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The lack of a cost-effective oxygen reduction reaction (ORR) catalyst is the key technical issue that restricts the large-scale application of proton-exchange membrane fuel cells (PEMFCs). Transition metal–Nx/C has exhibited promising potential to replace the precious Pt-based catalyst, and Mn–Nx/C is becoming the research hotspot as a result of the Fenton-inert property of Mn. To further improve the ORR activity of the Mn–Nx/C catalyst, a three-dimensional (3D) interconnected porous structure is constructed in two-dimensional Mn- and N-doped carbon nanosheets via a “porogen-in-resin” strategy. In combination with a post-adsorption treatment, the so-called 3D Mn–Nx/CS catalyst exhibits great ORR catalytic activity and stability under both acidic and alkaline conditions. The half-wave potential (E1/2) of 3D Mn–Nx/CS is high at 0.918 and 0.720 V under alkaline and acidic conditions, respectively. As a result, the practical PEMFC equipped with 3D Mn–Nx/CS possesses an open circuit voltage above 0.94 V and a maximum power density of 360 mW/cm2, showing a potential application prospect.

The CDK4/6 inhibitor palbociclib reduces tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Palbociclib in combination with anti-hormonal therapy brings significant benefit to breast cancer patients. In this study, novel combination approaches and underlying molecular/cellular mechanisms for palbociclib were explored in squamous cell lung cancer (SqCLC), the second most common subtype of non-small cell lung cancer. While approximate 20% lung patients benefit from immunotherapy, most SqCLC patients who receive platinum-doublet chemotherapy as first-line treatment, which often includes a taxane, are still in need of more effective combination therapies. Our results demonstrated enhanced cytotoxicity and anti-tumor effect with palbociclib plus taxanes at clinically achievable doses in multiple SqCLC models with diverse cancer genetic backgrounds. Comprehensive gene expression analysis revealed a sustained disruption of pRB-E2F signaling by combination that was accompanied with enhanced regulation of pleiotropic biological effects. These included several novel mechanisms such as abrogation of G2/M and mitotic spindle assembly checkpoints, as well as impaired induction of hypoxia-inducible factor 1 alpha (HIF-1α). The decrease in HIF-1α modulated a couple key angiogenic and anti-angiogenic factors, resulting in an enhanced anti-angiogenic effect. This preclinical work suggests a new therapeutic opportunity for palbociclib in lung and other cancers currently treated with taxane based chemotherapy as standard of care.

Serum -nerve growth factor (NGF) concentrations were determined in pregnant female, nonpregnant female, and male cynomolgus monkeys using a highly selective and sensitive immunoaffinity liquid chromatography–tandem mass spectrometry assay. NGF was significantly higher in pregnant monkeys than in nonpregnant female and male monkeys. NGF increased over pregnancy (mean NGF±SD: 541±448, 1590±520, and 3560±1430 pgml during the first, second, and third trimesters, respectively). These data will aid in further understanding the role of NGF during pregnancy.

Approximately 7% of non–small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. Although tumors harboring ALKfusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrate that patients with ROS1or RETfusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, we designed a combination of 3′ overexpression and fusion-specific detection strategies to detect ALK, ROS1 and RET fusion transcripts in NSCLC tumors. We validated the assay in 295 NSCLC specimens and showed that the assay is highly sensitive and specific. ALKresults were 100% concordant with fluorescence in situhybridization (FISH) (n= 52) and 97.8% concordant with IHC (n= 179) [sensitivity, 96.8% (95% CI 91.0%–98.9%); specificity, 98.8% (95% CI 93.6%–99.8%)]. For ROS1and RET, we also observed 100% concordance with FISH (n= 46 and n= 15, respectively). We identified seven ROS1and 14 RETfusion–positive tumors and confirmed the fusion status by RT-PCR and FISH. One RETfusion involved a novel partner, cutlike homeobox 1 gene (CUX1), yielding an in-frame CUX1-RET fusion. ROS1and RETfusions were significantly enriched in tumors without KRAS/EGFR/ALKalterations. ALK/ROS1/RET/EGFR/KRASalterations were mutually exclusive. As a single-tube assay, this test shows promise as a more practical and cost-effective screening modality for detecting rare but targetable fusions in NSCLC.

