Fermentation of Pleurotus ostreatus and Ganoderma lucidum mushrooms and their extracts by the gut microbiota of healthy and osteopenic women: potential prebiotic effect and impact of mushroom fermentation products on human osteoblasts.
Recent data have highlighted the role of the gut microbiota and its several metabolites in maintaining bone health. Thus, gut microbiota manipulation, e.g., by prebiotics, might offer a plausible target in the fight against bone degenerative diseases. This study aimed (a) to investigate the in vitro prebiotic potential of Ganoderma lucidum and Pleurotus ostreatus mushrooms in healthy and osteopenic women and (b) to explore the impact of mushroom fermentation products on human osteoblasts. G. lucidum LGAM 9720 and P. ostreatus IK 1123 lyophilized mushroom-powders (2% w/v) and their hot-water extracts (1% w/v) were fermented in a 24 h static batch culture model by using faecal inocula from healthy (n = 3) or osteopenic (n = 3) donors. Gut microbiota analysis (qPCR) and measurement of short chain fatty acids (SCFAs) were performed during fermentation, and 24 h-prebiotic indexes were calculated. Evaluation of the effects of fermentation products on bone metabolism parameters (OPG: osteoprotegerin; and RANKL: receptor activator of nuclear factor kappa B ligand) in osteoblast cultures was also performed. Our data suggest that the origin of the gut microbiota inoculum plays a major role in the viability of osteoblasts. The treatments using P. ostreatus mushroom-powder and G. lucidum mushroom-extract had positive effects based on gut microbiota and SCFA analyses. Both mushrooms exhibited lower RANKL levels compared to controls, whereas their extracts tended to enhance the osteoblastic activity. In conclusion, mushrooms that are rich in beta-glucans may exert beneficial in vitro effects on bone physiology by alterations in the gut microbiota and/or SCFA production.


Toxoplasmosis in female high school students, pregnant women and ruminants in Cyprus.
The protozoan parasite Toxoplasma gondii is important to human and animal health worldwide. This is the first study of prevalence of infection with T. gondii and associated risk factors in human populations and small ruminants in Cyprus. A random sample of 18 schools out of 46 participated: 1056 girls aged 16 to 18 years completed a questionnaire and were serologically tested for Toxoplasma between 2008 and 2011 (response rate 30%). In addition, infection with T. gondii laboratory results of 23 076 pregnant women tested between 2009 and 2014 were obtained from hospital records. Finally, 163 (out of 3123) farms were randomly sampled and blood samples from 515 sheep and 581 goats were obtained. Estimated seropositivity prevalence in female students was 6.5% (95% CI 4.3 to 8.7%) and 18% (95% CI 17 to 19%) in pregnant women. Overall, 40.1% of the ruminants tested were seropositive (95% CI 37.2% to 43.0%). Seropositivity differed according to geographical region in all three groups. Further studies are needed to investigate the differences between regions that lead to differing prevalence levels and patterns between ruminants and humans so that health education policies can be developed to help prevent infection and reduce environmental contamination.


Concurrent and enduring associations between married partners' shared beliefs and markers of aging.
Beliefs about aging are grounded in social experience. This study considered the extent to which married older adults' shared beliefs about aging and markers of aging maintain a concurrent and enduring association with their partners' beliefs about and markers of aging. Data from the 2010/2012 and 2014/2016 waves of the Health and Retirement Study provided measures of husbands' and wives' (3,779 couples) positive and negative beliefs about aging and internal (Cystatin C) and external (grip strength) markers of aging at 2 time points. Latent dyadic models parsed beliefs and markers into partners' individual and shared variances, which were connected both cross-sectionally and longitudinally. Longitudinal analysis showed that the cross-sectional associations between shared beliefs and markers of aging were stable over 4 years. Partners' shared beliefs and markers of aging were found to have enduring associations with each other over time. The enduring association between grip strength and future negative beliefs remained significant after accounting for partner selection and similarity in health. Model comparisons across marriage duration and emotional closeness showed partners' beliefs to be more similar in marriages that were either long established or emotionally close. In all groups, shared beliefs and markers of aging were associated with each other over time. The association between positive beliefs and future grip strength was stronger in long-established than in recent marriages. In summary, this study provides evidence that, within older couples, beliefs about aging are shaped in part through experiences of aging together. (PsycInfo Database Record (c) 2020 APA, all rights reserved).


Short report: characterization of enteroaggregative Escherichia coli isolates from Iranian children.
We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Iranian infants and children. To better understand the characteristics of EAEC in Iran, we analyzed EAEC isolates for the presence of pAA plasmid-borne factors. Ninety-eight E. coli strains that displayed the aggregative adherence (AA) pattern on HeLa cells were hybridized with the CVD432 (AA) probe and with genes encoding enteroaggregative heat-stable enterotoxin-1 and aggregative adherence fimbriae (AAF) I and II. Our data suggest that AAF/II is common in this population and that AAF/I and AAF/II can sometimes be detected in the same E. coli isolate. Surprisingly, we have found that AA probe-negative strains in Iran share virulence factors with AA probe-positive isolates and therefore may be more similar to probe-positive strains than previously believed.


Emissions of methyl halides and methane from rice paddies.
Methyl halide gases are important sources of atmospheric inorganic halogen compounds, which in turn are central reactants in many stratospheric and tropospheric chemical processes. By observing emissions of methyl chloride, methyl bromide, and methyl iodide from flooded California rice fields, we estimate the impact of rice agriculture on the atmospheric budgets of these gases. Factors influencing methyl halide emissions are stage of rice growth, soil organic content, halide concentrations, and field-water management. Extrapolating our data implies that about 1 percent of atmospheric methyl bromide and 5 percent of methyl iodide arise from rice fields worldwide. Unplanted flooded fields emit as much methyl chloride as planted, flooded rice fields.


Vitamin D deficiency and mortality in patients receiving dialysis: the Comprehensive Dialysis Study.
Although several studies have shown poorer survival among individuals with 25-hydroxy (OH) vitamin D deficiency, data on patients receiving dialysis are limited. Using data from the Comprehensive Dialysis Study (CDS), we tested the hypothesis that patients new to dialysis with low serum concentrations of 25-OH vitamin D would experience higher mortality and hospitalizations. The CDS is a prospective cohort study.We recruited participants from 56 dialysis units located throughout the United States. We obtained data on demographics, comorbidites, and laboratory values from the CDS Patient Questionnaire as well as the Medical Evidence Form (CMS form 2728). Participants provided baseline serum samples for 25-OH vitamin D measurements. We ascertained time to death and first hospitalization as well as number of first-year hospitalizations via the U.S. Renal Data System standard analysis files. We used Cox proportional hazards to determine the association between 25-OH vitamin D tertiles and survival and hospitalization. For number of hospitalizations in the first year, we used negative binomial regression. The analytic cohort was composed of 256 patients with Patient Questionnaire data and 25-OH vitamin D concentrations. The mean age of participants was 62 (±14.0) years, and mean follow-up was 3.8 years. Patients with 25-OH vitamin D concentrations in the lowest tertile (<10.6 ng/mL) at the start of dialysis experienced higher mortality (adjusted hazard ratio 1.75, 95% confidence interval [CI] 1.03-2.97) as well as hospitalization (adjusted hazard ratio 1.76, 95% CI 1.24-2.49). Patients in the lower 2 tertiles (<15.5 ng/mL) experienced a higher rate of hospitalizations in the first year (incidence rate ratio 1.70 [95% CI 1.06-2.72] for middle tertile, 1.66 [95% CI 1.10-2.51] for lowest tertile). We found a sizeable increase in mortality and hospitalization for patients on dialysis with severe 25-OH vitamin D deficiency.


Inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor.
Resident pulmonary alveolar macrophages (PAM) play an important role in the maintenance of immunological homeostasis in the lung via downmodulation of local T cell responses in the steady state. The present study demonstrates that this pathway for T cell suppression is reversible via granulocyte/macrophage colony-stimulating factor (GM-CSF). Thus, freshly isolated PAM strongly inhibit mitogen-induced T cell proliferation, and pretreatment of the PAM with cytokine-rich lung-conditioned medium (LCM) generated by exposure of lung to bacterial lipopolysaccharide (LPS) abrogated this suppressive activity. LCM from lungs of normal and athymic nude mice exhibited identical activity. Moreover, the PAM-modulating activity of LCM was inhibited by blocking antibody specific for GM-CSF, and the activity of LCM could be reproduced by recombinant GM-CSF. This suggests that secretion of GM-CSF by mesenchymal cells and/or macrophages under stimulation from agents such as LPS provides a potential mechanism for upregulation of local T cell responsiveness during acute inflammation. In addition, experiments with a range of cytokines indicated that interleukin 4, transforming growth factor beta 1 and tumor necrosis factor alpha (TNF-alpha) exhibited weaker (but significant) modulatory effects on PAM, and (in the case of TNF-alpha) amplified the effects of GM-CSF.


