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https://www.readbyqxmd.com/read/29212158/rnascope-in-situ-hybridization-confirms-mrna-integrity-in-formalin-fixed-paraffin-embedded-cancer-tissue-samples
#1
Victoria Bingham, Leanne McIlreavey, Christine Greene, Edwina O'Doherty, Rebecca Clarke, Stephanie Craig, Manuel Salto-Tellez, Stephen McQuaid, Claire Lewis, Jacqueline James
Immunohistochemistry remains the overwhelming technique of choice for test biomarker evaluation in both clinical or research settings when using formalin-fixed, paraffin embedded tissue sections. However, validations can be complex with significant issues about specificity, sensitivity and reproducibility. The vast array of commercially available antibodies from many vendors may also lead to non-standard approaches which are difficult to cross-reference. In contrast mRNA detection, by in situ hybridization (ISH) with sequence specific probes, offers a realistic alternative, with less validation steps and more stringent and reproducible assessment criteria...
November 7, 2017: Oncotarget
https://www.readbyqxmd.com/read/29128114/saturated-or-unsaturated-fat-supplemented-maternal-diets-influence-omental-adipose-tissue-proteome-of-suckling-goat-kids
#2
Laura Restelli, Andreia T Marques, Giovanni Savoini, Guido Invernizzi, Michela Carisetti, Cristina Lecchi, Emoke Bendixen, Fabrizio Ceciliani
The aim of the present study was to investigate how maternal diet can influence the adipose tissue of goat kids. Omental adipose tissue proteomes of goat-kids from mothers fed with diet enriched with stearic acid (ST-kids), fish oil (FO-kids) and standard diets (CTRL) were determined by quantitative iTRAQ 2D-LC-MS/MS analysis. Twenty proteins were found to be differentially expressed in suckling kids' omental adipose tissue. Stearic acid induces changes in a higher number of proteins when compared to fish oil...
November 3, 2017: Research in Veterinary Science
https://www.readbyqxmd.com/read/29116140/valid-gene-expression-normalization-by-rt-qpcr-in-studies-on-hpdl-fibroblasts-with-focus-on-orthodontic-tooth-movement-and-periodontitis
#3
Christian Kirschneck, Sarah Batschkus, Peter Proff, Josef Köstler, Gerrit Spanier, Agnes Schröder
Meaningful, reliable and valid mRNA expression analyses by real-time quantitative PCR (RT-qPCR) can only be achieved, if suitable reference genes are chosen for normalization and if appropriate RT-qPCR quality standards are met. Human periodontal ligament (hPDL) fibroblasts play a major mediating role in orthodontic tooth movement and periodontitis. Despite corresponding in-vitro gene expression studies being a focus of interest for many years, no information is available for hPDL fibroblasts on suitable reference genes, which are generally used in RT-qPCR experiments to normalize variability between samples...
November 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29045498/novel-drug-targets-in-cell-wall-biosynthesis-exploited-by-gene-disruption-in-pseudomonas-aeruginosa
#4
Ayssar A Elamin, Susanne Steinicke, Wulf Oehlmann, Yvonne Braun, Hanaa Wanas, Eduard A Shuralev, Carmen Huck, Marko Maringer, Manfred Rohde, Mahavir Singh
For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria...
2017: PloS One
https://www.readbyqxmd.com/read/28802583/cytoskeleton-and-nuclear-lamina-affection-in-recessive-osteogenesis-imperfecta-a-functional-proteomics-perspective
#5
Assunta Gagliardi, Roberta Besio, Chiara Carnemolla, Claudia Landi, Alessandro Armini, Mona Aglan, Ghada Otaify, Samia A Temtamy, Antonella Forlino, Luca Bini, Laura Bianchi
Osteogenesis imperfecta (OI) is a collagen-related disorder associated to dominant, recessive or X-linked transmission, mainly caused by mutations in type I collagen genes or in genes involved in type I collagen metabolism. Among the recessive forms, OI types VII, VIII, and IX are due to mutations in CRTAP, P3H1, and PPIB genes, respectively. They code for the three components of the endoplasmic reticulum complex that catalyzes 3-hydroxylation of type I collagen α1Pro986. Under-hydroxylation of this residue leads to collagen structural abnormalities and results in moderate to lethal OI phenotype, despite the exact molecular mechanisms are still not completely clear...
