Biomimetic sensors of molecularly-imprinted polymers for chlorpromazine determination

A new man-tailored biomimetic sensor for Chlorpromazine host-guest interactions and potentiometric transduction is presented. The artificial host was imprinted within methacrylic acid, 2-vinyl pyridine and 2-acrylamido-2-methyl-1-propanesulfonic acid based polymers. Molecularly imprinted particles were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. Slopes and detection limits ranged 51–67 mV/decade and 0.46–3.9 μg/mL, respectively, in steady state conditions. Sensors were independent from the pH of test solutions within 2.0–5.5. Good selectivity was observed towards oxytetracycline, doxytetracycline, ciprofloxacin, enrofloxacin, nalidixic acid, sulfadiazine, trimethoprim, glycine, hydroxylamine, cysteine and creatinine. Analytical features in flowing media were evaluated on a double-channel manifold, with a carrier solution of 5.0 × 10−2 mol/L phosphate buffer. Near-Nernstian response was observed over the concentration range 1.0 × 10−4 to 1.0 × 10−2 mol/L. Average slopes were about 48 mV/decade. The sensors were successfully applied to field monitoring of CPZ in fish samples, offering the advantages of simplicity, accuracy, automation feasibility and applicability to complex samples.

Molecularly-Imprinted Materials for Potentiometric Transduction: Application to the Antibiotic Enrofloxacin

Enrofloxacin (ENR) is an antimicrobial used both in humans and in food producing species. Its control is required in farmed species and their surroundings in order to reduce the prevalence of antibiotic resistant bacteria. Thus, a new biomimetic sensor enrofloxacin is presented. An artificial host was imprinted in specific polymers. These were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibited a near-Nernstian response. Slopes expressing mV/Δlog([ENR]/M) varied within 48–63. The detection limits ranged from 0.28 to 1.01 µg mL−1. Sensors were independent from the pH of test solutions within 4–7. Good selectivity was observed toward potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin, and tetracycline. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ± 0.7%), fast response, good sensitivity (47 mV/Δlog([ENR]/M), wide linear range (1.0 × 10−5–1.0 × 10−3 M), low detection limit (0.9 µg mL−1), and a stable baseline for a 5 × 10−2 M acetate buffer (pH 4.7) carrier. The sensors were used to analyze fish samples. The method offered the advantages of simplicity, accuracy, and automation feasibility. The sensing membrane may contribute to the development of small devices allowing in vivo measurements of enrofloxacin or parent-drugs.

Erythema induratum of Bazin and renal tuberculosis: report of an association

Erythema induratum of Bazin is a disease that usually affects women, in whom erythematous subcutaneous nodules and plaques appear on the posterior part of the lower extremities, some of which ulcerate. In many countries, tuberculosis is still the main etiologic factor. We report a case of a 40-year-old woman who presented a course of protracted and recurrent episodes over five years of cutaneous lesions on her legs. These tend to involute, but new crops appear at irregular intervals. It was painful, erythematous-violaceous nodules, some of which drained a reddish secretion. The histopathologic features of the lesions demonstrated inflammatory infiltration, with predominance of neutrophils in dermis and hypodermis, necrotizing vasculitis in the arterioles and septal fibrosis. There was no granuloma. The Ziehl-Neelsen stain did not revealed acid-fast bacilli, and the culture of biopsy specimen was negative. The tuberculin skin test was strongly positive (17 mm). The chest X-ray was normal. Few months later she presented adynamia and urinary complaints, such as polacyuria and dysuria. It has been done an urynalysis, which demonstrated acid pH urine, sterile pyuria and microscopic hematuria. It was then raised the diagnostic hypothesis of renal tuberculosis. The urine culture for M. tuberculosis was positive in two out of ten samples. The treatment was instituted with rifampin, isoniazid and pyrazinamide, with complete regression. This case illustrates a clear association between erythema induratum and renal tuberculosis, demonstrated by the remission of the cutaneous lesions after the treatment of the renal tuberculosis.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

Effect of sialic acid loss on dendritic cell maturation

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I(-/-) and ST6Gal.I(-/-) mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I(-/-) and ST6Gal.I(-/-) strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading.

