Biology subjects

Vaz, F. M.

Publications and source records attributed to Vaz, F. M..

14 recordsLinked to original sources

bioRxiv2026

Novel GC-MS/MS Strategy for Fructose Quantification and Stable Isotope Tracing: Development, Validation, and SIM vs MRM Comparison

High dietary fructose consumption is a major contributor to the development of obesity and related cardiometabolic diseases, highlighting the need for accurate assessment of fructose metabolism in humans. Stable isotope tracer approaches, such as 13C6-fructose, require highly sensitive and specific analytical methods to quantify both concentrations and isotopic enrichments. In this study, we developed and validated a robust gas chromatography-triple quadrupole mass spectrometry (GC-QQQ)-based method for the simultaneous measurement of unlabeled and 13C6-fructose in human plasma. The method employs oximation and per-acetate derivatization, and demonstrates high specificity and accuracy. Intra- and inter-assay precision were below 10%, with no detectable carry-over, and a lower limit of quantification (LLOQ) of 0.1 nmol/mL for concentration and 0.02 molar percent excess (MPE%) for enrichment and no interference from glucose. We further compared data acquisition using multiple reaction monitoring (MRM) and selected ion monitoring (SIM). MRM showed superior performance at the low concentrations and enrichment levels characteristic of clinical plasma samples, resulting in improved sensitivity and lower LLOQs compared to SIM. Overall, this validated method provides a sensitive and reliable approach for fructose tracer studies in humans. Its application will facilitate robust investigations into fructose metabolism, and its role in metabolic dysregulation and obesity-related disease.

biochemistry↗

bioRxiv2026

The Lipidome of iPSC-Derived Retinal Organoids and RPE Partially Resembles that of the Human Retina

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

molecular biology↗

bioRxiv2026

Chronic exposure to low-concentration urban PM2.5 accelerates maladaptive repair after ischemic injury via mitochondrial dysfunction and lysosomal stress.

BackgroundFine particulate matter (PM2.5), airborne particles with an aerodynamic diameter [≤]2.5 m that can penetrate deep into the lungs and enter the circulation, is increasingly recognized as a risk factor for chronic kidney disease (CKD) with long-term exposure. We previously demonstrated that high-dose PM2.5 exposure prior to ischemia-reperfusion injury (IRI) aggravates acute kidney injury (AKI). Here, we investigated how prolonged, low-concentration urban PM2.5 exposure (<15 {micro}g/m3) affects kidney repair after AKI. MethodsSix-week-old mice underwent bilateral IRI or sham surgery, followed by six months of exposure to either filtered air or ambient PM2.5 exposure in a unique exposome chamber. Kidneys were analyzed using pathomics, electron and super-resolution microscopy, immunohistochemistry, transcriptomics, and LC-MS lipidomics/metabolomics. Complementary in vitro hypoxia-reoxygenation and PM2.5 exposure experiments were performed in proximal tubular epithelial cells. ResultsLong-term PM2.5 exposure had minimal effects in sham-operated mice, including no significant changes in body weight or kidney function. Despite preserved kidney function, IRI+PM2.5 mice exhibited reduced weight gain, a marked expansion of the interstitial area, attributable to enhanced fibrosis and inflammatory responses, microvascular rarefaction, and endothelial-to-mesenchymal transition, consistent with maladaptive repair features. Proximal tubules displayed mitochondrial injury, glycolytic reprogramming, lipid accumulation, and a senescent phenotype. Energy Dispersive X-ray (EDX) microscopy confirmed PM2.5-derived elements within proximal tubules lysosomes, accompanied by lysosomal stress. Transcriptional signature-based drug screening identified nicotinamide as a compound capable of reversing PM2.5-induced metabolic alterations; in vitro validation confirmed restoration of mitochondrial function. ConclusionsTogether, these findings show that chronic post-AKI exposure to PM2.5 at levels currently considered safe by regulatory bodies drives maladaptive repair and accelerates CKD progression through mitochondrial dysfunction, lysosomal stress senescence in proximal tubules, due to local PM2.5 element accumulation. Translational StatementAcute kidney injury frequently progresses to chronic kidney disease due to maladaptive repair, yet environmental drivers of this transition remain underrecognized. Using a controlled exposome chamber, we demonstrate that chronic exposure to low, real-world concentrations of urban PM2.5 during post-ischemic recovery results in the accumulation of PM2.5-derived elements within proximal tubular lysosomes, leading to organelle dysfunction, metabolic reprogramming, lipid accumulation, and a senescence-like phenotype. Importantly, transcriptomics-based drug repurposing identified nicotinamide as a candidate compound capable of reversing metabolic dysfunction in injured proximal tubular cells subjected to hypoxia-reoxygenation and PM2.5 exposure, an effect validated in vitro.

