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We take up the challenge of developing an international network with capacity to survey the world's scientists on an ongoing basis, providing rich datasets regarding the opinions of scientists and scientific sub-communities, both at a time and also over time. The novel methodology employed sees local coordinators, at each institution in the network, sending survey invitation emails internally to scientists at their home institution. The emails link to a '10 second survey', where the participant is presented with a single statement to consider, and a standard five-point Likert scale. In June 2023, a group of 30 philosophers and social scientists invited 20,085 scientists across 30 institutions in 12 countries to participate, gathering 6,807 responses to the statement Science has put it beyond reasonable doubt that COVID-19 is caused by a virus. The study demonstrates that it is possible to establish a global network to quickly ascertain scientific opinion on a large international scale, with high response rate, low opt-out rate, and in a way that allows for significant (perhaps indefinite) repeatability. Measuring scientific opinion in this new way would be a valuable complement to currently available approaches, potentially informing policy decisions and public understanding across diverse fields.

An affective variant of the Stop-Signal task was used to study the interaction between emotion and response inhibition (RI) in healthy young participants. The task involved the covert presentation of emotional faces as go stimuli, as well as a manipulation of motivation and affect by inducing a negative mood through the assignment of unfair punishment. In the literature on emotion and RI, there are contrasting findings reflecting the variability in the method used to calculate the RI latency, namely the Stop-Signal Reaction Time (SSRT). In fact, previous studies found both facilitatory and detrimental effects of affective manipulations over RI. However, they did not use the most robust SSRT estimation approach, namely the integration, casting some doubts on the reliability of the inferences. For these reasons, the present research draws attention on how the effect of the emotional manipulation may be due to a biased SSRT estimation. Specifically, the focus of our study was on how the effect of emotion on the SSRT may vary according to different estimation procedures, the mean and two variants of the integration method. We predicted that the effect of the emotional manipulation in the SST would depend on the SSRT estimation method employed. Indeed, a significant effect of emotion was only found when SSRT was estimated with the mean method. We conclude that the mean method should be avoided in the study of emotion and RI because it overestimates SSRT. Rather, the integration approach should be used for future research in this field, while also factoring in information about the participants' strategy in emotional contexts that require greater effortful control and offer a challenge to self-regulation both in health and disease.

Music has been reported to facilitate motor performance. However, there is no data on the effects of different acoustic environmental stimuli on manual dexterity. The present observational study aimed at investigating the effects of background music and noise on a manual dexterity task in young, middle-aged and elderly subjects. Sixty healthy, right-handed subjects aged between 18 and 80 years were enrolled. Twenty young (mean age: 22±2 years), 20 middle-aged (mean age: 55±8 years) and 20 elderly (mean age: 72±5 years) subjects performed the Nine Hole Peg Test (NHPT) in four different acoustic environments: silence (noise < 20dBA), classical music at 60dBA, rock music at 70 dBA, and a noise stimulus at 80dBA. Performance was recorded using an optical motion capture system and retro-reflective markers (SMART DX, 400, BTS). Outcome measures included the total test time and peg-grasp, peg-transfer, peg-in-hole, hand-return, and removing phases times. Normalized jerk, mean and peak of velocity during transfer and return phases were also computed. No differences were found for NHPT phases and total times, normalized jerk, peak of velocity and mean velocity between four acoustic conditions (p>0.05). Between-group differences were found for NHPT total time, where young subjects revealed better performance than elderly (p˂0.001) and middle-aged (p˂0.001) groups. Music and noise stimuli in the considered range of intensity had no influence on the execution of a manual dexterity task in young, middle-aged and elderly subjects. These findings may have implications for working, sportive and rehabilitative activities.

BackgroundPersonal Protective Equipment (PPE) Portraits is a hybridized art and medical intervention that lessens the alienating appearance of PPE through wearable, smiling headshot pictures. During the pandemic, the use of these portraits was expanded, but Canadian initiatives offered portraits only to immediate stakeholders. PPE Portraits Canada (PPC) aimed to provide PPE portraits to any Canadian healthcare institution and surveyed healthcare workers (HCW) regarding these portraits' impact.MethodsUniversity student volunteers founded PPC via online platforms and coast-to-coast collaborations that allowed any HCW nationwide to request a free portrait via an accessible online form. PPC has gathered feedback from participating HCWs directly via an anonymous and bilingual survey.Results70% of HCWs wore their portraits "always" or "usually", 69% of HCWs "definitely would" recommend their portrait, 89.5% of HCWs found that the PPE portraits made a difference in their experiences with patients and 74% found the same for their colleagues. The pre- and post-effect of the portraits, led to a 37.5% greater likelihood that HCWs felt "connected" or "very connected" to patients/residents. For the thematic analysis, 70% or more of the comments were rated as positive, with less than 5% of comments being rated as negative.ConclusionThis model's logistical framework can be expanded beyond PPE portraits to other initiatives with limited resources, allowing them to reach and positively impact diverse populations. HCW feedback was predominantly positive. The optimal design and impact of PPE portraits on patients and HCWs should be studied further to improve portrait adoption.

Missing data is a prevalent problem that requires attention, as most data analysis techniques are unable to handle it. This is particularly critical in Multi-Label Classification (MLC), where only a few studies have investigated missing data in this application domain. MLC differs from Single-Label Classification (SLC) by allowing an instance to be associated with multiple classes. Movie classification is a didactic example since it can be "drama" and "bibliography" simultaneously. One of the most usual missing data treatment methods is data imputation, which seeks plausible values to fill in the missing ones. In this scenario, we propose a novel imputation method based on a multi-objective genetic algorithm for optimizing multiple data imputations called Multiple Imputation of Multi-label Classification data with a genetic algorithm, or simply EvoImp. We applied the proposed method in multi-label learning and evaluated its performance using six synthetic databases, considering various missing values distribution scenarios. The method was compared with other state-of-the-art imputation strategies, such as K-Means Imputation (KMI) and weighted K-Nearest Neighbors Imputation (WKNNI). The results proved that the proposed method outperformed the baseline in all the scenarios by achieving the best evaluation measures considering the Exact Match, Accuracy, and Hamming Loss. The superior results were constant in different dataset domains and sizes, demonstrating the EvoImp robustness. Thus, EvoImp represents a feasible solution to missing data treatment for multi-label learning.

