RET 634 germline/gonadal mosaicism generating a second pathogenic amino acid change in multiple endocrine neoplasia type 2A.

Genetic testing for germline RET pathogenic variants, which cause the Multiple Endocrine Neoplasia Type 2 (MEN2) syndrome, has become crucial in managing patients with medullary thyroid carcinoma (MTC). Classically, RET heterozygous missense pathogenic variants are transmitted in a Mendelian autosomal dominant pattern, of which germline/gonadal mosaicism has never been reported. We report the novel occurrence of a MEN2A patient's family in which the siblings inherited three different RET 634 genotypes: wild type (p.Cys634), p.Cys634Gly or p.Cys634Arg heterozygous pathogenic variants. We hypothesized that germline/gonadal mosaicism, derived from an inherited + early somatic mutation in the mother or a double de novo mutation during maternal embryogenesis, led to this rare event in the RET gene. Exome analysis of the proband's deceased mother's paraffin-embedded thyroid tissue confirmed the three nucleotides in the same 634 codon position. For the first time, we describe germline/gonadal mosaicism in RET, generating a second pathogenic amino acid change in the same codon causing MEN2A. Our finding shows that RET parental mosaicism, confirmed by somatic exome sequencing, might explain discrepant genotype cases in siblings with inherited cancers.

Cosegregation of a novel mutation in the sixth transmembrane segment of the luteinizing/choriogonadotropin hormone receptor with two Brazilian siblings with severe testotoxicosis.

PURPOSE: Testotoxicosis is an autosomal dominant form of gonadotropin-independent precocious puberty caused by heterozygous constitutively activating mutations of the luteinizing hormone/choriogonadotropin receptor (LHCGR) gene. The aim of this study was to describe two Brazilian siblings with testotoxicosis, to confirm the molecular diagnosis, and to perform an in silico analysis of a novel mutation in the hot spot of the LHCGR gene. MATERIALS AND METHODS: Molecular analysis of the mutation on the LHCGR gene was performed by direct Sanger sequencing, followed by an in silico analysis using HOPE bioinformatics tool to predict a functional defect of the mutant. RESULTS: Both patients presented with gonadotropin-independent precocious puberty before the age of four years. Genetic analysis revealed a novel non-maternally inherited p.Asp578Val mutation of the LHCGR gene. An in silico analysis showed that the p.Asp578Val mutation disturbed amino acid physicochemical features regarding its size, charge, and hydrophobicity value. CONCLUSIONS: Clinical and hormonal profile of the siblings here evaluated was not different while compared to those patients previously described. An in silico mutation analysis reinforced the causative role of recurrent activating mutations in the intracellular loop and transmembrane helices of the LHCGR. The segregation of this mutation with the offsprings' phenotype indicated that it is causative.

Whole genome and exome sequencing realignment supports the assignment of KCNJ12, KCNJ17, and KCNJ18 paralogous genes in thyrotoxic periodic paralysis locus: functional characterization of two polymorphic Kir2.6 isoforms.

Next-generation sequencing (NGS) has enriched the understanding of the human genome. However, homologous or repetitive sequences shared among genes frequently produce dubious alignments and can puzzle NGS mutation analysis, especially for paralogous potassium channels. Potassium inward rectifier (Kir) channels are important to establish the resting membrane potential and regulating the muscle excitability. Mutations in Kir channels cause disorders affecting the heart and skeletal muscle, such as arrhythmia and periodic paralysis. Recently, a susceptibility muscle channelopathy-thyrotoxic periodic paralysis (TPP)-has been related to Kir2.6 channel (KCNJ18 gene). Due to their high nucleotide sequence homology, variants found in the potassium channels Kir2.6 and Kir2.5 have been mistakenly attributable to Kir2.2 polymorphisms or mutations. We aimed at elucidating nucleotide misalignments by performing realignment of whole exome sequencing (WES) and whole genome sequencing (WGS) reads to specific Kir2.2, Kir2.5, and Kir2.6 cDNA sequences using BWA-MEM/GATK pipeline. WES/WGS reads correctly aligned 26.9/43.2, 37.6/31.0, and 35.4/25.8 % to Kir2.2, Kir2.5, and Kir2.6, respectively. Realignment was able to reduce over 94 % of misalignments. No putative mutations of Kir2.6 were identified for the three TPP patients included in the cohort of 36 healthy controls using either WES or WGS. We also distinguished sequences for a single Kir2.2, a single Kir2.5 sequence, and two Kir2.6 isoforms, which haplotypes were named RRAI and QHEV, based on changes at 39, 40, 56, and 249 residues. Electrophysiology records on both Kir2.6_RRAI and _QHEV showed typical rectifying currents. In our study, the reduction of misalignments allowed the elucidation of paralogous gene sequences and two distinct Kir2.6 haplotypes, and pointed the need for checking the frequency of these polymorphisms in other populations with different genetic background.

