Biology subjects

Pulido, C.

Publications and source records attributed to Pulido, C..

11 recordsLinked to original sources

bioRxiv2026

ACB1801 enhances tumor immunogenicity by targeting glycolysis/ferroptosis vulnerability and activating STAT1-signaling to overcome anti-PD-1 resistance in MSS colorectal cancer

BackgroundImmune checkpoint blockade (ICB) therapies demonstrate low efficacy in microsatellite stable (MSS) colorectal cancer (CRC) due to an immune-desert tumor microenvironment (TME) characterized by low antigen presentation and limited tumor-infiltrating lymphocytes (TILs). Harmine, a natural small-molecule and its promising derivatives ACB1801 have shown anti-tumor potential in preclinical models; however, their potential to reprogram the TME and overcome ICB resistance in MSS CRC remains unexplored. This study investigates whether and how ACB1801 can reshape TME to sensitize MSS CRC to ICB therapies. MethodsWe used the CT26 MSS colorectal cancer mouse model to evaluate the ability of the harmine derivative ACB1801 to enhance the efficacy of anti-PD-1 therapy. To characterize its mode of action, we performed immune landscape analysis and transcriptomic profiling of both CD45- and CD45+ tumor-derived cells. In parallel, mechanistic studies were conducted in vitro using mouse and human MSS CRC cell lines. ResultsWe demonstrate that the harmine derivative ACB1801 enhances the effectiveness of anti-PD-1 therapy in an MSS CRC mouse model. Combination therapy significantly increased CD8+ T cell infiltration and reduced regulatory T-cell (Treg) density in the TME. Transcriptomic profiling of CRC cells isolated from tumors treated with either anti-PD-1 alone or in combination with ACB1801 revealed significant enrichment of metabolic pathways in the combination group, characterized by reduced glycolysis and enhanced ferroptosis signatures. These findings were supported by in vitro data showing that ACB1801 reduces tumor cell glycolytic activity and promotes ferroptotic vulnerability. Mechanistically, ACB1801 induced STAT1 signaling, promoted CXCL10 release, and enhanced major histocompatibility complex class I (MHC-I)-dependent antigen presentation on tumor cells, thereby increasing tumor susceptibility to anti-PD-1 therapy. ConclusionCollectively, our findings indicate that combination therapy with harmine derivatives and ICBs represents a promising strategy for treating MSS CRC patients.

cancer biology↗

bioRxiv2025

Neuromodulator control of energy reserves in dopaminergic neurons

The brain is a metabolically vulnerable organ as neurons have both high resting metabolic rates and the need for local rapid conversion of carbon sources to ATP during activity. Midbrain dopamine neurons are thought to be particularly vulnerable to metabolic perturbations, as a subset of these are the first to undergo degeneration in Parkinsons disease (PD), a neurodegenerative disorder long suspected to be in part driven by deficits in mid-brain bioenergetics (1). In skeletal muscle, energy homeostasis under varying demands is achieved in part by its ability to rely on glycogen as a fuel store, whose conversion to ATP is under hormonal regulatory control. In neurons however the absence of easily observable glycogen granules has cast doubt on whether this fuel store is operational, even though brain neurons express the key regulatory enzymes associated with building or burning glycogen (2). We show here that that in primary mid brain dopaminergic neurons, glycogen availability is under the control of dopamine auto receptors (D2R), such that dopamine itself provides a signal to store glycogen. We find that when glycogen stores are present, they provide remarkable resilience to dopamine nerve terminal function under extreme hypometabolic conditions, but loss of this dopamine derived signal, or impairment of access to glycogen, makes them hypersensitive to fuel deprivation. These data show that neurons can use an extracellular cue to regulate local metabolism and suggest that loss of dopamine secretion might make dopamine neurons particularly subject to neurodegeneration driven by metabolic stress. Significance StatementThis work demonstrates that a neurons metabolic resilience is actively shaped by local neuromodulation, providing a potential explanation for why different neuronal subtypes exhibit unequal vulnerability to metabolic stressors and why dopamine neurons become more vulnerable when they lose the capacity to release dopamine.

neuroscience↗

bioRxiv2024

Two strains of Toscana virus show different virulence and replication capacity in mice and cell culture models

Toscana virus (TOSV), belonging to Phenuiviridae family, is circulating in most Mediterranean countries and is transmitted to humans by infected female sand flies. While most infections are asymptomatic, TOSV is considered as a leading cause of meningitis and encephalitis in humans during summer. Three TOSV genotypes (named A, B, and C) have been identified, although no virus strain belonging to lineage C has been isolated so far. To date, the relationship between TOSV genetic diversity and viral pathogenicity or replication capacity remains unknown. This study aimed to compare two TOSV strains from either lineage A (TOSV-A) or B (TOSV-B) in several cell culture and two mouse models. We showed that TOSV-A replicated more efficiently in BSR and A549 cells while TOSV-B had a replication advantage in human induced pluripotent stem cells differentiated in neural cells and LL-5 sand fly cells. In vivo, we were unable to detect any virus in the brains of immunocompetent C57BL/6JRj mice infected with either strain of TOSV. On the contrary, we showed that TOSV-A disseminated to the central nervous system of 129/Sv ifnar -/- mice unlike TOSV-B, despite higher viremia of TOSV-B and a greater dissemination of this strain in other organs. The reasons for these differences are not yet known, although we showed that the presence of TOSV neutralizing antibodies in serum was slightly delayed in TOSV-A-infected mice. Altogether, the data presented in this study provide new avenues to study TOSV-induced pathogenesis and ultimately unveil molecular viral determinants modulating TOSV replication capacity. Author SummaryToscana virus is a sand fly-borne phlebovirus with tropism for the human central nervous system (CNS) and the causal agent of aseptic meningitis and encephalitis during summer in countries surrounding the Mediterranean Sea. The pathogenesis induced by TOSV is still poorly understood as well as the correlation between TOSV genetic diversity and viral pathogenicity or replication capacity. In this study, we characterised two TOSV strains belonging to two different genetic lineages (named A and B) in several in vitro, ex vivo and in vivo models of infection. We showed that the biological properties of these two strains is strikingly different and, importantly, we identified 129/Sv ifnar -/- mice as a suitable model to study the mechanism of TOSV invasion of the CNS and viral pathogenesis. Our comparative study also highlights the importance of strengthening our efforts to identify molecular viral determinants modulating TOSV-induced pathogenesis and vector transmission, in order to better characterise the epidemiological risk posed by circulating strains.

microbiology↗

bioRxiv2023

iATPSnFR2: a high dynamic range fluorescent sensor for monitoring intracellular ATP

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted super-folder GFP inserted between the ATP-binding helices of the{varepsilon} -subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a [~] 5-6 fold improvement in the dynamic range compared to the previous generation sensor, with excellent discrimination against other analytes and affinity variants varying from 4 M to 500 M. A chimeric version of this sensor fused to either the HaloTag protein or a suitably spectrally separated fluorescent protein, provides a ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting of the sensor to nerve terminals reveals previously uncharacterized single synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics. Significance StatementAdenosine triphosphate (ATP) is a key metabolite necessary for cellular life. Here we develop a next-generation genetically encoded ratiometric fluorescent ATP sensor that allows subcellular tracking of ATP levels in living cells. The large dynamic range makes it possible to follow the dynamics of this metabolite across cells and subcellular regions under different metabolic stressors. We expect that iATPSnFR2 will provide researchers with exciting new opportunities to study ATP dynamics with temporal and spatial resolution that has, until now, been unavailable.

biochemistry↗

bioRxiv2023

PKM2 diverts glycolytic flux in dependence on formate overflow

Throughout the metastatic cascade, cancer cells are faced with harsh metabolic environments and nutritional stresses which apply selection pressure leaving only the most metabolically resilient cells to survive and form metastases. Metabolic characterisation of such cell populations in vitro is currently challenging. Using galactose as a tool compound to mimic glycolytic limitation within the tumour microenvironment of primary and secondary neoplastic sites, we were able to uncover metabolic flexibility and plasticity of cancer cells in vitro. In contrast to the established idea that high glycolytic flux and expression of dimeric PKM2 redirects carbons towards anabolic routes such as the pentose phosphate pathway and serine synthesis pathway (SSP), we have discovered by using stable-isotope tracing that also glycolytic limitation results in metabolic rewiring. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near complete block of pyruvate kinase isozyme M2 (PKM2) to divert carbons towards SSP. Simultaneously, TCA cycle flux is sustained and oxygen consumption is increased, both supported by glutamine. Glutamine not only supports TCA cycle flux but also SSP via distinct mechanisms. Due to PKM2 block, malic enzyme exclusively supports TCA cycle flux while mitochondrial phosphoenolpyruvate carboxykinase supports SSP. Moreover, by using genetic modifications of different one-carbon (1C) cycle enzymes, we are able to reverse the PKM2 block suggesting a link between mitochondrial 1C cycle and pyruvate kinase. Thus we show that PKM2 inhibition acts as a branching point to direct glycolytic and glutamine carbons into distinct routes, overall supporting the metabolic plasticity and flexibility of cancer cells.

cancer biology↗

bioRxiv2021

Multi-hallmark long noncoding RNA maps reveal non-small cell lung cancer vulnerabilities

Long noncoding RNAs (lncRNAs) are widely dysregulated in cancer, yet their functional roles in cellular disease hallmarks remain unclear. Here we employ pooled CRISPR deletion to perturb all 831 lncRNAs in KRAS-mutant non-small cell lung cancer (NSCLC), and measure their contribution to proliferation, chemoresistance and migration across two cell backgrounds. Integrative analysis of this data outperforms conventional "dropout" screens in identifying cancer genes, while prioritising disease-relevant lncRNAs with pleiotropic and background-independent roles. Altogether 60 high-confidence oncogenic lncRNAs are active in NSCLC, the majority identified here for the first time, and which tend to be amplified and overexpressed in tumours. A follow-up antisense oligonucleotide (ASO) screen shortlisted two candidates, Cancer Hallmarks in Lung LncRNA (CHiLL 1&2), whose knockdown consistently suppressed cancer hallmarks in a variety of 2D and 3D tumour models. Molecular phenotyping reveals that CHiLL 1&2 control cellular-level phenotypes via distinct transcriptional networks converging on common oncogenic pathways. In summary, this work reveals a multi-dimensional functional lncRNA landscape underlying NSCLC that contains potential therapeutic vulnerabilities.

genomics↗

bioRxiv2020

Synaptic vesicle pools are a major hidden resting metabolic burden of nerve terminals

The human brain is a uniquely vulnerable organ as interruption in fuel supply leads to acute cognitive impairment on rapid time scales. The reasons for this vulnerability are not well understood, but nerve terminals are likely loci of this vulnerability as they do not store sufficient ATP molecules and must synthesize them on-demand during activity or suffer acute degradation in performance. The requirements for on-demand ATP synthesis however depends in part on the magnitude of resting metabolic rates. We show here that, at rest, synaptic vesicle (SV) pools are a major source of presynaptic basal energy consumption. This basal metabolism arises from SV-resident V-ATPases compensating for a hidden resting H+ efflux from the SV lumen. We show that this steady-state H+ efflux is 1) mediated by vesicular neurotransmitter transporters, 2) independent of the SV cycle, 3) accounts for ~half of the resting synaptic energy consumption and 4) contributes to nerve terminal intolerance of fuel deprivation. O_LINerve terminals consume large amounts of ATP even at rest C_LIO_LIA large fraction of the resting metabolic burden arises from the V-ATPase C_LIO_LISteady-state V-ATPase activity is driven by a synaptic vesicle H+ leak C_LIO_LIThis H+ leak is mediated vesicular neurotransmitter transporters C_LI

neuroscience↗

bioRxiv2020

Hamster and ferret experimental infection with intranasal low dose of a single strain of SARS-CoV-2

