Elisabete Fernandes

A bacteriophage detection tool for viability assessment of Salmonella cells.

Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.

New trends in guided nanotherapies for digestive cancers: A systematic review.

Digestive tract tumors are among the most common and deadliest malignancies worldwide, mainly due to late diagnosis and lack of efficient therapeutics. Current treatments essentially rely on surgery associated with (neo)adjuvant chemotherapy agents. Despite an upfront response, conventional drugs often fail to eliminate highly aggressive clones endowed with chemoresistant properties, which are responsible for tumor recurrence and disease dissemination. Synthetic drugs also present severe adverse systemic effects, hampering the administration of biologically effective dosages. Nanoencapsulation of chemotherapeutic agents within biocompatible polymeric or lipid matrices holds great potential to improve the pharmacokinetics and efficacy of conventional chemotherapy while reducing systemic toxicity. Tagging nanoparticle surfaces with specific ligands for cancer cells, namely monoclonal antibodies or antibody fragments, has provided means to target more aggressive clones, further improving the selectivity and efficacy of nanodelivery vehicles. In fact, over the past twenty years, significant research has translated into a wide array of guided nanoparticles, providing the molecular background for a new generation of intelligent and more effective anti-cancer agents. Attempting to bring awareness among the medical community to emerging targeted nanopharmaceuticals and foster advances in the field, we have conducted a systematic review about this matter. Emphasis was set on ongoing preclinical and clinical trials for liver, colorectal, gastric and pancreatic cancers. To the best of our knowledge this is the first systematic and integrated overview on this field. Using a specific query, 433 abstracts were gathered and narrowed to 47 manuscripts when matched against inclusion/exclusion criteria. All studies showed that active targeting improves the effectiveness of the nanodrugs alone, while lowering its side effects. The main focus has been on hepatocarcinomas, mainly by exploring glycans as homing molecules. Other ligands such as peptides/small proteins and antibodies/antibody fragments, with affinity to either tumor vasculature or tumor cells, have also been widely and successfully applied to guide nanodrugs to gastrointestinal carcinomas. Conversely, few solutions have been presented for pancreatic tumors. To this date only three nanocomplexes have progressed beyond pre-clinical stages: i) PK2, a galactosamine-functionalized polymeric-DOX formulation for hepatocarcinomas; ii) MCC-465, an anti-(myosin heavy chain a) immunoliposome for advanced stage metastatic solid tumors; and iii) MBP-426, a transferrin-liposome-oxaliplatin conjugate, also for advanced stage tumors. Still, none has been approved for clinical use. However, based on the high amount of pre-clinical studies showing enthusiastic results, the number of clinical trials is expected to increase in the near future. A more profound understanding about the molecular nature of chemoresistant clones and cancer stem cell biology will also contribute to boost the field of guided nanopharmacology towards more effective solutions.

Overexpression of tumour-associated carbohydrate antigen sialyl-Tn in advanced bladder tumours

Little is known on the expression of the tumour‐associated carbohydrate antigen sialyl‐Tn (STn), in bladder cancer. We report here that 75% of the high‐grade bladder tumours, presenting elevated proliferation rates and high risk of recurrence/progression expressed STn. However, it was mainly found in non‐proliferative areas of the tumour, namely in cells invading the basal and muscle layers. STn was also found in tumour‐adjacent mucosa, which suggests its dependence on a field effect of the tumour. Furthermore, it was not expressed by the normal urothelium, demonstrating the cancer‐specific nature of this antigen. STn expression correlated with that of sialyltransferase ST6GalNAc.I, its major biosynthetic enzyme. The stable expression of ST6GalNAc.I in the bladder cancer cell line MCR induced STn expression and a concomitant increase of cell motility and invasive capability. Altogether, these results indicate for the first time a link between STn expression and malignancy in bladder cancer. Hence, therapies targeting STn may constitute new treatment approaches for these tumours.

Selection and characterization of a multivalent Salmonella phage and its production in a nonpathogenic Escherichia coli strain

ABSTRACT We report the selection and amplification of the broad-host-range Salmonella phage phi PVP-SE1 in an alternative nonpathogenic host. The lytic spectrum and the phage DNA restriction profile were not modified upon replication in Escherichia coli Bl21, suggesting the possibility of producing this phage in a nonpathogenic host, contributing to the safety and easier approval of a product based on this Salmonella biocontrol agent.

Response of high-risk of recurrence/progression bladder tumours expressing sialyl-Tn and sialyl-6-T to BCG immunotherapy

Background:High risk of recurrence/progression bladder tumours is treated with Bacillus Calmette-Guérin (BCG) immunotherapy after complete resection of the tumour. Approximately 75% of these tumours express the uncommon carbohydrate antigen sialyl-Tn (Tn), a surrogate biomarker of tumour aggressiveness. Such changes in the glycosylation of cell-surface proteins influence tumour microenvironment and immune responses that may modulate treatment outcome and the course of disease. The aim of this work is to determine the efficiency of BCG immunotherapy against tumours expressing sTn and sTn-related antigen sialyl-6-T (s6T).Methods:In a retrospective design, 94 tumours from patients treated with BCG were screened for sTn and s6T expression. In vitro studies were conducted to determine the interaction of BCG with high-grade bladder cancer cell line overexpressing sTn.Results:From the 94 cases evaluated, 36 had recurrence after BCG treatment (38.3%). Treatment outcome was influenced by age over 65 years (HR=2.668; (1.344–5.254); P=0.005), maintenance schedule (HR=0.480; (0.246–0.936); P=0.031) and multifocallity (HR=2.065; (1.033–4.126); P=0.040). sTn or s6T expression was associated with BCG response (P=0.024; P<0.0001) and with increased recurrence-free survival (P=0.001). Multivariate analyses showed that sTn and/or s6T were independent predictive markers of recurrence after BCG immunotherapy (HR=0.296; (0.148–0.594); P=0.001). In vitro studies demonstrated higher adhesion and internalisation of the bacillus to cells expressing sTn, promoting cell death.Conclusion:s6T is described for the first time in bladder tumours. Our data strongly suggest that BCG immunotherapy is efficient against sTn- and s6T-positive tumours. Furthermore, sTn and s6T expression are independent predictive markers of BCG treatment response and may be useful in the identification of patients who could benefit more from this immunotherapy.

Method for bacteriophage isolation against target Campylobacter strains

Aims:  Poultry meat is considered a major source of Campylobacter. This micro‐aerobic bacterium is commonly responsible for foodborne illness. This work focuses on the isolation of Campylobacter coli lytic bacteriophages (phages) against target C. coli strains.

Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics.

Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC.

P53 and Cancer-Associated Sialylated Glycans Are Surrogate Markers of Cancerization of the Bladder Associated with Schistosoma haematobium Infection

Background Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers. Methodology/Principal Findings Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%), cell-surface cancer-associated glycan sialyl-Tn (sTn) and sialyl-Lewisa/x (sLea/sLex), involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed in the healthy urothelium. Conclusion/Significance This preliminary study suggests that p53 and sialylated glycans are surrogate biomarkers of bladder cancerization associated with S. haematobium, highlighting a missing link between infection and cancer development. Eggs of S. haematobium express sLea and sLex antigens in mimicry of human leukocytes glycosylation, which may play a role in the colonization and disease dissemination. These observations may help the early identification of infected patients at a higher risk of developing bladder cancer and guide the future development of non-invasive diagnostic tests.

Detection of BCG bacteria using a magnetoresistive biosensor: A step towards a fully electronic platform for tuberculosis point-of-care detection

Tuberculosis is one of the major public health concerns. This highly contagious disease affects more than 10.4 million people, being a leading cause of morbidity by infection. Tuberculosis is diagnosed at the point-of-care by the Ziehl-Neelsen sputum smear microscopy test. Ziehl-Neelsen is laborious, prone to human error and infection risk, with a limit of detection of 104 cells/mL. In resource-poor nations, a more practical test, with lower detection limit, is paramount. This work uses a magnetoresistive biosensor to detect BCG bacteria for tuberculosis diagnosis. Herein we report: i) nanoparticle assembly method and specificity for tuberculosis detection; ii) demonstration of proportionality between BCG cell concentration and magnetoresistive voltage signal; iii) application of multiplicative signal correction for systematic effects removal; iv) investigation of calibration effectiveness using chemometrics methods; and v) comparison with state-of-the-art point-of-care tuberculosis biosensors. Results present a clear correspondence between voltage signal and cell concentration. Multiplicative signal correction removes baseline shifts within and between biochip sensors, allowing accurate and precise voltage signal between different biochips. The corrected signal was used for multivariate regression models, which significantly decreased the calibration standard error from 0.50 to 0.03log10 (cells/mL). Results show that Ziehl-Neelsen detection limits and below are achievable with the magnetoresistive biochip, when pre-processing and chemometrics are used.

Protein glycosylation in gastric and colorectal cancers: Toward cancer detection and targeted therapeutics

Glycosylation is the most frequent and structurally complex posttranslational modification in cell-surface and secreted proteins. Glycans are major orchestrators of biological processes, namely, by controlling protein folding and key biological functions such as cell adhesion, migration, signaling and immune recognition. Altered glycosylation is considered a hallmark of malignant transformations that decisively contributes to disease outcome. This review comprehensively summarizes the main findings related with gastrointestinal cancers and the decisive impact of aberrant glycosylation on tumor biology toward more aggressive phenotypes. Particular emphasis is given to alterations in O-glycosylation, namely, the overexpression of immature O-glycans, and the sialylated Lewis antigens sialyl-LeA and sialyl-LeX, frequently implicated in lymphohematogenous metastasis. We further discuss how recent contributions from glycoproteomics and glycoengineering fields have broadened our understanding of the human O-glycoproteome and its implications for cancer research. Finally, we address the tremendous potential of glycans in the context of targeted therapeutics (selective inhibition of glycosylation pathways, immunotherapy) and discuss the need to include glycomics/glycoproteomics in holistic panomics models toward true precision medicine settings.