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Li Zhang, Zhihong Yang, Jocelyn Trottier, Olivier Barbier, Li Wang
: Bile acids (BAs) play critical physiological functions in cholesterol homeostasis and deregulation of BA metabolism causes cholestatic liver injury. Maternally expressed gene 3 (MEG3) was recently shown as a potential tumor suppressor, however its basic hepatic function remains elusive. Using RNA pull-down with biotin-labeled sense or anti-sense MEG3RNA followed by mass spectrometry, we identified RNA binding protein polypyrimidine tract-binding protein 1 (PTBP1) as a MEG3 interaction protein and validated their interaction by RNA immunoprecipitation (RIP)...
October 22, 2016: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
Wanhui You, Stéphane Pien, Ueli Grossniklaus
Epigenetic control of plant development via histone modifications is involved in different processes ranging from embryonic development, vegetative development, flowering time control, floral organ development, to pollen tube growth. The identification of an increasing number of epigenetically regulated processes was greatly advanced by methods allowing the survey of genome-wide histone modifications and chromatin-protein interactions. However, genome-wide approaches are too broad to access in detail a large number of histone modifications taking place at a single locus...
2017: Methods in Molecular Biology
Arvind Y M Sundaram, Timothy Hughes, Shea Biondi, Nathalie Bolduc, Sarah K Bowman, Andrew Camilli, Yap C Chew, Catherine Couture, Andrew Farmer, John P Jerome, David W Lazinski, Andrew McUsic, Xu Peng, Kamran Shazand, Feng Xu, Robert Lyle, Gregor D Gilfillan
BACKGROUND: ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo "library preparation" to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures...
October 21, 2016: BMC Genomics
Yuxin Shu, Yan Lu, Xiaojuan Pang, Wei Zheng, Yahong Huang, Jiahong Li, Jianguo Ji, Can Zhang, Pingping Shen
Peroxisome proliferator-activating receptor γ (PPARγ), a transcription factor, is involved in many important biological processes, including cell terminal differentiation, survival and apoptosis. However, the role of PPARγ, which regulates tumour promoter and oncogene expression, is not well understood in hepatocellular carcinoma (HCC). In the present study, based on evidence from clinical samples that phosphorylation of PPARγ at Ser84 is up-regulated in human liver tumours, we confirmed that phosphorylation of PPARγ was also significantly increased in an HCC mouse model and was increased by Mitogen-activated protein kinase (MEK)/ Extracellular-signal-regulated kinases (ERK) kinase...
October 19, 2016: Oncotarget
Heba M Ismail, Athanasios Didangelos, Tonia L Vincent, Jeremy Saklatvala
Objectives Mechanical injury to cartilage predisposes to osteoarthritis (OA). Wounding the articular cartilage surface causes rapid activation of MAP kinases and NFκB mimicking the response to inflammatory cytokines. Here, we identify the upstream signalling mechanisms involved. Methods Cartilage was injured by dissecting it from the articular surface of porcine metacarpophalangeal joints (MCP) or by avulsing murine proximal femoral epiphyses. Protein phosphorylation was assayed by Western blotting of cartilage lysates...
October 21, 2016: Arthritis & Rheumatology
Michael Chalick, Oded Jacobi, Edward Pichinuk, Christian Garbar, Armand Bensussan, Alan Meeker, Ravit Ziv, Tania Zehavi, Nechama I Smorodinsky, John Hilkens, Franz-Georg Hanisch, Daniel B Rubinstein, Daniel H Wreschner
Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM...
2016: PloS One
Atsushi Matsuda, Haruhiko Asakawa, Tokuko Haraguchi, Yasushi Hiraoka
The fission yeast Schizosaccharomyces pombe is a useful experimental system for studying the organization of chromosomes within the cell nucleus. S. pombe has a small genome that is organized into three chromosomes. The small size of the genome and the small number of chromosomes are advantageous for cytological and genome-wide studies of chromosomes; however, the small size of the nucleus impedes microscopic observations owing to limits in spatial resolution during imaging. Recent advances in microscopy, such as super-resolution microscopy, have greatly expanded the use of S...
October 21, 2016: Yeast
Daniil A Maksimov, Petr P Laktionov, Stepan N Belyakin
Analysis of gene expression regulation typically requires identification of genomic sites bound by regulatory proteins. For this purpose, chromatin immunoprecipitation (ChIP) and Dam identification (DamID) methods can be applied to cell lines, whole organisms, or enriched cell populations. In this work, we present modifications to the experimental DamID protocol, as well as a custom data processing algorithm, that allow to confidently identify genomic sites enriched with the proteins of interest. This algorithm is implemented in Perl and is also available as executable files, thereby making DamID analysis relatively straightforward...
