Discovery, adaptation and transcriptional activity of two tick promoters: Construction of a dual luciferase reporter system for optimization of RNA interference in rhipicephalus (boophilus) microplus cell lines.

Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vector of bovine babesiosis and anaplasmosis. Commercial promoters were evaluated for transcriptional activity driving luciferase expression in the tick cell lines. The human phosphoglycerate kinase (PGK) promoter resulted in detectable firefly luciferase activity within 2 days post-transfection of the R. microplus cell line BME26, with maximal activity at 5 days post-transfection. Several other promoters were weaker or inactive in the tick cells, prompting identification and assessment of transcriptional activity of the homologous ribosomal protein L4 (rpL4, GenBank accession no.: KM516205) and elongation factor 1α (EF-1α, GenBank accession no.: KM516204) promoters cloned from R. microplus. Evaluation of luciferase expression driven by various promoters in tick cell culture resulted in selection of the R. microplus rpL4 promoter and the human PGK promoter driving transcription of sequences encoding modified firefly and NanoLuc® luciferases for construction of a dual luciferase reporter system for use in tick cell culture.

Relationship between growth hormone in vivo bioactivity, the insulin-like growth factor-I system and bone mineral density in young, physically fit men and women.

CONTEXT: Bone mineral density (BMD) is influenced by growth factors, such as growth hormone (GH) and insulin-like growth factor-I (IGF-I). The in vivo bioassay for GH (bioGH) provides a more physiologically relevant measurement than an in vitro immunoassay, since bioGH is quantified on a biological outcome. OBJECTIVE: To determine if bioGH and components of the IGF-I system were associated with BMD in age-matched men (M; n=41, 19.1+/-0.2 year, 70+/-3 kg, 163+/-25 cm) and women (W; n=39, 18.6+/-0.3 year, 66+/-3 kg, 141+/-15 cm). DESIGN: Blood was analyzed for growth-related hormones [bioGH, immunoreactive growth hormone (iGH), IGF-I and associated binding proteins], and BMD was measured by pDXA, pQCT, and central DXA (spine, hip). For the bioGH assay, hypophysectomizied female Sprague-Dawley rats were injected with a s.c. bolus of either a GH standard or unknown (each subject's plasma) in four daily injections. The tibia was then examined for epiphyseal growth plate width from which bioGH concentrations were extrapolated. RESULTS: M had greater (P<0.05) calcaneal BMD when measured by pDXA (M: 1.27+/-0.02; W: 1.14+/-0.02 g/cm2), while pQCT-assessed BMD at the tibia was not different (M: 777+/-16; W: 799+/-16 g/cm2). bioGH was similar between M (5388+/-800 microg/L) and W (4282+/-643 microg/L) and was not correlated with BMD. The only BMD-related biomarkers in women were acid-labile subunit (ALS; r=0.40) and IGFBP-3 (r=0.42) with DXA-measured spine and femoral neck BMD, and ALS (r=0.47) with pQCT-assessed tibial BMD and cortical thickness, respectively. CONCLUSION: Although bioGH was not associated with BMD, IGF-I and associated binding proteins (IGFBP-3 and ALS) emerged as correlates in W only.

Effects of intensified military field training on jumping performance.

A sensitive, reliable, field-expedient test may be valuable for monitoring interventions during periods of anticipated physical performance decline. The purpose of this study was to determine the capabilities of unloaded jumping tests for detecting decrements in physical performance following eight days of military sustained operations. Twenty-nine U. S. Marines (24 +/- 1 y; 180 +/- 6 cm; 82.5 +/- 8.2 kg) performed 1, 5 and 30 repetition(s) of unloaded countermovement jumps (UJ) before and after eight days of sustained operations (SUSOPS). Jump performance data was collected simultaneously using a switch mat (SM) and a linear position transducer (LPT). Jump height (m) and power (W) were highest using 1 UJ and declined 4.9 and 8.9%, respectively after SUSOPS. Jump power (JP) declined progressively over 30 UJ (20%). Five UJ offered no advantages over 1 UJ and was inadequate to examine changes in muscle fatigability (pre: 1294 +/- 138 W; post: 1250 +/- 165 W). The SM and a LPT were in agreement and had a high correlation (r = 0.92). One UJ was a sensitive, easy to implement test for monitoring the collective impact of high physical, nutritional, cognitive, and environmental stress on an individuals' physical performance before and after 8 days of SUSOPS, suggesting decrements in physical performance associated with overreaching can be detected by simply administered field-expedient jumping tests.