Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery.

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification remains highly challenging. Here, we perform CRISPR scanning on the essential maintenance methylation machinery-DNMT1 and its partner UHRF1-with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. In contrast to non-covalent DNMT1 inhibition, activity-based selection implicates numerous regions outside the catalytic domain in DNMT1 function. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function, exhibiting increased DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our study highlights the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, and more broadly, introduces new analytical tools that further refine the CRISPR scanning framework.

A Review: The Prospect of Inhaled Insulin Therapy via Vibrating Mesh Technology to Treat Diabetes.

Background: Inhaled insulin has proven to be viable and, in some aspects, a more effective alternative to subcutaneous insulin. Past and present insulin inhaler devices have not found clinical or commercial success. Insulin inhalers create a dry powder or soft mist insulin aerosol, which does not provide the required uniform particle size or aerosol volume for deep lung deposition. Methods: The primary focus of this review is to investigate the potential treatment of diabetes with a wet insulin aerosol. Vibrating mesh nebulisers allow the passive inhalation of a fine wet mist aerosol for the administration of drugs to the pulmonary system in higher volumes than other small-volume nebulisers. Results: At present, there is a significant focus on vibrating mesh nebulisers from the pharmaceutical and biomedical industries for the systemic administration of pharmaceuticals for non-traditional applications such as vaccines or the treatment of diabetes. Systemic drug administration using vibrating mesh nebulisers leads to faster-acting pharmaceuticals with a reduction in drug latency. Conclusions: Systemic conditions such as diabetes, require the innovative development of custom vibrating mesh devices to provide the desired flow rates and droplet size for effective inhaled insulin administration.

Interaction Between the a3 Region of Factor VIII and the TIL'E' Domains of the von Willebrand Factor.

The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two ß-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the ß-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the ß-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.

Prevalence of Exercise-Induced Arterial Hypoxemia in Distance Runners at Sea Level.

PURPOSE: It has been reported that ~50% of endurance-trained men demonstrate exercise-induced arterial hypoxemia (EIAH) during heavy exercise. However, this often-cited prevalence rate comes from a single study using a cohort of 25 highly trained men who completed maximal cycle ergometry. As arterial oxyhemoglobin saturation (SpO2) during maximal exercise is reported to be significantly lower during treadmill versus cycle ergometry in the same subjects, we hypothesized that the prevalence of EIAH would be greater than previously reported (and commonly referenced) in a larger cohort of highly endurance-trained men during maximal treadmill running. METHODS: Data from 124 highly trained male distance runners (V˙O2max range = 60.3-84.7 mL·kg·min) were retrospectively examined from previously published studies completed in the Indiana University Human Performance Laboratory. Subjects completed a constant speed, progressive-grade treadmill exercise test to volitional exhaustion, and arterial oxyhemoglobin saturation (SaO2ear) in all subjects was estimated using the same oximeter (Hewlett Packard 47201A). RESULTS: Using similar inclusion criteria as previously published for highly trained (V˙O2max > 68 mL·kg·min) and for EIAH (SaO2ear ≤ 91%), 55 of 79 subjects (70%) exhibited exercise-induced arterial desaturation. Across all 124 subjects, 104 (84%) demonstrated at least moderate EIAH (SaO2ear ≤ 93%) during maximal treadmill exercise. SaO2ear was significantly yet weakly correlated with V˙E/V˙O2 (P < 0.01, r = 0.28) and V˙E/V˙CO2 (P < 0.001, r = 0.33) but not with V˙O2max. CONCLUSION: These results indicate that the prevalence of EIAH in highly trained men during maximal treadmill exercise at sea level is greater compared with previously suggested data, with exercise mode perhaps playing a factor in the number of athletes who experience EIAH.

Runners maintain locomotor-respiratory coupling following isocapnic voluntary hyperpnea to task failure.

