Biology subjects

Valverde, A.

Publications and source records attributed to Valverde, A..

9 recordsLinked to original sources

bioRxiv2026

Clamp the LAMP: a photoelectrochemical platform for KRAS mutation detection via wild-type blocking

KRAS mutations are among the most prevalent oncogenic alterations in colorectal, lung, and pancreatic cancer, yet their detection remains analytically challenging in the presence of an overwhelming wild-type (WT) background. Here, we report a photoelectrochemical (PEC) genotyping platform that integrates clamp-inhibited loop-mediated isothermal amplification (C-LAMP) with enzyme-free singlet oxygen (1O2)-driven PEC transduction for mutation-selective KRAS detection. Locked nucleic acid (LNA) clamp probes selectively suppress WT amplification during isothermal amplification, enriching mutant alleles and enabling single-nucleotide variant (SNV) discrimination with high selectivity. Amplified products are magnetically captured and transduced into photocurrent via visible-light-induced 1O2 redox cycling, eliminating enzymatic reporters and reducing background interference. The C-LAMP/PEC platform achieves a limit of detection of 35 copies {micro}L-1 (58 aM) and a minimum detectable variant allele frequency (VAF) of 4.8% in heterogeneous mutant/WT genomic DNA mixtures. Analytical performance was validated in cancer cell lines and in patient-derived fresh frozen tissues, showing complete concordance with Nanopore sequencing and droplet digital PCR (ddPCR) within the evaluated cohort (n = 16). This work introduces a robust and modular PEC biosensing strategy that combines molecular WT suppession with enzyme-free photoelectrochemistry, offering an economically competitive and instrumentation-simplified approach for clinically relevant KRAS mutation analysis toward decentralized testing.

bioengineering↗

bioRxiv2025

Non-enzymatic isothermal strand displacement and amplification (NISDA) does not enable sensitive nucleic acid quantification.

Enzyme-free isothermal amplification methods offer a promising alternative to enzymatic assays for nucleic acid detection, particularly in low-resource settings. The nonenzymatic isothermal strand displacement and amplification (NISDA) assay was recently introduced as a highly sensitive, enzyme-free detection strategy. Here, we attempted to replicate its reported performance. Despite extensive testing, we failed to replicate the reported sensitivity and could only produce detectable signals at extremely high target concentrations ([≥]1 x 1011 copies/{micro}L). Our results highlight the critical importance of independent validation in the development of nonenzymatic diagnostic assays.

molecular biology↗

bioRxiv2024

Hampered AMPK-ULK1 cascade in Alzheimer disease (AD) instigates mitochondria dysfunctions and AD-related alterations that are alleviated by metformin

BackgroundMitochondrial structure and function alterations are key pathological features in Alzheimers disease (AD) brains. The adenosine monophosphate-activated protein kinase (AMPK) and its downstream effector Unc-51 like autophagy activating kinase 1 (ULK1) represent a key node controlling mitochondria health, the alteration of which likely contribute to AD development. MethodsWe designed this study to investigate AMPK-ULK1 activation state in post-mortem human sporadic AD brains, in 3xTgAD mice that recapitulate most of human AD features, and in neuronal cells expressing the amyloid precursor protein with the familial Swedish mutation (APPswe). We examined the impact of the pharmacological and genetic modulation of AMPK-ULK1 cascade on mitochondria structure and functions in APPswe cells. We evaluated the potential beneficial impact of AMPK-ULK1 activation by Metformin (Met) on mitochondria defects, as well as on early- and late-stage AD-related alterations in vivo and ex vivo. ResultsAt first, we show that AMPK-ULK1 cascade is defective in murine and human AD brains as well as in APPswe cells. We then report that Met administration to 3xTgAD mice alleviates the alterations of neuronal mitochondria structure and function and we consolidate these results in cells using both pharmacological and genetic tools to modulate AMPK-ULK1 cascade. In mice brains, Met reduces the early accumulation of APP C-terminal fragments (APP-CTFs) as well as the amyloid beta (A{beta}) burden present in aged mice. Mechanistically, we show that Met increases the localization of APP-CTFs within cathepsin D-positive lysosomal compartments in vivo and enhances cathepsin D activity in vitro. The reduction of A{beta} load by Met occurs through an increased recruitment of Iba1+ cells to A{beta} plaques and an enhancement of the phagocytic activity of microglia. Accordingly, in symptomatic 3xTgAD mice, Met alleviates microgliosis and astrogliosis, modulates microglia morphology, reduces peripheral proinflammatory cytokines levels, and regulates the expression of a set of inflammatory genes. In addition, Met normalizes dendritic spines shape in organotypic hippocampal slice cultures modeling AD and improves learning performance of 3xTgAD mice. ConclusionsOur study demonstrates potential therapeutic benefits of targeting AMPK-ULK1 cascade to reverse both early and late AD-related alterations, deserving further investigation in fundamental research and in human clinical studies.