Necrotic and dying cells release damage-associated molecular patterns (DAMPs) that can initiate sterile inflammatory responses in the heart. Although macrophages are essential for myocardial repair and regeneration, the effect of DAMPs on macrophage activation remains unclear. To address this gap in knowledge we studied the effect of necrotic cardiac myocyte extracts on primary peritoneal macrophage (PPM) cultures in vitro. We first performed unbiased transcriptomic profiling with RNA-sequencing of PPMs cultured for up to 72 hours in the presence and absence of: 1) necrotic cell extracts (NCEs) from necrotic cardiac myocytes in order to mimic the release of DAMPs; 2) lipopolysaccharide (LPS), which is known to polarize macrophages towards a classically activated phenotype and 3) Interleukin-4 (IL-4), which is known to promote polarization of macrophages towards an alternatively activated phenotype. NCEs provoke changes in differential gene expression (DEGs) that had considerable overlap with LPS-induced changes, suggesting that NCEs promote macrophage polarization towards a classically activated phenotype. Treating NCEs with proteinase-K abolished the effects of NCEs on macrophage activation, whereas NCE treatment with DNase and RNase did not affect macrophage activation. Stimulation of macrophage cultures with NCEs and LPS resulted in a significant increase in macrophage phagocytosis and interleukin-1β secretion, whereas treatment with IL-4 had no significant effect on phagocytosis and interleukin-1β. Taken together, our findings suggest that proteins released from necrotic cardiac myocytes are sufficient to skew the polarization of macrophages towards a classically activated phenotype.

IntroductionDuring COVID-19 pandemic, Internal Medicine Units (IMUs) accounted for about 70% of patients hospitalized. Although a large body of data has been published regarding the so-called first wave of the pandemic, little is known about the characteristics and predictors of worse outcomes of patients managed in IMUs during the second wave.MethodsWe prospectively assessed demographics, comorbidities, treatment and outcomes, including ventilation support (VS) and death, in patients admitted to our IMU for SARS-CoV-2 between October 13th, 2020 and January 21st, 2021. Clinical evolution and biochemical testing 1, 7 and 14 days after COVID-19 diagnosis were recorded.ResultsWe studied 120 patients (M/F 56/64, age 71±14.5 years) admitted to our IMU. Most of them had at least one comorbidity (80%). Patients who died were older, more frequently underweight, affected by malignant neoplasms and on statin therapy compared to patients eventually discharged. Both worse outcome groups (VS and death) presented higher neutrophils, ferritin, IL-6 and lower total proteins levels than controls. Age was significantly associated with mortality but not with VS need. The multivariate analysis showed age and gender independent association of mortality with underweight, malignancy and antibiotics use at the admission. With regard to biochemical parameters, both unfavourable outcomes were positively associated with high WBC count, neutrophils, blood urea nitrogen and low serum total proteins.ConclusionsOur study identified inflammation, underweight, malignancy and a marked catabolic state as the main predictors for worse outcomes in COVID-19 patients admitted to IMU during the so-called second wave of the pandemic.

PurposeTo compare the inter-observer variability of apparent diffusion coefficient (ADC) values of prostate lesions measured by 2D-region of interest (ROI) with and without specific measurement instruction.MethodsForty lesions in 40 patients who underwent prostate MR followed by targeted prostate biopsy were evaluated. A multi-reader study (10 readers) was performed to assess the agreement of ADC values between 2D-ROI without specific instruction and 2D-ROI with specific instruction to place a 9-pixel size 2D-ROI covering the lowest ADC area. The computer script generated multiple overlapping 9-pixel 2D-ROIs within a 3D-ROI encompassing the entire lesion placed by a single reader. The lowest mean ADC values from each 2D-small-ROI were used as reference values. Inter-observer agreement was assessed using the Bland-Altman plot. Intraclass correlation coefficient (ICC) was assessed between ADC values measured by 10 readers and the computer-calculated reference values.ResultsTen lesions were benign, 6 were Gleason score 6 prostate carcinoma (PCa), and 24 were clinically significant PCa. The mean±SD ADC reference value by 9-pixel-ROI was 733 ± 186 (10-6 mm2/s). The 95% limits of agreement of ADC values among readers were better with specific instruction (±112) than those without (±205). ICC between reader-measured ADC values and computer-calculated reference values ranged from 0.736-0.949 with specific instruction and 0.349-0.919 without specific instruction.ConclusionInterobserver agreement of ADC values can be improved by indicating a measurement method (use of a specific ROI size covering the lowest ADC area).

The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods' synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.

The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.

The current COVID-19 pandemic has led the world to an unprecedented global shortage of ventilators, and its sharing has been proposed as an alternative to meet the surge. This study outlines the performance of a preformed novel interface called 'ACRA', designed to split ventilator outflow into two breathing systems. The 'ACRA' interface was built using medical use approved components. It consists of four unidirectional valves, two adjustable flow-restrictor valves placed on the inspiratory limbs of each unit, and one adjustable PEEP valve placed on the expiratory limb of the unit that would require a greater PEEP. The interface was interposed between a ventilator and two lung units (phase I), two breathing simulators (phase II) and two live pigs with heterogeneous lung conditions (phase III). The interface and ventilator adjustments tested the ability to regulate individual pressures and the resulting tidal volumes. Data were analyzed using Friedman and Wilcoxon tests test (p < 0.05). Ventilator outflow splitting, independent pressure adjustments and individual tidal volume monitoring were feasible in all phases. In all experimental measurements, dual ventilation allowed for individual and tight adjustments of the pressure, and thus volume delivered to each paired lung unit without affecting the other unit's ventilation-all the modifications performed on the ventilator equally affected both paired lung units. Although only suggested during a dire crisis, this experiment supports dual ventilation as an alternative worth to be considered.

BackgroundPatients with heart failure (HF) with recovered ejection fraction (HFrecEF) are a recently identified cohort that are phenotypically and biologically different from HFrEF and HFpEF patients. Whether there are unique phenotypes among HFrecEF patients is not known.MethodsWe studied all patients at a large medical center, who had an improvement in LVEF from ≤ 35% to ≥ 50% (LVrecEF) between January 1, 2005 and December 31, 2013. We identified a set of 11 clinical variables and then performed unsupervised clustering analyses to identify unique clinical phenotypes among patients with LVrecEF, followed by a Kaplan-Meier analysis to identify differences in survival and the proportion of LVrecEF patients who maintained an LVEF ≥ 50% during the study period.ResultsWe identified 889 patients with LVrecEF who clustered into 7 unique phenotypes ranging in size from 37 to 420 patients. Kaplan-Meier analysis demonstrated significant differences in mortality across clusters (logrank pConclusionThere is significant clinical heterogeneity among patients with LVrecEF. Clinical outcomes are distinct across phenotype clusters as defined by clinical cardiac characteristics and co-morbidities. Clustering algorithms may identify patients who are at high risk for recurrent HF, and thus be useful for guiding treatment strategies for patients with LVrecEF.

