Androgen receptor phosphorylation at serine 515 by Cdk1 predicts biochemical relapse in prostate cancer patients

<br>Background:Prostate cancer cell growth is dependent upon androgen receptor (AR) activation, which is regulated by specific kinases. The aim of the current study is to establish if AR phosphorylation by Cdk1 or ERK1/2 is of prognostic significance.</br> <br>Methods: Scansite 2.0 was utilised to predict which AR sites are phosphorylated by Cdk1 and ERK1/2. Immunohistochemistry for these sites was then performed on 90 hormone-naive prostate cancer specimens. The interaction between Cdk1/ERK1/2 and AR phosphorylation was investigated in vitro using LNCaP cells.</br><br>Results:Phosphorylation of AR at serine 515 (pAR(S515)) and PSA at diagnosis were independently associated with decreased time to biochemical relapse. Cdk1 and pCdk1(161), but not ERK1/2, correlated with pAR(S515). High expression of pAR(S515) in patients with a PSA at diagnosis of ≤20 ng ml(-1) was associated with shorter time to biochemical relapse (P=0.019). This translated into a reduction in disease-specific survival (10-year survival, 38.1% vs 100%, P<0.001). In vitro studies demonstrated that treatment with Roscovitine (a Cdk inhibitor) caused a reduction in pCdk1(161) expression, pAR(S515)expression and cellular proliferation.</br> <br>Conclusion: In prostate cancer patients with PSA at diagnosis of ≤20 ng ml(-1), phosphorylation of AR at serine 515 by Cdk1 may be an independent prognostic marker.</br&gt

Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation

ABSTRACT

Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells

Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by >30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing >50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy

Search for the neutral Higgs bosons of the minimal supersymmetric standard model in pp collisions at root s=7 TeV with the ATLAS detector

A search for neutral Higgs bosons of the Minimal Supersymmetric Standard Model (MSSM) is reported. The analysis is based on a sample of proton-proton collisions at a centre-of-mass energy of 7TeV recorded with the ATLAS detector at the Large Hadron Collider. The data were recorded in 2011 and correspond to an integrated luminosity of 4.7 fb-1 to 4.8 fb-1. Higgs boson decays into oppositely-charged muon or τ lepton pairs are considered for final states requiring either the presence or absence of b-jets. No statistically significant excess over the expected background is observed and exclusion limits at the 95% confidence level are derived. The exclusion limits are for the production cross-section of a generic neutral Higgs boson, φ, as a function of the Higgs boson mass and for h/A/H production in the MSSM as a function of the parameters mA and tan β in the mhmax scenario for mA in the range of 90GeV to 500 GeV. Copyright CERN

Social Networking Technology, Social Network Composition, and Reductions in Substance Use Among Homeless Adolescents

Peer-based prevention programs for homeless youth are complicated by the potential for reinforcing high-risk behaviors among participants. The goal of this study is to understand how homeless youth could be linked to positive peers in prevention programming by understanding where in social and physical space positive peers for homeless youth are located, how these ties are associated with substance use, and the role of social networking technologies (e.g., internet and cell phones) in this process. Personal social network data were collected from 136 homeless adolescents in Los Angeles, CA. Respondents reported on composition of their social networks with respect to: home-based peers and parents (accessed via social networking technology; e.g., the internet, cell phone, texting), homeless peers and agency staff (accessed face-to-face) and whether or not network members were substance-using or non-substance-using. Associations between respondent’s lifetime cocaine, heroin, and methamphetamine use and recent (previous 30 days) alcohol and marijuana use were assessed by the number of non-substance-using versus substance-using ties in multivariate linear regression models. 43% of adolescents reported a non-substance-using home-based tie. More of these ties were associated with less recent alcohol use. 62% of adolescents reported a substance-using homeless tie. More of these ties were associated with more recent marijuana use as well as more lifetime heroin and methamphetamine use. For homeless youth, who are physically disconnected from positive peers, social networking technologies can be used to facilitate the sorts of positive social ties that effective peer-based prevention programs require

Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity

A before-after implementation trial of smoking cessation guidelines in hospitalized veterans

Abstract Background Although most hospitalized smokers receive some form of cessation counseling during hospitalization, few receive outpatient cessation counseling and/or pharmacotherapy following discharge, which are key factors associated with long-term cessation. US Department of Veterans Affairs (VA) hospitals are challenged to find resources to implement and maintain the kind of high intensity cessation programs that have been shown to be effective in research studies. Few studies have applied the Chronic Care Model (CCM) to improve inpatient smoking cessation. Specific objectives The primary objective of this protocol is to determine the effect of a nurse-initiated intervention, which couples low-intensity inpatient counseling with sustained proactive telephone counseling, on smoking abstinence in hospitalized patients. Key secondary aims are to determine the impact of the intervention on staff nurses' attitudes toward providing smoking cessation counseling; to identify barriers and facilitators to implementation of smoking cessation guidelines in VA hospitals; and to determine the short-term cost-effectiveness of implementing the intervention. Design Pre-post study design in four VA hospitals Participants Hospitalized patients, aged 18 or older, who smoke at least one cigarette per day. Intervention The intervention will include: nurse training in delivery of bedside cessation counseling, electronic medical record tools (to streamline nursing assessment and documentation, to facilitate prescription of pharmacotherapy), computerized referral of motivated inpatients for proactive telephone counseling, and use of internal nursing facilitators to provide coaching to staff nurses practicing in non-critical care inpatient units. Outcomes The primary endpoint is seven-day point prevalence abstinence at six months following hospital admission and prolonged abstinence after a one-month grace period. To compare abstinence rates during the intervention and baseline periods, we will use random effects logistic regression models, which take the clustered nature of the data within nurses and hospitals into account. We will assess attitudes of staff nurses toward cessation counseling by questionnaire and will identify barriers and facilitators to implementation by using clinician focus groups. To determine the short-term incremental cost per quitter from the perspective of the VA health care system, we will calculate cessation-related costs incurred during the initial hospitalization and six-month follow-up period. Trial number NCT00816036http://deepblue.lib.umich.edu/bitstream/2027.42/112349/1/13012_2009_Article_190.pd

A Combination of Nutriments Improves Mitochondrial Biogenesis and Function in Skeletal Muscle of Type 2 Diabetic Goto–Kakizaki Rats

BACKGROUND: Recent evidence indicates that insulin resistance in skeletal muscle may be related to reduce mitochondrial number and oxidation capacity. However, it is not known whether increasing mitochondrial number and function improves insulin resistance. In the present study, we investigated the effects of a combination of nutrients on insulin resistance and mitochondrial biogenesis/function in skeletal muscle of type 2 diabetic Goto-Kakizaki rats. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that defect of glucose and lipid metabolism is associated with low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle of the diabetic Goto-Kakizaki rats. The treatment of combination of R-alpha-lipoic acid, acetyl-L-carnitine, nicotinamide, and biotin effectively improved glucose tolerance, decreased the basal insulin secretion and the level of circulating free fatty acid (FFA), and prevented the reduction of mitochondrial biogenesis in skeletal muscle. The nutrients treatment also significantly increased mRNA levels of genes involved in lipid metabolism, including peroxisome proliferator-activated receptor-alpha (Ppar alpha), peroxisome proliferator-activated receptor-delta (Ppar delta), and carnitine palmitoyl transferase-1 (Mcpt-1) and activity of mitochondrial complex I and II in skeletal muscle. All of these effects of mitochondrial nutrients are comparable to that of the antidiabetic drug, pioglitazone. In addition, the treatment with nutrients, unlike pioglitazone, did not cause body weight gain. CONCLUSIONS/SIGNIFICANCE: These data suggest that a combination of mitochondrial targeting nutrients may improve skeletal mitochondrial dysfunction and exert hypoglycemic effects, without causing weight gain

PABPN1 gene therapy for oculopharyngeal muscular dystrophy

International audienceOculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment

Molecular Biomarker Analyses Using Circulating Tumor Cells

Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.Using spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.Our data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs