Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 µM insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 µM dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

[Zero Emissions. A shared responsibility. Gas capture and recycling project at the Cruces University Hospital (Spain).] / Emisiones Zero. Una responsabilidad compartida. Proyecto captura de gases y reciclado en el Hospital Universitario de Cruces.

OBJECTIVE: The use of volatile anesthetics plays an important role in the production of greenhouse gases and other environmental pollutants that negatively affect global health. Programs to reduce anesthesia contaminants have been shown to be effective and reduce costs. For this reason, we conducted a study to implementing a Zero Emissions Program for zero carbon dioxide emissions derived from anesthetic gases used in the operating room, as recommended by the Green Deal of the European Union by 2030 and be climate neutral in 2050, maintaining satisfaction and current clinical results. METHODS: A Zero Emissions Program was implemented within the Zero safety programs of the Cruces University Hospital in order to produce zero emissions of carbon dioxide derived from the anesthetic gases used in the operating rooms. The contribution of anesthetic gases to carbon dioxide production before and after implementation of program was determined. Data analysis was conducted descriptively to analyze program effectiveness. RESULTS: The implementation of a Zero Emissions Program allowed us to achieve a reduction in emissions to zero. CONCLUSIONS: Anesthesiologists must understand that minimizing our harmful impact on environmental health sustainability is not only desirable, but ethically necessary. A way to contribute to this ethical responsibility is Zero Emissions Programs which are effective in reducing emissions to zero, probably improving our impact on planet health.

OBJETIVO: El uso de anestésicos volátiles juega un papel importante en la producción de gases de efecto invernadero y otros contaminantes ambientales que afectan negativamente a la salud mundial. Se ha demostrado que los programas para reducir los contaminantes de la anestesia en el medio ambiente son eficaces y también reducen los costes. Por este motivo nos planteamos como objetivo implementar un Programa de Emisiones Zero para producir cero emisiones de dióxido de carbono derivados de los gases anestésicos utilizados en el quirófano, como recomienda el Pacto Verde de la Unión Europea, para 2030 y ser climáticamente neutros en 2050, manteniendo la satisfacción y los resultados clínicos actuales. METODOS: Se implementó un Programa de Emisiones Zero dentro de los programas Zero de seguridad del Hospital Universitario de Cruces (Barakaldo) con la finalidad de producir cero emisiones de dióxido de carbono derivado de los gases anestésicos utilizados en los quirófanos. Se determinó la contribución de los gases anestésicos a la producción de dióxido de carbono previo y posterior a la implementación del programa. El análisis de los datos se llevó a cabo de forma descriptiva para analizar la efectividad del programa. RESULTADOS: La implementación de un Programa de Emisiones de Zero nos permitió conseguir una disminución de las emisiones a cero. CONCLUSIONES: Los anestesiólogos debemos comprender que minimizar nuestro impacto nocivo en la sostenibilidad de la salud ambiental no es solo deseable, sino éticamente necesario. Una de las formas de contribuir con esta responsabilidad ética es con la implementación de Programas de Emisiones Zero que son eficaces en la reducción a cero de estas emisiones con lo que mejoraremos nuestro impacto en la salud del planeta.

[Aggressive multifocal extensive cutaneous epithelioid haemagioendothelioma. A case report]. / Hemangioendotelioma epitelioide cutáneo extenso multifocal agresivo. Informe de un caso.

Epithelioid haemangioendothelioma is a rare vascular tumor, first described in 1975 by Dail and Liebow as a bronchioloalveolar carcinoma. Although it usually behaves like a low-grade neoplasm, cases have been reported in which the tumor shows a high grade of malignancy, spreading rapidly throughout the body. We present the case of a 41-year-old man with dermatosis in the left thigh with rapid extension to the abdomen; the initial differential diagnoses were metastatic carcinoma versus lymphoma. When the histopathology was re-examined, a diagnosis of skin epithelioid hemangioendothelioma was confirmed and treatment with radiotherapy was initiated. This tumour rarely affects the skin; there are only a few previously reported cases.

In vitro Anticancer Activity of the Polar Fraction From the Lophocereus schottii Ethanolic Extract.

