant change in roots and leaves of the tolerant (SE) or sensitive (SW) genotypes upon drought stress are shown in Supplementary Table 2.

br Data br Experimental design materials and methods br


Experimental design, materials and methods

This work was supported by grant no. 14-50-00068 from the Russian Science Foundation.

The proteomic profiling of atypical lung carcinoid (AC) tissue is presented, together with the largest collection of proteins identified from a large-cell neuroendocrine carcinoma (LCNEC) so far. A total of 1260 and 2436 proteins were identified in the AC and LCNEC samples, respectively, with FDR <1%. Noteworthy, these data have been obtained using archival formalin-fixed paraffin-embedded (FFPE) samples, thus reinforcing the opportunity and reliability of employing such samples for clinical proteomic studies. Data files can be found at PeptideAtlas repository at
Experimental design, materials and methods
Proteins were extracted from FFPE tissues as published [1], and quantified using the EZQ Protein Quantification Kit (Molecular Probes, Eugene, OR, USA). Detergent Removal Spin Columns (Pierce, Rockford, IL, USA) were used to remove SDS, and eluates were subjected to in solution reduction, alkylation and trypsin digestion as previously described [2]. ZipTip Pipette Tips (Millipore, Billerica, MA, USA) were employed to purify peptide mixtures.
An LTQ Orbitrap Velos (Thermo Scientific, San Jose, CA, USA) interfaced with an UltiMate 3000 RSLCnano LC system (Dionex, Sunnyvale, CA, USA), set up in a data dependent MS/MS mode as described elsewhere [3], was used for MS analysis. Concentration, desalting, and separation of peptide mixtures was performed as in [2], but using a 270min gradient from 1 to 50% eluent B (0.2% formic order hoechst 33342 in 95% ACN) in eluent A (0.2% formic acid in 5% ACN). The gradient was as follows: 1–10% Solvent B (3min), 10–23% B (230min), 23–50% B (37min). Peptide identification was performed using Proteome Discoverer (version 1.4; Thermo Scientific) as described previously [2].

This work was financed by Sardegna Ricerche through the program “Progetto Strategico Biotecnologie”, by AIRCIG 2013 14696 to GR, and by grant 997/2010.0414 from Fondazione Banco di Sardegna to GF.

Value of the data

Here, we present FTS data featuring multidrug-binding nature of the transcriptional regulator StAcrR. The top sixteen binders of StAcrR, as shown in Table S1, were identified by the FTS analysis from a library of 320 unique ligands. Human enolase 1, a negative control protein (Fig. S1), did not bind the top six binders of StAcrR, while implying their specific interactions with StAcrR. The remaining compounds were not tested against human enolase 1. Theoretical data for Table S2 were obtained from National Center for Biotechnology Information PubChem Compound Database ( and include chemical properties such as topological polar surface (Å3), number of hydrogen bond donors/acceptors, XLogP3, and molecular weight (g/mol) of the top six StAcrR FTS hits, dequalinium chloride, proflavine, ethidium bromide and rhodamine 6G. The latter three molecules were identified as the StAcrR binders employing a fluorescence polarization experimental approach (see our original publication [1]).

Experimental design, materials and methods
The FTS assay was run in 384-well PCR plates using an Echo550 acoustic transfer robot (Labcyte, Sunnyvale, CA) for dispensing a dimethyl sulfoxide stock of ligands to assay plates that contain 10μl of a mixture of StAcrR (1μg) and 2.5×SYPRO Orange fluorescence dye (Invitrogen, Carlsbad, CA) in 100mM HEPES buffer pH 7.5 and 150mM NaCl. Thermal scanning (from 10 to 80°C at 1.5°C min−1 ramp rate) on a real-time PCR machine CFX384 (Bio-Rad Laboratories, Hercules, CA) was coupled with fluorescence detection every 10s.
A test molecule, dequalinium, bound at 40μM that prompted us to screen a 320-molecule subset of the Spectrum library (Micro Source Discovery, Gaylordsville, CT, hereafter referred as SPC2-ECH008), dequalinium belongs to. The best binders were selected based on ΔTm, reduction of the background reading, and shape of the melting curve. The top six hits were further subjected to a dose-dependent response analysis using their 2.5, 5, 10, 25, 50, 75 and 100μM concentrations. Human enolase 1 (1.2μg in 10μl assay mixture) was used as a negative control protein and tested against the top six binders at 10, 25 and 50 100μM concentrations. FTS data were analyzed with the in-house ExcelFTS software.

