A rare biofilm dispersion strategy demonstrated by Staphylococcus aureus under oxacillin stress.

Staphylococcus aureus (S. aureus), a versatile Gram-positive bacterium, is implicated in a spectrum of infections, and its resilience is often attributed to biofilm formation. This study investigates the effect of sub-inhibitory doses of oxacillin on biofilm formation by methicillin-resistant S. aureus (MRSA). Specifically, it examines how these doses influence biofilms' development, maturation, and dispersal. The biofilm's zenith reached 48 h of incubation, followed by a noteworthy decline at 96 h and a distinctive clearance zone around biofilm-positive cells exposed to oxacillin. Scanning electron micrographs unveiled an intriguing active biofilm dispersal mechanism, a rarity in this species. Among 180 isolates, only three carrying the elusive icaD gene exhibited this phenomenon. icaD gene was absent in their counterparts. Notably, the icaD gene emerges as a distinctive marker, crucial in regulating biofilm dispersion and setting these isolates apart. The captivating interplay of oxacillin, biofilm dynamics, and genetic signatures disintegrate novel dimensions in understanding MRSA's adaptive strategies and underscores the importance of the icaD gene in engineering biofilm resilience.

School-level intra-cluster correlation coefficients and autocorrelations for children's accelerometer-measured physical activity in England by age and gender.

BACKGROUND: Randomised, cluster-based study designs in schools are commonly used to evaluate children's physical activity interventions. Sample size estimation relies on accurate estimation of the intra-cluster correlation coefficient (ICC), but published estimates, especially using accelerometry-measured physical activity, are few and vary depending on physical activity outcome and participant age. Less commonly-used cluster-based designs, such as stepped wedge designs, also need to account for correlations over time, e.g. cluster autocorrelation (CAC) and individual autocorrelation (IAC), but no estimates are currently available. This paper estimates the school-level ICC, CAC and IAC for England children's accelerometer-measured physical activity outcomes by age group and gender, to inform the design of future school-based cluster trials. METHODS: Data were pooled from seven large English datasets of accelerometer-measured physical activity data between 2002-18 (> 13,500 pupils, 540 primary and secondary schools). Linear mixed effect models estimated ICCs for weekday and whole week for minutes spent in moderate-to-vigorous physical activity (MVPA) and being sedentary for different age groups, stratified by gender. The CAC (1,252 schools) and IAC (34,923 pupils) were estimated by length of follow-up from pooled longitudinal data. RESULTS: School-level ICCs for weekday MVPA were higher in primary schools (from 0.07 (95% CI: 0.05, 0.10) to 0.08 (95% CI: 0.06, 0.11)) compared to secondary (from 0.04 (95% CI: 0.03, 0.07) to (95% CI: 0.04, 0.10)). Girls' ICCs were similar for primary and secondary schools, but boys' were lower in secondary. For all ages, combined the CAC was 0.60 (95% CI: 0.44-0.72), and the IAC was 0.46 (95% CI: 0.42-0.49), irrespective of follow-up time. Estimates were higher for MVPA vs sedentary time, and for weekdays vs the whole week. CONCLUSIONS: Adequately powered studies are important to evidence effective physical activity strategies. Our estimates of the ICC, CAC and IAC may be used to plan future school-based physical activity evaluations and were fairly consistent across a range of ages and settings, suggesting that results may be applied to other high income countries with similar school physical activity provision. It is important to use estimates appropriate to the study design, and that match the intended study population as closely as possible.

Gender differences in the distribution of children's physical activity: evidence from nine countries.

