Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation.

Pectin methylesterases (PMEs) modify homogalacturonan's chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55-70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties.

TMT-Based Quantitative Proteomics Analysis Reveals Differentially Expressed Proteins between Different Sources of hMSCs.

Mesenchymal stem cells (MSCs) are an attractive therapeutic tool for tissue engineering and regenerative medicine owing to their regenerative and trophic properties. The best-known and most widely used are bone marrow MSCs, which are currently being harvested and developed from a wide range of adult and perinatal tissues. MSCs from different sources are believed to have different secretion potentials and production, which may influence their therapeutic effects. To confirm this, we performed a quantitative proteomic analysis based on the TMT technique of MSCs from three different sources: Wharton's jelly (WJ), dental pulp (DP), and bone marrow (BM). Our analysis focused on MSC biological properties of interest for tissue engineering. We identified a total of 611 differentially expressed human proteins. WJ-MSCs showed the greatest variation compared with the other sources. WJ produced more extracellular matrix (ECM) proteins and ECM-affiliated proteins and proteins related to the inflammatory and immune response processes. BM-MSCs expressed more proteins involved in osteogenic, adipogenic, neuronal, or muscular differentiation and proteins involved in paracrine communication. Compared to the other sources, DP-MSCs overexpressed proteins involved in the exocytosis process. The results obtained confirm the existence of differences between WJ, DP, and BM-MSCs and the need to select the MSC origin according to the therapeutic objective sought.

Impact of Rhamnolipids (RLs), Natural Defense Elicitors, on Shoot and Root Proteomes of Brassica napus by a Tandem Mass Tags (TMTs) Labeling Approach.

The rapeseed crop is susceptible to many pathogens such as parasitic plants or fungi attacking aerial or root parts. Conventional plant protection products, used intensively in agriculture, have a negative impact on the environment as well as on human health. There is therefore a growing demand for the development of more planet-friendly alternative protection methods such as biocontrol compounds. Natural rhamnolipids (RLs) can be used as elicitors of plant defense mechanisms. These glycolipids, from bacteria secretome, are biodegradable, non-toxic and are known for their stimulating and protective effects, in particular on rapeseed against filamentous fungi. Characterizing the organ responsiveness to defense-stimulating compounds such as RLs is missing. This analysis is crucial in the frame of optimizing the effectiveness of RLs against various diseases. A Tandem Mass Tags (TMT) labeling of the proteins extracted from the shoots and roots of rapeseed has been performed and showed a differential pattern of protein abundance between them. Quantitative proteomic analysis highlighted the differential accumulation of parietal and cytoplasmic defense or stress proteins in response to RL treatments with a clear effect of the type of application (foliar spraying or root absorption). These results must be considered for further use of RLs to fight specific rapeseed pathogens.

The bovine uterine fluid proteome is more impacted by the stage of the estrous cycle than the proximity of the ovulating ovary in the periconception period.

Uterine secretions provide a suitable environment for sperm selective migration during a couple of days preceding ovulation and for early embryo development before implantation. Our goal was to identify and quantify proteins in the bovine uterine fluid during the periovulatory period of the estrous cycle. Genital tracts with normal morphology were collected from adult cyclic Bos taurus females in a local slaughterhouse and classified into pre-ovulatory or post-ovulatory stages of cycle (around days 19-21 and 0-5 of cycle, respectively; n = 8 cows per stage) based on ovarian morphology. Proteins from uterine fluid collected from the utero-tubal junction to the base of each horn (four pools of two cows per condition) were analyzed by nanoLiquid Chromatography coupled with tandem Mass Spectrometry (nanoLC-MS/MS). A total of 1214 proteins were identified, of which 91% were shared between all conditions. Overall, 57% of proteins were predicted to be secreted and 17% were previously reported in uterine extracellular vesicles. Paired comparisons between uterine horns ipsilateral and contralateral to ovulation evidenced 12 differentially abundant proteins, including five at pre-ovulatory stage. Furthermore, 35 proteins differed in abundance between pre- and post-ovulatory stages, including 21 in the ipsilateral side of ovulation. Functional analysis of identified proteins demonstrated roles in binding, metabolism, cellular detoxification and the immune response. This study provides a valuable database of uterine proteins for functional studies on sperm physiology and early embryo development.

Terceirização no âmbito da saúde pública: reflexões e parâmetros / Outsourcing in the field of public health: reflections and parameters

A saúde é direito fundamental, pressuposto da dignidade da pessoa humana, e possui papel de destaque na complexa realidade social que integramos, a ponto de o Estado (gênero) assumir a obrigação constitucional de garanti-la a todos. Nesse contexto e para a máxima efetividade do direito à saúde, possibilitou-se à iniciativa privada participar de forma complementar do sistema público encarregado de assegurá-lo ­ com preferência a entidades sem fins lucrativos, como é o caso do chamado terceiro setor. Contudo, em sintonia com o constitucionalmente previsto e a bem de sua eficácia jurídico-sanitária, as hipóteses de parcerias do setor público com o privado necessitam observar determinados parâmetros. Com o propósito de colaborar para o debate, após reflexão e sem qualquer pretensão de ordem ex professo, sugestões de diretrizes foram propostas neste trabalho. Para o alcance desse resultado, utilizou-se da fenomenologia ou método de Husserl, pautado por evolutivo esclarecimento de ideias e de apreensão de essências.

Health is a fundamental right, presupposition of the dignity of the human person, and has a prominent role in the complex social reality that we integrate, to the point that the State (gender) assumes the constitutional obligation to guarantee it to all. In this context and to guarantee the maximum effectiveness of the right to health, the State has made possible for the private sector to participate in a complementary way in the public system in charge of ensuring health. The State has given preference for non-profit entities, as is the case of the socalled third sector. However, in line with what is constitutionally foreseen and for the good of its legal-sanitary effectiveness, the hypotheses of public-private partnerships need to observe certain parameters. Seeking to collaborate with the debate, after reflecting on it and without any pretension of an ex professo order, this article suggests some guidelines. To achieve this result, it makes use of Husserl's phenomenology or method, guided by an evolutionary clarification of ideas and apprehension of essences.

Evaluation of Placental Transfer and Tissue Distribution of cis- and Trans-Permethrin in Pregnant Rats and Fetuses Using a Physiological-Based Pharmacokinetic Model.

Biomonitoring studies have highlighted the exposure of pregnant women to pyrethroids based on the measurement of their metabolites in urine. Pyrethroids can cross the placental barrier and be distributed in the fetus as some pyrethroids were also measured in the meconium of newborns. Prenatal exposure to pyrethroids is suspected to alter the neurodevelopment of children, and animal studies have shown that early life exposure to permethrin, one of the most commonly used pyrethroid in household applications, can alter the brain development. This study aimed to characterize the fetal permethrin exposure throughout gestation in rats. We developed a pregnancy physiologically based pharmacokinetic (pPBPK) model that describes the maternal and fetal kinetics of the cis- and trans- isomers of permethrin during the whole gestation period. Pregnant Sprague-Dawley rats were exposed daily to permethrin (50 mg/kg) by oral route from the start of gestation to day 20. Permethrin isomers were quantified in the feces, kidney, mammary gland, fat, and placenta in dams and in both maternal and fetal blood, brain, and liver. Cis- and trans-permethrin were quantified in fetal blood and tissues, with higher concentrations for the cis-isomer. The pPBPK model was fitted to the toxicokinetic maternal and fetal data in a Bayesian framework. Several parameters were adjusted, such as hepatic clearances, partition coefficients, and intestinal absorption. Our work allowed to estimate the prenatal exposure to permethrin in rats, especially in the fetal brain, and to quantitatively estimate the placental transfer. These transfers could be extrapolated to humans and be incorporated in a human pPBPK model to estimate the fetal exposure to permethrin from biomonitoring data.

Reproducibility of the classification of proximal humeral ossification in idiopathic scoliosis / Reprodutibilidade da classificação da ossificação do úmero proximal na escoliose idiopática / Reproducibilidad de la clasificación de la osificación del húmero proximal en la escoliosis idiopática

ABSTRACT Objective To evaluate the intra- and interobserver reproducibility of the classification of proximal humeral ossification in patients with idiopathic scoliosis. Methods Observational, descriptive, cross-sectional study. Radiographs (anteroposterior) of 29 patients diagnosed with idiopathic scoliosis were chosen and investigated at the Spine Surgery Outpatient Clinic of the Department of Orthopedics and Traumatology of the Hospital Getúlio Vargas, Recife, Pernambuco. These radiographs were evaluated and classified by 9 spinal orthopedic surgeons at two moments, with a minimum interval of 30 days, for analysis of the intraobserver and interobserver reproducibility of the classification of proximal humeral ossification used as a predictor of peak growth velocity. Results Intraobserver reproducibility achieved moderate to substantial concordance with the kappa index. In the analysis of interobserver reproducibility using the Fleiss' kappa method, a reasonable to moderate concordance was observed, except for in stage 5, in which concordance ranged from substantial to almost perfect. Conclusion In this study, the reproducibility of the Li classification in most of the stages was reasonable to moderate for the interobserver assessment and moderate to substantial for the intraobserver assessment, which differs from the current literature. Level of evidence III; Diagnostic studies - Investigation of a diagnostic test.