INTRODUCTION: Evidence has long suggested that mammalian ventilatory and locomotor rhythms are linked, yet determinants and implications of locomotor-respiratory coupling (LRC) continue to be investigated. Anecdotally, respiratory muscle fatigue seen at the end of heavy exercise may result in an uncoupling of movement-ventilation rhythms; however, there is no scientific evidence to substantiate this claim. PURPOSE: We sought to determine whether or not fatigue of the respiratory muscles alters locomotor-respiratory coupling patterns typically observed in highly trained individuals while running. A related query was to examine the relationship between the potential changes in LRC and measures of running economy. METHOD: Twelve male distance runners ran at four submaximal workloads (68-89 % VO2peak) on two separate days while LRC was quantified. One LRC trial served as a control (CON), while the other was performed following an isocapnic voluntary hyperpnea to task failure to induce respiratory muscle fatigue (FT+). LRC was assessed as stride-to-breathing frequency ratios (SF/fB) and degree of LRC (percentage of breaths occurring during the same decile of the step cycle). RESULT: Hyperpnea resulted in significant declines in maximal voluntary inspiratory (MIP) and expiratory (MEP) mouth pressures (ΔMIP = -10 ± 12 cm H2O; ΔMEP = -6 ± 9 cm H2O). There were no differences in minute ventilation between CON and FT+ (CON, all speeds pooled = 104 ± 25 L min(-1); FT+ pooled = 106 ± 23 L min(-1)). Stride frequency was not different between trials; however, breathing frequency was significantly greater during FT+ compared to CON at all speeds (CON pooled = 47 ± 10 br min(-1); FT+ pooled = 52 ± 9 br min(-1)), resulting in smaller corresponding SF/fB. Yet, the degree of LRC was the same during CON and FT+ (CON pooled = 63 ± 15 %; FT+ pooled = 64 ± 18 %). CONCLUSION: The results indicate that trained runners are able to continue entraining breath and step cycles, despite marked changes in exercise breathing frequency, after a fatiguing hyperpnea challenge.

Characterization of the microstructure of GaP films grown on {111} Si by liquid phase epitaxy.

The development of a cost-effective Si based platform on which III-V's can be grown is of great interest. This work investigates the morphology of gallium phosphide (GaP) films grown on {111} silicon (Si) substrates by means of liquid phase epitaxy in a tin (Sn) - based solvent bath. Two types of single-crystal {111} Si substrates were used; the first type was oriented exactly along the ⟨111⟩ surface (no-miscut) and the second was miscut by 4°. The growth rate of the GaP films was found to be markedly different for the two types of substrates; the GaP films on the miscut Si substrate grew ∼4 times faster than those on the no-miscut substrate. The GaP films grew epitaxially on both types of substrates, but contained Si and Sn as inclusions. In the case of the no-miscut substrate, a number of large Sn particles were incorporated at the GaP/Si interface. As a result, these interfacial Sn particles affected the strain state of the GaP films dramatically, which, in turn, manifested itself in the form of a duplex microstructure that consists of strained and strain-free regions.

Ventilatory patterns differ between maximal running and cycling.

To determine the effect of exercise mode on ventilatory patterns, 22 trained men performed two maximal graded exercise tests; one running on a treadmill and one cycling on an ergometer. Tidal flow-volume (FV) loops were recorded during each minute of exercise with maximal loops measured pre and post exercise. Running resulted in a greater VO2peak than cycling (62.7±7.6 vs. 58.1±7.2mLkg(-1)min(-1)). Although maximal ventilation (VE) did not differ between modes, ventilatory equivalents for O2 and CO2 were significantly larger during maximal cycling. Arterial oxygen saturation (estimated via ear oximeter) was also greater during maximal cycling, as were end-expiratory (EELV; 3.40±0.54 vs. 3.21±0.55L) and end-inspiratory lung volumes, (EILV; 6.24±0.88 vs. 5.90±0.74L). Based on these results we conclude that ventilatory patterns differ as a function of exercise mode and these observed differences are likely due to the differences in posture adopted during exercise in these modes.

Ground contact time as an indicator of metabolic cost in elite distance runners.