BACKGROUND: Physical activity in childhood is thought to influences health and development. Previous studies have found that boys are typically more active than girls, yet the focus has largely been on differences in average levels or proportions above a threshold rather than the full distribution of activity across all intensities. We thus examined differences in the distribution of physical activity between girls and boys in a multi-national sample of children. METHODS: We used the harmonised International Children Accelerometry Database (ICAD), including waist-worn accelerometry data from 15,461 individuals (Boys: 48.3%) from 9 countries. Employing Generalised Additive Models of Location, Shape, and Scale (GAMLSS) we investigated gender differences in the distribution of individuals, including comparisons of variability (SD) and average physical activity levels (mean and median) and skewness. We conducted this analysis for each activity intensity (Sedentary, Light, and Moderate-to-Vigorous (MVPA)) and a summary measure (counts per minute (CPM)). RESULTS: Sizable gender differences in the distribution of activity were found for moderate to vigorous activity and counts per minute, with boys having higher average levels (38% higher mean volumes of MVPA, 20% higher CPM), yet substantially more between-person variability (30% higher standard deviation (SD) for MVPA, 17% higher SD for CPM); boys' distributions were less positively skewed than girls. Conversely, there was little to no difference between girls and boys in the distribution of sedentary or light-intensity activity. CONCLUSIONS: Inequality in activity between girls and boys was driven by MVPA. The higher mean volumes of MVPA in boys occurred alongside greater variability. This suggests a need to consider the underlying distribution of activity in future research; for example, interventions which target gender inequality in MVPA may inadvertently lead to increased inequality within girls.

Cross-sectional and longitudinal associations of active travel, organised sport and physical education with accelerometer-assessed moderate-to-vigorous physical activity in young people: the International Children's Accelerometry Database.

BACKGROUND: Physical activity (PA) declines during childhood. Important sources of PA are active travel, organised sport and physical education (PE), but it is unclear how these domain-specific PA sources contribute to (changes in) daily moderate-to-vigorous PA (MVPA) in young people. This study aimed to examine (1) the cross-sectional association between domain-specific physical activity (i.e., active travel, organised sport and PE) and daily minutes in accelerometer-assessed MVPA; and (2) the longitudinal association between domain-specific physical activity at baseline and change in daily minutes in MVPA. METHODS: Participants (baseline age 11.3 ± .1.2 years) were drawn from three studies in the International Children's Accelerometry Database. The contribution of self-reported standardised active travel, organised sport and PE to accelerometer-measured daily minutes in MVPA was examined using linear regression. In cross-sectional analyses, MVPA was regressed on each PA domain in separate models, adjusted for study, age, sex, maternal education, season, and monitor wear time. In longitudinal analyses, change in MVPA was regressed on each of the baseline PA domains, additionally adjusting for changes in season and wear time, follow-up duration, and baseline MVPA. R-squared was used to compare variance explained by each PA domain. RESULTS: In the cross-sectional analyses (n = 3871), organised sport (standardised ß = 3.81, 95% confidence interval [95%CI] = 3.06, 4.56) and active travel (ß = 3.46, 95%CI = 2.73, 4.19) contributed more to daily MVPA than PE (ß = 0.82, 95%CI = -0.02, 1.66). Compared to the base model which included only covariates (R2 = 21.5%), organised sport (absolute change: + 1.9%) and active travel (+ 1.7%) models explained more of the variance than the PE model (± < 0.1%). Associations followed a similar pattern in the longitudinal analyses (n = 2302), but none of the PA domains predicted change in MVPA (organised sport: standardised ß = 0.85, 95%CI = -0.03, 1.72; active travel: ß = 0.68, 95%CI = -0.14, 1.50; PE: ß = 0.02, 95%CI = -0.87, 0.91). CONCLUSIONS: A multi-sectoral approach covering a wide range of PA domains should be promoted to minimise the age-related decline in MVPA during childhood.

Incidence of biofilm formation among MRSA and MSSA clinical isolates from hospitalized patients in Israel.