RESUMO Objetivo Avaliar a reprodutibilidade intra e interobservadores da classificação de ossificação do úmero proximal em pacientes portadores de escoliose idiopática. Métodos Estudo observacional, descritivo e transversal. Foram selecionadas radiografias (anteroposteriores) de 29 pacientes com diagnóstico de escoliose idiopática acompanhados no Ambulatório de Cirurgia da Coluna do Departamento de Ortopedia do Hospital Getúlio Vargas, Recife, Pernambuco. Essas radiografias foram avaliadas e classificadas por nove cirurgiões ortopedistas de coluna em dois momentos, com intervalo mínimo de 30 dias, para análise da reprodutibilidade intraobservador e interobservador da classificação de ossificação do úmero proximal utilizada como preditor do pico de velocidade de crescimento. Resultados A reprodutibilidade intraobservador com o índice Kappa atingiu concordância moderada a substancial. Já na análise da reprodutibilidade interobservador com o método de kappa de Fleiss constatou-se concordância de razoável a moderada, exceto no estágio 5, no qual a concordância variou de substancial a quase perfeita. Conclusões Neste estudo, a reprodutibilidade da classificação de Li, na maioria dos estágios foi razoável a moderada na avaliação interobservador e moderada à substancial na avaliação intraobservador, o que diverge da literatura atual. Nível de evidência III; Estudos diagnósticos - Investigação de um exame para diagnóstico.

RESUMEN Objetivo Evaluar la reproducibilidad intra e interobservador de la clasificación de osificación del húmero proximal en pacientes con escoliosis idiopática. Métodos Estudio observacional, descriptivo y transversal. Se seleccionaron radiografías (anteroposteriores) de 29 pacientes con diagnóstico de escoliosis idiopática en la Clínica de Cuidados Ambulatorios de Cirugía de Columna del Departamento de Ortopedia del Hospital Getúlio Vargas, Recife, Pernambuco. Estas radiografías fueron evaluadas y clasificadas por nueve cirujanos ortopedistas de columna, en dos momentos, con un intervalo mínimo de 30 días, para analizar la reproducibilidad intraobservador e interobservador de la clasificación de osificación del húmero proximal utilizada como predictor de la velocidad máxima de crecimiento. Resultados La reproducibilidad intraobservador con el índice Kappa alcanzó concordancia de moderada a sustancial de la reproducibilidad intraobservador. En el análisis de la reproducibilidad interobservador con el método de kappa de Fleiss, se encontró concordancia de razonable a moderada, excepto en la etapa 5, en que la concordancia varió de sustancial a casi perfecta. Conclusiones En este estudio, la reproducibilidad de la clasificación de Li en la mayoría de las etapas fue de razonable a moderada en la evaluación interobservador y de moderada a sustancial en la evaluación intraobservador, lo que difiere de la literatura actual. Nivel de Evidencia III; Estudios de Diagnóstico - Investigación de una prueba de diagnóstico.

Physiological and Biochemical Traits of Two Major Arabidopsis Accessions, Col-0 and Ws, Under Salinity.

Salinity affects plant growth and development as shown with the glycophyte model plant, Arabidopsis thaliana (Arabidopsis). Two Arabidopsis accessions, Wassilewskija (Ws) and Columbia (Col-0), are widely used to generate mutants available from various Arabidopsis seed resources. However, these two ecotypes are known to be salt-sensitive with different degrees of tolerance. In our study, 3-week-old Col-0 and Ws plants were treated with and without 150 mM NaCl for 48, 72, or 96 h, and several physiological and biochemical traits were characterized on shoots to identify any specific traits in their tolerance to salinity. Before salt treatment was carried out, a different phenotype was observed between Col-0 and Ws, whose main inflorescence stem became elongated in contrast to Col-0, which only displayed rosette leaves. Our results showed that Col-0 and Ws were both affected by salt stress with limited growth associated with a reduction in nutrient uptake, a degradation of photosynthetic pigments, an increase in protein degradation, as well as showing changes in carbohydrate metabolism and cell wall composition. These traits were often more pronounced in Col-0 and occurred usually earlier than in Ws. Tandem Mass Tags quantitative proteomics data correlated well with the physiological and biochemical results. The Col-0 response to salt stress was specifically characterized by a greater accumulation of osmoprotectants such as anthocyanin, galactinol, and raffinose; a lower reactive oxygen detoxification capacity; and a transient reduction in galacturonic acid content. Pectin degradation was associated with an overaccumulation of the wall-associated kinase 1, WAK1, which plays a role in cell wall integrity (CWI) upon salt stress exposure. Under control conditions, Ws produced more antioxidant enzymes than Col-0. Fewer specific changes occurred in Ws in response to salt stress apart from a higher number of different fascilin-like arabinogalactan proteins and a greater abundance of expansin-like proteins, which could participate in CWI. Altogether, these data indicate that Col-0 and Ws trigger similar mechanisms to cope with salt stress, and specific changes are more likely related to the developmental stage than to their respective genetic background.

