Markers of bone metabolism during 14 days of bed rest in young and older men

OBJECTIVE: We aimed at comparing markers of bone metabolism during unloading in young and older men, and to assess countermeasure effectiveness. METHODS: 16 older (60\ub12 years) and 8 younger men (23\ub13 years) underwent bed rest (BR) for 14 days. A subgroup of the Older performed cognitive training during BR and supplemented protein and potassium bicarbonate afterwards. Biochemical markers of bone and calcium/phosphate metabolism were assessed. RESULTS: At baseline urinary NTX and CTX were greater in younger than in older subjects (P0.17). P1NP was greater in young than in older subjects (P<0.001) and decreased during BR in the Young (P<0.001). Sclerostin increased during BR across groups (P=0.016). No systematic effects of the countermeasure were observed. CONCLUSION: In men, older age did not affect control of bone metabolism, but bone turnover was reduced. During BR formation markers were reduced only in younger men whereas resorption markers increased to a comparable extent. Thus, we assume that older men are not at an elevated, and possibly even at a reduced risk to lose bone when immobilize

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

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

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

<p>Abstract</p> <p>Background</p> <p>Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient.</p> <p>Results</p> <p>The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing - thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing - thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior.</p> <p>Conclusions</p> <p>The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing - thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.</p

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

Bone metabolic responses to bed rest in young and older men

It is well known that immobilization, through reduction of musculoskeletal forces, leads to bone loss. This is well established for clinical conditions, such as stroke, spinal cord injury as well as for spaceflight and disuse models, such as experimental bed rest. Age is another condition that predisposes to bone loss, which is intimately linked to fractures in the elderly population. Bone turn-over is known to be reduced in the elderly, and animal studies suggest ablunted response of bont to mechanical stimuli at old age, and that old age might 'shield' against immobilization-reduced bone loss. Whether this applies also to humans is currently unknown. The aim of the present study therefore was to compare bone metabolic responses to bed rest in a group of young (23.4 (SD2.9) Years, n=7) and older men (59.1 (SD 2.5) Years, n=8). It was hypothesized that bone metabolic responeses would be generally blunted in older men

Markers of bone matebolism during 14 days of bed rest in young and older men

Objective: We aimed at comparing markers of bone metabolism during unloading in young and older men, and toassess countermeasure effectiveness. Methods: 16 older (60±2 years) and 8 younger men (23±3 years) underwentbed rest (BR) for 14 days. A subgroup of the Older performed cognitive training during BR and supplemented proteinand potassium bicarbonate afterwards. Biochemical markers of bone and calcium/phosphate metabolism were assessed.Results: At baseline urinary NTX and CTX were greater in younger than in older subjects (P0.17). P1NP was greater in young than in older subjects (P<0.001) and decreasedduring BR in the Young (P<0.001). Sclerostin increased during BR across groups (P=0.016). No systematic effects ofthe countermeasure were observed. Conclusion: In men, older age did not affect control of bone metabolism, but boneturnover was reduced. During BR formation markers were reduced only in younger men whereas resorption markersincreased to a comparable extent. Thus, we assume that older men are not at an elevated, and possibly even at a reducedrisk to lose bone when immobilized.Fil: Buehlmeier, J.. Institute of Aerospace Medicine; Alemania. Universitat Bonn; AlemaniaFil: Frings Meuthen, P.. Institute of Aerospace Medicine; AlemaniaFil: Mohorko, N.. University of Primorska; EsloveniaFil: Lau, P.. Institute of Aerospace Medicine; AlemaniaFil: Mazzucco, S.. University Trieste; ItaliaFil: Ferretti, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Biolo, G.. University Trieste; ItaliaFil: Pisot, R.. University of Primorska; EsloveniaFil: Simunic, B.. University of Primorska; EsloveniaFil: Rittweger, J.. Institute of Aerospace Medicine; Alemani

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