Targeting Neutrophil Extracellular Trap Formation: Exploring Promising Pharmacological Strategies for the Treatment of Preeclampsia.

Neutrophils, which constitute the most abundant leukocytes in human blood, emerge as crucial players in the induction of endothelial cell death and the modulation of endothelial cell responses under both physiological and pathological conditions. The hallmark of preeclampsia is endothelial dysfunction induced by systemic inflammation, in which neutrophils, particularly through the formation of neutrophil extracellular traps (NETs), play a pivotal role in the development and perpetuation of endothelial dysfunction and the hypertensive state. Considering the potential of numerous pharmaceutical agents to attenuate NET formation (NETosis) in preeclampsia, a comprehensive assessment of the extensively studied candidates becomes imperative. This review aims to identify mechanisms associated with the induction and negative regulation of NETs in the context of preeclampsia. We discuss potential drugs to modulate NETosis, such as NF-κß inhibitors, vitamin D, and aspirin, and their association with mutagenicity and genotoxicity. Strong evidence supports the notion that molecules involved in the activation of NETs could serve as promising targets for the treatment of preeclampsia.

Protocol for ovariectomy and estradiol replacement in mice.

Ovariectomy, involving the surgical removal of ovaries, and estradiol replacement facilitate the understanding of sexual dimorphism-related physiological changes, encompassing reproductive biology, metabolism, and hormone-related diseases. In this study, we present a protocol for conducting ovariectomy and estradiol replacement in mice. We describe steps for performing sham and ovariectomy operations, outline preoperative preparations, and provide details on postoperative care, including analgesia administration and the removal of surgical clips. Additionally, we elaborate on the procedures for performing vehicle and estradiol injections. For complete details on the use and execution of this protocol, please refer to Luengo-Mateos et al.1.

Hypothalamic free fatty acid receptor-1 regulates whole-body energy balance.

OBJECTIVE: Free fatty acid receptor-1 (FFAR1) is a medium- and long-chain fatty acid sensing G protein-coupled receptor that is highly expressed in the hypothalamus. Here, we investigated the central role of FFAR1 on energy balance. METHODS: Central FFAR1 agonism and virogenic knockdown were performed in mice. Energy balance studies, infrared thermographic analysis of brown adipose tissue (BAT) and molecular analysis of the hypothalamus, BAT, white adipose tissue (WAT) and liver were carried out. RESULTS: Pharmacological stimulation of FFAR1, using central administration of its agonist TUG-905 in diet-induced obese mice, decreases body weight and is associated with increased energy expenditure, BAT thermogenesis and browning of subcutaneous WAT (sWAT), as well as reduced AMP-activated protein kinase (AMPK) levels, reduced inflammation, and decreased endoplasmic reticulum (ER) stress in the hypothalamus. As FFAR1 is expressed in distinct hypothalamic neuronal subpopulations, we used an AAV vector expressing a shRNA to specifically knockdown Ffar1 in proopiomelanocortin (POMC) neurons of the arcuate nucleus of the hypothalamus (ARC) of obese mice. Our data showed that knockdown of Ffar1 in POMC neurons promoted hyperphagia and body weight gain. In parallel, these mice developed hepatic insulin resistance and steatosis. CONCLUSIONS: FFAR1 emerges as a new hypothalamic nutrient sensor regulating whole body energy balance. Moreover, pharmacological activation of FFAR1 could provide a therapeutic advance in the management of obesity and its associated metabolic disorders.

High-calorie diets uncouple hypothalamic oxytocin neurons from a gut-to-brain satiation pathway via κ-opioid signaling.

