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BackgroundAlthough physical activity (PA) has been recognized as a favourable factor in the prevention of various diseases, including certain forms of cancer, the relationship between PA and gastric cancer (GC) is not yet fully understood. This study aims to provide data from a pooled analysis of case-control studies within the Stomach cancer Pooling (StoP) Project to estimate the association between leisure-time PA and the occurrence of GC.MethodsSix case-control studies from StoP project collected data on leisure-time PA, for a total of 2,343 cases and 8,614 controls. Subjects were classified into three leisure-time PA categories, either none/low, intermediate or high, based on study-specific tertiles. We used a two-stage approach. Firstly, we applied multivariable logistic regression models to obtain study-specific odds ratios (ORs) and corresponding 95% confidence intervals (CIs) then, we used a random-effect models to obtain pooled effect estimates. We performed stratified analyses according to demographic, lifestyle and clinical covariates.ResultsThe meta-analysis showed ORs of GC with no significant differences between intermediate vs low and high vs low PA level (OR 1.05 [95%CI 0.76-1.45]; OR 1.23 [95%CI 0.78-1.94], respectively). GC risk estimates did not strongly differ across strata of selected covariates except for age ≤ 55 years old (high vs low level: OR 0.72 [95%CI 0.55-0.94]) and for control population-based studies (high vs low level: OR 0.79 [95%CI 0.68-0.93]).ConclusionsNo association was found between leisure time PA and GC, apart from a slight suggestion of decreased risk below age 55 and in control population-based studies. These results may reflect specific characteristics of GC at a younger age, or the presence of a cohort effect mediating and interacting with socioeconomic determinants of GC The different distribution of PA levels among hospitalized controls could have led to an underestimated effect of PA on GC risk.

BackgroundOne of the three main targets of the World Health Organization (WHO) End TB Strategy (2015-2035) is that no tuberculosis (TB) patients or their households face catastrophic costs (defined as exceeding 20% of the annual household income) because of the disease. Our study seeks to determine, as a baseline, the magnitude and main drivers of the costs associated with TB disease for patients and their households and to monitor the proportion of households experiencing catastrophic costs in Brazil.MethodsA national cross-sectional cluster-based survey was conducted in Brazil in 2019-2021 following WHO methodology. TB patients of all ages and types of TB were eligible for the survey. Adult TB patients and guardians of minors (ResultsWe interviewed 603 patients, including 538 (89%) with drug-sensitive (DS) and 65 (11%) with drug-resistant (DR) TB. Of 603 affected households, 48.1% (95%CI: 43-53.2) experienced costs above 20% of their annual household income during their TB episode. The proportion was 44.4% and 78.5% among patients with DS- and DR-TB, respectively. On average, patients incurred costs of US$1573 (95%CI: 1361.8-1785.0) per TB episode, including pre-diagnosis and post-diagnosis expenses. Key cost drivers were post-diagnosis nutritional supplements (US$317.6, 95%CI: 232.7-402.6) followed by medical costs (US$85.5, 95%CI: 54.3-116.5) and costs of travel for clinic visits during treatment (US$79.2, 95%CI: 61.9-96.5). In multivariate analysis, predictors of catastrophic costs included positive HIV status (aOR = 3.0, 95%CI:1.1-8.6) and self-employment (aOR = 2.7, 95%CI:1.1-6.5); high education was a protective factor (aOR = 0.1, 95%CI:0.0-0.9).ConclusionsAlthough the services offered to patients with TB are free of charge in the Brazilian public health sector, the availability of free diagnosis and treatment services does not alleviate patients' financial burden related to accessing TB care. The study allowed us to identify the costs incurred by patients and suggest actions to mitigate their suffering. In addition, this study established a baseline for monitoring catastrophic costs and fostering a national policy to reduce the costs to patients for TB care in Brazil.

BackgroundAlthough physical activity (PA) has been recognized as a favourable factor in the prevention of various diseases, including certain forms of cancer, the relationship between PA and gastric cancer (GC) is not yet fully understood. This study aims to provide data from a pooled analysis of case-control studies within the Stomach cancer Pooling (StoP) Project to estimate the association between leisure-time PA and the occurrence of GC.MethodsSix case-control studies from StoP project collected data on leisure-time PA, for a total of 2,343 cases and 8,614 controls. Subjects were classified into three leisure-time PA categories, either none/low, intermediate or high, based on study-specific tertiles. We used a two-stage approach. Firstly, we applied multivariable logistic regression models to obtain study-specific odds ratios (ORs) and corresponding 95% confidence intervals (CIs) then, we used a random-effect models to obtain pooled effect estimates. We performed stratified analyses according to demographic, lifestyle and clinical covariates.ResultsThe meta-analysis showed ORs of GC with no significant differences between intermediate vs low and high vs low PA level (OR 1.05 [95%CI 0.76-1.45]; OR 1.23 [95%CI 0.78-1.94], respectively). GC risk estimates did not strongly differ across strata of selected covariates except for age ≤ 55 years old (high vs low level: OR 0.72 [95%CI 0.55-0.94]) and for control population-based studies (high vs low level: OR 0.79 [95%CI 0.68-0.93]).ConclusionsNo association was found between leisure time PA and GC, apart from a slight suggestion of decreased risk below age 55 and in control population-based studies. These results may reflect specific characteristics of GC at a younger age, or the presence of a cohort effect mediating and interacting with socioeconomic determinants of GC The different distribution of PA levels among hospitalized controls could have led to an underestimated effect of PA on GC risk.

