Biology subjects

Silva, T. P.

Publications and source records attributed to Silva, T. P..

5 recordsLinked to original sources

bioRxiv2026

An integrated single cell and spatial omics atlas of human prenatal development

Single cell genomics has enabled analysis of human prenatal development at unprecedented resolution. However, most studies have relied on dissociated tissues during restricted windows of development, limiting insights into how spatially distributed networks of cells, and multicellular niches emerge and adapt to distinct organ microenvironments in situ. Moreover, existing human developmental atlases have not yet been harmonised, and we thus lack a comprehensive catalogue of known cell types in the developing human body. Here, we introduce the Human Developmental Cell Atlas (HDCA), a unified structural, cellular and molecular resource for prenatal human development. The HDCA integrates published and unpublished single cell/nucleus RNAseq atlases across prenatal organs, and includes a newly generated, spatially resolved, multimodal cell atlas of intact human embryos. Spanning 4-22 post conceptional weeks, capturing embryonic and early to mid fetal stages, the HDCA contains [~]4.6 million cells/nuclei which resolve into [~]450 cell types, explorable with a bespoke web portal. For a global overview of the human embryos multicellular communities, we applied unsupervised deep learning to our intact human embryo spatial data, charting 114 tissue niches that are structural and signalling hubs for the cellular interactions of the embryo. Guided by these niches, we profiled cellular networks over space and time, not examinable using single-organ atlases. In so doing, we revealed tissue-specific fibroblast patterning from previously undescribed mesenchyme progenitors, early diversification of organ-specific blood capillaries and lymphatic vasculature, emergence of neural crest cell fates, the formation of placode-and neural crest-derived peripheral sensory neurons, and how tissue niches guide peripheral neuron maturation and axonal migration. The HDCA thus serves as a comprehensive step towards a comprehensive understanding of human prenatal development, and a template towards unravelling the biology of congenital disorders.

developmental biology↗

bioRxiv2021

Transcriptome profiling of human pluripotent stem cell-derived cerebellar organoids reveals fastercommitment under dynamic conditions

Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to recapitulate cerebellar development in vitro is still limited. We presented a reproducible and scalable production of cerebellar organoids by using the novel Vertical-Wheel single-use bioreactors, in which functional cerebellar neurons were obtained. Here, we evaluate the global gene expression profiles by RNA sequencing (RNA-seq) across cerebellar differentiation, demonstrating a faster cerebellar commitment in this novel dynamic differentiation protocol. Furthermore, transcriptomic profiles suggest a significant enrichment of extracellular matrix (ECM) in dynamic-derived cerebellar organoids, which can better mimic the neural microenvironment and support a consistent neuronal network. Thus, an efficient generation of organoids with cerebellar identity was achieved for the first time in a continuous process using a dynamic system without the need of organoids encapsulation in ECM-based hydrogels, allowing the possibility of large-scale production and application in high-throughput processes. The presence of factors that favors angiogenesis onset was also detected in dynamic condition, which can enhance functional maturation of cerebellar organoids. We anticipate that large-scale production of cerebellar organoids may help developing models for drug screening, toxicological tests and studying pathological pathways involved in cerebellar degeneration.

bioengineering↗

bioRxiv2020

Whole slide imaging to understand Candida biofilm growth and response to antifungals

New strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyze fungal biofilm growth.

microbiology↗

bioRxiv2019

CImP: Cellular Imprinting Proteomics applied to ocular disorders elicited by Congenital Zika virus Syndrome

IMPORTANCEOcular complications in infants with Congenital Zika Syndrome (CZS) have been reported. However, the molecular mechanisms underlying of eye dysfunctions are presently unknown.\n\nOBJECTIVEA method (termed Cellular Imprinting Proteomics, CImP) for the identification and quantification of the ocular surface proteome using a minimally invasive membrane filter device is described. Moreover, The CImP method was applied to profile the molecular alterations in the eyes of infants exposed to Zika virus (ZIKV) infection during gestation.\n\nDESIGN, SETTINGS AND PARTICIMPANTSThe CImP method was applied to a cohort divided into three conditions: 1) Ctrl (infants with no infectious diseases, n=5). 2) Zikv (infants exposed to ZIKV gestation, with no microcephaly, n=5). 3) ZikvCZS (infants exposed to ZIKV, with microcephaly, n=3). All conditions were age and sex-matched. An improved impression cytology method was used to capture the outermost ocular surface cells. The number of impression cytology membrane collected was: Ctrl (12), Zikv (14) and ZikvCZS (8). Proteins were extracted and analysed using mass spectrometry-based proteomics technology followed by statistical analysis. Parallel reaction monitoring was performed to validate the expression of specific protein markers.\n\nRESULTSUsing the CImP method, 2209 proteins were identified on the membrane-captured conjunctiva epithelial cells. Modulation of neutrophil degranulation, cell death, ocular and neurodevelopment pathways are reported in infants with CZS compared to matched controls. Moreover, the molecular pattern of ocular surface cells retrieved from infants infected during the gestation but with no CZS was different from matched controls.\n\nCONCLUSIONS AND PERSPECTIVESMolecular alterations in the ocular cell surface associated to ZIKV infection with and without CZS complications are reported for the first time. We predict that this method will be introduced successfully in the study of several neurological diseases with the aim to identify novel diagnostic and therapeutic biomarkers.

biochemistry↗

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↗