Efficient activity screening of new glucuronoyl esterases using a pNP-based assay.

Glucuronoyl esterases (CE15, EC 3.1.1.117) catalyze the hydrolysis of ester bonds between lignin and carbohydrates in lignocellulose. They are widespread within fungi and bacteria, and are subjects to research interest due to their potential applicability in lignocellulose processing. Identifying new and relevant glucuronoyl esterase candidates is challenging because available model substrates poorly represent the natural substrate, which leads to inefficient screening for the activity. In this study, we demonstrate how fifteen novel, fungal, putative glucuronoyl esterases from family CE15 were expressed and screened for activity towards a commercially available, colorimetric assay based on the methyl-ester of 4-O-methyl-aldotriuronic acid linked to para-nitrophenol (methyl ester-UX-ß-pNP) and coupled with the activity of GH67 (α-glucuronidase) and GH43 (ß-xylosidase) activity. The assay provides easy means for accurately establishing activity and determining specific activity of glucuronoyl esterases. Out of the fifteen expressed CE15 proteins, seven are active and were purified to determine their specific activity. The seven active enzymes originate from Auricularia subglabra (3 proteins), Ganoderma sinensis (2 proteins) and Neocallimastix californiae (2 proteins). Among the CE15 proteins not active towards the screening substrate (methyl ester-UX-ß-pNP) were proteins originating from Schizophyllum commune, Podospora anserina, Trametes versicolor, and Coprinopsis cinerea. It is unexpected that CE15 proteins from such canonical lignocellulose degraders do not have the anticipated activity, and these observations call for deeper investigations.

Evaporative light scattering detection based reversed-phase ultra-high-performance liquid chromatography method to quantify intermediates and end products of biodiesel production.

A fast method based on reversed-phase ultra-high-performance liquid chromatography using evaporative light scattering detection (RP-UHPLC-ELSD) was developed for monitoring the intermediates and end products of biodiesel production. Gradient elution of water, acetonitrile, and a mixture of acetonitrile:2-propanol:n-hexane was used. With a minimal and easy sample preparation, fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), free fatty acids (FFA), monoacylglycerols (MAG), diacylglycerols (DAG), and triacylglycerols (TAG) were successfully separated. The developed method was used to monitor an ethylic enzymatic transesterification of soybean oil and to characterize the ester content of methyl and ethyl biodiesel. The ester content obtained was compared with the reference method by gas chromatography and flame ionization detector (GC-FID), with similar results for both methyl and ethyl biodiesel. The presented method is a simple and fast alternative, a 17 min run, to monitor the transesterification process, simultaneously quantifying all the analytes produced in the reaction: biodiesel and its intermediates. Limits of detection (LOD, between 29 and 307 ng) and quantification (LOQ, between 48 and 614 ng), linearity (R2>0.99), precision (between 0.30 and 6.58%), and accuracy (between 81.6 and 119.9%) were determined for the twenty-one compounds.

Predicting the mechanical response of the vaginal wall in ball burst tests based on histology.

A histologically motivated (HM) coefficient that establishes a link between tissue's microstructure and material model through histological data is used in the prediction of the mechanical properties of vaginal tissue that is subjected to multiaxial loading conditions. Therefore, the material parameters were based on an HM coefficient obtained from tensile testing and histological data of comparable tissues. Uniaxial tensile test data and histological data were collected from three groups of sheep at different time points in their life cycle, including virgins, pregnant, and parous ewes. From this data, a correlation between material parameters and histological data was obtained. Spherical indentation (ball burst [BB]) tests were then performed in specimens with similar tissue structure. The histological data of these samples were used in conjunction with the correlations already established for the uniaxial samples data, to define the material parameters of the BB samples. Mechanical properties of the BB specimens were predicted through basic histology and using finite element modeling (FEM) simulations, without direct mechanical measurements. The predicted force and displacement values of the FEM simulation displayed a good correlation with the experimental (BB) testing results. No fitting of the BB results was performed. In this way, the use of uniaxial tests coupled with useful histological information offers a promising approach to predicting macroscopic material behavior under multiaxial loading conditions in biomechanics.