neuroscience↗

bioRxiv2024

Myeloid-derived Suppressor Cells Mitigate Inflammation in Periodontal Disease

Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of immature, immune suppressive myeloid cells. However, their role in periodontal disease (PD), a microbially induced inflammatory oral disease, remains understudied. Here we show that gingiva (gums) from PD patients exhibit significantly higher levels of MDSC markers including Granulocytic (G)-MDSC and Monocytic (M)-MDSC subsets as well as CD4+ T cells and CD19+ B cells. Gingival MDSC subsets exhibit potent immunoregulatory activity as marked by attenuated autologous CD4+ T cells proliferation and IFN{gamma} production. In a murine model of ligature-induced periodontitis (LIP), we noticed time-dependent gingival MDSC infiltration, which correlates with CD4+ T cell and CD19+ B cell infiltration. To test whether MDSC confer immunoregulatory function in vivo, we adoptively transferred G-MDSCs and M-MDSCs in mice. Interestingly, we observed significant reduction in inflammatory marker expression (IL6, TNF-, and IL-1{beta}), infiltration of CD4+ T cells, and concomitant increase in MDSC-derived immune suppressive molecules, ARG1 and IL-10, and CD4+CD25+FoxP3+ Tregs compared to mock. Conversely, depletion of MDSC using anti-Gr1 antibody resulted in marked induction of periodontal inflammation, reduced Treg population, and significantly higher alveolar bone loss. These findings, for the first time, suggest an anti-inflammatory and osteoprotective function of MDSCs in PD and offer a promising target to treat unresolved periodontal inflammation.

immunology↗

bioRxiv2023

Global Profiling of Differentiating Macrophages Identifies Novel Functional Long Non-coding RNAs Regulating Polarization and Innate Immune Responses

Macrophages (M{varphi}) are functionally dynamic immune cells that bridge innate and adaptive immune responses. However, the underlying epigenetic mechanisms that control the macrophage plasticity and innate immune functions are not well-elucidated. Here we performed transcriptome profiling of differentiating M1M{varphi} and M2M{varphi} and identified thousands of previously known and novel lncRNAs. We characterized three M{varphi}-enriched lncRNAs (LRRC75A-As1, GAPLINC and AL139099.5) with novel functions in M{varphi} differentiation, polarization and innate immunity. Knockdown of LRRC75A-As1, and GAPLINC downregulated M{varphi} differentiation markers CDw93 and CD68, and skewed macrophage polarization by decreasing M1 markers but had no significant impact on M2 markers. LRRC75A-As1, and GAPLINC RNAi in M{varphi} attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion supporting their functional role in potentiating innate immune functions. Mechanistically, lncRNA knockdown perturbed the expression of multiple cytoskeleton signaling thereby impairing M{varphi} migration suggesting their critical role in regulating macrophage polarity and motility. Together, our results show that M{varphi} acquire a unique repertoire of lncRNAs to shape differentiation, polarization and innate immune functions.

immunology↗

bioRxiv2023

Long noncoding RNA AL109754.1 Regulates Myeloid Dendritic Cell Differentiation and Potentiates TLR signaling