A Tandem Mass Tags (TMTs) labeling approach highlights differences between the shoot proteome of two Arabidopsis thaliana ecotypes, Col-0 and Ws.

Arabidopsis has become a powerful model to study morphogenesis, plant growth, development but also plant response to environmental conditions. Over 1000 Arabidopsis genomes are available and show natural genetic variations. Among them, the main reference accessions Wassilewskija (Ws) and Columbia (Col-0), originally growing at contrasted altitudes and temperatures, are widely studied, but data contributing to their molecular phenotyping are still scarce. A global quantitative proteomics approach using isobaric stable isotope labeling (Tandem Mass Tags, TMT) was performed on Ws and Col-0. Plants have been hydroponically grown at 16 h/8 h (light/dark cycle) at 23°C day/19°C night for three weeks. A TMT labeling of the proteins extracted from their shoots has been performed and showed a differential pattern of protein abundance between them. These results have allowed identifying several proteins families possibly involved in the differential responses observed for Ws and Col-0 during plant development and upon environmental changes. In particular, Ws and Col-0 mainly differ in photosynthesis, cell wall-related proteins, plant defense/stress, ROS scavenging enzymes/redox homeostasis and DNA/RNA binding/transcription/translation/protein folding.

Acute pathophysiological myocardial changes following intra-cardiac electrical shocks using a proteomic approach in a sheep model.

Implantable cardioverter-defibrillators (ICD) are meant to fight life-threatening ventricular arrhythmias and reduce overall mortality. Ironically, life-saving shocks themselves have been shown to be independently associated with an increased mortality. We sought to identify myocardial changes at the protein level immediately after ICD electrical shocks using a proteomic approach. ICD were surgically implanted in 10 individuals of a healthy male sheep model: a control group (N = 5) without any shock delivery and a shock group (N = 5) with the delivery of 5 consecutive shocks at 41 J. Myocardial tissue samples were collected at the right-ventricle apex near to the lead coil and at the right ventricle basal free wall region. Global quantitative proteomics experiments on myocardial tissue samples were performed using mass spectrometry techniques. Proteome was significantly modified after electrical shock and several mechanisms were associated: protein, DNA and membrane damages due to extreme physical conditions induced by ICD-shock but also due to regulated cell death; metabolic remodeling; oxidative stress; calcium dysregulation; inflammation and fibrosis. These proteome modifications were seen in myocardium both "near" and "far" from electrical shock region. N-term acetylated troponin C was an interesting tissular biomarker, significantly decreased after electrical shock in the "far" region (AUC: 0.93). Our data support an acute shock-induced myocardial tissue injury which might be involved in acute paradoxical deleterious effects such as heart failure and ventricular arrhythmias.

[D-Leu1]MC-LR and MC-LR: A Small-Large Difference: Significantly Different Effects on Phaseolus vulgaris L. (Fabaceae) Growth and Phototropic Response after Single Contact during Imbibition with Each of These Microcystin Variants.

[D-Leu1]MC-LR and MC-LR, two microcystins differing in one amino acid, constitute a sanitary and environmental problem owing to their frequent and concomitant presence in water bodies of the Americas and their association with human intoxication during recreational exposure to cyanobacterial bloom. Present in reservoirs used for irrigation as well, they can generate problems in the development of crops such as Phaseolus vulgaris, of nutritional and economic interest to the region. Although numerous works address the toxic effects of MC-LR, information on the toxicity of [D-Leu1]MC-LR is limited. Our objective was to study the toxic effects of [D-Leu1]MC-LR and MC-LR (3.5 µg/ml) on P. vulgaris after a single contact at the imbibition stage. Our findings indicate that 10 days post treatment, [D-Leu1]MC-LR generates morphological and physiological alterations more pronounced than those caused by MC-LR. In addition to the alterations produced by [D-Leu1]MC-LR in the development of seedlings and the structure of the leaves, roots and stems, we also found alterations in leaf stomatal density and conductivity, a longer delay in the phototropic response and a decrease in the maximum curvature angles achieved with respect to that observed for MC-LR. Our findings indicate that these alterations are linked to the greater inhibition of phosphatase activity generated by [D-Leu1]MC-LR, rather than to oxidative damage. We observed that 30 days after treatment with MC-LR, plants presented better development and recovery than those treated with [D-Leu1]MC-LR. Further studies are required on [D-Leu1]MC-LR and MC-LR toxicity and their underlying mechanisms of action.

