Rapid LC-MS/MS quantification of the major benzodiazepines and their metabolites on dried blood spots using a simple and cost-effective sample pretreatment.

BACKGROUND: Dried blood spots (DBS) sampling has gained popularity in the bioanalytical community as an alternative to conventional plasma sampling, as it provides numerous benefits in terms of sample collection and logistics. The aim of this work was to show that these advantages can be coupled with a simple and cost-effective sample pretreatment, with subsequent rapid LC-MS/MS analysis for quantitation of 15 benzodiazepines, six metabolites and three Z-drugs. For this purpose, a simplified offline procedure was developed that consisted of letting a 5-µl DBS infuse directly into 100 µl of MeOH, in a conventional LC vial. RESULTS: The parameters related to the DBS pretreatment, such as extraction time or internal standard addition, were investigated and optimized, demonstrating that passive infusion in a regular LC vial was sufficient to quantitatively extract the analytes of interest. The method was validated according to international criteria in the therapeutic concentration ranges of the selected compounds. CONCLUSION: The presented strategy proved to be efficient for the rapid analysis of the selected drugs. Indeed, the offline sample preparation was reduced to a minimum, using a small amount of organic solvent and consumables, without affecting the accuracy of the method. Thus, this approach enables simple and rapid DBS analysis, even when using a non-DBS-dedicated autosampler, while lowering the costs and environmental impact.

The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase.

Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have only been successful with a molar excess of chaperones over their polypeptide substrates. Here we describe a stable, misfolded luciferase species that can be efficiently renatured by substoichiometric amounts of bacterial Hsp70-Hsp40-NEF. The reactivation rates increased with substrate concentration and followed saturation kinetics, thus allowing the determination of apparent V(max)' and K(m)' values for a chaperone-mediated renaturation reaction for the first time. Under the in vitro conditions used, one Hsp70 molecule consumed five ATPs to effectively unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to the native state, a process with an ATP cost a thousand times lower than expected for protein degradation and resynthesis.

Influence of the world's most challenging mountain ultra-marathon on energy cost and running mechanics

PURPOSE: To examine the effects of the world's most challenging mountain ultra-marathon (Tor des Géants(®) 2012) on the energy cost of three types of locomotion (cycling, level and uphill running) and running kinematics. METHODS: Before (pre-) and immediately after (post-) the competition, a group of ten male experienced ultra-marathon runners performed in random order three submaximal 4-min exercise trials: cycling at a power of 1.5 W kg(-1) body mass; level running at 9 km h(-1) and uphill running at 6 km h(-1) at an inclination of +15 % on a motorized treadmill. Two video cameras recorded running mechanics at different sampling rates. RESULTS: Between pre- and post-, the uphill-running energy cost decreased by 13.8 % (P = 0.004); no change was noted in the energy cost of level running or cycling (NS). There was an increase in contact time (+10.3 %, P = 0.019) and duty factor (+8.1 %, P = 0.001) and a decrease in swing time (-6.4 %, P = 0.008) in the uphill-running condition. CONCLUSION: After this extreme mountain ultra-marathon, the subjects modified only their uphill-running patterns for a more economical step mechanics.

Inter-limb coordination and energy cost in swimming.

OBJECTIVES: This study investigated the relationship between inter-arm coordination and the energy cost of locomotion in front crawl and breaststroke and explored swimmers' flexibility in adapting their motor organization away from their preferred movement pattern. DESIGN: Nine front-crawlers performed three 300-m in front crawl and 8 breaststrokers performed three 200-m in breaststroke at constant submaximal intensity and with 5-min rests. Each trial was performed randomly in a different coordination pattern: freely chosen, 'maximal glide' and 'minimal glide'. Two underwater cameras videotaped frontal and side views to analyze speed, stroke rate, stroke length and inter-limb coordination. METHODS: In front crawl, inter-arm coordination was quantified by the index of coordination (IdC) and the leg beat kicks were counted. In breaststroke, four time gaps quantified the arm to leg coordination (i.e., time between leg and arm propulsions; time between beginning, 90° flexion and end of arm and leg recoveries). The energy cost of locomotion was calculated from gas exchanges and blood lactate concentration. RESULTS: In both front crawl and breaststroke, the freely chosen coordination corresponded to glide pattern and showed the lowest energy cost (12.8 and 17.1Jkg(-1)m(-1), respectively). Both front-crawlers and breaststrokers were able to reach 'maximal glide' condition (respectively, +35% and +28%) but not 'minimal glide' condition for front crawl. CONCLUSIONS: The freely chosen pattern appeared more economic because more trained. When coordination was constrained, the swimmers had higher coordination flexibility in breaststroke than in front crawl, suggesting that breaststroke coordination was easier to regulate by changing glide time.

A high-mobility, low-cost phenotype defines human effector-memory CD8+ T cells.

