MESENCHYMAL STEM CELL-MEDIATED ENDOCHONDRAL OSSIFICATION UTILISING MICROPELLETS AND BRIEF CHONDROGENIC PRIMING

With limited autologous and donor bone graft availability, there is an increasing need for alternative graft substitutes. We have previously shown that chondrogenically priming mesenchymal stem cell (MSC) pellets for 28 d in vitro will reproducibly result in endochondral bone formation after in vivo implantation. However, pellet priming time for clinical applications is quite extensive. A micropellet (\xce\xbcpellet)-fibrin construct was developed and coupled, with a shorter priming period, determined by an in vitro time course experiment. In vitro data showed expression of chondrogenic genes and matrix production after 7 d of chondrogenic priming, indicating that briefer priming could possibly be used to induce bone formation in vivo. 7 and 28 d primed pellet, pellet-fibrin and \xce\xbcpellet-fibrin constructs were cultured for in vitro analysis and implanted subcutaneously for 8 weeks into nude mice. \xce\xbcpellet-fibrin constructs, cultured in vitro for 7 or 28 d, produced comparable bone to standard pellets in vivo. MSC-mediated bone formation was achieved following only 7 d of in vitro priming. Bone formation in vivo appeared to be influenced by overall matrix production pre-implantation. Given this short priming time and the injectable nature of the \xce\xbcpellet-fibrin constructs, this approach might be further developed as an injectable bone substitute, leading to a minimally-invasive treatment option, which would allow for tailored filling of bone defects

Collagen type X is essential for successful mesenchymal stem cell-mediated cartilage formation and subsequent endochondral ossification

In tissue engineering, endochondral ossification (EO) is often replicated by chondrogenically differentiating mesenchymal stromal cells (MSCs) in vitro and achieving bone formation through in vivo implantation. The resulting marrow-containing bone constructs are promising as a treatment for bone defects. However, limited bone formation capacity has prevented them from reaching their full potential. This is further complicated since it is not fully understood how this bone formation is achieved. Acellular grafts derived from chondrogenically differentiated MSCs can initiate bone formation; however, which component within these decellularised matrices contribute to bone formation has yet to be determined. Collagen type X (COLX), a hypertrophy-associated collagen found within these constructs, is involved in matrix organisation, calcium binding and matrix vesicle compartmentalisation. However, the importance of COLX during tissue-engineered chondrogenesis and subsequent bone formation is unknown. The present study investigated the importance of COLX by shRNA-mediated gene silencing in primary MSCs. A significant knock-down of COLX disrupted the production of extracellular matrix key components and the secretion profile of chondrogenically differentiated MSCs. Following in vivo implantation, disrupted bone formation in knock-down constructs was observed. The importance of COLX was confirmed during both chondrogenic differentiation and subsequent EO in this tissue engineered setting

