A transmitochondrial sodium gradient controls membrane potential in mammalian mitochondria.

Eukaryotic cell function and survival rely on the use of a mitochondrial H+ electrochemical gradient (Δp), which is composed of an inner mitochondrial membrane (IMM) potential (ΔΨmt) and a pH gradient (ΔpH). So far, ΔΨmt has been assumed to be composed exclusively of H+. Here, using a rainbow of mitochondrial and nuclear genetic models, we have discovered that a Na+ gradient equates with the H+ gradient and controls half of ΔΨmt in coupled-respiring mammalian mitochondria. This parallelism is controlled by the activity of the long-sought Na+-specific Na+/H+ exchanger (mNHE), which we have identified as the P-module of complex I (CI). Deregulation of this mNHE function, without affecting the canonical enzymatic activity or the assembly of CI, occurs in Leber's hereditary optic neuropathy (LHON), which has profound consequences in ΔΨmt and mitochondrial Ca2+ homeostasis and explains the previously unknown molecular pathogenesis of this neurodegenerative disease.

Urinary extracellular vesicles as a monitoring tool for renal damage in patients not meeting criteria for chronic kidney disease.

Background: Current definition of chronic kidney disease (CKD) identifies only advanced stages, but effective management demands early detection. Urinary albumin-to-creatinine ratio (ACR) 30 mg/g is a cut-off point for CKD clinical diagnosis. Patients with lower values (normoalbuminuria) and eGFR > 60 mL/min/1.73 m2 are considered at no increased cardiorenal risk. However, higher incidence of renal function decline and cardiovascular events have been shown within the normoalbuminuria range. Novel subclinical indicators may help to identify higher-risk patients. Urinary extracellular vesicles (uEVs) are sentinels of renal function non-invasively. Here we aimed to approach the early assessment of cardiorenal risk by investigating the protein cargo of uEVs. Methods: Hypertensive patients were classified in control group (C) with ACR < 10 mg/g, and high-normal group (HN) with ACR 10-30 mg/g. Isolated uEVs were characterized by western blotting and electron microscopy and the protein cargo was analyzed by untargeted proteomics (LC-MS/MS) in a first discovery cohort. Protein confirmation was performed in a different cohort by ExoView. Immunohistochemistry of human kidney biopsies was also performed to evaluate the potential of uEVs to reflect renal damage. Results: HN albuminuria does not affect the uEVs concentration, size, or tetraspanin profile. Among >6200 uEVs proteins identified, 43 define a panel significantly altered in HN patients without variation in urine, mostly annotated in the tubule (39 out of 43). The tubular transporter long-chain fatty acid transport protein 2 (SLC27A2) and the apical membrane protein amnionless (AMN) confirmed their alteration in HN patients evidencing impaired tubular reabsorption. SLC27A2 showed tubular expression and significantly reduced levels in patients with diagnostic criteria for CKD. Conclusions: Alterations in the EV-mediated molecular profile are evident before pathological ACR levels are reached. Direct quantitation of SLC27A2 and AMN in uEVs helps identifying normoalbuminuric subjects with higher cardiorenal risk in early monitoring of CKD.

Remodeling p38 signaling in muscle controls locomotor activity via IL-15.

Skeletal muscle has gained recognition as an endocrine organ releasing myokines upon contraction during physical exercise. These myokines exert both local and pleiotropic health benefits, underscoring the crucial role of muscle function in countering obesity and contributing to the overall positive effects of exercise on health. Here, we found that exercise activates muscle p38γ, increasing locomotor activity through the secretion of interleukin-15 (IL-15). IL-15 signals in the motor cortex, stimulating locomotor activity. This activation of muscle p38γ, leading to an increase locomotor activity, plays a crucial role in reducing the risk of diabetes and liver steatosis, unveiling a vital muscle-brain communication pathway with profound clinical implications. The correlation between p38γ activation in human muscle during acute exercise and increased blood IL-15 levels highlights the potential therapeutic relevance of this pathway in treating obesity and metabolic diseases. These findings provide valuable insights into the molecular basis of exercise-induced myokine responses promoting physical activity.

Influence of Rare-Earth Doping Content and Type on Phase Transformation and Transport Properties in Highly Doped CeO2.

