In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: Definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif {psi}KXE/D, where {psi} represents a large hydrophobic amino acid, and X represnts any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process

Look, the World is Watching How We Treat Migrants! The Making of the Anti-Trafficking Legislation during the Ma Administration

Employing the spiral model, this research analyses how anti-human trafficking legislation was promulgated during the Ma Ying-jeou (Ma Yingjiu) presidency. This research found that the gov- ernment of Taiwan was just as accountable for the violation of mi- grants’ human rights as the exploitive placement agencies and abusive employers. This research argues that, given its reliance on the United States for political and security support, Taiwan has made great ef- forts to improve its human rights records and meet US standards for protecting human rights. The reform was a result of multilevel inputs, including US pressure and collaboration between transnational and domestic advocacy groups. A major contribution of this research is to challenge the belief that human rights protection is intrinsic to dem- ocracy. In the same light, this research also cautions against Taiwan’s subscription to US norms since the reform was achieved at the cost of stereotyping trafficking victimhood, legitimising state surveillance, and further marginalising sex workers

Sumoylation inhibits α-synuclein aggregation and toxicity

Sumoylation of α-synuclein decreases its rate of aggregation and its deleterious effects in vitro and in vivo

Phosphorylation of human Argonaute proteins affects small RNA binding

Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5′-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5′ phosphate of the small RNA

Household secondhand smoke exposure of elementary schoolchildren in Southern Taiwan and factors associated with their confidence in avoiding exposure: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Exposure to household Secondhand Smoke (SHS) poses a major health threat to children after an indoor smoking ban was imposed in Taiwan. This study aimed to assess the household SHS exposure in elementary school children in southern Taiwan and the factors associated with their avoidance of SHS exposure before and after the implementation of Taiwan's new Tobacco Hazards Prevention Act in 2009.</p> <p>Methods</p> <p>In this cross-sectional school-based study, data on household SHS exposure, avoidance of SHS and related variables was obtained from the 2008 and 2009 Control of School-aged Children Smoking Study Survey. A random sample of 52 elementary schools was included. A total of 4450 3-6 graders (aged 8-13) completed the questionnaire. Regression models analyzed factors of children's self-confidence to avoid household SHS exposure.</p> <p>Results</p> <p>Over 50% of children were found to have lived with a family member who smoked in front of them after the new law enacted, and 35% of them were exposed to household SHS more than 4 days a week. Having a positive attitude toward smoking (β = -0.05 to -0.06) and high household SHS exposure (β = -0.34 to -0.47) were significantly associated with a lower avoidance of SHS exposure. Comparing to girls, boys had lower scores in their knowledge of tobacco hazards; and this factor was significantly related to their SHS avoidance (β = 0.13-0.14).</p> <p>Conclusions</p> <p>The intervention program should enhance school children do actively avoid exposure to SHS in home settings, and more importantly, provide tobacco hazard knowledge to male students to avoid exposure to household SHS for themselves. The results also provide further evidence that Tobacco Hazards Prevention Act should perhaps be extended to the family environment in order to protect children from the hazards of household SHS exposure.</p

The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85

The Slp65 adaptor molecule is important for B-cell receptor signalling. Here, the interactomes of SLP65 in resting and activated B cells are resolved by mass spectrometry. A number of SLP65 interacting partners, including CIN85, continuously associate with SLP65 in a stimulation-independent manner. CIN85 recruits SLP65 to the membrane and it is required for SLP65 phosphorylation

Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer

<p>Abstract</p> <p>Background</p> <p>A cross-talk between different receptor tyrosine kinases (RTKs) plays an important role in the pathogenesis of human cancers.</p> <p>Methods</p> <p>Both NIH-Met5 and T24-Met3 cell lines harboring an inducible human c-Met gene were established. C-Met-related RTKs were screened by RTK microarray analysis. The cross-talk of RTKs was demonstrated by Western blotting and confirmed by small interfering RNA (siRNA) silencing, followed by elucidation of the underlying mechanism. The impact of this cross-talk on biological function was demonstrated by Trans-well migration assay. Finally, the potential clinical importance was examined in a cohort of 65 cases of locally advanced and metastatic bladder cancer patients.</p> <p>Results</p> <p>A positive association of Axl or platelet-derived growth factor receptor-alpha (PDGFR-α) with c-Met expression was demonstrated at translational level, and confirmed by specific siRNA knock-down. The transactivation of c-Met on Axl or PDGFR-α <it>in vitro </it>was through a <it>ras</it>- and Src-independent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. In human bladder cancer, co-expression of these RTKs was associated with poor patient survival (<it>p </it>< 0.05), and overexpression of c-Met/Axl/PDGFR-α or c-Met alone showed the most significant correlation with poor survival (<it>p </it>< 0.01).</p> <p>Conclusions</p> <p>In addition to c-Met, the cross-talk with Axl and/or PDGFR-α also contributes to the progression of human bladder cancer. Evaluation of Axl and PDGFR-α expression status may identify a subset of c-Met-positive bladder cancer patients who may require co-targeting therapy.</p

Proteomics Characterization of Cytoplasmic and Lipid-Associated Membrane Proteins of Human Pathogen Mycoplasma fermentans M64

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data