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This study aimed to determine the relationship between age of first exposure (AFE) to repetitive head impacts through contact/collision sports and patient-reported outcomes in community rugby players.We recruited community rugby players older than 18 yr with at least 1 yr of contact rugby participation to complete an online survey. Participants completed the Brief Symptom Inventory-18 (BSI-18), Short-Form Health Survey 12 (SF-12), and Satisfaction with Life Scale (SWLS) via Qualtrics. We used generalized linear models to examine the association between AFE (continuous) and patient-reported outcomes by sex, while controlling for cumulative years contact/collision sport history, age, and concussion history (yes/no). In addition, we used Mann-Whitney U tests to compare patient-reported outcomes between AFE12 and AFE ≥12.A total of 1037 rugby players (31.6 ± 11.3 yr (range, 18-74 yr), 59.1% men) participated in this study. Whether analyzed continuously or dichotomously at age 12 yr, younger AFE was not associated with worse patient-reported outcomes for either men or women. Positive concussion history was a significant predictor of worse BSI-18 subscores, SF-12 subscores, and SWLS in women and worse BSI-18 subscores in men. Cumulative contact/collision sport history was a significant predictor of better BSI-18 Depression and SF-12 (Mental Component Summary) subscores in men only. In men and women, older age was a significant predictor of better BSI-18 Depression, Anxiety, and GSI subscores; better SWLS (in men only); and better SF-12 Mental Component Summary, but worse SF-12 (Physical Component Summary).Younger AFE to contact/collision sport is not associated with worse patient-reported outcomes in early adult rugby players. Concussion history was predictive of worse patient-reported outcomes. more...

To determine (1) the association between lifetime diagnosed concussion and lower extremity musculoskeletal injury (LE-MSI) among community rugby union players and (2) the sex specific risk of LE-MSI given concussion history among males and females.Retrospective survey.1037 (59.0% male, (612/1037), age: 31.6 ± 11.3 years) rugby players (10.1 ± 8.1 years played) completed an online survey to ascertain injury history. A chi-squared test of association was performed between concussion and LE-MSI; significant outcomes were followed-up with an odds ratio. A binary logistic regression with any LE-MSI (yes/no) as the outcome and concussion (yes/no) and sex (male/female) as predictors was performed to determine if there was a sex by concussion interaction.There was an overall significant association between concussion and any LE-MSI(χ(1) = 13.055, p 0.001, OR = 2.30 [95%CI: 1.45, 3.65]). Both male (OR = 2.21) and females (OR = 2.49) had significant associations for concussion and LE-MSI, but there were no differences between sex for risk of LE-MSI (RCommunity rugby players with a history of concussion are2× more likely to also experience an LE-MSI than those without a history of concussion. There were no differences in the odds of LE-MSI between males and females with a history of diagnosed concussion. In line with current World Rugby injury prevention programs, future research should aim to reduce LE-MSI incidence to maximize player safety and wellness through targeted injury prevention and teams should utilize a conservative return to play protocols following concussion. more...

IntroductionConcussions have been associated with an increased risk of lower-extremity musculoskeletal injury (LE-MSI) in athletes and US Army soldiers, creating an added economic, physical and social burden. Yet, there is a paucity of evidence on this relationship among Reserve Officers’ Training Corps (ROTC) cadets, a group which engages in activities with high-injury risk and will subsequently commission as active duty officers. This study aimed to examine the association between concussions and LE-MSI in ROTC cadets.Methods125 (83 were male) Army and Air Force ROTC cadets (19.8±2.0 years) from two large state universities’ Army and Air Force ROTC programmes participated in this study. Cadets completed a reliable injury history questionnaire to ascertain the following variables of interest: (1) any concussion history, (2) reported concussions, (3) undiagnosed concussions, and (4) potentially unrecognised concussion history and LE-MSI history (eg, ankle sprain, knee sprain or muscle strain). Data were analysed using a χ2test for association and binary logistic regression to determine ORs.ResultsCadets with any concussion history (n=42) had a significantly (p=0.035) higher association with LE-MSI (OR 2.47, 95% CI 1.05 to 5.83) than those without. Cadets who had a reported concussion (n=33) had a significantly (p=0.026) higher association with LE-MSI (OR 2.95, 95% CI 1.11 to 7.84) compared to cadets without.ConclusionsROTC cadets with a history of diagnosed concussion were more likely to have suffered an LE-MSI than cadets without a concussion history. ROTC cadre should be aware of this relationship and incorporate injury prevention protocols. more...

