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Brain dynamics is highly non-stationary, permanently subject to ever-changing external conditions and continuously monitoring and adjusting internal control mechanisms. Finding stationary structures in this system, as has been done recently, is therefore of great importance for understanding fundamental dynamic trade relationships. Here we analyse electroencephalographic recordings (EEG) of 13 subjects with unresponsive wakefulness syndrome (UWS) during rest and while being influenced by different acoustic stimuli. We compare the results with a control group under the same experimental conditions and with clinically healthy subjects during overnight sleep. The main objective of this study is to investigate whether a stationary correlation pattern is also present in the UWS group, and if so, to what extent this structure resembles the one found in healthy subjects. Furthermore, we extract transient dynamical features via specific deviations from the stationary interrelation pattern. We find that (i) the UWS group is more heterogeneous than the two groups of healthy subjects, (ii) also the EEGs of the UWS group contain a stationary cross-correlation pattern, although it is less pronounced and shows less similarity to that found for healthy subjects and (iii) deviations from the stationary pattern are notably larger for the UWS than for the two groups of healthy subjects. The results suggest that the nervous system of subjects with UWS receive external stimuli but show an overreaching reaction to them, which may disturb opportune information processing.

Analysing the effect water temperature has on Daphnia magna is essential in anticipating the impact climate change will have on this freshwater zooplanktonic keystone species. While many authors have followed this line of research, few have covered an extensive temperature range or complex temperature change scenarios. Global warming is mostly associated with increased extreme temperature events, such as heat waves, as well as earlier and more intense thermal stratification. Both of these events may directly influence D. magna fitness, especially in those populations performing diel vertical migration (DVM). We analysed the effect water temperatures, ranging from 11 to 29°C, have on the filtration capacity (FC) of D. magna, to anticipate the effects of acclimation, temperature change rate (TCR) and potential reversibility of responses to such conditions. Results show that sudden temperature changes have an immediate negative impact on the FC of D. magna and is more severe at higher temperatures and higher TCRs. However, D. magna individuals have shown themselves to be capable of quasi-acclimating to temperatures ranging from 11 to 25°C in around a week and achieving much higher FCs; albeit never reaching the optimal FC achieved at 20°C. That said, 29°C is lethal for D. magna individuals within approximately five days. Finally, non-optimal temperature acclimated individuals can recover maximal FC within 2-4 days of the optimal long-term acclimation temperature (20°C) being re-established, thus proving temperature responses to be reversible.

Cultural evolutionary theory provides a framework for explaining change in population-level distributions. A consistent finding in the literature is that multiple transmission episodes shape a distribution of cultural traits to become more compressible, i.e., a set of derived traits are more compressed than their ancestral forms. Importantly, this amplification of compressible patterns can become manifest in two ways, either via the homogenisation of variation or through the organisation of variation into structured and specialised patterns. Using a novel, large-scale dataset from Reddit Place, an online collaborative art project, we investigate the emergence and evolution of compressible patterns on a 1000x1000 pixel canvas. Here, all Reddit users could select a coloured pixel, place it on the canvas, and then wait for a fixed period before placing another pixel. By analysing all 16.5 million pixel placements by over 1 million individuals, we found that compression follows a quadratic trajectory through time. From a non-structured state, where individual artworks exist relatively independently from one another, Place gradually transitions to a structured state where pixel placements form specialised, interdependent patterns.

OBJECTIVE:We evaluated the clinical potential of a novel robotic system for autonomous performance of waterjet wound debridement. SUMMARY BACKGROUND DATA:Within the last decade, waterjet wound debridement has proven to be a valid alternative to the conventional approach using sharp spoons and scalpel. METHODS:The DLR MIRO robot using the DLR MICA instrument for robotic surgery was adapted for actuation of an ERBEJET 2 flexible endoscopic waterjet probe. Waterjet debridement of various wound shapes and sizes using a porcine skin model was compared between this novel robotic system and a control group of human medical professionals with regard to wound area cleaned by the waterjet, off-target area, and procedural time. RESULTS:After the wound area was registered in the robotic system, it automatically generated a cleaning path and performed debridement based on generated surface model. While the robotic system demonstrated a significant advantage for the covered wound area (p = 0.031), the average off-target area was not significantly different from human controls. Human participants had high variability in cleaning quality across users and trials, while the robotic system provided stable results. Overall procedural time was significantly lower in trials performed by humans. CONCLUSIONS:Robotic waterjet wound debridement is a promising new technological approach compared to the current clinical standard of interventional wound therapy, providing higher efficiency and quality of wound cleaning compared to human performance. Additional trials on more complicated wound shapes and in vivo tissue are necessary to more thoroughly evaluate the clinical potential of this technology.

The hepatic Na+/taurocholate co-transporting polypeptide (NTCP in man, Ntcp in animals) is the high-affinity receptor for the hepatitis B (HBV) and hepatitis D (HDV) viruses. Species barriers for human HBV/HDV within the order Primates were previously attributed to Ntcp sequence variations that disable virus-receptor interaction. However, only a limited number of primate Ntcps have been analysed so far. In the present study, a total of 11 Ntcps from apes, Old and New World monkeys were cloned and expressed in vitro to characterise their interaction with HBV and HDV. All Ntcps showed intact bile salt transport. Human NTCP as well as the Ntcps from the great apes chimpanzee and orangutan showed transport-competing binding of HBV derived myr-preS1-peptides. In contrast, all six Ntcps from the group of Old World monkeys were insensitive to HBV myr-preS1-peptide binding and HBV/HDV infection. This is basically predetermined by the amino acid arginine at position 158 of all studied Old World monkey Ntcps. An exchange from arginine to glycine (as present in humans and great apes) at this position (R158G) alone was sufficient to achieve full transport-competing HBV myr-preS1-peptide binding and susceptibility for HBV/HDV infection. New World monkey Ntcps showed higher sequence heterogeneity, but in two cases with 158G showed transport-competing HBV myr-preS1-peptide binding, and in one case (Saimiri sciureus) even susceptibility for HBV/HDV infection. In conclusion, amino acid position 158 of NTCP/Ntcp is sufficient to discriminate between the HBV/HDV susceptible group of humans and great apes (158G) and the non-susceptible group of Old World monkeys (158R). In the case of the phylogenetically more distant New World monkey Ntcps amino acid 158 plays a significant, but not exclusive role.

Group I introns are pre-mRNA introns that do not require the spliceosome for their removal. Instead, they fold into complex three-dimensional structures and catalyze two transesterification reactions, thereby excising themselves and joining the flanking exons. These catalytic RNAs (ribozymes) have been modified previously to work in trans, whereby the ribozymes can recognize a splice site on a substrate RNA and replace the 5'- or 3'-portion of the substrate. Here we describe a new variant of the group I intron ribozyme from Tetrahymena that recognizes two splice sites on a substrate RNA, removes the intron sequences between the splice sites, and joins the flanking exons, analogous to the action of the spliceosome. This 'group I spliceozyme' functions in vitro and in vivo, and it is able to mediate a growth phenotype in E. coli cells. The intron sequences of the target pre-mRNAs are constrained near the splice sites but can carry a wide range of sequences in their interior. Because the splice site recognition sequences can be adjusted to different splice sites, the spliceozyme may have the potential for wide applications as tool in research and therapy.

How does a non-coding RNA evolve in cells? To address this question experimentally we evolved a trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles of evolution in E.coli cells. Sequence variation was introduced during the evolution by mutagenic and recombinative PCR, and increasingly active ribozymes were selected by their repair of an mRNA mediating antibiotic resistance. The most efficient ribozyme contained four clustered mutations that were necessary and sufficient for maximum activity in cells. Surprisingly, these mutations did not increase the trans-splicing activity of the ribozyme. Instead, they appear to have recruited a cellular protein, the transcription termination factor Rho, and facilitated more efficient translation of the ribozyme's trans-splicing product. In addition, these mutations affected the expression of several other, unrelated genes. These results suggest that during RNA evolution in cells, four mutations can be sufficient to evolve new protein interactions, and four mutations in an RNA molecule can generate a large effect on gene regulation in the cell.

