Effect of an 11-Week Resistance Training Program on Arterial Stiffness in Young Women.

ABSTRACT: Morgan, B, Mirza, AM, Gimblet, CJ, Ortlip, AT, Ancalmo, J, Kalita, D, Pellinger, TK, Walter, JM, and Werner, TJ. Effect of an 11-week resistance training program on arterial stiffness in young women. J Strength Cond Res 37(2): 315-321, 2023-The current investigation was conducted to determine the effect of 2 resistance training models on indices of arterial stiffness in young, healthy women. Twenty-four women, untrained college students, aged 18-22 years were randomized into 1 of 3 groups: control (CON) group ( n = 8), high-intensity (HI) resistance exercise group ( n = 8), and high-volume (HV) resistance exercise group ( n = 8). Subjects randomized to resistance training groups were required to perform strength training exercises 3-5 days a week for 11 weeks. The exercise regimen consisted of 2-3 sets of 3-8 repetitions (80-90% of 1 repetition maximum [1RM]) for the HI group and 3-4 sets of 10-15 repetitions (50-70% of 1RM) for the HV group. All subjects were instructed to continue their normal diet and avoid cardiovascular exercise during the study. After the intervention, there was a significant increase in carotid femoral pulse wave velocity (PWV) (6.39 ± 0.73 to 8.40 ± 2.31 m·s -1 ; p < 0.05) and carotid radial PWV (9.77 ± 1.74 to 12.58 ± 2.09 m·s -1 ; p < 0.05) in the CON group alone. Both the HI and HV groups increased their maximum squat (36.6 ± 7.9 vs. 41.3 ± 31.8 percent change; p < 0.05), bench press (34.4 ± 12.6 vs. 23.4 ± 11.1 percent change; p < 0.05), and seated row (22.0 ± 12.6 vs. 21.9 ± 12.5 percent change; p < 0.05), respectively. Our findings support the use of resistance training exercise without undue impact on vascular compliance in otherwise healthy women.

Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9.

CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week. In addition, CRISPR-Cas can be used to consolidate multiple causal alleles into a single strain, bypassing the laborious traditional methods using marked constructs, or mating. These tools compress the engineering timeline sixfold or more, greatly increasing the productivity of the strain engineer.

Challenging the workhorse: Comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.

For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO-based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild-type eukaryotic micro-organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry-relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.

CRISPR-Cas-Assisted Multiplexing (CAM): Simple Same-Day Multi-Locus Engineering in Yeast.

Demands on the industrial and academic yeast strain engineer have increased significantly in the era of synthetic biology. Installing complex biosynthetic pathways and combining point mutations are tedious and time-consuming using traditional methods. With multiplex engineering tools, these tasks can be completed in a single step, typically achieving up to sixfold compression in strain engineering timelines. To capitalize on this potential, a variety of yeast CRISPR-Cas methods have been developed, differing largely in how the guide RNA (gRNA) reagents that direct the Cas9 nuclease are delivered. However, in nearly all reported protocols, the time savings of multiplexing is offset by multiple days of cloning to prepare the required reagents. Here, we discuss the advantages and opportunities of CRISPR-Cas-assisted multiplexing (CAM), a same-day, cloning-free method for multi-locus engineering in yeast. J. Cell. Physiol. 231: 2563-2569, 2016. © 2016 Wiley Periodicals, Inc.

Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas.

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated a similar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory.

Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.

Use of evidence-based practice among athletic training educators, clinicians, and students, part 1: perceived importance, knowledge, and confidence.

