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The AE765, AK692, AO233 and AO234 antibodies recognize human actin in PAF-fixed HEK cells by immunofluorescence. Phalloidin, which recognizes polymerized actin, is more adequate to label actin filamentous structures. The AK423 antibody recognizes human actin in methanol-fixed HEK cells, but in these conditions the actin network is poorly preserved.

The AK423 antibody, raised against Dictyostelium actin, recognizes human actin by western blot; AE765, AK692, AO233 and AO234 do not.

The recombinant antibodies AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 detect by western blot the spike S protein from SARS-CoV-2.

Inpatients with cancer are likely to experience care dependency and would benefit from nurses' evaluation of such dependency. The Care Dependency Scale (CDS) is a 15-item instrument widely used to assess patients' dependency in different populations and settings. This study aimed to test the psychometric properties of the proxy Italian version of the CDS in inpatients with cancer.A multicentre cross-sectional cross-validation study was conducted between February 2016 and October 2017 in a convenience sample of 517 adult patients with cancer admitted to four hospitals in Italy. The sample was randomly divided into two subsamples. The factor structure of the CDS based on previous studies was tested on a subsample through confirmatory factor analyses (CFAs). The best fitting model was cross-validated through CFA on the second subsample and on the total sample.The sample mean age was 65.68 years, 51.5% were male. The CFAs performed on the first subsample (n = 258), on the second subsample (n = 259) and on the total sample yielded acceptable fit indexes. The factors Physical care dependency and Psychosocial care dependency with a second-order factor were confirmed. Reliability in terms of internal consistency and inter-rater reliability were satisfactory.The Care Dependency Scale is a tool able to assess the level of care dependency in patients with cancer with adequate validity and reliability. The Care Dependency Scale can help to distinguish between physical and psychosocial needs and to create a base for personalized patient care.

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

Background and ObjectivesAgeism toward older adults, prevalent in contemporary societies, seems to be internalized during childhood and consolidated during adolescence. Although several instruments have been developed to measure adolescents’ ageism, they present a number of limitations. The study aimed at developing a new instrument, the Adolescents’ Ageism Toward Older Adults Scale, and testing its psychometric properties.Research Design and MethodsA three-phase process was followed: The instrument’s items were developed empirically from focus groups of adolescents; its content validity was evaluated; and finally, its psychometric properties were tested through a multicenter cross-sectional study involving 575 adolescents.ResultsExploratory factor analysis of the new scale retained 33 items grouped into 6 factors: moodiness, traditionalism, physical deterioration, antitechnologism, sageness, and sociability. Confirmatory factor analysis evidenced a second-order factor structure. Omega coefficients measuring internal consistency were above the cutoff of 0.60 for the 6 factors except for antitechnologism. Intraclass correlation coefficients for the entire scale and for the 6 factors were greater than 0.70, again excepting antitechnologism.Discussion and ImplicationsThe Adolescents’ Ageism Toward Older Adults Scale is a promising measure of adolescents’ negative and positive attitudes toward older adults. Its validation highlighted some criticalities that can be resolved by a few modifications. Further testing of the scale should be conducted after these modifications.

Background: Remote virtual rehabilitation aroused growing interest in the last decades, and its role has gained importance following the recent spread of COVID19 pandemic. The advantages of virtual reality (VR), augmented reality (AR), gamification, and telerehabilitation have been demonstrated in several medical fields. In this review, we searched the literature for studies using these technologies for orthopedic rehabilitation and analyzed studies’ quality, type and field of rehabilitation, patients’ characteristics, and outcomes to describe the state of the art of VR, AR, gamification, and telerehabilitation for orthopedic rehabilitation. Methods: A comprehensive search on PubMed, Medline, Cochrane, CINAHL, and Embase databases was conducted. This review was performed according to PRISMA guidelines. Studies published between 2015 and 2020 about remote virtual rehabilitations for orthopedic patients were selected. The Methodological Index for Non-Randomized Studies (MINORS) and Cochrane Risk-of-Bias assessment tool were used for quality assessment. Results: 24 studies (9 randomized controlled trials (RCTs) and 15 non-randomized studies) and 2472 patients were included. Studies mainly concern telerehabilitation (56%), and to a lesser extent VR (28%), AR (28%), and gamification (16%). Remote virtual technologies were used following knee and hip arthroplasty. The majority of included patients were between 40 and 60 years old and had a university degree. Remote virtual rehabilitation was not inferior to face-to-face therapy, and physical improvements were demonstrated by increased clinical scores. Orthopedic virtual remote rehabilitation decreased costs related to transports, hospitalizations, and readmissions. Conclusion: The heterogeneity of included studies prevented a meta-analysis of their results. Age and social context influence adaptability to technology, and this can modify compliance to treatment and outcomes. A good relationship between patient and physiotherapist is essential for treatment compliance and new technologies are useful to maintain clinical interactions remotely. Remote virtual technologies allow the delivery of high-quality care at reduced costs. This is a necessity given the growing demand for orthopedic rehabilitation and increasing costs related to it. Future studies need to develop specific and objective methods to evaluate the clinical quality of new technologies and definitively demonstrate advantages of VR, AR, gamification, and telerehabilitation compared to face-to face orthopedic rehabilitation.

Background and objectives: Our research aimed to evaluate the quality of reporting of randomized controlled trials (RCTs) linked to rotator cuff (RC) tears. The present study analyzed factors connected to the quality of the RCTs and trends in the quality of reporting through time. Materials and Methods: The online databases used to search all RCTs on the topic of RC surgery completed until March 2020 were PubMed and Ovid (MEDLINE). The quality of reporting was evaluated using the modified Coleman methodology score (MCMS) and the consolidated standards of reporting trials (CONSORT). Results: The online search found 957 articles. Finally, 183 studies were included in the quantitative synthesis. A total of 97 (53%) of 183 studies had a level of evidence I and 86 (47%) of 183 studies had a level of evidence II, according to the Oxford Center of Evidence Based Medicine (EBM). A statistically significant difference in MCMS between articles written before 2010 and articles written after 2010 was found. Articles written after 2010 had, on average, the highest Coleman score. The average number of CONSORT checklist items for each article across all analyzed RCTs was 21.67. The 37 studies completed up to 2010 averaged a number of checklist items of 19.97 and the studies completed between 2011 and 2019 averaged a number of checklist items of 22.10. A statistically significant difference in the number of checklist items between articles written before 2010 and articles written after 2010 was found. Articles written after 2010 had on average more checklist items. However, low correlation (0.26) between the number of checklist items for each article and the respective Coleman score was found. On the other hand, articles with the CONSORT diagram had a significantly high Coleman score. Conclusions: An improvement in the quantity and quality of RCTs relating to RC surgery over the analyzed period was found.