The selenoprotein glutathione peroxidase-2 (GPx2) appears to have a dual role in carcinogenesis. While it protected mice from colon cancer in a model of inflammation-triggered carcinogenesis (azoxymethane and dextran sodium sulfate treatment), it promoted growth of xenografted tumor cells. Therefore, we analyzed the effect of GPx2 in a mouse model mimicking sporadic colorectal cancer (azoxymethane-treatment only). GPx2-knockout (KO) and wild-type (WT) mice were adjusted to an either marginally deficient (-Se), adequate (+Se), or supranutritional (++Se) selenium status and were treated six times with azoxymethane (AOM) to induce tumor development. In the -Se and ++Se groups, the number of tumors was significantly lower in GPx2-KO than in respective WT mice. On the +Se diet, the number of dysplastic crypts was reduced in GPx2-KO mice. This may be explained by more basal and AOM-induced apoptotic cell death in GPx2-KO mice that eliminates damaged or pre-malignant epithelial cells. In WT dysplastic crypts GPx2 was up-regulated in comparison to normal crypts which might be an attempt to suppress apoptosis. In contrast, in the +Se groups tumor numbers were similar in both genotypes but tumor size was larger in GPx2-KO mice. The latter was associated with an inflammatory and tumor-promoting environment as obvious from infiltrated inflammatory cells in the intestinal mucosa of GPx2-KO mice even without any treatment and characterized as low-grade inflammation. In WT mice the number of tumors tended to be lowest in +Se compared to -Se and ++Se feeding indicating that selenium might delay tumorigenesis only in the adequate status. In conclusion, the role of GPx2 and presumably also of selenium depends on the cancer stage and obviously on the involvement of inflammation.

The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor.

BACKGROUND: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. METHODS AND RESULTS: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p

