Biology subjects

Gaitas, A.

Publications and source records attributed to Gaitas, A..

11 recordsLinked to original sources

bioRxiv2026

Micromachined Thermocouple Microprobe for Real-Time Thermometry of Hippocampal Slices during High-Frequency Stimulation

Neuronal activity is energetically demanding and generates heat as a metabolic byproduct. Here we investigated localized temperature changes in mouse hippocampal brain slices during intense synaptic activation. Using fast-response thermocouple microprobes placed in the tissue, we measured real-time temperature fluctuations during high-frequency (100 Hz) electrical stimulation of Schaffer collateral inputs. Stimulation induced a local temperature increase (peak {Delta}T = 0.54 {+/-} 0.07 {degrees}C; n = 4). In contrast, when action potentials were blocked with tetrodotoxin (TTX), the same stimulation produced no measurable temperature rise. These results demonstrate that high-frequency neural firing and synaptic transmission can transiently elevate local brain tissue temperature, presumably due to increased metabolic activity and adenosine triphosphate (ATP) consumption. The hippocampus tolerated these small temperature shifts, but even modest heating could influence neuronal function. Our findings provide direct experimental evidence linking synaptic activity to localized brain heat production, and underscore the importance of considering thermogenesis under stimulation conditions that give rise to forms of synaptic plasticity.

bioengineering↗

bioRxiv2025

Non-invasive Biomechanical Characterization of Embryos Using Microfluidic Cantilevers

Embryonic development is intricately regulated by mechanical properties such as stiffness, which influence developmental viability and implantation success - factors critical in assisted reproductive technologies (ART). Traditional embryo evaluation relies predominantly on morphology, lacking quantitative mechanical parameters that could enhance selection accuracy. Recent studies indicate that the stiffness (elasticity) of the zona pellucida (ZP) - the glycoprotein-rich extracellular matrix surrounding mammalian oocytes and embryos - correlates with embryo quality and developmental potential. However, current biomechanical characterization techniques - including micropipette aspiration, atomic force microscopy (AFM), microtactile sensors, and MEMS-based systems - either pose risks of mechanical damage or involve complex, time-consuming procedures unsuitable for clinical settings. Here, we introduce a novel approach leveraging fluidic force microscopy cantilevers to non-invasively evaluate embryo biomechanics. Our proof-of-concept study demonstrates rapid, precise stiffness profiling of intact mouse embryos (specifically ZP elasticity) in under one minute per embryo. Using gentle microsuction attachment with no chemical adhesives or rigid immobilization, the method preserves embryo integrity while providing reproducible elasticity measurements. This method combines the precision of AFM with minimal invasiveness, offering a promising new quantitative biomechanical indicator to augment clinical embryo assessment and paving the way for broader applications in reproductive biology.

biophysics↗

bioRxiv2023

Circulating exosomes from Alzheimer's disease suppress VE-cadherin expression and induce barrier dysfunction in recipient brain microvascular endothelial cell

BackgroundBlood-brain barrier (BBB) breakdown is a component of the progression and pathology of Alzheimers disease (AD). BBB dysfunction is primarily caused by reduced or disorganized tight junction or adherens junction proteins of brain microvascular endothelial cell (BMEC). While there is growing evidence of tight junction disruption in BMECs in AD, the functional role of adherens junctions during BBB dysfunction in AD remains unknown. Exosomes secreted from senescent cells have unique characteristics and contribute to modulating the phenotype of recipient cells. However, it remains unknown if and how these exosomes cause BMEC dysfunction in AD. ObjectivesThis study aimed to investigate the potential roles of AD circulating exosomes and their RNA cargos in brain endothelial dysfunction in AD. MethodsWe isolated exosomes from sera of five cases of AD compared with age- and sex-matched cognitively normal controls using size-exclusion chromatography technology. We validated the qualities and particle sizes of isolated exosomes with nanoparticle tracking analysis and atomic force microscopy. We measured the biomechanical natures of the endothelial barrier of BMECs, the lateral binding forces between live BMECs, using fluidic force miscopy. We visualized the paracellular expressions of the key adherens junction protein VE-cadherin in BMEC cultures and a 3D BBB model that employs primary human BMECs and pericytes with immunostaining and evaluated them using confocal microscopy. We also examined the VE-cadherin signal in brain tissues from five cases of AD and five age- and sex-matched cognitively normal controls. ResultsWe found that circulating exosomes from AD patients suppress the paracellular expression levels of VE-cadherin and impair the barrier function of recipient BMECs. Immunostaining analysis showed that AD circulating exosomes damage VE-cadherin integrity in a 3D model of microvascular tubule formation. We found that circulating exosomes in AD weaken the BBB depending on the RNA cargos. In parallel, we observed that microvascular VE-cadherin expression is diminished in AD brains compared to normal controls. ConclusionUsing in vitro and ex vivo models, our study illustrates that circulating exosomes from AD patients play a significant role in mediating the damage effect on adherens junction of recipient BMEC of the BBB in an exosomal RNA-dependent manner. This suggests a novel mechanism of peripheral senescent exosomes for AD risk.

