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[This corrects the article DOI: 10.1371/journal.pone.0276751.].

Both common pain and anxiety problems are widespread, debilitating and often begin in childhood-adolescence. Twin studies indicate that this co-occurrence is likely due to shared elements of risk, rather than reciprocal causation. A joint genome-wide investigation and pathway/network-based analysis of adolescent anxiety and pain problems can identify genetic pathways that subserve shared etiopathogenetic mechanisms. Pathway-based analyses were performed in the independent samples of: The Quebec Newborn Twin Study (QNTS; 246 twin pairs and 321 parents), the Longitudinal Study of Child Development in Quebec (QLSCD; n = 754), and in the combined QNTS and QLSCD sample. Multiple suggestive associations (p

Patients on dialysis are at risk of severe course of SARS-CoV-2 infection. Understanding the neutralizing activity and coverage of SARS-CoV-2 variants of vaccine-elicited antibodies is required to guide prophylactic and therapeutic COVID-19 interventions in this frail population. By analyzing plasma samples from 130 hemodialysis and 13 peritoneal dialysis patients after two doses of BNT162b2 or mRNA-1273 vaccines, we found that 35% of the patients had low-level or undetectable IgG antibodies to SARS-CoV-2 Spike (S). Neutralizing antibodies against the vaccine-matched SARS-CoV-2 and Delta variant were low or undetectable in 49% and 77% of patients, respectively, and were further reduced against other emerging variants. The fraction of non-responding patients was higher in SARS-CoV-2-naïve hemodialysis patients immunized with BNT162b2 (66%) than those immunized with mRNA-1273 (23%). The reduced neutralizing activity correlated with low antibody avidity. Patients followed up to 7 months after vaccination showed a rapid decay of the antibody response with an average 21- and 10-fold reduction of neutralizing antibodies to vaccine-matched SARS-CoV-2 and Delta variant, which increased the fraction of non-responders to 84% and 90%, respectively. These data indicate that dialysis patients should be prioritized for additional vaccination boosts. Nevertheless, their antibody response to SARS-CoV-2 must be continuously monitored to adopt the best prophylactic and therapeutic strategy.

BackgroundUtilization of Emergency Medical Services (EMS) declined during COVID-19 pandemic, but most of the studies analyzed components of the EMS system individually. The study aimed to evaluate the indirect impact of COVID-19 pandemic on the utilization of all the components of the EMS system of Tuscany Region (Italy) during the first pandemic wave.MethodsAdministrative data from the health care system of Tuscany were used. Changes in utilization for out-of-hospital emergency calls and emergency vehicle dispatched, emergency department (ED) visits, and patients being admitted from the ED to an inpatient hospital bed (hospitalizations from ED) during the first pandemic wave were analyzed in relation with corresponding periods of the previous two years. Percentage changes and 95%CI were calculated with Poisson models. Standardized Ratios were calculated to evaluate changes in in-hospital mortality and hospitalizations requiring ICU.ResultsSignificant declines were observed in the utilization of all the EMS considered starting from the week in which the first case of COVID-19 was diagnosed in Italy till the end of the first pandemic wave. During the epidemic peak, the maximum decreases were observed: -33% for the emergency calls, -45% for the dispatch of emergency vehicles, -71% for ED admissions. Furthermore, a decline of 37% for hospitalizations from ED was recorded. Significant decreases in ED admissions for life threatening medical conditions were observed: acute cerebrovascular disease (-36%, 95% CI: -43, -29), acute myocardial infarction (-42%, 95% CI: -52, -31) and renal failure (-42%, 95% CI: -52, -31). No significant differences were found between the observed and the expected in-hospital mortality and hospitalizations requiring ICU during the epidemic peak.ConclusionAll the components of the EMS showed large declines in their utilization during COVID-19 pandemic; furthermore, major reductions were observed for admissions for time-dependent and life-threatening conditions. Efforts should be made to ensure access to safe and high-quality emergency care during pandemic.

Despite new antivirals are being approved against SARS-CoV-2 they suffer from significant constraints and are not indicated for hospitalized patients, who are left with few antiviral options. Repurposed drugs have previously shown controversial clinical results and it remains difficult to understand why certain trials delivered positive results and other trials failed. Our manuscript contributes to explaining the puzzle: this might have been caused by a suboptimal drug exposure and, consequently, an incomplete virus suppression, also because the drugs have mostly been used as add-on monotherapies. As with other viruses (e.g., HIV and HCV) identifying synergistic combinations among such drugs could overcome monotherapy-related limitations. In a cell culture model for SARS-CoV-2 infection the following stringent criteria were adopted to assess drug combinations: 1) identify robust, synergistic antiviral activity with no increase in cytotoxicity, 2) identify the lowest drug concentration inhibiting the virus by 100% (LIC100) and 3) understand whether the LIC100 could be reached in the lung at clinically indicated drug doses. Among several combinations tested, remdesivir with either azithromycin or ivermectin synergistically increased the antiviral activity with no increase in cytotoxicity, improving the therapeutic index and lowering the LIC100 of every one of the drugs to levels that are expected to be achievable and maintained in the lung for a therapeutically relevant period of time. These results are consistent with recent clinical observations showing that intensive care unit admission was significantly delayed by the combination of AZI and RDV, but not by RDV alone, and could have immediate implications for the treatment of hospitalized patients with COVID-19 as the proposed "drug cocktails" should have antiviral activity against present and future SARS-CoV-2 variants without significant overlapping toxicity, while minimizing the onset of drug resistance. Our results also provide a validated methodology to help sort out which combination of drugs are most likely to be efficacious in vivo, based on their in vitro activity, potential synergy and PK profiles.

Objectives of the studyDemographic changes alongside medical advances have resulted in older adults accounting for an increasing proportion of emergency hospital admissions. Current measures of illness severity, limited to physiological parameters, have shortcomings in this cohort, partly due to patient complexity. This study aimed to derive and validate a risk score for acutely unwell older adults which may enhance risk stratification and support clinical decision-making.MethodsData was collected from emergency admissions in patients ≥65 years from two UK general hospitals (April 2017- April 2018). Variables underwent regression analysis for in-hospital mortality and independent predictors were used to create a risk score. Performance was assessed on external validation. Secondary outcomes included seven-day mortality and extended hospital stay.ResultsDerivation (n = 8,974) and validation (n = 8,391) cohorts were analysed. The model included the National Early Warning Score 2 (NEWS2), clinical frailty scale (CFS), acute kidney injury, age, sex, and Malnutrition Universal Screening Tool. For mortality, area under the curve for the model was 0.79 (95% CI 0.78-0.80), superior to NEWS2 0.65 (0.62-0.67) and CFS 0.76 (0.74-0.77) (P30 days.ConclusionsOur simple validated model (Older Persons' Emergency Risk Assessment [OPERA] score) predicts in-hospital mortality and prolonged length of stay and could be easily integrated into electronic hospital systems, enabling automatic digital generation of risk stratification within hours of admission. Future studies may validate the OPERA score in external populations and consider an impact analysis.

BACKGROUND:Cancer, chronic heart failure (CHF), and chronic obstructive pulmonary disease (COPD) in the advanced stages have similar symptom burdens and survival rates. Despite these similarities, the majority of the attention directed to improving the quality of end-of-life (EOL) care has focused on cancer. AIM:To assess the extent to which the quality of EOL care received by cancer, CHF, and COPD patients in the last month of life is diagnosis-sensitive. METHODS:This is a retrospective observational study based on administrative data. The study population includes all Tuscany region residents aged 18 years or older who died with a clinical history of cancer, CHF, or COPD. Decedents were categorized into two mutually exclusive diagnosis categories: cancer (CA) and cardiopulmonary failure (CPF). Several EOL care quality outcome measures were adopted. Multivariable generalized linear model for each outcome were performed. RESULTS:The sample included 30,217 decedents. CPF patients were about 1.5 times more likely than cancer patients to die in an acute care hospital (RR 1.59, 95% C.I.: 1.54-1.63). CPF patients were more likely to be hospitalized or admitted to the emergency department (RR 1.09, 95% C.I.: 1.07-1.10; RR 1.15, 95% C.I.: 1.13-1.18, respectively) and less likely to use hospice services (RR 0.08, 95% C.I.: 0.07-0.09) than cancer patients in the last month of life. CPF patients had a four- and two-fold higher risk of intensive care unit admission or of undergoing life-sustaining treatments, respectively, than cancer patients (RR 3.71, 95% C.I.: 3.40-4.04; RR 2.43, 95% C.I.: 2.27-2.60, respectively). CONCLUSION:The study has highlighted the presence of significant differences in the quality of EOL care received in the last month of life by COPD and CHF compared with cancer patients. Further studies are needed to better elucidate the extent and the avoidability of these diagnosis-related differences in the quality of EOL care.

[This corrects the article DOI: 10.1371/journal.pone.0200584.].

BACKGROUND:Acute kidney injury (AKI) is assoicated with high mortality and measures to improve risk stratification and early identification have been urgently called for. This study investigated whether an electronic clinical prediction rule (CPR) combined with an AKI e-alert could reduce hospital-acquired AKI (HA-AKI) and improve associated outcomes. METHODS AND FINDINGS:A controlled before-and-after study included 30,295 acute medical admissions to two adult non-specialist hospital sites in the South of England (two ten-month time periods, 2014-16); all included patients stayed at least one night and had at least two serum creatinine tests. In the second period at the intervention site a CPR flagged those at risk of AKI and an alert was generated for those with AKI; both alerts incorporated care bundles. Patients were followed-up until death or hospital discharge. Primary outcome was change in incident HA-AKI. Secondary outcomes in those developing HA-AKI included: in-hospital mortality, AKI progression and escalation of care. On difference-in-differences analysis incidence of HA-AKI reduced (odds ratio [OR] 0.990, 95% CI 0.981-1.000, P = 0.049). In-hospital mortality in HA-AKI cases reduced on difference-in-differences analysis (OR 0.924, 95% CI 0.858-0.996, P = 0.038) and unadjusted analysis (27.46% pre vs 21.67% post, OR 0.731, 95% CI 0.560-0.954, P = 0.021). Mortality in those flagged by the CPR significantly reduced (14% pre vs 11% post intervention, P = 0.008). Outcomes for community-acquired AKI (CA-AKI) cases did not change. A number of process measures significantly improved at the intervention site. Limitations include lack of randomization, and generalizability will require future investigation. CONCLUSIONS:In acute medical admissions a multi-modal intervention, including an electronically integrated CPR alongside an e-alert for those developing HA-AKI improved in-hospital outcomes. CA-AKI outcomes were not affected. The study provides a template for investigations utilising electronically generated prediction modelling. Further studies should assess generalisability and cost effectiveness. TRIAL REGISTRATION:Clinicaltrials.org NCT03047382.

Older adult patients with fragility hip fractures constitute a population at high risk for complications, in particular pressure ulcers. The aim was to evaluate the incidence of pressure ulcers and potential predictive factors.A prospective multicentric prognostic cohort study in orthopedic wards in three Italian public hospitals. Participants were all consecutive patients 65 years of age or older diagnosed with a fragility hip fracture. Outcomes were incidence of pressure ulcers. The exposure variables were grouped into three macro areas in order to facilitate reading: "intrinsic" variables, "extrinsic" variables and variables linked to the organization of patient care. One thousand eighty-three older adult patients with fragility hip fractures were enrolled from October 1st, 2013 to January 31st, 2015, and pressure ulcers developed in 22.7%. At multivariate analysis, the following were found to be risk factors: age> 80 years (odds ratio (OR) 1.03; p = 0.015), the length of time a urinary catheter was used (OR 1.013; p

BACKGROUND AND OBJECTIVES:Hypocitraturia has been associated with metabolic acidosis and mineral disorders. The aim of this study was to investigate the occurrence of urinary acidification defects underlying hypocitraturia. MATERIALS AND METHODS:This retrospective observational study included 67 patients (32 men), aged 40.7±15.1 years with hypocitraturia (

Lysosomal Storage Disorders (LSDs) are a group of metabolic syndromes, each one due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and overall about 75% of the patients present neurological impairment. Enzyme Replacement Therapy, although determining some systemic clinical improvements, is ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB). With the aim to deliver the therapeutic enzymes across the BBB, we here assayed biodegradable and biocompatible PLGA-nanoparticles (NPs) in two murine models for LSDs, Mucopolysaccharidosis type I and II (MPS I and MPS II). PLGA-NPs were modified with a 7-aminoacid glycopeptide (g7), yet demonstrated to be able to deliver low molecular weight (MW) molecules across the BBB in rodents. We specifically investigated, for the first time, the g7-NPs ability to transfer a model drug (FITC-albumin) with a high MW, comparable to the enzymes to be delivered for LSDs brain therapy. In vivo experiments, conducted on wild-type mice and knockout mouse models for MPS I and II, also included a whole series of control injections to obtain a broad preliminary view of the procedure efficiency. Results clearly showed efficient BBB crossing of albumin in all injected mice, underlying the ability of NPs to deliver high MW molecules to the brain. These results encourage successful experiments with enzyme-loaded g7-NPs to deliver sufficient amounts of the drug to the brain district on LSDs, where exerting a corrective effect on the pathological phenotype.

BACKGROUND:The expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (Gαgust) and α-transducin (Gαtran) G protein subunits. This study tested whether Gαtran and Gαgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract. RESULT:In the stomach, Gαgust and Gαtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, Gαgust and Gαtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of Gαtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P

The karyotype is shaped by different chromosome rearrangements during species evolution. However, determining which rearrangements are responsible for karyotype changes is a challenging task and the combination of a robust phylogeny with refined karyotype characterization, GS measurements and bioinformatic modelling is necessary. Here, this approach was applied in Heterotaxis to determine what chromosome rearrangements were responsible for the dysploidy variation. We used two datasets (nrDNA and cpDNA, both under MP and BI) to infer the phylogenetic relationships among Heterotaxis species and the closely related genera Nitidobulbon and Ornithidium. Such phylogenies were used as framework to infer how karyotype evolution occurred using statistical methods. The nrDNA recovered Ornithidium, Nitidobulbon and Heterotaxis as monophyletic under both MP and BI; while cpDNA could not completely separate the three genera under both methods. Based on the GS, we recovered two groups within Heterotaxis: (1) "small GS", corresponding to the Sessilis grade, composed of plants with smaller genomes and smaller morphological structure, and (2) "large GS", corresponding to the Discolor clade, composed of plants with large genomes and robust morphological structures. The robust karyotype modeling, using both nrDNA phylogenies, allowed us to infer that the ancestral Heterotaxis karyotype presented 2n = 40, probably with a proximal 45S rDNA on a metacentric chromosome pair. The chromosome number variation was caused by ascending dysploidy (chromosome fission involving the proximal 45S rDNA site resulting in two acrocentric chromosome pairs holding a terminal 45S rDNA), with subsequent descending dysploidy (fusion) in two species, H. maleolens and H. sessilis. However, besides dysploidy, our analysis detected another important chromosome rearrangement in the Orchidaceae: chromosome inversion, that promoted 5S rDNA site duplication and relocation.

The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft.

To estimate the effects of cycling promotion on major non-communicable diseases (NCDs) and costs from the public healthcare payer's perspective.Health impact assessment and economic evaluation using a dynamic model over a ten-year period and according to two cycling promotion scenarios.Cycling to work or school in Florence, Italy.All individuals aged 15 and older commuting to work or school in Florence.The primary outcome measures were changes in NCD incidence and healthcare direct costs for the Tuscany Regional Health Service (SST) due to increased cycling. The secondary outcome was change in road traffic accidents.Increasing cycling modal share in Florence from 7.5% to about 17% (Scenario 1) or 27% (Scenario 2) could decrease the incidence of type 2 diabetes by 1.2% or 2.5%, and the incidence of acute myocardial infarction (AMI) and stroke by 0.6% or 1.2%. Within 10 years, the number of cases that can be prevented is 280 or 549 for type 2 diabetes, 51 or 100 for AMI, and 51 or 99 for stroke in Scenario 1 or Scenario 2, respectively. Average annual discounted savings for the SST are estimated to amount to €400,804 or €771,201 in Scenario 1 or Scenario 2, respectively. In Florence, due to the high use of vulnerable motorized vehicles (such as scooters, mopeds, and motorcycles), road traffic accidents are expected to decline in both our scenarios. Sensitivity analyses showed that health benefits and savings for the SST are substantial, the most sensitive parameters being the relative risk estimates of NCDs and active commuting.Effective policies and programs to promote a modal shift towards cycling among students and workers in Florence will contribute to reducing the NCD burden and helping long-term economic sustainability of the SST.

The HAVCR2 gene encodes TIM-3, an immunoglobulin superfamily member expressed by exhausted CD8+ T cells during chronic viral infection. We investigated whether genetic variation at HAVCR2 modulates the susceptibility to HIV-1 acquisition; specifically we focused on a 3' UTR variant (rs4704846, A/G) that represents a natural selection target. We genotyped rs4704846 in three independent cohorts of HIV-1 exposed seronegative (HESN) individuals with different geographic origin (Italy and Spain) and distinct route of exposure to HIV-1 (sexual and injection drug use). Matched HIV-1 positive subjects and healthy controls were also analyzed. In all case-control cohorts the minor G allele at rs4704846 was more common in HIV-1 infected individuals than in HESN, with healthy controls showing intermediate frequency. Results from the three association analyses were combined through a random effect meta-analysis, which revealed no heterogeneity among samples (Cochrane's Q, p value = 0.89, I2 = 0) and yielded a p value of 6.8 ×10(-4). The minor G allele at rs4704846 was found to increase HAVCR2 expression after in vitro HIV-1 infection. Thus, a positively selected polymorphism in the 3' UTR, which modulates HAVCR2 expression, is associated with the susceptibility to HIV-1 infection. These data warrant further investigation into the role of TIM-3 in the prevention and treatment of HIV-1/AIDS.

p-Aminobenzoic acid (pABA) plays important roles in a wide variety of metabolic processes. Herein we report the synthesis, theoretical calculations, crystallographic investigation, and in vitro determination of the biological activity and phytotoxicity of the pABA salt, 2-hydroxyethylammonium p-aminobenzoate (HEA-pABA). The ability of neutral and anionic forms of pABA to interact with TIR1 pocket was investigated by calculation of molecular electrostatic potential maps on the accessible surface area, docking experiments, Molecular Dynamics and Quantum Mechanics/Molecular Mechanics calculations. The docking study of the folate precursor pABA, its anionic form and natural auxin (indole-3-acetic acid, IAA) with the auxin receptor TIR1 revealed a similar binding mode in the active site. The phytotoxic evaluation of HEA-pABA, pABA and 2-hydroxyethylamine (HEA) was performed on the model plant Arabidopsis thaliana ecotype Col 0 at five different concentrations. HEA-pABA and pABA acted as potential auxin-like regulators of root development in Arabidopsis thaliana (0.1 and 0.2 mM) and displayed an agravitropic root response at high concentration (2 mM). This study suggests that HEA-pABA and pABA might be considered as potential new regulators of plant growth.

