Biology subjects

Gallardo, F.

Publications and source records attributed to Gallardo, F..

14 recordsLinked to original sources

bioRxiv2025

SG33, a vaccine strain of myxoma virus with oncolytic potential, exploits macropinocytosis and clathrin-mediated endocytosis for entry into pancreatic cancer cells.

Oncolytic viruses are being investigated as therapeutic agents in cancer, yet their mechanisms of entry into tumor cells remain incompletely understood. We previously showed that SG33, a veterinary vaccinal strain derived from a pathogenous myxoma virus, displays oncolytic activity in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we investigated the entry pathways of SG33 into primary PDAC-derived cultures. We found that macropinocytosis, an endocytic process frequently upregulated in PDAC, contributes to SG33 uptake. Moreover, SG33 infection itself induced macropinocytosis in a subset of primary PDAC cultures. Mechanistic studies revealed that phosphatidylserine exposed on the viral envelope promotes SG33 internalization through apoptotic mimicry. In PDAC cultures lacking detectable macropinocytosis, SG33 employed clathrin-mediated endocytosis as an alternative entry route. These findings provide the first insights into the entry mechanisms of SG33 into PDAC-derived cells and indicate that this virus can utilize distinct endocytic pathways depending on the cellular context. ImportancePancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, with limited therapeutic options. Oncolytic virotherapy is emerging as a promising strategy to overcome treatment resistance, yet the mechanisms by which candidate viruses enter cancer cells remain poorly defined. Here, we characterize the entry route of SG33, a derivative of myxoma virus with potent oncolytic activity in PDAC models. We show that SG33 exploits multiple endocytic pathways, including macropinocytosis (MPC), induces MPC through apoptotic mimicry, and also uses clathrin-mediated endocytosis (CME). These findings provide the first evidence that SG33 has evolved a flexible cell entry strategy, which could enhance its efficacy in the heterogeneous context of PDAC. Understanding the molecular determinants of viral entry is essential for the rational design of improved oncolytic virotherapies. Our study is aligned with this objective and highlights SG33 as a promising candidate to expand the toolbox of virotherapeutic agents against aggressive cancers.

cancer biology↗

bioRxiv2025

Use of ANCHOR viruses to validate radiative, physical and chemical decontamination systems in operational conditions.

ANCHOR tagged viruses provide a high-performance platform for the rapid evaluation of disinfection methods across chemical, radiative, and physical modalities. This study highlights the robustness of Vaccinia virus (VACV) and human Adenovirus type 5 (hAdV5) as environmental persistence models, demonstrating their suitability for testing disinfection protocols. ANCHOR imaging capability allows real-time visualization of disinfection efficacy, enabling rapid calibration and cost-effective validation of new disinfection systems. This technology provides essential support for the development and optimization of disinfection solutions in public areas up to the improvement of safety protocols in high-containment laboratories. The increasing number of BSL-4 laboratories worldwide underscores the critical needs for efficient and reliable decontamination systems and methods to test them in operational conditions to ensure biohazard safety. HighlightsO_LIANCHOR-tagged viruses provide a rapid and cost-effective platform for evaluating disinfection methods across chemical, physical, and radiative modalities. C_LIO_LIHuman Adenovirus type 5 (hAdV5) and Vaccinia virus (VACV) serve as robust environmental persistence models for testing viral decontamination protocols. C_LIO_LIThe use of ANCHOR viruses enables rapid real-time visualization of disinfection efficacy, improving safety validation in high-containment laboratories and public health applications. C_LI

microbiology↗

bioRxiv2025

Exonic enhancers are a widespread class of dual-function regulatory elements

Exonic enhancers (EEs) occupy an under-appreciated niche in gene regulation. By integrating transcription factor binding, chromatin accessibility, and high-throughput enhancer-reporter assays, we demonstrate that many protein-coding exons possess enhancer activity across species. These EEs exhibit characteristic epigenomic signatures, form long-range interactions with gene promoters, and can be altered by both nonsynonymous and synonymous variants. CRISPR-mediated inactivation demonstrated the involvement of EEs in the cis-regulation of host and distal gene expression. Through large-scale cancer genome analyses, we reveal that EE mutations correlate with dysregulated target-gene expression and clinical outcomes, highlighting their potential relevance in disease. Evolutionary comparisons show that EEs exhibit both strong sequence constraint and lineage-specific plasticity, suggesting that they serve ancient regulatory functions while also contributing to species divergence. Our findings redefine the landscape of functional elements by establishing EEs as a component of gene regulation, while revealing how coding regions can simultaneously fulfil both protein-coding and cis-regulatory roles.

