A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells

We have developed a system for producing biotinylated recombinant proteins in mammalian cells. The expression construct consists of an inducible tetracycline response element (TRE) that drives expression of a bicistronic cassette comprising a biotin acceptor peptide (BioTag) fused to either terminus of the target protein, the gene for Escherichia coli biotin ligase (BirA), and an intervening internal ribosome entry site (IRES). By either transient or stable transfection of Chinese hamster ovary (CHO) Tet-On cells, we successfully expressed, detected, and immobilized biotinylated human Itch, a pleiotropic multi-domain ubiquitin-protein ligase, as well as Gla-RTK, a putative vitamin K-dependent receptor tyrosine kinase. The biotinylation of recombinant Itch in transiently transfected CHO Tet-On cells required biotin supplementation and coexpression of BirA, occurred quantitatively and specifically on the lysine residue of the BioTag, and enabled detection of Itch by Western blot in as little as 10ng of total lysate protein. Stably selected clones were rapidly pre-screened for doxycycline (dox)-inducible BirA expression by ELISA, and subsequently screened for dox-inducible expression of biotinylated Itch. Biotinylated Gla-RTK was detectable in as little as 5ng of total lysate protein from transiently transfected CHO Tet-On cells, and exhibited pronounced tyrosine phosphorylation. In stable clones however, constitutive phosphorylation was prevented by reducing the expression level of Gla-RTK through the titration of dox. These results demonstrate the utility of this system for the expression of 'difficult' proteins, particularly those that are cytotoxic or those that may require lower expression levels to ensure appropriate post-translational modification.     

Spinal loading during manual materials handling in a kneeling posture.

Stooped, restricted, kneeling, and other awkward postures adopted during manual materials handling have frequently been associated with LBP onset. However, lift assessment tools have focused on materials handling performed in an upright, or nearly upright standing posture. Unfortunately, many of the tools designed to analyze standing postures are not easily adapted to jobs requiring restricted postures. Therefore, the objective of this study was to evaluate spinal loading during manual materials handing in kneeling postures and determine if those loads can be predicted using simple regression. An EMG-driven biomechanical model, previously validated for upright lifting, was adapted for use in kneeling tasks. Subjects knelt under a 1.07m ceiling and lifted luggage of six weights (6.8, 10.9, 15.0, 19.1, 23.1 and, 27.2kgf) to one of four destination heights (0, 25.4, 53.3, 78.7cm). Spine loading was significantly affected by both destination height and load weight. Destination height increased compression, AP shear and lateral shear by an average of 14.5, 3.7 and 6.6N respectively per cm height increase. Load weight increased compression, AP shear and lateral shear by an average of 83.8, 27.0 and 13.1N respectively per kgf lifted. Regression equations were developed to predict peak spine loading using subject height, load weight and destination height with R(2) values of 0.62, 0.51 and 0.57 for compression, AP and lateral shear respectively.     

The effects of ten weeks of resistance and combined plyometric/sprint training with the Meridian Elyte athletic shoe on muscular performance in women

The purpose of this investigation was to examine the combined effects of resistance and sprint/plyometric training with or without the Meridian Elyte athletic shoe on muscular performance in women. Fourteen resistance-trained women were randomly assigned to one of 2 training groups: (a) an athletic shoe (N = 6) (AS) group or (b) the Meridian Elyte (N = 8) (MS) group. Training was performed for 10 weeks and consisted of resistance training for 2 days per week and 2 days per week of sprint/plyometric training. Linear periodized resistance training consisted of 5 exercises per workout (4 lower body, 1 upper body) for 3 sets of 3-12 repetition maximum (RM). Sprint/plyometric training consisted of 5-7 exercises per workout (4-5 plyometric exercises, 40-yd and 60-yd sprints) for 3-6 sets with gradually increasing volume (8 weeks) followed by a 2-week taper phase. Assessments for 1RM squat and bench press, vertical jump, broad jump, sprint speed, and body composition were performed before and following the 10-week training period. Significant increases were observed in both AS and MS groups in 1RM squat (12.0 vs. 14.6 kg), bench press (6.8 vs. 7.4 kg), vertical jump height (3.3 vs. 2.3 cm), and broad jump (17.8 vs. 15.2 cm). Similar decreases in peak 20-, 40-, and 60-m sprint times were observed in both groups (20 m: 0.14 vs. 0.11 seconds; 40 m: 0.29 vs. 0.34 seconds; 60 m: 0.45 vs. 0.46 seconds in AS and MS groups, respectively). However, when sprint endurance (the difference between the fastest and slowest sprint trials) was analyzed, there was a significantly greater improvement at 60 m in the MS group. These results indicated that similar improvements in peak sprint speed and jumping ability were observed following 10 weeks of training with either shoe. However, high-intensity sprint endurance at 60 m increased to a greater extent during training with the Meridian Elyte athletic shoe.     

High-content imaging analysis of the knockdown effects of validated siRNAs and antisense oligonucleotides

High-content imaging (HCI) provides researchers with a powerful tool for understanding cellular processes. Although phenotypic analysis generated through HCI is a potent technique to determine the overall cellular effects of a given treatment, it frequently produces complex data sets requiring extensive interpretation. The authors developed statistical analyses to decrease the time spent to determine the outcome of each HCI assay and to better understand complex phenotypic changes. To test these tools, the authors performed a comparison experiment between 2 types of oligonucleotide-mediated gene silencing (OMGS), antisense oligonucleotides (ASOs), and short, double-stranded RNAs (siRNAs). Although similar in chemical structure, these 2 methods differ in cellular mechanism of action and off-target effects. Using a library of 50 validated ASOs and siRNAs to the same targets, the authors characterized the differential effects of these 2 technologies using a HeLa cell G2-M cell cycle assay. Although knockdown of a variety of targets by ASOs or siRNAs affected the cell cycle profile, few of those targets were affected by both ASOs and siRNAs. Distribution analysis of population changes induced through target knockdown led to the identification of targets that, when inhibited, could affect the G2-M transition in the cell cycle in a statistically significant manner. The distinctly different mechanisms of action of these 2 forms of gene silencing may help define the use of these treatments in both clinical and research environments.