Understanding the pathogenesis of the SARS-CoV-2 infection is key to develop preventive and therapeutic strategies against COVID-19, in the case of severe illness but also when the disease is mild. The use of appropriate experimental animal models remains central in the in-vivo exploration of the physiopathology of infection and antiviral strategies. This study describes SARS-CoV-2 intra-nasal infection in ferrets and hamsters with low doses of low-passage SARS-CoV-2 clinical French isolate UCN19, describing infection levels, excretion, immune responses and pathological patterns in both animal species. Individual infection with 103 pfu SARS-CoV-2 induced a more severe disease in hamsters than in ferrets. Viral RNA was detected in the lungs of hamsters but not of ferrets and in the brain (olfactive and/or spinal bulbs) of both species. Overall, the clinical disease remained mild, with serological responses detected from 7 days and 10 days post inoculation in hamsters and ferrets respectively. Virus became undetectable and pathology resolved within 14 days. The kinetics and levels of infection can be used in ferrets and hamsters as experimental models for understanding the pathogenicity of SARS-CoV-2, and testing the protective effect of drugs.

microbiology↗

bioRxiv2020

How bank vole-PUUV interactions influence the eco-evolutionary processes driving nephropathia epidemica epidemiology: An experimental and genomic approach.

In Europe, Puumala virus (PUUV) is responsible for nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFSR). Despite the presence of its reservoir, the bank vole, on most of French territory, the geographic distribution of NE cases is heterogeneous and NE endemic and non-endemic areas have been reported. In this study we analyzed whether bank vole-PUUV interactions could partly shape these epidemiological differences. We performed crossed-experimental infections using wild bank voles from French endemic (Ardennes) and non-endemic (Loiret) areas, and two French PUUV strains isolated from these areas. The serological response and dynamics of PUUV infection were compared between the four cross-infection combinations. We showed that the serological response and the presence of PUUV in excretory organs were more important in bank voles infected with the PUUV endemic strain. Moreover, the within-host viral diversity in excretory organs was higher than in other non-excretory organs for the NE endemic cross-infection, but not for the NE non-endemic cross-infection. Altogether, our results showed that genetically different PUUV strains, and in a lesser extent their interaction with sympatric bank voles, could affect virus replication and diversity. This could impact PUUV excretion/transmission between rodents and to humans, and in turn at least partly shape NE epidemiology in France.

evolutionary biology↗

bioRxiv2020

Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters

Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-COV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria.

neuroscience↗

bioRxiv2017

Bank Vole Immunoheterogeneity May Limit Nephropatia Epidemica Emergence In A French Non-Endemic Region

Ecoevolutionary processes affecting hosts, vectors and pathogens are important drivers of zoonotic disease emergence. In this study, we focused on nephropathia epidemica (NE), which is caused by Puumala hantavirus (PUUV) whose natural reservoir is the bank vole, Myodes glareolus. Despite the continuous distribution of the reservoir in Europe, PUUV occurence is highly fragmented. We questioned the possibility of NE emergence in a French region that is considered to be NE-free but that is adjacent to a NE-endemic region. We first confirmed the epidemiology of these two regions using serological and virological surveys. We used bank vole population genetics to demonstrate the absence of spatial barriers that could have limited dispersal, and consequently, the spread of PUUV into the NE-free region. We next tested whether regional immunoheterogeneity could impact PUUV chances to establish, circulate and persist in the NE-free region. Immune responsiveness was phenotyped both in the wild and during experimental infections, using serological, virological and immune related gene expression assays. We showed that bank voles from the NE-free region were sensitive to experimental PUUV infection. We observed high levels of immunoheterogeneity between individuals and also between regions. In natural populations, antiviral gene expression (Tnf and Mx2 genes) reached higher levels in bank voles from the NE-free region. During experimental infections, anti-PUUV antibody production was higher in bank voles from the NE endemic region. Altogether, our results indicated a lower susceptibility to PUUV for bank voles from this NE-free region, what might limit PUUV circulation and persistence, and in turn, the risk of NE.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC=\"FIGDIR/small/130252_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (25K):\norg.highwire.dtl.DTLVardef@f90a69org.highwire.dtl.DTLVardef@1a9e0dorg.highwire.dtl.DTLVardef@17e96b8org.highwire.dtl.DTLVardef@1d936e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