The genetic landscape of mitochondrial diseases in the next-generation sequencing era: a Portuguese cohort study.

Introduction: Rare disorders that are genetically and clinically heterogeneous, such as mitochondrial diseases (MDs), have a challenging diagnosis. Nuclear genes codify most proteins involved in mitochondrial biogenesis, despite all mitochondria having their own DNA. The development of next-generation sequencing (NGS) technologies has revolutionized the understanding of many genes involved in the pathogenesis of MDs. In this new genetic era, using the NGS approach, we aimed to identify the genetic etiology for a suspected MD in a cohort of 450 Portuguese patients. Methods: We examined 450 patients using a combined NGS strategy, starting with the analysis of a targeted mitochondrial panel of 213 nuclear genes, and then proceeding to analyze the whole mitochondrial DNA. Results and Discussion: In this study, we identified disease-related variants in 134 (30%) analyzed patients, 88 with nuclear DNA (nDNA) and 46 with mitochondrial DNA (mtDNA) variants, most of them being pediatric patients (66%), of which 77% were identified in nDNA and 23% in mtDNA. The molecular analysis of this cohort revealed 72 already described pathogenic and 20 novel, probably pathogenic, variants, as well as 62 variants of unknown significance. For this cohort of patients with suspected MDs, the use of a customized gene panel provided a molecular diagnosis in a timely and cost-effective manner. Patients who cannot be diagnosed after this initial approach will be further selected for whole-exome sequencing. Conclusion: As a national laboratory for the study and research of MDs, we demonstrated the power of NGS to achieve a molecular etiology, expanding the mutational spectrum and proposing accurate genetic counseling in this group of heterogeneous diseases without therapeutic options.

HR/MS-based lipidome analysis of rat brain modulated by tolcapone.

Lipids play key roles in the body, influencing cellular regulation, function, and signalling. Tolcapone, a potent catechol-O-methyltransferase (COMT) inhibitor described to enhance cognitive performance in healthy subjects, was previously shown to impact fatty acid ß-oxidation and oxidative phosphorylation. However, its impact on the brain lipidome remains unexplored. Hence, this study aimed to assess how tolcapone affects the lipidome of the rat pre-frontal cortex (PFC), a region of the brain highly relevant to tolcapone therapeutic effect, while evaluating its influence on operant behaviour. Tolcapone at 20 mg/kg was chronically administered to Wistar rats during a behavioural task and an untargeted liquid chromatography high-resolution mass spectrometry (LC-HR/MS) approach was employed to profile lipid species. The untargeted analysis identified 7227 features, of which only 33% underwent statistical analysis following data pre-processing. The results revealed an improved cognitive performance and a lipidome remodelling promoted by tolcapone. The lipidomic analysis showed 32 differentially expressed lipid species in tolcapone-treated animals (FC ≥ 1.2, p-value ≤ 0.1), and among these several triacylglycerols, cardiolipins and N-acylethanolamine (NAE 16:2) were found upregulated whereas fatty acids, hexosylceramides, and several phospholipids including phosphatidylcholines and phosphatidylethanolamines were downregulated. These preliminary findings shed light on tolcapone impact on lipid pathways within the brain. Although tolcapone improved cognitive performance and literature suggests the significance of lipids in cognition, this study did not conclusively establish that lipids directly drove or contributed to this outcome. Nevertheless, it underscores the importance of lipid modulation and encourages further exploration of tolcapone-associated mechanisms in the central nervous system (CNS).

Oral Health-Related Quality of Life in Celiac Portuguese Children: a cross-sectional study.

PURPOSE: Celiac Disease (CD) presents a wide variety of clinical signs and symptoms, including oral manifestations. This study pretended to characterize Oral Health-Related Quality of Life (OHRQoL) and reported oral manifestations in children with CD. METHODS: Target-population were children with CD. An online questionnaire, applied to children's parents, collected information about OHRQoL (using the Early Childhood Oral Health Impact Scale-ECOHIS), oral health behaviours, and history of oral manifestations. Data analysis included descriptive statistics, Mann-Whitney, Kruskall-Wallis, and Spearman correlation tests (α = 0.05). RESULTS: The sample included 146 celiac patients, with a mean age of 10.5 years (sd = 4.1). Mean ECOHIS score was 5.2 (sd = 6.8). The most frequently reported oral manifestations were recurrent aphthous stomatitis (46.6%), dental caries (45.2%) and dental opacity (39%). About one third of the participants mentioned improvements in oral health when a gluten-free diet was introduced. Most of the reported oral manifestations had a significant association with the ECOHIS score (p < 0.05). CONCLUSIONS: The OHRQoL of children was good, however oral manifestations had a negative impact on OHRQoL. The most reported oral manifestations were recurrent aphthous stomatitis, dental caries, and dental opacities. Oral health professionals must be aware about the heterogeneity of the disease, to recognize oral manifestations associated and their importance in the early diagnosis to reduce complications and to an improvement in the OHRQoL of these patients.