Microflow Nanoprecipitation of Positively Charged Gastroresistant Polymer Nanoparticles of Eudragit® RS100: A Study of Fluid Dynamics and Chemical Parameters.
The objective of the present work was to produce gastroresistant Eudragit® RS100 nanoparticles by a reproducible synthesis approach that ensured mono-disperse nanoparticles under the size of 100 nm. Batch and micromixing nanoprecipitation approaches were selected to produce the demanded nanoparticles, identifying the critical parameters affecting the synthesis process. To shed some light on the formulation of the targeted nanoparticles, the effects of particle size and homogeneity of fluid dynamics, and physicochemical parameters such as polymer concentration, type of solvent, ratio of solvent to antisolvent, and total flow rate were studied. The physicochemical characteristics of resulting nanoparticles were studied applying dynamic light scattering (DLS) particle size analysis and electron microscopy imaging. Nanoparticles produced using a micromixer demonstrated a narrower and more homogenous distribution than the ones obtained under similar conditions in conventional batch reactors. Besides, fluid dynamics ensured that the best mixing conditions were achieved at the highest flow rate. It was concluded that nucleation and growth events must also be considered to avoid uncontrolled nanoparticle growth and evolution at the collection vial. Further, rifampicin-encapsulated nanoparticles were prepared using both approaches, demonstrating that the micromixing-assisted approach provided an excellent control of the particle size and polydispersity index. Not only the micromixing-assisted nanoprecipitation promoted a remarkable control in the nanoparticle formulation, but also it enhanced drug encapsulation efficiency and loading, as well as productivity. To the best of our knowledge, this was the very first time that drug-loaded Eudragit® RS100 nanoparticles (NPs) were produced in a continuous fashion under 100 nm (16.5 ± 4.3 nm) using microreactor technology. Furthermore, we performed a detailed analysis of the influence of various fluid dynamics and physicochemical parameters on the size and uniformity of the resulting nanoparticles. According to these findings, the proposed methodology can be a useful approach to synthesize a myriad of nanoparticles of alternative polymers.


O-acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall.
Radioactive labelling of the amino sugars in gonococcal peptidoglycan was followed by treatment with Chalaropsis muramidase and TLC separation of the products. Even after very brief periods of labelling (0.5 min) the peptidoglycan was already cross-linked to some 80% of the final value and little change occurred within 2 min. The remaining cross-linking was achieved only over a period of about one generation time. Streptomycete endopeptidase was used to show the extent to which new chains were cross-linked to old. Even at the earliest times many cross-linked units contained new material in both moieties and by 3 min there was little distinction in relative labelling, indicating that in Neisseria gonorrhoeae most newly synthesized glycan chains are cross-linked to other new chains rather than to pre-existing peptidoglycan. A model is proposed in which newly polymerized monomer units are predestined either towards dimer formation with other new chains, which are then rapidly O-acetylated and not further cross-linked, or towards the formation of trimers and higher oligomers, the latter being a slower process. Although significant O-acetylation of peptidoglycan was detectable even at the earliest times, efforts to detect O-acetylated lipid intermediates were unsuccessful. The chief lipid intermediate found was apparently the disaccharide-peptide unit linked to undecaprenol.


Effects of relationship education on maintenance of couple relationship satisfaction.
Couple relationship education (RE) is the provision of structured education intended to promote healthy couple relationships, and prevent future relationship distress. There is a well-replicated finding that 9-20 hours of curriculum-based RE produces short-term improvements in couple communication and relationship satisfaction, but that established finding does not test whether RE helps couples maintain high relationship satisfaction. The current paper summarizes 17 published studies evaluating RE that have follow up assessments of at least 1 year, of which 14 studies found RE helped maintenance of relationship satisfaction. Couples with elevations of modifiable risk factors benefit substantially from RE, while benefits for couples with low risk have not yet been reliably demonstrated. Couples with elevations on risk factors not readily modified by current forms of RE are likely to show little or no benefit. Future research needs to clarify the mediators of RE effects, and how those mediators are moderated by couple risk profiles.


Impact of ET-1 and sex in glomerular hyperfiltration in humanized sickle cell mice.
Hyperfiltration, highly prevalent early in sickle cell disease (SCD), is in part driven by an increase in ultrafiltration coefficient (Kf). The increase in Kf may be due to enlarged filtration surface area and/or increased glomerular permeability (Palb). Previous studies have demonstrated that endothelin-1 (ET-1) contributes to Palb changes in models of diabetes and SCD. Thus, we performed longitudinal studies of renal function to determine the relationship between ET-1 and glomerular size and Palb that may contribute to hyperfiltration in humanized sickle cell (HbSS) and control (HbAA) mice at 8-32 weeks of age. HbSS mice were characterized by significant increases in plasma and glomerular ET-1 expression in both sexes although this increase was significantly greater in males. HbSS glomeruli of both males and females presented with a progressive and significant increase in glomerular size, volume, and Kf During the onset of hyperfiltration, plasma and glomerular ET-1 expression were associated with a greater increase in glomerular size and Kf in HbSS mice, regardless of sex. The pattern of Palb augmentation during the hyperfiltration was also associated with an increase in glomerular ET-1 expression, in both male and female HbSS mice. However, the increase in Palb was significantly greater in males and delayed in time in females. Additionally, selective endothelin A receptor (ETA) antagonist prevented hyperfiltration in HbSS, regardless of sex. These results suggest that marked sex disparity in glomerular hyperfiltration may be driven, in part, by ET-1-dependent ultra-structural changes in filtration barrier components contributing to glomerular hyperfiltration in HbSS mice.


Evaluation of molecular chaperons Hsp72 and neuropeptide Y as characteristic markers of adaptogenic activity of plant extracts.
We have previously demonstrated that ADAPT-232, a fixed combination of adaptogenic substances derived from Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract stimulated the expression and release of neuropeptide Y (NPY) and molecular chaperone Hsp72 from isolated human neurolgia cells. Both of these mediators of stress response are known to play an important role in regulation of neuroendocrine system and immune response. We further demonstrated that ADAPT-232 induced release of Hsp70 is mediated by NPY, suggesting an existence of NPY-mediated pathway of activation of Hsp72 release into the blood circulation system. The objective of this study was to determine whether this pathway is common for adaptogens and whether NPY and/or Hsp72 can be considered as necessary specific biomarkers for adaptogenic activity. The release of NPY and Hsp72 from neuroglia cells in response to treatment with various plant extracts (n=23) including selected validated adaptogens, partly validated adaptogens, claimed but negligibly validated adaptogens and some other plant extracts affecting neuroendocrine and immune systems but never considered as adaptogens was measured using high throughput ELISA techniques. We demonstrated that adaptogens, e.g. R. rosea, S. chinensis and E. senticosus stimulate both NPY and Hsp70 release from neuroblastoma cells, while tonics and stimulants have no significant effect on NPY in this in vitro test. In the groups of partly validated adaptogens the effect of Panax ginseng and Withania somnifera was not statistically significant both on NPY and Hsp70 release, while the activating effect of Bryonia alba and Rhaponticum cartamoides was significant only on Hsp70. In contrast, all tested non-adaptogens, such as antiinflammatoty plant extracts Matricaria recutita, Pelargonium sidoides, Hedera helix and Vitis vinifera significantly inhibit Hsp70 release and have no influence on NPY release from neuroblastoma cells. These experiments were further validated using primary human neurons and confirmed that adaptogens activate the release of both NPY and Hsp70, while tested non adaptogens were inactive in NPY assay and inhibit the release of Hsp70. Taken together, our data demonstrates for the first time that neuropeptide Y and heat shock protein Hsp70 can be used as molecular biomarkers for adaptogenic activity.


Short-term starvation of immune deficient Drosophila improves survival to gram-negative bacterial infections.
Primary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-kappaB) pathway. Since the TIR-NF-kappaB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions. We used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-kappaB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-kappaB protein Dif, usually needed for responses against Gram-positive bacteria. Our results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity.


Outcome of Revision Shoulder Arthroplasty After Resurfacing Hemiarthroplasty in Patients with Glenohumeral Osteoarthritis.
Patients are often treated with a resurfacing hemiarthroplasty in the expectation that the bone-preserving design facilitates revision should the need for a revision arthroplasty arise. The aim of this study was to report the outcome of patients with glenohumeral osteoarthritis who underwent revision shoulder arthroplasty after resurfacing hemiarthroplasty. We reviewed all patients with osteoarthritis reported to the Danish Shoulder Arthroplasty Registry from 2006 to 2013. There were 1,210 primary resurfacing hemiarthroplasties, of which 107 cases (9%) required a revision surgical procedure, defined as the removal or exchange of the humeral component or the addition of a glenoid component. The Western Ontario Osteoarthritis of the Shoulder (WOOS) index was used to evaluate outcome at 1 year. The median WOOS of revision arthroplasty after failed resurfacing hemiarthroplasty was 62 points (interquartile range, 40 to 88 points). Of the 80 cases that had follow-up, 33 (41%) had an unacceptable outcome, defined as a WOOS of ≤50 points. Of the 107 cases that required a revision surgical procedure, 11 arthroplasties (10%) required a further revision surgical procedure. The resurfacing hemiarthroplasty was revised to a stemmed hemiarthroplasty (n = 39), anatomic total shoulder arthroplasty (n = 31), or reverse shoulder arthroplasty (n = 30). In 7 cases, the revision arthroplasty design was unknown. The median WOOS of patients who underwent revision stemmed hemiarthroplasty (48 points) was significantly inferior (p = 0.002) to that of patients who underwent primary stemmed hemiarthroplasty (75 points); the median WOOS of patients who underwent revision anatomic total shoulder arthroplasty (74 points) was also significantly inferior (p = 0.007) to that of patients who underwent primary anatomic total shoulder arthroplasty (93 points). However, the median WOOS of patients who underwent revision reverse shoulder arthroplasty (68 points) was not significantly different (p = 0.66) from that of patients who underwent primary reverse shoulder arthroplasty (77 points) used in the treatment of osteoarthritis. The outcome of revision shoulder arthroplasty after failed resurfacing hemiarthroplasty was variable and, in many cases, disappointing. It is important that resurfacing hemiarthroplasty is used for the correct indications and with adequate technique and skill. When resurfacing hemiarthroplasty is used in the treatment of osteoarthritis, revision cannot be counted upon as a fallback. Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.


