Biology subjects

Ferreira, M.

Publications and source records attributed to Ferreira, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Nitrogen metabolic map of Escherichia coli mediated by multi-functional aminotransferases

Nitrogen is a key element of organic molecules essential to all life. Unlike extensively characterized carbon metabolic maps, it remains elusive how assimilated nitrogen flows through metabolic networks. Here we determined the nitrogen metabolic map of Escherichia coli and used it to construct an enzyme-constrained metabolic model that includes multi-functionality of aminotransferase enzymes responsible for nitrogen transfer reactions. We characterized substrate specificities of sixteen E. coli aminotransferase enzymes by evaluating 2,528 reactions, uncovering 56 previously unrecognized activities. The cellular concentrations of these aminotransferase enzymes were quantified and used to estimate their catalytic rates across all enzyme-substrate pairs, leading to improved predictions of nitrogen flows in E. coli. This work advances our fundamental understanding of the nitrogen metabolic map critical for metabolism and growth.

biochemistry↗

bioRxiv2025

Reference Gene Validation in Energy Cane Under Smut Infection and Insights into Antioxidant Gene Expression

Sporisorium scitamineum is the causal agent of smut disease affecting sugarcane and energy cane crop production. Despite the frequent use of traditional reference genes for gene expression normalization by RT-qPCR, their stability can vary across genotypes and stress conditions. Here, we evaluated the stability of seven commonly used reference genes (GAPDH, SAMDC, UBQ, 25S rRNA, TUB, SARMp1, and ACAD) and two additional candidates (EF5A-1 and SDH1-1) identified from RNA-Seq data in two contrasting energy cane genotypes (Vertix 1, smut-susceptible; Vertix 2, smut-resistant). Stability was assessed at three infection time points (12 hours post-inoculation, 48 hpi, and 5 days post-inoculation) separately and also on a combined dataset comprising all time points, under mock and smut-inoculated conditions. Gene stability, evaluated through multiple approaches (coefficient of variation, RefFinder, GeNorm) and consolidated using RankAggreg, revealed genotype and condition-dependent variability, with no universal reference gene set for most conditions. GAPDH, commonly used in sugarcane studies, showed the highest variation in our experiments, reinforcing the need for genotype and condition-specific validation. The best-ranked reference genes were used to quantify ROS-related defense responses. Vertix 2 (Vx2) exhibited coordinated induction of SOD, POX5, and CATB, whereas Vertix 1 (Vx1) showed delayed or reduced activation of these genes alongside a compensatory up-regulation of GSTt3. By validating reliable endogenous genes from literature and RNA-Seq data, this study ensures accurate gene expression quantification under biotic stress and highlights antioxidant mechanisms as contributors to smut resistance in S. scitamineum-energy cane interactions.

plant biology↗

bioRxiv2025

A Core Pattern of Cerebellar and Brainstem Degeneration and Reduced Cerebrocerebellar Structural Covariance in Spinocerebellar Ataxia Type 3 (SCA3): MRI Volumetrics from ENIGMA-Ataxia

ObjectiveSpinocerebellar ataxia type 3 (SCA3) is a rare, inherited neurodegenerative disease. Here, we profile the spatial spread of atrophy across the whole brain, determine whether brain degeneration preferentially maps onto specific functional networks, and investigate the relationship between cerebellar and cerebral anatomical changes. MethodsWhole-brain grey and white matter (GM and WM) voxel-based morphometry was performed on 408 individuals with SCA3 (82 pre-ataxic) and 293 controls. The SCA3 cohort was stratified by ataxia severity to investigate disease progression, with cerebellar GM atrophy mapped onto a task-based functional atlas. Volume was correlated with disease duration and intensity. Cerebrocerebellar volumetric covariance was assessed to determine whether atrophy was coupled between infra- and supratentorial regions. ResultsThe pattern of atrophy is spatially consistent but progressive in magnitude across the disease course. The greatest atrophy was found in the pons, cerebellar WM, and cerebellar peduncles; correlations with disease severity and duration were also strongest in these regions. Cerebellar GM atrophy was greatest in functional regions associated with motor execution and planning, attention, and emotional processing. Sparse cerebral cortical atrophy appears only in the most severe disease subgroup, while striatal atrophy begins in the earliest stages. Reduced cerebrocerebellar structural covariance is observed in SCA3 participants versus controls. InterpretationWhile cerebellar and brainstem atrophy become more severe, the pattern of atrophy remains largely consistent as SCA3 progresses. Cerebellar GM degeneration occurs in regions associated with motor, cognitive, and affective control, in line with clinical presentation. Cerebellar atrophy is not directly mirrored by cerebral changes.

