New insights into enterocin CRL35: mechanism of action and immunity revealed by heterologous expression in Escherichia coli

The role of the class IIa bacteriocin membrane receptor protein remains unclear, and the following two different mechanisms have been proposed: the bacteriocin could interact with the receptor changing it to an open conformation or the receptor might act as an anchor allowing subsequent bacteriocin insertion and membrane disruption. Bacteriocin-producing cells synthesize an immunity protein that forms an inactive bacteriocin–receptor–immunity complex. To better understand the molecular mechanism of enterocin CRL35, the peptide was expressed as the suicidal probe EtpM-enterocin CRL35 in Escherichia coli, a naturally insensitive microorganism since it does not express the receptor. When the bacteriocin is anchored to the periplasmic face of the plasma membrane through the bitopic membrane protein, EtpM, E. coli cells depolarize and die. Moreover, co-expression of the immunity protein prevents the deleterious effect of EtpM-enterocin CRL35. The binding and anchoring of the bacteriocin to the membrane has demonstrated to be a sufficient condition for its membrane insertion. The final step of membrane disruption by EtpM-enterocin CRL35 is independent from the receptor, which means that the mannose PTS might not be involved in the pore structure. In addition, the immunity protein can protect even in the absence of the receptor.Fil: Barraza, Daniela Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Ríos Colombo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Galván, Adriana Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Acuña, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Minahk, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

The case for class II bacteriocins: A biophysical approach using “suicide probes” in receptor-free hosts to study their mechanism of action

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called “suicide probes”. The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.Fil: Ríos Colombo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Dupuy, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Gonzalez, Claudio Fabricio. University of Florida; Estados UnidosFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

Association of obesity and long-term mortality in patients with acute myocardial infarction with and without diabetes mellitus: results from the MONICA/KORA myocardial infarction registry

Background Paradoxically, beneficial effects of overweight and obesity on survival have been found in patients after cardiovascular events such as acute myocardial infarction (AMI). This obesity paradox has not been analyzed in AMI patients with diabetes even though their cardiovascular morbidity and mortality is increased compared to their counterparts without diabetes. Therefore, the objective of this long-term study was to analyze the association between body mass index (BMI) and all-cause mortality in AMI patients with and without diabetes mellitus. Methods Included in the study were 1190 patients with and 2864 patients without diabetes, aged 28-74 years, recruited from a German population-based AMI registry. Patients were consecutively hospitalized between 1 January 2000 and 31 December 2008 with a first ever AMI and followed up until December 2011. Data collection comprised standardized interviews and chart reviews. To assess the association between BMI and long-term mortality from all causes, Cox proportional hazards models were calculated adjusted for risk factors, co-morbidities, clinical characteristics, in-hospital complications as well as medical and drug treatment. Results AMI patients of normal weight (BMI 18.5-24.9 kg/m2) had the highest long-term mortality rate both in patients with and without diabetes with 50 deaths per 1000 person years and 26 deaths per 1000 person years, respectively. After adjusting for a selection of covariates, a significant, protective effect of overweight and obesity on all-cause mortality was found in AMI patients without diabetes (overweight: hazard ratio (HR) 0.73, 95% confidence interval (CI) 0.58-0.93; p=0.009; obesity: HR 0.64, 95% CI 0.47-0.87; p=0.004). In contrast, an obesity paradox was not found in AMI patients with diabetes. However, stratified analyses showed survival benefits in overweight AMI patients with diabetes who had been prescribed statins prior to AMI (HR 0.51, 95% CI 0.29-0.89, p=0.018) or four evidence-based medications at hospital-discharge (HR 0.52, 95% CI 0.34-0.80, p=0.003). Conclusion In contrast to AMI patients without diabetes, AMI patients with diabetes do not experience a survival benefit from an elevated BMI. To investigate the underlying reasons for these findings, further studies stratifying their samples by diabetes status are needed. &nbsp

Evaluation of efficacy and safety of itraconazole oral solution for the treatment of oropharyngeal candidiasis in AIDS patients

This study was a non-comparative multicenter clinical trial to evaluate the efficacy and tolerability of itraconazole oral solution 200 mg/day (100 mg twice a day in the fasting state) for the treatment of oropharyngeal candidiasis in AIDS patients. We included 50 patients who were treated and followed for up to 3 weeks after ending therapy in the analysis. Mycological cures at the end of therapy occurred in 20/50 patients (40%), but colonization by Candida sp. was recorded in 42/50 (84%) by the end of follow-up. A high rate of clinical response was observed in 46/50 (92%), and the response was sustained for up to 21 days after stopping therapy in 24/46 patients (52%). Clinical relapses were documented among 22 patients, but all causative fungal organisms associated with a relapse were susceptible to itraconazole. There were many patients with persistence or recurrence of Candida, but without mucositis. Relapse of Candida mucositis was significantly related to low levels of CD4 lymphocytes exhibited by symptomatic patients. The drug was well tolerated by all but 1 patient. We conclude that itraconazole oral solution (100 mg bid for 7-14 days) is a well tolerated and effective treatment for suppressing the symptoms of oropharyngeal candidiasis in AIDS patients. Patients with severe immunosuppression may relapse and require frequent cycles of treatment or longterm suppressive therapy.Federal University of Paraná Clinical HospitalAliança HospitalFederal University of São Paulo DIPA Special Mycology LaboratoryJanssen-Cilag Farmacêutica do BrasilUNIFESP, DIPA Special Mycology LaboratorySciEL

