Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

Analysis of differential gene expression by display of 3' end restriction fragments of cDNAs.

We have developed an approach to study changes in gene expression by selective PCR amplification and display of 3' end restriction fragments of double-stranded cDNAs. This method produces highly consistent and reproducible patterns, can detect almost all mRNAs in a sample, and can resolve hidden differences such as bands that differ in their sequence but comigrate on a gel. Bands corresponding to known cDNAs move to predictable positions on the gel, making this a powerful approach to correlate gel patterns with cDNA data bases. Applying this method, we have examined differences in gene expression patterns during T-cell activation. Of a total of 700 bands that were evaluated in this study, as many as 3-4% represented mRNAs that are upregulated, while approximately 2% were down-regulated within 4 hr of activation of Jurkat T cells. These and other results suggest that this approach is suitable for the systematic, expeditious, and nearly exhaustive elucidation of subtle changes in the patterns of gene expression in cells with altered physiologic states.

TCR{alpha} Genes Direct MHC Restriction in the Potent Human T Cell Response to a Class I-Bound Viral Epitope

The underlying generic properties of {alpha}β TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR β-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ β-chain was associated with distinct TCR {alpha}-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR {alpha}-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR {alpha}-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.

A rough cluster analysis of shopping orientation data

This paper describes the application of a new technique, rough clustering, to the problem of market segmentation. Rough clustering produces different solutions to k-means analysis because of the possibility of multiple cluster membership of objects. Traditional clustering methods generate extensional descriptions of groups, that show which objects are members of each cluster. Clustering techniques based on rough sets theory generate intensional descriptions, which outline the main characteristics of each cluster. In this study, a rough cluster analysis was conducted on a sample of 437 responses from a larger study of the relationship between shopping orientation (the general predisposition of consumers toward the act of shopping) and intention to purchase products via the Internet. The cluster analysis was based on five measures of shopping orientation: enjoyment, personalization, convenience, loyalty, and price. The rough clusters obtained provide interpretations of different shopping orientations present in the data without the restriction of attempting to fit each object into only one segment. Such descriptions can be an aid to marketers attempting to identify potential segments of consumers.

Reconstruction of an unsteady flow from incomplete boundary data:Part I. Theory

Purpose – To propose and investigate a stable numerical procedure for the reconstruction of the velocity of a viscous incompressible fluid flow in linear hydrodynamics from knowledge of the velocity and fluid stress force given on a part of the boundary of a bounded domain. Design/methodology/approach – Earlier works have involved the similar problem but for stationary case (time-independent fluid flow). Extending these ideas a procedure is proposed and investigated also for the time-dependent case. Findings – The paper finds a novel variation method for the Cauchy problem. It proves convergence and also proposes a new boundary element method. Research limitations/implications – The fluid flow domain is limited to annular domains; this restriction can be removed undertaking analyses in appropriate weighted spaces to incorporate singularities that can occur on general bounded domains. Future work involves numerical investigations and also to consider Oseen type flow. A challenging problem is to consider non-linear Navier-Stokes equation. Practical implications – Fluid flow problems where data are known only on a part of the boundary occur in a range of engineering situations such as colloidal suspension and swimming of microorganisms. For example, the solution domain can be the region between to spheres where only the outer sphere is accessible for measurements. Originality/value – A novel variational method for the Cauchy problem is proposed which preserves the unsteady Stokes operator, convergence is proved and using recent for the fundamental solution for unsteady Stokes system, a new boundary element method for this system is also proposed.

Effect of maternal protein restriction during pregnancy and postweaning high-fat feeding on diet-induced thermogenesis in adult mouse offspring

Purpose: Prenatal undernutrition followed by postweaning feeding of a high-fat diet results in obesity in the adult offspring. In this study, we investigated whether diet-induced thermogenesis is altered as a result of such nutritional mismatch. Methods: Female MF-1 mice were fed a normal protein (NP, 18 % casein) or a protein-restricted (PR, 9 % casein) diet throughout pregnancy and lactation. After weaning, male offspring of both groups were fed either a high-fat diet (HF; 45 % kcal fat) or standard chow (C, 7 % kcal fat) to generate the NP/C, NP/HF, PR/C and PR/HF adult offspring groups (n = 7-11 per group). Results: PR/C and NP/C offspring have similar body weights at 30 weeks of age. Postweaning HF feeding resulted in significantly heavier NP/HF offspring (P <0.01), but not in PR/HF offspring, compared with their chow-fed counterparts. However, the PR/HF offspring exhibited greater adiposity (P <0.01) v the NP/HF group. The NP/HF offspring had increased energy expenditure and increased mRNA expression of uncoupling protein-1 and β-3 adrenergic receptor in the interscapular brown adipose tissue (iBAT) compared with the NP/C mice (both at P <0.01). No such differences in energy expenditure and iBAT gene expression were observed between the PR/HF and PR/C offspring. Conclusions: These data suggest that a mismatch between maternal diet during pregnancy and lactation, and the postweaning diet of the offspring, can attenuate diet-induced thermogenesis in the iBAT, resulting in the development of obesity in adulthood. © 2014 Springer-Verlag Berlin Heidelberg.

