Screening, diagnosis and follow-up of Brugada syndrome in children: a Dutch expert consensus statement.

Brugada syndrome (BrS) is a rare inherited arrhythmia syndrome. Affected children may experience life-threatening symptoms, mainly during fever. The percentage of SCN5A variant carriers in children is higher than in adults. Current diagnostic and follow-up policies for children with (a family history of) BrS vary between centres. Here, we present a consensus statement based on the current literature and expert opinions to standardise the approach for all children with BrS and those from BrS families in the Netherlands. In summary, BrS is diagnosed in patients with a spontaneous type 1 electrocardiogram (ECG) pattern or with a Shanghai score ≥ 3.5 including ≥ 1 ECG finding. A sodium channel-blocking drug challenge test should only be performed after puberty with a few exceptions. A fever ECG is indicated in children with suspected BrS, in children with a first-degree family member with definite or possible BrS according to the Shanghai criteria with a SCN5A variant and in paediatric SCN5A variant carriers. In-hospital rhythm monitoring during fever is indicated in patients with an existing type 1 ECG pattern and in those who develop such a pattern. Genetic testing should be restricted to SCN5A. Children with BrS and children who carry an SCN5A variant should avoid medication listed at www.brugadadrugs.org and fever should be suppressed. Ventricular arrhythmias or electrical storms should be treated with isoproterenol infusion.

Essential oils from Ocimum basilicum cultivars: analysis of their composition and determination of the effect of the major compounds on Haemonchus contortus eggs.

The continuous use of synthetic anthelmintics against gastrointestinal nematodes (GINs) has resulted in the increased resistance, which is why alternative methods are being sought, such as the use of natural products. Plant essential oils (EOs) have been considered as potential products for the control of GINs. However, the chemical composition and, consequently, the biological activity of EOs vary in different plant cultivars. The aim of this study was to evaluate the anthelmintic activity of EOs from cultivars of Ocimum basilicum L. and that of their major constituents against Haemonchus contortus. The EOs from 16 cultivars as well the pure compound linalool, methyl chavicol, citral and eugenol were used in the assessment of the inhibition of H. contortus egg hatch. In addition, the composition of three cultivars was simulated using a combination of the two major compounds from each. The EOs from different cultivars showed mean Inhibition Concentration (IC50) varying from 0.56 to 2.22 mg/mL. The cultivar with the highest egg-hatch inhibition, Napoletano, is constituted mainly of linalool and methyl chavicol. Among the individual compounds tested, citral was the most effective (IC50 0.30 mg/mL). The best combination of compounds was obtained with 11% eugenol plus 64% linalool (IC50 0.44 mg/mL), simulating the Italian Large Leaf (Richters) cultivar. We conclude that different cultivars of O. basilicum show different anthelmintic potential, with cultivars containing linalool and methyl chavicol being the most promising; and that citral or methyl chavicol isolated should also be considered for the development of new anthelmintic formulations.

Correction to: Assessment of individual tumor buds using keratin immunohistochemistry: moderate interobserver agreement suggests a role for machine learning.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Assessment of individual tumor buds using keratin immunohistochemistry: moderate interobserver agreement suggests a role for machine learning.

Tumor budding is a promising and cost-effective biomarker with strong prognostic value in colorectal cancer. However, challenges related to interobserver variability persist. Such variability may be reduced by immunohistochemistry and computer-aided tumor bud selection. Development of computer algorithms for this purpose requires unequivocal examples of individual tumor buds. As such, we undertook a large-scale, international, and digital observer study on individual tumor bud assessment. From a pool of 46 colorectal cancer cases with tumor budding, 3000 tumor bud candidates were selected, largely based on digital image analysis algorithms. For each candidate bud, an image patch (size 256 × 256 µm) was extracted from a pan cytokeratin-stained whole-slide image. Members of an International Tumor Budding Consortium (n = 7) were asked to categorize each candidate as either (1) tumor bud, (2) poorly differentiated cluster, or (3) neither, based on current definitions. Agreement was assessed with Cohen's and Fleiss Kappa statistics. Fleiss Kappa showed moderate overall agreement between observers (0.42 and 0.51), while Cohen's Kappas ranged from 0.25 to 0.63. Complete agreement by all seven observers was present for only 34% of the 3000 tumor bud candidates, while 59% of the candidates were agreed on by at least five of the seven observers. Despite reports of moderate-to-substantial agreement with respect to tumor budding grade, agreement with respect to individual pan cytokeratin-stained tumor buds is moderate at most. A machine learning approach may prove especially useful for a more robust assessment of individual tumor buds.

