Trypsin-induced proteome alteration during cell subculture in mammalian cells

<p>Abstract</p> <p>Background</p> <p>It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions.</p> <p>Methods</p> <p>In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting.</p> <p>Results</p> <p>36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization.</p> <p>Conclusions</p> <p>In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.</p

DTNBP1, a schizophrenia susceptibility gene, affects kinetics of transmitter release

Schizophrenia is one of the most debilitating neuropsychiatric disorders, affecting 0.5–1.0% of the population worldwide. Its pathology, attributed to defects in synaptic transmission, remains elusive. The dystrobrevin-binding protein 1 (DTNBP1) gene, which encodes a coiled-coil protein, dysbindin, is a major susceptibility gene for schizophrenia. Our previous results have demonstrated that the sandy (sdy) mouse harbors a spontaneously occurring deletion in the DTNBP1 gene and expresses no dysbindin protein (Li, W., Q. Zhang, N. Oiso, E.K. Novak, R. Gautam, E.P. O'Brien, C.L. Tinsley, D.J. Blake, R.A. Spritz, N.G. Copeland, et al. 2003. Nat. Genet. 35:84–89). Here, using amperometry, whole-cell patch clamping, and electron microscopy techniques, we discovered specific defects in neurosecretion and vesicular morphology in neuroendocrine cells and hippocampal synapses at the single vesicle level in sdy mice. These defects include larger vesicle size, slower quantal vesicle release, lower release probability, and smaller total population of the readily releasable vesicle pool. These findings suggest that dysbindin functions to regulate exocytosis and vesicle biogenesis in endocrine cells and neurons. Our work also suggests a possible mechanism in the pathogenesis of schizophrenia at the synaptic level

Scientific Publications in Nephrology and Urology Journals from Chinese Authors in East Asia: A 10-Year Survey of the Literature

BACKGROUND: Diseases of the kidneys and genitourinary tract are common health problems that affect people of all ages and demographic backgrounds. In this study, we compared the quantity and quality of nephrological and urological articles published in international journals from the three major regions of China: the mainland (ML), Hong Kong (HK), and Taiwan (TW). METHODS: Nephrological and urological articles originating from ML, TW, and HK that were published in 61 journals from 1999-2008 were retrieved from the PubMed database. We recorded the numbers of total articles, clinical trials, randomized controlled trials, case reports, impact factors (IF), citations, and articles published in the leading general-medicine journals. We used these data to compare the quantity and quality of publication output from the three regions. RESULTS: The total number of articles increased significantly from 1999 to 2008 in the three regions. The number of articles from ML has exceeded that from HK since 2004, and surpassed that from TW in 2008. Publications from TW had the highest accumulated IF, total citations of articles, and the most articles published in leading general-medicine journals. However, HK publications had the highest average IF. Although ML produced the largest quantity of articles, it exhibited the lowest quality among the three regions. CONCLUSION: The number of nephrological and urological publications originating from the three major regions of China increased significantly from 1999 to 2008. The annual number of publications by ML researchers exceeded those from TW and HK. However, the quality of articles from TW and HK was higher than that from ML

Targeting DNA-PKcs and ATM with miR-101 Sensitizes Tumors to Radiation

Radiotherapy kills tumor-cells by inducing DNA double strand breaks (DSBs). However, the efficient repair of tumors frequently prevents successful treatment. Therefore, identifying new practical sensitizers is an essential step towards successful radiotherapy. In this study, we tested the new hypothesis: identifying the miRNAs to target DNA DSB repair genes could be a new way for sensitizing tumors to ionizing radiation.HERE, WE CHOSE TWO GENES: DNA-PKcs (an essential factor for non-homologous end-joining repair) and ATM (an important checkpoint regulator for promoting homologous recombination repair) as the targets to search their regulating miRNAs. By combining the database search and the bench work, we picked out miR-101. We identified that miR-101 could efficiently target DNA-PKcs and ATM via binding to the 3'- UTR of DNA-PKcs or ATM mRNA. Up-regulating miR-101 efficiently reduced the protein levels of DNA-PKcs and ATM in these tumor cells and most importantly, sensitized the tumor cells to radiation in vitro and in vivo.These data demonstrate for the first time that miRNAs could be used to target DNA repair genes and thus sensitize tumors to radiation. These results provide a new way for improving tumor radiotherapy