October 21, 2016: Chromosome Research
Oihane Irazoki, Jesús Aranda, Timo Zimmermann, Susana Campoy, Jordi Barbé
In addition to its role in DNA damage repair and recombination, the RecA protein, through its interaction with CheW, is involved in swarming motility, a form of flagella-dependent movement across surfaces. In order to better understand how SOS response modulates swarming, in this work the location of RecA and CheW proteins within the swarming cells has been studied by using super-resolution microscopy. Further, and after in silico docking studies, the specific RecA and CheW regions associated with the RecA-CheW interaction have also been confirmed by site-directed mutagenesis and immunoprecipitation techniques...
2016: Frontiers in Microbiology
Qian-Qian Luo, Zhong-Ming Qian, Yu-Fu Zhou, Meng-Wan Zhang, Dang Wang, Li Zhu, Ya Ke
The inconsistent of responses of IRP1 and HIF-1 alpha to hypoxia and the similar tendencies in the changes of IRP1 and pCREB contents led us to hypothesize that pCREB might be involved in the regulation of IRP1 under hypoxia. Here, we investigated the role of pCREB in IRP1 expression in HepG2 cells under hypoxia using quantitative PCR, western blot, immunofluorescence, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). We demonstrated that 1) Hypoxia increased pCREB levels inside of the nucleus; 2) Putative CREs were found in the IRP1 gene; 3) Nuclear extracts of HepG2 cells treated with hypoxia could bind to CRE1 and CRE3, and 100-fold competitor of putative CREs could abolish the binding activity to varying degrees; 4) pCREB was found in the CRE1 and CRE3 DNA-protein complexes of EMSA; 5) CRE1 and CRE3 binding activity of IRP1 depended on CREB activation but not on HIF-1; 6) Increased IRP1 expression under hypoxia could be prevented by LY294002; 7) ChIP assays demonstrated that pCREB binds to IRP1 promoter; and 8) HIF-1 and/or HIF-2 siRNA had no effect on the expression of pCREB and IRP1 proteins in cells treated with hypoxia for 8 hours...
2016: International Journal of Biological Sciences
Chengxing Zhu, Jiong Yu, Qiaoling Pan, Jinfeng Yang, Guangshu Hao, Yingjie Wang, Lanjuan Li, Hongcui Cao
Human placenta-derived mesenchymal stem cells (hPMSCs) reside in a physiologically low-oxygen microenvironment. Hypoxia influences a variety of stem cell cellular activities, frequently involving hypoxia-inducible factor-2 alpha (HIF-2α). This research showed that hPMSCs cultured in hypoxic conditions (5% O2) exhibited a more naïve morphology and had a higher proliferative capability and higher HIF-2α expression than hPMSCs cultured in normoxic conditions (21% O2). Similar to the hypoxic cultures, hPMSCs over-expressing HIF-2α showed higher proliferative potential and higher expression of CCND1 (CyclinD1), MYC (c-Myc), POU5F1 (Oct4) and the components of the MAPK/ERK pathway...
October 21, 2016: Scientific Reports
Akira Katsube, Hisamitsu Hayashi, Hiroyuki Kusuhara
OBJECTIVE: ATP-binding cassette transporter A1 (ABCA1) exerts an atheroprotective action through the biogenesis of high-density lipoprotein in hepatocytes and prevents the formation of foam cells from macrophages. Controlling ABCA1 is a rational approach to improving atherosclerotic cardiovascular disease. Although much is known about the regulatory mechanism of ABCA1 synthesis, the molecular mechanism underpinning its degradation remains to be clearly described. APPROACH AND RESULTS: ABCA1 possesses potential sites of phosphorylation by serine/threonine-protein kinase Pim-1 (Pim-1)...
October 20, 2016: Arteriosclerosis, Thrombosis, and Vascular Biology
Aitor Garzia, Cindy Meyer, Pavel Morozov, Marcin Sajek, Thomas Tuschl
Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP, key steps in the PAR-CLIP procedure must be optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data analysis pipeline to three distinct RBPs targeting different annotation categories of cellular RNAs...
