Blockage of the Early Events of Mitogenic Signaling by Interferon-gamma in Macrophages in Response to Colony-Stimulating Factor-1

Inhibition of cell proliferation is an important biologic funcphorbol ester, as measured by early gene expression, DNA tion of interferons (IFNs), which has been exploited in therasynthesis and cell proliferation. Although activation, phospeutic treatment of certain hematologic malignancies. Howphorylation, and turnover of the CSF-1 receptor and CSF-1- ever, the molecular mechanism was not clear. We have induced increase in diacylglycerol production remained norrecently shown that IFNs (/ and ) inhibit protein kinase mal, IFN- blocked CSF-1- stimulated activation of mitogen-C (PKC)-dependent (such as PDGF and phorbol ester) but activated protein kinases, Raf-1 kinase, increase in GTP-not PKC-independent (such as epidermal growth factor) actibound Ras and tyrosine phosphorylation, and activation of vation of Raf-1 and mitogen-activated protein kinases protein kinase C  (PKC-). PKC- was required for CSF-1- (MAPK/ERKs) in fibroblasts (Xu et al, Mol Cell Biol 14:8018, induced mitogenic signaling and a primary target for IFN-- 1994), suggesting a novel mechanism by which IFNs execute induced inhibition. Interestingly, although phorbol myristate their antiproliferative function. Monocytes/macrophages acetate stimulated Ras activation, PKC- did not appear to are primary targets in vivo for IFN-, the major activity of be an upstream activator of Ras. These studies clearly indimacrophage-activating factor. In the present study, mechacated that IFN- specifically inhibits PKC- activation, renism of IFN-- induced antiproliferative action in macrosulting in blockage of the early events of mitogenesis in phages in response to colony-stimulating factor- 1 (CSF-1) macrophages in response to CSF-1.     

Rare LRP6 Variants Identified in Spina Bifida Patients

Several single‐nucleotide variants (SNVs) in low‐density lipoprotein receptor‐related protein 6 (Lrp6) cause neural tube defects (NTDs) in mice. We therefore examined LRP6 in 192 unrelated infants from California with the NTD, spina bifida, and found four heterozygous missense SNVs, three of which were predicted to be deleterious, among NTD cases and not in 190 ethnically matched nonmalformed controls. Parents and siblings could not be tested because of the study design. Like Crooked tail and Ringleschwanz mouse variants, the p.Tyr544Cys Lrp6 protein failed to bind the chaperone protein mesoderm development and impaired Lrp6 subcellular localization to the plasma membrane of MDCK II cells. Only the p.Tyr544Cys Lrp6 variant downregulated canonical Wnt signaling in a TopFlash luciferase reporter in vitro assay. In contrast, three Lrp6 mutants (p.Ala3Val, p.Tyr544Cys, and p.Arg1574Leu) increased noncanonical Wnt/planar cell polarity (PCP) signaling in an Ap1‐luciferase assay. Thus, LRP6 variants outside of YWTD repeats could potentially predispose embryos to NTDs, whereas Lrp6 modulation of Wnt/PCP signaling would be more essential than its canonical pathway role in neural tube closure.     

Career and Family Aspirations of Female Athletic Trainers Employed in the National Collegiate Athletic Association Division I Setting

Context:Female athletic trainers (ATs) tend to depart the profession of athletic training after the age of 30. Factors influencing departure are theoretical. Professional demands, particularly at the collegiate level, have also been at the forefront of anecdotal discussion on departure factors.Objective:To understand the career and family intentions of female ATs employed in the collegiate setting.Design:Qualitative study.Setting:National Collegiate Athletic Association Division I.Results:Our participants indicated a strong desire to focus on family or to start a family as part of their personal aspirations. Professionally, many female ATs were unsure of their longevity within the Division I collegiate setting or even the profession itself, with 2 main themes emerging as factors influencing decisions to depart: family planning persistence and family planning departure. Six female ATs planned to depart the profession entirely because of conflicts with motherhood and the role of the AT. Only 3 female ATs indicated a professional goal of persisting at the Division I setting regardless of their family or marital status, citing their ability to maintain work-life balance because of support networks. The remaining 17 female ATs planned to make a setting change to balance the roles of motherhood and AT because the Division I setting was not conducive to parenting.Conclusions:Our results substantiate those of previous researchers, which indicate the Division I setting can be problematic for female ATs and stimulate departure from the setting and even the profession.Key Words: retention, attrition, work-life balance

Key Points

• Female athletic trainers decided to depart the Division I setting because the required hours of the job limited the time available for parenting.
• Female athletic trainers working in the Division I setting who were able to persist after having a family credit strong support networks and the development of effective work-life balance strategies.

Traditionally, working women endure more challenges balancing career demands and family responsibilities than working men, often because of their mothering philosophies and traditional gender stereotypes.¹ Surprisingly, gender differences have not been found in the occurrence of conflicts between work and life in the athletic training profession.^2,3 This finding is perplexing because female athletic trainers (ATs) continue to depart from the profession.⁴ Hypothetically, the decline in the number of female ATs in the profession has been linked to the desire to strike a balance among work responsibilities, personal interests, and family obligations.^1–3,5Concerns about work-life balance (WLB) and time for parenting have been found to influence decisions to persist within the collegiate levels, as the job responsibilities often include long hours (>40 h/wk) and travel, which can limit time spent at home with family.^1–3,5 It is an unfortunate reality that female ATs make up only approximately 28% of the full-time collegiate staff.⁵ This is especially concerning when the National Athletic Trainers'' Association indicates that more than 50% of its members are female.⁶ A relationship appears to exist between balancing professional responsibilities with parenthood and retention factors, especially for those who leave the collegiate clinical setting to work in clinical settings more favorable to family life.Female ATs in the National Collegiate Athletic Association Division I setting experience great challenges in maintaining WLB because of the demands of the setting.¹ In a recent study,¹ the primary reasons female ATs continued in the Division I setting were enjoyment of the job and atmosphere, increased autonomy, positive athlete dynamics, and the social support network. It is important for female ATs to have support at work and home to persist in the collegiate or athletic training clinical setting. However, long work hours and the inability to find WLB can stress this support network. Mazerolle and colleagues^2,3 first proposed that motherhood plausibly could lead to departure from the profession as the result of a myriad of factors but mostly because of a lack of time and control over work schedules. Further investigations have supported this theory and also have found that other reasons for leaving the profession are WLB concerns, supervisory and coach conflicts, caring for children, and role overload.^1,4,5Fulfillment of WLB is an important retention factor for female coaches within the collegiate setting,⁷ thus providing some supporting evidence to the suppositions that motherhood can be a mediating factor in the retention of female ATs in the collegiate setting. Additional support can be garnered from Mazerolle et al,² who found that only 22 female ATs with children were employed at the collegiate setting, a statistic supported by Kahanov et al,⁵ who reported that only about a quarter of all full-time ATs at the collegiate setting were female.Concerns about retention, particularly of female ATs, have become an increasingly popular topic within the athletic training literature, with attention focused on the collegiate clinical setting. This setting not only is one of the largest employment settings for the AT⁶ but is recognized as a time-intensive, demanding work environment.^2,3,8,9 Moreover, data suggest women are leaving this particular clinical setting to find a more family-friendly work environment, which may or may not be in the profession of athletic training.^10,11 Additionally, 2 recent studies^10,11 suggest that female athletic training students intend to pursue careers in athletic training, but as highlighted by Kahanov and Eberman,⁴ women are rapidly departing the profession for a variety of reasons. The emigration of female ATs from the profession has been theoretically associated with the desire to attain balance among family commitments, personal time, and work responsibilities.^1,2 Difficulties maintaining WLB and sufficient time for parenting shape decisions to continue at the collegiate level.^1,2Because of the concerning trend of female AT attrition, the purpose of our study was to understand the perspectives of female ATs, regardless of marital status, and to evaluate career and family intentions. Our objective was to gain a more thorough understanding of female ATs'' professional goals as they may be influenced by family planning. Our research questions included, “What factors influence the career intentions of female ATs regarding career longevity?” and “Do female ATs have intentions to remain in the NCAA Division I setting?”     

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.Complexities of natural biological systems make it difficult to understand and define precisely the roles of individual genes and their integrated functions. The use of model organisms with a relatively small number of genes enables the isolation of core biological processes from their complex regulatory networks for extensive characterization. However, even the simplest natural microbes contain many genes of unknown function, as well as genes that can be singly or simultaneously deleted without any noticeable effect on growth rate in a laboratory setting (Hutchison et al. 1999; Glass et al. 2006; Posfai et al. 2006). Ill-defined genes and those mediating functional redundancies both compound the challenge of understanding even the simplest life forms.Toward generating a minimal cell where every gene is essential for the axenic viability of the organism, we are pursuing strategies to reduce the 1-Mb genome of Mycoplasma mycoides JCVI-syn1.0 (Gibson et al. 2010). Because we can (1) introduce this genome into yeast and maintain it as a plasmid (Benders et al. 2010; Karas et al. 2013a); and (2) “transplant” the genome from yeast into mycoplasma recipient cells (Lartigue et al. 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering. Here we further exploit distinctive features of yeast for this purpose.Methods for serially replacing genomic regions with selectable markers are limited by the number of available markers. One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al. 2001). We have previously used a self-excising marker (Noskov et al. 2010) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 ∆1-6) (Karas et al. 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming. When applied to poorly characterized genes with the potential to interact with other genes, some paths for multigene knockout may lead to dead ends that result from synergistic mutant phenotypes. When a dead end is reached, sequentially returning to a previous genome in an effort to find a detour to a viable higher-order multimutant may be prohibitively time-consuming.An alternative approach to multigene engineering, available in yeast, is to prepare a set of single mutants and combine the deletions into a single strain via cycles of mating and meiotic recombination (Fig. 1A; Pinel et al. 2011; Suzuki et al. 2011, 2012). With a green fluorescent protein (GFP) reporter gene inserted in each deletion locus, the enrichment of higher-order yeast deletion strains in the meiotic population can be accomplished using flow cytometry. Here we apply this method to the JCVI-syn1.0 ∆1-6 exogenous, bacterial genome harbored in yeast to nonsequentially assemble deletions for genes predicted to be individually deletable based on biological knowledge or transposon-mediated disruption data. The functional identification of simultaneously deletable regions is expected to accelerate the effort to construct a minimal genome.Open in a separate windowFigure 1.Progressive clustering of deleted genomic segments. (A) Scheme of the method. Light blue oval represents a bacterial cell. Black ring or horizontal line denotes a bacterial genome, with the orange box indicating the yeast vector used as a site for linearization and recircularization. Gray shape denotes a yeast cell. Green dot in the genome indicates a deletion replaced with a GFP marker. (B) Map of deleted regions. Orange box indicates the yeast vector sequence used for genome linearization and recircularization. Green boxes indicate regions deleted in multimutant mycoplasma strains. Blue boxes denote restriction modification (RM) systems that are also deleted in the strains. (C) Pulsed-gel electrophoresis result for deleted genomes. The starting strain was the JCVI-syn1.0 ∆1–6 strain (1062 kb). Two strains were analyzed for each design of simultaneous deletion (962 kb for eight-deletion or 974 kb for seven-deletion genome). Ladder is a set of yeast chromosomes (New England BioLabs). (D) GFP-RFP ratio sorting result. Standard sorting was compared with sorting based on a GFP-RFP ratio (Methods).     

Experimental comparisons of passive and powered ankle-foot orthoses in individuals with limb reconstruction

Background

Ankle-foot orthoses (AFO) are commonly prescribed to provide functional assistance for patients with lower limb injuries or weakness. Their passive mechanical elements can provide some energy return to improve walking ability, but cannot restore plantar flexor push-off. Powered AFOs provide an assistive torque about the ankle to address the limitations of passive devices, but current designs have yet to be implemented on a large scale clinically. Purpose: To compare passive AFOs to a new untethered, powered AFO design in a clinical population with lower limb reconstruction.

Methods

A crossover study design, conducted on three individuals with lower limb reconstruction, compared gait mechanics at a standardized speed (based on leg length) in 4 AFO conditions: 1. None (shoes only), 2. Blue Rocker (BR, Allard, USA), 3. Intrepid Dynamic Exoskeletal Orthosis (IDEO), and 4. PowerFoot Orthosis (PFO BionX Medical Technologies, Inc.). The PFO was a custom, battery-powered device whose damping and power were capable to being tuned to meet patient needs. Subjects performed biomechanical gait analysis and metabolic testing at slow, moderate and fast speeds. Dependent variables included total limb power (calculated using a unified deformable segment model), mechanical work, mechanical efficiency, ankle motion, net metabolic cost across three speeds, and performance measures were calculated. Effect sizes (d) were calculated and d?>?0.80 denoted a large effect.

Results

Net positive work (d > 1.17) and efficiency (d > 1.43) were greatest in the PFO. There were large effects for between limb differences in positive work for all conditions except the PFO (d?=?0.75). The PFO normalized efficiency between the affected and unaffected limbs (d?=?0.50), whereas efficiency was less on the affected limb for all other conditions (d > 1.69). Metabolic rate was not consistently lowest in any one AFO condition across speeds. Despite some positive results of the PFO, patient preferred their daily use AFO (2 IDEO, 1 BR). All participants indicated that mass and size were concerns with using the PFO.

Conclusions

A novel PFO resulted in more biomimetic mechanical work and efficiency than commercially-available and custom passive AFO models. Although the powered AFO provided some biomechanical benefits, further improvements are warranted to improve patient satisfaction.

     

Leadership Identity Development Through Reflection and Feedback in Team-Based Learning Medical Student Teams

Problem: Studies on leadership identity development through reflection with Team-Based Learning (TBL) in medical student education are rare. We assumed that reflection and feedback on the team leadership process would advance the progression through leadership identity development stages in medical students within the context of classes using TBL. Intervention: This study is a quasi-experimental design with pretest–posttest control group. The pretest and posttest were reflection papers of medical students about their experience of leadership during their TBL sessions. In the intervention group, TBL and a team-based, guided reflection and feedback on the team leadership process were performed at the end of all TBL sessions. In the other group, only TBL was used. The Stata 12 software was used. Leadership Identity was treated both as a categorical and quantitative variable to control for differences in baseline and gender variables. Chi-square, t tests, and linear regression analysis were performed. Context: The population was a cohort of 2015–2016 medical students in a TBL setting at Tehran University of Medical Sciences, School of Medicine. Teams of four to seven students were formed by random sorting at the beginning of the academic year (intervention group n = 20 teams, control group n = 19 teams). Outcome: At baseline, most students in both groups were categorized in the Awareness and Exploration stage of leadership identity: 51 (52%) in the intervention group and 59 (55%) in the control group: uncorrected χ²(3) = 15.6, design-based F(2.83, 108) = 4.87, p = .003. In the posttest intervention group, 36 (36%) were in exploration, 33 (33%) were in L-identified, 20 (20%) were in Leadership Differentiated, and 10 (10%) were in the Generativity. None were in the Awareness or Integration stages. In the control group, 3 (20%) were in Awareness, 56 (53%) were in Exploration, 35 (33%) were in Leader Identified, 13 (12%) were in Leadership Differentiated. None were in the Generativity and Integration stages. Our hypothesis was supported by the data: uncorrected χ²(4) = 18.6, design-based F(3.77, 143) = 4.46, p = .002. The mean of the leadership identity in the pretest, intervention group equaled 1.93 (SD = 0.85) and the pretest, control group mean was 2.36 (SD = 0.86), p = .004. The mean of the posttest, intervention group was 3.04 (SD = 0.98) and posttest, control group mean was 2.54 (SD = 0.74), T = ?4.00, design df = 38, p < .001, and adjusted on baseline and gender T = ?8.97, design df = 38, p < .001. Lessons Learned: Reflection and feedback on the team leadership process in TBL advances the progression in stages of leadership identity development in medical students. Although the TBL strategy itself could have an impact on leadership identity development, this study demonstrates that when a reflection and feedback on leadership intervention are added, there is much greater impact.     

Cognitive Functional Therapy (CFT) for chronic non-specific neck pain

This case report presents the effect of Cognitive Functional Therapy (CFT) in a patient with chronic non-specific neck pain. The patient believed that pain signified tissue damage, and demonstrated pain catastrophizing, hypervigilance, stress sensitivity, and movement impairment of the neck, during extension and rotation. The CFT intervention integrated a cognitive approach with manual therapy and active exercises to encourage the patient to trust her neck again. One month after the first appointment, the patient had recovered confidence, and the pain and disability had disappeared almost entirely.