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Reproductive and genetic medicine are evolving rapidly, and new technologies are already impacting current practices. This includes technologies that can identify a couples’ risk of having a child with a genetic disorder. Responsible implementation of new technologies requires evaluation of safety and ethics. Valuable insights for shaping governance processes are provided by various stakeholders involved, including healthcare professionals. Their willingness to adopt these technologies and guide the necessary systemic changes is required for the successful implementation of these technologies. In this study, twenty-one semi-structured interviews were conducted with professionals from different disciplines in the field of reproductive and genetic healthcare in the Netherlands. Three emerging technologies were discussed: expanded carrier screening (ECS), non-invasive prenatal diagnosis (NIPD) and germline genome editing (GGE). By probing stakeholders’ views, we explored how culture, structure and practice in healthcare is being shaped by innovations and changing dynamics in genetic and reproductive medicine. The general consensus was that the implementation of reproductive genetic technologies nationwide is a slow process in Dutch healthcare. A “typical Dutch approach” emerged that is characterized by restrictive legislation, broad support for people living with disabilities, values of an egalitarian society and limited commercialisation. Different scenarios for embedding ECS in future practice were envisioned, while implementation of NIPD in clinical practice was considered obvious. Views on GGE varied among stakeholders. Previous implementation examples in the Netherlands suggest introduction of new technology involves an organized collective learning process, with pilot studies and stepwise implementation. In addition, introducing and scaling up new technologies is complex due to perceived barriers from the legislative framework and the complex relationship between the government and stakeholders in this area. This paper describes how the international trends and advances of technologies are expected to manifest itself in a national setting.

EU legislation prohibits clinical trials that modify germ line 'genetic identity'. 'Genetic identity' however, is left undefined. This study aims to identify the use of the term 'genetic identity' in academic literature, and investigate its relevance for debates on genetic modification. A total of 616 articles that contained the term were identified. Content analysis revealed that the term was used in various and contradicting ways and a clear understanding of the term is lacking. This review demonstrates that the EU legislation is open to interpretation, because of the diversity of meaning with which 'genetic identity' is currently used. Because of the diversity of meaning with which 'genetic identity' is used and understood, further reflection is needed. This requires further medical, legal, ethical and social debate and a coordinated response at both a European and a global level.

PURPOSE:Quantitative computed tomography (QCT) provides important prognostic information of coronary atherosclerosis. We investigated intraobserver and interobserver QCT reproducibility in asymptomatic individuals, patients with acute chest pain without acute coronary syndrome (ACS), and patients with acute chest pain and ACS. METHODS:Fifty patients from each cohort, scanned between 01/02/2010-14/11/2013 and matched according to age and gender, were retrospectively assessed for inclusion. Patients with no coronary artery disease, previous coronary artery bypass graft surgery, and poor image quality were excluded. Coronary atherosclerosis was measured semi-automatically by 2 readers. Reproducibility of minimal lumen area (MLA), minimal lumen diameter (MLD), area stenosis, diameter stenosis, vessel remodeling, plaque eccentricity, plaque burden, and plaque volumes was assessed using concordance correlation coefficient (CCC), Bland-Altman, coefficient of variation, and Cohen's kappa. RESULTS:A total of 84 patients (63 matched) were included. Intraobserver and interobserver reproducibility estimates were acceptable for MLA (CCC = 0.94 and CCC = 0.91, respectively), MLD (CCC = 0.92 and CCC = 0.86, respectively), plaque burden (CCC = 0.86 and CCC = 0.80, respectively), and plaque volume (CCC = 0.97 and CCC = 0.95, respectively). QCT detected area and diameter stenosis ≥50%, positive remodeling, and eccentric plaque with moderate-good intraobserver and interobserver reproducibility (kappa: 0.64-0.66, 0.69-0.76, 0.46-0.48, and 0.41-0.62, respectively). Reproducibility of plaque composition decreased with decreasing plaque density (intraobserver and interobserver CCC for dense calcium (>0.99; 0.98), fibrotic (0.96; 0.93), fibro-fatty (0.95; 0.91), and necrotic core tissue (0.89; 0.84). Reproducibility generally decreased with worsening clinical risk profile. CONCLUSIONS:Semi-automated QCT of coronary plaque morphology is reproducible, albeit with some decline in reproducibility with worsening patient risk profile.

General practitioners (GPs) are increasingly called upon to identify patients at risk for hereditary cancers, and their genetic competencies need to be enhanced. This article gives an overview of a research project on how to build effective educational modules on genetics, assessed by randomized controlled trials (RCTs), reflecting the prioritized educational needs of primary care physicians. It also reports on an ongoing study to investigate long-term increase in genetic consultation skills (1-year follow-up) and interest in and satisfaction with a supportive website on genetics among GPs. Three oncogenetics modules were developed: an online Continuing Professional Development (G-eCPD) module, a live genetic CPD module, and a "GP and genetics" website (huisartsengenetica.nl) providing further genetics information applicable in daily practice. Three assessments to evaluate the effectiveness (1-year follow-up) of the oncogenetic modules were designed: 1.An online questionnaire on self-reported genetic competencies and changes in referral behaviour, 2.Referral rates from GPs to clinical genetics centres and 3.Satisfaction questionnaire and visitor count analytics of supportive genetics website. The setting was Primary care in the Netherlands and three groups of study participants were included in the reported studies:. Assessment 1. 168 GPs responded to an email invitation and were randomly assigned to an intervention or control group, evaluating the G-eCPD module (n = 80) or the live module (n = 88). Assessment 2. Referral rates by GPs were requested from the clinical genetics centres, in the northern and southern parts of the Netherlands (Amsterdam and Maastricht), for the two years before (2010 [n = 2510] and 2011 [n = 2940]) and the year after (2012 [n = 2875]) launch of the oncogenetics CPD modules and the website. Assessment 3. Participants of the website evaluation were all recruited online. When they visited the website during the month of February 2013, a pop-up invitation came up. Of the 1350 unique visitors that month, only 38 completed the online questionnaire. Main outcomes measure showed long-term (self-reported) genetic consultation skills (i.e. increased genetics awareness and referrals to clinical genetics centres) among GPs who participated in the oncogenetic training course, and interest in and satisfaction with the supportive website. 42 GPs (52%) who previously participated in the G-eCPD evaluation study and 50 GPs (57%) who participated in the live training programme responded to the online questionnaire on long-term effects of educational outcome. Previous RCTs showed that the genetics CPD modules achieved sustained improvement of oncogenetic knowledge and consultation skills (3-months follow-up). Participants of these RCTs reported being more aware of genetic problems long term; this was reported by 29 GPs (69%) and 46 GPs (92%) participating in the G-eCPD and live module evaluation studies, respectively (Chisquare test, p