In this article, I explore the performative nature of ghost bike memorials across the United States. Ghost bikes, flat-white painted immobile bicycles, are installed roadside to mark the location o... more...

To provide a summary of the evidence on the comparative effectiveness of two surgical treatment strategies, sentinel node biopsy (SNB) and elective neck dissection (END), in patients with T1-T2 oral cancer and clinically negative (cN0) neck, in terms of overall survival (OS), disease-free survival (DFS) and neck recurrence rates (NRRs).A systematic review was performed by including studies published up to April 2019. Meta-analysis was performed to compare NRRs between SNB and END. A narrative summary of the results was generated for OS, DFS and morbidity outcomes. The certainty of evidence was assessed according to the GRADE methodology.No randomized studies were retrieved. Five observational studies were included in the comparative effectiveness analysis and four observational studies were included in the comparative morbidity analysis. The pooled risk ratio showed no differences in NRRs between SNB and END (10.5% vs 11.6%; pooled RR 1.09; 95% CI 0.67-1.76). No differences in OS or DFS between the two treatments were found. SNB appears to be associated with a lower rate of postoperative complications and lower shoulder dysfunction than END. Conversely, the results of the quality of life (QoL) questionnaires are not sufficient to advocate a particular strategy.Our review highlights the lack of well conducted and randomized studies comparing SNB to END, leading to poor clinical evidence. Although our findings suggest no significant differences in OS, DFS and NRR between the two strategies, the certainty of our evidence is too low to make it useful for clinical decision making. more...

Background In the last decade of the past century, primary Laparoscopic Heller-Dor (LHD) for Achalasia progressively became the procedure of choice in the new millennium. The aim of this study was to assess the long-term outcome of LHD to treat Achalasia at a single high-volume institution during the past 25 years. Methods 1000 patients underwent LHD from 1992-2017 by 6 staff surgeons alternatively. Patients who had already been treated with surgical or endoscopic myotomy were ruled out. Symptoms were collected and scored using a detailed questionnaire; barium-swallow, endoscopy, manometry were performed, before and after surgery while, 24-hour pH monitoring were performed 6 months after surgery. Results LHD was the primary treatment for 1000 patients (M:F=536:464); the median age was 46 (IQR 36-54), 183 (18.3%) had a history of endoscopic treatments (pneumatic dilation or botox injections, or both). The surgical procedure was completed laparoscopically in all but 7 patients (0.7%) and there was one perioperative death for heart attack. There were 25 perforations (2.5%): 22 were recognized and repaired during the operation, 3 were detected by postoperative contrast swallow. The outcome was positive in 902 patients (90.2%). In patients who had a previous treatment the failures were 25/183 (13.7%) while in the primary treatment group the failures were 73/817 (8.9%) (p=0.055). All the 98 patients whose LHD failed subsequently underwent one or more endoscopic pneumatic dilations, which ameliorated their recurrent symptoms in all but 11 patients (10 of whom required reoperation). The overall success rate of the combination of LHD and endoscopic dilations (where necessary) was 98.4%. At univariate analysis, manometric pattern (p=0.001), sigmoid megaesophagus (p=0.003) and a chest pain score (p=0.002) were the only factors predictive of a positive final results. At multivariate analysis, these three factors were independently associated to good outcome. Postoperative 24-hour pH-monitoring was abnormal in 50/590 patients (8.5%) Two patients developed an esophageal cancer during the follow-up time. Conclusions In a university tertiary referral center LHD can durably relieve achalasia symptoms. Preoperative manometric pattern, a presence of a sigmoid esophagus and the chest pain score represent the strongest predictor of outcome. more...

This data article is related to the research article entitled “Silver nanoparticles alter epithelial basement membrane integrity, cell adhesion molecule expression and TGF-beta secretion”, available in the journal Nanomedicine: Nanotechnology, Biology, and Medicine [1]. This Data in Brief consists of data that describe changes in the expression of basement membrane (BM)-associated genes and proteins in three non-transformed epithelial cell lines following acute (6 h) and chronic (24 h plus 7-day chase) exposure to silver nanoparticles (AgNPs). Human BEAS2B (lung), MCF10AI (breast), and CCD-18Co (colon) cultured epithelia were analyzed for protein expression by LC-MS/MS and for gene expression by pathway-focused QRT-PCR arrays of 168 focal adhesion, integrin, and extracellular matrix (ECM) genes known to be localized to the plasma membrane, the BM/ECM, or secreted into the extracellular space. Ingenuity pathway analysis (IPA) of combined gene and protein expression datasets was then used to predict canonical pathways affected by AgNP exposure. Keywords: Silver nanoparticles, Epithelial cells, Basement membrane, Extracellular matrix, TGF-Beta, Intracellular signalling pathway analysis more...