neuroscience↗

bioRxiv2023

Identification of common sequence motifs shared exclusively among selectively packed exosomal pathogenic microRNAs during rickettsial infections

We previously reported that microRNA (miR)23a and miR30b are selectively sorted into rickettsia-infected, endothelial cell-derived exosomes (R-ECExos). Yet, the mechanism remains unknown. The number of cases of spotted fever rickettsioses has been increasing in recent years, and infections with these bacteria cause life-threatening diseases by targeting brain and lung tissues. Therefore, the aim of the present study is to continue to dissect the molecular mechanism underlying R-ECExos-induced barrier dysfunction of normal recipient microvascular endothelial cells (MECs), depending on their exosomal RNA cargos. Rickettsiae are transmitted to human hosts by the bite of an infected tick into the skin. In the present study we demonstrate that treatment with R-ECExos, which were derived from spotted fever group R parkeri infected human dermal MECs, induced disruptions of the paracellular adherens junctional protein VE-cadherin and breached the paracellular barrier function in recipient pulmonary MECs (PMECs) in an exosomal RNA-dependent manner. Similarly, we did not detect different levels of miRs in parent dermal MECs following rickettsial infections. However, we demonstrated that the microvasculopathy-relevant miR23a-27a-24 cluster and miR30b are selectively enriched in R-ECExos. Bioinformatic analysis revealed that common sequence motifs are shared exclusively among the exosomal, selectively-enriched miR23a cluster and miR30b at different levels. Taken together, these data warrant further functional identification and characterization of a single, bipartition, or tripartition among ACA, UCA, and CAG motifs that guide recognition of microvasculopathy-relevant miR23a-27a-24 and miR30b, and subsequently results in their selective enrichments in R-ECExos.

molecular biology↗

bioRxiv2022

Exosomally targeting microRNA23a ameliorates microvascular endothelial barrier dysfunction following rickettsial infection

Spotted fever group rickettsioses caused by Rickettsia (R) are devastating human infections, which mainly target microvascular endothelial cells (EC) and can induce lethal EC barrier dysfunction in the brain and lungs. Our previous evidence reveals that exosomes (Exos) derived from rickettsial-infected ECs, namely R-ECExos, can induce disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs). However, the underlying mechanism remains elusive. Given that we have observed that microRNA23a (miR23a), a negative regulator of endothelial ZO-1 mRNA, is selectively sorted into R-ECExos, the aim of the present study was to characterize the potential functional role of exosomal miR23a delivered by R-ECExos in normal recipient BMECs. We demonstrated that EC-derived Exos (ECExos) have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase-abundant environment. miR23a in ECExos impairs normal recipient BMEC barrier function, directly targeting TJ protein ZO-1 mRNAs. In separate studies using a traditional in vitro model and a novel single living-cell biomechanical assay, our group demonstrated that miR23a anti-sense oligonucleotide-enriched ECExos ameliorate R-ECExo-provoked recipient BMEC dysfunction in association with stabilization of ZO-1 in a dose-dependent manner. These results suggest that Exo-based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation.

