Biology subjects

Neves, L. X.

Publications and source records attributed to Neves, L. X..

5 recordsLinked to original sources

bioRxiv2026

Efficient plasmid-based rescue of T7 RNA polymerase-driven calicivirus reverse genetics systems in mammalian cells using vaccinia virus RNA capping enzymes

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either on the production of in vitro-transcribed RNA or plasmid-based rescue either from a mammalian promoter, or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for Murine Norovirus (MNV) reverse genetics. Addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells, and viral polyprotein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LFcells, viral titres increased 100-1000-fold compared over standard BSR-T7 cells. This system offers a robust, high-throughput means of assessing viral mutants.

microbiology↗

bioRxiv2025

Single-cell phenotypic analysis and multiplet detection through incorporation of microscopy data into cellenONE-based single-cell proteomic data analysis

The reliability of single-cell proteomics (SCP) is intrinsically linked to the fidelity of cell isolation; however, the identification of co-isolated cells (doublets or multiplets) remains a persistent challenge for single-cell (sc-) omics. While single-cell transcriptomics has established probabilistic frameworks for doublet detection, these methods are ill-suited for the sparser throughput of SCP datasets. This work presents scpImaging, a novel computational pipeline that repurposes the latent microscopy data routinely generated during cellenONE-based sample preparation to provide deterministic quality control (QC) and high-content phenotypic profiling. We demonstrate that standard proteomic quality metrics for SCP (e.g., peptide count, signal intensity) paradoxically favour retaining doublets. In contrast, scpImaging applies machine-learning-based cell segmentation (Cellpose-SAM) to identify and exclude these artefacts with high precision. Furthermore, the framework integrates morphological metrics generated using the open-source CellProfiler, with proteomic abundance data, enabling joint analysis that links cell shape and texture to molecular cell state. Provided as an open-source R package, scpImaging offers a scalable, automated solution for enhancing data integrity and adding a phenotypic dimension to SCP experiments without increasing experimental cost or complexity.

systems biology↗

bioRxiv2025

Viral protease-mediated polyprotein processing in human astroviruses

Positive-sense RNA viruses often encode large polyproteins that are proteolytically processed by viral and host proteases into functional replication proteins. Astroviruses infect intestinal and neuronal cells across diverse human and animal hosts and also rely on polyprotein cleavage for replication. In this study, we mapped the cleavage sites of the nonstructural polyproteins of classical human astrovirus 1 (HAstV1) and neurotropic astrovirus strain MLB2 using complementary N-terminomics of infected cells and analyses of untagged overexpressed polyproteins. Notably, we identified two adjacent cleavage sites at the N-terminus of HAstV1 and MLB2 proteases, as well as a similar dual cleavage site at the C-terminus of the MLB2 protease. We also demonstrated processing of the hypervariable region and VPg in both astrovirus strains. This allowed us to define the boundaries of individual protein products and identify conserved and divergent processing features between classical and non-classical astroviruses. Additionally, we characterized several polyprotein precursors and evaluated the replication properties of cleavage-deficient mutant replicons, revealing the critical role of polyprotein processing for functional replication complex formation. Understanding the dynamics of polyprotein processing is essential for interpreting the stages of viral infection and identifying new drug targets and antiviral strategies.

microbiology↗

bioRxiv2023

Deacetylation by sirtuins is important for Aspergillus fumigatus pathogenesis and virulence

Protein acetylation is a crucial post-translational modification that controls gene expression and a variety of biological processes. Sirtuins, a prominent class of NAD+-dependent lysine deacetylases, serve as key regulators of protein acetylation and gene expression in eukaryotes. In this study, six single knockout strains of fungal pathogen Aspergillus fumigatus were constructed, in addition to a strain lacking all predicted sirtuins (SIRTKO). Phenotypic assays suggest that sirtuins are involved in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. AfsirE deletion resulted in attenuation of virulence, as demonstrated in murine and Galleria infection models. The absence of AfSirE leads to altered acetylation status of proteins, including histones and non-histones, resulting in significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

microbiology↗

bioRxiv2021

Wiring multiple microenvironment proteomes uncovers the biology in head and neck cancer

The poor prognosis of head and neck cancer (HNC) is associated with the presence of metastasis within the lymph nodes (LNs). Herein, the proteome of 140 multisite samples from a 59-HNC patient cohort, including primary and matched LN-negative or -positive tissues, saliva, and blood cells, reveals insights into the biology and potential metastasis biomarkers that may assist in clinical decision making. Protein profiles are strictly associated with immune modulation across datasets, and this provides the basis for investigating immune markers associated with metastasis. The proteome of LN metastatic cells recapitulates the proteome of the primary tumor sites. Conversely, the LN microenvironment proteome highlights the candidate prognostic markers. By integrating prioritized peptide, protein, and transcript levels with machine learning models, we identified a nodal metastasis signature in the blood and saliva. In summary, we present the deepest proteome characterization wiring multiple sampling sites in HNC, thus providing a promising basis for understanding tumoral biology and identifying metastasis-associated signatures.

cancer biology↗