Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.

The spatial and temporal distributions of proteins are critical to protein function, but cannot be directly assessed by measuring protein bundance. Here we describe a mass spectrometry-based proteomics strategy, Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently protein turnover rates and subcellular localization in the same experiment. Applying the method, we find that unfolded protein response (UPR) has different effects on protein turnover dependent on their subcellular location in human AC16 cells, with proteome-wide slowdown but acceleration among stress response proteins in the ER and Golgi. In parallel, UPR triggers broad differential localization of proteins including RNA-binding proteins and amino acid transporters. Moreover, we observe newly synthesized proteins including EGFR that show a differential localization under stress than the existing protein pools, reminiscent of protein trafficking disruptions. We next applied SPLAT to an induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) model of cancer drug cardiotoxicity upon treatment with the proteasome inhibitor carfilzomib. Paradoxically, carfilzomib has little effect on global average protein half-life, but may instead selectively disrupt sarcomere protein homeostasis. This study provides a view into the interactions of protein spatial and temporal dynamics and demonstrates a method to examine protein homeostasis regulations in stress and drug response.

Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.

The functions of proteins depend on their spatial and temporal distributions, which are not directly measured by static protein abundance. Under endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) pathway remediates proteostasis in part by altering the turnover kinetics and spatial distribution of proteins. A global view of these spatiotemporal changes has yet to emerge and it is unknown how they affect different cellular compartments and pathways. Here we describe a mass spectrometry-based proteomics strategy and data analysis pipeline, termed Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently the changes in protein turnover and subcellular distribution in the same experiment. Investigating two common UPR models of thapsigargin and tunicamycin challenge in human AC16 cells, we find that the changes in protein turnover kinetics during UPR varies across subcellular localizations, with overall slowdown but an acceleration in endoplasmic reticulum and Golgi proteins involved in stress response. In parallel, the spatial proteomics component of the experiment revealed an externalization of amino acid transporters and ion channels under UPR, as well as the migration of RNA-binding proteins toward an endosome co-sedimenting compartment. The SPLAT experimental design classifies heavy and light SILAC labeled proteins separately, allowing the observation of differential localization of new and old protein pools and capturing a partition of newly synthesized EGFR and ITGAV to the ER under stress that suggests protein trafficking disruptions. Finally, application of SPLAT toward human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) exposed to the cancer drug carfilzomib, identified a selective disruption of proteostasis in sarcomeric proteins as a potential mechanism of carfilzomib-mediated cardiotoxicity. Taken together, this study provides a global view into the spatiotemporal dynamics of human cardiac cells and demonstrates a method for inferring the coordinations between spatial and temporal proteome regulations in stress and drug response.

Senescence in human AC16 cardiac cells is associated with thymidine kinase induction and histone loss.

AC16 cells are a transformed human cardiac cell line commonly used to study cardiomyocyte biology. We show that reduced proliferation and senescence markers can be robustly induced in AC16 cells cultured in low serum condition and treated with (i) low-dose doxorubicin, (ii) UV 254 nm, or (iii) H 2 O 2 exposure for up to 48 hours. Increased p21 (CDKN1A) and H2A.X variant histone (H2AX) levels serve as reliable molecular markers upon all three treatment conditions, but the up-regulation of another common senescence marker, p16 (CDKN2A) was not observed. A proteomics screen further shows that the loss of histones and the increased expression of thymidine kinases (TK1) are prominent features of AC16 cells under doxorubicin induced senescence.

Co-culture with normal and senescent AC16 cardiomyocytes modulates fibrotic response in primary human ventricular fibroblasts.

Transwell co-culture with human AC16 cardiomyocyte-like cells modifies the response of primary human ventricular fibroblasts to TGF-ß stimulation. Fibrotic response markers including collagen I (COL1A1) and ɑ-smooth muscle actin (ACTA2) are amplified in the presence of AC16 cells, whereas others including periostin (POSTN) and fibronectin (FN1) are suppressed. Similar modulation is observed when the ventricular fibroblasts are co-cultured with AC16 cells under baseline and induced senescence conditions. Given that the response to TGF-ß stimulation is commonly measured to study fibrotic signaling and drug treatments in vitro, the results here suggest that the effect of cellular crosstalk should be more broadly considered.

Latin American perceptions of fear and exaggeration transmitted by the media with regard to COVID-19: frequency and association with severe mental pathologies.