ObjectiveTo determine the relationship between exposure to repetitive head impacts (RHI) through collision sports prior to the age of 12 and quality of life measures in community rugby players.BackgroundIt is suggested that RHI incurred before age 12 may be associated with later life neurologic impairments. However, research on age of first exposure (AFE) to collision sports and psychological outcomes has not be explored in rugby, a sport which participants often continue in community settings beyond college.Design/MethodsIndividuals over 18 years old who currently or previously played contact rugby completed an online questionnaire. To assess quality of life and psychological status, participants completed the Brief-Symptoms Inventory 18 (BSI-18), Short Form 12 (SF-12), and Satisfaction with Life Survey (SWLS). Participants were dichotomized into AFE to collision sports (12); AFE to rugby was not used since most participated in other collision sports prior to rugby. Data were not normally distributed; therefore, a Mann-Whitney U test was performed to compare outcomes between AFE groups.Results1,037 rugby players (31.6 + 11.3 years, 59.1% male) participated in this study. There were no significant differences between AFE 12 groups on all outcomes: BSI-18 Somatization (U = 97,286, p = 0.307), BSI-18 Depression (U = 100,267, p = 0.778), BSI-18 Anxiety (U = 98,851, p = 0.531), SF-12 Physical (U = 94,413, p = 0.241), SF-12 Mental (U = 96,517, p = 0.512), SWLS (U = 98,866, p = 0.537). Mean scores for all outcomes were: BSI-18 Somatization (2.33 + 2.99), BSI-18 Depression (4.20 + 4.91), BSI-18 Anxiety (3.32 + 3.75), SF-12 Physical (52.40 + 7.25), SF-12 Mental (46.20 + 11.45), SWLS (24.86 + 6.31).ConclusionsConsistent with recent cohort studies, there was no observed difference on three common measures of psychological well-being and quality of life in rugby players based upon AFE to collision sports. However, later life potential consequences of RHI in rugby players remains to be elucidated. more...

ObjectiveTo examine sex differences between concussion and lower-extremity musculoskeletal injury (LE-MSI) in community male and female rugby players.BackgroundThere is an ∼2x elevated risk of post-concussion subsequent MSI in high school through professional athletes. However, the effect of sex on risk is inconsistent and sparse, and rugby provides an ideal population as it’s the only collision sport with the same rules for both sexes.Design/Methods1,037 rugby players (31.6 + 11.3 years, 59.1% male), with at least one year of rugby playing experience, participated in this study, completing an online injury history questionnaire to ascertain concussion (yes/no) and LE-MSI (yes/no) history. A chi-squared test was performed to determine the association between concussion and any LE-MSI; significant findings were followed up with a post hoc odds ratio test. A binary logistic regression with any LE-MSI (yes/no) as the outcome and concussion (yes/no) and sex (male/female) as predictors was performed to determine if there was a sex by concussion interaction.ResultsThere was a significant association between concussion and any LE-MSI for all groups (Overall: ?(1) =13.06, p < 0.001, OR = 2.30 [95% CI: 1.45–3.65]; Males: ?(1) =7.43 p = 0.006, OR = 2.21 [95% CI: 1.24–3.96]; and Females: ?(1) = 5.78, p = 0.016, OR = 2.48 [95% CI: 1.16–5.31]). However, there were no differences for risk of LE-MSI between males and females (p = 0.99, R2 = 0.024).ConclusionsBoth male and female community rugby players had a 2x greater risk of LE-MSI, given a history of concussion compared to those without a history of concussion, which aligns with previous studies focused on collegiate athletes. However, there was no difference in risk of LE-MSI between sexes, contrary to smaller, but more controlled studies. Future research should investigate the potential physiological mechanisms for increased risk of LE-MSI. more...

International audience; Single crystals of transition metal carbides from group IV, TiC 1-x and ZrC 1-x , have been irradiated at room temperature (RT) with 1.2 MeV gold ions for various fluences in the range 2 × 10 14 –3 × 10 16 ions/cm 2 and polycrystals for fluences between 2 × 10 13 and 4 × 10 15 ions/cm 2 . The irradiated samples were characterized by micro Raman spectroscopy. For this purpose, the evolution of main parameters of Raman spectra for both types of samples (band positions, shifts, and intensities, and optical over acoustical band area ratios) versus ion fluence are reported, and discussed. All acoustic and optical bands increase and broaden with fluence. Yet no amorphization is achieved under the highest fluence for both carbides. From the lowest fluence up to 10 15 ions/cm 2 , point defects like interstitials and vacancies are created. The increasing number of carbon vacancies involves a local variation in stoichiometry accompanied by carbon release. For fluence above 10 15 ions/cm 2 , extended defects like dislocation loops appear, as seen by TEM observations. more...

Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods. more...

Debilitating heart conditions, notably dilated and hypertrophic cardiomyopathies (CM), are associated with point mutations in metavinculin, a larger isoform of the essential cytoskeletal protein vinculin. Metavinculin is co-expressed with vinculin at sub-stoichiometric ratios in cardiac tissues. Cardiomyopathy mutations in the metavinculin tail domain (MVt) occur within the extra 68-residue insert that differentiates it from the vinculin tail domain (Vt). Vt binds actin filaments (F-actin) and promotes vinculin dimerization to bundle F-actin into thick fibers. While MVt binds to F-actin in a similar manner to Vt, MVt is incapable of F-actin bundling and inhibits Vt-mediated F-actin bundling. We performed F-actin co-sedimentation and negative-stain EM experiments to dissect the coordinated roles of metavinculin and vinculin in actin fiber assembly and the effects of three known metavinculin CM mutations. These CM mutants were found to weakly induce the formation of disordered F-actin assemblies. Notably, they fail to inhibit Vt mediated F-actin bundling, and instead promote formation of large assemblies embedded with linear bundles. Computational models of MVt bound to F-actin suggest that MVt undergoes a conformational change licensing the formation of a protruding sub-domain incorporating the insert, which sterically prevents dimerization and bundling of F-actin by Vt. Sub-domain formation is destabilized by CM mutations, disrupting this inhibitory mechanism. These findings provide new mechanistic insights into the ability of metavinculin to tune actin organization by vinculin and suggest that dysregulation of this process by CM mutants could underlie their malfunction in disease. more...

Extracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer. The negative regulatory factor (Nef) encoded by simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) plays a critical role in the progression to AIDS and impairs endosomal trafficking. Whether HIV-1 Nef can be loaded into EVs has been the subject of controversy, and nothing is known about the connection between SIV Nef and EVs. We find that both SIV and HIV-1 Nef proteins are present in affinity-purified EVs derived from cultured cells, as well as in EVs from SIV-infected macaques. Nef-positive EVs were functional, i.e., capable of membrane fusion and depositing their content into recipient cells. The EVs were able to transfer Nef into recipient cells. This suggests that Nef readily enters the exosome biogenesis pathway, whereas HIV virions are assembled at the plasma membrane. It suggests a novel mechanism by which lentiviruses can influence uninfected and uninfectable, i.e., CD4-negative, cells., IMPORTANCE Extracellular vesicles (EVs) transfer biologically active materials from one cell to another, either within the adjacent microenvironment or further removed. EVs also package viral RNAs, microRNAs, and proteins, which contributes to the pathophysiology of infection. In this report, we show that both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) incorporate the virus-encoded Nef protein into EVs, including EVs circulating in the blood of SIV-infected macaques and that this presents a novel mechanism of Nef transfer to naive and even otherwise non-infectable cells. Nef is dispensable for viral replication but essential for AIDS progression in vivo. Demonstrating that Nef incorporation into EVs is conserved across species implicates EVs as novel mediators of the pathophysiology of HIV. It could help explain the biological effects that HIV has on CD4-negative cells and EVs could become biomarkers of disease progression. more...

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. more...

Hyaluronic acid (HA) is a polyanionic natural polymer occurring as a linear polysaccharide composed of glucuronic acid and N-acetylglucosamine repeats. Hyaluronic acid has a wide range of applications with its excellent physicochemical properties such as biodegradability, biocompatibility, non-toxicity, non-immunogenicity and serves as an excellent tool in biomedical applications such as osteoarthritis surgery, ocular surgery, plastic surgery, tissue engineering and drug delivery. This work provides an overview on hyaluronic acid, its chemistry and biochemistry and its medical applications. more...

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with the human malignancy Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. KSHV establishes lytic infection of monocytes in vivo, which may represent an important cellular reservoir during KS disease progression. KS tumors consist of latently infected endothelial cells; however, lytic phase gene products are important for KS onset. Early KS lesion progression is driven by proinflammatory cytokines supplied by immune cell infiltrates including T cells and monocytes. KSHV-infected monocytes may supply the lytic viral products and the inflammatory milieu conducive to KS tumor progression. To establish successful infection, KSHV extensively modulates the host immune system. KSHV antigens activate both innate and adaptive immune responses including KSHV-specific T cells, but lifelong infection is still established. Programmed death ligand 1 (PD-L1) is a prosurvival cell surface protein that suppresses T-cell-mediated killing. PD-L1 is variably present on various tumor cells and is a targetable marker for cancer treatment. We show that KSHV infection of human monocytes increases PD-L1 expression and transcription in a dose-dependent manner. We also saw evidence of lytic gene expression in the KSHV-infected monocytes. Intact KSHV is needed for full PD-L1 response in human monocytes. KSHV induces a general proinflammatory cytokine milieu including interleukins 1α, 1β, and 6, which have been implicated in early KS lesion progression. KSHV-mediated PD-L1 increase may represent a novel mechanism of KSHV-mediated immune modulation to allow for virus survival and eventually malignant progression., IMPORTANCE KSHV is the etiologic agent of Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. Programmed death ligand 1 (PD-L1) is an immunosuppressive cell surface marker that inhibits T cell activation. We report that KSHV infection of primary human monocytes upregulates PD-L1 transcription and protein expression. Analysis of the cytokine and chemokine milieu following KSHV infection of monocytes revealed that KSHV induces interleukins 1α, 1β, and 6, all of which have been implicated in KS development. Our work has identified another potential immune evasion strategy for KSHV and a potential target for immunotherapy of KSHV-derived disease. more...

Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology, motility, and force transduction. Vinculin is distinct from metavinculin in its ability to bundle filamentous actin (F-actin). To elucidate the molecular basis for these differences, we employed computational and experimental approaches. Results from these analyses suggest that the C terminus of both vinculin and metavinculin form stable interactions with the F-actin surface. However, the metavinculin tail (MVt) domain contains a 68 amino acid insert, with helix 1 (H1) sequestered into a globular subdomain, which protrudes from the F-actin surface and prevents actin bundling by sterically occluding actin filaments. Consistent with our model, deletion and selective point mutations within the MVt H1 disrupt this protruding structure, and facilitate actin bundling similar to vinculin tail (Vt) domain. more...

Silver nanoparticles (AgNPs) are widely used in consumer and pharmaceutical products due to their antipathogenic properties. However, safety concerns have been raised due to their bioactive properties. While reports have demonstrated AgNPs can embed within the extracellular matrix, their effects on basement membrane (BM) production, integrin engagement, and tissue-integrity are not well-defined. This study analyzed the effects of AgNPs on BM production, composition and integrin/focal adhesion interactions in representative lung, esophageal, breast and colorectal epithelia models. A multidisciplinary approach including focused proteomics, QPCR arrays, pathway analyses, and immune-based, structural and functional assays was used to identify molecular and physiological changes in cell adhesions and the BM induced by acute and chronic AgNP exposure. Dysregulated targets included CD44 and transforming growth factor-beta, two proteins frequently altered during pathogenesis. Results indicate AgNP exposure interferes with BM and cell adhesion dynamics, and provide insight into the mechanisms of AgNP-induced disruption of epithelial physiology. more...

ObjectiveTo examine the association between concussions and lower extremity musculoskeletal injury (LE-MSI) rates in Reserve Officer Training Corps (ROTC) cadets.BackgroundConcussions have been associated with an increased risk for LE-MSI among high school, collegiate, and professional athletes as well as U.S. Army Soldiers. However, there is a paucity evidence on this relationship among U.S. Army ROTC cadets, future U.S. Army Officers, and a group similar to student-athletes in regards to physical activity levels.Design/MethodsA modified reliable injury questionnaire (ICC = 0.92) was used to identify the total number of reported concussions, intentionally unreported concussions, and potentially unrecognized concussions (e.g., memory loss not diagnosed as a concussion) as well as LE-MSI (e.g., muscle strains, ACL rupture) a cadet had suffered. A chi-square analysis was performed to identify the association between concussion and LE-MSI and any concussive injury and LE-MSI.Results47 cadets (19.9 ± 1.3 years) were recruited from one Army ROTC program. There was not a significant association between reported concussions and LE-MSI (Χ(1) = 3.122, p = 0.077). There was not a significant association between any concussive injury (reported, unreported, or potentially unrecognized) and LE-MSI (Χ(1) = 3.590, p = 0.058). The reported concussion history was 38.3% (18/47), any concussive history was 46.8% (22/47), and 68.1% (32/47) reported history of LE-MSI.ConclusionsPreliminary results showed that there was no statistically significant association between concussion and LE-MSI among ROTC cadets at this university. Future research is warranted on a larger cohort of cadets to determine if this relationship exists since cadets will soon commission, potentially risking injury while serving on active duty, causing limited duty days, reduced Department of Defense readiness, and increased healthcare costs. Cadets showed a high incidence of concussion and LE-MSK injury, and future research should target reducing these injuries among ROTC cadets prior to commissioning. more...

Background The independent prognostic impact of diabetes mellitus ( DM ) and prediabetes mellitus (pre‐ DM ) on survival outcomes in patients with chronic heart failure has been investigated in observational registries and randomized, clinical trials, but the results have been often inconclusive or conflicting. We examined the independent prognostic impact of DM and pre‐ DM on survival outcomes in the GISSI ‐HF (Gruppo Italiano per lo Studio della Sopravvivenza nella Insufficienza Cardiaca‐Heart Failure) trial. Methods and Results We assessed the risk of all‐cause death and the composite of all‐cause death or cardiovascular hospitalization over a median follow‐up period of 3.9 years among the 6935 chronic heart failure participants of the GISSI ‐ HF trial, who were stratified by presence of DM (n=2852), pre‐ DM (n=2013), and non‐ DM (n=2070) at baseline. Compared with non‐ DM patients, those with DM had remarkably higher incidence rates of all‐cause death (34.5% versus 24.6%) and the composite end point (63.6% versus 54.7%). Conversely, both event rates were similar between non‐ DM patients and those with pre‐ DM . Cox regression analysis showed that DM , but not pre‐ DM , was associated with an increased risk of all‐cause death (adjusted hazard ratio, 1.43; 95% CI , 1.28–1.60) and of the composite end point (adjusted hazard ratio, 1.23; 95% CI , 1.13–1.32), independently of established risk factors. In the DM subgroup, higher hemoglobin A1c was also independently associated with increased risk of both study outcomes (all‐cause death: adjusted hazard ratio, 1.21; 95% CI , 1.02–1.43; and composite end point: adjusted hazard ratio, 1.14; 95% CI , 1.01–1.29, respectively). Conclusions Presence of DM was independently associated with poor long‐term survival outcomes in patients with chronic heart failure. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 00336336. more...

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2. more...

Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize. more...

Background: Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo. Methods: HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP). Results: VL-17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced. Conclusions: Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD. more...

Extracellular signaling is a mechanism that higher eukaryotes have evolved to facilitate organismal homeostasis. Recent years have seen an emerging interest in the role of secreted microvesicles, termed extracellular vesicles (EV) or exosomes in this signaling network. EV contents can be modified by the cell in response to stimuli, allowing them to relay information to neighboring cells, influencing their physiology. Here we show that the tumor virus Kaposi’s Sarcoma-associated herpesvirus (KSHV) hijacks this signaling pathway to induce cell proliferation, migration, and transcriptome reprogramming in cells not infected with the virus. KSHV-EV activates the canonical MEK/ERK pathway, while not alerting innate immune regulators, allowing the virus to exert these changes without cellular pathogen recognition. Collectively, we propose that KSHV establishes a niche favorable for viral spread and cell transformation through cell-derived vesicles, all while avoiding detection., Author summary The role of extracellular vesicles (EV) has received considerable attention in recent years. The contents of these cell-derived vesicles have been shown to be modulated upon challenge by a virus or neoplastic transformation, and can influence the behavior of recipient cells. Here we demonstrate that purified EV from an AIDS-associated, virus-driven lymphoma induces unique cellular signaling, motility, and gene expression reprogramming in recipient endothelial cells. This was accomplished without activation of innate immune activation, even after prolonged exposure to these EV. Collectively our results point toward a model in which tumor-derived EV condition neighboring cell physiology, while avoiding detection by immune regulators. more...

Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses. more...

To perform quantitative live cell imaging, investigators require fluorescent reporters that accurately report protein localization and levels, while minimally perturbing the cell. Yet, within the biochemically distinct environments of cellular organelles, popular fluorescent proteins (FPs), including EGFP, can be unreliable for quantitative imaging, resulting in the underestimation of protein levels and incorrect localization. Specifically, within the secretory pathway, significant populations of FPs misfold and fail to fluoresce due to non-native disulphide bond formation. Furthermore, transmembrane FP-fusion constructs can disrupt organelle architecture due to oligomerizing tendencies of numerous common FPs. Here, we describe a powerful set of bright and inert FPs optimized for use in multiple cellular compartments, especially oxidizing environments and biological membranes. Also, we provide new insights into the use of red FPs in the secretory pathway. Our monomeric ‘oxFPs’ finally resolve long-standing, underappreciated and important problems of cell biology and should be useful for a number of applications. Quantitative live cell imaging of protein trafficking suffers from misfolding and inappropriate disulphide bond formation of fluorescent proteins in the secretory pathway. Here, the authors present an optimized collection of fluorescent proteins suitable for use in oxidizing subcellular compartments. more...

Several single phase hollandite ceramics having Ba2+ xCs+ y(C = Al,Fe)3+ 2x+y Ti4+ 8−2x−y O16 composition have been synthesized and irradiated under external electron beams simulating the β-irradiation of radioactive cesium. These samples have been characterized by 57Fe transmission Mossbauer spectroscopy. Mossbauer results show that the irradiation modifies the local arrangements of Ba cations around Fe3+ ions by atomic displacement of Ba ions. more...

The phase behavior of the binary system consisting of the supercritical fluid carbon dioxide (CO2) and the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]) was studied experimentally. A synthetic method was used to measure its phase behavior. Bubble-point pressures of the system CO2 + [bmim][BF4] are reported for carbon dioxide concentrations ranging from (10.22 to 60.17) mole % and within a temperature range of (278.47 to 368.22) K. The CO2 + [bmim][BF4] binary system has a two-phase liquid-vapor region extending up to very high pressures. Most likely, the type of fluid-phase behavior is type III according to the classification of Scott and Van Konynenburg. The experimental results obtained were compared with the available phase behavior data of the binary system CO2 + 1-hexyl-3-methylimidazolium tetrafluoroborate ([hmim][BF4]) to investigate the effect of the length of the alkyl group on the phase behavior of this type of system. A larger alkyl group leads to lower bubble-point pressures and, therefore, to higher solubilities of carbon dioxide in the imidazolium-based ionic liquid more...

The occurrence of neonatal sandbar sharks Carcharhinus plumbeus in the Adriatic Sea is reported; however, nursery areas still need to be identified.

A newborn baby boy presented with a full thickness defect of the scalp, skull, and dura measuring 6 x 7 cm caused by aplasia cutis congenita. Full thickness loss is extremely rare and to our knowledge this case is the twenty-first reported. It has an established mortality of up to 55%. An encephalocele forced us to do an emergency operation with a single large scalp flap based on the supratrochlear and superficial temporal arteries. After revision the child is now 9 months old and progressing well. There are several ways to treat these rare and delicate cases. more...

This is the protocol for a review and there is no abstract. The objectives are as follows: To assess the effectiveness and cost-effectiveness of inpatient specialist palliative care in acute hospitals for adults with advanced illness and their unpaid caregivers. more...

The right length of the myotomy on the gastric side for esophageal achalasia is still a debated issue. We aimed to investigate the final outcome after classic myotomy (CM) as compared with a longer myotomy on the gastric side (LM) in two cohorts of achalasia patients. Forty-four achalasia patients who underwent laparoscopic Heller-Dor were considered; patients with a sigmoid-shaped esophagus were excluded. Symptoms were scored using a detailed questionnaire for dysphagia, regurgitation, and chest pain. Barium swallow, endoscopy, and esophageal manometry were performed before and 6 months after the surgical treatment; 24-hour pH-monitoring was also performed 6 months after the procedure. CM was defined as a gastric myotomy length in the range of 1.5-2.0 cm, while LM was 2.5-3 cm in length. The surgical treatment (CM or LM) was adopted in two consecutive cohorts. Treatment failure was defined as a postoperative symptom score10th percentile of the preoperative score (i.e.8). Of the 44 patients representing the study population, 20 had CM and 24 had LM. The patients' demographic and clinical parameters (age, sex, symptom score, duration of symptoms, esophageal diameter, and manometric pattern) were similar in the two groups. The median follow up was 24 months (interquartile range 12-39). One patient in each group was classified as a treatment failure. After the treatment, there was a significant decrease in both groups' symptom score, and resting and residual pressure (P0.01), with no statistically significant differences between the two groups in terms of postoperative symptom score, resting and residual pressure, or total and abdominal lower esophageal sphincter length and esophageal diameter. Extending the length of the myotomy on the gastric side does not seem to change the final outcome of the laparoscopic Heller-Dor procedure. more...