The main objective of this paper is to assess the efficiency of the European Union's public sector from a quality of governance approach, employing a two-step methodology. In the first stage, EU states' efficiency scores are determined using Data Envelopment Analysis. Once quantifying public efficiency, the second step of the methodology examines the determinants of efficiency using a quantile regression estimation technique, with an emphasis on demographic features, corruption, economic freedom, and governmental digitalization. The novelty is provided by the assessment of the public sector's efficiency and efficacy from a broader approach in order to determine the efficiency scores of European Union countries, as well as the key factors that may impact the public sector's performance. The main findings, namely that governance quality can be considered an important resource in analysing public performance and that human resources, freedom, democracy, corruption, and digitalization have an impact on efficiency, are important considerations not only for policymakers but also for society, researchers, and the academic community. Reform measures should strive to improve both the technical and democratic components of public institutions in order to more effectively and transparently allocate public resources, while taking into consideration local and national peculiarities.

BackgroundIn patients with COVID-19 and baseline soluble urokinase plasminogen receptor plasma (suPAR) levels ≥ 6ng/mL, early administration of anakinra, a recombinant interleukin-1 receptor antagonist, may prevent disease progression and death. In case of suPAR testing unavailability, the Severe COvid Prediction Estimate (SCOPE) score may be used as an alternative in guiding treatment decisions.MethodsWe conducted a monocenter, retrospective cohort study, including patients with SARS-CoV2 infection and respiratory failure. Patients treated with anakinra (anakinra group, AG) were compared to two control groups of patients who did not receive anakinra, respectively with ≥ 6 ng/mL (CG1) and < 6 ng/mL (CG2) baseline suPAR levels. Controls were manually paired by age, sex, date of admission and vaccination status and, for patients with high baseline suPAR, propensity score weighting for receiving anakinra was applied. Primary endpoint of the study was disease progression at day 14 from admission, as defined by patient distribution on a simplified version of the 11-point World Health Organization Clinical Progression Scale (WHO-CPS).ResultsBetween July, 2021 and January, 2022, 153 patients were included, among which 56 were treated with off-label anakinra, 49 retrospectively fulfilled prescriptive criteria for anakinra and were assigned to CG1, and 48 presented with suPAR levels < 6ng/mL and were assigned to CG2. At day 14, when comparing to CG1, patients who received anakinra had significantly reduced odds of progressing towards worse clinical outcome both in ordinal regression analysis (OR 0.25, 95% CI 0.11-0.54, pConclusionThis real-word, retrospective cohort study confirmed the safety and the efficacy of suPAR-guided, early use of anakinra in hospitalized COVID-19 patients with respiratory failure.

Objective The aim of this retrospective observational study is to analyse clinical, serological and radiological predictors of outcome in patients with COVID-19 pneumonia treated with tocilizumab, providing clinical guidance to its use in real-life. Method This is a retrospective, monocentric observational cohort study. All consecutive patients hospitalized between February the 11th and April 14th 2020 for severe COVID-19 pneumonia at Reggio Emilia AUSL and treated with tocilizumab were enrolled. The patient’s clinical status was recorded every day using the WHO ordinal scale for clinical improvement. Response to treatment was defined as an improvement of one point (from the status at the beginning of tocilizumab treatment) during the follow-up on this scale. Bivariate association of main patients’ characteristics with outcomes was explored by descriptive statistics and Fisher or Kruskal Wallis tests (respectively for qualitative or quantitative variables). Each clinically significant predictor was checked by a loglikelihood ratio test (in univariate logistic models for each of the considered outcomes) against the null model. Results A total of 173 patients were included. Only hypertension, the use of angiotensin-converting enzyme inhibitors, PaO2/FiO2, respiratory rate and C-reactive protein were selected for the multivariate analysis. In the multivariable model, none of them was significantly associated with response. Conclusions Evaluating a large number of clinical variables, our study did not find new predictors of outcome in COVID19 patients treated with tocilizumab. Further studies are needed to investigate the use of tocilizumab in COVID-19 and to better identify clinical phenotypes which could benefit from this treatment.