UNLABELLED: Differences in running economy at common speeds have been demonstrated between male and female distance runners, as well as between middle-distance (MD) and long-distance (LD) specialists. Whether measures of foot ground contact time (tc), known to be proportional to the mass-specific cost of locomotion, follows the same running economy relationships in these groups is unknown. PURPOSE: This study examined if differences in tc and selected gait kinematic variables exist between elite male and female distance runners, as well as between elite MD and LD specialists, as running speed increases. METHODS: Twelve male and six female elite distance runners completed multiple 30-s trials on a treadmill at common competitive racing velocities. Wireless triaxial 10-g accelerometers, sampling at 1024 Hz, were securely attached to the laces of each shoe. Values of tc, swing time, stride length, and stride frequency were determined from accelerometric output corresponding to foot strike and toe-off events obtained from a minimum of 20 consecutive steps of each foot. A proportional estimate of metabolic cost was obtained by using 1/tc. RESULTS: Women displayed shorter tc, swing time, and stride length with greater stride frequency compared with men at common speeds; however, these differences were largely negated by normalizing to standing height. At common speeds, women demonstrated smaller measures of tc compared with men, suggesting an increased metabolic cost, paralleling published oxygen uptake data. MD specialists displayed smaller increases in 1/tc as speed increased, compared with LD specialists. CONCLUSIONS: Elite distance runners demonstrate ground contact measures that suggest that known differences in running economy between sexes and event specialties may be a result of differences in running gait.

Shaken baby syndrome and a triple-dose strategy for its prevention.

OBJECTIVES: Inflicted traumatic brain injury associated with Shaken Baby Syndrome (SBS) is a leading cause of injury mortality and morbidity in infants. A triple-dose SBS prevention program was implemented with the aim to reduce the incidence of SBS. The objectives of this study were to describe the epidemiology of SBS, the triple-dose prevention program, and its evaluation. METHODS: Descriptive and spatial epidemiologic profiles of SBS cases treated at Children's Hospital, London Health Sciences Centre, from 1991 to 2010 were created. Dose 1 (in-hospital education): pre-post impact evaluation of registered nurse training, with a questionnaire developed to assess parents' satisfaction with the program. Dose 2 (public health home visits): process evaluation of additional education given to new parents. Dose 3 (media campaign): a questionnaire developed to rate the importance of factors on a 7-point Likert scale. These factors were used to create weights for statistical modeling and mapping within a geographic information system to target prevention ads. RESULTS: Forty-three percent of severe infant injuries were intentional. A total of 54 SBS cases were identified. The mean age was 6.7 months (standard deviation, 10.9 months), with 61% of infant males. The mean Injury Severity Score was 26.3 (standard deviation, 5.5) with a 19% mortality rate. Registered nurses learned new information on crying patterns and SBS, with a 47% increase in knowledge posttraining (p < 0.001). Over 10,000 parents were educated in-hospital, a 93% education compliance rate. Nearly all parents (93%) rated the program as useful, citing "what to do when the crying becomes frustrating" as the most important message. Only 6% of families needed to be educated during home visits. Locations of families with a new baby, high population density, and percentage of lone parents were found to be the most important factors for selecting media sites. The spatial analysis revealed six areas needed to be targeted for ad locations. CONCLUSIONS: SBS is a devastating intentional injury that often results in poor outcomes for the child. Implementing a triple-dose prevention program that provides education on crying patterns, coping strategies, and the dangers of shaking is key to SBS prevention. The program increased knowledge. Parents rated the program as useful. The media campaign allowed us to extend the primary prevention beyond new parents to help create a cultural change in the way crying, the primary trigger for SBS, is viewed. Targeting our intervention increased the likelihood that our message was reaching the population in greatest need.

Evolution of the courtship display of Melittobia (Hymenoptera: Eulophidae).

Melittobia, a polyphagous pupal parasitoid of many solitary bees and wasps, has a complex courtship display, the evolutionary history of which is poorly understood. The current phylogeny of this genus suggests the complexity of the courtship has increased over time. We test this hypothesis using Bayesian and Maximum likelihood techniques to construct a molecular phylogeny of Melittobia-the first for this genus using modern techniques--using two intergenic regions (Internal Transcribed Spacer regions 1 and 2) and Cytochrome Oxidase 1 (CO1), and reconstruct the ancestral state of each major node in the phylogeny for five behaviors coincidental to major shifts in display complexity. We show that species-groups identified solely on behavioral characters are reliable, although the relationships among groups are different than those previously hypothesized. We also show that behaviors traditionally used in the courtship studies do not support a hypothesis of increasing display complexity over time.

