Low frequency of AIP mutations in patients with young-onset sporadic pituitary macroadenomas.

PURPOSE: Mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene cause familial isolated pituitary adenomas (FIPA). AIP mutations have also been found in patients with apparently sporadic pituitary adenomas, particularly in young patients with large adenomas. The aim of this study was to determine the frequency of AIP germline mutations in patients with young-onset sporadic pituitary macroadenomas. METHODS: The AIP gene was sequenced in 218 Portuguese patients with sporadic pituitary macroadenomas diagnosed before the age of 40 years. RESULTS: Heterozygous rare sequence variants in AIP were identified in 18 (8.3%) patients. However, only four (1.8%) patients had pathogenic or likely pathogenic variants. These consisted of two already known mutations (p.Arg81* and p.Leu115Trpfs*41) and two novel mutations (p.Glu246*, p.Ser53Thrfs*36). All four patients had GH-secreting adenomas diagnosed between the ages of 14 and 25 years. The frequency of AIP pathogenic or likely pathogenic variants in patients under the age of 30 and 18 years was 3.4% and 5.0%, respectively. CONCLUSION: The frequency of AIP mutations in this cohort was lower than in other studies. Previous reports may have overestimated the contribution of AIP mutations due to the inclusion of genetic variants of uncertain significance. The identification of novel AIP mutations expands the known spectrum of genetic causes of pituitary adenomas and may help understand the role of AIP mutations in the molecular mechanisms underlying pituitary tumorigenesis.

Effects of age, weight, and housing system on prevalence of dead on arrival and carcass condemnation causes in laying hens.

Causes of condemnation and dead on arrival (DoA) birds were studied at the slaughterhouse and effects of age, BW, and housing system were investigated. A total of 1,156 (0.18%) birds were found DoA and 20,754 carcasses out of 629,331 (3.30%) were condemned during postmortem inspection. The main reasons for condemnation were peritonitis, septicemia, salpingitis, emaciation, and tumors. The mean percentages of condemnation by septicemia and emaciation differed according to age and BW groups and were more common in batches of younger (≤87 wk) and lighter (≤1.88 kg) birds. Moreover, peritonitis and ascites differed significantly between age groups, occurring more frequently in batches of older hens. The presence of tumors of the reproductive system was more frequently observed in older and heavier hens. This result raises the possibility of tumors being correlated with the higher number of prior ovulatory events. The total condemnation rate was lower in hens from organic systems, followed by free-range, and differed significantly from barns and cage systems. Carcasses with ascites and peritonitis were found more commonly in hens from cages and barns and both differed significantly from organic systems. Salpingitis was statistically more prevalent in barns, presenting differences from organic and free-range systems. Monitoring condemnation causes of end-of-lay hens at slaughter provided a better understanding of health and welfare issues in different housing systems and allowed to identify potential welfare problems, which can be used to improve management and welfare on farms.

Cytotoxic activity of poly-ɛ-caprolactone lipid-core nanocapsules loaded with lycopene-rich extract from red guava (Psidium guajava L.) on breast cancer cells.