A new approach to distance measurements between two spin labels in the >10 nm range.

ESR spectroscopy can be efficiently used to acquire the distance between two spin labels placed on a macromolecule by measuring their mutual dipolar interaction frequency, as long as the distance is not greater than ∼10 nm. Any hope to significantly increase this figure is hampered by the fact that all available spin labels have a phase memory time (Tm), restricted to the microseconds range, which provides a limited window during which the dipolar interaction frequency can be measured. Thus, due to the inverse cubic dependence of the dipolar frequency over the labels' separation distance, evaluating much larger distances, e.g. 20 nm, would require to have a Tm that is ∼200 microsecond, clearly beyond any hope. Here we propose a new approach to greatly enhancing the maximum measured distance available by relying on another type of dipole interaction-mediated mechanism called spin diffusion. This mechanism operates and can be evaluated during the spin lattice relaxation time, T1 (commonly in the milliseconds range), rather than only during Tm. Up until recently, the observation of spin diffusion in solid electron spin systems was considered experimentally impractical. However, recent developments have enabled its direct measurement by means of high sensitivity pulsed ESR that employs intense short magnetic field gradients, thus opening the door to the subsequent utilization of these capabilities. The manuscript presents the subject of spin diffusion, the ways it can be directly measured, and a theoretical discussion on how intramolecular spin-pair distance, even in the range of 20-30 nm, could be accurately extracted from spin diffusion measurements.

Analysis of genetic diversity of Hyptis pectinata (L.) Poit. plants using ISSR markers.

Hyptis pectinata, popularly known as 'sambacaitá' or 'canudinho', is a medicinal and aromatic species widely used in the Brazilian Northeast. In Sergipe, the excessive extraction of natural resources may reduce the genetic variability of native plants. Thus, molecular markers have frequently been applied to the characterization of genetic diversity as the basis for germplasm conservation and breeding programs. The objective of the present study was to evaluate the genetic diversity of H. pectinata plants collected in different municipalities of the State of Sergipe using ISSR molecular markers. Thirty-four primers were tested, nine of which were selected for providing reproducible and analyzable amplification products, resulting in 67 polymorphic bands. The expected heterozygosity ranged from 0.32 to 0.45, with a mean of 0.39. Polymorphism information content was of 0.49, which classifies the markers as moderately informative. A dendrogram was constructed using unweighted pair group method with arithmetic mean, forming three clusters: Cluster I (79 plants); Cluster II (4 plants); and Cluster III (2 plants). Jaccard's similarity coefficients ranged from 0.06 to 0.98. The plants SAM-117 and SAM-119 presented greater similarity. Conversely, SAM-107 and SAM-171 were the most genetically distant. In general, H. pectinata plants collected in the State of Sergipe presented low to moderate genetic diversity.

Assessment of genetic diversity of a native population of Eplingiella fruticosa: a plant with therapeutic potential.

Eplingiella fruticosa (Salzm. ex Benth.) Harley & J.F.B. Pastore, ex Hyptis fruticosa Salzm. ex Benth. is an aromatic and medicinal plant of the family Lamiaceae, found mainly in regions with intense anthropic activity. Information on the genetic diversity of this species is scarce. However, it can be assessed using molecular markers that identify the level of diversity among phenotypically identical individuals. The present study aimed to characterize the genetic diversity of a native population of E. fruticosa from the State of Sergipe using ISSR molecular markers. Samples of 100 plants were collected in 11 municipalities of the State of Sergipe and analyzed using eight ISSR primers, resulting in 72 informative bands. The cluster analysis obtained using the neighbor joining method resulted in three groups: Group I consisted of 50 plants, mainly from the municipalities of Areia Branca, Estância, Japaratuba, Moita Bonita, Pirambu, and Salgado; Group II was formed by 21 plants, with nine representatives from the municipality of Itaporanga d'Ajuda and 13 representatives from other municipalities; Group III was composed by 29 plants, being represented mainly by the municipalities of Malhada dos Bois and São Cristóvão. The smallest genetic distance occurred between plants EPF94 and EPF96 (0.250), and the greatest distance occurred between plants EPF50 and EPF96 (0.9778). The Shannon index had a mean value of 0.42, and diversity was considered moderate. Heterozygosity had a mean value of 0.267 and was considered low. Polymorphic information content (0.253) was considered moderately informative. Genetic diversity of E. fruticosa plants was intermediate, and the results of the present study can assist in the conservation and use of the genetic resources of this species.

