Enhanced Patch-Clamp Technique to Study Antimicrobial Peptides and Viroporins, Inserted in a Cell Plasma Membrane with Fully Inactivated Endogenous Conductances

Many short peptides selectively permeabilize the bacteria plasma membrane, leading to their lyses and death: they are therefore a source of antibacterial molecules, and inspiration for novel and more selective drugs. Another class of short (<100 residues) membrane proteins called viroporins, because they are coded by viral genes, permeabilizes the membrane of susceptible cells during infection of by most animal viruses. The permeabilization leads to host cell lyses and the release of the virus mass, replicated at host cell expense, to propagate the infection. Detailed knowledge of the permeabilization properties of these proteins would allow to design, for instance, selective blockers of these pores, that would contrast the spread of the viral infection. In this chapter, the patch-clamp technique is employed to study the mechanism of membrane permeabilization induced by the pore-forming peptides, under strict physiological conditions. This goal is achieved by recording the ion current through the channels formed by these peptides, once inserted in a cell plasma membrane. To avoid contamination by the cell membrane currents, all the endogenous current sources must be blocked. It has been found that the photoreceptor rod outer segment mechanically isolated from the retina of low vertebrates (OS) was the most suitable cell to carry on the above studies, because it was possible to fully block all its endogenous currents without using any drug (such as TTX, TEA, dihydropyridines, etc.), that could obstruct the peptide pores or interfere with the pore formation. The peptides were applied to (and removed from) the extracellular OS side in ~50 ms with a computer-controlled microperfusion system, in which every perfusion parameter (as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles) was controlled by a user-friendly interface. This system allowed rapid application and removal of ions, drugs and peptides on the cells with a controlled timing, so that the ion channel characteristics (as its selectivity, blockade and gating) and the dynamics of pore formation could be precisely assessed. On the basis of the electrophysiological recordings obtained with representative peptides and with selected analogs, as alamethicin F50/5, the cecoprine-mellitin hybrid peptide, and a 20-aminoacid long fragment of the viroporin poliovirus 2B, it will be shown that the membrane pore formation occurs according to the barrel and stave, toroidal, and carpet model, respectively, that are the most widely-accepted mechanisms of membrane permeabilization. When recording large currents (produced for instance by high concentrations of peptides and/or highly permeable peptides), it is necessary to minimize series resistance, to reduce time constant of charging the cell membrane capacitance and error in membrane potential control. A second problem arises from the asymmetry of the plasma membrane: it is possible that the permeabilization properties of a particular peptide could be different depending upon the side of the membrane to which it is applied. For example, it is conceivable that viroporins are optimized to insert in the intracellular face of the plasma membrane, because they are synthesized in host cell cytosol. These two problems could be circumvented by widening the patch pipette shank, through the calibrated combination of heat and air pressure. These pipettes dramatically reduce series resistance, and allow at the same time to insert pulled quartz or plastic tubes very close to the pipette tip, making it possible the delivery of large molecules to the cytosol with a controlled timing. Finally, it is presented here a simple procedure to consistently attain seals with conventional or pressure polished pipettes, made from just one glass type, on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes made with many different lipid mixtures

The Partners of My Partners: Shared Collaborative Experience and Team Performance in Surgical Teams

When teams in organizations are assembled to perform contingent tasks, team members carry with them experiences of prior interaction with partners in different teams. Focal team members share collaborative experiences to the extent that they worked with common external prior partners. Extending current research on team effectiveness, we investigate how shared collaborative experience (SCE) affects team performance. Consistent with the established understanding of team processes as carrying both a teamwork and a taskwork component, we conceptualize SCE as having two distinct dimensions that we call SCE extent and SCE diversity. We posit that high SCE extent increases the ability of teams to refine their teamwork processes, increasing their performance through enhanced coordination and reflexivity. We argue that high SCE diversity hinders the ability of teams to form a shared understanding of task demands, thus undermining team performance. Furthermore, we investigate the contingent effect of task complexity on the relationship between SCE and performance. We argue that the benefits of implicit coordination and the drawbacks of experience diversity decrease as tasks become more complex and require more explicit coordination and wider repertoires of responses. These predictions find support in an analysis of 1343 robot-assisted surgery operations performed by 114 surgeons during a four-year period in a private university hospital. By explicitly recognizing how team members benefit from the network of their shared prior partners, our study contributes to developing a new approach to study the effectiveness of temporary teams in organizations

Persistent high plasma levels of sCD163 and sCD14 in adult patients with measles virus infection

