Biology subjects

Ocampo, A.

Publications and source records attributed to Ocampo, A..

17 recordsLinked to original sources

bioRxiv2025

An integrated single-cell and spatial transcriptomic atlas of thyroid cancer progression identifies prognostic fibroblast subpopulations

Thyroid cancer progression from curable well-differentiated thyroid carcinoma to highly lethal anaplastic thyroid carcinoma is distinguished by tumor cell de-differentiation and recruitment of a robust stromal infiltrate. Combining an integrated thyroid cancer single-cell sequencing atlas with spatial transcriptomics and bulk RNA-sequencing, we define stromal cell subpopulations and tumor-stromal cross-talk occurring across the histologic and mutational spectrum of thyroid cancer. We identify distinct inflammatory and myofibroblastic cancer-associated fibroblast (iCAF and myCAF) populations and perivascular-like populations. The myCAF population is only found in malignant samples and is associated with tumor cell invasion, BRAFV600E mutation, lymph node metastasis, and disease progression. Tumor-adjacent myCAFs abut invasive tumor cells with a partial epithelial-to-mesenchymal phenotype. Tumor-distant iCAFs infiltrate inflammatory autoimmune thyroid lesions and anaplastic tumors. In summary, our study provides an integrated atlas of thyroid cancer fibroblast subtypes and spatial characterization at sites of tumor invasion and de-differentiation, defining the stromal reorganization central to disease progression.

cancer biology↗

bioRxiv2024

Loss of H3K9 trimethylation leads to premature aging

Aging is the major risk factor for most human diseases and represents a major socio-economical challenge for modern societies. Despite its importance, the process of aging remains poorly understood. Epigenetic dysregulation has been proposed as a key driver of the aging process. Modifications in transcriptional networks and chromatin structure might be central to age-related functional decline. A prevalent feature described during aging is the overall reduction in heterochromatin, specifically marked by the loss of repressive histone modification, Histone 3 lysine 9 trimethylation (H3K9me3). However, the role of H3K9me3 in aging, especially in mammals, remains unclear. Here we show using a novel mouse strain, (TKOc), carrying a triple knockout of three methyltransferases responsible for H3K9me3 deposition, that the inducible loss of H3K9me3 in adulthood results in premature aging. TKOc mice exhibit reduced lifespan, lower body weight, increased frailty index, multi-organ degeneration, transcriptional changes with significant upregulation of transposable elements, and accelerated epigenetic age. Our data strongly supports the concept that the loss of epigenetic information directly drives the aging process. These findings reveal the importance of epigenetic regulation in aging and suggest that interventions targeting epigenetic modifications could potentially slow down or reverse age-related decline. Understanding the molecular mechanisms underlying the process of aging will be crucial for developing novel therapeutic strategies that can delay the onset of age-associated diseases and preserve human health at old age specially in rapidly aging societies.

physiology↗

bioRxiv2024

Neuron-specific partial reprogramming in the dentate gyrus impacts mouse behavior and ameliorates age-related decline in memory and learning.

Age-associated neurodegenerative disorders represent significant challenges due to progressive neuronal decline and limited treatments. In aged mice, partial reprogramming, characterized by pulsed expression of reprogramming factors, has shown promise in improving function in various tissues, but its impact on the aging brain remains poorly understood. Here we investigated the impact of in vivo partial reprogramming on mature neurons in the dentate gyrus of young and aged mice. Using two different approaches - a neuron-specific transgenic reprogrammable mouse model and neuron-specific targeted lentiviral delivery of OSKM reprogramming factors - we demonstrated that in vivo partial reprogramming of mature neurons in the dentate gyrus, a neurogenic niche in the adult mouse brain, can influence animal behavior, and ameliorate age-related decline in memory and learning. These findings underscore the potential of in vivo partial reprogramming as an important therapeutic intervention to rejuvenate the neurogenic niche and ameliorate cognitive decline associated with aging or neurodegeneration.

neuroscience↗

bioRxiv2024

Peptide Coated Polycaprolactone-Benzalkonium Chloride Nanocapsules for Targeted Drug Delivery to the Pancreatic β-Cell