Radiographical analysis of flexibility of idiopatic scoliosis in prono and supino / Análise radiográfica da flexibilidade da escoliose idiopática em prono e supino / Análisis radiográfico de la flexibilidad de la escoliosis idiopática en prono y supino

ABSTRACT Objective To determine if there is a statistically significant difference in the flexibility of the curves in the adolescent idiopathic scoliosis (AIS) by using lateral inclination radiographs in supine or prone decubitus. Methods We evaluated 19 patients with AIS, waiting for surgery. Radiographs of the patients were performed in orthostatic anteroposterior incidences and right and left lateral inclinations in prone and supine decubitus. The comparison between prone and supine decubitus was performed through the flexibility rates of the curves measured in each position. Results The mean flexibility rates measured in lateral inclination radiographs with the patient in the supine position were 54.4% ± 38.8% in the proximal thoracic curve, 45.8% ± 15.6% in the main thoracic curve, and 80.5% ± 20.7% in the thoracolumbar / lumbar curve. When the lateral inclination radiographs were performed with the patient in the prone position, we observed mean flexibility rates of 66.4% ± 34.3% in the proximal thoracic curve, 50.1% ± 12.8% in the main thoracic curve, and 80.6% ± 19.0% in the thoracolumbar / lumbar curve. Conclusion This present study did not find a statistically significant difference between the flexibility rates of the curves in the prone and supine positions, suggesting that the two radiographic methods analyzed are similar in the evaluation of the flexibility of the curves in adolescent idiopathic scoliosis. Level of evidence II; Development of diagnostic criteria in consecutive patients (with "gold" reference standard applied).

RESUMO Objetivo Determinar se há diferença estatisticamente significativa na flexibilidade das curvas na escoliose idiopática do adolescente (EIA), ao utilizar radiografias em inclinação lateral em decúbito supino ou prono. Métodos Foram avaliados 19 pacientes com EIA, em espera de cirurgia. Os pacientes realizaram radiografias em incidências anteroposterior em ortostase, inclinações laterais direita e esquerda em decúbito prono e supino. Realizou-se a comparação entre o decúbito prono e supino através das taxas de flexibilidade das curvas aferidas em cada posição. Resultados A taxa de flexibilidade média aferida, quando realizadas as inclinações laterais com o paciente em posição supino, foi de 54,4% ± 38,8% na curva torácica proximal, 45,8% ± 15,6% na curva torácica principal e 80,5% ± 20,7% na curva toracolombar/lombar. Quando realizadas as inclinações laterais com o paciente em posição prono, observou-se taxa de flexibilidade média de 66,4% ± 34,3% na curva torácica proximal, 50,1% ± 12,8% na curva torácica principal e 80,6% ± 19,0% na curva toracolombar/lombar. Conclusão O presente estudo não encontrou diferença com significância estatística nas taxas de flexibilidade das curvas nas posições prono e supino, sugerindo que os dois métodos radiográficos analisados são semelhantes na avaliação da flexibilidade das curvas na escoliose idiopática do adolescente. Nível de evidência II; Desenvolvimento de critérios diagnósticos em pacientes consecutivos (com padrão de referência "ouro" aplicado).

RESUMEN Objetivo Determinar si hay diferencia estadísticamente significativa en la flexibilidad de las curvas en la escoliosis idiopática del adolescente (EIA), al utilizar radiografías en inclinación lateral en decúbito supino o prono. Métodos Se evaluaron 19 pacientes con EIA, en espera de cirugía. Los pacientes realizaron radiografías en incidencias anteroposterior en ortostasis, inclinaciones laterales derecha e izquierda en decúbito prono y supino. Se realizó la comparación entre el decúbito prono y supino a través de las tasas de flexibilidad de las curvas medidas en cada posición. Resultados La tasa de flexibilidad promedio medida, cuando se realizaron las inclinaciones laterales con el paciente en posición supino, fue de 54,4% ± 38,8% en la curva torácica proximal, 45,8% ± 15,6% en la curva torácica principal, y 80,5% ± 20,7% en la curva toracolumbar/lumbar. Cuando se realizaron las inclinaciones laterales con el paciente en posición prono, se observó tasa de flexibilidad promedio de 66,4% ± 34,3% en la curva torácica proximal, 50,1% ± 12,8% en la curva torácica principal, y 80,6% ± 19,0% en la curva toracolumbar/lumbar. Conclusión El presente estudio no encontró diferencia con significancia estadística en las tasas de flexibilidad de las curvas en las posiciones prono y supino, sugiriendo que los dos métodos radiográficos analizados son semejantes en la evaluación de la flexibilidad de las curvas en la escoliosis idiopática del adolescente. Nivel de evidencia II; Desarrollo de criterios diagnósticos en pacientes consecutivos (con el estándar de referencia "oro" aplicado).

An untargeted liquid chromatography-mass spectrometry-based workflow for the structural characterization of plant polyesters.

