Selection and validation of reference genes for quantitative real-time PCR studies during Saccharomyces cerevisiae alcoholic fermentation in presence of sulfite

Sulfur dioxide is extensively used during industrial fermentations and contributes to determine the harsh conditions of winemaking together with low pH, high sugar content and increasing ethanol concentration. Therefore the presence of sulfite has to be considered in yeast gene expression studies to properly understand yeast behavior in technological environments such as winemaking. A reliable expression pattern can be obtained only using an appropriate reference gene set that is constitutively expressed regardless of perturbations linked to the experimental conditions. In this work we tested 15 candidate reference genes suitable for analysis of gene expression during must fermentation in the presence of sulfite. New reference genes were selected from a genome-wide expression experiment, obtained by RNA sequencing of four Saccharomyces cerevisiae wine strains grown in enological conditions. Their performance was compared to that of the most common genes used in previous studies. The most popular software based on different statistical approaches (geNorm, NormFinder and BestKeeper) were chosen to evaluate expression stability of the candidate reference genes. Validation was obtained using other wine strains by comparing normalized gene expression data with transcriptome quantification both in the presence and absence of sulfite. Among 15 reference genes tested ALG9, FBA1, UBC6 and PFK1 appeared to be the most reliable while ENO1, PMA1, DED1 and FAS2 were the worst. The most popular reference gene ACT1, widely used for S. cerevisiae gene expression studies, showed a stability level markedly lower than those of our selected reference genes. Finally, as the expression of the new reference gene set remained constant over the entire fermentation process, irrespective of the perturbation due to sulfite addition, our results can be considered also when no sulfite is added to the must. \ua9 2015 Elsevier B.V

The hidden interplay between sex and COVID-19 mortality: the role of cardiovascular calcification

Recent clinical and demographical studies on COVID-19 patients have demonstrated that men experience worse outcomes than women. However, in most cases, the data were not stratified according to gender, limiting the understanding of the real impact of gender on outcomes. This study aimed to evaluate the disaggregated in-hospital outcomes and explore the possible interactions between gender and cardiovascular calcifications. Data was derived from the sCORE-COVID-19 registry, an Italian multicentre registry that enrolled COVID-19 patients who had undergone a chest computer tomography scan on admission. A total of 1683 hospitalized patients (mean age 67 +/- 14 years) were included. Men had a higher prevalence of cardiovascular comorbidities, a higher pneumonia extension, more coronary calcifications (63% vs.50.9%, p<0.001), and a higher coronary calcium score (391 +/- 847 vs. 171 +/- 479 mm(3), p<0.001). Men experienced a significantly higher mortality rate (24.4% vs. 17%, p=0.001), but the death event tended to occur earlier in women (15 +/- 7 vs. 8 +/- 7 days, p= 0.07). Non-survivors had a higher coronary, thoracic aorta, and aortic valve calcium score. Female sex, a known independent predictor of a favorable outcome in SARS-CoV2 infection, was not protective in women with a coronary calcification volume greater than 100 mm(3). There were significant differences in cardiovascular comorbidities and vascular calcifications between men and women with SARS-CoV2 pneumonia. The differences in outcomes can be at least partially explained by the different cardiovascular profiles. However, women with poor outcomes had the same coronary calcific burden as men. The presumed favorable female sex bias in COVID-19 must therefore be reviewed in the context of comorbidities, especially cardiovascular ones

Brivaracetam as add‐on treatment in focal epilepsy: A real‐world time‐based analysis

The study assessed the clinical response to add-on brivaracetam (BRV) in real-world practice by means of time-to-baseline seizure count methodology. Patients with focal epilepsy who were prescribed add-on BRV were identified. Primary endpoint was the time-to-baseline seizure count defined as the number of days until each patient experienced the number of focal seizures that occurred in the 90 days before BRV initiation. Subgroup analysis was performed according to levetiracetam (LEV) status (naive vs prior use). Three-hundred eighty-seven patients were included. The overall median time-to-baseline seizure count was 150 (95% confidence interval [CI] = 130-175) days. The median time-to-baseline seizure count was 198 (lower limit of 95% CI = 168) days for LEV-naive patients, 126 (95% CI = 105-150) days for patients with prior LEV use and withdrawal due to insufficient efficacy, and 170 (95% CI = 128-291) days for patients who discontinued LEV due to adverse events (P = .002). The number of prior antiseizure medications (adjusted hazard ratio [adjHR] = 1.07, 95% CI = 1.02-1.13, P = .009) and baseline monthly seizure frequency (adjHR = 1.004, 95% CI = 1.001-1.008, P = .028) were independently associated with the primary endpoint. Add-on BRV improved seizure control in LEV-naive and LEV-prior patients. The time-to-baseline seizure count represents an informative endpoint alongside traditional study outcomes and designs

Brivaracetam as add-on treatment in patients with post-stroke epilepsy: real-world data from the BRIVAracetam add-on First Italian netwoRk Study (BRIVAFIRST)

