Differential effects of coverslip-induced hypoxia and cobalt chloride mimetic hypoxia on cellular stress, metabolism, and nuclear structure.

Hypoxia has profound effects on cell physiology, both in normal or pathological settings like cancer. In this study, we asked whether a variant of coverslip-induced hypoxia that recapitulates the conditions found in the tumor microenvironment would elicit similar cellular responses compared to the well established model of cobalt chloride-induced hypoxia. Comparable levels of nuclear HIF-1α were observed after 24 h of coverslip-induced hypoxia or cobalt chloride treatment in CAL-27 oral squamous carcinoma cells. However, cellular stress levels assessed by reactive oxygen species production and lipid droplet accumulation were markedly increased in coverslip-induced hypoxia compared to cobalt chloride treatment. Conversely, mitochondrial ATP production sharply decreased after coverslip-induced hypoxia but was preserved in the presence of cobalt chloride. Coverslip-induced hypoxia also had profound effects in nuclear organization, assessed by changes in nuclear dry mass distribution, whereas these effects were much less marked after cobalt chloride treatment. Taken together, our results show that coverslip-induced hypoxia effects on cell physiology and structure are more pronounced than mimetic hypoxia induced by cobalt chloride treatment. Considering also the simplicity of coverslip-induced hypoxia, our results therefore underscore the usefulness of this method to recapitulate in vitro the effects of hypoxic microenvironments encountered by cells in vivo.

Evolution of attack in handball when playing 7 vs. 6 with empty goal between 2020 and 2023: coaches' perception vs. observational results.

Introduction: Recently, several studies on the 7 vs. 6 "empty goal" (EG) in handball have produced different and even contradictory results. The aim of the present study was to investigate the behavior of teams and players in the 7 vs. 6 EG attack in the European (Euro) and World Championships (WCh) between 2020 and 2023 and characterize the coaches' perceptions. Methods: A mixed-methods approach was used, consisting of the following: (i) an observational methodology and instrument developed and validated to collect observational data on player and team behavior; and (ii) a developed and validated questionnaire to coaches on their perceptions of the 7 vs. 6 game. Observational data were collected during the Euro 2020 and 2022 games (n = 62) and the WCh 2021 and 2023 games (n = 70). A total of 132 games and 391 situations of 7 vs. 6 attacking sequences were observed. In total, 156 coaches participated (146 men), with a mean age 42.33 ± 11.87 years, 19 nationalities, and with 12.77 ± 9.45 years of experience. Results and discussion: The choice of 7 vs. 6 offensive play was mostly made in the second half (>73%). The effectiveness of 7 vs. 6 offensive sequences was higher in the top six teams than in the team's ranked 7th to 12th (Euro 2020 51.6%-50.0%; WCh 2021 52.0%-50.0%; Euro 2022 53.1%-41.7%; WCh 2023 50.0%-43.8%). Some patterns of association were found (p < 0.05 and with values >±1.96): (i) scoring a goal with a breakthrough shot was significantly associated with the effectiveness of the 7 vs. 6 attack (Euro 2020 2.61; WCh 2021 2.87; Euro 2022 2.68; WCh 2023 2.32); (ii) teams in the top six significantly used 7 vs. 6 when they were winning (Euro 2020 2.17; WCh 2021 3.52; Euro 2022 5.88; WCh 2023 2.54); and (iii) teams in the bottom six used it when they were losing by at least four goals (Euro 2020 7.56; Euro 2022 6.64; WCh 2023 4.37) or when they were winning by four goals or more (WCh 2021 2.58). Coaches that agree with the possibility of playing 7 vs. 6 (74.4%), rarely or never do so (55.6%) because it brings little or no advantage (52.6%). The results of the analysis confirmed the perception of the coaches, the low use of 7 vs. 6, the low advantage associated with it, and the influence of the result and the moment of the game on its use.

Real-time phase retrieval in division of aperture microscopy with the transport of intensity equation.