genomics↗

bioRxiv2025

Tracking Replicating HPV Genomes in Proliferating Keratinocytes

Human papillomavirus (HPV) genomes replicate and partition as minichromosomes alongside host chromatin during persistent infection. However, it is difficult to monitor genome dynamics in living cells because the small and compact genome will not easily tolerate expression cassettes. Here, we use ANCHOR technology to detect HPV18 genomes in living cells. We incorporated the cis element from ANCHORTM technology into the late region of the HPV18 genome and expressed the ParB-GFP protein from an HPV18-dependent replicon. The replicon contains the HPV18 replication origin and viral transcriptional enhancer element and can replicate stably in keratinocytes when complemented by the HPV18 genome. This small replicon expresses the neomycin resistance gene in both bacteria and eukaryotic cells and has minimal prokaryotic elements that could induce innate immunity. This molecular tool enables us to indirectly monitor the presence of the virus by detecting these fluorescent proteins in live cells and allows for real-time tracking of replicating HPV18-ANCH3 genomes in proliferating keratinocytes to inform on models of HPV genome maintenance, tethering, and amplification. Here, we visualize partitioning of the viral DNA in dividing cells and show that HPV18-ANCH genomes are distributed somewhat equally to daughter cells by random attachment to host chromosomes. ImportanceIn persistent HPV infection, the viral genome is maintained at a constant copy number, replicates in synchrony with host DNA during S-phase, and is partitioned into daughter cells. The exact method by which HPVs partition to daughter cells is not well understood and the elucidation of such mechanisms may reveal relevant pharmacological targets to combat persistent HPV infection.

microbiology↗

bioRxiv2025

Diverse transcriptomic and mutational patterns but limited functional pathway alterations in patient-derived SS cells

Structured AbstractO_ST_ABSBackgroundC_ST_ABSEradication of SS is hampered by its genetic and molecular heterogeneity. A better understanding of the putative commonalities underlying SS oncogenicity may help to provide more efficient therapeutic strategies against this devastating disease. PurposeThe present work analyzes the whole transcriptome of different patient-derived SS cells to identify expression patterns, functional programs and expressed gene mutations that may provide clues on new therapeutic options for SS patients MethodsMononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of SS patients (n=7). Selected pathway-based compounds and the MALT1 inhibitor MI2 were used for in vitro drug sensitivity testing. SS cells viability was evaluated using CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated the usefulness of MI2 using patient-derived SS cells xenotransplanted (PDX) into Nod Scid Gamma mice. ResultsIn vitro data indicated that cell lines and primary malignant SS cells all display different sensitivities against specific pathway inhibitors. However, MALT1 inhibition led to a robust effect in vitro that was partially reproduced in the in vivo NSG mice xenograft model. ConclusionOur investigations revealed the actual possibility of inhibiting the downstream TCR signaling complex form by CARD11, BCL10 and MALT1 in SS therapy. Key PointsPatient-derived SS cells are transcriptionally and mutationally heterogeneous but share some common pathway alterations. Inhibition of MALT1 reduces NF-{kappa}B signaling and cell growth in cell lines and patient-derived SS cells.

cancer biology↗

bioRxiv2025

In vitro and in vivo Antiviral Activity of the Acyclic Nucleoside Phosphonate Prodrug LAVR-289 against Poxvirus and African Swine Fever Virus Replication

Poxviruses are double-stranded DNA viruses including relevant zoonotic pathogens with high morbidity. Although African swine fever virus (ASFV) belongs to the Asfarviridae family and is not strictly classified as a member of the Poxviridae, both fall within the same class of Pokkesviricetes that replicate in the cytoplasm, and some poxviruses pose potential biological warfare threats. Among compounds targeting these viruses, acyclic nucleoside phosphonate prodrugs are nucleoside analogues inhibitors of viral DNA polymerases that have been identified as promising agents. However, some limitations related to their toxicity and the rapid emergence of resistance highlight the need for new antiviral molecules. In this study, the new nucleoside analogue LAVR-289 was shown to effectively inhibit the viral replication by intervening early in the viral replication step, targeting a specific domain of the poxvirus DNA polymerase. Using monkeypox virus models, the subcutaneous or oral administration of LAVR-289 demonstrates protective efficacy in infected animal models without toxicity or behavioral modification. The stability in vivo, long shelf-life and efficacy make LAVR-289 a promising candidate for further development and stockpiling as a medical countermeasure against dsDNA virus outbreaks. Its broad-spectrum efficacy is a real asset in a context of recurrent viral epidemics, risk of bioterrorism and emergence of resistance strains in the population. HighlightsO_LILAVR-289 is a unique acyclic nucleoside phosphonate prodrug targeting viral DNA polymerases. C_LIO_LILAVR-289 displays antiviral activity against dsDNA viruses, ASFV and poxviruses. C_LIO_LIFirst report of in vivo evaluation of LAVR-289 against MPXV by subcutaneous and oral administration. C_LIO_LILAVR-289 reduces clinical signs and increase survival in animal models. C_LI

microbiology↗

bioRxiv2025

LAVR-289, a New Orally Bioavailable Inhibitor of Adenovirus Replication in vitro and in vivo.

Adenoviruses are responsible for a range of pathologies, including respiratory infections in children, accounting for 5-10% of such cases. Although most adenovirus infections are self-resolving, they can cause serious illness, particularly in immunocompromised individuals. There is currently no approved treatment for adenovirus infections, although various therapeutic approaches are under investigation, including nucleoside analog inhibitors of replication. However, these treatments have shown limited efficacy. In this study, we report on the antiviral activity of LAVR-289, a broad-spectrum acyclonucleoside phosphonate exhibiting potent in vitro efficacy against several adenovirus serotypes, comparable to that of brincidofovir. LAVR-289 specifically inhibits viral replication, blocking the formation of viral replication centers and preventing late protein expression without affecting viral entry or delivery of viral genomes to the nucleus. In vivo using immunocompromised Syrian hamsters infected with HAdV-C6, oral administration of LAVR-289 resulted in 100% animal survival. These results suggest that LAVR-289 holds promise as a potential therapy for adenovirus infections, particularly in immunocompromised patients. HighlightsO_LILAVR-289 is a unique acyclic nucleoside phosphonate prodrug. C_LIO_LILAVR-289 displays antiviral activity against Adenovirus with an EC50 of about 100 nM against HAdV-C5 ANCHOR. C_LIO_LILAVR-289 inhibits viral replication by targeting viral DNA polymerase, preventing DBP clustering and replication center formation. C_LIO_LIIn immunosuppressed Syrian hamsters, LAVR-289 is well tolerated and ensures 100% survival while effectively stopping virus replication. C_LIO_LILAVR-289's broad-spectrum activity positions it as a promising treatment for immunocompromised patients facing multiple viral infections. C_LI

microbiology↗

bioRxiv2025

LAVR-289, a new acyclo-nucleoside phosphonate having broad-spectrum activity against herpesviruses.

Human herpesviruses are latent opportunistic large dsDNA viruses that can have deleterious effect in immunocompromised patients by triggering life-threatening infections. Over the past years, different antivirals have been developed against a variety of herpesviruses, including Acyclovir for herpes simplex viruses (HSV1 and 2) and ganciclovir and letermovir for human cytomegalovirus (hCMV). However, broad-spectrum inhibitors of herpesvirus infections are still missing. Here we report the efficacy of LAVR-289, a new acyclic nucleoside analog, on a broad variety of herpesviruses from human and animal origin. LAVR-289 displays nanomolar efficiency in vitro, is active on viral strains resistant to gold standard antivirals and ex vivo on reconstituted human skin infected with HSV1. Combined with its anti-poxvirus and anti-adenovirus activity, LAVR-289 can become the next gold standard for the management of opportunistic virus infections in immunocompromised patient.

microbiology↗

bioRxiv2022

Tecovirimat is highly efficient on the Monkeypox virus lineage responsible for the international 2022 outbreak

The ongoing monkeypox virus (MPXV) outbreak is the largest ever recorded outside of Africa. Genomic analysis revealed a divergent phylogenetic lineage within clade 3, and atypical clinical presentations have been noted. We report the sequencing and isolation of the virus from the first clinical case diagnosed in France in May 2022. We tested the in vitro effect of tecovirimat (ST-246), a FDA approved drug, against this novel strain, showing efficacy at the nanomolar range. In comparison, cidofovir showed activity at micromolar concentrations. These results and the safety profile of tecovirimat strongly support its use in clinical care of severe forms for the 2022 MPXV outbreak.

microbiology↗

bioRxiv2021

ANCHOR, a technical approach to monitor single-copy locus localization in planta

RESUMEGene expression is governed by several layers of regulation which in addition to genome organization, local chromatin structure, gene accessibility and the presence of transcription factors also includes gene positioning. Although basic mechanisms are expected to be conserved in Eukaryotes, surprisingly little information on the role of gene positioning is available in plant cells, mainly due to the lack of a highly resolutive approach. In this manuscript, we adapted the use of the ANCHOR system to perform real-time single-locus detection in planta. ANCHOR is a DNA-labelling tool derived from the partitioning system. We demonstrate its suitability to monitor a single-locus in planta and used this approach to track chromatin mobility during cell differentiation in Arabidopsis root epidermal cells. Finally, we discuss the potential of this approach to investigate the role of gene positioning during transcription and DNA repair in plants.

plant biology↗

bioRxiv2020

Infestation dynamics between parasitic Antarctic fish leeches (Piscicolidae) and their crocodile icefish hosts (Channichthyidae)

An understanding of host-parasite interactions represents an important, but often overlooked, axis for predicting how marine biodiversity may be impacted by continued environmental change over the next century. For host and parasite communities in the Southern Ocean, investigations of many major groups of parasites have largely been limited to taxonomic and phylogenetic studies, creating an urgent need for the collection of baseline ecological data if we are to detect changes in host-parasite interactions in the future. Here, we survey three species of Crocodile icefish (Notothenioidei: Channichthyidae) collected from two island archipelagos in Antarcticas South Scotia Arc region for evidence of leech infestations. Specifically, we report on infestation prevalence and intensity of three leech species (Trulliobdella bacilliformis, Trulliobdella capitis, and Nototheniobdella sawyeri) on the host fish species Chaenocephalus aceratus, Champsocephalus gunnari, and Chionodraco rastrospinosus. Additionally, we characterize spatial patterns of relative abundances of each leech species across the Elephant and South Orkney Islands, size distribution of parasitized fish, and patterns of host and attachment site specificity. Our results reveal high levels of attachment area fidelity for each leech species. These results suggest skin thickness and density of the vascular network constrain leech attachment sites and further suggest trophic transmission to be an important axis of parasitization. We also demonstrate that, while leech species appear to be clustered spatially, this clustering does not appear to be correlated with fish biomass. This study illuminates the complex interactions among fish hosts and leech parasites in the Southern Ocean and lays the groundwork for future studies of Antarctic marine leech ecology that can aid in forecasting how host-parasite interactions may shift in the face of ongoing climate change.

ecology↗

bioRxiv2018

Biopurification system as a source of pesticide-tolerant bacteria able to degrade the commonly used pesticides chlorpyrifos and iprodione