Dendritic cells (DCs) are key antigen presentation cells (APC) that bridge innate and adaptive immune functions to contain the pathogenic threats. Long noncoding RNAs (lncRNAs) are implicated in functional regulation of various biological processes including inflammation and immunity. However, the knowledge on myeloid DC expressed lncRNA repertoire and their regulatory functions is limited. In this study, we have reconnoitered the time-kinetics of lncRNA expression profiles during monocyte-to-DC differentiation and their roles in shaping DC functions. Our RNA-seq data identified thousands of differentially expressed lncRNAs associated with primary human monocyte-to-DC differentiation in vitro. We selected two lncRNAs viz., AL109754.1 and AC093278.2 that were enriched during DC differentiation. Knockdown of AL109754.1 but not AC093278 affects DCs differentiation as observed by marked reduction of surface markers CD1a, CD93 and CD209. These DCs also exhibit significant reduction in the expression of TLR 2, 4, 5, 7 and 9, suggesting that AL109754.1 expression is critical in maintaining TLR expression in DCs. Furthermore, reduced phosphorylation of NF-{kappa}B, IRF3 and IRF7 in AL109754.1 knockdown DCs treated with TLR agonists further substantiate their role in potentiating TLR signaling. Mechanistically, AL109754.1 knockdown DC showed significant downregulation of multiple NF-{kappa}B-induced genes and time-dependent inhibition of pro-inflammatory cytokine (IL-1{beta}, IL-6, IL-8 and TNF) secretion upon challenge with TLR 4, 5, or 7 agonists. Overall, this study characterized novel functions of AL109754.1 that regulates DC differentiation, and TLR-dependent innate immune activation.

immunology↗

bioRxiv2023

Dynamic Changes in Macrophage Polarization during the Resolution Phase of Periodontal Disease

Periodontal inflammation is largely governed by infiltration of myeloid cells, in particular macrophages. Polarization of M{varphi} within the gingival tissues is a well-controlled axis and has considerable consequences for how M{varphi} participate in inflammatory and resolution (tissue repair) phases. We hypothesize that periodontal therapy may instigate a pro-resolution environment favoring M2 M{varphi} polarization and contribute towards resolution of inflammation post-therapy. We aimed to evaluate the markers of macrophage polarization before and after periodontal therapy. Gingival biopsies were excised from human subjects with generalized severe periodontitis, undergoing routine non-surgical therapy. A second set of biopsies were excised after 4-6 weeks to assess the impact of therapeutic resolution at the molecular level. As controls, gingival biopsies were excised from periodontally healthy subjects, undergoing crown lengthening. Total RNA was isolated from gingival biopsies to evaluate pro- and anti-inflammatory markers associated with macrophage polarization by RT-qPCR. Mean periodontal probing depths, CAL and BOP reduced significantly after therapy and corroborated with the reduced levels of periopathic bacterial transcripts after therapy. Compared to heathy and treated biopsies, higher load of Aa and Pg transcripts were observed in disease. Lower expression of M1M{varphi} markers (TNF-, STAT1) were observed after therapy as compared to diseased samples. Conversely, M2M{varphi} markers (STAT6, IL-10) were highly expressed in post-therapy as opposed to pre-therapy, which correlated with clinical improvement. These findings corroborated with murine ligature-induced periodontitis and resolution model, comparing the respective murine M{varphi} polarization markers (M1 M{varphi}: cox2, iNOS2 and M2 M{varphi}: tgm2 and arg1). Our findings suggest that imbalance in M1 and M2 polarized macrophages by assessment of their markers can provide relevant clinical information on the successful response of periodontal therapy and can be used to target non-responders with exaggerated immune responses.