cell biology↗

bioRxiv2022

AFM Microfluidic Cantilevers as Weight Sensors for Single Cell Mass Measurements

Reliably measuring small mass changes at the single-cell level is challenging. In this manuscript, we report the use of microfluidic cantilevers in liquid with sub-nanogram scale weight sensing capability for the measurement of cellular mass changes of living single cells. With this instrumentation, we were able to perform fast mass measurements within 3 minutes. We show results of mass measurements of polystyrene and metal beads of various sizes (smallest weight measured at 280 {+/-} 95 pg) and live single-cell mass measurements in a physiologically relevant environment. We also performed finite element analysis to simulate and optimize the structural design and materials of cantilevers. Our simulation results indicate that using polymer materials, such as SU8 and polyimide, could improve the minimal detectable mass by 3-fold compared to conventional silicon cantilevers. The simulations also suggest that smaller dimensions of length, width, and thickness would improve the mass detection capability of microfluidic cantilevers.

biophysics↗

bioRxiv2021

Host EPAC1 modulates rickettsial adhesion to vascular endothelial cells via regulation of ANXA2 Y23 phosphorylation

Recently we have identified that endothelial surface annexin A2 (ANAX2) functions as a receptor for spotted fever group rickettsial adhesin outer membrane protein B (OmpB), which binds to the endothelial cell (EC) surface. Moreover, we reported that intracellular cAMP receptor EPAC1 modulates ANXA2 tyrosine (Y) 23 phosphorylation, and inactivation of EPAC1 suppresses ANXA2 expression on the EC luminal surface by downregulating Y23 phosphorylation. Since we reported that EPAC1 plays a critical role in the initial step to successfully establish rickettsial infection of ECs, this work aims to answer the following: (a) What is the mechanism underlying the regulatory role of EPAC1 in ECs during the initial step of bacterial infection? (b) Is the EPAC1-ANXA2 signaling pathway involved in the regulation of rickettsial adhesion to ECs? In the present study, an established system that is anatomically-based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae-EC adhesion. We reveal that the deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. In addition, single living brain microvascular EC study that employs FluidFM and site-directed mutagenesis provides evidence that supports our finding that EPAC1 governs rickettsial adhesion to EC surfaces via regulation of ANXA2 Y23 phosphorylation.

pathology↗

bioRxiv2020

Endothelial exosome plays functional role during rickettsial infection

Spotted fever group rickettsioses (SFRs) are devastating human infections. Vascular endothelial cells (ECs) are the primary targets of infection. Edema resulting from EC barrier dysfunction occurs in the brain and lungs in most cases of lethal SFR, but the underlying mechanisms remain unclear. The aim of the study is to explore the potential role of Rickettsia (R)-infected, EC-derived exosomes (Exos) during infection. Using size-exclusion chromatography (SEC), we purified Exos from conditioned, filtered, bacteria-free media collected from R-infected human umbilical vein ECs (HUVECs) (R-ECExos) and plasma of R-infected mice (R-plsExos). We observed that rickettsial infection increases the release of heterogeneous plsExos, but endothelial exosomal size, morphology, and production were not significantly altered following infection. Compared to normal plsExos and ECExos, both R-plsExos and R-ECExos induced dysfunction of recipient normal brain microvascular Ecs (BMECs). The effect of R-plsExos on mouse recipient BMEC barrier function is dose-dependent. The effect of R-ECExos on human recipient BMEC barrier function is dependent on exosomal RNA cargo. Next-generation sequencing analysis and stem-loop quantitative reverse transcription PCR (RT-qPCR) validation revealed that R infection triggered the selective enrichment of endothelial exosomal mir-23a and mir-30b, which target the endothelial barrier. To our knowledge, this is the first report on the functional role of extracellular vesicles following infection by obligately intracellular bacteria. ImportanceSpotted fever group rickettsioses are devastating human infections. Vascular endothelial cells are the primary targets of infection. Edema resulting from endothelial barrier dysfunction occurs in the brain and lungs in most cases of lethal rickettsioses, but the underlying mechanisms remain unclear. The aim of the study is to explore the potential role of Rickettsia-infected, endothelial cell-derived exosomes during infection. We observed that rickettsial infection increases the release of heterogeneous plasma Exos, but endothelial exosomal size, morphology, and production were not significantly altered following infection. Rickettsia-infected, endothelial cell-derived exosomes induced dysfunction of recipient normal brain microvascular endothelial cells. The effect is dependent on exosomal RNA cargo. Next-generation sequencing analysis revealed that rickettsial infection triggered the selective enrichment of endothelial exosomal mir-23a and mir-30b, which target the endothelial barrier. To our knowledge, this is the first report on the functional role of extracellular vesicles following infection by obligately intracellular bacteria.

