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- New Quantum-Dot-Based Fluorescent Immunosensor for Cancer Biomarker DetectionPublication . Sousa, Mariana P.; Piloto, Ana Margarida L.; Pereira, Ana Cláudia; Schmitt, Fernando; Fernandes, Ruben; Moreira, Felismina T. C.Cancer antigen 15-3 (CA 15-3) is a biomarker for breast cancer used to monitor response to treatments and disease recurrence. The present work demonstrates the preparation and application of a fluorescent biosensor for ultrasensitive detection of the cancer antigen CA 15-3 protein tumor marker using mercaptopropionic-acid-functionalized cadmium telluride (CdTe@MPA) quantum dots (QDs) conjugated with CA 15-3 antibodies. First, the QDs were synthesized by the hydrothermal route, resulting in spherical nanoparticles up to 3.50 nm in diameter. Subsequently, the QD conjugates were characterized by Fourier transform infrared spectroscopy (FTIR), UV absorption, and fluorescence. The interaction between the conjugates and the protein was studied by fluorescence spectroscopy in buffer and in 10-fold diluted commercial human serum. Calibration in spiked serum samples gave a detection limit of 0.027 U/mL, 1000-fold lower than the clinical limit for CA 15-3 (25 U/mL to 30 U/mL), indicating that this is an ultrasensitive technique. In addition, a rapid response was obtained within 10 min. The biosensor was selective in the presence of the interfering serum proteins BSA, CEA, and CA-125, with a maximum interference of 2% for BSA. The percent recovery was close to 100% with maximum relative standard deviation (RSD%) values of 1.56. Overall, the developed CA 15-3 biosensor provides a simple and sensitive method for ultrasensitive monitoring of breast cancer, as well as the ability to detect other molecules of interest in human serum matrices.
- Antimicrobial resistance in bacterial strains isolated from different types of Portuguese cheesePublication . Amador, P.; Fernandes, Ruben; Tato-Costa, J.; Teles-Grilo, L.; Brito, L.; Prudêncio, CristinaThe present work aimed to study an eventual antimicrobial resistance and the presence of β-lactamases in enterobacteria isolated from cheese. β-lactams are among the most common antibiotics used for bacterial infections. Rates of bacterial resistance to antimicrobial agents are increasing world wide and the presence of β-lactamases is among one of the most common resistance mechanisms. In the present study we used twenty cheese samples, corresponding to six different types of Portuguese cheese, collected from local retail establishments. Samples were screened for the presence of β-lactams resistant Enterobacteriaceae in selective media with high concentration values of ampicilin. Colonies presumed to be resistant to ampicilin were further confirmed by antibiogram testing against other 13 different antibiotics, including six β-lactams. A total of 160 isolates were recovere from the selective media. The presence of β-lactamases on colonies was detected by adding nitrocefin to the cell biomass. After antibiogram testing about 40 isolates were confirmed to be resistant to, at least, one β -lactam subclass. The results obtained in this study point out to the presence, in different cheese samples, of Enterobacteriaceae resistant to β-lactams and the presence of β-lactamases. These results suggest that cheese may be a source of potential resistance transfer to the normal gastrointestinal flora of the consumer,which may be a cause of serious public health problems.
- Protein structural alterations in diabetic cavernosal tissue - their role in erectile dysfunction progressionPublication . Castela, A.; Soares, R.; Gomes, P.; Coelho, Pedro; Fernandes, RubenErectile dysfunction (ED) is one of the most prevalent complications in diabetic men. Hyperglicemia contributes to increased oxidative stress (OS) in diabetic corpus cavernosum (CC), promoting alterations in cavernosal cellular components. However, it remains unclear the mechanisms by which OS induces modifi cations in diabetic penile tissue with the progression of diabetes and its role in the devel opment of ED. We intended to evaluate/quantify CC protein structural modifi cation caused by OS in an early and late stage of diabetes.
- Relação entre os hábitos alimentares e actividade física com o excesso de peso em adolescentes portuguesesPublication . Prudêncio, Cristina; Dores, Artemisa Rocha; Fernandes, Ruben; Peixoto, Vanda; Pereira, Ana Cláudia; Borges, CristinaA obesidade é uma patologia que tem aumentado drasticamente nos últimos anos, sendo que a mesma se tem manifestado de forma particularmente assustadora em crianças e adolescentes.
- Prevalence of antibiotic resistance genes in multidrug-resistant enterobacteriaceae on Portuguese Livestock ManurePublication . Amador, Paula; Fernandes, Rúben; Prudêncio, Cristina; Duarte, IsabelThe exposure of both crop fields and humans to antibiotic-resistant bacteria in animal excreta is an emergent concern of the One Health initiative. This study assessed the contamination of livestock manure from poultry, pig, dairy farms and slaughterhouses in Portugal with resistance determinants. The resistance profiles of 331 Enterobacteriaceae isolates to eight β-lactam (amoxicillin, cefoxitin, cefotaxime, cefpirome, aztreonam, ceftazidime, imipenem and meropenem) and to five non-β-lactam antibiotics (tetracycline (TET), trimethoprim/sulfamethoxazole (SXT), ciprofloxacin (CIP), chloramphenicol (CHL) and gentamicin) was investigated. Forty-nine integron and non-β-lactam resistance genes were also screened for. Rates of resistance to the 13 antibiotics ranged from 80.8% to 0.6%. Multidrug resistance (MDR) rates were highest in pig farm samples (79%). Thirty different integron and resistance genes were identified. These were mainly associated with resistance to CHL (catI and catII), CIP (mainly, qnrS, qnrB and oqx), TET (mainly tet(A) and tet(M)) and SXT (mostly dfrIa group and sul3). In MDR isolates, integron presence and non-β-lactam resistance to TET, SXT and CHL were positively correlated. Overall, a high prevalence of MDR Enterobacteriaceae was found in livestock manure. The high gene diversity for antibiotic resistance identified in this study highlights the risk of MDR spread within the environment through manure use.
- Comparison of two methods for Streptococcus agalactiae clinical diagnosis: growth on Granada and polymerase chain reaction detectionPublication . Corrêa, K.; Fernandes, Ruben; Gomes, A.; Vieira, Mónica; Ferraz, Ricardo; Prudêncio, CristinaGroup B Streptococcus (GBS) or Streptococcus agalactiae is one of the most common causes of life-threatening infections in newborns suchd as sepsis, pneumonia or meningitis due to its vertical transmissi on mother-child mech-anism.
- Oxidative stress genes involved in the virulence-dependent susceptibility to antibiotics in Pseudomonas aeruginosaPublication . Coelho, Pedro Barata; Fernandes, Ruben; Silva, Carina; Oliveira, Marco; Veiga, Marlene; Sá, Sara; Vieira, André; Guedes, Carla; Baylina, PilarPseudomonas aeruginosa is a Gram-negative opportunistic pathogen which rarely causes disease in healthy people. P. aeruginosa, in particular strain PAO1 is also a biological model for studying virulence and bacterial social traits, such as quorum sensing, SOS response among other. Antibiotic response is dependent, among several other factors, to the response to environmental stress conditions. The present study aims to understand the role of 10 PAO1 oxidative gene mutants in the response to antibiotic stress in elastase, protease and pyocyanin-dependent virulence factors. PAO1 was stressed to several antibiotics (penicilins, cephalosporins, macrolides, and quinolones), and the virulence proteins were measured by means of spectroscopic methods. Viability was measured by means of Erythrosin B. PAO1 GGT, GLO1, RubA2, GSH A mutants were the most susceptible to the production of virulence-dependent factors.
- Avoiding the interference of doxorubicin with MTT measurements on the MCF-7 breast cancer cell linePublication . Luís, Carla; Castaño-Guerrero, Yuselis; Soares, Raquel; Sales, Goreti; Fernandes, RúbenDoxorubicin (DOXO) is an adjuvant chemotherapy agent and is also commonly used in cell biology research. Cytotoxic assays in cell culture are frequently used in order to stablish drug concentrations that are useful for controlling cell proliferation. One common cytotoxic method used is 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). Our present research aims to support future studies in engaging MTT assay using DOXO that exhibits a strong red coloration and fluorescence, and so it is assumed that DOXO may interfere with commonly used colorimetric assays such as MTT. The interference of DOXO in the MTT determination was evaluated in a Breast Cancer cell line Michigan Cancer Foundation-7 (MCF-7). The interference was evaluated by means of spectroscopic methods in particular spectrophometry and fluorescence spectroscopy of MTT and DOXO. We postulate that the medium and the MTT reagent itself can interfere on the metabolic activity method, so in order to achieve better results, DMEM was replaced by a neutral buffer like Phosphate-buffered saline (PBS). This protocol may be extremely useful in future studies involving DOXO.
- Hábitos e comportamentos associados ao uso de antibióticos: A autonomia individual versus a saúde da comunidadePublication . Prudêncio, Cristina; Ferraz, Ricardo; Vieira, Mónica; Ribas, Cláudia; Ribas, Jorge; Fonte, Rosália; Fernandes, RubenA resistência a drogas é um dos graves problemas de saúde atual embora possa ser considerada uma resposta natural devida à pressão seletiva da droga. Esta resposta é no entanto exacerbada por vários fatores que incluem, abuso, subutilização ou má utilização, má adesão ao tratamento ou má qualidade das drogas disponíveis. Trata-se de um dos maiores desafios que enfrentam atualmente os profissionais de Saúde e a população em geral. A auto-medicação é um dos fatores que mais contribui para a resistência a antibióticos. No presente trabalho apresentamos um estudo preliminar onde foram avaliados os hábitos e comportamentos associados ao uso de antibióticos num grupo de Pais.
- Bloodstream infections caused by multidrugresistant Enterobacteriaceae: report from two portuguese hospitalsPublication . Fernandes, Ruben; Vieira, Mónica; Ferraz, Ricardo; Prudêncio, Cristina