Impairment of 3000-m run time at altitude is influenced by arterial oxyhemoglobin saturation.

UNLABELLED: The decline in maximal oxygen uptake (ΔVO(2)max) with acute exposure to moderate altitude is dependent on the ability to maintain arterial oxyhemoglobin saturation (SaO2). PURPOSE: This study examined if factors related to ΔVO(2)max at altitude are also related to the decline in race performance of elite athletes at altitude. METHODS: Twenty-seven elite distance runners (18 men and 9 women, VO(2)max = 71.8 ± 7.2 mL·kg(-1)·min(-1)) performed a treadmill exercise at a constant speed that simulated their 3000-m race pace, both in normoxia and in 16.3% O2 (∼2100 m). Separate 3000-m time trials were completed at sea level (18 h before altitude exposure) and at 2100 m (48 h after arrival at altitude). Statistical significance was set at P ≤ 0.05. RESULTS: Group 3000-m performance was significantly slower at altitude versus sea level (48.5 ± 12.7 s), and the declines were significant in men (48.4 ± 14.6 s) and women (48.6 ± 8.9 s). Athletes grouped by low SaO2 during race pace in normoxia (SaO2 < 91%, n = 7) had a significantly larger ΔVO(2) in hypoxia (-9.2 ± 2.1 mL·kg(-1)·min(-1)) and Δ3000-m time at altitude (54.0 ± 13.7 s) compared with athletes with high SaO2 in normoxia (SaO2 > 93%, n = 7, ΔVO(2) = -3.5 ± 2.0 mL·kg(-1)·min(-1), Δ3000-m time = 38.9 ± 9.7 s). For all athletes, SaO2 during normoxic race pace running was significantly correlated with both ΔVO(2) (r = -0.68) and Δ3000-m time (r = -0.38). CONCLUSIONS: These results indicate that the degree of arterial oxyhemoglobin desaturation, already known to influence ΔVO(2)max at altitude, also contributes to the magnitude of decline in race performance at altitude.

Hip fractures in long-term care: is the excess explained by the age and gender distribution of the residents?

Introduction. This study compares hip fracture rates in Long Term Care (LTC) residents with those in the community to determine if their high rate of fracturing reflects the extreme age and predominantly female nature of that population. Methods. Hospital discharge data in London Ontario (population 350,000) and Statistics Canada data were used to correct the hip fracture rate in the LTC setting for age and gender. Results. The risk of hip fracture is 1.8 times greater in LTC than in the community for people of similar age and gender. The rate in women is 1.5 times higher whereas in men it is 4.3 times higher. In the oldest residents, the risk in men exceeds that of women in LTC. Conclusion. The high hip fracture rate in LTC is not just a reflection of the age and predominantly female nature of this population. The oldest men in LTC are a particularly high risk group, deserving more attention.

Adaptation or constraint? Reference-dependent scatter in honey bee dances.

The waggle dance of the honey bee is used to recruit nest mates to a resource. Dancer bees, however, may indicate many directions within a single dance bout; we show that this scatter in honey bee dances is strongly dependent on the sensory modality used to determine a reference angle in the dance. Dances with a visual reference are more precise than those with a gravity reference. This finding undermines the idea that scatter is introduced into dances, which the bees could perform more precisely, in order to spread recruits out over resource patches. It also calls into question reported interspecific differences that had been interpreted as adaptations of the dance to different habitats. Our results support a non-adaptive hypothesis: that dance scatter results from sensory and performance constraints, rather than modulation of the scatter by the dancing bee. However, an alternative adaptive hypothesis cannot be ruled out.

Does Imprecision in The Waggle Dance Fit Patterns Predicted by The Tuned-Error Hypothesis?

The waggle dance of the honey bee is used to recruit nest mates to a resource, though direction indicated for a resource may vary greatly within a single dance. Some authors suggest that this variation exits as an adaptation to distribute recruits across a patch of flowers, and that, due to the variation's inverse relationship with distance, the shape of the recruit distribution will remain constant for resources at different distances. In this study, we test this hypothesis by examining how variation in the indication of direction and distance changes with respect to distance. We find that imprecision in the communication of direction does not diminish rapidly enough to accommodate an adaptive-error hypothesis, and we also find that variation in the indication of distance has a positive relationship with the distance of a resource from the hive.