In vitro gut modeling is a useful approach to investigate some factors and mechanisms of the gut microbiota independent of the effects of the host. This study tested the use of immobilized fecal microbiota to develop different designs of continuous colonic fermentation models mimicking elderly gut fermentation. Model 1 was a three-stage fermentation mimicking the proximal, transverse and distal colon. Models 2 and 3 were based on the new PolyFermS platform composed of an inoculum reactor seeded with immobilized fecal microbiota and used to continuously inoculate with the same microbiota different second-stage reactors mounted in parallel. The main gut bacterial groups, microbial diversity and metabolite production were monitored in effluents of all reactors using quantitative PCR, 16S rRNA gene 454-pyrosequencing, and HPLC, respectively. In all models, a diverse microbiota resembling the one tested in donor's fecal sample was established. Metabolic stability in inoculum reactors seeded with immobilized fecal microbiota was shown for operation times of up to 80 days. A high microbial and metabolic reproducibility was demonstrated for downstream control and experimental reactors of a PolyFermS model. The PolyFermS models tested here are particularly suited to investigate the effects of environmental factors, such as diet and drugs, in a controlled setting with the same microbiota source.

Although body weight (BW) and body surface area (BSA) are utilized to establish the appropriate dosage of anticancer drugs, their distribution in cancer patients is poorly studied, making it challenging to predict the amount of drug use and related costs of BW or BAS-dosed regimens. This study investigates the distribution of BW and BSA in adults with selected cancers who initiated systemic anticancer treatment in the eastern Emilia-Romagna region hospitals between 2011 and 2021. BW and BSA were collected at the first cycle of each new treatment line, with multiple measurements for patients receiving various treatments or treating for other primary malignancies. Results were grouped by sex, tumor site and treatment setting, and the normal distribution hypothesis was tested for each group. Both linear regression model and quantile regression at the 50th, 25th and 75th percentiles were run to explore the factors influencing BSA. The analysis included 20,634 treatment lines and the corresponding BW and BSA measures from a sample of 13,036 patients. The average BW was 68.05kg (64.20kg for females and 75.07kg for males) and the average BSA was 1.76m2 (1.66m2 for females and 1.87m2 for males). In women, the highest BW was in breast and colon groups, while in men, it was associated with prostate and rectum cancers. The model indicated significant association between BSA, age, sex and tumor localization. Notably, stomach and lung cancers were linked to lower BSA for both sexes (for females -0.081 and -0.041m2 respectively compared to those with breast cancer). Advanced settings were related to lower BSA than neoadjuvant treatment, especially for stomach cancer patients, who experienced a weight loss of 3 to 6kg as therapy progressed. The regression models for predicting BSA can assist regulatory bodies in determining reimbursement for new chemotherapy drugs and help hospitals forecast drug utilization and expenditure more accurately.

In recent years, energy-storage systems have become increasingly important, particularly in the context of increasing efforts to mitigate the impacts of climate change associated with the use of conventional energy sources. Renewable energy sources are an environmentally friendly source of energy, but by their very nature, they are not able to supply the required amount of energy in a uniform distribution. This study evaluated the economic efficiency of short-term electrical energy storage technology based on the principle of high-speed flywheel mechanism using vacuum with the help of an innovative approach based on life cycle cost analysis (LCC). The innovative potential of high-speed flywheel energy storage systems (FESS) can be seen in increasing the reliability of the electricity transmission system with the possibility of providing control power to compensate for residual loads caused by volatile renewable power sources and power sinks. Based on the research conducted, the LCC method was selected in this study as the most appropriate method to evaluate the economic efficiency of a high-speed FESS used to compensate for short-term fluctuations in an upgraded electric transmission system. As a result, the adjusted LCC per MWh values were compared with the average intra-hour margin realisable in the Intra-Day OTE Market, while the margin calculation also considered the efficiency of the inertial storage. Under the modelled technical and economic conditions, it was found that a high-speed FESS project that can compensate for short-term fluctuations in the electricity transmission system can be economically efficient in the Czech Republic.

Microbial communities in terrestrial geothermal systems often contain chemolithoautotrophs with well-characterized distributions and metabolic capabilities. However, the extent to which organic matter produced by these chemolithoautotrophs supports heterotrophs remains largely unknown. Here we compared the abundance and activity of peptidases and carbohydrate active enzymes (CAZymes) that are predicted to be extracellular identified in metagenomic assemblies from 63 springs in the Central American and the Andean convergent margin (Argentinian backarc of the Central Volcanic Zone), as well as the plume-influenced spreading center in Iceland. All assemblies contain two orders of magnitude more peptidases than CAZymes, suggesting that the microorganisms more often use proteins for their carbon and/or nitrogen acquisition instead of complex sugars. The CAZy families in highest abundance are GH23 and CBM50, and the most abundant peptidase families are M23 and C26, all four of which degrade peptidoglycan found in bacterial cells. This implies that the heterotrophic community relies on autochthonous dead cell biomass, rather than allochthonous plant matter, for organic material. Enzymes involved in the degradation of cyanobacterial- and algal-derived compounds are in lower abundance at every site, with volcanic sites having more enzymes degrading cyanobacterial compounds and non-volcanic sites having more enzymes degrading algal compounds. Activity assays showed that many of these enzyme classes are active in these samples. High temperature sites (> 80°C) had similar extracellular carbon-degrading enzymes regardless of their province, suggesting a less well-developed population of secondary consumers at these sites, possibly connected with the limited extent of the subsurface biosphere in these high temperature sites. We conclude that in < 80°C springs, chemolithoautotrophic production supports heterotrophs capable of degrading a wide range of organic compounds that do not vary by geological province, even though the taxonomic and respiratory repertoire of chemolithoautotrophs and heterotrophs differ greatly across these regions.

[This corrects the article DOI: 10.1371/journal.pone.0226089.].

Introduction This paper describes how mortality among hospitalised COVID-19 patients changed during the first three waves of the epidemic in Italy. Methods This 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. Results Of 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). Conclusions Hospitalisation 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.