Targeting of current therapies to treat or prevent loss of pancreatic islet {beta}-cells in Type 1 Diabetes (T1D) may provide improved efficacy and reduce off target effects. Current efforts to target the {beta}-cell are limited by a lack of {beta}-cell specific targets and the inability to test multiple targeting moieties with the same delivery vehicle. Here we fabricate a novel tailorable polycaprolactone nanocapsule (NC) where multiple different targeting peptides can be interchangeably attached for {beta}-cell specific delivery. Incorporation of a cationic surfactant in the NC shell allows for the attachment of Exendin-4 and an antibody for ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) for {beta}-cell specific targeting. The average NC size ranges from 250-300nm with a polydispersity index under 0.2. The NCs are non-toxic, stable in media culture, and can be lyophilized and reconstituted. NCs coated with targeting peptide were taken up by human cadaveric islet {beta}-cells and human stem cell-derived {beta}-like cells (sBC) in vitro with a high level of specificity. Furthermore, NCs successfully delivered both hydrophobic and hydrophilic cargo to human {beta}-cells. Finally, Exendin-4 coated NCs were stable and targeted the mouse pancreatic islet {beta}-cell in vivo. Our unique NC design allows for the interchangeable coating of targeting peptides for future screening of targets with improved cell specificity. The ability to target and deliver thera-peutics to human pancreatic {beta}-cells opens avenues for improved therapies and treatments to help the delay onset, prevent, or reverse T1D.

bioengineering↗

bioRxiv2024

Comprehensive evaluation of lifespan-extending molecules in C. elegans

The nematode C. elegans has long served as a gold-standard model organism in aging research, particularly since the discovery of long-lived mutants in conserved aging pathways including daf-2 (IGF1) and age-1 (PI3K). Its short lifespan and small size make it highly suitable for high throughput experiments. While numerous molecules have been tested for their effects on C. elegans lifespan, consensus is still lacking regarding the most effective and reproducible compounds. Confounding effects, especially those related to drug-bacteria interactions, remain a contentious issue in the literature. In this study, we evaluated 16 of the most frequently reported lifespan-extending molecules in C. elegans, examining their effects on lifespan with two different diets (live and UV-killed OP50). In addition, we assessed the compounds impact on bacterial growth, their effects on various nematode strains, and the impact of the starting age of treatment. Our findings first confirmed robust lifespan extension with many, but not all, of the 16 tested compounds from the literature, and revealed that some of them could be combined to get synergistic effects. Additionally, we showed that some of these compounds also extend lifespan in the fly D. melanogaster, demonstrating a conserved effect across species. Finally, by expanding our screen to a broader pool of molecules, we identified novel lifespan-extending compounds in C. elegans.

pharmacology and toxicology↗

bioRxiv2024

In vivo reprogramming of Caenorhabditis elegans leads to heterogeneous effects on lifespan

In the last decade, cellular reprogramming of fully differentiated cells to pluripotent stem cells has become of great interest. Importantly, cellular reprogramming by expression of Oct4, Sox2, Klf4, and cMyc (OSKM) can ameliorate age-associated phenotypes in multiple tissues and extend lifespan in progeroid and aged wild-type mice. Surprisingly, the effects of in vivo reprogramming have not been deeply investigated in any other model organisms. Here, for the first time, we induce in vivo reprogramming in C. elegans using a heat-inducible system at multiple developmental and adult stages. Similar to mice, expression of the reprogramming factors leads to premature death with different levels of toxicity at distinct developmental stages and aging. In vivo reprogramming in C. elegans might represent a valuable tool to improve our understanding of development and in vivo reprogramming.

cell biology↗

bioRxiv2024

Comparative analysis of mouse strains for in vivo reprogramming

In vivo reprogramming through the forced expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) has demonstrated great potential for reversing age-associated phenotypes, as the combination of these transcription factors actively promote cell regeneration and rejuvenation in various tissues and organs. However, continuous in vivo OSKM expression raised safety concerns due to loss of cell identity, decrease in body weight, and premature death. Although cyclic short-term or targeted expression of the reprogramming factors can mitigate some of these detrimental effects in mice, systemic rejuvenation of wild type mice has remained elusive potentially due to these current technical limitations. To improve the fundamental understanding of in vivo reprogramming, we conducted a comparative analysis across multiple reprogrammable mouse strains, tissues, and expression methods, presenting a comprehensive atlas of formerly established strains. In addition, we developed novel reprogrammable mouse strains by avoiding OSKM expression in specific organs, in dividing cells, or implementing chimeric expression approaches within specific cells, thereby offering safer strategies to induce in vivo reprogramming and fully harness its potential. We hope that these new tools will become valuable resources for future research in this very exciting field of research with potential implications to human health.

molecular biology↗

bioRxiv2024

Unravelling the ageing-reversal potency of stem cell-derived extracellular vesicles in a rat model of premature cardiac senescence

Cardiosphere-derived cells (CDCs) and the extracellular vesicles they release (CDC-EVs) have demonstrated to induce rejuvenation and to improve cardiac structure and function, but their efficacy may vary among donors and there is still a lack of adequate potency assays. We aimed to identify parameters that could easily predict the ageing-reversal potency of CDC-EVs. CDCs derived from cardiac tissue of 34 human donors (age range: 3-months to 81-years old) were characterized in terms of their phenotypical and biological properties. The anti-senescent activity of the CDC-EVs was assessed in vitro using a predesigned matrix assay with several ageing-related markers. We found that while CDC-donors chronological age was not determinant, the degree of CDC senescence in culture showed a strong correlation to most CD[C]s bioactive properties. However, CDC senescence alone was insufficient to predict CDC-EV in vitro potency. Thus, we scored the potency of the CDC-EVs based on their performance in the newly designed in vitro matrix assay and classified them as more-(P-EVs) and less-potent (NP-EVs). We then tested P- and NP-EVs in a rat model of premature cardiac ageing and found that only the P-EVs reduced senescence-associated GLB1 gene expression in the heart and tended to protect it from hypertrophy development and fibrosis. On the contrary, NP-EVs failed to induce any improvements and negatively affected cardiac hypertrophy, fibrosis, and perfusion. In conclusion, despite CDC-EV anti-ageing potency cannot be predicted by the chronological age of the donors or CDC senescence as surrogate markers, we propose an in vitro potency assay that could be used to evaluate allogenic EV suitability before its use in the treatment of cardiac ageing.

cell biology↗

bioRxiv2024

Impaired glycolysis in aged endothelial cells is associated with reduced neovascularisation upon tissue ischemia

The vascular system experiences an age-associated decline in tissue perfusion and response to ischemic diseases. The factors driving this age-associated neovascularization decline remain unclear. While old endothelial cells (ECs) adopt a pro-angiogenic gene expression profile, we observed a stark reduction in the proliferative capacity of old ECs, while migratory capabilities remain intact. This is paralleled by a drastic decline in glycolytic capacity and ATP production, which likely act as limiting factors to neovascularization. These findings may provide new strategies to restore EC function in aging, thereby improving organ resilience and extending health- and lifespan.

molecular biology↗

bioRxiv2023

Initiation phase cellular reprogramming ameliorates DNA damage in the ERCC1 mouse model of premature aging.

Unlike aged somatic cells, which exhibit a decline in molecular fidelity and eventually reach a state of replicative senescence, pluripotent stem cells can indefinitely replenish themselves while retaining full homeostatic capacity. The conferment of beneficial-pluripotency related traits via in vivo partial cellular reprogramming (IVPR) significantly extends lifespan and restores aging phenotypes in mouse models. Although the phases of cellular reprogramming are well characterized, details of the rejuvenation processes are poorly defined. To understand whether epigenetic reprogramming can ameliorate DNA damage, we created reprogrammable accelerated aging mouse model with an ERCC1 mutation. Importantly, using enhanced partial reprogramming by combining small molecules with the Yamanaka factors, we observed potent reversion of DNA damage, significant upregulation of multiple DNA damage repair processes, and restoration of the epigenetic clock. In addition, we present evidence that pharmacological inhibition of ALK5 and ALK2 receptors in TGFb pathway is able to phenocopy some benefits including epigenetic clock restoration suggesting a role in the mechanism of rejuvenation by partial reprogramming.

cell biology↗

bioRxiv2022

ERCC1 mice, unlike other premature aging models, display accelerated epigenetic age

Over the last decades, several premature aging mouse models have been developed to study aging and identify interventions that can delay age-related diseases. Yet, it is still unclear whether these models truly recapitulate natural aging. Here, we analyzed DNA methylation in multiple tissues of four previously reported mouse models of premature aging (ERCC1, LAKI, POLG and XPG). We estimated DNA methylation (DNAm) age of these samples using the Horvath clock. The most pronounced increase in DNAm age could be observed in ERCC1 mice, a strain which exhibits a deficit in DNA nucleotide excision repair. In line with these results, we detected an increase in epigenetic age in fibroblasts isolated from patients with progeroid syndromes associated with mutations in DNA excision repair genes. These findings highlight ERCC1 as a particularly attractive mouse model to study aging in mammals and suggest a strong connection between DNA damage and epigenetic dysregulation during aging.