immunology↗

bioRxiv2023

LncRNA MALAT1/microRNA-30b axis regulate macrophage polarization and function

IntroductionMacrophages (M{varphi}) can polarize towards the proinflammatory M1 or proresolving M2 phenotype to control diverse biological processes such as inflammation, and tissue regeneration. Noncoding RNAs play critical roles in numerous biological pathways; however, their functional interaction in the regulation of M{varphi} polarization and immune responses remain unclear. ObjectivesTo examine relationship between lncRNA (MALAT1) and microRNA (miR-30b) in shaping macrophage polarization and immune functions. MethodsExpression of MALAT1 and miR-30b was examined in differentiating M1/M2 M{varphi}, human and murine inflamed gingival biopsies by RT-qPCR. MALAT1 and miR-30b direct interaction was examined by dual luciferase assays. Impact of MALAT1 knockdown and miR-30b overexpression was examined on macrophage polarization markers, bacterial phagocytosis, antigen uptake/processing and cytokine profiles. ResultsMALAT1 expression displays a time-dependent induction during M{varphi} differentiation and, upon challenge with TLR4 agonist (E. coli LPS). Knockdown of MALAT1 enhanced the expression of M2M{varphi} markers without affecting the M1M{varphi} markers, suggesting that MALAT1 favors the M1 phenotype by suppressing M2 polarization. MALAT1 knockdown M{varphi} exhibit reduced antigen uptake and processing, bacterial phagocytosis, and bactericidal activity, strongly supporting its critical role in regulating innate immune functions. Consistent with this, MALAT1 knockdown showed impaired cytokine secretion upon challenge with LPS. Importantly, MALAT1 exhibit an antagonistic expression pattern with all five members of the miR-30 family during M2M{varphi} differentiation. Dual-luciferase assays validated a novel sequence on MALAT1 that interacts with miR-30b, a microRNA that promotes the M2 phenotype. Phagocytosis and antigen processing assays unequivocally demonstrated that MALAT1 and miR-30b are functionally antagonistic. In human subjects with periodontal disease and murine model of ligature-induced periodontitis, we observed higher levels of MALAT1, and downregulation of miR-30b that correlates with higher M1M{varphi} markers expression in gingival tissues suggesting a pro-inflammatory function of MALAT1. ConclusionMALAT1/miR-30b antagonistic interaction shapes M{varphi} polarization in vitro and in inflamed gingival biopsies.

immunology↗

bioRxiv2019

Clinical evaluation of postoperative analgesia and serum biochemical changes of paracetamol compared to meloxicam and carprofen in bitches undergoing ovariohysterectomy

BackgroundIn veterinary medicine, the administration of nonsteroidal anti-inflammatory analgesics (NSAIDs) for the control of postsurgical pain in dogs and cats is common given the anti-inflammatory, analgesic, and antipyretic effects of these drugs. This study compared the serum biochemical changes and postoperative analgesic effects of paracetamol, meloxicam, and carprofen in bitches submitted to an ovariohysterectomy using the Dynamic Interactive Visual Analog Scale (DIVAS) and Pain Scale of the University of Melbourne (UMPS) scoring systems.\n\nMethodsThirty bitches of different breeds underwent elective ovariohysterectomies and were randomly assigned to one of three treatment groups: a paracetamol group [15 mg kg-1 intravenous (IV)], a carprofen group (4 mg kg-1 IV), and a meloxicam group (0.2 mg kg-1 IV). All treatments were administered 30 minutes prior to surgery. Paracetamol was administered every 8 hours postoperatively for 48 hours total, while carprofen and meloxicam were intravenously administered every 24 hours. An evaluation of post-surgical pain was done with the DIVAS and the UMPS. The first post-surgical pain measurement was performed 1 hour after surgery and then 2, 4, 6, 8, 12, 16, 20, 24, 36, and 48 hours after surgery.\n\nResultsAll groups exhibited a gradual reduction in pain throughout the postoperative period in both scales; however, neither scale significantly differed between the three treatment groups (P > 0.05) during the 48 postoperative hours.\n\nConclusionsParacetamol was as effective as meloxicam and carprofen for post-surgical analgesia in bitches subjected to elective ovariohysterectomy. The present study demonstrates that paracetamol may be considered a tool for the effective treatment of acute perioperative pain in dogs. Furthermore, this drug led to no adverse reactions or changes in the parameters assessed in the present study, indicating its safety.

pharmacology and toxicology↗