The transport of intensity equation (TIE) allows to recover the phase of a microscopy sample from differently focused intensity measures along the axial direction of its optical field. In the present work, we propose a cost-effective technique for snapshot phase retrieval with TIE. The optics of a commercially available camera is replaced with a doublet system consisting of a microscope objective and a lenslet array with an extra lens mask attached to it. The system allows to obtain, in real-time and with no mechanical shift of either the sample or the sensor, the in-focus as well as a defocused image of the sample. From these two sub-aperture images, the intensity derivative term in TIE can then be approximated after image rectification. Phase is then retrieved for static as well as dynamic samples over the common view area. Validation experiments are presented.

Automated resolution of the spiral torsion spring inverse design problem.

Many mechanical applications take advantage of spiral torsion springs due to their robustness, compactness, and simplicity. Brand-new manufacturing methods allow to create spiral springs with unconventional geometries and materials that suit a wider range of uses demanding either linearity or nonlinearity. Designing a spiral torsion spring with a nonlinear desired torque curve may be a great challenge, due to their many degrees-of-freedom (length, width, thickness, arbor, and barrel diameters, etc.) and the complexity of the geometrical and mechanical requirements to ensure their manufacturability, system compatibility, operation safety and reliability; and the solution is never unique. This manuscript proposes and validates an innovative methodology for the resolution of this inverse design problem based on the application of a nonlinear restrained global optimization algorithm. This algorithm is adjusted to converge, out of the infinity of designs that match the desired torque curve and hold all the functional and manufacturing constraints, to a design solution that minimizes strip mass. The methodology is built on a formulation for the calculation of the torque curve of a generalized spiral spring, with or without coiling and with any along-the-length cross-section, already published by the authors.

Contributions of viral oncogenes of HPV-18 and hypoxia to oxidative stress and genetic damage in human keratinocytes.

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the development of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.

HRpI System Based on Wavenet Controller with Human Cooperative-in-the-Loop for Neurorehabilitation Purposes.

There exist several methods aimed at human-robot physical interaction (HRpI) to provide physical therapy in patients. The use of haptics has become an option to display forces along a given path so as to it guides the physiotherapist protocol. Critical in this regard is the motion control for haptic guidance to convey the specifications of the clinical protocol. Given the inherent patient variability, a conclusive demand of these HRpI methods is the need to modify online its response with neither rejecting nor neglecting interaction forces but to process them as patient interaction. In this paper, considering the nonlinear dynamics of the robot interacting bilaterally with a patient, we propose a novel adaptive control to guarantee stable haptic guidance by processing the causality of patient interaction forces, despite unknown robot dynamics and uncertainties. The controller implements radial basis neural network with daughter RASP1 wavelets activation function to identify the coupled interaction dynamics. For an efficient online implementation, an output infinite impulse response filter prunes negligible signals and nodes to deal with overparametrization. This contributes to adapt online the feedback gains of a globally stable discrete PID regulator to yield stiffness control, so the user is guided within a perceptual force field. Effectiveness of the proposed method is verified in real-time bimanual human-in-the-loop experiments.

Validez diagnóstica de cinco escalas de predicción clínica para la trombosis venosa profunda (TVP)

Introducción: la trombosis venosa profunda (TVP) es una entidad común que afecta principalmente el sistema venoso profundo de los miembros inferiores, para el cual se han desarrollado múltiples escalas de predicción clínica, las cuales han sido construidas y validadas en pacientes ambulatorios y hospitalizados. Objetivos: validar cinco escalas de predicción clínica para TVP en pacientes atendidos en un centro de tercer nivel en la sabana de Bogotá, Colombia. Métodos: se llevó a cabo un estudio de corte transversal con análisis de prueba diagnóstica en sujetos con sospecha de TVP, incluyendo aquellos que contaran con la realización de ecografía Doppler venosa de miembros inferiores. Se calculó el rendimiento de cinco escalas de predicción clínica para TVP (Wells clásico y modificado, Oudega, CEBI y Constans) para pacientes ambulatorios u hospitalizados, individualizando la población en la que fueron validadas. Resultados: ingresaron al análisis 974 pacientes, de estos 485 (49,7 %) presentaron TVP. La escala de Constans tuvo un mejor rendimiento diagnóstico entre los pacientes hospitalizados y ambulatorios, con un área bajo la curva ROC de 0,73 (95 % 0,70-0,78) al compararla con Wells clásico, Wells modificado, Oudega y CEBI. Al comparar el rendimiento de Constans en ambos grupos de pacientes por separado, también se observó un mejor rendimiento con respecto a las demás escalas. Conclusión: la escala de Constans presenta un mejor rendimiento diagnóstico comparado con las demás escalas al ser aplicada en paciente hospitalizados y ambulatorios.