Genetic variability in Jatropha curcas L. from diallel crossing.

Physic nut (Jatropha curcas L.) presents high oilseed yield and low production cost. However, technical-scientific knowledge on this crop is still limited. This study aimed to evaluate and estimate the genetic variability of hybrids obtained from dialell crossing. Genetic variability was carried out using ISSR molecular markers. For genetic variability, nine primers were used, and six were selected with 80.7% polymorphism. Genetic similarity was obtained using the NTSYS pc. 2.1 software, and cluster analysis was obtained by the UPGMA method. Mean genetic similarity was 58.4% among hybrids; the most divergent pair was H1 and H10 and the most similar pair was H9 and H10. ISSR PCR markers provided a quick and highly informative system for DNA fingerprinting, and also allowed establishing genetic relationships of Jatropha hybrids.

Genetic diversity of native populations of Croton tetradenius Baill. using ISSR markers.

Brazil has about 300 Croton species in different types of vegetation. Croton tetradenius Baill., which is endemic to the Northeast region and predominant in the Caatinga vegetation, stands out among the several species of this genus. Considering the importance of knowing the genetic variability of a species, the objective of this study was to analyze the genetic diversity of the genotypes of natural populations of C. tetradenius in the State of Sergipe, using ISSR molecular markers. Forty individuals were sampled in four natural populations of the State of Sergipe, Brazil. Thirteen primers were used for DNA amplification using ISSR-PCR, totaling 77 amplified fragments, of which 94.8% were polymorphic. Results of the cluster analysis obtained by the Jaccard's similarity index, using the UPGMA method, resulted in the formation of six distinct clusters. Analysis of molecular variance (AMOVA), used to estimate the genetic variability among populations, revealed significant genetic variance (P < 0.01) between and within the studied populations, and most of the genetic diversity was found (87%) within populations. According to the Jaccard's similarity index, none of the studied plants was genetically identical. CTE210 and CTE305 presented high similarity index (0.76), while CTE105 presented low similarity index (<0.16) with all related individuals. ISSR markers were efficient and allowed the formation of a molecular profile, and had sufficient polymorphism to estimate the genetic variability between the accessions of the studied populations.

[Management of chronic pain using extended release tilidine : Quality of life and implication of comedication on tilidine metabolism]. / Management von chronischem Schmerz mit retardiertem Tilidin : Lebensqualität und Bedeutung der Komedikation für den Tilidinmetabolismus.

BACKGROUND AND OBJECTIVES: The synthetic opioid tilidine is often used in chronic pain treatment. However, the activation via metabolism in patients with concomitant medication and reduced liver or kidney function is not thoroughly investigated. We therefore studied pain treatment efficacy, health-related quality of live and the metabolism of tilidine in patients with chronic pain. METHODS AND MATERIALS: In all, 48 patients, who were on a stable dose of oral prolonged release tilidine for at least 7 days, were included in this observational multicenter study. Liver and kidney function were assessed in routine blood samples, concentrations of tilidine, nortilidine and bisnortilidine were determined using a validated LC/MS/MS method. Comedication was registered and patients experience with regard to quality of life, pain, gastrointestinal symptoms and adverse events was assessed in standardised questionnaires. RESULTS: On average a daily dose of 180 mg tilidine was taken. Dose normalized plasma concentrations of the active metabolite nortilidine ranged between 1.6 ng/ml and 76.5 ng/ml (mean 29.2 ± 25.1 ng/ml). Ratios between tilidine and nortilidine were on average 0.28 (median = 0.13, standard deviation = 0.67). Patients were on 1 to 14 different concomitant medications. About 66% of the patients had sufficient pain treatment. Almost no opioid-induced constipation was observed. Only few patients had decreased kidney or liver function which did not result in elevated nortilidine concentrations. CONCLUSION: Pain treatment using tilidine resulted in variable nortilidine concentrations which are obviously not strongly influenced by comedication or reduced liver or kidney function. Only a few side effects were observed with almost no opioid-induced constipation.

