Biology subjects

Pinto, M.

Publications and source records attributed to Pinto, M..

13 recordsLinked to original sources

bioRxiv2026

CRISPR-Cas9 Editing of Myeloid Lectins Reveals Target-Dependent Transcriptional Remodeling in Tumor-Associated Macrophages

Tumor-associated macrophages (TAMs) integrate multiple microenvironmental signals, including lectin-mediated recognition of tumor-associated glycans. Although several myeloid lectins have been implicated in tumor-macrophage interactions, their contributions to immunosuppressive TAM phenotypes remain poorly defined. We implemented a CRISPR-Cas9 ribonucleoprotein (RNP) workflow in primary human monocytes to enable comparative loss-of-function interrogation of five potentially immunosuppressive lectins we previously reported. CRISPR-Cas9 RNP delivery conditions were benchmarked in freshly isolated monocytes using CD14 as a technical target, followed by sgRNA editing efficiency screening for CLEC4E, CLEC6A, CD209, SELL, and SIGLEC9 in HeLa S3 cells. Selected guides were applied to primary monocytes before co-culture in a 3D model with BT474 breast cancer cells. Editing was quantified by Sanger-sequencing deconvolution, while co-culture-derived CD45+ macrophages were isolated by fluorescence-activated cell sorting and characterized by bulk RNA sequencing. All five lectins were successfully perturbed, although gene editing efficiency remained target- and donor-dependent. Transcriptomic analysis revealed target-specific reprogramming and recurrent immunometabolic responses. mTORC1 signaling was consistently enriched, together with the remodeling of cholesterol homeostasis, fatty acid metabolism, and oxidative phosphorylation. CLEC6A deletion achieved attenuation of hypoxia-, angiogenesis-, and TNF/NF-kB-associated programs. CD209 and SIGLEC9 deletion similarly reduced hypoxia- and angiogenesis-associated signatures while promoting lipid and oxidative metabolism. CLEC4E perturbation induced broad remodeling of lipid and oxidative metabolism and stress-response, despite comparatively limited editing efficiency. Comparative lectin perturbation thus identified CLEC6A as the strongest candidate for further mechanistic investigation, while CLEC4E showed broad transcriptional remodeling despite lower editing efficiency, supporting its prioritization for improved genetic interrogation. Responses to CD209 and SIGLEC9 targeting further support tumor glycan-myeloid signaling as a regulator of TAM state. Overall, lectin perturbation redirected TAM transcriptional states through distinct immunometabolic programs rather than a uniform pro-inflammatory polarization, highlighting lectin-dependent immunometabolic regulation as a promising axis for further investigation.

cancer biology↗

bioRxiv2026

Myeloid Lectin Profiling Identifies SYK as a Targetable Signaling Node for Remodeling Immunosuppressive Tumor-Associated Macrophages in Breast Cancer

Immunosuppressive tumor microenvironments (TMEs) are common in breast cancer (BC), where tumor-promoting myeloid-driven inflammation contributes to immune dysfunction and poor immunotherapy response. Lectins expressed by tumor-associated macrophages (TAMs) act as glycan-sensing immunoregulatory receptors driving immunosuppression, but whether specific myeloid lectins define immunosuppressive TAM states, or their signaling is pharmacologically targetable remain unclear. We used an orthogonal prioritization approach combining immunosuppressive stratification of the SCAN-B cohort (3207 patients), differential expression, single-cell myeloid localization, and immune-related upstream-regulator inference. Immunosuppressive tumors were enriched for Basal-like and HER2-enriched subtypes, with worse survival. This identified a 12-lectin panel, which was probed in a human 3D immunosuppressive TME model combining BC spheroids, fibroblasts, and blood-derived macrophages under agitation. Flow cytometry confirmed high expression of eight lectins, with four upregulated in TAMs. Survival analyses associated CLEC4E/Mincle, CLEC6A/Dectin-2, and CD209/DC-SIGN with poorer outcomes. As CLEC4E and CLEC6A converge on FcR{gamma}/SYK signaling, this shared node was selected for pathway-level pharmacological perturbation. R406 reduced SYK phosphorylation and induced transcriptional remodeling in TAMs, with attenuation of immunosuppressive macrophage features, reduced CD204/CD206, increased HLA-DR, and remodeling of the soluble-factor profile. These findings define a myeloid lectin framework associated with BC immunosuppression and identify SYK as a candidate targetable node for remodeling TAMs.

