RESUMEN
We describe a non-chromatographic, ligand-free platform for the efficient purification of recombinant human lactoferrin (LF). The platform consists of a [metal:chelator] complex precipitate in the presence of osmotically active polyethylene glycol 6000 (PEG-6000). Purification is achieved in three stages. Following formation of the complex, LF is captured under neutral conditions by the aggregated complexes (Step I), a washing step follows (Step II) and then, (Step III) LF is extracted in pure form with 100 mM tribasic Na citrate buffer (pH 7). Of the four complexes investigated, [bathophenanthroline (batho)3:Fe2+] was determined to be the most efficient. LF is recovered with high yield (â¼90%, by densitometry) and purity (≥97%, by SDS polyacrylamide gel electrophoresis (SDS-PAGE)) from an artificial contamination background comprising E. coli lysate proteins. Purified LF is demonstrated to be monomeric by dynamic light scattering (DLS); to preserve its native secondary structure by circular dichroism (CD) spectroscopy; and, as apo-LF, to efficiently inhibit bacterial growth. Process yield is not affected by a 45-fold increase in LF concentration from 0.2 to 9 mg/mL. We provide evidence that protein capture relies on [cation:π] interactions between the lysine and arginine residues of LF with the fully aromatic [(batho)3:Fe2+] complexes. The use of [metal:chelator] complex aggregates is demonstrated to provide an economical and efficient avenue for LF purification.
Asunto(s)
Lactoferrina , Humanos , Lactoferrina/aislamiento & purificación , Lactoferrina/química , Fenantrolinas/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/química , Electroforesis en Gel de Poliacrilamida , Dicroismo Circular , Polietilenglicoles/química , Hierro/químicaRESUMEN
BACKGROUND: Nutrigenetics indicates that individual genetic variability results in altered health outcomes necessitating personalized nutrition adaptation. Registered dietitians are recognized as the clinical nutrition experts, but their knowledge and attitudes regarding nutrigenetics has not been delineated. METHODS: This cross sectional online survey was conducted in a convenience sample of 169 national nutrition conference attendees. The survey queried demographics, knowledge, and attitudes towards nutrigenetics and information on training in nutrigenetics. RESULTS: The majority of participants were registered dietitians and female, 45% of whom held advanced degrees. Personalized nutrition was perceived by 93.5% of participants as highly important or important; however, 94% of respondents indicated they are not sufficiently knowledgeable in personalized nutrition and only 9.5% had received training in nutrigenetics. The mean nutrigenetics knowledge score was 6.89 ± 1.67 (out of a possible 12). A multivariate regression model of knowledge score identified education as the only independent predictor of this outcome. CONCLUSION: Personalized nutrition is a rapidly developing field that incorporates genetic data into clinical practice. Dietitians recognize the importance of advanced studies to acquire knowledge in nutrigenetics. Only by acquiring the necessary knowledge can dietitians accurately translate this nutrigenetics into clinical practice.
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Conocimientos, Actitudes y Práctica en Salud , Nutrigenómica , Nutricionistas , Adulto , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas y CuestionariosRESUMEN
BACKGROUND: Dietitians and other health care professionals must be able to translate findings from clinical trials into best treatment practices, a skill termed "knowledge translation". This skill requires knowledge of treatment guidelines as well as the science underpinning treatment recommendations. Unsatisfactory knowledge translation of medical nutrition therapy (MNT) has been documented. METHODS: Individuals registered to attend a leading national nutrition conference were asked to participate in an online cross-sectional survey. Participants were asked to provide demographic and professional information, answer questions on nutrition knowledge and to choose a clinical action plan in response to dietitian-designed case vignettes describing research outcomes. Responses were compared by profession and participation in research activities. RESULTS: Of 3000 registered conference attendees, 299 individuals replied: 79.0% dietitians, 93.3% female, with a mean household income matching the 5th decile of income, 60.7% indicated a single employment setting, 20.7% reported participating in research. Almost 74% of respondents indicated that they would make clinical recommendations based on findings of an in vitro study. In one vignette, a patient with a disease previously not encountered by the respondent required a clinical treatment plan. Only 53% of participants chose to seek formal nutrition guidelines. Fewer than 15% of participants could identify the pathway for fat during weight loss. Differences in knowledge translation skills by research participation were not detected. CONCLUSIONS: Our findings reveal a deficit in knowledge translation proficiency in a convenience sample of dietitians and other health professionals, highlighting the need to develop these skills.
