Biology subjects

Acevedo, C.

Publications and source records attributed to Acevedo, C..

11 recordsLinked to original sources

bioRxiv2025

Disruption of the Lacunar Canalicular Network in Type 2 Diabetes: Impaired Osteocyte Connectivity in Zucker Diabetic Rats

Type 2 diabetes affects multiple organ systems, including the skeletal system. Diabetes reduces bones mechanical properties and impacts bone cells, such as osteocytes, which are crucial to preserving bone health. Osteocytes maintain bone health through the lacunar canalicular network (LCN), a highly interconnected system vital for remodeling, mechanotransduction, and nutrient transport. Yet the specific impact diabetes has on this network has remained unclear. Here, we used confocal laser scanning microscopy combined with advanced connectomics modeling to achieve high-resolution, three-dimensional reconstructions of the LCN in Zucker Diabetic Sprague Dawley rats, a polygenic model that closely mimics human type 2 diabetes. Diabetes profoundly disrupted LCN connectivity in the femoral mid-cortex, with canalicular and node density reduced by 21% and 30%, respectively. Additionally, we observed a 30-40% increase in lacunar density and highly connected nodes. These architectural shifts impair bone permeability, diminishing mechanosensitivity and compromising nutrient and oxygen transport. Our findings uncover a previously unrecognized mechanism of skeletal fragility in diabetes and highlight the LCN as a promising therapeutic target.

physiology↗

bioRxiv2025

Disruption of Marrow Microenvironments in Chronic Lymphocytic Leukemia by High-Resolution Synchrotron Micro-Computed Tomography

Chronic lymphocytic leukemia (CLL) is associated with increased fracture risk unexplained by standard bone density scans, suggesting underlying microstructural alterations. To investigate this, we used high-resolution synchrotron micro-computed tomography (SR{micro}CT) on bone marrow biopsies from 17 CLL patients, who were stratified into low and high infiltration groups using objective, data-driven clustering. To our knowledge, this is the first report to quantify these changes. High CLL marrow infiltration was associated with a 40.3% reduction in lacuna density and a 101% increase in the normalized adipose surface area-to-volume ratio, a metric indicating greater structural fragmentation. Both changes correlated with leukemic infiltration percentage and showed partial reversal after therapy in a longitudinal case. Furthermore, high CLL burden significantly altered the morphological distributions of the remaining lacunar osteocyte (p < 0.001). We identify a novel marrow remodeling phenotype in CLL characterized by osteocyte depletion and adipose disruption. These changes likely contribute to skeletal fragility and represent potential microstructural biomarkers for assessing marrow health more accurately than conventional imaging.

physiology↗

bioRxiv2025

Genome-Wide Interrogation of SARS-CoV-2 RNA-Protein Interactions Uncovers Hidden Regulatory Sites

The global impact of the COVID-19 pandemic underscores the critical need for a comprehensive understanding of SARS-CoV-2 replication mechanisms. While the central roles of the RNA dependent RNA polymerase (NSP12), primase protein (NSP8), and nucleocapsid protein (N) in the virus life cycle are extensively studied, the precise nature of their interactions with the full-length viral RNA genome remain incompletely characterized. In this study, we sought to address this knowledge gap by employing enhanced crosslinking and immunoprecipitation (eCLIP) to map the binding sites of NSP8, NSP12, and N proteins across the SARS-CoV-2 genome at early stages of viral RNA and protein synthesis and late stages of virion assembly. Our findings revealed interactions of NSP8 and NSP12 to the 5 and 3 untranslated regions (UTRs) of both positive and negative sense RNA, regions known to regulate viral replication, transcription, and translation. We identified a surprising and essential NSP12 binding site within the RNA sequence encoding the conserved Y1 domain of NSP3, which regulates RNA abundance upstream of the site. Additionally, we found that N protein interacts with the 5 UTR and influences translation efficiency. Finally, we report a novel regulatory function of N protein in modulating ribosomal frameshifting proximal to the frameshift element, a crucial process for maintaining viral protein stoichiometry. Our results provide a detailed molecular map of SARS-CoV-2 protein-RNA interactions, revealing potential therapeutic targets for attenuating viral fitness and informing the development of next-generation antiviral strategies.

molecular biology↗

bioRxiv2024

Intestinal Epithelial PTPN2 Limits Pathobiont Colonization by Immune-Directed Antimicrobial Responses