Necromechanics: Death-induced changes in the mechanical properties of human tissues.

After the death phenomenon, the rigor mortis development, characterized by body stiffening, is one of the most evident changes that occur in the body. In this work, the development of rigor mortis was assessed using a skinfold caliper in human cadavers and in live people to measure the deformation in the biceps brachii muscle in response to the force applied by the device. Additionally, to simulate the measurements with the finite element method, a two-dimensional model of an arm section was used. As a result of the experimental procedure, a decrease in deformation with increasing postmortem time was observed, which corresponds to an increase in rigidity. As expected, the deformations for the live subjects were higher. The finite element method analysis showed a correlation between the c1 parameter of the neo-Hookean model in the 4- to 8-h postmortem interval. This was accomplished by adjusting the c1 material parameter in order to simulate the measured experimental displacement. Despite being a preliminary study, the obtained results show that combining the proposed experimental procedure with a numerical technique can be very useful in the study of the postmortem mechanical modifications of human tissues. Moreover, the use of data from living subjects allows us to estimate the time of death paving the way to establish this process as an alternative to the existing techniques. This solution constitutes a portable, non-invasive method of estimating the postmortem interval with direct quantitative measurements using a skinfold caliper. The tools and methods described can be used to investigate the subject and to gain epidemiologic knowledge on rigor mortis phenomenon.

Occurrence and characterization of Campylobacter spp. isolates in dogs, cats and children / Ocorrência e caracterização de isolados de Campylobacter spp. em cães, gatos e crianças

To improve the understanding of implications of Campylobacter spp. infections in pets and children of different environments were analysed 160 faecal samples from children and 120 from pets (103 dogs and 17 cats). Campylobacter spp. were detected in 6.87% of the children and in 18.3% of the dogs and cats. From 33 stool samples positive for Campylobacter spp., 57.6% were identified as C. jejuni, and 33.4% were identified as C. coli. More than 50% of the isolates in pets were resistant to ceftiofur, sulphazotrim, norfloxacin and tetracycline. In humans, most of the isolates were resistant to amoxicillin, cefazolin, ceftiofur, erythromycin and norfloxacin. From 19 isolates of C. jejuni, 11 isolates from children and 5 from dogs contained two to four of the virulence genes flaA, pldA, cadF or ciaB. We found an association between the presence of virulence genes and diarrhoea. Furthermore, an association was observed between the presence of Campylobacter spp. and diarrhoea in dewormed pets with blood picture suggestive of bacterial infection, and the therapeutic use of antibiotics was associated with more positive detection of Campylobacter spp. in the faeces of pets. Our data indicate that virulent strains of Campylobacter spp. can be risk factor to diarrhoea in animals, and that high resistance to antimicrobial agents is common in pets.(AU)

Com o objetivo de melhorar o entendimento das infecções por Campylobacter spp. em cães, gatos e crianças no Brasil, foram avaliadas 160 amostras fecais de crianças e 120 swabs retais de pets (103 cães e 17 gatos). Do total das amostras das crianças, 6,87% foram positivas para Campylobacter spp. e em cães e gatos a positividade foi de 18,3%. Das 33 amostras positivas para Campylobacter spp., 57,6% foram identificadas como C. jejuni e 33,4% foram identificadas como C. coli. Mais de 50% das amostras isoladas de pets foram resistentes a ceftiofur, sulphazotrim, norfloxacina e tetraciclina. Em crianças, a maioria das amostras foi resistente a amoxilina, cefazolina, ceftiofur, eritromicina e norfloxacina. De 19 isolados de C. jejuni, 11 isolados de crianças e cinco (5) de cães tinham dois (2) dos quatro (4) genes de virulência flaA, pldA, cadF or ciaB. Associação positiva entre a presença de Campylobacter spp. e diarreia em cães e gatos foi observada em animais desverminados e com hemograma sugestivo de infecção bacteriana. Também houve associação positiva entre a presença dos genes de virulência e a ocorrência de diarreia, e entre o uso de antibióticos e a positividade para Campylobacter spp. em suabes fecais de pets. Os dados desse trabalho indicam que cepas virulentas de Campylobacter spp. são fatores de risco para diarreia em cães e a resistência antimicrobiana é comum em isolados de cães(AU)

Uniaxial mechanical behavior of the human female bladder.