Predictors of psychotherapy dropout in patients with borderline personality disorder: A systematic review.

INTRODUCTION: Borderline personality disorder (BPD) is a highly debilitating psychiatric condition. Despite the expansion of new BPD specific forms of psychotherapy in the last few decades, high dropout rates have been reported in these treatments. Treatment discontinuation is associated with poor patient outcomes, inefficient resource utilization and the demoralization of healthcare providers. METHODS: In order to identify predictors of psychotherapy dropout among patients with BPD, a systematic search of Medline, the Cochrane Library, PsycInfo and PsycArticles was conducted. Studies included were randomized-controlled trials in which patients diagnosed with BPD were exposed to a therapeutic intervention consisted of an evidence-based psychotherapy. The quality of evidence in the studies was assessed through the use of revised Cochrane risk of bias tool. RESULTS: Six articles, incorporating four types of psychotherapy programmes, were included. Overall, the studies present low risk of attrition and reporting bias and unclear risk of selection, performance and detection bias. Patients with weaker therapeutic alliance scores and higher hostility presented with higher dropout rates. In contrast, better mindfulness skills and greater performance in specific neuropsychological domains, such as memory and executive control, were identified as predictive of lower risk of dropout. Sociodemographic variables and treatment history did not influence treatment retention. CONCLUSIONS: Factors that influence discontinuation should be taken into consideration in future treatment programmes, in an effort to optimize retention. Qualitative assessments of patients' reasons for dropping out may also help guide adjustments.

Whole-plant flint corn silage inclusion in total mixed rations for pre- and postweaning dairy calves.

Assuming that acetic acid plays a minor role in the development of ruminal epithelium of preweaning dairy calves, the fiber supply for growing calves has been neglected. More research has been done on including starch and nonfibrous carbohydrates in solid feed for preweaning calves. Accordingly, the fiber requirement of these calves is not well known, as diet recommendations vary greatly. Hence, elucidating the effects of including fiber from long particle sizes in the diet may be essential for helping calves overcome the transition challenge during weaning. Forty-five Holstein calves were used in a randomized block design, considering sex, birth date, and weight at 28 d of age, when the supply of the total mixed ration (TMR) with the inclusion of corn silage started. Three TMR with increasing whole-plant flint corn silage content (0, 10, or 20% on a dry matter basis) were compared: 0CS, 10CS, or 20CS, respectively. During the first 28 d of life, the calves were managed homogeneously and were fed 6 L/d of whole milk, a commercial calf starter pelleted, and water ad libitum. Next, the solid diet was changed to the respective solid feed treatment. Calves were gradually weaned from 52 to 56 d of age but were evaluated for an additional 14 d postweaning. Feed intake was measured daily, while body weight and metabolic indicators of intermediate metabolism were evaluated weekly. Ruminal fluid was collected at 6, 8, and 10 wk of age. Behavioral analysis was conducted on wk 7 (preweaning) and 10 (postweaning). There was a quadratic effect for dry matter intake from wk 7 to 10, with higher intake for the 10CS diet than the 0CS and 20CS diets. Consequently, the 10CS diet also promoted greater average daily gain at wk 8 and 9 compared with the 0CS and 20CS diets. However, the final body weight was not affected by the different solid diets. Silage inclusion in calves' diet positively affected time spent ruminating and chewing pre- and postweaning. Including 10% of whole-plant flint corn silage in the diets of young dairy calves is a strategy to increase total solid intake and decrease acidosis risk by increasing pH and ruminating activity around weaning.

Integral valorisation of tomato by-products towards bioactive compounds recovery: Human health benefits.