Introduction: Aging is the strongest risk factor for most chronic diseases. The rising burden of an aging population and non-communicable diseases (NCDs), contributes to escalating costs for society. Several non-pharmaceutical interventions can lower the risk of NCDs, including common mental disorders (CMDs), and may slow down biological aging, as evidenced by outcome markers such as epigenetic clocks. However, a comprehensive overview of whether and which non-pharmaceutical interventions may impact human epigenetic aging is missing. Synthesizing evidence of interventions on epigenetic aging that can be adopted by a wider population is key to guide healthy aging initiatives and to reduce the burden of NCDs and CMDs. This scoping review will identify and assess non-pharmaceutical interventions aimed to slow down epigenetic aging, including their intervention components, and the mode used for intervention delivery., Methods and Analysis: This protocol will include single- and multicomponent intervention studies that target individuals ≥ 18 years of age and use epigenetic clocks as primary or secondary outcomes. Five electronic databases will be searched for studies between July 2011 until December 2023. The final search will include the search terms adults, non-pharmaceutical interventions, epigenetic aging and their respective synonyms. We will include randomized controlled trials, non-randomized controlled studies, cohort studies, and case-control studies. Additionally, the reference list of other reviews will be screened for relevant articles. Study selection is carried out based on the defined eligibility criteria by two authors. Quality and risk of bias for the included studies will be assessed using the Critical Appraisal Skills Programme (CASP) checklist. Data extraction will include generic key information such as the research question and results, the intervention components, and specific epigenetic outcome measures used. Further data regarding the delivery mode of the treatment protocol will be collected., Ethics and Dissemination: This scoping review will summarize the characteristics of non-pharmaceutical intervention studies on epigenetic aging. This review will be the first step to formally identify key intervention components and delivery modes to guide future research on healthy aging interventions. The results will be disseminated through a peer-reviewed publication and presented at relevant conferences. This review will synthesize information from available publications and does not require further ethical approval., Registration Details: Open Science Framework https://doi.org/10.17605/OSF.IO/FEHNB., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: AL, JM, MJ, and TK are affiliated with the Centre for Digital Health Interventions, a joint initiative of the Institute for Implementation Science in Health Care, University of Zurich, the Department of Management, Technology, and Economics at ETH Zurich, and the Institute of Technology Man-agement and School of Medicine at the University of St.Gallen. The Centre for Digital Health In-terventions is funded in part by CSS, a Swiss health insurer, MavieNext, an Austrian healthcare provider, and MTIP, a Swiss digital health investor. TK is also a co-founder of Pathmate Technol-ogies, a university spin-off company that creates and delivers digital clinical pathways. However, CSS, MavieNext, MTIP, or Pathmate Technologies were not involved in this protocol. All other authors declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Liebich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Screening for mental health problems has been shown to be effective to detect depression and initiate treatment in primary care. Current guidelines recommend periodic screening for depression and anxiety. This study examines the association of patient sociodemographic factors and clinic characteristics on mental health screening in primary care., Design: In this retrospective cohort study, electronic medical record (EMR) data from a 14-month period from 10/15/2021 to 12/14/2022 were analyzed. Data were retrieved from 18 primary care clinics from the Corewell Health healthcare system in West Michigan. The main outcome was documentation of any Patient Health Questionnaire (PHQ-4/PHQ-9/GAD-7) screening in the EMR within the 14-month period at patient level. General linear regression models with logit link function were used to assess adjusted odds ratio (aOR) of having a documented screening., Results: In total, 126,306 unique patients aged 16 years or older with a total of 291,789 encounters were included. The prevalence of 14-month screening was 79.8% (95% CI, 79.6-80.0). Regression analyses revealed higher screening odds for patients of smaller clinics (<5,000 patients, aOR 1.88; 95% CI 1.80-1.98 vs. clinics >10.000 patients), clinics in areas with mental health provider shortages (aOR 1.69; 95% CI 1.62-1.77), frequent visits (aOR 1.80; 95% CI, 1.78-1.83), and having an annual physical / well child visit encounter (aOR 1.52; 95% CI, 1.47-1.57). Smaller positive effect sizes were also found for male sex, Black or African American race, Asian race, Latinx ethnicity (ref. White/Caucasians), and having insurance through Medicaid (ref. other private insurance)., Discussion: The 14-month mental health screening rates have been shown to be significantly lower among patients with infrequent visits seeking care in larger clinics and available mental health resources in the community. Introducing and incentivizing mandatory mental health screening protocols in annual well visits, are viable options to increase screening rates., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Müller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Physical inactivity and sedentary behavior are significant risk factors for various non-communicable diseases. Bangkok, Thailand's capital, is one of the fastest-growing metropolitans in Southeast Asia. Few studies have investigated the epidemiology of physical activity and sedentary behavior among Bangkok residents. This study aims to investigate the prevalence of combined physical activity and sedentary behavior patterns among Bangkok residents and examine relationships between participants' characteristics and the combined movement patterns., Methods: We analyzed data from the nationally representative 2021 Health Behavior Survey conducted by the Thailand National Statistical Office. The Global Physical Activity Questionnaire was used to assess physical activity and sedentary behavior. 'Sufficiently active' was defined as meeting the World Health Organization's guidelines for aerobic physical activity (≥150 minutes of moderate-to-vigorous physical activity per week). 'Low sedentary time' was defined as sitting for ≤7 hours per day. Participants were categorized into one of four movement patterns: highly active/low sedentary, highly active/highly sedentary, low active/low sedentary, and low active/highly sedentary. Multinomial logistic regression was used to identify the factors associated with each group of four movement patterns., Results: Among the 3,137 individuals included in the study, the majority were categorized as highly active/highly sedentary (64.8%), followed by highly active/low sedentary (17.9%) and low active/highly sedentary (14.3%). Only a few (3.0%) of participants were categorized as being low active/low sedentary. Compared to males, female participants had a significantly higher likelihood of belonging to the highly active/low sedentary (AOR = 1.69, 95%CI: 1.25, 2.28) or highly active/highly sedentary (AOR = 1.51, 95%CI: 1.19, 1.93) group, rather than the low active/high sedentary group. Compared to unemployed/retired participants, those in labor-intensive occupations had a significantly higher likelihood of being in the highly active/low sedentary group (AOR = 1.89, 95%CI: 1.22, 2.94). Compared to participants with no chronic physical conditions, participants who reported multimorbidity had a significantly lower likelihood of being in the highly active/low sedentary group (AOR = 0.60, 95%CI: 0.37, 0.98)., Conclusion: This study provides valuable insights into the patterns of physical activity and sedentary behavior among residents of Bangkok using up-to-date data. The majority belonged to the highly active/highly sedentary group, followed by the highly active/low sedentary group. Correlates such as sex, occupation, and chronic conditions were associated with these patterns. Targeted interventions in recreational activities, workplaces, and urban areas, including screen time control measures, movement breaks and improved built environments, are crucial in reducing sedentary behavior and promoting physical activity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Topothai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background/aims: The COVID-19 pandemic situation poses new challenges for research. Ethical issues might arise if especially vulnerable individuals for severe COVID-19 course expose themselves because of participation in studies to a higher risk of infection for study purposes. How is the feasibility and acceptance of self-organized blood sample collections to measure anti-SARS-CoV-2 Spike IgG antibodies in persons with a high risk for a severe COVID-19 disease progression?, Methods: Persons with a high risk for a severe COVID-19 disease progression (immunocompromised, oncology patients or over 80 years old) were recruited between January and September 2021 to send in blood samples (at least 500 μl) 1 month and 6 months after second COVID-19 vaccination. Participants were given the choice of drawing capillary or venous blood themselves or having blood drawn by health professionals belonging to either the study's own research team or the personnel found in local practices or clinics. Participants were surveyed via a telephone interview in December 2021 and January 2022 about their choice of blood sampling methods and influence of blood collection choice upon study participation., Results: Data from 360 participants was collected via telephone follow-up. First blood samples were collected by the participants themselves (35.8%), local practices or clinics (31.9%) and the research team (22.5%). Second blood samples were mostly collected in local practices or clinics (35.6%) followed by participants themselves (25.9%) and the research team (11.5%). Blood samples were not collected in 2.5% and 19.1% of persons during first and second blood draw, respectively. Only 2% of blood samples did not reach the laboratory or were not analyzable. About one-fourth (26%) of participants stated that they would not have participated in the study if it would have been required to travel to the university hospital to give their blood sample., Conclusions: Participants were able to self-organize blood collection, making use of several different blood sample methods. Nearly all blood samples were analyzable when self-collected and sent in by post. One-fourth of the participants would not have participated in the study if required to give their blood sample in the study location., Trial Registration: German Clinical Trial Registry, DRKS00021152., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Schröder et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Surface compatibility with blood is critical both for scientific investigations on hemostasis and clinical applications. Regarding in vitro and ex vivo investigations, minimal alteration in physiological hemostasis is of particular importance to draw reliable conclusions on the human coagulation system. At the same time, artificial coagulation activation must be avoided, which is relevant for the patient, for example to prevent stent graft occlusion. The aim was to evaluate the advantages and disadvantages of antithrombotic and antifouling surface coatings in the context of their suitability for ex vivo incubation and the study of coagulation properties., Methods: We investigated the impact of different protocols for surface coating of synthetic material and different anticoagulants on hemostasis and platelet activation in ex vivo human whole blood. Blood samples from healthy donors were incubated in coated microtubes on a rotating wheel at 37°C. Two protocols for surface coating were analyzed for hemostatic parameters and metabolic status, a heparin-based coating (CHC, Corline Heparin Conjugate) without further anticoagulation and a passivating coating (MPC, 2-methacryloyloxethyl phosphorylcholine) with added anticoagulants (enoxaparin, ENOX; or fondaparinux, FPX). Employing the MPC-based coating, the anticoagulants enoxaparin and fondaparinux were compared regarding their differential effects on plasmatic coagulation by thrombelastometry and on platelet activation by flowcytometry and platelet function assays., Results: Using the CHC coating, significant coagulation cascade activation was observed, whereas parameters remained mostly unchanged with MPC-based protocols. Extended incubation caused significantly elevated levels of the soluble membrane attack complex. Neither ENOX nor FPX caused a relevant impairment of platelet function or activation capacity and thrombelastometric parameters remained unchanged with both protocols. For translational purposes, we additionally modeled endotoxemia with the MPC-based protocols by incubating with lipopolysaccharide plus/minus thrombin. While coagulation parameters remained unchanged, elevated Interleukin 8 and Matrix Metalloproteinase 9 demonstrated preserved immune cell responsiveness., Conclusions: The MPC-based protocols demonstrated better hemocompatibility compared to CHC, and ENOX and FPX proved useful for additional anticoagulation. Furthermore, this simple-to-use whole blood model may be useful for experimental analyses of the early coagulatory and immunological response without decalcification., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Spiegelburg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Established research has documented the pervasive influence of incentives (i.e., food, sex, money) on animal and human behavior. Additionally, motivational theories postulating intra-individually stable preferences for specific types of incentives (i.e., motives) highlight that effects of a given incentive are highly dependent on the motive disposition of the individual. Indeed, also research on motor performance has documented the interactive effects of motives and motive-specific incentives on motor outcomes. However, the majority of this research has relied on correlational designs focusing on the effects of the achievement motive, with few studies addressing the role of the affiliation and power motive. In order to extend findings in this domain, we tested whether a fit between individuals' power (affiliation) motive and incentives of competition (cooperation) would improve motor performance. Following baseline measures, participants performed a dart-throwing task as part of a dyadic performance (i.e., cooperative) or a one-on-one competition scenario. In the dyadic performance scenario, a stronger affiliation motive did not translate to better performance. However, in the one-on-one competition scenario a stronger power motive was associated with better performance. Results highlight the role of the power motive in predicting motor performance, particularly in competitive situations., Competing Interests: The authors have declared that no competing interests exist.

This mixed-methods study aims to inform the development of a 'Park Prescription' intervention, including face-to-face counseling on physical activity and park use and providing weekly structured exercise sessions in the park to promote physical activity. Participants aged 40-65 years were recruited from regional health screening events in Singapore where they completed a questionnaire (N = 97) and consented to focus group (FG) participation (N = 16). The questionnaire assessed current park use, and the type, duration, and intensity of park-based activities that would be of interest. FGs explored the barriers and facilitators of physical activity (in parks). Short interviews (N = 16) with 'doers', i.e., people already engaging in park-based physical activity, identified motivational factors and ways to overcome common barriers. Participants acknowledged the health benefits of parks and valued them because of their pleasant landscapes, greenery and facilities. However, few participants engaged in physical activity at the parks, because they were too busy or too tired. Participants mostly indicated doing informal activities, such as walking, cycling or playing traditional Asian games when using the parks for exercise. A variety of low-to-moderate intensity park-based activities such as walking, cycling or aerobics were of interest to participants who expressed the willingness to engage in structured exercise sessions on weekday evenings or weekend mornings. Strategies to increase physical activity in parks included: encourage planning, create social support, identify alternatives for bad weather, improve proximity/accessibility to parks and park safety. The effectiveness of the Park Prescription intervention in promoting physical activity, park use, as well as physical and mental well-being will be tested in a one-year Randomized Controlled Trial., Competing Interests: The authors have declared that no competing interests exist.