Objectives Unintentional injuries such as falls, are particularly frequent in early childhood. To date, epidemiological studies in this field have been carried out using routine data sources or registries and many studies were observational studies with a cross-sectional design. The aims of the study are to describe unintentional injuries in the first two years of life in the Piccolipiù birth cohort, and to investigate the association between mother and children characteristics and the First Event of Raised surface Fall (FERF). Methods This longitudinal observational study included 3038 children from an Italian birth cohort. Data on socio-demographic factors, socio-economic indicators, maternal health and lifestyle characteristics and child’s sleeping behavior, obtained from questionnaires completed at birth, 12 and 24 months of age, were considered in the analyses as potential risk factors of FERF. Time of occurrence of FERF was analyzed using the Kaplan-Meier method. The multivariable analysis for time to event was carried out using a Cox proportional hazards model. Results Falls from raised surfaces are the leading cause of unintentional injuries in the cohort with 610 (21.1%) and 577 (20.0%) cases among children during the first and second year of life, respectively. An increased risk of FERF was associated with several risk factors: maternal psychological distress (HR 1.41, 95%CI 1.10–1.81), maternal alcohol intake (HR 1.26, 95%CI 1.10–1.45), and child’s sleeping problems (HR 1.28, 95%CI 1.09–1.51). Children with older aged mothers (HR 0.98, 95%CI 0.96–0.99) and living in northern Italy (HR 0.64, 95%CI 0.55–0.75) had a lower risk of FERF. Conclusion The results of the study suggest that a higher risk of FERF is associated with socio-demographic factors, maternal characteristics and child sleeping behavior that could hinder parent empowerment.

SPATS1 (spermatogenesis-associated, serine-rich 1) is an evolutionarily conserved, testis-specific protein that is differentially expressed during rat male meiotic prophase. Some reports have suggested a link between SPATS1 underexpression/mutation and human pathologies such as male infertility and testicular cancer. Given the absence of functional studies, we generated a Spats1 loss-of-function mouse model using CRISPR/Cas9 technology. The phenotypic analysis showed no overt phenotype in Spats1-/- mice, with both males and females being fertile. Flow cytometry and histological analyses did not show differences in the testicular content and histology between WT and knockout mice. Moreover, no significant differences in sperm concentration, motility, and morphology, were observed between WT and KO mice. These results were obtained both for young adults and for aged animals. Besides, although an involvement of SPATS1 in the Wnt signaling pathway has been suggested, we did not detect changes in the expression levels of typical Wnt pathway-target genes in mutant individuals. Thus, albeit Spats1 alteration might be a risk factor for male testicular health, we hereby show that this gene is not individually essential for male fertility and spermatogenesis in mouse.

There is a growing need to understand how species respond to habitat changes and the potential key role played by natal dispersal in population dynamics, structure and gene flow. However, few studies have explored differences in this process between conspecifics living in natural habitats and those inhabiting landscapes highly transformed by humans, such as cities. Here, we investigate how individual traits and social characteristics can influence the natal dispersal decisions of burrowing owls (Athene cunicularia) living in urban and rural areas, as well as the consequences in terms of reproductive success and apparent survival. We found short dispersal movements among individuals, with differences between urban and rural birds (i.e., the former covering shorter distances than the latter), maybe because of the higher conspecific density of urban compared to rural areas. Moreover, we found that urban and rural females as well as bold individuals (i.e., individuals with shorter flight initiation distance) exhibited longer dispersal distances than their counterparts. These dispersal decisions have effects on individual fitness. Individuals traveling longer distances increased their reproductive prospects (productivity during the first breeding attempt, and long term productivity). However, the apparent survival of females decreased when they dispersed farther from their natal territory. Although further research is needed to properly understand the ecological and evolutionary consequences of dispersal patterns in transformed habitats, our results provide information about the drivers and the consequences of the restricted natal movements of this species, which may explain its population structuring through restricted gene flow between and within urban and rural areas.

INTRODUCTION:Lysosomal storage diseases (LSDs) are rare inherited metabolic diseases characterized by an abnormal accumulation of various toxic materials in the cells as a result of enzyme deficiencies leading to tissue and organ damage. Among clinical manifestations, cardiac diseases are particularly important in Pompe glycogen storage diseases (PD), in glycosphingolipidosis Fabry disease (FD), and mucopolysaccharidoses (MPS). Here, we evaluated the occurrence of aortopathy in knock out (KO) mouse models of three different LSDs, including PD, FD, and MPS IIIB. METHODS:We measured the aortic diameters in 15 KO male mice, 5 for each LSD: 5 GLA-/- mice for FD, 5 NAGLU-/- mice for MPS IIIB, 5 GAA-/- mice for PD, and 15 wild type (WT) mice: 5 for each strain. In order to compare the aortic parameters between KO and WT mice deriving from the same colonies, different diameters were echocardiographically measured: aortic annulus, aortic sinus, sino-tubular junction, ascending aorta, aortic arch and descending aorta. Storage material content and aortic defects of the KO mice were also analyzed by histology, when available. RESULTS:Compared to their correspondent WT mice: GAA-/- mice showed greater diameters of ascending aorta (1.61mm vs. 1.11mm, p-value = 0.01) and descending aorta (1.17mm vs 1.02mm, p-value 0.04); GLA-/- mice showed greater diameters of aortic annulus (1.35mm vs. 1.22mm, p-value = 0.01), sinus of Valsalva (1.6mm vs. 1.38mm, p-value

In virtual reality, visual speed is usually underestimated relative to locomotor speed. Here we investigated how physical activity and fitness affect perceived visual speed when running in a treadmill-mediated virtual environment. Thirty healthy participants (ten sedentary individuals, ten team sport players and ten expert runners) ran on a treadmill at two different speeds (8, 12km/h) in front of a moving virtual scene. Participants were asked to match the speed of the visual scene to their running speed (i.e. treadmill speed), indicating for each trial whether the scene was moving slower or faster than the treadmill. The speed of the visual scene was adjusted according to the participant's response using a staircase until visual and running speeds were perceived as equivalent. More sedentary participants underestimated visual speed relative to their actual running speed. Specifically, visual speed had to exceed running speed to be perceived as equivalent. The underestimation of visual speed was speed-dependent, and it was significantly larger for sedentary participants than for team sports players and expert runners. The volume of physical activity per week was found to be the best predictor of visual speed perception for both running speeds, while the perceived effort constituted a good predictor only at 8km/h. Physical fitness, on the other hand turned out to be a poor predictor of visual speed perception. Therefore, in order to enhance users' engagement and their adherence to physical activity programs, the development of "personalized" treadmill-mediated virtual environments should take into account users' personal characteristics to provide the most natural and engaging feedback possible.