Summary Introduction: The deep vein thrombosis (DVT) is a common entity that mainly affects the deep venous system of the lower limbs, for which multiple clinical prediction scales have been developed, which have been constructed and validated in outpatients and inpatients. Objetives: We aimed to validated five clinical prediction scores for the diagnosis of lower limb DVT in patients from La Sabana de Bogota, Colombia. Methods: A cross-sectional study with analysis of a diagnostic test was carried out in patiens with suspected deep vein thrombosis, including those who had venous Doppler ultrasound of the lower limbs for suspected DVT. The performance of five clinical prediction scales for DVT (classic and modified Wells, Oudega, CEBI and Constans) for outpatients and inpatients was calculated in those scores who are validated in both populations and only in ambulatory or hospitalized patients for those that are specific scores. Results: Nine hundred seventy-four patients were entered into the analysis, of which 485 (49.7%) presented DVT. The Constans scale had a better diagnostic performance among inpatients and outpatients with an area under the ROC curve of 0.73 (95% 0.70-0.78) when compared with classic Wells, modified Wells, Oudega and CEBI. When we compared Constans performance in both groups of patients separately, we observed better performance with respect to the other scores. Conclusion: The Constans scale presents a better diagnostic performance compared to the other scales when applied to inpatients and outpatients.

HSFs drive transcription of distinct genes and enhancers during oxidative stress and heat shock.

Reprogramming of transcription is critical for the survival under cellular stress. Heat shock has provided an excellent model to investigate nascent transcription in stressed cells, but the molecular mechanisms orchestrating RNA synthesis during other types of stress are unknown. We utilized PRO-seq and ChIP-seq to study how Heat Shock Factors, HSF1 and HSF2, coordinate transcription at genes and enhancers upon oxidative stress and heat shock. We show that pause-release of RNA polymerase II (Pol II) is a universal mechanism regulating gene transcription in stressed cells, while enhancers are activated at the level of Pol II recruitment. Moreover, besides functioning as conventional promoter-binding transcription factors, HSF1 and HSF2 bind to stress-induced enhancers to trigger Pol II pause-release from poised gene promoters. Importantly, HSFs act at distinct genes and enhancers in a stress type-specific manner. HSF1 binds to many chaperone genes upon oxidative and heat stress but activates them only in heat-shocked cells. Under oxidative stress, HSF1 localizes to a unique set of promoters and enhancers to trans-activate oxidative stress-specific genes. Taken together, we show that HSFs function as multi-stress-responsive factors that activate distinct genes and enhancers when encountering changes in temperature and redox state.

Computational multifocus fluorescence microscopy for three-dimensional visualization of multicellular tumor spheroids.

SIGNIFICANCE: Three-dimensional (3D) visualization of multicellular tumor spheroids (MCTS) in fluorescence microscopy can rapidly provide qualitative morphological information about the architecture of these cellular aggregates, which can recapitulate key aspects of their in vivo counterpart. AIM: The present work is aimed at overcoming the shallow depth-of-field (DoF) limitation in fluorescence microscopy while achieving 3D visualization of thick biological samples under study. APPROACH: A custom-built fluorescence microscope with an electrically focus-tunable lens was developed to optically sweep in-depth the structure of MCTS. Acquired multifocus stacks were combined by means of postprocessing algorithms performed in the Fourier domain. RESULTS: Images with relevant characteristics as extended DoF, stereoscopic pairs as well as reconstructed viewpoints of MCTS were obtained without segmentation of the focused regions or estimation of the depth map. The reconstructed images allowed us to observe the 3D morphology of cell aggregates. CONCLUSIONS: Computational multifocus fluorescence microscopy can provide 3D visualization in MCTS. This tool is a promising development in assessing the morphological structure of different cellular aggregates while preserving a robust yet simple optical setup.

Mutation of putative phosphorylation sites in the free fatty acid receptor 1: Effects on signaling, receptor phosphorylation, and internalization.