This study aims to assess the potential supply of the ecosystem service (ES) nutrient regulation on two spatial scales, the federal German state of Schleswig-Holstein (regional) and the Bornhöved Lakes District (local), exemplarily for the nutrient nitrogen. The methodology was developed using the ES matrix approach, which can be applied to evaluate and map ES at different geospatial units such as land use/land cover classes. A Bayesian Belief Network (BBN) was constructed in order to include additional spatial information on environmental characteristics in the assessment. The integration of additional data, which describes site-specific characteristics such as soil type and slope, resulted in shifted probability distributions for the nutrient regulation ES potential. The focal objective of the study was of methodological nature: to test the application of a BBN as an integrative modelling approach combining the information from the ES matrix with additional data sets. In the process, both study areas were assessed with a regional differentiation with regard to the predominant landscape types. For both study areas, regional differences could be detected. Furthermore, the results indicate a spatial mismatch between ES demand and supply of the nutrient regulation potential. Land management and agricultural practices seem not to be in harmony with the spatial patterns of the environmental characteristics in the study areas. The assessment on the local scale, which comprised higher resolution input data, emphasized these circumstances even more clearly., Competing Interests: The authors have declared that no competing interests exist.

Grunting in tennis is a widespread phenomenon and whether it influences opponents' predictions of ball trajectory-and if so, why-is subject of ongoing debate. Two alternative hypotheses have been proposed to explain why grunting may impede opponents' predictions, referred to as the distraction account (i.e., grunts capture attentional resources necessary for anticipation) and the multisensory integration account (i.e., auditory information from the grunt systematically influences ball trajectory prediction typically assumed to rely on visual information). To put these competing hypotheses to test, in the current study we presented tennis players with a series of temporally occluded video clips of tennis rallies featuring experimentally amplified, attenuated, or muted grunting sounds. Participants were asked to predict the ball landing position. Results indicated that higher grunt intensities yielded judgments of longer ball trajectories whereas radial prediction errors were not affected. These results are clearly at odds with the distraction account of grunting, predicting increased prediction errors after higher intensity grunts. In contrast, our findings provide strong support for the multisensory integration account by demonstrating that grunt intensity systematically influences judgments of ball trajectory., Competing Interests: The authors have declared that no competing interests exist.

Executive dysfunctions frequently occur in patients with Major Depressive Disorder and have been shown to improve during effective antidepressant treatment. However, the time course of improvement and its relationship to treatment outcome is unknown. The aim of the study was to assess the test performance and clinical outcome by repetitive assessments of executive test procedures during antidepressant treatment. Executive test performance was assessed in 209 -patients with Major Depressive Disorder (mean age 39.3 ± 11.4 years) and 84 healthy controls five times in biweekly intervals from baseline to week 8. Patients were treated by a defined treatment algorithm within the early medication change study (EMC trial; ClinicalTrials.gov NCT00974155), controls did not receive any intervention. Cognitive domains were processing speed, cognitive flexibility, phonemic and semantic verbal fluency. Intelligence was assessed at baseline. Depression severity was tested once a week by the Hamilton Depression Rating Scale (HAMD17). 130 patients (62%) showed executive dysfunctions in at least one of four tests at baseline. Linear mixed regression models revealed that the course of depression severity was associated to the course of cognitive flexibility (p = 0.004) and semantic verbal fluency (p = 0.020). Cognitive flexibility and semantic verbal fluency may be candidates easily to apply for therapy response prediction in clinical routine, which should be tested in further prospective studies., Trial Registration: ClinicalTrials.gov NCT00974155 EudraCT: 2008-008280-96.

[This corrects the article DOI: 10.1371/journal.pone.0187618.].

Retinal prostheses that are currently used to restore vision in patients suffering from retinal degeneration are not adjusted to the changes occurring during the remodeling process of the retina. Recent studies revealed abnormal rhythmic activity in the retina of genetic mouse models of retinitis pigmentosa. Here we describe this abnormal activity also in a pharmacologically-induced (MNU) mouse model of retinal degeneration. To investigate how this abnormal activity affects the excitability of retinal ganglion cells, we recorded the electrical activity from whole mounted retinas of rd10 mice and MNU-treated mice using a microelectrode array system and applied biphasic current pulses of different amplitude and duration to stimulate ganglion cells electrically. We show that the electrical stimulation efficiency is strongly reduced in degenerated retinas, in particular when abnormal activity such as oscillations and rhythmic firing of bursts of action potentials can be observed. Using a prestimulus pulse sequence, we could abolish rhythmic retinal activity. Under these conditions, the stimulation efficiency was enhanced in a few cases but not in the majority of tested cells. Nevertheless, this approach supports the idea that modified stimulation protocols could help to improve the efficiency of retinal prostheses in the future.

We prospectively studied the consequences of extensive antibiotic treatment on faecal carriage of antibiotic-resistant enterobacteria in a cohort of children with cystic fibrosis (CF) and a cohort of children with cancer compared to healthy children with no or low antibiotic exposure. The study was conducted in Norway in a low resistance prevalence setting. Sixty longitudinally collected faecal samples from children with CF (n = 32), 88 samples from children with cancer (n = 45) and 127 samples from healthy children (n = 70) were examined. A direct MIC-gradient strip method was used to detect resistant Enterobacteriaceae by applying Etest strips directly onto agar-plates swabbed with faecal samples. Whole genome sequencing (WGS) data were analysed to identify resistance mechanisms in 28 multidrug-resistant Escherichia coli isolates. The prevalence of resistance to third-generation cephalosporins, gentamicin and ciprofloxacin was low in all the study groups. At inclusion the prevalence of ampicillin-resistant E. coli and trimethoprim-sulfamethoxazole-resistant E. coli in the CF group compared to healthy controls was 58.6% vs. 28.4% (p = 0.005) and 48.3% vs. 14.9% (p = 0.001), respectively, with a similar prevalence at the end of the study. The prevalence of resistant enterobacteria was not significantly different in the children with cancer compared to the healthy children, not even at the end of the study when the children with cancer had been treated with repeated courses of broad-spectrum antibiotics. Children with cancer were mainly treated with intravenous antibiotics, while the CF group mainly received peroral treatment. Our observations indicate that the mode of administration of antibiotics and the general level of antimicrobial resistance in the community may have an impact on emergence of resistance in intestinal enterobacteria during antibiotic treatment. The WGS analyses detected acquired resistance genes and/or chromosomal mutations that explained the observed phenotypic resistance in all 28 multidrug-resistant E. coli isolates examined.

Mice are extensively used for experimental bone-healing studies. However, there are few established nondestructive in vivo techniques for longitudinal fracture-healing analysis in mice, including in vivo micro-computed tomography (μCT) and radiography. Importantly, none of the established methods can discriminate between non-mineralized fibrous tissue and cartilage in the soft fracture callus. Therefore, the objective was to establish high-resolution in vivo magnetic resonance imaging (MRI) for the longitudinal assessment of soft callus formation during bone healing in mice. C57BL/6J mice received a femur osteotomy stabilized using an external fixator and were randomly assigned to five groups. Group 1 mice were scanned three times longitudinally during fracture healing using an optimized MRI scanning protocol to establish an algorithm to characterize the different fracture-callus tissues. Mice of groups 2-4 were scanned once on day 10, 14 or 21, respectively, euthanized after scanning and their femurs subjected to ex vivo μCT and histomorphometric analysis to compare the data assessed by MRI with μCT and histology. Control group 5 mice were not scanned. After 28 days, mice of groups 1 and 5 were euthanized and the fracture-healing outcome was evaluated by bending-test, μCT and histology to determine whether the repeated anesthesia, handling and the MRI measurements themselves influenced fracture healing. The callus-tissue values determined by MRI were mostly comparable to those obtained by μCT and histomorphometric analysis. However, at time points characterized by small relative bone or cartilage areas, MRI measurements were weakly comparable to histomorphometric data, possibly due to the inferior spatial resolution. Importantly, at the early and intermediate phases of healing, cartilage and fibrous-tissue values obtained by MRI were highly accurate. Furthermore, repeated anesthesia, handling and MRI scans did not impact bone healing. Therefore, we demonstrated the feasibility of high-resolution in vivo MRI for longitudinal assessment of soft callus formation during murine endochondral fracture healing.