cancer biology↗

bioRxiv2025

From muscle fibres to gears: How fibre rotation and shape change impact muscle function

Skeletal muscle architectural design strongly affects force-generating capacity and excursion range, and its functional importance to animal and human movement is well founded. Traditionally, this structure-function relation has been inferred from architecture measurements with muscles at rest (from anatomical dissections or medical imaging techniques such as ultrasonography or magnetic resonance imaging) and muscle function tested under quasi-steady force conditions (e.g., isometric). A contemporary view recognises that, during active, dynamic contractions, muscles undergo load- and velocity-dependent changes in architecture and three-dimensional shape while remaining near-constant volume. Consequently, whole-muscle length change and velocity can become decoupled from fascicle length change and velocity, such that the whole muscle cannot be treated as a simple linear velocity transmission system but instead as a geared system where the input fascicle length change and velocity differ from the output whole muscle length change and velocity. This phenomenon is described by the concept of muscle gearing and can be quantified as the ratio of the velocities or displacement of the muscle to the fascicle. Gear ratios are not fixed; instead, gear shifts with force and velocity, allowing the whole-muscle output to better match the mechanical demands of the task. Variable gearing is thought to emerge from load-dependent changes in fascicle angle and three-dimensional muscle deformations under volume-preserving and tissue constraints. Variable gearing has important functional implications, as it influences fascicle operating length and shortening velocity for a given task and thereby shifts where fascicles operate along their intrinsic force-length and force-velocity relationships. Through these effects, along with changes in the projection of fascicle force onto the muscle line of action as pennation varies, variable gearing can broaden whole-muscle force and power output across movement conditions. Accordingly, muscle gearing should be considered an integral component of a comprehensive framework for understanding dynamic muscle function in both normal and pathological states. This review aims to (i) provide a brief historical overview of how the muscle structure-function relation has been understood from resting muscle structure measurements and modelling and steady-force contractions to observations during dynamic, time-varying active contractions, and (ii) synthesise evidence on muscle gearing, including its measurement, functional consequences, and current understanding of the factors that influence it by reviewing findings from animal and human studies and raising hypotheses to explain the variation in gearing identified across muscle, species, and contraction conditions. Given the challenges in experimentally isolating the contribution of individual factors, we also employ a previously validated three-dimensional finite-element muscle model to interrogate the mechanical phenomena underpinning variable gearing and for the first time report effects from muscle activation. Finally, we briefly discuss the potential role of gearing in the observed changes in muscle function with ageing, injury, and exercise training. Overall, muscle gearing is a distinctive feature of skeletal muscles with important functional implications, and our modelling demonstrates that it is an emergent property of the physics of muscle contraction.