In this work a shared pressure-controlled ventilation device for two patients is considered. By the use of different valves incorporated to the circuit, the device enables the restriction of possible cross contamination and the individualization of tidal volumes, driving pressures, and positive end expiratory pressure PEEP. Possible interactions in the expiratory dynamics of different pairs of patients are evaluated in terms of the characteristic exhalatory times. These characteristic times can not be easily established using simple linear lumped element models. For this purpose, a 1D model using the Hydraulic and Mechanical libraries in Matlab Simulink was developed. In this sense, experiments accompany this study to validate the model and characterize the different valves of the circuit. Our results show that connecting two patients in parallel to a ventilator always resulted in delays of time during the exhalation. The size of this effect depends on different parameters associated with the patients, the circuit and the ventilator. The dynamics of the exhalation of both patients is determined by the ratios between patients exhalatory resistances, compliances, driving pressures and PEEPs. Adverse effects on exhalations became less noticeable when respiratory parameters of both patients were similar, flow resistances of valves added to the circuit were negligible, and when the ventilator exhalatory valve resistance was also negligible. The asymmetries of driving pressures, compliances or resistances exacerbated the possibility of auto-PEEP and the increase in relaxation times became greater in one patient than in the other. In contrast, exhalatory dynamics were less sensitive to the ratio of PEEP imposed to the patients.

Declining grip strength is an indicator of cognitive loss in older individuals but it has not been explored people younger than 65 years old. The purpose of this study was to investigate the relationship between grip strength and specific cognitive tests known to decline with mild cognitive impairment in young and middle-aged adults. Declines in cognitive performance in middle-aged adults may provide evidence that these changes occur earlier than previously reported. A cross sectional design was used to compare differences between young and middle-aged healthy adults and to investigate associations between cognitive and grip strength measures within groups. Healthy young (20-30 years old) and middle-aged (45-65 years old) adults completed five cognitive tests including the Stroop, California Verbal Learning Test, Symbol Digit Modalities Test, Trail Making Tests and the Controlled Oral Word Association Test. All participants completed right and left maximum grip strength measures. Middle-aged adults performed significantly worse on right and left grip strength and the Stroop test (p

P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.

Human Metapneumovirus (hMPV) is responsible for acute respiratory infections in humans, with clinical and epidemiological relevance in pediatric, elderly, and immunocompromised populations. These features are largely unknown in Córdoba, Argentina and in adults in general. Hence, our goal was to broadly characterize hMPV infection in patients of all ages hospitalized with acute respiratory infections in Córdoba, Argentina, including epidemiology, clinical features and genetic diversity. Nasopharyngeal secretions were obtained from 795 patients during 2011-2013, 621 patients were 0-25 years old and 174 were 26-85 years old. HMPV was assayed by RT-PCR and other respiratory viruses by indirect immunofluorescence. Local strains were identified by sequence analysis. Human Metapneumovirus was detected in 20.3% (161/795) patients, 13.1% as single infections and 7.2% in co-infections, more frequently with Respiratory Syncytial Virus. HMPV circulated during late winter and spring in all age patients, but mainly in children under 4 years old in 71.4% (115/161) and adults between 26 and 59 years old in 12.4% (20/161). The most prevalent diagnosis was mild acute respiratory infection in 59.6% (96/161) and bronchiolitis in 9.3% (15/161). Local strains were clustered within A2 subtype; they presented 73-100% identities among them, showing a high degree of homology compared to isolations from neighboring countries. We demonstrate that hMPV circulated among all age patients with respiratory infection during 2011-2013 in Córdoba, contributing to the understanding of this virus, its diagnosis and patient handling in local health-care centers.

In recent years, metabarcoding has become a key tool to describe microbial communities from natural and artificial environments. Thanks to its high throughput nature, metabarcoding efficiently explores microbial biodiversity under different conditions. It can be performed on environmental (e)DNA to describe so-called total microbial community, or from environmental (e)RNA to describe active microbial community. As opposed to total microbial communities, active ones exclude dead or dormant organisms. For what concerns Fungi, which are mostly filamentous microorganisms, the relationship between DNA-based (total) and RNA-based (active) communities is unclear. In the present study, we evaluated the consequences of performing metabarcoding on both soil and wood-extracted eDNA and eRNA to delineate molecular operational taxonomic units (MOTUs) and differentiate fungal communities according to the environment they originate from. DNA and RNA-based communities differed not only in their taxonomic composition, but also in the relative abundances of several functional guilds. From a taxonomic perspective, we showed that several higher taxa are globally more represented in either "active" or "total" microbial communities. We also observed that delineation of MOTUs based on their co-occurrence among DNA and RNA sequences highlighted differences between the studied habitats that were overlooked when all MOTUs were considered, including those identified exclusively by eDNA sequences. We conclude that metabarcoding on eRNA provides original functional information on the specific roles of several taxonomic or functional groups that would not have been revealed using eDNA alone.

Should females alter their reproductive strategy when attacked by pathogens? Two hypotheses provide opposite predictions. Terminal reproductive investment theory predicts that reproduction should increase when the risk of death increases. However, physiological trade-offs between reproduction and immune function might be expected to produce a decrease in reproduction during a robust immune response. There is evidence for both hypotheses. We examine whether age determines the effect of an immune challenge on reproductive strategy in long-winged females of the Texas field cricket, Gryllus texensis, when fed an ecologically valid (i.e. limited) diet. The limited diet reduced reproductive output. However, even under resource-limited conditions, immune challenge had no effect on the reproductive output of young or middle-aged females. Both reproductive output and immune function (lysozyme-like activity and phenoloxidase (PO) activity) increased with age, which is contrary to both hypotheses. We hypothesize that PO activity is pleiotropic and represents an investment in both reproduction and immune function. Three proPO genes (identified in a published RNA-seq dataset (transcriptome)) were expressed either in the fat body or the ovaries (supporting the hypothesis that PO is bifunctional). The possible bifunctionality of PO suggests that it may not be an appropriate immune measure for studies on immune/reproductive trade-offs. This study also suggests that the threshold for terminal reproductive investment may not decrease prior to senescence in some species.

OBJECTIVE:Fetal exposure to an intrauterine environment affected by maternal obesity and excessive gestational weight gain increases the likelihood of infants born large for gestational age and childhood obesity. This study examined behavioural factors and lifestyle practices associated with women's perceived attainability of meeting the 2009 Institute of Medicine (IOM) weight gain guidelines. METHODS:Cross-sectional data were collected from pregnant (n = 320) and postpartum (n = 1179) women who responded to the validated Canadian Electronic Maternal (EMat) health survey. Consenting women completed the survey through REDCap™ a secure, web-based data capture platform. Multiple logistic regression analyses were used to evaluate correlates associated with meeting or not meeting IOM recommendations. Odds ratios (ORs) were adjusted for relevant behavioural and sociodemographic covariates. RESULTS:There were no significant differences between adjusted and unadjusted ORs for self-efficacy, barriers, and facilitators to weight gain during pregnancy. Women who reported worry regarding weight gain were significantly less likely to meet IOM guidelines (OR = 0.48, 95% CI = 0.33-0.69). Perceived controllability of behaviour was significantly associated with meeting IOM guidelines. An internal locus of control for weight gain was associated with an increased odds of meeting guidelines when women perceived to be in control of their weight gain (OR = 1.75, 95% CI = 1.29-2.37), healthy and exercised (OR = 1.91, 95% CI = 1.34-2.71), and when no barriers to healthy weight gain were perceived (OR = 1.43, 95% CI = 1.04-1.95); whereas, an external locus of control in which women viewed weight gain as beyond their control, was associated with a significantly reduced odds of achieving guidelines (OR = 0.58, 95% CI = 0.39-0.88). CONCLUSIONS:Self-efficacy and perceived controllability of behaviour are key factors to consider when developing pregnancy-specific interventions to help women achieve guideline-concordant weight gain and ensure the downstream health of both mother and infant.

INTRODUCTION:In the WHO European Region, endemic transmission of measles and rubella had been interrupted by 37 and 42 of the 53 member states (MSs), respectively, by 2018. Sixteen MSs are still endemic for measles, 11 for rubella and nine for both diseases, the latter including Italy. Elimination is documented by each country's National Verification Committee (NVC) through an annual status update (ASU). OBJECTIVE:By analysing data used to produce the ASUs, we aimed to describe the advances made by Italy towards elimination of measles and rubella. Moreover, we propose a set of major interventions that could facilitate the elimination process. METHODS:A total of 28 indicators were identified within the six core sections of the ASU form and these were evaluated for the period 2013-2018. These indicators relate to the incidence of measles/rubella; epidemiological investigation of cases; investigation of outbreaks; performance of the surveillance system; population immunity levels; and implementation of supplemental immunization activities (SIAs). RESULTS:From 2013 to 2018, epidemiological and laboratory analyses of measles cases in Italy improved substantially, allowing timely investigation in 2017 and 2018 of most outbreak and sporadic cases and identification of the majority of genotypic variants. Moreover, since 2017, vaccination coverage has increased significantly. Despite these improvements, several areas of concern emerged, prompting the following recommendations: i) improve outbreak monitoring; ii) strengthen the MoRoNet network; iii) increase the number of SIAs; iv) reinforce vaccination services; v) maintain regional monitoring; vi) design effective communication strategies; vii) foster the role of general practitioners and family paediatricians. CONCLUSIONS:The review of national ASUs is a crucial step to provide the NVC with useful insights into the elimination process and to guide the development of targeted interventions. Against this background, the seven recommendations proposed by the NVC have been shared with the Italian Ministry of Health and the Technical Advisory Group on measles and rubella elimination and have been incorporated into the new Italian Elimination Plan 2019-2023 as a technical aid to facilitate the achievement of disease elimination goals.

Although most countries in the WHO European Region were verified in 2017 as having interrupted endemic measles transmission, nine countries were still endemic. Among these, Italy accounted for the second highest number of measles cases reported in Europe in 2017. The elimination of measles is verified at national level by each country's National Verification Committee (NVC) through the production of an Annual Status Update (ASU). Since in Italy decentralization has led to an inhomogeneous implementation of immunization strategies among the 21 administrative Regions, the Italian NVC proposed that measles elimination should also be documented at the subnational level through regional ASUs and Synthetic Regional Reports (SRRs). The regional ASUs and the SRRs for 2014, 2015 and 2016 were produced and appraised by the NVC to evaluate the Regions' performances in each individual year as well as over the whole period. A specific analysis of vaccination coverage, including official immunization data for 2017, was performed. Moreover, the measles epidemic of 2017 was examined. Firstly, in the period 2014-2016, low immunization rates were registered in most Regions. Sixty-three per cent of southern Regions reported rates below the national mean and an overall low-quality performance. The approval of Italy's mandatory vaccination law in 2017 resulted in a marked increase in vaccination coverage; however, this increase was not homogeneous among Regions. Secondly, more than 50% of Regions did not report any supplemental immunization activity (SIA) for the period 2014-2016. Thirdly, from 2014 to 2016, fewer than one-third of Regions improved their reporting of outbreaks. Finally, over the study period, only two Regions reached the target required by the WHO for measles laboratory investigations. In countries with decentralized health policies, subnational monitoring can help identify local barriers to measles elimination. In Italy it has highlighted the need for further SIAs and a stronger surveillance system.

In Sub-Saharan Africa, where burden, impact, and incidence of acute respiratory infections (ARI) are the highest in the world, conversely, the epidemiology of influenza-associated severe acute respiratory infections (SARI) is incompletely known. The aim of this study was to describe the clinical and epidemiological features of influenza-associated SARI in hospitalized children in Maputo city, Mozambique. Nasopharyngeal and oropharyngeal swabs were collected from children aged 0-14 years old who met the case definition for SARI in two hospitals in Maputo city after their parents or legal representative consented to participate. A structured questionnaire was used to collect clinical and demographic data. Typing and subtyping of influenza were performed by real-time PCR. From January 2014 to December 2016, a total of 2,007 eligible children were recruited, of whom 1,997 (99.5%) were screened for influenza by real-time PCR. The median age of participants was 16.9 months (IQR: 7.0-38.9 months) and 53.9% (1076/1991) were male. A total of 77 were positive for influenza, yielding a frequency of 3.9% (77/1,991), with the highest frequency being reported in the age group 1-5 years old. Cases of influenza peaked twice each year, during which, its frequency reached up to 60%-80%. Among all influenza confirmed cases, 33.7% (26/77), 35.1% (27/77) and 28.6% (22/77) were typed as influenza A/H3N2, A/H1N1pdm09, and B, respectively. This represents the first report of influenza in urban/sub urban setting in Mozambique and the first evidence of distribution of strains of influenza in the country. Our data showed that frequency of influenza was lower than reported in a rural setting in Mozambique and the frequency of seasonal (A/H1N1pdm09) and (A/H3N2) subtypes were similar in children with SARI.

[This corrects the article DOI: 10.1371/journal.pone.0180249.].

Animal studies have suggested that maternal weight-related factors during pregnancy can program offspring physical activity in a sex-dependent manner. However, there is limited evidence in humans. The purpose of this study was to investigate the association between maternal gestational weight gain (GWG) and offspring total physical activity (TPA) level and to determine whether these associations are moderated by sex of offspring or maternal pre-pregnancy weight status.We studied 56 boys (mean age = 3.7 years, standard deviation (SD) 0.5) and 57 girls (mean age = 3.5±0.5 years) enrolled in licensed childcare centers. TPA was objectively measured using Actical® accelerometers. Information on pre-pregnancy body mass index (BMI), GWG, and other maternal factors were collected with a maternal health questionnaire. Associations between GWG, as a continuous variable or categorically (inadequate, adequate, and excessive), and offspring TPA were analysed using linear mixed models to take into account the intraclass correlation between the clusters (childcare centers). Models were adjusted for gestational age, accelerometer weartime, socioeconomic status, and pre-pregnancy BMI status.We found a significant sex interaction (P-value = 0.009). In boys, greater GWG was associated with decreased offspring TPA (β = -3.2 counts⋅1000-1/d, 95% confidence intervals (CI) = -6.4-0.02, P-value = 0.049). In girls born to mothers categorized as overweight or obese, the association between the GWG and TPA followed an inverted U-shape curve (β for GWG squared = -0.1 counts⋅1000-1/d, 95% CI = (-0.2 --0.04), P-value = 0.005). In contrast, a U-shaped curve was found in girls born to mothers classified as lean (pre-pregnancy BMI