somatostatin receptor br Experimental design materials and methods

Experimental design, materials and methods
The list of medulloblastoma cell lines was compiled from Cellosaurus, the controlled vocabulary of cell lines developed by the Swiss Institute of Bioinformatics [4], and the review by Xu et al. [5]. The relative percentage of citations for each cell line was determined separately for each database and reported as the mean percentage for three databases. The time of the first publication was used to determine the number of years the cell line has been available for, and the difference with the current year (2016) was used to normalise the citation data.

The research was supported by an EPSRC Doctoral Prize award hosted by the University of Nottingham (DP2014/DI).

These data include:

Experimental design, materials and methods

The acknowledgements for these studies are presented in Shah et al. [1]. This project was supported by NSFIOS-1147047 (A.K.S., PI).

The data used in this analysis has been generated after a detailed structural examination of membrane proteins. This structural data provides solid evidence for the utility and various roles of perturbed helices in membrane proteins. See Figs. 1–17 and Tables 1–5.

Experimental design, materials and methods
Structural analysis of membrane protein structures was performed after they were downloaded from the Orientation of Proteins in Membrane (OPM) database [9]. The identification of secondary structures was carried out using Assignment of Secondary Structures in Proteins (ASSP) [10] and non-bonded interactions were identified using MolBridge [11]. Next, we identified geometries of helical fragments using Helanal-Plus [2] and computed the backbone torsion angles (φ–ψ). Multiple sequence alignment of protein sequences was carried out using ClustalΩ [12].
We prepared datasets of somatostatin receptor belonging to Sodium Calcium family of transporters as mentioned in [1] to examine conservation of kinks in functionally important helices. A dataset of proteins belonging to Heme Copper Oxidase (HCO) superfamily was created to gain insights about the presence of the π-helix in each protein (Table 3). To understand the variation if any in the π-helix within different types of HCOs, we analyzed two crystal structures from the A-type, one from B-type and three crystal structures from the C-type HCOs along with proteins representing each Nitric Oxide Reductase (Table 3). The presence of the unusually long π-helix in Cytochrome-c-oxidase (PDB ID: 1v55) defined by ASSP was reconfirmed by its identification using DSSP – a program based on hydrogen bond energetics for secondary structure identification ( (Table 4).


Experimental design, materials and methods
The animals subjected to surgery were euthanized after collecting aqueous humor 3, 6, 12, 24, 48, 168, and 720h after surgery (n=4–6), and six control animals were euthanized without surgery. ELISA and immunohistochemical analysis were conducted as described previously [1]. Dunnett׳s test was used for statistical analyses, and P values less than 0.05 were considered significant.

Conflict of interest

Here, we share methods and experimental data that relate to the article entitled \”Construction of a compatible Gateway-based co-expression vector set for expressing multiprotein complexes in E. coli\” by L. Salim, C. Feger and D. Busso [1]. We describe the cloning procedure not only for elaborating each one of the vector but to clone gene target as well. We give a detailed snapshot of the cloning area and describe the restriction site(s) to be used to modify the set. We share the protocols used to quantify protein expression level by imaging and by fluorescent measurement. Finally, we detailed experimental data (image and quantification) for all combinations tested for the 33 (co)-expression plasmids constructed.