molecular biology↗

bioRxiv2025

Integrated Multi-Omics Mapping of Mitochondrial Dysfunction and Substrate Preference in Barth Syndrome Cardiac Tissue

Barth syndrome (BTHS) is a rare X-linked recessively inherited disorder caused by variants in the TAFAZZIN gene, leading to impaired conversion of monolysocardiolipin (MLCL) into mature cardiolipin (CL). Accumulation of MLCL and CL deficiency are diagnostic markers for BTHS. Clinically, BTHS includes cardiomyopathy, skeletal myopathy, neutropenia, and growth delays. Severely affected patients may require early cardiac transplants due to unpredictable cardiac phenotypes. The pathophysiological mechanisms of BTHS are poorly understood, and treatments remain symptomatic. This study analyzed heart samples from five pediatric male BTHS patients (5 months-15 years) and compared them to tissues from 24 non-failing donors (19-71 years) using an integrated omics method combining metabolomics, lipidomics, and proteomics. The analysis confirmed changes in diagnostic markers (CL and MLCL), severe mitochondrial alterations, metabolic shifts, and elevated heart-failure markers. It also revealed significant interindividual differences among BTHS patients. With this study describe a powerful analytical tool for the in-depth analysis of metabolic disorders and a solid foundation for the understanding of BTHS disease phenotypes in cardiac tissues.

molecular biology↗

bioRxiv2024

In vivo human uterine temperature, pH, and uterine fluid composition analysis

Study questionWhat are the temperature, pH and uterine fluid composition in the human uterus three days following a positive LH test or ovum pick-up? Summary answerThe mean uterine temperature was 36.94{+/-}0.26{degrees}C, the mean uterine pH was 6.76{+/-}0.22, and the concentrations of 37 components in aspirated uterine fluid were successfully determined. What is known alreadyEmbryo culture conditions in the laboratory impact key outcomes of IVF/ICSI treatments, such as the quality of the embryos and the live birth rate after treatment, and child outcomes, such as birth weight. Currently used conditions, including temperature, pH, and culture medium composition, are largely derived from clinical experience and experimental studies using animal models. Limited studies have been performed to determine the natural human preimplantation embryo environment in vivo during the physiologically relevant time of the menstrual cycle. This type of fundamental knowledge is required for evidence-based optimization of the in vitro embryo culture environment and improving IVF/ICSI outcomes. Study design, size, durationIn this cross-sectional study, conducted between April 2015 and March 2016, temperature and pH were measured in the human uterine cavity on the third day following a positive LH test or ovum pick-up, and uterine fluid was simultaneously aspirated for composition analysis. Uterine temperature was measured in fifty eight women, uterine pH was determined in fifty three women, and twenty two samples of aspirated uterine fluid were analysed for the concentrations of thirty-seven components. Participants/materials, setting, methodsThis study involved 61 healthy reproductive-aged women: 53 without ovarian stimulation and 8 who underwent ovarian stimulation. We measured uterine temperature using a probe inserted into the uterine cavity directly, and uterine pH after inserting a probe through the outer sheath of an IVF catheter. Uterine fluid was then aspirated using this outer IVF catheter and a 10 ml syringe, and subsequently analysed with a Cobas 8000 chemistry analyser and ultra-performance liquid chromatography-tandem mass spectrometry. Main results and the role of chanceThe mean uterine temperature on the third day following a positive LH test or ovum pick-up was 36.94 {+/-} 0.26{degrees}C and correlated with the womens core body temperature. The mean pH in the uterine cavity was pH 6.76 {+/-} 0.22, clearly lower than the standard pH used for human preimplantation embryo culture in vitro (pH 7.3 {+/-} 0.1). Concentrations of important energy sources were 0.8 {+/-} 0.02 mM pyruvate, 5.1 {+/-} 1.78 mM glucose and 6.60 {+/-} 1.12 mM lactate. Glutamic acid (1162 {+/-} 183 M), glycine (955 {+/-} 156 M) and alanine (513 {+/-} 82 M) were the most abundant amino acids in uterine fluid. Limitations, reasons for cautionIn absence of a preimplantation embryo, synergistic influences on the uterine environment may be overlooked. Single centre and specific population limitations may hinder broader generalization of the results. Uterine fluid likely contains additional components. Wider implications of the findingsThe in vivo uterine characteristics identified in this study are foundational to develop an in vivo evidence-based culture medium for human embryos. Further research is necessary to evaluate whether such a medium can improve human preimplantation embryo development and quality, thereby increasing cumulative live birth rates and improving child outcomes.