Free fatty acid receptor 1 phosphorylation sites were studied using mutants, including a) a mutant with T215V in the third intracellular loop (3IL), b) another with changes in the carboxyl terminus (C-term): T287V, T293V, S298A, and c) a mutant with all of these changes (3IL/C-term). Agonist-induced increases in intracellular calcium were similar between cells expressing wild-type or mutant receptors. In contrast, agonist-induced FFA1 receptor phosphorylation was reduced in mutants compared to wild type. Phorbol ester-induced FFA1 receptor phosphorylation was rapid and robust in cells expressing the wild-type receptor and essentially abolished in the mutants. Agonist-induced ERK 1/2 phosphorylation and receptor internalization were decreased in cells expressing the mutant receptors compared to those expressing the wild-type receptor. Our data suggest that the identified sites might participate in receptor phosphorylation, signaling, and internalization.

The LPA3 Receptor: Regulation and Activation of Signaling Pathways.

The lysophosphatidic acid 3 receptor (LPA3) participates in different physiological actions and in the pathogenesis of many diseases through the activation of different signal pathways. Knowledge of the regulation of the function of the LPA3 receptor is a crucial element for defining its roles in health and disease. This review describes what is known about the signaling pathways activated in terms of its various actions. Next, we review knowledge on the structure of the LPA3 receptor, the domains found, and the roles that the latter might play in ligand recognition, signaling, and cellular localization. Currently, there is some information on the action of LPA3 in different cells and whole organisms, but very little is known about the regulation of its function. Areas in which there is a gap in our knowledge are indicated in order to further stimulate experimental work on this receptor and on other members of the LPA receptor family. We are convinced that knowledge on how this receptor is activated, the signaling pathways employed and how the receptor internalization and desensitization are controlled will help design new therapeutic interventions for treating diseases in which the LPA3 receptor is implicated.

Abscés cervical profund associat a col·locació de mascareta laríngia / Absceso cervical profundo asociado a la colocación de mascarilla laríngea / Deep neck abscess associated to the placement of a laryngeal mask

INTRODUCCIÓ: Els abscessos parafaringi I retrofaringi són infeccions profundes del coll que solen associar-se a l'antecedent d'infecció de vies respiratòries altes. Ocasionalment poden ser causats per traumatismes com els que comporten algunes manipulacions mèdiques, per exemple, la col·locació de mascareta laríngia, molt utilitzada en cirurgia pediàtrica. CAS CLÍNIC: Es presenta el cas d'una nena de 6 anys amb torticoli de 8 dies d'evolució I febre de 24 hores, sense cap altra simptomatologia. La pacient havia estat intervinguda quirúrgicament sota anestèsia general amb col·locació de mascareta laríngia 36 hores abans de l'inici del quadre, sense incidències. En l'exploració, destaca una contractura cervical bilateral amb flexió del cap a la dreta, I a l'analítica es troba leucocitosi amb predomini de neutròfils I augment de la proteïna C reactiva. Es fa una ressonància magnètica cervical en què s'observa un abscés d'extensió parafaríngia I retrofaríngia, I s'indica una punció percutània ecoguiada de l'àrea abscessificada, que resulta positiva per a S. pyogenes. S'ingressa la pacient amb antibioteràpia endovenosa I s'aconsegueix la millora clínica I radiològica de l'abscés. COMENTARIS: Els abscessos cervicals profunds s'han de considerar davant de simptomatologia obstructiva I inflamatòria de la via aèria I el tracte digestiu superior, I símptomes locals o dolor al moviment cervical. El diagnòstic es basa en les troballes radiològiques, analítiques I microbiològiques, I cal instaurar antibioteràpia endovenosa empírica amb cobertura per a estafilococs, estreptococs I anaerobis. Tot I que aquests abscessos en la majoria de casos són secundaris a l'extensió d'infeccions del tracte respiratori superior, hi ha altres causes que cal considerar