Assessment of the repellent effect of Lippia alba essential oil and major monoterpenes on the cattle tick Rhipicephalus microplus.

The control of Rhipicephalus microplus (Ixodida: Ixodidae) is achieved using synthetic acaricides. However, resistant tick populations are widespread around the world. Plant essential oils can act as repellents, keeping ticks away from hosts and decreasing the selection pressure on synthetic acaricides. The aim of this study was to evaluate the in vitro repellent effect of Lippia alba essential oil on R. microplus larvae. Leaves from two L. alba genotypes maintained under the same agronomic and environmental conditions were collected. Essential oil was extracted by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). The major monoterpenes detected in the chemical analysis were commercially acquired and tested. For the repellency test, a glass rod was vertically fixed to measure active climbing of approximately 30 R. microplus larvae aged 14-21 days in response to essential oils and monoterpenes. Repellency was evaluated at 1 h, 3 h and 5 h after treatment. Variation in repellent action was detected between the genotypes. The major monoterpenes identified in the essential oils (limonene and carvone) showed low repellent effects in comparison with intact essential oils. Thus, the present results showed that L. alba essential oil contains bioactive compounds with great repellent activity against ticks that varies according to the plant genotype.

Analysis of genetic diversity of Laeliinae (Orchidaceae) in the State of Sergipe using ISSR markers.

The Orchidaceae represent one of the largest and most diverse families on the planet. However, this family is constantly threatened by predators and by the advancement of urban centers over its natural habitats. The objective of this study was to use inter-simple sequence repeat markers to evaluate the genetic diversity between orchid accessions of the Laeliinae subtribe, which comprise part of the Orchidaceae study collection at the Department of Agronomic Engineering of the Federal University of Sergipe. DNA was extracted from each specimen by using an adapted 2% cetyltrimethyl ammonium bromide protocol. Similarity between individuals was calculated using the Jaccard method. Clustering was carried out by the unweighted pair group method with arithmetic mean method, with resampling and 10,000 bootstraps. Eighty-seven fragments were obtained, all of which were polymorphic, revealing high variability between accessions. The mean similarity was 35.77% between Encyclia sp individuals, and 35.90% between specimens of Cattleya tigrina. For Epidendrum secundum, a relationship between geographic and genetic distances was observed, and the accession collected in the southern part of the State of Sergipe (Serra de Itabaiana National Park) was more divergent than that of the other parts of the state. The data generated in this study will guide further research aimed at the ex situ conservation of these materials.

Genetic diversity of Lippia sidoides Cham. and L. gracilis Schauer germplasm.

The conservation of plants in germplasm banks ensures the characterization and availability of these resources for future generations. The present study used DNA markers to obtain genetic information about germplasm collections of Lippia sidoides and L. gracilis, which are maintained in an Active Germplasm Bank (AGB). Genetic variability of samples in the AGB was assessed using 12 combinations of amplified fragment length polymorphism (AFLP) primers (EcoRI/MseI). Twenty simple sequence repeat primers designed for L. alba were tested to determine their transferability in L. sidoides and L. gracilis. The AFLP markers generated 789 markers. The assessed loci exhibited a moderate Shannon diversity index (I = 0.42) in both species, suggesting that the conserved accessions possess an intermediate level of genetic diversity. Twelve microsatellite loci amplified satisfactorily, and nine loci were polymorphic in each species. A total of 23, 22, and 36 alleles, with an average of 2.5, 2.4, and 3.27 alleles per locus were identified for L. sidoides and L. gracilis accessions in the AGB, and Lippia sp sampled plants, respectively. Analyses of genetic structure permitted the identification of three different groups using both sets of markers, of which two were representative of L. sidoides. The information generated in this study may help to create, expand, and maintain collections of these species and may assist in genetic-breeding programs.

