Targeted next-generation sequencing helps to decipher the genetic and phenotypic heterogeneity of hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is mainly associated with myosin, heavy chain 7 (MYH7) and myosin binding protein C, cardiac (MYBPC3) mutations. In order to better explain the clinical and genetic heterogeneity in HCM patients, in this study, we implemented a target-next generation sequencing (NGS) assay. An Ion AmpliSeq Custom Panel for the enrichment of 19 genes, of which 9 of these did not encode thick/intermediate and thin myofilament (TTm) proteins and, among them, 3 responsible of HCM phenocopy, was created. Ninety-two DNA samples were analyzed by the Ion Personal Genome Machine: 73 DNA samples (training set), previously genotyped in some of the genes by Sanger sequencing, were used to optimize the NGS strategy, whereas 19 DNA samples (discovery set) allowed the evaluation of NGS performance. In the training set, we identified 72 out of 73 expected mutations and 15 additional mutations: the molecular diagnosis was achieved in one patient with a previously wild-type status and the pre-excitation syndrome was explained in another. In the discovery set, we identified 20 mutations, 5 of which were in genes encoding non-TTm proteins, increasing the diagnostic yield by approximately 20%: a single mutation in genes encoding non-TTm proteins was identified in 2 out of 3 borderline HCM patients, whereas co-occuring mutations in genes encoding TTm and galactosidase alpha (GLA) altered proteins were characterized in a male with HCM and multiorgan dysfunction. Our combined targeted NGS-Sanger sequencing-based strategy allowed the molecular diagnosis of HCM with greater efficiency than using the conventional (Sanger) sequencing alone. Mutant alleles encoding non-TTm proteins may aid in the complete understanding of the genetic and phenotypic heterogeneity of HCM: co-occuring mutations of genes encoding TTm and non-TTm proteins could explain the wide variability of the HCM phenotype, whereas mutations in genes encoding only the non-TTm proteins are identifiable in patients with a milder HCM status

Neutrophil–lymphocyte ratio is associated with worse outcomes in patients with cirrhosis: insights from the PRO-LIVER Registry

Background Liver cirrhosis (LC) is a leading global cause of morbidity and mortality, with inflammation playing a key role in disease progression and clinical complications of LC. The Neutrophil/Lymphocyte Ratio (NLR), a readily available marker of systemic inflammation, has been linked to short-term adverse outcomes in LC, but data on long-term follow-up are limited. This study aimed to investigate the relationship between NLR and long-term all-cause mortality in an unselected cohort of LC patients. Methods Data were gathered from the Italian multicenter observational study "PRO-LIVER". Patients with available data to calculate NLR at baseline were included. Baseline clinical determinants of NLR and the association of NRL with all-cause mortality at 2-year follow-up were evaluated. Results From the overall cohort (n = 753), 506 patients with LC (31% female, mean age 64.8 +/- 11.9 years) were included in the analysis. Median value of NLR was 2.42 (Interquartile Range [IQR]: 1.61-3.52). At baseline, patients with NLR >= 2.42 were more likely to have Child-Pugh B or C, hepatocellular carcinoma (HCC), or portal vein thrombosis (PVT). After a median follow-up of 21 months, 129 patients died: 44 (17%) with NLR < 2.42 and 85 (34%) with NLR >= 2.42 (p < 0.001). At multiple-adjusted Cox regression analysis, NLR >= 2.42 was independently associated with all-cause mortality (HR: 1.65; 95% CI: 1.12-2.44; p = 0.012), along with age, Child-Pugh C class, HCC and PVT. Conclusions NLR is associated with long-term all-cause mortality in LC. NLR may serve as a potentially easily available tool to aid risk refinement in LC. Trial registration numberClinicalTrials.gov Identifier: NCT01470547

Determination of phenolic content, antioxidant activity, and brine shrimp toxicity of the aerial part extracts from Sinapis alba and Sinapis arvensis (Brassicaceae) growing wild in Sicily (Italy).