CONTEXT: Although evidence-based practice (EBP) has become more prevalent, athletic trainers' perceptions of importance and knowledge of these concepts and their confidence in EBP are largely unknown. OBJECTIVE: To assess perceived importance and knowledge of and confidence in EBP concepts in athletic trainers in various roles and with different degree levels. DESIGN: Cross-sectional study. SETTING: Online survey instrument. PATIENTS OR OTHER PARTICIPANTS: The survey was sent to 6702 athletic training educators, clinicians, and postprofessional students. A total of 1209 completed the survey, for a response rate of 18.04%. MAIN OUTCOME MEASURE(S): Demographic information and perceived importance and knowledge of and confidence in the steps of EBP were obtained. One-way analysis of variance, a Kruskal-Wallis test, and an independent-samples t test were used to determine differences in scores among the demographic variables. RESULTS: Athletic trainers demonstrated low knowledge scores (64.2% ± 1.29%) and mild to moderate confidence (2.71 ± 0.55 out of 4.0). They valued EBP as moderately to extremely important (3.49 ± 0.41 out of 4.0). Perceived importance scores differed among roles (clinicians unaffiliated with an education program scored lower than postprofessional educators, P = .001) and highest educational degree attained (athletic trainers with terminal degrees scored higher than those with bachelor's or master's degrees, P < .001). Postprofessional athletic training students demonstrated the highest total EBP knowledge scores (4.65 ± 0.91), whereas clinicians demonstrated the lowest scores (3.62 ± 1.35). Individuals with terminal degrees had higher (P < .001) total knowledge scores (4.31 ± 1.24) than those with bachelor's (3.78 ± 1.2) or master's degrees (3.76 ± 1.35). Postprofessional educators demonstrated greater confidence in knowledge scores (3.36 ± 0.40 out of 4.0) than did those in all other athletic training roles (P < .001). CONCLUSIONS: Overall knowledge of the basic EBP steps remained low across the various athletic trainers' roles. The higher level of importance indicated that athletic trainers valued EBP, but this value was not reflected in the knowledge of EBP concepts. Individuals with a terminal degree possessed higher knowledge scores than those with other educational preparations; however, EBP knowledge needs to increase across all demographics of the profession.

Use of evidence-based practice among athletic training educators, clinicians, and students, part 2: attitudes, beliefs, accessibility, and barriers.

CONTEXT: Successful implementation of evidence-based practice (EBP) within athletic training is contingent upon understanding the attitudes and beliefs and perceived barriers toward EBP as well as the accessibility to EBP resources of athletic training educators, clinicians, and students. OBJECTIVE: To assess the attitudes, beliefs, and perceived barriers toward EBP and accessibility to EBP resources among athletic training educators, clinicians, and students. DESIGN: Cross-sectional study. SETTING: Online survey instrument. PATIENTS OR OTHER PARTICIPANTS: A total of 1209 athletic trainers participated: professional athletic training education program directors (n = 132), clinical preceptors (n = 266), clinicians (n = 716), postprofessional athletic training educators (n = 24) and postprofessional students (n = 71). MAIN OUTCOME MEASURE(S): Likert-scale items (1 = strongly disagree, 4 = strongly agree) assessed attitudes and beliefs and perceived barriers, whereas multipart questions assessed accessibility to resources. Kruskal-Wallis H tests (P ≤ .05) and Mann-Whitney U tests with a Bonferroni adjustment (P ≤ .01) were used to determine differences among groups. RESULTS: Athletic trainers agreed (3.27 ± 0.39 out of 4.0) that EBP has various benefits to clinical practice and disagreed (2.23 ± 0.42 out of 4.0) that negative perceptions are associated with EBP. Benefits to practice scores (P = .002) and negative perception scores (P < .001) differed among groups. With respect to perceived barriers, athletic trainers disagreed that personal skills and attributes (2.29 ± 0.52 out of 4.0) as well as support and accessibility to resources (2.40 ± 0.40 out of 4.0) were barriers to EBP implementation. Differences were found among groups for personal skills and attributes scores (P < .001) and support and accessibility to resources scores (P < .001). Time (76.6%) and availability of EBP mentors (69.6%) were the 2 most prevalent barriers reported. Of the resources assessed, participants were most unfamiliar with clinical prediction rules (37.6%) and Cochrane databases (52.5%); direct access to these 2 resources varied among participants. CONCLUSIONS: Athletic trainers had positive attitudes toward the implementation of EBP within didactic education and clinical practice. However, accessibility and resource use remained low for some EBP-related resources. Although the perceived barriers to implementation are minimal, effective integration of EBP within athletic training will present challenges until these barriers dissolve.

Designing synthetic regulatory networks capable of self-organizing cell polarization.

How cells form global, self-organized structures using genetically encoded molecular rules remains elusive. Here, we take a synthetic biology approach to investigate the design principles governing cell polarization. First, using a coarse-grained computational model, we searched for all possible simple networks that can achieve polarization. All solutions contained one of three minimal motifs: positive feedback, mutual inhibition, or inhibitor with positive feedback. These minimal motifs alone could achieve polarization under limited conditions; circuits that combined two or more of these motifs were significantly more robust. With these design principles as a blueprint, we experimentally constructed artificial polarization networks in yeast, using a toolkit of chimeric signaling proteins that spatially direct the synthesis and degradation of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)). Circuits with combinatorial motifs yielded clear foci of synthetic PIP(3) that can persist for nearly an hour. Thus, by harnessing localization-regulated signaling molecules, we can engineer simple molecular circuits that reliably execute spatial self-organized programs.