Global perspectives on animal welfare: Europe.
Effective implementation and enforcement of legislation is essential to ensure animal welfare. In the World Organisation for Animal Health (OIE) European Region the well-established body of national and European Union laws in existence is growing continuously. The growth is due to various factors, such as new technology in animal farming and experimentation, exploitation of wildlife, new understanding of animal needs, and increasing public awareness and concern. The latter, in particular, determines the need for new animal welfare legislation to regulate and discipline the 'use' of animals for different purposes, such as food production, companionship, work and leisure. This paper intends to provide an overview of the more relevant activities carried out by the Council of Europe and the European Union in the field of animal welfare. The authors identify eLearning as a tool to harmonise the interpretation and the implementation of animal welfare legislation.


Risk practices for animal and human anthrax in Bangladesh: an exploratory study.
From August 2009 to October 2010, International Centre for Diarrheal Disease Research, Bangladesh and the Institute of Epidemiology, Disease Control and Research together investigated 14 outbreaks of anthrax which included 140 animal and 273 human cases in 14 anthrax-affected villages. Our investigation objectives were to explore the context in which these outbreaks occurred, including livestock rearing practices, human handling of sick and dead animals, and the anthrax vaccination program. Field anthropologists used qualitative data-collection tools, including 15 hours of unstructured observations, 11 key informant interviews, 32 open-ended interviews, and 6 group discussions in 5 anthrax-affected villages. Each cattle owner in the affected communities raised a median of six ruminants on their household premises. The ruminants were often grazed in pastures and fed supplementary rice straw, green grass, water hyacinth, rice husk, wheat bran, and oil cake; lactating cows were given dicalcium phosphate. Cattle represented a major financial investment. Since Islamic law forbids eating animals that die from natural causes, when anthrax-infected cattle were moribund, farmers often slaughtered them on the household premises while they were still alive so that the meat could be eaten. Farmers ate the meat and sold it to neighbors. Skinners removed and sold the hides from discarded carcasses. Farmers discarded the carcasses and slaughtering waste into ditches, bodies of water, or open fields. Cattle in the affected communities did not receive routine anthrax vaccine due to low production, poor distribution, and limited staffing for vaccination. Slaughtering anthrax-infected animals and disposing of butchering waste and carcasses in environments where ruminants live and graze, combined with limited vaccination, provided a context that permitted repeated anthrax outbreaks in animals and humans. Because of strong financial incentives, slaughtering moribund animals and discarding carcasses and waste products will likely continue. Long-term vaccination coverage for at-risk animal populations may reduce anthrax infection.


Probabilistic sparse matching for robust 3D/3D fusion in minimally invasive surgery.
Classical surgery is being overtaken by minimally invasive and transcatheter procedures. As there is no direct view or access to the affected anatomy, advanced imaging techniques such as 3D C-arm computed tomography (CT) and C-arm fluoroscopy are routinely used in clinical practice for intraoperative guidance. However, due to constraints regarding acquisition time and device configuration, intraoperative modalities have limited soft tissue image quality and reliable assessment of the cardiac anatomy typically requires contrast agent, which is harmful to the patient and requires complex acquisition protocols. We propose a probabilistic sparse matching approach to fuse high-quality preoperative CT images and nongated, noncontrast intraoperative C-arm CT images by utilizing robust machine learning and numerical optimization techniques. Thus, high-quality patient-specific models can be extracted from the preoperative CT and mapped to the intraoperative imaging environment to guide minimally invasive procedures. Extensive quantitative experiments on 95 clinical datasets demonstrate that our model-based fusion approach has an average execution time of 1.56 s, while the accuracy of 5.48 mm between the anchor anatomy in both images lies within expert user confidence intervals. In direct comparison with image-to-image registration based on an open-source state-of-the-art medical imaging library and a recently proposed quasi-global, knowledge-driven multi-modal fusion approach for thoracic-abdominal images, our model-based method exhibits superior performance in terms of registration accuracy and robustness with respect to both target anatomy and anchor anatomy alignment errors.


Prevalence and correlates of low fundamental movement skill competency in children.
To describe the demographic and health-related characteristics of school-aged children with low competency in fundamental movement skills (FMS). Cross-sectional representative school-based survey of Australian elementary and high school students (n = 6917) conducted in 2010. Trained field staff measured students' height, weight, and assessed FMS and cardiorespiratory endurance (fitness). Information on students' demographics and physical activity was collected by questionnaire. Overall, the prevalence of students with low motor skill competency was high. Girls with low socioeconomic status (SES) were twice as likely to be less competent in locomotor skills compared with high SES peers. Among boys, there was a strong association between low competency in FMS and the likelihood of being from non-English-speaking cultural backgrounds. There was a clear and consistent association between low competency in FMS and inadequate cardiorespiratory fitness. For boys, there was a clear association between low competency in object-control skills and not meeting physical activity recommendations. Conversely, the odds of being inactive were double among girls who had low competency in locomotor skills. Low competency in FMS is strongly associated with lower cardiorespiratory fitness and physical activity levels in children and adolescents. The characteristics of students with competency in FMS differ by gender and skills types and show that interventions need to target girls from low SES backgrounds and boys from non-English-speaking cultural backgrounds. The high prevalence of low competency in FMS among Grade 4 students indicates that FMS interventions need to start during the preschool and early school years.


Thrombelastography-Based Dosing of Enoxaparin for Thromboprophylaxis in Trauma and Surgical Patients: A Randomized Clinical Trial.
Prophylactic enoxaparin is used to prevent venous thromboembolism (VTE) in surgical and trauma patients. However, VTE remains an important source of morbidity and mortality, potentially exacerbated by antithrombin III or anti-Factor Xa deficiencies and missed enoxaparin doses. Recent data suggest that a difference in reaction time (time to initial fibrin formation) greater than 1 minute between heparinase and standard thrombelastogram (TEG) is associated with a decreased risk of VTE. To evaluate the effectiveness of TEG-adjusted prophylactic enoxaparin dosing among trauma and surgical patients. This randomized clinical trial, conducted from October 2012 to May 2015, compared standard dosing (30 mg twice daily) with TEG-adjusted enoxaparin dosing (35 mg twice daily) for 185 surgical and trauma patients screened for VTE at 3 level I trauma centers in the United States. The incidence of VTE, bleeding complications, anti-Factor Xa deficiency, and antithrombin III deficiency. Of the 185 trial participants, 89 were randomized to the control group (median age, 44.0 years; 55.1% male) and 96 to the intervention group (median age, 48.5 years; 74.0% male). Patients in the intervention group received a higher median enoxaparin dose than control patients (35 mg vs 30 mg twice daily; P < .001). Anti-Factor Xa levels in intervention patients were not higher than levels in control patients until day 6 (0.4 U/mL vs 0.21 U/mL; P < .001). Only 22 patients (11.9%) achieved a difference in reaction time greater than 1 minute, which was similar between the control and intervention groups (10.4% vs 13.5%; P = .68). The time to enoxaparin initiation was similar between the control and intervention groups (median [range] days, 1.0 [0.0-2.0] vs 1.0 [1.0-2.0]; P = .39), and the number of patients who missed at least 1 dose was also similar (43 [48.3%] vs 54 [56.3%]; P = .30). Rates of VTE (6 [6.7%] vs 6 [6.3%]; P > .99) were similar, but the difference in bleeding complications (5 [5.6%] vs 13 [13.5%]; P = .08) was not statistically significant. Antithrombin III and anti-Factor Xa deficiencies and hypercoagulable TEG parameters, including elevated coagulation index (>3), maximum amplitude (>74 mm), and G value (>12.4 dynes/cm2), were prevalent in both groups. Identified risk factors for VTE included older age (61.0 years vs 46.0 years; P = .04), higher body mass index (calculated as weight in kilograms divided by height in meters squared; 30.6 vs 27.1; P = .03), increased Acute Physiology and Chronic Health Evaluation II score (8.5 vs 7.0; P = .03), and increased percentage of missed doses per patient (14.8% vs 2.5%; P = .05). The incidence of VTE was low and similar between groups; however, few patients achieved a difference in reaction time greater than 1 minute. Antithrombin III deficiencies and hypercoagulable TEG parameters were prevalent among patients with VTE. Low VTE incidence may be due to an early time to enoxaparin initiation and an overall healthier and less severely injured study population than previously reported. clinicaltrials.gov Identifier: NCT00990236.