microbiology↗

bioRxiv2020

Establishing correlation of axial and vertical force of cardiac artificial tissue using a novel micromachined sensor

Cardiomyocytes iPSC (iPSC-CMs) have great potential for cell therapy, drug assessment, and for understanding the pathophysiology and genetic underpinnings of cardiac diseases. Contraction forces are one of the most important characteristics of cardiac function and are predictors of healthy and diseased states. Cantilever techniques, such as atomic force microscopy, measure the vertical force of a single cell, while systems designed to more closely resemble the physical heart function, such as cardiac tissue on posts, measure the axial force. One important question is how do these two force measurements correlate? By establishing a correlation of the axial and vertical force we will be one step closer in being able to use single cell iPSC instead of more elaborate human engineered tissue or animal heart tissue as models. A novel micromachined sensor for measuring force contractions of artificial tissue has been developed. Using this novel sensor a correlation between axial force and vertical force is experimentally established. This finding supports the use of vertical measurements as an alternative to tissue measurements.

bioengineering↗

bioRxiv2020

EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation

Coagulopathy is associated with both inflammation and infection, including infection with the novel SARS-CoV-2 (COVID-19). Endothelial cells (ECs) fine tune hemostasis via cAMP-mediated secretion of von Willebrand factor (vWF), which promote the process of clot formation. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a key role in stabilizing ECs and suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed an increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1-/- phenotype. EPAC1 regulated TNF-triggered vWF secretion from human umbilical vein endothelial cells (HUVECs) in a phosphoinositide 3-kinases (PI3K)/endothelial nitric oxide synthase (eNOS)-dependent manner. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role of EPAC1 in vWF secretion and shed light on potential development of new strategies to controlling thrombosis during inflammation. Key PointPI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.

cell biology↗

bioRxiv2017

Exchange protein directly activated by cAMP plays a critical role in regulation of vascular fibrinolysis

RationaleTo maintain vascular patency, endothelial cells (ECs) actively regulate hemostasis. Among the myriad of pathways by which they control both fibrin formation and fibrinolysis is EC expression of annexin A2 (ANXA2) in a heterotetrameric complex with S100A10 [(ANXA2-S100A10)2]. This complex is a well-recognized endothelial surface platform for the activation of plasminogen by tissue plasminogen activator. A noteworthy advance in this field came about when it was shown that the cAMP pathway is linked to the regulation of (ANXA2-S100A10)2 in ECs.\n\nObjectiveThese findings prompted us to determine whether a druggable target, namely the exchange protein directly activated by cAMP (EPAC) pathway, plays a role in vascular luminal fibrinolysis.\n\nMethods and ResultsTaking advantage of our Epac1-null mouse model, we found that depletion of Epac1 results in fibrin deposition, fibrinolytic dysfunction, and decreased endothelial surface ANXA2 in mice, which are similar to phenomena discovered in ANXA2-null and S100A10-null mice. We observed upregulation of EPAC1 and downregulation of fibrin in endocardial tissues beneath atrial mural thrombi in humans. Of note, our thrombosis model revealed that dysfunction of fibrinolysis in EPAC1-null mice can be ameliorated by recombinant ANXA2. Furthermore, we demonstrated that suppression of EPAC1 using a small-molecule inhibitor (ESI09) reduces the expression of ANXA2 in lipid rafts and impedes ANXA2 association with S100A10. Endothelial apical surface expression of both ANXA2 and S100A10 were markedly decreased in ESI09-treated ECs, which was corroborated by results from a nanoforce spectroscopy study. Moreover, inactivation of EPAC1 decreases tyrosine 23 phosphorylation of ANXA2 in the cell membrane compartment.\n\nConclusionsOur data reveal a novel role for EPAC1 in vascular fibrinolysis, by showing that EPAC1 is responsible for the translocation of ANXA2 to the EC surface. This process promotes conversion of plasminogen to plasmin, thereby enhancing local fibrinolytic activity.

cell biology↗