Me parece que la descripci n autoetnogr fica que elabora

Me parece que la descripción autoetnográfica que elabora Monsiváis de la noche disidente de los años 90 tiene, en muchos sentidos, el mismo tono de la realizada por Novo para las primeras décadas del siglo xx. Hay en ambos un tono mordaz pero quieto, una agudeza que se fija en los detalles y en los cuadros generales que se desarrollan TW37 su alrededor, una mirada solitaria y una lengua seductora. Los dos describen un mundo sórdido hecho de acomodos siempre frágiles, de silencios y pactos, de renuncias y pérdidas. Monsiváis escribe que “gracias a la impudicia o la tradición de no-tener-nada-que-perder, los homosexuales instituyen zonas de estridencia y provocación que, en rigor, son los primeros espacios de resistencia” (Monsiváis 1998: 34). La resistencia surge como un resultado no planeado del afán de tener ciertos lugares: las zonas de estridencia y provocación que instituyeron los homosexuales. Pero siempre son un margen, más o menos amplio, más o menos clandestino y secreto, perseguido o tolerado; un borde o un deslinde del mundo real y normal.
También los sujetos se ubican en esa marginalidad estridente y provocadora. Monsiváis se pregunta:
La interiorización de los insultos es una forma de trazar el margen en la subjetividad misma, como lo hace Novo con sus autoescarnios, y sus contemporáneos con el choteo y autochoteo. La única manera de lograrlo, como antes lo dijimos, es estableciendo una distancia que permita recibir el insulto, interiorizarlo y luego desdeñarlo por irreal. Lo real regresa de nuevo, pero esta vez de la mano de los escritos de Monsiváis: ¿por qué la intensidad hiriente de las agresiones las haría parcialmente irreales?, ¿la irrealidad está del lado de las agresiones o del agredido?, ¿no podría pensarse que los insultos dan realidad, no la querida, pero alguna al menos, a quien los recibe, y que solo se puede insultar a alguien real? No habría insultos para fantasmas, aunque se recomiende lanzarles improperios a las apariciones y otras presencias semejantes, si llegaran a cruzarse en nuestro camino. En ese caso, ¿por qué huirían al escuchar los insultos? Tal vez podríamos hipotetizar que lo hacen justamente porque eso los hace reales. Los fantasmas agredidos no huirían de los insultos, sino de la realidad que implican.
Monsiváis, sin pensar en aparecidos, escribe, paso siguiente, que: “lo intensamente real de los gays se centra en el coito, en el diálogo con los iguales centrado obsesivamente en el sexo. Al ser tan costosa en lo psíquico y lo social la disidencia, acrecientan su significado y su valor los actos sexuales y el idioma del ghetto’ (Monsiváis 1998: 38). ¿Qué sería lo real del coito o más bien qué tipo de intenso real daría el coito? El sexo sería el lugar de una igualdad disidente, pero también el sedimento del idioma del gueto que sirve ante todo para hablar de sexo, como el de Novo servía para burlarse de sí mismo y de los otros homosexuales.
El texto sobre la noche popular solo describe colectivos y multitudes urbanas; el de Novo, un personaje histórico y famoso. Monsiváis ve en la noche popular tipos de sujetos que deambulan, sacudidos a Plectonemic winding las crisis recurrentes del país, pero indemnes en sus deseos. Monsiváis y Novo describen el sexo proletario; ambos hablan de soldados y de hombres de clases populares, pero los ven de lejos, atractivos y peligrosos a la vez. Dice Monsiváis que Novo:
Varias décadas después, muchos ya pueden hablar y son dignos de la palabra, en diversos sentidos. Pero esos hombres y mujeres a quienes ambos autores rodean y miran, a quienes describen de alguna manera, permanecen en silencio. La comunidad que se ha formado es también un límite: entre clases sociales, entre mundos de vida, entre palabras y derechos adquiridos. Monsiváis escribe en su texto sobre la vida nocturna capitalina: “Me detengo al borde del extravío de las identificaciones, y reconozco lo convincente de la voz, cualquiera sea su origen” (Monsiváis 2010b). Ha presenciado un show travesti y está confuso acerca de la identidad de la artista. Se detiene en ese borde que atribuye al otro y no a sus propias categorías de visibilidad e inteligibilidad. De Novo dice que “la provocación es un gran instrumento de salud mental, y por eso todo lo ostenta, su relación con los choferes de autobuses, con los luchadores, con los soldados” (Monsiváis 2008: 59). Ambos autores, cada uno a su manera, son creadores de una alteridad sexual, nocturna, apenas descriptible, pero siempre muda. Esos hombres no hablan. Si la práctica de sí que antes mencionamos requería de la distancia, de carácter retórico, del sujeto con respecto a sí mismo, las prácticas colectivas de constitución de comunidades y redes requieren de esta distancia social en relación con el otro popular. El coleccionista describe sus objetos de maneras diversas: los más raros y lejanos solo a grandes rasgos. En este ejercicio histórico, algunos hablan y otros callan definitivamente.