The aims of this study were to produce poly-ɛ-caprolactone lipid-core nanocapsules containing lycopene-rich extract from red guava (LEG), to characterize those nanoparticles and to evaluate their cytotoxic effects on human breast cancer cells. Lipid-core nanocapsules containing the extract (nanoLEG) were produced by the method of interfacial deposition of the preformed polymer. The nanoparticles were characterized by Dynamic Light Scattering (DLS), Polydispersity Index, Zeta Potential, pH, Encapsulation Efficiency, Nanoparticle Tracking Analysis (NTA), Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM). Cell viability was evaluated by the MTT dye reduction method in the human breast cancer MCF-7 cell line and inhibition of ROS and NF-κB was assayed in living human microglial cell line (HMC3) by time-lapse images microscopy. A hemolytic activity assay was carried out with sheep blood. Data showed that nanoparticles average size was around 200 nm, nanoparticles concentration/mL was around 0.1 µM, negative zeta potential, pH < 5.0 and spherical shape, with low variation during a long storage period (7 months) at 5 °C, indicating stability of the system and protection against lycopene degradation. The percentage of encapsulation varied from 95% to 98%. The nanoLEG particles significantly reduced the viability of the MCF-7 cells after 24 h (61.47%) and 72 h (55.96%) of exposure, even at the lowest concentration tested (6.25-200 µg/ml) and improved on the cytotoxicity of free LEG to MCF-7. NanoLEG inhibited LPS-induced NF-kB activation and ROS production in microglial cells. The particles did not affect the membrane integrity of sheep blood erythrocytes at the concentrations tested (6.25-200 µg/mL). Thus, the formulation of lipid-core nanocapsules with a polysorbate 80-coated poly-ɛ-caprolactone wall was efficiently applied to stabilize the lycopene-rich extract from red guava, generating a product with satisfactory physico-chemical and biological properties for application as health-promoting nanotechnology-based nutraceutical, emphasizing its potential to be used as a cancer treatment.

Evaluation of Hygienic Quality of Food Served in Universities Canteens of Northem Portugal.

Mass catering services have increased in the last years since people need to eat out mainly by work or study reasons. Microbiological quality of foodstuffs (n = 156) was evaluated in 20 food establishment (cafes and canteens) of two universities of northern Portugal. Overall, data revealed a high level of microbiological quality of foods served. No safety risks for consumers were detected since Clostridium spp., Listeria monocytogenes and Salmonella spp. were not detected. Among food types, hot meals displayed better microbiological results than cold foods (p < 0.05) as expected. Regarding hot meals, no differences were observed among different types (p > 0.05). Among cold meals, salads displayed the highest microbiological counts for hygiene indicators as well for food foodborne pathogens such as Staphylococcus aureus, Escherichia coli and Bacillus cereus. Although the risk of foodborne disease is scarce since counts were low. In cafes' meals, higher counts were observed than in canteens' meals which indicates that monitoring measures should be improved to avoid potential foodborne outbreaks related to the ready-to-eat products (salads, sandwiches and pastry). Results could be used as microbiological guidelines for canteens. Results indicated that proper food handling and adequate conservation of fresh foods along the food chain is essential in mass catering services to guarantee the food safety.

Efficacy of LISTEX P100 at Different Concentrations for Reduction of Listeria monocytogenes Inoculated in Sashimi.

Sushi restaurants have become quite popular in Europe, with an increase in the consumption of the sashimi speciality. Pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus have been reported in this kind of food. Controlling the presence and multiplication of L. monocytogenes is a challenge for food safety management systems owing to its ubiquitous presence and psychrotrophic growth. Bacteriophages have been used as pathogenic biocide agents for decades. The bacteriophage P100, present in LISTEX P100, was used in this study to understand the possibility of implementing a new critical control point for L. monocytogenes in sashimi preparation and sale. Different concentrations of LISTEX P100 were used for reduction of L. monocytogenes in inoculated samples at 3 and 22°C. The reduction in initial counts of 2 log CFU/g was effective in the first 24 h with the 8-log PFU/g inoculation. Promising results were obtained in assays with the 6-log CFU/g initial counts and the 8-log PFU/g inoculation, at 22°C, achieving a maximum reduction of 4.44 log CFU/g, compared with the control group. These results seem to confirm that bacteriophages can be an option in reducing the population of the L. monocytogenes pathogenic bacteria in sashimi, mainly in takeaway sales.

A chemometrics approach applied to Fourier transform infrared spectroscopy (FTIR) for monitoring the spoilage of fresh salmon (Salmo salar) stored under modified atmospheres.