Oxytocin-expressing paraventricular hypothalamic neurons (PVNOT neurons) integrate afferent signals from the gut, including cholecystokinin (CCK), to adjust whole-body energy homeostasis. However, the molecular underpinnings by which PVNOT neurons orchestrate gut-to-brain feeding control remain unclear. Here, we show that mice undergoing selective ablation of PVNOT neurons fail to reduce food intake in response to CCK and develop hyperphagic obesity on a chow diet. Notably, exposing wild-type mice to a high-fat/high-sugar (HFHS) diet recapitulates this insensitivity toward CCK, which is linked to diet-induced transcriptional and electrophysiological aberrations specifically in PVNOT neurons. Restoring OT pathways in diet-induced obese (DIO) mice via chemogenetics or polypharmacology sufficiently re-establishes CCK's anorexigenic effects. Last, by single-cell profiling, we identify a specialized PVNOT neuronal subpopulation with increased κ-opioid signaling under an HFHS diet, which restrains their CCK-evoked activation. In sum, we document a (patho)mechanism by which PVNOT signaling uncouples a gut-brain satiation pathway under obesogenic conditions.

Psychobiotic Effects on Anxiety Are Modulated by Lifestyle Behaviors: A Randomized Placebo-Controlled Trial on Healthy Adults.

Psychobiotics are modulators of the Microbiota-Gut-Brain Axis (MGBA) with promising benefits to mental health. Lifestyle behaviors are established modulators of both mental health and the MGBA. This randomized placebo-controlled clinical trial (NCT04823533) on healthy adults (N = 135) tested 4 weeks of probiotic supplementation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175). We assessed effects on wellbeing, quality of life, emotional regulation, anxiety, mindfulness and interoceptive awareness. We then analyzed if lifestyle behaviors modulated probiotic effectiveness. Results showed no significant effects of probiotic intake in whole sample outcomes. Correlational analyses revealed Healthy Behaviors were significantly correlated with wellbeing across scales. Moreover, the linear mixed-effects model showed that the interaction between high scores in Healthy Behaviors and probiotic intake was the single significant predictor of positive effects on anxiety, emotional regulation, and mindfulness in post-treatment outcomes. These findings highlight the relevance of controlling for lifestyle behaviors in psychobiotic and mental health research.

Estradiol regulates leptin sensitivity to control feeding via hypothalamic Cited1.

Until menopause, women have a lower propensity to develop metabolic diseases than men, suggestive of a protective role for sex hormones. Although a functional synergy between central actions of estrogens and leptin has been demonstrated to protect against metabolic disturbances, the underlying cellular and molecular mechanisms mediating this crosstalk have remained elusive. By using a series of embryonic, adult-onset, and tissue/cell-specific loss-of-function mouse models, we document an unprecedented role of hypothalamic Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 (Cited1) in mediating estradiol (E2)-dependent leptin actions that control feeding specifically in pro-opiomelanocortin (Pomc) neurons. We reveal that within arcuate Pomc neurons, Cited1 drives leptin's anorectic effects by acting as a co-factor converging E2 and leptin signaling via direct Cited1-ERα-Stat3 interactions. Together, these results provide new insights on how melanocortin neurons integrate endocrine inputs from gonadal and adipose axes via Cited1, thereby contributing to the sexual dimorphism in diet-induced obesity.

CPT1A in AgRP neurons is required for sex-dependent regulation of feeding and thirst.

BACKGROUND: Fatty acid metabolism in the hypothalamus has an important role in food intake, but its specific role in AgRP neurons is poorly understood. Here, we examined whether carnitinea palmitoyltransferase 1A (CPT1A), a key enzyme in mitochondrial fatty acid oxidation, affects energy balance. METHODS: To obtain Cpt1aKO mice and their control littermates, Cpt1a(flox/flox) mice were crossed with tamoxifen-inducible AgRPCreERT2 mice. Food intake and body weight were analyzed weekly in both males and females. At 12 weeks of age, metabolic flexibility was determined by ghrelin-induced food intake and fasting-refeeding satiety tests. Energy expenditure was analyzed by calorimetric system and thermogenic activity of brown adipose tissue. To study fluid balance the analysis of urine and water intake volumes; osmolality of urine and plasma; as well as serum levels of angiotensin and components of RAAS (renin-angiotensin-aldosterone system) were measured. At the central level, changes in AgRP neurons were determined by: (1) analyzing specific AgRP gene expression in RiboTag-Cpt1aKO mice obtained by crossing Cpt1aKO mice with RiboTag mice; (2) measuring presynaptic terminal formation in the AgRP neurons with the injection of the AAV1-EF1a-DIO-synaptophysin-GFP in the arcuate nucleus of the hypothalamus; (3) analyzing AgRP neuronal viability and spine formations by the injection AAV9-EF1a-DIO-mCherry in the arcuate nucleus of the hypothalamus; (4) analyzing in situ the specific AgRP mitochondria in the ZsGreen-Cpt1aKO obtained by breeding ZsGreen mice with Cpt1aKO mice. Two-way ANOVA analyses were performed to determine the contributions of the effect of lack of CPT1A in AgRP neurons in the sex. RESULTS: Changes in food intake were just seen in male Cpt1aKO mice while only female Cpt1aKO mice increased energy expenditure. The lack of Cpt1a in the AgRP neurons enhanced brown adipose tissue activity, mainly in females, and induced a substantial reduction in fat deposits and body weight. Strikingly, both male and female Cpt1aKO mice showed polydipsia and polyuria, with more reduced serum vasopressin levels in females and without osmolality alterations, indicating a direct involvement of Cpt1a in AgRP neurons in fluid balance. AgRP neurons from Cpt1aKO mice showed a sex-dependent gene expression pattern, reduced mitochondria and decreased presynaptic innervation to the paraventricular nucleus, without neuronal viability alterations. CONCLUSIONS: Our results highlight that fatty acid metabolism and CPT1A in AgRP neurons show marked sex differences and play a relevant role in the neuronal processes necessary for the maintenance of whole-body fluid and energy balance.

Perspectives on Inequity and Health Disparities in Chile and Their Relationship to Microbial Ecology.

Among countries in the Organisation for Economic Cooperation and Development (OECD), Chile stands out as having important inequalities in income distribution, dietary quality, access to urban green spaces, and health outcomes. People in lower socioeconomic groups consistently show higher rates of noncommunicable chronic diseases and are being hit the hardest by the COVID-19 pandemic. These chronic conditions are increasingly considered to be shaped, or affected by, the human gut microbiome. Moreover, inequity as an overarching concept might also be associated with microbial patterns and if so, this may represent a novel pathway through which to address health and other disparities. Focusing on the case of Chile, our goal is to contribute to a critical discussion and motivate researchers and policymakers to consider the role of the microbiome in social equity in future endeavors.

Influence of the Corporate Image of Nursing Homes on the Loyalty of Residents' Family Members.

This study analyzes the influence of the corporate image of nursing homes on the decisions made by family members as to whether their elderly relatives will stay in the same nursing home. An empirical study was conducted considering 566 residents' family members with the capacity to decide whether said residents will remain in the same nursing home, using a binary regression model with a logistic link function (i.e., logit). For the first time in the nursing home sector, these results show the specific variables of the corporate image that influence family members when deciding whether their elders will stay in the same nursing home. In order of importance, these variables are the level of trust conveyed by the nursing home, the investment made in the facilities, price-quality ratio, emotional connection to the nursing home, and the promotion of the nursing home's services. The study also highlights the importance of other personal factors in family members' decisions to keep their elders in the same nursing home, such as the family members' employment situations (higher loyalty among those employed by third parties) and the determining factors involved in the relative's choice of nursing home (higher loyalty among those whose choice was mainly based on humane and dignified treatment). This study offers a discussion of the theoretical contributions this research brings to academia as well as managerial implications for the industry. We believe that one future line of research should be continued after the COVID-19 pandemic comes to an end to compare the results and observe whether the most influential variables on family members' loyalty remain the same as data for this study was collected from November 2019 to February 2020.

