Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n=67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

The equine DNAH3 gene: SNP discovery and exclusion of an involvement in recurrent airway obstruction (RAO) in European Warmblood horses

Recurrent airway obstruction is one of the most common airway diseases affecting mature horses. Increased bronchoalveolar mucus, neutrophil accumulation in airways, and airway obstruction are the main features of this disease. Mucociliary clearance is a key component of pulmonary defense mechanisms. Cilia are the motile part of this system and a complex linear array of dynein motors is responsible for their motility by moving along the microtubules in the axonemes of cilia and flagella. We previously detected a QTL for RAO on ECA 13 in a half-sib family of European Warmblood horses. The gene encoding DNAH3 is located in the peak of the detected QTL and encodes a dynein subunit. Therefore, we analysed this gene as a positional and functional candidate gene for RAO. In a mutation analysis of all 62 exons we detected 53 new polymorphisms including 7 non-synonymous variants. We performed an association study using 38 polymorphisms in a cohort of 422 animals. However, after correction for multiple testing we did not detect a significant association of any of these polymorphisms with RAO (P>0.05). Therefore, it seems unlikely that variants at the DNAH3 gene are responsible for the RAO QTL in European Warmblood horses.

Animal models for peri-implant mucositis and peri-implantitis

The treatment of infectious diseases affecting osseointegrated implants in function has become a demanding issue in implant dentistry. Since the early 1990s, preclinical data from animal studies have provided important insights into the etiology, pathogenesis and therapy of peri-implant diseases. Established lesions in animals have shown many features in common with those found in human biopsy material. The current review focuses on animal studies, employing different models to induce peri-implant mucositis and peri-implantitis.

Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage.

Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs). The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning.

Comportamiento de coníferas bajo riego en Dique Yaucha : Mendoza, Argentina

La provincia de Mendoza, que se ubica en el Centro-Oeste de la República Argentina, posee una extensión de 150.830 km2. Prácticamente todas las actividades agropecuarias y forestales están concentradas en el 3% de su territorio que es posible irrigar. Al oeste, en el límite con la República de Chile, está la cadena montañosa que forma parte de la región fitogeográfica del Desierto Andino que se extiende por más de 500 km, con un ancho promedio de 100 km. El objetivo de este trabajo fue evaluar la supervivencia y el comportamiento de distintas especies de coníferas en zonas del piedemonte mendocino. Para ello se instaló una parcela experimental en el Dique Yaucha, ubicado en el Departamento de San Carlos a 34°00' S y 69°07' O, a una altura de 1213 msnm. Las especies del ensayo fueron: Pinus pinea L., Pinus halepensis Mill. y Cupressus arizonica Greene. Se llevaron plantas de aproximadamente 0,70 m de altura, que se instalaron a una distancia de plantación definitiva de 3 x 3 m y se regaron superficialmente por surcos. Se tomaron periódicamente datos dasométricos de diámetro altura de pecho (DAP) de todas las plantas, altura total de los árboles promedio de cada especie, registrándose además las fallas producidas y el estado sanitario. Los resultados obtenidos a la edad de 17 años son: Pinus pinea: diámetro promedio 17 cm, altura 6,60 m y 22,5% de fallas; Pinus halepensis: diámetro promedio 16 cm, altura 8,60 m y 12,2% de fallas; Cupressus arizonica: diámetro promedio 20 cm, altura 7,75 m y 18,1% de fallas. Es de destacar que P. pinea presenta el 6% de fustes bifurcados a baja altura. Todas las especies tienen un buen estado sanitario, no registrándose hasta la fecha plagas o enfermedades que hayan afectado el desarrollo del ensayo.

Comportamiento de procedencias de Pinus nigra bajo riego en Dique Yaucha : Mendoza, Argentina

