Biology subjects

Alonso, A. M.

Publications and source records attributed to Alonso, A. M..

9 recordsLinked to original sources

bioRxiv2026

Histone H2A.X contributes to Gene Expression Regulation and Cell-Cycle Progression in Toxoplasma gondii Tachyzoites

Histone H2A.X is a conserved variant classically associated with the DNA damage response (DDR), where its phosphorylation ({gamma}H2A.X) marks double-strand breaks and recruits repair factors. H2A.X is absent in many parasitic protozoans such as Plasmodium, Trypanosoma and Leishmania. In Toxoplasma gondii, the obligate intracellular parasite, causative agent of toxoplasmosis, however, its presence raises the question of whether it contributes to genome stability. To tackle this, we generated a T. gondii knockout of H2A.X (RH{Delta}H2A.X) and evaluated its phenotype. Although H2A.X is phosphorylated in response to DNA damage in T. gondii as in other systems, RH{Delta}H2A.X parasites showed no increased sensitivity to genotoxic agents, indicating that H2A.X is dispensable for canonical DDR. Instead, loss of H2A.X caused reduced gliding motility, increased host cell invasion and clustering of extracellular parasites after egress. Despite no defects in replication rate, RH{Delta}H2A.X showed delayed mitotic progression with abnormal vacuole morphology. Transcriptomic profiling revealed a moderate yet coordinated reprogramming of protein-coding genes. Specifically, H2A.X loss altered the expression of key apical secretory and virulence factors, especially ROP18, while inducing the transcriptional activation of mitotic and DNA replication machinery. Differential enrichment analysis further revealed that upregulated genes were preferentially associated with S phase, whereas downregulated genes were enriched during mitosis and cytokinesis. This is consistent with the delayed mitotic progression and spindle abnormalities observed by microscopy and flow cytometry and suggests a cell cycle phase-specific mechanism of H2A.X-dependent transcriptional control. Rather than a driver of DDR, these findings suggest that T. gondii H2A.X operates as a chromatin organizer that coordinates cell division fidelity and virulence gene expression.

cell biology↗

bioRxiv2025

Matrix Metalloproteinase (MMP) inhibition rescues endothelial glycocalyx damage and reduces neutrophil infiltration in sepsis-associated acute kidney injury

Endothelial glycocalyx (eGlx), a carbohydrate-rich endothelial coat, maintains vascular homeostasis, and its disruption contributes to several vascular diseases. We previously identified matrix metalloproteinase (MMP) 2 and 9-mediated eGlx shedding as a key mechanism in human glomerular endothelial cell (GEnC) damage and kidney dysfunction in diabetes. Here, we sought to determine whether this mechanism contributes to renal and systemic microvascular endothelial cell injury in sepsis-AKI. Sepsis-AKI was induced in mice by lipopolysaccharide (LPS) injection. Confocal microscopy demonstrated glomerular and peritubular eGlx damage, and increased circulating SDC4 demonstrated systemic eGlx shedding. These were associated with enhanced mRNA expression of eGlx components (syndecans 1 and 4) and endothelial inflammatory markers, Intercellular and Vascular Cell Adhesion Molecules (ICAM and VCAM) in the kidney. Additionally, sepsis-AKI markers, serum creatinine and urea, were increased. We found that renal and circulating MMP9, but not MMP2, levels were raised in sepsis-AKI. Moreover, MMP9 staining colocalises with neutrophil, endothelial and mesangial staining in glomeruli in sepsis-AKI. Treatment with MMP2 and 9 inhibitor 1, 1h before LPS injection, prevented glomerular, peritubular and systemic eGlx damage in sepsis-AKI. MMP inhibition also attenuated glomerular leucocyte numbers in sepsis-AKI. We confirmed increased MMP9 activity, shedding of eGlx syndecan 1 and elevated angiopoietin 2 levels, an endothelial damage marker, in sepsis-AKI in humans. Moreover, we used human glomerular endothelial cells (GEnC) in vitro to demonstrate that human sepsis-AKI serum directly damages GEnC eGlx. Our studies confirm that MMP-mediated eGlx damage is a key contributor to renal and systemic microvascular dysfunction in sepsis-AKI and represents a potential therapeutic target for protecting the microvasculature.

cell biology↗

bioRxiv2025

Core microRNAs regulate neural crest delamination and condensation in the developing trigeminal ganglion