September 7, 2017: Journal of Proteomics
https://www.readbyqxmd.com/read/28800011/rna-in-situ-hybridization-for-epstein-barr-virus-and-cytomegalovirus-comparison-with-in-situ-hybridization-and-immunohistochemistry
#6
Catherine J Roe, Momin T Siddiqui, Diane Lawson, Cynthia Cohen
The RNAscope utilizes in situ hybridization (RISH) technology to detect single RNA molecules in a variety of tissue samples, including formalin fixed paraffin embedded (FFPE) tissues. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are found in association with neoplastic tissues and inflammatory lesions, and immunohistochemistry (IHC) or other techniques (ISH) are utilized to identify them. We compared the RNAscope RISH to ISH and IHC in the detection of EBV and CMV respectively to determine RNAscope utility in a clinical setting...
August 9, 2017: Applied Immunohistochemistry & Molecular Morphology: AIMM
https://www.readbyqxmd.com/read/28752912/functional-analysis-of-the-cyclophilin-ppib-role-in-bacterial-cell-division
#7
Aggeliki Skagia, Chrysoula Zografou, Anastasia Venieraki, Costas Fasseas, Panagiotis Katinakis, Maria Dimou
Escherichia coli PpiB is a peptidyl-prolyl cis/trans isomerase (PPIase, EC: 5.2.1.8) with chaperone activity. Here, we show that the ΔppiB deletion strain and the PpiB over-expression wild-type strain are both characterized by defects in cell division involving milder or severe cell filamentation, respectively. Using various PpiB mutants, we show that the PPIase activity of PpiB is necessary for the observed cell filamentation, whereas other structural features apart from the active site are also important for this phenotype...
September 2017: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
https://www.readbyqxmd.com/read/28682833/cross-platform-comparison-of-computer-assisted-image-analysis-quantification-of-in-situ-mrna-hybridization-in-investigative-pathology
#8
Timothy R Holzer, Jeffrey C Hanson, Erin M Wray, Jason A Bailey, Kim R Kennedy, Patrick R Finnegan, Aejaz Nasir, Kelly M Credille
Although availability of automated platforms has proliferated, there is no standard practice for computer-assisted generation of scores for mRNA in situ hybridization (ISH) visualized by brightfield microscopic imaging on tissue sections. To address this systematically, an ISH for peptidylprolyl isomerase B (PPIB) (cyclophilin B) mRNA was optimized and applied to a tissue microarray of archival non-small cell lung carcinoma cases, and then automated image analysis for PPIB was refined across 4 commercially available software platforms...
July 4, 2017: Applied Immunohistochemistry & Molecular Morphology: AIMM
https://www.readbyqxmd.com/read/28534145/alterations-in-gene-expression-after-gamma-hydroxybutyric-acid-intake-a-pilot-study
#9
Lena-Maria Mehling, Annika Spottke, Anna Heidbreder, Peter Young, Burkhard Madea, Cornelius Hess, Cornelius Courts
Gamma-hydroxybutyric acid (GHB) acts as an agonist of the GABAB receptor, where GHB induces a depressant effect in the central nervous system. Besides its therapeutic application, GHB is also used as a date rape drug. However, the detection of GHB ingestion proves to be difficult due to its narrow detection window. The aim of this pilot study was to assess differential gene expressions after GHB intake to identify potential biomarkers for the detection of GHB intake. To this aim, alteration in gene expression of ALDH5A1, AKR7A2, EREG, and PEA15 was investigated via quantitative PCR (qPCR)...
May 22, 2017: International Journal of Legal Medicine
https://www.readbyqxmd.com/read/28385890/ziploc-ing-the-structure-2-0-endoplasmic-reticulum-resident-peptidyl-prolyl-isomerases-show-different-activities-toward-hydroxyproline
#10
Yoshihiro Ishikawa, Kazunori Mizuno, Hans Peter Bächinger
Extracellular matrix proteins are biosynthesized in the rough endoplasmic reticulum (rER), and the triple-helical protein collagen is the most abundant extracellular matrix component in the human body. Many enzymes, molecular chaperones, and post-translational modifiers facilitate collagen biosynthesis. Collagen contains a large number of proline residues, so the cis/trans isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl cis/trans isomerases (PPIases) play an important role in the zipper-like triple-helix formation in collagen...