physiology↗

bioRxiv2025

EEG reveals how space acts as a late heuristic of time

To compensate for its sensory intangibility, humans often rely on spatial metaphors, gestures, and visual tools to represent the passage of time. These spatial tools, i.e. heuristics, range from everyday practices--such as directional hand gestures to indicate past or future events--to more abstract scientific conceptualizations, such as the "curving of space-time" in the theory of relativity. Despite this widespread spatialization of time, it remains unclear to what extent space is an inherent component of the brains representation of time and its role in monitoring temporal durations. Here, we combine EEG-behavioral methods and neural network models of optimal decision-making to show that space is a late compensatory mechanism of time representation recruited when faster and non-spatial timekeeping mechanisms are suboptimally engaged. EEG analyses reveal a cascade-like process: spatial engagement in timekeeping follows the insufficient non-spatial encoding of time intervals, leading to delayed decisions on their length and slower response selection. Computational modelling further indicates that trial-by-trial fluctuations in the spatialization of time are explained by stochastic variations in the activity of the dopaminergic/noradrenergic (DA/NE) system and its interaction with the anterior cingulate cortex. These findings provide the first clear evidence of when, why, and how the brain recruits spatial mechanisms in the service of temporal processing and demonstrate that non-spatial and spatial timekeeping systems can be dissociated at both behavioural and electrophysiological levels.

neuroscience↗

bioRxiv2025

Analyzing the morphological variation of the Galapagos mistletoePhoradendron berteroanum (Santalaceae) using herbarium specimens

The hemiparasitic mistletoe Phoradendron berteroanum in the Galapagos shows remarkable morphological variation suggesting either the presence of clinal or abrupt geographical variation. To find patterns of the morphological variation of P. berteroanum. Eight vegetative and flower traits were measured from 68 herbarium specimens. Univariate and multivariate analyses comparing the traits by altitude, island, host and vegetation cover were performed. It was found that leaf size and internode length were larger at mid-elevations, while the number of floral segments decreased with altitude. Flower number varied by host, notably higher on Scalesia baurii from Pinzon Island. No evidence of unique island populations was found, so there is no morphological support for considering more than one species of mistletoe in the Galapagos. These results, however, reveal how P. berteroanum respond to local environmental conditions across the Galapagos.

plant biology↗

bioRxiv2025

Museums and Zoos: Rapid genetic identification of rare species and practical applications for conservation and systematics in a biodiverse country

AbstractObtaining genetic information from rare species is challenging for scientists, but it is crucial for understanding animal evolutionary history and informing conservation management initiatives. We present the first example of a collaborative local network that includes zoos and natural history collections to investigate the evolution, systematics, and conservation concerns of olingos (genus Bassaricyon, Procyonidae, Carnivora, Mammalia). We sequenced the entire (1,146 base pairs) cytochrome b gene to phylogenetically identify individuals that have been victims of wildlife trafficking. Unexpectedly, we detected an individual specimen belonging to Bassaricyon medius orinomus (western lowland olingo), which may represent a new geographical record for this taxon in Ecuador. Through our practical experiences, we describe how local collaboration is possible and crucial for promoting wildlife genetic research in the Global South and contributing to protecting the last populations of rare mammals. We also discuss the significance of wild animals under human care as a valuable genetic resource for scientific research, conservation strategies, and informed wildlife management decisions.

genetics↗

bioRxiv2024

The widely used cymoxanil fungicide impairs respiration in Saccharomyces cerevisiae via cytochrome c oxidase inhibition

Cymoxanil (CYM) is a synthetic acetamide fungicide that has been widely used to combat downy mildew diseases in grapevine cultures and late blight diseases in tomato and potato caused by the oomycetes Plasmopara viticola and Phytophthora infestans, respectively. Despite its extensive application, the biochemical mode of action of CYM remains elusive. Previous reports indicate that CYM affects growth, DNA and RNA synthesis in Phytophthora and inhibits cell growth, biomass production and respiration rate in the well-characterized fungal model Saccharomyces cerevisiae. We therefore used this model to further characterize the effect of CYM on mitochondria. We found that CYM inhibits oxygen consumption in whole cells after 3 h of exposure, which persists over time. Using isolated mitochondria, we demonstrated that CYM specifically inhibits cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking algorithms, we propose that CYM acts by blocking the interaction of cytochrome c (cyt c) with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that can be instrumental to drive informed management of the use of this fungicide.

biochemistry↗

bioRxiv2023

Extracellular communication between brain cells through functional transfer of Cre mRNA

1In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo overtime. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEVs transfer at physiological levels which will shed light on the role of bdEVs in neural communication within the brain and beyond.