INTRODUCCIÓN: Los abscesos retrofaríngeo y parafaríngeo son infecciones profundas del cuello que suelen asociarse al antecedente de infección de vías respiratorias altas. Ocasionalmente pueden ser causados por traumatismos como los que resultan de algunos procedimientos médicos, por ejemplo, la colocación de la mascarilla laríngea, muy utilizada en cirugía pediátrica. CASO CLÍNICO: Se presenta el caso de una niña de 6 años con tortícolis de 8 días de evolución y fiebre de 24 horas, sin otra sintomatología. La paciente había sido intervenida quirúrgicamente bajo anestesia general con colocación de mascarilla laríngea 36 horas antes del inicio del cuadro, sin incidencias. A la exploración, destaca una contractura cervical bilateral con flexión de la cabeza hacia la derecha, y en la analítica se encuentra leucocitosis con predominio neutrofílico y ligero aumento de proteína C reactiva. Se realiza una resonancia magnética cervical donde se observa un absceso de extensión parafaríngea y retrofaríngea, y se practica una punción percutánea ecoguiada de la zona abscesificada, que resulta positiva para S. pyogenes. La paciente ingresa con antibioterapia endovenosa y se consigue mejoría clínica y radiológica del absceso. COMENTARIO: Los abscesos cervicales profundos deben considerarse ante sintomatología obstructiva e inflamatoria de la vía aérea y del tracto digestivo superior, y síntomas locales o dolor al movimiento del cuello. El diagnóstico se basa en los hallazgos radiológicos, analíticos y microbiológicos y se debe instaurar antibioterapia endovenosa empírica con cobertura para estafilococos, estreptococos y anaerobios. Aunque en la mayoría de casos estos abscesos son secundarios a la extensión de infecciones del tracto respiratorio superior, existen otras causas que deben considerarse

INTRODUCTION: Retropharyngeal and parapharyngeal abscesses are deep neck infections that are usually associated with superior airway infections in children. However, they could also be caused by injuries secondary to medical procedures such as the placement of laryngeal masks, which are frequently used in pediatric surgery. CASE REPORT: A 6-year-old female presented to the emergency room with an 8-day history of torticollis and 24 hours of fever with no other associated symptomatology. She had undergone surgery under general anesthesia using a laryngeal mask 36 hours prior, without immediate complications. In the physical examination, the patient had bilateral cervical contracture with right bending. The blood examination showed leukocytosis with predominance of neutrophils and increase of C-reactive protein. Magnetic resonance imaging showed an abscess with parapharyngeal and retropharyngeal extension, and a percutaneous ultrasound-guided puncture of the abscessed area was performed, which resulted positive for S. pyogenes. The patient received intravenous antibiotic therapy and achieved clinical and radiological resolution of the abscess. COMMENTS: Deep neck abscesses should be considered in children with obstructive and inflammatory symptomatology of the airway and upper digestive tract and also local symptoms as neck pain. Diagnosis is based on radiological, analytical and microbiological findings and empirical intravenous antibiotics, with coverage for staphylococcus, streptococcus and anaerobics. Although in most cases these abscesses are secondary to the spread of upper respiratory tract infections, other causes should be considered

Heat Shock Factor 2 Protects against Proteotoxicity by Maintaining Cell-Cell Adhesion.

Maintenance of protein homeostasis, through inducible expression of molecular chaperones, is essential for cell survival under protein-damaging conditions. The expression and DNA-binding activity of heat shock factor 2 (HSF2), a member of the heat shock transcription factor family, increase upon exposure to prolonged proteotoxicity. Nevertheless, the specific roles of HSF2 and the global HSF2-dependent gene expression profile during sustained stress have remained unknown. Here, we found that HSF2 is critical for cell survival during prolonged proteotoxicity. Strikingly, our RNA sequencing (RNA-seq) analyses revealed that impaired viability of HSF2-deficient cells is not caused by inadequate induction of molecular chaperones but is due to marked downregulation of cadherin superfamily genes. We demonstrate that HSF2-dependent maintenance of cadherin-mediated cell-cell adhesion is required for protection against stress induced by proteasome inhibition. This study identifies HSF2 as a key regulator of cadherin superfamily genes and defines cell-cell adhesion as a determinant of proteotoxic stress resistance.

Receptor tyrosine kinase activation induces free fatty acid 4 receptor phosphorylation, ß-arrestin interaction, and internalization.

