Tratamento da Litíase Renal com Ureterorrenoscopia: Experiência de um centro

Introduction: In this study, we evaluated the initial results of this procedure in our hospital, aiming to evaluate retrograde intrarenal surgery efficacy and safety and possible success predictors of this technique. Material and Methods: After collecting data from the medical records and imaging studies of all patients undergoing retrograde intrarenal surgery in 2014 e 2015 at Centro Hospitalar de São João, and applying our exclusion criteria, we analyzed the data of 138 patients (total of 179 retrograde intrarenal surgery). The primary outcomes of our study were the immediate success rate, assessed by the surgeon’s perception intraoperatively, and postoperative success rate, assessed by image control. Residual lithiasis was considered significant in the presence of calculi > 3 mm. Results: The overall success rate was 67.0%, considering the surgeon’s perception. Considering the image control, the success rate was 66.7% for calculi smaller than 150 mm2 and located outside the ICG, but smaller in other locations or bigger calculi. In the univariate analysis, stone burden, calculi number and location were statistically significant predictors of retrograde intrarenal surgery success. Conclusion: Location in the ICG was considered a predictor of retrograde intrarenal surgery failure and, in this location, RIRS was more effective for calculi < 150 mm2; this differenced was not encountered for calculi outside the ICG. It is a safe intervention, which allows a staged use.Introdução: No presente estudo, analisámos os resultados iniciais desta técnica no nosso centro, com o objetivo de avaliar a eficácia e segurança da cirurgia intra-renal retrógrada no tratamento de cálculos renais e fatores preditores do seu sucesso. Material e Métodos: Após a recolha dos dados dos processos clínicos e exames de imagem dos doentes submetidos a cirurgia intra-renal retrógrada para tratamento de cálculos renais em 2014 e 2015 no Centro Hospitalar de São João, e aplicação dos critérios de exclusão, analisámos os dados relativos a 138 doentes (total de 179 cirurgia intra-renal retrógrada). Os nossos desfechos primários foram a taxa de sucesso imediata, avaliada pela perceção do cirurgião, e a taxa de sucesso pós-operatória, avaliada pela imagiologia de controlo, considerando como litíase residual significativa cálculos > 3 mm. Resultados: A taxa de sucesso global foi de 67,0% tendo em conta a perceção do cirurgião. Considerando o controlo imagiológico, a taxa de sucesso foi de 66,7% nos cálculos < 150 mm2 fora do grupo calicial inferior, mas menor nas restantes localizações ou carga litiásica maior. Na análise univariada, a carga litiásica, o número de cálculos e a localização foram estatisticamente significativos como preditores do sucesso da cirurgia intra- renal retrógrada. Não foram registadas complicações major. Conclusão: A localização no grupo calicial inferior foi considerada um preditor do insucesso da cirurgia intra-renal retrógrada, sendo que neste grupo calicial a eficácia foi maior nos cálculos com <150 mm2; esta diferença não se verificou nos cálculos fora desta localização. É uma intervenção segura, permitindo a sua utilização seriada

Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

Despite the emergence of non-O157 shiga toxin-producing Escherichia coli (STEC) infections, E. coli O157 serotype is still the most commonly identified STEC in world. It causes high morbidity and mortality, and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain as the gold standard.In here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157.Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. Then, the method was optimized for the detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1×10-2 to 1×102 CFU per 25g or ml of food. Samples were then pre-enriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrixes with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% CI 82.83 - 100), a sensitivity of 97.22% (95% CI, 83.79 - 99.85) and an accuracy of 98,33% (CI 95%, 83.41 - 99.91). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.This work was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (FCT), project PIC/IC/82815/2007. C.A. acknowledges FCT for individual postdoctoral fellowship SFRH/BPD/74480/2010. We also acknowledge Biomode S.A. for providing some supplies for this project

Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fm.2018.12.009.Listeria monocytogenes is one of the most important foodborne pathogens due to the high hospitalization and mortality rates associated to an outbreak. Several new molecular methods that accelerate the identification of L. monocytogenes have been developed, however conventional culture-based methods still remain the gold standard. In this work we developed a novel Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH) method for the specific detection of L. monocytogenes. The method was based on an already existing PNA probe, LmPNA1253, coupled with a novel blocker probe in a 1:2 ratio. The method was optimized for the detection of L. monocytogenes in food samples through an evaluation of several rich and selective enrichment broths. The best outcome was achieved using One Broth Listeria in a two-step enrichment of 24h plus 18h. For validation in food samples, ground beef, ground pork, milk, lettuce and cooked shrimp were artificially contaminated with two ranges of inoculum: a low level (0.22CFU/25g or mL) and a high level (210CFU/25g or mL). The PNA-FISH method performed well in all types of food matrices, presenting an overall accuracy of 99% and a detection limit of 0.5CFU/25g or mL of food sample.Nuno R. da Silva acknowledges the financial support byFundac¸ ão para a Ciência e Tecnologia (FCT) of the Portuguese’s Min-istry for Science, Technology and Higher Education (MCTES), in theframework of the POCH – Programa Operacional Capital Humano,co-funded by the European Social Fund (SFRH/BD/111825/2015).This work was also supported by FCT under the scope ofthe strategic funding of UID/BIO/04469 unit and COMPETE2020 (POCI-01-0145-FEDER-006684) and BioTecNorte opera-tion (NORTE-01-0145-FEDER-000004) funded by the EuropeanRegional Development Fund under the scope of Norte2020 – Pro-grama Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Una revisión de los discos de Fortios

We have used EDXRF, Micro-PIXE and optical microscopy (metallographic analysis), complemented with SEM-EDS, to first determine the elemental content, and second, to identify the process used to join the components (disk, peripheral rod and tab) of several Iron Age gold buttons. These have a very similar typology and were found at three archaeological sites in the South-Western part of the Iberian Peninsula. A set of 35 buttons from Castro dos Ratinhos (7), Outeiro da Cabeça (23) and Fortios (5) were analyzed and the results published in Trabajos de Prehistoria (Soares et al. 2010). Recently Perea et al. (2016) have published analyses of other 4 gold buttons from Fortios with the same purpose, but only using one technique, SEM-EDS. As they only analysed the rough surface layer, the results are neither effective nor reliable, taking into account the constraints associated with the technique, namely the small depth reached (< 2 ?m) by the incident beam and, consequently, its sensitivity to the topography of the analyzed surface. Despite these constraints, they have accepted uncritically their results and, at the same time, question our own analyses and results and the interpretation we have made. Here we discuss the approach of Perea et al. in order to determine not only the elemental content of the Fortios gold buttons, but also to identify the joining process used in their manufacture.Hemos empleado fluorescencia de rayos X por energía dispersiva (EDXRF), emisión de rayos X inducida por partículas (Micro-PIXE) y microscopía (análisis metalográfico), complementada con microscopia electrónica de barrido y espectroscopia de rayos X en energía dispersiva (SEM-EDS), primero para determinar el contenido elemental y segundo para identificar el proceso empleado para unir los componentes (disco, anillo periférico y presilla) de varios de los discos de oro de la Edad del Hierro. Su tipología es muy similar y fueron encontrados en tres yacimientos arqueológicos de la zona suroccidental de la Península Ibérica. Una serie de 35 discos del Castro dos Ratinhos (7), Outeiro da Cabeça (23) y Fortios (5) fueron analizados y sus resultados publicados en Trabajos de Prehistoria (Soares et al. 2010). Recientemente Perea et al. (2016) han publicado análisis de otros 4 discos de oro de Fortios con el mismo propósito pero usando solo SEM-EDS. Al haber analizado solo la capa superficial irregular, los resultados no son reales, ni fiables, considerando las limitaciones asociadas con la técnica, especialmente la escasa profundidad alcanzada (< 2 ?m) por el haz incidente y, consecuentemente, su sensibilidad a la topografía de la superficie analizada. A pesar de esas limitaciones, han aceptado acríticamente sus resultados y, a la vez, han puesto en cuestión nuestros análisis y resultados y las interpretaciones que hemos hecho. Aquí discutimos el enfoque de Perea et al. para determinar no solo el contenido elemental de los discos de oro de Fortios, sino también para identificar el proceso de unión empleado en su manufactura

Estudio de botones de oro de la primera Edad del Hierro del Sudoeste de la Península Ibérica. Identificación de un taller metalúrgico de oro

Early Iron Age gold buttons from Castro dos Ratinhos, Fortios and Outeiro da Cabeça were analysed by conventional EDXRF, Micro-PIXE, SEM-EDS and Optical Microscopy. EDXRF results point out to a rather homogeneous alloy composition throughout all the analysed buttons. PIXE microanalyses show that all the button components (disk, tab and peripheral grooved decorated rod) have the same alloy composition. PIXE and SEM-EDS microanalyses, supplemented with optical microscopy characterization, show the absence of chemical composition differences between distinct components and joining zones, suggesting that no solder had been applied, i.e. that a partial melting/solid state diffusion process had been used for the welding of button components. Finally, the noticeable similar compositions together with the use of the same welding process and the very similar artefact typologies suggest that those small gold treasures could be interpreted as the result of the work of a single metallurgical workshop, probably located somewhere in the South-Western Iberian Peninsula.Botones de oro pertenecientes a la primera Edad del Hierro, procedentes de Castro dos Ratinhos, Fortios e Outeiro da Cabeça (Portugal), fueron analizados por EDXRF y Micro-PIXE. Los resultados de los análisis por EDXRF mostraron una composición similar en todos los botones, independientemente de su procedencia. Por otra parte, los microanálisis por PIXE permitieron verificar que los componentes soldados de cada botón (disco, presilla y cordón exterior) tienen la misma composición química. Además de eso, las áreas de soldadura fueron estudiadas mediante Micro-PIXE, SEM-EDS y posterior análisis metalográfico por microscopia óptica de reflexión. Estos análisis permitieron comprobar la ausencia de soldaduras en las zonas de unión de estos componentes, lo que nos permite concluir que debe haber tenido lugar un proceso de fusión parcial y de difusión en estado sólido para unir los componentes de estos botones. La gran semejanza en la composición química, junto a la presencia del mismo tipo de soldadura y tipologías similares, nos sugiere que todas estas piezas fueron resultado del trabajo de un mismo artesano-joyero, cuyo taller se encontraría localizado en el sudoeste de la Península Ibérica

Benefits of spine stabilization with biodegradable scaffolds in spinal cord injured rats

Spine stabilization upon spinal cord injury (SCI) is a standard procedure in clinical practice, but rarely employed in experimental models. Moreover, the application of biodegradable biomaterials for this would come as an advantage as it would eliminate the presence of a nondegradable prosthesis within the vertebral bone. Therefore, in the present work, we propose the use of a new biodegradable device specifically developed for spine stabilization in a rat model of SCI. A 3D scaffold based on a blend of starch with polycaprolactone was implanted, replacing delaminated vertebra, in male Wistar rats with a T8-T9 spinal hemisection. The impact of spinal stabilization on the locomotor behavior was then evaluated for a period of 12 weeks. Locomotor evaluation—assessed by Basso, Beatie, and Bresnahan test; rotarod; and open field analysis—revealed that injured rats subjected to spine stabilization significantly improved their motor performance, including higher coordination and rearing activity when compared with SCI rats without stabilization. Histological analysis further revealed that the presence of the scaffolds not only stabilized the area, but also simultaneously prevented the infiltration of the injury site by connective tissue. Overall, these results reveal that SCI stabilization using a biodegradable scaffold at the vertebral bone level leads to an improvement of the motor deficits and is a relevant element for the successful treatment of SCI.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (Doctoral fellowship to Nuno Silva, SFRH/BD/40684/2007; Ciência 2007 Program to António Salgado; Grant N PTDC/SAU-BMA/114059/2009) and the Foundation Calouste de Gulbenkian to funds attributed to A.J. Salgado under the scope of the The Gulbenkian Programme to Support Cutting Edge Research in the Life Sciences