Rare-earth doped CeO2 materials find extensive application in high-temperature energy conversion devices such as solid oxide fuel cells and electrolyzers. However, understanding the complex relationship between structural and electrical properties, particularly concerning rare-earth ionic size and content, remains a subject of ongoing debate, with conflicting published results. In this study, we have conducted comprehensive long-range and local order structural characterization of Ce1-xLnxO2-x/2 samples (x ≤ 0.6; Ln = La, Nd, Sm, Gd, and Yb) using X-ray and neutron powder diffraction, Raman spectroscopy, and electron diffraction. The increase in the rare-earth dopant content leads to a progressive phase transformation from a disordered fluorite structure to a C-type ordered superstructure, accompanied by reduced ionic conductivity. Samples with low dopant content (x = 0.2) exhibit higher ionic conductivity in Gd3+ and Sm3+ series due to lower lattice cell distortion. Conversely, highly doped samples (x = 0.6) exhibit superior conductivity for larger rare-earth dopant cations. Thermogravimetric analysis confirms increased water uptake and proton conductivity with increasing dopant concentration, while the electronic conductivity remains relatively unaffected, resulting in reduced ionic transport numbers. These findings offer insights into the relationship between transport properties and defect-induced local distortions in rare-earth doped CeO2, suggesting the potential for developing new functional materials with mixed ionic oxide, proton, and electronic conductivity for high-temperature energy systems.

An innovative environmental tool to evaluate the sustainability of anthropogenic processes: the tetrahedron approach.

The Tetrahedron approach is a new environmental tool adapted to assess the sustainability of anthropogenic processes. This tool is based on a four-step methodology that includes (a) the identification of critical parameters, (b) evaluation through the Tetrahedron Parameter Global Evaluator, (c) construction of a tetrahedron diagram based on the final scores and (d) quantitative estimation of the global sustainability. The Tetrahedron incorporates various aspects of sustainability, including economic, social and environmental factors, and provides a comprehensive framework for evaluating the impact of human activities. This article presents the methodology and application of the Tetrahedron in determining the sustainability of five case studies: CO2 capture, unconventional methanol production, the Solvay process, CO2-alcoholic fermentation process strategy and the CO2-Rumen fermentation process strategy. The results demonstrate the Tetrahedron as an effective and reliable tool to quantify the sustainability of anthropogenic processes and to promote sustainable practices across various industries and sectors. The Tetrahedron offers several advantages over other environmental assessment tools, including holistic approach, simplicity and flexibility.

Dissociative photoionization of acetaldehyde in the 10.2-19.5 eV VUV range.

The valence-shell dissociative photoionization of acetaldehyde has been investigated by means of the photoion photoelectron coincidence technique in conjunction with tuneable synchrotron radiation. The experimental results consist of threshold photoelectron spectra for the parent ion and for each fragment ion in the 10.2-19.5 eV photon energy range, along with (ion, e) kinetic energy coincidence diagrams obtained from measurements at fixed photon energies. The results are complemented by high-level ab initio calculations of potential energy curves as a function of the C-H bond distance. The nudged elastic band (NEB) method has been employed to connect the parent ion Franck-Condon region to the formation of the HCO+, CH3+ and CH4+ ion fragments. Appearance energies have been determined for six fragment ions with an improved accuracy, including two fragmentation channels, which to the best of our knowledge have not been reported previously, i.e. the formation of CH2CO+, lying at 13.10 ± 0.05 eV, and the formation of CH2+ at 15.1 ± 0.1 eV. Based on both experimental and theoretical results, the dissociation dynamics following ionization of acetaldehyde into the different fragmentation channels are discussed.

Concurrent validity of the Global Physical Activity Questionnaire to accelerometry in Hispanic/Latino adults: The Hispanic Community Health Study/Study of Latinos (HCHS/SOL).

OBJECTIVES: The validity of the Global Physical Activity Questionnaire has not been thoroughly evaluated among Hispanics/Latinos. In this cross-sectional study, we assessed the concurrent validity and correlates of discordance of the Global Physical Activity Questionnaire by comparing it to accelerometry in estimating sedentary behavior, moderate-to-vigorous physical activity, and meeting United States physical activity guidelines by sociodemographic, behavioral, and health characteristics. DESIGN: The Hispanic Community Health Study/Study of Latinos is a 4-site cohort study of United States adults aged 18-74 years enrolled from 2008 to 2011. METHODS: Participants (n = 11,873) completed the Global Physical Activity Questionnaire and wore an accelerometer for 1 week. Lin's concordance and Pearson correlations assessed concurrent validity between self-reported and accelerometry-assessed measures of sedentary behavior and moderate-to-vigorous physical activity. Kappa coefficients assessed agreement of meeting physical activity guidelines. Linear and logistic regression models identified correlates of discordance. RESULTS: The overall Lin's concordance and Pearson correlations between the Global Physical Activity Questionnaire and accelerometry estimates were 0.10 (95 % confidence interval 0.09, 0.12) and 0.24 (0.21, 0.27) for sedentary behavior, and 0.04 (0.03, 0.05) and 0.18 (0.15, 0.22) for moderate-to-vigorous physical activity, respectively. Agreement was poor for meeting the physical activity guideline classifications (Kappa coefficients: 0.12 to 0.26). Over a 16-hour day, sedentary behavior was under-reported by 3.8 h and moderate-to-vigorous physical activity was over-reported by 1.9 h. CONCLUSIONS: The concurrent validity of the Global Physical Activity Questionnaire in measuring moderate-to-vigorous physical activity and sedentary behavior when compared to accelerometry was poor among Hispanic/Latino adults.