June 2, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28261567/mycobacterium-tuberculosis-peptidyl-prolyl-isomerases-are-immunogenic-alter-cytokine-profile-and-aid-in-intracellular-survival
#11
Saurabh Pandey, Deeksha Tripathi, Mohd Khubaib, Ashutosh Kumar, Javaid A Sheikh, Gaddam Sumanlatha, Nasreen Z Ehtesham, Seyed E Hasnain
Mycobacterium tuberculosis (M. tb) has two peptidyl-prolyl isomerases (Ppiases) PpiA and PpiB, popularly known as cyclophilin A and cyclophilin B. The role of cyclophilins in processes such as signaling, cell surface recognition, chaperoning, and heat shock response has been well-documented. We present evidence that M. tb Ppiases modulate the host immune response. ELISA results revealed significant presence of antibodies to M. tb Ppiases in patient sera as compared to sera from healthy individuals. Treatment of THP-1 cells with increasing concentrations of rPpiA, induced secretion of pro-inflammatory cytokines TNF-α and IL-6...
2017: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/28242392/two-novel-mutations-in-the-ppib-gene-cause-a-rare-pedigree-of-osteogenesis-imperfecta-type-ix
#12
Yu Jiang, Jingxin Pan, Dongwei Guo, Wei Zhang, Jie Xie, Zishui Fang, Chunmiao Guo, Qun Fang, Weiying Jiang, Yibin Guo
BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic skeletal disorder characterized by increased bone fragility and vulnerability to fractures. PPIB is identified as a candidate gene for OI-IX, here we detect two pathogenic mutations in PPIB and analyze the genotype-phenotype correlation in a Chinese family with OI. METHODS: Next-generation sequencing (NGS) was used to screen the whole exome of the parents of proband. Screening of variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations...
February 27, 2017: Clinica Chimica Acta; International Journal of Clinical Chemistry
https://www.readbyqxmd.com/read/28116328/molecular-spectrum-and-differential-diagnosis-in-patients-referred-with-sporadic-or-autosomal-recessive-osteogenesis-imperfecta
#13
Jose A Caparros-Martin, Mona S Aglan, Samia Temtamy, Ghada A Otaify, Maria Valencia, Julián Nevado, Elena Vallespin, Angela Del Pozo, Carmen Prior de Castro, Lucia Calatrava-Ferreras, Pilar Gutierrez, Ana M Bueno, Belen Sagastizabal, Encarna Guillen-Navarro, Maria Ballesta-Martinez, Vanesa Gonzalez, Sarenur Y Basaran, Ruksan Buyukoglan, Bilge Sarikepe, Cecilia Espinoza-Valdez, Francisco Cammarata-Scalisi, Victor Martinez-Glez, Karen E Heath, Pablo Lapunzina, Victor L Ruiz-Perez
BACKGROUND: Osteogenesis imperfecta (OI) is a heterogeneous bone disorder characterized by recurrent fractures. Although most cases of OI have heterozygous mutations in COL1A1 or COL1A2 and show autosomal dominant inheritance, during the last years there has been an explosion in the number of genes responsible for both recessive and dominant forms of this condition. Herein, we have analyzed a cohort of patients with OI, all offspring of unaffected parents, to determine the spectrum of variants accounting for these cases...
January 2017: Molecular Genetics & Genomic Medicine
https://www.readbyqxmd.com/read/28058638/functional-analysis-of-the-two-cyclophilin-isoforms-of-sinorhizobium-meliloti
#14
Eirini-Evangelia Thomloudi, Aggeliki Skagia, Anastasia Venieraki, Panagiotis Katinakis, Maria Dimou
The nitrogen fixing Sinorhizobium meliloti possesses two genes, ppiA and ppiB, encoding two cyclophilin isoforms which belong to the superfamily of peptidyl prolyl cis/trans isomerases (PPIase, EC: 5.2.1.8). Here, we functionally characterize the two proteins and we demonstrate that both recombinant cyclophilins are able to isomerise the Suc-AAPF-pNA synthetic peptide but neither of them displays chaperone function in the citrate synthase thermal aggregation assay. Furthermore, we observe that the expression of both enzymes increases the viability of E...