BACKGROUND:TNBC is an aggressive subset of breast cancer (BC) without specific target therapy. METHODS:This observational, retrospective study included 45 cases of TNBC. The aim of this study was to evaluate the expression of the AR, E-cadherin and Ki-67 in relation to histological type, time to relapse and overall survival (OS). Immunohistochemistry (IHC) was carried out on formalin-fixed paraffin-embedded tumor samples obtained from patients defined TNBC. RESULTS:The AR was positive (IHC >10%) in 26.6%. E-cadherin (CDH1) expression was considered positive if the score was ≥ 2. This expression was negative in 53.3% cases. The Ki-67 index was ≥ 20% in 37.7%. Univariate analyses showed that AR, CDH1 and Ki-67 are significantly associated with OS. Multivariate analysis showed that AR and Ki-67 expression are independent variables associated with OS. The statistical analysis showed that patients with AR negative and Ki-67 positive expression have a significant correlation with poor outcome. CONCLUSIONS:Our data suggest that the combination of AR and E-cadherin expression as well as Ki-67 status might be useful prognostic markers in TNBC. Hence, these molecular determinants could play an interesting role to classify subgroups of TNBC.

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as "CASQ1-couplonopathy".

Hypertrophic cardiomyopathy (HCM), the most common genetic heart disease, is characterized by heterogeneous phenotypic expression. Body mass index has been associated with LV mass and heart failure symptoms in HCM. The aim of our study was to investigate whether regional (trunk, appendicular, epicardial) fat distribution and extent could be related to hypertrophy severity and pattern in HCM.Cardiovascular magnetic resonance was performed in 32 subjects with echocardiography-based diagnosis of HCM (22M/10F, 57.2±12.6 years) characterized by predominant hypertrophy at the interventricular septum (IVS). Regional fat distribution was assessed by dual-energy X-ray absorptiometry.Gender differences were detected in maximum IVS thickness (M: 18.3±3.8 mm vs. F: 14.3±4 mm, p = 0.012), right ventricle (RV) systolic function (M: 61.3±6.7%; F: 67.5±6.3%, p = 0.048), indexed RV end-diastolic (M: 64.8±16.3 ml/m2; F: 50.7±15.5 ml/m2, p = 0.04) and end-systolic volumes (M: 24.3±8.3 ml/m2; F: 16.7±7.4 ml/m2, p = 0.04). After adjusting for age and gender, maximum IVS thickness was associated with truncal fat (Tr-FAT) (β = 0.43, p = 0.02), but not with either appendicular or epicardial fat. Epicardial fat resulted independently associated with NT-proBNP levels (β = 0.63, p = 0.04). Late Gadolinium Enhancement-positive subjects displayed greater maximum IVS thickness (p = 0.02), LV mass index (p = 0.015) and NT-proBNP levels (p = 0.04), but no associations with fat amount or distribution were observed.Truncal, but not appendicular or epicardial fat amount, seems to be related with maximum IVS thickness, the hallmark feature in our cohort of HCM patients. Further prospective researches are needed to assess a potential causative effect of central adiposity on HCM phenotype.

Aggressive tumors induce tumor-supporting changes in the benign parts of the prostate. One factor that has increased expression outside prostate tumors is hemoxygenase-1 (HO-1). To investigate HO-1 expression in more detail, we analyzed samples of tumor tissue and peritumoral normal prostate tissue from rats carrying cancers with different metastatic capacity, and human prostate cancer tissue samples from primary tumors and bone metastases. In rat prostate tumor samples, immunohistochemistry and quantitative RT-PCR showed that the main site of HO-1 synthesis was HO-1+ macrophages that accumulated in the tumor-bearing organ, and at the tumor-invasive front. Small metastatic tumors were considerably more effective in attracting HO-1+ macrophages than larger non-metastatic ones. In clinical samples, accumulation of HO-1+ macrophages was seen at the tumor invasive front, almost exclusively in high-grade tumors, and it correlated with the presence of bone metastases. HO-1+ macrophages, located at the tumor invasive front, were more abundant in bone metastases than in primary tumors. HO-1 expression in bone metastases was variable, and positively correlated with the expression of macrophage markers but negatively correlated with androgen receptor expression, suggesting that elevated HO-1 could be a marker for a subgroup of bone metastases. Together with another recent observation showing that selective knockout of HO-1 in macrophages reduced prostate tumor growth and metastatic capacity in animals, the results of this study suggest that extratumoral HO-1+ macrophages may have an important role in prostate cancer.

Vespula germanica is a social wasp that has become established outside its native range in many regions of the world, becoming a major pest in the invaded areas. In the present work we analyze social communication processes used by V. germanica when exploiting un-depleted food sources. For this purpose, we investigated the arrival pattern of wasps at a protein bait and evaluated whether a forager recruited conspecifics in three different situations: foragers were able to return to the nest (full communication), foragers were removed on arrival (communication impeded), or only one forager was allowed to return to the nest (local enhancement restricted). Results demonstrated the existence of recruitment in V. germanica, given that very different patterns of wasp arrivals and a higher frequency of wasp visits to the resource were observed when communication flow between experienced and naive foragers was allowed. Our findings showed that recruitment takes place at a distance from the food source, in addition to local enhancement. When both local enhancement and distant recruitment were occurring simultaneously, the pattern of wasp arrival was exponential. When recruitment occurred only distant from the feeder, the arrival pattern was linear, but the number of wasps arriving was twice as many as when neither communication nor local enhancement was allowed. Moreover, when return to the nest was impeded, wasp arrival at the bait was regular and constant, indicating that naive wasps forage individually and are not spatially aggregated. In conclusion, this is the first study to demonstrate recruitment in V. germanica at a distance from the food source by modelling wasps' arrival to a protein-based resource. In addition, the existence of correlations when communication was allowed and reflected in tandem arrivals indicates that we were not in the presence of random processes.

We tested for interactions between body mass index (BMI) and common genetic variants affecting serum urate levels, genome-wide, in up to 42569 participants. Both stratified genome-wide association (GWAS) analyses, in lean, overweight and obese individuals, and regression-type analyses in a non BMI-stratified overall sample were performed. The former did not uncover any novel locus with a major main effect, but supported modulation of effects for some known and potentially new urate loci. The latter highlighted a SNP at RBFOX3 reaching genome-wide significant level (effect size 0.014, 95% CI 0.008-0.02, Pinter= 2.6 x 10-8). Two top loci in interaction term analyses, RBFOX3 and ERO1LB-EDARADD, also displayed suggestive differences in main effect size between the lean and obese strata. All top ranking loci for urate effect differences between BMI categories were novel and most had small magnitude but opposite direction effects between strata. They include the locus RBMS1-TANK (men, Pdifflean-overweight= 4.7 x 10-8), a region that has been associated with several obesity related traits, and TSPYL5 (men, Pdifflean-overweight= 9.1 x 10-8), regulating adipocytes-produced estradiol. The top-ranking known urate loci was ABCG2, the strongest known gout risk locus, with an effect halved in obese compared to lean men (Pdifflean-obese= 2 x 10-4). Finally, pathway analysis suggested a role for N-glycan biosynthesis as a prominent urate-associated pathway in the lean stratum. These results illustrate a potentially powerful way to monitor changes occurring in obesogenic environment.

Implantation of rat prostate cancer cells into the normal rat prostate results in tumor-stimulating changes in the tumor-bearing organ, for example growth of the vasculature, an altered extracellular matrix, and influx of inflammatory cells. To investigate this response further, we compared prostate morphology and the gene expression profile of tumor-bearing normal rat prostate tissue (termed tumor-instructed/indicating normal tissue (TINT)) with that of prostate tissue from controls. Dunning rat AT-1 prostate cancer cells were injected into rat prostate and tumors were established after 10 days. As controls we used intact animals, animals injected with heat-killed AT-1 cells or cell culture medium. None of the controls showed morphological TINT-changes. A rat Illumina whole-genome expression array was used to analyze gene expression in AT-1 tumors, TINT, and in medium injected prostate tissue. We identified 423 upregulated genes and 38 downregulated genes (p

In order to grow, tumors need to induce supportive alterations in the tumor-bearing organ, by us named tumor instructed normal tissue (TINT) changes. We now examined if the nature and magnitude of these responses were related to tumor size and aggressiveness. Three different Dunning rat prostate tumor cells were implanted into the prostate of immune-competent rats; 1) fast growing and metastatic MatLyLu tumor cells 2) fast growing and poorly metastatic AT-1 tumor cells, and 3) slow growing and non-metastatic G tumor cells. All tumor types induced increases in macrophage, mast cell and vascular densities and in vascular cell-proliferation in the tumor-bearing prostate lobe compared to controls. These increases occurred in parallel with tumor growth. The most pronounced and rapid responses were seen in the prostate tissue surrounding MatLyLu tumors. They were, also when small, particularly effective in attracting macrophages and stimulating growth of not only micro-vessels but also small arteries and veins compared to the less aggressive AT-1 and G tumors. The nature and magnitude of tumor-induced changes in the tumor-bearing organ are related to tumor size but also to tumor aggressiveness. These findings, supported by previous observation in patient samples, suggest that one additional way to evaluate prostate tumor aggressiveness could be to monitor its effect on adjacent tissues.

Although significant advances in H2 photoproduction have recently been realized in fresh water algae (e.g. Chlamydomonas reinhardtii), relatively few studies have focused on H2 production and hydrogenase adaptations in marine or halophilic algae. Salt water organisms likely offer several advantages for biotechnological H2 production due to the global abundance of salt water, decreased H2 and O2 solubility in saline and hypersaline systems, and the ability of extracellular NaCl levels to influence metabolism. We screened unialgal isolates obtained from hypersaline ecosystems in the southwest United States and identified two distinct halophilic strains of the genus Tetraselmis (GSL1 and QNM1) that exhibit both robust fermentative and photo H2-production activities. The influence of salinity (3.5%, 5.5% and 7.0% w/v NaCl) on H2 production was examined during anoxic acclimation, with the greatest in vivo H2-production rates observed at 7.0% NaCl. These Tetraselmis strains maintain robust hydrogenase activity even after 24 h of anoxic acclimation and show increased hydrogenase activity relative to C. reinhardtii after extended anoxia. Transcriptional analysis of Tetraselmis GSL1 enabled sequencing of the cDNA encoding the FeFe-hydrogenase structural enzyme (HYDA) and its maturation proteins (HYDE, HYDEF and HYDG). In contrast to freshwater Chlorophyceae, the halophilic Tetraselmis GSL1 strain likely encodes a single HYDA and two copies of HYDE, one of which is fused to HYDF. Phylogenetic analyses of HYDA and concatenated HYDA, HYDE, HYDF and HYDG in Tetraselmis GSL1 fill existing knowledge gaps in the evolution of algal hydrogenases and indicate that the algal hydrogenases sequenced to date are derived from a common ancestor. This is consistent with recent hypotheses that suggest fermentative metabolism in the majority of eukaryotes is derived from a common base set of enzymes that emerged early in eukaryotic evolution with subsequent losses in some organisms.

Coffee, one of the most popular beverages in the world, contains many different physiologically active compounds with a potential impact on people's health. Despite the recent attention given to the genetic basis of its consumption, very little has been done in understanding genes influencing coffee preference among different individuals. Given its markedly bitter taste, we decided to verify if bitter receptor genes (TAS2Rs) variants affect coffee liking. In this light, 4066 people from different parts of Europe and Central Asia filled in a field questionnaire on coffee liking. They have been consequently recruited and included in the study. Eighty-eight SNPs covering the 25 TAS2R genes were selected from the available imputed ones and used to run association analysis for coffee liking. A significant association was detected with three SNP: one synonymous and two functional variants (W35S and H212R) on the TAS2R43 gene. Both variants have been shown to greatly reduce in vitro protein activity. Surprisingly the wild type allele, which corresponds to the functional form of the protein, is associated to higher liking of coffee. Since the hTAS2R43 receptor is sensible to caffeine, we verified if the detected variants produced differences in caffeine bitter perception on a subsample of people coming from the FVG cohort. We found a significant association between differences in caffeine perception and the H212R variant but not with the W35S, which suggests that the effect of the TAS2R43 gene on coffee liking is mediated by caffeine and in particular by the H212R variant. No other significant association was found with other TAS2R genes. In conclusion, the present study opens new perspectives in the understanding of coffee liking. Further studies are needed to clarify the role of the TAS2R43 gene in coffee hedonics and to identify which other genes and pathways are involved in its genetics.

Accumulation of fat at ectopic sites has been gaining attention as pivotal contributor of insulin resistance, metabolic syndrome and related cardiovascular complications. Intermuscular adipose tissue (IMAT), located between skeletal muscle bundles and beneath muscle fascia, has been linked to physical inactivity, ageing and body mass index, but little is known about its relationship with the other AT compartments, in particular with increasing age. To address this issue, erector spinae IMAT, epicardial (EAT), intraabdominal (IAAT) and abdominal subcutaneous adipose tissue (SAT) were simultaneously measured by Magnetic Resonance Imaging (MRI) and related to waist circumference measurements and age in 32 sedentary subjects without cardiovascular disease (18 men; 14 women; mean age 48.5 ± 14 years). Fasting glucose, triglycerides and HDL-cholesterol were also assessed. We observed that, after dividing individuals according to age (≤ or > 50 years), IMAT and EAT depots were significantly more expanded in older subjects (63.2 ± 8.3 years) than in the younger ones (38.4 ± 5.2 years) (p < 0.001). Overall, both IMAT and EAT showed stronger positive associations with increasing age (β = 0.63 and 0.67, respectively, p < 0.001 for both) than with waist circumference (β = 0.55 and 0.49, respectively, p < 0.01 for both) after adjusting for gender. In addition, the gender-adjusted associations of IMAT and EAT with waist circumference and IAAT were significant in individuals ≤ 50 years only (p