IntroductionThis paper describes how mortality among hospitalised COVID-19 patients changed during the first three waves of the epidemic in Italy.MethodsThis prospective cohort study used the Kaplan-Meier method to analyse the time-dependent probability of death of all of the patients admitted to a COVID-19 referral centre in Milan, Italy, during the three consecutive periods of: 21 February-31 July 2020 (first wave, W1), 1 August 2020-31 January 2021 (second wave, W2), and 1 February-30 April 2021 (third wave, W3). Cox models were used to examine the association between death and the period of admission after adjusting for age, biological sex, the time from symptom onset to admission, disease severity upon admission, obesity, and the comorbidity burden.ResultsOf the 2,023 COVID-19 patients admitted to our hospital during the study period, 553 (27.3%) were admitted during W1, 838 (41.5%) during W2, and 632 (31.2%) during W3. The crude mortality rate during W1, W2 and W3 was respectively 21.3%, 23.7% and 15.8%. After adjusting for potential confounders, hospitalisation during W2 or W3 was independently associated with a significantly lower risk of death than hospitalisation during W1 (adjusted hazard ratios [AHRs]: 0.75, 95% confidence interval [CI] 0.59-0.95, and 0.58, 95% CI 0.44-0.77). Among the patients aged >75 years, there was no significant difference in the probability of death during the three waves (AHRs during W2 and W3 vs W1: 0.93, 95% CI 0.65-1.33, and 0.88, 95% CI 0.59-1.32), whereas those presenting with critical disease during W2 and W3 were at significantly lower risk of dying than those admitted during W1 (AHRs 0.61, 95% CI 0.43-0.88, and 0.44, 95% CI 0.28-0.70).ConclusionsHospitalisation during W2 and W3 was associated with a reduced risk of COVID-19 death in comparison with W1, but there was no difference in survival probability in patients aged >75 years.

[This corrects the article DOI: 10.1371/journal.pone.0241056.].

The use of renal biopsy through laparoscopy is increasingly present both in human and veterinary medicine. However, both techniques require skill and training to make the operator capable to do it. The learning curve allows the quantitative and qualitative assessment of the number of attempts and minimum time for the surgical procedure. The objective included establish the learning curve for laparoscopy-guided kidney biopsy procedures in dog and pig corpses. Six dogs and six pigs corpses weighing less than 10 kg were used for this study. All corpses underwent kidney biopsy performed through laparoscopy. Twenty-four operators, two per animal, performed 20 renal biopsies each (10 for each kidney), with 480 collection-procedures in total. Duration and difficulty of the procedure and the biopsy sample quality were evaluated and statistical analysis was performed using a mixed regression model with a random effect of individuals and multivariate analysis of data. There were 91.5% of the samples that were adequate for evaluation. There was no significant difference in the number of glomeruli or cortex percentage considering the attempts in either species, demonstrating the operator's ability since first collection. Swine samples showed higher amounts of renal cortex than canine samples. The procedure duration was shorter as more attempts were performed in dogs and pigs. From the fourth repetition, the professional reached a plateau for the variable related to 'collection', and from the second, the professional presented uniform duration for 'sample storage'. Operators of the swine model acquired more agility than the dog ones. The variable 'difficulty' decreased as more repetitions were performed, reaching a plateau in the sixth attempt. Seven renal biopsies laparoscopy-guided are required for an operator to be considered 'capable' to perform the procedure in the referred species included. The learning curve for image-guided kidney biopsy procedures improves the implementation of this technique and benefits patients that undergo this procedure.

Cell therapy is poised to play a larger role in medicine, most notably for immuno-oncology. Despite the recent success of CAR-T therapeutics in the treatment of blood tumors and the rapid progress toward improved versions of both CAR- and TCR-Ts, important analytical aspects of preclinical development and manufacturing of engineered T cells remain immature. One limiting factor is the absence of robust multivariate assays to disentangle key parameters related to function of engineered effector cells, especially in the peptide-MHC (pMHC) target realm, the natural ligand for TCRs. Here we describe an imaging-based primary T cell assay that addresses several of these limitations. To our knowledge, this assay is the first quantitative, high-content assay that separates the key functional parameters of time- and antigen-dependent T cell proliferation from cytotoxicity. We show that the assay sheds light on relevant biology of CAR- and TCR-T cells, including response kinetics and the influence of effector:target ratio.

IntroductionAs Novel psychoactive substances (NPS) are conceived to mimic the effects of common illicit drugs, they represent a serious public health challenge due to the spike in intoxications and fatalities that have been linked to their use. This study aims to provide epidemiological data on NPS use in the USA, determining lifetime prevalence of use and defining demographic, socioeconomic, drug use patterns and mental health correlates.MethodsThis study uses secondary data from the US National Survey on Drug Use and Health (NSDUH), which is a large cross-sectional population-based survey carried out annually in the USA. We analysed data from 2007-14 (N = 307,935) using bivariate descriptive analysis and binary logistic regression to calculate prevalence and determine factors underlying NPS consumption. Adjusted odds ratios (OR) with 95% CI's were calculated for a set of selected independent variables.Results and discussionOur analysis NSDUH from 2007-14 highlights an increase in NPS use among adults, especially among white young men aged 18 to 25. Although the level of education of NPS users was relatively higher as compared to non-users, NPS users seemed to have a less wealthy situation. However, socioeconomic vulnerability appeared to be less important than mental health issues as a correlate to NPS use. NPS users seem to have followed a pattern of polysubstance use throughout their life, which involves both traditional illicit drugs and classic synthetic drugs. As NPS use seemed to be more prevalent among people having mental health issues, the rise in their use may have a negative impact on population mental health outcomes.ConclusionFurther comparative research on trends in NPS use and potential public health responses would be instrumental for developing appropriate health interventions, including drug checking, education for users and training for healthcare professionals working both within emergency wards and in/outpatient addiction and mental health services.