Cell wall localized heterogeneous polyesters are widespread in land plants. The composition of these polyesters, such as cutin, suberin, or more plant-specific forms such as the flax seed coat lignan macromolecule, can be determined after total hydrolysis of the ester linkages. The main bottleneck in the structural characterization of these macromolecules, however, resides in the determination of the higher order monomer sequences. Partial hydrolysates of the polyesters release a complex mixture of fragments of different lengths, each present in low abundance and therefore are challenging to structurally characterize. Here, a method is presented by which liquid chromatography-mass spectrometry (LC-MS) profiles of such partial hydrolysates are searched for pairs of related fragments. LC-MS peaks that show a mass difference corresponding to the addition of one or more macromolecule monomers were connected in a network. Starting from the lowest molecular weight peaks in the network, the annotation of the connections as the addition of one or more polyester monomers allows the prediction of consecutive and increasingly complex adjacent peaks. Multi-stage MS (MSn) experiments further helped to reject, corroborate, and sometimes refine the structures predicted by the network. As a proof of concept, this procedure was applied to partial hydrolysates of the flax seed coat lignan macromolecule, and allowed to characterize 120 distinct oligo-esters, consisting of up to six monomers, and containing monomers and linkages for which incorporation in the lignan macromolecule had not been described before. These results showed the capacity of the approach to advance the structural elucidation of complex plant polyesters.

Determination of maternal and foetal distribution of cis- and trans-permethrin isomers and their metabolites in pregnant rats by liquid chromatography tandem mass spectrometry (LC-MS/MS).

We developed a method to quantify cis-permethrin and trans-permethrin and their metabolites in several biological matrices in pregnant rats and foetuses using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The objective was to quantify cis-permethrin and trans-permethrin in faeces, kidney, mammary gland, fat and placenta in mothers and in both maternal and foetal blood, brain and liver. The metabolites cis-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (cis-DCCA), trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (trans-DCCA) and 3-phenoxybenzoic acid (3-PBA) were measured in blood, liver and urine. Sample preparation was performed by liquid-liquid extraction. A purification step was not carried out except for the more complex biological samples (fat, mammary glands and faeces). Validation parameters including specificity, linearity, matrix effect, limits of quantification (LOQs), accuracy and precision were evaluated. The recoveries of target compounds ranged from 47 to 136%. LOQs were in the range 4 to 80 ng/mL for permethrin isomers and 4 to 800 ng/mL for their respective metabolites. Intra- and inter-batch precision and accuracy in matrix were better than 15%. The validated method was applied in a preliminary toxicokinetic study in pregnant rats with oral dosing of 50 mg/kg permethrin. In pregnant rats, permethrin isomers and their metabolites were quantified in all requested matrices except maternal liver and blood for trans-permethrin and cis-DCCA respectively. In foetuses, cis- and trans-permethrin were also quantified, demonstrating that the method is suitable for the analysis of foetal distribution of permethrin in toxicokinetic studies.

Cytological Approaches Combined With Chemical Analysis Reveals the Layered Nature of Flax Mucilage.

The external seed coat cell layer of certain species is specialized in the production and extrusion of a polysaccharide matrix called mucilage. Variations in the content of the released mucilage have been mainly associated with genetically regulated physiological modifications. Understanding the mucilage extrusion process in crop species is of importance to gain deeper insight into the complex cell wall biosynthesis and dynamics. In this study, we took advantage of the varying polysaccharide composition and the size of the flax mucilage secretory cells (MSCs) to study mucilage composition and extrusion in this species of agricultural interest. We demonstrate herein that flax MSCs are structured in four superimposed layers and that rhamnogalacturonans I (RG I) are firstly synthesized, in the upper face, preceding arabinoxylan and glucan synthesis in MSC lower layers. Our results also reveal that the flax mucilage release originates from inside MSC, between the upper and deeper layers, the latter collaborating to trigger polysaccharide expansion, radial cell wall breaking and mucilage extrusion in a peeling fashion. Here, we provide evidence that the layer organization and polysaccharide composition of the MSCs regulate the mucilage release efficiency like a peeling mechanism. Finally, we propose that flax MSCs may represent an excellent model for further investigations of mucilage biosynthesis and its release.

Metabolic markers for the yield of lipophilic indole alkaloids in dried woad leaves (Isatis tinctoria L.).

The pharmacologically active dichloromethane extracts of dried woad leaves (Isatis tinctoria L.), and the methanol extracts of comparable fresh leaves of the same plants, were analyzed by LC-MSn. The fresh leaf metabolite profile revealed a complex pattern of indolic compounds. Besides the known indigo precursors, isatan A, isatan B and indican, seven previously unreported indole derivatives were characterized: acetylindican, malonylindican, two dioxindole glucosides, dioxindole malonylglucoside, 6-hydroxyindole-3-carboxylic acid 6-O-glucoside and 6-hydroxyindole-3-carboxylic acid glucose ester. The integration of 122 compounds in fresh leaves and of five selected compounds (indoxyl, isatin, indigo, indirubin, and tryptanthrin) in dried leaves, formed the input data for a stepwise modelling procedure generating five predictive linear models. The structure of the predictive models and a cross validation provide evidence that the models could predict well or moderately well the accumulation of the selected lipophilic compounds, and were simple enough to be used in a woad cultivation program. PLS regression models relating each of the five selected dry leaf indolics to the fresh leaf metabolome were then fitted in order to deduct potential precursors and mechanisms leading to the formation of these lipophilic indolics in drying woad leaves. The models suggested glucobrassicin, isatan A and isatan B as the main candidate precursors of these compounds, besides a minor contribution of other fresh leaf indolics, including malonylindican, actylindican and dioxindole malonylglucoside. Dioxindole malonylglucoside was identified here as isatan C. The models further suggested that the accumulation of phenylpropanoid antioxidants in woad leaves has a negative impact on the formation of indoxyl, isatin, indigo, indirubin and tryptanthrin.

Urinary Metabolomics Profiles Associated to Bovine Meat Ingestion in Humans.

SCOPE: The impact of meat consumption on human health is widely examined in nutritional epidemiological studies, especially due to the connection between the consumption of red and processed meat and the risk of colon cancer. Food questionnaires do not assess the exposure to different methods of meat cooking. This study aimed to identify biomarkers of the acute ingestion of bovine meat cooked with two different processes. METHODS AND RESULTS: Non-targeted UPLC-MS metabolite profiling was done on urine samples obtained from 24 healthy volunteers before and 8 h after the ingestion of a single meal composed of intrinsically 15 N labelled bovine meat, either cooked at 55 °C for 5 min or at 90 °C for 30 min. A discriminant analysis extension of independent components analysis was applied to the mass spectral data. After meat ingestion, the urinary excretion of 1-methylhistidine, phenylacetylglutamine, and short- and medium-chained acylcarnitines was observed. 15 N labelling was detected in these metabolites, thus confirming their origin from ingested meat. However, no difference was observed in urinary metabolomic profiles according to the meat cooking process used. CONCLUSION: Meat ingestion led to the excretion of several nitrogen-containing compounds, but although a metabolic signature was detected for meat ingestion, the impact of the cooking process was not detectable at the level of urinary metabolic signature in our experimental conditions.

Importance of Stem Cell Transplantation in Cleft Lip and Palate Surgical Treatment Protocol.

Cleft lip and palate is a congenital malformation that requires a multidisciplinary treatment that evolves pediatrician, obstetrics, fetal medicine, genetics, plastic surgery, orthodontics, speech therapist, nursery, and psychology. Actually, the authors believe that it could be possible to ad protocols to use stem cells.The intrauterine diagnosis leads to preborn parental orientation and better parental collaboration to accept a precocious multidisciplinary treatment. After birth the authors' protocol is: orthodontic devices, phonoaudiology, and surgical procedures.The authors' cleft lip and palate reconstructive surgery protocol demands several steps and begins at 4 to 6-month old with rhinocheiloplasty and soft palate closure at the same moment. The treatment sequence involves the hard palate surgery (8-18 months after the first surgical step), alveoloplasty (after 10 years old), and secondary rhinoplasty (after 14 years old).New ideas to use stem cells and blood from the umbilical cord and also blood from placenta are discussed to improve final surgical results. Maternal stem cells are easy to collect, there are no damage to the patient and mother, it is autologous and it could be very useful in the authors' protocol.Nine patients with clef lip and palate were operated and had stem cells from umbilical cord blood and placenta blood injected into the bone and soft tissue during the primary procedure (rhinocheiloplasty).The stem cells activity into soft tissue and bone were evaluated. Preliminary results have shown no adverse results and improvement at the inflammatory response. A treatment protocol with stem cells was developed. It had a long time follow-up of 10 years.

Tutela da saúde pública: novas perspectivas e a construtiva atuação do Ministério Público / Public health care new perspectives and the constructive performance of the Ministério Público

