CHD5 is down-regulated through promoter hypermethylation in gastric cancer

<p>Abstract</p> <p>Background</p> <p>Nonhistone chromosomal proteins in concert with histones play important roles in the replication and repair of DNA and in the regulation of gene expression. The deregulation of these proteins can contribute to the development of a variety of diseases such as cancer. As a nonhistone chromosomal protein, chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as the product of a novel tumor suppressor gene (TSG), promoting the transcription of p19^{<it>ink4a </it>}and p16^<it>arf</it>. The inactivation of CHD5 was achieved partly through genetic deletion since it is located in 1p36, a region frequently deleted in human tumors. In this study, we aim to study the involvement of CHD5 in gastric cancer, the second most common cancer worldwide.</p> <p>Methods</p> <p>CHD5 expression in a panel of gastric cancer cells were determined by quantitative RT-PCR. The methylation of CHD5 was evaluated by methylation specific PCR and bisulfite genome sequencing. The effect of CHD5 on growth of gastric cancer cells was tested by colony formation assay.</p> <p>Results</p> <p>CHD5 expression was down-regulated in all of gastric cancer cell lines used (100%, 7/7) and significantly restored after pharmacological demethylation. Methylation of CHD5 promoter was detected in all of seven gastric cancer cell lines and in the majority of primary gastric carcinoma tissues examined (73%, 11/15). Finally, ectopic expression of CHD5 in gastric cancer cells led to a significant growth inhibition.</p> <p>Conclusion</p> <p>CHD5 was a TSG epigenetically down-regulated in gastric cancer.</p

Dynamics of Natural Killer Cells Cytotoxicity in Microwell Arrays with Connecting Channels

Natural killer (NK) cells serve an important role in immune system by recognizing and killing the potentially malignant cells without antigen sensitization, and could be promising in cancer therapy. We have designed and fabricated microwell arrays with microchannel connections in polydimethylsiloxane (PDMS) substrates to study the interaction dynamics of NK-92MI cells with MCF7 breast cancer cells using time-lapse imaging by fluorescence microscopy for 15 h. Although cell seeding density was the same, NK cell cytotoxicity was found to be higher in larger microwells, which is manifested as increased target death ratio from 13.7 ± 3.1 to 46.3 ± 3.3% and shorter triggering time of first target lysis from 502 ± 49 to 391 ± 63 min in 150 μm × 150 μm microwells comparing to 50 μm × 50 μm wells in 15 h. Mirochannel connection between adjacent microwells of the same size increased the overall target death ratio by &gt;10%, while connection between microwells of different sizes led to significantly increased target death ratio and delayed first target lysis in smaller microwells. Our findings reveal unique cell interaction dynamics, such as initiation and stimulation, of NK cell cytotoxicity in a confined microenvironment, which is different from population-based study, and the results could lead to a better understanding of the dynamics of NK cell cytotoxicity

Repo-Man recruits PP1γ to chromatin and is essential for cell viability

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1α and -γ are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1γ is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1γ onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1α and -γ in vivo. When overexpressed, Repo-Man can also recruit PP1α to chromatin. Mutating Repo-Man's PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference–induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1γ and is required for the recruitment of PP1 to chromatin

NOPdb: Nucleolar Proteome Database

The Nucleolar Proteome Database (NOPdb) archives data on >700 proteins that were identified by multiple mass spectrometry (MS) analyses from highly purified preparations of human nucleoli, the most prominent nuclear organelle. Each protein entry is annotated with information about its corresponding gene, its domain structures and relevant protein homologues across species, as well as documenting its MS identification history including all the peptides sequenced by tandem MS/MS. Moreover, data showing the quantitative changes in the relative levels of ∼500 nucleolar proteins are compared at different timepoints upon transcriptional inhibition. Correlating changes in protein abundance at multiple timepoints, highlighted by visualization means in the NOPdb, provides clues regarding the potential interactions and relationships between nucleolar proteins and thereby suggests putative functions for factors within the 30% of the proteome which comprises novel/uncharacterized proteins. The NOPdb () is searchable by either gene names, nucleotide or protein sequences, Gene Ontology terms or motifs, or by limiting the range for isoelectric points and/or molecular weights and links to other databases (e.g. LocusLink, OMIM and PubMed)

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments

Laboratório de indicadores de Governança Pública: uma proposta para mensurar a efetividade dos gastos na Segurança Pública Municipal

Anais do 35º Seminário de Extensão Universitária da Região Sul - Área temática: EducaçãoPressões por maior transparência e accountability tem sido o mote de muitas mudanças no setor público. No entanto, parece existir uma dificuldade de colocar tais conceitos em prática na área de segurança pública. Este trabalho apresenta algumas iniciativas do Laboratório de Indicadores de Governança Pública, do CESFI-UDESC, na criação de indicadores de efetividade dos gastos dos municípios do Estado de Santa Catarina, em segurança pública. São apresentados no trabalho o que foi feito até o momento e quais os desafios na mensuração das ações de políticas públicas para esta ár

Investigation on the direct and bystander effects in HeLa cells exposed to very low α-radiation using electrical impedance measurement

The impact of radiation-induced bystander effect (RIBE) is still not well understood in radiotherapy. RIBEs are biological effects expressed by nonirradiated cells near or far from the irradiated cells. Most radiological studies on cancer cells have been based on biochemical characterization. However, biophysical investigation with label-free techniques to analyze and compare the direct irradiation effect and RIBE has lagged. In this work, we employed an electrical cell-indium tin oxide (ITO) substrate impedance system (ECIIS) as a bioimpedance sensor to evaluate the HeLa cells’ response. The bioimpedance of untreated/nonirradiated HeLa (N-HeLa) cells, α-particle (Am-241)-irradiated HeLa (I-HeLa) cells, and bystander HeLa (B-HeLa) cells exposed to media from I-HeLa cells was monitored with a sampling interval of 8 s over a period of 24 h. Also, we imaged the cells at times where impedance changes were observed. Different radiation doses (0.5 cGy, 1.2 cGy, and 1.7 cGy) were used to investigate I-HeLa and B-HeLa cells’ radiation-dose-dependence. By analyzing the changes in absolute impedance and cell size/number with time, compared to N-HeLa cells, B-HeLa cells mimicked the I-HeLa cells’ damage and modification of proliferation rate. Contrary to the irradiated cells, the bystander cells’ damage rate and proliferation rate enhancements have an inverse radiation-dose-response. Also, we report multiple RIBEs in HeLa cells in a single measurement and provide crucial insights into the RIBE mechanism without any labeling procedure. Unambiguously, our results have shown that the time-dependent control of RIBE is important during α-radiation-based radiotherapy of HeLa cells

BING, a novel antimicrobial peptide isolated from Japanese medaka plasma, targets bacterial envelope stress response by suppressing cpxR expression

Antimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target

Differential Actions of Chlorhexidine on the Cell Wall of Bacillus subtilis and Escherichia coli

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of Gram-positive and Gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli