Unusual N-acetylated sugars have been observed on the O-antigens of some Gram-negative bacteria and on the S-layers of both Gram-positive and Gram-negative bacteria. One such sugar is 3-acetamido-3,6-dideoxy-α-d-galactose or Fuc3NAc. The pathway for its production requires five enzymes with the first step involving the attachment of dTMP to glucose-1-phosphate. Here we report a structural and biochemical characterization of a bifunctional enzyme from Shewanella denitificans thought to be involved in the biosynthesis of dTDP-Fuc3NAc. On the basis of a bioinformatics analysis, the enzyme, hereafter referred to as FdtD, has been postulated to catalyze the third and fifth steps in the pathway, namely a 3,4-keto isomerization and an N-acetyltransferase reaction. For the X-ray analysis reported here, the enzyme was crystallized in the presence of dTDP and CoA. The crystal structure shows that FdtD adopts a hexameric quaternary structure with 322 symmetry. Each subunit of the hexamer folds into two distinct domains connected by a flexible loop. The N-terminal domain adopts a left-handed β-helix motif and is responsible for the N-acetylation reaction. The C-terminal domain folds into an antiparallel flattened β-barrel that harbors the active site responsible for the isomerization reaction. Biochemical assays verify the two proposed catalytic activities of the enzyme and reveal that the 3,4-keto isomerization event leads to inversion of configuration about the hexose C-4' carbon.

It has become increasingly apparent within the last several years that unusual N-formylated sugars are often found on the O-antigens of such Gram negative pathogenic organisms as Francisella tularensis, Campylobacter jejuni, and Providencia alcalifaciens, amongst others. Indeed, in some species of Brucella, for example, the O-antigen contains 1,2-linked 4-formamido-4,6-dideoxy-α-D-mannosyl groups. These sugars, often referred to as N-formylperosamine, are synthesized in pathways initiating with GDP-mannose. One of the enzymes required for the production of N-formylperosamine, namely WbkC, was first identified in 2000 and was suggested to function as an N-formyltransferase. Its biochemical activity was never experimentally verified, however. Here we describe a combined structural and functional investigation of WbkC from Brucella melitensis. Four high resolution X-ray structures of WbkC were determined in various complexes to address its active site architecture. Unexpectedly, the quaternary structure of WbkC was shown to be different from that previously observed for other sugar N-formyltransferases. Additionally, the structures revealed a second binding site for a GDP molecule distinct from that required for GDP-perosamine positioning. In keeping with this additional binding site, kinetic data with the wild type enzyme revealed complex patterns. Removal of GDP binding by mutating Phe 142 to an alanine residue resulted in an enzyme variant displaying normal Michaelis-Menten kinetics. These data suggest that this nucleotide binding pocket plays a role in enzyme regulation. Finally, by using an alternative substrate, we demonstrate that WbkC can be utilized to produce a trideoxysugar not found in nature.

Background Occupational stress in resident physicians has profound implications for wellness, professionalism, and patient care. This observational pilot trial measured psychological and physiological stress biomarkers before, during, and after the start of anesthesia residency. Methods Eighteen physician interns scheduled to begin anesthesia residency were recruited for evaluation at three time points: baseline (collected remotely before residency in June 2013); first month visit 1 (July); and follow-up visit 2 (residency month 3–5, Sept–Nov). Validated scales were used to measure stress, anxiety, resilience, and wellness at all 3 time points. During visits 1 and 2, we measured resting heart rate variability, responses to laboratory mental stress (hemodynamic, catecholamine, cortisol, and interleukin-6) and chronic stress indices (C-reactive protein, 24 h ambulatory heart rate and blood pressure, 24 h urinary cortisol and catecholamines, overnight heart rate variability). Results Thirteen interns agreed to participate (72% enrollment). There were seven men and six women, ages 27–33 years. The mean ± SD of all study variables are reported. Conclusion The novelty of our report is the prospective design in a defined cohort of residents newly exposed to the similar occupational stress of the operating environment. Because of the paucity of literature specific to the measures and stress conditions in this investigation, no data were available to generate a priori definition of primary outcomes and a data analytic plan. These findings will allow power analysis for future design of trials examining occupational stress and stress-reducing interventions. Given the importance of physician burnout in our country, the impact of chronic stress on resident wellness requires further study.

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Perception of limb and body positions is known as proprioception. Sensory feedback, especially from proprioceptive receptors, is essential for motor control. Aging is associated with a decline in position sense at proximal joints, but there is inconclusive evidence of distal joints being equally affected by aging. In addition, there is initial evidence that physical activity attenuates age-related decline in proprioception. Our objectives were, first, to establish wrist proprioceptive acuity in a large group of seniors and compare their perception to young adults, and second, to determine if specific types of training or regular physical activity are associated with preserved wrist proprioception. We recruited community-dwelling seniors (n = 107, mean age, 70 ± 5 years, range, 65–84 years) without cognitive decline (Mini Mental State Examination-brief version ≥13/16) and young adult students (n = 51, mean age, 20 ± 1 years, range, 19–26 years). Participants performed contralateral and ipsilateral wrist position sense matching tasks with a bimanual wrist manipulandum to a 15° flexion reference position. Systematic error or proprioceptive bias was computed as the mean difference between matched and reference position. The respective standard deviation over five trials constituted a measure of random error or proprioceptive precision. Current levels of physical activity and previous sport, musical, or dance training were obtained through a questionnaire. We employed longitudinal mixed effects linear models to calculate the effects of trial number, sex, type of matching task and age on wrist proprioceptive bias and precision. The main results were that relative proprioceptive bias was greater in older when compared to young adults (mean difference: 36% ipsilateral, 88% contralateral, p < 0.01). Proprioceptive precision for contralateral but not for ipsilateral matching was smaller in older than in young adults (mean difference: 38% contralateral, p < 0.01). Longer years of dance training were associated with smaller bias during ipsilateral matching (p < 0.01). Other types of training or physical activity levels did not affect bias or precision. Our findings demonstrate that aging is associated with a decline in proprioceptive bias in distal arm joints, but age does not negatively affect proprioceptive precision. Further, specific types of long-term dance related training may attenuate age-related decline in proprioceptive bias.

Serious adverse events of syncope have been reported with single pulse transcranial magnetic stimulation (sp-TMS) in both healthy adults and adolescents post stroke [3,5,[7][8][9]. Despite these reports, the physiological mechanisms leading to syncope during sp-TMS administration are not clearly understood. A syncopal event is classically identified by a sudden, large reduction in blood supply to neural tissue "cerebral hypoperfusion" to ultimately cause a loss of consciousness [2]. Common symptoms preceding syncope include lightheadedness, nausea, diaphoresis, and/or visual changes. Syncopal events may occur via an irregular autonomic nervous system (ANS) response to emotional or administered stimuli, which is commonly known as neurocardiogenic syncope [2,4]. Neurocardiogenic syncope can result from transient mean arterial pressure (MAP) and heart rate (HR) changes in response to precipitating factors such as anxiety in combination with a mildly noxious, novel stimulus [4]. Although literature is sparse, children with perinatal stroke appear to be at an additional increased risk for autonomic dysfunction [1] and autonomic immaturity in typically-developing pediatric populations increases the risk for neurocardiogenic syncope [4]. We aimed to determine if sp-TMS caused immediate cardiovascular changes in in children and adolescents with congenital hemiparesis due to perinatal stroke.Seven males (12±4 yrs, average±sd) and eight females (14±5 yrs) completed an experimental session as part of a larger clinical trial (). A 70-mm-diameter Magstim figureof-eight coil (Magstim, Whitland, U.K.) with a Magstim 200 2 unit delivered stimulation for

Purpose: To determine if acute slow breathing at 6 breaths/min would improve baroreflex sensitivity (BRS) and heart rate variability (HRV), and lower blood pressure (BP) in adults after stroke.Methods: Twelve individuals completed two randomized study visits where they performed a 15-min bout of breathing exercises at 6 breaths/min (slow) and at 12 breaths/min (control). Continuous BP and heart rate (HR) were measured throughout, and BRS, BRS response to elevations in blood pressure (BRSup), BRS response to depressions in blood pressure (BRSdown), and HRV were calculated and analyzed before (pre), during, and after (post) breathing exercises.Results: BRS increased from pre to post slow breathing by 10% (p = 0.012), whereas BRSup increased from pre to during slow breathing by 30% ( p = 0.04). BRSdown increased from pre to post breathing for both breathing conditions (p < 0.05). HR (control: Δ − 4 ± 4; slow: Δ − 3 ± 4 beats/min, time, p < 0.01) and systolic BP (control: Δ − 0.5 ± 5; slow: Δ − 6.3 ± 8 mmHg, time, p < 0.01) decreased after both breathing conditions. Total power, low frequency power, and standard deviation of normal inter-beat intervals (SDNN) increased during the 6-breaths/min condition (condition × time, p < 0.001), whereas high frequency increased during both breathing conditions (time effect, p = 0.009).Conclusions: This study demonstrated that in people post-stroke, slow breathing may increase BRS, particularly BRSup, more than a typical breathing space; however, paced breathing at either a slow or typical breathing rate appears to be beneficial for acutely decreasing systolic BP and HR and increasing HRV.

Ventilation/carbon dioxide production (VE/VCO2slope) is used clinically to determine cardiorespiratory fitness and morbidity in heart failure (HF). Previously, we demonstrated that lower lean leg mass is associated with high VE/VCO2slope during exercise in HF. In healthy individuals, we evaluated 1) whether VE/VCO2slope differed between lean and overweight women and 2) the relationship between lean leg mass and VE/VCO2slope in overweight sedentary (OWS), overweight trained (OWTR) and lean, trained (LTR) women.MethodsGas exchange and ventilation were collected during a treadmill peak oxygen uptake test (VO2peak) in 40 women [26 OWS (29 ± 7 yrs., mean ± SD), 7 OWTR (33 ± 5 yrs) and 7 LTR (26 ± 6 yrs)]. Body composition was measured by dual X-ray absorptiometry.ResultsVO2peak was highest in LTR (46.6 ± 8 ml/kg/min) compared with OWTR (38.1 ± 4.9 ml/kg/min) and OWS women (25.3 ± 4.8 ml/kg/min, p < 0.05). Lean leg mass was highest in OWTR and lowest in LTR women (p < 0.05). VE/VCO2slope was similar between groups (p > 0.05). Higher lean leg mass was associated with lower VE/VCO2slope in overweight women (OWS + OWTR: r = −0.55, p < 0.001), contrasting with higher VE/VCO2slope in LTR women (r = 0.86, p < 0.001).ConclusionsThese findings suggest VE/VCO2slope may not differentiate between low and high cardiorespiratory fitness in healthy individuals and muscle mass may play a role in determining the VE/VCO2slope, independent of disease.