LRRK2 Knockout Confers Resistance in HEK-293 Cells to Rotenone-Induced Oxidative Stress, Mitochondrial Damage, and Apoptosis

Leucine-rich repeat kinase 2 (LRRK2) has been linked to dopaminergic neuronal vulnerability to oxidative stress (OS), mitochondrial impairment, and increased cell death in idiopathic and familial Parkinson’s disease (PD). However, how exactly this kinase participates in the OS-mitochondria-apoptosis connection is still unknown. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 LRRK2 knockout (KO) in the human embryonic kidney cell line 293 (HEK-293) to evaluate the cellular response to the mitochondrial inhibitor complex I rotenone (ROT), a well-known OS and cell death inducer. We report successful knockout of the LRRK2 gene in HEK-293 cells using CRISPR editing (ICE, approximately 60%) and flow cytometry (81%) analyses. We found that HEK-293 LRRK2 WT cells exposed to rotenone (ROT, 50 μM) resulted in a significant increase in intracellular reactive oxygen species (ROS, +7400%); oxidized DJ-1-Cys106-SO3 (+52%); phosphorylation of LRRK2 (+70%) and c-JUN (+171%); enhanced expression of tumor protein (TP53, +2000%), p53 upregulated modulator of apoptosis (PUMA, +1950%), and Parkin (PRKN, +22%); activation of caspase 3 (CASP3, +8000%), DNA fragmentation (+35%) and decreased mitochondrial membrane potential (ΔΨm, −58%) and PTEN induced putative kinase 1 (PINK1, −49%) when compared to untreated cells. The translocation of the cytoplasmic fission protein dynamin-related Protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of the outer membrane 20 (TOM20). Outstandingly, HEK-293 LRRK2 KO cells treated with ROT showed unaltered OS and apoptosis markers. We conclude that loss of LRRK2 causes HEK-293 to be resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. Our data support the hypothesis that LRRK2 acts as a proapoptotic kinase by regulating mitochondrial proteins (e.g., PRKN, PINK1, DRP1, and PUMA), transcription factors (e.g., c-JUN and TP53), and CASP3 in cells under stress conditions. Taken together, these observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.Committee for Development and Research-UdeA” (Comité para el Desarrollo y la Investigacion-CODI, Universidad de Antioquia-UdeA) grants (#2017- 15829)The Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER)Grants PI15/02015 and PI18/00337. The APC was funded by Committee for Development and Research-UdeA

Rnd3 Expression is Necessary to Maintain Mitochondrial Homeostasis but Dispensable for Autophagy

Autophagy is a highly conserved process that mediates the targeting and degradation of intracellular components to lysosomes, contributing to the maintenance of cellular homeostasis and to obtaining energy, which ensures viability under stress conditions. Therefore, autophagy defects are common to different neurodegenerative disorders. Rnd3 belongs to the family of Rho GTPases, involved in the regulation of actin cytoskeleton dynamics and important in the modulation of cellular processes such as migration and proliferation. Murine models have shown that Rnd3 is relevant for the correct development and function of the Central Nervous System and lack of its expression produces several motor alterations and neural development impairment. However, little is known about the molecular events through which Rnd3 produces these phenotypes. Interestingly we have observed that Rnd3 deficiency correlates with the appearance of autophagy impairment profiles and irregular mitochondria. In this work, we have explored the impact of Rnd3 loss of expression in mitochondrial function and autophagy, using a Rnd3 KO CRISPR cell model. Rnd3 deficient cells show no alterations in autophagy and mitochondria turnover is not impaired. However, Rnd3 KO cells have an altered mitochondria oxidative metabolism, resembling the effect caused by oxidative stress. In fact, lack of Rnd3 expression makes these cells strictly dependent on glycolysis to obtain energy. Altogether, our results demonstrate that Rnd3 is relevant to maintain mitochondria function, suggesting a possible relationship with neurodegenerative diseases.MINECO (SAF 2013-49176-C2-1- R)Conselleria d’ Educació, Investigació, Cultura i Esport (AICO/2016/047)FUSP-CEU-UCH (FUSP-PPC-19- 28A751CC)Instituto de Salud Carlos III (ISCIII) (PI18/ 00337)WKZ Fonds (R4376)ReumaNederland (16-1-2-1)Nicolas Monardes regional Ministry of Health contrac