Background: Caregivers represent the core of patients’ care in hospital structures, in the process of care and self-care after discharge. We aim to identify the factors that affect the strain of caring for orthopedic patients and how these factors are related to the quality of life of caregivers. We also want to evaluate the role of caregivers in orthopedic disease, focusing attention on the patient–caregiver dyad. Methods: A comprehensive search on PubMed, Cochrane, CINAHL and Embase databases was conducted. This review was reported following PRISMA statement guidance. Studies were selected, according to inclusion and exclusion criteria, about patient–caregiver dyads. For quality assessment, we used the MINORS and the Cochrane Risk of BIAS assessment tool. Results: 28 studies were included in the systematic review; in these studies, 3034 dyads were analyzed. Caregivers were not always able to bear the difficulties of care. An improvement in strain was observed after behavioral interventions from health-care team members; Conclusions: The role of the caregiver can lead to a deterioration of physical, cognitive and mental conditions. The use of behavioral interventions increased quality of life, reducing the strain in caregivers of orthopedic patients. For this reason, it is important to consider the impact that orthopedic disease has on the strain of the caregiver and to address this topic.

Background Patients in palliative care are the population cohort that most frequently develop pressure injuries, severely impacting their quality of life. Data from prospective studies on the prevalence and incidence of pressure injuries in hospices are lacking. Aim To describe the point prevalence and cumulative incidence of pressure injuries in patients admitted to residential hospices, and to analyze their predictive factors over time. Design Multicentre prospective longitudinal observational study. Setting/participants Adult patients (n = 992) enrolled in 13 Italian residential hospices, with a minimum sample of 280 for each macro-region (North, center, South/Islands). Methods Assessments including the Karnofsky Performance Status, Braden, Edmonton Symptom Assessment System Revised scales and pressure injury staging according to National Pressure Ulcer Advisory Panel were conducted at least every four days, from admission to patients’ death/discharge. Results The 7,967 observations recorded provided prevalence and incidence rates of 34.1% and 26.5%, respectively. The logistic regression model showed non-cancer disease (OR = 2.39, 95%CI = 1.65–3.47), age >80 (OR = 2.01, 95%CI = 1.49–2.71), Braden score ‘at risk’ (OR = 1.92, 95%CI = 1.17–3.14), urinary catheter (OR = 1.96, 95%CI = 1.40–2.75), drowsiness (OR = 1.41, 95%CI = 1.02–1.95) and artificial nutrition (OR = 1.47, 95%CI = 1.01–2.14) as the variables associated with pressure injury at admission. The generalized estimating equations models, built on the timeframes for observation groups, revealed male gender (OR = 1.68, 95%CI = 1.01–2.79) and Braden score ‘at risk’ (OR = 4.45, 95%CI = 1.74–11.34) as predictive factors of a new pressure injury developed up to three weeks before a patient's death, while in the last ten days of life these predictors were replaced by diagnosis of cancer (OR = 1.80, 95%CI = 1.11–2.91), worsening pain (OR = 1.65, 95%CI = 1.10–2.49), drowsiness (OR = 1.79, 95%CI = 1.25–2.57) and dyspnea (OR = 1.48, 95%CI = 1.01–2.18). Conclusions The high incidence and prevalence of pressure injuries confirm the importance of palliative care nurses continuously focusing on prevention and management strategies. In the last three weeks of a patient's life, the predictive power of the Braden scale for a new pressure injury is not confirmed, throwing doubt on the effectiveness of aimed interventions at modifying risk factors. Along the different disease trajectories, pressure injuries developed during the instability/worsening phases of illness, occurring before hospice admission for non-cancer patients and in the end-of-life phase for cancer patients. Despite continuous provision of appropriate interventions, most of the new pressure injuries were detected during the last ten days of a patient's’ life and assessed as 'unavoidable'. These results are crucial to guiding palliative care nursing plans during the different phases of illness, and to predicting care needs, possible management strategies (‘wound management’ vs. ‘wound palliation’), and resource utilization.

Background: Advanced cancer patients often die in hospital after receiving needless, aggressive treatment. Although palliative care improves symptom management, barriers to accessing palliative care services affect its utilisation, and such disparities challenge the equitable provision of palliative care. This study aimed to identify which factors are associated with inequitable palliative care service utilisation among advanced cancer patients by applying the Andersen Behavioural Model of Health Services Use.Material and methods: This was a retrospective cohort study using administrative healthcare data. A total of 13,656 patients residing in the Lazio region of Italy, who died of an advanced cancer-related cause-either in hospital or in a specialised palliative care facility-during the period of 2012-2016 were included in the study. Potential predictors of specialised palliative service utilisation were explored by grouping the following factors: predisposing factors (i.e., individuals' characteristics), enabling factors (i.e., systemic/structural factors) and need factors (i.e., type/severity of illness).Results: The logistic hierarchical regression showed that older patients (odds ratio [OR] = 1.45

Vaccine hesitancy represents an increasingly important global health issue. Nurses, together with other healthcare professionals, can play a key role in increasing vaccination uptake. The aim of this article is to analyze the main elements of positive communication styles used in vaccination campaigns and initiatives. The most important components in style, construction and contents used in several vaccination campaigns were evaluated through an original instrument, specifically developed for the present study. The most relevant aspects of nursing expertise and competencies were mixed with foundations of mass communication theory, in order to develop a framework made by 13 items, useful to evaluate different aspects of immunization campaigns. A multi-professional project was developed, to obtain a deep integration between nurse expertise in health promotion and other groups with similar public health orientation. The Authors further evaluated sociological instruments and theories from other fields, as mass communication theory and the social/behavioral approach. An integrative review was performed, about the following main themes: vaccination adherence basis; nurse role in health promotion; health education competences; multi-professionals integration in public health; positive communication style; efficacy evaluation. Key elements were collected, to prepare an original instrument to be used in evaluating several communication campaigns. The evaluation framework (13 items) was prepared and applied to vaccine communication in different institutional settings in the last five years. Main components are: elements taken from main communication models (persuasive communication models); nurse competencies in health education; public health expertise (from an expert panel). The main findings of this study regard the usefulness of evaluating main aspects of communication in the vaccine field. Nurses' knowledge and competencies in health promotion and health education add relevant meanings and cues to act against the causes for not vaccinating. It shows that is worth analyzing the main aspects of communication techniques and evaluating innovative methods to increase population confidence levels in public health.

The recombinant antibodies AC650, AC653, AC656 and AD460 detect by immunofluorescence the human CD8α protein.

The recombinant antibodies AC650, AC653, AC656 and AD460 detect the human CD8α protein by flow cytometry.