Household food insecurity: a systematic review of the measuring instruments used in epidemiological studies.

OBJECTIVE: To conduct a systematic review aimed at identifying and characterizing the experience-based household food security scales and to synthesize their psychometric properties. DESIGN: Search in the MEDLINE, LILACS and SciELO databases, using the descriptors ('food insecurity' OR 'food security') AND ('questionnaires' OR 'scales' OR 'validity' OR 'reliability'). There was no limitation on the period of publication. All articles had their titles and abstracts analysed by two reviewers. The studies of interest were read in their entirety and the relevant information extracted using a standard form. RESULTS: The initial bibliographic search identified 299 articles. Of these, the 159 that seemed to meet the criteria for inclusion were read fully. After consultation of the bibliographic references of these articles, twenty articles and five documents were added, as they satisfied the previously determined criteria for inclusion. Twenty-four different instruments were identified; all were brief and of easy application. The majority were devised in the USA. Forty-seven references reported results of psychometric studies. The instruments that presented the highest number of psychometric studies were the Core Food Security Measurement/Household Food Security Survey Module (CFSM/HFSSM) and the Self-Perceived Household Food Security Scale. CONCLUSIONS: There are a number of structured scales available in the literature for characterization of household food insecurity. However, despite some psychometric studies already existing about the majority of the instruments, it is observed that, except for the studies of the CFSM/HFSSM, these are still restricted to appraisal of a few aspects of reliability and validity.

Sustainable extraction of proteins from lime peels using ultrasound, deep eutectic solvents, and pressurized liquids, as a source of bioactive peptides.

The aim of this work was to develop, for the first time, sustainable strategies, based on the use of Ultrasound-Assisted Extraction, Natural Deep Eutectic Solvents, and Pressurized Liquid Extraction, to extract proteins from lime (Citrus x latifolia) peels and to evaluate their potential to release bioactive peptides. PLE showed the largest extraction of proteins (66-69%), which were hydrolysed using three different enzymes (Alcalase 2.4 L FG, Alcalase®PURE 2.4 L, and Thermolysin). The in vitro antioxidant and antihypertensive activities of released peptides were evaluated. Although all hydrolysates showed antioxidant and antihypertensive activity, the hydrolysate obtained with Thermolysin showed the most significant values. Since the Total Phenolic Content in all hydrolysates was low, peptides were likely the main contributors to these bioactivities. Hydrolysates were analyzed by UHPLC-QTOF-MS and a total of 98 different peptides were identified. Most of these peptides were rich in amino acids associated with antioxidant activity.

COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors.

BACKGROUND: The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored. RESULTS: By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells. CONCLUSIONS: Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

Successive extraction using natural deep eutectic solvents and pressurized liquids for a greener and holistic recovery of proteins from pomegranate seeds.

