A novel high-throughput screen for identifying lipids that stabilise membrane proteins in detergent based solution

Membrane proteins have a range of crucial biological functions and are the target of about 60% of all prescribed drugs. For most studies, they need to be extracted out of the lipid-bilayer, e.g. by detergent solubilisation, leading to the loss of native lipids, which may disturb important protein-lipid/bilayer interactions and thus functional and structural integrity. Relipidation of membrane proteins has proven extremely successful for studying challenging targets, but the identification of suitable lipids can be expensive and laborious. Therefore, we developed a screen to aid the high-throughput identification of beneficial lipids. The screen covers a large lipid space and was designed to be suitable for a range of stability assessment methods. Here, we demonstrate its use as a tool for identifying stabilising lipids for three membrane proteins: a bacterial pyrophosphatase (Tm-PPase), a fungal purine transporter (UapA) and a human GPCR (A(2A)R). A(2A)R is stabilised by cholesteryl hemisuccinate, a lipid well known to stabilise GPCRs, validating the approach. Additionally, our screen also identified a range of new lipids which stabilised our test proteins, providing a starting point for further investigation and demonstrating its value as a novel tool for membrane protein research. The pre-dispensed screen will be made commercially available to the scientific community in future and has a number of potential applications in the field.Peer reviewe

Strategies for successful isolation of a eukaryotic transporter

The isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643-672 (OsBOR3 ). While both express well as C-terminal GFP fusion proteins in Saccharomyces cerevisiae, the full length protein isolates poorly in the detergent dodecyl-β-d-maltoside (DDM). The OsBOR3 isolated in DDM in large quantities but was contaminated with GFP tagged protein, indicated incomplete protease removal of the tag. Addition of the reducing agent dithiothreitol (DTT) had no effect on isolation. Detergent screening indicated that the neopentyl glycol detergents, LMNG, UDMNG and DMNG conferred greater stability on the OsBOR3 than DDM. Isolation of OsBOR3 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3 in DMNG + DTT resulted in protein sample that does not contain any detectable GFP but elutes at a higher retention volume than that seen for protein isolated in either DDM or LMNG. Mass spectrometry confirmed that the LMNG and DMNG purified protein is OsBOR3 indicating that the DMNG isolated protein is monomer compared to the dimer isolated using LMNG. This was further supported by single particle electron microscopic analysis revealing that the DMNG protein particles are roughly half the size of the LMNG protein particles. [Abstract copyright: Copyright © 2019 Elsevier Inc. All rights reserved.

Dissecting the cytochrome P450 OleP substrate specificity: evidence for a preferential substrate

The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein–substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP–DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound

Efficacy of very low-calorie ketogenic diet with the Pronokal® method in obese women with polycystic ovary syndrome: a 16-week randomized controlled trial

Objective: The aim of this study isto assess the efficacy of a very low-calorie ketogenic diet (VLCKD) method vs a Mediterranean low-calorie diet (LCD) in obese polycystic ovary syndrome (PCOS) women of a reproductive age.Design: Randomized controlled open-label trial was performed in this study. The treatment period was 16 weeks; VLCKD for 8 weeks then LCD for 8 weeks, according to the Pronokal (R) method (experimental group; n = 15) vs Mediterranean LCD for 16 weeks (control group; n = 15). Ovulation monitoring was carried out at baseline and after 16 weeks, while a clinical exam, bioelectrical impedance analysis (BIA), anthropometry, and biochemical analyses were performed at baseline, at week 8, and at week 16.Results: BMI decreased significantly in both groups and to a major extent in the experimental group (-13.7% vs -5.1%, P = 0.0003). Significant differences between the experimental and the control groups were also observed in the reduction of waist circumference (-11.4% vs -2.9%), BIA-measured body fat (-24.0% vs -8.1%), and free testosterone (-30.4% vs -12.6%) after 16 weeks (P = 0.0008, P = 0.0176, and P = 0.0009, respectively). Homeostatic model assessment for insulin resistance significantly decreased only in the experimental group (P = 0.0238) but without significant differences with respect to the control group (-23% vs -13.2%, P > 0.05). At baseline, 38.5% of participants in the experimental group and 14.3% of participants in the control group had ovulation, which increased to 84.6% (P = 0.031) and 35.7% (P > 0.05) at the end of the study, respectively.Conclusion: In obese PCOS patients, 16 weeks of VLCKD protocol with the Pronokal (R) method was more effective than Mediterranean LCD in reducing total and visceral fat, and in ameliorating hyperandrogenism and ovulatory dysfunction.Significance statements: To the best of our knowledge, this is the first randomized controlled trial on the use of the VLCKD method in obese PCOS. It demonstrates the superiority of VLCKD with respect to Mediterranean LCD in reducing BMI with an almost selective reduction of fat mass and a unique effect of VLCKD in reducing visceral adiposity, insulin resistance, and in increasing SHBG with a consequent reduction of free testosterone. Interestingly, this study also demonstrates the superiority of the VLCKD protocol in improving ovulation, whose occurrence increased by 46.1% in the group treated by the VLCKD method against a rise of 21.4% in the group treated by Mediterranean LCD. This study extends the therapeutic approach possibilities in obese PCOS women