developmental biology↗

bioRxiv2024

Plasma triacylglycerol length and saturation level mark healthy aging groups in humans

Complex lipids, essential components in biological processes, exhibit conserved age-related changes that alter membrane properties, cellular functions, and are implicated as biomarkers and contributors to longevity and age-related diseases. While physical activity alleviates age-related comorbidities and physical impairments, comprehensive exploration of the underlying biological mechanisms, particularly at the level of complex lipids, remains limited. However, clinical studies suggest that physical activity may counteract these age-related lipidomic changes, presenting a promising avenue for intervention. We performed lipidomic profiling of plasma from an extensively characterized cohort of young and aged individuals. Annotating 1446 unique lipid species across 24 lipid classes we found the most prominent difference in older adults was an accumulation of triacylglycerols (TGs), with lower physical activity levels associated with higher TG levels in plasma and reduced physical functionality. Remarkably, lipid species in the TG class did not accumulate uniformly. Rather, our study unveiled a negative correlation between higher physical activity levels and TGs with shorter chain length and more double bonds in this demographic. Overall, our research highlights that plasma TG length and saturation level can help mark healthy aging groups in humans. These findings deepen our understanding of how aging affects complex lipids, and the influence of physical activity on this process.

developmental biology↗

bioRxiv2023

Tracer-based lipidomics identifies novel disease-specific biomarkers in mitochondrial β-oxidation disorders

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and possible influence on cellular lipid homeostasis are, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore new disease markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two new candidate disease markers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second biomarker is an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. These are hypothesized to be degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two new disease markers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.

molecular biology↗

bioRxiv2023

DHA shortage causes the early degeneration of photoreceptors and RPE in mice with peroxisomal β-oxidation deficiency

PurposePatients deficient in peroxisomal {beta}-oxidation, which is essential for the synthesis of docosahexaenoic acid (DHA, C22:6n-3) and breakdown of very long-chain polyunsaturated fatty acids (VLC-PUFAs), both important components of photoreceptor outer segments, present with retinopathy. The representative mouse model lacking the central enzyme of this pathway, multifunctional protein 2 (Mfp2-/-), also develops early onset retinal decay and cell-autonomous retinal pigment epithelium (RPE) degeneration, accompanied by reduced plasma and retinal DHA levels. In this study, we investigated whether DHA supplementation can rescue the retinal degeneration of Mfp2-/- mice. MethodsMfp2+/- breeding pairs and their offspring were fed a 0.12% DHA or control diet during gestation, lactation and until sacrifice. Offspring were analysed for retinal function via electroretinograms, for lipid composition of neural retina and plasma with lipidome analysis and gas chromatography respectively, and histologically using retinal sections and RPE flatmounts at the age of 4, 8 and 16 weeks. ResultsDHA supplementation to Mfp2-/- mice restored retinal DHA levels and prevented photoreceptor shortening, impaired functioning and death until 8 weeks. In addition, rescue of retinal DHA levels temporarily improved the ability of the RPE to phagocytose outer segments and delayed the RPE dedifferentiation. However, despite the initial rescue of retinal integrity, DHA supplementation could not prevent retinal degeneration at 16 weeks. ConclusionsWe reveal that the shortage of systemic supply of DHA is pivotal for the early retinal degeneration in Mfp2-/- mice. Furthermore, we unveil that adequate retinal DHA levels are essential for both photoreceptor and RPE homeostasis.

cell biology↗

bioRxiv2022

Acyl-CoA dehydrogenase substrate promiscuity limits the potential for development of substrate reduction therapy in disorders of valine and isoleucine metabolism