Diet triggers specific responses of hypothalamic astrocytes in time and region dependent manner.

Hypothalamic astrocytes are particularly affected by energy-dense food consumption. How the anatomical location of these glial cells and their spatial molecular distribution in the arcuate nucleus of the hypothalamus (ARC) determine the cellular response to a high caloric diet remains unclear. In this study, we investigated their distinctive molecular responses following exposure to a high-fat high-sugar (HFHS) diet, specifically in the ARC. Using RNA sequencing and proteomics, we showed that astrocytes have a distinct transcriptomic and proteomic profile dependent on their anatomical location, with a major proteomic reprogramming in hypothalamic astrocytes. By ARC single-cell sequencing, we observed that a HFHS diet dictates time- and cell- specific transcriptomic responses, revealing that astrocytes have the most distinct regulatory pattern compared to other cell types. Lastly, we topographically and molecularly characterized astrocytes expressing glial fibrillary acidic protein and/or aldehyde dehydrogenase 1 family member L1 in the ARC, of which the abundance was significantly increased, as well as the alteration in their spatial and molecular profiles, with a HFHS diet. Together, our results provide a detailed multi-omics view on the spatial and temporal changes of astrocytes particularly in the ARC during different time points of adaptation to a high calorie diet.

The 4E approach to the human microbiome: Nested interactions between the gut-brain/body system within natural and built environments.

The complexity of the human mind and its interaction with the environment is one of the main epistemological debates throughout history. Recent ideas, framed as the 4E perspective to cognition, highlight that human experience depends causally on both cerebral and extracranial processes, but also is embedded in a particular sociomaterial context and is a product of historical accumulation of trajectory changes throughout life. Accordingly, the human microbiome is one of the most intriguing actors modulating brain function and physiology. Here, we present the 4E approach to the Human Microbiome for understanding mental processes from a broader perspective, encompassing one's body physiology and environment throughout their lifespan, interconnected by microbiome community structure and dynamics. We review evidence supporting the approach theoretically and motivates the study of the global set of microbial ecosystem networks encountered by a person across their lifetime (from skin to gut to natural and built environments). We furthermore trace future empirical implementation of the approach. We finally discuss novel research opportunities and clinical interventions aimed toward developing low-cost/high-benefit integrative and personalized bio-psycho-socio-environmental treatments for mental health and including the brain-gut-microbiome axis.

Olfactomedin 2 deficiency protects against diet-induced obesity.

BACKGROUND AND AIMS: Olfactomedin 2 (OLFM2; also known as noelin 2) is a pleiotropic protein that plays a major role in olfaction and Olfm2 null mice exhibit reduced olfactory sensitivity, as well as abnormal motor coordination and anxiety-related behavior. Here, we investigated the possible metabolic role of OLFM2. METHODS: Olfm2 null mice were metabolically phenotyped. Virogenetic modulation of central OLFM2 was also performed. RESULTS: Our data showed that, the global lack of OLFM2 in mice promoted anorexia and increased energy expenditure due to elevated brown adipose tissue (BAT) thermogenesis and browning of white adipose tissue (WAT). This phenotype led to resistance to high fat diet (HFD)-induced obesity. Notably, virogenetic overexpression of Olfm2 in the lateral hypothalamic area (LHA) induced weight gain associated with decreased BAT thermogenesis. CONCLUSION: Overall, this evidence first identifies central OLFM2 as a new molecular actor in the regulation of whole-body energy homeostasis.

Differences in Survival of Patients With COPD According to the New GesEPOC 2021 Classification of Phenotypes.

Introduction: Chronic obstructive pulmonary disease (COPD) is a respiratory pathology with high prevalence, morbidity and mortality. The Spanish COPD guideline (GesEPOC) recommends individualizing treatment according to phenotypes. The phenotype classification was updated in 2021. This study aimed to determine the survival of patients by this new classification and compare the predictive capacity of mortality compared to the previous version. Methods: This observational study of COPD patients involved prospective follow-up for 6 years. Demographic and clinical data were collected at the beginning and evolutionary data at the end of the study. Patients were classified according to GesEPOC 2017 and GesEPOC 2021. Univariate survival analysis and multivariate analysis identified mortality risk factors. Results: Of the 273 patients, 243 (89.0%) were male. Ninety-three patients (34.1%) died during follow-up. Regarding phenotypes, 190 patients (69.6%) were non-exacerbators, 69 (25.3%) belonged to the non-eosinophilic exacerbator phenotype and 14 (5.1%) were of the eosinophilic exacerbator phenotype. Compared with non-exacerbator patients, those with the non-eosinophilic exacerbator phenotype had lower survival (p = 0.009). Risk factors independently associated with mortality were older age (p < 0.001), non-eosinophilic exacerbator phenotype (p = 0.017) and a high Charlson index score (p < 0.001). The new classification presented a worse ability to predict mortality than the previous version (area under the curve 0.632 vs 0.566, p = 0.018). Conclusion: Patients with the non-eosinophilic exacerbator phenotype had worse prognoses. This phenotype, advanced age and high comorbidity were mortality risk factors. The GesEPOC 2021 classification predicts mortality worse than the 2017 version. These data must be considered for more individualized management of COPD patients.

Introducción: La enfermedad obstructiva crónica (EPOC) es una patología respiratoria con elevada prevalencia y alta morbimortalidad. La guía española de la EPOC (GesEPOC) recomienda individualizar el tratamiento según fenotipos. En su última actualización en 2021, se ha actualizado la clasificación de fenotipos. Se realiza este estudio para conocer la supervivencia de los pacientes sobre esta nueva clasificación y para comparar la capacidad predictiva de mortalidad con respecto a la versión previa. Métodos: Estudio observacional de pacientes con EPOC con un seguimiento prospectivo durante 6 años. Se recogieron datos demográficos y clínicos al inicio y datos evolutivos al final del estudio. Se clasificó a los pacientes según GesEPOC 2017 y GesEPOC 2021. Se realizó un análisis univariante de supervivencia y un análisis multivariante para identificar factores de riesgo de mortalidad. Resultados: Del los 273 pacientes, 243 (89,0%) eran varones. Fallecieron 93 sujetos (34,1%) durante el seguimiento. En cuanto a los fenotipos, 190 pacientes (69,6%) eran no agudizadores, 69 (25,3%) pertenecían al fenotipo agudizador no eosinofílico, y 14 (5,1%) eran del fenotipo agudizador eosinofílico. Comparando con los enfermos no agudizadores, los del fenotipo agudizador no eosinofílico tuvieron una menor supervivencia (p = 0,009). Los factores de riesgo independientemente asociados a la mortalidad fueron la edad avanzada (p < 0,001), el fenotipo agudizador no eosinofílico (p = 0,017) y una puntuación elevada en el índice de Charlson (p < 0,001). La nueva clasificación presentó una peor capacidad para predecir mortalidad en comparación con la versión previa (área bajo curva 0,632 vs 0,566, p = 0,018). Conclusión: Los pacientes del fenotipo agudizador no eosinofílico tenían peor pronóstico. Este fenotipo, junto con la edad avanzada y la elevada comorbilidad, fueron factores de riesgo de mortalidad. La clasificación GesEPOC 2021 predice peor la mortalidad con respecto a la versión de 2017. Es importante tener estos datos en cuenta para ofrecer un manejo más individualizado a los pacientes con EPOC.

Effect of abutment finish lines on the mechanical behavior and marginal fit of screw-retained implant crowns: An in vitro study.

STATEMENT OF PROBLEM: The design of the implant-abutment connection has been widely researched, but the impact of different crown-abutment geometries remains unclear. PURPOSE: The purpose of this in vitro study was to evaluate the effect of different crown-abutment margin geometries on the mechanical behavior and fit of screw-retained implant-supported single-crown restorations by using mechanical static and fatigue tests and mastication simulation. MATERIAL AND METHODS: A total of 45 cobalt-chromium premolar-shaped metal frameworks were fabricated for single-unit implant-supported screw-retained restorations on stock abutments and internal hexagon Ø4.25×11-mm cylindrical implants. They were divided into 3 groups according to margin geometry: S, shoulder; C, chamfer; and F, feather-edge. Three static load until fracture and 24 dynamic load tests were performed by using the International Organization for Standardization 14801:2016 standard (ISO 14801:2016) (number of cycles limit: 5×106 cycles, frequency: 6 Hz). The ProFatigue software program was used to optimize the procedure (S, n=12 specimens; C, n=7 specimens; and F, n=5 specimens). Six additional specimens from each group were subjected to a mastication simulation (limit number of cycles: 1×106 cycles, cyclic loading from Pmin=30 N to Pmax=300 N, frequency: 6 Hz). Results from the fatigue tests were reported descriptively, and the Fisher exact test was used to analyze the difference in failure modes. Data from maximum misfit were evaluated by photogrammetry and statistically analyzed with the Anderson-Darling test and the Kruskal-Wallis and Dunn multiple comparison tests (α=.05). RESULTS: The fatigue limit was 456 N for group S, 512 N for group C, and 514 N for group F. The mean ±standard deviation misfit was 2.6 ±0.1 µm for group S, 3.8 ±1.1 µm for group C, and 3.6 ±0.8 µm for group F. Differences in misfit between groups S and C and between groups S and F were statistically significant (P<.05). CONCLUSIONS: Crown-abutment connections with chamfer and feather-edge margins showed better mechanical behavior, while shoulder margin exhibited better fit. However, high levels of fit were achieved for all the evaluated geometries.

Development and validation of the food allergy severity score.

BACKGROUND: The heterogeneity and lack of validation of existing severity scores for food allergic reactions limit standardization of case management and research advances. We aimed to develop and validate a severity score for food allergic reactions. METHODS: Following a multidisciplinary experts consensus, it was decided to develop a food allergy severity score (FASS) with ordinal (oFASS) and numerical (nFASS) formats. oFASS with 3 and 5 grades were generated through expert consensus, and nFASS by mathematical modeling. Evaluation was performed in the EuroPrevall outpatient clinic cohort (8232 food reactions) by logistic regression with request of emergency care and medications used as outcomes. Discrimination, classification, and calibration were calculated. Bootstrapping internal validation was followed by external validation (logistic regression) in 5 cohorts (3622 food reactions). Correlation of nFASS with the severity classification done by expert allergy clinicians by Best-Worst Scaling of 32 food reactions was calculated. RESULTS: oFASS and nFASS map consistently, with nFASS having greater granularity. With the outcomes emergency care, adrenaline and critical medical treatment, oFASS and nFASS had a good discrimination (receiver operating characteristic area under the curve [ROC-AUC]>0.80), classification (sensitivity 0.87-0.92, specificity 0.73-0.78), and calibration. Bootstrapping over ROC-AUC showed negligible biases (1.0 × 10-6 -1.23 × 10-3 ). In external validation, nFASS performed best with higher ROC-AUC. nFASS was strongly correlated (R 0.89) to best-worst scoring of 334 expert clinicians. CONCLUSION: FASS is a validated and reliable method to measure severity of food allergic reactions. The ordinal and numerical versions that map onto each other are suitable for use by different stakeholders in different settings.

Synaptotagmin-13 Is a Neuroendocrine Marker in Brain, Intestine and Pancreas.

Synaptotagmin-13 (Syt13) is an atypical member of the vesicle trafficking synaptotagmin protein family. The expression pattern and the biological function of this Ca2+-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage, allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.