Fetida Cave is an active sulfuric acid cave influenced by seawater, showing abundant microbial communities that organize themselves under three main different morphologies: water filaments, vermiculations and moonmilk deposits. These biofilms/deposits have different cave distribution, pH, macro- and microelement and mineralogical composition, carbon and nitrogen content. In particular, water filaments and vermiculations had circumneutral and slightly acidic pH, respectively, both had abundant organic carbon and high microbial diversity. They were rich in macro- and microelements, deriving from mineral dissolution, and, in the case of water filaments, from seawater composition. Vermiculations had different color, partly associated with their mineralogy, and unusual minerals probably due to trapping capacities. Moonmilk was composed of gypsum, poor in organic matter, had an extremely low pH (0-1) and low microbial diversity. Based on 16S rRNA gene analysis, the microbial composition of the biofilms/deposits included autotrophic taxa associated with sulfur and nitrogen cycles and biomineralization processes. In particular, water filaments communities were characterized by bacterial taxa involved in sulfur oxidation and reduction in aquatic, aphotic, microaerophilic/anoxic environments (Campylobacterales, Thiotrichales, Arenicellales, Desulfobacterales, Desulforomonadales) and in chemolithotrophy in marine habitats (Oceanospirillales, Chromatiales). Their biodiversity was linked to the morphology of the water filaments and their collection site. Microbial communities within vermiculations were partly related to their color and showed high abundance of unclassified Betaproteobacteria and sulfur-oxidizing Hydrogenophilales (including Sulfuriferula), and Acidiferrobacterales (including Sulfurifustis), sulfur-reducing Desulfurellales, and ammonia-oxidizing Planctomycetes and Nitrospirae. The microbial community associated with gypsum moonmilk showed the strong dominance (>60%) of the archaeal genus Thermoplasma and lower abundance of chemolithotrophic Acidithiobacillus, metal-oxidizing Metallibacterium, Sulfobacillus, and Acidibacillus. This study describes the geomicrobiology of water filaments, vermiculations and gypsum moonmilk from Fetida Cave, providing insights into the microbial taxa that characterize each morphology and contribute to biogeochemical cycles and speleogenesis of this peculiar seawater-influenced sulfuric acid cave.

We apply mixed logit regression to investigate patients' choice of non-emergency outpatient cardiovascular specialists in Tuscany, Italy. We focused on the effects of travel time and waiting time. Results reveal that patients prefer clinics nearby and with shorter waiting times. Differences in patient choice depend on age and socioeconomic conditions, thus confirming equity concerns in the access of non-acute services. Our results could be used to optimize the allocation of resources, reduce inequities and increase the efficiency and responsiveness of outpatient systems considering patient preferences.

Robust preclinical testing is essential to predict clinical safety and efficacy and provide data to determine safe dose for first-in-man studies. There are a growing number of examples where the preclinical development of drugs failed to adequately predict clinical adverse events in part due to their assessment with inappropriate preclinical models. Preclinical investigations of T cell receptor (TCR)-based immunotherapies prove particularly challenging as these biologics are human-specific and thus the conventional testing in animal models is inadequate. As these molecules harness the full force of the immune system, and demonstrate tremendous potency, we set out to design a preclinical package that would ensure adequate evaluation of these therapeutics. Immune Mobilising Monoclonal TCR Against Cancer (ImmTAC) molecules are bi-specific biologics formed of an affinity-enhanced TCR fused to an anti-CD3 effector function. ImmTAC molecules are designed to activate human T lymphocytes and target peptides within the context of a human leukocyte antigen (HLA), thus require an intact human immune system and peptidome for suitable preclinical screening. Here we draw upon the preclinical testing of four ImmTAC molecules, including IMCgp100, the first ImmTAC molecule to reach the clinic, to present our comprehensive, informative and robust approach to in vitro preclinical efficacy and safety screening. This package comprises a broad range of cellular and molecular assays using human tissues and cultured cells to test efficacy, safety and specificity, and hence predict human responses in clinical trials. We propose that this entirely in vitro package offers a potential model to be applied to screening other TCR-based biologics.

We investigated how visual and kinaesthetic/efferent information is integrated for speed perception in running. Twelve moderately trained to trained subjects ran on a treadmill at three different speeds (8, 10, 12 km/h) in front of a moving virtual scene. They were asked to match the visual speed of the scene to their running speed-i.e., treadmill's speed. For each trial, participants indicated whether the scene was moving slower or faster than they were running. Visual speed was adjusted according to their response using a staircase until the Point of Subjective Equality (PSE) was reached, i.e., until visual and running speed were perceived as equivalent. For all three running speeds, participants systematically underestimated the visual speed relative to their actual running speed. Indeed, the speed of the visual scene had to exceed the actual running speed in order to be perceived as equivalent to the treadmill speed. The underestimation of visual speed was speed-dependent, and percentage of underestimation relative to running speed ranged from 15% at 8km/h to 31% at 12km/h. We suggest that this fact should be taken into consideration to improve the design of attractive treadmill-mediated virtual environments enhancing engagement into physical activity for healthier lifestyles and disease prevention and care.