The number of patients using palliative care services, particularly residential hospices, is increasing. Policymakers are urging these services to reflect on the most effective organizational strategies for meeting patients' complex care needs.To analyze the predictive power of staffing, structure and process indicators towards optimal control of patients' clinically significant symptoms over time.Secondary analysis of data from a multicentre prospective longitudinal observational study (PRELUdiHO) collected between November 2017 and September 2018.Adult patients (n = 992) enrolled in 13 Italian residential hospices.Two generalized estimating equations logistic models were built, both with number of hospice beds and length of stay as independent variables as well as, in one case, patient-to-healthcare worker ratios, and, in the other, health professionals' qualification levels. Dependent variables were six not clinically significant (score4) symptoms: pain, nausea, shortness of breath, feeling sad, feeling nervous, and 'how you feel overall', according to the Edmonton Symptom Assessment System revised (ESAS-r) scale.The generalized estimating equations indicators on staff revealed the following 'optimal' model: Patient-to-Physician ratio (5.5:1-6.5:1); Patient-to-Nurse ratio (1.5:1-2.7:1); Patient-to-Nurse-Assistant ratio (4.1:1-6.3:1); with the most balanced staff composition including 19% physicians, 23% nurse assistants, and 58% registered nurses; hospice beds (12-25); length of stay (median = 12 days). This model predicted an up to four times greater likelihood of controlling all six ESAS-r symptoms over time. The generalized estimating equations model on the educational level of physicians and registered nurses showed that it was significantly associated with optimal patients' symptom control during the entire hospice stay.This study showed the exact skill-mix composition and proportions of palliative care team able to ensure optimal control of patients' symptoms. The added value of physicians and nurses with a qualification in palliative care in terms of better patient outcomes reaffirmed the importance of education in guaranteeing quality care. Hospices with 12-25 beds, and recruitment methods guaranteeing at least 12-day stay ensured the most propitious organizational environment for optimal management of clinically significant symptoms. The transferability of these results mainly depends on whether the skills of health professionals in our `ideal' model are present in other contexts. Our results provide policymakers and hospice managers with specific, evidence-based information to support decision-making processes regarding hospice staffing and organization. Further prospective studies are needed to confirm the positive impact of this 'optimal' organizational framework on patient outcomes.

This study aimed to: (1) to assess the Quality of Life (QoL) of pediatric patients who had undergone Hematopoietic Stem Cell Transplantation (HSCT) and their parents; (2) to explore how patients and parents perceived a nurse counseling intervention; and (3) to explore how health professionals consider the nurse counselor's role.This was a pilot descriptive study using quantitative and qualitative data.Parents' QoL was compromised in the domains of Social Functioning, Physical and Emotional roles. Patients' QoL was poor in all the domains except in Behavior and Family Cohesion. Patients and their parents considered the counseling as an effective help. Seven categories emerged from the analysis of qualitative data: support and taking charge, empathy, active communication and listening, evasion, sharing, awareness, and organizational availability. Regarding health professionals, five categories emerged from the analysis of the interviews and a focus group: support and taking charge, recovery in self-expression, support in relationship with patients and family, connecting role, and difficult integration within the team.The nurse counselor could empower patients and parents to improve their attitudes towards care pathway. The nurse counselor can be an important resource in the context of HSCT, even if greater integration within the team is required.Questo studio è stato condotto allo scopo di: (1) valutare la Qualità di Vita (QdV) dei pazienti pediatrici che sono stati sottoposti a trapianto di cellule staminali ematopoietiche e dei loro genitori; (2) esplorare come i pazienti ed i loro genitori hanno vissuto un intervento infermieristico di counselling; e (3) esplorare come i professionisti sanitari considerano il ruolo dell’infermiere counselor.Questo è uno studio pilota a scopo descrittivo che utilizza dati qualitativi e quantitativi.La QdV dei genitori è risultata compromessa nei domini relativi al Funzionamento Sociale ed i ruoli Fisico ed Emotivo. La QdV dei pazienti è risultata scarsa in tutti i domini ad eccezione del Comportamento e della Coesione familiare. I pazienti ed i loro genitori hanno ritenuto che l’intervento di counseling fosse un aiuto concreto. Dall’analisi dei dati qualitativi sono emerse sette categorie: supporto e presa in carico, empatia, comunicazione e ascolto attivo, evasione, condivisione, consapevolezza, e disponibilità organizzativa. Per quanto riguarda i professionisti sanitari, sono emerse cinque categorie dall’analisi delle interviste e del focus group: supporto e presa in carico, guarigione attraverso l’espressione del sé, supporto nella relazione con i pazienti e la famiglia, ruolo di connessione ed integrazione difficile nell’equipe.L’infermiere counselor potrebbe consentire a pazienti e genitori di migliorare il loro atteggiamento nei confronti del percorso assistenziale. L’infermiere counselor può essere una importante risorsa nel contesto trapiantologico. È necessaria una maggiore integrazione di questa figura all’interno dell’equipe.

International literature reports that nursing students feel unprepared when facing patients and families within dying care. They consider their curricula inadequate in teaching end-of-life care and promoting the attitudes required to care for dying patients. Findings of recent studies exploring nursing students' attitudes towards care of the dying patient are often contradictory.To explore Italian nursing students' attitudes towards caring for dying patients.A multicenter cross-sectional study was conducted.The Bachelor's Degree in Nursing courses of four Universities of the Lazio Region.The sample included 1193 students.Data were collected between September 2017 and March 2018 using the Italian version of FATCOD-B-I. The differences between the mean scores were compared through t-test or ANOVA. Associations between scores and participant characteristics were evaluated through generalized linear regression.The mean score of FATCOD-B-I was 115.3 (SD = 9.1). Higher scores were significantly associated with training in palliative care (p 0.0001) and experience with terminally ill patients (p 0.0001). Students manifested more negative attitudes when they perceived patients losing hope of recovering, and patient's family members interfering with health professionals' work. Uncertainties emerged around knowledge of opioid drugs, decision-making, concepts of death and dying, management of mourning, and relational aspects of patient care.Italian nursing students seem to have more positive attitudes towards care of dying patients than most other countries. They believe that caring for a terminal patient is a formative, useful experience but they do not feel adequately prepared in practice. Deeper palliative care education, integrated with practical training, would prepare students better, enabling them to discover their own human and professional capacity to relieve suffering.

Acute myeloid leukemia (AML) is a heterogeneous disease where selected subgroups of patients, linked by the presence of biological and clinical high-risk features, are candidates to receive allogenic hematopoietic stem cell transplantation HSCT) as post-remission consolidation treatment. The achievement of morphological complete remission (CR) before HSCT is an important pre-requisite to optimize the chance of successful post-transplant outcome. Minimal residual disease (MRD) assessment by quantitative polymerase chain reaction (q-PCR) has been shown to increase the ability to monitor therapy response in AML, improving prognostic accuracy and allowing to refine transplant strategies. Although MRD assessment was shown to have potential benefit when measured after induction and consolidation therapy courses, its role before HSCT remains to be fully elucidated. In order to contribute to better clarify this issue, we conducted a q-PCR I-BFM-AML collaborative study to measure MRD in bone marrow samples collected within 5 weeks prior to HSCT of 108 pediatric AML patients harboring one of the main recurrent AML aberrancies t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1q22)/t(16;16)(p13.1;q22); CBFB-MYH11, t(9;11)(p22;q23); KMT2A-MLLT3 or FLT3-ITD. Sixty patients underwent HSCT in first complete remission (CR1) with an overall survival (OS) of 84% versus 54% for the 48 transplanted in CR2 achieved after an initial relapse. Sixty patients showed q-MRD negativity (defined as a value lower than 2.1x10-4 calculated by ROC curve analysis with respect to diagnosis or relapse), whereas in 48 patients we detected q-MRD levels >2.1x10-4. Five-year OS after HSCT was 83% for patients with q-MRD negativity, while that of patients with q-MRD above the cutoff was 57% (p=0.012). As regards, cumulative incidence of relapse (CIR), q-MRD above the cutoff was associated with a high risk of recurrence (26% versus 10% for patients with q-MRD 2-log versus 73% for q-MRD2.1x10-4 and 2-log, HR). This combined stratification by q-MRD resulted into a better subdivision of the OS probability, which was 83%, 69% and 39% for LR, IR and HR respectively (p=0.004). Finally, a multivariate Cox regression model revealed that, together with CR status at time of the allograft (CR2, hazard ratio 4.4, p=0.001), q-MRD was an independent factor (hazard ratio 0.5, p=0.001) predicting HSCT outcome. In conclusion, this study supports the role of q-MRD pre-HSCT as a useful prognostic tool in childhood AML, able to provide information to tailor transplant strategies involving conditioning regimen intensity and graft-versus-host disease prophylaxis. Disclosures Reinhardt: AbbVie: Consultancy; Novartis: Consultancy, Other: Institutional Research Funding; Jazz: Consultancy, Other: Institutional Research Funding; Celgene: Consultancy, Other: Institutional Research Funding; bluebird bio: Consultancy; Roche: Consultancy, Other: Institutional Research Funding; Biotest: Other: Institutional Research Funding; Novo Nordisk: Other: Institutional Research Funding; Behring: Other: Institutional Research Funding. Merli:Bellicum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; SOBI: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria; Sanofi-Genzyme: Honoraria; Atara Therapeutics: Honoraria.

Background and Objectives: Sleep disorders are one of the most common complaints of patients with rotator cuff (RC) tears. However, potential correlations between the treatment of RC tears and the causal factors of sleep disorders are still under discussion. The aim of this review is to evaluate quality of sleep in patients before and after surgery for RC tears and to identify which factors affected patients’ sleep. Materials and Methods: A systematic review was conducted. To provide high quality of the review, the included studies were evaluated with the standardized tool “Quality Assessment Tool for Quantitative Studies” developed by the Effective Public Health Practice Project. Results: The search strategy yielded 78 articles. After duplicate removal and titles, abstracts and full-texts review, four studies were included in the systematic review. Concerning shoulder function, the most frequently reported scale was the Simple Shoulder Test (SST). Regarding sleep quality, the most frequently reported score was the Pittsburgh Sleep Quality Index (PSQI). Conclusion: We found that the majority of patients with RC tears had a sleep disturbance, especially before surgery with a general improvement in sleep quality post-operatively. Moreover, sleep quality was correlated with pain and it also seems that factors as comorbidities, obligatory position during night time, preoperative and prolonged postoperative use of narcotics and psychiatric issues may play an important role in sleep quality.

Objectives The Care Dependency Scale is a theory-based, comprehensive tool widely used in low-intensity care settings to evaluate patients’ dependency. This study aimed to test the psychometric properties of the Care Dependency Scale in intensive care units. Research methodology/design A multicentre cross-sectional validation study was conducted. Exploratory factor analysis and confirmatory factor analysis were performed using a Maximum Likelihood robust estimator with Geomin oblique rotation. Setting Adult patients admitted to intensive care units of four Italian hospitals. Results The sample included 453 patients (mean age = 68 years, 62% male). The exploratory factor analysis, conducted on a subsample of 227 patients, revealed a two-factor structure (Physical care dependency and Psychosocial care dependency) with good fit indexes. The confirmatory factor analysis was conducted on another subsample of 226 patients and a second-order factor was specified. The model tested yielded adequate fit indexes. Concurrent and known-groups validity, and reliability, were also adequate. Conclusion The Care Dependency Scale is a multidimensional, valid and reliable tool able to assess the care dependency of critically ill patients. It can help to distinguish between physical and psychosocial needs and to create a base for patient-customised and holistic care.

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine‐rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.

Patients with advanced cancer are likely to face increasing levels of care dependence. Adult patients who become care dependent on others can experience this condition as one of suffering and humiliation. The nurse-patient relationship plays a key role in the experience of dependence. Understanding patients’ and nurses’ perceptions of care dependence is crucial to addressing the impact it has on the lives of both. The aim of this study is to explore the experiences of patients with cancer and nurses caring for them. A multicentre qualitative study was conducted in Italy using semi-structured interviews with patients with advanced cancer admitted to 3 hospitals, and 9 focus groups with nurses working in oncology wards of 2 hospitals. Data were analysed with inductive content analysis. Thirty-two patients and 44 nurses participated in the study. Three common themes were identified: within dependence, the relationship is a lifeline; dependence is influenced by internal and external factors and dependence generates changes. Dependence impacts on patients’ and nurses’ lives and implies a process of personal maturing for both. Patients learn the humility to ask for help by exposing their vulnerability. Nurses become aware that a trusting relationship helps patients to accept dependence, and they learn to self-transcend in order to build it. Striving to build positive relationships implies a change in nurses’ and patients’ lives. In this way, they come to understand important aspects of life and find meaning in difficult situations. Further studies should explore also homecare settings and patients’ families.

Background Worldwide, nursing students have reported a preference for working in intensive care, paediatrics, and operating theatres after graduation, disregarding psychiatry and geriatrics. Many factors can influence student choices. Educators need to know students' preferences and influencing factors in order to plan appropriate interventions to orient future nurses towards the clinical areas that are most in need of trained and motivated nurses. Objectives To identify career preferences and student-related factors that influence the career intentions of students attending Italian nursing schools. Design A cross sectional design. Setting The study was conducted in 14 Italian nursing schools. Participants Students enrolled on a three-year undergraduate nursing program were invited to participate. Methods A questionnaire was used to collect sociodemographic data and clinical area preferences. A multivariate binary logistic regression was performed to identify the student characteristics influencing career choices. Results 1534 students were enrolled in the study. Students preferred working in paediatrics, emergency departments and operating theatres, and these preferences were consistent in all of the three years. Psychiatry and geriatrics were the clinical areas least preferred in all the three years. Age, gender, nationality, and university attended were the factors that predicted students' preferences for specific clinical areas. Conclusions In line with international literature, students attending Italian nursing schools expressed preferences for working in some clinical areas and to disregard others. Nursing curricula and internships need to be reviewed in terms of declared and hidden curriculum in order to enable students to view all areas of practice as equally valuable.

As a practice, nursing centers on patients' bodies and how they live the experience of illness and disability through their bodies (embodiment). International nursing studies conducted on the topic of body care primarily include theoretical studies, whereas empirical studies remain sparse. The aim of this study was to describe nurses' perceptions of the role of their bodies and the bodies of patients during body care. This study used a descriptive phenomenological study based on Husserl's philosophical perspective. A purposive sample of 11 Italian registered nurses representing the highest level of nursing education, different clinical experience levels, and different clinical specialties were interviewed. Data were collected between July 2015 and September 2015. Open-ended interviews were analyzed using Giorgi's descriptive phenomenological method. Lincoln and Guba's strategies were used to ensure the trustworthiness of the data. In addition to the overarching theme, "Body care is the heart of nursing," 4 major themes with 13 subthemes emerged from the data. The 4 major themes were as follows: (1) "Body care encompasses the essence of person"; (2) "Body care touches the heart of person"; (3) "The body generates opposite strategies of care"; and (4) "In time, the body 'nourishes' the helping relationship." Nurses considered body and embodiment concepts central to nursing. Body care is treated as an experience with extraordinary emotional contents, allowing nurses to discover the essentials of human nature.

Context The hidden curriculum is a learning dimension made up of culturally acquired, unintended lessons. Although nurse educators are not fully aware of it, through the hidden curriculum students are prepared for adult professional roles, internalising professional values and developing a professional identity. However, academic nursing education has paid relatively scarce attention to it. The objective was to map the nursing education literature about the hidden curriculum and to identify and verify to which of the four areas suggested by Hafferty (institutional policies, resource allocation decisions, institutional slang and evaluation) it refers. Methods A scoping study was conducted. In July 2018, a search was performed in MEDLINE/PubMed, Scopus, EBSCO/ Cumulative Index to Nursing and Allied Health Literature (CINAHL) and Cochrane Library databases, without time or language restrictions. Two authors independently performed the selection of studies, which followed the preferred reporting items for systematic reviews and meta-analysis (PRISMA) flowchart. Data charting was conducted with both an analytical and a narrative approach. Results The study included 18 articles, 13 of which were published during the last 5 years. A total of 12 were research articles, 10 of which used qualitative methodology. Regarding the area of analysis, all four areas could be identified within nursing education literature. The most widely explored area proved to be institutional policies, mentioned by 15 articles, predominantly to highlight the negative effect of the hidden curriculum. Some relational aspects, attributed to the hidden curriculum within nursing literature, belong to the informal curriculum. Conclusions The hidden curriculum in nursing education remains a largely overlooked topic. It appears to be a broader concept than that theorised by Hafferty, often also encompassing the informal curriculum. Furthermore, the literature reviewed mostly highlights the negative consequences of the hidden curriculum, such as the difficulty of transmitting professional values and ethics. Conversely, future researchers should concentrate on its positive consequences as a way to limit the loss of professional values.

Background: The application of virtual and augmented reality technologies to orthopaedic surgery training and practice aims to increase the safety and accuracy of procedures and reducing complications and costs. The purpose of this systematic review is to summarise the present literature on this topic while providing a detailed analysis of current flaws and benefits. Methods: A comprehensive search on the PubMed, Cochrane, CINAHL, and Embase database was conducted from inception to February 2021. The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were used to improve the reporting of the review. The Cochrane Risk of Bias Tool and the Methodological Index for Non-Randomized Studies (MINORS) was used to assess the quality and potential bias of the included randomized and non-randomized control trials, respectively. Results: Virtual reality has been proven revolutionary for both resident training and preoperative planning. Thanks to augmented reality, orthopaedic surgeons could carry out procedures faster and more accurately, improving overall safety. Artificial intelligence (AI) is a promising technology with limitless potential, but, nowadays, its use in orthopaedic surgery is limited to preoperative diagnosis. Conclusions: Extended reality technologies have the potential to reform orthopaedic training and practice, providing an opportunity for unidirectional growth towards a patient-centred approach.

The Forgotten Joint Score-12 (FJS-12) is a valid patient-reported outcome measures (PROMs) used to assess prosthesis awareness during daily activities after total hip arthroplasty (THA). The minimum clinically important difference (MCID) can be defined as the smallest change or difference that is evaluated as beneficial and could change the patient’s clinical management. The patient acceptable symptom state (PASS) is considered the minimum PROMs cut-off value that corresponds to a patient’s satisfactory state of health. Despite the validity and reliability of the FJS-12 having been already demonstrated, the MCID and the PASS of this score have not previously been defined. Patients undergoing THA from January 2019 to October 2019 were assessed pre-operatively and six months post-surgery using the FJS-12, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the Oxford Hip Score (OHS). Pre-operative and follow-up questionnaires were completed by 50 patients. Both distribution-based approaches and anchor approaches were used to estimate MCID. The aim of this paper was to assess the MCID and PASS values of FJS-12 after total hip replacement. The FJS-12 MCID from baseline to 6 months post-operative follow-up was 17.5. The PASS calculated ranged from 69.8 to 91.7.

Background Understanding students' experiences with regard to older adults is important for educators in developing specific educational strategies to encourage future healthcare professionals to consider the geriatric field as a career choice. Objectives The study explored Italian university healthcare students' experiences with older people and their perceptions of them at the beginning of their course. Design A qualitative descriptive design. Settings A Faculty of Medicine in Italy. Participants A sample of 15 students enrolled in healthcare courses was included. Methods Semistructured interviews were conducted. The data were analyzed using inductive content analysis. Results Six categories were identified. The young Italian students' first experiences with older adults were with their grandparents, representing their first models of aging. Grandparents were sources of emotional support. Older adults were seen as custodians of collective memory and as a guide for younger generations. The intergenerational relationship was characterized by respect and reciprocity. Young adults recognize the frailty and vulnerability of older adults and view intergenerational solidarity as a way to protect them. They imagined their own old age as characterized by strong family relationships and by active aging, due to their personal experience with their grandparents. Conclusions This study revealed mainly positive experiences and attitudes of healthcare students toward older adults. The students' relationships with their grandparents contributed greatly to this outcome.

