Percorrer por autor "Esteves, Ana Cristina"
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- An analysis of protein patterns present in the saliva of diabetic patients using pairwise relationship and hierarchical clusteringPublication . Soares, Airton; Esteves, Eduardo; Rosa, Nuno; Esteves, Ana Cristina; Lins, Anthony; Bastos-Filho, Carmelo J. A.Molecular diagnosis is based on the quantification of RNA, proteins, or metabolites whose concentration can be correlated to clinical situations. Usually, these molecules are not suitable for early diagnosis or to follow clinical evolution. Large-scale diagnosis using these types of molecules depends on cheap and preferably noninvasive strategies for screening. Saliva has been studied as a noninvasive, easily obtainable diagnosis fluid, and the presence of serum proteins in it enhances its use as a systemic health status monitoring tool. With a recently described automated capillary electrophoresis-based strategy that allows us to obtain a salivary total protein profile, it is possible to quantify and analyze patterns that may indicate disease presence or absence. The data of 19 persons with diabetes and 58 healthy donors obtained by capillary electrophoresis were transformed, treated, and grouped so that the structured values could be used to study individuals’ health state. After Pairwise Relationships and Hierarchical Clustering analysis were observed that amplitudes of protein peaks present in the saliva of these individuals could be used as differentiating parameters between healthy and unhealthy people. It indicates that these characteristics can serve as input for a future computational intelligence algorithm that will aid in the stratification of individuals that manifest changes in salivary proteins.
- COVID-19 salivary protein profile: unravelling molecular aspects of SARS-CoV-2 infectionPublication . Esteves, Eduardo; Mendes, Vera M.; Manadas, Bruno; Lopes, Rafaela; Bernardino, Liliana; Correia, Maria José; Barros, Marlene; Esteves, Ana Cristina; Rosa, NunoCOVID-19 is the most impacting global pandemic of all time, with over 600 million infected and 6.5 million deaths worldwide, in addition to an unprecedented economic impact. Despite the many advances in scientific knowledge about the disease, much remains to be clarified about the molecular alterations induced by SARS-CoV-2 infection. In this work, we present a hybrid proteomics and in silico interactomics strategy to establish a COVID-19 salivary protein profile. Data are available via ProteomeXchange with identifier PXD036571. The differential proteome was narrowed down by the Partial Least-Squares Discriminant Analysis and enrichment analysis was performed with FunRich. In parallel, OralInt was used to determine interspecies Protein-Protein Interactions between humans and SARS-CoV-2. Five dysregulated biological processes were identified in the COVID-19 proteome profile: Apoptosis, Energy Pathways, Immune Response, Protein Metabolism and Transport. We identified 10 proteins (KLK 11, IMPA2, ANXA7, PLP2, IGLV2-11, IGHV3-43D, IGKV2-24, TMEM165, VSIG10 and PHB2) that had never been associated with SARS-CoV-2 infection, representing new evidence of the impact of COVID-19. Interactomics analysis showed viral influence on the host immune response, mainly through interaction with the degranulation of neutrophils. The virus alters the host’s energy metabolism and interferes with apoptosis mechanisms.
- Effect of γ-aminobutyric acid (GABA) on the metabolome of two strains of Lasiodiplodia theobromae isolated from grapevinePublication . Salvatore, Maria Michela; Félix, Carna; Lima, Fernanda; Ferreira, Vanessa; Duarte, Ana Sofia; Salvatore, Francesco; Alves, Artur; Esteves, Ana Cristina; Andolfi, AnnaThe effect of γ-aminobutyric acid (GABA) on the metabolome of two strains of Lasiodiplodia theobromae isolated from grapevine that hold a different degree of virulence to the host plant (LA-SOL3 (more virulent), LA-SV1 (less virulent)) was investigated. The culture filtrates and crude extracts from the two strains grown in the presence and absence of 10 mM of GABA were tested for phytotoxicity on tomato plant cuttings and leaves, respectively. Considering the opportunistic nature of this fungus for humans, crude extracts were also tested for cytotoxicity on mammalian cell lines. We found that culture filtrates and crude extracts have a decreased toxicity in the presence of GABA. Metabolomic analysis, conducted on both strains at both growth conditions, revealed the production of several compounds, such as indole-3-carboxylic acid (ICA, which is the main compound produced by L. theobromae), 3-indolecarboxyaldehyde, (3R,4S)-botryodiplodin, (R)-mellein. Finally, data demonstrate that GABA both induces a decrease in the amount of ICA, and a diversification of the metabolites produced by L. theobromae.
- Molecular techniques and target selection for the identification of Candida spp. in oral samplesPublication . Magalhães, Joana; Correia, Maria José; Silva, Raquel M.; Esteves, Ana Cristina; Alves, Artur; Duarte, Ana SofiaCandida species are the causative agent of oral candidiasis, with medical devices being platforms for yeast anchoring and tissue colonization. Identifying the infectious agent involved in candidiasis avoids an empirical prescription of antifungal drugs. The application of high-throughput technologies to the diagnosis of yeast pathogens has clear advantages in sensitivity, accuracy, and speed. Yet, conventional techniques for the identification of Candida isolates are still routine in clinical and research settings. Molecular approaches are the focus of intensive research, but conversion into clinic settings requires overcoming important challenges. Several molecular approaches can accurately identify Candida spp.: Polymerase Chain Reaction, Microarray, High-Resolution Melting Analysis, Multi-Locus Sequence Typing, Restriction Fragment Length Polymorphism, Loop-mediated Isothermal Amplification, Matrix Assisted Laser Desorption Ionization-mass spectrometry, and Next Generation Sequencing. This review examines the advantages and disadvantages of the current molecular methods used for Candida spp. Identification, with a special focus on oral candidiasis. Discussion regarding their application for the diagnosis of oral infections aims to identify the most rapid, affordable, accurate, and easy-to-perform molecular techniques to be used as a point-of-care testing method. Special emphasis is given to the difficulties that health care professionals need to overcome to provide an accurate diagnosis.
- P8 - Marine fungi exhibit antimicrobial activity against human oral pathogensPublication . Correia, Bruna L.; Devesas, Daniela; Noites, Rita; Gomes, Ana T. P. C.; Esteves, Ana Cristina; Alves, Artur; Duarte, Ana SofiaThe emergence of resistance to antibiotics and antimycotics has become a challenge in the treatment of infectious diseases, including infections of the oral cavity. Marine fungi are a source of novel biologically active compounds, namely in what concerns the development of antimicrobial and anticancer solutions. Our study aimed to test the antimicrobial activity and the cytotoxicity of the extracts of the two recent identified species of marine fungi, Penicillum lusitanum and Aspergillus affinis. Candida spp. and Enterococcus faecalis isolated from oral pathologies were included to evaluate the antimicrobial potential of the marine fungi by the disk diffusion assay. The cytotoxicity of the effective concentrations of the extract was tested using the Vero cell line (ECACC 88020401, African Green Monkey Kidney cells, GMK clone), according to the ISO 10993-5. The extracts of P. lusitanum and A. affinis were active against C. albicans and E. faecalis, respectively. Penicillum lusitanum active extracts are non-cytotoxic, in contrast to A. affinis extracts that showed high cytotoxic effects on Vero cells, for all concentrations tested. The results on the biological characterization of the P. lusitanumextract are promising and support the development of new disinfecting solutions that may be used during root canal therapy cleaning and shaping.
- Population wide testing pooling strategy for SARS-CoV-2 detection using salivaPublication . Esteves, Eduardo; Mendes, Ana Karina; Barros, Marlene; Figueiredo, Cátia; Andrade, Joana; Capelo, Joana; Novais, António; Rebelo, Carla; Soares, Rita; Nunes, Ana; Ferreira, André; Lemos, Joana; Duarte, Ana Sofia; Silva, Raquel M.; Bernardino, Liliana Inácio; Correia, Maria José; Esteves, Ana Cristina; Rosa, NunoSARS-CoV-2 pandemic has forced frequent testing of populations. It is necessary to identify the most cost-effective strategies for the detection of COVID-19 outbreaks. Nasopharyngeal samples have been used for SARS-CoV-2 detection but require a healthcare professional to collect the sample and cause discomfort and pain to the individual. Saliva has been suggested as an appropriate fluid for the diagnosis of COVID-19. We have investigated the possibility of using pools of saliva samples to detect SARS-CoV-2 in symptomatic and asymptomatic patients. Two hundred and seventy-nine saliva samples were analyzed through RT-PCR of Envelope, Nucleocapsid and Open Reading Frame 1ab genes. Reproducibility assays showed an almost perfect agreement as well as high sensitivity (96.6%), specificity (96.8%), positive predicted value (96.6%), and negative predicted value (96.8%). The average Cycle Threshold of the genes detected was 29.7. No significant differences (p > 0.05) were detected when comparing the cycle threshold average of two consecutive reactions on the same positive saliva samples. Saliva samples have a higher median viral load (32.6) than in nasopharyngeal samples (28.9), although no significant differences were detected (p > 0.05). Saliva-pool samples allowed effective SARS-CoV-2 screening, with a higher sensibility (96.9%) on 10-sample pools than in 20-sample pools (87.5%). Regardless of pools size specificity was high (99.9%) and an almost perfect agreement was observed. Our strategy was successfully applied in population wide testing of more than 2000 individuals, showing that it is possible to use pooled saliva as diagnostic fluid for SARS-CoV-2 infection.