AIM: To assess the biofilm-producing capacities of Staphylococcus aureus strains isolated from hospitalized patients in Israel. METHODS AND RESULTS: A total of 16 S. aureus (80 MRSA and 83 MSSA) from screening (nasal swab) and clinical samples (blood and wounds) were characterized. Biofilm-producing capacities were determined using two different biofilm detection assays: Congo Red agar (CRA) and microtiter plate (MtP). In addition, a real-time PCR analysis was performed to detect the presence of biofilm-associated genes (icaA and icaD) and mecA gene. The two assays showed similar biofilm production pattern (28.2% agreement). MRSA strains tended to be greater biofilm-producers than MSSA strains. The presence of mecA was associated with biofilm production (p = 0.030). Additionally, bacteria isolated from blood samples produced less biofilm compared to those from other sources. Finally, no association was found between icaA and icaD presence and biofilm production. CONCLUSION: This study supports earlier assumptions that biofilm formation depends strongly on environmental conditions. SIGNIFICANCE AND IMPACT OF STUDY: This study significantly improved our knowledge on the biofilm production capacity of S. aureus strains in Israel. Moreover, it revealed an association between the mecA gene and biofilm production. Finally, this study underscores the importance of further research to evaluate risk factors for biofilm production.

Association of change in the school travel mode with changes in different physical activity intensities and sedentary time: A International Children's Accelerometry Database Study.

Our aim was to assess the association between changes in active travel to school and changes in different intensities of physical activity (i.e. moderate - MPA and vigorous - VPA) and time spent sedentary (SED) among adolescents and assess the moderating effect of children's sex, age and weight status. Data from six cohort studies in the International Children's Accelerometry Database were used (4108 adolescents aged 10-13y at baseline, with 1.9±0.7y of follow-up). Participants self-reported travel mode to school at baseline and follow-up. Mutually exclusive categories of change were created using passive (e.g. by car) or active (cycling or walking) forms of transport (active/active, passive/active, active/passive, passive/passive). Multilevel linear regression analyses assessed associations with change in accelerometer-assessed time spent MPA, VPA and SED, adjusting for potential confounders. The moderation of sex, age and weight status was tested though the inclusion of interaction terms in the regression models. Relative to those remaining in active travel (active/active), participants classified as passive/active increased VPA (B: 2.23 min/d; 95%CI: 0.97-3.48), while active/passive (MPA: -5.38min/d; -6.77 to -3.98; VPA: -2.92min/d; -4.06 to -1.78) and passive/passive (MPA: -4.53min/d; -5.55 to -3.50; VPA: -2.84min/d; -3.68 to -2.01) decreased MPA and VPA. There were no associations with SED. An interaction was observed, age group moderated the association with change in VPA: among 12-13y-olds a greater increase in VPA was observed for the passive/active group compared to active/active. Promoting active travel to school can be a strategy to attenuate the decline in physical activity through adolescence.

Tracking of total sedentary time and sedentary patterns in youth: a pooled analysis using the International Children's Accelerometry Database (ICAD).

BACKGROUND: To gain more understanding of the potential health effects of sedentary time, knowledge is required about the accumulation and longitudinal development of young people's sedentary time. This study examined tracking of young peoples' total and prolonged sedentary time as well as their day-to-day variation using the International Children's Accelerometry Database. METHODS: Longitudinal accelerometer data of 5991 children (aged 4-17y) was used from eight studies in five countries. Children were included if they provided valid (≥8 h/day) accelerometer data on ≥4 days, including ≥1 weekend day, at both baseline and follow-up (average follow-up: 2.7y; range 0.7-8.2). Tracking of total and prolonged (i.e. ≥10-min bouts) sedentary time was examined using multilevel modelling to adjust for clustering of observations, with baseline levels of sedentary time as predictor and follow-up levels as outcome. Standardized regression coefficients were interpreted as tracking coefficients (low: < 0.3; moderate: 0.3-0.6; high: > 0.6). RESULTS: Average total sedentary time at study level ranged from 246 to 387 min/day at baseline and increased annually by 21.4 min/day (95% confidence interval [19.6-23.0]) on average. This increase consisted almost entirely of prolonged sedentary time (20.9 min/day [19.2-22.7]). Total (standardized regression coefficient (B) = 0.48 [0.45-0.50]) and prolonged sedentary time (B = 0.43 [0.41-0.45]) tracked moderately. Tracking of day-to-day variation in total (B = 0.04 [0.02-0.07]) and prolonged (B = 0.07 [0.04-0.09]) sedentary time was low. CONCLUSION: Young people with high levels of sedentary time are likely to remain among the people with highest sedentary time as they grow older. Day-to-day variation in total and prolonged sedentary time, however, was rather variable over time.