The Urinary Glycopeptide Profile Differentiates Early Cardiorenal Risk in Subjects Not Meeting Criteria for Chronic Kidney Disease.

Early diagnosis and treatment of chronic kidney disease (CKD) is a worldwide challenge. Subjects with albumin-to-creatinine ratio (ACR) ≥ 30 mg/g and preserved renal function are considered to be at no cardiorenal risk in clinical practice, but prospective clinical studies evidence increased risk, even at the high-normal (HN) ACR range (10-30 mg/g), supporting the need to identify other molecular indicators for early assessment of patients at higher risk. Following our previous studies, here we aim to stratify the normoalbuminuria range according to cardiorenal risk and identify the glycoproteins and N-glycosylation sites associated with kidney damage in subclinical CKD. Glycoproteins were analyzed in urine from hypertensive patients within the HN ACR range compared to control group (C; ACR < 10 mg/g) by mass spectrometry. A different cohort was analyzed for confirmation (ELISA) and sex perspective was evaluated. Patients' follow-up for 8 years since basal urine collection revealed higher renal function decline and ACR progression for HN patients. Differential N-glycopeptides and their N -glycosylation sites were also identified, together with their pathogenicity. N-glycosylation may condition pathological protein deregulation, and a panel of 62 glycoproteins evidenced alteration in normoalbuminuric subjects within the HN range. Haptoglobin-related protein, haptoglobin, afamin, transferrin, and immunoglobulin heavy constant gamma 1 (IGHG1) and 2 (IGHG2) showed increased levels in HN patients, pointing to disturbed iron metabolism and tubular reabsorption and supporting the tubule as a target of interest in the early progression of CKD. When analyzed separately, haptoglobin, afamin, transferrin, and IGHG2 remained significant in HN, in both women and men. At the peptide level, 172 N-glycopeptides showed differential abundance in HN patients, and 26 showed high pathogenicity, 10 of them belonging to glycoproteins that do not show variation between HN and C groups. This study highlights the value of glycosylation in subjects not meeting KDIGO criteria for CKD. The identified N-glycopeptides and glycosylation sites showed novel targets, for both the early assessment of individual cardiorenal risk and for intervention aimed at anticipating CKD progression.

Non-adiabatic dynamics of photoexcited cyclobutanone: Predicting structural measurements from trajectory surface hopping with XMS-CASPT2 simulations.

Over the years, theoretical calculations and scalable computer simulations have complemented ultrafast experiments, as they offer the advantage of overcoming experimental restrictions and having access to the whole dynamics. This synergy between theory and experiment promises to yield a deeper understanding of photochemical processes, offering valuable insights into the behavior of complex systems at the molecular level. However, the ability of theoretical models to predict ultrafast experimental outcomes has remained largely unexplored. In this work, we aim to predict the electron diffraction signals of an upcoming ultrafast photochemical experiment using high-level electronic structure calculations and non-adiabatic dynamics simulations. In particular, we perform trajectory surface hopping with extended multi-state complete active space with second order perturbation simulations for understanding the photodissociation of cyclobutanone (CB) upon excitation at 200 nm. Spin-orbit couplings are considered for investigating the role of triplet states. Our simulations capture the bond cleavage after ultrafast relaxation from the 3s Rydberg state, leading to the formation of the previously observed primary photoproducts: CO + cyclopropane/propene (C3 products), ketene, and ethene (C2 products). The ratio of the C3:C2 products is found to be about 1:1. Within 700 fs, the majority of trajectories transition to their electronic ground state, with a small fraction conserving the initial cyclobutanone ring structure. We found a minimal influence of triplet states during the early stages of the dynamics, with their significance increasing at later times. We simulate MeV-ultrafast electron diffraction (UED) patterns from our trajectory results, linking the observed features with specific photoproducts and the underlying structural dynamics. Our analysis reveals highly intense features in the UED signals corresponding to the photochemical processes of CB. These features offer valuable insights into the experimental monitoring of ring opening dynamics and the formation of C3 and C2 photoproducts.