- A protein profiling strategy for periodontal disease applications: the Perio-SalivaPRINTPublication . Rosa, Nuno; Esteves, Eduardo; Esteves, Ana Cristina; Fernandes, Gustavo; Correia, Maria; Siqueira, Walter L.; Barros, MarleneObjectives: It is known that several clinical situations have characteristic molecular deregulations. Some molecular data underlying these deregulations can be found in saliva and have been annotated in databases (SalivaTecDB). Strategies are needed to identify the phenotypes characteristic of these deregulations. Our group has developed a strategy that allows the establishment of saliva protein profiles reflecting different conditions (health and disease). These profiles can be integrated to clinical data (SalivaPRINT Toolkit). The present work aims to identify the Periodontal Diseases (PD)-specific protein profiles. Methods: Unstimulated whole saliva was collected from a group of healthy subjects and a group of PD patients (with gingivitis, periodontitis or periimplantitis). Salivary proteins were separated by the Experion™ automated capillary electrophoresis. The protein profiles of each condition were integrated with the corresponding protein data retrieved from our in-house database (SalivaTecDB). Results: The strategy used enabled the determination of a total protein profile from saliva characteristic of each PDs -the Perio-SalivaPrint. The use of the SalivaPrint Toolkit allowed the identification of molecular weight ranges altered in PD. Using SalivaTecDB we were able to suggest proteins potentially involved in the underlying dysregulated mechanisms of the disease. Conclusions: This approach enabled the determination of a Perio-SalivaPrint – protein profiles specific for gingivitis, periodontitis or periimplantitis - that could empower the use of saliva as a simple and less expensive diagnostic and monitoring fluid. The strategy presented could be an important tool for future applications in the early diagnostic/ screening of Periodontal Disease patients with applications in chairside monitoring.
- SalivaPrint as a non-invasive diagnostic toolPublication . Esteves, Eduardo; Cruz, Igor; Esteves, Ana Cristina; Barros, Marlene; Rosa, NunoCurrently, the molecular diagnosis is based on the quantification of RNA, proteins and metabolites because they present changes in their quantity related to clinical situations. The same molecules are not generally suitable for early diagnosis or to follow clinical evolution, making necessary strategies to evaluate the complete molecular scenario. There are already experimental strategies that allow the determination of total protein profiles from saliva samples (the SalivaPrint). The goal of this work is to identify a profile of saliva proteins (similar to a fingerprint) and, using computational methods, identify how this profiles changes with age and gender. So far it has been possible to collect 79 samples as well as the metadata associated with each sample using an electronic questionnaire developed by us. A total protein profile was obtained and their association with gender was verified using statistical methods. Currently we are developing the Python scripts for automatic data acquiring and normalization. Total protein profiles annotation on a database (SalivaPrintDB) and their integration with the factors that affects them using machine learning strategies can empower the use of the approach proposed on this work as a tool for monitoring the individual's health status.
- Secondary metabolites produced by macrophomina phaseolina isolated from eucalyptus globulusPublication . Salvatore, Maria Michela; Félix, Carina; Lima, Fernanda; Ferreira, Vanessa; Naviglio, Daniele; Salvatore, Francesco; Duarte, Ana Sofia; Alves, Artur; Andolfi, Anna; Esteves, Ana CristinaIn the course of investigations on the role of secondary metabolites in plant‐microbe interactions, the production of secondary metabolites by Macrophomina phaseolina isolates from Eucalyptus globulus, was studied. This fungus is responsible for several plant diseases which affect crop productivity and industry. Although secondary metabolites may play a role in disease development, there are very few reports on M. phaseolina metabolomics and, as far as we know, isolates from eucalypts have not been investigated for secondary metabolites production. In the present paper, metabolites typical of fungi, from the family Botryosphaeriaceae, were identified for the first time as products of M. phaseolina. Furthermore, the isolate under examination was grown in the presence and absence of host stem tissue, and metabolite profiles were compared. Five products are reported for the first time in this species and azelaic acid was exclusively produced in the presence of eucalypt stem. Finally, phytotoxicity and cytotoxicity tests of culture filtrates and crude organic extracts were also performed. Key Contribution: Lipophilic metabolites produced by M. phaseolina might play a role in the plant‐fungus interactions responsible for serious diseases of E. globulus.
- A step forward to implement saliva as diagnostic fluidPublication . Esteves, Eduardo; Fernandes, Mónica; Rosa, Nuno; Esteves, Ana Cristina; Correia, Maria; Barros, Marlene