IntroductionThe rapid rise in trade and use of NPS and the lack of information concerning their potential toxicity pose serious challenges to public health authorities across the world. Policy measures towards NPS taken so far have a special focus on their legal status, while the implementation of a public health strategy seems to be still missing. The aim of this study is to perform a general assessment of NPS-related policy (including regulatory measures and public health strategies) implemented by six European countries: Portugal, the Netherlands, Czech Republic, Poland, the United Kingdom and Sweden.MethodsSix EU countries were included in this scoping review study. Drug policies (including legal responses and public health strategies) were analysed. UNODC drug policy classification system was used as a benchmark, while path dependency approach was used for data analysis; a net of inter-dependencies between international, EU and national policies was highlighted.Results and discussionThe countries included in this study can be placed in a wide spectrum according to their formulation of drug policy, from Portugal and the UK that have specific legal responses to NPS but have differently focused on harm reduction strategies at one end, to Sweden whose drug-free society goal is not translated into a specific regulation of NPS at the other end.ConclusionThe findings of the study reveal limited development towards harmonisation of national drug policies-particularly with regard to NPS. To tackle the challenge presented by NPS, EU Member states have formulated legislation and public health strategies independently. National approaches to NPS are therefore in line with their already existing drug policies, reflecting cultural values towards substance abuse and national political interests, while the homogenization at an international level has so far mostly been focused on law enforcement and drugs use preventive strategies.

Agro-biodiversity is seriously threatened worldwide and strategies to preserve it are dramatically required. We propose here a methodological approach aimed to identify areas with a high level of agro-biodiversity in which to set or enhance in situ conservation of plant genetic resources. These areas are identified using three criteria: Presence of Landrace diversity, Presence of wild species and Agro-ecosystem ecological diversity. A Restrictive and an Additive prioritization strategy has been applied on the entire Italian territory and has resulted in establishing nationwide 53 and 197 agro-biodiversity hotspots respectively. At present the strategies can easily be applied at a European level and can be helpful to develop conservation strategies everywhere.

PURPOSE:We aimed to investigate biomarkers and predictive factors for visual and anatomical outcome in patients with naïve diabetic macular edema (DME) who underwent small gauge pars plana vitrectomy (PPV) with internal limiting membrane (ILM) peeling as a first line treatment. DESIGN:Multicenter, retrospective, interventional study. PARTICIPANTS:120 eyes from 120 patients with naïve DME treated with PPV and ILM peeling with a follow up of 24 months. METHODS:Change in baseline best corrected visual acuity (BCVA) and central subfoveal thickness (CST) 1, 6, 12 and 24 months after surgery. Predictive value of baseline BCVA, CST, optical coherence tomography (OCT) features (presence of subretinal fluid (SRF) and photoreceptor damage), and time between DME diagnosis and surgery. Additional treatment for DME needed. Intra- and post-operative complications (cataract rate formation, increased intraocular pressure). MAIN OUTCOME MEASURES:The correlation between baseline characteristics and BCVA response (mean change from baseline; categorized improvement ≥5 or ≥10; Early Treatment Diabetic Retinopathy Study (ETDRS) letters) 12 and 24 months after surgery. RESULTS:Mean BCVA was 0.66 ± 0.14 logMAR, 0.52 ± 0.21 logMAR, and 0.53 ± 0.21 logMAR (p

Fruits of the sweet cherry (Prunus avium L.) accumulate a range of antioxidants that can help to prevent cardiovascular disease, inflammation and cancer. We tested the in vitro antioxidant activity of 18 sweet cherry cultivars collected from 12 farms in the protected geographical indication region of Marostica (Vicenza, Italy) during two growing seasons. Multiple targeted and untargeted metabolomics approaches (NMR, LC-MS, HPLC-DAD, HPLC-UV) as well as artificial simplified phytocomplexes representing the cultivars Sandra Tardiva, Sandra and Grace Star were then used to determine whether the total antioxidant activity reflected the additive effects of each compound or resulted from synergistic interactions. We found that the composition of each cultivar depended more on genetic variability than environmental factors. Furthermore, phenolic compounds were the principal source of antioxidant activity and experiments with artificial simplified phytocomplexes indicated strong synergy between the anthocyanins and quercetins/ascorbic acid specifically in the cultivar Sandra Tardiva. Our data therefore indicate that the total antioxidant activity of sweet cherry fruits may originate from cultivar-dependent interactions among different classes of metabolite.