neuroscience↗

bioRxiv2025

Discovery and characterisation of gene by environment and epistatic genetic effects in a vertebrate model

Phenotypic variation arises from the interplay between genetic and environmental factors. However, disentangling these interactions for complex traits remains challenging in observational cohorts such as human biobanks. Instead, model organisms where genetic (G) and environmental (E) variation can be controlled offer a valuable complement to human studies in the analysis of higher-order genetic effects such as GxE interactions, dominance, and epistasis. Here, we utilized 76 medaka strains of the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel, to compare heart rate plasticity across temperatures. An F2 segregation analysis identified 16 quantitative trait loci (QTLs), with many exhibiting dominance, GxE, GxG, and GxGxE interactions. We experimentally validated four candidate genes using gene editing, revealing their temperature-sensitive impact on heart function. Finally, we devised simulations to assess how GWAS discovery power is influenced by the choice of statistical models, showing that the apparent additivity in human GWAS is to be expected given study design and sample sizes of current studies. This work demonstrates the value of controlled model organism studies for dissecting the genetics of complex traits and provides guidance on the design of genetic association studies.

genetics↗

bioRxiv2025

Extracellular matrix mechanical cues (dys)regulate metabolic redox homeostasis due to impaired autophagic flux

Extracellular matrix (ECM) stiffness is increasingly recognized as a critical regulator of cellular behavior, governing processes such as proliferation, differentiation, and metabolism. Neurodegenerative diseases are characterized by mitochondrial dysfunction, oxidative stress, impaired autophagy, and progressive softening of the brain tissue, yet research into how mechanical cues influence cellular metabolism in this context remains scarce. In this study, we evaluated the long-term effects of brain-compliant, soft ECM on mitochondrial bioenergetics, redox balance, and autophagic capacity in neuronal cells. Using human neuroblastoma (SH-SY5Y) and mouse hippocampal (HT22) cell lines, as well as primary mouse neurons, we observed that prolonged exposure to soft ECM resulted in mitochondrial bioenergetic dysfunction, redox imbalance, and disrupted autophagic flux. These findings were consistently validated across both human and mouse neuronal cells. Our data indicate a decreased maximal autophagic capacity in cells exposed to long-term soft ECM, potentially due to an imbalance in autophagosome formation and degradation, as demonstrated by decreased LC3 II levels following chloroquine-induced autophagic flux inhibition. This impairment in autophagy was coupled with increased cellular oxidative stress, further indicating metabolic alterations. These findings emphasize the critical role of ECM stiffness in regulating neuronal cell metabolism and suggest that prolonged exposure to soft ECM may mimic key aspects of neurodegenerative disease pathology, thereby enhancing the physiological relevance of in vitro models. This study underscores the necessity for further research into ECM mechanics as a contributing factor in neurodegenerative disease progression and as a potential target for therapeutic strategies.

cell biology↗

bioRxiv2024

FEHAT: Efficient, Large scale and Automated Heartbeat Detection in Medaka Fish Embryos.

High resolution imaging of model organisms allows the quantification of important physiological measurements. In the case of fish with transparent embryos, these videos can visualise key physiological processes, such as heartbeat. High throughput systems can provide enough measurements for the robust investigation of developmental processes as well as the impact of system perturbations on physiological state. However, few analytical schemes have been designed to handle thousands of high-resolution videos without the need for some level of human intervention. We developed a software package, named FEHAT, to provide a fully automated solution for the analytics of large numbers of heart rate imaging datasets obtained from developing Medaka fish embryos in 96 well plate format imaged on an Acquifer machine. FEHAT uses image segmentation to define regions of the embryo showing changes in pixel intensity over time, followed by the classification of the most likely position of the heart and Fourier Transformations to estimate the heart rate. Here we describe some important features of the FEHAT software, showcasing its performance across a large set of medaka fish embryos and compare its performance to established, less automated solutions. FEHAT provides reliable heart rate estimates across a range of temperature-based perturbations and can be applied to tens of thousands of embryos without the need for any human intervention.

bioinformatics↗

bioRxiv2024

Multi-omics data and model integration reveal the main mechanisms associated with respiro-fermentative metabolism and ethanol stress responses in Kluyveromyces marxianus