Establishment of the first WHO Erythropoietin antibody reference panel: Report of an international collaborative study.
A panel of 9 fully human monoclonal antibodies against human erythropoietin (EPO) with defined characteristics (non-neutralizing, neutralizing, various isotypes, affinities) representative of those evident in antibody-mediated pure red cell aplasia (PRCA) and non-PRCA patients were formulated and lyophilized. The panel was evaluated in a multi-centre international collaborative study comprising eighteen different laboratories using different assay platforms including those in routine use. These included binding assays, some based on use of novel technologies and neutralization assays predominantly employing EPO responsive cell-lines. Results showed that detection and titre varied depending on antibody characteristics and the method used. Only selective assay platforms were capable of detecting the diverse repertoire of EPO antibodies in the panel indicating that some clinically relevant antibodies are likely to be missed in some assays. Importantly, the clinical samples from PRCA patients were distinguished as antibody-positive and the healthy donor serum as antibody negative across all different platforms tested. For neutralization, data was generally consistent across the assays for the different samples regardless of the cell-line and the assay conditions. The heterogeneity in data from the study clearly indicated the need for reference standards for consistency in detecting and measuring EPO antibodies across different assay platforms for monitoring the safety and efficacy of erythropoiesis stimulating agents. Therefore, the WHO ECBS at its meeting in October'15 established the EPO antibody panel, available from NIBSC, to facilitate decision-making on assay selection for testing antibodies against human EPO, for evaluating assay performance of antibody assays for clinical use, for assay validation and for standardization.


Diagnostic strategy for occult hepatitis B virus infection.
In 2008, the European Association for the study of the liver (EASL) defined occult hepatitis B virus infection (OBI) as the "presence of hepatitis B virus (HBV) DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing hepatitis B surface antigen (HBsAg) negative by currently available assays". Several aspects of occult HBV infection are still poorly understood, including the definition itself and a standardized approach for laboratory-based detection, which is the purpose of this review. The clinical significance of OBI has not yet been established; however, in terms of public health, the clinical importance arises from the risk of HBV transmission. Consequently, it is important to detect high-risk groups for occult HBV infection to prevent transmission. The main issue is, perhaps, to identify the target population for screening OBI. Viremia is very low or undetectable in occult HBV infection, even when the most sensitive methods are used, and the detection of the viral DNA reservoir in hepatocytes would provide the best evaluation of occult HBV prevalence in a defined set of patients. However, this diagnostic approach is obviously unsuitable: blood detection of occult hepatitis B requires assays of the highest sensitivity and specificity with a lower limit of detection < 10 IU/mL for HBV DNA and < 0.1 ng/mL for HBsAg.


Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease.
Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.


Phenotypic effects of null and haploinsufficiency of acid-labile subunit in a family with two novel IGFALS gene mutations.
IGF-I deficiency may result from impairment of GH secretion or action, or from defects in IGF-I synthesis, transport, or action. Complete deficiency of the acid-labile subunit (ALS), previously described in two male patients, the only known inherited alteration in IGF-I transport, is characterized by severe circulating IGF-I and IGF binding protein (IGFBP)-3 deficiency with only mild growth retardation. Our objective was to study the characterization, at biochemical and molecular levels, of the cause for severe circulating IGF-I and IGFBP-3 deficiency in a male patient with mild growth retardation. We report an adolescent male with delayed growth and pubertal development (Tanner stage I, -2.00 sd score for height at the age of 15.3 yr), profound circulating IGF-I and IGFBP-3 deficiency, and poor response to GH treatment. The index case, as well as one of his brothers, and his sister were found to be compound heterozygotes for two novel IGFALS gene mutations: C540R, a missense point mutation; and S195_197Rdup, a 9-bp duplication. The parents and youngest brother were found to be carriers for one of these two mutations. The three affected siblings had marked reduction of IGF-I and IGFBP-3 levels, undetectable serum levels of ALS, inability to form ternary complexes, and moderate insulin resistance. All of them attained a normal near-adult height (between -1.0 and -0.5 sd score), which was nonetheless lower than that of their heterozygous brother. The IGF system was only modestly affected in the heterozygous carriers. This study confirms the critical role of ALS in forming ternary complexes and the maintenance of normal levels of IGF-I and IGFBP-3. Insulin resistance, pubertal delay in male patients, and poor GH responsiveness seem to be frequent findings in ALS deficiency. However, haploinsufficiency of the IGFALS gene has no discernible clinical effects with only modest impact on the IGF system.


Prognostic factors and therapeutic strategies for differentiated carcinomas of the thyroid.
Differentiated thyroid carcinoma originates from thyroid follicular cells and is the most prominent malignancy of the endocrine organs. There are two histological types of differentiated carcinoma, namely, papillary and follicular carcinoma. According to reports from Western countries, papillary carcinoma comprises 85.3% of thyroid malignancies in whites, and 72.3% in blacks. In Japan, a previous study showed that the prevalence of papillary carcinoma was 78.4% based on material registered between 1977 and 1986, but according to recent findings reported in 2004 by Japanese Society of Thyroid Surgeons (JSTS), papillary carcinoma accounted for as much as 93% of all thyroid carcinomas. Papillary carcinoma frequently metastasizes to the regional lymph node and shows multicentricity in the thyroid gland. It usually shows a typical ultrasonographic appearance and can be rather easily diagnosed by fine needle aspiration biopsy (FNAB). Follicular carcinoma accounts for 10.9-20.5% of the patients in the United States. In Japan, the prevalence of follicular carcinoma was reported to be 17.2%, but it decreased to 5% in a report by JSTS in 2004. This carcinoma is only occasionally diagnosed preoperatively, because it is hard to discriminate follicular carcinoma from benign adenoma on imaging studies and cytologic findings. In contrast to papillary carcinoma, follicular carcinoma more often metastasizes to distant organs than regional lymph nodes. In Japan, the prevalence of papillary carcinoma increased and that of follicular carcinoma decreased between reports from 1977 to 1986 and that in 2004, which may be because follicular variant of papillary carcinoma was classified into follicular carcinoma in the previous results. Generally, these carcinomas show an indolent character, but when the lesion dedifferentiates and becomes undifferentiated carcinoma, it displays very rapid growth with an adverse prognosis and is regarded even as the most aggressive malignancy among human solid carcinomas. Furthermore, cases showing certain characteristics are likely to be constantly progressive and even become life-threatening. Such cases should be regarded as "high-risk" requiring careful and extensive surgical treatment and postoperative follow-up. Indeed, it is most important for physicians to correctly distinguish high-risk cases from those with an indolent character, although how to evaluate the biological characteristics of thyroid carcinoma and how to identify high-risk cases remains highly controversial. In this review, the methods of distinguishing high-risk cases and the appropriate therapeutic strategies for papillary and follicular carcinomas predominantly based on our experience are emphasized and our proposals for therapies including surgical treatment are demonstrated.


Whole genome sequencing and phylogenetic analysis of strains of the agent of Lyme disease Borrelia burgdorferi from Canadian emergence zones.
Lyme disease is emerging in southern Canada due to range expansion of the tick vector, followed by invasion of the agent of Lyme disease Borrelia burgdorferi sensu stricto. Strain diversity, as determined by Multi Locus Sequence Typing, occurs in this zone of emergence, and this may have its origins in adaptation to ecological niches, and have phenotypic consequences for pathogenicity and serological test performance. Sixty-four unique strains were cultured from ticks collected in southern Canada and the genomes sequenced using the Illumina MiSeq platform. A maximum likelihood phylogenetic tree of the chromosome revealed two large clades with multiple subclades. Consistent with previous studies on this species, the clades were not geographically defined, and some Canadian strains were highly divergent from previously sequenced US strains. There was evidence for recombination in the chromosome but this did not affect the phylogeny. Analysis of chromosomal genes indicated that these are under intense purifying selection. Phylogenies of the accessory genome and chromosome were congruent. Therefore strain differences identified in the phylogeny of chromosomal genes likely act as a proxy for genetic determinants of phenotypic differences amongst strains that are harboured in the accessory genome. Further studies on health implications of strain diversity are needed.


Single-cell RNA-seq: advances and future challenges.
Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here-each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.


[Catamenial pneumothorax. Case report and review of the literature].
Catamenial pneumothorax is a clinical variant of spontaneous pneumothorax. Its etiology is attributed either, generally, to endometriosis involving the diaphragm, or to prostaglandin-induced bronchiolar constriction. Clinically, it coincides with the onset of menstruation and resolves during the subsequent days. Treatment is currently based upon progestational agents and antigonadotrophins.


Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose-binding proteins.
Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.


Benchmarking and analysis of protein docking performance in Rosetta v3.2.
RosettaDock has been increasingly used in protein docking and design strategies in order to predict the structure of protein-protein interfaces. Here we test capabilities of RosettaDock 3.2, part of the newly developed Rosetta v3.2 modeling suite, against Docking Benchmark 3.0, and compare it with RosettaDock v2.3, the latest version of the previous Rosetta software package. The benchmark contains a diverse set of 116 docking targets including 22 antibody-antigen complexes, 33 enzyme-inhibitor complexes, and 60 'other' complexes. These targets were further classified by expected docking difficulty into 84 rigid-body targets, 17 medium targets, and 14 difficult targets. We carried out local docking perturbations for each target, using the unbound structures when available, in both RosettaDock v2.3 and v3.2. Overall the performances of RosettaDock v2.3 and v3.2 were similar. RosettaDock v3.2 achieved 56 docking funnels, compared to 49 in v2.3. A breakdown of docking performance by protein complex type shows that RosettaDock v3.2 achieved docking funnels for 63% of antibody-antigen targets, 62% of enzyme-inhibitor targets, and 35% of 'other' targets. In terms of docking difficulty, RosettaDock v3.2 achieved funnels for 58% of rigid-body targets, 30% of medium targets, and 14% of difficult targets. For targets that failed, we carry out additional analyses to identify the cause of failure, which showed that binding-induced backbone conformation changes account for a majority of failures. We also present a bootstrap statistical analysis that quantifies the reliability of the stochastic docking results. Finally, we demonstrate the additional functionality available in RosettaDock v3.2 by incorporating small-molecules and non-protein co-factors in docking of a smaller target set. This study marks the most extensive benchmarking of the RosettaDock module to date and establishes a baseline for future research in protein interface modeling and structure prediction.


