Biology subjects

Alonso, S.

Publications and source records attributed to Alonso, S..

14 recordsLinked to original sources

bioRxiv2026

Dengue virus 2 lacking N153 glycosylation displayed enhanced recognition by neutralizing antibodies

Deglycosylated (N153Q) dengue virus (DENV) mutant shows attenuated infection in a mouse model, mainly due to its increased antibody susceptibility. This is consistent with the neutralization assay showing human monoclonal antibodies 2D22 and C10 are more potent towards the mutant than the wild-type (WT) virus. Here, we compared the cryoEM structures of WT and mutant viruses complexed with these Fabs (2.7-3.2 [A] resolution). We observed increased occupancies for both Fabs on the mutant virus, suggesting higher accessibility of epitopes that were previously blocked by glycosylation. Using biolayer interferometry, we showed although the Fabs have slower binding rate to the mutant than WT virus, they also have slower dissociation rate. The slow dissociation rate might contribute to higher Fab occupancies, as once bound, they remain associated with the virus. N153Q mutant might be a good vaccine candidate, as important epitopes are made more accessible for stimulating highly potent antibodies.

biophysics↗

bioRxiv2025

Concurrent genetic and non-genetic resistance mechanisms to KRAS inhibition in CRC

KRAS is mutationally activated in 45-50% of colorectal cancer (CRC) cases, and while KRAS-targeted therapies have shown some clinical promise, upfront and acquired resistance limit their efficacy. To explore the acute response and mechanisms underlying KRAS inhibitor resistance, we used targeted exome sequencing and single-cell spatial transcriptomics to analyze patient-matched pre-treatment, on-treatment, and progression biopsies from patients treated with combined KRASG12C and EGFR inhibition. Acquired genetic events were identified in most patients at progression but were often subclonal and coexisted with transcriptional adaptive states. Mesenchymal, YAP, and fetal-like transcriptional signatures predominated in resistant tumors, while tumor cell-intrinsic inflammatory programs were induced in the early treatment phase. Single-cell spatial analysis revealed significant intratumoral heterogeneity, with diverse adaptive states predominating in different zones of individual tumors. Using human and murine organoid models, we show that these drug-induced inflammatory programs are cancer-cell autonomous and precede the emergence of regenerative fetal-like programs associated with drug resistance. We uncover TBK1 as a promising target to abrogate the early inflammatory adaptive phase and enhance responses to KRAS inhibition.

cancer biology↗

bioRxiv2025

Microbiota promote enhanced CD39 expression in γδ intraepithelial lymphocytes through the activation of TCR and IL-15 signaling

Intraepithelial lymphocytes expressing the {gamma}{delta} T cell receptor ({gamma}{delta} IEL) provide continuous surveillance of the intestinal epithelium. We report that mice harboring a microbiota-specific hyperproliferative {gamma}{delta} IEL ({gamma}{delta}HYP) phenotype also upregulate the expression of the ectonucleotidase CD39, a marker of regulatory {gamma}{delta} T cells. Enhanced TCR and IL-15 signaling correlates with a progression from a naive-like CD39neg {gamma}{delta} IEL to a more mature, tissue-adapted CD39hi IEL population. We found that TCR{gamma}{delta} activation drives CD122-mediated CD39 upregulation on {gamma}{delta}HYP IELs and increased mucosal IL-15 further amplifies CD39 expression in these cells. Further investigation revealed that CD39 induction requires sustained exposure to the {gamma}{delta}HYP-associated microbiota. Moreover, CD39hi {gamma}{delta} IELs exhibit a reduced capacity to produce pro-inflammatory cytokine, which may explain the lack of histopathology in {gamma}{delta}HYP mice. Overall, our study identifies a previously unappreciated mechanism by which an altered microbiota amplifies CD39 expression on {gamma}{delta}HYP IELs, leading to the expansion of {gamma}{delta} IELs with regulatory potential.

immunology↗

bioRxiv2024

In vitro discovery of a therapeutic lead for HFMD from a library screen of rocaglates/aglains

The lack of an effective antiviral treatment for enteroviruses, including the human enterovirus A71 (EV-A71), has resulted in an immense global healthcare burden associated with hand-foot-and-mouth disease (HFMD). Rocaglates and aglains belong to a family of compounds produced by Aglaia genus plants. Since the initial discovery of rocaglates in 1982, various rocaglates and aglains have been synthesized and extensively studied as anticancer and antiviral agents. Here, we report our studies towards the discovery of a novel aglain derivative as an EV-A71 inhibitor and work to decipher its antiviral effect. From an immunofluorescence-based phenotypic screen of a library of 296 rocaglate and aglain derivatives, we identified a lead aglain derivative which effectively suppressed EV-A71 replication by 2.3 log fold at a non-cytotoxic concentration. Further validation revealed inhibition of EV-A71 across multiple cell types and a pan-enterovirus inhibitory spectrum against other enteroviruses. Subsequent mechanistic investigation revealed interference with EV-A71 intracellular post-entry events including viral RNA transcription and translation. Findings from this study have established a strong foundation for development of aglain scaffolds as much needed antiviral agents for HFMD, paving the way for future medicinal chemistry optimization and in vivo studies.

microbiology↗

bioRxiv2024

Targeted Time-Varying Functional Connectivity

To elucidate the neurobiological basis of cognition, which is dynamic and evolving, various methods have emerged to characterise time-varying functional connectivity (FC) and track the temporal evolution of functional networks. However, given a selection of regions, many of these methods are based on modelling all possible pairwise connections, diluting a potential focus of interest on individual connections. This is the case with the hidden Markov model (HMM), which relies on region-by-region covariance matrices across all pairs of selected regions, assuming that fluctuations in FC occur across all investigated connections; that is, that all connections are locked to the same temporal pattern. To address this limitation, we introduce Targeted Time-Varying FC (T-TVFC), a variant of the HMM that explicitly models the temporal fluctuations between two sets of regions in a targeted fashion, rather than across the entire connectivity matrix. In this study, we apply T-TVFC to both simulated and real-world data. Specifically, we investigate thalamocortical connectivity, hypothesizing distinct temporal signatures compared to corticocortical networks. Given the thalamuss role as a critical hub, thalamocortical connections might contain unique information about cognitive processing that could be overlooked in a coarser representation. We tested these hypotheses on high-field functional magnetic resonance data from 60 participants engaged in a reasoning task with varying complexity levels. Our findings demonstrate that the time-varying interactions captured by T-TVFC contain task-related information not detected by more traditional decompositions.

neuroscience↗

bioRxiv2023

Enterovirus-A71 exploits Rab11 to recruit chaperones for virus morphogenesis

Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. A siRNA screen in EV-A71 infected-motor neurons identified small GTPase Rab11a as a pro-viral host factor. Rab11a and Rab11b isoforms were interchangeably exploited by strains from major EV-A71 genogroups and Coxsackievirus 16, another major causative agent of HFMD. We showed that Rab11 did not play a role in viral entry, IRES-mediated protein translation, or viral genome replication, although it co-localized with replication organelles. GTPase-defective Rab11 mutants had no effect on EV-A71 replication, ruling out Rab11 involvement in intracellular trafficking of viral or host components. Instead, reduced VP2:VP0 ratio in siRab11-treated cells supported a role in provirion maturation. Co-immunoprecipitation and mass spectrometry revealed chaperones as Rab11 top interacting partners during infection. Among which, CCT8 subunit of the chaperone complex TRiC/CCT was found to interact with viral structural proteins specifically. Together, this study describes a novel, unconventional role for Rab11 during viral infection, where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.

microbiology↗

bioRxiv2023

Single shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad and durable systemic and mucosal immune responses

Current COVID-19 vaccines face certain limitations, which include waning immunity, immune escape by SARS-CoV-2 variants, limited CD8+ cellular response, and poor induction of mucosal immunity. Here, we engineered a Clec9A-RBD antibody construct that delivers the Receptor Binding Domain (RBD) from SARS-CoV-2 spike protein to conventional type 1 dendritic cells (cDC1). We showed that single dose immunization with Clec9A-RBD induced high RBD-specific antibody titers with a strong T-helper 1 (TH1) isotype profile and exceptional durability, whereby antibody titers were sustained for at least 21 months post-vaccination. Uniquely, affinity maturation of the antibody response was observed over time, as evidenced by enhanced neutralization potency and breadth across the sarbecovirus family. Consistently and remarkably, RBD-specific T-follicular helper cells and germinal center B cells were still detected at 12 months post-immunization. Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the immune sera was also measured over time with comparable efficacy against ancestral SARS-CoV-2 and variants, including Omicron. Furthermore, Clec9A-RBD immunization induced a durable poly-functional TH1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants, including Omicron, and with robust CD8+ T cell signature. Lastly, Clec9A-RBD single dose systemic immunization primed effectively RBD-specific cellular and humoral mucosal immunity in lung. Taken together, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal immune responses against rapidly evolving SARS-CoV2 variants.

immunology↗

bioRxiv2023

Towards stability of dynamic FC estimates in neuroimaging and electrophysiology: solutions and limits

Time-varying functional connectivity methods are used to map the spatiotemporal organization of brain activity. However, their estimation can be unstable, in the sense that different runs of the inference may yield different solutions. But to draw meaningful relations to behaviour, estimates must be robust and reproducible. Here, we propose two solutions using the Hidden Markov Model (HMM) as a descriptive model of time-varying FC. The first, best-ranked HMM, involves running the inference multiple times and selecting the best model based on a quantitative measure combining fitness and model complexity. The second, hierarchical clustered HMM, generates stable aggregated state timeseries by applying hierarchical clustering to the state timeseries obtained from multiple runs. Experimental results on fMRI and MEG data demonstrate that these approaches substantially improve the stability of time-varying FC estimations. Overall, hierarchical clustered HMM is preferred when the inference variability is high, while the best-ranked HMM performs better otherwise.

neuroscience↗

bioRxiv2022

A benchmarking of human Y-chromosomal haplogroup classifiers from whole-genome and whole-exome sequence data

The non-recombinant region of the Y chromosome (NRY) contains a great number of polymorphic markers that allows to accurately reconstruct pedigree relationships and retrieve ancestral information from study samples. The analysis of NRY is typically implemented in anthropological, medical, and forensic studies. High-throughput sequencing (HTS) has profoundly increased the identification of genetic markers in the NRY genealogy and has prompted the development of automated NRY haplogroup classification tools. Here, we present a benchmarking study of five command-line tools for NRY haplogroup classification. The evaluation was done using empirical short-read HTS data from 50 unrelated donors using paired data from whole-genome sequencing (WGS) and whole-exome sequencing (WES) experiments. Besides, we evaluate the performance of the top-ranked tool in the classification of data of third generation HTS obtained from a subset of donors. Our findings demonstrate that WES can be an efficient approach to infer the NRY haplogroup, albeit generally providing a lower level of genealogical resolution than that recovered by WGS. Among the tools evaluated, YLeaf offers the best performance for both WGS and WES applications. Finally, we demonstrate that YLeaf is able to correctly classify all samples sequenced with nanopore technology from long noisy reads.

genomics↗

bioRxiv2021

A transmissible γδ intraepithelial lymphocyte hyperproliferative phenotype is associated with the intestinal microbiota and confers protection against acute infection.

Intraepithelial lymphocytes expressing the {gamma}{delta} T cell receptor ({gamma}{delta} IELs) serve as a first line of defense against luminal microbes. Although the presence of an intact microbiota is dispensable for {gamma}{delta} IEL development, several microbial factors contribute to the maintenance of this sentinel population. However, whether specific commensals influence population of the {gamma}{delta} IEL compartment under homeostatic conditions has yet to be determined. We identified a novel {gamma}{delta} IEL hyperproliferative phenotype that arises early in life and is characterized by expansion of multiple V{gamma} subsets. Horizontal transfer of this hyperproliferative phenotype to mice harboring a phenotypically normal {gamma}{delta} IEL compartment was prevented following antibiotic treatment, thus demonstrating that the microbiota is both necessary and sufficient for the observed increase in {gamma}{delta} IELs. Further, we identified a group of unique gut bacteria represented by 5 amplicon sequence variants (ASV) which are strongly associated with {gamma}{delta} IEL expansion. Using intravital microscopy, we find that hyperproliferative {gamma}{delta} IELs also exhibit increased migratory behavior leading to enhanced protection against bacterial infection. These findings reveal that transfer of a specific group of commensals can regulate {gamma}{delta} IEL homeostasis and immune surveillance, which may provide a novel means to reinforce the epithelial barrier.

immunology↗

bioRxiv2021

Somatic hypomethylation of pericentromeric SST1 repeats and tetraploidization in human colorectal cancer cells

