Biology subjects

Valenzuela, C.

Publications and source records attributed to Valenzuela, C..

12 recordsLinked to original sources

bioRxiv2026

Monosynaptic connections link functionally similar regions in human cortex

The functional organization of human cortex has been mapped in considerable detail1-5, yet the axonal connections linking these areas remain largely unknown. This gap precludes inferring either the computational pathways through cortical networks or the principles governing why each area connects to its particular target regions. Here, in patients undergoing intracranial monitoring for epilepsy, we used concurrent electrical stimulation and functional magnetic resonance imaging (es-fMRI), validated previously against tracer studies in nonhuman primates6, to map anatomical connectivity of cortical sites across the whole brain, and combined these maps with task and resting-state fMRI in the same individuals. Es-fMRI revealed four main findings. First, connectivity followed an asymmetric functional-similarity principle: connected sites tended to share similar functional profiles, but, because connectivity is sparse, most pairs of functionally similar sites were not connected. Second, although connectivity also declines with distance7, the functional profile explained three times as much variance in connectivity as distance did, and long-range connections were the most functionally specific. Third, es-fMRI revealed direct monosynaptic links between established functional regions, including between the fusiform face area (FFA)2 and the temporoparietal junction (TPJ)3 for social cognition. Fourth, resting-state functional connectivity (rsFC) of a stimulation site was only weakly correlated with that sites es-fMRI connectivity. Together, these results provide a first principled link between anatomical connectivity and functional organization in the human cortex, and establish es-fMRI as a scalable approach to building a tracer-grade connectome of the human brain.

neuroscience↗

bioRxiv2025

IQM-22110 as a selective KV4.3/KChIP3 modulator. Molecular determinants of the KChIP3 binding site

The goal of the present study was to discover novel KChIP ligands as research tools for modulating the KV4.3/KChIP channels. By employing a multidisciplinary approach, combining medicinal chemistry and electrophysiology studies, a novel KV4.3/KChIP modulator (IQM-22110) was successfully identified. IQM-22110 has emerged from the combination of our prior knowledge regarding the (phenylacetamido)benzoic acid moiety as an effective scaffold for KChIP3 ligands and a virtual screening of a focused chemical library. Guided by docking studies--which indicated that incorporating an additional aromatic ring could enhance binding affinity--IQM-22110 was selected for synthesis and identified as a potent KChIP3 ligand. Its electrophysiological effects on KV4.3/KChIP3 currents indicate that IQM-22110 binds to a high affinity site in KV4.3/KChIP3 channels that it is not present in KV4.3/KChIP2 or KV4.3. To the best of our knowledge, here we describe the first KChIP3 ligand that selectively modulates KV4.3/KChIP3 versus KV4.3/KChIP2 and KV4.3 alone channels. Given that KChIP2 is primarily expressed in heart, our findings might pave the way for the development of KV4.3/KChIP3 blockers with reduced cardiac side effects. Computational and site-directed mutagenesis studies allowed the identification of IQM-22110s binding site on KChIP3. Knowledge gained from our structural and functional studies with this novel KChIP3 ligand could establish the basis for drug discovery programs fostering treatments for diseases in which KV4.3/KChIPs channels are involved.

pharmacology and toxicology↗

bioRxiv2025

Provision of Preferred Nutrients to Macrophages Enables Salmonella to Replicate Intracellularly Without Relying on Type III Secretion Systems

Intracellular survival and replication within macrophages are key virulence determinants of Salmonella enterica serovar Typhimurium. This phenomenon is traditionally attributed to the activity of its two Type III Secretion Systems (T3SS) and their associated effectors. A critical challenge for these bacteria is acquiring nutrients from inside the host cell. Thus, they modulate the metabolism of host cells to replicate. Given the metabolic plasticity of macrophages, a key unresolved question is how their metabolic heterogeneity shapes intracellular Salmonella replication. By using human primary macrophages and live-cell imaging to monitor bacterial dynamics at the single-cell level, we revealed that Salmonella does not replicate in all infected cells. However, supplementation with specific carbon sources used by Salmonella during infection accelerated bacterial replication and increased the proportion of macrophages showing replicative bacteria. Remarkably, this occurred even in the absence of functional T3SSs, as a {Delta}prgH/{Delta}ssaV double mutant was able to replicate in a subset of infected cells under favorable nutrient conditions. These phenotypes are further amplified in macrophages with higher glycolytic activity, such as the murine RAW 264.7 cell line. Further analyses demonstrated that enhanced Salmonella replication is not strictly dependent on host glycolytic activity but is instead driven by the ability of the host cell to take up the nutrients Salmonella prefers for its replication early during infection. In summary, our findings suggest that the dependence of Salmonella on its T3SSs for intracellular replication can be bypassed when host cells provide optimal access to key nutrients and highlight the impact of metabolic heterogeneity in shaping intracellular bacterial replication during infection of macrophages.

microbiology↗

bioRxiv2025

Plasmidome-defensome interactions drive adaptation of 'Fervidacidithiobacillus caldus' in natural and industrial extreme acidic environments

Plasmids are major drivers of microbial evolution, enabling horizontal gene transfer (HGT) and facilitating adaptation through the dissemination of relevant functional genes and traits. However, little is known about plasmid diversity and function in extremophiles. Fervidacidithiobacillus caldus, a meso-thermo-acidophilic sulfur oxidizer, is a key player in sulfur cycling in natural and industrially engineered acidic environments. Here, we present a comprehensive analysis of the plasmidome, and associated anti-mobile genetic element (anti-MGE) defense systems (defensome), across genomes of this species and metagenomes from diverse natural and industrial settings harboring F. caldus. We identified >30 distinct plasmids, representing five consistent replication-mobilization families. Plasmids ranged in size between 2.5-65 kb, with gene content and plasmid modularity scaling with element size and copy numbers inversely correlating with size. Plasmids carried variable numbers of hypothetical proteins and transposases, with annotated cargo genes reflecting functional differentiation by habitat. Defensome profiling revealed over 50 anti-MGE systems in sequenced F. caldus isolates, including diverse restriction-modification systems, CRISPR-Cas types IV-A and V-F, and widespread abortive infection and composite defense systems such as Wadjet, Gabija, and Zorya. In environmental populations, an inverse relationship was observed between defensome complexity and plasmidome abundance and diversity, underscoring a pivotal role of the host defensome in modulating persistence, compatibility, and overall plasmid diversity across F. caldus populations. Yet, other plasmids appeared decoupled from both host abundance and defensome complexity, suggesting potential host shifts, environmental persistence, or differential replication under suboptimal growth conditions for the host. Altogether, these findings reveal a modular, adaptive plasmidome shaped by selective pressures and host-plasmid-defensome interactions and positions plasmids as key contributors to adaptation, gene flow, and functional innovation in this extreme acidophile. ImportancePlasmids are key vehicles of gene exchange and adaptation in bacteria, yet their roles in extremophilic systems remain poorly understood. This study provides the first integrated view of the plasmidome and defense systems in Fervidacidithiobacillus caldus, a sulfur-oxidizing acidophile relevant to both natural biogeochemical cycling and industrial bioleaching. We uncover a rich plasmid diversity structured into modular families with variable cargo and backbone features and reveal their coexistence with complex anti-MGE defense repertoires. By combining genomic and metagenomic approaches, we expose principles of plasmid compatibility, persistence, and habitat-specific adaptation. These insights expand current knowledge of mobile genetic elements in extreme environments and provide a foundation for plasmid-based vector design and synthetic biology in acidophiles, with direct implications for biomining and environmental remediation in extreme environments.

genomics↗

bioRxiv2025

SifA-mediated Remodeling of the Salmonella-Containing Vacuole Prevents Bacterial Dormancy by Promoting Nutrient Accessibility