Cancer is an increasingly common disease and is considered one of the main causes of death in the world. Lophocereus schottii (L. schottii) is a cactus used in Mexico in traditional medicine for cancer treatment. This study aimed to determine the effect of the ethanolic extract and the polar and nonpolar fractions of L. schottii in murine L5178Y lymphoma cells in vitro, analyzing their effect on the proliferative activity of splenocytes, and establishing the effective concentration 50 (EC50) of the polar fraction. In addition, the secondary metabolites present in the extracts were determined by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). The study establishes that the three extracts of L. schottii have a cytotoxic effect on L5178Y cells and on the splenocytes stimulated with ConA. Additionally, the polar fraction has a significantly greater effect being three times more effective than cyclophosphamide on inhibiting the viability of L5178Y cells. Secondary metabolites present are mainly flavonoids and alkaloids, but there are also some terpenoids and sterols. Ultimately, polar fraction can be considered an anticancer substance, since its EC50 of 15 µg/mL is within the parameters established by the National Cancer Institute.

Patrones clínicos e histopatológicos de dermatosis en pacientes con infección por COVID-19 en la Unidad Médica de Alta Especialidad del Bajío, IMSS-T1, de León, Guanajuato / Clinical and histopathological patterns of dermatosis in COVID-19 positive patients treated in the Unidad Médica de Alta Especialidad del Bajío, IMSS-T1, de León, Guanajuato

INTRODUCCIÓN: La enfermedad COVID-19, además de presentar síntomas respiratorios, puede afectar otros órganos como la piel. Al momento, se han descrito cinco variantes clínicas de manifestaciones cutáneas por COVID-19. Pocos reportes abordan el tema de la gravedad de las dermatosis cutáneas de COVID-19 y el pronóstico. OBJETIVO: Describir patrones clínicos e histológicos de dermatosis en pacientes con COVID-19. Pacientes y MÉTODOS: Es una cohorte para pacientes del IMSS-T1 en León, Guanajuato, México, entre septiembre 2020 y enero 2021. Identificamos pacientes con dermatosis asociada a COVID-19 desde su ingreso hospitalario y aquellos que la desarrollaron durante su estancia. Se les invitó a participar para evaluación clínica y toma de biopsia que fueron descritas por un patólogo experto. RESULTADOS: La frecuencia de las dermatosis por COVID-19 fue de 15,7%. Los que desarrollaron las lesiones durante su estancia hospitalaria presentaron mayor morbi-mortalidad (p = 0,001). Las lesiones vaso-oclusivas fueron las más diagnosticadas y asociadas con mayor mortalidad (p = 0,003). Histológicamente el hallazgo más común fue trombosis superficial y profunda (58%). CONCLUSIONES: Los pacientes que desarrollaron las lesiones durante su hospitalización y aquellos con lesiones vaso-oclusivas tuvieron la mayor morbi-mortalidad. Las lesiones vaso-oclusivas pueden ser un factor de mal pronóstico en pacientes con COVID-19.

BACKGROUND: COVID-19 disease, besides presenting respiratory manifestations, can affect other organs such as kidneys, gastrointestinal system, heart, and skin. So far, five clinical variants of dermatoses have been described. Few reports discuss the severity associated with the cutaneous manifestations of COVID-19 and the prognosis. AIM: To describe the clinical and histopathological patterns of dermatoses in patients with COVID-19 infection. PATIENTS AND METHODS: Prospective cohort study conducted in patients admitted to "IMSS T1" in Leon, Guanajuato, Mexico from September 2020 to January 2021. We identified those with COVID-19 dermatosis from the moment they were admitted; and those who developed them during hospitalization. Patients were invited to participate for a clinical evaluation and biopsy. The biopsies were described by an expert pathologist. RESULTS: The frequency of dermatological lesions was 15.7%. Those who developed dermatosis during their hospital stay presented higher mortality (p = 0.001) and severity of COVID-19 (p = 0.001) Vasoocclusive lesions were the most frequent in the hospital setting, and were associated to higher mortality (p = 0.003). The most frequent histopathological feature was superficial and deep thrombosis (58%). CONCLUSIONS: Patients who developed dermatologic lesions during hospitalization and those with vaso-occlusive dermatoses had higher morbi-mortality. Vaso-occlusive lesions could be considered as a poor prognostic factor.

Plastic Antibody of Polypyrrole/Multiwall Carbon Nanotubes on Screen-Printed Electrodes for Cystatin C Detection.

This work reports the design of a novel plastic antibody for cystatin C (Cys-C), an acute kidney injury biomarker, and its application in point-of-care (PoC) testing. The synthetic antibody was obtained by tailoring a molecularly imprinted polymer (MIP) on a carbon screen-printed electrode (SPE). The MIP was obtained by electropolymerizing pyrrole (Py) with carboxylated Py (Py-COOH) in the presence of Cys-C and multiwall carbon nanotubes (MWCNTs). Cys-C was removed from the molecularly imprinted poly(Py) matrix (MPPy) by urea treatment. As a control, a non-imprinted poly(Py) matrix (NPPy) was obtained by the same procedure, but without Cys-C. The assembly of the MIP material was evaluated in situ by Raman spectroscopy and the binding ability of Cys-C was evaluated by the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) electrochemical techniques. The MIP sensor responses were measured by the DPV anodic peaks obtained in the presence of ferro/ferricyanide. The peak currents decreased linearly from 0.5 to 20.0 ng/mL of Cys-C at each 20 min successive incubation and a limit of detection below 0.5 ng/mL was obtained at pH 6.0. The MPPy/SPE was used to analyze Cys-C in spiked serum samples, showing recoveries <3%. This device showed promising features in terms of simplicity, cost and sensitivity for acute kidney injury diagnosis at the point of care.

Toward a broader view of mechanisms of drug cardiotoxicity.

Cardiotoxicity, defined as toxicity that affects the heart, is one of the most common adverse drug effects. Numerous drugs have been shown to have the potential to induce lethal arrhythmias by affecting cardiac electrophysiology, which is the focus of current preclinical testing. However, a substantial number of drugs can also affect cardiac function beyond electrophysiology. Within this broader sense of cardiotoxicity, this review discusses the key drug-protein interactions known to be involved in cardiotoxic drug response. We cover adverse effects of anticancer, central nervous system, genitourinary system, gastrointestinal, antihistaminic, anti-inflammatory, and anti-infective agents, illustrating that many share mechanisms of cardiotoxicity, including contractility, mitochondrial function, and cellular signaling.

[Clinical and histopathological patterns of dermatosis in COVID-19 positive patients treated in the Unidad Médica de Alta Especialidad del Bajío, IMSS-T1, de León, Guanajuato]. / Patrones clínicos e histopatológicos de dermatosis en pacientes con infección por COVID-19 en la Unidad Médica de Alta Especialidad del Bajío, IMSS-T1, de León, Guanajuato.

BACKGROUND: COVID-19 disease, besides presenting respiratory manifestations, can affect other organs such as kidneys, gastrointestinal system, heart, and skin. So far, five clinical variants of dermatoses have been described. Few reports discuss the severity associated with the cutaneous manifestations of COVID-19 and the prognosis. AIM: To describe the clinical and histopathological patterns of dermatoses in patients with COVID-19 infection. PATIENTS AND METHODS: Prospective cohort study conducted in patients admitted to "IMSS T1" in Leon, Guanajuato, Mexico from September 2020 to January 2021. We identified those with COVID-19 dermatosis from the moment they were admitted; and those who developed them during hospitalization. Patients were invited to participate for a clinical evaluation and biopsy. The biopsies were described by an expert pathologist. RESULTS: The frequency of dermatological lesions was 15.7%. Those who developed dermatosis during their hospital stay presented higher mortality (p = 0.001) and severity of COVID-19 (p = 0.001) Vasoocclusive lesions were the most frequent in the hospital setting, and were associated to higher mortality (p = 0.003). The most frequent histopathological feature was superficial and deep thrombosis (58%). CONCLUSIONS: Patients who developed dermatologic lesions during hospitalization and those with vaso-occlusive dermatoses had higher morbi-mortality. Vaso-occlusive lesions could be considered as a poor prognostic factor.

Solid-state NMR spectroscopy for disentangling structural and motional features of lyophilized ripened cheese water-soluble extracts related to antimicrobial activity.