Suspended sediments produced from dredging activities, or added to the sediment budget via river runoff, are a concern for marine resource managers. Understanding the impact of suspended sediments on critical life history stages of keystone species like corals is fundamental to effective management of coastlines and reefs. Coral embryos (Acropora tenuis and A. millepora) and larvae (A. tenuis, A. millepora and Pocillopora acuta) were subjected to a range of suspended sediment concentrations of different sediment types (siliciclastic and carbonate) to assess concentration-response relationships on ecologically relevant endpoints, including survivorship and ability to metamorphose. Embryos were subjected to short (12 h) suspended sediment exposures from ages of 3-12 hours old or a long (30 h) exposure at 6 hours old. Neither the survivorship nor metamorphosis function of embryos were significantly affected by realistic sediment exposures to ~1000 mg L-1. However, some embryos exhibited a previously undescribed response to dynamically suspended sediments, which saw 10% of the embryos form negatively buoyant cocoons at siliciclastic suspended sediment concentrations ≥35 mg L-1. Scanning electron and optical microscopy confirmed the presence of a coating on these embryos, possibly mucus with incorporated sediment particles. Cocoon formation was common in embryos but not in larvae, and occurred more often after exposure to siliciclastic rather than carbonate sediments. Once transferred into sediment-free seawater, functional ~36-h-old embryos began emerging from the cocoons, coinciding with cilia development. Ciliated (> 36-h-old) larvae exposed to suspended sediments for 60 h were also observed to secrete mucus and were similarly unaffected by suspended sediment concentrations to ~800 mg L-1. This study provides evidence that mucous secretion and cilia beating effectively protect coral embryos and larvae from suspended sediment and that these mechanisms may enhance their chances of successful recruitment.

Inshore coral reefs are experiencing the combined pressures of excess nutrient availability associated with coastal activities and warming seawater temperatures. Both pressures are known to have detrimental effects on the early life history stages of hard corals, but studies of their combined effects on early demographic stages are lacking. We conducted a series of experiments to test the combined effects of nutrient enrichment (three levels) and elevated seawater temperature (up to five levels) on early life history stages of the inshore coral Acropora tenuis, a common species in the Indo-Pacific and Red Sea. Gamete fertilization, larval survivorship and larval settlement were all significantly reduced as temperature increased, but only fertilization was further affected by simultaneous nutrient enrichment. Combined high temperatures and nutrient enrichment affected fertilization in an additive manner, whereas embryo abnormalities increased synergistically. Higher than normal temperatures (32°C) increased coral juvenile growth rates 1.6-fold, but mortality also increased by 50%. The co-occurrence of nutrient enrichment with high temperatures reduced juvenile mortality to 36%, ameliorating temperature stress (antagonistic interaction). Overall, the types of effect (additive vs synergistic or antagonistic) and their magnitude varied among life stages. Gamete and embryo stages were more affected by temperature stress and, in some cases, also by nutrient enrichment than juveniles. The data suggest that coastal runoff events might exacerbate the impacts of warming temperatures on fertilization if these events co-occur during corals spawning. The cumulative impacts of simultaneous exposure to nutrient enrichment and elevated temperatures over all early life history stages increases the likelihood for failure of larval supply and recruitment for this coral species. Our results suggest that improving the water quality of river discharges into coastal areas might help to enhance the thermal tolerances of early life history stages in this common coral species.

Widespread contamination of nearshore marine systems, including the Great Barrier Reef (GBR) lagoon, with agricultural herbicides has long been recognised. The fate of these contaminants in the marine environment is poorly understood but the detection of photosystem II (PSII) herbicides in the GBR year-round suggests very slow degradation rates. Here, we evaluated the persistence of a range of commonly detected herbicides in marine water under field-relevant concentrations and conditions. Twelve-month degradation experiments were conducted in large open tanks, under different light scenarios and in the presence and absence of natural sediments. All PSII herbicides were persistent under control conditions (dark, no sediments) with half-lives of 300 d for atrazine, 499 d diuron, 1994 d hexazinone, 1766 d tebuthiuron, while the non-PSII herbicides were less persistent at 147 d for metolachlor and 59 d for 2,4-D. The degradation of herbicides was 2-10 fold more rapid in the presence of a diurnal light cycle and coastal sediments; apart from 2,4-D which degraded more slowly in the presence of light. Despite the more rapid degradation observed for most herbicides in the presence of light and sediments, the half-lives remained > 100 d for the PS II herbicides. The effects of light and sediments on herbicide persistence were likely due to their influence on microbial community composition and its ability to utilise the herbicides as a carbon source. These results help explain the year-round presence of PSII herbicides in marine systems, including the GBR, but more research on the transport, degradation and toxicity on a wider range of pesticides and their transformation products is needed to improve their regulation in sensitive environments.

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.

In this pilot study for the first time, ancient DNA has been extracted from bone remains of Salmo trutta. These samples were from a stratigraphic succession located in a coastal cave of Calabria (southern Italy) inhabited by humans from upper Palaeolithic to historical times. Seven pairs of primers were used to PCR-amplify and sequence from 128 to 410 bp of the mtDNA control region of eleven samples. Three haplotypes were observed: two (ADcs-1 and MEcs-1) already described in rivers from the Italian peninsula; one (ATcs-33) belonging to the southern Atlantic clade of the AT Salmo trutta mtDNA lineage (sensu Bernatchez). The prehistoric occurrence of this latter haplotype in the water courses of the Italian peninsula has been detected for the first time in this study. Finally, we observed a correspondence between frequency of trout remains and variation in haplotype diversity that we related with ecological and demographic changes resulting from a period of rapid cooling known as the Younger Dryas.