Background and aims: Measles is an infectious disease that represents a serious public health problem worldwide, being associated with increased susceptibility to secondary infections, especially in the respiratory and gastrointestinal tracts. The aim of this study was to evaluate sCD163 and sCD14 levels in measles virus (MV) infected patients, as markers of immune activation, in order to better understand their role in the pathogenesis of the disease. TNF-α plasma levels were also evaluated. Methods: sCD163, sCD14 and TNF-α were measured by ELISA in plasma samples of 27 MV infected patients and 27 healthy donors (HD) included as controls. Results: At the time of hospital admission, sCD163 and sCD14 levels were significantly higher in MV infected patients than in HD, while a decrease in TNF-α levels were found even if without statistical significance. sCD163 and sCD14 levels were significantly decreased after two months from acute infection compared to hospital admission although they remained significantly higher compared to HD. TNF-α levels increased significantly during the follow-up period. Considering clinical parameters, sCD163 levels positively correlated with aspartate aminotransferase, white blood cell count and neutrophils rate, while negatively correlated with the lymphocyte percentage. sCD14 levels positively correlated with the neutrophil and lymphocyte percentages. Conclusions. These results indicate that, despite the resolution of symptoms, an important macrophage/ monocyte activation persists in measles patients, even after two months from infection

Changes in inflammatory biomarkers in HCV-infected patients undergoing direct acting antiviral-containing regimens with or without interferon

Background and aims Increased levels of chemokine interferon-gamma (IFN-γ)-inducible protein-10 (CXCL10), soluble CD163 (sCD163) and soluble CD14 (sCD14) have been reported in HCV infection. The aim of this study was to compare, sCD163 and sCD14 levels in HCV-infected patients undergoing direct acting antiviral (DAA)-containing regimens with or without interferon (IFN). Methods sCD163, sCD14 and CXCL10 were longitudinally measured by ELISA in 159 plasma samples from 25 HCV-infected patients undergoing IFN-based treatment plus telaprevir or boceprevir and 28 HCV infected subjects treated with DAA IFN-free regimens. Twenty-five healthy donors (HD) were included as controls. Results At baseline CXCL10, sCD163 and sCD14 levels were higher in HCV-infected patients than in HD. CXCL10 and sCD163 levels were significantly decreased in responder (R) patients who achieved sustained virological response (SVR), with both IFN-based and IFN-free regimens, while they were persistently elevated in non-responders (NR) patients who stopped IFN-based treatments because of failure or adverse events. Conversely, sCD14 levels were apparently unchanged during therapy, but at the end of treatment the levels reached normal ranges. Comparing the two regimens, the extent of CXCL10 reduction was more pronounced in patients undergoing DAA IFN-free therapies, whereas sCD163 and sCD14 reduction was similar in the two groups. Interestingly, only in IFN-based regimens baseline sCD163 levels were significantly higher in NR than in R patients, while in the IFN-free treatment group also patients with highsCD163 plasma levels obtained SVR. At the end of therapy, even if the biomarkers were largely decreased, their levels remained significantly higher compared to HD. Only in the early fibrosis stages, sCD163 values tended to normalize. Conclusions These results indicate that IFN-free regimens including newer DAA induce an early and marked decrease in circulating inflammatory biomarkers. However, the full normalization of biomarkers was not obtained, especially in patients with advanced fibrosis, thus underlying the need for a treatment in the early stages of HCV infection

A dual perspective on geostationary satellite monitoring using DSLR RGB and sCMOS sloan filters

This paper outlines a multi-system approach for ground-based optical observations and the characterization of satellites in geostationary orbit. This multi-system approach is based on an in-depth analysis of the key factors to consider for light curve analysis of Earth’s orbiting satellites. Light curves have been observed in different spectral bands using two different systems. The first system is specialized for astronomical observations and consists of a telescope equipped with an sCMOS camera and Sloan photometric filters. In contrast, the second system is a more cost-effective solution designed for professional non-astronomical applications, incorporating DSLR cameras equipped with RGB channels associated with a Bayer mask and photographic lenses. This comparative analysis aims to highlight the differences and advantages provided by each system, stressing their respective performance characteristics. The observed light curves will be presented as a function of the phase angle, which depends on the relative positions of the observer, the object, and the Sun. This angle plays an important role in optimizing the visibility of Earth’s orbiting satellites. Finally, multiband observations of different satellites will be compared to seek an associated spectral signature, which may allow the identification of structurally similar objects through optical observations