This systematic review aimed to assess the advantages of biosensors in detecting biomarkers for the early diagnosis of osteoarthritis (OA). OA is the most prevalent musculoskeletal disease and is a leading cause of disability and pain worldwide. The diagnosis of OA could be performed through clinical examinations and imaging only during the late stages of the disease. Biomarkers could be used for the diagnosis of the disease in the very early stages. Biosensors could detect biomarkers with high accuracy and low costs. This paper focuses on the biosensors mainly adopted to detect OA markers (electrochemical, optical, Quartz crystal microbalance, molecular and wearable biosensors). A comprehensive search on PubMed, Cochrane, CINAHL and Embase databases was conducted from the inception to November 2020. The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were used to improve the reporting of the review. The Methodological Index for Non-Randomized Studies (MINORS) was used for quality assessment. From a total amount of 1086 studies identified, only 19 articles were eligible for this study. The main advantages of the biosensors reported were accuracy, limited cost and ease of use, compared to traditional methods (ELISA). Otherwise, due to the lack of data and the low level of evidence of the papers included, it was impossible to find significant results. Therefore, further high-quality studies are required.

Purpose To investigate the degree of concordance on fatigue assessment between children and adolescents with cancer and their parents, and its changes over time. Method Multicentre longitudinal study. Results Data from 134 dyads were analysed. The mean age of patients was 11.7 years; caregivers had a mean age of 44.1 years. Almost 90% of patients already reported mild or moderate fatigue at the time of diagnosis, decreasing to 69.7% after one year. Concordance on the total fatigue improved over time for the total sample, moving from moderate at the time of diagnosis to good concordance after one year. Conclusions This was the first study with a longitudinal design investigating concordance between paediatric self-reports and parent proxy reports on fatigue. It showed how concordance between proxies and patients changed over time reaching a good level after one year from the cancer diagnosis.

Introduction Decision-making on artificial nutrition and hydration for terminal cancer patients can be influenced by nurses’ knowledge, attitudes, and behavioural intentions. A comprehensive 57-item questionnaire including six sections on the Knowledge, Attitudes, and Behavioural Intentions in Providing Artificial Nutrition and Hydration for Terminal Cancer Patients, has been developed and used in Taiwan. However, it needs further psychometric testing and adaptation to other cultures. This study aimed at cross-culturally adapting the above questionnaire to the Italian cultural context and testing its psychometric properties. Materials and Methods The questionnaire was translated into Italian and cross-culturally adapted following the recommendations of Beaton. A panel of ten experts assessed content validity. A multicentre cross-sectional study was conducted with 411 nurses to test its psychometric properties. Dimensionality and construct validity were assessed through exploratory and confirmatory factor analyses. Reliability was estimated by composite omega and traditional methods such as the Kunder Richardson formula-20 and Cronbach's α coefficients. Results The overall content validity index was .85. Confirmatory factor analysis was conducted for the knowledge section and the four attitudes sections, while preliminary analysis for the behavioural intentions section yielded non-acceptable results. The internal consistency for the scales was adequate (ranging .64 - .93). Conclusion This study constituted a notable advancement in the psychometric testing of the tool. It provides evidence that the Italian version of the questionnaire has acceptable psychometric characteristics for the sections regarding Knowledge and Attitudes.

Historically, the practical work of nursing has been based on a relationship and contact with the human body (Collie re, 2001; Draper, 2014; Lawler, 2006; Pupulim & Sawada, 2005; Sakalys, 2006; Wolf, 2014). Nurses interact with the patient's body so frequently that they touch the patient more than other healthcare professionals do (McCann & McKenna, 1993; Melia, 2014; Ribeiro Souza & Ordones do Nascimento Brandao, 2013). The human body is nursing's "site of labor" (Wolkowitz, 2002, p. 497), and nursing benefits from "privileged access" to the body (Lawler, 2006, p. 11) in states, places, and circumstances in which the body has the same need: to be assisted (Melia, 2014; Twigg, Wolkowitz, Cohen, & Nettleton, 2011; Wilde, 2003).In the literature, the human body is interpreted as a physical, concrete, and material entity to heal, treat, and medicate (body) and as a "lived body," which refers to the sensations, emotions, and feelings experienced through the body, in other words, a way of being and living "in," "with," and "through" one's own body (embodiment; Draper, 2014; James & Hockey, 2007; Lawler, 2006; Mariotti de Santana & Bessa Jorge, 2007; Moreno Altamirano, 2010; Sakalys, 2006; Wilde, 1999; Yakhlef, 2010).Scientific reflection has embraced the Cartesian dualism that sees the body-object as a physical entity, the object of disease treatment, and the body-subject as a subjective experience of illness (Carnevale, 1995; Carr, 2000; Lakoff & Johnson, 1999; Sakalys, 2006; Wilde, 2003; Yakhlef, 2010). This perspective has been questioned by modern antireductionist and antipositivist perspectives, such as that of Merleau-Ponty (Benner, 2000; Thomas, 2005). He contended that a person is an indissoluble unity of the "I" who thinks, his or her body, and his or her embodiment; we do not "have" a body, "we are" a body (Merleau-Ponty, 1945). Consequently, the meanings that we give to body and embodiment are linked to social rules and cultural beliefs (Lawler, 2006; Vannini & Waskul, 2012).However, dichotomous thinking has been strongly established in scientific research, creating a hierarchy in which the body has become the subject of detailed clinical studies, whereas embodiment has a marginal role (Holmes, 1994; Madjar, 1997). This is due to the biomedical model's preference for investigation methods applicable to the body and the objectively measurable knowledge that these methods generate, instead of patients' embodiment (Draper, 2014).Despite the increase in scientific literature and the recognition of the importance of body and embodiment in nursing over the last two decades, international theoretical and empirical research of these concepts is still neglected (Draper, 2014). The terms body and embodiment, although conceptually different, are used interchangeably and are little known and developed within the nursing discipline; researchers in the nursing field have taken very sporadic theoretical interest in the concepts (Sakalys, 2006; Thomas, 2005). Furthermore, the recognition of people's embodiment in the body as an explicit focus of the care process is rare in nursing theories (Lawler, 2006; Sakalys, 2006). Consequently, nurses learn how to work on the body through medicine, science, and practice, but they receive little instruction about the meaning of the "lived body" and the privilege of caring for it (Draper, 2014). Although both concepts are understood and defined differently in various disciplines (philosophy, anthropology, sociology, etc.), the specific contribution of nursing science is missing. The nursing field could provide a coherent representation of body and embodiment as the fundamental conditions of a person, reflecting his or her multiple dimensions and levels of understanding (Sakalys, 2006).Although body care and attention to embodiment are the central focus of nursing practice, these concepts have not yet been openly, explicitly, and theoretically accepted in the nursing discipline. …

MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H2O2-induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis.