Type I inhibitors bind to the ATP

Type I inhibitors: bind to the ATP binding site through the formation of hydrogen bonds to the kinase “hinge” residues and through hydrophobic interactions in and around the region occupied by the adenine ring of ATP [40].
Type II inhibitors: act on the inactive (unphosphorylated) conformation of enzyme, by stabilizing the inactive conformation. Type I and II work as ATP competitive inhibitors and target ATP binding site [41].
Type III inhibitors: they are noncompetitive ATP inhibitors, they bind to the allosteric site of the protein distant from ATP pocket. They don\’t require any typical hinge binding motifs and can bind the enzyme regardless it\’s activation state [42].

Synthetic strategies
A plenty of synthetic methods (Fig. 1) have been outlined for the synthesis of pyrazolo[3,4-d]pyrimidines. 5-Amino-4-cyanopyrazole I has been used in the number of reactions to attain the synthesis of desired compounds through different routes. Reaction of I with formamide at 180 °C for 24 h afforded the corresponding compounds (route a) [43,44]. Two step reaction in which compound I is reacted with N,N-dimethylphosgeniminium chloride in dichloromethane under reflux for 2 h gave the corresponding dimethylcarbamimidic chloride derivative, which was then cyclized with hydrochloric STA9090 to obtain the 4-chloro derivative of target compound (route b) [45]. Moreover, Rashad et al. [46] reported that treatment of compounds I with a mixture of hydrochloric acid/acetic acid (1:3) under reflux, gave pyrazolo[3,4-d]pyrimidine-4-one derivatives (route c). In addition, it was reported that treatment of I with triethyl orthoformate and acetic anhydride under reflux for 24 h. afforded the corresponding pyrazolo[3,4-d]pyrimidine derivatives at good yield (route d) [47,48].
Meanwhile, concentrated sulfuric acid mediated hydrolysis of compound I gave the corresponding 5-amino-4-pyrazolcarboxamide II derivative which underwent oxidative cyclization with various substituted aromatic aldehydes in the presence of equimolar molecular iodine as a mild Lewis acid and oxidant under neutral conditions in boiling acetonitrile to give a new series of pyrazolo[3,4-d]pyrimidine derivatives (route e) [12,49]. On the other hand, a microwave-assisted condensation employing 5-amino-4-pyrazolcarboxamide derivative II in formamide smoothly afforded the corresponding analogous 1H-pyrazolo[3,4-d]pyrimidines (route f) [50]. Pyrimidine derivatives can also used as a starting material for the synthesis of the corresponding pyrazolo[3,4-d]pyrimidines. It is reported that chlorination of barbituic acid using POCl3 introduced the corresponding tri-chloro pyrimidine derivative III[51,52] that can be converted to pyrazolo[3,4-d]pyrimidines via reaction with 1-benzyl-4-hydrazinylpiperidine dihydrochloride in presence of tri-ethyl amine at 78 °C (route g) [52,53]. Soth et al. [54] carried out the reaction of tert-butyl carbazate (route h) with 4-chloro-5- cyano pyrimidine IV in ethyl amine to afford the target derivative. Also, the title compounds pyrazolo[3,4-d]pyrimidine were reported to be prepared by reacting 4-amino-6-aryl-2-phenyl pyrimidine-5-carbonitrile V derivatives and hydrazine hydrate in EtOH under reflux (route i) [55].