cell biology↗

bioRxiv2021

The first Miocene fossils from coastal woodlands in the southern East African Rift

The Miocene is a key time in the evolution of African mammals and their ecosystems witnessing the origin of the African apes and the isolation of eastern coastal forests through an expanding biogeographic arid corridor. Until recently, however, Miocene sites from the southeastern regions of the continent were unknown. Here we report discovery of the first Miocene fossil teeth from the shoulders of the Urema Rift in Gorongosa National Park, Mozambique, at the southern East African Rift System. We provide the first 1) radiometric age determinations of the fossiliferous Mazamba Formation, 2) reconstructions of past vegetation in the region based on pedogenic carbonates and fossil wood, and 3) description of fossil teeth from the southern rift. Gorongosa is unique in the East African Rift System in combining marine invertebrates, marine vertebrates, terrestrial mammals, and fossil woods in coastal paleoenvironments. The Gorongosa fossil sites offer the first evidence of persistent woodlands and forests on the coastal margins of southeastern Africa during the Miocene, and an exceptional assemblage of fossil vertebrates including new species. Further work will allow the testing of hypotheses positing the formation of a northeast-southwest arid corridor isolating species on the eastern coastal forests from those elsewhere in Africa. BriefThe Miocene is a key time in the evolution of African mammals and their ecosystems encompassing hominine origins and the establishment of an arid corridor that isolated eastern Africas coastal forests. Until now, however, Miocene sites from southeastern Africa have been unknown. We report the discovery of the first Miocene fossil sites from Gorongosa National Park, Mozambique, and show that these sites formed in coastal settings. We provide radiometric ages for the fossiliferous sediments, reconstructions of past vegetation based on stable isotopes and fossil wood, and a description of the first fossil teeth from the region. Gorongosa is the only paleontological site in the East African Rift that combines fossil woods, marine invertebrates, marine vertebrates, and terrestrial mammals. Gorongosa offers the first evidence of persistent woodlands and forests on the coastal margins of southeastern Africa during the Miocene.

paleontology↗

bioRxiv2021

Microbial consortiums of putative degraders of low-density polyethylene-associated compounds in the ocean

Polyethylene (PE) is one of the most abundant plastics in the ocean. The development of a biofilm on PE in the ocean has been reported, yet whether some of the biofilm-forming organisms can biodegrade this plastic in the environment remains unknown. Via metagenomics analysis, we taxonomically and functionally analysed three biofilm communities using low-density polyethylene (LDPE) as their sole carbon source for two years. Several of the taxa that increased in relative abundance over time were closely related to known degraders of alkane and other hydrocarbons. Alkane degradation has been proposed to be involved in PE degradation, and most of the organisms increasing in relative abundance over time harboured genes encoding proteins essential in alkane degradation, such as the genes alkB and CYP153, encoding an alkane monooxygenase and a cytochrome P450 alkane hydroxylase. Weight loss of PE sheets when incubated with these communities and chemical and electron microscopic analyses provided evidence for alteration of the PE surface over time. Taken together, these results provide evidence for the utilization of LDPE-associated compounds by the prokaryotic communities. This study identifies a group of genes potentially involved in the degradation of the LDPE polymeric structure and/or associated plastic additives in the ocean and describes a phylogenetically diverse community of plastic biofilm-dwelling microbes with the potential of utilizing LDPE-associated compounds as carbon and energy source. ImportanceLow-density polyethylene (LDPE) is one of the most used plastics worldwide and a large portion of it ends up in the ocean. Very little is known about its fate in the ocean and whether it can be biodegraded by microorganisms. By combining 2-year incubations with metagenomics, respiration measurements and LDPE surface analysis, we identified bacteria and associated genes and metabolic pathways potentially involved in LDPE biodegradation. After two years of incubation, two of the microbial communities exhibited a very similar taxonomic composition mediating changes to the LDPE pieces they were incubated with. We provide evidence that there are plastic-biofilm dwelling bacteria in the ocean, that might have the potential to degrade LDPE-associated compounds, and that alkane degradation pathways might be involved.