The aim of this work was to investigate the potential of Fourier transform infrared spectroscopy (FTIR) to detect and predict the bacterial load of salmon fillets (Salmo salar) stored at 3, 8 and 30°C under three packaging conditions: air packaging (AP) and two modified atmospheres constituted by a mixture of 50%N2/40%CO2/10%O2 with lemon juice (MAPL) and without lemon juice (MAP). Fresh salmon samples were periodically examined for total viable counts (TVC), specific spoilage organisms (SSO) counts, pH, FTIR and sensory assessment of freshness. Principal components analysis (PCA) allowed identification of the wavenumbers potentially correlated with the spoilage process. Linear discriminant analysis (LDA) of infrared spectral data was performed to support sensory data and to accurately identify samples freshness. The effect of the packaging atmospheres was assessed by microbial enumeration and LDA was used to determine sample packaging from the measured infrared spectra. It was verified that modified atmospheres can decrease significantly the bacterial load of fresh salmon. Lemon juice combined with MAP showed a more pronounced delay in the growth of Brochothrix thermosphacta, Photobacterium phosphoreum, psychrotrophs and H2S producers. Partial least squares regression (PLS-R) allowed estimates of TVC and psychrotrophs, lactic acid bacteria, molds and yeasts, Brochothrix thermosphacta, Enterobacteriaceae, Pseudomonas spp. and H2S producer counts from the infrared spectral data. For TVC, the root mean square error of prediction (RMSEP) value was 0.78logcfug-1 for an external set of samples. According to the results, FTIR can be used as a reliable, accurate and fast method for real time freshness evaluation of salmon fillets stored under different temperatures and packaging atmospheres.

Feather conditions and clinical scores as indicators of broilers welfare at the slaughterhouse.

The objective of this study was to evaluate the welfare of 64 different broiler farms on the basis of feather conditions and clinical scores measures collected at the slaughterhouse. A 3-point scale (0, 1 or 2) was used to classify dirty feathers, footpad dermatitis and hock burns measures, and a 2-point scale (present or absent) was used to classify breast burns, breast blisters and breast ulcer measures. Flocks were allocated into three body weight (BW) classes (A, B, C): class A (light) ≥1.43 and ≤1.68kg, class B (medium) ≥1.69 and ≤1.93kg; class C (heavy) ≥1.94 and ≤2.41kg. The absence of hock burns was more common in class A, while mild hock burns was more common in class B flocks. Breast ulcer was observed in class C flocks. The association observed for mild hock burns, breast burns and severe footpad dermatitis can indicate a simultaneous occurrence of these painful lesions. Very dirty feathers and severe footpad dermatitis relationship suggest litter humidity to be the common underlying cause. In conclusion, it was shown that clinical indicators can be used at the slaughterhouse to identify welfare problems. In the studied flocks, footpad dermatitis, feather conditions and hock burns were the main restrictions for good welfare and should be considered significant welfare indicators of the on-farm rearing conditions.

MicroRNA-124 loaded nanoparticles enhance brain repair in Parkinson's disease.

Modulation of the subventricular zone (SVZ) neurogenic niche can enhance brain repair in several disorders including Parkinson's disease (PD). Herein, we used biocompatible and traceable polymeric nanoparticles (NPs) containing perfluoro-1,5-crown ether (PFCE) and coated with protamine sulfate to complex microRNA-124 (miR-124), a neuronal fate determinant. The ability of NPs to efficiently deliver miR-124 and prompt SVZ neurogenesis and brain repair in PD was evaluated. In vitro, miR-124 NPs were efficiently internalized by neural stem/progenitors cells and neuroblasts and promoted their neuronal commitment and maturation. The expression of Sox9 and Jagged1, two miR-124 targets and stemness-related genes, were also decreased upon miR-124 NP treatment. In vivo, the intracerebral administration of miR-124 NPs increased the number of migrating neuroblasts that reached the granule cell layer of the olfactory bulb, both in healthy and in a 6-hydroxydopamine (6-OHDA) mouse model for PD. MiR-124 NPs were also able to induce migration of neurons into the lesioned striatum of 6-OHDA-treated mice. Most importantly, miR-124 NPs proved to ameliorate motor symptoms of 6-OHDA mice, monitored by the apomorphine-induced rotation test. Altogether, we provide clear evidences to support the use of miR-124 NPs as a new therapeutic approach to boost endogenous brain repair mechanisms in a setting of neurodegeneration.

