Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.     

Thermal comfort modelling of body temperature and psychological variations of a human exercising in an outdoor environment

Human thermal comfort assessments pertaining to exercise while in outdoor environments can improve urban and recreational planning. The current study applied a simple four-segment skin temperature approach to the COMFA (COMfort FormulA) outdoor energy balance model. Comparative results of measured mean skin temperature ( [`(T)]\nolimits<sub>Msk</sub> \mathop{{\bar{T}}}\nolimits_{{Msk}} ) with predicted [`(T)]\nolimits_sk \mathop{{\bar{T}}}\nolimits_{{sk}} indicate that the model accurately predicted [`(T)]\nolimits<sub>sk</sub> \mathop{{\bar{T}}}\nolimits_{{sk}} , showing significantly strong agreement (r = 0.859, P < 0.01) during outdoor exercise (cycling and running). The combined 5-min mean variation of the [`(T)]\nolimits_sk \mathop{{\bar{T}}}\nolimits_{{sk}} RMSE was 1.5°C, with separate cycling and running giving RMSE of 1.4°C and 1.6°C, respectively, and no significant difference in residuals. Subjects’ actual thermal sensation (ATS) votes displayed significant strong rank correlation with budget scores calculated using both measured and predicted [`(T)]\nolimits<sub>sk</sub> \mathop{{\bar{T}}}\nolimits_{{sk}} (r <sub>s</sub>  = 0.507 and 0.517, respectively, P < 0.01). These results show improved predictive strength of ATS of subjects as compared to the original and updated COMFA models. This psychological improvement, plus [`(T)]\nolimits_sk \mathop{{\bar{T}}}\nolimits_{{sk}} and T _c validations, enables better application to a variety of outdoor spaces. This model can be used in future research studying linkages between thermal discomfort, subsequent decreases in physical activity, and negative health trends.     

Improved predictive ability of climate–human–behaviour interactions with modifications to the COMFA outdoor energy budget model

The purpose of this paper is to implement current and novel research techniques in human energy budget estimations to give more accurate and efficient application of models by a variety of users. Using the COMFA model, the conditioning level of an individual is incorporated into overall energy budget predictions, giving more realistic estimations of the metabolism experienced at various fitness levels. Through the use of VO₂ reserve estimates, errors are found when an elite athlete is modelled as an unconditioned or a conditioned individual, giving budgets underpredicted significantly by −173 and −123 W m⁻², respectively. Such underprediction can result in critical errors regarding heat stress, particularly in highly motivated individuals; thus this revision is critical for athletic individuals. A further improvement in the COMFA model involves improved adaptation of clothing insulation (I _cl), as well clothing non-uniformity, with changing air temperature (T _a) and metabolic activity (M _act). Equivalent T _a values (for I _cl estimation) are calculated in order to lower the I _cl value with increasing M _act at equal T _a. Furthermore, threshold T _a values are calculated to predict the point at which an individual will change from a uniform I _cl to a segmented I _cl (full ensemble to shorts and a T-shirt). Lastly, improved relative velocity (v _r) estimates were found with a refined equation accounting for the degree angle of wind to body movement. Differences between the original and improved v _r equations increased with higher wind and activity speeds, and as the wind to body angle moved away from 90°. Under moderate microclimate conditions, and wind from behind a person, the convective heat loss and skin temperature estimates were 47 W m⁻² and 1.7°C higher when using the improved v _r equation. These model revisions improve the applicability and usability of the COMFA energy budget model for subjects performing physical activity in outdoor environments. Application is possible for other similar energy budget models, and within various urban and rural environments.     