microbiology↗

bioRxiv2019

Virulence and antibiotic resistance plasticity of Arcobacter butzleri: insights on the genomic diversity of an emerging human pathogen

Arcobacter butzleri is a food and waterborne bacteria and an emerging human pathogen, frequently displaying a multidrug resistant character. Still, no comprehensive genome-scale comparative analysis has been performed so far, which has limited our knowledge on A. butzleri diversification and pathogenicity. Here, we performed a deep genome analysis of A. butzleri focused on decoding its core- and pan-genome diversity and specific genetic traits underlying its pathogenic potential and diverse ecology. In total, 49 A. butzleri strains (collected from human, animal, food and environmental sources) were screened.\n\nA. butzleri (genome size 2.07-2.58 Mbp) revealed a large open pan-genome with 7474 genes (about 50% being singletons) and a small core-genome with 1165 genes. The core-genome is highly diverse ([≥]55% of the core genes presenting at least 40/49 alleles), being enriched with genes associated with housekeeping functions. In contrast, the accessory genome presented a high proportion of loci with an unknown function, also being particularly overrepresented by genes associated with defence mechanisms. A. butzleri revealed a plastic virulome (including newly identified determinants), marked by the differential presence of multiple adaptation-related virulence factors, such as the urease cluster ureD(AB)CEFG (phenotypically confirmed), the hypervariable hemagglutinin-encoding hecA, a putative type I secretion system (T1SS) harboring another agglutinin potentially related to adherence and a novel VirB/D4 T4SS likely linked to interbacterial competition and cytotoxicity. In addition, A. butzleri harbors a large repertoire of efflux pumps (EPs) (ten \"core\" and nine differentially present) and other antibiotic resistant determinants. We provide the first description of a genetic determinant of macrolides resistance in A. butzleri, by associating the inactivation of a TetR repressor (likely regulating an EP) with erythromycin resistance. Fluoroquinolones resistance correlated with the Thr-85-Ile substitution in GyrA and ampicillin resistance was linked to an OXA-15-like {beta}-lactamase. Remarkably, by decoding the polymorphism pattern of the porin- and adhesin-encoding main antigen PorA, this study strongly supports that this pathogen is able to exchange porA as a whole and/or hypervariable epitope-encoding regions separately, leading to a multitude of chimeric PorA presentations that can impact pathogen-host interaction during infection. Ultimately, our unprecedented screening of short sequence repeats detected potential phase-variable genes related to adaptation and host/environment interaction, such as lipopolysaccharide modification and motility/chemotaxis, suggesting that phase variation likely modulate A. butzleri key adaptive functions.\n\nIn summary, this study constitutes a turning point on A. butzleri comparative genomics revealing that this human gastrointestinal pathogen is equipped with vast virulence and antibiotic resistance arsenals, which, coupled with its remarkable core- and pan-genome diversity, opens a multitude of phenotypic fingerprints for environmental/host adaptation and pathogenicity.\n\nIMPACT STATEMENTDiarrhoeal diseases are the most common cause of human illness caused by foodborne hazards, but the surveillance of diarrhoeal diseases is biased towards the most commonly searched infectious agents (namely Campylobacter jejuni and C. coli). In fact, other less studied pathogens are frequently found as the etiological agent when refined non-selective culture conditions are applied. A hallmark example is the diarrhoeal-causing Arcobacter butzleri which, despite being also associated with extra-intestinal diseases, such as bacteremia in humans and mastitis in animals, and displaying high rates of antibiotic resistance, has not yet been profoundly investigated regarding its epidemiology, diversity and pathogenicity. To overcome the general lack of knowledge on A. butzleri comparative genomics, we provide the first comprehensive genome-scale analysis of A. butzleri focused on exploring the intraspecies virulome content and diversity, resistance determinants, as well as how this pathogen shapes its genome towards ecological adaptation and host invasion. The unveiled scenario of A. butzleri rampant diversity and plasticity reinforces the pathogenic potential of this food and waterborne hazard, while opening multiple research lines that will certainly contribute to the future development of more robust species-oriented diagnostics and molecular surveillance of A. butzleri.\n\nDATA SUMMARYA. butzleri raw sequence reads generated in the present study were deposited in the European Nucleotide Archive (ENA) (BioProject PRJEB34441). The assembled contigs (.fasta and .gbk files), the nucleotide sequences of the predicted transcripts (CDS, rRNA, tRNA, tmRNA, misc_RNA) (.ffn files) and the respective amino acid sequences of the translated CDS sequences (.faa files) are available at http://doi.org/10.5281/zenodo.3434222. Detailed ENA accession numbers, as well as the draft genome statistics are described in Table S1.