This work describes a novel and sustainable strategy for the recovery of proteins by successive extractions using natural deep eutectic solvents (NADES) and pressurized liquid extraction (PLE). This strategy was applied to the valorisation of pomegranate seeds. Nine different NADES were screened and that constituted by choline chloride and acetic acid was chosen due to its best performance. A Response Surface Methodology was employed to optimize other conditions in this extraction step: time, temperature, amount of sample, and HIFU amplitude. Protein recovery, under optimal conditions, was 13.3 g of proteins/100 g of milled and dried defatted seeds. Proteins were next characterized by their separation using RP-HPLC, SDS-PAGE, isoelectrofocusing electrophoresis, and by the evaluation of their digestibility and antioxidant properties. Comparison of these results with those from extracts obtained by other techniques supported the interest of combining the extraction using acid NADES with PLE, under alkaline conditions. The successive extraction by both methodologies enabled to double the total recovery of proteins. The analysis of samples by UHPLC-MS/MS, after a simulated gastrointestinal digestion, and de novo identification revealed the presence of 19 peptides in the NADES hydrolysate, while the successive extraction by PLE enabled to observe 15 additional peptides. Additional 83 peptides were found by database searching against Punica granatum and by homology with other organisms. Differences between peptides and the proteins in both hydrolysates confirmed the different protein selectivity of both strategies and the potential of NADES for extracting larger proteins and PLE for the extraction of smaller ones. Some phenolic compounds, amino acids, and fatty acids were also co-extracted with proteins in both extractions.

Modelling the simultaneous chiral separation of a group of drugs by electrokinetic chromatography using mixtures of cyclodextrins.

Two mixtures of neutral cyclodextrins (CDs) were used in Electrokinetic Chromatography (EKC) to model and optimize the simultaneous enantiomeric separation of a group of seven drugs. Heptakis(2,6-di-O-methyl)-ß-CD (DM-ß-CD) combined with methyl-γ-CD (M-γ-CD) or with carboxyethyl-γ-CD (CE-γ-CD) was employed in a 25 mM formate buffer at pH 3.0 to have the drugs studied positively charged. Dubsky's model was applied to calculate the enantiomer effective electrophoretic mobilities for each combination of CDs at different averaged molar fractions and total CDs concentrations. The most adequate averaged molar fraction and total CDs concentration in terms of the simultaneous enantiomeric separation of the drug mixture were predicted by the model and results were experimentally corroborated. The model also foresaw interesting effects, derived from the combination of DM-ß-CD with M-γ-CD or with CE-γ-CD, on the individual chiral separation of some of the drugs studied. The observed reversal of the migration order for some compounds when changing the total CDs concentration was also predicted and the model showed its potential even at concentrations out of the experimental range of CD concentrations experimentally employed. The use of an averaged molar fraction of 0.8 for DM-ß-CD at a total CDs concentration of 40 mM in the DM-ß-CD/CE-γ-CD system predicted by the model enabled the simultaneous enantiomeric separation of six of the drugs studied (except verapamil) with resolutions ranging from 0.6 to 4.0.

Impact of the use of pressurized liquids on the extraction and functionality of proteins and bioactives from brewer's spent grain.

A green methodology based on pressurized liquids (PLE) to extract proteins and obtain highly active extracts from brewer's spent grain (BSG) is proposed. Box-Behnken experimental design was employed to study the effect of extraction parameters on the protein content (PC), the total phenolic content (TPC), and the antioxidant activity of extracts. Results were compared with those obtained by conventional alkaline extraction assisted with ultrasounds (UAE). The selection of PLE conditions enabled to tailor the PC and TPC of extracts. PLE extracted 36 % more proteins than UAE. PLE extracts showed higher antioxidant, cholesterol esterase inhibition, and ACE inhibitory activities than UAE extract. HPLC-MS/MS enabled to observe that the extraction technique and experimental conditions significantly affected to the kind and amount of extracted proteins, and released peptides, and phenolic compounds. A higher ratio of hydrophobic peptides was observed in PLE extracts, which justified their higher bioactivity.

Ecotoxicity evaluation of tetramethrin and analysis in agrochemical formulations using chiral electrokinetic chromatography.

The separation of the four isomers of tetramethrin was performed for the first time using a cyclodextrin-micellar electrokinetic chromatography methodology. Using sodium deoxycholate and 2-hydroxypropyl-ß-CD as chiral selectors, tetramethrin isomers were separated with resolution values of 1.7 and 1.1 for trans- and cis-isomers, respectively, in analysis times lower than 12.5 min. Once developed and optimized, the analytical method was applied to the analysis of an antiparasitic commercial formulation and to the evaluation of the stability and ecotoxicity of tetramethrin. Using measured concentrations, the stability was assessed at enantiomeric level and the ecotoxicological parameters on Daphnia magna were determined. Tetramethrin presents toxicity on aquatic microinvertebrates, with EC50 (t = 72 h) of 1.8 mg/L. The acute toxicity of tetrametrin was attributed to the trans-1 enantiomer. The first evidence of oxidative stress-mediated mode of action for tetramethrin on Daphnia magna is reported in the present work.