FFA4 (Free Fatty Acid receptor 4, previously known as GPR120) is a G protein-coupled receptor that acts as a sensor of long-chain fatty acids, modulates metabolism, and whose dysfunction participates in endocrine disturbances. FFA4 is known to be phosphorylated and internalized in response to agonists and protein kinase C activation. In this paper report the modulation of this fatty acid receptor by activation of receptor tyrosine kinases. Cell-activation with growth factors (insulin, epidermal growth factor, insulin-like growth factor-I, and platelet-derived growth factor) increases FFA4 phosphorylation in a time- and concentration-dependent fashion. This effect was blocked by inhibitors of protein kinase C and phosphoinositide 3-kinase, suggesting the involvement of these kinases in it. FFA4 phosphorylation did not alter agonist-induced FFA4 calcium signaling, but was associated with decreased ERK 1/2 phosphorylation. In addition, insulin, insulin-like growth factor-I, epidermal growth factor, and to a lesser extent, platelet-derived growth factor, induce receptor internalization. This action of insulin, insulin-like growth factor I, and epidermal growth factor was blocked by inhibitors of protein kinase C and phosphoinositide 3-kinase. Additionally, cell treatment with these growth factors induced FFA4-ß-arrestin coimmunoprecipitation. Our results evidenced cross-talk between receptor tyrosine kinases and FFA4 and suggest roles of protein kinase C and phosphoinositide 3-kinase in such a functional interaction.

Using a variant of coverslip hypoxia to visualize tumor cell alterations at increasing distances from an oxygen source.

Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid-containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development.

Percepciones estudiantiles sobre las propiedades del examen clínico objetivo estructurado en Uruguay. Disponiendo un cuestionario válido para analizar la factibilidad del nuevo formato durante la transición curricular / Student perceptions on properties of the objective structured clinical examination in Uruguay. The use of a valid questionnaire to analyse the feasibility of the new format during curriculum transition

INTRODUCCIÓN: Aunque el formato de examen clínico objetivo estructurado (ECOE) se aplica en Uruguay desde 2004 proveyendo medidas confiables de rendimiento, las percepciones sobre sus propiedades y el grado de satisfacción estudiantiles no han sido exploradas. OBJETIVO: La siguiente experiencia estuvo dirigida a evaluar la validez aparente del ECOE como aporte al estudio de su factibilidad local. MATERIAL Y MÉTODOS: Al final del ciclo propedéutico, la subcohorte cursando en el Hospital Universitario contestó un cuestionario de 28 ítems dirigido a explorar las percepciones sobre las propiedades del ECOE en los potenciales factores «diseño» y «validez aparente». Tras analizar su confiabilidad, se depuró el formulario para proveer una herramienta más breve y fiable. RESULTADOS: El cuestionario original mostró una confiabilidad aceptable (alfa de Cronbach = 0,70), dominando las opiniones de «acuerdo/total acuerdo» sobre autenticidad de los estímulos, dinámica, pertinencia y justicia del examen. Los estudiantes percibieron obstáculos organizativos durante la prueba, manifestando desacuerdo en incluir alguna estación dirigida a evaluar solamente habilidades para la comunicación y requiriendo instancias de devolución (feedback) personalizada. La versión depurada del cuestionario original provee medidas consistentes sobre las percepciones estudiantiles y resulta una herramienta útil que podría aplicarse extensamente. Se discuten los aportes de esta experiencia al estudio de la factibilidad del nuevo formato durante la transición curricular. CONCLUSIONES: El ECOE es bien evaluado por los estudiantes al final del ciclo propedéutico, lo que apoya su validez. Deben aprovecharse sus potenciales formativos proveyendo feedback efectivo a estudiantes, tutores clínicos e institución

INTRODUCTION: Although an objective structured clinical examination (OSCE) format has been applied in Uruguay since 2004, and providing reliable performance measures, perceptions of it properties and level of student satisfaction have not been determined. OBJECTIVE: To evaluate the face validity of OSCE format as a contribution to its local feasibility study. MATERIAL AND METHODS: At the end of the introductory clinical course, the sub-cohort enrolled at the University Hospital responded to a 28-item questionnaire aimed at exploring perceptions about the properties of the OSCE about the potential factors 'design' and 'apparent validity'. After analysing the reliability of the original questionnaire, the questionnaire was refined in an attempt to provide a shorter and more reliable tool. RESULTS: The original questionnaire showed good internal consistency (Cronbach Alpha = 0.70), with a dominance of 'agreement/total agreement' opinions on authenticity of the stimuli, dynamic, relevance, and equity of the test. Students perceived organisational obstacles during the test, expressing disagreement to include some stations only aimed at assessing communication skills, and requiring personalised feedback sessions. The refined version of the questionnaire provides consistent measures on student perceptions and is a useful tool that can be widely applied. A discussion is presented on the contributions of this experience to a comprehensive feasibility study of the new format during curriculum transition. CONCLUSIONS: The OSCE is well evaluated by students at the end of the propaedeutic course, supporting its validity. Educational potentials of the new format should be exploited, providing effective feedback to students, clinical teachers, and institutions

Peptidoglycan Remodeling Enables Escherichia coli To Survive Severe Outer Membrane Assembly Defect.

Gram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCE In Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show that Escherichia coli cells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.

Type 2 Diabetes Alters Intracellular Ca2+ Handling in Native Endothelium of Excised Rat Aorta.

An increase in intracellular Ca2+ concentration ([Ca2+]i) plays a key role in controlling endothelial functions; however, it is still unclear whether endothelial Ca2+ handling is altered by type 2 diabetes mellitus, which results in severe endothelial dysfunction. Herein, we analyzed for the first time the Ca2+ response to the physiological autacoid ATP in native aortic endothelium of obese Zucker diabetic fatty (OZDF) rats and their lean controls, which are termed LZDF rats. By loading the endothelial monolayer with the Ca2+-sensitive fluorophore, Fura-2/AM, we found that the endothelial Ca2+ response to 20 µM and 300 µM ATP exhibited a higher plateau, a larger area under the curve and prolonged duration in OZDF rats. The "Ca2+ add-back" protocol revealed no difference in the inositol-1,4,5-trisphosphate-releasable endoplasmic reticulum (ER) Ca2+ pool, while store-operated Ca2+ entry was surprisingly down-regulated in OZDF aortae. Pharmacological manipulation disclosed that sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity was down-regulated by reactive oxygen species in native aortic endothelium of OZDF rats, thereby exaggerating the Ca2+ response to high agonist concentrations. These findings shed new light on the mechanisms by which type 2 diabetes mellitus may cause endothelial dysfunction by remodeling the intracellular Ca2+ toolkit.

The Fluorescent D-Amino Acid NADA as a Tool to Study the Conditional Activity of Transpeptidases in Escherichia coli.

The enzymes responsible for the synthesis of the peptidoglycan (PG) layer constitute a fundamental target for a large group of antibiotics. The family of ß-lactam antibiotics inhibits the DD-transpeptidase (TPase) activity of the penicillin binding proteins (PBPs), whereas its subgroup of carbapenems can also block the TPase activity of the LD-TPases. D-Ala fluorescent probes, such as NADA, are incorporated into the PG presumably by TPases in Escherichia coli and can be used to study conditions that are required for their function. Of all LD-TPases of E. coli, only LdtD was able to incorporate NADA during exponential growth. Overproduction of LdtD caused NADA to be especially inserted at mid cell in the presence of LpoB-activated PBP1b and the class C PBP5. Using the NADA assay, we could confirm that LpoB activates PBP1b at mid cell and that CpoB regulates the activity of PBP1b in vivo. Overproduction of LdtD was able to partly compensate for the inhibition of the cell division specific class B PBP3 by aztreonam. We showed that class A PBP1c and the class C PBP6b cooperated with LdtD for NADA incorporation when PBP1b and PBP5 were absent, respectively. Besides, we proved that LdtD is active at pH 7.0 whereas LdtE and LdtF are more active in cells growing at pH 5.0 and they seem to cooperate synergistically. The NADA assay proved to be a useful tool for the analysis of the in vivo activities of the proteins involved in PG synthesis and our results provide additional evidence that the LD-TPases are involved in PG maintenance at different conditions.

Structural Analysis of the Interaction between the Bacterial Cell Division Proteins FtsQ and FtsB.

Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the ß domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.IMPORTANCE In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.