Intensidade e duração dos esforços físicos em aulas de Educação Física

OBJETIVO: Avaliar a intensidade e a duração dos esforços físicos em aulas de Educação Física no ensino fundamental e médio. MÉTODOS: Estudo transversal de base escolar por meio de observação de 218 aulas de Educação Física, incluindo um total de 272 estudantes (avaliados três vezes cada um). O estudo foi realizado em Pelotas, RS, de agosto a setembro de 2009. Para a intensidade dos esforços, foram utilizados acelerômetros e adotados os pontos de corte (em counts por minuto): atividades sedentárias (0 a 100), leves (101 a 2.000), moderadas (2.001 a 4.999), vigorosas (5.000 a 7.999) e muito vigorosas (&gt; 8.000). RESULTADOS: O tempo médio de duração das aulas foi de 35,6 minutos (dp 6,0). A proporção média de tempo das aulas em atividades físicas de intensidade moderada a vigorosa foi de 32,7% (dp 25,2). Os meninos (44,1%) envolveram-se significativamente mais em atividades físicas moderadas a vigorosas do que as meninas (21,0%; p < 0,01). Estudantes que se envolvem em atividade física fora das aulas tiveram maior participação em atividades físicas moderadas a vigorosas nas aulas de Educação Física. CONCLUSÕES: Além de o tempo da aula de Educação Física ser reduzido, os estudantes praticam atividades físicas de intensidade moderada a vigorosa um terço da aula, com pouca contribuição significativa para o nível de atividade física dos estudantes.OBJECTIVE: To evaluate the intensity and duration of physical efforts in Physical Education classes in primary and secondary school. METHODS: School-based cross-sectional study carried out by means of the observation of 218 Physical Education classes, including a total of 272 students (each one of the students was evaluated three times). The study was carried out in the city of Pelotas (Southern Brazil), between August and December 2009. In order to evaluate the intensity of the efforts, accelerometers were used and the following cut-off points were adopted (in counts per minute): sedentary activities (0-100), light activities (101-2,000), moderate (2,001-4,999), vigorous (5,000-7,999), and very vigorous activities (&gt;8000). RESULTS: The mean duration of the classes was 35.6 minutes (SD 6.0). The mean proportion of time spent in moderate to vigorous physical activity was 32.7% (SD 25.2). Boys (44.1%) were involved significantly more in moderate to vigorous physical activity as compared to girls (21.0%; pOBJETIVO: Evaluar la intensidad y la duración de los esfuerzos físicos en clases de Educación Física en la enseñanza básica y media. MÉTODOS: Estudio transversal de base escolar por medio de observación de 218 clases de Educación Física, incluyendo un total de 272 estudiantes (evaluados tres veces cada uno). El estudio fue realizado en Pelotas, Sur de Brasil, de agosto a septiembre del año 2009. Para la intensidad de los esfuerzos, se utilizaron acelerómetros y se adoptaron los puntos de corte (en counts por minuto): actividades sedentarias (0 a 100), leves (101 a 2.000), moderadas (2.001 a 4.999), vigorosas (5.000 a 7.999) y muy vigorosas (&gt; 8.000). RESULTADOS: El tiempo promedio de duración de las clases fue de 35,6 minutos (de 6,0). La proporción promedio de tiempo de las clases en actividades físicas de intensidad moderada a vigorosa fue de 32,7% (desviación estándar 25,2). Los niños varones (44,1%) se involucraron significativamente en actividades físicas moderadas a vigorosas con relación a las niñas (21,0%;

HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available

BACKGROUND: HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties. CONCLUSION/SIGNIFICANCE: This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a "compare-exchange" sorting algorithm on an available peptide pool. Such a "third party"-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems

Altered regulation of Prox1-gene-expression in liver tumors

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Plasma triglyceride concentrations are rapidly reduced following individual bouts of endurance exercise in women

It is known that chronic endurance training leads to improvements in the lipoprotein profile, but less is known about changes that occur during postexercise recovery acutely. We analyzed triglyceride (TG), cholesterol classes and apolipoproteins in samples collected before, during and after individual moderate- and hard-intensity exercise sessions in men and women that were isoenergetic between intensities. Young healthy men (n = 9) and young healthy women (n = 9) were studied under three different conditions with diet unchanged between trials: (1) before, during and 3 h after 90 min of exercise at 45% VO2peak (E45); (2) before, during and 3 h after 60 min of exercise at 65% VO2peak (E65), and (3) in a time-matched sedentary control trial (C). At baseline, high-density lipoprotein cholesterol (HDL-C) was higher in women than men (P < 0.05). In men and in women, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), HDL-C, apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), and LDL peak particle size were unaltered by exercise either during exertion or after 3 h of recovery. In women, but not in men, average plasma TG was significantly reduced below C at 3 h postexercise by approximately 15% in E45 and 25% in E65 (P < 0.05) with no significant difference between exercise intensities. In summary, plasma TG concentration rapidly declines following exercise in women, but not in men. These results demonstrate an important mechanism by which each individual exercise session may incrementally reduce the risk for cardiovascular disease (CVD) in women

Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships