Biology subjects

Alvarez, R.

Publications and source records attributed to Alvarez, R..

13 recordsLinked to original sources

bioRxiv2026

Ultrafast X-Rays Capture Retinal Traversing Conical Intersection in Rhodopsin

G-protein-coupled receptors of the Rhodopsin family are crucial medicinal targets, transmitting signals across biomembranes. While light absorption by visual rhodopsin is well studied, its activation via retinal cofactor dynamics remains unclear. Here we use a free-electron laser to show that time-resolved X-ray solution scattering captures the retinal cis-trans isomerization as it passes the conical intersection of excited and ground-state photoproduct energy surfaces. Femtosecond-scale nuclear changes occur due to resonant photon absorption, with all-atom simulations revealing ultrafast amino acid movements that initiate transmembrane helix shifts. Ligand-free opsin measurements confirm that light activation is unaffected by non-resonant processes, showing the photonic energy is directly transmitted within the protein. Our method unveils how cofactor dynamics activate rhodopsin, free of constraints from crystal lattice packing or cryotrapping photointermediates.

biophysics↗

bioRxiv2025

A TARGETED COMBINATION THERAPY ACHIEVES EFFECTIVE PANCREATIC CANCER REGRESSION AND PREVENTS TUMOR RESISTANCE

Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest cancer survival rates. Recent studies using RAS(ON) inhibitors as single agents have opened the door to more efficacious therapies. Here, we demonstrate that genetic ablation of three independent nodes involved in downstream (RAF1), upstream (EGFR) and orthogonal (STAT3) KRAS signaling pathways leads to complete and permanent disappearance of orthotopic PDACs induced by KRAS/TP53 mutations. Likewise, a combination of RAS(ON) (RMC-6236/daraxonrasib), EGFR family (afatinib) and STAT3 (SD36) selective inhibitors/degraders induced the effective regression of these orthotopic tumors with no evidence of tumor resistance for over 200 days post-treatment. This combination therapy also led to significant regression of genetically engineered mouse tumors as well patient-derived tumor xenografts (PDX) in the absence of tumor relapses. Finally, this combination therapy was well tolerated by the animals. These results should guide the development of clinical trials that could benefit PDAC patients.

cancer biology↗

bioRxiv2025

A large global soil carbon sink informed by repeated soil samplings

Partitioning the terrestrial carbon sink between vegetation and soil is crucial for predicting future climate change, but the role of soils remains poorly quantified. Here, we compiled 3,099 soil organic carbon time series spanning five decades. We found a global soil organic carbon sink of 1.83 {+/-} 0.9 (mean {+/-} SE) petagrams per year from 1992 to 2020, driven by extratropical young forests, boreal old forests, and grasslands, while trends in tropical ecosystems remain uncertain. Our findings suggest the net land sink resides almost exclusively belowground as soil carbon, emphasizing the global opportunity of soil conservation and restoration for climate mitigation.

ecology↗

bioRxiv2024

Synthesis and pharmacological characterization of UVI3502, a novel cannabinoid receptor 1 (CB1) antagonist/inverse agonist

The endocannabinoid (eCB) system regulates several brain functions and is implicated in neurological disorders. The pharmacological blockade of cannabinoid receptors has a therapeutic potential for various cognitive deficits, but also produces severe psychiatric side effects. Hence, new cannabinoid compounds that potentiate therapeutic effects, while minimizing toxicity, are required. In this study, we synthesized and characterized a novel antagonist/inverse agonist of CB1 receptors. UVI3502 showed affinity for two [3H]CP55,940 binding sites (IC50Hi 0.47 {+/-} 1.94 nM and IC50Lo 1470 {+/-} 1.80 nM). Subsequent binding assays performed in CB1 and CB2 overexpressing membranes determined that the low affinity binding site corresponded to CB1, but the high-affinity binding site of UVI3502 did not correspond to CB2 and the possibility of it corresponding to GPR55 was analyzed. The affinity of UVI3502 for CB1 receptors was further confirmed with neuroanatomical specificity by autoradiography in key brain areas, in which functional [35S]GTP{gamma}S assays demonstrated that UVI3502 behaved as an antagonist/inverse agonist of CB1 receptors, blocking the stimulation evoked by potent cannabinoid receptor agonist CP55,940 and decreasing basal [35S]GTP{gamma}S binding. The in silico characterization of the binding to CB1 receptor through molecular docking and molecular dynamics suggests that this activity is explained by the planar and rigid structure of UVI3502, which is optimal for interactions with the inactive state of the receptor. These results indicate that UVI3502 is a novel antagonist/inverse agonist of CB1 receptors, making it a compelling candidate for pharmacologically blocking cannabinoid receptors in the central nervous system. Significance StatementUVI3502 is a novel antagonist/inverse agonist of CB1 receptors, with almost no affinity for CB2 receptors and an additional high-affinity binding site for a third, cannabinoid-like receptor, potentially GPR55. In relevant brain areas for learning and memory processes with a high expression of CB1, UVI3502 blocks the stimulation evoked by the cannabinoid receptor agonist CP55,940, rendering it as an interesting compound for the pharmacological blockade of cannabinoid receptors in the central nervous system.

pharmacology and toxicology↗

bioRxiv2024

Minimized Sample Consumption for Time-Resolved Serial Crystallography Applied to the Redox Cycle of Human NQO1

Sample consumption for serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) remains a major limitation preventing broader use of this powerful technology in macromolecular crystallography. This drawback is exacerbated in the case of time-resolved (TR)-SFX experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. Thus, in order to reduce the limitation of sample consumption, here we demonstrate the implementation of segmented droplet generation in conjunction with a mix-and-inject approach for TR studies on NAD(P)H:quinone oxidoreductase 1 (NQO1). We present the design and application of mix-and-inject segmented droplet injectors for the Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument at the European XFEL (EuXFEL) with a synchronized droplet injection approach that allows liquid phase protein crystal injection. We carried out TR-crystallography experiments with this approach for a 305 ms and a 1190 ms time point in the reaction of NQO1 with its coenzyme NADH. With this successful TR-SFX approach, up to 97% of the sample has been conserved compared to continuous crystal suspension injection with a gas dynamic virtual nozzle. Furthermore, the obtained structural information for the reaction of NQO1 with NADH is an important part of the future elucidation of the reaction mechanism of this crucial therapeutic enzyme.

biochemistry↗

bioRxiv2022

IL-13-programmed airway tuft cells produce PGE2, which promotes CFTR-dependent mucociliary function

Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single cell RNA sequencing of airway epithelium and mouse modeling, we find that IL-13 expands and programs airway tuft cells towards eicosanoid metabolism, and that tuft cell deficiency leads to a reduction in airway prostaglandin E2 (PGE2) concentration. Allergic airway epithelia bear a signature of prostaglandin E2 activation, and PGE2 activation leads to CFTR-dependent ion and fluid secretion and accelerated mucociliary transport. Together these data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.

immunology↗

bioRxiv2020

Platelet miRNA bio-signature discriminates between dementia with Lewy bodies and Alzheimer disease

Dementia with Lewy bodies (DLB) is one of the most common causes of degenerative dementia after Alzheimers disease (AD) and presents pathological and clinical overlap with both AD and Parkinsons disease (PD). Consequently, only one in three DLB cases is diagnosed correctly. Platelets, previously related to neurodegeneration, contain microRNAs (miRNAs) whose analysis may provide disease biomarkers. Here, we profiled the whole platelet miRNA transcriptome from DLB patients and healthy controls. Differentially expressed miRNAs were further validated in three consecutive studies from 2017 to 2019 enrolling 162 individuals, including DLB, AD, and PD patients, and healthy controls. Results comprised a 7-miRNA biosignature, showing the highest diagnostic potential for the differentiation between DLB and AD. Additionally, compared to controls, two miRNAs were down-regulated in DLB, four miRNAs were up-regulated in AD, and two miRNAs were down-regulated in PD. Predictive target analysis identified three disease-specific clusters of pathways as a result of platelet-miRNA deregulation. Our cross-sectional study assesses the identification of a novel, highly specific and sensitive platelet-associated miRNA-based bio-signature, which distinguishes DLB from AD. The paper explainedO_ST_ABSProblemC_ST_ABSDementia with Lewy bodies (DLB) presents pathological and clinical overlap with both Alzheimers (AD) and Parkinsons disease (PD), which impairs its correct diagnosis. Although numerous papers report peripheral biomarkers for AD, well-established biomarkers for DLB distinguishing it from AD are still missing. Platelet miRNA transcriptome was analyzed in several works, but their putative role as disease biomarkers for neurological disorders has not been assessed. It would be of paramount importance to establish a blood-based bio-signature as a minimally invasive mean for DLB diagnosis, improving differentiation of DLB patients from controls and AD. ResultsOur study revealed that platelet miRNAs might be promising biomarkers for the correct diagnosis of DLB stratifying patients in comparison with overlapping disorders, especially AD, and may help to highlight possible disease-related processes. In this cross-sectional study, which includes 162 individuals (DLB, AD, PD and healthy controls), platelet-associated miRNA content was disease group-specific. Three different miRNA sets together with their predicted targeted pathways were defined. ImpactThis study suggests that platelet miRNA may serve as DLB biomarker allowing the correct diagnosis and stratification in an easily-applied manner in clinical settings, and may help to highlight possible disease-related processes.

neuroscience↗

bioRxiv2019

Deep metagenomics examines the oral microbiome during dental caries, revealing novel taxa and co-occurrences with host molecules

Dental caries is the most common chronic infectious disease globally. The microbial communities associated with caries have mainly been examined using relatively low-resolution 16S rRNA gene amplicon sequencing and/or using downstream analyses that are unsound for the compositional nature of the data provided by sequencing. Additionally, the relationship between caries, oral microbiome composition, and host immunological markers has not been explored. In this study, the oral microbiome and a panel of 38 host markers was analyzed across the saliva from 23 children with dentin caries and 24 children with healthy dentition. Metagenomic sequencing, followed by investigation using tools designed to be robust for compositional data, illustrated that several Prevotella spp. were prevalent in caries, while Rothia spp. were associated with the health. The contributional diversity (extent to which multiple taxa contribute to each pathway) of functional pathways present in the oral microbiome was decreased in the caries group. This decrease was especially noticeable in several pathways known to impede caries pathogenesis, including arginine and branched-chain amino acid biosynthesis. 10 host immunological markers were found to be significantly elevated in the saliva of the caries group, and microbe-metabolite co-occurrence analysis provided an atlas of relationships contributing to the bi-directional influence between the oral microbiome and the host immune system. Finally, 527 metagenome-assembled genomes were obtained from the metagenomics data, representing 151 species. 23 taxa were novel genera/species and a further 20 taxa were novel species. This study thus serves as a model analysis pipeline that will tremendously expand our knowledge of the oral microbiome and its relationship to dental caries once applied to large populations.

microbiology↗

bioRxiv2019

Enhancing Myocardial Repair with CardioClusters

BackgroundCellular therapy to treat heart failure is an ongoing focus of intense research and development, but progress has been frustratingly slow due to limitations of current approaches. Engineered augmentation of established cellular effectors overcomes impediments, enhancing reparative activity with improved outcomes relative to conventional techniques. Such next generation implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, as previously reported by our group, prompted design of a three-dimensional (3D) structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell losses upon delivery. MethodsThree distinct populations of human cardiac interstitial cells including mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and c-Kit+ cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed CardioClusters. Biological consequences of CardioCluster formation were assessed by multiple assays including single cells RNA-Seq transcriptional profiling. Protective effects of CardioClusters in vitro were measured using cell culture models for oxidative stress and myocardial ischemia in combination with freshly isolated neonatal rat ventricular myocytes. Long-term impact of adoptively transferred CardioClusters upon myocardial structure and function in a xenogenic model of acute infarction using NODscid mice was assessed over a longitudinal time course of 20-weeks. ResultsCardioCluster design enables control over composite cell types, cell ratios, size, and preservation of structural integrity during delivery. Profound changes for biological properties of CardioClusters relative to constituent parental cell populations include enhanced expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines. The CardioCluster 3D microenvironment maximizes cellular interaction while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster delivery improves cell retention following intramyocardial injection with preservation of long-term cardiac function relative to monolayer-cultured cells when tested in an experimental murine infarction model followed for up to 20 weeks post-challenge. CardioCluster-treated hearts show increases in capillary density, preservation of cardiomyocyte size, and reduced scar size indicative of blunting pathologic infarction injury. ConclusionsCardioClusters are a novel next generation development and delivery approach for cellular therapeutics that potentiate beneficial activity and enhance protective effects of human cardiac interstitial cell mixed populations. CardioClusters utilization in this preclinical setting establishes fundamental methodologic and biologic insights, laying the framework for optimization of CardioCluster design to provide greater efficacy in cell-based therapeutic interventions intended to mitigate cardiomyopathic damage.

cell biology↗

bioRxiv2019

Adaptation Within Embryonic and Neonatal Heart Environment Reveals Alternative Fates for Adult c-Kit+ Cardiac Interstitial Cells

Cardiac interstitial cells (CIC) perform essential roles in myocardial biology through preservation of homeostasis as well as response to injury or stress. Studies of murine CIC biology reveal remarkable plasticity in terms of transcriptional reprogramming and ploidy state with important implications for function. Despite over a decade of characterization and in vivo utilization of adult c-Kit+ CIC (cCIC), adaptability and functional responses upon delivery to adult mammalian hearts remain poorly understood. Limitations of characterizing cCIC biology following in vitro expansion and adoptive transfer into the adult heart were circumvented by delivery of the donated cells into early cardiogenic environments of embryonic, fetal, and early postnatal developing hearts. These three developmental stages were permissive for retention and persistence, enabling phenotypic evaluation of in vitro expanded cCICs after delivery as well as tissue response following introduction to the host environment. Embryonic blastocyst environment prompted cCIC integration into trophectoderm as well as persistence in amniochorionic membrane. Delivery to fetal myocardium yielded cCIC perivascular localization with fibroblast-like phenotype, similar to cCICs introduced to postnatal P3 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast-like phenotype of exogenously transferred cCICs in fetal and postnatal cardiogenic environments is consistent with inability to contribute directly toward cardiogenesis and lack of functional integration with host myocardium. In contrast, cCICs incorporation into extra-embryonic membranes is consistent with fate of polyploid cells in blastocysts. These findings provide insight into cCIC biology, their inherent predisposition toward fibroblast fates in cardiogenic environments, and remarkable participation in extra-embryonic tissue formation.

cell biology↗

bioRxiv2019

The Autoimmune Susceptibility Gene, PTPN2, Restricts Expansion of a Novel Mouse Adherent-Invasive E. coli

Inflammatory bowel diseases (IBD) involve genetic and environmental factors that play major roles in disease pathogenesis. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase the risk of IBD and are associated with altered microbiome population dynamics in IBD. Moreover, expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in the pathogenesis of IBD as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. The aim of this study was to identify if PTPN2 deficiency disturbs the composition of the intestinal microbiome to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2 we identified increased abundance of a novel adherent-invasive E. coli (AIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages to the IBD associated AIEC, LF82. Furthermore, we confirmed this novel mouse AIEC (mAIEC) caused disease when administered to germ-free and mice lacking segmented-filamentous bacteria (SFB). Moreover, mAIEC infection increased severity of and prevented recovery from dextran-sodium sulfate (DSS)-induced colitis. mAIEC genome sequence analysis showed >90% similarity to LF82. Interestingly, mAIEC contained distinct attachment genes not found in LF82 thereby also demonstrating the novelty of this AIEC. We show here for the first time that an IBD susceptibility gene, PTPN2, plays a key role in modulating the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD.

microbiology↗

bioRxiv2018

Identification of the bacterial biosynthetic gene clusters of the oral microbiome illuminates the unexplored social language of bacteria during health and disease

Small molecules are the primary communication media of the microbial world. Recent bioinformatics studies, exploring the biosynthetic gene clusters (BGCs) which produce many small molecules, have highlighted the incredible biochemical potential of the signaling molecules encoded by the human microbiome. Thus far, most research efforts have focused on understanding the social language of the gut microbiome, leaving crucial signaling molecules produced by oral bacteria, and their connection to health versus disease, in need of investigation. In this study, a total of 4,915 BGCs were identified across 461 genomes representing a broad taxonomic diversity of oral bacteria. Sequence similarity networking provided a putative product class for over 100 unclassified novel BGCs. The newly identified BGCs were cross-referenced against 254 metagenomes and metatranscriptomes derived from individuals with either good oral health, dental caries, or periodontitis. This analysis revealed 2,473 BGCs, which were differentially represented across the oral microbiomes associated with health versus disease. Co-abundance network analysis identified numerous inverse correlations between BGCs and specific oral taxa. These correlations were present in health, but greatly reduced in dental caries, which may suggest a defect in colonization resistance. Finally, corroborating mass spectrometry identified several compounds with homology to products of the predicted BGC classes. Together, these findings greatly expand the number of known biosynthetic pathways present in the oral microbiome and provide an atlas for experimental characterization of these abundant, yet poorly understood, molecules and socio-chemical relationships, which impact the development of caries and periodontitis, two of the worlds most common chronic diseases.\n\nIMPORTANCEThe healthy oral microbiome is symbiotic with the human host, importantly providing colonization resistance against potential pathogens. Dental caries and periodontitis are two of the worlds most common and costly chronic infectious diseases, and are caused by a localized dysbiosis of the oral microbiome. Bacterially produced small molecules, often encoded by BGCs, are the primary communication media of bacterial communities, and play a crucial, yet largely unknown, role in the transition from health to dysbiosis. This study provides a comprehensive mapping of the BGC repertoire of the human oral microbiome and identifies major differences in health compared to disease. Furthermore, BGC representation and expression is linked to the abundance of particular oral bacterial taxa in health versus dental caries and periodontitis. Overall, this study provides a significant insight into the chemical communication network of the healthy oral microbiome, and how it devolves in the case of two prominent diseases.

microbiology↗

bioRxiv2017

Whole-exome sequencing of plasma cell-free DNA portrays the somatic mutation landscape of refractory metastatic colorectal cancer and enables the discovery of mutated KDR/VEGFR2 receptors as modulators of anti-angiogenic therapies.

The non-invasive detection of cancer mutations is a breakthrough in oncology. Here, we applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens including VEGFR inhibitors. Using WES-cfDNA, we could detect 73% (54/74) of the somatic mutations uncovered by WES-tumor including a variety of mutation types: frameshift (indels), missense, noncoding (splicing), and nonsense mutations. Additionally, WES-cfDNA discovered 14 high-confidence somatic mutations not identified by WES-tumor. Importantly, in the absence of the tumor specimen, WES-cfDNA could identify 68 of the 88 (77.3%) total mutations that could be identified by both techniques. Of tumor biology relevance, we identified the novel KDR/VEGFR2 L840F somatic mutation, which we showed was a clonal mutation event in this tumor. Comprehensive in vitro and in vivo functional assays confirmed that L840F causes strong resistance to anti-angiogenic drugs, whereas the KDR/VEGFR2 hot-spot mutant R1032Q confers sensitivity to cabozantinib. Moreover, we found a 1-3% of recurrent KDR somatic mutations across large and non-overlapping cancer sequencing projects, and the majority of these mutations were located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK, suggesting a functional role.\n\nIn summary, the current study highlights the capability of exomic sequencing of cfDNA from plasma of cancer patients as a powerful platform for somatic landscape analysis and discovery of resistance-associated cancer mutations. Because of its advantage to generate results highly concordant to those of tumor sequencing without the hurdle of conventional tumor biopsies, we anticipate that WES-cfDNA will become frequently used in oncology. Moreover, our study identified for the first-time KDR/VEGFR2 somatic mutations as potential genetic biomarkers of response to anti-angiogenic cancer therapies and will serve as reference for further studies on the topic.

cancer biology↗