TEC4SEA - A Modular Platform for Research, Test and Validation of Technologies Supporting a Sustainable Blue Economy

This paper presents the TEC4SEA research infrastructure created in Portugal to support research, development, and validation of marine technologies. It is a multidisciplinary open platform, capable of supporting research, development, and test of marine robotics, telecommunications, and sensing technologies for monitoring and operating in the ocean environment. Due to the installed research facilities and its privileged geographic location, it allows fast access to deep sea, and can support multidisciplinary research, enabling full validation and evaluation of technological solutions designed for the ocean environment. It is a vertically integrated infrastructure, in the sense that it possesses a set of skills and resources which range from pure conceptual research to field deployment missions, with strong industrial and logistic capacities in the middle tier of prototype production. TEC4SEA is open to the entire scientific and enterprise community, with a free access policy for researchers affiliated with the research units that ensure its maintenance and sustainability. The paper describes the infrastructure in detail, and discusses associated research programs, providing a strategic vision for deep sea research initiatives, within the context of both the Portuguese National Ocean Strategy and European Strategy frameworks.

An Exact Schedulability Test for Global FP Using State Space Pruning

Presented at 23rd International Conference on Real-Time Networks and Systems (RTNS 2015). 4 to 6, Nov, 2015, Main Track. Lille, France.

Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

An immunofluorescence test for diagnosis of ophthalmic herpes in a mouse corneal model

Herpes simplex virus type 1 (HSV-1) ophthalmic disease is the most common cause of corneal blindness in humans world-wide. Current culture techniques for HSV take several days and commercially available HSV laboratory based diagnostic techniques vary in sensitivity. Our study was conducted to evaluate the use of a quicker and simpler method to herpes ophthalmic diagnosis. Corneal smears were made by firm imprints of infected mouse eyes to glass slides, after smears were fixated with cold acetone, and an indirect immunofluorescence (IIF) method was performed using monoclonal antibodies in a murine model of ophthalmic herpes. Eye swabs from infected mice were inoculated in Vero cells for virus isolation. Cytology and histology of the eye were also performed, using hematoxylin-eosin routine. Mouse eyes were examined by slit-lamp biomicroscopy for evidence of herpetic disease at various times postinoculation. We made a comparative evaluation of sensitivity, specificity and speed of methods for laboratory detection of HSV. Our results indicate that this IIF method is quick, sensitive, specific and can be useful in the diagnosis of ophthalmic herpes as demonstrated in an animal model.

Are coffee silverskin extracts safe for topical use? An in vitro and in vivo approach

Recent changes in regulatory requirements and social views on animal testing have incremented the development of reliable alternative tests for predicting skin and ocular irritation potential of products based on new raw materials. In this regard, botanical ingredients used in cosmetic products are among those materials, and should be carefully reviewed concerning the potential presence of irritant constituents. In particular, cosmetic products used on the face, in vicinity of the eyes or that may come in contact with mucous membranes, should avoid botanical ingredients that contain, or are suspected to contain, such ingredients. In this study, we aimed to evaluate the effect of a new cosmetic ingredient, namely, coffee silverskin (CS), with an in vitro skin and ocular irritation assay using reconstructed human epidermis, EpiSkin™, and human corneal epithelial model, SkinEthics™ HCE, and an in vivo assay. Three different extracts of CS were evaluated. The histology of the models after extracts applications was analysed. The in vitro results demonstrated that extracts were not classified as irritant and the histological analyses proved that extracts did not affect both models structure. The content of caffeine, 5-hydroxymethyl furfural and chlorogenic acid was quantified after the epidermal assay. The in vivo test carried out with the most promising extract (hydroalcoholic) showed that, with respect to irritant effects, these extracts can be regarded as safe for topical application.