Genetic diversity analysis of Varronia curassavica Jacq. accessions using ISSR markers.

Varronia curassavica Jacq. is a medicinal and aromatic plant from Brazil with significant economic importance. Studies on genetic diversity in active germplasm banks (AGB) are essential for conservation and breeding programs. The aim of this study was to analyze the genetic diversity of V. curassavica accessions of the AGB of Medicinal and Aromatic Plants of the Federal University of Sergipe (UFS), using inter-simple sequence repeat molecular markers. Twenty-four primers were tested, and 14 were polymorphic and informative, resulting in 149 bands with 97.98% polymorphism. The UPGMA dendrogram divided the accessions into Clusters I and II. Jaccard similarity coefficients for pair-wise comparisons of accessions ranged between 0.24 and 0.78. The pairs of accessions VCUR-001/VCUR-503, VCUR-001/VCUR-504, and VCUR-104/VCUR-501 showed relatively low similarity (0.24), and the pair of accessions VCUR-402/VCUR- 403 showed medium similarity (0.78). Twenty-eight accessions were divided into three distinct clusters, according to the STRUCTURE analysis. The genetic diversity of V. curassavica in the AGB of UFS is low to medium, and it requires expansion. Accession VCUR-802 is the most suitable for selection in breeding program of this species, since it clearly represents all of the diversity present in the AGB.

Molecular characterization of patchouli (Pogostemon spp) germplasm.

Patchouli [Pogostemon cablin (Blanco) Benth.] is an aromatic, herbaceous plant belonging to the Lamiaceae family native to Southeast Asia. Its leaves produce an essential oil regularly used by the perfume and cosmetics industries. However, since patchouli from the Philippines and India were described and named Pogostemon patchouli, there has been a divergence in the identity of these species. The objective of the current study was to study the genetic diversity of patchouli accessions in the Active Germplasm Bank of Universidade Federal de Sergipe using microsatellite and inter simple sequence repeat markers. The results of both types of molecular markers showed that there are two well-defined clusters of accessions that harbor exclusive alleles. It was observed that these two clusters are genetically distant, suggesting that they belong to two different species. Based on the results, two accessions were classified as Pogostemon heyneanus and the remaining accessions were classified as P. cablin.

Analysis of genetic diversity of a native population of Myrcia lundiana Kiaersk. plants using ISSR markers.

Myrcia lundiana Kiaersk. is a tree of the family Myrtaceae found in tropical and subtropical areas of the southern hemisphere that produces essential oil. The aim of this study was to characterize the genetic diversity of M. lundiana plants from a native population of Parque Nacional de Itabaiana, using inter-simple sequence repeat molecular markers. Thirty-five primers were tested, 20 of which were polymorphic, resulting in 135 polymorphic and informative bands. Results of the cluster analysis, obtained using the unweighted pair group method with arithmetic mean, grouped plants into three clusters: Cluster I - MLU001, MLU002, MLU003, MLU004, MLU005, MLU006, MLU018, MLU019, MLU020, MLU021, MLU022; MLU008, MLU011, MLU012, MLU014, MLU015, MLU017, MLU026, and MLU028; Cluster II - MLU007, MLU009, MLU010, MLU013, and MLU016; and Cluster III - MLU023, MLU024, MLU025, and MLU027. Jaccard similarity coefficients for pair-wise comparisons of plants ranged between 0.15 and 0.87. MLU014 and MLU015 presented low genetic diversity, with a similarity index of 0.87. Conversely, MLU007 and MLU019 presented high diversity, with a similarity index of 0.15. According to the structure analysis, three distinct clusters were formed. Genetic diversity of M. lundiana plants was intermediate, and expansion of its genetic diversity is necessary. MLU026 and MLU028 are the most suitable for selection in breeding programs, since they clearly represent all of the diversity present in these plants. Moreover, these results provide important information on the existing genetic variability, highlighting the importance of Parque Nacional de Itabaiana for the conservation of this species.