Somatic DNA hypomethylation and aneuploidy are hallmarks of cancer, and there is evidence for a causal relationship between them in knockout mice, but not in human cancer. The non-mobile pericentromeric repetitive elements SST1 are hypomethylated in about 17% of human colorectal cancers (CRC) with some 5-7% exhibiting a more severe age-independent demethylation. Tetraploidy is a common and early event in solid tumors generating subsequent aneuploidy. We compared the relative frequency of chromosomal variations during culture of randomly selected single cell clones of diploid LS174T human CRC cells differing in their levels of SST1 demethylation. Diploid cells underwent frequent genome reduplication events generating tetraploid clones that correlated with SST1 demethylation. In primary CRC, severe SST1 hypomethylation was significantly associated with global genomic hypomethylation and mutations in TP53. This work uncovers the association of the naturally occurring demethylation of the SST1 pericentromeric repeat with the onset of spontaneous tetraploidization in human CRC cells in culture, and with TP53 mutations in primary CRCs. Altogether, our findings provide further support for an oncogenic pathway linking somatic epigenetic and genetic alterations in a subset of human CRC.

cancer biology↗

bioRxiv2020

Reduced placental transfer of antibodies against microbial and vaccine antigens in HIV-infected women in Mozambique

Antibody transplacental transfer is essential for conferring protection in newborns against infectious diseases. This transfer may be affected by gestational age and maternal infections, although the effects are not consistent across studies. We measured total IgG and IgG subclasses by quantitative suspension array technology against fourteen pathogens and vaccine antigens, including target of maternal immunization, in 341 delivering HIV- and HIV+ mother-infant pairs from southern Mozambique. Maternal antibody levels were the main determinant of cord antibody levels. HIV broadly reduced the placental transfer and cord levels of IgG and IgG1, but also IgG2 to half of the antigens. Plasmodium falciparum exposure and prematurity were negatively associated with cord antibody levels and placental transfer but this was antigen-subclass dependent. These findings suggest maternal infections may impact the efficacy of maternal immunization and confirm the lower transfer of antibodies as one of the causes underlying increased susceptibility to infections in HIV-exposed infants.

immunology↗

bioRxiv2020

HflX controls hypoxia-induced non-replicating persistence in slow growing mycobacteria

GTPase HflX is highly conserved in prokaryotes and is a ribosome splitting factor during heat shock in E. coli. Here we report that HflX produced by slow growing M. tuberculosis and M. bovis BCG is a GTPase that plays a critical role in the pathogens transition to a non-replicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model, that precipitated entry into dormancy. The KO displayed the hallmarks of dormant mycobacteria including phenotypic drug resistance, altered morphology, low intracellular ATP and up-regulated dormancy dos regulon. KO-infected mice displayed increased bacterial burden during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic phase. Unlike fast-growing mycobacteria, BCG HlfX was not involved in antibiotic resistance under normoxia. Proteomics, pull-down and ribo-sequencing supported that mycobacterial HflX is a ribosome binding protein that controls the translational activity of the cell. Collectively, our study provides further insights into the mechanisms deployed by mycobacteria to adapt to their hypoxic microenvironment.

microbiology↗

bioRxiv2018

Structure mapping of dengue and Zika viruses reveals new functional long-range interactions

Dengue and Zika are clinically important members of the Flaviviridae family that utilizes an 11kb positive strand RNA for genome regulation. While structures have been mapped primarily in the UTRs, much remains to be learnt about how the rest of the genome folds to enable function. Here, we performed secondary structure and pair-wise interaction mapping on four dengue serotypes and four Zika strains in their native virus particles and infected cells. Comparative analysis of SHAPE reactivities across serotypes nominated potentially functional regions that are highly structured, show structure conservation, and low synonymous mutation rates, including a structure associated with ribosome pausing. Pair-wise interaction mapping by SPLASH further reveals new pair-wise interactions, in addition to the known circularization sequence. 40% of pair-wise interactions form alternative structures, suggesting extensive structural heterogeneity. Analysis of shared pair-wise interactions between serotypes revealed macro-organization whereby interactions are preserved at their physical locations, beyond their sequence identities. In addition, structure mapping of virus genomes released in solution-as well as inside host cells-showed that other helicases, in addition to the ribosome, play a role in unwinding viral structures inside cells. Mutational experiments that disrupt in cell and in virion pair-wise interactions result in virus attenuation, demonstrating their importance during the virus life-cycle.

genomics↗