Erratum: Semiâ automated pulmonary nodule interval segmentation using the NLST data

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144604/1/mp12905_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144604/2/mp12905.pd

Diabetes and Pre-Diabetes as Determined by Glycated Haemoglobin A1c and Glucose Levels in a Developing Southern Chinese Population

BACKGROUND: The American Diabetes Association and World Health Organization have recently adopted the HbA1c measurement as one method of diagnostic criteria for diabetes. The change in diagnostic criteria has important implications for diabetes treatment and prevention. We therefore investigate diabetes using HbA1c and glucose criteria together, and assess the prevalent trend in a developing southern Chinese population with 85 million residents. METHODS: A stratified multistage random sampling method was applied and a representative sample of 3590 residents 18 years of age or above was obtained in 2010. Each participant received a full medical check-up, including measurement of fasting plasma glucose, 2-hour post-load plasma glucose, and HbA1c. Information on history of diagnosis and treatment of diabetes was collected. The prevalence of diabetes obtained from the present survey was compared with the data from the survey in 2002. RESULTS: The prevalence of diabetes based on both glucose and HbA1c measurements was 21.7% (95% CI: 17.4%-26.1%) in 2010, which suggests that more than 1 in 5 adult residents were suffering from diabetes in this developing population. Only 12.9% (95% CI: 8.3%-17.6%) of diabetic residents were aware of their condition. The prevalence of pre-diabetes was 66.3% (95% CI: 62.7%-69.8%). The prevalence of diabetes and pre-diabetes which met all the three diagnostic thresholds (fast plasma glucose, 2 hour post-load plasma glucose, and HbA1c) was 3.1% and 5.2%, respectively. Diabetes and pre-diabetes as determined by HbA1c measurement had higher vascular risk than those determined by glucose levels. The prevalence of diabetes increased from 2.9% (95% CI: 2.0%-3.7%) in 2002 to 13.8% (95% CI: 10.2%-17.3%) in 2010 based on the same glucose criteria. CONCLUSIONS: Our results show that the diabetes epidemic is accelerating in China. The awareness of diabetes is extremely low. The glucose test and HbA1c measurement should be used together to increase detection of diabetes and pre-diabetes

Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics

Women with endometriosis have higher comorbidities: Analysis of domestic data in Taiwan

AbstractEndometriosis, defined by the presence of viable extrauterine endometrial glands and stroma, can grow or bleed cyclically, and possesses characteristics including a destructive, invasive, and metastatic nature. Since endometriosis may result in pelvic inflammation, adhesion, chronic pain, and infertility, and can progress to biologically malignant tumors, it is a long-term major health issue in women of reproductive age. In this review, we analyze the Taiwan domestic research addressing associations between endometriosis and other diseases. Concerning malignant tumors, we identified four studies on the links between endometriosis and ovarian cancer, one on breast cancer, two on endometrial cancer, one on colorectal cancer, and one on other malignancies, as well as one on associations between endometriosis and irritable bowel syndrome, one on links with migraine headache, three on links with pelvic inflammatory diseases, four on links with infertility, four on links with obesity, four on links with chronic liver disease, four on links with rheumatoid arthritis, four on links with chronic renal disease, five on links with diabetes mellitus, and five on links with cardiovascular diseases (hypertension, hyperlipidemia, etc.). The data available to date support that women with endometriosis might be at risk of some chronic illnesses and certain malignancies, although we consider the evidence for some comorbidities to be of low quality, for example, the association between colon cancer and adenomyosis/endometriosis. We still believe that the risk of comorbidity might be higher in women with endometriosis than that we supposed before. More research is needed to determine whether women with endometriosis are really at risk of these comorbidities