GROWTH AND DEVELOPMENT SURVEILLANCE: ANALYSIS OF RECORDS IN THE CHILD HEALTH HANDBOOK

La finalidad del estudio fue analizar el registro de datos acerca de acompañamiento de crecimiento y desarrollo de niños de menos de un año por la tarjeta de salud de niños. Estudio transversal con abordaje cuantitativo, cuyo material empírico fue obtenido en 116 tarjetas, entre diciembre de 2012 y marzo de 2013, en João Pessoa – Paraíba. Para análisis, fue utilzado el test chi cuadrado y el exato de Fisher. Ninguna tarjeta contenía registros acerca de la salud bucal y del número en el Sistema Único de Salud; 109 (94%) no presentaban datos del prenatal; y 115 (99,1%) no traían informaciones de las acciones de la Primera Semana Salud Integral. Hubo significativa asociación entre el número de consultas y edad de los niños y datos antropométricos, con mayor porcentaje de fallas en los registros de aquellos que tuvieron entre una y seis consultas. Se espera que los profesionales de salud hagan una reflexión acerca de su proceso de trabajo cuanto a la valoración de la Tarjeta de Salud del Niño.The objective in this study was to analyze the records of growth and development data for children under one year of age in the child health handbook. Cross-sectional study with quantitative approach. The empirical material was collected in 116 handbooks between December 2012 and March 2013 in João Pessoa – Paraíba, Brazil. The chi-square test and Fisher’s exact test were used for analysis. None of the handbooks contained oral health records and the number of the Unified Health System card, 109 (94%) did not present prenatal data and 115 (99.1%) did not contain information on the actions of the First Integral Health Week. A significant association was found between the number of consults and the children’s age and anthropometric data, with a higher percentage of missing records for children who took part in one to six consults. The health professionals are expected to reflect on their work process regarding the valuation of the Child Health Handbook.Objetivou analisar o registro de dados sobre vigilância do crescimento e desenvolvimento de crianças menores de um ano na caderneta de saúde da criança. Estudo transversal com abordagem quantitativa. O material empírico foi coletado em 116 cadernetas, entre dezembro de 2012 e março de 2013, em João Pessoa – Paraíba. Para análise, utilizou-se o teste qui-quadrado e o exato de Fisher. Nenhuma caderneta continha registros acerca da saúde bucal e do número do cartão do Sistema Único de Saúde, 109 (94%) não apresentavam dados do pré-natal e 115 (99,1%) não tinham informações referentes às ações da Primeira Semana Saúde Integral. Houve associação significativa entre o número de consultas e idade das crianças e dados antropométricos, com maior porcentagem de falhas nos registros daquelas que tiveram entre uma e seis consultas. Espera-se que os profissionais de saúde reflitam sobre seu processo de trabalho quanto à valorização da Caderneta de Saúde da Criança