The multivariate physical activity signature associated with metabolic health in children and youth: An International Children's Accelerometry Database (ICAD) analysis.

There is solid evidence for an association between physical activity and metabolic health outcomes in children and youth, but for methodological reasons most studies describe the intensity spectrum using only a few summary measures. We aimed to determine the multivariate physical activity intensity signature associated with metabolic health in a large and diverse sample of children and youth, by investigating the association pattern for the entire physical intensity spectrum. We used pooled data from 11 studies and 11,853 participants aged 5.8-18.4 years included in the International Children's Accelerometry Database. We derived 14 accelerometry-derived (ActiGraph) physical activity variables covering the intensity spectrum (from 0-99 to ≥8000 counts per minute). To handle the multicollinearity among these variables, we used multivariate pattern analysis to establish the associations with indices of metabolic health (abdominal fatness, insulin sensitivity, lipid metabolism, blood pressure). A composite metabolic health score was used as the main outcome variable. Associations with the composite metabolic health score were weak for sedentary time and light physical activity, but gradually strengthened with increasing time spent in moderate and vigorous intensities (up to 4000-5000 counts per minute). Association patterns were fairly consistent across sex and age groups, but varied across different metabolic health outcomes. This novel analytic approach suggests that vigorous intensity, rather than less intense activities or sedentary behavior, are related to metabolic health in children and youth.

A closer look at the relationship among accelerometer-based physical activity metrics: ICAD pooled data.

BACKGROUND: Accelerometers are widely used to assess child physical activity (PA) levels. Using the accelerometer data, several PA metrics can be estimated. Knowledge about the relationships between these different metrics can improve our understanding of children's PA behavioral patterns. It also has significant implications for comparing PA metrics across studies and fitting a statistical model to examine their health effects. The aim of this study was to examine the relationships among the metrics derived from accelerometers in children. METHODS: Accelerometer data from 24,316 children aged 5 to 18 years were extracted from the International Children's Accelerometer Database (ICAD) 2.0. Correlation coefficients between wear time, sedentary behavior (SB), light-intensity PA (LPA), moderate-intensity PA (MPA), vigorous-intensity PA (VPA), moderate- and vigorous-intensity PA (MVPA), and total activity counts (TAC) were calculated. RESULTS: TAC was approximately 22X103 counts higher (p < 0.01) with longer wear time (13 to 18 h/day) as compared to shorter wear time (8 to < 13 h/day), while MVPA was similar across the wear time categories. MVPA was very highly correlated with TAC (r = .91; 99% CI = .91 to .91). Wear time-adjusted correlation between SB and LPA was also very high (r = -.96; 99% CI = -.96, - 95). VPA was moderately correlated with MPA (r = .58; 99% CI = .57, .59). CONCLUSIONS: TAC is mostly explained by MVPA, while it could be more dependent on wear time, compared to MVPA. MVPA appears to be comparable across different wear durations and studies when wear time is ≥8 h/day. Due to the moderate to high correlation between some PA metrics, potential collinearity should be addressed when including multiple PA metrics together in statistical modeling.

Substituting prolonged sedentary time and cardiovascular risk in children and youth: a meta-analysis within the International Children's Accelerometry database (ICAD).

