Biology subjects

Vasconcelos, E. J. R.

Publications and source records attributed to Vasconcelos, E. J. R..

8 recordsLinked to original sources

bioRxiv2026

Direct RNA sequencing reveals selective remodeling of the host m6A epitranscriptome during Leishmania infection

N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA, yet its role in host responses to protozoan infection remains poorly explored. Here, we provide the first characterization of the host m6A epitranscriptome during Leishmania amazonensis infection, integrating clinical data, in vitro macrophage models, and direct RNA sequencing. Analysis of publicly available transcriptomic datasets from patients with cutaneous and visceral leishmaniasis, validated in an independent cohort of patients with cutaneous leishmaniasis, revealed coordinated remodeling of the m6A regulatory machinery, characterized by significant upregulation of the writer METTL3 and downregulation of the eraser ALKBH5 and the reader YTHDF3. These changes were recapitulated in RAW264.7 macrophages infected in vitro, where METTL3 protein levels increased progressively between 2 and 8 hours post-infection (hpi), while ALKBH5 abundance declined, resulting in elevated global m6A levels detectable as early as the first post-internalization time point. Transcriptome-wide mapping by direct RNA sequencing at 8 hpi identified 1,998 m6A-modified transcripts harboring 7,132 high-confidence sites in non-infected macrophages, compared with 2,956 modified transcripts and 13,495 sites in infected cells. Beyond this quantitative expansion, infection was associated with increased modification occupancy at shared sites, a greater prevalence of multi-site and multi-region methylation, and selective remodeling of immune-related and immunometabolic transcripts, including Tlr4, Ccl2, Hmox1, Socs3, Rela, Hk1, Hk2, and Dicer1. Altogether, these findings establish that Leishmania infection drives extensive yet selective remodeling of the host m6A landscape and position the host epitranscriptome as a previously unrecognized regulatory axis in Leishmania-macrophage interactions, with implications for the development of host-directed therapeutic strategies. Impact StatementN6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA, and emerging evidence links epitranscriptomic remodeling to host responses against diverse pathogens. However, whether m6A regulates host-macrophage responses during Leishmania infection remains unknown. Using Oxford Nanopore Direct RNA sequencing, we provided the first transcriptome-wide map of host m6A modifications during Leishmania amazonensis infection. Infection selectively remodels the m6A landscape of macrophages, targeting immune-regulatory and immunometabolic transcripts, establishing the host epistranscriptome as a previously unrecognized regulatory layer in Leishmania-macrophage interactions.

microbiology↗

bioRxiv2026

Conventional dendritic cells type I with an enhanced type-I-IFN signaling underpin anti-tumor immune responses in brain metastases

Brain metastases are associated with poor prognosis. A better understanding of anti-tumor immune responses in the context of immune specialized microenvironment of the brain is required to develop improved therapeutic strategies for this disease. We demonstrate that the conventional dendritic cells type 1 (cDC1) gene signature positively correlates with a prolonged BrM-dependent survival in melanoma and breast cancer patients. Furthermore, intracranial anti-tumor immune responses in preclinical BrM models consistently rely on cDC1s for tumor growth control, BrM-dependent survival and maintenance of the intra-tumoral CD8+ T cell pool, in contrast to variable, cancer type-dependent cDC1 roles in extracranial tumors. This is underpinned by tumor site-specific cDC1 molecular profiles with distinct Toll like receptor repertoires, upregulation of co-stimulatory molecules and IL-12, and enhanced type-I-IFN signaling in intracranial cDC1s, with the latter driving increased cDC1 activation. cDC1s also promote the conversion of progenitor exhausted CD8+ T cells to transient effectors, which is further enhanced by immune checkpoint blockade therapy. These findings pinpoint cDC1s as a major cell population of interest in the development of future immunotherapies for BrM. Significance statementCharacteristics of immune cells and tumor microenvironment in brain metastases (BrM) are distinct to extracranial tumor sites and characterized by special immunological barriers and suppressive stroma. Conventional dendritic cells type 1 (cDC1s), which are key regulators of anti-tumor immunity, remain largely unexplored in this context. This study deepens our understanding of how anti-tumor immune responses differ between BrM and extracranial tumors by revealing BrM-specific phenotype and function of cDC1s, with the latter being critical for anti-tumor immunity in BrM even in cancer models where extracranial cDC1s demonstrate pro-tumorigenic function, highlighting cDC1s as universal regulators of intracranial anti-tumor immunity. Our study suggests that tumor site-specific augmentation of cDC1s should be pursued to enhance the efficacy of immunotherapies in BrM.

cancer biology↗

bioRxiv2026

Cell-autonomous restoration of splicing homeostasis and RP11 phenotype in patient-derived RPE and retinal organoids by PRPF31.AAV gene therapy

Mutations in PRPF31 gene cause retinitis pigmentosa type 11 (RP11) through haploinsufficiency, impairing spliceosome assembly and triggering progressive retinal degeneration. While gene augmentation holds therapeutic promise, key questions remain regarding the mechanistic basis of rescue and its therapeutic efficacy across all primarily affected human retinal cell types and disease stages. Here, we utilised patient-specific induced pluripotent stem cells (iPSCs)-derived retinal pigment epithelium (RPE) cells and three-dimensional retinal organoids (ROs) to determine the therapeutic mechanism of AAV-mediated PRPF31 delivery. Using the ShH10(Y445F) serotype to ensure robust dual targeting of RPE and photoreceptors, we demonstrated that PRPF31 transduction restores nuclear localisation, reorganises SC35+ nuclear speckles and enhances p-SF3B1 active spliceosome foci. Transcriptomic and proteomic profiling revealed a global rescue of splicing activity and upregulation of phagocytosis, protein aggregate clearance pathways, and mitochondrial proteins. These molecular shifts facilitated the clearance of proteotoxic cytoplasmic aggregates and reversed key functional deficits; specifically, they reinforced RPE apical-basal polarity, restored phagocytic capacity and normal ciliary morphology and incidence, and boosted light-evoked activity in photoreceptors. Combining gene therapy with rapamycin-mediated-autophagy activation conferred no additive benefit, identifying the restoration of splicing homeostasis as the critical therapeutic driver. Notably, substantial phenotypic rescue is achievable in mature RPE, supporting a broad clinical window for intervention. Collectively, these data provide a systems-level validation of ShH10(Y445F)-PRPF31 gene therapy and establish a mechanistic framework for its clinical translation in RP11.

molecular biology↗

bioRxiv2024

Translational activity of 80S monosomes varies dramatically across different tissues

SummaryTranslational regulation at the stage of initiation can impact the number of ribosomes translating each mRNA molecule. However, the translational activity of single 80S ribosomes (monosomes) on mRNA is less well understood, even though these 80S monosomes represent the dominant ribosomal complexes in vivo. Here, we used cryo-EM to determine the translational activity of 80S monosomes across different tissues in Drosophila melanogaster. We discovered that while head and embryo 80S monosomes are highly translationally active, testis and ovary 80S monosomes are translationally inactive. RNA-Seq analysis of head monosome- and polysome-translated mRNAs, revealed that head 80S monosomes preferentially translate mRNAs with TOP motifs, short 5-UTRs, short ORFs and are enriched for uORFs. Overall, these findings highlight that regulation of translation initiation, and that protein synthesis is mostly performed by monosomes in head and embryo, whilst polysomes are the main source of protein production in testis and ovary. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/600330v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@12881e5org.highwire.dtl.DTLVardef@6804dborg.highwire.dtl.DTLVardef@116fa63org.highwire.dtl.DTLVardef@19095d6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LID. melanogaster 80S monosomes and polysomes purified from different tissues exhibit different translational activities C_LIO_LIHead and 0-2 hr embryo 80S monosomes are highly translationally active C_LIO_LITestis and ovary 80S monosomes are translationally inactive C_LIO_LIHead 80S monosomes preferentially translate mRNAs with TOP motifs, short 5-UTRs, short ORFs and are enriched for uORFs C_LIO_LIHead polysomes preferentially translate mRNAs with neuronal functions C_LI

molecular biology↗

bioRxiv2023

Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin: In vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo Vs in vitro and in vivo Vs cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). miRNA cargo was similar between all three groups (426 common cargo) differences between in vivo and cloned (8 miRNAs), and in vivo and in vitro (6 miRNAs) observed. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changes to the proteomic content of target cells (96, and 85 respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy. SIGNIFICANCE STATEMENTDuring the peri-implantation period of pregnancy in mammals, the endometrium acts as a biosensor for the developmental competency of the embryo. However, the mechanism by which biosensor capability of the endometrium is established, remains elusive. In this study, we show that embryos of different developmental competencies have distinct microRNA cargo contained in their extracellular vesicles (EVs). Exposure of the endometrium to these conceptuses alters the transcriptional response of the endometrium during the process of pregnancy recognition. This differential response is mediated in part, by the delivery and action of the these differentially abundant microRNAs into EVs. Here we propose differences in EV-mediated miRNA cargo are responsible in part for this biosensor capability of the endometrium.

developmental biology↗

bioRxiv2020

Endometrium-on-a-chip reveals the endometrial transcriptome, and protein content of secretome are altered by changes in circulating concentrations of insulin and glucose in vitro.

The molecular interactions between the maternal environment and developing embryo that are key for early pregnancy success are known to be influenced by factors such as the metabolic status. We are, however, limited in our understanding of the mechanism by which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success. Here we report for the first time the use of endometrium-on-a-chip microfluidics approach to produce a multi-cellular endometrium in vitro, that is exposed to glucose and insulin concentrations associated with maternal metabolic stressors. Following isolation of endometrial cells (epithelial and stromal) from the uteri of non-pregnant cows in early-luteal phase (Day 4-7 approximately) epithelial cells were seeded into the upper chamber (4-6 104 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 104 cells/mL). Three different concentration of glucose 1) 0.5 mM 2) 5.0 mM or 3) 50 mM or insulin 1) Vehicle, 2) 1 ng/mL or 3) 10 ng/mL were performed in the endometrial cells at a flow rate of 1{micro}L/min for 72 hr to mimic the rate of secretion in vivo. Quantitative differences in the transcriptomic response of the cells and the secreted proteome of in vitro-derived uterine luminal fluid (ULF) were determined by RNA-sequencing and TMT respectively. Changes in maternal glucose altered 21 and 191 protein coding genes in epithelial and stromal cells respectively (p<0.05). While there was a dose-dependent quantitative change in protein secretome (1 and 23 proteins). Insulin resulted in limited transcriptional changes including insulin-like binding proteins that were cell specific (5, 12, and 20) but altered the quantitative secretion of 196 proteins including those involved in extracellular matrix-receptor interaction and proteoglycan signaling in cancer. Collectively, these highlight the potential mechanism by which changes to maternal glucose and insulin alter uterine function.

molecular biology↗

bioRxiv2020

LncRNAs interacting with the translation machinery contribute to human neuronal differentiation.

LncRNAs are less conserved, yet more tissue and developmental-stage specific than mRNAs and are particularly enriched in the nervous system of Drosophila melanogaster, mouse and human. The function of cytoplasmic lncRNAs and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of SH-SH5Y cells. We discovered 237 cytoplasmic lncRNAs upregulated during early neuronal differentiation, most of which are associated with polysome complexes. The majority are cytoplasmically enriched and are intergenic or anti-sense. In addition, we find 45 small ORFs in lncRNAs to be actively translated, 17 specifically upon differentiation. 11 of these smORFs exhibit high sequence conservation across Hominidae suggesting they are under strong selective constraint with putative function in this clade. We discover LINC01116 is induced upon differentiation and contains an 87 codon smORF, which we detect as translated, with increased ribosome profiling signal upon differentiation. The LINC01116 peptide exhibits a cytoplasmic distribution and is detected in neurites. Knockdown of LINC01116 results in significant reduction of neurite length in differentiated cells indicating it contributes to neuronal differentiation. Our findings indicate lncRNAs are a source of non-canonical peptides and contribute to neuronal function.

molecular biology↗

bioRxiv2018

In silico identification of GPI-anchored proteins in Paracoccidioides

Glycosylphosphatidylinositol-anchored proteins (GPI-proteins) are widely found in eukaryotic organisms. In fungi, GPI-proteins are thought to be involved in diverse cellular mechanisms such as cell wall biosynthesis and cell wall remodeling, adhesion, antigenicity, and virulence. The conserved structural domains of GPI-protein allow the utilization of in silico prediction approach to identify this class of proteins using a genome-wide analysis. We used different previously characterized algorithms to search for genes that encode predicted GPI-proteins in the genome of P. brasiliensis and P. lutzii, thermal dimorphic fungi that causes paracoccidioidomycosis (PCM). By using these methods, 98 GPI-proteins were found in P. brasiliensis with orthologs in P. lutzii. A series of 28 GPI-proteins were classified in functional categories (such as glycoside hydrolases, chitin-processing proteins, and proteins involved in the biogenesis of the cell wall). Furthermore, 70 GPI-proteins exhibited homology with hypothetical conserved proteins of unknown function. These data will be an important resource for the future analysis of GPI-proteins in Paracoccidioides spp.

microbiology↗