This study aims to translate and validate Early Trauma Inventory Self Report -Short Form (ETISR-SF) to Brazilian Portuguese. 253 adult subjects answered the ETISR-SF, Beck Anxiety Inventory (BAI), Fagerström Test for Nicotine Dependence (FTND), Patient Health Questionnaire (PHQ-9) and Fast Alcohol Screening Test (FAST). The instrument showed good internal consistency (0.83). Correlations with the PHQ-9 and BAI were moderate (r=0.26-0.47) and showed the expected associations with psychiatric constructs. No associations were found for FTND and FAST. Confirmatory Factor Analysis revealed that a correlated four-factor model as well as a second order model subsuming four lower order components presented the best model fit. Test-retest reliability was also excellent (ICC=0.78-0.90). ETISR-SF is suitable for assessing traumatic experiences in a Brazilian community sample. Given the importance of trauma as a public health problem, tools such as ETISR-SF may help clinicians/ researchers to better evaluate and measure such events and further advance clinical care of trauma victims.

Social Anxiety Disorder (SAD) is prevalent and rarely diagnosed due to the difficulty in recognizing its symptoms as belonging to a disorder. Therefore, the evaluation/screening scales are of great importance for its detection, with the most used being the Liebowitz Social Anxiety Scale (LSAS). Thus, this study proposed to evaluate the psychometric properties of internal consistency and convergent validity, as well as the confirmatory factorial analysis and reliability of the self-reported version of the LSAS (LSAS-SR), translated into Brazilian Portuguese, in a sample of the general population (N = 413) and in a SAD clinical sample (N = 252). The convergent validity with specific scales for the evaluation of SAD and a general anxiety scale presented correlations ranging from 0.21 to 0.84. The confirmatory factorial analysis did not replicate the previously indicated findings of the literature, with the difficulty being in obtaining a consensus factorial structure common to the diverse cultures in which the instrument was studied. The LSAS-SR presented excellent internal consistency (α = 0.90-0.96) and test-retest reliability (Intraclass Correlation Coefficient = 0.81; Pearson's = 0.82). The present findings support those of international studies that attest to the excellent psychometric properties of the LSAS-SR, endorsing its status as the gold standard.

ObjectiveThe aim of the present study was to comprehensively evaluate systemic and local inflammation as well as progression of vascular inflammation in normal and mechanically injured vessels in a large animal model of mild hypercholesterolemia. Our aim was also to test the effect of high-dose statin treatment on these processes.MethodsPigs were kept for 120 days on a standard diet (SD, n=7), high-cholesterol diet (HCD, n=7) or high-cholesterol diet with Atorvastatin starting after 50 days (STATIN, n=7). Left carotid artery balloon injury was conducted in all groups after 60 days of diet treatment. Biochemical analysis together with evaluation of blood and tissue markers of vascular injury and inflammation were performed in all groups at the end of experiment.ResultsHCD compared to SD induced systemic inflammation demonstrated by increased number of circulating monocytes and lymphocytes. HCD compared to SD induced also local inflammation demonstrated by adipocyte hypertrophy and infiltration of T-lymphocytes in abdominal white adipose tissue, activation of hepatic stellate cells with infiltration of T- and B-lymphocytes and macrophages in the liver and increased macrophage content in lung parenchyma. These changes were accompanied by increased Intima/Media thickness, stenosis, matrix deposition and activated T-cell infiltrates in injured but not in uninjured contralateral carotid artery as we previously reported. The treatment with high-dose statin attenuated all aspects of systemic and local inflammation as well as pathological changes in injured carotid artery.ConclusionsDiet related mild hypercholesterolemia induce systemic and local inflammation in the liver, lung and adipose tissue that coincide with enhanced inflammation of injured vessel but is without deleterious effect on uninjured vessels. High dose statin attenuated systemic and local inflammation and protected injured vessels. However, finding exact role of reduced systemic and remote inflammation in vascular protection requires further studies.

