Validation of an opto-electronic instrument for the measurement of weighted countermovement jump execution velocity.

The purpose of this study was to analyse the reliability and validity of an opto-electronic sensor system (Velowin) compared to a linear velocity transducer (T-Force System) considered as the gold standard. Mean velocity (MV) and peak velocity (PV) generated in the Smith machine bar placed on the shoulders in counter-movement jump exercise (CMJ) were analysed. The study was conducted with a sample of 21 men with experience in resistance training. Five measurements were analysed for CMJ exercise in concentric phase using a progressive loading increase. Three jumps were made per load with a 3-4 min recovery between loads. The analysis of the variance confirmed that there were no significant differences (p > 0.05) in the execution velocity between Velowin and T-Force with each of the loads. The reliability analysis showed, with each of the loads, high values of the intraclass correlation coefficient (ICC = 0.95-0.99) and a 'substantial' Lin´s concordance coefficient in MV (CCC ≥0.96) and between 'substantial' (CCC = 0.98) and 'almost perfect' (CCC = 0.99) in PV. These results confirm the reliability and validity of the Velowin device is reliable for measuring the execution velocity in loaded CMJ exercise.

Validation of an opto-electronic instrument for the measurement of execution velocity in squat exercise.

The purpose of this study was to analyse the reliability and validity of an opto-electronic sensor system (Velowin) for assessment of the bar-velocity in the deep squat exercise. Mean velocity, mean propulsive velocity and peak velocity generated in the deep squat exercise performed in the Smith machine bar were analysed compared to a linear velocity transducer considered as the gold standard. The study was conducted with a sample of 26 men with experience in resistance training. Six measurements were analysed for squat exercise in concentric phase using a progressive loading increase. Three consecutive repetitions were performed per load with a 3-4 min recovery between loads. Analysis of variance confirmed that there were no significant differences (p > 0.05) for the velocity variables between Velowin and T-Force for each of the loads. The reliability analysis showed high values of the intraclass correlation coefficient (ICC = 0.94-0.99), an 'almost perfect' Lin's concordance coefficient (CCC = 0.99) and a low coefficient of variation (CV <3.4%) for each of the loads and velocities. These results confirm the reliability and validity of the Velowin device for measuring the execution velocity in deep squat exercise.

Bioreactor-scale cell performance and protein production can be substantially increased by using a secretion signal that drives co-translational translocation in Pichia pastoris.

Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae α-factor mating pheromone. For comparison, the commonly used full-length α-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5 L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved.

Perceived Classroom Responsibility Climate Questionnaire: A new scale.

BACKGROUND: Given the lack of measuring instruments, we aimed to develop and validate a scale to assess the classroom responsibility climate fostered by the teacher and the students in different school subject lessons. METHOD: 810 students from all secondary education years agreed to participate. Data were obtained from three different subjects: Mathematics (n = 240; 118 female, 13.10 ± 1.07 years), Spanish Language and Literature (n = 318; 169 female 13.37 ± 1.03 years) and Physical Education (n = 252, 112 female, 13.23 ± .99 years). The first version of the questionnaire underwent a three-phase refining process: a) Assessment by experts (n = 8), b) Pilot study one (n = 100) to assess comprehensive adequacy, and c) Pilot study two (n = 300), an exploratory factor analysis. RESULTS: The instrument consists of two factors: climate generated by the teacher and by the students. Confirmatory factor analyses showed that all fit indices were acceptable. CONCLUSION: The instrument presented adequate convergent, discriminant and concurrent validity and completes other existing tools, being suitable for use in the different secondary school subjects to assess the responsibility climate produced by the teacher and students.

Maturation-driven transport and AP-1-dependent recycling of a secretory cargo in the Golgi.

Golgi cisternal maturation has been visualized by fluorescence imaging of individual cisternae in the yeast Saccharomyces cerevisiae, but those experiments did not track passage of a secretory cargo. The expectation is that a secretory cargo will be continuously present within maturing cisternae as resident Golgi proteins arrive and depart. We tested this idea using a regulatable fluorescent secretory cargo that forms ER-localized aggregates, which dissociate into tetramers upon addition of a ligand. The solubilized tetramers rapidly exit the ER and then transit through early and late Golgi compartments before being secreted. Early Golgi cisternae form near the ER and become loaded with the secretory cargo. As predicted, cisternae contain the secretory cargo throughout the maturation process. An unexpected finding is that a burst of intra-Golgi recycling delivers additional secretory cargo molecules to cisternae during the early-to-late Golgi transition. This recycling requires the AP-1 adaptor, suggesting that AP-1 can recycle secretory cargo proteins as well as resident Golgi proteins.

An improved secretion signal enhances the secretion of model proteins from Pichia pastoris.

BACKGROUND: Proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal. RESULTS: We engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal. CONCLUSIONS: The improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.