The tomato processing industry is one of the world's most important markets. This industry aims to optimise production, minimise energy costs and waste streams while ensuring high-quality products. This sector produces substantial amounts of by-products frequently disposed of as waste rather than reintroducing them with a new intent into the supply chain. However, these by-products are rich in bioactive compounds (BC), including carotenoids, fibre, which exhibit antioxidant, anti-inflammatory and chemopreventive properties, and cardiovascular protection. Reusing these compounds is favourable to reducing the environmental impact and enables the development of added-value products with various possible uses such as food and feed additives, nutraceuticals, cosmeceuticals, etc. This review summarises relevant issues towards the recovery and valorisation of BC from industrial tomato by-products within a circular economy context.

Exploring the response of a key Mediterranean gorgonian to heat stress across biological and spatial scales.

Understanding the factors and processes that shape intra-specific sensitivity to heat stress is fundamental to better predicting the vulnerability of benthic species to climate change. Here, we investigate the response of a habitat-forming Mediterranean octocoral, the red gorgonian Paramuricea clavata (Risso, 1826) to thermal stress at multiple biological and geographical scales. Samples from eleven P. clavata populations inhabiting four localities separated by hundreds to more than 1500 km of coast and with contrasting thermal histories were exposed to a critical temperature threshold (25 °C) in a common garden experiment in aquaria. Ten of the 11 populations lacked thermotolerance to the experimental conditions provided (25 days at 25 °C), with 100% or almost 100% colony mortality by the end of the experiment. Furthermore, we found no significant association between local average thermal regimes nor recent thermal history (i.e., local water temperatures in the 3 months prior to the experiment) and population thermotolerance. Overall, our results suggest that local adaptation and/or acclimation to warmer conditions have a limited role in the response of P. clavata to thermal stress. The study also confirms the sensitivity of this species to warm temperatures across its distributional range and questions its adaptive capacity under ocean warming conditions. However, important inter-individual variation in thermotolerance was found within populations, particularly those exposed to the most severe prior marine heatwaves. These observations suggest that P. clavata could harbor adaptive potential to future warming acting on standing genetic variation (i.e., divergent selection) and/or environmentally-induced phenotypic variation (i.e., intra- and/or intergenerational plasticity).

Living in endemic area for infectious diseases accelerates epigenetic age.

Inflammaging is a low-grade inflammatory state generated by the aging process that can contribute to frailty and age-related diseases in the elderly. However, it can have distinct effects in the elderly living in endemic areas for infectious diseases. An increased inflammatory response may confer protection against infectious agents in these areas, although this advantage can cause accelerating epigenetic aging. In this study, we evaluated the inflammatory profile and the epigenetic age of infected and noninfected individuals from an endemic area in Brazil. The profile of cytokines, chemokines and growth factors analyzed in the sera of the two groups of individuals showed similarities, although infected individuals had a higher concentration of these mediators. A significant increase in IL-1ra, CXCL8, CCL2, CCL3 and CCL4 production was associated with leprosy infection. Notably, elderly individuals displayed distinct immune responses associated with their infection status when compared to adults suggesting an adaptive remodelling of their immune responses. Epigenetic analysis also showed that there was no difference in epigenetic age between the two groups of individuals. However, individuals from the endemic area had a significant accelerated aging when compared to individuals from São Paulo, a non-endemic area in Brazil. Moreover, the latter cohort was also epigenetically aged in relation to an Italian cohort. Our data shows that living in endemic areas for chronic infectious diseases results in remodelling of inflammaging and acceleration of epigenetic aging in individuals regardless of their infectious status. It also highlights that geographical, genetic and environmental factors influence aging and immunosenescence in their pace and profile.

Lactate modulates cardiac gene expression in mice during acute physical exercise.

The aim of the present study was to verify the role of lactate as a signaling molecule in cardiac tissue under physiological conditions. C57BL6/J male mice were submitted to acute running bouts on a treadmill at different exercise intensities (30, 60, and 90% of maximal speed - Smax) under the effect of two doses (0.5 and 5 mM) of α-cyano-4-hydroxycynnamate (CINN), a blocker of lactate transporters. Cardiac lactate levels, activity of the enzymes of glycolytic [hexokinase (HK) and lactate dehydrogenase (LDH)] and oxidative metabolism [citrate synthase (CS)], and expression of genes also related to metabolism [LDH, nuclear factor erythroid 2-related factor 2 (NRF-2), cytochrome oxidase IV (COX-IV), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)] were evaluated. Elevated cardiac lactate levels were observed after high intensity running at 90% of Smax, which were parallel to increased activity of the HK and CS enzymes and mRNA levels of PGC-1α and COX-IV. No changes were observed in cardiac lactate levels in mice running at lower exercise intensities. Interestingly, prior intraperitoneal administration (15 min) of CINN (0.5 mM) significantly reduced cardiac lactate concentration, activities of HK and CS, and mRNA levels of PGC-1α and COX-IV in mice that ran at 90% of Smax. In addition, cardiac lactate levels were significantly correlated to both PGC-1α and COX-IV cardiac gene expression. The present study provides evidence that cardiac lactate levels are associated to gene transcription during an acute bout of high intensity running exercise.