Effet des charges sur les chaussées en période de restriction des charges - volet terrain

Dans un contexte climatique rigoureux comme celui du Québec, l’interaction entre la charge et le climat a une grande influence sur la performance des structures de chaussées flexibles (Doré et Zubeck, 2009). Pendant le dégel printanier, avec la fonte de la glace, la chaussée s’affaiblit et cet affaiblissement la rend vulnérable à la sollicitation par le trafic lourd ce qui accélère divers phénomènes de dégradation, notamment l’endommagement par fatigue et l’orniérage structural (Farcette, 2010). Afin de minimiser les effets des charges lourdes sur une chaussée affaiblie lors du printemps, les administrations routières choisissent souvent de limiter les charges par essieu ou par véhicule lors du dégel. L’objectif de ce projet est de développer un outil d’aide pour la gestion des restrictions de charge en période de dégel en fonction des données recueillies par les stations de météo routière. Deux sections expérimentales composées des mêmes matériaux mais avec des épaisseurs d’enrobés bitumineux différentes situées au Site Expérimental Routier de l’Université Laval (SERUL) ont été utilisées pour ce projet. Pour bien interpréter le comportement des structures, des jauges de déformations verticales et horizontales, des jauges de contraintes, des jauges de teneur en eau et des thermistances ont été installées dans chaque couche. Pour solliciter mécaniquement la chaussée, un déflectomètre à masse tombante (FWD) a été utilisé. Les résultats obtenus ont permis de de bien comprendre les mécanismes d’affaiblissement de la chaussée durant la période de dégel. Ils ont aussi montré que l’application d’une période de restriction de charge pendant la période de dégel permettait d’avoir un gain sur la durée de vie de la chaussée, cette période de restriction est donc justifiée et efficace. Néanmoins, pour une meilleure gestion du réseau routier, de nouveaux critères pour mieux déterminer la période de restriction de charges sont proposés.

Haemodynamics of aerobic and resistance blood flow restriction exercise in young and older adults

PURPOSE: Light-load blood flow restriction exercise (BFRE) may provide a novel training method to limit the effects of age-related muscle atrophy in older adults. Therefore, the purpose of this study was to compare the haemodynamic response to resistance and aerobic BFRE between young adults (YA; n = 11; 22 ± 1 years) and older adults (OA; n = 13; 69 ± 1 years). METHOD: On two occasions, participants completed BFRE or control exercise (CON). One occasion was leg press (LP; 20 % 1-RM) and the other was treadmill walking (TM; 4 km h(-1)). Haemodynamic responses (HR, [Formula: see text], SV and BP) were recorded during baseline and exercise. RESULT: At baseline, YA and OA were different for some haemodynamic parameters (e.g. BP, SV). The relative responses to BFRE were similar between YA and OA. Blood pressures increased more with BFRE, and also for LP over TM. [Formula: see text] increased similarly for BFRE and CON (in both LP and TM), but with elevated HR and reduced SV (TM only). CONCLUSION: While BFR conferred slightly greater haemodynamic stress than CON, this was lower for walking than leg-press exercise. Given similar response magnitudes between YA and OA, these data support aerobic exercise being a more appropriate BFRE for prescription in older adults that may contribute to limiting the effects of age-related muscle atrophy.

PPM-HDA: privacy-preserving and multifunctional health data aggregation with fault tolerance

Wireless body area networks (WBANs), as a promising health-care system, can provide tremendous benefits for timely and continuous patient care and remote health monitoring. Owing to the restriction of communication, computation and power in WBANs, cloud-assisted WBANs, which offer more reliable, intelligent, and timely health-care services for mobile users and patients, are receiving increasing attention. However, how to aggregate the health data multifunctionally and efficiently is still an open issue to the cloud server (CS). In this paper, we propose a privacy-preserving and multifunctional health data aggregation (PPM-HDA) mechanism with fault tolerance for cloud-assisted WBANs. With PPM-HDA, the CS can compute multiple statistical functions of users' health data in a privacy-preserving way to offer various services. In particular, we first propose a multifunctional health data additive aggregation scheme (MHDA⁺) to support additive aggregate functions, such as average and variance. Then, we put forward MHDA^⊕ as an extension of MHDA⁺ to support nonadditive aggregations, such as min/max, median, percentile, and histogram. The PPM-HDA can resist differential attacks, which most existing data aggregation schemes suffer from. The security analysis shows that the PPM-HDA can protect users' privacy against many threats. Performance evaluations illustrate that the computational overhead of MHDA⁺ is significantly reduced with the assistance of CSs. Our MHDA^⊕ scheme is more efficient than previously reported min/max aggregation schemes in terms of communication overhead when the applications require large plaintext space and highly accurate data.

High body mass index is not a barrier to physical activity: Analysis of international rugby players' anthropometric data

Recent data indicate that levels of overweight and obesity are increasing at an alarming rate throughout the world. At a population level (and commonly to assess individual health risk), the prevalence of overweight and obesity is calculated using cut-offs of the Body Mass Index (BMI) derived from height and weight. Similarly, the BMI is also used to classify individuals and to provide a notional indication of potential health risk. It is likely that epidemiologic surveys that are reliant on BMI as a measure of adiposity will overestimate the number of individuals in the overweight (and slightly obese) categories. This tendency to misclassify individuals may be more pronounced in athletic populations or groups in which the proportion of more active individuals is higher. This differential is most pronounced in sports where it is advantageous to have a high BMI (but not necessarily high fatness). To illustrate this point we calculated the BMIs of international professional rugby players from the four teams involved in the semi-finals of the 2003 Rugby Union World Cup. According to the World Health Organisation (WHO) cut-offs for BMI, approximately 65% of the players were classified as overweight and approximately 25% as obese. These findings demonstrate that a high BMI is commonplace (and a potentially desirable attribute for sport performance) in professional rugby players. An unanswered question is what proportion of the wider population, classified as overweight (or obese) according to the BMI, is misclassified according to both fatness and health risk? It is evident that being overweight should not be an obstacle to a physically active lifestyle. Similarly, a reliance on BMI alone may misclassify a number of individuals who might otherwise have been automatically considered fat and/or unfit.