molecular biology↗

bioRxiv2022

Chemical reprogramming ameliorates cellular hallmarks of aging and extends lifespan

The dedifferentiation of somatic cells into a pluripotent state by cellular reprogramming coincides with a reversal of age-associated molecular hallmarks. Although transcription factor induced cellular reprogramming has been shown to ameliorate these aging phenotypes in human cells and extend health and lifespan in mice, translational applications of this approach are still limited. More recently, chemical reprogramming via small molecule cocktails have demonstrated a similar ability to induce pluripotency in vitro, however, its potential impact on aging is unknown. Here, we demonstrated that partial chemical reprogramming is able to improve key drivers of aging including genomic instability and epigenetic alterations in aged human cells. Moreover, we identified an optimized combination of two reprogramming molecules sufficient to induce the amelioration of additional aging phenotypes including cellular senescence and oxidative stress. Importantly, in vivo application of this two-chemical combination significantly extended C. elegans lifespan. Together, these data demonstrate that improvement of key drivers of aging and lifespan extension is possible via chemical induced partial reprogramming, opening a path towards future translational applications.

cell biology↗

bioRxiv2022

Development of a novel aging clock based on chromatin accessibility

The establishment of aging clocks based on age-associated changes in DNA methylation has highlighted the strong link between epigenetic alterations and aging. However, the connection between DNA methylation changes at clock sites and their effect on cellular function remains unclear. We hypothesize that chromatin accessibility, a readout that integrates multiple epigenetic mechanisms, may connect epigenetic changes with downstream biological effects. To investigate this hypothesis, we generated chromatin accessibility profiles from peripheral blood mononuclear cells (PBMCs) of 157 human donors and construct a novel aging clock with a median absolute error on prediction of 5.69 years. Moreover, by comparing our chromatin accessibility data to matched transcriptomic profiles, we show that the genomic sites selected for the prediction of age based on chromatin accessibility undergo transcriptional changes during aging. This chromatin accessibility clock could therefore be used to investigate the direct effect of aged epigenetic states on cellular function.

genomics↗

bioRxiv2022

A scalable approach for continuous time Markov models with covariates

Existing methods for fitting continuous time Markov models (CTMM) in the presence of covariates suffer from scalability issues due to high computational cost of matrix exponentials calculated for each observation. In this paper we propose an optimization technique for CTMM which uses a stochastic gradient descent algorithm combined with differentiation of the matrix exponential using a Pade approximation which we show then makes fitting large scale data feasible. We compare two methods for computing standard errors, one using Pade expansion, and the other using power series expansion of the matrix exponential. Through simulations we find improved performance relative to existing CTMM methods, and we demonstrate the method on the large-scale multiple sclerosis NO.MS dataset.

bioinformatics↗

bioRxiv2022

In vivo reprogramming leads to premature death due to hepatic and intestinal failure.

SUMMARYThe induction of cellular reprogramming by forced expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC (OSKM) has been shown to allow the dedifferentiation of somatic cells and ameliorate age-associated phenotypes in multiple tissues and organs. Yet to date, the benefits of in vivo reprogramming are limited by the occurrence of detrimental side-effects. Here, using complementary genetic approaches, we demonstrated that continuous in vivo induction of the reprogramming factors leads to hepatic and intestinal dysfunction resulting in decreased body weight and premature death. By generating a novel transgenic reprogrammable mouse strain, which avoids OSKM expression in both liver and intestine, we drastically reduced the early lethality and adverse effects associated with in vivo reprogramming. This new reprogramming mouse allows safe and long-term continuous induction of OSKM and might enable a better understanding of in vivo reprogramming as well as maximize its potential effects on rejuvenation and regeneration.

molecular biology↗

bioRxiv2021

Identification of CRF66_BF, a new HIV-1 circulating recombinant form of South American origin