Serum calprotectin as new biomarker for disease severity in idiopathic pulmonary fibrosis: a cross-sectional study in two independent cohorts

Background: Non-invasive biomarkers for the assessment of disease severity in idiopathic pulmonary fibrosis (IPF) are urgently needed. Calprotectin belongs to the S-100 proteins produced by neutrophils, which likely contribute to IPF pathogenesis. Calprotectin is a well-established biomarker in inflammatory bowel diseases. In this cross-sectional study, we aimed to establish the potential role of calprotectin as a biomarker in IPF. Specifically, we hypothesised that patients with IPF have higher serum calprotectin levels compared with healthy controls, and that calprotectin levels are associated with disease severity. Methods: Blood samples were obtained from healthy volunteers (n=26) and from two independent IPF cohorts (derivation cohort n=26, validation cohort n=66). Serum calprotectin levels were measured with a commercial kit adapted for that purpose and compared between healthy controls and patients with IPF. Clinical parameters, including forced vital capacity, diffusing capacity for carbon monoxide (DLCO) and the Composite Physiologic Index (CPI), were correlated with calprotectin serum levels. Results: The IPF derivation cohort showed increased serum calprotectin levels compared with healthy controls (2.47 +/- 1.67 vs 0.97 +/- 0.53 mu g/mL, p<0.001). In addition, serum calprotectin levels correlated with DLCO% predicted (r=-0.53, p=0.007) and with CPI (r=0.66, p=0.007). These findings were confirmed in an independent IPF validation cohort. Conclusion: Serum calprotectin levels are significantly increased in patients with IPF compared with healthy controls and correlate with DLCO and CPI. Calprotectin might be a potential new biomarker for disease severity in IPF

Chemical composition and anti-inflammatory effect of Phellodendron amurense Rupr. stem bark extract

In many diseases inflammation and oxidative stress coexist and are therapeutic targets. Phellodendron amurense Rupr. has anti-inflammatory and antioxidant effects, but the mechanisms are not completely elucidated. Therefore, first P. amurense stem bark extract was analysed regarding chemical compounds such as total polyphenol content (TPC), polyphenols (gallic acid, 4-hydroxybenzoic acid, caffeic acid and ferulic acid) and alkaloids (berbamine, jatrorrhizine, palmatine, and berberine). Quantitative determination of alkaloids revealed that berberine had the highest concentration of 2.44±0.22 mg/g. In vitro antioxidant activity was significant. Then, thein vivo anti-inflammatory and antioxidative effects of P. amurense extract were studied before and after acute experimental rat inflammation induction in order to find some mechanisms of these effects. The P. amurense stem bark extract had a good anti-inflammatory activity by reducing nitric oxide, 3-nitrotyrosine and NF-kB, and an antioxidant activity by lowering oxidants and increasing antioxidants. This study provides scientific knowledge and could contribute to the development of novel drugs based on P. amurense stem bark extract for the treatment of inflammatory diseases and prevention of oxidative stress

Coenzyme Q10 modulates sulfide metabolism and links the mitochondrial respiratory chain to pathways associated to one carbon metabolism