Introduction: Accelerometers are commonly used to assess physical activity. Consumer activity trackers have become increasingly popular today, such as the Fitbit. This study aimed to compare the average number of steps per day using the wrist-worn Fitbit Flex and waist-worn ActiGraph (wGT3X-BT) in free-living conditions., Methods: 104 adult participants (n = 35 males; n = 69 females) were asked to wear a Fitbit Flex and an ActiGraph concurrently for 7 days. Daily step counts were used to classify inactive (<10,000 steps) and active (≥10,000 steps) days, which is one of the commonly used physical activity guidelines to maintain health. Proportion of agreement between physical activity categorizations from ActiGraph and Fitbit Flex was assessed. Statistical analyses included Spearman's rho, intraclass correlation (ICC), median absolute percentage error (MAPE), Kappa statistics, and Bland-Altman plots. Analyses were performed among all participants, by each step-defined daily physical activity category and gender., Results: The median average steps/day recorded by Fitbit Flex and ActiGraph were 10193 and 8812, respectively. Strong positive correlations and agreement were found for all participants, both genders, as well as daily physical activity categories (Spearman's rho: 0.76-0.91; ICC: 0.73-0.87). The MAPE was: 15.5% (95% confidence interval [CI]: 5.8-28.1%) for overall steps, 16.9% (6.8-30.3%) vs. 15.1% (4.5-27.3%) in males and females, and 20.4% (8.7-35.9%) vs. 9.6% (1.0-18.4%) during inactive days and active days. Bland-Altman plot indicated a median overestimation of 1300 steps/day by the Fitbit Flex in all participants. Fitbit Flex and ActiGraph respectively classified 51.5% and 37.5% of the days as active (Kappa: 0.66)., Conclusions: There were high correlations and agreement in steps between Fitbit Flex and ActiGraph. However, findings suggested discrepancies in steps between devices. This imposed a challenge that needs to be considered when using Fibit Flex in research and health promotion programs.

Background: Due to the aging of the population, society includes a growing proportion of older individuals prone to chronic morbidity. This study aimed to investigate the adverse effects of single and multiple chronic morbidity on psychosocial health and whether these effects are more pronounced in individuals who are non-partnered or living alone., Materials and Methods: Baseline data from the 'Lifelines Cohort Study' collected between 2006 and 2013 in the Netherlands were used. Individuals aged 50+ (n = 25,214) were categorized according to their health status (healthy, single chronic morbidity, multiple chronic morbidity), relationship status (partnered, non-partnered), and living arrangement (living with someone, living alone). Analyses of covariance (ANCOVA) were performed to study the main- and the interaction-effects on mental health and role functioning as assessed with the RAND-36., Results: Irrespective of having chronic morbidity, having a partner was associated with better mental health when partners shared a home. Individuals with single and especially multiple chronic morbidity had impaired role functioning. Having a partner mitigated the adverse effects of multimorbidity on role functioning, but only in individuals who shared a home with their partner. Non-partnered individuals with multimorbidity and those not sharing a home with their partner demonstrated impaired role functioning., Conclusions: The results demonstrate that multimorbidity negatively affects role functioning, but not the mental health, of middle-aged and older individuals. Sharing a home with a partner can mitigate these adverse effects, while other combinations of relationship status and living arrangement do not. Offering intervention to those individuals most vulnerable to impaired functioning may relieve some of the increasing pressure on the health care system. An individual's relationship status along with one's living arrangement could foster the identification of a target group for such interventions attempting to sustain physical functioning or to adapt daily goals., Competing Interests: The authors have declared that no competing interests exist.

Sexual reproduction is a critical process in the life-cycle of plants and very sensitive to environmental perturbations. To better understand the effect of high temperature on plant reproduction, we cultivated tomato (Solanum lycopersicum) plants in continuous mild heat. Under this condition we observed a simultaneous reduction in pollen viability and appearance of anthers with pistil-like structures, while in a more thermotolerant genotype, both traits were improved. Ectopic expression of two pistil-specific genes, TRANSMITTING TISSUE SPECIFIC and TOMATO AGAMOUS LIKE11, in the anthers confirmed that the anthers had gained partial pistil identity. Concomitantly, expression of the B-class genes TOMATO APETALA3, TOMATO MADS BOX GENE6 (TM6) and LePISTILLATA was reduced in anthers under continuous mild heat. Plants in which TM6 was partially silenced reacted hypersensitively to temperature elevation with regard to the frequency of pistilloid anthers, pollen viability and pollen quantity. Taken together, these results suggest that high-temperature-induced down-regulation of tomato B-class genes contributes to anther deformations and reduced male fertility. Improving our understanding of how temperature perturbs the molecular mechanisms of anther and pollen development will be important in the view of maintaining agricultural output under current climate changes., Competing Interests: The authors have declared that no competing interests exist.

Assessment of heart function in zebrafish larvae using electrocardiography (ECG) is a potentially useful tool in developing cardiac treatments and the assessment of drug therapies. In order to better understand how a measured ECG waveform is related to the structure of the heart, its position within the larva and the position of the electrodes, a 3D model of a 3 days post fertilisation (dpf) larval zebrafish was developed to simulate cardiac electrical activity and investigate the voltage distribution throughout the body. The geometry consisted of two main components; the zebrafish body was modelled as a homogeneous volume, while the heart was split into five distinct regions (sinoatrial region, atrial wall, atrioventricular band, ventricular wall and heart chambers). Similarly, the electrical model consisted of two parts with the body described by Laplace's equation and the heart using a bidomain ionic model based upon the Fitzhugh-Nagumo equations. Each region of the heart was differentiated by action potential (AP) parameters and activation wave conduction velocities, which were fitted and scaled based on previously published experimental results. ECG measurements in vivo at different electrode recording positions were then compared to the model results. The model was able to simulate action potentials, wave propagation and all the major features (P wave, R wave, T wave) of the ECG, as well as polarity of the peaks observed at each position. This model was based upon our current understanding of the structure of the normal zebrafish larval heart. Further development would enable us to incorporate features associated with the diseased heart and hence assist in the interpretation of larval zebrafish ECGs in these conditions., Competing Interests: Competing Interests: Ferenc Müller is academic editor at PLOS ONE. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Background: The purpose of this study was to examine the impact of combined cardiorespiratory fitness and waist-to-height ratio in the form of a fit-fat index on incident diabetes risk. Additionally, the independent predictive performance of cardiorespiratory fitness, waist-to-height ratio, and body mass index also were estimated and compared., Methods: This was a prospective cohort study of 10,381 men who had a normal electrocardiogram and no history of major chronic disease at baseline from 1979 to 2005. Random survival forest models and traditional Cox proportional hazards models were used to predict diabetes at 5-, 10-, and 15-year incidence horizons., Results: Overall, 4.8% of the participants developed diabetes. Receiver operating characteristic curve analyses for incidence risk demonstrated good discrimination using random survival forest models across fitness and fatness measures; Cox models were poor to fair. The differences between fitness and fatness measures across horizons were clinically negligible. Smoothed random survival forest estimates demonstrated the impact of each fitness and fatness measure on incident diabetes was intuitive and graded., Conclusions: Although fitness and fatness measures showed a similar discriminative ability in predicting incident diabetes, unique to the study was the ability of the fit-fat index to demonstrate a better indication of incident risk when compared to fitness or fatness alone. A single index combining cardiorespiratory fitness and waist-to-height ratio may be more useful because it can indicate improvements in either or both of the measures.