Influence of season and type of restaurants on sashimi microbiota.

BACKGROUND: In recent years, an increase in the consumption of Japanese food in European countries has been verified, including in Portugal. These specialities made with raw fish, typical Japanese meals, have been prepared in typical and on non-typical restaurants, and represent a challenge to risk analysis on HACCP plans. The aim of this study was to evaluate the influence of the type of restaurant, season and type of fish used on sashimi microbiota. METHODS: Sashimi samples (n = 114) were directly collected from 23 sushi restaurants and were classified as Winter and Summer Samples. They were also categorized according to the type of restaurant where they were obtained: as typical or non-typical. The samples were processed using international standards procedures. RESULTS: A middling seasonality influence was observed in microbiota using mesophilic aerobic bacteria, psychrotrophic microorganisms, Lactic acid bacteria, Pseudomonas spp., H2S positive bacteria, mould and Bacillus cereus counts parameters. During the Summer Season, samples classified as unacceptable or potentially Hazardous were observed. Non-typical restaurants had the most cases of Unacceptable/potentially hazardous samples 83.33%. These unacceptable results were obtained as a result of high values of pathogenic bacteria like Listeria monocytogenes and Staphylococcus aureus No significant differences were observed on microbiota counts from different fish species. CONCLUSION: The need to implement more accurate food safety systems was quite evident, especially in the warmer season, as well as in restaurants where other kinds of food, apart from Japanese meals, was prepared.

Influência de diferentes tipos de micro-organismos na contagem bacteriana total por citometria de fluxo do leite cru refrigerado / Influence of different types of microorganisms in total bacterial count by flow cytometry of refrigerated raw milk

Contagens bacterianas de 179 amostras de leite cru refrigerado, provenientes de diferentes bacias leiteiras de Minas Gerais, foram realizadas pelos métodos de citometria de fluxo e contagem padrão em placas. O estudo das associações entre a contagem bacteriana total e as contagens bacterianas dos diferentes grupos de micro-organismos foi realizado pela correlação de Pearson. Houve uma forte correlação positiva e significativa entre os valores de contagem de psicrotróficos (r = 0,67) e mesófilos (r = 0,70), determinados pelo método de referência, e os valores da contagem bacteriana total, determinados pela citometria de fluxo. Foi possível estabelecer uma curva de calibração para o citômetro de fluxo utilizando equações de regressão linear. As equações para a contagem bacteriana obtida por citometria de fluxo em função da contagem de mesófilos e psicrotróficos foram: log (CBI) = 0,701 log mesófilos + 1,9091 e log (CBI) = 0,4703 log psicrotróficos + 3,2784, respectivamente. Não foi possível estabelecer curvas de calibrações para termófilos e termodúricos, pois as correlações obtidas entre as contagens desses micro-organismos e a contagem bacteriana total, determinada por citometria de fluxo, foram fracas (r = 0,30 e 0,27, respectivamente).(AU)

Total Bacterial count was performed in 179 raw milk samples from different dairy farms in Minas Gerais State, Brazil, through flow cytometry and standard plate count, that was regarded as the reference method. There was significant strong correlation between the values of psychrotrophic and mesophilic counts determined by the reference method with the values of bacterial count determined by flow cytometry (r = 0.67 and 0.70, respectively), which allowed to establish linear regression equations for bacterial count to calibrate the flow cytometry and estimate the count of mesophilic (log bacterial individual count (BIC) by flow cytometry = 0.701 log mesophilic + 1.9091) and psychrotrophic (log BIC by flow cytometry = 0.4703 log psychrotrophic + 3.2784) bacteria. It was not possible to establish calibration curves for thermophiles and thermodurics due to the weak to moderate correlation coefficients found among these microorganism counts and total bacterial count determined by flow cytometry (r = 0.30 and 0.27, respectively).(AU)