Dissemination of patient blood management practices in Swiss intensive care units: a cross-sectional survey

BACKGROUND Patient blood management (PBM) promotes the routine detection and treatment of anaemia before surgery, optimising the management of bleeding disorders, thus minimising iatrogenic blood loss and pre-empting allogeneic blood utilisation. PBM programmes have expanded from the elective surgical setting to nonsurgical patients, including those in intensive care units (ICUs), but their dissemination in a whole country is unknown. METHODS We performed a cross-sectional, anonymous survey (10 October 2018 to 13 March 2019) of all ordinary medical members of the Swiss Society of Intensive Care Medicine and the registered ICU nurses from the 77 certified adult Swiss ICUs. We analysed PBM-related interventions adopted in Swiss ICUs and related them to the spread of PBM in Swiss hospitals. We explored blood test ordering policies, blood-sparing strategies and red blood cell-related transfusion practices in ICUs. RESULTS A total of 115 medical doctors and 624 nurses (response rates 27% and 30%, respectively) completed the surveys. Hospitals had implemented a PBM programme according to 42% of physicians, more commonly in Switzerland's German-speaking regions (Odds Ratio [OR] 3.39, 95% confidence interval [CI] 1.23-9.35; p = 0.018) and in hospitals with more than 500 beds (OR 3.91, 95% CI 1.48-10.4; p = 0.006). The PBM programmes targeted the detection and correction of anaemia before surgery (79%), minimising perioperative blood loss (94%) and optimising anaemia tolerance (98%). Laboratory tests were ordered in 70.4% by the intensivist during morning rounds; the nurses performed arterial blood gas analyses autonomously in 48.4%. Blood-sparing techniques were used by only 42.1% of nurses (263 of 624, missing: 6) and 47.0% of physicians (54 of 115). Approximately 60% of respondents used an ICU-specific transfusion guideline. The reported haemoglobin threshold for the nonbleeding ICU population was 70 g/l and, therefore, was at the lower limit of current guidelines. CONCLUSIONS Based on this survey, the estimated proportion of the intensivists working in hospitals with a PBM initiative is 42%, with significant variability between regions and hospitals of various sizes. The risk of iatrogenic anaemia is relevant due to liberal blood sample collection practices and the underuse of blood-sparing techniques. The reported transfusion threshold suggests excellent adherence to current international ICU-specific transfusion guidelines

Enhanced endocannabinoid-mediated modulation of rostromedial tegmental nucleus drive onto dopamine neurons in sardinian alcohol-preferring rats

The progressive predominance of rewarding effects of addictive drugs over their aversive properties likely contributes to the transition from drug use to drug dependence. By inhibiting the activity of DA neurons in the VTA, GABA projections from the rostromedial tegmental nucleus (RMTg) are well suited to shift the balance between drug-induced reward and aversion. Since cannabinoids suppress RMTg inputs to DA cells and CB1 receptors affect alcohol intake in rodents, we hypothesized that the endocannabinoid system, by modulating this pathway, might contribute to alcohol preference. Here we found that RMTg afferents onto VTA DA neurons express CB1 receptors and display a 2-arachidonoylglycerol (2-AG)-dependent form of short-term plasticity, that is, depolarization-induced suppression of inhibition (DSI). Next, we compared rodents with innate opposite alcohol preference, the Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats. We found that DA cells from alcohol-naive sP rats displayed a decreased probability of GABA release and a larger DSI. This difference was due to the rate of 2-AG degradation. In vivo, we found a reduced RMTg-induced inhibition of putative DA neurons in sP rats that negatively correlated with an increased firing. Finally, alcohol failed to enhance RMTg spontaneous activity and to prolong RMTg-induced silencing of putative DA neurons in sP rats. Our results indicate functional modifications of RMTg projections to DA neurons that might impact the reward/aversion balance of alcohol attributes, which may contribute to the innate preference observed in sP rats and to their elevated alcohol intak

Nanocrystals for Improved Drug Delivery of Dexamethasone in Skin Investigated by EPR Spectroscopy