Cranial neural crest cells (NCCs) undergo dynamic processes during embryonic development, including delamination from the neural tube by epithelial-to-mesenchymal transition (EMT), migration to the periphery, condensation via mesenchymal-to-epithelial transition (MET) and differentiation into structures like the trigeminal ganglion. Here, we identify and characterize the function of a core set of miRNAs involved in these transitions during the formation of the trigeminal ganglion in the chick embryo. We further identify putative targets of miRNAs involved in neural crest EMT and MET. Notably, introducing MET-involved miRNAs into trunk NCCs endows these cells with the ability to condense and differentiate into neurons in vivo in a manner reminiscent of cranial rather than trunk NCCs. Our findings shed light on the intricate regulatory networks governing NCC behavior, positioning miRNAs as key regulatory elements required for migratory transitions and axial level specific differentiation capabilities.

developmental biology↗

bioRxiv2024

A bioinformatic approach for the prediction andfunctional classification of Toxoplasma gondii longnon-coding RNAs

Long non-coding RNAs (lncRNAs) have emerged as significant players in diverse cellular processes, including cell differentiation. Advancements in computational methodologies have facilitated the prediction of lncRNA functions, enabling insights even in non-model organisms like pathogenic parasites, in roles such as parasite development, antigenic variation, and epigenetics. In this work, we focus on the apicomplexan Toxoplasma gondii differentiation process, where the infective stage, tachyzoite, can develop into the cysted stage, bradyzoite, under stress conditions. Using a publicly available transcriptome dataset, we predicted lncRNA sequences associated with this differentiation process. Notably, a substantial proportion of these predicted lncRNAs exhibited stage-specific expression, particularly at the bradyzoite stage. Furthermore, co-expression patterns between coding transcripts and lncRNAs suggest their involvement in shared processes, such as bradyzoite development. TglncRNA loci analysis revealed their potential influence on the expression of nearby coding genes, including subtelomeric genes unique to the T. gondii genome. Finally, with a k-mer analysis approach, we identified functional relationships between characterized lncRNAs from model organisms like Homo sapiens and predicted T. gondii lncRNAs. Our perspective led to the identification of a T. gondii lncRNA potentially mediating DNA damage repair pathways, shedding light on the adaptive mechanisms of T. gondii in response to stress conditions.

bioinformatics↗

bioRxiv2022

Intra-islet glucagon signalling regulates pulsatile insulin secretion and glucose homeostasis

BackgroundType 2 diabetes (T2D) is characterised by the loss of pulsatile insulin secretion. We studied mice with {beta}-cell specific loss of the glucagon receptor (Gcgr fl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor signalling on pan-islet calcium oscillations and insulin pulsatility. MethodsFrequently sampled intravenous glucose tolerance tests were conducted on Gcgr {beta}-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for {beta}-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in {beta}-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. ResultsGcgr {beta}-cell-/- mice exhibited impaired glucose tolerance following intraperitoneal glucose challenge (control 12.7mmol/L {+/-}0.6 vs. Gcgr {beta}-cell-/- 15.4mmol/L {+/-}0.0 at 15 min, p=0.002); fasting glycaemia was not different to controls. In vitro, Gcgr {beta}-cell-/- islets showed profound loss of synchronised calcium waves in response to glucose which was only partially rescued in vivo. First-phase insulin pulsatility on peripheral blood sampling (n=5) was significantly disordered in Gcgr {beta}-cell-/- mice (burst mass Gcgr {beta}-cell-/- 0.30 {+/-}0.03 versus 0.84 {+/-}0.23 for controls p=0.04). Diet induced obesity and hyperglycaemia resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n=8). ConclusionThese data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised calcium oscillations and support a possible therapeutic role for glucagonergic agents to restore the insulin pulsatility lost in T2D.

physiology↗

bioRxiv2021

A novel RAB11-containing adaptor complex anchoring myosin-5 to secretory vesicles