Lactate modulates cardiac gene expression in mice during acute physical exercise

The aim of the present study was to verify the role of lactate as a signaling molecule in cardiac tissue under physiological conditions. C57BL6/J male mice were submitted to acute running bouts on a treadmill at different exercise intensities (30, 60, and 90% of maximal speed - Smax) under the effect of two doses (0.5 and 5 mM) of α-cyano-4-hydroxycynnamate (CINN), a blocker of lactate transporters. Cardiac lactate levels, activity of the enzymes of glycolytic [hexokinase (HK) and lactate dehydrogenase (LDH)] and oxidative metabolism [citrate synthase (CS)], and expression of genes also related to metabolism [LDH, nuclear factor erythroid 2-related factor 2 (NRF-2), cytochrome oxidase IV (COX-IV), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)] were evaluated. Elevated cardiac lactate levels were observed after high intensity running at 90% of Smax, which were parallel to increased activity of the HK and CS enzymes and mRNA levels of PGC-1α and COX-IV. No changes were observed in cardiac lactate levels in mice running at lower exercise intensities. Interestingly, prior intraperitoneal administration (15 min) of CINN (0.5 mM) significantly reduced cardiac lactate concentration, activities of HK and CS, and mRNA levels of PGC-1α and COX-IV in mice that ran at 90% of Smax. In addition, cardiac lactate levels were significantly correlated to both PGC-1α and COX-IV cardiac gene expression. The present study provides evidence that cardiac lactate levels are associated to gene transcription during an acute bout of high intensity running exercise.

The burden of heat-related mortality attributable to recent human-induced climate change.

Climate change affects human health; however, there have been no large-scale, systematic efforts to quantify the heat-related human health impacts that have already occurred due to climate change. Here, we use empirical data from 732 locations in 43 countries to estimate the mortality burdens associated with the additional heat exposure that has resulted from recent human-induced warming, during the period 1991-2018. Across all study countries, we find that 37.0% (range 20.5-76.3%) of warm-season heat-related deaths can be attributed to anthropogenic climate change and that increased mortality is evident on every continent. Burdens varied geographically but were of the order of dozens to hundreds of deaths per year in many locations. Our findings support the urgent need for more ambitious mitigation and adaptation strategies to minimize the public health impacts of climate change.

A rapid screening method to evaluate acidifying activity by lactic acid bacteria.

The determination of pH in fermented milk is an important parameter for monitoring the production of acid by lactic acid bacteria (LAB). In this work, a colorimetric method is proposed that allows a fast determination of LAB acidification ability by evaluation of pH drop in whey fermentation. The proposed method uses spectrophotometry to measure the pH change by bacteria and uses bromocresol purple as a pH indicator dye. The absorbance at 430 nm of a buffer solution with bromocresol purple was found to be correlated with pH values. This colorimetric assay was linear within the pH range of 4.6-7.0. Upon regression analysis, linear equation y = -0.1267× + 0.9196 was obtained having r2 value of 0.9927. The assay was validated by the use of LAB fermentation in sweet whey and comparison to the values obtained by glass electrode/pH meter. Estimation of acidification activity of LAB in whey was found to be similar in both methods (r = 0.801, p > 0.05). The proposed procedure presents a viable alternative to the measurement of pH by the standard method and allows the simultaneous and fast screening of LAB acidifying activity.

Systolic dysfunction by two-dimensional speckle tracking echocardiography in dogs with parvoviral enteritis.