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Personal de Salud , Fenómenos Fisiológicos de la Nutrición , Investigación Biomédica Traslacional , Adulto , Estudios Transversales , Femenino , Humanos , Masculino , Evaluación Nutricional , Encuestas y CuestionariosRESUMEN
Irisin is a novel secreted myokine, encoded by the fibronectin type III domain-containing protein 5 (FNDC5) precursor gene. Irisin plays a role in the female reproductive system in pregnancy and in embryonic development, and is associated with fetal size. It is expressed in the ovary, placenta and neonatal cord serum. We studied whether maternal and neonatal FNDC5 genetic polymorphisms are associated with preterm birth (PTB). Blood for DNA analysis was collected from Israeli mothers (nâ¯=â¯315) and from umbilical veins of their respected idiopathic preterm (24-36â¯weeks) and control term (>37â¯weeks) newborns (nâ¯=â¯161). Genotypes of maternal and neonatal FNDC5 polymorphisms (rs726344 and rs1746661) were determined by restriction fragment length polymorphism analysis. Genotype-phenotype associations were analyzed using SPSS program. The Frequency of FNCD5 rs726344 G alleles in the Israeli cohort is 82%. We found significant FNCD5 rs726344 genotype frequencies control and PTB groups. Women bearing the FNDC5 rs726344 GG genotype had 2.18 fold ([CI] 1.193-4.008, pâ¯=â¯0.01) higher chance to deliver at term compared to both AG and AA genotypes (adjusting to age, gravidity, parity, weight percentile per gestational age and gender of newborn). Neonates carrying the FNDC5 rs726344 GG genotype had 2.24 fold ([CI] 0.979-5.134, pâ¯=â¯0.05) higher chance to be born at term compared to either AG or AA genotypes (adjusting to parity, previous abortions and weight percentile per gestational age). There was no significant association of the rs1746661 polymorphism with PTB. Thus, we determined FNDC5 polymorphisms frequencies in the Israeli population and demonstrated that maternal and neonatal FNDC5 rs726344 polymorphism is significantly associated with increased risk for PTB.
Asunto(s)
Fibronectinas/genética , Nacimiento Prematuro/genética , Adulto , Alelos , Estudios de Cohortes , Femenino , Fibronectinas/sangre , Fibronectinas/metabolismo , Frecuencia de los Genes/genética , Estudios de Asociación Genética/métodos , Predisposición Genética a la Enfermedad/genética , Humanos , Recién Nacido , Israel , Masculino , Neuropéptidos/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
INTRODUCTION: Pancreatic pathologies are characterized by a progressive fibrosis process. Pancreatic stellate cells (PSC) play a crucial role in pancreatic fibrogenesis. Endoplasmic reticulum (ER) stress emerges as an important determinant of fibrotic remodeling. Overload of fatty acids (FA), typical to obesity, may lead to lipotoxic state and cellular stress. AIM: To study the effect of different lipolytic challenges on pancreatic ER stress and PSC activation. METHODS: Primary PSCs were exposed to different FAs, palmitate (pal) and oleate (ole), at pathophysiological concentrations typical to obese state, and in acute caerulein-induced stress (cer). PSC activation and differentiation were analyzed by measuring fat accumulation (oil-red staining and quantitation), proliferation (cells count) and migration (wound- healing assay). PSC differentiation markers (α-sma, fibronectin, tgf-ß and collagen secretion), ER stress unfolded protein response and immune indicators (Xbp1, CHOP, TNF-α, IL-6) were analyzed at the transcript and protein expression levels (quantitative RT-PCR and western blotting). RESULTS: PSC exposure to pal and ole FAs (500µM) increased significantly fat accumulation. Proliferation and migration analysis demonstrated that ole FA retained PSC activation, while exposure to pal FA significantly halted proliferation rate and delayed migration. Cer significantly augmented PSC differentiation markers α- sma, fibronectin and collagen, and ER stress and inflammation markers including Xbp1, CHOP, TNF-α and IL-6. The ole FA treatment significantly elevated PSC differentiation markers α-sma, fibronectin and collagen secretion. PSC ER stress was demonstrated following pal treatment with significant elevation of Xbp1 splicing and CHOP levels. CONCLUSION: Exposure to pal FA halted PSC activation and differentiation and elevated ER stress markers, while cer and ole exposure significantly induced activation, differentiation and fibrosis. Thus, dietary FA composition should be considered and optimized to regulate PSC activation and differentiation in pancreatic pathologies.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Ácidos Grasos/farmacología , Células Estrelladas Pancreáticas/metabolismo , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Técnica del Anticuerpo Fluorescente , Masculino , Células Estrelladas Pancreáticas/citología , Células Estrelladas Pancreáticas/efectos de los fármacos , Empalme del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
PURPOSE: Preterm birth (PTB) is a complex trait with strong genetic background, whose etiology is not fully understood. It was recently suggested that pregnancy duration is affected by fetal genetic variation even more than by the maternal genome. Vitamin D receptor (VDR) is involved in embryonic implantation and fertility. We studied the association between both maternal and neonatal vitamin D receptor (VDR) genetic variation and PTB. METHODS: Maternal and fetal (umbilical cord) DNA was isolated from Jewish Israeli idiopathic preterm newborns (24-36 weeks, n = 146) and control term newborns (>37 weeks, n = 229). Maternal and fetal VDR polymorphisms (FokI, ApaI, BsmI, TaqI) were analyzed by restriction fragment length polymorphism analysis. Using SPSS analysis to correlate VDR genotypes with phenotypic variation: pregnancy duration, preterm birth and spontaneous miscarriages, adjusted for gravidity, parity and gender of newborn. RESULTS: Women homozygous to VDR ApaI (AA) genotype had significant twofold increase risk for PTB [OR 1.973, (CI) 1.183-3.289, p = 0.009] compared to heterozygous women. Male newborns had significant (p < 0.05) 1.73-fold increase of PTB. Women with history of previous (≥1) spontaneous miscarriage had a significant increased risk for PTB if their newborn carried either of the VDR BsmI homozygous (BB or bb) genotypes compared to the heterozygous (Bb) genotype [OR 6.857, (CI) 1.273-36.934, p = 0.018 and OR 9.231, (CI) 1.753-48.618, p = 0.008, respectively], or VDR ApaI homozygous (AA or aa) genotype compared to heterozygous (Aa) genotype [OR 4.33, (CI) 1.029-18.257, p = 0.046 and OR 7.2, (CI) 1.34-38.917, p = 0.021, respectively]. CONCLUSIONS: We show association between maternal and fetal VDR genotype variants with PTB.
Asunto(s)
Predisposición Genética a la Enfermedad , Polimorfismo de Longitud del Fragmento de Restricción/genética , Nacimiento Prematuro/genética , Receptores de Calcitriol/genética , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Recién Nacido , Israel , Judíos , Masculino , Polimorfismo Genético , Embarazo , Riesgo , Nacimiento a Término/sangre , Nacimiento a Término/genéticaRESUMEN
AIM: Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3) is an essential enzyme in hydrolysis of dietary fat. Dietary fat, especially polyunsaturated fatty acids (PUFA), regulate pancreatic lipase (PNLIP); however, the molecular mechanism underlying this regulation is mostly unknown. As PUFA are known to regulate expression of proliferator-activated receptor gamma (PPARγ), and as we identified in-silico putative PPARγ binding sites within the putative PNLIP promoter sequence, we hypothesized that PUFA regulation of PNLIP might be mediated by PPARγ. MATERIALS AND METHODS: We used in silico bioinformatics tools, reporter luciferase assay, PPARγ agonists and antagonists, PPARγ overexpression in exocrine pancreas AR42J and primary cells to study PPARγ regulation of PNLIP. RESULTS: Using in silico bioinformatics tools we mapped PPARγ binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARγ dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARγ agonists and reduced by PPARγ antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARγ significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARγ agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARγ antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. CONCLUSION: PPARγ transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP.
Asunto(s)
Lipasa/genética , Lipasa/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Páncreas Exocrino/metabolismo , Páncreas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Biología Computacional/métodos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Páncreas/efectos de los fármacos , Páncreas Exocrino/efectos de los fármacos , Pioglitazona , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , Ratas , Alineación de Secuencia , Tiazolidinedionas/farmacología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
UNLABELLED: Leptin (LEP) and leptin receptor (LEPR) are suggested to play a role in female reproduction and especially in pregnancy Both LEP and LEPR are synthesized by the pregnant female and embryo. The link between genetic polymorphisms of LEP and LEPR and preterm birth (PTB) is unknown. We studied maternal and neonatal LEP and LEPR genetic polymorphisms and the association with PTB. Blood for DNA analysis was collected from Israeli mothers and from venous umbilical of their respected idiopathic preterm newborns (24-36weeks, n=102) and control term newborns (>37weeks, n=158). Genotypes of maternal and neonatal LEP (rs7799039) and LEPR (rs1137101) polymorphisms were analyzed by restriction fragment length polymorphism analysis. Genotype-phenotype association was assayed using SPSS program. We found a significant independent increased risk of PTB for women and neonates bearing the homozygous AA form of LEP genotype; where women carrying AA LEP genotype had 2.53 fold ([CI] 1.367-4.685, p=0.03) and 2.38 fold ([CI] 1.150-4.915, p=0.019) increased risk for PTB compared to AG and GG genotypes, respectively. Neonates carrying the LEP AA genotype had a significant 2.8 fold increased risk for PTB compared to the AG genotype (CI11.040-7.577, p=0.042). Maternal LEPR polymorphism was significantly associated with severe PTB; where women carrying the AA and AG genotypes had a significant 4.32 and 4.76 fold increased risk for severe PTB compared to women carrying the GG genotype (CI=1.090-17.112 and 1.332-17.027, respectively p=0.035). IN CONCLUSION: maternal and neonatal LEP and LEPR polymorphisms are significantly associated with increased risk for PTB.
Asunto(s)
Leptina/genética , Receptores de Leptina/genética , Adulto , Estudios de Cohortes , Femenino , Frecuencia de los Genes/genética , Humanos , Recién Nacido , Polimorfismo de Nucleótido Simple/genética , Embarazo , Nacimiento Prematuro/genéticaRESUMEN
Members of the NIMA-related kinases (NRK) family are recently emerging as central regulators of various aspects of the cell cycle. However, the cellular roles of the mammalian NRK, Nek7, remain obscure. We show here that the endogenous Nek7 protein is enriched at the centrosome in a microtubule-independent manner. Overexpression of wt or kinase-defective Nek7 resulted in cells of rounder appearance, and higher proportions of multinuclear and apoptotic cells. Down-regulation of Nek7 using a small interfering RNA approach resulted in a significant increase in mitotic cells presenting multipolar spindle phenotype. These results suggest a role for Nek7 in regulating proper spindle assembly and mitotic progression.