Present work comprises the use of different solid-state Nuclear Magnetic Resonance strategies for characterizing structural and motional aspects of the peptide matrix that compose a set of four lyophilized Mexican cheese aqueous soluble extracts, each with a controlled ripening. Heteronuclear dipolar coupling modulation schemes allowed to characterize local mobility and structural homogeneity of cheeses' peptide segments in the solid-state as a function of ripening. Results suggest that ripened samples with certain local flexibility but important structural homogeneity present efficient microbial inhibition against tested bacterial strains, whilst high local rigidity of peptides within ripened cheese soluble fractions could partially explain the observed lack of antimicrobial activity. The present study attempts to propose novel observables for lyophilized cheese water soluble extracts that could be partially associated to their ripening-dependent antimicrobial activities, whereas said observables shall contribute to the better targeting, design and optimization of solid-state natural food bio-preservatives.

Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles.

Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such as Staphylococcus aureus and their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. S. aureus produce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show that S. aureus secretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-ß response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.

Activation of sarcolipin expression and altered calcium cycling in LMNA cardiomyopathy.

Cardiomyopathy caused by A-type lamins gene (LMNA) mutations (LMNA cardiomyopathy) is associated with dysfunction of the heart, often leading to heart failure. LMNA cardiomyopathy is highly penetrant with bad prognosis with no specific therapy available. Searching for alternative ways to halt the progression of LMNA cardiomyopathy, we studied the role of calcium homeostasis in the evolution of this disease. We showed that sarcolipin, an inhibitor of the sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) was abnormally elevated in the ventricular cardiomyocytes of mutated mice compared with wild type mice, leading to an alteration of calcium handling. This occurs early in the progression of the disease, when the left ventricular function was not altered. We further demonstrated that down regulation of sarcolipin using adeno-associated virus (AAV) 9-mediated RNA interference delays cardiac dysfunction in mouse model of LMNA cardiomyopathy. These results showed a novel role for sarcolipin on calcium homeostasis in heart and open perspectives for future therapeutic interventions to LMNA cardiomyopathy.

Dual Transcriptomic and Molecular Machine Learning Predicts all Major Clinical Forms of Drug Cardiotoxicity.

Computational methods can increase productivity of drug discovery pipelines, through overcoming challenges such as cardiotoxicity identification. We demonstrate prediction and preservation of cardiotoxic relationships for six drug-induced cardiotoxicity types using a machine learning approach on a large collected and curated dataset of transcriptional and molecular profiles (1,131 drugs, 35% with known cardiotoxicities, and 9,933 samples). The algorithm generality is demonstrated through validation in an independent drug dataset, in addition to cross-validation. The best prediction attains an average accuracy of 79% in area under the curve (AUC) for safe versus risky drugs, across all six cardiotoxicity types on validation and 66% on the unseen set of drugs. Individual cardiotoxicities for specific drug types are also predicted with high accuracy, including cardiac disorder signs and symptoms for a previously unseen set of anti-inflammatory agents (AUC = 80%) and heart failures for an unseen set of anti-neoplastic agents (AUC = 76%). Besides, independent testing on transcriptional data from the Drug Toxicity Signature Generation Center (DToxS) produces similar results in terms of accuracy and shows an average AUC of 72% for previously seen drugs and 60% for unseen respectively. Given the ubiquitous manifestation of multiple drug adverse effects in every human organ, the methodology is expected to be applicable to additional tissue-specific side effects beyond cardiotoxicity.

Erythropoiesis and Red Cell Indices Undergo Adjustments during Pregnancy in Response to Maternal Body Size but not Inflammation.

