Biology subjects

Caputo, M.

Publications and source records attributed to Caputo, M..

11 recordsLinked to original sources

bioRxiv2026

Decoding EGFR ligand bias through an endocytic organelle platform

How growth factor receptors decode ligand identity into distinct cellular responses remains a fundamental question in cell signaling. Here, we identify a receptor-proximal mechanism that links ligand-specific EGFR activation to distinct endocytic and biological outputs. We show that EGF, but not TGF, selectively engages a RAC1-PLC{gamma}2-IP3R signaling axis that supports EGFR non-clathrin endocytosis (NCE). PLC{gamma}2, but not PLC{gamma}1, localizes to RTN3-dependent PM-ER contact sites, where it generates localized Ca{superscript 2} signals required for completion of NCE, mitochondrial activation and cell motility. This specificity requires the RAC-binding interface of PLC{gamma}2 and is associated with RAC1-dependent formation of CTxB-positive PM regions, indicating that spatial organization contributes to signaling specificity. TGF fails to efficiently assemble the EGFR-associated organelle platform and instead favors clathrin-dependent EGFR uptake, prolonged proliferative signaling, greater organoid yield, and reduced migration compared with EGF. Together, our findings identify the RAC1-PLC{gamma}2 axis as the key determinant that decodes EGFR ligand bias by coupling receptor trafficking to the metabolic program that supports cell migration.

cell biology↗

bioRxiv2026

Scale-dependent feedback between sociality and space use in a long-lived marine predator

Spatial and social behaviours in animals are intertwined, yet the causal direction of this feedback--and how it varies across scales--remains largely unresolved. Using long-term data from false killer whales (Pseudorca crassidens) in the main Hawaiian Islands, we developed and applied a scale-explicit analytical framework to test how social reliance and resource ephemerality govern top-down versus bottom-up processes linking movement and sociality. Movements, association networks, genetic relatedness, and isotopic niches reveal that strong social bonds drive bottom-up emergence of short-term intra-group movements, while ephemeral and likely island-associated prey landscapes impose top-down constraints on inter-group dynamics across scales. These complementary processes generate persistent, fine-scale fidelity within some groups and relatively well-differentiated feeding niches among them. Our findings highlight a general mechanism by which life-history strategies and environmental stochasticity jointly determine the scale and direction of feedback between space use and sociality--shaping population structure and connectivity in mobile social predators.

ecology↗

bioRxiv2024

Altered inflammatory state and mitochondrial function identified by transcriptomics in paediatric congenital heart patients prior to surgical repair

ObjectiveCongenital heart disease (CHD) remains the most common birth defect, with surgical intervention required in complex cases. Right ventricle (RV) function is known to be a major predictor in sustained cardiac health in these patients, thus by elucidating divergent profiles between CHD and control through tissue analysis this study aims to identify new avenues of investigation into the mechanisms surrounding reduced RV function. Approach & ResultsTranscriptomic profiling, in silico cellular deconvolution and functional network analysis was conducted on RV biopsies obtained from CHD and control paedatric patients. Analysis identified an increase in mitochondrial dysfunction genes RPPH1 and RMPR (padj = 4.67E-132, 2.23E-107, respectively), Cytotoxic T cell markers CD8a, LAGE3 and CD49a (p = 0.0006, p < 0.0001, p = 0.0118, respectively) and proinflammatory marker Caspase1 (p=0.0055) in CHD compared to control. Gene set enrichment identified mitochondrial dysfunctional pathways, predominately changes to the oxidative phosphorylation processes. Negative regulation of mitochondrial functions and metabolism was identified in functional network analysis, with dysregulation of mitochondrial complex formation. Histological analysis confirmed an increase in cellular bodies with the CHD RV tissue, and positive staining for both CD45 and CD8 in CHD RV tissue, which was absent in control. Deconvolution of bulk RNAseq data suggests a reduction in CD4+ T cells (p = 0.0067) and an increase in CD8+ T cells (p = 0.0223). Network analysis identified positive regulation of the immune system and cytokine signalling clusters within the inflammation functional network as were lymphocyte activation and leukocyte differentiation. ConclusionsUtilizing RV tissue from paediatric patients undergoing CHD cardiac surgery this study identifies dysfunctional mitochondrial pathways and an increase in inflammatory T cell presence prior to reparative surgery.