Kluyveromyces marxianus is a yeast capable of fermenting sugars into ethanol and growing at high temperatures (>37{o}C). However, it is less tolerant to ethanol than Saccharomyces cerevisiae, which limits its application in second-generation ethanol production. Since the mechanisms of ethanol stress response are still poorly described, especially compared to S. cerevisiae, we used an integrative multi-omics approach, combining transcriptomics, coexpression networks, gene regulation, and genome-scale metabolic modelling to gain insights about these mechanisms. Through metabolic modelling, we predicted the occurrence of a respiro-fermentative metabolism and its onset as the dilution rate increased. From gene coexpression networks, we detected that the protein quality control system is a main mechanism involved in the ethanol stress response. Further, we identified key regulators in the ethanol stress response, such as HAP3, MET4, and SNF2, and assessed how disturbances in their gene expression affect cellular metabolism. We also found that amino acid metabolism, membrane lipid metabolism, and ergosterol exhibit increased metabolic flux under the explored conditions. These findings provide useful cues to develop and implement genetic and metabolic engineering strategies to enhance ethanol tolerance.

systems biology↗

bioRxiv2024

Meningioma transcriptomic landscape demonstrates novel subtypes with regional associated biology and patient outcome.

Meningiomas, the most common intracranial tumor, though mostly benign can be recurrent and fatal. WHO grading does not always identify high risk meningioma and better characterizations of their aggressive biology is needed. To approach this problem, we combined 13 bulk RNA-Seq datasets to create a dimension-reduced reference landscape of 1298 meningiomas. Clinical and genomic metadata effectively correlated with landscape regions which led to the identification of meningioma subtypes with specific biological signatures. Time to recurrence also correlated with the map location. Further, we developed an algorithm that maps new patients onto this landscape where nearest neighbors predict outcome. This study highlights the utility of combining bulk transcriptomic datasets to visualize the complexity of tumor populations. Further, we provide an interactive tool for understanding the disease and predicting patient outcome. This resource is accessible via the online tool Oncoscape, where the scientific community can explore the meningioma landscape.

cancer biology↗

bioRxiv2023

lista-GEM: the genome-scale metabolic reconstruction of Lipomyces starkeyi

Oleaginous yeasts cultivation in low-cost substrates is an alternative for more sustainable production of lipids and oleochemicals. Lipomyces starkeyi accumulates high amounts of lipids from different carbon sources, such as glycerol, and glucose and xylose (lignocellulosic sugars). Systems metabolic engineering approaches can further enhance its capabilities for lipid production, but no genome-scale metabolic networks have been reconstructed and curated for L. starkeyi. Herein, we propose lista-GEM, the first genome-scale metabolic model of L. starkeyi. We reconstructed the model using two high-quality models of oleaginous yeasts as templates and further curated the model to reflect the metabolism of L. starkeyi. We simulated phenotypes and predicted flux distributions in good accordance with experimental data. We also predicted targets to improve lipid production in glucose, xylose, and glycerol. The phase plane analysis indicated that the carbon availability affected lipid production more than oxygen availability. We found that the maximum lipid production in glucose and xylose required more oxygen than glycerol. Enzymes related to lipid synthesis in the endoplasmic reticulum were the main targets to improve lipid production: stearoyl-CoA desaturase, fatty-acyl-CoA synthase, diacylglycerol acyltransferase, and glycerol-3-phosphate acyltransferase. The glycolytic genes encoding pyruvate kinase, enolase, phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase were predicted as targets for overexpression. Pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase, adenylate kinase, inorganic diphosphatase, and triose-phosphate isomerase were predicted only when glycerol was the carbon source. Therefore, we demonstrated that lista-GEM provides multiple metabolic engineering targets to improve lipid production by L. starkeyi using carbon sources from agricultural and industrial wastes. HighlightsO_LILipomyces starkeyi can accumulate high amounts of lipids from carbon sources found in agricultural and industrial wastes. C_LIO_LIWe reconstructed lista-GEM, the first genome-scale metabolic model of L. starkeyi. C_LIO_LISimulated phenotypes were in line with experimental results of L. starkeyi. C_LIO_LIWe identified key gene targets for improving lipid production using metabolic engineering. C_LI

bioinformatics↗

bioRxiv2023

Trunk neural crest gene MOXD1 affects embryonic development

Ectoderm-derived neural crest is a transient structure arising during early embryogenesis in vertebrates. Neural crest consists of four derivatives based on their anterior- to posterior location along the body axis; cranial, vagal, trunk and sacral, respectively. We recently showed that trunk neural crest-specific gene MOXD1 functions as a tumor suppressor in trunk neural crest-derived childhood cancer form neuroblastoma and is essential for proper development of healthy adrenal glands. However, the role of MOXD1 during early embryogenesis is not known. Here, we conditionally knocked out MOXD1 in trunk neural crest cells before they become lineage-committed, using a CRISPR/Cas9 approach in chick embryos. Assessment of embryo growth showed that knockout of MOXD1 delayed development with knockout embryos being smaller. RNA sequencing of trunk-derived neural crest cells from control and knockout embryos showed enrichment of genes connected to gland development, copper ion metabolism and neuroblastoma progression. In conclusion, MOXD1 is important during early and prolonged embryonic development with effects on gland formation, possibly mediated via its role in copper metabolism.