Intensive use of pesticides applied simultaneously in field to improve the effectiveness of pest control increase the environmental contamination, affecting the soil and water quality. Some of the commonly used pesticides are the insecticide chlorpyrifos and the fungicide iprodione; being thus critically essential to develop bioremediation methods to remove these contaminants by tolerant-bacteria. In this study we selected and characterized different pesticides-tolerant bacteria isolated from a biomixture of a biopurification system that had received continuous applications of a mixture of the pesticides chlorpyrifos and iprodione. Out of the 10 isolated bacterial colonies, only six strains presented adequate growth in presence of the both pesticides at 100 mg L-1. Biochemical and enzymatic characterization using API ZYM showed that all isolates (100%) were positive for esterase, leucine aminopeptidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. According to the molecular level study of the 16S ribosomal gene and MALDI TOF/TOF MS, it was possible to determine that the isolated bacteria belong to the genera Pseudomonas, Rhodococcus and Achromobacter. Bacterial growth decreased proportionally (R2 > 0.96) as been as both pesticide concentrations increased from 10 to 100 mg L-1. Achromobacter sp. strain C1 showed the best chlorpyrifos removal (between 56-29%) after 120 h of incubation. On the other hand, the highest iprodione removal (between 91.2-98.9%) was observed for the Pseudomonas sp. strain C9, which was not detected after 48 h of incubation. According with their identification and ability to remove the contaminants, Achromobacter sp. strain C1 and Pseudomonas sp. strain C9 appear as promising microorganisms for their use in the treatment of matrices contaminated with chlorpyrifos, iprodione or their mixture. The results of this study will help to improve current technologies for the biodegradation of this commonly used insecticide and fungicide, in order to give a response to the problem of contamination by pesticides.

microbiology↗

bioRxiv2018

Real-time visualization and quantification of human Cytomegalovirus replication in living cells using the ANCHOR DNA labeling technology.

Human cytomegalovirus (HCMV) induces latent life-long infections in all human populations. Depending on geographic area and socio-economic conditions between 30 to nearly 100% of individuals are affected. The biology of this virus is difficult to explore due to its extreme sophistication and the lack of pertinent animal model. Here we present the first application of the ANCHOR DNA labeling system to a herpes virus, allowing real time imaging and direct monitoring of HCMV infection and replication in human living cells. The ANCHOR system is composed of a protein (OR) which specifically binds to a short, non-repetitive DNA target sequence (ANCH) and spreads onto neighboring sequences due to protein oligomerization. If OR protein is fused to GFP, this accumulation results in a site specific fluorescent focus. We have created a recombinant ANCHOR-HCMV harboring an ANCH target sequence and the gene encoding the cognate OR-GFP fusion protein. Infection of permissive cells with ANCHOR-HCMV enables visualization of the nearly complete viral cycle until cell fragmentation and death. Quantitative analysis of infection kinetics and of viral DNA replication revealed cell-type specific behavior of HCMV and sensitivity to inhibitors. Our results show that the ANCHOR technology is a very efficient tool for the study of complex DNA viruses and new highly promising biotechnology applications.\n\nIMPORTANCEThe ANCHOR technology is to date the most powerful tool to follow and quantify the replication of HCMV in living cells and to gain new insights into its biology. This technology is applicable to virtually any DNA virus or virus presenting a dsDNA phase, paving the way to infection imaging in various cell lines or even in animal models and opening fascinating fundamental and applied prospects. Associated to high content automated microscopy, this technology permitted rapid, robust and precise determination of Ganciclovir IC50 and IC90 on HCMV replication, with minimal hands-on investment. To search for new antiviral activities, the experiment is easy to up-grade towards efficient and cost-effective screening of large chemical libraries. The simple infection of permissive cells with ANCHOR-viruses in the presence of a compound of interest may even provide a first estimation about the stage of the viral cycle this molecule is acting upon.

microbiology↗

bioRxiv2017

Real-time chromatin dynamics at the single gene levelduring transcription activation

Genome dynamics relate to regulation of gene expression, the most fundamental process in biology. Yet we still do not know whether the very process of transcription drives spatial organization and chromatin conformation at specific gene loci. To address this issue, we have optimized the ANCHOR/ParB DNA labeling system for real-time imaging and quantitative analysis of the dynamics of a single-copy transgene in human cells. Transcription of the transgene under the control of the endogenous Cyclin D1 promoter was induced by addition of 17{beta}-estradiol. Motion of the ANCHOR3-tagged DNA locus was recorded in the same cell prior to and during appearance of nascent mRNA visualized using the MS2 system. We found that transcription initiation resulted in rapid confinement of the mRNA-producing gene. The confinement was maintained even upon inhibition of pol2 elongation. It did not occur when recruitment of pol2 or transcription initiation was blocked by anti-estrogens or Triptolide. These results suggest that preinitiation complex formation and concomitant reorganization of the chromatin domain constrains freedom of movement of an induced genes promoter within minutes. Confined diffusion reflects assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.

cell biology↗