Anticancer activity


Photochemical reduction, UV irradiation and ultrasonic fields are widely used physiochemical methods [1] for synthesis of nanoparticles. However, the uses of non-biodegradable compounds as reducing agents in the synthesis of nanoparticles are potentially hazardous to the environment and biological system. Recently, bio-green method using medicinal plant extract has gained the unique importance due to less time requirement in the synthesis of nanoparticles. Furthermore, the use of plant extracts also reduces the cost of microorganism\’s isolation and culture media by enhancing the cost competitive feasibility over nanoparticles synthesis by microorganisms [2]. Nanotechnology is an interdisciplinary approach in biochemical applications and focusing on synthesis of nanoparticles having improved antimicrobial and antioxidant properties against the degenerative diseases and cancer [3]. Currently, research trends in natural and, synthetic antioxidant led the screening and identification of new antioxidants from the plant sources. Synthetic antioxidant is reported to have various properties such as antiallergencity, anticarcinogenicity, antiaging activity and anti-mutagenicity [4]. Antioxidant activity in plant extract is due to the redox potential of phytochemicals [5], which can play an important role in quenching singlet and triplet oxygen, decomposing the peroxides or neutralizing the free radicals. Therefore, it is assumed that higher antioxidant activity of nanoparticles is might be due to the preferential adsorption of the antioxidant material from the extract onto the surface of the nanoparticles. Different parameters like surface area, particle size and surface reactivity determine the toxicity of the nanoparticles in plant extracts. Jacob et al. [6] reported the cytotoxicity of the silver nanoparticles on cancer cells. Due to the microbial resistance, metallic nanoparticles have gained more attention from researchers [7] and among other metals silver are of more importance due to its vital role as antimicrobial agent [8]. Comparative to others, Silver nanoparticles are widely used in commercial products due to their antimicrobial properties.

Banks generate additional cash for the economy by

Banks generate additional cash for the economy by turning deposits into loans. Our study shows that deposits have a positive association with bank liquidity. Bonner et al. (2013) also had similar findings. However, Alger and Alger (1999); Dinger (2009) and Kashyap et al. (2002) found a negative relationship between deposits and bank liquidity. It is noteworthy that these studies were not conducted in an Indian context. This finding implies that with an increase in deposits, banks should also increase their liquidity holding so that a bank run can be avoided in case of high deposit withdrawal.

Uncertainty in global market has made organizations more aware and prone to adopt change on a continuous basis. The complexity of the business operations requires that there must be an effective participation from every level of the organization. In addition to land, labor, and capital, human resource is also an important asset of organization. It plays a key role in the smooth running of the organization and achieving its goals. In this ever-changing and competitive global market, human resource can become the competitive advantage of the organization, if managed effectively. This is evident from literature that leaders have significant rotenone influence on subordinates. The significance of leadership style increases in the services sector, as it has direct impact on economic development. Therefore, the development and growth in services sector ensures the overall growth of national economy, especially in a developing country. The banking sector of Pakistan is growing gradually that has given rise to intense rotenone among banks. Therefore, in order to stay ahead of competitors, banks can manage their human resources effectively by employing different leadership styles in this regard. According to Saari and Judge (2004), use of transformational leadership can boost up employees’ morale and result in job satisfaction. As the banking sector of Pakistan is facing different problems like high turnover, lack of commitment and job stress among employees (Asrar-ul-Haq, 2015), the importance of effective leadership has increased. Therefore, the purpose of this study is to examine the impact of managers’ leadership styles on employee performance.

Problem statement
Asian countries for its performance (Rehman & Raoof, 2010). The competition in the banking sector is increasing day by day. Today, the high pressure on the economy of Pakistan and changing monetary policies has increased the importance and challenges of this sector. In addition, the long work hours, stress, employees’ lack of commitment, job dissatisfaction and high turnover in banks have intensified the need for effective leadership. In order to maintain the growth and achieve higher objectives, the top management in the banks needs to understand the problems and make strategies to satisfy, retain, and motivate employees to exert extra efforts. In other words, it needs such leadership in its branches that can achieve organizational goals efficiently and effectively. Leaders should have the ability to motivate its employees to exert extra efforts to achieve higher goals. Moreover, the existing leadership (managers) should adopt such leadership styles that help to augment subordinates’ satisfaction, their efforts and performance. According to the Full Range of Leadership (FRL) model by Bass and Avolio (1994), the most effective leadership styles are transformational and transactional leadership styles, if adopted collectively, to motivate subordinates, influence their behaviors and attitudes and improve their performance. Although FRL model has been validated in numerous settings to measure the impact of both transformational and transactional leadership styles, yet the researchers are unable to reach some final conclusion that what types of leadership styles should be used in which settings. It might be due to the difference of culture (organizational as well as national). Leadership is not the same thing across cultures (Bhagat & Steers, 2009) and leadership styles may be perceived differently in different settings. Therefore, there is an acute need to study this concept in Pakistani context to examine the universality of Full Range Leadership model. Further, it will help the banking leadership to know that how their subordinate perceive their leadership styles and how it impacts their performance. It can help them change their leadership styles according to achieve higher goals (Fig. 1).