This work was supported by grants from Ministerio de Ciencia e Innovacion, Spain, and the ERDF (RTI2018-093503-B-100); the Muscular Dystrophy Association (MDA-602322); the University of Granada (grant reference 'UNETE', UCE-PP2017-06) (L.C.L.) and the National Institute of Health (NIH, United States) P01 HD080642-01 (C.M.Q.). A.H.-G. and P.G.-G. are `FPU fellows' from the Ministerio de Universidades, Spain. E.B.-C. was supported by the Junta de Andalucia. U.B.A. was supported by the Erasmus+ Program.Abnormalities of one carbon, glutathione and sulfide metabolisms have recently emerged as novel pathomechanisms in diseases with mitochondrial dysfunction. However, the mechanisms underlying these abnormalities are not clear. Also, we recently showed that sulfide oxidation is impaired in Coenzyme Q10 (CoQ10) deficiency. This finding leads us to hypothesize that the therapeutic effects of CoQ10, frequently administered to patients with primary or secondary mitochondrial dysfunction, might be due to its function as cofactor for sulfide:quinone oxidoreductase (SQOR), the first enzyme in the sulfide oxidation pathway. Here, using biased and unbiased approaches, we show that supraphysiological levels of CoQ10 induces an increase in the expression of SQOR in skin fibroblasts from control subjects and patients with mutations in Complex I subunits genes or CoQ biosynthetic genes. This increase of SQOR induces the downregulation of the cystathionine β-synthase and cystathionine γ-lyase, two enzymes of the transsulfuration pathway, the subsequent downregulation of serine biosynthesis and the adaptation of other sulfide linked pathways, such as folate cycle, nucleotides metabolism and glutathione system. These metabolic changes are independent of the presence of sulfur aminoacids, are confirmed in mouse models, and are recapitulated by overexpression of SQOR, further proving that the metabolic effects of CoQ10 supplementation are mediated by the overexpression of SQOR. Our results contribute to a better understanding of how sulfide metabolism is integrated in one carbon metabolism and may explain some of the benefits of CoQ10 supplementation observed in mitochondrial diseases.Spanish GovernmentEuropean Union (EU) RTI2018-093503-B-100Muscular Dystrophy Association MDA-602322University of Granada UCE-PP2017-06United States Department of Health & Human Services National Institutes of Health (NIH) - USA P01 HD080642-01Junta de AndaluciaErasmus+ Progra

SISTEMA MHEALTH PARA EL REGISTRO ELECTRÓNICO DE LA ATENCIÓN DE PERSONAS EN CONDICIÓN DE POSTRACIÓN EN EL HOGAR

La mSalud permite dar acceso y continuidad a la atención y cuidado de salud a personas que por su condición o lejanía de otra manera no podrían tener, acercando al equipo de salud al punto del cuidado donde la persona requiere la atención. Este estudio experimental randomizado, cuanti cualitativo, longitudinal, tiene por propósito diseñar un registro electrónico móvil para el cuidado domiciliario del paciente postrado. En este paper se da a conocer la primera de 6 fases que contempla el estudio, la de determinación de requerimientos de información, permitiendo reconocer la necesidad de sistematizar procesos clínicos y administrativos, así como representar el flujo de trabajo de los clínicos y representar el cuidado de las personas en el hogar. </p

ERS International Congress 2021: highlights from the Interstitial Lung Diseases Assembly

This article provides an overview of scientific highlights in the field of interstitial lung disease (ILD), presented at the virtual European Respiratory Society Congress 2021. A broad range of topics was discussed this year, ranging from translational and genetic aspects to novel innovations with the potential to improve the patient pathway. Early Career Members summarise a selection of interesting findings from different congress sessions, together with the leadership of Assembly 12 - Interstitial Lung Disease. © The authors 2022

Physiological lentiviral vectors for the generation of improved CAR-T cells

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms.However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%–50%of the treated patients.MostCAR-Tcells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of theirmoderate andTCR-like expression profile. Compared with CAR-T cells generated with human elongation factor a (EF1a)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-a) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo.Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing ofAW-CAR-T cells in good manufacturing practice (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.Spanish ISCIII Health Research FundEuropean Commission PI15/02015 PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA 2016000073332-TRA PI-57069 PA IDI-Bio326 CARTPI-0001-201 PECART-0031-2020 Red RANTECAR CAR-T 2019 00400200101918 PLEC2021-008094 PI-0014-2016 PEER-0286-2019Spanish Government PLEC2021-008094 00123009/SNEO-20191072Nicolas Monardes contracts from regional Ministry of Health 0006/2018 C2-0002-2019German Research Foundation (DFG) FPU16/05467 FPU17/02268 FPU17/04327 MCI DIN2018-010180Fundacion Andaluza Progreso y SaludGerman Research Foundation (DFG) PEJ-2018-001760-AJunta de Andalucia PE-0223-2018Biomedicine Programme of the University of Granada (Spain