Hyphal fungi grow rapidly by apical extension, providing a notorious example of polarized growth. The continuous supply of secretory vesicles necessary to meet the demands of the extending tip and the long intracellular distances existing between the tip and the basal septum, often localized > 100 {micro}m away from the former, impose the need of efficient networks of intracellular traffic involving exquisite cooperation between microtubule- and actin-mediated transport. In Aspergillus nidulans kinesin-1 conveys secretory vesicles to the hyphal tip, where they are transferred to myosin-5, which focuses them at the growing apex, thereby determining cell shape. This relay mechanism and the central role played by myosin-5 in hyphal morphogenesis suggested that the mechanisms anchoring secretory vesicles to this motor should involve specific adaptor(s) ensuring the robustness of actomyosin-dependent transport. Secretory vesicles are charged with RAB11, a regulatory GTPase that determines the Golgi to post-Golgi identity transition. By using a combination of shotgun proteomics, GST-RAB pull-down assays, in vitro reconstitution experiments, targeted reverse genetics and multidimensional fluorescence microscopy with endogenously tagged proteins we show that RAB11, the master regulator of fungal exocytosis, mediates myosin-5 engagement both by contacting the motor and by recruiting UDS1, a homologue of an as yet uncharacterized Schizosaccharomyces protein upregulated during mitosis, which we demonstrate to be a novel RAB11 effector. Analytical ultracentrifugation determined that UDS1 is an elongated dimer and negative-stain electron microscopy showed that, in agreement, UDS1 is rod-shaped. UDS1 does not contact myosin-5 directly, but rather recruits the coiled-coil HMSV, which bridges RAB11/UDS1 to myosin-5. An HMSV-scaffolded complex containing UDS1 and myosin-5 is present in cells, and a RAB11-UDS1-HMSV complex can be reconstituted in vitro in a RAB nucleotide state-dependent manner. In the absence of UDS1/HMSV the steady state levels of myosin-5 at the apical vesicle supply center diminish markedly, such that microtubule-dependent transport spreading vesicles across the apical dome predominates over apex-focused actin-mediated transport. As a consequence, RAB11 and chitin-synthase B (a cargo of the RAB11 pathway) are not focused at the apex, being distributed instead across the apical dome. Therefore, the RAB11 effector UDS1/HMSV cooperates with the GTPase to adapt secretory vesicles to myosin-5, which is required for the apical targeting of RAB11 cargoes and thus for the normal morphology of the hyphae.

cell biology↗

bioRxiv2020

SARS-CoV-2 codon usage bias downregulates host expressed genes with similar codon usage

Severe acute respiratory syndrome is quickly spreading throughout the world and was declared as a pandemic by the World Health Organisation (WHO). The pathogenic agent is a new coronavirus (SARS-CoV-2) that infects pulmonary cells with great effectiveness. In this study we focus on the codon composition for the viral proteins synthesis and its relationship with the proteins synthesis of the host. Our analysis reveals that SARS-CoV-2 preferred codons have poor representation of G or C nucleotides in the third position, a characteristic which could conduct to an unbalance in the tRNAs pools of the infected cells with serious implications in host protein synthesis. By integrating this observation with proteomic data from infected cells, we observe a reduced translation rate of host proteins associated with highly expressed genes, and that they share the codon usage bias of the virus. The functional analysis of these genes suggests that this mechanism of epistasis contributes to understand some deleterious collateral effect as result of the viral replication. In this manner, our finding contribute to the understanding of the SARS-CoV-2 pathogeny and could be useful for the design of a vaccine based on the live attenuated strategy.

genomics↗

bioRxiv2018

A molecular inversion probe and sequencing-based microsatellite instability assay for high throughput cancer diagnostics and Lynch syndrome screening

BackgroundClinical guidelines recommend microsatellite instability (MSI) and BRAF V600E testing of all colorectal cancers (CRCs) to screen for Lynch syndrome (LS), a hereditary predisposition to cancer. MSI is also associated with response to immunotherapy. However, uptake of MSI testing is poor and current assays are not suitable for high throughput diagnostics.\n\nWe aimed to develop a cheap and scalable sequencing assay for MSI classification, which is robust to variables in clinical samples and simultaneously tests for BRAF V600E to streamline the LS screening pipeline.\n\nMethods24 short (7-12bp) microsatellites and the BRAF V600E locus were amplified in multiplex using single molecule molecular inversion probes (smMIPs) and sequenced using the Illumina MiSeq platform. Reads were aligned to reference genome hg19. An MSI classifier was trained from 98 CRCs and validated in 99 independent CRCs collected in pathology laboratories in Edinburgh, Spain and Newcastle.\n\nResultsThe smMIP-based MSI assay has 100% accuracy for MSI status relative to MSI Analysis System (Promega). MSI classification is reproducible (100% concordance) and is robust to sample variables, detecting less than 5% MSI-high content in template DNA and giving reliable classification from sequencing only 75 DNA molecules per marker. BRAF V600E was detected with mutant allele frequencies down to 1.7%.\n\nConclusionsOur short microsatellite, smMIP-based, MSI assay provides a cheap and fully automatable assessment of MSI status and BRAF mutation. It is readily scalable to high throughput cancer diagnostics, and is suitable both as a companion diagnostic for immunotherapy and for streamlined LS screening.

cancer biology↗

bioRxiv2018

A novel panel of short mononucleotide repeats linked to informative polymorphisms enabling effective high volume low cost discrimination between mismatch repair deficient and proficient tumours

Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7-12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI.

genomics↗