Salmonella exploits a range of intracellular niches within host cells, adapting to different microenvironments that support its survival and replication. This niche diversity is mediated by bacterial effectors injected via the two type 3 secretion systems (T3SS). Salmonella resides either within membrane-bound Salmonella containing vacuoles (SCV) or they grow rapidly within the cytosol upon SCV rupture. Recently, we identified a third intracellular subpopulation, dormant Salmonella within modified vacuoles in epithelial cells, which can survive for up to 7 days in vitro. To explore how bacterial effectors influence the balance of these three subpopulations, we constructed a panel of mutants lacking genes encoding key effectors and examined their intracellular behaviors at 6 and 24 hours post-infection (hpi). Deletion of the T3SS-2 effector SifA significantly increased the dormant subpopulation at later infection time points, identifying SifA as the first known effector regulating bacterial dormancy in epithelial cells. SifA-induced Salmonella-induced filaments (SIFs) characterize the mature Salmonella-containing vacuole (SCV), and we observed that SIFs were needed for intravacuolar growth of Salmonella. We could show that SIF formation was critical for nutrient acquisition within the SCV; inhibition of SIF formation resulted in a higher proportion of dormant bacteria, akin to the effect of reduced glucose availability during infection. Both bacterial and host glycolysis pathways were required to prevent dormancy, as proper nutrient scavenging through SIF-mediated modification of the SCV is essential for maintaining a replicative, non-dormant state. These results underscore the importance of nutrient access, facilitated by reprogramming host endomembrane trafficking, for Salmonella to avoid dormancy and sustain active replication within its intracellular niche. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/631959v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d4e9cdorg.highwire.dtl.DTLVardef@419d70org.highwire.dtl.DTLVardef@1c75fb0org.highwire.dtl.DTLVardef@5b5d59_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

RvD1 and LXA4 inhibitory effects on cardiac voltage-gated potassium channels

AimsThe resolution of inflammation is modulated by specialized pro-resolving lipid mediators (SPMs), which can be modified in some cardiovascular diseases. Among them, RvD1 and LXA4 prevent atrial fibrillation (AF) remodeling in the atria and cardiac hypertrophy, respectively in animal models. However, little is known about their electrophysiological effects on cardiac voltage-gated (VG) ion channels. Methods and resultsWe used the patch-clamp technique in heterologous systems and cardiomyocytes to assess the acute effect of RvD1, and LXA4, on VG potassium currents. In silico simulations were used to predict the effect of current modulation on the atrial and ventricular action potentials (AP). RvD1 and LXA4 reduced IKs (channel KV7.1/KCNE1) in COS-7 cells and guinea-pig cardiomyocytes without modifying its voltage dependence; RvD1 was more potent than LXA4. In heterologous systems, RvD1 was also tested on IKur (channel KV1.5), Ito (channel KV4.3/KChIP2), IKr (channel KV11.1), and IK1 (channel Kir2.1) with the largest inhibitory effect on IKs and IKr. In simulations RvD1 prolonged repolarization significantly in both atrial and ventricular myocytes. ConclusionThe results provide a comprehensive evaluation of RvD1 and LXA4 on cardiac human potassium channels, at pathophysiological relevant concentrations, being RvD1 more potent than LXA4. The predicted effects on the action potential suggest that, along with their antiinflammatory action, RvD1 may reverse AF-induced electrical remodeling in the atria by direct modulation of K+ currents. The same action might instead contribute to ventricular functional remodeling; however, direct evidence for this is missing.

pharmacology and toxicology↗

bioRxiv2024

Novel insights into IKur modulation by Lgi3-4: Implications in atrial fibrillation