Up-regulation of endothelial delta-like 4 expression correlates with vessel maturation in bladder cancer.
Angiogenesis and vascular endothelial growth factor (VEGF) expression are associated with a poor outcome in bladder cancer. To understand more about the mechanisms, we studied the role of delta-like 4 (DLL4), an endothelial-specific ligand of the Notch signaling pathway, in bladder cancer angiogenesis. The expression of DLL4, CD34, and VEGF were studied in a cohort of 60 bladder tumors and 10 normal samples using quantitative PCR. In situ hybridization was used to study the pattern of DLL4 expression in 22 tumor and 9 normal samples. Serial sections were also stained for CD34 and alpha-smooth muscle actin (alpha-SMA) using conventional immunohistochemistry. The expression of DLL4 was significantly up-regulated in superficial (P < 0.01) and invasive (P < 0.05) bladder cancers. DLL4 expression significantly correlated with CD34 (P < 0.001) and VEGF (P < 0.001) expression. The in situ hybridization studies showed that DLL4 was highly expressed within bladder tumor vasculature. Additionally, DLL4 expression significantly correlated with vessel maturation as judged by periendothelial cell expression of alpha-SMA, 98.7% of DLL4-positive tumor vessels coexpressed alpha-SMA, compared with 64.5% of DLL4-negative tumor vessels (P < 0.001). High DLL4 expression may have prognostic value in superficial and invasive bladder. DLL4 expression is associated with vascular differentiation in bladder cancer; thus, targeting DLL4 may be a novel antiangiogenic therapy.


Projected prevalences of age-related eye diseases.
To examine projections of age-related eye diseases in the United States for health care planning. Review of published scientific articles, census data, and unpublished research findings. The numbers of cases of all age-related eye diseases are expected to rise in the coming years. These projections are primarily based upon population projections, but give little consideration to changes in health behaviors, racial/ethnic differences, environmental exposures, and changes in health care practices that may influence estimates of costs of actual health care burden. Ongoing monitoring of trends in eye disease distribution is needed rather than projections based on old data that may be inadequate for generating reliable prediction models. There is a perpetual need to train new researchers with expertise in epidemiology, as the exigency for current prevalence estimates is crucial to maximize optimal visual health in the population.


Synergistic effect of heat and solar UV on DNA damage and water disinfection of E. coli and bacteriophage MS2.
The response of a representative virus and indicator bacteria to heating, solar irradiation, or their combination, was investigated in a controlled solar simulator and under real sun conditions. Heating showed higher inactivation of Escherichia coli compared to the bacteriophage MS2. Heating combined with natural or simulated solar irradiation demonstrated a synergistic effect on the inactivation of E. coli, with up to 3-log difference for 50 °C and natural sun insolation of 2,000 kJ m(-2) (compared to the sum of the separate treatments). Similar synergistic effect was also evident when solar-UV induced DNA damage to E. coli was assessed using the endonuclease sensitive site assay (ESS). MS2 was found to be highly resistant to irradiation and heat, with a slightly synergistic effect observed only at 59 °C and natural sun insolation of 5,580 kJ m(-2). Heat treatment also hindered light-dependent recovery of E. coli making the treatment much more effective.


Reduction of diabetes-induced renal oxidative stress by a cantaloupe melon extract/gliadin biopolymers, oxykine, in mice.
Oxidative stress is implicated as an important mechanism by which diabetes causes nephropathy. Oxykine is the cantaloupe melon extract rich in vegetal superoxide dismutase covered by polymeric films of wheat matrix gliadin. In this study, we examined whether chronic oral administration of oxykine could prevent the progression of diabetic nephropathy induced by oxidative stress using preclinical rodent model of type 2 diabetes. We used female db/db mice and their non-diabetic db/m littermates. The mice were divided into the following three groups: non-diabetic db/m; diabetic db/db, and diabetic db/db treated with oxykine. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments. Histological and 8-OHdG immunohistochemical studies were preformed on 12 weeks from the beginning of treatment. After 12 weeks of treatment, the levels of blood glucose and the body weight were not significantly different between the oxykine-treated group and the non-treated db/db group, however both groups kept significantly high levels rather than db/m mice. The relative mesangial area calculated by mesangial area/total glomerular area ratio was significantly ameliorated in the oxykine treated group compared with non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with oxykine. The 8-OHdG immunoreactive cells in the glomeruli of non-treated db/db mice were more numerous than that of oxykine-treated db/db mice. In this study, treatment of oxykine ameliorated the progression and acceleration of diabetic nephropathy for rodent model of type 2 diabetes. These results indicated that the oxykine reduced the diabetes-induced oxidative stress and renal mesangial cell injury. In conclusion, oxykine might be a novel approach for the prevention of diabetes nephropathy.


Enhancing the flotation recovery of copper minerals in smelter slags from Namibia prior to disposal.
Namibia Custom Smelters (NCS) process a range of copper concentrates in their three furnaces, namely; top submerged lance, copper converter and reverberatory furnaces, in order to produce mattes and fayalitic slags. The copper content of the slags range between 0.8 to 5 wt. % and this is considered too high for disposal to the environment. Currently, the slags are sent to a milling and flotation plant for liberation and recovery of residual copper. The copper recoveries realized in the plant are much lower than expected and it has been postulated that some copper minerals may be occurring in forms that are more difficult to float like oxides or fine disseminations in the gangue matrix. Mineralogical analysis of the slag samples was done using X-Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) techniques. The analysis did not reveal the presence of copper oxide minerals, however most scans showed copper sulphide minerals as free grains and some finely disseminated in fayalite gangue. In the first phase of the present experimental studies, the slags were milled to 75% passing 45 microns, which is the degree of milling done in the existing plant mill-float circuit. A range of commercial flotation reagents that include xanthates, dithiophosphates, mercaptobenzothiazole, thionocarbamates, fatty acids, sulphides and sulphates were used in the flotation test-work. The copper recoveries obtained in the mill-float stage were between 70 - 80%. In the second phase of the study, the flotation tailings were further milled to 90% passing 45 microns and floated. The cumulative copper recoveries increased markedly to over 90%, which represents a significant improvement in comparison to the recoveries obtained from the mill-float process. Sodium alkyl dithiophosphate, mercaptobenzothiazole (FC7245) was found to be the secondary flotation reagent that gave the best copper recoveries.


Sample size calculations for clustered binary data.
In this paper we propose a sample size calculation method for testing on a binomial proportion when binary observations are dependent within clusters. In estimating the binomial proportion in clustered binary data, two weighting systems have been popular: equal weights to clusters and equal weights to units within clusters. When the number of units varies cluster by cluster, performance of these two weighting systems depends on the extent of correlation among units within each cluster. In addition to them, we will also use an optimal weighting method that minimizes the variance of the estimator. A sample size formula is derived for each of the estimators with different weighting schemes. We apply these methods to the sample size calculation for the sensitivity of a periodontal diagnostic test. Simulation studies are conducted to evaluate a finite sample performance of the three estimators. We also assess the influence of misspecified input parameter values on the calculated sample size. The optimal estimator requires equal or smaller sample sizes and is more robust to the misspecification of an input parameter than those assigning equal weights to units or clusters.


Single origin of sex chromosomes and multiple origins of B chromosomes in fish genus Characidium.
Chromosome painting with DNA probes obtained from supernumerary (B) and sex chromosomes in three species of fish genus Characidium (C. gomesi, C. pterostictum and C. oiticicai) showed a close resemblance in repetitive DNA content between B and sex chromosomes in C. gomesi and C. pterostictum. This suggests an intraspecific origin for B chromosomes in these two species, probably deriving from sex chromosomes. In C. oiticicai, however, a DNA probe obtained from its B chromosome hybridized with the B but not with the A chromosomes, suggesting that the B chromosome in this species could have arisen interspecifically, although this hypothesis needs further investigation. A molecular phylogenetic analysis performed on nine Characidium species, with two mtDNA genes, showed that the presence of heteromorphic sex chromosomes in these species is a derived condition, and that their origin could have been unique, a conclusion also supported by interspecific chromosome painting with a CgW probe derived from the W chromosome in C. gomesi. Summing up, our results indicate that whereas heteromorphic sex chromosomes in the genus Characidium appear to have had a common and unique origin, B chromosomes may have had independent origins in different species. Our results also show that molecular phylogenetic analysis is an excellent complement for cytogenetic studies by unveiling the direction of evolutionary chromosome changes.


How can brain activity and hypnosis inform each other?
Applying P. Teitelbaum's conception of the method of physiological psychology as interlocking stages of analysis and synthesis, the authors examine how studies of brain function can advance the understanding of hypnosis as a psychological process. They consider the exploration of animal analogues of human hypnosis, the study of brain injury in humans as a window on mechanisms underlying hypnosis, the distinct questions that brain-imaging studies of hypnosis can address, and the potential for comparative and developmental approaches to hypnosis as close allies to psychophysiological approaches. They conclude both that hypnosis is a powerful technique for studying the brain mechanisms of conscious experience and that underlying brain mechanisms may shed new light on longstanding theoretical debates about hypnosis.