immunology↗

bioRxiv2023

Phospholipid Scramblase-1 is required for efficient neurotransmission and synaptic vesicle retrieval at cerebellar synapses

Structural phospholipids are asymmetrically distributed at the plasma membrane, with phosphatidylethanolamine and phosphatidylserine (PS) virtually absent from the outer leaflet. This asymmetric lipid distribution is transiently altered during specific biological processes including calcium-regulated exocytosis. However, the impact of this transient remodeling of membrane asymmetry on presynaptic function remains unknown. PhosphoLipid SCRamblase 1 (PLSCR1), a protein that randomizes phospholipid distribution between the two leaflets of the plasma membrane in response to calcium activation is an ideal candidate to alter this asymmetry. We therefore set out to determine the role of PLSCR1 in both neurotransmitter release and synaptic vesicle recycling by combining electron microscopy, optical live cell imaging of pHluorin probes and electrophysiology in cerebellar granule cells (GrC) from Plscr1 knock-out mice (Plscr1-/-). We report that PLSCR1 is expressed in GrCs and that PLSCR1-dependent PS egress occurred at synapses in response to neuron stimulation. Furthermore, synaptic transmission is impaired at GrC Plscr1-/- synapses and both PS egress and synaptic vesicle endocytosis are inhibited in Plscr1-/- cultured neurons, demonstrating that PLSCR1 controls phospholipid asymmetry remodeling and synaptic vesicle retrieval following neurotransmitter release. Altogether, our data reveal a key role for PLSCR1 in synaptic vesicle recycling and provide the first evidence that phospholipid scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.

cell biology↗

bioRxiv2023

Regulation of BACH1 by hemin improves cardiac function in a mouse model of myocardial infarction

AimsThe BTB and CNC homology 1 (BACH1) transcription factor is a repressor of heme oxygenase-1 (HMOX1), a pivotal enzyme involved in antioxidant response and iron recycling. Here we investigated whether pharmacological modulation of the BACH1 by hemin impacts on antioxidant responses and reparative angiogenesis in a mouse model of myocardial infarction (MI). Methods and resultsIn vitro studies on vascular cells showed hemin treatment downregulates BACH1 gene and protein expression and upregulates HMOX1. This axis was confirmed to be modulated in the murine infarcted heart, with BACH1 being upregulated, and HMOX1 downregulated compared to sham. Treatment with hemin every 3 days for 28 days post-MI significantly decreased BACH1 and increased HMOX1 protein expression, though no decrease in oxidative stress markers was detected. Hemin treated mice showed increases in both capillary and arteriole density, and reduced iron accumulation compared with controls. Furthermore, echocardiology measurements showed hemin treatment induced significant improvements in left ventricular wall thickness, and cardiac function as indicated by increased ejection fraction, fractional shortening, and stroke volume measurements. ConclusionHemin has therapeutic potential to improve revascularisation and cardiac function in the heart post-MI.

molecular biology↗

bioRxiv2023

Single-cell T cell receptor sequencing of paired tissue and blood samples reveals clonal expansion of CD8+ effector T cells in patients with calcific aortic valve disease

Calcific aortic valve disease (CAVD) is a complex cardiovascular pathology, culminating in aortic stenosis, heart failure and premature mortality, with no comprehensive treatment strategy, except valve replacement. While T cells have been identified within the valve, their contribution to pathogenesis remains unclear. To elucidate the heterogenous phenotype of the immune populations present within patients with CAVD, deep phenotypic screens of paired valve and peripheral blood cells were conducted via flow cytometry (n=20) and immunohistochemistry (n=10). Following identification of a significant population of memory T cells; specifically, CD8+ T cells within the valve, single cell RNA sequencing and paired single T cell receptor sequencing was conducted on a further 4 patients on CD45+ CD3+, CD4+ or CD8+ T cells. Through unsupervised clustering, 7 T cell populations were identified within the blood and 10 identified within the valve. Tissue resident memory (TRM) T cells were detected for the first time within the valve, exhibiting a highly cytotoxic, activated, and terminally differentiated phenotype. This pan-pro-inflammatory signal was differentially identified in T cells originating from the valve, and not observed in the blood, indicative of an adaptive, local not-systemic inflammatory signature in CAVD patients. T cell receptor analysis identified hyperexpanded clones within the CD8+ T cell central memory (TCM) population, with TRM cells comprising the majority of large and medium clonal expansion within the entire T cell population. Clonal interaction network analysis demonstrated the greatest proportion of clones originating from CD8+ T cell effector memory (TEM) and CD4+ naive / TCM populations and ending in the CD8+ TRM and CD8+ TCM clusters, suggesting a clonal expansion and predicted trajectory of T cells towards a tissue resident, cytotoxic environment within the valve. CDR3 epitope predictive analysis identified 7 potential epitope targets, of which GALNT4 and CR1L have previously been implicated in a cardiovascular context as mediators of inflammation. Taken together, the data identified T cell sub-populations within the context of CAVD and further predicted possible epitopes responsible for the clonal expansion of the valvular T cells, which may be important for propagating inflammation in CAVD.