developmental biology↗

bioRxiv2023

tRNASer overexpression induces adaptive mutations in NSCLC tumors

tRNAs are a driving force of genome evolution in Yeast and Bacteria. Their deregulation is frequently observed in tumors with Serine tRNAs being often overexpressed. This has important functional consequences, such as increased metabolism and tumor growth. In yeast, time and chemical stimulus boost alterations in the genome driven by tRNA deregulation. Therefore, we hypothesized that tRNA deregulation may contribute to the increased genome instability observed in tumors. To study the effect of tRNA deregulation in tumors, we overexpressed tRNA-Ser-AGA-2-1 in a NSCLC cell line, H460. This cell line and a Mock (control) were xenografted in nude mice and collected at 3 timepoints: T1-Naive; T2-Treated once with cisplatin/vehicle and; T3) treated twice with cisplatin/vehicle. These tumors were characterized by WES, RNAseq and Mass Spectrometry and the data obtained was integrated. The tumor mutation burden was increased in T3 tRNASerOE tumors, regardless of treatment. Although in T1 Mock and tRNASer tumors have a similar number of variants, in T2&3, tRNASerOE tumors display two times more variants than Mock tumors regardless of treatment. Interestingly, tRNASerOE exclusive variants favor proliferation and therapy resistance, which is in line with the phenotypes observed and supported by RNAseq and proteomics data. In conclusion, tRNASerOE increases the tumor mutation burden and the variants detected favor tumor growth, proving tRNA deregulation is enough to induce adaptive mutations in the genome.

cancer biology↗

bioRxiv2023

Accurate prediction of in vivo protein abundances by coupling constraint-based modeling and machine learning

Quantification of how different environmental cues affect protein allocation can provide important insights for understanding cell physiology. While absolute quantification of proteins can be obtained by resource-intensive mass-spectrometry-based technologies, prediction of protein abundances offers another way to obtain insights into protein allocation. Here we present CAMEL, a framework that couples constraint-based modelling with machine learning to predict protein abundance for any environmental condition. This is achieved by building machine learning models that leverage static features, derived from protein sequences, and condition-dependent features predicted from protein-constrained metabolic models. Our findings demonstrate that CAMEL results in excellent prediction of protein allocation in E. coli (average Pearson correlation of at least 0.9), and moderate performance in S. cerevisiae (average Pearson correlation of at least 0.5). Therefore, CAMEL outperformed contending approaches without using molecular read-outs from unseen conditions and provides a valuable tool for using protein allocation in biotechnological applications.

bioinformatics↗

bioRxiv2023

Joint testing of rare variant burden scores using non-negative least squares

Gene-based burden tests are a popular and powerful approach for analysis of exome-wide association studies. These approaches combine sets of variants within a gene into a single burden score that is then tested for association. Typically, a range of burden scores are calculated and tested across a range of annotation classes and frequency bins. Correlation between these tests can complicate the multiple testing correction and hamper interpretation of the results. We introduce a new method called the Sparse Burden Association Test (SBAT) that tests the joint set of burden scores under the assumption that causal burden scores act in the same effect direction. The method simultaneously assesses the significance of the model fit and selects the set of burden scores that best explain the association at the same time. Using simulated data, we show that the method is well calibrated and highlight some scenarios where the test outperforms existing gene-based tests. We apply the method to 73 quantitative traits from the UK Biobank which further illustrates the power of the method. This test is implemented in the REGENIE software.

genetics↗

bioRxiv2023

A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells

Cellular lineage histories along with their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have limited barcode diversity and poor single-cell lineage coverage, thus precluding their use in tissues composed of millions of cells. Here, we developed DARLIN, an improved Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) to enhance insertion events over 30 CRISPR target sites, stably integrated into 3 distinct genomic loci. DARLIN is inducible, has an estimated ~1018 lineage barcodes across tissues, and enables detection of usable barcodes in ~60% of profiled single cells. Using DARLIN, we examined fate priming within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we adapted a method to jointly profile DNA methylation, chromatin accessibility, gene expression, and lineage information in single cells. DARLIN will enable widespread high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