Potential of light-harvesting proton pumps for bioenergy applications.

Concerns about the security and longevity of traditional energy sources have increased interest in alternative methods of energy production, particularly those which utilize abundantly available solar energy. Solar energy can be harvested either indirectly through the conversion of plant or algal byproducts into biofuels or directly using engineered microorganisms. Here we summarize the main features of light-harvesting proton pumps, which may provide a relatively simple way to boost the efficiency of energy-limited biological processes in fuel production. This family of proton pumps, which includes bacteriorhodopsin and proteorhodopsin, directly uses light energy to create a proton motive force (pmf) which can be used by other enzymes to facilitate active transport, regulate transmembrane proteins, or to generate ATP and NADH.

An assessment of burnout in undergraduate athletic training education program directors.

CONTEXT: Athletic training education program directors (ATEPDs) often manage their time among students, program administration, and patient care. OBJECTIVE: To assess the level of burnout in ATEPDs and to determine the relationship between burnout and various demographics of ATEPDs. DESIGN: Cross-sectional study. SETTING: Public and private colleges and universities nationwide. PATIENTS OR OTHER PARTICIPANTS: Two hundred forty-nine ATEPDs of undergraduate athletic training education programs accredited by the Commission on Accreditation of Athletic Training Education. INTERVENTION(S): We administered the Maslach Burnout Inventory (MBI) to all participants. MAIN OUTCOME MEASURE(S): The MBI consisted of 21 items assessing 3 characteristics of burnout: emotional exhaustion, depersonalization, and personal accomplishment. Another component of the survey requested demographic information about the ATEPDs. We used univariate, multivariate, and factorial analyses of variance with the alpha level set a priori at .05. We also calculated Pearson product moment correlation coefficients. RESULTS: Women had greater emotional exhaustion than men (20.67 +/- 9.43 and 16.47 +/- 9.64, respectively) (P = .001). The difference between tenure-status groups for emotional exhaustion was significant (P = .014), with tenure-track ATEPDs scoring higher on emotional exhaustion than tenured ATEPDs. Pearson product moment correlation coefficients revealed a weak negative relationship among emotional exhaustion and age (r = -0.263, P < .001), years of program director experience (r = -0.157, P = .013), and years at current job (r = -0.162, P = .010), indicating that as ATEPDs aged, gained more experience, and stayed in their current jobs, their emotional exhaustion scores decreased. There was also a weak negative relationship between age and depersonalization (r = -0.171, P = .007). There was a weak positive relationship between years at current job and personal accomplishment (r = 0.197, P = .002). CONCLUSIONS: We found that ATEPDs experienced a moderate form of emotional exhaustion burnout and low depersonalization and personal accomplishment burnout, with women experiencing greater emotional exhaustion than males. Additionally, ATEPDs in tenure-track positions experienced greater emotional exhaustion than tenured ATEPDs. The ATEPDs need to obtain healthy coping strategies early within their directorships to manage components related to burnout.

Experimental test of connector rotation during DNA packaging into bacteriophage phi29 capsids.

The bacteriophage phi29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.

Light-powering Escherichia coli with proteorhodopsin.

Proteorhodopsin (PR) is a light-powered proton pump identified by community sequencing of ocean samples. Previous studies have established the ecological distribution and enzymatic activity of PR, but its role in powering cells and participation in ocean energy fluxes remains unclear. Here, we show that when cellular respiration is inhibited by depleting oxygen or by the respiratory poison azide, Escherichia coli cells expressing PR become light-powered. Illumination of these cells with light coinciding with PR's absorption spectrum creates a proton motive force (pmf) that turns the flagellar motor, yielding cells that swim when illuminated with green light. By measuring the pmf of individual illuminated cells, we quantify the coupling between light-driven and respiratory proton currents, estimate the Michaelis-Menten constant (Km) of PR (10(3) photons per second/nm2), and show that light-driven pumping by PR can fully replace respiration as a cellular energy source in some environmental conditions. Moreover, sunlight-illuminated PR+ cells are less sensitive to azide than PR- cells, consistent with PR+ cells possessing an alternative means of maintaining cellular pmf and, thus, viability. Proteorhodopsin allows Escherichia coli cells to withstand environmental respiration challenges by harvesting light energy.