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Among 108 reported in the literature, 17 are BF1 intersubtype recombinant, most of which are of South American origin. Among these, all 5 identified in the Southern Cone and neighboring countries, except Brazil, derive from a common recombinant ancestor related to CRF12_BF, which circulates widely in Argentina, as deduced from coincident breakpoints and clustering in phylogenetic trees. In a HIV-1 molecular epidemiological study in Spain, we identified a phylogenetic cluster of 20 samples from 3 separate regions which were of F1 subsubtype, related to the Brazilian strain, in protease-reverse transcriptase (Pr-RT) and of subtype B in integrase. Remarkably, 14 individuals from this cluster (designated BF9) were Paraguayans and only 4 were native Spaniards. HIV-1 transmission was predominantly heterosexual, except for a subcluster of 6 individuals, 5 of which were men who have sex with men. Ten additional database sequences, from Argentina (n=4), Spain (n=3), Paraguay (n=1), Brazil (n=1), and Italy (n=1), branched within the BF9 cluster. To determine whether it represents a new CRF, near full-length genome (NFLG) sequences were obtained for 6 viruses from 3 Spanish regions. Bootscan analyses showed a coincident BF1 recombinant structure, with 5 breakpoints, located in p17gag, integrase, gp120, gp41-rev overlap, and nef, which was identical to that of two BF1 recombinant viruses from Paraguay previously sequenced in NFLGs. Interestingly, none of the breakpoints coincided with those of CRF12_BF. In a maximum likelihood phylogenetic tree, all 8 NFLG sequences grouped in a strongly supported clade segregating from previously identified CRFs and from the CRF12_BF family clade. These results allow us to identify a new HIV-1 CRF, designated CRF66_BF. Through a Bayesian coalescent analysis, the most recent common ancestor of CRF66_BF was estimated around 1984 in South America, either in Paraguay or Argentina. Among Pr-RT sequences obtained by us from HIV-1-infected Paraguayans living in Spain, 14 (20.9%) of 67 were of CRF66_BF, suggesting that CRF66_BF may be one of the major HIV-1 genetic forms circulating in Paraguay. CRF66_BF is the first reported non-Brazilian South American HIV-1 CRF_BF unrelated to CRF12_BF.

microbiology↗

bioRxiv2020

High concordance between hippocampal transcriptome of the intraamygdala kainic acid model and human temporal lobe epilepsy

ObjectivePharmacoresistance and the lack of disease-modifying actions of current anti-seizure drugs persist as major challenges in the treatment of epilepsy. Experimental models of chemoconvulsant-induced status epilepticus remain the models of choice to discover potential anti-epileptogenic drugs but doubts remain as to the extent to which they model human pathophysiology. The aim of the present study was to compare the molecular landscape of the intraamygdala kainic acid model of status epilepticus in mice with findings in resected brain tissue from patients with drug-resistant temporal lobe epilepsy (TLE). MethodsStatus epilepticus was induced via intraamygdala microinjection of kainic acid in C57BL/6 mice and gene expression analysed via microarrays in hippocampal tissue at acute and chronic time-points. Results were compared to reference datasets in the intraperitoneal pilocarpine and intrahippocampal kainic acid model and to human resected brain tissue (hippocampus and cortex) from patients with drug-resistant TLE. ResultsIntraamygdala kainic acid injection in mice triggered extensive dysregulation of gene expression which was [~]3-fold greater shortly after status epilepticus (2729 genes) when compared to epilepsy (412). Comparison to samples of patients with TLE revealed a particular high correlation of gene dysregulation during established epilepsy. Pathway analysis found suppression of calcium signalling to be highly conserved across different models of epilepsy and patients. CREB was predicted as one of the main up-stream transcription factors regulating gene expression during acute and chronic phases and inhibition of CREB reduced seizure severity in the intraamygdala kainic acid model. SignificanceOur findings suggest the intraamygdala kainic acid model faithfully replicates key molecular features of human drug-resistant temporal lobe epilepsy and provides potential rationale target approaches for disease-modification through new insights into the unique and shared gene expression landscape in experimental epilepsy. Key point boxO_LIMore genes show expression changes shortly following intraamygdala kainic acid-induced status epilepticus when compared to established epilepsy. C_LIO_LIThe intraamygdala kainic acid mouse model mimics closely the gene expression landscape in the brain of patients with temporal lobe epilepsy. C_LIO_LISupressed calcium signalling in the brain as common feature across experimental models of epilepsy and patients with temporal lobe epilepsy. C_LIO_LICREB is a major up-stream transcription factor during early changes following status epilepticus and once epilepsy is established. C_LI

neuroscience↗