During human pregnancy, iron requirements gradually increase, leading to higher amounts of erythropoietin (EPO) and reticulocytes, and changes in erythrocyte size and density. Women with pregestational obesity experience "obesity hypoferremia" during pregnancy, which alters iron homeostasis. In this study we aimed to describe the relationship between EPO and iron nutrition status during nonanemic pregnancy, and to explore whether obesity and inflammation influence erythropoiesis and red cell indices. We conducted a secondary analysis of a cohort followed throughout pregnancy. Participants were nonanemic women assigned to two study groups based on pregestational body mass index (pgBMI): adequate weight (AW, n = 53) or obesity (Ob, n = 40). All received a multivitamin supplement. At gestational ages (GA) 13, 21, 28 and 34, we measured hemoglobin and red cell indices with an ACT-5DIFF hematology counter, and reticulocyte percentage by manual cell counting. EPO, interleukin (IL-6) and markers of iron status, i.e., hepcidin, serum transferrin receptor (sTfr) and ferritin, were measured by ELISA. Bivariate correlations showed that EPO was positively associated with pgBMI, GA, sTfr and IL-6, but negatively associated with hepcidin, ferritin and hemoglobin, and unrelated to iron intake. Generalized linear models adjusted for confounding factors showed that EPO and erythrocyte concentrations were significantly higher in women in the Ob group, while mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and red cell distribution width (RDW) were lower; reticulocytes and mean corpuscular hemoglobin concentration (MCHC) were not different. Differences were not altered when controlling for inflammation (IL-6). These changes suggest that, in addition to altering iron metabolism, a larger maternal body size during pregnancy results in higher erythropoiesis without increasing hemoglobin, which is exhibited in the latter being distributed among more and smaller erythrocytes.

An ultrasensitive Cystatin C renal failure immunosensor based on a PPy/CNT electrochemical capacitor grafted on interdigitated electrode.

An interdigitated immunosensor for Cystatin C detection based on polypyrrole/carbon nanotube electrochemical capacitor is described. Cystatin C (CysC) is powerful biomarker for early acute renal failure and one predictive for cardiovascular risk, sepsis, cancer and death. Recently, electrochemical immunosensors based on interdigitated electrodes (IDE) have been successfully focused on development of point-of-care testing, due to their miniaturization facilities and higher sensitivity as compared with the screen-printed electrochemical sensing. Herein, a polypyrrole/carbon nanotube nanoyhibrid film was grafted on two gold fingers by electropolymerization obtaining a supercapacitor. Anti-CysC antibodies were immobilized on the IDE by covalent entrapment via ethylenediamine bifunctional agent, followed by glycine blocking in acid and alkaline medium. Under low frequency, capacitive effect of antigen-antibody interaction were observed by double layer capacitance, and analytical responses of this IDE immunosensor to CysC serum were obtained by changes on phase angle a linear range up to 300 ng/mL. The cutoff was calculated for serum samples showing a total reducing of non-specific binding at approximately 28 ng/mL CysC. This immunosensor based on interdigitated electrode (IDE) is a potential tools as portable device,with possibility to use as a practical and rapid test for CysC diagnostic in samples of serum.

Chaste: Cancer, Heart and Soft Tissue Environment.

Chaste (Cancer, Heart And Soft Tissue Environment) is an open source simulation package for the numerical solution of mathematical models arising in physiology and biology. To date, Chaste development has been driven primarily by applications that include continuum modelling of cardiac electrophysiology ('Cardiac Chaste'), discrete cell-based modelling of soft tissues ('Cell-based Chaste'), and modelling of ventilation in lungs ('Lung Chaste'). Cardiac Chaste addresses the need for a high-performance, generic, and verified simulation framework for cardiac electrophysiology that is freely available to the scientific community. Cardiac chaste provides a software package capable of realistic heart simulations that is efficient, rigorously tested, and runs on HPC platforms. Cell-based Chaste addresses the need for efficient and verified implementations of cell-based modelling frameworks, providing a set of extensible tools for simulating biological tissues. Computational modelling, along with live imaging techniques, plays an important role in understanding the processes of tissue growth and repair. A wide range of cell-based modelling frameworks have been developed that have each been successfully applied in a range of biological applications. Cell-based Chaste includes implementations of the cellular automaton model, the cellular Potts model, cell-centre models with cell representations as overlapping spheres or Voronoi tessellations, and the vertex model. Lung Chaste addresses the need for a novel, generic and efficient lung modelling software package that is both tested and verified. It aims to couple biophysically-detailed models of airway mechanics with organ-scale ventilation models in a package that is freely available to the scientific community. Chaste is designed to be modular and extensible, providing libraries for common scientific computing infrastructure such as linear algebra operations, finite element meshes, and ordinary and partial differential equation solvers. This infrastructure is used by libraries for specific applications, such as continuum mechanics, cardiac models, and cell-based models. The software engineering techniques used to develop Chaste are intended to ensure code quality, re-usability and reliability. Primary applications of the software include cardiac and respiratory physiology, cancer and developmental biology.

A single T cell epitope drives the neutralizing anti-drug antibody response to natalizumab in multiple sclerosis patients.

Natalizumab (NZM), a humanized monoclonal IgG4 antibody to α4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS)1,2, but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation3,4. To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4+ T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to α4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment.

Microtubule cytoskeleton regulates Connexin 43 localization and cardiac conduction in cardiomyopathy caused by mutation in A-type lamins gene.

Mutations in the lamin A/C gene (LMNA) cause an autosomal dominant inherited form of dilated cardiomyopathy associated with cardiac conduction disease (hereafter referred to as LMNA cardiomyopathy). Compared with other forms of dilated cardiomyopathy, mutations in LMNA are responsible for a more aggressive clinical course owing to a high rate of malignant ventricular arrhythmias. Gap junctions are intercellular channels that allow direct communication between neighboring cells, which are involved in electrical impulse propagation and coordinated contraction of the heart. For gap junctions to properly control electrical synchronization in the heart, connexin-based hemichannels must be correctly targeted to intercalated discs, Cx43 being the major connexin in the working myocytes. We here showed an altered distribution of Cx43 in a mouse model of LMNA cardiomyopathy. However, little is known on the molecular mechanisms of Cx43 remodeling in pathological context. We now show that microtubule cytoskeleton alteration and decreased acetylation of α-tubulin lead to remodeling of Cx43 in LMNA cardiomyopathy, which alters the correct communication between cardiomyocytes, ultimately leading to electrical conduction disturbances. Preventing or reversing this process could offer a strategy to repair damaged heart. Stabilization of microtubule cytoskeleton using Paclitaxel improved intraventricular conduction defects. These results indicate that microtubule cytoskeleton contributes to the pathogenesis of LMNA cardiomyopathy and that drugs stabilizing the microtubule may be beneficial for patients.

Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4.

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.

N-acetyl cysteine alleviates oxidative stress and protects mice from dilated cardiomyopathy caused by mutations in nuclear A-type lamins gene.

Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscular and electrical dysfunction of the heart, often leading to heart failure-related disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. We showed here an increase in oxidative stress levels in the hearts of mice carrying LMNA mutation, associated with a decrease of the key cellular antioxidant glutathione (GHS). Oral administration of N-acetyl cysteine, a GHS precursor, led to a marked improvement of GHS content, a decrease in oxidative stress markers including protein carbonyls and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our novel results provide therapeutic insights into LMNA cardiomyopathy.

From ionic to cellular variability in human atrial myocytes: an integrative computational and experimental study.

Variability refers to differences in physiological function between individuals, which may translate into different disease susceptibility and treatment efficacy. Experiments in human cardiomyocytes face wide variability and restricted tissue access; under these conditions, computational models are a useful complementary tool. We conducted a computational and experimental investigation in cardiomyocytes isolated from samples of the right atrial appendage of patients undergoing cardiac surgery to evaluate the impact of variability in action potentials (APs) and subcellular ionic densities on Ca2+ transient dynamics. Results showed that 1) variability in APs and ionic densities is large, even within an apparently homogenous patient cohort, and translates into ±100% variation in ionic conductances; 2) experimentally calibrated populations of models with wide variations in ionic densities yield APs overlapping with those obtained experimentally, even if AP characteristics of the original generic model differed significantly from experimental APs; 3) model calibration with AP recordings restricts the variability in ionic densities affecting upstroke and resting potential, but redundancy in repolarization currents admits substantial variability in ionic densities; and 4) model populations constrained with experimental APs and ionic densities exhibit three Ca2+ transient phenotypes, differing in intracellular Ca2+ handling and Na+/Ca2+ membrane extrusion. These findings advance our understanding of the impact of variability in human atrial electrophysiology. NEW & NOTEWORTHY Variability in human atrial electrophysiology is investigated by integrating for the first time cellular-level and ion channel recordings in computational electrophysiological models. Ion channel calibration restricts current densities but not cellular phenotypic variability. Reduced Na+/Ca2+ exchanger is identified as a primary mechanism underlying diastolic Ca2+ fluctuations in human atrial myocytes.