developmental biology↗

bioRxiv2022

Inter-individual variation in human cortical cell type abundance and expression

Single cell transcriptomic studies have identified a conserved set of neocortical cell types from small post-mortem cohorts. We extend these efforts by assessing cell type variation across 75 adult individuals undergoing epilepsy and tumor surgeries. Nearly all nuclei map to one of 125 robust cell types identified in middle temporal gyrus, but with varied abundances and gene expression signatures across donors, particularly in deep layer glutamatergic neurons. A minority of variance is explainable by known factors including donor identity and small contributions from age, sex, ancestry, and disease state. Genomic variation was significantly associated with variable expression of 150-250 genes for most cell types. Thus, human individuals display a highly consistent cellular makeup, but with significant variation reflecting donor characteristics, disease condition, and genetic regulation. One-Sentence SummaryInter-individual variation in human cortex is greatest for deep layer excitatory neurons and largely unexplainable by known factors.

neuroscience↗

bioRxiv2022

Mucosal administration of anti-bacterial antibody provides long-term cross-protection against Pseudomonas aeruginosa respiratory infection

Bacterial respiratory infections, associated with acute, sometimes recurrent infections and with chronic respiratory diseases, are a major threat for human health. Mucosal administration of therapeutic antibodies (Ab), using the airways as a delivery route, has a tremendous opportunity to benefit to patients with respiratory infections, with remarkable preclinical achievements in both viral and bacterial respiratory infection models and ongoing clinical developments. The primary mode of action of anti-infective Ab delivered through the airways is pathogen neutralization and to a lesser extent, Fc-mediated direct recruitment of immune effectors to facilitate their elimination. Using a mouse model of acute pneumonia induced by P. aeruginosa, a bacterium frequently associated with multidrug resistance and a high rate of recurrence, we characterized an immunomodulatory mode of action of anti-bacterial Ab. Beyond the rapid and efficient containment of the primary infection, the anti-infective Ab delivered through the airways harnessed adaptive immunity to provide a long-term response, preventing from a secondary pathogen infection. This effect is specific and dependent on the Ab dose, intensity of infection and antigen expression by the pathogen upon primary infection. As shown by adoptive transfer experiments, it is mediated by a sustained and protective humoral immune response. Interestingly, the long-lasting response protected partially against secondary infections due to heterologous P. aeruginosa strains. Overall, our findings suggest that mucosal delivery of Ab through the airways offers a dual advantage: a rapid onset of action to neutralize respiratory bacteria and a long-term protection against secondary infections, thereby opening novel perspectives for the development of anti-infective antibody delivered to the lung mucosa, to treat respiratory infections.

immunology↗

bioRxiv2022

Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis.

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PCM1. To study the requirement for centriolar satellites, we generated mice lacking PCM1. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia, as well as variable expressivity of other ciliopathy features including cystic kidneys. Pcm1-/- multiciliated ependymal cells and PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromized early ciliogenesis. We show these molecular cascades are maintained in vivo, and we suggest that the cellular threshold to trigger ciliogenesis varies between cell types. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, inducing the departure of CP110 and CEP97 to initiate ciliogenesis.

cell biology↗

bioRxiv2021

Epithelial-mesenchymal plasticity induced by discontinuous exposure to TGFβ1 promotes tumour growth

Transitions between epithelial and mesenchymal cellular states (EMT/MET) contribute to cancer progression. We hypothesize that EMT followed by MET promotes cell population heterogeneity favouring tumour growth. We developed an EMT model by on/off exposure of epithelial EpH4 cells (E-cells) to TGF{beta}1 that mimics phenotypic EMT (M-cells) and MET. We aimed at understanding whether phenotypic MET is accompanied by molecular and functional reversion back to epithelia, by using RNA sequencing, Immunofluorescence (IF), proliferation, wound healing, focus formation and mamosphere formation assays, as well as cell-xenografts in nude mice. Phenotypic reverted-epithelial cells (RE-cells), obtained after MET induction, presented pure epithelial morphology and proliferation rate resembling E-cells. However, RE transcriptomic profile and IF staining of epithelial and mesenchymal markers revealed a unique and heterogeneous mixture of cell-subpopulations, with high self-renewal ability fed by oxidative phosporylation. RE-cells heterogeneity is stably maintained for long periods after TGF{beta}1 removal, both in vitro and in large derived tumours in nude mice. Overall, we show that phenotypic reverted-epithelial cells (RE-cells) do not return to the molecular and functional epithelial state, present mesenchymal features related with aggressiveness and cellular heterogeneity that favour tumour growth in vivo. This work strengthens epithelial cells reprogramming and cellular heterogeneity fostered by inflammatory cues as a tumour-growth promoting factor in vivo.

cell biology↗