Herramientas de trabajo colaborativo para la dinamización de la competencia transversal responsabilidad ética, medioambiental y profesional

[EN] This article analyzes the experience of using different collaborative work tools to dynamize the tasks that help to acquire and improve key competence ethical, environmental and professional responsibility. The selected tools are integrated in the learning management system (LMS), PoliformaT, of the Universitat Politècnica de València. In particular, we have worked with the forum and the wiki, and the blog which is integrated in Poliblogs. In this experience three Bachelor degrees and a Master degree of engineering and life sciences have been involved: Bachelor's Degree in Environmental Sciences, Bachelor's Degree in Rural and Agrifood Engineering, Bachelor's Degree in Mechanical Engineering and Master's Degree in Design Engineering. The results show that the students value the experience satisfactorily thanks to the virtual interaction with other colleagues and they highlight the extra motivation offered by the use of these tools. It is noteworthy that to obtain good results there are two key elements: 1) clear rules of operation and evaluation and 2) high participation of the teacher as moderator.[ES] En el presente artículo se analiza la experiencia de uso de distintas herramientas de trabajo colaborativo para dinamizar las tareas que ayudan a adquirir y mejorar la competencia transversal responsabilidad ética, medioambiental y profesional. Las herramientas seleccionadas están integradas en la plataforma de gestión del aprendizaje, poliformat, de la Universitat Politècnica de València. En particular hemos trabajado con el foro y la wiki, además de con el blog integrado en Poliblogs. En esta experiencia han participado tres titulaciones de Grado y un Máster de ingeniería y ciencias de la vida: Grado en Ciencias Ambientales, Grado en Ingeniería Agroalimentaria y del Medio Rural, Grado en Ingeniería Mecánica, y Máster Universitario en Ingeniería del Diseño. Los resultados obtenidos muestran que el alumnado valora satisfactoriamente la experiencia gracias a la interacción virtual con otros compañeros y destacan la motivación extra que les ofrece el uso de estas herramientas. Es destacable que para la obtención de buenos resultados hay dos elementos clave: 1) Normas claras de funcionamiento y evaluación y 2) Alta participación del profesor como moderador.Sebastiá Frasquet, MT.; Asensio Cuesta, S.; Gasch Molina, MI.; Pascual Seva, N.; Vargas Colás, MD. (2017). Herramientas de trabajo colaborativo para la dinamización de la competencia transversal responsabilidad ética, medioambiental y profesional. En In-Red 2017. III Congreso Nacional de innovación educativa y de docencia en red. Editorial Universitat Politècnica de València. 1256-1266. https://doi.org/10.4995/INRED2017.2017.6827OCS1256126

The role of pathologists in the diagnosis of occupational lung diseases: an expert opinion of the European Society of Pathology Pulmonary Pathology Working Group

Occupational lung/thoracic diseases are a major global public health issue. They comprise a diverse spectrum of health conditions with complex pathology, most of which arise following chronic heavy workplace exposures to various mineral dusts, metal fumes, or following inhaled organic particulate reactions. Many occupational lung diseases could become irreversible; thus accurate diagnosis is mandatory to minimize dust exposure and consequently reduce damage to the respiratory system. Lung biopsy is usually required when exposure history is inconsistent with imaging, in case of unusual or new exposures, in case of unexpected malignancy, and in cases in which there are claims for personal injury and legal compensation. In this paper, we provide an overview of the most frequent occupational lung diseases with a focus on pathological diagnosis. This is a paper that summarizes the expert opinion from a group of European pathologists, together with contributions from other specialists who are crucial for the diagnosis and management of these diseases. Indeed, tight collaboration of all specialists involved in the workup is mandatory as many occupational lung diseases are misdiagnosed or go unrecognized. This document provides a guide for pathologists in practice to facilitate the accurate diagnosis of occupational lung disease. The review article reports relevant topics discussed during an educational course held by expert pathologists, active members of the Pulmonary Pathology Working Group of the European Society of Pathology. The course was endorsed by the University of Padova as a “winter school” (selected project in the call for “Shaping a World-class University” 2022)