Genetic structure and domestication history of the grape.
The grape is one of the earliest domesticated fruit crops and, since antiquity, it has been widely cultivated and prized for its fruit and wine. Here, we characterize genome-wide patterns of genetic variation in over 1,000 samples of the domesticated grape, Vitis vinifera subsp. vinifera, and its wild relative, V. vinifera subsp. sylvestris from the US Department of Agriculture grape germplasm collection. We find support for a Near East origin of vinifera and present evidence of introgression from local sylvestris as the grape moved into Europe. High levels of genetic diversity and rapid linkage disequilibrium (LD) decay have been maintained in vinifera, which is consistent with a weak domestication bottleneck followed by thousands of years of widespread vegetative propagation. The considerable genetic diversity within vinifera, however, is contained within a complex network of close pedigree relationships that has been generated by crosses among elite cultivars. We show that first-degree relationships are rare between wine and table grapes and among grapes from geographically distant regions. Our results suggest that although substantial genetic diversity has been maintained in the grape subsequent to domestication, there has been a limited exploration of this diversity. We propose that the adoption of vegetative propagation was a double-edged sword: Although it provided a benefit by ensuring true breeding cultivars, it also discouraged the generation of unique cultivars through crosses. The grape currently faces severe pathogen pressures, and the long-term sustainability of the grape and wine industries will rely on the exploitation of the grape's tremendous natural genetic diversity.


Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages.
The rate of uptake of oxidized low density lipoprotein (LDL) by mouse peritoneal macrophages is similar to that of acetyl LDL; but only approximately 50% of the internalized oxidized LDL is ultimately degraded, in contrast to the near-complete degradation seen with acetyl LDL. The objectives of this study were to determine if this was due to increased surface binding of oxidized LDL, different uptake pathways for oxidized LDL and acetyl LDL, lysosomal dysfunction caused by oxidized LDL, or resistance of oxidized LDL to hydrolysis by lysosomal proteinases. LDL binding studies at 4 degrees C showed that the increased cell association with oxidized LDL could not be explained by differences in cell-surface binding. Immunofluorescence microscopy confirmed intracellular accumulation of apoB-immunoreactive material in macrophages incubated with oxidized LDL, but not with acetyl LDL. The scavenger receptor ligand polyinosinic acid inhibited both the cell association and degradation of oxidized LDL in macrophages by greater than 75%, suggesting a common uptake pathway for degraded LDL and nondegraded LDL. Studies in THP-1 cells also did not reveal more than one specific uptake pathway for oxidized LDL. LDL derivatized by incubation with oxidized arachidonic acid (under conditions that prevented oxidation of the LDL itself) showed inefficient degradation, similar to oxidized LDL. When macrophages were incubated with oxidized LDL together with acetyl 125I-LDL, the acetyl LDL was degraded normally, excluding lysosomal dysfunction as the explanation for the accumulation of oxidized LDL. Generation of trichloroacetic acid-soluble products from oxidized 125I-LDL by exposure to cathepsins B and D was less than that observed with native 125I-LDL. LDL modified by exposure to reactive products derived from oxidized arachidonic acid was also degraded more slowly than native 125I-LDL by cathepsins. In contrast, acetyl 125I-LDL was degraded more rapidly by cathepsins than native 125I-LDL, and aggregated LDL and malondialdehyde-modified LDL were degraded at the same rate as native 125I-LDL. It is concluded that the intracellular accumulation of oxidized LDL in macrophages can be explained at least in part by the resistance of oxidatively modified apolipoprotein B to cathepsins. This resistance to cathepsins does not appear to be due to aggregation of oxidized LDL, but may be a consequence of modification of apolipoprotein B by lipid peroxidation products.


Systematic review and meta-analysis of the adverse health effects of ambient PM2.5 and PM10 pollution in the Chinese population.
As the largest developing country, China has some of the worst air quality in the world. Heavy smog in January 2013 led to unprecedented public concern about the health impact of exposure to particulate matter. Conducting health impact assessments of particulate matter has thus become an urgent task for public health practitioners. Combined estimates of the health effects of exposure to particulate matter from quantitative reviews could provide vital information for epidemiology-based health impact assessments, but estimates for the Chinese population are limited. On December 31, 2013, we systematically searched the PubMed, Web of Science, and China National Knowledge Infrastructure databases using as keywords names of 127 major cities in Mainland China, Hong Kong, and Taiwan. From among the 1464 articles identified, 59 studies were manually screened. Random-effects or fixed-effects models were used to combine their risk estimates, the funnel plots with Egger test were performed to evaluate the publication bias and Meta regression were run to explore the association between exposure to particulate matter with aerodynamic diameters less than 10 and 2.5 µm (PM10 and PM2.5) and the resulting health effects by the Comprehensive Meta Analysis. In terms of short-term effects, the combined excess risks of total non-accidental mortality, mortality due to cardiovascular disease, and mortality due to respiratory disease were 0.36% (95% confidence interval [95%CI]: 0.26%, 0.46%), 0.36% (95%CI: 0.24%, 0.49%), and 0.42% (95%CI: 0.28%, 0.55%), for each 10 μg/m(3) increase in PM10. A 10 μg/m(3) increase in PM2.5 was associated with a 0.40% (95%CI: 0.22%, 0.59%) increase in total non-accidental mortality, a 0.63% (95%CI: 0.35%, 0.91%) increase in mortality due to cardiovascular disease, and a 0.75% (95%CI: 01.39%, 1.11%) increase in mortality due to respiratory disease. For constituent-specific mortality, increases of 0.40-3.11% were associated with an increase of 10 ng/m(3) for nickel in PM. The summary estimate ranges of hospital utilization were 0.08% ~ 0.72% and -0.58% ~ 1.32% for a 10 μg/m(3) increase in PM10 and PM2.5. In terms of long-term effects, a 10 μg/m(3) increase of PM10 corresponded to 23-67% increase in the risk of mortality. Short exposures to PM10 and PM2.5 are associated with increases in mortality, but evidence of constituent-associated health effects, long-term effects and morbidity in China is still inadequate.


Spontaneous regression of malignant lymphoma of the breast.
A complete spontaneous regression of diffuse large B cell lymphoma involving the right breast, confirmed by aspiration cytology, is reported. The patient visited a hospital due to the rapid growth of a tumor in the right breast. Five years previously she underwent a craniotomy for a brain tumor, diagnosed as B-cell malignant lymphoma, and received several courses of irradiation to the brain. Analysis of the breast tumor cells obtained by aspiration revealed lymphoma cells morphologically, which were similar to the tumor cells in the brain expressing CD20. While waiting for further examination, the tumor regressed rapidly and was not palpable after 20 days. An excisional biopsy of the breast exhibited no definite malignant lymphoma cells among a diffuse population of CD45RO and CD8-positive small lymphocytes. Nucleotide sequencing of HCDR3s of the brain tumor and breast tumor cells showed a completely matched sequence, revealing the breast mass to be a metastatic lesion from the tumor of the brain. Although there was no tumorous lesion, the patient received additional chemotherapy and has shown no sign of recurrence in the breast for 7 years. We were able to confirm that the breast lymphoma shown in the aspiration cytology was a metastatic one, which was not proven histologically prior to chemotherapy, and regard the present case as a malignant lymphoma of the breast showing spontaneous regression. The present case shows a rare occurrence of spontaneous regression of diffuse large B cell malignant lymphoma after aspiration and suggests that CD8-positive T cells might be related to the regression.


Epigenome analysis reveals TBX5 as a novel transcription factor involved in the activation of rheumatoid arthritis synovial fibroblasts.
In this study, we analyzed the methylation status of human promoters in rheumatoid arthritis synovial fibroblasts (RASF). Differentially methylated genes between RASF and osteoarthritis synovial fibroblasts (OASF) were identified by methylated DNA immunoprecipitation and hybridization to human promoter tiling arrays. The methylation status was confirmed by pyrosequencing. Gene and protein expression of differentially methylated genes was evaluated with real-time PCR, Western blot, and immunohistochemistry. Chromatin immunoprecipitation was used to measure the gene promoter-associated acetylation and methylation of histones. Transcription factor-specific targets were identified with microarray and luciferase assays. We found that the transcription factor T-box transcription factor 5 (TBX5) was less methylated in rheumatoid arthritis (RA) synovium and RASF than in osteoarthritis (OA) samples. Demethylation of the TBX5 promoter in RASF and RA synovium was accompanied by higher TBX5 expression than in OASF and OA synovium. In RA synovium, TBX5 expression was primarily localized to the synovial lining. In addition, the TBX5 locus was enriched in activating chromatin marks, such as histone 4 lysine 4 trimethylation and histone acetylation, in RASF. In our functional studies, we observed that 790 genes were differentially expressed by 2-6-fold after overexpression of TBX5 in OASF. Bioinformatic analysis of these genes revealed that the chemokines IL-8, CXCL12, and CCL20 were common targets of TBX5 in OASF. Taken together, our data show that TBX5 is a novel inducer of important chemokines in RASF. Thus, we conclude that RASF contribute to the inflammatory processes operating in the pathogenesis of RA via epigenetic control of TBX5.