Nanocrystals represent an improvement over the traditional nanocarriers for dermal application, providing the advantages of 100% drug loading, a large surface area, increased adhesion, and the potential for hair follicle targeting. To investigate their advantage for drug delivery, compared to a base cream formulation, dexamethasone (Dx), a synthetic glucocorticoid frequently used for the treatment of inflammatory skin diseases, was covalently linked with the paramagnetic probe 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) to DxPCA. To investigate the penetration efficiency between these two vehicles, electron paramagnetic resonance (EPR) spectroscopy was used, which allows the quantification of a spin-labeled drug in different skin layers and the monitoring of the drug release. The penetration behavior in excised healthy and barrier-disrupted porcine skin was monitored by EPR, and subsequently analyzed using a numerical diffusion model. As a result, diffusion constants and free energy values in the different layers of the skin were identified for both formulations. Dx-nanocrystals showed a significantly increased drug amount that penetrated into viable epidermis and dermis of intact (factor 3) and barrier-disrupted skin (factor 2.1) compared to the base cream formulation. Furthermore, the observed fast delivery of the spin-labeled drug into the skin (80% DxPCA within 30 min) and a successive release from the aggregate unit into the viable tissue makes these nanocrystals very attractive for clinical applications

Case report: Primary Ewing sarcoma of the ureter, an exceptional finding of unique manifestation of disease

Ewing sarcoma (ES) is the second most common malignant bone tumor in children and has also been described in adults with highly aggressive behavior. ES belongs to the small round blue cell tumor family and presents the distinctive translocation of FET-ETS family genes (85% with EWSR1), generating gene fusions. Extraskeletal ES mainly occurs in soft tissues; the urogenital tract is rarely affected, and ureteral localization is an exceptional event with only 4 cases described in the literature. Here we report the first Italian case of primary ES of the ureter, a 24-year-old young man with lower back pain and a narrowed left ureteral lumen on CT scan. ES of the urogenital tract is an almost unique condition with a nonspecific clinical presentation and a challenging diagnosis for pathologists. We encourage awareness of these exceptional events in the differential diagnosis of ureteral lesions in young patients

In vivo systems to study class II bacteriocins toxicity and immunity

Class II bacteriocins are membrane-active peptides that act over a narrowspectrum of bacterial targets and have a great potential application as antibioticsin medical sciences. They act on the cytoplasmic membrane dissipating thetransmembrane potential by forming pores. There is solid evidence thatmembrane receptor proteins are necessary for their function, however the preciserole of this receptor and the nature of the pore remain elusive. The most acceptedmodel suggest that bacteriocins bind the receptor to change its conformation,creating a channel that remains open. Nonetheless, several studies support asecond model in which the bacteriocin is able to disrupt the membrane itself andthe receptor might act just as an anchor allowing the subsequent bacteriocininsertion to form the pore. In order to reveal whether or not the pore structureinvolves the specific receptor, we designed chimeric peptides fusing themembrane protein EtpM with different class II bacteriocins. We chose E. coli as areceptor-free expression host. The fusion EtpM-bacteriocin anchors eachbacteriocin to the membrane and kills the expressing host cell, even in theabsence of the specific receptor. These results are in line with the second model inwhich the pore is formed through a receptor-independent interaction with the lipidbilayer. The effect of these interactions was also analyzed, through a fluorophorethat changes its fluorescence intensity according to transmembrane potential.On the other hand, an immunity protein protects the producer strain against itsown bacteriocin. For antimicrobials under investigation for clinical applications, thepotential emergence of resistant pathogens and the study of immune mechanismsare a primary concern. Though no direct in vitro interaction bacteriocin-immunityhas been reported before, by using an in vivo system, we present evidence thatthis binding might occur, not in aqueous solution but in a membrane inserted .Fil: Ríos Colombo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Galván, Adriana Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Navarro, Silvia Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Lanza, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Barraza, Daniela Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Acuña, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Fernandez de Ullivarri, Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Minahk, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaXLVII Reunión Anual de la Sociedad Argentina de BiofísicaLa PlataArgentinaSociedad Argentina de Biofísic

Long-term effects of preeclampsia on maternal cardiovascular health and postpartum utilization of primary care: an observational claims data study

Purpose: Preeclampsia occurs in up to 15% of pregnancies and constitutes a major risk factor for cardiovascular disease. This observational cohort study aimed to examine the association between preeclamptic pregnancies and cardiovascular outcomes as well as primary and specialized care utilization after delivery. Methods: Using statutory claims data we identified women with singleton live births between 2010 and 2017. Main outcomes included the occurrence of either hypertension or cardiovascular disease after one or more preeclamptic pregnancies, number of contacts to a general practitioner or cardiologist after delivery and prescribed antihypertensive medication. Data were analyzed using Cox proportional hazard regression models adjusted for maternal age, diabetes, dyslipidemia, and obesity. Results: The study cohort consisted of 181,574 women with 240,698 births. Women who experienced preeclampsia once had an increased risk for cardiovascular (hazard ratio, HR = 1.29) or hypertensive (HR = 4.13) events. In women affected by recurrent preeclampsia, risks were even higher to develop cardiovascular disease (HR = 1.53) or hypertension (HR = 6.01). In the following years after delivery, general practitioners were seen frequently, whereas cardiologists were consulted rarely (0.3 and 2.4%). Conclusion: Women affected by preeclampsia experience an increased risk of developing chronic hypertension and cardiovascular disease, especially those with recurrent preeclampsia. Future medical guidelines should take this potential risk into account