[Psychomotor development in late preterms at two years of age: a comparison with full-term newborn infants using two different instruments]. / Desarrollo psicomotor en prematuros tardios a los dos años de edad: comparacion con recien nacidos a termino mediante dos herramientas diferentes.

INTRODUCTION: Late preterm infants currently constitute 70% of preterm infant births. They present greater comorbidity, including neurodevelopment disorders, which may not manifest until the school age. AIM: To identify the existence of difficulties in the neurodevelopment at the age of two years. SUBJECTS AND METHODS: The psychomotor development was performed at two years of age in late preterm infants and term control group born at our center between January and September 2014, with Brunet-Lezine Revised test and Ages and Stages Questionnaires (ASQ-3) questionnaire. RESULTS: 88 children were included. Late preterm infants had lower scores in the language area and postural developmental. Girls achieved better results than males at global developmental age, oculo-motor coordination, language area and sociability. The ASQ-3 questionnaire detected differences in communication and socio-individual. Prematurity and male sex were identified as an independent risk factor to present a developmental disorder, prematurity for language impairment and male sex for younger developmental age and language impairment. The correlation between language assessment with the Brunet-Lezine Revised test and the ASQ-3 questionnaire was good, with a Pearson correlation coefficient of 0.7 (p < 0.001), showing the usefulness of the questionnaire. CONCLUSIONS: Late preterm infants have a lower developmental age in the language area at two years. Prematurity and male sex are risk factors for developmental disorder. Language assessment with the ASQ-3 questionnaire may be a useful tool to detect disorders and intervene early.

TITLE: Desarrollo psicomotor en prematuros tardios a los dos años de edad: comparacion con recien nacidos a termino mediante dos herramientas diferentes.Introduccion. Los prematuros tardios constituyen actualmente el 70% de los nacimientos prematuros. Presentan mayor comorbilidad, incluyendo las alteraciones del neurodesarrollo, que pueden no manifestarse hasta la escolarizacion. Objetivo. Identificar dificultades en el desarrollo neurologico a los dos años de edad. Sujetos y metodos. Se valoro el desarrollo psicomotor a los dos años de los prematuros tardios y del grupo control a termino nacidos en nuestro centro entre enero y septiembre del año 2014 mediante la escala de Brunet-Lezine revisada y el cuestionario de edades y etapas para la deteccion de trastornos del neurodesarrollo Ages and Stages Questionnaires (ASQ-3). Resultados. Se incluyo a 88 niños. Los prematuros tardios presentaron puntuaciones inferiores en el lenguaje y el desarrollo postural. Las niñas obtuvieron resultados superiores en la edad de desarrollo global, la coordinacion oculomotriz, el lenguaje y la sociabilidad. El cuestionario ASQ-3 detecto las diferencias en comunicacion y socioindividuales. Se identificaron como factores de riesgo para presentar alteracion del desarrollo la prematuridad, para alteracion del lenguaje, y el sexo masculino, para menor edad de desarrollo y alteracion del lenguaje. La correlacion entre la valoracion del lenguaje con la escala de Brunet-Lezine revisada y el cuestionario ASQ-3 fue buena, con un coeficiente de correlacion de Pearson de 0,7 (p < 0,001), lo que muestra la utilidad del cuestionario. Conclusiones. Los prematuros tardios presentan menor desarrollo del lenguaje a los dos años. La prematuridad y el sexo masculino son factores de riesgo para presentar alteracion. La valoracion del lenguaje con el cuestionario ASQ-3 puede ser util para detectar alteraciones.

Estimation of the percentage of transgenic Bt maize in maize flour mixtures using perfusion and monolithic reversed-phase high-performance liquid chromatography and chemometric tools.