February 2017: World Journal of Microbiology & Biotechnology
https://www.readbyqxmd.com/read/27868330/structural-and-functional-analysis-of-cyclophilin-ppib-mutants-supports-an-in%C3%A2-vivo-function-not-limited-to-prolyl-isomerization-activity
#15
Aggeliki Skagia, Eleni Vezyri, Markezina Sigala, Areti Kokkinou, Michael Karpusas, Anastasia Venieraki, Panagiotis Katinakis, Maria Dimou
Escherichia coli cyclophilin PpiB is a peptidyl-prolyl cis/trans isomerase (PPIase, EC: 5.2.1.8), involved in the negative modulation of various bacterial processes, such as swimming and swarming motility and biofilm formation ability. In this study, we show that PpiB possesses also a chaperone function as it can prevent the thermal denaturation of citrate synthase even with essentially eliminated PPIase activity. We demonstrate, using active site mutations, that the PPIase activity of PpiB is required in all processes, except for the negative effect on swimming, indicating a possible isomerase-independent function...
January 2017: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
https://www.readbyqxmd.com/read/27795319/an-intracellular-peptidyl-prolyl-cis-trans-isomerase-is-required-for-folding-and-activity-of-the-staphylococcus-aureus-secreted-virulence-factor-nuclease
#16
Richard E Wiemels, Stephanie M Cech, Nikki M Meyer, Caleb A Burke, Andy Weiss, Anastacia R Parks, Lindsey N Shaw, Ronan K Carroll
Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc...
January 1, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/27606620/reconfiguring-the-ar-tif2-protein-protein-interaction-hcs-assay-in-prostate-cancer-cells-and-characterizing-the-hits-from-a-lopac-screen
#17
Ashley T Fancher, Yun Hua, Daniel P Camarco, David A Close, Christopher J Strock, Paul A Johnston
The continued activation of androgen receptor (AR) transcription and elevated expression of AR and transcriptional intermediary factor 2 (TIF2) coactivator observed in prostate cancer (CaP) recurrence and the development of castration-resistant CaP (CRPC) support a screening strategy for small-molecule inhibitors of AR-TIF2 protein-protein interactions (PPIs) to find new drug candidates. Small molecules can elicit tissue selective effects, because the cells of distinct tissues express different levels and cohorts of coregulatory proteins...
October 2016: Assay and Drug Development Technologies
https://www.readbyqxmd.com/read/27448823/evaluation-and-validation-of-reference-gene-stability-during-marek-s-disease-virus-mdv-infection
#18
Sabari Nath Neerukonda, Upendra K Katneni, Sergey Golovan, Mark S Parcells
Quantitative RT-PCR (qRT-PCR) is widely used in the study of relative gene expression in general, and has been used in the field of Marek's disease (MD) research to measure transcriptional responses to infection and/or vaccination. Studies in the past have either employed cellular β-actin (BACT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference genes, although the stability of their expression in the context of Marek's disease virus (MDV) infection has never been investigated. In the present study, we compared the stability of five reference genes (BACT, 28S RNA, 18S RNA, GAPDH, Peptidyl-prolyl-isomerase B [PPIB], a...
October 2016: Journal of Virological Methods
https://www.readbyqxmd.com/read/27306110/cyclophilin-ppib-is-involved-in-motility-and-biofilm-formation-via-its-functional-association-with-certain-proteins
#19
Aggeliki Skagia, Chrysoula Zografou, Eleni Vezyri, Anastasia Venieraki, Panagiotis Katinakis, Maria Dimou
PpiB belongs to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), which catalyze the rate-limiting protein folding step at peptidyl-prolyl bonds and control several biological processes. In this study, we show that PpiB acts as a negative effector of motility and biofilm formation ability of Escherichia coli. We identify multicopy suppressors of each ΔppiB phenotype among putative PpiB prey proteins which upon deletion are often characterized by analogous phenotypes. Many putative preys show similar gene expression in wild-type and ΔppiB genetic backgrounds implying possible post-translational modifications by PpiB...
August 2016: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
https://www.readbyqxmd.com/read/27003577/high-level-soluble-expression-and-one-step-purification-of-ibdv-vp2-protein-in-escherichia-coli
#20
Dawei Jiang, Yunchao Liu, Aiping Wang, Gaiping Zhang, Guoyu Yang, Yumei Chen, Pengchao Ji, Chang Liu, Yapeng Song, Yunfang Su, Guoqiang Wang, Jucai Wang, Baolei Zhao, Ruiguang Deng
OBJECTIVES: To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli. RESULTS: Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni-NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV...
June 2016: Biotechnology Letters
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