Herbicides are detected year-round in marine waters, including those of the World Heritage listed Great Barrier Reef (GBR). The few previous studies that have investigated herbicide persistence in seawater generally reported half-lives in the order of months, and several studies were too short to detect significant degradation. Here we investigated the persistence of eight herbicides commonly detected in the GBR or its catchments in standard OECD simulation flask experiments, but with the aim to mimic natural conditions similar to those found on the GBR (i.e., relatively low herbicide concentrations, typical temperatures, light and microbial communities). Very little degradation was recorded over the standard 60 d period (Experiment 1) so a second experiment was extended to 365 d. Half-lives of PSII herbicides ametryn, atrazine, diuron, hexazinone and tebuthiuron were consistently greater than a year, indicating high persistence. The detection of atrazine and diuron metabolites and longer persistence in mercuric chloride-treated seawater confirmed that biodegradation contributed to the breakdown of herbicides. The shortest half-life recorded was 88 d for growth-regulating herbicide 2,4-D at 31°C in the dark, while the fatty acid-inhibitor metolachlor exhibited a minimum half-life of 281 d. The presence of moderate light and elevated temperatures affected the persistence of most of the herbicides; however, the scale and direction of the differences were not predictable and were likely due to changes in microbial community composition. The persistence estimates here represent some of the first appropriate data for application in risk assessments for herbicide exposure in tropical marine systems. The long persistence of herbicides identified in the present study helps explain detection of herbicides in nearshore waters of the GBR year round. Little degradation of these herbicides would be expected during the wet season with runoff and associated flood plumes transporting a high proportion of the original herbicide from rivers into the GBR lagoon.

The impact of cardiac surgery using cardiopulmonary bypass (CPB) on the respiratory mucociliary function is unknown. This study evaluated the effects of CPB and interruption of mechanical ventilation on the respiratory mucociliary system.Twenty-two pigs were randomly assigned to the control (n = 10) or CPB group (n = 12). After the induction of anesthesia, a tracheostomy was performed, and tracheal tissue samples were excised (T0) from both groups. All animals underwent thoracotomy. In the CPB group, an aorto-bicaval CPB was installed and maintained for 90 minutes. During the CPB, mechanical ventilation was interrupted, and the tracheal tube was disconnected. A second tracheal tissue sample was obtained 180 minutes after the tracheostomy (T180). Mucus samples were collected from the trachea using a bronchoscope at T0, T90 and T180. Ciliary beat frequency (CBF) and in situ mucociliary transport (MCT) were studied in ex vivo tracheal epithelium. Mucus viscosity (MV) was assessed using a cone-plate viscometer. Qualitative tracheal histological analysis was performed at T180 tissue samples.CBF decreased in the CPB group (13.1 ± 1.9 Hz vs. 11.1 ± 2.1 Hz, p < 0.05) but not in the control group (13.1 ± 1 Hz vs. 13 ± 2.9 Hz). At T90, viscosity was increased in the CPB group compared to the control (p < 0.05). No significant differences were observed in in situ MCT. Tracheal histology in the CPB group showed areas of ciliated epithelium loss, submucosal edema and infiltration of inflammatory cells.CPB acutely contributed to alterations in tracheal mucocilliary function.

Due to anthropogenic factors, the franciscana dolphin, Pontoporia blainvillei, is the most threatened small cetacean on the Atlantic coast of South America. Four Franciscana Management Areas have been proposed: Espiritu Santo to Rio de Janeiro (FMA I), São Paulo to Santa Catarina (FMA II), Rio Grande do Sul to Uruguay (FMA III), and Argentina (FMA IV). Further genetic studies distinguished additional populations within these FMAs. We analyzed the population structure, phylogeography, and demographic history in the southernmost portion of the species range. From the analysis of mitochondrial DNA control region sequences, 5 novel haplotypes were found, totalizing 60 haplotypes for the entire distribution range. The haplotype network did not show an apparent phylogeographical signal for the southern FMAs. Two populations were identified: Monte Hermoso (MH) and Necochea (NC)+Claromecó (CL)+Río Negro (RN). The low levels of genetic variability, the relative constant size over time, and the low levels of gene flow may indicate that MH has been colonized by a few maternal lineages and became isolated from geographically close populations. The apparent increase in NC+CL+RN size would be consistent with the higher genetic variability found, since genetic diversity is generally higher in older and expanding populations. Additionally, RN may have experienced a recent split from CL and NC; current high levels of gene flow may be occurring between the latter ones. FMA IV would comprise four franciscana dolphin populations: Samborombón West+Samborombón South, Cabo San Antonio+Buenos Aires East, NC+CL+Buenos Aires Southwest+RN and MH. Results achieved in this study need to be taken into account in order to ensure the long-term survival of the species.

Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1) to the risk of coronary artery disease (CAD). Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS)-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging.

Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

Honey bees (Apis mellifera L.) are bioindicators of environmental pollution levels. During their wide-ranging foraging activity, these hymenopterans are exposed to pollutants, thus becoming a useful tool to trace the environmental contaminants as heavy metals, pesticides, radionuclides and volatile organic compounds. In the present work we demonstrate that bees can also be used as active samplers of airborne particulate matter. Worker bees were collected from hives located in a polluted postmining area in South West Sardinia (Italy) that is also exposed to dust emissions from industrial plants. The area is included in an official list of sites of national interest for environmental remediation, and has been characterized for the effects of pollutants on the health of the resident population. The head, wings, hind legs and alimentary canal of the bees were investigated with Scanning Electron Microscopy coupled with X-ray spectroscopy (SEM-EDX). The analyses pointed to specific morphological and chemical features of the particulate, and resulted into the identification of three categories of particles: industry-, postmining-, and soil-derived. With the exception of the gut, all the analyzed body districts displayed inorganic particles, mostly concentrated in specific areas of the body (i.e. along the costal margin of the fore wings, the medial plane of the head, and the inner surface of the hind legs). The role of both past mining activities and the industrial activity close to the study area as sources of the particulate matter is also discussed. We conclude that honey bees are able to collect samples of the main airborne particles emitted from different sources, therefore could be an ideal tool for monitoring such a kind of pollutants.

Photosystem II (PSII) herbicides have been detected in nearshore tropical waters such as those of the Great Barrier Reef and may add to the pressure posed by runoff containing sediments and nutrients to threatened seagrass habitats. There is a growing number of studies into the potential effects of herbicides on seagrass, generally using large experimental setups with potted plants. Here we describe the successful development of an acute 12-well plate phytotoxicity assay for the PSII herbicide Diuron using isolated Halophila ovalis leaves. Fluorescence images demonstrated Diuron affected the entire leaf surface evenly and responses were not influenced by isolating leaves from the plant. The optimum exposure duration was 24 h, by which time the inhibition of effective quantum yield of PSII (∆F/F(m)') was highest and no deterioration of photosystems was evident in control leaves. The inhibition of ∆F/F(m)' by Diuron in isolated H. ovalis leaves was identical to both potted and hydroponically grown plants (with leaves remaining attached to rhizomes), indicating similar reductions in photosynthetic activity in these acute well-plate assays. The sensitivity of the assay was not influenced by irradiance (range tested 40 to 400 μmol photons m(-2) s(-1)). High irradiance, however, caused photo-oxidative stress in H. ovalis and this generally impacted in an additive or sub-additive way with Diuron to damage PSII. The bioassay using isolated leaves is more rapid, uses far less biological material and does not rely on specialised aquarium facilities in comparison with assays using potted plants. The development and validation of this sensitive bioassay will be useful to reliably screen and monitor the phytotoxicity of existing and emerging PSII herbicides and contribute to risk assessments and water quality guideline development in the future.

Recent reports highlight the potential for integrase-defective lentiviral vectors (IDLV) to be developed as vaccines due to their ability to elicit cell-mediated and humoral immune responses after intramuscular administration. Differently from their integrase-competent counterpart, whose utility for vaccine development is limited by the potential for insertional mutagenesis, IDLV possess a mutation in their integrase gene that prevents genomic integration. Instead, they are maintained as episomal DNA circles that retain the ability to stably express functional proteins. Despite their favorable profile, it is unknown whether IDLV elicit immune responses after intranasal administration, a route that could be advantageous in the case of infection with a respiratory agent. Using influenza as a model, we constructed IDLV expressing the influenza virus nucleoprotein (IDLV-NP), and tested their ability to generate NP-specific immune responses and protect from challenge in vivo. We found that administration of IDLV-NP elicited NP-specific T cell and antibody responses in BALB/c mice. Importantly, IDLV-NP was protective against homologous and heterosubtypic influenza virus challenge only when given by the intranasal route. This is the first report demonstrating that IDLV can induce protective immunity after intranasal administration, and suggests that IDLV may represent a promising vaccine platform against infectious agents.

Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae.

Vulvovaginal candidiasis (VVC) is among the most prevalent vaginal diseases. Candida albicans is still the most prevalent species associated with this pathology, however, the prevalence of other Candida species, such as C. glabrata, is increasing. The pathogenesis of these infections has been intensely studied, nevertheless, no consensus has been reached on the pathogenicity of VVC. In addition, inappropriate treatment or the presence of resistant strains can lead to RVVC (vulvovaginal candidiasis recurrent). Immunomodulation therapy studies have become increasingly promising, including with the β-glucans. Thus, in the present study, we evaluated microbicidal activity, phagocytosis, intracellular oxidant species production, oxygen consumption, myeloperoxidase (MPO) activity, and the release of tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), IL-1β, and IL-1Ra in neutrophils previously treated or not with β-glucan. In all of the assays, human neutrophils were challenged with C. albicans and C. glabrata isolated from vulvovaginal candidiasis. β-glucan significantly increased oxidant species production, suggesting that β-glucan may be an efficient immunomodulator that triggers an increase in the microbicidal response of neutrophils for both of the species isolated from vulvovaginal candidiasis. The effects of β-glucan appeared to be mainly related to the activation of reactive oxygen species and modulation of cytokine release.

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134-197 fmol STX cell(-1)) was similar to the parent cultures (169-206 fmol STX cell(-1)), however cultures grown with single bacterial types contained less toxin (134-146 fmol STX cell(-1)) than offspring or parent cultures grown with more complex mixed bacterial communities (152-176 fmol STX cell(-1)). Specific toxin production rate (fmol STX day(-1)) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell(-1) day(-1)) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.

Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes.In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity.Our screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases.

Research has demonstrated that people who embrace different ideological orientations often show differences at the level of basic cognitive processes. For instance, conservatives (vs. liberals) display an automatic selective attention for negative (vs. positive) stimuli, and tend to more easily form illusory correlations between negative information and minority groups. In the present work, we further explored this latter effect by examining whether it only involves the formation of explicit attitudes or it extends to implicit attitudes. To this end, following the typical illusory correlation paradigm, participants were presented with members of two numerically different groups (majority and minority) each performing either a positive or negative behaviour. Negative behaviors were relatively infrequent, and the proportion of positive and negative behaviors within each group was the same. Next, explicit and implicit (i.e., IAT-measured) attitudes were assessed. Results showed that conservatives (vs. liberals) displayed stronger explicit as well as implicit illusory correlations effects, forming more negative attitudes toward the minority (vs. majority) group at both the explicit and implicit level.

Many infectious agents infiltrate the host at the mucosal surfaces and then spread systemically. This implies that an ideal vaccine should induce protective immune responses both at systemic and mucosal sites to counteract invasive mucosal pathogens. We evaluated the in vivo systemic and mucosal antigen-specific immune response induced in mice by intramuscular administration of an integrase defective lentiviral vector (IDLV) carrying the ovalbumin (OVA) transgene as a model antigen (IDLV-OVA), either alone or in combination with sublingual adjuvanted OVA protein. Mice immunized intramuscularly with OVA and adjuvant were compared with IDLV-OVA immunization. Mice sublingually immunized only with OVA and adjuvant were used as a positive control of mucosal responses. A single intramuscular dose of IDLV-OVA induced functional antigen-specific CD8+ T cell responses in spleen, draining and distal lymph nodes and, importantly, in the lamina propria of the large intestine. These results were similar to those obtained in a prime-boost regimen including one IDLV immunization and two mucosal boosts with adjuvanted OVA or vice versa. Remarkably, only in groups vaccinated with IDLV-OVA, either alone or in prime-boost regimens, the mucosal CD8+ T cell response persisted up to several months from immunization. Importantly, following IDLV-OVA immunization, the mucosal boost with protein greatly increased the plasma IgG response and induced mucosal antigen-specific IgA in saliva and vaginal washes. Overall, intramuscular administration of IDLV followed by protein boosts using the sublingual route induced strong, persistent and complementary systemic and mucosal immune responses, and represents an appealing prime-boost strategy for immunization including IDLV as a delivery system.

Common bean diversity within and between Mesoamerican and Andean gene pools was compared in 89 landraces from America and 256 landraces from Europe, to elucidate the effects of bottleneck of introduction and selection for adaptation during the expansion of common bean (Phaseolus vulgaris L.) in Europe. Thirteen highly polymorphic nuclear microsatellite markers (nuSSRs) were used to complement chloroplast microsatellite (cpSSRs) and nuclear markers (phaseolin and Pv-shatterproof1) data from previous studies. To verify the extent of the introduction bottleneck, inter-gene pool hybrids were distinguished from "pure" accessions. Hybrids were identified on the basis of recombination of gene pool specific cpSSR, phaseolin and Pv-shatterproof1 markers with a Bayesian assignments based on nuSSRs, and with STRUCTURE admixture analysis. More hybrids were detected than previously, and their frequency was almost four times larger in Europe (40.2%) than in America (12.3%). The genetic bottleneck following the introduction into Europe was not evidenced in the analysis including all the accessions, but it was significant when estimated only with "pure" accessions, and five times larger for Mesoamerican than for Andean germplasm. The extensive inter-gene pool hybridization generated a large amount of genotypic diversity that mitigated the effects of the bottleneck that occurred when common bean was introduced in Europe. The implication for evolution and the advantages for common bean breeding are discussed.

ObjectiveA correlation between glucose control and 25(OH)D metabolism has been suggested by previous studies. However, this correlation has not yet been evaluated considering the impact of chronic complications of type 2 diabetes, especially the presence of nephropathy. Thus, the aim of this study was to determine the correlation between A1C and 25(OH)D in a well characterized cohort of type 2 diabetic patients.Research design and methodsWe cross-sectionally examined the association between A1C and serum 25(OH) D in 715 type 2 diabetic patients attending our clinic during the years 2011-2012. The average age was 68±12 years (range 26-94 years). The relation between A1C and serum 25(OH)D levels was modelled by multiple linear regression analyses.ResultsSerum 25(OH)D levels were inversely associated with A1C levels (r = -0.116, p = .003). This relation maintains its independence in the multivariate analysis after adjusting for age, sex, A1C, BMI, treatment and duration of diabetes and nephropathy.ConclusionsIn type 2 diabetic patients, high A1C levels are associated with low concentrations of serum 25(OH)D independently of duration of diabetes, diabetic treatment and nephropathy. Future studies are needed to clarify the biological relation between glucose control and vitamin D metabolism in type 2 diabetes.

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.