Biology subjects

Palmisano, G.

Publications and source records attributed to Palmisano, G..

23 records · Page 2Linked to original sources

bioRxiv2020

New insights into Trypanosoma cruzi evolution and genotyping based on system-wide protein expression profiles (PhyloQuant)

The etiological agent of Chagas disease, Trypanosoma cruzi, is subdivided into seven genetic subdivisions termed discrete typing units (DTUs), TcI-TcVI and Tcbat. The relevance of T. cruzi genetic diversity to the variable clinical course of the disease, virulence, pathogenicity, drug resistance, transmission cycles and ecological distribution justifies the concerted efforts towards understanding the population structure of T. cruzi strains. In this study, we introduce a novel approach termed phyloquant to infer the evolutionary relationships and assignment of T. cruzi strains to their DTUs based on differential protein expression profiles evidenced by bottom up large scale mass spectrometry-based quantitative proteomic features. Mass spectrometry features analyzed using parsimony (MS1, iBAQ and LFQ) showed a close correlation between protein expression and T. cruzi DTUs and closely related trypanosome species. Although alternative topologies with minor differences between the three MS features analyzed were demonstrated, we show congruence to well accepted evolutionary relationships of T. cruzi DTUs; in all analyses TcI and Tcbat were sister groups, and the parental nature of genotype TcII and the hybrid genotypes TcV/TcVI were corroborated. Character mapping of genetic distance matrices based on phylogenetics and phyloquant clustering showed statistically significant correlations. We propose the first quantitative shotgun proteomics approach as a complement strategy to the genetic-based assignment of T. cruzi strains to DTUs and evolutionary inferences. Moreover, this approach allows for the identification of differentially regulated and strain/DTU/species-specific proteins, with potential application in the identification of strain/DTU specific biomarkers and candidate therapeutic targets. In addition, the correlation between multi-gene protein expression and divergence of trypanosome species was evaluated, adding another level to understand the genetic subdivisions among T. cruzi DTUs.

evolutionary biology↗

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↗

bioRxiv2019

Simultaneous proteomics and three PTMomics characterization of pro-inflammatory cytokines stimulated INS-1E cells using TiO2 enrichment strategy

Diverse protein post-translational modifications (PTMs) in proteins form complex combinatorial patterns to regulate the protein function and biological processes in a fine-tuning manner. Reversible phosphorylation, cysteines (Cys) modification, and N-linked glycosylation are essentially involved in cellular signaling pathways of pro-inflammatory cytokines, which can induce beta cell death and diabetes. Here we developed a novel mass spectrometry-based proteomic strategy (termed TiCPG) for the simultaneous comprehensive characterization of the proteome and three post-translational modifications (PTMomes) by applying TiO2 enrichment of peptides with reversibly modified Cysteine (rmCys), Phosphorylation, and sialylated N-linked (SAN-) Glycosylation from low amount of sample material with largely minimized sample loss. We applied this TiCPG strategy to quantitatively study the change of the three PTMs in {beta}-cell-like INS-1E cells subject to pro-inflammatory cytokines stimulation. It enabled efficient enrichment and quantitative analysis of 8346 rmCys sites, 10321 phosphosites and 1906 SAN-glycosylation sites from 5853 proteins. Significant regulation was found on 100 proteins at the total protein level, while much higher degree of regulation was identified on 3025 peptides with PTMs from 1490 proteins. The three PTMs were co-regulated in proteins, but demonstrated differential spatial and temporal patterns related to protein cellular localization and function in the time course of cytokines stimulation, and they were extensively involved in essential signaling pathways related to pro-inflammatory cytokine mediated {beta}-cell apoptosis, such as the inducible NO synthase (NOS2) signaling pathway, Overall, the TiCPG strategy is proved as a straight forward and powerful tool for multiple PTMomics studies.

biochemistry↗

bioRxiv2017

Advanced Protection Against Marine Biofouling Using Solar Light

We undertook prolonged testing of a new xerogel coating (AquaSun) to protect from marine biofouling a surface probe immersed in the seawater of Abu Dhabi. Electron microscopy, Raman mapping, and photoluminescence experiments coupled to extensive analyses of marine water before and after 122 days of testing showed excellent action against biofouling when irradiating the probe with visible light. Considering the high chemical and mechanical stability and the low cost of the sol-gel coating, the technology has significant potential en route to replacing conventional antifouling and foul release coatings with a single product of broad applicability.

bioengineering↗