immunology↗

bioRxiv2021

Label-free screening of brain tissue myelin content using phase imaging with computational specificity (PICS)

Inadequate myelination in the central nervous system is associated with neurodevelopmental complications. Thus, quantitative, high spatial resolution measurements of myelin levels are highly desirable. We used spatial light interference microcopy (SLIM), a highly sensitive quantitative phase imaging (QPI) technique, to correlate the dry mass content of myelin in piglet brain tissue with dietary changes and gestational size. We combined SLIM micrographs with an AI classifying model that allows us to discern subtle disparities in myelin distributions with high accuracy. This concept of combining QPI label-free data with AI for the purpose of extracting molecular specificity has recently been introduced by our laboratory as phase imaging with computational specificity (PICS). Training on nine thousand SLIM images of piglet brain tissue with the 71-layer transfer learning model Xception, we created a two-parameter classification to differentiate gestational size and diet type with an accuracy of 82% and 80%, respectively. To our knowledge, this type of evaluation is impossible to perform by an expert pathologist or other techniques.

bioinformatics↗

bioRxiv2020

The SARS-CoV-2 spike protein disrupts the cooperative function of human cardiac pericytes - endothelial cells through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease

Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known whether heart vascular pericytes (PCs) are infected by SARS-CoV-2, and if the S protein alone provokes PC dysfunction. Here, we aimed to investigate the effects of the S protein on primary human cardiac PC signalling and function. Results show, for the first time, that cardiac PCs are not permissive to SARS-CoV-2 infection in vitro, whilst a recombinant S protein alone elicits functional alterations in PCs. This was documented as: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors responsible for EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation and rescued PC function in the presence of the S protein. In conclusion, our findings suggest that circulating S protein prompts vascular PC dysfunction, potentially contributing to establishing microvascular injury in organs distant from the site of infection. This mechanism may have clinical and therapeutic implications. Clinical perspectiveO_LISevere COVID-19 manifests as a microvascular syndrome, but whether SARS-CoV-2 infects and damages heart vascular pericytes (PCs) remains unknown. C_LIO_LIWe provide evidence that cardiac PCs are not infected by SARS-CoV-2. Importantly, we show that the recombinant S protein alone elicits cellular signalling through the CD147 receptor in cardiac PCs, thereby inducing cell dysfunction and microvascular disruption in vitro. C_LIO_LIThis study suggests that soluble S protein can potentially propagate damage to organs distant from sites of infection, promoting microvascular injury. Blocking the CD147 receptor in patients may help protect the vasculature not only from infection, but also from the collateral damage caused by the S protein. C_LI

cell biology↗

bioRxiv2020

Contractile reprogramming of cardiac pericytes by MEK inhibition promotes arteriologenesis of the ischemic heart