October 17, 2016: Methods: a Companion to Methods in Enzymology
Jing Chen, Sudhirkumar Shinde, Prabal Subedi, Celina Wierzbicka, Börje Sellergren, Stefan Helling, Katrin Marcus
Selective enrichment techniques are essential for mapping of protein posttranslational modifications (PTMs). Phosphorylation is one of the PTMs which continues to be associated with significant analytical challenges. Particularly problematic are tyrosine-phosphorylated peptides (pY-peptides) resulting from tryptic digestion which commonly escape current chemo- or immuno- affinity enrichments and hence remain undetected. We here report on significant improvements in this regard using pY selective molecularly imprinted polymers (pY-MIPs)...
October 11, 2016: Journal of Chromatography. A
Uday B Maachani, Uma Shankavaram, Tamalee Kramp, Philip J Tofilon, Kevin Camphausen, Anita T Tandle
Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo...
October 14, 2016: Oncotarget
Weijie Li, Shicai Dong, Wei Wei, Guangxiu Wang, Anling Zhang, Peiyu Pu, Zhifan Jia
The transcriptional coactivator with PDZ-binding motif (TAZ) is one of the important downstream effectors of Hippo pathway. In this study, the potential implication of TAZ in gliomagenesis was explored. TAZ expression was identified to be upregulated in glioma specimens and positively correlated with tumor grade. Meanwhile, its expression in nucleus was increased more significantly with the ascending order of tumor grade. Knocking down TAZ inhibited glioma cell proliferation, invasion and promoted apoptosis...
October 13, 2016: Oncotarget
Siroon Bekkering, Inge van den Munckhof, Tim Nielen, Evert Lamfers, Charles Dinarello, Joost Rutten, Jacqueline de Graaf, Leo A B Joosten, Mihai G Netea, Marc E R Gomes, Niels P Riksen
BACKGROUND AND AIMS: We have recently reported that monocytes can undergo functional and transcriptional reprogramming towards a long-term pro-inflammatory phenotype after brief in vitro exposure to atherogenic stimuli such as oxidized LDL. This process is termed 'trained immunity', and is mediated by epigenetic remodeling and a metabolic switch towards increased aerobic glycolysis. We hypothesize that trained immunity contributes to atherogenesis. Therefore, we investigated the inflammatory phenotype and epigenetic remodeling of monocytes from patients with and without established atherosclerosis...
October 12, 2016: Atherosclerosis
Jawed Iqbal, Mairaj Ahmed Ansari, Binod Kumar, Dipanjan Dutta, Arunava Roy, Leela Chikoti, Gina Pisano, Sujoy Dutta, Shahrooz Vahedi, Mohanan Valiya Veettil, Bala Chandran
IFI16 (gamma-interferon-inducible protein 16), a predominantly nuclear protein involved in transcriptional regulation, also functions as an innate immune response DNA sensor and induces the IL-1β and antiviral type-1 interferon-β (IFN-β) cytokines. We have shown that IFI16, in association with BRCA1, functions as a sequence independent nuclear sensor of episomal dsDNA genomes of KSHV, EBV and HSV-1. Recognition of these herpesvirus genomes resulted in IFI16 acetylation, BRCA1-IFI16-ASC-procaspase-1 inflammasome formation, cytoplasmic translocation, and IL-1β generation...
October 2016: PLoS Pathogens
S Y Zhu, P L Wang, C S Liao, Y Q Yang, C Y Yuan, S Wang, W L Dissanayaka, B C Heng, C F Zhang
BACKGROUND AND OBJECTIVE: The goal of periodontal therapy is to regenerate/reconstruct the damaged supporting tissues of diseased teeth and to facilitate recovery of their physiological functions. Combination of stem cell transplantation and gene therapy offers a viable method for accelerating periodontal repair and regeneration. In this study, the role of the ephrinB2/EphB4 signaling pathway in regulating osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and crosstalk between PDLSCs and pre-osteoblasts within co-culture was investigated through ephrinB2 transgenic expression in PDLSCs...
October 20, 2016: Journal of Periodontal Research
Demin Cai, Mengjie Yuan, Haoyu Liu, Shifeng Pan, Wenqiang Ma, Jian Hong, Ruqian Zhao
Betaine serves as an animal and human nutrient which has been heavily investigated in glucose and lipid metabolic regulation, yet the underlying mechanisms are still elusive. In this study, feeding sows with betaine-supplemented diets during pregnancy and lactation increased cholesterol content and low-density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SR-BI) gene expression, but decreasing bile acids content and cholesterol-7a-hydroxylase (CYP7a1) expression in the liver of weaning piglets...
October 18, 2016: Nutrients
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