A saúde é direito fundamental, garantidor da adequada formação, desenvolvimento e preservação da vida, pressuposto da dignidade da pessoa humana, com capacidade de influir para o qualificado desfrute dos demais direitos. Tanto assim que as ações e os serviços sanitários, na complexa realidade social e no campo constitucional, são reconhecidos como de relevância pública. Essas circunstâncias obrigam o Estado (gênero) a atuar diligentemente em prol da gestão, prestação e regulação desse direito, a fim de que possa ser assegurado de modo universal, equitativo e integral a todos. Ademais, justifica ser eficazmente protegido e operacionalizado mediante políticas públicas, a bem do interesse de cada indivíduo e da coletividade. Essa conjuntura, somada à necessidade de receber enfoque democrático, obriga que a tutela da saúde seja trabalhada através de postura transdisciplinar para a prevalência de justa resposta sanitária, em proximidade com a verdade e sem que o sistema jurídico e o microssistema jurídico-sanitário percam suas identidades no contato indispensável com outros sistemas. Tais aspectos reforçam a indispensabilidade de o Ministério Público funcionar como colaborador capaz de construtivamente proteger o direito à saúde. Todavia, a atuação da instituição em prol da saúde permanece aquém das obrigações constitucionais que lhe foram destinadas e abaixo das expectativas prevalentes no ambiente social. Por isso, não escapa de críticas e necessita adotar providências, no intuito de, concretamente, assegurar-lhe eficácia jurídica e social. Este trabalho objetiva contribuir para que a saúde pública e sua tutela através do Ministério Público sejam melhor compreendidas, organizadas, planejadas, estruturadas e executadas ainda no campo extrajudicial. Utiliza da fenomenologia, de fatores histórico-axiológicos, do rigor linguístico e do perspectivismo de José Ortega Y Gasset para alcançar esses propósitos. Propõe que o direito à saúde e as políticas correspondentes sejam tutelados pelo Ministério Público a partir: i) de seu atuar conjuntivo e cooperado; ii) da prática da humanização cidadã; iii) de postura baseada na ressignificação e reinicialização de seus atos; iv) do desempenho de funções setoriais e regionais; v) do reconhecimento e valorização de seus membros, quando intervirem de modo operoso; vi) da racionalização de seu agir; vii) da implantação e alimentação de banco de dados nacional e estaduais; viii) da definição de posições amparadas em ponderação criteriosa; ix) do funcionamento norteado pela resolutividade orientadora do sistema público de saúde e x) da busca de auxílio na biomimética, quando possível

Health is a fundamental right, guarantor of adequate formation, development and preservation of life, a prerequisite for the dignity of the human person, with the capacity to influence the qualified enjoyment of other rights. So much so that health actions and services, in the complex social reality and in the constitutional field, are recognized as of public relevance. These circumstances oblige the State (gender) to act diligently for the management, provision and regulation of this right, so that it can be ensured in a universal, equitable and integral manner to all. In addition, they justify being effectively protected and operationalized through public policies, in the interests of each individual and the community. This conjuncture, coupled with the need to receive a democratic approach, obliges health care to be worked through transdisciplinary posture to the prevalence of a just health response, in close proximity to the truth, and without the legal system and the legal-sanitary micro-system losing their identities in the indispensable contact with other systems. These aspects reinforce the indispensability of the Ministério Público (Public Prosecution Service) to function as a collaborator capable of constructively protecting it. However, the institution's action in favor of health remains below of the constitutional obligations and down of expectations prevalent in the social environment. Therefore, it does not escape criticism and needs to take measures, in order to concretely ensure legal and social effectiveness. This work aims to contribute to public health and its your protection through the Ministério Público are better understood, organized, planned, structured and executed still in the extrajudicial field. It uses phenomenology, historical-axiological factors, linguistic rigor and the perspectivism of José Ortega Y Gasset to achieve these purposes. Proposes that the right to health and the corresponding policies be protect by the Ministério Público from: i) its your conjunctive and cooperative activity; ii) the practice of citizen humanization; iii) of posture based on the re-signification and reinitialization of its acts; iv) the performance of sectoral and regional functions; v) recognition and appreciation of its members, when they intervene in an efficient way; vi) the rationalization of its action; vii) the implantation and feeding of national and state database; (viii) the definition of positions supported by careful balancing; ix) the activity guided by resolutiveness advisor of the public health system and x) the search for biomimetic assistance, when possible

Structural and dynamical characterization of the pH-dependence of the pectin methylesterase-pectin methylesterase inhibitor complex.

Pectin methylesterases (PMEs) catalyze the demethylesterification of pectin, one of the main polysaccharides in the plant cell wall, and are of critical importance in plant development. PME activity generates highly negatively charged pectin and mutates the physiochemical properties of the plant cell wall such that remodeling of the plant cell can occur. PMEs are therefore tightly regulated by proteinaceous inhibitors (PMEIs), some of which become active upon changes in cellular pH. Nevertheless, a detailed picture of how this pH-dependent inhibition of PME occurs at the molecular level is missing. Herein, using an interdisciplinary approach that included homology modeling, MD simulations, and biophysical and biochemical characterizations, we investigated the molecular basis of PME3 inhibition by PMEI7 in Arabidopsis thaliana Our complementary approach uncovered how changes in the protonation of amino acids at the complex interface shift the network of interacting residues between intermolecular and intramolecular. These shifts ultimately regulate the stability of the PME3-PMEI7 complex and the inhibition of the PME as a function of the pH. These findings suggest a general model of how pH-dependent proteinaceous inhibitors function. Moreover, they enhance our understanding of how PMEs may be regulated by pH and provide new insights into how this regulation may control the physical properties and structure of the plant cell wall.