Atividade antimicrobiana e sinérgica das frações das folhas de Schinopsis brasiliensis Engl. frente a clones multirresistentes de Staphylococcus aureus / Antimicrobial and synergic activity of fractions from the leaves of Schinopsis brasiliensis Engl. against Staphylococcus aureus multiresistant clones

No presente estudo objetivou-se avaliar a atividade antimicrobiana e sinérgica de 4 frações das folhas de Schinopsis brasiliensis Engl (F1', F2', F1" e F2") frente às cepas Staphylococcus aureus MRSA multirresistentes. Os métodos utilizados foram poços de difusão em ágar, concentração mínima inibitória (CMI) - diluição em ágar, e bioautografia. Nos resultados bioautográficos observou-se três halos de inibição relacionados, no mínimo, à quatro constituintes ativos; sendo dois deles isolados das folhas (galato de metila e ácido gálico). A F2" (200∝g/mL) apresentou halos de inibição de 16 e 19mm frente as cepas de S. aureus multirresistente e Klebsiella pneumoniae, e CMI 100∝g/mL, respectivamente. Quanto as análises das associações das frações F1" ou F2" (25 e 50∝g/mL) com a tetraciclina e oxacilina, mostraram ações aditiva e sinérgica para a F2" (50∝g/mL), embora não suficiente para que a CMI atingisse valores inferiores a 2 e 4∝g/mL, necessário para serem classificadas como cepas sensíveis a oxacilina e tetraciclina, respectivamente. "Assim, conclui-se que a F2" das folhas de S. brasiliensis apresentou potencial antimicrobiano frente às cepas de S. aureus MRSA multirresistentes e que as associações das frações com os antibióticos testados não apresentaram benefícios não justificando o uso concomitante.

The aim of this study was to evaluate the antimicrobial and synergic activity of 4 leaf fractions of Schinopsis brasiliensis Engl (F1', F2', F1" and F2") against multidrug-resistant Staphylococcus aureus strains. The used methods were agar well diffusion, minimum inhibitory concentration (MIC) - agar dilution, and bioautography. The bioautographic results showed three inhibition zones that corresponded to at least four active compounds, two of which (methyl gallate and gallic acid) have already been isolated from leaves. The F2" (200∝g/mL) fraction showed inhibition zones of 16 mm and 19 mm against S. aureus multidrug-resistant and Klebsiella pneumoniae strains and a MIC value of 100∝g/mL, respectively. The analyses of associations of fraction F1" or F2" (25 and 50∝g/mL) with tetracycline and oxacillin showed additive and synergistic action for F2" (50∝g/mL), although it was not enough to decrease the MIC values to less than 2 and 4∝g/mL, necessary to classify the strains as susceptible to oxacillin and tetracycline, respectively. Thus, it was concluded that F2" from the leaves of S. brasiliensis showed antimicrobial potential against multidrug-resistant MRSA strains, and the associations of the fractions with the tested antibiotics showed no benefits, not justifying their concomitant use.

Estado de frescor e qualidade higiênica do pescado vendido numa cidade do interior de Portugal / Freshness and hygienic quality of fish sold in a Portuguese town