The estimation of the percentage of transgenic Bt maize in maize flour mixtures has been achieved in this work by high-performance liquid chromatography using perfusion and monolithic columns and chemometric analysis. Principal component analysis allowed a preliminary study of the data structure. Then, linear discriminant analysis was used to develop decision rules to classify samples in the established categories (percentages of transgenic Bt maize). Finally, linear regression (LR) and multivariate regression models (namely, principal component analysis regression (PCR), partial least squares regression (PLS-1), and multiple linear regression (MLR)) were assayed for the prediction of the percentages of transgenic Bt maize present in a maize flour mixture. Using the relative areas of the protein peaks, MLR provided the best models and was able to predict the percentage of transgenic Bt maize in flour mixtures with an error of ±5.3%, ±2.3%, and ±3.8% in the predictions of Aristis Bt, DKC6575, and PR33P67, respectively.

Assessing the clinical and molecular diagnosis of inherited forms of impaired sensitivity to thyroid hormone from a single tertiary center.

AIM: Resistance to thyroid hormone (RTH), characterized by persistent hyperthyroxinemia with non-suppressed thyrotropin (TSH), is mostly caused by mutations in thyroid hormone receptor beta gene (THRB). Two differential diagnoses should be considered due to similar clinical and laboratory findings: TSH-producing pituitary adenoma (TPA) and Familial Dysalbuminemic Hyperthyroxinemia (FDH). The aim of this study is to describe our single tertiary center experience in the molecular diagnosis of RTH in Brazilian patients, analyzing their clinical and laboratory characteristics and the most common differential diagnosis. SUBJECTS AND METHODS: We enrolled 30 subjects with clinical and laboratory features of RTH. Patient´s evaluations included clinical examination, thyroid hormone profile and imaging tests. Sequencing analysis for THRB hot spot region was conducted on all patients, and those without mutations in beta isoform of the thyroid hormone receptor (TRß) (non-TR-RTH) were investigated for albumin gene (ALB) mutation. RESULTS: Seventeen patients presented mutations in TRß (RTHß); six were non-TR-RTH, three had a diagnosis of FDH with a mutation in ALB, and four were diagnosed with TPA. Two characteristics were different to what is commonly described in the literature: higher serum TSH levels in RTHß patients when compared to the non-TR-RTH group, but this difference did not extend to free T4 (FT4) level; also the percentage of non-TR-RTH was higher than what was reported in other series. CONCLUSION: In the present series, most cases were RTHß with higher levels of TSH. We described three novel mutations in THRB (p.M313V, p.R320G and p.R438P) and the first patients with FDH molecular diagnosis (p.R242H) documented in Brazil.

Development of a perfusion ion-exchange chromatography method for the separation of soybean proteins and its application to cultivar characterization.

A perfusion ion-exchange chromatography method has been designed, for the first time, for the separation of soybean proteins and its application to the characterization of soybean cultivars. For that purpose, the gradient, the mobile phase composition (buffer concentration, buffer pH, and elution salt), and the temperature were optimized. The method consisted of a two-step gradient (0% B for 2 min and from 0 to 50% B in 10 min) being mobile phase A a 2 0mM borate buffer (pH 9) and mobile phase B a 20 mM borate buffer (pH 9) containing 1M sodium chloride. The procedure used for the preparation of sample solutions was significantly simpler than that proposed by other authors and basically consisted of dissolving in water. This method enabled the separation of soybean proteins from a soybean protein isolate in 11 peaks in about 9 min. The method was used to separate soybean proteins in different commercial soybeans. In general, the 11 peaks yielded by the soybean protein isolate were also observed in the chromatograms of all soybeans. However, the area percentages of every peak in every soybean enabled the differentiation between soybeans. Moreover, the method was also used to separate soybean proteins in the proteic fractions obtained from every soybean. Multivariate methods were used for patterns recognition and the classification of samples.

A cross-platform metabolomics workflow for volume-restricted tissue samples: application to an animal model for polycystic kidney disease.