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor used in first-line combination antiretroviral therapy (cART). It is usually administered with nucleoside reverse transcriptase inhibitors (NRTI), many of which are substrates of OCT uptake solute carriers (SLC22A) and MATE (SLC47A), P-gp (MDR1, ABCB1), BCRP (ABCG2), or MRP2 (ABCC2) efflux transporters. The aim of this study was to evaluate the inhibitory potential of efavirenz towards these transporters and investigate its effects on the pharmacokinetics and tissue distribution of a known Oct/Mate substrate, lamivudine, in rats. Accumulation and transport assays showed that efavirenz inhibits the uptake of metformin by OCT1-, OCT2- and MATE1-expressing MDCK cells and reduces transcellular transport of lamivudine across OCT1/OCT2- and MATE1-expressing MDCK monolayers. Only negligible inhibition of MATE2-K was observed in HEK-MATE2-K cells. Efavirenz also reduced the efflux of calcein from MDCK-MRP2 cells, but had a rather weak inhibitory effect on Hoechst 33342 accumulation in MDCK-MDR1 and MDCK-BCRP cells. An in vivo pharmacokinetic interaction study in male Wistar rats revealed that intravenous injection of efavirenz or the control Oct/Mate inhibitor cimetidine significantly reduced the recovery of lamivudine in urine and greatly increased lamivudine retention in the renal tissue. Co-administration with efavirenz or cimetidine also increased the AUC0-∞ value and reduced total body clearance of lamivudine. These data suggest that efavirenz is a potent inhibitor of OCT/Oct and MATE/Mate transporters. Consequently, it can engage in drug-drug interactions that reduce renal excretion of co-administered substrates and enhance their retention in the kidneys, potentially compromising therapeutic safety.

High lipid content of oocytes and embryos in domestic animals is one of the well-known factors associated with poor cryosurvival. Herein, we wanted to determine whether the use of delipidated estrous sheep serum during in vitro maturation (IVM) of ovine oocytes reduces the cytoplasmic lipid droplets content and improves embryo development and cryotolerance after vitrification. Cumulus oocytes complexes (COCs) were matured in vitro for 24 h in medium supplemented with whole or delipidated estrous sheep serum prior to vitrification. Neutral lipid present in lipid droplets of COCs, cleavage rate, embryo development rate on Day 6 and Day 8, and hatching rate on Day 8, were compared among experimental groups. Endoplasmic reticulum stress genes were evaluated in in vitro matured COCs under different lipid conditions prior to vitrification. The lipid droplets' content (mean fluorescence intensity) of oocytes cultured with IVM media supplemented with delipidated serum was lower than COCs matured with whole serum (7.6 ± 1.7 vs. 22.8 ± 5.0 arbitrary units, respectively; P< 0.05). Despite IVM treatment, oocytes subjected to vitrification showed impaired competence compared with the non-vitrified groups (P

PURPOSE:To define normal values of the cardiac time intervals obtained by tissue Doppler imaging (TDI) M-mode through the mitral valve (MV). Furthermore, to evaluate the association of the myocardial performance index (MPI) obtained by TDI M-mode (MPITDI) and the conventional method of obtaining MPI (MPIConv), with established echocardiographic and invasive measures of systolic and diastolic function. METHODS:In a large community based population study (n = 974), where all are free of any cardiovascular disease and cardiovascular risk factors, cardiac time intervals, including isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), and ejection time (ET) were obtained by TDI M-mode through the MV. IVCT/ET, IVRT/ET and the MPI ((IVRT+IVCT)/ET) were calculated. We also included a validation population (n = 44) of patients who underwent left heart catheterization and had the MPITDI and MPIConv measured. RESULTS:IVRT, IVRT/ET and MPI all increased significantly with increasing age in both genders (p

It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

Traffic is often acknowledged as a threat to biodiversity, but its effects have been mostly studied on roads subjected to high traffic intensity. The impact of lower traffic intensity such as those affecting protected areas is generally neglected, but conservation-oriented activities entailing motorized traffic could paradoxically transform suitable habitats into ecological traps. Here we questioned whether roadside-nesting bee-eaters Merops apiaster perceived low traffic intensity as a stressor eliciting risk-avoidance behaviors (alarm calls and flock flushes) and reducing parental care. Comparisons were established within Doñana National Park (Spain), between birds exposed to either negligible traffic (ca. 0-10 vehicles per day) or low traffic intensity (ca. 10-90 vehicles per day) associated to management and research activities. The frequencies of alarm calls and flock flushes were greater in areas of higher traffic intensity, which resulted in direct mortality at moderate vehicle speeds (≤ 40 km/h). Parental feeding rates paralleled changes in traffic intensity, but contrary to our predictions. Indeed, feeding rates were highest in traffic-exposed nests, during working days and traffic rush-hours. Traffic-avoidance responses were systematic and likely involved costs (energy expenditure and mortality), but vehicle transit positively influenced the reproductive performance of bee-eaters through an increase of nestling feeding rates. Because the expected outcome of traffic on individual performance can be opposed when responses are monitored during mating (i.e. negative effect by increase of alarm calls and flock flushes) or nestling-feeding period (i.e. at least short-term positive effect by increase of nestling feeding rates), caution should be taken before inferring fitness consequences only from isolated behaviors or specific life history stages.

Titanium dioxide (TiO2) nanofibres are a novel fibrous nanomaterial with increasing applications in a variety of fields. While the biological effects of TiO2 nanoparticles have been extensively studied, the toxicological characterization of TiO2 nanofibres is far from being complete. In this study, we evaluated the toxicity of commercially available anatase TiO2 nanofibres using TiO2 nanoparticles (NP) and crocidolite asbestos as non-fibrous or fibrous benchmark materials. The evaluated endpoints were cell viability, haemolysis, macrophage activation, trans-epithelial electrical resistance (an indicator of the epithelial barrier competence), ROS production and oxidative stress as well as the morphology of exposed cells. The results showed that TiO2 nanofibres caused a cell-specific, dose-dependent decrease of cell viability, with larger effects on alveolar epithelial cells than on macrophages. The observed effects were comparable to those of crocidolite, while TiO2 NP did not decrease cell viability. TiO2 nanofibres were also found endowed with a marked haemolytic activity, at levels significantly higher than those observed with TiO2 nanoparticles or crocidolite. Moreover, TiO2 nanofibres and crocidolite, but not TiO2 nanoparticles, caused a significant decrease of the trans-epithelial electrical resistance of airway cell monolayers. SEM images demonstrated that the interaction with nanofibres and crocidolite caused cell shape perturbation with the longest fibres incompletely or not phagocytosed. The expression of several pro-inflammatory markers, such as NO production and the induction of Nos2 and Ptgs2, was significantly increased by TiO2 nanofibres, as well as by TiO2 nanoparticles and crocidolite. This study indicates that TiO2 nanofibres had significant toxic effects and, for most endpoints with the exception of pro-inflammatory changes, are more bio-active than TiO2 nanoparticles, showing the relevance of shape in determining the toxicity of nanomaterials. Given that several toxic effects of TiO2 nanofibres appear comparable to those observed with crocidolite, the possibility that they exert length dependent toxicity in vivo seems worthy of further investigation.

The reduction of insulin-like growth factor 1 (IGF-1) plasma levels is associated with the degree of liver dysfunction and mortality in cirrhotic patients. However, little research is available on the recovery of the IGF-1 level and its prognostic role after liver transplantation (LT).From April 2010 to May 2011, 31 patients were prospectively enrolled (25/6 M/F; mean age±SEM: 55.2±1.4 years), and IGF-1 serum levels were assessed preoperatively and at 15, 30, 90, 180 and 365 days after transplantation. The influence of the donor and recipient characteristics (age, use of extended criteria donor grafts, D-MELD and incidence of early allograft dysfunction) on hormonal concentration was analyzed. The prognostic role of IGF-1 level on patient survival and its correlation with routine liver function tests were also investigated.All patients showed low preoperative IGF-1 levels (mean±SEM: 29.5±2.1), and on postoperative day 15, a significant increase in the IGF-1 plasma level was observed (102.7±11.7 ng/ml; p65 years) or extended criteria donor grafts. An inverse correlation between IGF-1 and bilirubin serum levels at day 15 (r = -0.3924, p = 0.0320) and 30 (r = -0.3894, p = 0.0368) was found. After multivariate analysis, early (within 15 days) IGF-1 normalization [Exp(b) = 3.913; p = 0.0484] was the only prognostic factor associated with an increased 3-year survival rate.IGF-1 postoperative levels are correlated with the graft's quality and reflect liver function. Early IGF-1 recovery is associated with a higher 3-year survival rate after LT.

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.

AIM:To report a single-centre experience with the novel Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) technique and systematically review the related literature. METHODS:Since January 2013, patients with extended primary or secondary liver tumors whose future liver remnant (FLR) was considered too small to allow hepatic resection were prospectively assessed for the ALPPS procedure. A systematic literature search was performed using PubMed, Scopus and the Cochrane Library Central. RESULTS:Until July 2014 ALPPS was completed in 9 patients whose mean age was 60 ± 8 years. Indications for surgical resection were metastases from colorectal cancer in 3 cases, perihilar cholangiocarcinoma in 3 cases, intrahepatic cholangiocarcinoma in 2 cases and hepatocellular carcinoma without chronic liver disease in 1 case. The calculated FLR volume was 289 ± 122 mL (21.1 ± 5.5%) before ALPPS-1 and 528 ± 121 mL (32.2 ± 5.7%) before ALLPS-2 (p < 0.001). The increase in FLR between the two procedures was 96 ± 47% (range: 24-160%, p < 0.001). Additional interventions were performed in 4 cases: 3 patients underwent Roux-en-Y hepaticojejunostomy, and one case underwent wedge resection of a residual tumor in the FLR. The average time between the first and second step of the procedure was 10.8 ± 2.9 days. The average hospital stay was 24.1 ± 13.3 days. There was 1 postoperative death due to hepatic failure in the oldest patient of this series who had a perihilar cholangiocarcinoma and concomitant liver fibrosis; 11 complications occurred in 6 patients, 4 of whom had grade III or above disease. After a mean follow-up of 17.1 ± 8.5 months, the overall survival was 89% at 3-6 and 12 months. The recurrence-free survival was 100%, 87.5% and 75% at 3-6-12 months respectively. The literature search yielded 148 articles, of which 22 articles published between 2012 and 2015 were included in this systematic review. CONCLUSION:The ALPPS technique effectively increased the resectability of otherwise inoperable liver tumors. The postoperative morbidity in our series was high in accordance with the data from the systematic review. Age, liver fibrosis and presence of biliary stenting were predisposing factors for postoperative morbidity and mortality.

Mutations in the lamin A/C gene (LMNA) were associated with dilated cardiomyopathy (DCM) and, recently, were related to severe forms of arrhythmogenic right ventricular cardiomyopathy (ARVC). Both genetic and phenotypic overlap between DCM and ARVC was observed; molecular pathomechanisms leading to the cardiac phenotypes caused by LMNA mutations are not yet fully elucidated. This study involved a large Italian family, spanning 4 generations, with arrhythmogenic cardiomyopathy of different phenotypes, including ARVC, DCM, system conduction defects, ventricular arrhythmias, and sudden cardiac death. Mutation screening of LMNA and ARVC-related genes PKP2, DSP, DSG2, DSC2, JUP, and CTNNA3 was performed. We identified a novel heterozygous mutation (c.418_438dup) in LMNA gene exon 2, occurring in a highly conserved protein domain across several species. This newly identified variant was not found in 250 ethnically-matched control subjects. Genotype-phenotype correlation studies suggested a co-segregation of the LMNA mutation with the disease phenotype and an incomplete and age-related penetrance. Based on clinical, pedigree, and molecular genetic data, this mutation was considered likely disease-causing. To clarify its potential pathophysiologic impact, functional characterization of this LMNA mutant was performed in cultured cardiomyocytes expressing EGFP-tagged wild-type and mutated LMNA constructs, and indicated an increased nuclear envelope fragility, leading to stress-induced apoptosis as the main pathogenetic mechanism. This study further expands the role of the LMNA gene in the pathogenesis of cardiac laminopathies, suggesting that LMNA should be included in mutation screening of patients with suspected arrhythmogenic cardiomyopathy, particularly when they have ECG evidence for conduction defects. The combination of clinical, genetic, and functional data contribute insights into the pathogenesis of this form of life-threatening arrhythmogenic cardiac laminopathy.