BackgroundThe development of collateral arteries after a myocardial infarction (MI) was intensively studied, while the mechanism by which pericytes (PCs) contribute to arteriologenesis remains unexplored. This study aimed to 1) investigate if cardiac PCs gain functional features of contractile vascular smooth muscle cells (VSMCs) in vitro, and 2) determine if this potential can be evoked pharmacologically to encourage heart arteriologenesis in vivo. MethodsPCs were immunosorted as CD31neg/CD34pos cells from human and mouse hearts. Contractile reprogramming was induced by either depletion of growth factors or addition of PD0325901, a clinically available MEK inhibitor. Next generation RNA-Sequencing was performed in naive and differentiated human PCs to assess the whole-transcriptome profile. Three in vivo studies were conducted in C57BL6/J mice to determine: 1) the ability of human PCs to promote arteriole formation when implanted subcutaneously within PD0325901-containing Matrigel plugs, 2) the effect of orally administered PD0325901 on the arteriole density of normoperfused hearts, and 3) the possibility of promoting capillary formation and muscularization of the infarcted heart through the same pharmacological approach. ResultsRemoval of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) from the culture medium induced the differentiation of PCs into contractile VSMC-like cells. Because both growth factors induce the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling, we attempted to induce PC differentiation in vitro and in vivo using PD0325901. RNA-sequencing revealed that differentiated PCs were enriched in transcripts associated with smooth muscle contraction and biological function. PD0325901-treated PCs rapidly acquired antigenic and functional features of contractile VSMCs in vitro. Moreover, human PCs formed new arterioles when implanted subcutaneously within PD0325901-containing Matrigel plugs in mice. Oral administration of PD0325901 for two weeks increased the density and expression of contractile proteins in small-calibre arterioles of the murine heart, thereby increasing myocardial perfusion. Similarly, PD0325901 induced reparative arteriologenesis and capillarization, reduced the scar, and improved left ventricular performance in a murine model of MI. ConclusionWe propose a novel method to promote the heart vascularization through the pharmacological modulation of resident mural cells. This novel approach could have an immediate impact on the treatment of coronary artery disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIHuman myocardial pericytes have intrinsic vascular plasticity that can be pharmacologically evoked using PD0325901, a clinically available MEK inhibitor. C_LIO_LIIn mice, the pharmacological inhibition of ERK1/2 signalling, by the oral administration of PD0325901 for 2 weeks, encouraged the heart arteriologenesis through pericyte differentiation. C_LIO_LIIn a preclinical mouse model of myocardial infarction, the oral administration of PD0325901 for 2 weeks induced reparative arteriologenesis and capillarization, reduced the scar, and improved left ventricular performance. C_LI What are the clinical implications?O_LIThis novel drug-based therapeutic approach is readily available to all patients. C_LIO_LITherefore, it could have an immediate clinical impact for the treatment of coronary artery disease and other heart conditions associated with deficient coronary vascularization. C_LI

cell biology↗

bioRxiv2020

Maternal phylogenetic relationships and genetic variation among rare similar phenotype donkey breeds

Maternal inheritance is an indispensable aspect in donkey rare breed population biodiversity management and breeding programs. It is a challenge to characterize breeds genetic inheritance using morphology and historical records, we study mtDNA, to overcome those limitations. The mitochondrial DNA (mtDNA) sequencing is a highly informative system to investigate maternal lineages and breed linkage such as molecular evolution and phylogenetic relationships. Martina Franca, Ragusano, Pantesco and Catalonian donkey mtDNA sequencing analyses were used to study intraspecific genetic diversity and population structure, and to reconstruct phylogenetic relations among these geographically isolated breeds.A wide lost in variability among all breeds emerged. In this scenario, the primeval haplotypes, higher haplogroups variability and larger number of maternal lineages are preserved in Martina Franca and Ragusano. Accordingly, a putative pivotal role in the phyletic relationship is likely for such breeds.Given the level of endangerment undergone by these breeds, some actions are necessary to ensure their longtime survival and conservation. Improving the reproduction and management of existing populations, clarifying their historic interactions by studying the genetic status of their populations, extending and improving monitoring maternal lineages represent valid options.View Full Text

zoology↗

bioRxiv2020

Quantifying myelin content in brain tissue using color spatial light interference microscopy (cSLIM)

Deficient myelination of the brain is associated with neurodevelopmental delays, particularly in high-risk infants, such as those born small in relation to their gestational age (SGA). New methods are needed to further study this condition. Here, we employ Color Spatial Light Interference Microscopy (cSLIM), which uses a brightfield objective and RGB camera to generate pathlength-maps with nanoscale sensitivity in conjunction with a regular brightfield image. Using tissue sections stained with Luxol Fast Blue, the myelin structures were segmented from a brightfield image. Using a binary mask, those portions were quantitatively analyzed in the corresponding phase maps. We first used the CLARITY method to remove tissue lipids and validate the sensitivity of cSLIM to lipid content. We then applied cSLIM to brain histology slices. These specimens are from a previous MRI study, which demonstrated that appropriate for gestational age (AGA) piglets have increased internal capsule myelination (ICM) compared to small for gestational age (SGA) piglets and that a hydrolyzed fat diet improved ICM in both. The identity of samples was blinded until after statistical analyses.

neuroscience↗