Background and AimsLoss of activity of the inflammatory bowel disease (IBD) susceptibility gene, protein tyrosine phosphatase non-receptor type 2 (PTPN2), is associated with altered microbiome composition in both human subjects and mice. Further, expansion of the bacterial pathobiont, adherent- invasive E. coli (AIEC), is strongly linked to IBD pathogenesis. The mechanism by which intestinal epithelial cells (IEC) maintain equilibrium between commensal microbiota and immune cells to restrict invading pathobionts is poorly understood. Here, we investigated the role of IEC-specific PTPN2 in regulating AIEC colonization. MethodsTamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice (Ptpn2{Delta}IEC) and control Ptpn2fl/fl mice were infected with either non-invasive E. coli K12, or fluorescent-tagged mAIEC (mAIECred) for four consecutive days or administered PBS. Subsequently, bacterial colonization in mouse tissues was quantified. mRNA and protein expression were assayed in intestinal epithelial cells (IECs) or whole tissue lysates by PCR and Western blot. Tissue cytokine expression was determined by ELISA. Intestinal barrier function was determined by in vivo administration of 4 kDa FITC-dextran (FD4) or 70kDa Rhodamine-B dextran (RD70) fluorescent probes. Confocal microscopy was used to determine the localization of tight-junction proteins. ResultsPtpn2{Delta}IEC mice exhibited increased mAIECred - but not K12 - bacterial load in the distal colon compared to infected Ptpn2fl/fl mice. The higher susceptibility to mAIECred infection was associated with altered levels of antimicrobial peptide (AMPs). Ileal RNA expression of the alpha-defensin AMPs, Defa5 and Defa6, as well as MMP7, was significantly lower in Ptpn2{Delta}IEC vs. Ptpn2fl/fl mice, after mAIECred but not K12 infection. Further, we observed increased tight junction-regulated permeability determined by elevated in vivo FD4 but not RD70 permeability in Ptpn2{Delta}IEC-K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2{Delta}IEC mAIEC-infected mice. Further, Ptpn2{Delta}IEC mice displayed lower IL-22, IL-6, IL-17A cytokine expression post mAIEC infection compared to Ptpn2fl/fl controls. Recombinant IL-22 reversed the FD4 permeability defect and reduced bacterial burden in Ptpn2{Delta}IEC mice post mAIEC challenge. ConclusionOur findings highlight that intestinal epithelial PTPN2 is crucial for mucosal immunity and gut homeostasis by promoting anti-bacterial defense mechanisms involving coordinated epithelial-immune responses to restrict pathobiont colonization.

immunology↗

bioRxiv2023

Whole-bone toughness is linked to canal and osteocyte lacunae deficits in the ZDSD type 2 diabetic rat model

Type 2 diabetes mellitus (T2DM) is associated with an increased fracture risk independent of bone mass. The exact origin of this increased fracture risk is still not well understood. Using a polygenic diabetic rat model, synchrotron radiation micro-computed tomography (SRCT), and in situ scanning electron microscope (SEM) fracture toughness, we related the changes at the microscale to toughness and material properties of diabetic rat femurs. The diabetic rat model (ZDSD) displayed overnight fasting hyperglycemia and an increased AGEs content. Additionally, we measured the impairment of post-yield properties and toughness in diabetic rats. The cortical geometry and porosity were also affected in this ZDSD model. We measured a decrease in osteocyte lacunar density associated with a decreased lacunar volume. Moreover, we found decreased canal density while maintaining a similar canal diameter. These results indicate that diabetes impairs bone remodeling, affecting bone microstructure. Because canals and lacunae are also linked with extrinsic toughening mechanisms, we attribute the decreased toughness largely to these microstructural changes. In conclusion, we showed that changes in lacunae and canal density, combined with AGEs accumulation, decreased toughness in T2DM rat bone.

bioengineering↗

bioRxiv2022

Intervertebral disc cell chondroptosis elicits neutrophil response in Staphylococcus aureus spondylodiscitis

ObjectiveTo understand the pathophysiology of spondylodiscitis due to Staphylococcus aureus, an emerging infectious disease of the intervertebral disc (IVD) and vertebral body with a high complication rate, by combining clinical insights and experimental approaches. DesignClinical data and histological material of nine patients suffering from S. aureus spondylodiscitis were retrospectively collected at a single center. To mirror the clinical findings experimentally, we developed a novel porcine ex vivo model mimicking acute S. aureus spondylodiscitis and assessed the interaction between S. aureus and IVD cells within their native environment. In addition, the inflammatory features underlying this interaction were assessed in primary human IVD cells. Finally, mirroring the clinical findings, we assessed primary human neutrophils for their ability to respond to secreted inflammatory modulators of IVD cells upon S. aureus challenge. ResultsAcute S. aureus spondylodiscitis in patients was characterized by tissue necrosis and neutrophil infiltration. Additionally, the presence of empty IVD cells lacunae was observed. This was mirrored in the ex vivo porcine model, where S. aureus induced extensive IVD cell death, leading to empty lacunae. Concomitant engagement of the apoptotic and pyroptotic cell death pathways was observed in primary human IVD cells, resulting in cytokine release. Among the released cytokines, functionally intact neutrophil-priming as well as broad pro- and anti-inflammatory cytokines known for their involvement in IVD degeneration were found. ConclusionsIn patients as well as ex vivo in a novel porcine model, S. aureus spondylodiscitis infection caused IVD cell death, resulting in empty lacunae, which was accompanied by release of inflammation markers and recruitment of neutrophils. These findings offer valuable insights into the important role of inflammatory IVD cell death during the onset of spondylodiscitis and potential future therapeutic approaches.

