Keratin Dynamics and Spatial Distribution in Wild-Type and K14 R125P Mutant Cells-A Computational Model.

Keratins are one of the most abundant proteins in epithelial cells. They form a cytoskeletal filament network whose structural organization seriously conditions its function. Dynamic keratin particles and aggregates are often observed at the periphery of mutant keratinocytes related to the hereditary skin disorder epidermolysis bullosa simplex, which is due to mutations in keratins 5 and 14. To account for their emergence in mutant cells, we extended an existing mathematical model of keratin turnover in wild-type cells and developed a novel 2D phase-field model to predict the keratin distribution inside the cell. This model includes the turnover between soluble, particulate and filamentous keratin forms. We assumed that the mutation causes a slowdown in the assembly of an intermediate keratin phase into filaments, and demonstrated that this change is enough to account for the loss of keratin filaments in the cell's interior and the emergence of keratin particles at its periphery. The developed mathematical model is also particularly tailored to model the spatial distribution of keratins as the cell changes its shape.

Debriefing or Feedback: Exploring the Impact of Two Post-Scenario Discussion Methods in the Acquisition and Retention of Non-Technical Skills.

INTRODUCTION: There is a paucity of quantitative studies objectively comparing debriefing and feedback as methods for post-scenario discussion and its impact on healthcare teams' acquisition and retention of non-technical skills. The main purpose of this study is to provide some insight on this research question, using a sample of medical students. A secondary objective explores students' opinion and preference on the post-scenario discussion. MATERIAL AND METHODS: Forty-five medical students were distributed among 15 teams, and randomly allocated to two groups. Each team participated in three different simulated scenarios, with similar levels of difficulty and opportunities to apply specific non-technical skills: leadership, communication, and task management. To assess the acquisition and retention of skills, scenarios occurred on days one (baseline), two (acquisition) and 20 (retention). Team performance was objectively evaluated by an observer, using scenario recordings. Students individually assessed different aspects of debriefing and feedback. RESULTS: Both debriefing and feedback groups showed similar overall increase in objective scores, with significant increase between days one and two (acquisition), and a smaller increase between days two and 20 (retention). Students indicated debriefing as the preferred discussion method. CONCLUSION: Debriefing and feedback are effective post-scenario discussion methods, promoting acquisition and retention of non-technical skills, by undergraduate students. Allying debriefing reflexive practice with feedback directive style, and shifting appropriately between facilitation and instruction, can be a good compromise to achieve a timely and educationally meaningful discussion.

Introdução: Há uma escassez de estudos quantitativos comparando objetivamente o debriefing e o feedback como métodos de discussão pós-cenário e o seu impacto na aquisição e retenção de competências não-técnicas pelas equipas de saúde. O objetivo principal deste estudo é explorar esta questão de investigação, usando uma amostra de estudantes de medicina. Adicionalmente, foi analisada a opinião e preferência dos estudantes sobre o método de discussão pós-cenário. Material e Métodos: Quarenta e cinco estudantes de medicina foram distribuídos em 15 equipas e alocados aleatoriamente a dois grupos. Cada equipa participou em três cenários de simulação diferentes, com níveis de dificuldade semelhantes e as mesmas oportunidades para aplicar as seguintes competências não-técnicas específicas: liderança, comunicação e gestão de tarefas. Para avaliar a aquisição e retenção de competências, os cenários decorreram nos dias um (linha de base), dois (aquisição) e 20 (retenção). O desempenho de cada equipa foi avaliado objetivamente por um observador, através da análise das gravações dos cenários e de uma checklist. Os estudantes foram ainda convidados a avaliar individualmente a condução do debriefing e do feedback. Resultados: Ambos os grupos (debriefing e feedback) demonstraram um incremento semelhante nas pontuações objetivas, com um aumento acentuado entre os dias um e dois (aquisição) e um aumento ligeiro entre os dias dois e 20 (retenção). Os estudantes indicaram o debriefing como método de discussão preferencial. Conclusão: O debriefing e o feedback são métodos eficazes de discussão pós-cenário, promovendo a aquisição e retenção de competências não-técnicas por estudantes pré-graduados. A aliança da prática reflexiva do debriefing com o estilo diretivo de feedback, alternando apropriadamente entre facilitação e instrução, é um compromisso aceitável para alcançar uma discussão educacionalmente significativa num tempo limitado.

How abundant are superoxide and hydrogen peroxide in the vasculature lumen, how far can they reach?

Paracrine superoxide (O2•-) and hydrogen peroxide (H2O2) signaling critically depends on these substances' concentrations, half-lives and transport ranges in extracellular media. Here we estimated these parameters for the lumen of human capillaries, arterioles and arteries using reaction-diffusion-advection models. These models considered O2•- and H2O2 production by endothelial cells and uptake by erythrocytes and endothelial cells, O2•- dismutation, O2•- and H2O2 diffusion and advection by the blood flow. Results show that in this environment O2•- and H2O2 have half-lives <60. ms and <40. ms, respectively, the former determined by the plasma SOD3 activity, the latter by clearance by endothelial cells and erythrocytes. H2O2 concentrations do not exceed the 10 nM scale. Maximal O2•- concentrations near vessel walls exceed H2O2's several-fold when the latter results solely from O2•- dismutation. Cytosolic dismutation of inflowing O2•- may thus significantly contribute to H2O2 delivery to cells. O2•- concentrations near vessel walls decay to 50% of maximum 12 µm downstream from O2•- production sites. H2O2 concentrations in capillaries decay to 50% of maximum 22 µm (6.0 µm) downstream from O2•- (H2O2) production sites. Near arterioles' (arteries') walls, they decay by 50% within 6.0 µm (4. µm) of H2O2 production sites. However, they reach maximal values 50 µm (24 µm) downstream from O2•- production sites and decrease by 50% over 650 µm (500 µm). Arterial/olar endothelial cells might thus signal over a mm downstream through O2•--derived H2O2, though this requires nM-sensitive H2O2 transduction mechanisms.

A mathematical model for the dependence of keratin aggregate formation on the quantity of mutant keratin expressed in EGFP-K14 R125P keratinocytes.

We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture.

Intratumoral VEGF nanotrapper reduces gliobastoma vascularization and tumor cell mass.

Glioblastoma multiforme (GBM) is the most aggressive and invasive malignant brain cancer. GBM is characterized by a dramatic metabolic imbalance leading to increased secretion of the pro-angiogenic factor VEGF and subsequent abnormal tumor vascularization. In 2009, FDA approved the intravenous administration of bevacizumab, an anti-VEGF monoclonal antibody, as a therapeutic agent for patients with GBM. However, the number of systemic side effects and reduced accessibility of bevacizumab to the central nervous system and consequently to the GBM tumor mass limited its effectiveness in improving patient survival. In this study, we combined experimental and computational modelling to quantitatively characterize the dynamics of VEGF secretion and turnover in GBM and in normal brain cells and simultaneous monitoring of vessel growth. We showed that sequestration of VEGF inside GBM cells, can be used as a novel target for improved bevacizumab-based therapy. We have engineered the VEGF nanotrapper, a cargo system that allows cellular uptake of bevacizumab and inhibits VEGF secretion required for angiogenesis activation and development. Here, we show the therapeutic efficacy of this nanocargo in reducing vascularization and tumor cell mass of GBM in vitro and in vivo cancer models.

Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers.

Keratin intermediate filaments are the principal structural element of epithelial cells. Their importance in providing bulk cellular stiffness is well recognized, but their role in the mechanics of cell cortex is less understood. In this study, we therefore compared the cortical stiffness of three keratinocyte lines: primary wild type cells (NHEK2), immortalized wild type cells (NEB1) and immortalized mutant cells (KEB7). The cortical stiffness was measured by lateral indentation of cells with AOD-steered optical tweezers without employing any moving mechanical elements. The method was validated on fixed cells and Cytochalasin-D treated cells to ensure that the observed variations in stiffness within a single cell line were not a consequence of low measurement precision. The measurements of the cortical stiffness showed that primary wild type cells were significantly stiffer than immortalized wild type cells, which was also detected in previous studies of bulk elasticity. In addition, a small difference between the mutant and the wild type cells was detected, showing that mutation of keratin impacts also the cell cortex. Thus, our results indicate that the role of keratins in cortical stiffness is not negligible and call for further investigation of the mechanical interactions between keratins and elements of the cell cortex.

Kinetic and thermodynamic parameters, and partial characterization of the crude extract of tannase produced by Saccharomyces cerevisiae CCMB 520.