Introduction: The COVID-19 pandemic contributed to the spread of abundant misinformation by the media, which caused fear and concern. Objective: To determine the association between the pathologies of the mental sphere and the perceptions of fear and exaggeration transmitted by the media with respect to COVID-19 in Latin America. Methodology: The present study has an analytical cross-sectional design that is based on a validated survey to measure fear and exaggeration transmitted by the media and other sources (Cronbach's α: 0.90). We surveyed more than 6,000 people, originally from 12 Latin American countries, who associated this perceived exaggeration with stress, depression, and anxiety (measured through DASS-21, Cronbach's α: 0.96). Results: Social networks (40%) or television (34%) were perceived as the sources that exaggerate the magnitude of the events. In addition, television (35%) and social networks (28%) were perceived as the sources that generate much fear. On the contrary, physicians and health personnel are the sources that exaggerated less (10%) or provoked less fear (14%). Through a multivariate model, we found a higher level of global perception that was associated with whether the participant was older (p = 0.002), had severe or more serious anxiety (p = 0.033), or had stress (p = 0,037). However, in comparison with Peru (the most affected country), there was a lower level of perception in Chile (p < 0.001), Paraguay (p = 0.001), Mexico (p < 0.001), Ecuador (p = 0.001), and Costa Rica (p = 0.042). All of them were adjusted for gender and for those having severe or major depression. Conclusion: There exists an association between some mental pathologies and the perception that the media does not provide moderate information.

Sex and gender differences in acute stroke care: metrics, access to treatment and outcome. A territorial analysis of the Stroke Code System of Catalonia.

INTRODUCTION: Previous studies have reported differences in the management and outcome of women stroke patients in comparison with men. We aim to analyze sex and gender differences in the medical assistance, access to treatment and outcome of acute stroke patients in Catalonia. PATIENTS AND METHODS: Data were obtained from a prospective population-based registry of stroke code activations in Catalonia (CICAT) from January/2016 to December/2019. The registry includes demographic data, stroke severity, stroke subtype, reperfusion therapy, and time workflow. Centralized clinical outcome at 90 days was assessed in patients receiving reperfusion therapy. RESULTS: A total of 23,371 stroke code activations were registered (54% men, 46% women). No differences in prehospital time metrics were observed. Women more frequently had a final diagnosis of stroke mimic, were older and had a previous worse functional situation. Among ischemic stroke patients, women had higher stroke severity and more frequently presented proximal large vessel occlusion. Women received more frequently reperfusion therapy (48.2% vs 43.1%, p < 0.001). Women tended to present a worse outcome at 90 days, especially for the group receiving only IVT (good outcome 56.7% vs 63.8%; p < 0.001), but not for the group of patients treated with IVT + MT or MT alone, although sex was not independently associated with clinical outcome in logistic regression analysis (OR 1.07; 95% CI, 0.94-1.23; p = 0.27) nor in the analysis after matching using the propensity score (OR 1.09; 95% CI, 0.97-1.22). DISCUSSION AND CONCLUSION: We found some differences by sex in that acute stroke was more frequent in older women and the stroke severity was higher. We found no differences in medical assistance times, access to reperfusion treatment and early complications. Worse clinical outcome at 90 days in women was conditioned by stroke severity and older age, but not by sex itself.

Neurocognitive and emotional status after one-year of mindfulness-based intervention in patients with relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is the most common disabling neurological disease among young adults. The diagnosis of this disease usually leads to uncertainty, stress, and anxiety, which in turn can lead to negative outcomes. Consequently, the development of healthy lifestyle strategies can lead to better emotional management of perceived stress. Due to the importance of the patient's emotional response during the development and management of the disease and the repercussions of the cognitive deficits, the objective of this study is to develop a mindfulness-based intervention to provide to patients a tool for stress management. This study offers data on neuropsychological, functional and emotional outcomes in a sample of 30 patients with remitting relapsing form of MS after one year of intervention compared to 30 controls. We tested a model of intervention with a program of Mindfulness-Based Cognitive Therapy, based on an adapted Jon Kabat-Zinn's MBSR program. Results showed cognitive and emotional benefits after one year following this program. Discussion about the opportunities of these types of intervention in neurological diseases is provided in light of results obtained. This study represents pioneering research in MS and psychological treatment of this neurological autoimmune disease and it offers positive results of applying mindfulness-based intervention in this population.

Bottlenecks in the Acute Stroke Care System during the COVID-19 Pandemic in Catalonia.

INTRODUCTION: The COVID-19 pandemic resulted in significant healthcare reorganizations, potentially striking standard medical care. We investigated the impact of the COVID-19 pandemic on acute stroke care quality and clinical outcomes to detect healthcare system's bottlenecks from a territorial point of view. METHODS: Crossed-data analysis between a prospective nation-based mandatory registry of acute stroke, Emergency Medical System (EMS) records, and daily incidence of COVID-19 in Catalonia (Spain). We included all stroke code activations during the pandemic (March 15-May 2, 2020) and an immediate prepandemic period (January 26-March 14, 2020). Primary outcomes were stroke code activations and reperfusion therapies in both periods. Secondary outcomes included clinical characteristics, workflow metrics, differences across types of stroke centers, correlation analysis between weekly EMS alerts, COVID-19 cases, and workflow metrics, and impact on mortality and clinical outcome at 90 days. RESULTS: Stroke code activations decreased by 22% and reperfusion therapies dropped by 29% during the pandemic period, with no differences in age, stroke severity, or large vessel occlusion. Calls to EMS were handled 42 min later, and time from onset to hospital arrival increased by 53 min, with significant correlations between weekly COVID-19 cases and more EMS calls (rho = 0.81), less stroke code activations (rho = -0.37), and longer prehospital delays (rho = 0.25). Telestroke centers were afflicted with higher reductions in stroke code activations, reperfusion treatments, referrals to endovascular centers, and increased delays to thrombolytics. The independent odds of death increased (OR 1.6 [1.05-2.4], p 0.03) and good functional outcome decreased (mRS ≤2 at 90 days: OR 0.6 [0.4-0.9], p 0.015) during the pandemic period. CONCLUSION: During the COVID-19 pandemic, Catalonia's stroke system's weakest points were the delay to EMS alert and a decline of stroke code activations, reperfusion treatments, and interhospital transfers, mostly at local centers. Patients suffering an acute stroke during the pandemic period had higher odds of poor functional outcome and death. The complete stroke care system's analysis is crucial to allocate resources appropriately.

Stakeholders' perceptions of humanized birth practices and obstetric violence in Chile: A scoping review. / Stakeholders' perceptions of humanized birth practices and obstetric violence in Chile: A scoping review.

INTRODUCTION: Chile has an incipient policy regarding humanized birth practices. Obstetric violence is becoming an issue in the public discussion, as brought up by women. Despite this advancement, no initiatives were observed to overcome the conflict. Questions arise from the different points of view of the main stakeholders involved. These questions help identify strategies contributing to the development of health policies that consider influencing actors. OBJECTIVES: To identify stakeholders' perceptions of humanized care in childbirth and obstetric violence. METHODS: We conducted a scoping review that included articles and analysis of texts reflecting the scientific communities' point of view. We included statements from governmental, social, professional, and political actors as expressed in institutional websites. Moreover, we performed a qualitative inductive, thematic content analysis. RESULTS: We included seventy documents. The scientific community is visualized as aligned with ministerial recommendations for personalized childbirth. Several researchers analyze the difficulties for its improvement due to the historical, socio-cultural, and economic construction of the predominantly biomedical model for birthing. Convergence is observed among the scientific community and other stakeholders in recognition of humanized birth benefits and the need to overcome institutional obstacles within the health sector. However, the progress of the proposed change is slow, and health professionals' resistance to address women's complaints towards obstetric violence and claim of quality care is observed. This discussion finds its reflection in a parliamentary discussion. CONCLUSIONS: The stakeholders' analysis reflects areas of conflict and consensus, as well as the diverse interacting dimensions that hinder the advance of humanized care in childbirth. This broad analysis strategy contributes to identifying critical aspects to be addressed in the development of integral and effective health policies.

Percepción de actores involucrados acerca del parto humanizado y la violencia obstétrica en Chile: una revisión panorámica / Stakeholders' perceptions of humanized birth practices and obstetric violence in Chile: a scoping review

INTRODUCCIÓN: Chile tiene una política incipiente en relación con prácticas de parto humanizado y la violencia obstétrica es una materia de creciente discusión pública levantada por las mujeres. A pesar de este avance, no se han observado iniciativas para superar el conflicto. Surge la pregunta acerca de los diferentes puntos de vista de los principales actores involucrados, con el fin de identificar estrategias que contribuyan al desarrollo de políticas públicas de salud que consideren a los actores influyentes. OBJETIVO: Identificar la percepción de actores acerca del parto humanizado y la violencia obstétrica. MÉTODO: Se condujo una revisión panorámica (scoping review) que incluyó la descripción y el análisis de artículos y textos de análisis que reflejaran la visión de la comunidad científica y declaraciones de actores del sector gubernamental, social, profesional y político expresados en sitios web institucionales. Se desarrolló un análisis de contenido temático cualitativo, de naturaleza inductiva. RESULTADOS: Se incluyeron setenta documentos. La comunidad científica se visualiza alineada con las recomendaciones ministeriales de parto personalizado. Varios investigadores analizan las dificultades de implementación debido a la construcción histórica, sociocultural y económica del modelo biomédico de atención del parto predominante. Se observa convergencia entre la comunidad científica y otros actores en torno al reconocimiento de los beneficios del parto humanizado, así como acerca de la necesidad de superar obstáculos institucionales del sector de salud. No obstante, el proceso de cambio propuesto es lento y se observan resistencias entre profesionales de la salud para abordar las quejas de las mujeres frente a la violencia obstétrica y su demanda de atención de calidad. Esta situación se refleja a nivel parlamentario. CONCLUSIONES: El análisis de actores identifica áreas de conflicto y de consenso, así como diversas dimensiones interactuantes que obstaculizan el avance hacia la humanización de la atención del parto. Esta estrategia amplia de análisis contribuye a la identificación de aspectos críticos a ser abordados para un desarrollo de políticas sanitarias integrales y efectivas.

INTRODUCTION: Chile has an incipient policy regarding humanized birth practices. Obstetric violence is becoming an issue in the public discussion, as brought up by women. Despite this advancement, no initiatives were observed to overcome the conflict. Questions arise from the different points of view of the main stakeholders involved. These questions help identify strategies contributing to the development of health policies that consider influencing actors. OBJECTIVES: To identify stakeholders' perceptions of humanized care in childbirth and obstetric violence. METHODS: We conducted a scoping review that included articles and analysis of texts reflecting the scientific communities' point of view. We included statements from governmental, social, professional, and political actors as expressed in institutional websites. Moreover, we performed a qualitative inductive, thematic content analysis. RESULTS: We included seventy documents. The scientific community is visualized as aligned with ministerial recommendations for personalized childbirth. Several researchers analyze the difficulties for its improvement due to the historical, socio-cultural, and economic construction of the predominantly biomedical model for birthing. Convergence is observed among the scientific community and other stakeholders in recognition of humanized birth benefits and the need to overcome institutional obstacles within the health sector. However, the progress of the proposed change is slow, and health professionals' resistance to address women's complaints towards obstetric violence and claim of quality care is observed. This discussion finds its reflection in a parliamentary discussion. CONCLUSIONS: The stakeholders' analysis reflects areas of conflict and consensus, as well as the diverse interacting dimensions that hinder the advance of humanized care in childbirth. This broad analysis strategy contributes to identifying critical aspects to be addressed in the development of integral and effective health policies.

Incidence of Vibrio cholerae in Fresh Fish and Ceviche in Guadalajara, Mexico.

The incidence of Vibrio cholerae O1 and non-Ol was determined in fresh fish and ceviche, a marinated raw fish dish ready for consumption. Fresh red snapper ( Lutjanus purpureous ) and mackerel ( Scomberomorus sierra ) were obtained from distribution centers, and ceviche from street vendors and small open restaurants in Guadalajara, Mexico. In addition to V. cholerae , the fish samples were tested for aerobic plate count (APC), total volatile nitrogen (TVN), trimethylamine (TMA), and the ceviche for APC, coliforms, and pH. V. cholerae O1 and non-O1 was isolated from 10% and 26% ofthe fish respectively. The mean data for the fish samples were in the region of: APC, 106 CFU/g of fish; more than 25 mg of TVN per 100 g of fish, but less than 5 mg of nitrogen as TMA per 100 g. Eleven percent of the ceviche obtained from street vendors and 6% obtained from restaurants were positive for V. cholerae O1. The mean APC and coliform counts were 6.6 and 4.8 log CFU/g of fish respectively, and the pH of the ceviche ranged from 3.0 to 4.5. All the strains of V. cholerae O1 isolated during this study were identified as biotype El Tor, serotypes Inaba and Ogawa. For both fresh fish and ceviche, the frequency of isolation of V. cholerae was highest during the summer months.