PRÁCTICA EDUCATIVA DE ENFERMERAS EN LA ATENCIÓN PRIMARIA A LA SALUD, PARA EL DESARROLLO INFANTIL SALUDABLE

El objetivo de ese estudio fue identificar la concepción de educación en salud que nortea la práctica educativa de enfermeras que trabajan con Atención Primaria a la Salud, para la promoción del desarrollo infantil saludable. Los datos fueron obtenidos de septiembre de 2012 a enero de 2013, por medio de entrevista realizada con 10 enfermeras que actuan en la Estrategia Salud de la Familia en municipio de João Pessoa, Paraíba. El material empírico fue tratado con base en el análisis categorial temático. Se percibió que hay enfermeras que desconocen la esencia del proceso de educación en salud, manteniendo concepciones biologicistas, en cuanto otras afirman desarrollar práctica educativa solamente durante la consulta de puericultura, pero no contempla el estímulo del desarrollo neuropsicomotor. Delante de eso, surge la necesidad de despertar el pensar/actuar de esas enfermeras, para ampliar el concepto de educación en salud y implementarlas en la vigilancia del desarrollo infantil para cambiar sus prácticas.  O objetivo desse estudo foi identificar a concepção de educação em saúde que norteia a prática educativa de enfermeiras que atuam na Atenção Primária à Saúde, visando à promoção do desenvolvimento infantil saudável. Os dados foram coletados de setembro de 2012 a janeiro de 2013, por meio de entrevista realizada com 10 enfermeiras que atuam na Estratégia Saúde da Família no município de João Pessoa-Paraíba. O material empírico foi tratado a partir de análise categorial temática. Percebeu-se que há enfermeiras que desconhecem a essência do processo de educação em saúde, mantendo concepções biologicistas, enquanto outras referem desenvolver prática educativa somente durante a consulta de puericultura, porém, não contempla a estimulação do desenvolvimento neuropsicomotor. Diante disso, surge a necessidade de despertar o pensar/agir dessas enfermeiras, a fim de ampliar o conceito de educação em saúde e implementá-las na vigilância do desenvolvimento infantil para transformar suas práticas.The purpose of the present study is to identify the concept of health education that guides the educational practice of nurses who work in Primary Health Care, aimed at promoting children’s healthy development. The data were collected from September 2012 to January 2013, through interviews with 10 nurses who work in the Family Health Strategy, in the city of João Pessoa-Paraíba. Thematic analysis was the technique used in the treatment of the empirical material. Some nurses were unaware of the importance of the health education process, keeping biologicist views of this process, while others reported developing educational practices only during child care consultation, which however did not include stimulation of neuropsychomotor development. Therefore, it is necessary to awaken the thinking/acting of these nurses, in order to broaden the concept of health education for including child development monitoring in their practices

Estimulação de crianças com síndrome congênita pelo Zika vírus no domicílio: desafios do cuidador

RESUMOObjetivo: Identificar os desafios enfrentados por cuidadores quanto ao estímulo do desenvolvimento de crianças com SíndromeCongênita pelo Zika vírus no domicílio.Método: Estudo qualitativo, realizado com dez cuidadores de crianças com a síndrome e cinco profissionais de saúde que atuam numserviço especializado de referência à criança/adolescente com deficiência de um município pernambucano. A coleta ocorreu entrenovembro/2018 e março/2019 através de entrevista semiestruturada, e os dados interpretados pela análise temática.Resultados: Os desafios enfrentados pelos cuidadores são permeados pelo medo, insegurança e falta de apoio para a realização doestímulo no domicílio. Os profissionais reconhecem a situação, contudo, enfatizam a importância da continuidade do estímulo parapreservar as capacidades destas crianças.Conclusões: Torna-se necessário sensibilizar as equipes de saúde para apoiar as famílias nos desafios, e para o cuidado integral àcriança com a Síndrome do Zika, a fim de alcançar o máximo possível o seu desenvolvimento.Palavras-chave: Saúde da criança. Zika vírus. Deficiências do desenvolvimento. Relações profissional-família. Enfermagempediátrica

Mamíferos Marinhos

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