Electrosprayed whey protein-based nanocapsules for beta-carotene encapsulation

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2019.126157.In this work an electrohydrodynamic process (electrospray) was used to produce beta-carotene loaded nanocapsules based on whey protein isolate (WPI). WPI solutions were prepared in aqueous solutions with different concentrations of ethanol (5, 10 and 15%) which were used for beta-carotene solubilization. Different electrospray conditions were tested and the morphology and molecular organization of the nanocapsules were studied on dried and hydrated state. The size of the dried nanocapsules ranged between 227 and 283nm. After hydration, there was a significant increase in the mean size of the nanocapsules, being the sizes higher for nanocapsules produced with increasing concentrations of ethanol. Results, obtained from the reactivity of free sulfhydryl groups and fluorescence analysis, showed that the increase of ethanol concentration had a destabilizing effect on the protein unfolding. Electrosprayed WPI-based nanocapsules can be used for the encapsulation of ?-carotene answering the industrial demand for novel encapsulation technologies to protect sensitive bioactive compounds.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and by BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. The author Rui M. Rodrigues thanks to FCT the financial grant with SFRH/BD/110723/2015. This work was supported by the project ARMAdilhas seletivas para eliminação da VESPA velutina. Medida 6 – Investigação e Desenvolvimento (n° 5894057). Instituto de Financiamento de Agricultura e Pescas, I.P.info:eu-repo/semantics/publishedVersio

Application of fluorescence in situ hybridisation using peptide nucleic acid probes in gastric samples for detection of Helicobacter pylori clarithromycin resistance

Microorganisms are responsible for several infectious diseases that can cause severe problems to patients and their treatment success is seriously correlated with the fast detection of the infectious agent. Some of the standard methods used, such as culturing methods are fastidious and time-consuming and do not give any information about the antibiotic resistance profile. Therefore, molecular methods have been developed during the last several years in order to overcome these shortcomings. In this work a new genotypic method that permits the identification of the microorganism in clinical samples in a prompt way is proposed. This technique is based on Fluorescence in situ hybridization with PNA probes that are synthetic molecules, complementary to a specific rRNA sequence of the microorganism. Methods: A set of PNA probes were designed concerning H. pylori point mutations regarding clarithromycin resistance which is the main problem of gastric diseases treatment failure. An additional probe concerning susceptibility was also designed. After hybridization conditions optimization, probes were applied to H. pylori smears to achieve their practical sensitivity and specificity. At the end they were applied to gastric biopsies in a retrospective study for method validation in real samples. E-test and PCR-sequencing were used to evaluate the results. Results: The probes concerning clarithromycin resistance hybridized only with the resistant strains that had the corresponding point mutations and as such presented 100% sensitivity (95% CI, 79.9-100) and 100% specificity (95% CI, 71.6-100). Results also showed that it is possible to discriminate susceptible from resistant H. pylori strains in gastric biopsy samples since it was presented similar results between the 3 tests used. Overall, the PNA-FISH method was in full agreement with PCR-sequencing although it was a little bit lower when compared to E-test that it was used as gold standard method in this retrospective study (86%). Conclusion: PNA-FISH proved to be an important in situ method for detection of microorganisms in clinical samples in a more prompt way than the standard methods. Due to high H. pylori probes sensitivity and specificity it is proved the applicability of PNA-FISH methodology to clinical material, thus overcoming the need of culturing steps and/or PCR/sequencing procedures and enabling rapid initiation of appropriate antibiotic therapy until culture confirmation several days later

PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori

<p>Abstract</p> <p>Background</p> <p>Triple therapy is the gold standard treatment for <it>Helicobacter pylori </it>eradication from the human stomach, but increased resistance to clarithromycin became the main factor of treatment failure. Until now, fastidious culturing methods are generally the method of choice to assess resistance status. In this study, a new genotypic method to detect clarithromycin resistance in clinical samples, based on fluorescent <it>in situ </it>hybridization (FISH) using a set of peptide nucleic acid probes (PNA), is proposed.</p> <p>Results</p> <p>The set of probes targeting the point mutations responsible for clarithromycin resistance was applied to <it>H. pylori </it>suspensions and showed 100% sensitivity and specificity (95% CI, 79.9-100 and 95% CI, 71.6-100 respectively). This method can also be amenable for application to gastric biopsy samples, as resistance to clarithromycin was also detected when histological slides were tested.</p> <p>Conclusions</p> <p>The optimized PNA-FISH based diagnostic method to detect <it>H. pylori </it>clarithromycin resistance shown to be a very sensitive and specific method for the detection of clarithromycin resistance in the <it>H. pylori </it>smears and also proved to be a reliable method for the diagnosis of this pathogen in clinical samples and an alternative to existing plating methods.</p