Hydroxytyrosol (HT), a phenolic compound mainly derived from olives, has been proposed as a nutraceutical useful in prevention or treatment of degenerative diseases. In the present study we have evaluated the ability of HT to counteract the appearance of osteoarthritis (OA) features in human chondrocytes. Pre-treatment of monolayer cultures of chondrocytes with HT was effective in preventing accumulation of reactive oxidant species (ROS), DNA damage and cell death induced by H2O2 exposure, as well as the increase in the mRNA level of pro-inflammatory, matrix-degrading and hypertrophy marker genes, such as iNOS, COX-2, MMP-13, RUNX-2 and VEGF. HT alone slightly enhanced ROS production, but did not enhance cell damage and death or the expression of OA-related genes. Moreover HT was tested in an in vitro model of OA, i.e. three-dimensional micromass cultures of chondrocytes stimulated with growth-related oncogene α (GROα), a chemokine involved in OA pathogenesis and known to promote hypertrophy and terminal differentiation of chondrocytes. In micromass constructs, HT pre-treatment inhibited the increases in caspase activity and the level of the messengers for iNOS, COX-2, MMP-13, RUNX-2 and VEGF elicited by GROα. In addition, HT significantly increased the level of SIRT-1 mRNA in the presence of GROα. In conclusion, the present study shows that HT reduces oxidative stress and damage, exerts pro-survival and anti-apoptotic actions and favourably influences the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features.

Background and objectivesHypertension represents a major cause of cardiovascular morbidity and mortality worldwide but its prevalence has been shown to vary in different countries. The reasons for such differences are still matter of debate, the relative contributions given by environmental and genetic factors being still poorly defined. We estimated the current prevalence, distribution and determinants of hypertension in isolated Sardinian populations and also investigated the environmental and genetic contribution to hypertension prevalence taking advantage of the characteristics of such populations.Methods and resultsAn epidemiological survey with cross-sectional design was carried out measuring blood pressure in 9845 inhabitants of 10 villages of Ogliastra region between 2002 and 2008. Regression analysis for assessing blood pressure determinants and variance component models for estimating heritability were performed. Overall 38.8% of this population had hypertension, its prevalence varying significantly by age, sex and among villages taking into account age and sex structure of their population. About 50% of hypertensives had prior cardiovascular disease. High blood pressure was independently associated with age, obesity related factors, heart rate, total cholesterol, alcohol consumption, low education and smoking status, all these factors contributing more in women than in men. Heritability was 27% for diastolic and 36% for systolic blood pressure, its contribution being significantly higher in men (57%) than in women (46%). Finally, the genetic correlation between systolic and diastolic blood pressure was 0.74, indicating incomplete pleiotropy.ConclusionGenetic factors involved in the expression of blood pressure traits account for about 30% of the phenotypic variance, but seem to play a larger role in men; comorbidities and environmental factors remain of predominant importance, but seem to contribute much more in women.

BackgroundBone metastasis represents an increasing clinical problem in advanced renal cell carcinoma (RCC) as disease-related survival improves. There are few data on the natural history of bone disease in RCC.Patients and methodsData on clinicopathology, survival, skeletal-related events (SREs), and bone-directed therapies for 398 deceased RCC patients (286 male, 112 female) with evidence of bone metastasis were statistically analyzed.ResultsMedian time to bone metastasis was 25 months for patients without bone metastasis at diagnosis. Median time to diagnosis of bone metastasis by MSKCC risk was 24 months for good, 5 months for intermediate, and 0 months for poor risk. Median number of SREs/patient was one, and 71% of patients experienced at least one SRE. Median times to first, second, and third SRE were 2, 5, and 12 months, respectively. Median survival was 12 months after bone metastasis diagnosis and 10 months after first SRE. Among 181 patients who received zoledronic acid (ZOL), median time to first SRE was significantly prolonged versus control (n = 186) (3 months vs 1 month for control; PConclusionsRCC patients with bone metastasis are at continuous risk of SREs, and in this survey ZOL effectively reduced this risk.

Breast cancer brain metastases (BCBM) are a challenging consequence of advanced BC. Nanoparticle agents, including liposomes, have shown enhanced delivery to solid tumors and brain. We compared pharmacokinetics (PK) and efficacy of PEGylated liposomal doxorubicin (PLD) with non-liposomal doxorubicin (NonL-doxo) in an intracranial model of BC.Athymic mice were inoculated intracerebrally with MDA-MB-231-BR-luciferase-expressing cells. Tumor-bearing mice were administered PLD or NonL-doxo at 6 mg/kg IV × 1 and were euthanized prior to and 0.083, 1, 3, 6, 24, 72 and 96 h post-treatment. Samples were processed to measure sum total doxorubicin via HPLC. PLD and NonL-doxo were administered IV weekly as single agents (6 mg/kg) or in combination (4.5 mg/kg) with the PARP inhibitor, ABT-888, PO 25 mg/kg/day. Efficacy was assessed by survival and bioluminescence.Treatment with PLD resulted in approximately 1,500-fold higher plasma and 20-fold higher intracranial tumor sum total doxorubicin AUC compared with NonL-doxo. PLD was detected at 96 h; NonL-doxo was undetectable after 24 h in plasma and tumor. Median survival of PLD-treated animals was 32 days (d, [CI] 31-38), which was significantly longer than controls (26d [CI 25-28]; p = 0.0012) or NonL-doxo treatment (23.5d [CI 18-28], p = 0.0002). Combination treatment with PLD/ABT-888 yielded improved survival compared to NonL-doxo/ABT-888 (35d [CI 31-38] versus 29.5d [CI 25-34]; p = 0.006).PLD provides both PK and efficacy advantage over NonL-doxo in the treatment of an in vivo model of BCBM. The results provide preclinical rationale to translate findings into early phase trials of PLD, with or without ABT-888, for patients with BCBM.

BACKGROUND: Caesarean delivery (CD) rates are commonly used as an indicator of quality in obstetric care and risk adjustment evaluation is recommended to assess inter-institutional variations. The aim of this study was to evaluate whether the Ten Group classification system (TGCS) can be used in case-mix adjustment. METHODS: Standardized data on 15,255 deliveries from 11 different regional centers were prospectively collected. Crude Risk Ratios of CDs were calculated for each center. Two multiple logistic regression models were herein considered by using: Model 1- maternal (age, Body Mass Index), obstetric variables (gestational age, fetal presentation, single or multiple, previous scar, parity, neonatal birth weight) and presence of risk factors; Model 2- TGCS either with or without maternal characteristics and presence of risk factors. Receiver Operating Characteristic (ROC) curves of the multivariate logistic regression analyses were used to assess the diagnostic accuracy of each model. The null hypothesis that Areas under ROC Curve (AUC) were not different from each other was verified with a Chi Square test and post hoc pairwise comparisons by using a Bonferroni correction. RESULTS: Crude evaluation of CD rates showed all centers had significantly higher Risk Ratios than the referent. Both multiple logistic regression models reduced these variations. However the two methods ranked institutions differently: model 1 and model 2 (adjusted for TGCS) identified respectively nine and eight centers with significantly higher CD rates than the referent with slightly different AUCs (0.8758 and 0.8929 respectively). In the adjusted model for TGCS and maternal characteristics/presence of risk factors, three centers had CD rates similar to the referent with the best AUC (0.9024). CONCLUSIONS: The TGCS might be considered as a reliable variable to adjust CD rates. The addition of maternal characteristics and risk factors to TGCS substantially increase the predictive discrimination of the risk adjusted model.