Background: Contributors to long-term mortality in patients with community-acquired pneumonia (CAP) remain unclear, with little attention paid to pneumonia etiology. We examined long-term survival, causes of death, and risk factors for long-term mortality in adult patients who had been hospitalized for CAP, with emphasis on demographic, clinical, laboratory, and microbiological characteristics., Methods: Two hundred and sixty-seven consecutive patients admitted in 2008-2011 to a general hospital with CAP were prospectively recruited and followed up. Patients who died during hospital stay were excluded. Demographic, clinical, and laboratory data were collected within 48 hours of admission. Extensive microbiological work-up was performed to establish the etiology of CAP in 63% of patients. Mortality data were obtained from the Norwegian Cause of Death Registry. Cox regression models were used to identify independent risk factors for all-cause mortality., Results: Of 259 hospital survivors of CAP (median age 66 years), 79 (30.5%) died over a median of 1,804 days (range 1-2,520 days). Cumulative 5-year survival rate was 72.9% (95% CI 67.4-78.4%). Standardized mortality ratio was 2.90 for men and 2.05 for women. The main causes of death were chronic obstructive pulmonary disease (COPD), vascular diseases, and malignancy. Independent risk factors for death were the following (hazard ratio, 95% CI): age (1.83 per decade, 1.47-2.28), cardiovascular disease (2.63, 1.61-4.32), COPD (2.09, 1.27-3.45), immunocompromization (1.98, 1.17-3.37), and low serum albumin level at admission (0.75 per 5 g/L higher, 0.58-0.96), whereas active smoking was protective (0.32, 0.14-0.74); active smokers were younger than non-smokers (P < 0.001). Microbial etiology did not predict mortality., Conclusions: Results largely confirm substantial comorbidity-related 5-year mortality after hospitalization for CAP and the impact of several well-known risk factors for death, and extend previous findings on the prognostic value of serum albumin level at hospital admission. Pneumonia etiology had no prognostic value, but this remains to be substantiated by further studies using extensive diagnostic microbiological methods in the identification of causative agents of CAP.

The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint.

One major aim of the neurological rehabilitation of patients with severe disorders of consciousness (DOC) is to enhance patients' arousal and ability to communicate. Mobilization into a standing position by means of a tilt table has been shown to improve their arousal and awareness. However, due to the frequent occurrence of syncopes on a tilt table, it is easier to accomplish verticalization using a tilt table with an integrated stepping device. The objective of this randomized controlled clinical trial was to evaluate the effectiveness of a tilt table therapy with or without an integrated stepping device on the level of consciousness. A total of 50 participants in vegetative or minimally conscious states 4 weeks to 6 month after injury were treated with verticalization during this randomized controlled trial. Interventions involved ten 1-hour sessions of the specific treatment over a 3-week period. Blinded assessors made measurements before and after the intervention period, as well as after a 3-week follow-up period. The coma recovery scale-revised (CRS-R) showed an improvement by a median of 2 points for the group receiving tilt table with integrated stepping (Erigo). The rate of recovery of the group receiving the conventional tilt table therapy significantly increased by 5 points during treatment and by an additional 2 points during the 3-week follow-up period. Changes in spasticity did not significantly differ between the two intervention groups. Compared to the conventional tilt table, the tilt table with integrated stepping device failed to have any additional benefit for DOC patients. Verticalization itself seems to be beneficial though and should be administered to patients in DOC in early rehabilitation. Trial Registration: Current Controlled Trials Ltd (www.controlled-trials.com), identifier number ISRCTN72853718.

Objective: Epidemiological studies relating maternal 25-hydroxyvitamin D (25OHD) with gestational diabetes mellitus (GDM) and mode of delivery have shown controversial results. We examined if maternal 25OHD status was associated with plasma glucose concentrations, risks of GDM and caesarean section in the Growing Up in Singapore Towards healthy Outcomes (GUSTO) study., Methods: Plasma 25OHD concentrations, fasting glucose (FG) and 2-hour postprandial glucose (2HPPG) concentrations were measured in 940 women from a Singapore mother-offspring cohort study at 26-28 weeks' gestation. 25OHD inadequacy and adequacy were defined based on concentrations of 25OHD ≤75nmol/l and >75nmol/l respectively. Mode of delivery was obtained from hospital records. Multiple linear regression was performed to examine the association between 25OHD status and glucose concentrations, while multiple logistic regression was performed to examine the association of 25OHD status with risks of GDM and caesarean section., Results: In total, 388 (41.3%) women had 25OHD inadequacy. Of these, 131 (33.8%), 155 (39.9%) and 102 (26.3%) were Chinese, Malay and Indian respectively. After adjustment for confounders, maternal 25OHD inadequacy was associated with higher FG concentrations (β = 0.08mmol/l, 95% Confidence Interval (CI) = 0.01, 0.14), but not 2HPPG concentrations and risk of GDM. A trend between 25OHD inadequacy and higher likelihood of emergency caesarean section (Odds Ratio (OR) = 1.39, 95% CI = 0.95, 2.05) was observed. On stratification by ethnicity, the association with higher FG concentrations was significant in Malay women (β = 0.19mmol/l, 95% CI = 0.04, 0.33), while risk of emergency caesarean section was greater in Chinese (OR = 1.90, 95% CI = 1.06, 3.43) and Indian women (OR = 2.41, 95% CI = 1.01, 5.73)., Conclusions: 25OHD inadequacy is prevalent in pregnant Singaporean women, particularly among the Malay and Indian women. This is associated with higher FG concentrations in Malay women, and increased risk of emergency caesarean section in Chinese and Indian women.

Green tea catechins inhibit the function of organic anion transporting polypeptides (OATPs) that mediate the uptake of a diverse group of drugs and endogenous compounds into cells. The present study was aimed at investigating the effect of green tea and its most abundant catechin epigallocatechin gallate (EGCG) on the transport activity of several drug transporters expressed in enterocytes, hepatocytes and renal proximal tubular cells such as OATPs, organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and P-glycoprotein (P-gp). Uptake of the typical substrates metformin for OCTs and MATEs and bromosulphophthalein (BSP) and atorvastatin for OATPs was measured in the absence and presence of a commercially available green tea and EGCG. Transcellular transport of digoxin, a typical substrate of P-gp, was measured over 4 hours in the absence and presence of green tea or EGCG in Caco-2 cell monolayers. OCT1-, OCT2-, MATE1- and MATE2-K-mediated metformin uptake was significantly reduced in the presence of green tea and EGCG (P < 0.05). BSP net uptake by OATP1B1 and OATP1B3 was inhibited by green tea [IC50 2.6% (v/v) and 0.39% (v/v), respectively]. Green tea also inhibited OATP1B1- and OATP1B3-mediated atorvastatin net uptake with IC50 values of 1.9% (v/v) and 1.0% (v/v), respectively. Basolateral to apical transport of digoxin was significantly decreased in the presence of green tea and EGCG. These findings indicate that green tea and EGCG inhibit multiple drug transporters in vitro. Further studies are necessary to investigate the effects of green tea on prototoypical substrates of these transporters in humans, in particular on substrates of hepatic uptake transporters (e.g. statins) as well as on P-glycoprotein substrates.