As part of a project aimed at the valorization of taxa included in the Brassicaceae family belonging to the spontaneous flora of Sicily, our research team has recently focused on studying the taxa of Sinapis L., utilized as food plants and in traditional medicine as sources of bioactive compounds with application in pharmaceutical, nutraceutical and cosmetic fields. In Italy, the genus is represented by Sinapis arvensis L., S. pubescens L., and S. alba L., the latter consisting of three infra-specific subdivisions namely subsp. alba, subsp. dissecta (Lag.) Bonnier, subsp. mairei (H.Lindb.) Maire. All these specific and intraspecific taxa are present in Sicily. Particularly, current research is focused on the aerial parts (leaves, flowers, and stems) of Sinapis alba (white mustard) and Sinapis arvensis (wild mustard). The total phenolic and flavonoid content of the 70% methanol extracts from the aerial parts of the selected species has been spectrophotometrically quantified. Moreover, the antioxidant properties were determined by in vitro methods based on different mechanisms: the primary antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power assays, while the ferrous ion chelating activity assay estimated the secondary antioxidant properties. Finally, Artemia salina lethality bioassay was performed for the preliminary toxicity assessment. The total phenolic content was found to be higher in S. alba leaf, flower, and stem extracts (68.53 ± 2.57, 69.33 ± 1.83 and 47.01 ± 1.56 mg GAE/g extract, respectively) than those of S. arvensis. The same trend was also observed for the flavonoid content (32.94 ± 0.91, 29.93 ± 0.67 and 17.36 ± 0.41 mg QE/g extract, respectively). Concerning the antioxidant properties, all the extracts of the two species showed mild to moderate DPPH radical scavenging activity compared to the standard butylated hydroxytoluene (BHT); among the extracts, S. alba flower extract displayed the best activity, reaching 95% inhibition at the highest concentration tested (IC50 reducing power assay, both species showed mild to moderate reducing power, compared to the standard BHT being S. alba extracts more active than those from S. arvensis. In the ferrous ion chelating activity assay, S. arvensis extracts displayed good secondary antioxidant properties, higher than those from S. alba, with flower extract being the most active (IC50 = 0.15 ± 0.09 mg/mL) followed by stem and leaf extracts (IC50 = 0.22 ± 0.005 and 0.37 ± 0.08 mg/mL, respectively). The results of the antioxidant tests indicate that the extracts of the two Sinapis taxa possess antioxidant properties based on different mechanisms: the best primary antioxidant activity was observed for S. alba extracts, whereas those from S. arvensis displayed better secondary antioxidant properties. At last, the results of A. salina lethality bioassay showed the absence of toxicity against brine shrimp larvae for all the extracts. Overall, the obtained results improve the knowledge of the Sinapis taxa, also indicating S. alba and S. arvensis, belonging to the spontaneous flora of Sicily, as safe sources of antioxidant compounds

Platelet function in health and disease: from molecular mechanisms, redox considerations to novel therapeutic opportunities

increased oxidative stress appears to be of fundamental importance in the pathogenesis and development of several disease processes. indeed, it is well known that reactive oxygen species (ROS) exert critical regulatory functions within the vascular wall, and it is, therefore, plausible that platelets represent a relevant target for their action. platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated. In addition to already well-defined platelet regulatory factors, ROS may participate in the regulation of platelet activation. It is already established that enhanced ROS release from the vascular wall can indirectly affect platelet activity by scavenging nitric oxide (NO), thereby decreasing the antiplatelet properties of endothelium. on the other hand, recent data suggest that platelets themselves generate ROS, which may evoke pro-thrombotic responses, triggering many biological processes participating in atherosclerosis initiation, progression, and complication. that oxidative stress may alter platelet function is conceivable when considering that antioxidants play a role in the prevention of cardiovascular disease, although the precise mechanism accounting for changes attributable to antioxidants in atherosclerosis remains unknown. It is possible that the effects of antioxidants may be a consequence of their enhancing or promoting the antiplatelet effects of NO derived from both endothelial cells and platelets. this review focuses on current knowledge regarding ROS-dependent regulation of platelet function in health and disease, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance. antioxid. redox signal. 17, 1447-1485

Remission, minimal disease activity, and acceptable symptom state in juvenile idiopathic arthritis: Defining criteria based on the juvenile arthritis disease activity score

To determine cutoff values for defining remission, minimal disease activity, and parent and child acceptable symptom state in juvenile idiopathic arthritis (JIA) using the Juvenile Arthritis Disease Activity Score (JADAS).For the selection of cutoff values, data from a clinical database including 609 children with JIA were used. Optimal cutoff values were determined against external criteria by calculating the 75th percentile of cumulative score distribution and through receiver operating characteristic curve analysis. External criteria included formal definitions of inactive disease and minimal disease activity, subjective rating of remission by physicians, parents, and children, and rating of acceptable symptom state by parents and children. The choice of cutoffs was made based on clinical and statistical grounds. Cross-validation was performed using 4 JIA patient samples that included a total of 1,323 patients, and was based on assessment of construct, discriminant, and predictive validity.With all versions of the JADAS, the cutoff score for classifying a patient as having inactive disease was 1, whereas the cutoff for classification of minimal disease activity was 2 for oligoarticular JIA and 3.8 for polyarticular JIA. Cutoffs for physicians', parents', and children's subjective rating of remission ranged from 2 to 2.3. Cutoffs for acceptable symptom state ranged from 3.2 to 5.4 for parents and from 3 to 4.5 for children. Results of cross-validation analyses strongly supported the selected cutoff values.Cutoff values for classifying various disease states in JIA using the JADAS were developed. In cross-validation analyses, they proved to have good construct and discriminant validity and ability to predict disease outcome