Plasma total antioxidant capacity is associated with dietary intake and plasma level of antioxidants in postmenopausal women

Increased plasma total antioxidant capacity (TAC) has been associated with a high consumption of fruits and vegetables. However, limited information is available on whether plasma TAC reflects the dietary intake of antioxidants and the levels of individual antioxidants in plasma. By using three different assays, the study aimed to determine if plasma TAC can effectively predict dietary intake of antioxidants and plasma antioxidant status. Forty overweight and apparently healthy postmenopausal women were recruited. Seven-day food records and 12-h fasting blood samples were collected for dietary and plasma antioxidant assessments. Plasma TAC was determined by vitamin C equivalent antioxidant capacity (VCEAC), ferric-reducing ability of plasma (FRAP) and oxygen radical absorbance capacity (ORAC) assays. TAC values determined by VCEAC were highly correlated with FRAP (r=0.79, P<.01) and moderately correlated with ORAC (r=0.34, P<.05). Pearson correlation analyses showed that plasma TAC values by VCEAC and ORAC had positive correlation with plasma uric acid (r=0.56 for VCEAC; r=0.49 for ORAC) and total phenolics (r=0.63 for VCEAC; r=0.36 for ORAC). However, TAC measured by FRAP was correlated only with uric acid (r=0.69). After multivariate adjustment, plasma TAC determined by VCEAC was positively associated with dietary intakes of γ-tocopherol (P<.001), β-carotene (P<.05), anthocyanidins (P<.05), flavones (P<.05), proanthocyanidins (P<.01) and TAC (P<.05), as well as with plasma total phenolics (P<.05), α-tocopherol (P<.001), β-cryptoxanthin (P<.05) and uric acid (P<.05). The findings indicate that plasma TAC measured by VCEAC reflects both dietary and plasma antioxidants and represents more closely the plasma antioxidant levels than ORAC and FRAP.     

Finger loop inhibitors of the HCV NS5b polymerase. Part II. Optimization of tetracyclic indole-based macrocycle leading to the discovery of TMC647055

Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 μM) and promising pharmacokinetic profiles in rats and dogs. TMC647055 is currently being evaluated in the clinic.     

Validation of an electronic jump mat to assess stretch-shortening cycle function

The purpose of this investigation was to determine the concurrent validity of a commonly used electronic switch mat (ESM), or jump mat, compared with force plate (FP) data. The efficiency of collection and accuracy of data are paramount to athlete and player field testing for the strength and conditioning coach who often has access only to a jump mat. Ten subjects from 5 different sporting backgrounds completed 3 squat jumps (SJs), 3 countermovement jumps (CMJs), and 3 drop jumps (DJs). The jumps were performed on an AMTI FP operating at 1,000 Hz with an ESM positioned on top of the platform. All the subjects were experienced with the protocols involved with jump testing. The resulting absolute errors between FP and ESM data were 0.01, 0.02, and 0.01 m for CMJ, SJ, and DJ heights, respectively. However, the coefficient of variation for the DJ contact time (CT) was 57.25%, CMJ (r = 0.996), and SJ (r = 0.958) heights correlated very strongly with force platform data, and DJ data were not as strong (r = 0.683). Confidence interval tests revealed bias toward CMJ and SJ (p < 0.05). The jump mat can accurately calculate the CMJ height, SJ height, and reactive strength index for all the 3 jump protocols. However, the faster CTs and rapid movements involved in a DJ may limit its reliability when giving measures of CT, flight time, and height jumped for DJs. Strength and conditioning coaches can use such a jump mat device with the confidence that it is accurately producing valid measurements of their athlete's performance for CMJ and SJ slow SSC protocols.     

PAK1 Kinase Promotes Cell Motility and Invasiveness through CRK-II Serine Phosphorylation in Non-Small Cell Lung Cancer Cells

The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells.     

Integration potential of mouse and human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a multipotent cell population which has been described to exert renoprotective and regenerative effects in experimental models of kidney injury. Several lines of evidence indicate that MSCs also have the ability to contribute to nephrogenesis, suggesting that the cells can be employed in stem cell-based applications aimed at de novo renal tissue generation. In this study we re-evaluate the capacity of mouse and human bone marrow-derived MSCs to contribute to the development of renal tissue using a novel method of embryonic kidney culture. Although MSCs show expression of some genes involved in renal development, their contribution to nephrogenesis is very limited in comparison to other stem cell types tested. Furthermore, we found that both mouse and human MSCs have a detrimental effect on the ex vivo development of mouse embryonic kidney, this effect being mediated through a paracrine action. Stimulation with conditioned medium from a mouse renal progenitor population increases the ability of mouse MSCs to integrate into developing renal tissue and prevents the negative effects on kidney development, but does not appear to enhance their ability to undergo nephrogenesis.