Tannase can be used in different industrial sectors such as in food (juices and wine) and pharmaceutical production (trimethoprim) because it catalyses the hydrolysis of hydrolysable tannins. The aim of the current study is to assess the tannase found in the crude extract of Saccharomyces cerevisiae CCMB 520, and to set its catalytic and thermodynamic properties. The enzyme was optimally active at pH 6.0 and temperature 30 °C. Tannase was activated by Na+, Ca2+, K+ at 5 × 10-3 mol/L. The half-life at 30 °C was 3465.7 min. The activation energy was 40.32 kJ/mol. The Gibbs free energy, enthalpy and entropy at 30 °C were 85.40, 48.10 and -0.12 kJ/mol K, respectively. Our results suggest that the tannase found in the crude extract of S. cerevisiae is an attractive enzyme for industrial applications, such as for beverage manufacturing and gallic acid production, due its catalytic and thermodynamic properties (heat-stable and resistant to metal ions).

Keto analogue and amino acid supplementation and its effects on ammonemia and performance under thermoneutral conditions.

Alterations of cerebral function, fatigue and disturbance in cognitive-motor performance can be caused by hyperammonemia and/or hot environmental conditions during exercise. Exercise-induced hyperammonemia can be reduced through supplementation with either amino acids or combined keto analogues and amino acids (KAAA) to improve exercise tolerance. In the present study, we evaluated KAAA supplementation on ammonia metabolism and cognitive-motor performance after high-intensity exercise under a low heat stress environment. Sixteen male cyclists received a ketogenic diet for 2 d and were divided into two groups, KAAA (KEx) or placebo (CEx) supplementation. The athletes performed a 2 h cycling session followed by a maximum test (MAX), and blood samples were obtained at rest and during exercise. Cognitive-motor tasks were performed before and after the protocol, and the exhaustion time was used to evaluate physical performance. The hydration status was also evaluated. The CEx group showed a significant increase (∼ 70%) in ammonia concentration at MAX, which did not change in the KEx group. The non-supplemented group showed a significant increase in uremia. Both the groups had a significant increase in blood urate concentrations at 120 min, and an early significant increase from 120 min was observed in the CEx group. There was no change in the glucose concentrations of the two groups. A significant increase in lactate was observed at the MAX moment in both groups. There was no significant difference in the exhaustion times between the groups. No changes were observed in the cognitive-motor tasks after the protocol. We suggest that KAAA supplementation decreases ammonia concentration during high-intensity exercise but does not affect physical or cognitive-motor performances under a low heat stress environment.

Antioxidant, antinociceptive, and anti-inflammatory properties of the ethanolic extract of Combretum duarteanum in rodents.

The antioxidant, antinociceptive, and anti-inflammatory activities of the ethanolic extract from leaves of Combretum duarteanum (EEC) were assessed in rodents through in vitro tests. The antioxidant activity was investigated by using thiobarbituric acid reactive species (TBARS), hydroxyl radical-scavenging, and scavenging activity of nitric oxide assays. The antinociceptive activity was investigated by using acetic acid-induced writhing, formalin, and hot-plate tests in mice. The anti-inflammatory activity was assessed in rats by using the carrageenan-induced hind-paw edema test and arachidonic acid-induced paw edema test. EEC possesses a strong antioxidant potential according to the TBARS, nitric oxide, and hydroxyl radical-scavenging assays; it also presented scavenger activity in all in vitro tests. After intraperitoneal injection, EEC (100, 200, and 400 mg/kg) significantly reduced the number of writhes (38.1%, 90.6%, and 97.8%, respectively) in a writhing test and the number of paw licks during phase 1 (30.5% and 69.5%, higher doses) and phase 2 (38.1%, 90.6%, and 97.8%, all doses) of a formalin test when compared with the control group. Naloxone (1.5 mg/kg, intraperitoneally) antagonized the antinociceptive action of EEC (400 mg/kg), and this finding suggests participation of the opioid system. Administration of 200 and 400 mg/kg (intraperitoneally) of EEC exhibited an anti-inflammatory activity in the carrageenin test, which was based on interference with prostaglandin synthesis. This finding was confirmed by the arachidonic acid test. Together, these results indicate that properties of EEC might be further explored in the search for newer tools to treat painful inflammatory conditions, including those related to pro-oxidant states.