Aim To develop and psychometrically test the Italian-language Nurse Caring Behaviours Scale, a short measure of nurse caring behaviour as perceived by inpatients. Background Patient perceptions of nurses’ caring behaviours are a predictor of care quality. Caring behaviours are culture-specific, but no measure of patient perceptions has previously been developed in Italy. Moreover, existing tools show unclear psychometric properties, are burdensome for respondents, or are not widely applicable. Design Instrument development and psychometric testing. Method Item generation included identifying and adapting items from existing measures of caring behaviours as perceived by patients. A pool of 28 items was evaluated for face validity. Content validity indexes were calculated for the resulting 15-item scale; acceptability and clarity was pilot tested with 50 patients. To assess construct validity, a sample of 2001 consecutive adult patients admitted to a hospital in 2014 completed the scale and was split into two groups. Reliability was evaluated using nonlinear Structural Equation Modeling coefficients. Measurement invariance was tested across sub-samples. Results Item 15 loaded poorly in the exploratory factor analysis (n=983) and was excluded from the final solution, positing a single latent variable with 14 indicators. This model fitted the data moderately. The confirmatory factor analysis (n=1018) returned similar results. Internal consistency was excellent in both sub-samples. Full scalar invariance was reached and no significant latent mean differences were detected across sub-samples. Conclusion The new instrument shows reasonable psychometric properties and is a promising short and widely applicable measure of inpatient perceptions of nurse caring behaviours. This article is protected by copyright. All rights reserved.

Background Breast cancer is the most common cancer in women in the world, and it is also the leading cause of cancer deaths among women. Nevertheless, breast cancer survival has increased as a result of improvements in early diagnosis and therapy, for example, oral endocrine therapy. Despite the importance of adherence to endocrine therapy, its trend appears complex and multidimensional and therefore has many loopholes and missing information. Objective The study aims to explore the experiences of adherence to endocrine therapy in women with breast cancer and their perceptions of the challenges they face in adhering to their medication prescribed. Methods The study used a qualitative exploratory design, with face-to-face semistructured interviews. Data were analyzed using framework analysis in accordance with Ritchie and Spencer's approach. Results The sample included 27 women. Seven themes were identified: the different faces of adherence, fear of the drug, adherence stimulates the balance of the experience of illness, adherence influences the future of disease, adherence requires attention to the person, knowledge seeking, and "forgetfulness" activates the search for functional strategies. Conclusions This study shows that adherence assumes different connotations that are mainly influenced by the type of relationship established with health professionals the attention paid to the person, the information received, and the influence that the drug has on the disease. Implications for practice It appears that fear has a strong influence on the behaviors involved in taking the therapy. The only way to overcome irrational fear is to improve the patient's knowledge.

International audience; Dictyostelium discoideum is a widely used model to study molecular mechanisms controlling cell adhesion, cell spreading on a surface, and phagocytosis. In this study we isolated and characterize a new mutant created by insertion of a mutagenic vector in the heretofore uncharacterized spdA gene. SpdA-ins mutant cells produce an altered, slightly shortened version of the SpdA protein. They spread more efficiently than WT cells when allowed to adhere to a glass substrate, and phagocytose particles more efficiently. On the contrary, a functional spdA knockout mutant where a large segment of the gene was deleted phagocytosed less efficiently and spread less efficiently on a substrate. These phenotypes were highly dependent on the cellular density, and were most visible at high cell densities, where secreted quorum-sensing factors inhibiting cell motility, spreading and phagocytosis are most active. These results identify the involvement of SpdA in the control of cell spreading and phagocytosis. The underlying molecular mechanisms, as well as the exact link between SpdA and cell spreading, remain to be established.

Pseudomonas aeruginosa can cause acute lung infections in intubated patients or chronic infections in patients with cystic fibrosis (CF). In both situations, P. aeruginosa accumulates specific mutations, in particular in the lasR quorum-sensing regulator gene. Using a Dictyostelium discoideum amoeba model, we assessed whether these mutations affect bacterial virulence. Among a collection of clinical isolates from 16 CF patients, initial isolates were fully virulent in 15 patients, but for late isolates collected several years later, virulence was decreased in eight patients. No significant correlation between genetic inactivation of lasR and decreased virulence was observed. Among strains isolated from ten colonized intubated patients, all initial isolates were fully virulent. Despite the accumulation of lasR-inactivating mutations in strains collected over a 3-week period, no decrease in virulence was observed in eight of 10 patients. In one intubated patient, the virulent initial strain was replaced a few days later with a different, less virulent, strain. We observed a gradual decrease in bacterial virulence in only one intubated patient. We conclude that adaptation of P. aeruginosa to chronically infected CF patients can lead to a slow and gradual loss of virulence, as measured in a Dictyostelium model system. However, loss of virulence is not caused predominantly by mutations in lasR. During short-term colonization of intubated patients for up to 20 days, a decrease in virulence was exceptional, despite the accumulation of lasR mutations.

Bacterial ingestion and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. However, only few proteins implicated in intracellular bacterial killing have been identified to date. We used Dictyostelium discoideum, a phagocytic bacterial predator, to study intracellular killing. In a random genetic screen we identified Kil2, a type V P-ATPase as an essential element for efficient intracellular killing of Klebsiella pneumoniae bacteria. Interestingly, kil2 knockout cells still killed efficiently several other species of bacteria, and did not show enhanced susceptibility to Mycobacterium marinum intracellular replication. Kil2 is present in the phagosomal membrane, and its structure suggests that it pumps cations into the phagosomal lumen. The killing defect of kil2 knockout cells was rescued by the addition of magnesium ions, suggesting that Kil2 may function as a magnesium pump. In agreement with this, kil2 mutant cells exhibited a specific defect for growth at high concentrations of magnesium. Phagosomal protease activity was lower in kil2 mutant cells than in wild-type cells, a phenotype reversed by the addition of magnesium to the medium. Kil2 may act as a magnesium pump maintaining magnesium concentration in phagosomes, thus ensuring optimal activity of phagosomal proteases and efficient killing of bacteria.

Dictyostelium amoebae have been used as a host model to measure virulence of a wide range of bacterial pathogens. The simple protocol described here takes advantage of the ability of Dictyostelium to grow and form plaques on a lawn of nonpathogenic bacteria but not on virulent bacteria. This assay can be modulated to measure the virulence of different bacterial pathogens. By adjusting various parameters such as cell numbers or media, a more quantitative measure of bacterial virulence can also be obtained. The entire procedure takes about 5 h to compete, and up to 2 further weeks may be required for plaques to form on the bacterial lawn.