Fatty acid oxidation flux predicts the clinical severity of VLCAD deficiency.

PURPOSE: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid ß-oxidation (LC-FAO) and is included in many newborn screening (NBS) programs worldwide. Patients may present with hypoketotic hypoglycemia, cardiomyopathy, and/or myopathy, but clinical severity varies widely and the clinical outcome is unpredictable. We investigated predictive markers that may determine clinical severity. METHODS: We developed a clinical severity score (CSS), which was determined for 13 Dutch patients with VLCADD, all of whom were diagnosed before the introduction of VLCADD in NBS to prevent bias from early diagnosis. In cultured skin fibroblasts from these patients, we measured LC-FAO flux (the rate of oleate oxidation), VLCAD activity, and acylcarnitine profiles following palmitate loading. RESULTS: The strongest correlation (r = 0.93; P < 0.0001) was observed between LC-FAO flux and the CSS. VLCAD activity measurement and the C14/C16-to-acylcarnitine ratio correlated much less. A median LC-FAO flux of 6% of control values (range 5.6-6.8%) was associated with cardiomyopathy (P < 0.01), and 32.4% (range 5.6-50.5%) was associated with myopathy (P < 0.05). CONCLUSION: Our results demonstrate a very strong correlation between LC-FAO flux in fibroblasts and the clinical severity of VLCADD. LC-FAO flux measurements may thus predict whether patients are likely to develop symptoms.

Development of a novel set of microsatellite markers for Lippia alba (Verbenaceae).

Microsatellite primers were developed and optimized for Lippia alba to characterize the L. alba germplasm bank of Universidade de São Paulo. A genomic library enabled the design of 9 microsatellite primers. Six of the 9 primers yielded polymorphic products, which defined 2 groups in the bank. The data provide support to characterize germplasm banks, genetic breeding programs for L. alba, and other genetic diversity studies and classifications of species in the genus Lippia.

Molecular and chemical characterization of vetiver, Chrysopogon zizanioides (L.) Roberty, germplasm.

Due to the economic interests in vetiver, Chrysopogon zizanioides (L.) Roberty, molecular and chemical studies are essential to generate information for its sustainable exploitation. The aim of this study was to undertake a molecular and chemical characterization of vetiver accessions of the active germplasm bank of the Universidade Federal de Sergipe. The molecular characteristics of the accessions were studied using amplified fragment length polymorphism markers, with a total of 14 primer combinations that generated 442 loci, allowing us to observe that these accessions have similar genomes. The vetiver accessions were divided into three distinct groups, where accession UFS-VET005 was the most differentiated and accession UFS-VET004 had the lowest essential oil content (0.70%). The content of the chemical constituents of the essential oils was observed to vary, with a predominance of khusimol, which ranged from 18.97 to 25.02%. It was possible to divide the vetiver accessions into two groups based on chemical composition, and these groups do not correlate with the molecular grouping. Therefore, it is necessary to perform molecular and chemical analyses to characterize vetiver accessions.

Cardiopulmonary Exercise Testing in Children and Adolescents With Dystrophinopathies: A Pilot Study.

PURPOSE: To determine exercise response during cardiopulmonary exercise testing in children and adolescents with dystrophinopathies. METHODS: Exercise response on the cardiopulmonary exercise test (CPET) was compared with a standard care test protocol. RESULTS: Nine boys (aged 10.8 ± 4.7 years) with Becker muscular dystrophy (n = 6) and Duchenne muscular dystrophy (n = 3) were included. The feasibility of the CPET was similar to a standard care test protocol, and no serious adverse events occurred. In 67% of the subjects with normal or only mildly impaired functional capacity, the CPET could be used to detect moderate to severe cardiopulmonary exercise limitations. CONCLUSIONS: The CPET seems to be a promising outcome measure for cardiopulmonary exercise limitations in youth with mild functional limitations. Further research with larger samples is warranted to confirm current findings and investigate the additional value of the CPET to longitudinal follow-up of cardiomyopathy and the development of safe exercise programs for youth with dystrophinopathies.