Making Sense of Censored Covariates: Statistical Methods for Studies of Huntington's Disease.

The landscape of survival analysis is constantly being revolutionized to answer biomedical challenges, most recently the statistical challenge of censored covariates rather than outcomes. There are many promising strategies to tackle censored covariates, including weighting, imputation, maximum likelihood, and Bayesian methods. Still, this is a relatively fresh area of research, different from the areas of censored outcomes (i.e., survival analysis) or missing covariates. In this review, we discuss the unique statistical challenges encountered when handling censored covariates and provide an in-depth review of existing methods designed to address those challenges. We emphasize each method's relative strengths and weaknesses, providing recommendations to help investigators pinpoint the best approach to handling censored covariates in their data.

Evidence for widespread translation of 5' untranslated regions.

Ribosome profiling experiments support the translation of a range of novel human open reading frames. By contrast, most peptides from large-scale proteomics experiments derive from just one source, 5' untranslated regions. Across the human genome we find evidence for 192 translated upstream regions, most of which would produce protein isoforms with extended N-terminal ends. Almost all of these N-terminal extensions are from highly abundant genes, which suggests that the novel regions we detect are just the tip of the iceberg. These upstream regions have characteristics that are not typical of coding exons. Their GC-content is remarkably high, even higher than 5' regions in other genes, and a large majority have non-canonical start codons. Although some novel upstream regions have cross-species conservation - five have orthologues in invertebrates for example - the reading frames of two thirds are not conserved beyond simians. These non-conserved regions also have no evidence of purifying selection, which suggests that much of this translation is not functional. In addition, non-conserved upstream regions have significantly more peptides in cancer cell lines than would be expected, a strong indication that an aberrant or noisy translation initiation process may play an important role in translation from upstream regions.

In-gel protein digestion using acidic methanol produces a highly selective methylation of glutamic acid residues.

Mass-tolerant open search methods allow the high-throughput analysis of modified peptides by mass spectrometry. These techniques have paved the way to unbiased analysis of post-translational modifications in biological contexts, as well as of chemical modifications produced during the manipulation of protein samples. In this work, we have analyzed in-depth a wide variety of samples of different biological origin, including cells, extracellular vesicles, secretomes, centrosomes and tissue preparations, using Comet-ReCom, a recently improved version of the open search engine Comet-PTM. Our results demonstrate that glutamic acid residues undergo intensive methyl esterification when protein digestion is performed using in-gel techniques, but not using gel-free approaches. This effect was highly specific to Glu and was not found for other methylable residues such as Asp.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

A Sample Preparation Procedure for Isobaric Labeling-Based Single-Cell Proteomics.

Mass spectrometry-based proteomics has traditionally been limited by the amount of input material for analysis. Single-cell proteomics has emerged as a challenging discipline due to the ultra-high sensitivity required. Isobaric labeling-based multiplex strategies with a carrier proteome offer an approach to overcome the sensitivity limitations. Following this as the basic strategy, we show here the general workflow for preparing cells for single-cell mass spectrometry-based proteomics. This protocol can also be applied to manually isolated cells when large cells, such as cardiomyocytes, are difficult to isolate properly with conventional fluorescence-activated cell sorting (FACS) sorter methods.

Cox7a1 controls skeletal muscle physiology and heart regeneration through complex IV dimerization.

The oxidative phosphorylation (OXPHOS) system is intricately organized, with respiratory complexes forming super-assembled quaternary structures whose assembly mechanisms and physiological roles remain under investigation. Cox7a2l, also known as Scaf1, facilitates complex III and complex IV (CIII-CIV) super-assembly, enhancing energetic efficiency in various species. We examined the role of Cox7a1, another Cox7a family member, in supercomplex assembly and muscle physiology. Zebrafish lacking Cox7a1 exhibited reduced CIV2 formation, metabolic alterations, and non-pathological muscle performance decline. Additionally, cox7a1-/- hearts displayed a pro-regenerative metabolic profile, impacting cardiac regenerative response. The distinct phenotypic effects of cox7a1-/- and cox7a2l-/- underscore the diverse metabolic and physiological consequences of impaired supercomplex formation, emphasizing the significance of Cox7a1 in muscle maturation within the OXPHOS system.