The Xpert® MTB/RIF (Xpert) is an automated molecular test for simultaneous detection of tuberculosis (TB) and rifampicin resistance, recommended by the World Health Organization as the preferred diagnostic method for individuals presumed to have multi-drug resistant TB (MDR-TB) or HIV-associated TB. We describe the performance of Xpert and key lessons learned during two years of implementation under routine conditions in 33 projects located in 18 countries supported by Médecins Sans Frontières across varied geographic, epidemiological and clinical settings.Xpert was used following three strategies: the first being as the initial test, with microscopy in parallel, for all presumptive TB cases; the second being only for patients at risk of MDR-TB, or with HIV- associated TB, or presumptive paediatric TB; and the third being as the initial test for these high-risk patients plus as an add-on test to microscopy in others. Routine laboratory data were collected, using laboratory registers. Qualitative data such as logistic aspects, human resources, and tool acceptance were collected using a questionnaire.In total, 52,863 samples underwent Xpert testing from April 2011 to December 2012. The average MTB detection rate was 18.5%, 22.3%, and 11.6% for the three different strategies respectively. Analysis of the results on samples tested in parallel showed that using Xpert as add-on test to microscopy would have increased laboratory TB confirmation by 49.7%, versus 42.3% for Xpert replacing microscopy. The main limitation of the test was the high rate of inconclusive results, which correlated with factors such as defective modules, cartridge version (G3 vs. G4) and staff experience. Operational and logistical hurdles included infrastructure renovation, basic computer training, regular instrument troubleshooting and maintenance, all of which required substantial and continuous support.The implementation of Xpert was feasible and significantly increased TB detection compared to microscopy, despite the high rate of inconclusive results. Xpert implementation was accompanied by considerable operational and logistical challenges. To further decentralize diagnosis, simpler, low-cost TB technologies well-suited to low-resource settings are still urgently needed.

The essential role of autophagy in muscle homeostasis has been clearly demonstrated by phenotype analysis of mice with muscle-specific inactivation of genes encoding autophagy-related proteins. Ambra1 is a key component of the Beclin 1 complex and, in zebrafish, it is encoded by two paralogous genes, ambra1a and ambra1b, both required for normal embryogenesis and larval development. In this study we focused on the function of Ambra1, a positive regulator of the autophagic process, during skeletal muscle development by means of morpholino (MO)-mediated knockdown and compared the phenotype of zebrafish Ambra1-depleted embryos with that of Ambra1gt/gt mouse embryos. Morphological analysis of zebrafish morphant embryos revealed that silencing of ambra1 impairs locomotor activity and muscle development, as well as myoD1 expression. Skeletal muscles in ATG-morphant embryos displayed severe histopathological changes and contained only small areas of organized myofibrils that were widely dispersed throughout the cell. Double knockdown of ambra1a and ambra1b resulted in a more severe phenotype whereas defects were much less evident in splice-morphants. The morphants phenotypes were effectively rescued by co-injection with human AMBRA1 mRNA. Together, these results indicate that ambra1a and ambra1b are required for the correct development and morphogenesis of skeletal muscle.

Invasive species can take advantage of resources unexploited by natives (opportunism hypothesis) or they can exploit the same resources but more aggressively or efficiently (competition hypothesis), thus impacting native species. However, invasive species tend to exploit anthropogenic habitats that are inefficiently used by natives such as urban environments. Focusing on the ring-necked parakeet (Psittacula krameri), one of the most invasive birds worldwide, we combined observations of interspecific aggressions, species-specific cavity-nest preferences and the spatial distribution of the native cavity-nesting vertebrate community to determine the invasion process as well as its potential impacts on native species in a Mediterranean city. Our results support the competition hypothesis, suggesting that ring-necked parakeets are outcompeting native species sharing nest-site preferences. Parakeets initiated and won most interspecific aggressions, which were directed towards competitors but also towards predators. This behaviour could explain the spatial arrangement of natives, with most bird species breeding close to parakeets possibly to take advantage of their effective antipredatory behaviour. However, temporal and spatial patterns of segregation suggest that a threatened bat species is negatively affected by parakeets. This demonstrates that common species gain benefits and threatened ones (in this study, a bat and possibly a falcon) lose nest sites due to invaders. Therefore, the conservation status of the native species that pay the costs of competition with invaders should be considered. This scenario of winners and losers may, however, shift towards more losers if the ring-necked parakeet population continues to grow, thus requiring close monitoring and control/eradication programs to avoid further impacts.

The dominant criterion to determine when an introduced species is established relies on the maintenance of a self-sustaining population in the area of introduction, i.e. on the viability of the population from a demographic perspective. There is however a paucity of demographic studies on introduced species, and establishment success is thus generally determined by expert opinion without undertaking population viability analyses (PVAs). By means of an intensive five year capture-recapture monitoring program (involving >12,000 marked individuals) we studied the demography of five introduced passerine bird species in southern Spain which are established and have undergone a fast expansion over the last decades. We obtained useful estimates of demographic parameters (survival and reproduction) for one colonial species (Ploceus melanocephalus), confirming the long-term viability of its local population through PVAs. However, extremely low recapture rates prevented the estimation of survival parameters and population growth rates for widely distributed species with low local densities (Estrilda troglodytes and Amandava amandava) but also for highly abundant yet non-colonial species (Estrilda astrild and Euplectes afer). Therefore, determining the establishment success of introduced passerine species by demographic criteria alone may often be troublesome even when devoting much effort to field-work. Alternative quantitative methodologies such as the analysis of spatio-temporal species distributions complemented with expert opinion deserve thus their role in the assessment of establishment success of introduced species when estimates of demographic parameters are difficult to obtain, as is generally the case for non-colonial, highly mobile passerines.

Urbanization causes widespread endangerment of biodiversity worldwide. However, some species successfully colonize cities reaching higher densities than in their rural habitats. In these cases, although urban city dwellers may apparently be taking advantage of these new environments, they also face new ecological conditions that may induce behavioural changes. For example, the frequency of alternative reproductive behaviours such as extra-pair paternity and intraspecific brood parasitism might increase with breeding densities. Here, using a panel of 17 microsatellites, we tested whether increments in breeding densities such as those associated with urban invasion processes alter genetic monogamy in the burrowing owl Athene cunicularia. Our results show low rates of extra-pair paternity (1.47%), but relatively high levels of intraspecific brood parasitism (8.82%). However, we were not able to detect differences in the frequency at which either alternative reproductive behaviour occurs along a strong breeding density gradient. Further research is needed to properly ascertain the role of other social and ecological factors in the frequency at which this species presents alternative reproductive strategies. Meanwhile, our results suggest that genetic monogamy is maintained despite the increment in conspecific density associated with a recent urban invasion process.

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED(99) of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.

UNLABELLED: Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo.