Hip fracture types in men and women change differently with age.

BACKGROUND: Hip fractures are expensive and a frequent cause of morbidity and mortality in the elderly. In most studies hip fractures have been viewed as a unitary fracture but recently the two main types of fracture (intertrochanteric and subcapital) have been viewed as two fractures with a different etiology and requiring a different approach to prevention. The relative proportion of intertrochanteric fractures increases with age in women. In previous studies no particular pattern in men has been noted. In this study, we explored changes in the relative proportion of the two fracture types with age in the two genders. METHODS: Patients of 50 years and older, with a diagnosis of hip fracture, discharged from two local acute care hospitals over a 5 year period (n = 2150) were analyzed as a function of age and gender to explore the relative proportions of intertrochanteric and subcapital fractures, and the change in relative proportion in the two genders with age. RESULTS: Overall, for the genders combined, the proportion of intertrochanteric fractures increases with age (p = .007). In women this increase is significant (p < .001), but in men the opposite pattern is observed, with the proportion of intertrochanteric fractures falling significantly with age (p = .025). CONCLUSIONS: The pattern of hip fractures is different in men and women with aging. It is likely that the pattern difference reflects differences in type and rate of bone loss in the genders, but it is conjectured that the changing rate and pattern of falling with increasing age may also be important. The two main hip fracture types should be considered distinct and different and be studied separately in studies of cause and prevention.

Enhanced electrochemiluminescence and charge transport through films of metallopolymer-gold nanoparticle composites.

Water-soluble 4-(dimethylamino) pyridine (DMAP) stabilized gold nanoparticles (DMAP-AuNP) were synthesized by ligand exchange and phase transfer (toluene/water). The DMAP-AuNPs are positively charged with the core diameter of 4 +/- 1 nm. Metallopolymer-gold nanocomposites were prepared by mixing gold nanoparticles and [Ru(bpy)(2)PVP(10)](ClO(4))(2), in water at different mole ratios; bpy is 2,2'-bipyridyl and PVP is poly (4-vinylpyridine). The photoluminescence emission intensity of the metallopolymer decreases with increasing AuNP loading and approximately 57% of the emission intensity is quenched when the Au NP:Ru mole ratio is 14.8 x 10(-2). The rate of homogeneous charge transfer through thin layers of the nanocomposite deposited on glassy carbon electrodes increases with increasing nanoparticle loading. The homogeneous charge transport diffusion coefficient, D(CT), for the composite (AuNP:Ru mole ratio 13.2 x 10(-2)) is (2.8 +/- 0.8) x 10(-11) cm(2) s(-1) and is approximately 3-fold higher than that found for the pure metallopolymer. Significantly, despite the ability of the metal nanoparticles to quench the ruthenium-based emission, the electrochemiluminescence of the nanocomposite with a AuNP:Ru mole ratio of 4.95 x 10(-2) is approximately three times more intense than the parent metallopolymer. This enhancement arises from the increased rate of charge transport that leads to a greater number of excited states per unit time while minimizing the quenching effects. The implications of these findings for the design of electrochemiluminescent sensors are discussed.

One-step synthesis of stoichiometrically defined metal oxide nanoparticles at room temperature.

A great variety of metal oxide nanoparticles have been readily synthesized by using alkali metal oxides, M(2)O (M is Na or Li) and soluble metal salts (metal chlorides) in polar organic solutions, for example, methanol and ethanol, at room temperature. The oxidation states of the metals in the resulting metal oxides (Cu(2)O, CuO, ZnO, Al(2)O(3), Fe(2)O(3), Bi(2)O(3), TiO(2), SnO(2), CeO(2), Nb(2)O(5), WO(3), and CoFe(2)O(4)) range from 1 to 6 and remain invariable through the reactions where good control of stoichiometry is achieved. Metal oxide nanoparticles are 1-30 nm and have good monodispersivity and displayed comparable optical spectra. These syntheses are based on a general ion reaction pathway during which the precipitate occurs when O(2-) ions meet metal cations (M(n+)) in anhydrous solution and the reaction equation is M(n+) + n/2 O(2-) --> MO(n/2) (n=1-6).