The Importance of the Coach in Predicting Implicit Beliefs about Skill and Beliefs about the Causes of Success in Handball Players.

The objective of the present study was to determine the predictive capacity of the motivational climate generated by coaches and perceived by handball players on implicit beliefs about ability and beliefs about the causes of success in sport. The sample consisted of 444 youth handball players. These players completed the Beliefs about the Causes of Success in Sport Questionnaire, the Conceptions of the Nature of Athletic Ability Questionnaire, Version Two, and the Perceived Motivational Climate in Sport Questionnaire. The structural regression model showed that the mastery climate positively predicted the belief in incremental ability and that this in turn positively predicts both belief in athletic success through effort and ability. The results reflected the importance of the coach in the formative process of the player and the search for performance in sport.

GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris.

BACKGROUND: State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in P. pastoris. However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for P. pastoris. METHODS: With the GoldenPiCS system, we provide a flexible modular system for advanced strain engineering in P. pastoris based on Golden Gate cloning. For this purpose, we established a wide variety of standardized genetic parts (20 promoters of different strength, 10 transcription terminators, 4 genome integration loci, 4 resistance marker cassettes). RESULTS: All genetic parts were characterized based on their expression strength measured by eGFP as reporter in up to four production-relevant conditions. The promoters, which are either constitutive or regulatable, cover a broad range of expression strengths in their active conditions (2-192% of the glyceraldehyde-3-phosphate dehydrogenase promoter P GAP ), while all transcription terminators and genome integration loci led to equally high expression strength. These modular genetic parts can be readily combined in versatile order, as exemplified for the simultaneous expression of Cas9 and one or more guide-RNA expression units. Importantly, for constructing multigene constructs (vectors with more than two expression units) it is not only essential to balance the expression of the individual genes, but also to avoid repetitive homologous sequences which were otherwise shown to trigger "loop-out" of vector DNA from the P. pastoris genome. CONCLUSIONS: GoldenPiCS, a modular Golden Gate-derived P. pastoris cloning system, is very flexible and efficient and can be used for strain engineering of P. pastoris to accomplish pathway expression, protein production or other applications where the integration of various DNA products is required. It allows for the assembly of up to eight expression units on one plasmid with the ability to use different characterized promoters and terminators for each expression unit. GoldenPiCS vectors are available at Addgene.

An improved reversibly dimerizing mutant of the FK506-binding protein FKBP.

FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP.

Identification of a set of miRNAs differentially expressed in transiently TIA-depleted HeLa cells by genome-wide profiling.

BACKGROUND: T-cell intracellular antigen (TIA) proteins function as regulators of cell homeostasis. These proteins control gene expression globally at multiple levels in response to dynamic regulatory changes and environmental stresses. Herein we identified a micro(mi)RNA signature associated to transiently TIA-depleted HeLa cells and analyzed the potential role of miRNAs combining genome-wide analysis data on mRNA and miRNA profiles. RESULTS: Using high-throughput miRNA expression profiling, transient depletion of TIA-proteins in HeLa cells was observed to promote significant and reproducible changes affecting to a pool of up-regulated miRNAs involving miR-30b-3p, miR125a-3p, miR-193a-5p, miR-197-3p, miR-203a, miR-210, miR-371-5p, miR-373-5p, miR-483-5p, miR-492, miR-498, miR-503-5p, miR-572, miR-586, miR-612, miR-615-3p, miR-623, miR-625-5p, miR-629-5p, miR-638, miR-658, miR-663a, miR-671-5p, miR-769-3p and miR-744-5p. Some up-regulated and unchanged miRNAs were validated and previous results confirmed by reverse transcription and real time PCR. By target prediction of the miRNAs and combined analysis of the genome-wide expression profiles identified in TIA-depleted HeLa cells, we detected connections between up-regulated miRNAs and potential target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis suggest that target genes are related with biological processes associated to the regulation of DNA-dependent transcription, signal transduction and multicellular organismal development as well as with the enrichment of pathways involved in cancer, focal adhesion, regulation of actin cytoskeleton, endocytosis and MAPK and Wnt signaling pathways, respectively. When the collection of experimentally defined differentially expressed genes in TIA-depleted HeLa cells was intersected with potential target genes only 7 out of 68 (10%) up- and 71 out of 328 (22%) down-regulated genes were shared. GO and KEGG database analyses showed that the enrichment categories of biological processes and cellular pathways were related with innate immune response, signal transduction, response to interleukin-1, glomerular basement membrane development as well as neuroactive ligand-receptor interaction, endocytosis, lysosomes and apoptosis, respectively. CONCLUSION: All this considered, these observations suggest that individual miRNAs could act as potential mediators of the epigenetic switch linking transcriptomic dynamics and cell phenotypes mediated by TIA proteins.