Toxicity of accumulating substrates is a significant problem in several disorders of valine and isoleucine degradation notably short-chain enoyl-CoA hydratase (ECHS1 or crotonase) deficiency, 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, propionic acidemia (PA) and methylmalonic aciduria (MMA). Isobutyryl-CoA dehydrogenase (ACAD8) and short/branched-chain acyl-CoA dehydrogenase (SBCAD, ACADSB) function in the valine and isoleucine degradation pathways, respectively. Deficiencies of these acyl-CoA dehydrogenase (ACAD) enzymes are considered biochemical abnormalities with limited or no clinical consequences. We investigated whether substrate reduction therapy through inhibition of ACAD8 and SBCAD can limit the accumulation of toxic metabolic intermediates in disorders of valine and isoleucine metabolism. Using analysis of acylcarnitine isomers, we show that 2-methylenecyclopropaneacetic acid (MCPA) inhibited SBCAD, isovaleryl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase, but not ACAD8. MCPA treatment of wild-type and PA HEK-293 cells caused a pronounced decrease in C3-carnitine. Furthermore, deletion of ACADSB in HEK-293 cells led to an equally strong decrease in C3-carnitine when compared to wild-type cells. Deletion of ECHS1 in HEK-293 cells caused a defect in lipoylation of the E2 component of the pyruvate dehydrogenase complex, which was not rescued by ACAD8 deletion. MCPA was able to rescue lipoylation in ECHS1 KO cells, but only in cells with prior ACAD8 deletion. SBCAD was not the sole ACAD responsible for this compensation, which indicates substantial promiscuity of ACADs in HEK-293 cells for the isobutyryl-CoA substrate. Substrate promiscuity appeared less prominent for 2-methylbutyryl-CoA at least in HEK-293 cells. We suggest that pharmacological inhibition of SBCAD to treat PA should be investigated further.

biochemistry↗

bioRxiv2021

Work and oxygen consumption of isolated right ventricular papillary muscle in experimental pulmonary hypertension

Right-sided myocardial mechanical efficiency (work output/metabolic energy input) in pulmonary hypertension can be severely reduced. We determined the contribution of intrinsic myocardial determinants of efficiency using papillary muscle preparations from monocrotaline-induced pulmonary hypertensive (MCT-PH) rats. The hypothesis was tested that efficiency is reduced by mitochondrial dysfunction in addition to increased activation heat reported previously. Right ventricular (RV) muscle preparations were subjected to 5 Hz sinusoidal length changes at 37{degrees}C. Work and suprabasal oxygen consumption (VO2) were measured before and after cross-bridge inhibition by blebbistatin. Cytosolic cytochrome c concentration, myocyte cross-sectional area, proton permeability of the inner mitochondrial membrane (PP IMM), and monoamine oxidase (MAO)-A and glucose 6-phosphate dehydrogenase (G-6-PDH) activities and phosphatidylglycerol (PG) and cardiolipin (CL) contents were determined. Mechanical efficiency ranged from 23 to 11% in control (n = 6) and from 22 to 1% in MCT-PH (n = 15) and correlated with work (r2 = 0.68, P < 0.0001) but not with VO2 (r2 = 0.004, P = 0.7919). VO2 for cross-bridge cycling was proportional to work (r2 = 0.56, P = 0.0005). Blebbistatin-resistant VO2 (r2 = 0.32, P = 0.0167) and IMM PP (r2 = 0.36, P = 0.0110) correlated inversely with efficiency. Together, these variables explained the variance of efficiency (coefficient of multiple determination R2 = 0.79, P = 0.0001). Cytosolic cytochrome c correlated inversely with work (r2 = 0.28, P = 0.0391), but not with efficiency (r2 = 0.20, P = 0.0867). G-6-PDH, MAO-A and PG/CL increased in the RV wall of MCT-PH but did not correlate with efficiency. Reduced myocardial efficiency in MCT-PH is due to activation processes and mitochondrial dysfunction. The variance of work and the ratio of activation heat reported previously and blebbistatin-resistant VO2 are discussed. Key pointsO_LIMechanical efficiency of right ventricular myocardium is reduced in pulmonary hypertension. Increased energy use for activation processes has been demonstrated previously, but the contribution of mitochondrial dysfunction is unknown. C_LIO_LIWork and oxygen consumption is determined during work loops. Oxygen consumption for activation and cross-bridge cycling confirm the previous heat measurements. C_LIO_LICytosolic cytochrome c concentration, proton permeability of the mitochondrial inner membrane and phosphatidylglycerol/cardiolipin are increased in experimental pulmonary hypertension. C_LIO_LIMitochondrial dysfunction in right ventricular myocytes is related to reduced work and mechanical efficiency in experimental pulmonary hypertension. C_LIO_LIUpregulation of the pentose phosphate pathway and a potential gap in the energy balance suggest mitochondrial dysfunction in right ventricular overload is due to excessive production of reactive oxygen species. C_LI

physiology↗

bioRxiv2021

Barth syndrome cellular models have dysregulated respiratory chain complex I and mitochondrial quality control due to abnormal cardiolipin

