Biology subjects

Menendez, A.

Publications and source records attributed to Menendez, A..

9 recordsLinked to original sources

bioRxiv2026

Twin-prime editing enables endogenous protein visualization and mutant allele tracking

Precise analysis of endogenous protein behavior in mosaic tissues requires strategies that both modify native alleles and identify successfully edited cells at single-cell resolution. Here, we establish a CRISPR-based genome-editing platform that combines endogenous protein visualization with allele-specific mutant tracking in vivo. Using the chick neural tube and {beta}-catenin as a model system, we validate cytosine base editing, prime editing, and twin-prime editing by introducing stabilizing mutations that reproduce their expected cellular and morphological phenotypes. We then develop a twin-prime editing strategy that couples installation of a defined oncogenic mutation to simultaneous insertion of minimal peptide tags, thereby making productive editing directly observable at single-cell resolution without clonal selection. Comparative analysis of ALFA, V5, and split-GFP tags identifies split-GFP as the most reliable strategy for endogenous protein visualization in vivo, and fluorescence-based cell selection strongly enriches for the intended twin-prime editing product. Finally, we extend the approach to endogenous wild-type {beta}-catenin, identifying a functionally neutral insertion site that enables visualization at physiological, non-stabilized levels while preserving normal protein behavior. Together, these results establish twin-prime editing as a versatile platform for directly linking precise endogenous genome modification to protein visualization and allele-specific mutation analysis in intact vertebrate tissues.

cell biology↗

bioRxiv2026

A strong-to-weak interaction shift during microbiome succession is coupled to colonizer-dependent antimicrobial resistance

The outcome of ecological succession is often attributed to the characteristics of the invader or the resident community, but rarely to how the community's interaction network reorganizes during assembly. Here, we track intraspecific lineage dynamics and infer time-resolved community interaction networks using Dynamic Covariance Mapping during ecological invasion of the mouse gut by a chromosomally barcoded, spectinomycin-resistant Escherichia coli K12 colonizer. The network is initially dominated by strong, predominantly inhibitory interactions, but as community diversity recovers, the distribution of interaction strengths contracts toward zero, producing a community increasingly dominated by weak and near-neutral interactions. The dominant eigenvalue of the DCM-inferred interaction matrix moves toward marginal stability predicted for dynamically assembling ecological networks. This pattern replicates across eight independent mice in two experimental cohorts, at both inter- and intra-species resolution. The ecological transition coincides with the reproducible resurgence of Paenibacillaceae to high relative abundance and persistent coexistence with E. coli under continued spectinomycin pressure. Whole-genome sequencing of recovered Paenibacillus macerans isolates identifies recurrent mutations in ribosomal protein S5 region associated with spectinomycin binding and strongly implicating this variant in resistance. Strikingly, under antibiotic pressure but without E. coli K12 invasion, resident Paenibacillaceae never blooms, indicating that expansion of the resistant population depends on the ecological context established by the colonizer. These findings show that gut microbiome succession is accompanied by a reproducible transition from strong toward weak interactions and link this network reorganization to the colonizer-dependent ecological benefit of antimicrobial resistance.

ecology↗

bioRxiv2026

SOCS1 expression in prostate epithelial cells is essential for tissue homeostasis and tumor suppression

Suppressor of cytokine signaling 1 (SOCS1) negative regulates inflammatory cytokine production and attenuates oncogenic growth factor signaling pathways. Reduced SOCS1 protein expression in human prostate cancer correlates with greater disease severity. To define the physiological functions of SOCS1 functions in the prostate, we conditionally ablated Socs1 in prostate epithelial cells of C57BL/6 mice. These Socs1{Delta}PE mice exhibited normal prostate development, maturation and lobular architecture. However, adult Socs1{Delta}PEmice developed progressive epithelial hyperplasia and inflammatory cell infiltration that were temporally and spatially distinct. SOCS1-deficient prostate showed increased epithelial cell proliferation and elevated oxidative stress markers, and prostate organoids recapitulated this hyperplasia phenotype. Diet-induced obesity exacerbated both hyperplasia and inflammation in SOCS1-deficient prostate. Upon transurethral infection with uropathogenic Escherichia coli UPEC1677 expressing the genotoxin colibactin, Socs1{Delta}PE mice developed invasive prostate cancer with complete loss of lobular architecture, whereas control mice developed hyperplasia and pre-neoplastic lesions. In vitro, SOCS1-deficient prostate organoid-derived epithelial cells exhibited increased DNA damage following exposure to UPEC1677. Deletion of the colibactin biosynthetic gene clbP in UPEC1677 abolished its ability to induce DNA damage in SOCS1-deficient cells and to drive prostate cancer in vivo. Proteomic analysis of prostate organoids revealed dysregulation of basal and luminal epithelial lineage markers and signaling pathway proteins that could promote neoplasia in SOCS1-deficient cells. Collectively, these findings establish an essential, epithelial cell-intrinsic role for SOCS1 in maintaining prostate tissue homeostasis by restraining proliferation, regulating lineage plasticity, limiting inflammation and oxidative stress, and conferring protection against genotoxic injury and neoplastic transformation.

cancer biology↗

bioRxiv2023

SARS-CoV-2 spike antigen-specific B cell and antibody responses in pre-vaccination period COVID-19 convalescent males and females with or without post-covid condition

BackgroundFollowing SARS-CoV-2 infection a significant proportion of convalescent individuals develop the post-COVID condition (PCC) that is characterized by wide spectrum of symptoms encompassing various organs. Even though the underlying pathophysiology of PCC is not known, detection of viral transcripts and antigens in tissues other than lungs raise the possibility that PCC may be a consequence of aberrant immune response to the viral antigens. To test this hypothesis, we evaluated B cell and antibody responses to the SARS-CoV-2 antigens in PCC patients who experienced mild COVID-19 disease during the pre-vaccination period of COVID-19 pandemic. MethodsThe study subjects included unvaccinated male and female subjects who developed PCC or not (No-PCC) after clearing RT-PCR confirmed mild COVID-19 infection. SARS-CoV-2 D614G and omicron RBD specific B cell subsets in peripheral circulation were assessed by flow cytometry. IgG, IgG3 and IgA antibody titers toward RBD, spike and nucleocapsid antigens in the plasma were evaluated by ELISA. ResultsThe frequency of the B cells specific to D614G-RBD were comparable in convalescent groups with and without PCC in both males and females. Notably, in females with PCC, the anti-D614G RBD specific double negative (IgD-CD27-) B cells showed significant correlation with the number of symptoms at acute of infection. Anti-spike antibody responses were also higher at 3 months post-infection in females who developed PCC, but not in the male PCC group. On the other hand, the male PCC group also showed consistently high anti-RBD IgG responses compared to all other groups. ConclusionsThe antibody responses to the spike protein, but not the RBD-specific B cell responses diverge between convalescent males and females, and those who develop PCC or not. Our findings suggest that sex-related factors may also be involved in the development of PCC via modulating antibody responses to the SARS-CoV-2 antigens. Short SummaryPost-COVID Condition (PCC) is lingering illness that afflicts a significant proportion of COVID-19 patients from three months after clearing SARS-CoV-2 infection. Therapy for PCC is only palliative and the underlying disease mechanisms are unclear. The wide spectrum of PCC symptoms that can affect different organs and the detection of viral components in tissues distant from lungs raise the possibility that PCC may be associated with aberrant immune response due to presence of viral antigens. Therefore, we studied B cell and antibody responses to the spike and nucleoprotein antigens in PCC patients who cleared mild SARS-CoV-2 infection during the pre-vaccination COVID-19 pandemic period. We observed divergent patterns of immune reactivity to the spike protein in PCC males and females at different times post-infection, suggesting that the immune responses in PCC may also be influenced by sex-related factors.

immunology↗

bioRxiv2022

Intra- and inter-species interactions drive early phases of invasion in mice gut microbiota

The stability and dynamics of ecological communities are dictated by interaction networks typically quantified at the level of species.1-10 But how such networks are influenced by intra-species variation (ISV) is poorly understood.11-14 Here, we use ~500,000 chromosomal barcodes to track high-resolution intra-species clonal lineages of Escherichia coli invading mice gut with the increasing complexity of gut microbiome: germ-free, antibiotic-perturbed, and innate microbiota. By co-clustering the dynamics of intra-species clonal lineages and those of gut bacteria from 16S rRNA profiling, we show the emergence of complex time-dependent interactions between E. coli clones and resident gut bacteria. With a new approach, dynamic covariance mapping (DCM), we differentiate three phases of invasion in susceptible communities: 1) initial loss of community stability as E. coli enters; 2) recolonization of some gut bacteria; and 3) recovery of stability with E. coli coexisting with resident bacteria in a quasi-steady state. Comparison of the dynamics, stability and fitness from experimental replicates and different cohorts suggest that phase 1 is driven by mutations in E. coli before colonization, while phase 3 is by de novo mutations. Our results highlight the transient nature of interaction networks in microbiomes driven by the persistent coupling of ecological and evolutionary dynamics. One-Sentence SummaryHigh-resolution lineage tracking and dynamic covariance mapping (DCM) define three distinct phases during early gut microbiome invasion.

evolutionary biology↗

bioRxiv2022

Arbuscular mycorrhizae reduce stress-induced plasticity of plant functional traits. A meta-analysis study

We aimed at exploring the plant functional traits whose stress-induced plasticity is altered by the presence of AM fungi, considering the direction of their changes. We also sought for a coordinated variation of plant biomass and functional traits, during plant adaptation to environmental stressors, and the role of AM status on the variation. We performed a meta-analysis across 114 articles spanning 110 plant species or cultivars. We quantified the size effect of AM symbiosis on the stress-induced plasticity of several reported and calculated functional traits, and using linear mixed model analysis (LMM). Correlation between traits plasticity and total biomass variation were also performed through LMM. The literature search and further selection yielded seven functional traits, extracted from 114 laboratory studies, including 888 observations and 110 plant species/cultivars. Evidence for significant effects of predictor variables (type of stress, AM symbiosis and/or their interaction) on plasticity were found for three of these functional traits: leaf-area ratio (LAR), root mass fraction (RMF) and root-shoot (R:S) ratio. Our results provided evidence to accept the hypothesis that AM fungal inoculation may reduce the phenotypic plasticity of important plant functional traits leaf area ratio (LAR), root mass fraction (RMF) and root-shoot (R:S) ratio, by decreasing its magnitude. We also found a weak correlation between traits plasticity and total biomass variation. Although our literature search and data collection were intensive and our results robust, the scope of our conclusions is limited by the agronomical bias of plant species targeted by the meta-analysis. Further knowledge on non-cultivable plant species and better understanding of the mechanisms ruling resources allocation in plants would allow more generalized conclusions.

plant biology↗

bioRxiv2019

β-Catenin tumour-suppressor activity depends on its ability to promote Pro-N-Cadherin maturation

Neural stem cells (NSCs) form a pseudostratified, single-cell layered epithelium with a marked apico-basal polarity. In these cells, {beta}-Catenin associates with classic cadherins in order to form the apical adherens junctions (AJs). We previously reported that oncogenic forms of {beta}-Catenin (s{beta}-Catenin) maintain neural precursors as progenitors, while also enhancing their polarization and adhesiveness, thereby limiting their malignant potential. Here we show that {beta}-Catenin can bind to phosphorylated Pro-N-Cadherin, promoting the excision of the propeptide and its maturation into N-Cadherin in the trans-Golgi network (TGN). Moreover, {beta}-Catenin-assisted maturation of Pro-N-Cadherin is required for the formation of the AJs and for them to recruit other apical complex (AC) components like aPKC, and accordingly, to establish apico-basal polarity. Notably, we show that NSCs expressing unprocessed Pro-N-Cadherin invade the ventricle and they breach the basement membrane to invade the surrounding mesenchyme. Hence, we propose that the tumour-suppressor activity of s{beta}-Catenin depends on it promoting Pro-N-Cadherin processing.

cell biology↗

bioRxiv2018

E proteins differentially co-operate with proneural bHLH transcription factors to sharpen neurogenesis

Basic HLH proteins heterodimerize with class I HLH/E proteins to promote transcription. Here we show that E proteins differentially co-operate with proneural bHLH transcription factors sharpening their neurogeneic activity. We find that inhibiting BMP signaling or its target ID2, in the chick embryo spinal cord, impairs the neuronal production from progenitors expressing ATOH1/ASCL1, but less severely that from progenitors expressing NEUROG1/2/PTF1a. We define the mechanisms of this differential response as a dual co-operation of E proteins with proneural proteins. E proteins synergize with bHLH proteins when acting on CAGSTG motifs, thereby facilitating the neurogenic activity of ASCL1/ATOH1 which preferentially bind to such motifs. Conversely, E proteins restrict the strong neurogenic potential of NEUROG1/2 by directly inhibiting their preferential binding to CADATG motifs. Since we find this mechanism to be conserved in corticogenesis, we propose this dual co-operation of E proteins with bHLH proteins as a novel though general feature of their mechanism of action.

developmental biology↗

bioRxiv2017

PI3K regulates intraepithelial cell positioning through Rho GTP-ases in the developing neural tube.

SUMMARY STATEMENTDuring neural tube development, PI3K pathway promotes cell survival and provides the apical-basal navigation clues that define the final location of neurons in the epithelium.\n\nSUMMARYPhosphatidylinositol 3-kinases (PI3Ks) are signal transducers of many biological processes. Class 1A PI3Ks are hetero dimers formed by a regulatory and a catalytic subunit. We have used the developing chicken neural tube (NT) to study the roles played by PI3K during the process of cell proliferation and differentiation. Notably, we have observed that in addition to its well characterized anti apoptotic activity, PI3K also plays a crucial role in intra epithelial cell positioning, and unlike its role in survival that mainly depends on AKT, the activity in cell positioning is mediated by Rho GTPase family members. Additionally, we have observed that activating mutations of PI3K that are remarkably frequent in many human cancers, cause an unrestrained basal migration of the neuroepithelial cells that end up breaking through the basal membrane invading the surrounding mesenchymal tissue. The mechanism described in this work contribute not only to acquire a greater knowledge of the intraepithelial cell positioning process, but also give new clues on how activating mutations of PI3K contribute to cell invasion during the first stages of tumour dissemination.

cell biology↗