Avaliaram-se o estado de frescor e a qualidade higiênica do pescado disponível em alguns estabelecimentos comerciais de Vila Real, uma cidade do interior de Portugal. Vinte e três amostras foram submetidas a uma análise sensorial e a análises fisico-químicas - determinação do teor de azoto básico volátil total (ABVT), do índice de refração do humor aquoso e do pH. A avaliação da qualidade higiênica do pescado foi feita por meio de diferentes análises microbiológicas na superfície e na profundidade do músculo do pescado. O pescado fresco, considerado por todos os métodos de análise, próprio para consumo, apresentou estado de frescor entre bom e satisfatório. O ABVT foi considerado o método objetivo de eleição, sempre que surgiram dúvidas relativamente ao grau de frescor do pescado. O pescado próprio para consumo apresentou valores que variaram entre os 20,60 e 27,36mgN/100g e perfil microbiológico aceitável, com contagens de microrganismos psicrotróficos entre 5,41 e 6,03 UFC/cm² na superfície e entre 4,79 e 5,28 UFC/cm² no músculo

The freshness and hygienic quality of fresh fish sold in some fish markets of Vila Real, a Portuguese town, were evaluated. Twenty-three samples were examined by sensorial and physico-chemical analyses (total volatil basic nitrogen - TVBN, aqueous humour refraction rate and pH). The determination of hygienic quality of fish was carried out by different microbiological analysis on the surface and deepness of fish muscle. Fish was considered proper for consumption by all the methods used in the evaluation of fish freshness, and was classified between good and satisfactory. TVBN was considered the objective method of election, in case of doubt about fish freshness, and fish proper for consumption presented TVBN values between 20.60 and 27.36mgN/100g. The microbiological profile was considered acceptable with psychrotrophic microorganisms counts between 5.41 and 6.03 CFU/cm² on fish surface and between 4.79 and 5.28 CFU/g in muscle deepness

Molecular methods for the detection of mutations.

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

Association of p53 genomic instability with the glutathione S-transferase null genotype in gastric cancer in the Portuguese population.

AIMS: p53 gene mutations are the most common genetic changes known to occur in human cancer. In previous studies, the presence of alterations to the p53 gene has been linked to the null phenotype of the glutathione S-transferase mu gene (GSTM1). GSTM1 appears to be part of a protective mechanism against the development of cancers in which environmental chemical carcinogens are involved. To screen for such an association in stomach cancer, p53 allelic loss and genomic instability and GSTM1 genotypes were investigated in gastric tumour DNA samples from 113 patients. METHODS: The polymerase chain (PCR) reaction was used to amplify a (CA) repeat array in the p53 locus; electrophoresis, genotyping, and allele quantification were performed using an automated DNA sequencer and Genescan software. The presence of the GSTM1 gene was determined by means of a differential PCR in which multiple genes were co-amplified in the same reaction tube. RESULTS: Loss of heterozygosity (LOH) of the p53 gene was found in 36 of 87 informative cases and genomic instability was present in eight of 113 cases. Further analysis into histological subtypes and sites of tumours did not show any positive association with p53 loss. An association between the presence of LOH and the GSTM1 null genotype was not seen; however, all the samples with genomic instability of the p53 gene (eight of 113) also showed a GSTM1 null genotype. CONCLUSION: This study does not support the hypothesis of an association between LOH in the p53 gene and the GSTM1 null genotype, but suggests that the GSTM1 null genotype might influence p53 genomic instability.

[Auto-immune polyglandular syndrome in an adult with chronic active hepatitis]. / Síndrome poliglandular auto-imune num adulto com hepatite crónica activa.

We present a case report with IgA deficiency, pernicious anemia, chronic active hepatitis, insulino-dependent diabetes mellitus and hypogonadism. A diagnosis of Polyglandular Auto-immune Syndrome was done and we discuss the difficulty in including this particular case in any of the types of the Neufeld's classification of Polyglandular Auto-immune Disease.

[Pneumopyopericardium]. / Pneumopiopericárdio.

Pneumopyopericardium is a rare disease. The most common causes seems to be ulceration or carcinoma in the lower esophagus or upper stomach. We report a case of pneumopiopericardium secondary to a pneumonia in a 60 years-old-woman after the first course of chemotherapy for acute myeloid leukemia. We use this case as a basis for a review of the literature on similar cases. This syndrome can be recognised promptly because of its characteristic physical findings and radiographic features.