Biology subjects

Melo, R.

Publications and source records attributed to Melo, R..

4 recordsLinked to original sources

bioRxiv2026

Structure-Guided Computational Analysis of Linker effects in an scFv Targeting Guanylyl Cyclase C

Single-chain variable fragments (scFvs) are widely used in diagnostic and therapeutic applications. These antibody fragments comprise two antibody variable domains connected by a flexible peptide linker whose properties critically influence folding, stability, oligomeric state, and antigen-binding. Therefore, careful linker selection represents a key step in scFv design. Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface receptor expressed in gastrointestinal malignancies, including more than 90% of colorectal cancer (CRC) cases across all disease stages. Its restricted physiological expression pattern makes GUCY2C an attractive target for immunotherapy and precision oncology therapies. Here, we investigated the structural and functional consequences of incorporating alternative linker designs into an anti-GUCY2C scFv. Using molecular modeling, protein-protein docking, and molecular dynamics (MD) simulations, we evaluated the conformational stability, interdomain organization, and antigen-binding interactions of each construct. Our results provide a dynamic, structure-based assessment of how linker composition influences GUCY2C recognition and scFv structural behavior. Furthermore, this work establishes a computational framework for the rational optimization of GUCY2C-targeted antibody fragments.

bioinformatics↗

bioRxiv2026

Capturing Cardiomyocyte Cell-to-Cell Heterogeneity via Shotgun Single Cell Top-Down Proteomics

Individual cells exhibit distinct molecular landscapes shaped by proteins and the diverse functional repertoire of their corresponding proteoforms. These structurally diverse variants (e.g., post-translationally modified including truncated proteolyzed forms) collectively orchestrate cellular functions. However, resolving proteoform heterogeneity at single-cell (SC) resolution remains a significant analytical challenge. Here, we present a shotgun SC top-down proteomics (SC-TDP) strategy that enables direct, unbiased proteoform profiling from single cardiomyocytes. Across 13 individual cardiomyocytes isolated from mouse heart, we identified a total of 57 proteins represented by 165 distinct proteoforms, including phosphorylated, succinylated, trimethylated, truncated, amongst others. Notably, proteoform composition varied substantially among cells, revealing a previously unrecognized level of molecular heterogeneity among cardiomyocytes. Together, these findings establish SC-TDP as a powerful tool for uncovering the proteoform diversity at the SC level. Our strategy paves the way for defining functional heterogeneity in cardiac tissue with unprecedented molecular resolution, enabling direct examination of the proteoform landscape that underlies cellular identity and physiology.

cell biology↗

bioRxiv2019

Dynamical rearrangement of human epidermal growth factor receptor 2 upon antibody binding: effects on the dimerization

Human epidermal growth factor 2 (HER2) is a ligand-free tyrosine kinase receptor of the HER family that is overexpressed in some of the most aggressive tumours. Treatment of HER2+ breast cancers with the humanized monoclonal anti-HER2 antibody (Trastuzumab) revealed highly effective, encouraging the development of various HER2-specific antibodies, kinase inhibitors and dimerization inhibitors for cancer therapy. Although it is known that HER2 dimerization involves a specific region of its extracellular domain, the so-called \"dimerization arm\", the mechanism of dimerization inhibition remains uncertain. However, uncovering how antibody interactions lead to inhibition of HER2 dimerization is of key importance in understanding its role in tumour progression and therapy. Herein, we employed several computational modelling techniques for a molecular-level understanding of the interactions between HER and specific anti-HER2 antibodies, namely an antigen-binding (Fab) fragment (F0178) and a single chain variable fragment from Trastuzumab (scFv). Specifically, we investigated the effects of antibody-HER2 interactions on the key residues of \"dimerization arm\" from molecular dynamics (MD) simulations of unbound HER (in a total of 1 {micro}s), as well as scFv:HER2 and F0178:HER2 complexes (for a total of 2.5 {micro}s). A deep surface analysis of HER receptor revealed that the binding of specific anti-HER2 antibodies induced conformational changes both in the interfacial residues, which was expected, and in the ECDII, in particular at the \"dimerization arm\", which is critical in establishing protein-protein interface (PPI) interactions. Our results support and advance the knowledge on the already described trastuzumab effect on blocking HER2 dimerization through synergistic inhibition and/or steric hindrance. Furthermore, our approach offers a new strategy for fine-tuning target activity through allosteric ligands.\n\nAuthor summaryIncreasing insight into the genetics and molecular biology of diseases has resulted in the identification of a high number of potential molecular targets for drug discovery and development. Human Epidermal Growth Factor Receptor 2 (HER2) is one of the most relevant Epidermal Growth Factor Receptor (EGFR) members, whose overexpression has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. Thus, the development of novel approaches on anti-HER2 therapies is quiet relevant. Molecular modelling and simulation can be used to bring new perspectives, both in structure-based drug design and to provide atomistic information of the intermolecular coupling dynamics between inhibitors and receptors via interactive computographic software. Considering my interest in drug design and mechanisms of action using integrated in silico approaches, herein, I have used multiple methods to evaluate how HER2 coupling to its different partners could alter this functional mechanism. The results suggest that the antibodies fragments studied show different dynamic complexes with HER2 although both could contribute to downstream of the tumour cell receptor pathways. I do believe that future research breakthroughs with aid of chemo-bioinformatics will allow a more comprehensive perception on biomedicine.

bioinformatics↗

bioRxiv2019

Impression cytology is a non-invasive and effective method for ocular cell obtention from babies with Congenital Zika Syndrome: perspectives in OMIC studies.

IMPORTANCENoninvasive techniques for obtaining ocular surface cells (neuroepithelial) from babies with Congenital Zika Syndrome CZS - resulting from infection by zika virus (ZIKV) during gestational period (malformations include ocular abnormalities and microcephaly) - remain to be determined.\n\nOBJECTIVESThe aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from babies with CZS in satisfactory amounts and quality to enable the application in the context of genome approaches well as morphological and molecular evaluations.\n\nDESIGN, SETTINGS AND PARTICIPANTSIn this observational study, ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. Samples were collected with an authorized informed consent from both eyes of eight ZIKV infected babies according to the CZS diagnostic criteria (4 babies with positive PCR for Zika virus in gestation and presence of clinical signs which included ocular abnormalities and microcephaly and 4 babies with positive PCR for Zika virus during gestation but no clinical signs identified) and four unaffected babies (control samples / negative PCR, without clinical signs). Cells were used for microscopy analyses, transcriptomic and proteomic experiments and molecular procedure.\n\nMAIN OUTCOMES AND MEASURESThe microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures, including viability evaluation. In parallel, molecular approaches were performed.\n\nRESULTSOn impression cytology, a considerable amount of viable cells were captured. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed clear morphological alterations (cell keratinization, piknosis, karyolysis, anucleation and vacuolization). Genomic DNA and RNA were successfully isolated from all samples and allowed the establishment of transcriptomic and proteomic studies. Transcriptome analysis showed 8582 transcripts quantified in all samples and 63 differentially expressed genes in ocular cells from the exposed babies. Proteomics analysis allowed the identification of 2080, 2085 and 2086 high confident and unique proteins with at least one unique peptide in the unaffected, exposed to ZIKV and asymptomatic and CZS babies, respectively, being 2062 in common. Multivariate supervised analysis using the total quantitative protein features revealed a clear discrimination between the groups.\n\nCONCLUSIONS AND RELEVANCEOur method proved to be a suitable, fast, and non-invasive tool for detailed and precise morphological analyses with a perspective of application in OMIC studies for clinical and research studies of CZS.\n\nKey pointsO_ST_ABSQuestionC_ST_ABSAre the ocular surface cells of babies with Congenital Zika Syndrome viable to investigate the association between Zika virus infection during embryogenesis and ocular impairment?\n\nFindingsTo this date, this is the first study using an approach with perspectives in morphological, molecular and \"OMICs\" research from ocular samples captured by impression cytology of ZIKV infected babies during embryogenesis. The microscopic features of the conjunctival epithelial cells from all ZIKV infected babies showed clear morphological alterations.\n\nMeaningOcular cell surface capture offers a powerful model for studying the pathways involved in ocular diseases associated with ZIKV.

molecular biology↗