BACKGROUND: Evidence on the association between sitting for extended periods (i.e. prolonged sedentary time (PST)) and cardio-metabolic health is inconsistent in children. We aimed to estimate the differences in cardio-metabolic health associated with substituting PST with non-prolonged sedentary time (non-PST), light (LIPA) or moderate-to-vigorous physical activity (MVPA) in children. METHODS: Cross-sectional data from 14 studies (7 countries) in the International Children's Accelerometry Database (ICAD, 1998-2009) was included. Accelerometry in 19,502 participants aged 3-18 years, together with covariate and outcome data, was pooled and harmonized. Iso-temporal substitution in linear regression models provided beta coefficients (95%CI) for substitution of 1 h/day PST (sedentary time accumulated in bouts > 15 min) with non-PST, LIPA or MVPA, for each study, which were meta-analysed. RESULTS: Modelling substitution of 1 h/day of PST with non-PST suggested reductions in standardized BMI, but estimates were > 7-fold greater for substitution with MVPA (- 0.44 (- 0.62; - 0.26) SD units). Only reallocation by MVPA was beneficial for waist circumference (- 3.07 (- 4.47; - 1.68) cm), systolic blood pressure (- 1.53 (- 2.42; - 0.65) mmHg) and clustered cardio-metabolic risk (- 0.18 (- 0.3; - 0.1) SD units). For HDL-cholesterol and diastolic blood pressure, substitution with LIPA was beneficial; however, substitution with MVPA showed 5-fold stronger effect estimates (HDL-cholesterol: 0.05 (0.01; 0.10) mmol/l); diastolic blood pressure: - 0.81 (- 1.38; - 0.24) mmHg). CONCLUSIONS: Replacement of PST with MVPA may be the preferred scenario for behaviour change, given beneficial associations with a wide range of cardio-metabolic risk factors (including adiposity, HDL-cholesterol, blood pressure and clustered cardio-metabolic risk). Effect estimates are clinically relevant (e.g. an estimated reduction in waist circumference of ≈1.5 cm for 30 min/day replacement). Replacement with LIPA could be beneficial for some of these risk factors, however with substantially lower effect estimates.

Caspase-mediated cleavage of X-ray repair cross-complementing group 4 promotes apoptosis by enhancing nuclear translocation of caspase-activated DNase.

X-ray repair cross-complementing group 4 (XRCC4), a repair protein for DNA double-strand breaks, is cleaved by caspases during apoptosis. In this study, we examined the role of XRCC4 in apoptosis. Cell lines, derived from XRCC4-deficient M10 mouse lymphoma cells and stably expressing wild-type XRCC4 or caspase-resistant XRCC4, were established and treated with staurosporine (STS) to induce apoptosis. In STS-induced apoptosis, expression of wild-type, but not caspase-resistant, XRCC4 in XRCC4-deficient cells enhanced oligonucleosomal DNA fragmentation and the appearance of TUNEL-positive cells by promoting nuclear translocation of caspase-activated DNase (CAD), a major nuclease for oligonucleosomal DNA fragmentation. CAD activity is reportedly regulated by the ratio of two inhibitor of CAD (ICAD) splice variants, ICAD-L and ICAD-S mRNA, which, respectively, produce proteins with and without the ability to transport CAD into the nucleus. The XRCC4-dependent promotion of nuclear import of CAD in STS-treated cells was associated with reduction of ICAD-S mRNA and protein, and enhancement of phosphorylation and nuclear import of serine/arginine-rich splicing factor (SRSF) 1. These XRCC4-dependent, apoptosis-enhancing effects were canceled by depletion of SRSF1 or SR protein kinase (SRPK) 1. In addition, overexpression of SRSF1 in XRCC4-deficient cells restored the normal level of apoptosis, suggesting that SRSF1 functions downstream of XRCC4 in activating CAD. This XRCC4-dependent, SRPK1/SRSF1-mediated regulatory mechanism was conserved in apoptosis in Jurkat human leukemia cells triggered by STS, and by two widely used anti-cancer agents, Paclitaxel and Vincristine. These data imply that the level of XRCC4 expression could be used to predict the effects of apoptosis-inducing drugs in cancer treatment.

Biofilm-producing ability of Staphylococcus spp isolated from different foodstuff products.

BACKGROUND: In recent times, microbial-biofilm contamination has attracted considerable attention to the food industry. Pathogenic microorganisms can attach to food surfaces, grow on them, and form biofilm that cause an increase in the food safety risk. The mechanisms of biofilm formation have become an important issue in the food-processing industry, therefore, the aim of this study is to determine the biofilm formation and profiles of genes involved in biofilm production of staphylococci isolated from various foodstuff products. MATERIAL AND METHODS: This cross-sectional study was conducted at some grocery stores and confectionaries from September 2015 to October 2016 in different areas of Isfahan, Iran. Staphylococcus spp were isolated from different foodstuff samples including sweet pastries, cakes and similar baked goods, dairy products such as cheese and yogurt, meat products such as sausages, and hamburgers. Standard microbiological methods were used for identification of Staphylococcus spp isolates. Antibiotic susceptibility pattern was determined by the disc diffusion method and icaA/icaD genes have been investigated as PCR target because of their role in the expression of intercellular adhesions involved in biofilm formation by S. aureus. RESULTS: From a total of 194 different foodstuffs samples, 84 Staphylococcus spp were isolated. Out of the 84 Staphylococcus isolates, 95.2% (80/84) were positive to the ability of biofilm formation. Overall, 35.7% (30/84) and 26.2% (22/84) of Staphylococcus spp isolates were positive for icaA and icaD genes, respectively. CONCLUSION: The results of the present study indicate that the remarkable rate of biofilm formation with the emergence of antibiotic resistance still remains a significant risk for the food safety, especially in foodstuff samples.

Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling.

Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies.

Phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus isolates from clinical specimens, an Atomic Force Microscopic (AFM) study.

INTRODUCTION: Staphylococcus aureus is a major cause of nosocomial infections. Biofilm formation is an important factor for bacterial pathogenesis. Its mechanisms are complex and include of many genes depends on expression of icaADBC operon involved in the synthesis of a polysaccharide intercellular adhesion. AIM: The aim of study was to investigate biofilm forming ability of Staphylococcus aureus strains by phenotypic and genotypic methods. Also Atomic Force microscope (AFM) was used to visualize biofilm formation. METHODS: 140 Isolates were collected from clinical specimens of patients in Milad Hospital, Tehran and diagnosed by biochemical tests. The ability of strains to produce slime was evaluated by CRA method. For diagnosing of bacterial EPS, Indian ink staining were used and finally biofilm surface of 3 isolates observed by AFM. The prevalence of icaA and icaD genes was determined by PCR. RESULTS: By CRA method 15% of samples considered as positive slime producers, 44.28% as intermediate and 40.71% indicative as negative slime producers. 118 staphylococcus aureus strains showed a distinct halo transparent zone but 22 strains showed no halo zone. AFM analysis of Slime positive isolates showed a distinct and complete biofilm formation. In slime negative strain, there was not observed biofilm. The prevalence of icaA, icaD genes was 44.2% and 10% of the isolates had both genes simultaneously. CONCLUSION: There is a relationship between exopolysaccharide layer and biofilm formation of Staphylococcus aureus isolates. The presence of icaAD genes among isolates is not associated with in vitro formation of biofilm. AFM is a useful tool for observation of bacterial biofilm formation.

Weather and children's physical activity; how and why do relationships vary between countries?