BACKGROUND: Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. METHODOLOGY/PRINCIPAL FINDINGS: The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. CONCLUSIONS/SIGNIFICANCE: Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.

A new class of antiretrovirals, AntiViral-HyperActivation Limiting Therapeutics (AV-HALTs), has been proposed as a disease-modifying therapy to both reduce Human Immunodeficiency Virus Type 1 (HIV-1) RNA levels and the excessive immune activation now recognized as the major driver of not only the continual loss of CD4(+) T cells and progression to Acquired Immunodeficiency Syndrome (AIDS), but also of the emergence of both AIDS-defining and non-AIDS events that negatively impact upon morbidity and mortality despite successful (ie, fully suppressive) therapy. VS411, the first-in-class AV-HALT, combined low-dose, slow-release didanosine with low-dose hydroxycarbamide to accomplish both objectives with a favorable toxicity profile during short-term administration. Five dose combinations were administered as VS411 to test the AV-HALT Proof-of-Concept in HIV-1-infected subjects.Multinational, double-blind, 28-day Phase 2a dose-ranging Proof-of-Concept study of antiviral activity, immunological parameters, safety, and genotypic resistance in 58 evaluable antiretroviral-naïve HIV-1-infected adults. Randomization and allocation to study arms were carried out by a central computer system. Results were analyzed by ANOVA, Kruskal-Wallis, ANCOVA, and two-tailed paired t tests.VS411 was well-tolerated, produced significant reductions of HIV-1 RNA levels, increased CD4(+) T cell counts, and led to significant, rapid, unprecedented reductions of immune activation markers after 28 days despite incomplete viral suppression and without inhibiting HIV-1-specific immune responses. The didanosine 200 mg/HC 900 mg once-daily formulation demonstrated the greatest antiviral efficacy (HIV-1 RNA: -1.47 log(10) copies/mL; CD4(+) T cell count: +135 cells/mm(3)) and fewest adverse events.VS411 successfully established the Proof-of-Concept that AV-HALTs can combine antiviral efficacy with rapid, potentially beneficial reductions in the excessive immune system activation associated with HIV-1 disease. Rapid reductions in markers of immune system hyperactivation and cellular proliferation were obtained despite the fact that VS411 did not attain maximal suppression of HIV RNA, suggesting this effect was due to the HALT component.ITEudraCT 2007-002460-98.

Cognitive assessment in a clinical setting is generally made by pencil-and-paper tests, while computer-based tests enable the measurement and the extraction of additional performance indexes. Previous studies have demonstrated that in a research context exploration deficits occur also in patients without evidence of unilateral neglect at pencil-and-paper tests. The objective of this study is to apply a touchscreen-based cancellation test, feasible also in a clinical context, to large groups of control subjects and unilaterally brain-damaged patients, with and without unilateral spatial neglect (USN), in order to assess disturbances of the exploratory skills. A computerized cancellation test on a touchscreen interface was used for assessing the performance of 119 neurologically unimpaired control subjects and 193 patients with unilateral right or left hemispheric brain damage, either with or without USN. A set of performance indexes were defined including Latency, Proximity, Crossings and their spatial lateral gradients, and Preferred Search Direction. Classic outcome scores were computed as well. Results show statistically significant differences among groups (assumed p