INTRODUCTION/OBJECTIVES: Parvoviral enteritis (PVE) can cause either primary or secondary myocardial injury; the latter is associated with systemic inflammatory response syndrome and sepsis. Strain (St) and strain rate (SR) are relatively new speckle tracking echocardiographic (STE) variables used to assess myocardial function and are less influenced by preload and volume status than are conventional variables. The aim of this study was to evaluate systolic function in dogs with PVE using two-dimensional STE. ANIMALS: Forty-five client-owned dogs were included. MATERIALS AND METHODS: Dogs were classified into four groups: healthy (n = 9), PVE-mild (n = 15), PVE-severe (n = 13) and PVE-died (n = 8). Left ventricular global and segmental myocardial St and SR were assessed in radial, circumferential and longitudinal axes in the right parasternal transverse and apical 4-chamber views. In the circumferential and longitudinal axes, the value of each segment was determined separately at the endocardial and epicardial levels. RESULTS: Compared to healthy animals, all dogs with PVE showed significantly impaired St and SR values, mainly for PVE-severe and PVE-died groups. Moreover, the lowest SR value was observed in the circumferential axis at the mid-septal epicardial segment in the PVE-died group. For this variable, a cut-off value of 0.95 s-1 demonstrated 100% sensitivity and specificity for distinguishing between PVE-severe and PVE-died groups. CONCLUSIONS: In the present study, all dogs with PVE developed systolic dysfunction, which was more severe in non-survivors. Assessment of St and SR in dogs with PVE might be clinically useful for evaluating haemodynamic status and developing suitable therapeutic strategies to improve prognosis.

ω-Conotoxina MVIIC e células-tronco mesenquimais promovem recuperação motora em ratos Wistar após trauma medular agudo / ω-Conotoxin MVIIC and mesenchymal stem cells promote motor recovery in Wistar rats after acute spinal cord injury]

O objetivo deste estudo foi avaliar o efeito da ω-conotoxina MVIIC e das células-tronco mesenquimais (CTM) de forma isolada e sua associação nos ratos submetidos ao trauma medular agudo (TMA). Trinta Rattus novergicus, linhagem Wistar, três meses de idade, foram distribuídos igualmente em cinco grupos experimentais: controle negativo (CN), controle positivo (CP), ω-conotoxina MVIIC (MVIIC), células-tronco mesenquimais da medula óssea (CTM-MO) e associação (MVIIC + CTM-MO). O grupo CN foi submetido à laminectomia sem trauma medular, e os grupos CP, MVIIC, CTM-MO e MVIIC + CTM-MO foram submetidos ao trauma medular contusivo. O grupo CP recebeu, uma hora após o TMA, 10µL de PBS estéril, e os grupos MVIIC e MVIIC + CTM-MO receberam 10µL de PBS contendo 20pmol da ω-conotoxina MVIIC, todos por via intratecal. Os grupos CTM-MO e MVIIC + CTM-MO receberam, 24 horas após, 1x106 de CTM via intravenosa. Avaliou-se a recuperação da função locomotora até o sétimo dia pós-trauma. Os animais tratados com MVIIC + CTM-MO obtiveram recuperação motora após o trauma medular agudo (P<0,05). Conclui-se que essa associação apresentou efeito neuroprotetor com melhora na função locomotora em ratos Wistar.(AU)

The objective of this study was to evaluate the effect of isolated ω-conotoxin MVIIC and mesenchymal stem cells (MSCs) and its association in rats submitted to acute spinal cord injury (SCI). Thirty Rattus norvegicus, Wistar strain, three-month-old rats were randomly distributed in five experimental groups with six animals: negative control (CN), positive control (CP), ω-conotoxin MVIIC (MVIIC), bone marrow mesenchymal stem cells (CTM-MO) and the association (MVIIC + CTM-MO). The CN group underwent laminectomy without spinal cord trauma, and groups CP, MVIIC, CTM-MO and MVIIC + CTM-MO were submitted to contusive spinal cord trauma. The CP group received 10µl of PBS one hour after SCI, and groups MVIIC and MVIIC + CTM-MO received 10µl of PBS containing 20pmol of ω-conotoxin MVIIC, both intrathecally. Groups CTM-MO and MVIIC + CTM-MO received 1x106 of MSCs intravenously 24 hours later. The recovery of locomotor function was evaluated up to seven days post-injury. The animals treated with MVIIC + CTM-MO obtained motor recovery after SCI (P<0.05). It is concluded that this association showed neuroprotective effect with improvements in locomotor function in Wistar rats.(AU)