Maternal, social and abiotic environmental effects on growth vary across life stages in a cooperative mammal.
Resource availability plays a key role in driving variation in somatic growth and body condition, and the factors determining access to resources vary considerably across life stages. Parents and carers may exert important influences in early life, when individuals are nutritionally dependent, with abiotic environmental effects having stronger influences later in development as individuals forage independently. Most studies have measured specific factors influencing growth across development or have compared relative influences of different factors within specific life stages. Such studies may not capture whether early-life factors continue to have delayed effects at later stages, or whether social factors change when individuals become nutritionally independent and adults become competitors for, rather than providers of, food. Here, we examined variation in the influence of the abiotic, social and maternal environment on growth across life stages in a wild population of cooperatively breeding meerkats. Cooperatively breeding vertebrates are ideal for investigating environmental influences on growth. In addition to experiencing highly variable abiotic conditions, cooperative breeders are typified by heterogeneity both among breeders, with mothers varying in age and social status, and in the number of carers present. Recent rainfall had a consistently marked effect on growth across life stages, yet other seasonal terms only influenced growth during stages when individuals were growing fastest. Group size and maternal dominance status had positive effects on growth during the period of nutritional dependence on carers, but did not influence mass at emergence (at 1 month) or growth at independent stages (>4 months). Pups born to older mothers were lighter at 1 month of age and subsequently grew faster as subadults. Males grew faster than females during the juvenile and subadult stage only. Our findings demonstrate the complex ways in which the external environment influences development in a cooperative mammal. Individuals are most sensitive to social and maternal factors during the period of nutritional dependence on carers, whereas direct environmental effects are relatively more important later in development. Understanding the way in which environmental sensitivity varies across life stages is likely to be an important consideration in predicting trait responses to environmental change.


Emaciated mannequins: a study of mannequin body size in high street fashion stores.
There is concern that the body size of fashion store mannequins are too thin and promote unrealistic body ideals. To date there has been no systematic examination of the size of high street fashion store mannequins. We surveyed national fashion retailers located on the high street of two English cities. The body size of 'male' and 'female' mannequins was assessed by two blinded research assistants using visual rating scales. The average female mannequin body size was representative of a very underweight woman and 100% of female mannequins represented an underweight body size. The average male mannequin body size was significantly larger than the average female mannequin body size. Only 8% of male mannequins represented an underweight body size. The body size of mannequins used to advertise female fashion is unrealistic and would be considered medically unhealthy in humans.


Expanded HIV Testing Strategy Leveraging the Electronic Medical Record Uncovers Undiagnosed Infection Among Hospitalized Patients.
Routine HIV testing of hospitalized patients is recommended, but few strategies to expand testing in the hospital setting have been described. We assessed the impact of an electronic medical record (EMR) prompt on HIV testing for hospitalized patients. We performed a pre-post study at 3 hospitals in the Bronx, NY. We compared the proportion of admissions of patients 21-64 years old with an HIV test performed, characteristics of patients tested, and rate of new HIV diagnoses made by screening while an EMR prompt recommending HIV testing was inactive vs. active. The prompt appeared for patients with no previous HIV test or a high-risk diagnosis after their last HIV test. Among 36,610 admissions while the prompt was inactive, 9.5% had an HIV test performed. Among 18,943 admissions while the prompt was active, 21.8% had an HIV test performed. Admission while the prompt was active was associated with increased HIV testing among total admissions [adjusted odds ratio (aOR) 2.78, 95% confidence interval (CI): 2.62 to 2.96], those without a previous HIV test (aOR 4.03, 95% CI: 3.70 to 4.40), and those with a previous negative test (aOR 1.52, 95% CI: 1.37 to 1.68) (P < 0.0001 for all). Although the prompt was active, testing increased across all patient characteristics. New HIV diagnoses made by screening increased from 8.2/100,000 admissions to 37.0/100,000 admissions while the prompt was inactive and active, respectively (OR 4.51 95% CI: 1.17 to 17.45, P = 0.03). An EMR prompt for hospitalized patients was associated with a large increase in HIV testing, a diversification of patients tested, and an increase in diagnoses made by screening.


A new oxygen carrier for improved long-term storage of human pancreata before islet isolation.
Pancreas oxygenation during cold storage has been established in islet isolation and transplantation to prevent ischemic tissue damage using perfluorodecalin (PFD) as hyperoxygen carrier. However, studies in humans and pigs provided conflicting results about the efficiency of PFD for pancreas oxygenation. The aim of this study was to compare PFD with a newly developed oxygen carrier composed of perfluorohexyloctane and polydimethylsiloxane 5 (F6H8S5) for long-term storage of human pancreata. After 24-hr storage in preoxygenated PFD or F6H8S5, pancreata were processed using Liberase HI for pancreas dissociation and a Ficoll gradient for islet purification. Islet quality assessment was performed measuring glucose-stimulated insulin release, viability, islet ATP content, and posttransplant function in diabetic nude mice. Compared with PFD, F6H8S5 significantly increased the intrapancreatic partial oxygen pressure and islet ATP content. This corresponded to an increase of islet yield, recovery after culture, glucose stimulation index, viability, and improved graft function in diabetic nude mice. The present findings indicate clearly that F6H8S5 improves isolation outcome after prolonged ischemia compared with PFD. This observation seems to be related to the significant lipophilicity and almost pancreas-specific density of F6H8S5. Moreover, these characteristics facilitate pancreas shipment without using custom-made transport vessels as required for PFD.


Environmental impact assessment on the construction and operation of municipal solid waste sanitary landfills in developing countries: China case study.
An inventory of material and energy consumption during the construction and operation (C&O) of a typical sanitary landfill site in China was calculated based on Chinese industrial standards for landfill management and design reports. The environmental impacts of landfill C&O were evaluated through life cycle assessment (LCA). The amounts of materials and energy used during this type of undertaking in China are comparable to those in developed countries, except that the consumption of concrete and asphalt is significantly higher in China. A comparison of the normalized impact potential between landfill C&O and the total landfilling technology implies that the contribution of C&O to overall landfill emissions is not negligible. The non-toxic impacts induced by C&O can be attributed mainly to the consumption of diesel used for daily operation, while the toxic impacts are primarily due to the use of mineral materials. To test the influences of different landfill C&O approaches on environmental impacts, six baseline alternatives were assessed through sensitivity analysis. If geomembranes and geonets were utilized to replace daily and intermediate soil covers and gravel drainage systems, respectively, the environmental burdens of C&O could be mitigated by between 2% and 27%. During the LCA of landfill C&O, the research scope or system boundary has to be declared when referring to material consumption values taken from the literature; for example, the misapplication of data could lead to an underestimation of diesel consumption by 60-80%.


Polyploidy and the evolution of complex traits.
We explore how whole-genome duplications (WGDs) may have given rise to complex innovations in cellular networks, innovations that could not have evolved through sequential single-gene duplications. We focus on two classical WGD events, one in bakers' yeast and the other at the base of vertebrates (i.e., two rounds of whole-genome duplication: 2R-WGD). Two complex adaptations are discussed in detail: aerobic ethanol fermentation in yeast and the rewiring of the vertebrate developmental regulatory network through the 2R-WGD. These two examples, derived from diverged branches on the eukaryotic tree, boldly underline the evolutionary potential of WGD in facilitating major evolutionary transitions. We close by arguing that the evolutionary importance of WGD may require updating certain aspects of modern evolutionary theory, perhaps helping to synthesize a new evolutionary systems biology.


An elaborate sweep-stick code in rat barrel cortex.
In rat barrel cortex, feature encoding schemes uncovered during broadband whisker stimulation are hard to reconcile with the simple stick-slip code observed during natural tactile behaviors, and this has hindered the development of a generalized computational framework. By designing broadband artificial stimuli to sample the inputs encoded under natural conditions, we resolve this disparity while markedly increasing the percentage of deep layer neurons found to encode whisker movements, as well as the diversity of these encoded features. Deep layer neurons encode two main types of events, sticks and sweeps, corresponding to high angular velocity bumps and large angular displacements with high velocity, respectively. Neurons can exclusively encode sticks or sweeps, or they can encode both, with or without direction selectivity. Beyond unifying coding theories from naturalistic and artificial stimulation studies, these findings delineate a simple and generalizable set of whisker movement features that can support a range of perceptual processes.


Respiratory Abnormalities among Occupationally Exposed, Non-Smoking Brick Kiln Workers from Punjab, India.
Brick manufacturing industry is one of the oldest and fast-growing industries in India that employs a large section of people. Brick kiln workers are occupationally exposed to air pollutants. Nonetheless, only a few studies have so far been conducted on their respiratory health. To investigate the extent of respiratory impairment in brick kiln workers and to correlate it with the duration of exposure. A cross-sectional study was conducted. Spirometric parameters of 110 non-smoking male brick kiln workers aged 18-35 years in Patiala district, Punjab, India, were compared with an age-matched comparison group of 90 unexposed individuals. Brick kiln workers showed a significant (p<0.05) decline in forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), forced mid-expiratory flow rate (FEF25-75%) and peak expiratory flow rate (PEFR) compared with those of the comparison group. The extent of deterioration in lung function of brick kiln workers was associated with the duration of exposure. In workers with >8 years of exposure, the mean values of FEV1 (1.92 L), FVC (2.01 L), FEF25-75% (2.19 L/s) and PEFR (4.81 L/s) were significantly (p<0.05) lower than those recorded in workers with <8 years of exposure in whom the values were 2.01 L, 2.68 L, 2.71 L/s, and 5.76 L/s, respectively. There is a significant association between exposure to workplace pollutants and lung function deterioration among brick kiln workers.