Attitudes toward relevant aspects of medical practice: a cross-sectional study with a random sample of second- and sixth-year students

This was a cross-sectional study with two random samples: 50 second-year and 50 sixth-year undergraduate medical students. An open-ended questionnaire was applied, in addition to a scale known as the Instrument for evaluating medical students' attitudes towards key aspects of medical practice (Colares, 2002). The scale contains 52 questions on: 1. Psychological and emotional aspects of physical and mental illness; 2. managing situations related to death; 3. primary healthcare; 4. aspects related to mental illness; 5. the physician's contribution to scientific progress in medicine; and 6. other aspects of medical work and health policies. The attitudes are categorized as positive, negative, or conflictive. According to the findings, students had positive attitudes towards at least three of the six aspects. Second-year and sixth-year students differed significantly (chi² = 6.901, d.f. = 1, p < 0.05) in their attitudes toward factor 2 (managing situations related to death).Estudo transversal com duas amostras randomizadas de 50 alunos do segundo e 50 do sexto ano de graduação em Medicina. Foi aplicado um questionário com questões abertas e a escala Instrumento de avaliação de atitudes de estudantes de medicina frente a aspectos relevantes da prática médica (Colares, 2002). A escala contém 52 questões referentes à: 1. Aspectos psicológicos e emocionais nas doenças orgânicas e mentais; 2. Manejo de situações relacionadas à morte; 3. Atenção primária à saúde; 4. Aspectos relacionados à doença mental; 5. Contribuição do médico ao avanço científico da medicina; 6. Outros aspectos relacionados à atuação médica e às políticas de saúde. As atitudes são categorizadas em positivas, negativas e conflitantes. Observou-se que os estudantes apresentaram atitudes positivas frente a pelo menos três dos seis aspectos abordados; os alunos do segundo ano e do sexto ano apresentaram diferença estatisticamente significativa (chi² = 6,901, g.l. = 1, p < 0,05) nas atitudes relacionadas ao fator 2 (manejo de situações relacionadas à morte).Universidade Federal de São Paulo (UNIFESP)Secretaria de Estado da Saúde de São Paulo Instituto de SaúdeUNIFESP, Psiquiatra-EPMSciEL

Accuracy of Plateau Pressure and Stress Index to Identify Injurious Ventilation in Patients with Acute Respiratory Distress Syndrome.

BACKGROUND: Guidelines suggest a plateau pressure (PPLAT) of 30 cm H(2)O or less for patients with acute respiratory distress syndrome, but ventilation may still be injurious despite adhering to this guideline. The shape of the curve plotting airway pressure versus time (STRESS INDEX) may identify injurious ventilation. The authors assessed accuracy of PPLAT and STRESS INDEX to identify morphological indexes of injurious ventilation. METHODS: Indexes of lung aeration (computerized tomography) associated with injurious ventilation were used as a "reference standard." Threshold values of PPLAT and STRESS INDEX were determined assessing the receiver-operating characteristics ("training set," N = 30). Accuracy of these values was assessed in a second group of patients ("validation set," N = 20). PPLAT and STRESS INDEX were partitioned between respiratory system (Pplat,Rs and STRESS INDEX,RS) and lung (PPLAT,L and STRESS INDEX,L; esophageal pressure; "physiological set," N = 50). RESULTS: Sensitivity and specificity of PPLAT of greater than 30 cm H(2)O were 0.06 (95% CI, 0.002-0.30) and 1.0 (95% CI, 0.87-1.00). PPLAT of greater than 25 cm H(2)O and a STRESS INDEX of greater than 1.05 best identified morphological markers of injurious ventilation. Sensitivity and specificity of these values were 0.75 (95% CI, 0.35-0.97) and 0.75 (95% CI, 0.43-0.95) for PPLAT greater than 25 cm H(2)O versus 0.88 (95% CI, 0.47-1.00) and 0.50 (95% CI, 0.21-0.79) for STRESS INDEX greater than 1.05. Pplat,Rs did not correlate with PPLAT,L (R(2) = 0.0099); STRESS INDEX,RS and STRESS INDEX,L were correlated (R(2) = 0.762). CONCLUSIONS: The best threshold values for discriminating morphological indexes associated with injurious ventilation were Pplat,Rs greater than 25 cm H(2)O and STRESS INDEX,RS greater than 1.05. Although a substantial discrepancy between Pplat,Rs and PPLAT,L occurs, STRESS INDEX,RS reflects STRESS INDEX,L