immunology↗

bioRxiv2021

skater: An R package for SNP-based Kinship Analysis, Testing, and Evaluation

MotivationSNP-based kinship analysis with genome-wide relationship estimation and IBD segment analysis methods produces results that often require further downstream processing and manipulation. A dedicated software package that consistently and intuitively implements this analysis functionality is needed. ResultsHere we present the skater R package for SNP-based kinship analysis, testing, and evaluation with R. The skater package contains a suite of well-documented tools for importing, parsing, and analyzing pedigree data, performing relationship degree inference, benchmarking relationship degree classification, and summarizing IBD segment data. AvailabilityThe skater package is implemented as an R package and is released under the MIT license at https://github.com/signaturescience/skater. Documentation is available at https://signaturescience.github.io/skater.

bioinformatics↗

bioRxiv2021

Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients

Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

immunology↗

bioRxiv2020

Neutrophil and monocyte dysfunctional effector response towards bacterial challenge in critically-ill COVID-19 patients

COVID-19 displays diverse disease severities and symptoms. Elevated inflammation mediated by hypercytokinemia induces a detrimental dysregulation of immune cells. However, there is limited understanding of how SARS-CoV-2 pathogenesis impedes innate immune signaling and function against secondary bacterial infections. We assessed the influence of COVID-19 hypercytokinemia on the functional responses of neutrophils and monocytes upon bacterial challenges from acute and corresponding recovery COVID-19 ICU patients. We show that severe hypercytokinemia in COVID-19 patients correlated with bacterial superinfections. Neutrophils and monocytes from acute COVID-19 patients showed severely impaired microbicidal capacity, reflected by abrogated ROS and MPO production as well as reduced NETs upon bacterial challenges. We observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes leading to a suppressive autocrine and paracrine signaling during bacterial challenges. Our data provide insights into the innate immune status of COVID-19 patients mediated by their hypercytokinemia and its transient effect on immune dysregulation upon subsequent bacterial infections

immunology↗

bioRxiv2019

TGFβ regulation of perilacunar/canalicular remodeling is sexually dimorphic

1.Bone fragility is the product of defects in bone mass and bone quality, both of which show sex-specific differences. Despite this, the cellular and molecular mechanisms underpinning the sexually dimorphic control of bone quality remain unclear, limiting our ability to effectively prevent fractures, especially in postmenopausal osteoporosis. Recently, using male mice, we found that systemic or osteocyte-intrinsic inhibition of TGF{beta} signaling, achieved using the 9.6-kb DMP1 promoter-driven Cre recombinase (T{beta}RIIocy-/- mice), suppresses osteocyte perilacunar/canalicular remodeling (PLR) and compromises bone quality. Since systemic TGF{beta} inhibition more robustly increases bone mass in female than male mice, we postulated that sex-specific differences in bone quality could likewise result, in part, from dimorphic regulation of PLR by TGF{beta}. Moreover, since lactation induces PLR, we examined the effect of TGF{beta} inhibition on the female skeleton during lactation. In contrast to males, female mice that possess an osteocyte-intrinsic defect in TGF{beta} signaling were protected from TGF{beta}-dependent defects in PLR and bone quality. The expression of requisite PLR enzymes, the lacuno-canalicular network, and the flexural strength of female T{beta}RIIocy-/- bone was intact. With lactation, however, bone loss, and induction in PLR and osteocytic parathyroid hormone type I receptor (PTHR1) expression, were suppressed in T{beta}RIIocy-/- bone, relative to wild-type. Indeed, differential control of PTHR1 expression, by TGF{beta} and other factors, may contribute to dimorphism in PLR regulation in male and female T{beta}RIIocy-/- mice. These findings provide key insights into the sex-based differences in osteocyte PLR that underlie bone quality and highlight TGF{beta} signaling as a crucial regulator of lactation-induced PLR.

molecular biology↗

bioRxiv2019

Suppressed Osteocyte Perilacunar / Canalicular RemodelingPlays a Causal Role in Osteoarthritis

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. To determine if PLR suppression plays a causal role in OA, we ablated the PLR enzyme MMP13 in osteocytes, while leaving chondrocytic MMP13 intact. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, this osteocyte-intrinsic PLR defect was sufficient to reduce cartilage proteoglycan content and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, osteocyte PLR is a critical regulator of cartilage homeostasis. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and identify the causal role of suppressed perilacunar/canalicular remodeling in osteoarthritis.

physiology↗