Objective: Post-stroke epilepsy (PSE) is one of the most common causes of acquired epilepsy and accounts for about 10-15% of all newly diagnosed epilepsy cases. However, evidence about the clinical profile of antiseizure medications in the PSE setting is currently limited. Brivaracetam (BRV) is a rationally developed compound characterized by high-affinity binding to synaptic vesicle protein 2A. The aim of this study was to assess the 12-month effectiveness and tolerability of adjunctive BRV in patients with PSE treated in a real-world setting. Methods: This was a subgroup analysis of patients with PSE included in the BRIVAracetam add-on First Italian netwoRk Study (BRIVAFIRST). The BRIVAFIRST was a 12-month retrospective, multicentre study including adult patients prescribed adjunctive BRV. Effectiveness outcomes included the rates of seizure response (≥50% reduction in baseline seizure frequency), seizure-freedom, and treatment discontinuation. Safety and tolerability outcomes included the rate of treatment discontinuation due to adverse events (AEs) and the incidence of AEs. Results: Patients with PSE included in the BRIVAFIRST were 75 and had a median age of 57 (interquartile range, 42-66) years. The median daily doses of BRV at 3, 6, and 12 months from starting treatment were 100 (100-150) mg, 125 (100-200) mg and 100 (100-200) mg, respectively. At 12 months, 32 (42.7%) patients had a reduction in their baseline seizure frequency by at least 50%, and the seizure freedom rates was 26/75 (34.7%). During the 1-year study period, 10 (13.3%) patients discontinued BRV. The reasons of treatment withdrawal were insufficient efficacy in 6 (8.0%) patients and poor tolerability in 4 (5.3%) patients. Adverse events were reported by 13 (20.3%) patients and were rated as mild in 84.6% and moderate in 15.4% of cases. Significance: Adjunctive BRV was efficacious and generally well-tolerated when used in patients with PSE in clinical practice. Adjunctive BRV can be a suitable therapeutic option for patients with PSE

Adjunctive Brivaracetam in Older Patients with Focal Seizures: Evidence from the BRIVAracetam add‑on First Italian netwoRk Study (BRIVAFIRST)

BACKGROUND: The management of epilepsy in older adults has become part of daily practice because of an aging population. Older patients with epilepsy represent a distinct and more vulnerable clinical group as compared with younger patients, and they are generally under-represented in randomized placebo-controlled trials. Real-world studies can therefore be a useful complement to characterize the drug's profile. Brivaracetam is a rationally developed compound characterized by high-affinity binding to synaptic vesicle protein 2A and approved as adjunctive therapy for focal seizures in adults with epilepsy. OBJECTIVE: The aim of this study was to assess the 12-month effectiveness and tolerability of adjunctive brivaracetam in older patients (≥65 years of age) with epilepsy treated in a real-world setting. METHODS: The BRIVAFIRST (BRIVAracetam add-on First Italian netwoRk STudy) was a 12-month retrospective multicenter study including adult patients prescribed adjunctive brivaracetam. Effectiveness outcomes included the rates of seizure response (≥50% reduction in baseline seizure frequency), seizure freedom, and treatment discontinuation. Safety and tolerability outcomes included the rate of treatment discontinuation due to adverse events and the incidence of adverse events. Data were compared for patients aged ≥65 years of age ('older') vs those aged <65 years ('younger'). RESULTS: There were 1029 patients with focal epilepsy included in the study, of whom 111 (10.8%) were aged ≥65 years. The median daily dose of brivaracetam at 3 months was 100 [interquartile range, 100-175] mg in the older group and 100 [100-200] mg in the younger group (p = 0.036); it was 150 [100-200] mg in both groups either at 6 months (p = 0.095) or 12 months (p = 0.140). At 12 months, 49 (44.1%) older and 334 (36.4%) younger patients had a reduction in their baseline seizure frequency by at least 50% (p = 0.110), and the seizure freedom rates were 35/111 (31.5%) and 134/918 (14.6%) in older and younger groups, respectively (p < 0.001). During the 1-year study period, 20 (18.0%) patients in the older group and 245 (26.7%) patients in the younger group discontinued brivaracetam (p = 0.048). Treatment withdrawal because of insufficient efficacy was less common in older than younger patients [older: n = 7 (6.3%), younger: n = 152 (16.6%); p = 0.005]. Adverse events were reported by 24.2% of older patients and 30.8% of younger patients (p = 0.185); the most common adverse events were somnolence, nervousness and/or agitation, vertigo, and fatigue in both study groups. CONCLUSIONS: Adjunctive brivaracetam was efficacious, had good tolerability, and no new or unexpected safety signals emerged when used to treat older patients with uncontrolled focal seizures in clinical practice. Adjunctive brivaracetam can be a suitable therapeutic option in this special population

Sustained seizure freedom with adjunctive brivaracetam in patients with focal onset seizures

The maintenance of seizure control over time is a clinical priority in patients with epilepsy. The aim of this study was to assess the sustained seizure frequency reduction with adjunctive brivaracetam (BRV) in real-world practice. Patients with focal epilepsy prescribed add-on BRV were identified. Study outcomes included sustained seizure freedom and sustained seizure response, defined as a 100% and a >= 50% reduction in baseline seizure frequency that continued without interruption and without BRV withdrawal through the 12-month follow-up. Nine hundred ninety-four patients with a median age of 45 (interquartile range = 32-56) years were included. During the 1-year study period, sustained seizure freedom was achieved by 142 (14.3%) patients, of whom 72 (50.7%) were seizure-free from Day 1 of BRV treatment. Sustained seizure freedom was maintained for >= 6, >= 9, and 12 months by 14.3%, 11.9%, and 7.2% of patients from the study cohort. Sustained seizure response was reached by 383 (38.5%) patients; 236 of 383 (61.6%) achieved sustained >= 50% reduction in seizure frequency by Day 1, 94 of 383 (24.5%) by Month 4, and 53 of 383 (13.8%) by Month 7 up to Month 12. Adjunctive BRV was associated with sustained seizure frequency reduction from the first day of treatment in a subset of patients with uncontrolled focal epilepsy