Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays

To identify candidate genes relevant for prostate tumour prognosis and progression, we performed an exhaustive gene search in seven previously described genomic-profiling studies of 161 prostate tumours, and four expression profiling studies of 61 tumours. From the resulting list of candidate genes, six were selected for protein-expression analysis based on the availability of antibodies applicable to paraffinised tissue: fatty acid synthase (FASN), MYC, β-adrenergic receptor kinase 1 (BARK1, GRK2) the catalytic subunits of protein phosphatases PP1α (PPP1CA) and PP2A (PPP2CB) and metastasis suppressor NM23-H1. These candidates were analysed by immunohistochemistry (IHC) on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias (BPH) from 175 patients. In univariate analysis, expression of PP1α (P=0.001) was found to strongly correlate with Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score (P<0.001 each) in univariate as well as in multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterised by a complete loss of BARK1 protein (P=0.023). In conclusion, our study revealed novel molecular markers of potential diagnostic and therapeutic relevance for prostate carcinoma

PolyQ Repeat Expansions in ATXN2 Associated with ALS Are CAA Interrupted Repeats

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27–33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1–3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2

Identification of the CRE-1 Cellulolytic Regulon in Neurospora crassa

Background: In filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and b-galactosidase. Methodology/Principal Findings: Here we show that a strain carrying a deletion of cre-1 has increased cellulolytic activity and increased expression of cellulolytic genes during growth on crystalline cellulose (Avicel). Constitutive expression of cre-1 complements the phenotype of a N. crassa Dcre-1 strain grown on Avicel, and also results in stronger repression of cellulolytic protein secretion and enzyme activity. We determined the CRE-1 regulon by investigating the secretome and transcriptome of a Dcre-1 strain as compared to wild type when grown on Avicel versus minimal medium. Chromatin immunoprecipitation-PCR of putative target genes showed that CRE-1 binds to only some adjacent 59-SYGGRG-39 motifs, consistent with previous findings in other fungi, and suggests that unidentified additional regulatory factors affect CRE-1 binding to promoter regions. Characterization of 30 mutants containing deletions in genes whose expression level increased in a Dcre-1 strain under cellulolytic conditions identified novel genes that affect cellulase activity and protein secretion

Electrochemically synthesized polymers in molecular imprinting for chemical sensing

This critical review describes a class of polymers prepared by electrochemical polymerization that employs the concept of molecular imprinting for chemical sensing. The principal focus is on both conducting and nonconducting polymers prepared by electropolymerization of electroactive functional monomers, such as pristine and derivatized pyrrole, aminophenylboronic acid, thiophene, porphyrin, aniline, phenylenediamine, phenol, and thiophenol. A critical evaluation of the literature on electrosynthesized molecularly imprinted polymers (MIPs) applied as recognition elements of chemical sensors is presented. The aim of this review is to highlight recent achievements in analytical applications of these MIPs, including present strategies of determination of different analytes as well as identification and solutions for problems encountered

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected

Beneficial effects of parenteral GLP-1 delivery by cell therapy in insulin-deficient streptozotocin diabetic mice.

Parenteral delivery of long-Acting glucagon-like peptide-1 (GLP-1) mimetics has received much attention as a therapeutic option for diabetes. However, cell therapy-based GLP-1 treatments may provide a more physiological regulation of blood glucose. The present study assessed the effects of chronic GLP-1 delivery by cell therapy, using the GLP-1-secreting GLUTag cell line, in normoglycemic and streptozotocin-induced diabetic mice. GLUTag cell aggregates were transplanted into the subscapular region of mice. Over 30 days, cellular transplantation gave rise to encapsulated and well-vascularized growths, which contained immunoreactive GLP-1. Cell implantation was well tolerated and had no appreciable metabolic effects in normal mice. However, transplantation significantly (P<0.001) countered excessive food and fluid intake in diabetic mice and maintained normal body weight. Circulating glucose (P<0.01) and glucagon (P<0.05) were significantly reduced and plasma insulin and GLP-1 dramatically increased. This was associated with significantly (P<0.01) improved glucose tolerance in diabetic mice. Histological examination of the pancreata of these mice revealed elevations (P<0.001) in islet and β-cell area, with reduced (P<0.001) -cell area. Increased β-cell mass reflected the enhanced proliferation relative to apoptosis. These studies emphasize the potential of chronic GLP-1 delivery by cell therapy as a potential therapeutic option for diabetes

Extracellular matrix formation enhances the ability of streptococcus pneumoniae to cause invasive disease

Extent: 17p.During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100- fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.Claudia Trappetti, Abiodun D. Ogunniyi, Marco R. Oggioni and James C. Pato