genomics↗

bioRxiv2019

How to make “number lines” stable in the mind’s eye

Humans are prone to mentally organise the ascending series of integers according to reading habits, so that in western cultures small numbers are positioned to the left of larger ones on a mental number line (MNL, Dehaene et al., 1993). Despite 140 years since seminal observations made by Sir Francis Galton in human observers that experienced vivid MNLs (Galton, 1880a,b), the neural and functional mechanisms that give rise to MNLs remain elusive. Here, in a series of three experiments we show that spatially organised MNLs are not \"all or none\" phenomena and that MNLs get progressively less noisy and more stable in the minds eye, the more an observer fully combines left/right spatial codes with small/large magnitude codes to operate on numbers. These results show that conceptualising the ascending series of integers in spatial terms depends on contextual clues rather than being inherent to the semantic representation of numbers.

neuroscience↗

bioRxiv2018

Reconstructing the origins of the space-number association: spatial and number-magnitude codes must be used jointly to elicit spatially organised mental number lines.

In a series of recent studies we have pointed out that the use of contrasting left/right spatial codes, whether indirectly related to number magnitudes through response selection or directly associated to the same magnitudes to guide their spatial positioning on a mental number line, is crucial in eliciting space-number associations (Aiello, 2012; Fattorini et al., 2015; 2016; Pinto et al., 2018). Nonetheless, this conclusion is based on experiments in which spatial and number-magnitudes codes are used jointly during task performance. Here, in a series of unimanual Go/No-Go tasks with intermixed central numerical and pictorial targets, i.e. arrows pointing to the left or to the right, we explore whether spatial codes used in isolation inherently evoke the left-to-right representation of number magnitudes and, vice-versa, whether number-magnitude codes used in isolation inherently evoke the conceptual activation of left/right spatial codes. In a first series of experiments participants were asked to provide unimanual Go/N-Go responses based on instructions that activated only magnitude codes, e.g. \"push only if the number is lower than 5 and whenever an arrow appears\", or only spatial codes, e.g. \"push only when an arrow points to the left and whenever a number appears\". In a second series of experiments, the same numerical instructions were combined with the request of responding only to arrows in a specific colour, e.g. \"push when the number is lower than 5 and whenever a blue arrow appears\". At variance with a recent experiment by Shaki and Fischer (2018), in our experiments no constant association was present between a specific arrow colour and a specific arrow direction. The results of these experiments highlight no space-number congruency effects: e.g. no faster RTs to arrows pointing to the left rather than to the right when participants attend to numbers lower than 5 and, vice-versa, no faster RTs to numbers lower than 5 rather than higher, when participants attend to arrows pointing to the left. Based on these findings it must be concluded that neither space codes used in isolation can elicit a spatial representation of number magnitudes nor number-magnitude codes used in isolation can trigger the activation of spatial codes. Thus, spatial and numerical codes must be used jointly to evoke spatially organised mental number lines.

neuroscience↗