BackgroundThe European Union Bologna Process has laid the foundation for a common European competence-based educational framework. In many countries, nursing education is in transition from vocational to higher education, with many diverse systems. The competence-based approach provided by the project Tuning Educational Structures offers a common and coherent framework able to facilitate the implementation of the principles underpinning the Bologna Process reform.ObjectivesThis study aimed to ascertain the relevance that Italian nursing university lecturers attributed to the 40 competences of the Italian version of the nursing Bachelor's and Master's Degrees. These competences were developed through adoption of the Tuning Methodology in the nursing context.SettingThe study was conducted in the 4 universities of one region of Italy which offer nursing Bachelor's and Master's Degrees.ParticipantsA total of 164 Italian university nursing lecturers.MethodsUsing a four point scale, a cross sectional survey was conducted from March 2011 to April 2012. Participants evaluated each competence according to its relevance for Bachelor's or Master's Education. Frequency analysis was conducted.ResultsThe significance for each competence of Tuning was rated very high by Italian lecturers and appeared to overlap partially with the original European study. In Italy, the most relevant competences for Bachelor's Degree were the skills associated with the use of appropriate interventions, activities and skills in nursing and the skills associated with nursing practice and clinical decision-making. For Master's Degree, leadership, management and team competences were the most important.ConclusionsThe Tuning Nursing Project was accepted by the Italian lecturers. The competence-based approach was considered by Italian lectures as a support enabling to reflect on the current Italian nursing education cycles of study and to ensure shared visions and common approaches between Italian and European lecturers.

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca(2+) entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca(2+) influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca(2+) imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1(-/-)/Stim2(-/-) fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca(2+) elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides.

Multivesicular endosomes are present in virtually every eucaryotic cell, where they arise by intra-endosomal budding of the limiting endosomal membrane. Some genetic diseases such as Chediak-Higashi syndrome are characterized by enlarged membrane-filled endosomes. The same altered endosomal morphology can be observed in cells exposed to certain drugs, for example U18666A. The mechanisms involved are still poorly characterized, partially because this atypical budding event is particularly difficult to observe in mammalian cells. Taking advantage of the simplicity of the endosomal structure in Dictyostelium discoideum, we could visualize intraendosomal budding at the ultrastructural level. In this model organism, the drug U18666A was shown to stimulate intra-endosomal budding, while an inhibitor of PI 3-kinase activity was found to have no effect on this process. Inactivation of a Dictyostelium gene with similarity to the gene responsible for Chediak-Higashi syndrome did not alter the intra-endosomal budding or the accumulation of intra-endosomal membranes. Thus, although treatment with U18666A and inactivation of the Chediak-Higashi gene cause similar morphological defects in mammalian cells, observations in a different model reveal that their respective modes of action are different.

Bacterial sensing, ingestion, and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. The cellular mechanisms involved in intracellular killing, their relative importance, and their specificity towards different bacteria are however poorly defined. In this study, we used Dictyostelium discoideum, a phagocytic cell model amenable to genetic analysis, to identify new gene products involved in intracellular killing. A random genetic screen led us to identify the role of Vps13F in intracellular killing of Klebsiella pneumoniae. Vps13F knock‐out (KO) cells exhibited a delayed intracellular killing of K. pneumoniae, although the general organization of the phagocytic and endocytic pathway appeared largely unaffected. Transcriptomic analysis revealed that vps13F KO cells may be functionally similar to previously characterized fspA KO cells, shown to be defective in folate sensing. Indeed, vps13F KO cells showed a decreased chemokinetic response to various stimulants, suggesting a direct or indirect role of Vps13F in intracellular signaling. Overstimulation with excess folate restored efficient killing in vps13F KO cells. Finally, genetic inactivation of Far1, the folate receptor, resulted in inefficient intracellular killing of K. pneumoniae. Together, these observations show that stimulation of Dictyostelium by bacterial folate is necessary for rapid intracellular killing of K. pneumoniae.

Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified) sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

We show that Daphnia magna can be used to assess acute virulence of pathogens relevant to human health, such as Pseudomonas aeruginosa or Photorhabdus asymbiotica . Analysis of bacterial mutants suggests that P. aeruginosa uses similar mechanisms to infect Daphnia and other hosts.

Efficient sorting of proteins is essential to allow transport between intracellular compartments while maintaining their specific composition. During endocytosis, membrane proteins can be concentrated in endocytic vesicles by specific interactions between their cytoplasmic domains and cytosolic coat proteins. It is, however, unclear whether they can be excluded from transport vesicles and what the determinants for this sorting could be. Here, we show that in the absence of cytosolic sorting signals, transmembrane domains control the access of surface proteins to endosomal compartments. They act in particular by determining the degree of exclusion of membrane proteins from endocytic clathrin-coated vesicles. When cytosolic endocytosis signals are present, it is the combination of cytosolic and transmembrane determinants that ultimately controls the efficiency with which a given transmembrane protein is endocytosed.

Background Dictyostelium amoebae are frequently used to study the organization and function of the endocytic pathway, and specific protocols are essential to measure the dynamics of endocytic compartments and their internal pH. Findings We have revisited these classical protocols to measure more accurately endosomal pH, making use of a fluorescent probe (Oregon green) more adequate for very acidic pH values. This pH-sensitive probe was combined with a pH-insensitive marker, in order to visualize simultaneously endosome dynamics and pH changes. Finally, a flow cytometer was used to measure endosomal pH in individual cells. Conclusion Using these simple protocols the endosomal pH of endocytic compartments can be assessed accurately, revealing the extreme acidity of Dictyostelium lysosomes (pH

International audience; The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.

International audience; Proteins of the Chediak-Higashi/Beige (BEACH) family have been implicated in the function of lysosomes, as well as in signal transduction, but their molecular role is still poorly understood. In Dictyostelium, at least six members of the family can be identified. Here cells with mutations in two of these genes, LVSA and LVSB, were analyzed. Interestingly both mutants exhibited defects in the organization of the endocytic pathway, albeit at distinct stages. In lvsB mutant cells, the regulated secretion of lysosomal enzymes was enhanced, a phenotype reminiscent of the Chediak-Higashi syndrome. LvsA mutant cells exhibited alterations in the organization and function of the early endocytic and phagocytic pathway. The LvsA protein may participate in the signaling pathway, which links adhesion of a particle to the subsequent formation of a phagocytic cup. Further genetic analysis will be necessary to determine whether other members of the BEACH family of proteins are also involved in controlling the organization of the endocytic pathway.Proteins of the Chediak-Higashi/Beige (BEACH) family have been implicated in the function of lysosomes, as well as in signal transduction, but their molecular role is still poorly understood. In Dictyostelium, at least six members of the family can be identified. Here cells with mutations in two of these genes, LVSA and LVSB, were analyzed. Interestingly both mutants exhibited defects in the organization of the endocytic pathway, albeit at distinct stages. In lvsB mutant cells, the regulated secretion of lysosomal enzymes was enhanced, a phenotype reminiscent of the Chediak-Higashi syndrome. LvsA mutant cells exhibited alterations in the organization and function of the early endocytic and phagocytic pathway. The LvsA protein may participate in the signaling pathway, which links adhesion of a particle to the subsequent formation of a phagocytic cup. Further genetic analysis will be necessary to determine whether other members of the BEACH family of proteins are also involved in controlling the organization of the endocytic pathway.