MnFe2O4@CNT-N as novel electrochemical nanosensor for determination of caffeine, acetaminophen and ascorbic acid

For the first time, a glassy carbon electrode (GCE) modified with novel N-doped carbon nanotubes (CNT-N) functionalized with MnFe2O4 nanoparticles (MnFe2O4@CNT-N) has been prepared and applied for the electrochemical determination of caffeine (CF), acetaminophen (AC) and ascorbic acid (AA). The electrochemical behaviour of CF, AC and AA on the bare GCE, CNT-N/GCE and MnFe2O4@CNT-N/GCE were carefully investigated using cyclic voltammetry (CV) and square-wave voltammetry (SWV). Compared to bare GCE and CNT-N modified electrode, the MnFe2O4@CNT-N modified electrode can remarkably improve the electrocatalytic activity towards the oxidation of CF, AC and AA with an increase in the anodic peak currents of 52%, 50% and 55%, respectively. Also, the SWV anodic peaks of these molecules could be distinguished from each other at the MnFe2O4@CNT-N modified electrode with enhanced oxidation currents. The linear response ranges for the square wave voltammetric determination of CF, AC and AA were 1.0 × 10−6 to 1.1 × 10−3 mol dm−3, 1.0 × 10−6 to 1.0 × 10−3 mol dm−3 and 2.0 × 10−6 to 1.0 × 10−4 mol dm−3 with detection limit (S/N = 3) of 0.83 × 10−6, 0.83 × 10−6 and 1.8 × 10−6 mol dm−3, respectively. The sensitivity values at the MnFe2O4@CNT-N/GCE for the individual determination of AC, AA and CF and in the presence of the other molecules showed that the quantification of AA and CF show no interferences from the other molecules; however, AA and CF interfered in the determination of AC, with the latter molecule showing the strongest interference. Nevertheless, the obtained results show that MnFe2O4@CNT-N composite material acted as an efficient electrochemical sensor towards the selected biomolecules.

Comparison between E-test and CLSI broth microdilution method for antifungal susceptibility testing of Candida albicans oral isolates

Thirty Candida albicans isolated from oral candidosis patients and 30 C. albicans isolated from control individuals were studied. In vitro susceptibility tests were performed for amphotericin B, fluconazole, 5-flucytosine and itraconazole through the Clinical and Laboratorial Standards Institute (CLSI) reference method and E test system. The results obtained were analyzed and compared. MIC values were similar for the strains isolated from oral candidosis patients and control individuals. The agreement rate for the two methods was 66.67% for amphotericin B, 53.33% for fluconazole, 65% for flucytosine and 45% for itraconazole. According to our data, E test method could be an alternative to trial routine susceptibility testing due to its simplicity. However, it can not be considered a substitute for the CLSI reference method.

Diterpenos de Copaifera reticulata Ducke com atividade larvicida contra Aedes aegypti (L.) (Diptera, Culicidae)

The objective of this study was to evaluate the larvicidal activity of diterpenoids obtained from the oil-resin of Copaifera reticulata against Aedes aegypti larvae, the principal vector of dengue and urban yellow fever. Four diterpenes were obtained from oil-resin extraction with organic solvents and subsequent chromatographic and spectroscopic procedures allowed to isolation and identification of these compounds as 3-b-acetoxylabdan-8(17)-13-dien-15-oic acid (1), alepterolic acid (2), 3-b-hidroxylabdan-8(17)-en-15-oic acid (3), and ent-agatic acid (4). Each compound was previously dissolved in dimethylsulphoxide, and distilled water was added to obtain the desired concentrations. Twenty larvae of third instars were placed into plastic beckers, containing the solution test (25 mL), in a five repetitions scheme, and their mortality, indicated by torpor and darkening of the cephalic capsule, was recorded after 48h. Probit analyses were used to determine lethal concentrations (LC50 and LC90) and their respective 95% confidence intervals. This study showed that only diterpenoids 1 and 2 exhibited larvicidal properties with LC50 of 0.8 ppm and 87.3 ppm, respectively, revealing the former as the most toxic compound against third instars of Ae. aegypti. Therefore, this compound seems to be an interesting source for new metabolite to be exploited.

Epidemiological characterization, antimicrobial resistance and virulence mechanisms of human and animal streptococci

Dissertação para obtenção do Grau de Doutor em Biologia