T cells move randomly ("random-walk"), a characteristic thought to be integral to their function. Using migration assays and time-lapse microscopy, we found that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrate more efficiently in transwell assays, and that these same cells are characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu, but not when examined on endothelial cells. To assess the energy efficiency of cells crawling at a high frequency, we measured mRNA expression of genes key to mitochondrial energy metabolism (peroxisome proliferator-activated receptor gamma coactivator 1beta [PGC-1beta], estrogen-related receptor alpha [ERRalpha], cytochrome C, ATP synthase, and the uncoupling proteins [UCPs] UCP-2 and -3), quantified ATP contents, and performed calorimetric analyses. Together these assays indicated a high energy efficiency of the high crawling frequency CD8+ T-cell population, and identified differentially regulated heat production among nonlymphoid versus lymphoid homing CD8+ T cells.

Coordination pattern adaptability: energy cost of degenerate behaviors.

This study investigated behavioral adaptability, which could be defined as a blend between stability and flexibility of the limbs movement and their inter-limb coordination, when individuals received informational constraints. Seven expert breaststroke swimmers performed three 200-m in breaststroke at constant submaximal intensity. Each trial was performed randomly in a different coordination pattern: 'freely-chosen', 'maximal glide' and 'minimal glide'. Two underwater and four aerial cameras enabled 3D movement analysis in order to assess elbow and knee angles, elbow-knee pair coordination, intra-cyclic velocity variations of the center of mass, stroke rate and stroke length and inter-limb coordination. The energy cost of locomotion was calculated from gas exchanges and blood lactate concentration. The results showed significantly higher glide, intra-cyclic velocity variations and energy cost under 'maximal glide' compared to 'freely-chosen' instructional conditions, as well as higher reorganization of limb movement and inter-limb coordination (p<0.05). In the 'minimal glide' condition, the swimmers did not show significantly shorter glide and lower energy cost, but they exhibited significantly lower deceleration of the center of mass, as well as modified limb movement and inter-limb coordination (p<0.05). These results highlight that a variety of structural adaptations can functionally satisfy the task-goal.

Cost-effectiveness of a new combined immunosuppressive and anti-infectious regimen in kidney transplantation.

OBJECTIVES: The aims of this study were to assess the 1-year cost-effectiveness of a new combined immunosuppressive and anti-infectious regimen in kidney transplantation to prevent both rejection and infectious complications. METHODS: Patients (pts) transplanted from January 2000 to March 2003 (Group A) and treated with a conventional protocol were compared with pts submitted to a combined regimen including universal cytomegalovirus (CMV) prophylaxis between April 2003 and July 2005 (Group B). Costs were computed from the hospital accounting system for hospital stays, and official tariffs for outpatient visits. Patients with incomplete costs data were excluded from analysis. RESULTS: Fifty-three patients were analyzed in Group A, and 60 in Group B. Baseline characteristics including CMV serostatus were not significantly different between the two groups. Over 12 months after transplantation, acute rejections decreased from 41.5 percent in Group A to 6.7 percent in Group B (p &lt; .001), and CMV infections from 47 percent to 15 percent (p &lt; .001). Overall, readmissions decreased from 68 percent to 55 percent (p = .160), and average hospital days from 28 +/- 19 to 20 +/- 11 days (p &lt; .007). The average number of outpatient visits decreased from 49 +/- 10 to 39 +/- 8 (p &lt; .001). Average 1-year immunosuppressive and CMV prophylaxis costs (per patient) increased from CHF20,402 +/- 7,273 to 27,375 +/- 6,063 (p &lt; .001), graft rejection costs decreased from CHF4,595 +/- 10,182 to 650 +/- 3,167 (p = .005), CMV treatment costs from CHF2,270 +/- 6,161 to 101 +/- 326 (p = .008), and outpatient visits costs from CHF8,466 +/- 1'721 to 6,749 +/- 1,159 (p &lt; .001). Altogether, 1-year treatment costs decreased from CHF39'957 +/- 16,573 to 36,204 +/- 6,901 (p = .115). CONCLUSIONS: The new combined regimen administered in Group B was significantly more effective, and its additional costs were more than offset by savings associated with complications avoidance.

Availability, Cost or Culture ? : Obstacles to Childcare Services for Low-Income Families

Recent research has highlighted the existence of a social bias in the extent to which children have access to childcare. In general, children living in higher income households are more likely to be cared for in childcare centres. While the existence of a social bias in access to childcare services has been clearly demonstrated, we currently lack a clear explanation as to why this is the case. This paper uses a unique dataset based on survey data collected specifically to study patterns of childcare use in the Swiss canton of Vaud (N = 875). The paper exploits the variation in the way childcare is organised within the canton. Childcare is a municipal policy, as a result of which there are twenty-nine different systems in operation. Fees are progressive everywhere, but variation is substantial. Availability is also very different. This peculiar institutional setup provides an ideal situation to examine the determinants of childcare use by different income groups. Our findings suggest that differences in the fees charged to low-income households, as well as the degree of progressivity of the fee structure, are significant predictors of use, while availability seems to matter less.