Metabolic profiling provides an unbiased view of the physiological status of an organism as a "function" of the metabolic composition of a measured sample. Here, we propose a simple LC-MS based workflow for metabolic profiling of volume-restricted samples, namely individual 20 µm-thick histological sections of a mouse kidney. The main idea of this workflow is to re-use the material after an RPLC-MS run, namely using the volume remaining in the vial after injection, and then introducing a phase changing step to enable HILIC-MS analysis. To test the applicability of the workflow and its ability to extract valuable biological information, we applied it to an animal model of polycystic kidney disease (PKD).

Novel lincRNA Susceptibility Gene and Its Role in Etiopathogenesis of Thyrotoxic Periodic Paralysis.

Thyrotoxic periodic paralysis (TPP) is a life-threatening neuromuscular complication of thyrotoxicosis characterized by muscle weakness and hypokalemia and with an unclear etiopathogenesis. However, the 17q24.3 locus had been genetically linked to TPP, in which the genetic variant rs312691 (TC genotype) in long intergenic noncoding RNA (lincRNA) CTD-2378E21.1 is located downstream of inward-rectifier potassium (Kir) channel genes [KCNJ2 and its antisense KCNJ2 (AS-KCNJ2)]. A TPP patient with a suppressed thyroid-stimulating hormone level, a high free thyroxine level of (5.8 ng/dL), and low serum potassium level of (2 mEq/L) was evaluated for Kir channel expression during and after recovery from thyrotoxicosis. We observed that circulating lincRNA and Kir expression varied in accordance with thyroid status and TC genotype. To endorse this association of a lincRNA-rs312691 variant with a genetic risk of TPP, an additional series of 37 patients with TPP and 32 patients with thyrotoxic without paralysis (TWP) were assessed. We verified that the risk of minor allele C was greater in TPP than in TWP (odds ratio, 5.289; P = 0.0062), and protective major allele T was more frequent than observed in the 1000 genome controls (odds ratio, 11.90; P < 0.0001). AS-KCNJ2 was downregulated during thyrotoxicosis in the TWP controls carrying allele T and were upregulated in those with TPP with risk allele C. Moreover, KCNJ2 (Kir2.1) expression was reduced during thyrotoxicosis and restored in euthyroid status. We further excluded any other coding variant by performing targeted exome sequencing mutational screening in 17q24.3. Our data suggest that high lincRNA AS-KCNJ2 and CDT-2378E21.1 expression, possibly driven by the triiodothyronine regulatory mechanism, reduces the Kir2.1 expression observed during thyrotoxicosis. This finding could contribute to the understanding of the reduced inward-rectifying current observed during muscle weakness in genetically susceptible TPP patients.

DUOX2 Mutations Are Associated With Congenital Hypothyroidism With Ectopic Thyroid Gland.

Context: Thyroid dysgenesis (TD) is the leading cause of congenital hypothyroidism (CH). The etiology of TD remains unknown in ∼90% of cases, the most common form being thyroid ectopia (TE) (48% to 61%). Objective: To search for candidate genes in hypothyroid children with TE. Design, Setting, and Participants: We followed a cohort of 268 children with TD and performed whole-exome sequencing (WES) in three children with CH with TE (CHTE) and compared them with 18 thyroid-healthy controls. We then screened an additional 41 children with CHTE by Sanger sequencing and correlated the WES and Sanger molecular findings with in vitro functional analysis. Main Outcome Measures: Genotyping, mutation prediction analysis, and in vitro functional analysis. Results: We identified seven variants in the DUOX2 gene, namely G201E, L264CfsX57, P609S, M650T, E810X, M822V, and E1017G, and eight known variations. All children carrying DUOX2 variations had high thyroid-stimulating hormone levels at neonatal diagnosis. All mutations were localized in the N-terminal segment, and three of them led to effects on cell surface targeting and reactive oxygen species generation. The DUOX2 mutants also altered the interaction with the maturation factor DUOXA2 and the formation of a stable DUOX2/DUOXA2 complex at the cell surface, thereby impairing functional enzymatic activity. We observed no mutations in the classic genes related to TD or in the DUOX1 gene. Conclusion: Our findings suggest that, in addition to thyroid hormonogenesis, the DUOX2 N-terminal domain may play a role in thyroid development.

Patterns of care of brain tumor-related epilepsy. A cohort study done in Italian Epilepsy Center.