Barth syndrome (BTHS) is an X-linked genetic condition caused by defects in TAZ, which encodes a transacylase involved in the remodeling of the inner mitochondrial membrane phospholipid, cardiolipin (CL). As such, CL has been implicated in numerous mitochondrial functions, and the role of defective CL in the clinical pathology of BTHS is under intense investigation. We used untargeted proteomics, shotgun lipidomics, gene expression analysis, and targeted metabolomics to identify novel areas of mitochondrial dysfunction in a new model of TAZ deficiency in HEK293 cells. Functional annotation analysis of proteomics data revealed abnormal regulation of mitochondrial respiratory chain complex I (CI), driven by the reduced abundance of 6 CI associated proteins in TAZ-deficient HEK293 cells: MT-ND3, NDUFA5, NDUFAB1, NDUFB2, NDUFB4, and NDUFAF1. This resulted in reduced assembly and function of CI in TAZ-deficient HEK293 cells as well as BTHS patient derived lymphoblast cells. We also identified increased abundance of PARL, a rhomboid protein involved in the regulation of mitophagy and apoptosis, and abnormal downstream processing of PGAM5, another mediator of mitochondrial quality control, in TAZ-deficient cells. Lastly, we modulated CL via the phospholipase inhibitor bromoenol lactone and the CL targeted SS-peptide, SS-31, and showed that each is able to remediate abnormalities in CI abundance as well as PGAM5 processing. Thus, mitochondrial respiratory chain CI and PARL/PGAM5 regulated mitochondrial quality control, both of whose functions localize to the inner mitochondrial membrane, are dysregulated due to TAZ deficiency and are partially remediated via modulation of CL.

genetics↗

bioRxiv2020

Reduced ech-6 Expression Attenuates Fat-induced Premature Aging in C. elegans

Deregulated energy homeostasis represents a hallmark of aging and results from complex gene-by-environment interactions. Here, we discovered that reducing the expression of the gene ech-6 encoding enoyl-CoA hydratase remitted fat diet-induced deleterious effects on lifespan in Caenorhabditis elegans, while a basal expression of ech-6 was important for survival under normal dietary conditions. Lipidomics revealed that supplementation of fat in ech-6-silenced worms had marginal effects on lipid profiles, suggesting an alternative fat utilization for energy production. Transcriptomics further suggest a causal relation between the lysosomal pathway, energy production, and the longevity effect conferred by the interaction between ech-6 and high-fat diets. Indeed, enhancing energy production from endogenous fat by overexpressing lysosomal lipase lipl-4 recapitulated the lifespan effects of high-fat diets on ech-6-silenced worms. Collectively, these results reveal that the gene ech-6 modulates metabolic flexibility and may be a target for promoting metabolic health and longevity.

cell biology↗

bioRxiv2020

Aging selectively dampens oscillation of lipid abundance in white and brown adipose tissue

Lipid metabolism is under the control of the circadian system and circadian dysregulation has been linked to obesity and dyslipidemia. These factors and outcomes have also been associated to, or affected by, the process of aging. Here, we investigated whether murine white (WAT) and brown (BAT) adipose tissue lipids exhibit rhythmicity and if this is affected by aging. To this end, we have measured the 24 hour lipid profiles of WAT and BAT using a global lipidomics analysis of >1100 lipids. We observed rhythmicity in nearly all lipid classes including glycerolipids, glycerophospholipids, sterol lipids and sphingolipids. Overall, ~22% of the analyzed lipids were considered rhythmic in WAT and BAT. Despite a general accumulation of lipids upon aging the fraction of oscillating lipids decreased in both tissues to 14% and 18%, respectively. Diurnal profiles of lipids in BAT appeared to depend on the lipid acyl chain length and this specific regulation was lost in aged mice. Our study revealed how aging affects the rhythmicity of lipid metabolism and could contribute to the quest for targets that improve diurnal lipid homeostasis to maintain cardiometabolic health during aging.

biochemistry↗

bioRxiv2020

Metabolomics and lipidomics in C. elegans using a single sample preparation

Comprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand, for instance in research related to nutrition and aging. The nematode C. elegans is a key model organism in these fields, due to the large repository of available C. elegans mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, using a single sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for instance, glycolysis and the TCA cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