INTRODUCTION AND HYPOTHESIS: The objective of the present study was to investigate the tensile biomechanical properties of the human female bladder. METHODS: Tissue samples were obtained from 13 cadavers without pelvic floor dysfunctions. We performed uniaxial tensile tests to measure the stiffness and maximum stress of the bladder tissue. Correlations were calculated using the Pearson correlation coefficient. RESULTS: The bladder tissue stiffness ranged from 1 to 4.1 MPa (mean stiffness, 1.9 ± 0.2 MPa) and the maximum stress ranged from 0.5 to 2.6 MPa (mean maximum stress, 0.9 ± 0.1 MPa). There was a strong positive correlation between stiffness and maximum stress in the bladder tissue (ρ = 0.829, p < 0.001). Tissue from women younger than 50 years presented higher bladder stiffness than did tissue from older subjects (2.1 ± 0.2 versus 1.3 ± 0.1 MPa, p = 0.02). Maximum bladder stress, however, was not associated with age (1.0 ± 0.2 versus 0.7 ± 0.1 MPa, p = 0.349). In addition, body mass index and menopausal status were not associated with these biomechanical properties. CONCLUSIONS: Age may influence the uniaxial mechanical behavior of the human female bladder.

Vaginal tissue properties versus increased intra-abdominal pressure: a preliminary biomechanical study.

BACKGROUND/AIMS: This study aims to evaluate the pelvic floor (PF) tension response during simulated increased intra-abdominal pressure (IAP) and the vaginal biomechanical properties. METHODS: A 3-dimensional computational finite element model for PF was developed based on magnetic resonance imaging from a nulliparous healthy volunteer. The model was used to simulate an IAP of 90 cm H(2)O and to evaluate the PF stresses in the longitudinal and transversal axes. The vaginal samples were obtained from 15 non-prolapsed female cadavers. A uniaxial tensile test to obtain stiffness and maximum stress of vaginal tissue in the longitudinal and transversal axes was performed. RESULTS: The simulated IAP was associated with a similar PF stress state in the longitudinal and transversal axes. The stiffness and maximum stress in vaginal tissues presented a great variability between subjects. There was no difference in the vaginal tissue elasticity (6.2 ± 1.5 vs. 5.4 ± 1.1 MPa; p = 0.592) and maximum stress (2.3 ± 0.5 vs. 2.6 ± 0.9 MPa; p = 0.692) regarding the measurements in the longitudinal and transversal axes. CONCLUSION: The isotropic biomechanical behavior of vagina is in agreement with the PF stress state response during increased IAP.

Translation of biomechanics research to urogynecology.

INTRODUCTION: Pelvic floor (PF) dysfunctions represent a frequent and complex problem for women. The interaction between the vagina and its supportive structures, that are designed to support increases in abdominal pressure, can be considered a biomechanical system. Recent advances in imaging technology have improved the assessment of PF structures. The aim of this paper is to review the applications of biomechanics in urogynecology. METHODS: The available literature on biomechanics research in urogynecology was reviewed. RESULTS: Computational models have been demonstrated to be an effective tool to investigate the effects of vaginal delivery and PF dysfunctions. Biomechanical analysis of PF tissues provides a better understanding on PF dysfunctions etiology. These studies are also important for the development of synthetic prostheses utilized in PF surgery. CONCLUSION: An interdisciplinary and multidisciplinary collaborative research, involving bioengineers and clinicians, is crucial to improve clinical outcomes in patients with PF dysfunctions.