Lack of p38 activation in T cells increases IL-35 and protects against obesity by promoting thermogenesis.

Obesity is characterized by low-grade inflammation, energy imbalance and impaired thermogenesis. The role of regulatory T cells (Treg) in inflammation-mediated maladaptive thermogenesis is not well established. Here, we find that the p38 pathway is a key regulator of T cell-mediated adipose tissue (AT) inflammation and browning. Mice with T cells specifically lacking the p38 activators MKK3/6 are protected against diet-induced obesity, leading to an improved metabolic profile, increased browning, and enhanced thermogenesis. We identify IL-35 as a driver of adipocyte thermogenic program through the ATF2/UCP1/FGF21 pathway. IL-35 limits CD8+ T cell infiltration and inflammation in AT. Interestingly, we find that IL-35 levels are reduced in visceral fat from obese patients. Mechanistically, we demonstrate that p38 controls the expression of IL-35 in human and mouse Treg cells through mTOR pathway activation. Our findings highlight p38 signaling as a molecular orchestrator of AT T cell accumulation and function.

Analysis of Vascular Smooth Muscle Cells from Thoracic Aortic Aneurysms Reveals DNA Damage and Cell Cycle Arrest as Hallmarks in Bicuspid Aortic Valve Patients.

Thoracic aortic aneurysm (TAA) is mainly sporadic and with higher incidence in the presence of a bicuspid aortic valve (BAV) for unknown reasons. The lack of drug therapy to delay TAA progression lies in the limited knowledge of pathophysiology. We aimed to identify the molecular hallmarks that differentiate the aortic dilatation associated with BAV and tricuspid aortic valve (TAV). Aortic vascular smooth muscle cells (VSMCs) isolated from sporadic TAA patients with BAV or TAV were analyzed by mass spectrometry. DNA oxidative damage assay and cell cycle profiling were performed in three independent cohorts supporting proteomics data. The alteration of secreted proteins was confirmed in plasma. Stress phenotype, oxidative stress, and enhanced DNA damage response (increased S-phase arrest and apoptosis) were found in BAV-TAA patients. The increased levels of plasma C1QTNF5, LAMA2, THSB3, and FAP confirm the enhanced stress in BAV-TAA. Plasma FAP and BGN point to an increased inflammatory condition in TAV. The arterial wall of BAV patients shows a limited capacity to counteract drivers of sporadic TAA. The molecular pathways identified support the need of differential molecular diagnosis and therapeutic approaches for BAV and TAV patients, showing specific markers in plasma which may serve to monitor therapy efficacy.

Anti-sperm Antibody Positivity in Men with Varicocele: A Systematic Review and Meta-Analysis.

PURPOSE: Despite the significant role of varicocele in the pathogenesis of male infertility, its association with anti-sperm antibodies (ASA) remains controversial. This systematic review and meta-analysis (SRMA) aims to investigate the frequency of ASA positivity in men with varicocele. MATERIALS AND METHODS: This SRMA is conducted in accordance with the Meta-analysis of Observational Studies in Epidemiology guidelines. We investigated the frequency of ASA positivity in ejaculates or serum of men with varicocele as compared to men without varicocele (controls). A literature search was performed using the Scopus and PubMed databases following the Population Exposure Comparison Outcome, Study Design model. Data extracted from eligible studies were meta-analyzed and expressed as odds ratios (ORs) and confidence intervals (CIs). RESULTS: Out of 151 abstracts identified during the initial screening, 6 articles met the inclusion criteria and were included in the meta-analysis. Using mixed antiglobulin reaction (MAR) assay, 61 out of the 153 (39.8%) patients with varicocele tested positive for ASA in their ejaculates as compared to 22 out of the 129 control subjects (17%, OR=4.34 [95% CI: 1.09-17.28]; p=0.04). Using direct or indirect immunobead test, 30 out of 60 cases diagnosed with varicocele (50%) had shown ASA positivity in their ejaculates as compared to 16 out of 104 controls (15.4%, OR=3.57 [95% CI: 0.81-15.68]; p=0.09). Using enzyme-linked immunosorbent assay (ELISA), out of 89 varicocele patients, 33 (37.1%) tested positive for serum ASA as compared to 9 out of 57 participants in the control group (15.8%, OR=7.87 [95% CI: 2.39-25.89]; p<0.01). CONCLUSIONS: This SRMA indicates that ASA positivity is significantly higher among men with varicocele when tested by direct method (MAR) or indirect method (ELISA). This data suggests an immunological pathology in infertile men with varicocele and may have implications for the management of these patients.