La provincia de Mendoza, que se ubica en el Centro-Oeste de la República Argentina, posee una extensión de 150.830 km2. Prácticamente todas las actividades agropecuarias y forestales están concentradas en el 3% de su territorio que es posible irrigar. Al Oeste, en el límite con la República de Chile, está la cadena montañosa que forma parte de la región fitogeográfica del Desierto Andino que se extiende por más de 500 km, con un ancho promedio de 100 km. El objetivo de este trabajo fue evaluar la supervivencia y el comportamiento de distintas procedencias de Pinus nigra en zonas del piedemonte mendocino. Para ello se instaló una parcela experimental en el Dique Yaucha, ubicado en el Departamento de San Carlos a 34°00’ S y 69°07’ O, a una altura de 1.213 msnm. Las procedencias del ensayo fueron: Pinus nigra var. clusiana (Francia), Pinus nigra var. austríaca (Livo - Italia), Pinus nigra var. austríaca (Lasa - Italia), Pinus nigra var. knin (ex Yugoslavia) y Pinus nigra var. pyramidalis (Turquía). Se llevaron plantas de aproximadamente 0,30 m de altura que se instalaron a una distancia de plantación definitiva de 3 x 3 m y se regaron superficialmente por surcos. Se tomaron periódicamente datos dasométricos de diámetro altura de pecho (DAP) de todas las plantas, altura total de los árboles promedio de cada procedencia, registrándose además las fallas producidas y el estado sanitario. Los resultados obtenidos a la edad de 17 años son: Pinus nigra var. clusiana (Francia): diámetro promedio 12,1 cm, altura 5,2 m y 10% de fallas; Pinus nigra var. austríaca (Livo -Italia): diámetro promedio 13 cm, altura 4,6 m y 25% de fallas; Pinus nigra var. austríaca (Lasa - Italia): diámetro promedio 13,9 cm, altura 5,60 m y 0% de fallas; Pinus nigra var. knin (ex Yugoslavia): diámetro promedio 14,1cm, altura 6,2 m y 15% de fallas y Pinus nigra var. pyramidalis (Turquía): diámetro promedio 11,6 cm, altura 5,8 m y 40% de fallas. Las plantas presentan una gran variabilidad de formas desde porte abierto a muy fastigiado. Todas las procedencias presentan un buen estado sanitario, no registrándose hasta la fecha plagas o enfermedades que hayan afectado el desarrollo del ensayo.

Lesion, and haematologic and serum characteristics of Weddell seals (Leptonychotes weddellii) sampled in McMurdo Sound, Antarctica

We examined and collected biomedical samples from Weddell seals (Leptonychotes weddellii) during studies of post-breeding-season foraging behaviour of adults and movements of weaned pups as a complement to ongoing studies on the ecology and population dynamics of the McMurdo seals (Stewart et al. 2000, 2003). Here we report on Weddell seal health assessments conducted during the 1996/97, 1997/98 and 1998/99 breeding seasons at the Delbridge Islands (77.68°S, 166.50°E), McMurdo Sound, Antarctica. Our objectives were to compile baseline biomedical data for Weddell seals in McMurdo Sound, and to identify infectious and non-infectious diseases affecting the population. Development of such a database, including information on normal background morbidity and mortality, is an important first step in evaluating natural versus anthropogenic impacts on population health (Geraci et al. 1999; Reddy et al. 2001). These data will be integral to international studies of southern ocean pinnipeds that seek to evaluate the influence of biotic and abiotic factors on the ecology of these apex predators.

Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.

We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.

Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration

Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss.

O transplante pulmonar e hepático combinado na fibrose quística : a propósito de um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Climate Change and the Color Line

Climate change is estimated to be responsible for 400,000 deaths per year, mostly because of hunger and communicable diseases affecting children in the Global South. Using the sociology of W.E.B. Du Bois, I attempt to demonstrate how and why climate change occurs along the color line. I conclude by arguing why it is important to think about climate change as a human rights issue.

Evidências de validade do Millon Behavioral Medicine Diagnostic na avaliação de aderência terapêutica em pacientes com insuficiência cardíaca crônica

Chronic heart failure (CHF) is the final common pathway of most diseases affecting the heart, being responsible for a high level of mortality and hospitalization, as well as significant reduction in quality of life of those affected. Interventions that claim to optimize patient adherence to their medical regimen, and improve self-care behavior, have proven effective in preventing unplanned admissions and improves the outcome for patients, however, studies have shown the problem of non-adherence, and some psychological instruments have been used to show that traces indicate difficulties with treatment adherence. Having shown this, the aim of this work is to evaluate the evidence of validity of the Millon Behavioral Medicine Diagnostic (MBMD) in a population of patients with CHF. The study included individuals with CHF, males and females, between the age of 18 and 85 years, treated in a reference hospital in the city of NatalRN. A total of 120 patients answered, in addition to the MBMD, another questionnaire structured with sociodemographic aspects and clinical itens. The results indicated that the parameter of the MBMD reliability was satisfactory the most of extracted factors, and some scale. In terms of the population studied, we could verify that the disease was more prevalent in men, but women had the highest average in indicators related to negative health habits and depressed mood. Younger pacients and those who had no partner had the highest averages in groups of items that dealt with feelings of sadness and discouragement. Hasn’t been observed differences related to negative health habits and problematic adherence among patients in different functional classes. More studies in this research line, with a larger population and from other regions of the country, are needed in order to expand the data presented here

Étude de l'expression des microARNs et des enzymes de synthèse des corticostéroïdes dans le développement pulmonaire