We have used electron spin resonance spectroscopy to study the defects induced in yttria-stabilized zirconia (YSZ) single crystals by 2.5-MeV electron irradiations. Two paramagnetic centers are produced: the first one with an axial symmetry is similar to the trigonal Zr 3+ electron center (T center) found after X-ray irradiation or thermochemical reduction, whereas the second one is a new oxygen hole center with an axial symmetry different from the orthorhombic O - center induced by X-ray irradiation. At a fluence around 10 18 e/cm , both centers are bleached out near 600 K, like the corresponding X-ray induced defects. At a fluence around 10 19 e/cm 2 , defects are much more stable, since complete thermal bleaching occurs near 1000 K. Accordingly, ageing of as-irradiated samples shows that high-dose defects at more stable than the low-dose ones. more...

Natural quartz single crystals (α-SiO2) have been exposed to pulsed heavy ion beams (12C, 19F, 32S) with energies of 1 MeV amu−1 in the electronic slowing down regime. The simultaneous recording of the ion fluence and emitted photons with time-resolved spectroscopy experiments allowed the measurement of the “blue luminescence” time decay at 85 K as a function of the fluence at the various electronic stopping power, Se=(−dE/dx)e, of the ions. For all ions, regardless of fluence, the spectra are similar and have two broad bands centered at 1.60 and 2.75 eV with full widths at half maximum around 0.30 and 0.75 eV, respectively. Single-exponential time decay curves are found regardless of Se increasing from 1.4 keV nm−1 (12 MeV 12C) to 5.2 keV nm−1 (32 MeV 32S) across the amorphous track-formation threshold at 2.5±0.5 keV nm−1. At low damaged fractions (⩽22%), the decay-time constant ranges between 1.0 and 1.6 ms. The luminescence intensities at zero delay time approximately decrease in an exponential fashion... more...

The Philadelphia chromosome, t(9;22)(q34;q11) gives rise more frequently, in chronic myeloid leukaemia (CML), to two BCR/ABL chimeric transcripts differing only by the absence of 75 nucleotides and defined as b2a2 and b3a2 types, encoding two 210-kDa tyrosine kinase proteins differing only by the absence of 25 amino acids coded by the b3 exon. In the present study the two transcripts, detected by RT-PCR in 88 consecutive unselected CML patients, were correlated with haematological findings at diagnosis and with the megakaryocyte size and frequency by morphometric evaluation of 45 bone marrow biopsies. The secondary structure prediction and hydrophobicity of the b2a2 and b3a2 type BCR/ABL protein were also obtained. The prediction results for the b3 exon amino acids using GOR IV and NnPredict methods showed a short beta strand corresponding to the hydrophobic portion of the peptide. Significantly higher values were found in the platelet count of patients carrying b3a2 transcripts. The megakaryocyte size and frequency in bone marrow biopsies did not show significant differences between the two groups of patients. Stratifying the patients on the basis of white blood cell (WBC) count below or above 100x10(9)/l we still had, in both groups, a significant difference in the platelet count between the b2a2 and b3a2 patients. The possible relationships between the structure of b2a2 and b3a2 types of BCR/ABL fused protein and thrombopoiesis are discussed. more...

The room-temperature dc conductivity is used to monitor the damage and structural modifications induced by swift heavy ion irradiations in yttrium iron garnet (Y3Fe5O12 or YIG) epitaxial layers doped with calcium (CaYIG) or silicon (SiYIG), with a variable conductivity due to a variable degree of compensation, and amorphous YIG layers. Irradiations are performed with heavy ions in the 0.8–6 MeV amu−1 energy range, in the electronic slowing down regime, with an electronic stopping power ranging between 7 and 41 MeV μm−1 above the amorphous track formation threshold (4.5 MeV μm−1) in this low-ion velocity range. A conductivity decrease versus ion fluence is found in the case of the high-conductivity uncompensated epilayers whereas an increase occurs for the low-conductivity compensated ones, either p-type (CaYIG) or n-type (SiYIG). These results are discussed by considering the competing effects of disorder on the carrier density and mobility in the case of compensated and uncompensated semiconductors. In b... more...