BackgroundPatients with atrial fibrillation (AF) exhibit a reduction in the ultrarapid outward potassium current (IKur) conducted by KV1.5 channels. Ion channels are closely modulated by regulatory subunits, forming macromolecular complexes known as channelosomes. One such regulatory family is the leucine-rich glioma-inactivated protein family (Lgi1-4), which has been shown to interact with KV1, modifying their trafficking and/or biophysical properties in neurons. However, the expression and impact of these proteins in the heart is still unknown. We investigated the role of Lgi3-4 proteins in cardiac electrophysiology, focusing specifically on IKur, and their potential contribution to the pathophysiology of AF. MethodsWe used three complementary biological systems, including heterologous COS-7, HEK297 and CHO cells, AAV-mediated cardiac-specific Lgi4 gene transfer in mice (Lgi4 mice), and human samples from patients in sinus rhythm and AF. Our multidisciplinary approach included immunolocalization, patch clamping, surface ECG, transvenous catheter-mediated intracardiac stimulation, and molecular biology techniques. ResultsOnly Lgi3 and Lgi4 were expressed in the human heart. In human atrial tissue and heterologous cells, Lgi3 and Lgi4 interacted with KV1.5 channels. In HEK293 cells, Lgi3-4 impaired KV1.5/KV{beta} association, partially reversing the KV{beta}-induced N-type inactivation and reducing IKur amplitude. On surface ECG, the QRS interval was prolonged, and impulse conduction was impaired in cardiac-specific Lgi4 mice compared with control. In isolated ventricular cardiomyocytes from Lgi4 mice, early action potential repolarization was prolonged compared to control cardiomyocytes. These results correlated with the reduced KV1.5 membrane expression and IKur density observed in Lgi4 cardiomyocytes and HEK293 cells. Notably, Lgi4 protein expression was lower in atrial tissue from patients with AF than sinus rhythm patients. The reduction in Lgi4 protein levels in AF was also associated with an altered colocalization with KV1.5 channels, suggesting potential disruptions in their functional interactions. ConclusionsLgi3-4 proteins are new components of the KV1.5 channelosome. They modulate IKur by interfering with KV1.5 interaction with the KV{beta} subunit. Importantly, Lgi4 is dysregulated differently in paroxysmal versus permanent AF. The results improved the understanding of this most common type of arrhythmia and identified Lgi proteins as a new potential target for treatment. NOVELTY AND SIGNIFICANCEWhat is known? O_LILeucine-rich glioma-inactivated protein family (Lgi1-4) exert an important role in the nervous system and neurological diseases. In neurons, certain Lgi proteins interact with KV1 channels, modifying their trafficking and/or biophysical properties. C_LIO_LIIn cardiomyocytes, the activation of KV1.5 channels generates the ultrarapid outward potassium current (IKur), which is essential for the initial phase of human atrial repolarization, and it is dysregulated in AF. C_LIO_LIChanges in the properties or functional expression of some KV1.5 interacting proteins have crucial pathophysiological consequences. C_LI What new information does this article contribute? O_LIWe demonstrate that Lgi3-4 are novel components of KV1.5 channelosome, modulating IKur and hence human atrial electrophysiology. Lgi3-4 proteins decrease IKur by interfering with the interaction between KV1.5 and KV{beta} subunits. C_LIO_LIThe decrease in IKur in cardiac-specific mouse model expressing Lgi4 slows the early repolarization in the action potential, as well as produce electrophysiological changes in the surface ECG and the cardiac conduction system. C_LIO_LILgi4 is dysregulated differently in paroxysmal (PX) versus permanent (PM) AF, thus shedding light into the mechanisms underlying this cardiac arrhythmia. C_LI

physiology↗

bioRxiv2024

TYPE THREE SECRETION SYSTEM-INDUCED MECHANOPORATION OF VACUOLAR MEMBRANES

SO_SCPLOWUMMARYC_SCPLOWEndomembrane breaching is a crucial strategy employed by intracellular pathogens enclosed within vacuoles to access the nutrient-rich cytosol for intracellular replication. While bacteria use various mechanisms to compromise host membranes, the specific processes and factors involved are often unknown. Shigella flexneri, a major human pathogen, accesses the cytosol relying on the Type Three Secretion System (T3SS) and secreted effectors. Using in-cell correlative light and electron microscopy, we tracked the sequential steps of Shigella host cell entry. Moreover, we captured the T3SS, which projects a needle from the bacterial surface, in the process of puncturing holes in the vacuolar membrane. This initial puncture ensures disruption of the vacuolar membrane. Together this introduces the concept of mechanoporation via a bacterial secretion system as a crucial process for bacterial pathogen-induced membrane damage. GO_SCPLOWRAPHICALC_SCPLOW AO_SCPLOWBSTRACTC_SCPLOW O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/613840v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1669028org.highwire.dtl.DTLVardef@8259cborg.highwire.dtl.DTLVardef@1d522caorg.highwire.dtl.DTLVardef@1df0f2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Poly (ADP-ribose) polymerase-1 regulates HIV-1 replication in human CD4+ T cells

The cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) regulates multiple processes that are potentially implicated in HIV-1 infection. However, the role of PARP-1 in HIV-1 infection remains controversial, with reports indicating or excluding that PARP-1 influence early steps of the HIV-1 life cycle. Most of these studies have been conducted with Vesicular Stomatitis virus Glycoprotein G (VSV-G)-pseudotyped, single-round infection HIV-1; limiting our understanding of the role of PARP-1 in HIV-1 replication. Therefore, we evaluated the effect of PARP-1 deficiency or inhibition in HIV-1 replication in human CD4+ T cells. Our data showed that PARP-1 knockout increased viral replication in SUP-T1 cells. Similarly, a PARP-1 inhibitor that targets PARP-1 DNA-binding activity enhanced HIV-1 replication. In contrast, inhibitors affecting the catalytic activity of the enzyme were inactive. In correspondence with the pharmacological studies, mutagenesis analysis indicated that the DNA-binding domain was required for the PARP-1 anti-HIV-1 activity, but the poly-ADP-ribosylation activity was dispensable. Our results also demonstrated that PARP-1 acts at the production phase of the viral life cycle since HIV-1 produced in cells lacking PARP-1 was more infectious than control viruses. The effect of PARP-1 on HIV-1 infectivity required Env, as PARP-1 deficiency or inhibition did not modify the infectivity of Env-deleted, VSV-G-pseudotyped HIV-1. Furthermore, virion-associated Env was more abundant in sucrose cushion-purified virions produced in cells lacking the enzyme. However, PARP-1 did not affect Env expression or processing in the producer cells. In summary, our data indicate that PARP-1 antagonism enhances HIV-1 infectivity and increases levels of virion-associated Env. ImportanceDifferent cellular processes counteract viral replication. A better understanding of these interfering mechanisms will enhance our ability to control viral infections. We have discovered a novel, antagonist effect of the cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) in HIV-1 replication. Our data indicate that PARP-1 deficiency or inhibition augment HIV-1 infectivity in human CD4+ T cells, the main HIV-1 target cell in vivo. Analysis of the mechanism of action suggested that PARP-1 antagonism increases in the virus the amounts of the viral protein mediating viral entry to the target cells. These findings identify for the first time PARP-1 as a host factor that regulates HIV-1 infectivity, and could be relevant to better understand HIV-1 transmission and to facilitate vaccine development.

microbiology↗

bioRxiv2023

Shigella generates distinct IAM subpopulations during epithelial cell invasion to promote efficient intracellular niche formation

The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection- associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive "contact-IAMs" involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Finally, hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successive BCV disassembly. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.

microbiology↗

bioRxiv2023

Schlafen14 Regulates Gene Expression Depending on Codon Usage

Schlafen (SLFN) is a family of proteins upregulated by type I interferons with a regulatory role in translation. Intriguingly, SLFN14 associates with the ribosome and can degrade rRNA, tRNA, and mRNA in vitro, but a role in translation is still unknown. Ribosomes are important regulatory hubs during translation elongation of mRNAs rich in rare codons. Therefore, we evaluated the potential role of SLFN14 in the expression of mRNAs enriched in rare codons, using HIV-1 genes as a model. We found that SLFN14 regulates in a variety of cell types, including primary immune cells, the expression of HIV-1 and non-viral genes based on their codon adaptation index, a measurement of the synonymous codon usage bias; consequently, SLFN14 inhibited the replication of HIV-1. The potent inhibitory effect of SLFN14 on the expression of the rare codon-rich transcript HIV-1 Gag was minimized by codon optimization. Mechanistically, we found that the endoribonuclease activity of SLFN14 is required, and that ribosomal, but not messenger, RNA degradation is associated with the transcript selectivity of SLFN14. Therefore, we propose that SLFN14 impairs the expression of transcripts rich in rare codons, in a catalytic-dependent manner.

molecular biology↗

bioRxiv2019

SopB- and SifA-dependent shaping of the Salmonella-containing vacuole proteome in the social amoeba Dictyosteliumdiscoideum

The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-three secretion systems encoded in SPI-1 and SPI-2 (T3SSSPI-1 and T3SSSPI-2, respectively). Recently, we reported that S. Typhimurium requires T3SSSPI-1 and T3SSSPI-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the {Delta}sopB and {Delta}sifA mutant strains of S. Typhimurium, and we characterized the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae. ImportanceThe molecular mechanisms involved in Salmonella survival to predation by phagocytic amoebae, such as D. discoideum, remains poorly understood. Although we established that S. Typhimurium requires two specialized type-three secretion systems to survive in D. discoideum, no effector protein has been implicated in this process so far. Here, we confirmed the presence of a membrane-bound compartment containing S. Typhimurium in D. discoideum, and purified the D. discoideum SCV to characterize the associated proteome. In doing so, we established a key role for effector proteins SopB and SifA in remodeling the protein content of the SCV that ultimately allow the intracellular survival of S. Typhimurium in D. discoideum. We also discuss similarities and differences with the proteomes of the human SCV. These findings contribute to unravel the mechanisms used by Salmonella to survive in the environment exploiting phagocytic amoebae as a reservoir.

microbiology↗