IGF-1 receptor (IGF-1R) signaling regulates cell growth, transformation and survival. Haploinsufficiency of the IGF-1R is reported to paradoxically confer resistance to oxidative stress in vivo and in cells cultured from Igf1r(+/-) mice. In order to determine whether IGF-1R deficiency directly confers resistance to oxidative stress in specific cell types, an siRNA-mediated approach was applied to reduce IGF-1R in C2C12 myoblasts, NIH3T3 fibroblasts and MC3T3-E1 osteoblasts. Treating the IGF-1R deficient myoblasts with H2O2 resulted in significantly higher phosphorylation of Akt as compared to cells having normal expression of IGF-1R. Similar results were obtained with UV treatment, another inducer of oxidative stress. This enhanced activation of Akt was associated with reduced level of cleaved caspase-3 and PARP. Moreover, in the IGF-1R knockdown myoblasts, phosphorylation of the Akt substrate Bad was enhanced after peroxide treatment. However, in NIH-3T3 fibroblasts and MC3T3-E1 osteoblasts, the loss of IGF-1R by siRNA directed knockdown was associated with reduced levels of phosphorylated Akt on treatment with H2O2 or UV as compared to control cells and these cells showed more apoptosis. These results suggest a novel mechanism of cell type specific differential regulation of resistance to oxidative stress induced apoptosis by reduced levels of IGF-1R.

The development of phase contrast methods for diagnostic x-ray imaging is inspired by the potential of seeing the internal structures of the human body without the need to deposit any harmful radiation. An efficient class of x-ray phase contrast imaging and scatter correction methods share the idea of using structured illumination in the form of a periodic fringe pattern created with gratings or grids. They measure the scatter and distortion of the x-ray wavefront through the attenuation and deformation of the fringe pattern via a phase stepping process. Phase stepping describes image acquisition at regular phase intervals by shifting a grating in uniform steps. However, in practical conditions the actual phase intervals can vary from step to step and also spatially. Particularly with the advent of electromagnetic phase stepping without physical movement of a grating, the phase intervals are dependent upon the focal plane of interest. We describe a demodulation algorithm for phase stepping at arbitrary and position-dependent (APD) phase intervals without assuming a priori knowledge of the phase steps. The algorithm retrospectively determines the spatial distribution of the phase intervals by a Fourier transform method. With this ability, grating-based x-ray imaging becomes more adaptable and robust for broader applications.

BACKGROUND: Obesity is characterized by a low grade chronic inflammation state. Indeed circulating pro-inflammatory cytokines, such as TNF-α and IL-6, are elevated in obese subjects, while anti-inflammatory cytokines, such as IL-10, appear to be reduced. Cytokines profile improves after weight loss, but how visceral or subcutaneous fat loss respectively affect pro- or anti-inflammatory cytokines plasma levels has not been precisely assessed. Therefore in the present study we correlated changes in circulating cytokine profile with quantitative changes in visceral and subcutaneous adipose tissue depots measured by an ad hoc Magnetic Resonance Imaging (MRI) protocol before and after weight loss. MATERIALS AND METHODS: In 14 obese subjects, MRI determination of visceral and subcutaneous fat and plasma glucose, insulin, TNF-α IL-6, and IL-10 measurements were performed before and after a caloric restriction induced weight loss of at least 5% of the original body weight. RESULTS: Weight loss improved insulin sensitivity (QUICKI Index: 0.35±0.03 vs 0.37±0.04; P

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.

The GDI1 gene encodes αGDI, which retrieves inactive GDP-bound RAB from membranes to form a cytosolic pool awaiting vesicular release. Mutations in GDI1 are responsible for X-linked Intellectual Disability. Characterization of the Gdi1-null mice has revealed alterations in the total number and distribution of hippocampal and cortical synaptic vesicles, hippocampal short-term synaptic plasticity and specific short-term memory deficits in adult mice, which are possibly caused by alterations of different synaptic vesicle recycling pathways controlled by several RAB GTPases. However, interpretation of these studies is complicated by the complete ablation of Gdi1 in all cells in the brain throughout development. In this study, we generated conditionally gene-targeted mice in which the knockout of Gdi1 is restricted to the forebrain, hippocampus, cortex and amygdala and occurs only during postnatal development. Adult mutant mice reproduce the short-term memory deficit previously reported in Gdi1-null mice. Surprisingly, the delayed ablation of Gdi1 worsens the pre-synaptic phenotype at cortico-amygdala synaptic connections compared to Gdi1-null mice. These results suggest a pivotal role of αGDI via specific RAB GTPases acting specifically in forebrain regions at the pre-synaptic sites involved in memory formation.

Mutations in insulin/IGF-1 signaling pathway have been shown to lead to increased longevity in various invertebrate models. Therefore, the effect of the haplo-insufficiency of the IGF-1 receptor (Igf1r(+/-)) on longevity/aging was evaluated in C57Bl/6 mice using rigorous criteria where lifespan and end-of-life pathology were measured under optimal husbandry conditions using large sample sizes. Igf1r(+/-) mice exhibited reductions in IGF-1 receptor levels and the activation of Akt by IGF-1, with no compensatory increases in serum IGF-1 or tissue IGF-1 mRNA levels, indicating that the Igf1r(+/-) mice show reduced IGF-1 signaling. Aged male, but not female Igf1r(+/-) mice were glucose intolerant, and both genders developed insulin resistance as they aged. Female, but not male Igf1r(+/-) mice survived longer than wild type mice after lethal paraquat and diquat exposure, and female Igf1r(+/-) mice also exhibited less diquat-induced liver damage. However, no significant difference between the lifespans of the male Igf1r(+/-) and wild type mice was observed; and the mean lifespan of the Igf1r(+/-) females was increased only slightly (less than 5%) compared to wild type mice. A comprehensive pathological analysis showed no significant difference in end-of-life pathological lesions between the Igf1r(+/-) and wild type mice. These data show that the Igf1r(+/-) mouse is not a model of increased longevity and delayed aging as predicted by invertebrate models with mutations in the insulin/IGF-1 signaling pathway.

BACKGROUND: While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2-4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation. CONCLUSIONS: Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.