BACKGROUND: Globally most children do not engage in enough physical activity. Day length and weather conditions have been identified as determinants of physical activity, although how they may be overcome as barriers is not clear. We aim to examine if and how relationships between children's physical activity and weather and day length vary between countries and identify settings in which children were better able to maintain activity levels given the weather conditions they experienced. METHODS: In this repeated measures study, we used data from 23,451 participants in the International Children's Accelerometry Database (ICAD). Daily accelerometer-measured physical activity (counts per minute; cpm) was matched to local weather conditions and the relationships assessed using multilevel regression models. Multilevel models accounted for clustering of days within occasions within children within study-cities, and allowed us to explore if and how the relationships between weather variables and physical activity differ by setting. RESULTS: Increased precipitation and wind speed were associated with decreased cpm while better visibility and more hours of daylight were associated with increased cpm. Models indicated that increases in these variables resulted in average changes in mean cpm of 7.6/h of day length, -13.2/cm precipitation, 10.3/10 km visibility and -10.3/10kph wind speed (all p < 0.01). Temperature showed a cubic relationship with cpm, although between 0 and 20 degrees C the relationship was broadly linear. Age showed interactions with temperature and precipitation, with the associations larger among younger children. In terms of geographic trends, participants from Northern European countries and Melbourne, Australia were the most active, and also better maintained their activity levels given the weather conditions they experienced compared to those in the US and Western Europe. CONCLUSIONS: We found variation in the relationship between weather conditions and physical activity between ICAD studies and settings. Children in Northern Europe and Melbourne, Australia were not only more active on average, but also more active given the weather conditions they experienced. Future work should consider strategies to mitigate the impacts of weather conditions, especially among young children, and interventions involving changes to the physical environment should consider how they will operate in different weather conditions.

Harmonising data on the correlates of physical activity and sedentary behaviour in young people: Methods and lessons learnt from the international Children's Accelerometry database (ICAD).

BACKGROUND: Large, heterogeneous datasets are required to enhance understanding of the multi-level influences on children's physical activity and sedentary behaviour. One route to achieving this is through the pooling and co-analysis of data from multiple studies. Where this approach is used, transparency of the methodology for data collation and harmonisation is essential to enable appropriate analysis and interpretation of the derived data. In this paper, we describe the acquisition, management and harmonisation of non-accelerometer data in a project to expand the International Children's Accelerometry Database (ICAD). METHOD: Following a consultation process, ICAD partners were requested to share accelerometer data and information on selected behavioural, social, environmental and health-related constructs. All data were collated into a single repository for cataloguing and harmonisation. Harmonised variables were derived iteratively, with input from the ICAD investigators and a panel of invited experts. Extensive documentation, describing the source data and harmonisation procedure, was prepared and made available through the ICAD website. RESULTS: Work to expand ICAD has increased the number of studies with longitudinal accelerometer data, and expanded the breadth of behavioural, social and environmental characteristics that can be used as exposure variables. A set of core harmonised variables, including parent education, ethnicity, school travel mode/duration and car ownership, were derived for use by the research community. Guidance documents and facilities to enable the creation of new harmonised variables were also devised and made available to ICAD users. An expanded ICAD database was made available in May 2017. CONCLUSION: The project to expand ICAD further demonstrates the feasibility of pooling data on physical activity, sedentary behaviour and potential determinants from multiple studies. Key to this process is the rigorous conduct and reporting of retrospective data harmonisation, which is essential to the appropriate analysis and interpretation of derived data. These documents, made available through the ICAD website, may also serve as a guide to others undertaking similar projects.

Auxin-induced rapid degradation of inhibitor of caspase-activated DNase (ICAD) induces apoptotic DNA fragmentation, caspase activation, and cell death: a cell suicide module.

Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms.

Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is commonly found in Asia, especially among the Chinese ethnic group. Chromosome rearrangements are common among NPC patients. Although the mechanism underlying the chromosome rearrangements in NPC is unclear, various mechanisms including activation of caspase-activated DNase (CAD) were proposed to contribute to chromosome rearrangements in leukaemia. Activation of CAD can be initiated by multiple agents, including oxidative stress, which is well implicated in carcinogenesis. CAD is the main enzyme that causes DNA fragmentation during apoptosis, and CAD is also implicated in promoting cell differentiation. In view of the role of oxidative stress in carcinogenesis and CAD activation, and since CAD was suggested to contribute to chromosome rearrangement in leukaemia, we hypothesise that oxidative stress-induced CAD activation could be one of the mechanisms that leads to chromosome rearrangements in NPC. METHODS: SUNEI cells were treated with various concentrations of H2O2 for different period of time to ensure that cells undergo H2O2-induced MLL gene cleavage. Transfections with hCAD, mCAD, mutant hCAD, or cotransfection with hCAD and mICAD, and cotransfection with mutant hCAD and mICAD were performed. Gene expression was confirmed by Western blotting and MLL gene cleavage was assessed by inverse polymerase chain reaction (IPCR). RESULTS: Treatment with H2O2 clearly induces cleavages within the MLL gene which locates at 11q23, a common deletion site in NPC. In order to investigate the role of CAD, CAD was overexpressed in SUNE1 cells, but that did not result in significant changes in H2O2-induced MLL gene cleavage. This could be because CAD requires ICAD for proper folding. Indeed, by overexpressing ICAD alone or co-expressing ICAD with CAD, Western blotting showed that CAD was expressed. In addition, ICAD overexpression also suppressed H2O2-induced MLL gene cleavage, suggesting a possible role of CAD in initiating chromosome cleavage during oxidative stress. CONCLUSIONS: Oxidative stress mediated by H2O2 induces cleavage of the MLL gene, most likely via the caspase-activated DNase, CAD, and CAD expression requires ICAD. Since the MLL gene is located at 11q23, a common deletion site in NPC, thus stress-induced CAD activation may represent one of the mechanisms leading to chromosome rearrangement in NPC.

Painless hypoglossal palsy as an isolated symptom of spontaneous carotid dissection.

Spontaneous internal carotid artery dissection (sICAD) occurs annually in 2.5 to 3 per 100,000 presenting with signs of ischemic events in the majority of cases. In contrast, lower cranial nerve palsy due to peripheral nerve affection is seldom the presenting clinical sign. In symptomatic cases (>90%), sICAD is most commonly accompanied by local pain. We report a case of a 49-year old woman with a left sICAD presenting with isolated ipsilateral hypoglossal palsy as the sole clinical sign. Compared to other cases, local pain was absent and other cranial nerves were not affected. Further, sICAD could not be detected in repeated Doppler-/Duplex-sonography, but magnetic resonance imaging and MR-angiography only.

Emerging threats of high biofilm formation and antibiotic resistance in clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from Pakistan.

OBJECTIVES: This multicenter study, conducted from a One Health perspective, aimed to comprehensively examine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections and their biofilm-forming capabilities in Pakistan. Phylogenetic analysis of the study isolates was also performed. METHODS: A total of 150 MRSA isolates were screened from various clinical samples using Cefoxitin antibiotic discs. Genotypic confirmation was conducted through mecA, S. aureus-specific nuc, and 16S rRNA genes. Biofilm formation was assessed using Congo red agar (CRA) and slime layer quantification methods. The intercellular adhesion (ica) operon genes, specifically icaA and icaD, were detected. Phylogenetic analysis utilized the 16S rRNA sequences. Statistical associations between various parameters were determined using chi-square analysis. RESULTS: The presence of the mecA gene was observed in 131 out of 150 isolates (87.3%). CRA identified 28% and 40% of isolates as strong and moderate biofilm producers, respectively, while 9.3% were classified as non-biofilm producers. The slime layer assay exhibited higher sensitivity, classifying only 4.7% of isolates as non-biofilm producers. Biofilm-forming genes icaA and icaD were detected in 85.3% and 86.7% of the isolates, respectively. Antibiotic resistance was more prevalent among biofilm-forming isolates, particularly against ciprofloxacin, levofloxacin, erythromycin, trimethoprim-sulfamethoxazole, and fosfomycin. Ceftaroline demonstrated efficacy irrespective of biofilm-forming abilities. Conversely, non-biofilm producers exhibited complete susceptibility to clarithromycin and tigecycline. CONCLUSIONS: Clinical MRSA strains exhibit a substantial potential for biofilm formation, contributing to a resistant phenotype. Routine antibiotic testing in clinical settings that overlook the biofilm aspect may lead to the failure of empiric antibiotic therapy.