Le syndrome de détresse respiratoire du nouveau-né (SDR) est l’une des pathologies les plus fréquentes dont souffrent les bébés prématurés. Le SDR est causé par un déficit dans la synthèse du surfactant pulmonaire en raison de l’immaturité du poumon lors d’une naissance prématurée. Plusieurs éléments régulent le développement pulmonaire notamment les stéroïdes sexuels et les corticostéroïdes. Le sexe est aussi un élément régulateur du développement pulmonaire. En effet, les garçons sont plus atteints que les filles par le SDR. Ce dimorphisme sexuel est attribué aux androgènes. Le traitement anténatal aux glucocorticoïdes est prescrit aux femmes qui sont à risque d’accoucher prématurément. En effet, les corticostéroïdes favorisent la maturation pulmonaire anténatale. Également, il a été démontré que les microARNs sont primordiaux pour le développement pulmonaire. Ceci nous a conduit à étudier l’impact des androgènes sur le profil d’expression des microARNs lors de la transition du stade canaliculaire au stade sacculaire (jour gestationnel (JG)17.0 au JG18.0), période qui coïncide avec la montée de la synthèse et de la sécrétion du surfactant chez la souris. Tout d’abord, nous avons étudié la stabilité des gènes de normalisation (snoRNAs) afin de quantifier les microARNs par qPCR. Cette analyse a été effectuée avec 3 logiciels différents et sur plusieurs stades du développement notamment de la période pseudoglandulaire jusqu’au stade alvéolaire chez les deux sexes. On a identifié les meilleures combinaisons de gènes de normalisation les plus stables pour chaque stade du développement étudié ainsi que pour la période couvrant tous les stades étudiés. Ensuite nous avons analysé à GD17.0 et GD18.0 le profil d’expression des microARNs chez des fœtus mâles dont les mères ont été traitées au flutamide (anti-androgènes pure). Les résultats ont montré que 43 microARNs matures sont modulés par les androgènes à GD17.0 et 35 microARNs à GD18.0. Pour certains microARNs, nous avons identifié des cibles potentielles qui sont inversement modulées par les androgènes par rapport aux microARNs. Ces cibles sont impliquées dans plusieurs processus biologiques tels que le métabolisme des lipides et la prolifération cellulaire ainsi que dans des fonctions moléculaires tels que la liaison des facteurs de transcription. Des expériences de validation ont été effectuées par qPCR. Nos résultats ont montré que les androgènes régulent des processus qui peuvent être impliqués dans la maturation pulmonaire via la régulation des microARNs. En plus de l’intérêt porté aux androgènes dans la maturation pulmonaire, nous avons analysé l’expression d’enzymes de synthèse des corticostéroïdes dans le poumon fœtal humain. L’expression de l’enzyme 21-hydroxylase a été étudiée par qPCR et par immunobuvardage. Également la localisation de l’ARNm de cette enzyme clé de la synthèse des glucocorticoïdes, a été effectuée par hybridation in situ. L’ARNm de CYP21A2 a été détecté par qPCR dans les 34 échantillons analysés et dont les âges variaient entre 17 et 40 semaines de grossesse. Aucune corrélation, avec l’âge gestationnel ou le sexe, n’a été observée. Des niveaux significatifs de la protéine 21-hydroxylase ont été détectés dans nos échantillons. Nous avons investigué l’expression d’autres enzymes impliquées dans la voie de synthèse des glucocorticoïdes notamment CYP11B1, CYP11B2 et CYP17A1. Les ARNm des gènes CYP11B1, CYP11B2 n’ont pas été détectés dans nos échantillons, contrairement à CYP17A1 dont l’ARNm a été détecté dans tous nos tissus fœtaux analysés. La protéine de la 17α-hydroxylase a été détectée à de faibles niveaux. Nos résultats d’hybridation in situ ont montré que l’expression de CYP21A2 est localisée presqu’exclusivement dans l’épithélium pulmonaire distal. Nos résultats suggèrent que les produits de la 21-hydroxylase agiront via une action intracrine sur l’épithélium distal en activant le récepteur des glucocorticoïdes (GR). L’activation du récepteur des minéralocorticoïdes (MR) ne semble pas dépendre de produits de la 21-hydroxylase en raison des quantités importantes d’aldostérone circulante.

Accessing occupational exposure to fungi in a cork industry

Different forms of fungal diseases affecting the nose and paranasal sinuses are recognized, including invasive and non-invasive fungal rhinosinusitis. Penicillium glabrum complex is associated with respiratory diseases such as suberosis, a typical disease of cork industry workers. In addition, Chrysonilia sitophila has been described as causing occupational asthma, associated to prolonged exposure to high counts of spores. In this study we aimed to access fungal exposure in workers from one cork industry through the mycological analysis of their nasal exudate and the environmental fungal contamination of their surroundings as well.