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50≥ 0.03 and [I2]/IC50≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50values, translating into a range of [I1]/IC50and [I2]/IC50values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50values.

Anaplastic lymphoma kinase gene (ALK) fusions have been identified in approximately 5% of non-small-cell lung carcinomas (NSCLCs) and define a distinct subpopulation of patients with lung cancer who are highly responsive to ALK kinase inhibitors, such as crizotinib. Because of this profound therapeutic implication, the latest National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology recommend upfront ALK screening for all patients with NSCLC. The Food and Drug Administration–approved companion diagnostic test (ie, fluorescence in situhybridization) for identification of ALK-positive patients, however, is complex and has considerable limitations in terms of cost and throughput, making it difficult to screen many patients. To explore alternative screening modalities for detecting ALK fusions, we designed a combination of two transcript-based assays to detect for presence or absence of ALK fusions using NanoString’s nCounter technology. By using this combined gene expression and ALK fusion detection strategy, we developed a multiplexed assay with a quantitative scoring modality that is highly sensitive, reproducible, and capable of detecting low-abundant ALK fusion transcripts, even in samples with a low tumor cell content. In 66 archival NSCLC samples, our results were highly concordant to prior results obtained by fluorescence in situhybridization and IHC. Our assay offers a cost-effective, easy-to-perform, high-throughput, and FFPE-compatible screening alternative for detection of ALK fusions.

Recent regulatory guidance suggests that metabolites identified in human plasma should be present at equal or greater levels in one of the animal species used in safety assessments. In this report, a high-performance liquid chromatography-tandem mass spectrometry method is described whereby quantitative comparisons of exposures to metabolites between species can be obtained in the absence of authentic standards of the metabolites, calibration curves, and other attributes of standard bioanalytical methods. This novel method was tested using six drug-metabolite combinations. Plasma samples from animals are mixed with control plasma from humans and vice versa to remove possible differential effects of matrices. Through multiple ion monitoring-triggered enhanced product ion (EPI) scans, all metabolites were qualitatively confirmed, and daughter ions were selected for the most sensitive mass transitions to trigger EPI scans. Direct comparisons of metabolites in animal versus human plasma were achieved by calculating the peak area ratios of the metabolites versus an internal standard. Linearity of instrument responses was established by serial dilution. A statistical analysis demonstrated that experimentally measured ratios of the parent and metabolites in rat versus human correlated well with the nominal ratios of concentrations using linear regression with an average slope of 0.99 ± 0.08 (r = 0.994 ± 0.005). This analysis showed that if the experimentally determined ratio of mass spectrometer responses is ≥2.0, then the actual exposure ratio is unity or greater (p < 0.01). This method offers time- and resource-sparing advantages to ascertaining metabolite exposure comparisons between humans and laboratory animal species. A strategy for application of this approach within standard drug development processes is described.

Real-time PCR has become increasingly important in gene expression profiling research, and it is widely agreed that normalized data are required for accurate estimates of messenger RNA (mRNA) expression. With increased gene expression profiling in pre-clinical research and toxicogenomics, a need for reference genes in the rat has emerged, and the studies in this area have not yet been thoroughly evaluated. The purpose of our study was to evaluate a panel of rat reference genes for variation of gene expression in different tissue types. We selected 48 known target genes based on their putative invariability. The gene expression of all targets was examined in 11 types of rat tissues using TaqMan® low density array (LDA) technology. The variability of each gene was assessed using a two-step statistical model. The analysis of mean expression using multiple reference genes was shown to provide accurate and reliable normalized expression data. The least five variable genes from each specific tissue were recommended for future tissue-specific studies. Finally, a subset of investigated rat reference genes showing the least variation is recommended for further evaluation using the LDA platform. Our work should considerably enhance a researcher's ability to simply and efficiently identify appropriate reference genes for given experiments.