Epilepsy is the most common comorbidity in patients with brain tumors. STUDY AIMS: To define characteristics of brain tumor-related epilepsy (BTRE) patients and identify patterns of care. Nationwide, multicenter retrospective cohort study. Medical records of BTRE patients seen from 1/1/2010 to 12/31/2011, followed for at least one month were examined. Information included age, sex, tumor type/treatments, epilepsy characteristics, antiepileptic drugs (AEDs). Time to modify first AED due to inefficacy and/or toxicity was assessed with the Kaplan-Meier method and Cox proportional hazard models were used to identify predictors of treatment outcome. Enrolled were 808 patients (447 men, 361 women) from 26 epilepsy centers. Follow-up ranged 1 to 423 months (median 18 months). 732 patients underwent surgery, 483 chemotherapy (CT), 508 radiotherapy. All patients were treated with AEDs. Levetiracetam was the most common drug. 377 patients (46.7%) were still on first drug at end of follow-up, 338 (41.8%) needed treatment modifications (uncontrolled seizures, 229; side effects, 101; poor compliance, 22). Treatment discontinuation for lack of efficacy was associated with younger age, chemotherapy, and center with <20 cases. Treatment discontinuation for side effects was associated with female sex, enzyme-inducing drugs and center with > 20 cases. About one-half of patients with BTRE were on first AED at end of follow-up. Levetiracetam was the most common drug. A non enzyme-inducing AED was followed by a lower risk of drug discontinuation for SE.

Development of a perfusion reversed-phase high performance liquid chromatography method for the characterisation of maize products using multivariate analysis.

A perfusion reversed-phase high performance liquid chromatography (RP-HPLC) method has been designed to allow rapid (3.4 min) separations of maize proteins with high resolution. Several factors, such as extraction conditions, temperature, detection wavelength and type and concentration of ion-pairing agent were optimised. A fine optimisation of the gradient elution was also performed by applying experimental design. Commercial maize products for human consumption (flours, precocked flours, fried snacks and extruded snacks) were characterised for the first time by perfusion RP-HPLC and their chromatographic profiles allowed a differentiation among products relating the different technological process used for their preparation. Furthermore, applying discriminant analysis makes it possible to group the samples according with the technological process suffered by maize products, obtaining a good prediction in 92% of the samples.

Copper(II) interactions with non-steroidal anti-inflammatory agents. III--3-Methoxyanthranilic acid as a potential *OH-inactivating ligand: a quantitative investigation of its copper handling role in vivo.

Pharmacological activities of copper(II) complexes are a direct function of the nature of their ligands associated with the metal ion in vivo. Some of these, defined as *OH-inactivating ligands (G. Berthon, Agents Actions 39 (1993) 210-217), may act as specific "lures" for hydroxyl radicals at inflammatory sites and behave as pseudo-catalase-like agents. This property has been advanced for anthranilic acid (H. Miche, V. Brumas, G. Berthon, J. Inorg. Biochem. 68 (1997) 27-38). With a view to improve the chemical features required to render such inactive substances effective anti-inflammatory drugs through their association with copper(II), an in vitro investigation into copper(II) interactions with the anionic form of an anthranilic acid derivative, namely 3-methoxyanthranilate (Man), has been performed under experimental conditions pertaining in vivo. Copper(II)-Man complex equilibria have been determined using glass electrode potentiometry, then checked by UV-vis and mass spectrometries. Given the prime role of histidine as a copper(II) ligand in blood plasma, copper(II)-histidine-Man ternary equilibria have also been studied. Subsequent computer simulations of the distribution of copper(II) in the extracellular fluid revealed that Man can specifically mobilize Cu(II) ions under inflammatory conditions without affecting their distribution under normal physiological conditions. Thiobarbituric acid reactive substances (TBARS) tests conducted with respect to standardized copper-mediated Fenton-type reactions (P. Maestre, L. Lambs, J.P. Thouvenot, G. Berthon, Free Rad. Res. 20 (1994) 205-218) have shown that, like anthranilic acid, Man can effectively both increase the Fenton-like reactivity of copper and decrease the amount of TBARS detected in solution, i.e., act as a potential *OH-inactivating ligand.