Analysis of congenital disorder of glycosylation-Id in a yeast model system shows diverse site-specific under-glycosylation of glycoproteins

Asparagine-linked glycosylation is a common post translational modification of proteins in eukaryotes. Mutations in the human ALG3 gene cause changed levels and altered glycan structures on mature glycoproteins and are the cause of a severe congenital disorder of glycosylation (CDG-Id). Diverse glycoproteins are also under-glycosylated in Saccharomyces cerevisae alg3 mutants. Here we analyzed site-specific glycosylation occupancy in this yeast model system using peptide-N-glycosidase F to label glycosylation sites with an asparagine-aspartate conversion that creates a new endoproteinase AspN cleavage site, followed by proteolytic digestion, and detection of peptides and glycopeptides by LC-ESI-MS/MS. We used this analytical method to identify and measure site specific glycosylation occupancy in alg3 mutant and wild type yeast strains. We found decreased site specific N-glycosylation occupancy in the alg3 knockout strain preferentially at Asn-Xaa-Ser sequences located in secondary structural elements, features previously associated with poor glycosylation efficiency. Furthermore, we identified 26 previously experimentally unverified glycosylation sites. Our results provide insights into the underlying mechanisms of disease in CDG-Id, and our methodology will be useful in site specific glycosylation analysis in many model systems and clinical applications

Oligosaccharyltransferase Inhibition Induces Senescence in RTK-Driven Tumor Cells

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high throughput screen and lead compound optimization campaign that delivered a cell permeable inhibitor (NGI-1). NGI-1 targets the oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small cell lung cancer cells NGI-1 blocks cell surface localization and signaling of the EGFR glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or FGFR) for survival. In these cell lines OST inhibition causes cell cycle arrest accompanied by induction of p21, autofluorescence, and changes in cell morphology; all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor tyrosine kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells

Longitudinal Tracking of Human Fetal Cells Labeled with Super Paramagnetic Iron Oxide Nanoparticles in the Brain of Mice with Motor Neuron Disease

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival

Pre-Survey Text Messages (SMS) Improve Participation Rate in an Australian Mobile Telephone Survey: An Experimental Study

Mobile telephone numbers are increasingly being included in household surveys samples. As approach letters cannot be sent because many do not have address details, alternatives approaches have been considered. This study assesses the effectiveness of sending a short message service (SMS) to a random sample of mobile telephone numbers to increase response rates. A simple random sample of 9000 Australian mobile telephone numbers: 4500 were randomly assigned to be sent a pre-notification SMS, and the remaining 4500 did not have a SMS sent. Adults aged 18 years and over, and currently in paid employment, were eligible to participate. American Association for Public Opinion Research formulas were used to calculated response cooperation and refusal rates. Response and cooperation rate were higher for the SMS groups (12.4% and 28.6% respectively) than the group with no SMS (7.7% and 16.0%). Refusal rates were lower for the SMS group (27.3%) than the group with no SMS (35.9%). When asked, 85.8% of the pre-notification group indicated they remembered receiving a SMS about the study. Sending a pre-notification SMS is effective in improving participation in population-based surveys. Response rates were increased by 60% and cooperation rates by 79%

Bias of health estimates obtained from chronic disease and risk factor surveillance systems using telephone population surveys in Australia: Results from a representative face-to-face survey in Australia from 2010 to 2013

Background: Emerging communication technologies have had an impact on population-based telephone surveys worldwide. Our objective was to examine the potential biases of health estimates in South Australia, a state of Australia, obtained via current landline telephone survey methodologies and to report on the impact of mobile-only household on household surveys. Methods: Data from an annual multi-stage, systematic, clustered area, face-to-face population survey, Health Omnibus Survey (approximately 3000 interviews annually), included questions about telephone ownership to assess the population that were non-contactable by current telephone sampling methods (2006 to 2013). Univariable analyses (2010 to 2013) and trend analyses were conducted for sociodemographic and health indicator variables in relation to telephone status. Relative coverage biases (RCB) of two hypothetical telephone samples was undertaken by examining the prevalence estimates of health status and health risk behaviours (2010 to 2013): directory-listed numbers, consisting mainly of landline telephone numbers and a small proportion of mobile telephone numbers; and a random digit dialling (RDD) sample of landline telephone numbers which excludes mobile-only households. Results: Telephone (landline and mobile) coverage in South Australia is very high (97 %). Mobile telephone ownership increased slightly (7.4 %), rising from 89.7 % in 2006 to 96.3 % in 2013; mobile-only households increased by 431 % over the eight year period from 5.2 % in 2006 to 27.6 % in 2013. Only half of the households have either a mobile or landline number listed in the telephone directory. There were small differences in the prevalence estimates for current asthma, arthritis, diabetes and obesity between the hypothetical telephone samples and the overall sample. However, prevalence estimate for diabetes was slightly underestimated (RCB value of -0.077) in 2013. Mixed RCB results were found for having a mental health condition for both telephone samples. Current smoking prevalence was lower for both hypothetical telephone samples in absolute differences and RCB values: -0.136 to -0.191 for RDD landline samples and -0.129 to -0.313 for directory-listed samples. Conclusion: These findings suggest landline-based sampling frames used in Australia, when appropriately weighted, produce reliable representative estimates for some health indicators but not for all. Researchers need to be aware of their limitations and potential biased estimates.Emerging communication technologies have had an impact on population-based telephone surveys worldwide. Our objective was to examine the potential biases of health estimates in South Australia, a state of Australia, obtained via current landline telephone survey methodologies and to report on the impact of mobile-only household on household surveys