Objective: The Global Physical Activity Questionnaire (GPAQ) was originally designed to be interviewer-administered by the World Health Organization in assessing physical activity. The main aim of this study was to compare the psychometric properties of a self-administered GPAQ with the original interviewer-administered approach. Additionally, this study explored whether using different accelerometry-based physical activity bout definitions might affect the questionnaire's validity., Methods: A total of 110 participants were recruited and randomly allocated to an interviewer- (n = 56) or a self-administered (n = 54) group for test-retest reliability, of which 108 participants who met the wear time criteria were included in the validity study. Reliability was assessed by administration of questionnaires twice with a one-week interval. Criterion validity was assessed by comparing against seven-day accelerometer measures. Two definitions for accelerometry-data scoring were employed: (1) total-min of activity, and (2) 10-min bout., Results: Participants had similar baseline characteristics in both administration groups and no significant difference was found between the two formats in terms of validity (correlations between the GPAQ and accelerometer). For validity, the GPAQ demonstrated fair-to-moderate correlations for moderate-to-vigorous physical activity (MVPA) for self-administration (rs = 0.30) and interviewer-administration (rs = 0.46). Findings were similar when considering 10-min activity bouts in the accelerometer analysis for MVPA (rs = 0.29 vs. 0.42 for self vs. interviewer). Within each mode of administration, the strongest correlations were observed for vigorous-intensity activity. However, Bland-Altman plots illustrated bias toward overestimation for higher levels of MVPA, vigorous- and moderate-intensity activities, and underestimation for lower levels of these measures. Reliability for MVPA revealed moderate correlations (rs = 0.61 vs. 0.63 for self vs. interviewer)., Conclusions: Our findings showed comparability between both self- and interviewer-administration modes of the GPAQ. The GPAQ in general but especially the self-administered version may offer a relatively inexpensive method for measuring physical activity of various types and at different domains. However, there may be bias in the GPAQ measurements depending on the overall physical activity. It is advisable to incorporate accelerometers in future studies, particularly when measuring different intensities of physical activity.

Paired associative stimulation (PASLTP) of the human primary motor cortex (M1) can induce LTP-like plasticity by increasing corticospinal excitability beyond the stimulation period. Previous studies showed that two consecutive PASLTP protocols interact by homeostatic metaplasticity, but animal experiments provided evidence that LTP can be augmented by repeated stimulation protocols spaced by ~30 min. Here we tested in twelve healthy selected PASLTP responders the possibility that LTP-like plasticity can be augmented in the human M1 by systematically varying the interval between two consecutive PASLTP protocols. The first PASLTP protocol (PAS1) induced strong LTP-like plasticity lasting for 30-60 min. The effect of a second identical PASLTP protocol (PAS2) critically depended on the time between PAS1 and PAS2. At 10 min, PAS2 prolonged the PAS1-induced LTP-like plasticity. At 30 min, PAS2 augmented the LTP-like plasticity induced by PAS1, by increasing both magnitude and duration. At 60 min and 180 min, PAS2 had no effect on corticospinal excitability. The cumulative LTP-like plasticity after PAS1 and PAS2 at 30 min exceeded significantly the effect of PAS1 alone, and the cumulative PAS1 and PAS2 effects at 60 min and 180 min. In summary, consecutive PASLTP protocols interact in human M1 in a time-dependent manner. If spaced by 30 min, two consecutive PASLTP sessions can augment LTP-like plasticity in human M1. Findings may inspire further research on optimized therapeutic applications of non-invasive brain stimulation in neurological and psychiatric diseases.

Anti-mesothelin Pseudomonas exotoxin A-based recombinant immunotoxins (RITs) present a potential treatment modality for pancreatic ductal adenocarcinoma (PDAC). To study mechanisms of resistance, the sensitive PDAC cell line KLM-1 was intermittently exposed to the anti-mesothelin SS1-LR-GGS RIT. Surviving cells were resistant to various anti-mesothelin RITs (IC50s >1 μg/ml), including the novel de-immunized RG7787. These resistant KLM-1-R cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40 RIT as KLM-1, indicating resistance was specific to anti-mesothelin RITs. Mesothelin gene expression was partially down-regulated in KLM-1-R, resulting in 5-fold lower surface protein levels and decreased cellular uptake of RG7787 compared to KLM-1. Bisulfite sequencing analysis found that the mesothelin promoter region was significantly more methylated in KLM-1-R (59 ± 3.6%) compared to KLM-1 (41 ± 4.8%), indicating hypermethylation as a mechanism of mesothelin downregulation. The DNA methyltransferase inhibitor 5-azacytidine restored original mesothelin surface expression to more than half in KLM-1-R and increased sensitivity to RG7787 (IC50 = 722.4 ± 232.6 ng/ml), although cells remained significantly less sensitive compared to parental KLM-1 cells (IC50 = 4.41 ± 0.38 ng/ml). Mesothelin cDNA introduction in KLM-1-R led to 5-fold higher surface protein levels and significantly higher RG7887 uptake compared to KLM-1. As a result, the original sensitivity to RG7787 was fully restored (IC50 = 4.49 ± 1.11 ng/ml). A significantly higher RG7787 uptake was thus required to reach the original cytotoxicity in resistant cells, hinting that intracellular RIT trafficking is also a limiting factor. RNA deep sequencing analysis of KLM-1 and KLM-1-R cells supported our experimental findings; compared to KLM-1, resistant cells displayed differential expression of genes linked to intracellular transport and an expression pattern that matched a more general hypermethylation status. In conclusion, resistance to anti-mesothelin RITs in KLM-1 is linked to a methylation-associated down-regulation of mesothelin, while aberrations in RIT trafficking could also play a role.

In the widely used mouse model of retinal degeneration, rd1, the loss of photoreceptors leads to rhythmic electrical activity of around 10-16 Hz in the remaining retinal network. Recent studies suggest that this oscillation is formed within the electrically coupled network of AII amacrine cells and ON-bipolar cells. A second mouse model, rd10, displays a delayed onset and slower progression of degeneration, making this mouse strain a better model for human retinitis pigmentosa. In rd10, oscillations occur at a frequency of 3-7 Hz, raising the question whether oscillations have the same origin in the two mouse models. As rd10 is increasingly being used as a model to develop experimental therapies, it is important to understand the mechanisms underlying the spontaneous rhythmic activity. To study the properties of oscillations in rd10 retina we combined multi electrode recordings with pharmacological manipulation of the retinal network. Oscillations were abolished by blockers for ionotropic glutamate receptors and gap junctions. Frequency and amplitude of oscillations were modulated strongly by blockers of inhibitory receptors and to a lesser extent by blockers of HCN channels. In summary, although we found certain differences in the pharmacological modulation of rhythmic activity in rd10 compared to rd1, the overall pattern looked similar. This suggests that the generation of rhythmic activity may underlie similar mechanisms in rd1 and rd10 retina.

Purpose: The aim of the present study was to examine the biological differences between seminomas with occult and clinically apparent metastases at the time of diagnosis of the primary tumor to gain insight into the biology of these tumors and facilitate the identification of novel predictors of seminoma metastasis., Materials and Methods: Total RNA including small RNAs was isolated from testicular tumors of patients with pure seminoma presenting with lymphogenic metastasis (n = 5, clinical stage IIb/c) and occult metastasis (n = 5, clinical stage I). The regulation of biological processes was examined (1) throughout the mRNA transcriptome (whole genome microarrays, 8×60 K Array, Agilent with 4 samples/group) and (2) the miRNA transcriptome employing small RNA next generation sequencing (SOLID, Life Technologies with 5 samples/group). Protein coding genes (mRNAs) and small RNAs showing a significant (≥2-fold) difference between the groups were identified. Finally (3), we examined 95 candidate miRNAs in 36 apparent metastasized and another 5 occult metastasized seminoma using logistic regression analysis., Results: Among 19,596 genes, on average 12,894 mRNAs appeared expressed (65.8%, SD+/-2.4; range, 62.0-69.3%) and 16.99×106/13.94×106 small RNA reads were identified for apparent/occult metastasized seminoma. These reads on average convert into 9,901/9,675 small RNAs including 422/404 mature microRNAs. None of these mRNAs/small RNAs met our selection criteria for candidate genes. From 95 candidate miRNAs 44 appeared expressed, with 3 of them showing weak but significant (p = 0.05) differences among both groups., Conclusions: Occult and apparent metastasized seminomas are biologically almost indistinguishable and probably represent no separate tumor entities. These findings may simplify future research on seminoma metastasis.

Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common AGXT2 single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding AGXT2 SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the AGXT2 SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated AGXT2 SNP rs16899974, which was not significantly linked to the other AGXT2 SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [²H₆]-SDMA as substrate. In vitro, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). In silico modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of AGXT2 affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper-β-aminoisobutyric aciduria.

Background: Statins have anti-inflammatory and immunomodulatory properties in addition to lipid-lowering effects., Objectives: To report the 12-month extension of a phase II trial evaluating the efficacy, safety and tolerability of atorvastatin 40 mg/d added to interferon beta-1b (IFNB-1b) in relapsing-remitting multiple sclerosis (RRMS)., Methods: In the randomized, multicenter, parallel-group, rater-blinded core study, 77 RRMS patients started IFNB-1b. At month three they were randomized 1∶1 to receive atorvastatin 40 mg/d or not in addition to IFNB-1b until month 15. In the subsequent extension study, patients continued with unchanged medication for another 12 months. Data at study end were compared to data at month three of the core study., Results: 27 of 72 patients that finished the core study entered the extension study. 45 patients were lost mainly due to a safety analysis during the core study including a recruitment stop for the extension study. The primary end point, the proportion of patients with new lesions on T2-weighted images was equal in both groups (odds ratio 1.926; 95% CI 0.265-14.0007; p = 0.51). All secondary endpoints including number of new lesions and total lesion volume on T2-weighted images, total number of Gd-enhancing lesions on T1-weighted images, volume of grey and white matter, EDSS, MSFC, relapse rate, number of relapse-free patients and neutralizing antibodies did not show significant differences either. The combination therapy was well tolerated., Conclusions: Atorvastatin 40 mg/day in addition to IFNB-1b did not have any beneficial effects on RRMS compared to IFNB-1b monotherapy over a period of 24 months., Trial Registration: ClinicalTrials.gov NCT01111656.

Effective chemical compound toxicity screening is of paramount importance for safe cardiac drug development. Using mammals in preliminary screening for detection of cardiac dysfunction by electrocardiography (ECG) is costly and requires a large number of animals. Alternatively, zebrafish embryos can be used as the ECG waveform is similar to mammals, a minimal amount of chemical is necessary for drug testing, while embryos are abundant, inexpensive and represent replacement in animal research with reduced bioethical concerns. We demonstrate here the utility of pre-feeding stage zebrafish larvae in detection of cardiac dysfunction by electrocardiography. We have optimised an ECG recording system by addressing key parameters such as the form of immobilization, recording temperature, electrode positioning and developmental age. Furthermore, analysis of 3 days post fertilization (dpf) zebrafish embryos treated with known QT prolonging drugs such as terfenadine, verapamil and haloperidol led to reproducible detection of QT prolongation as previously shown for adult zebrafish. In addition, calculation of Z-factor scores revealed that the assay was sensitive and specific enough to detect large drug-induced changes in QTc intervals. Thus, the ECG recording system is a useful drug-screening tool to detect alteration to cardiac cycle components and secondary effects such as heart block and arrhythmias in zebrafish larvae before free feeding stage, and thus provides a suitable replacement for mammalian experimentation.

In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes), which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera. In this respect they are very similar to Spiegelmers (mirror image aptamers), which we had previously developed and for which it has been shown that they are non-toxic and non-immunogenic. Since we are also able to demonstrate that the hammerhead and DNAzyme Spiegelzymes can also hydrolyze mirror image oligonucleotide sequences, like they occur in Spiegelmers, in vivo, it seems reasonable to assume that Spiegelzymes may in principle be used as an antidote against Spiegelmers. Since the Spiegelzymes contain the same building blocks as the Spiegelmers, it can be expected that they will have similar favorable biological characteristics concerning toxicity and immunogenety. In trying to understand the mechanism of action of the Spiegelzymes described in this study, we have initiated for the first time a model building system with L-nucleic acids. The models for L-hammerhead ribozyme and L-DNAzyme interaction with the same L-RNA target will be presented.

Allelopathy, a phenomenon where compounds produced by one plant limit the growth of surrounding plants, is a controversially discussed factor in plant-plant interactions with great significance for plant community structure. Common mycorrhizal networks (CMNs) form belowground networks that interconnect multiple plant species; yet these networks are typically ignored in studies of allelopathy. We tested the hypothesis that CMNs facilitate transport of allelochemicals from supplier to target plants, thereby affecting allelopathic interactions. We analyzed accumulation of a model allelopathic substance, the herbicide imazamox, and two allelopathic thiophenes released from Tagetes tenuifolia roots, by diffusion through soil and CMNs. We also conducted bioassays to determine how the accumulated substances affected plant growth. All compounds accumulated to greater levels in target soils with CMNs as opposed to soils without CMNs. This increased accumulation was associated with reduced growth of target plants in soils with CMNs. Our results show that CMNs support transfer of allelochemicals from supplier to target plants and thus lead to allelochemical accumulation at levels that could not be reached by diffusion through soil alone. We conclude that CMNs expand the bioactive zones of allelochemicals in natural environments, with significant implications for interspecies chemical interactions in plant communities.

Biochemical purifications from mammalian cells and Xenopus oocytes revealed that vertebrate Mi-2 proteins reside in multisubunit NuRD (Nucleosome Remodeling and Deacetylase) complexes. Since all NuRD subunits are highly conserved in the genomes of C. elegans and Drosophila, it was suggested that NuRD complexes also exist in invertebrates. Recently, a novel dMec complex, composed of dMi-2 and dMEP-1 was identified in Drosophila. The genome of C. elegans encodes two highly homologous Mi-2 orthologues, LET-418 and CHD-3. Here we demonstrate that these proteins define at least three different protein complexes, two distinct NuRD complexes and one MEC complex. The two canonical NuRD complexes share the same core subunits HDA-1/HDAC, LIN-53/RbAp and LIN-40/MTA, but differ in their Mi-2 orthologues LET-418 or CHD-3. LET-418 but not CHD-3, interacts with the Krüppel-like protein MEP-1 in a distinct complex, the MEC complex. Based on microarrays analyses, we propose that MEC constitutes an important LET-418 containing regulatory complex during C. elegans embryonic and early larval development. It is required for the repression of germline potential in somatic cells and acts when blastomeres are still dividing and differentiating. The two NuRD complexes may not be important for the early development, but may act later during postembryonic development. Altogether, our data suggest a considerable complexity in the composition, the developmental function and the tissue-specificity of the different C. elegans Mi-2 complexes.

Hedgehog (Hh) genes play major roles in animal development and studies of their evolution, expression and function point to major differences among chordates. Here we focused on Hh genes in lampreys in order to characterize the evolution of Hh signalling at the emergence of vertebrates. Screening of a cosmid library of the river lamprey Lampetra fluviatilis and searching the preliminary genome assembly of the sea lamprey Petromyzon marinus indicate that lampreys have two Hh genes, named Hha and Hhb. Phylogenetic analyses suggest that Hha and Hhb are lamprey-specific paralogs closely related to Sonic/Indian Hh genes. Expression analysis indicates that Hha and Hhb are expressed in a Sonic Hh-like pattern. The two transcripts are expressed in largely overlapping but not identical domains in the lamprey embryonic brain, including a newly-described expression domain in the nasohypophyseal placode. Global alignments of genomic sequences and local alignment with known gnathostome regulatory motifs show that lamprey Hhs share conserved non-coding elements (CNE) with gnathostome Hhs albeit with sequences that have significantly diverged and dispersed. Functional assays using zebrafish embryos demonstrate gnathostome-like midline enhancer activity for CNEs contained in intron2. We conclude that lamprey Hh genes are gnathostome Shh-like in terms of expression and regulation. In addition, they show some lamprey-specific features, including duplication and structural (but not functional) changes in the intronic/regulatory sequences.