[CHROMagar KPC. Comparison with the method proposed by the Centers for Disease Control and Prevention (CDC, USA) for rectal screening and evaluation of false positive results].
Eighty one rectal swabs (RS) were cultured on CHROMagar KPC and the CDC method. Of the 81 samples, 9 were positive for KPC-producing Klebsiella pneumoniae on CHROMagar KPC, and 6 for the CDC method. CHROMagar KPC had two false positive (FP) results: 1 K. pneumoniae and 1 Acinetobacter sp. FP results on the CDC method were: 25 Acinetobacter spp., 2 Escherichia coli and 4 K. pneumoniae: CHROMagar KPC yielded a better recovery of KPC-producing bacteria and less FP results than CDC method. In order to evaluate FP results on CHROMagar KPC, 1247 RS were cultured and yielded 1021 negatives, 171 KPC-producing K. pneumoniae and 55 FP (4.4 %). Because of the FP results growing on CHROMagar KPC, KPC must be phenotypically confirmed in the bacteria isolated.


Molecular crowding shapes gene expression in synthetic cellular nanosystems.
The integration of synthetic and cell-free biology has made tremendous strides towards creating artificial cellular nanosystems using concepts from solution-based chemistry, where only the concentrations of reacting species modulate gene expression rates. However, it is known that macromolecular crowding, a key feature in natural cells, can dramatically influence biochemical kinetics via volume exclusion effects, which reduce diffusion rates and enhance binding rates of macromolecules. Here, we demonstrate that macromolecular crowding can increase the robustness of gene expression by integrating synthetic cellular components of biological circuits and artificial cellular nanosystems. Furthermore, we reveal how ubiquitous cellular modules, including genetic components, a negative feedback loop and the size of the crowding molecules can fine-tune gene circuit response to molecular crowding. By bridging a key gap between artificial and living cells, our work has implications for efficient and robust control of both synthetic and natural cellular circuits.


Sildenafil Enhances Quantity of Immature Neurons and Promotes Functional Recovery in the Developing Ischemic Mouse Brain.
Hypoxic-ischemic (HI) injury to the developing brain occurs in 1 out of 1,000 live births and remains a major cause of significant morbidity and mortality. A large number of survivors suffer from long-term sequelae including seizures and neurological deficits. However, the pathophysiological mechanisms of recovery after HI insult are not clearly understood, and preventive measures or clinical treatments are nonexistent or not sufficiently effective in the clinical setting. Sildenafil as a specific phosphodiesterase 5 inhibitor leads to increased levels of the second messenger cyclic guanosine monophosphate (cGMP) and promotes functional recovery and neurogenesis after ischemic injury to the adult brain. Here, we investigated the effect of sildenafil treatment on activation of intracellular signaling pathways, histological and neurogenic response including functional recovery after an ischemic insult to the developing brain. Nine-day-old C57BL/6 mice were subjected either to sham operation or underwent ligation of the right common carotid artery followed by hypoxia (8%) for 60 min. Animals were either administered sildenafil (10 mg/kg, i.p.) or vehicle 2 h after hypoxia. A subgroup of animals received multiple injections of 10 mg/kg daily on 5 consecutive days. Pups were either perfusion fixed at postnatal days 14 or 47 for immunohistochemical analysis, or brains were dissected 2, 6, 12, and 24 h after the end of hypoxia and analyzed for cGMP, pAkt, pGSK-3β, and β-catenin by means of ELISA or immunoblotting. In addition, behavioral studies using the wire hang test and elevated plus maze were conducted 21 and 38 days after HI injury. Based on cresyl violet staining, single or multiple sildenafil injections did not reveal any differences in injury scoring compared to sham animals. However, cerebral levels of cGMP were altered after sildenafil therapy. Treatment significantly increased numbers of immature neurons, as indicated by doublecortin immunoreactivity in the ipsilateral subventricular zone and striatum. In addition, animals treated with sildenafil after HI insult demonstrated improved functional recovery. pAkt, pGSK-3β, and β-catenin levels vary after HI injury but additional sildenafil treatment had no impact on protein expression compared to the level of sham controls. Here, we report that treatment with sildenafil after HI insult did not improve histological brain injury scores. Nevertheless, our results suggest involvement of the cGMP and PI3K/Akt/GSK-3β signaling pathway with promotion of a neurogenic response and reduction of neurological deficits. In summary, sildenafil may have a role in promoting recovery from HI injury in the developing brain.


Severity dependent distribution of impairments in PSP and CBS: Interactive visualizations.
Progressive supranuclear palsy (PSP) -Richardson's Syndrome and Corticobasal Syndrome (CBS) are the two classic clinical syndromes associated with underlying four repeat (4R) tau pathology. The PSP Rating Scale is a commonly used assessment in PSP clinical trials; there is an increasing interest in designing combined 4R tauopathy clinical trials involving both CBS and PSP. To determine contributions of each domain of the PSP Rating Scale to overall severity and characterize the probable sequence of clinical progression of PSP as compared to CBS. Multicenter clinical trial and natural history study data were analyzed from 545 patients with PSP and 49 with CBS. Proportional odds models were applied to model normalized cross-sectional PSP Rating Scale, estimating the probability that a patient would experience impairment in each domain using the PSP Rating Scale total score as the index of overall disease severity. The earliest symptom domain to demonstrate impairment in PSP patients was most likely to be Ocular Motor, followed jointly by Gait/Midline and Daily Activities, then Limb Motor and Mentation, and finally Bulbar. For CBS, Limb Motor manifested first and ocular showed less probability of impairment throughout the disease spectrum. An online tool to visualize predicted disease progression was developed to predict relative disability on each subscale per overall disease severity. The PSP Rating Scale captures disease severity in both PSP and CBS. Modelling how domains change in relation to one other at varying disease severities may facilitate detection of therapeutic effects in future clinical trials.


Hippo-mediated suppression of IRS2/AKT signaling prevents hepatic steatosis and liver cancer.
Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for liver cancer; therefore, its prevention is an important clinical goal. Ablation of phosphatase and tensin homolog (PTEN) or the protein kinase Hippo signaling pathway induces liver cancer via activation of AKT or the transcriptional regulators YAP/TAZ, respectively; however, the potential for crosstalk between the PTEN/AKT and Hippo/YAP/TAZ pathways in liver tumorigenesis has thus far remained unclear. Here, we have shown that deletion of both PTEN and SAV1 in the liver accelerates the development of NAFLD and liver cancer in mice. At the molecular level, activation of YAP/TAZ in the liver of Pten-/- Sav1-/- mice amplified AKT signaling through the upregulation of insulin receptor substrate 2 (IRS2) expression. Both ablation of YAP/TAZ and activation of the Hippo pathway could rescue these phenotypes. A high level of YAP/ TAZ expression was associated with a high level of IRS2 expression in human hepatocellular carcinoma (HCC). Moreover, treatment with the AKT inhibitor MK-2206 or knockout of IRS2 by AAV-Cas9 successfully repressed liver tumorigenesis in Pten-/- Sav1-/- mice. Thus, our findings suggest that Hippo signaling interacts with AKT signaling by regulating IRS2 expression to prevent NAFLD and liver cancer progression and provide evidence that impaired crosstalk between these 2 pathways accelerates NAFLD and liver cancer.


Vegetarian diet improves insulin resistance and oxidative stress markers more than conventional diet in subjects with Type 2 diabetes.
The aim of this study was to compare the effects of calorie-restricted vegetarian and conventional diabetic diets alone and in combination with exercise on insulin resistance, visceral fat and oxidative stress markers in subjects with Type 2 diabetes. A 24-week, randomized, open, parallel design was used. Seventy-four patients with Type 2 diabetes were randomly assigned to either the experimental group (n = 37), which received a vegetarian diet, or the control group (n = 37), which received a conventional diabetic diet. Both diets were isocaloric, calorie restricted (-500 kcal/day). All meals during the study were provided. The second 12 weeks of the diet were combined with aerobic exercise. Participants were examined at baseline, 12 weeks and 24 weeks. Primary outcomes were: insulin sensitivity measured by hyperinsulinaemic isoglycaemic clamp; volume of visceral and subcutaneous fat measured by magnetic resonance imaging; and oxidative stress measured by thiobarbituric acid reactive substances. Analyses were by intention to treat. Forty-three per cent of participants in the experimental group and 5% of participants in the control group reduced diabetes medication (P < 0.001). Body weight decreased more in the experimental group than in the control group [-6.2 kg (95% CI -6.6 to -5.3) vs. -3.2 kg (95% CI -3.7 to -2.5); interaction group × time P = 0.001]. An increase in insulin sensitivity was significantly greater in the experimental group than in the control group [30% (95% CI 24.5-39) vs. 20% (95% CI 14-25), P = 0.04]. A reduction in both visceral and subcutaneous fat was greater in the experimental group than in the control group (P = 0.007 and P = 0.02, respectively). Plasma adiponectin increased (P = 0.02) and leptin decreased (P = 0.02) in the experimental group, with no change in the control group. Vitamin C, superoxide dismutase and reduced glutathione increased in the experimental group (P = 0.002, P < 0.001 and P = 0.02, respectively). Differences between groups were greater after the addition of exercise training. Changes in insulin sensitivity and enzymatic oxidative stress markers correlated with changes in visceral fat. A calorie-restricted vegetarian diet had greater capacity to improve insulin sensitivity compared with a conventional diabetic diet over 24 weeks. The greater loss of visceral fat and improvements in plasma concentrations of adipokines and oxidative stress markers with this diet may be responsible for the reduction of insulin resistance. The addition of exercise training further augmented the improved outcomes with the vegetarian diet.