After the quasi-extinction of much of the European vertebrate megafauna during the last few centuries, many reintroduction projects seek to restore decimated populations. However, the future of numerous species depends on the management scenarios of metapopulations where the flow of individuals can be critical to ensure their viability. This is the case of the bearded vulture Gypaetus barbatus, an Old World, large body-sized and long-lived scavenger living in mountain ranges. Although persecution in Western Europe restrained it to the Pyrenees, the species is nowadays present in other mountains thanks to reintroduction projects. We examined the movement patterns of pre-adult non-breeding individuals born in the wild population of the Pyrenees (n = 9) and in the reintroduced populations of the Alps (n = 24) and Andalusia (n = 13). Most birds were equipped with GPS-GSM radio transmitters, which allowed accurate determination of individual dispersal patterns. Two estimators were considered: i) step length (i.e., the distance travelled per day by each individual, calculated considering only successive days); and ii) total dispersal distance (i.e., the distance travelled between each mean daily location and the point of release). Both dispersal estimators showed a positive relationship with age but were also highly dependent on the source population, birds in Andalusia and Alps moving farther than in Pyrenees. Future research should confirm if differences in dispersal distances are the rule, in which case the dynamics of future populations would be strongly influenced. In summary, our findings highlight that inter-population differences can affect the flow of individuals among patches (a key aspect to ensure the viability of the European metapopulation of the endangered bearded vulture), and thus should be taken into account when planning reintroduction programs. This result also raises questions about whether similar scenarios may occur in other restoration projects of European megafauna.

Reintroduction or reinforcement (RorR) of wild populations is a common conservation strategy. Many conservation projects involve the release of individuals of poorly studied species. This may lead to inefficient results or negative impacts on the conservation efforts. Here, we provide new insights into the conservation implications and potential consequences of a skew in the sex ratio of released birds and of the number of birds supplemented for the demography of a long-lived dimorphic bird species, the Andean condor (Vulturgryphus). We demonstrate that a RorR conservation program may be less effective in conserving a species if the sex ratios of the releases and the recipient populations are not considered. We also show that releases can reduce population declines but only if carried out over long periods (i.e., several decades). This can mean high costs for release programs and the added challenge of maintaining programs over time. If RorR programs are to be implemented, bearing in mind the importance of properly assessing their effectiveness, we urge conservation researchers and managers to consider the implications of sex ratio biases for wild populations, and particularly for dimorphic species with sexually despotic behaviour.

Obesity is associated with an increased risk of cardiomyopathy, and mechanisms linking the underlying risk and dietary factors are not well understood. We tested the hypothesis that dietary intake of saturated fat increases the levels of sphingolipids, namely ceramide and sphingomyelin in cardiac cell membranes that disrupt caveolae, specialized membrane micro-domains and important for cellular signaling. C57BL/6 mice were fed two high-fat diets: palmitate diet (21% total fat, 47% is palmitate), and MCT diet (21% medium-chain triglycerides, no palmitate). We established that high-palmitate feeding for 12 weeks leads to 40% and 50% increases in ceramide and sphingomyelin, respectively, in cellular membranes. Concomitant with sphingolipid accumulation, we observed a 40% reduction in systolic contractile performance. To explore the relationship of increased sphingolipids with caveolins, we analyzed caveolin protein levels and intracellular localization in isolated cardiomyocytes. In normal cardiomyocytes, caveolin-1 and caveolin-3 co-localize at the plasma membrane and the T-tubule system. However, mice maintained on palmitate lost 80% of caveolin-3, mainly from the T-tubule system. Mice maintained on MCT diet had a 90% reduction in caveolin-1. These data show that caveolin isoforms are sensitive to the lipid environment. These data are further supported by similar findings in human cardiac tissue samples from non-obese, obese, non-obese cardiomyopathic, and obese cardiomyopathic patients. To further elucidate the contractile dysfunction associated with the loss of caveolin-3, we determined the localization of the ryanodine receptor and found lower expression and loss of the striated appearance of this protein. We suggest that palmitate-induced loss of caveolin-3 results in cardiac contractile dysfunction via a defect in calcium-induced calcium release.

Cyclin-dependent kinase inhibitors (CDKi) have high potential applicability in anticancer therapy, but various aspects of their pharmacokinetics, especially their interactions with drug efflux transporters, have not yet been evaluated in detail. Thus, we investigated interactions of five CDKi (purvalanol A, olomoucine II, roscovitine, flavopiridol and SNS-032) with the ABCB1 transporter. Four of the compounds inhibited efflux of two ABCB1 substrates, Hoechst 33342 and daunorubicin, in MDCKII-ABCB1 cells: Olomoucine II most strongly, followed by roscovitine, purvalanol A, and flavopiridol. SNS-032 inhibited ABCB1-mediated efflux of Hoechst 33342 but not daunorubicin. In addition, purvalanol A, SNS-032 and flavopiridol lowered the stimulated ATPase activity in ABCB1 membrane preparations, while olomoucine II and roscovitine not only inhibited the stimulated ATPase but also significantly activated the basal ABCB1 ATPase, suggesting that these two CDKi are ABCB1 substrates. We further revealed that the strongest ABCB1 inhibitors (purvalanol A, olomoucine II and roscovitine) synergistically potentiate the antiproliferative effect of daunorubicin, a commonly used anticancer drug and ABCB1 substrate, in MDCKII-ABCB1 cells as well as in human carcinoma HCT-8 and HepG2 cells. We suggest that this pronounced synergism is at least partly caused by (i) CDKi-mediated inhibition of ABCB1 transporter leading to increased intracellular retention of daunorubicin and (ii) native cytotoxic activity of the CDKi. Our results indicate that co-administration of the tested CDKi with anticancer drugs that are ABCB1 substrates may allow significant dose reduction in the treatment of ABCB1-expressing tumors.

Age-dependent skewed sex ratios have been observed in bird populations, with adult males generally outnumbering females. This trend is mainly driven by higher female mortality, sometimes associated with anthropogenic factors. Despite the large amount of work on bird sex ratios, research examining the spatial stability of adult sex ratios is extremely scarce. The Andean condor (Vultur gryphus) is the only bird of prey with strong sexual dimorphism favouring males (males are 30% heavier than females). By examining data from most of its South-American range, we show that while the juvenile sex ratio is balanced, or even female-skewed, the sex ratio becomes increasing male-skewed with age, with adult males outnumbering females by >20%, and, in some cases by four times more. This result is consistent across regions and independent of the nature of field data. Reasons for this are unknown but it can be hypothesized that the progressive disappearance of females may be associated with mortality caused by anthropogenic factors. This idea is supported by the asymmetric habitat use by the two sexes, with females scavenging in more humanized areas. Whatever the cause, male-skewed adult sex ratios imply that populations of this endangered scavenger face higher risks of extinction than previously believed.