The ERAP1 gene encodes an aminopeptidase involved in antigen processing. A functional polymorphism in the gene (rs30187, Arg528Lys) associates with susceptibility to ankylosying spondylitis (AS), whereas a SNP in the interacting ERAP2 gene increases susceptibility to another inflammatory autoimmune disorder, Crohn's disease (CD). We analysed rs30187 in 572 Italian patients with CD and in 517 subjects suffering from multiple sclerosis (MS); for each cohort, an independent sex- and age-matched control group was genotyped. The frequency of the 528Arg allele was significantly higher in both disease cohorts compared to the respective control population (for CD, OR = 1.20 95%CI: 1.01-1.43, p = 0.036; for RRMS, OR = 1.26; 95%CI: 1.04-1.51, p = 0.01). Meta-analysis with the Wellcome Trust Cases Control Consortium GWAS data confirmed the association with MS (p(meta) = 0.005), but not with CD. In AS, the rs30187 variant has a predisposing effect only in an HLA-B27 allelic background. It remains to be evaluated whether interaction between ERAP1 and distinct HLA class I alleles also affects the predisposition to MS, and explains the failure to provide definitive evidence for a role of rs30187 in CD. Results herein support the emerging concept that a subset of master-regulatory genes underlay the pathogenesis of autoimmunity.

Intact synaptic function and plasticity are fundamental prerequisites to a healthy brain. Therefore, synaptic proteins are one of the major targets for drugs used as neuro-chemical therapeutics. Unfortunately, the majority of drugs is not able to cross the blood-brain barrier (BBB) and is therefore distributed within the CNS parenchyma. Here, we report the development of novel biodegradable Nanoparticles (NPs), made of poly-lactide-co-glycolide (PLGA) conjugated with glycopeptides that are able to cross the BBB and deliver for example Zn(2+) ions. We also provide a thorough characterization of loaded and unloaded NPs for their stability, cellular uptake, release properties, toxicity, and impact on cell trafficking. Our data reveal that these NPs are biocompatible, and can be used to elevate intracellular levels of Zn(2+). Importantly, by engineering the surface of NPs with antibodies against NCAM1 and CD44, we were able to selectively target neurons or glial cells, respectively. Our results indicate that these biodegradable NPs provide a potential new venue for the delivery Zn(2+) to the CNS and thus a means to explore the influence of altered zinc levels linked to neuropsychological disorders such as depression.

BACKGROUND:Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. METHODOLOGY/PRINCIPAL FINDINGS:In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. CONCLUSIONS/SIGNIFICANCE:Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.

During uncontrolled HIV disease, both TNF-related apoptosis inducing ligand (TRAIL) and TRAIL receptor expression are increased. Enhanced TRAIL sensitivity is due to TRAIL receptor up-regulation induced by gp120. As a result of successful antiretroviral therapy TRAIL is down-regulated, and there are fewer TRAIL-sensitive cells. In this setting, we hypothesized that all cells that contain virus, including those productively- and latently-infected, have necessarily been "primed" by gp120 and remain TRAIL-sensitive, whereas uninfected cells remain relatively TRAIL-resistant.We evaluated the immunologic and antiviral effects of TRAIL in peripheral blood lymphocytes collected from HIV-infected patients with suppressed viral replication. The peripheral blood lymphocytes were treated with recombinant TRAIL or an equivalent amount of bovine serum albumin as a negative control. Treated cells were then analyzed by quantitative flow cytometry, ELISPOT for CD4+ and CD8+ T-cell function, and limiting dilution microculture for viral burden. Alterations in the cytokine milieu of treated cells were assessed with a multiplex cytokine assay. Treatment with recombinant TRAIL in vitro reduced viral burden in lymphocytes collected from HIV-infected patients with suppressed viral load. TRAIL treatment did not alter the cytokine milieu of treated cells. Moreover, treatment with recombinant TRAIL had no adverse effect on either the quantity or function of immune cells from HIV-infected patients with suppressed viral replication.TRAIL treatment may be an important adjunct to antiretroviral therapy, even in patients with suppressed viral replication, perhaps by inducing apoptosis in cells with latent HIV reservoirs. The absence of adverse effect on the quantity or function of immune cells from HIV-infected patients suggests that there is not a significant level of "bystander death" in uninfected cells.