MCMURRAY, R. G., J. S. HARRELL, C. B. BRADLEY, S. DENG, and S. I. BANGDIWALA. Predicted maximal aerobic power in youth is related to age, gender, and ethnicity. Med. Sci. Sports Exerc., Vol. 34, No. 1, 2002, pp. 145–151.

BRADLEY, C. B., R. G. MCMURRAY, J. S. HARRELL, and S. DENG. Changes in common activities of 3rd through 10th graders the CHIC Study. Med. Sci. Sports Exerc., Vol. 32, No. 12, 2000, pp. 2071–2078.

Objective:This study examined the effects of physical activity, television viewing, video game play, socioeconomic status (SES), and ethnicity on body mass index (BMI). Research Methods and Procedures:The sample was 2389 adolescents, 10 to 16 years of age (12.7 ± 1.0 years); 1240 (52%) females and 1149 (48%) males; 77% white and 23% African American; from rural (77%) and urban (23%) settings. BMI and skinfolds were directly assessed. All other data were obtained from questionnaires. Results:Watching television on non‐school days was related to being overweight (p< 0.005). However, when BMI analyses were adjusted for ethnicity and SES, there were no significant effects of television viewing on BMI (p> 0.061). Increased hours of video game play enhanced the risk of being overweight for both genders when analyses were adjusted for ethnicity and SES (p< 0.019). In males, participation in as little as one high‐intensity physical activity 3 to 5 days a week decreased the ethnic‐ and SES‐adjusted relative risk of being overweight (RR = 0.646; CI: 0.427 to 0.977). For females, the ethnic‐ and SES‐adjusted relative risk for being overweight was not significantly altered by physical activity. The logistic analyses further indicated the influence of low SES and African American ethnicity overshadowed any direct effect of television or videos. Discussion:Because weight status of male adolescents appears to be more related to exercise habits than to television or video game habits, increased participation in high‐intensity exercise appears to be important. For females, neither videos nor exercise habits appear to be related to risk of being overweight. However, ethnicity and SES may be important factors that can influence body weight status, while television viewing may be of some importance. Thus, programs to reduce obesity in female adolescent should focus their efforts in lower SES communities.

Abstract: Previous studies on the influence of a rural/urban setting on the prevalence of cardiovascular disease risk factors in children have not sufficiently controlled for socioeconomic status, race, gender, and perhaps, may not have included a representative sample of rural and urban children. This study compared the cardiovascular disease risk factors and rate of obesity of children living in rural and urban settings. It also determined the magnitude of the effect of the rural/urban setting on cardiovascular disease risk factors and obesity when controlling for race, socioeconomic status, and gender. The subjects were 2,113 third‐ and fourth‐grade children; 962 from an urban setting and 1,151 from a rural setting. Height, weight, skinfolds, resting blood pressure, and total cholesterol levels were measured. Aerobic power (pV02max) was estimated from cycle ergomerty. Physical activity and smoking history were obtained from a questionnaire. Clustering analyses using adjustment for sample error indicated that total cholesterol, blood pressure, smoking, and physical activity levels of rural and urban children were not different (P>0.10); however, body mass index and sum of skinfolds was greater for rural youth (P<0.004). Logistic regression indicated that rural children had a 54.7 percent increased risk of obesity (P=0.0001). This study's results indicate that, in children, a rural setting is associated with obesity, but not with the major risk factors associated with cardiovascular disease.

ABSTRACTMacroautophagy (autophagy) is an essential cellular catabolic process required for survival under conditions of starvation. The role of autophagy in cancer is complex, context-dependent and at times contradictory, as it has been shown to inhibit, promote or be dispensable for tumor progression. In this study, we evaluated the contribution of the immune system to the reliance of tumors on autophagy by depleting autophagy-related 7 (ATG7) in murine tumor cells and grafting into immunocompetent versus immunodeficient hosts. Although loss of ATG7 did not affect tumor growth in vitro or in immunodeficient mice, our studies revealed that cancer cell reliance on autophagy was influenced by anti-tumor immune responses, including those mediated by CD8+T cells. Furthermore, we provide insights into possible mechanisms by which autophagy disruption can enhance anti-tumor immune responses and suggest that autophagy disruption may further benefit patients with immunoreactive tumors.