Effect of 14 days experimental horizontal bed rest on lipid and inflammatory profile.

Objectives: Physical inactivity reduces quality of life and implies higher healthcare costs. Experimental bed-Rest (BR) model allows to analyze metabolic consequences of physical inactivity. The PANGeA group set 14-day horizontal BR to evaluate the effect of bed confinement on body composition, lipid and inflammatory profile. Methods: 23 healthy male subjects were enrolled, divided in 'YOUNG'(n=7;18-25 years) and 'Olders'(n=16;55-65 years). Body composition was assessed by BIA and blood samples were collected at baseline (BDC) and after 14-day BR (BR14). Among the 'OLD', 8 subjects underwent daily specific visual-spatial brain-training during BR (OLDBT). Results: At BDC body mass index was similar in the two groups while OLD showed higher levels of total- (204±39mg/dl vs. 151±15mg/dl;P: 0,002) and LDL-cholesterol (137±34mg/dl vs. 89±12mg/dl;P:0,002). At BR14, a significant decrease of body mass, free-fat Mass (FFM), total body water (TBW), body cell mass (BCM) and muscular mass (MM) was observed. Moreover, OLD showed a total- and LDL-cholesterol reduction (13% and 16%;P: 0,002) while the reduction in the YOUNG was not significant (4.3% and 4%). HDL-cholesterol was significantly reduced only in YOUNG (-17%; P=0,065). Both groups showed a significant increase in serum TNF-a. Althout not significant OLDBT showed an increase in HDL-c (+10%) opposite to the trend observed in OLD without brain traning (OLDNO-BT -10%). Similarly, Total-C/HDL-c ratio was 7% reduced in Old [OLDBT -18%; OLDNO-BT +2%] and 15% increased in YOUNG (P:0, 03). Conclusion: As expected BR was associated with a reduction in body mass, FFM, TBW, BCM and MM. This 'catabolic state' was associated with an increase inflammatory response and, in YOUNG, with a worsened lipid profile. Conversely, OLD showed an apparent lipid profile improvement which seems to be further modulated by brain-training. Further analysis are needed to investigate whether: 1) these changes are associated with decreased cardiovascular risk. 2) Brain-training might have a role in preventing BR associated modifications

N-Glycosylation Is Required for Secretion and Mitosis in C. elegans

N-glycosylation of proteins is an essential process, and N-glucans serve as important beacons in protein folding and ER associated degradation. More importantly, N-glycosylation increases the structural repertoire of proteins because the addition of the N-glucan on proteins will serve as a base for further sugar additions in the Golgi apparatus, and hence complex three-dimensional structures can be build. N-glycosylation is mediated by the ER-resident OST complex, which is essential throughout eukaryotes. Partial knockdown of conserved OST complex members, such as C. elegans RIBO-1, led to an embryonic lethal phenotype. Although the ER morphology was not grossly altered in ribo-1(RNAi) oocytes and embryos, secretion of yolk and of the yolk receptor RME-2 was perturbed in those worms. Perhaps as a consequence of reduced arrival of N-glycosylated proteins at the plasma membrane, cytokinesis occurred less efficiently leading to multinuclear cells. Unexpectedly, we detected a chromosome segregation defect in ribo-1(RNAi) embryos suggesting an essential role of at least one N-glycosylated protein in metaphase-anaphase transition

The redox state regulates the conformation of Rv2466c to activate the antitubercular prodrug TP053

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin fold with a Cys19-Pro20-Trp21-Cys22 motif, and an insertion consisting of a four \u3b1-helical bundle and a short \u3b1-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in \u3b1-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle X-ray scattering showed that the fully reduced form of Rv2466c adopts a 'closed' compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an 'open' conformation, where tertiary structural changes in the \u3b1-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily