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Background: Different studies, mostly with limited cohorts, have suggested the effects of patients' characteristics on levodopa (LD) pharmacokinetics., Objective: We primarily aimed at investigating in a large population the relationship between patients' features and LD kinetic variables, to assess the main demographic and clinical predictors of LD clinical pharmacokinetics., Methods: The study was retrospective, based on data collected from subjects with parkinsonism on chronic LD undergoing LD therapeutic monitoring (TM). LD TM includes serial quantitative motor tests and blood samples to measure plasma drug concentrations after each subject's chronically taken first-morning LD dose intake., Results: Five hundred patients, 308 males (61.6%), mean (SD) age of 65 (10.1) years were included. Parkinsonian symptoms and LD therapy lasted 5.5 (4.5) and 3.4 (3.9) years, respectively. MDS-UPDRS part III "off" score was 28.8 (15.2). LD dose was 348.2 (187.1) mg/day. From multiple linear regression analysis, test dose, sex, type of LD decarboxylase inhibitor, weight and MDS-UPDRS part III score were linear predictors of both LD peak plasma concentration (Cmax) (R2 = 0.52) and area under the 3-h plasma concentration-time curve (AUC) (R2 = 0.71), while age was a further predictor only for AUC. Besides test dose, sex was the strongest independent contributing variable to LD AUC, which resulted 27% higher in females compared to males., Conclusion: This is the largest collection of data on the relationship between demographic and clinical-therapeutic variables and LD kinetics in patients with parkinsonian symptoms. As a main clinically practical finding, women might require a 25% reduced weight-normalized LD dose compared with men to achieve the same LD bioavailability.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A gene (GLA) mutations, resulting in loss of activity of the lysosomal hydrolase, α-galactosidase A (α-Gal A). As a result, the main glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), accumulate in plasma, urine, and tissues. Here, we propose a simple, fast, and sensitive method for plasma quantification of lyso-Gb3, the most promising secondary screening target for FD. Assisted protein precipitation with methanol using Phree cartridges was performed as sample pre-treatment and plasma concentrations were measured using UHPLC-MS/MS operating in MRM positive electrospray ionization. Method validation provided excellent results for the whole calibration range (0.25-100 ng/mL). Intra-assay and inter-assay accuracy and precision (CV%) were calculated as <10%. The method was successfully applied to 55 plasma samples obtained from 34 patients with FD, 5 individuals carrying non-relevant polymorphisms of the GLA gene, and 16 healthy controls. Plasma lyso-Gb3 concentrations were larger in both male and female FD groups compared to healthy subjects ( p < 0.001). Normal levels of plasma lyso-Gb3 were observed for patients carrying non-relevant mutations of the GLA gene compared to the control group ( p = 0.141). Dropping the lower limit of quantification (LLOQ) to 0.25 ng/mL allowed us to set the optimal plasma lyso-Gb3 cut-off value between FD patients and healthy controls at 0.6 ng/mL, with a sensitivity of 97.1%, specificity of 100%, and accuracy of 0.998 expressed by the area under the ROC curve (C.I. 0.992 to 1.000, p -value < 0.001). Based on the results obtained, this method can be a reliable tool for early phenotypic assignment, assessing diagnoses in patients with borderline GalA activity, and confirming non-relevant mutations of the GLA gene.

Introduction: Epilepsy is a chronic condition requiring consistent follow-up aimed at seizure control, and monitoring of anti-seizure medication (ASM) levels and side effects. Telemedicine (TM) offers invaluable support to patient follow-up, guaranteeing the prompt availability of a team of experts for persons with epilepsy (PWE) widely distributed across the country. Although many health institutions have endorsed the use of TM, robust data on effectiveness, safety and costs of TM applied to epilepsy are lacking. TELEmedicine for EPIlepsy Care (TELE-EPIC) will evaluate the effectiveness of video consultation (VC) via TM compared with usual care (UC) for the monitoring of PWE (TELE-EPIC&#95;RCT). Moreover, TELE-EPIC will apply an innovative Volumetric Absorptive Microsampling (VAMS) device for quantitation of ASM through finger prick blood sampling as an alternative to venipuncture sampling (TELE-EPIC&#95;VAMS)., Methods and Analysis: TELE-EPIC&#95;RCT is a multicentre, open, pragmatic two-arm randomised controlled trial prospectively including adult and paediatric outpatients with established diagnosis of epilepsy consecutively attending the Epilepsy Centres of Bologna and Rome, respectively. The primary outcome is the non-inferiority of VC on seizure control compared with UC after an 18-month follow-up. Secondary outcomes are adherence to treatment, ASM-related adverse events, quality of life, mood disorders, patient and caregiver satisfaction, safety and costs. TELE-EPIC&#95;VAMS is a cross-validation study for blood ASM quantitation through a novel, VAMS-based device, comparing (1) VAMS versus plasma samples (reference standard method); and (2) nurse-collected versus self-collected blood by VAMS device., Ethics and Dissemination: The study has been approved by the local ethics committee (349-2019-SPER-AUSLBO). Complete information about the state of project, relevant events and results will be regularly updated on the project's webpage on ClinicalTrials.gov. The project's results and data on the potential impact of TM in epilepsy will be disseminated on social media. A closeout meeting will be convened for the communication and dissemination of the project, highlighting its main achievements and impacts., Trial Registration Number: NCT04496310., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Two soils contaminated with potentially toxic metals (PTMs) contrasting in pH and mineralogy were remediated with CaEDTA, and changes in soil organic matter (SOM) composition were investigated. Previous studies showed no significant loss of SOM from CaEDTA-treated soils, but the results of our study reflected significant decreases (from 46 to 49%) in the free fraction of humic acids (HAs). Remediation affected the composition of the free HA fraction via disturbance of intermolecular bonds - an increase in phenolic and aromatic groups with a simultaneous decrease in carbohydrates - which was confirmed by FTIR spectroscopy in both soils. Because non-radical molecules such as carbohydrates were selectively removed, the concentration of free radicals in the free HA fraction increased in acidic soil. The bound fraction of HAs and fulvic acids (FAs) in SOM, which are important due to their stability and the permanent effects they have on the soil's physical properties, remained unchanged in both remediated soils. The effect of soil recultivation was observed only in the excitation emission matrix (EEM) fluorescence spectra of HAs. In terms of SOM, CaEDTA soil washing can be considered moderately conservative; however, the restoration of free humic fractions is likely to be a long-term process., (© 2021. The Author(s).)

The N 2 -fixing shrub Amorpha fruticosa L. is rapidly spreading in the dry riparian natural grasslands of Europe, altering ecosystem functions and depleting plant diversity. Alteration of the N cycle represents the key factor involved in invasions by N 2 -fixing plants with cascading effects on plant species richness. We hypothesized that A. fruticosa encroachment strongly impacts not only the N but also the C cycle and that the magnitude of such alterations may be modulated by soil characteristics. To test these hypotheses, we selected four river floodplains in North East of Italy and compared natural uninvaded grasslands with half invaded and completely invaded sites, based on A. fruticosa stand characteristic and relevant leaf traits and on soil properties related to soil texture and to C and N cycles. Soil organic matter mineralisation, ammonification and nitrification rates were determined. Soil nitrification increased remarkably with plant invasion while ammonification was significantly higher only in half invaded sites. Soil organic matter mineralisation, microbial biomass C sustained per soil organic C unit and nitrification positively correlated with stand age, regardless to the stage of the encroachment. Mineralisation and nitrification increased with soil organic C and total N in uninvaded and completely invaded sites, but decreased in half invaded sites. At the half invasion stage, trends in nitrification and CO 2 mineralisation were transitionally reverted and remediation may be facilitated by less pronounced changes in soil properties compared to completely invaded sites. Direct effects of plant invasion are modulated by the action of soil characteristics such as soil organic C and clay contents, with soils rich in organic C showing larger nitrification and mineralisation rates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: "Impulse Control Disorders" are behavioral conditions (e.g., gambling, hypersexuality), which are increasingly reported as reactions to dopamine agonists in Parkinson's disease. The Questionnaire for Impulsive-Compulsive Disorders in Parkinson's disease focuses only on 6 behaviors. Nonetheless, impulsivity could affect the entire range of human practices. Because of their heterogeneity and undefined boundaries, it is not clear what conditions should be considered as Impulse Control Disorders. This results in poorly standardized scientific literature and underdiagnosis., Objective: We aimed to create a comprehensive list of possible manifestations of drug-induced Impulse Control Disorders in Parkinson's disease and test it on pharmacosurveillance data., Methods: PubMed was used to identify reviews in English about Impulse Control Disorders in Parkinson's disease. Mentioned conditions were charted and translated to the lexicon of MedDRA, ICD-11, and DSM-5. The relevant MedDRA terms were used to test potential association with dopamine agonists on the FDA Adverse Event Reporting System., Results: 50 reviews published between 2001 and 2020 were identified. 66 conditions were collected as possible Impulse Control Disorders. Pathological gambling, shopping, eating and sexuality, dopamine dysregulation syndrome, hobbyism and punding were the most frequently mentioned, together with leisure activities, body-focused compulsivity, disruptive, impulse control and conduct disorders, and substance abuse. All these conditions were disproportionately reported with dopamine agonists, except for substance abuse., Conclusions: We defined a potential extended list of ICDs, which, along with its conversion to international taxonomies, can support the identification of drug-induced conditions in pharmacovigilance archives, as well as monitoring processes in clinical practice., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Brown carbon (BrC) is involved in atmospheric light absorption and climate forcing and can cause adverse health effects. Understanding the formation mechanisms and molecular structure of BrC is of key importance in developing strategies to control its environment and health impact. Structure determination of BrC is challenging, due to the lack of experiments providing molecular fingerprints and the sheer number of molecular candidates with identical mass. Suggestions based on chemical intuition are prone to errors due to the inherent bias. We present an unbiased algorithm, using graph-based molecule generation and machine learning, which can identify all molecular structures of compounds involved in biomass burning and the composition of BrC. We apply this algorithm to C 12 H 12 O 7 , a light-absorbing "test case" molecule identified in chamber experiments on the aqueous photo-oxidation of syringol, a prevalent marker in wood smoke. Of the 260 million molecular graphs, the algorithm leaves only 36,518 (0.01%) as viable candidates matching the spectrum. Although no unique molecular structure is obtained from only a chemical formula and a UV/vis absorption spectrum, we discuss further reduction strategies and their efficacy. With additional data, the method can potentially more rapidly identify isomers extracted from lab and field aerosol particles without introducing human bias.

Background: Purified cannabidiol (CBD) was administered to highly refractory patients with Dravet (DS) or Lennox-Gastaut (LGS) syndromes in an ongoing expanded access program (EAP). Herein, we report interim results on CBD safety and seizure outcomes in patients treated for a 12-month period. Material and Methods: Thirty centers were enrolled from December 2018 to December 2019 within the open-label prospective EAP up to a maximum of 25 mg/kg per day. Adverse effects and liver function tests were assessed after 2 weeks; 1, 3, and 6 months of treatment; and periodically thereafter. Seizure endpoints were the percentage of patients with ≥50 and 100% reduction in seizures compared to baseline. Results: A total of 93 patients were enrolled and included in the safety analysis. Eighty-two patients [27 (32.9%) DS, 55 (67.1%) LGS] with at least 3 months of treatment have been included in the effectiveness analysis; median previously failed antiseizure medications was eight. Pediatric and adult patients were uniformly represented in the cohort. At 3-month follow-up, compared to the 28-day baseline period, the percentage of patients with at least a 50% reduction in seizure frequency was 40.2% (plus 1.2% seizure-free). Retention rate was similar according to diagnosis, while we found an increased number of patients remaining under treatment in the adult group. CBD was mostly coadministered with valproic acid (62.2%) and clobazam (41.5%). In the safety dataset, 29 (31.2%) dropped out: reasons were lack of efficacy [16 (17.2%)] and adverse events (AEs) [12 (12.9%)], and one met withdrawal criteria (1.1%). Most reported AEs were somnolence (22.6%) and diarrhea (11.9%), followed by transaminase elevation and loss of appetite. Conclusions: CBD is associated with improved seizure control also in a considerable proportion of highly refractory patients with DS and LGS independently from clobazam use. Overall, CBD safety and effectiveness are not dose-related in this cohort., Competing Interests: FB has participated in clinical trials for GW Pharmaceuticals; received research grants, speaker fees or participated to advisory boards for Eisai, Cyberonics, UCB Pharma and Bial. MPC Canevini has participated advisory boards and/or received research fundings from UCB Pharma, Eisai, Italfarmaco, Cyberonics, Novartis, and the European Union. AC has received speaker fees by Eisai. CB has received speaker fees from Eisai, UCB Pharma, FB Health and Sandoz. Gd'O has served on the advisory board of Eisai. CG has received research grants and/or speaker fees from UCB Pharma, Eisai and Bial. TG received a speaker fee from GW Pharmaceuticals. RG has received consulting fees and speaker honoraria from Zogenix, Biomarin, UCB, Eisai, Novartis, GW Pharma, and Biocodex. OM has received consulting fees and speaker honoraria by Bial, Eisai, GW Pharmaceuticals and UCB Pharma. AL has received speaker's or consultancy fees from Eisai, Mylan, Sanofi, Bial, GW Pharmaceuticals and UCB Pharma. PP received speaker's fees from Eisai and UCB Pharma. AR received consulting fees from GW Pharmaceuticals. ER has received speaker fees and/or fundings and has participated in advisory boards for Eisai, Pfizer, GW Pharmaceuticals, UCB Pharma, Arvelle Therapeutics. NS has received grant support and fees for advisory board participation from GW Pharmaceuticals. PS developed within the framework of the DINOGMI Department of Excellence of MIUR 2018-2022 (legge 232 del 2016) and received speaker fees and participated at advisory boards for Biomarin, Zogenyx, GW Pharmaceuticals, Neuraxpharma; he also received research funding by ENECTA BV, GW Pharmaceuticals, Kolfarma srl., Eisai. ET has received speaker's fees from Eisai and Sandoz. AV received speaker's fees from Eisai, Italfarmaco, and GW Pharmaceuticals. MV received speaker's fees from Eisai and GW Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Iannone, Arena, Battaglia, Bisulli, Bonanni, Boni, Canevini, Cantalupo, Cesaroni, Contin, Coppola, Cordelli, Cricchiuti, De Giorgis, De Leva, De Rinaldis, d'Orsi, Elia, Galimberti, Morano, Granata, Guerrini, Lodi, La Neve, Marchese, Masnada, Michelucci, Nosadini, Pilolli, Pruna, Ragona, Rosati, Santucci, Spalice, Pietrafusa, Striano, Tartara, Tassi, Papa, Zucca, Russo, Mecarelli and The CBD LICE Italy Study Group.)

Background and Aim: Data on the clinical pharmacokinetics of cannabidiol (CBD) are scanty. We explored the effect of demographic and clinical variables on plasma concentrations of purified CBD in patients with Dravet (DS) and Lennox-Gastaut syndrome (LGS). Methods: The study design was an open, prospective, multicenter expanded access program (EAP). Venous blood samples were drawn from patients between 8 and 9 am, before the CBD morning dose, 12 h apart from the last evening dose, and then 2.5 h after their usual morning dose. Results: We collected 127 plasma samples (67-morning pre-dosing and 60 post-dosing) from 43 patients (24 females, 19 males), 27 with LGS and 16 with DS. Mean ± standard deviation age was 26 ± 15 years. Duration of CBD treatment averaged 4.2 ± 2.9 months at 13.2 ± 4.6 mg/kg/day. CBD median trough plasma concentration was 91 ng/ml; it doubled to 190 ng/ml 2.5 h post-dosing ( p < 0.001). Cannabidiol trough plasma concentrations were linearly related to daily doses (r = 0.564, p < 0.001). Median trough CBD plasma concentration-to-weight-adjusted dose ratio (C/D) was 32% higher ( p < 0.02) in plasma samples from subjects aged 18 and over than in those under 18. Sex and concomitant antiseizure medications (ASMs) were not associated with significant variations in CBD C/D, but caution is required due to the potential influence of confounders. Conclusion: These are the first data on CBD pharmacokinetics in children and adults with LGS or DS in a real-world setting. The most relevant finding was the higher CBD C/D in adults. In practice, reduced weight-normalized doses might be required with aging to achieve the same CBD plasma levels., Competing Interests: OM has received consulting fees and speaker honoraria by Bial, Eisai, GW Pharmaceuticals, and UCB Pharma. ER has received speaker fees and/or fundings and has participated in advisory boards for Eisai, Pfizer, GW Pharmaceuticals, UCB Pharma, Arvelle Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Contin, Mohamed, Santucci, Lodi, Russo, Mecarelli and CBD LICE Italy Study Group.)

Background: Differential diagnosis between Parkinson's disease (PD) and atypical parkinsonisms (APs) may be difficult at disease onset. The response to levodopa (LD) is a key supportive feature but its definition is largely empirical. Studies evaluating this issue by quantitative tests are scanty., Objective: We aimed to assess the utility of a subacute low LD dose kinetic-dynamic test in the differential diagnosis between PD and APs. It was applied at the baseline of a prospective follow-up in patients with parkinsonian signs within three years of disease motor onset ("BoProPark" cohort) and eventually diagnosed as PD or APs according to consensus criteria., Methods: Patients under at least 3-month LD therapy received a first morning fasting dose of LD/benserazide or carbidopa (100/25 mg) and underwent simultaneous serial assessments of plasma LD concentration and alternate finger tapping frequency up to 3 h. The main outcome was the extent of LD motor response, calculated by the area under the 3 h tapping effect-time curve (AUC&#95;ETap). A receiver operating characteristic (ROC) curve analysis was performed to establish the optimal AUC&#95;ETap cut-off to differentiate PD and APs., Results: The first 100 consecutive "BoProPark" patients were analyzed. Forty-seven patients were classified as possible, 37 as probable PD and 16 as APs. AUC&#95;ETap medians were similar in the PD subgroups but reduced to a third in APs (p < 0.001). The optimal AUC&#95;ETap cut-off value was >2186 [(tap/min) x min], with a sensitivity of 92% and a specificity of 75%. Accuracy of the test was 0.85 (95% CI 0.74-0.95), p < 0.0001., Conclusion: The estimation of 3 h AUC&#95;ETap after a subacute low LD dose proved a reliable, objective tool to assess LD motor response in our cohort of patients. AUC&#95;ETap value rounded to ≥2200 supports PD diagnosis, while lower values may alert to AP diagnoses.

We report the longest follow-up of clinical and biochemical features of two previously reported adult mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients treated with liver transplantation (LT), adding information on a third, recently transplanted, patient. All three patients overcame the early post-operative period and tolerated immunosuppressive therapy. Plasma nucleoside levels dramatically decreased, with evidence of clinical improvement of ambulation and neuropathy. Conversely, other features of MNGIE, as gastrointestinal dysmotility, low weight, ophthalmoparesis, and leukoencephalopathy were essentially unchanged. A similar picture characterized two patients treated with allogenic hematopoietic stem cell transplantation (AHSCT). In conclusion, LT promptly and stably normalizes nucleoside imbalance in MNGIE, stabilizing or improving some clinical parameters with marginal periprocedural mortality rate as compared to AHSCT. Nevertheless, restoring thymidine phosphorylase (TP) activity, achieved by both LT and AHSCT, does not allow a full clinical recovery, probably due to consolidated cellular damage and/or incomplete enzymatic tissue replacement.

Purpose: The purpose of the study was to investigate the potential correlation between plasma concentration of the newer antiseizure medication (ASM) perampanel (PMP) and both tolerability and seizure control in patients with epilepsy., Methods: The study design was multicenter, open, and prospective. Plasma samples were collected in the morning 12 h apart from once-a-day bedtime PMP dose. Perampanel tolerability was assessed on the day of drug monitoring by clinical examination and patients' interview. Response to PMP was defined as ≥50% reduction from baseline seizure frequency (pretreatment). The main outcomes were the comparisons of PMP plasma concentration-to-weight-adjusted dose ratio (C/D) [(μg/mL)/(mg/kg/day)] between patients with and without PMP-related adverse effects (AEs) and between responders and nonresponders., Results: Ninety-seven patients (54% men), mean ± SD age 36 ± 14 years were enrolled in the study. The mean PMP dose was 6.7 ± 2.3 mg, drug treatment averaged 46 ± 34 weeks. The mean plasma concentration was 360 ± 268 ng/mL (range: 37-1213 ng/mL). Forty patients (41%) reported at least one AE, mainly dizziness and behavioral changes. No significant difference was found in median PMP C/Ds between patients with (2.94) and without (2.76) AEs, otherwise comparable for clinical variables. Forty-four patients (45%) were responders, at a median PMP C/D of 3.10, similar to the value of 2.76 found in nonresponders. These two groups also overlapped for clinical characteristics., Conclusion: This is the first prospective real-life study to evaluate the relationship between PMP plasma concentrations, seizure control, and AEs. In line with the few real-world available data, we did not find any significant correlation between PMP plasma concentrations and both tolerability and seizure control., Competing Interests: Declaration of competing interest Dr. Bisulli has participated in clinical trials for GW; received research grants from Eisai; received support for travel to congresses from Cyberonics, Eisai, UCB and Bial; participated in advisory boards for Eisai and UCB; has received speaker honoraria from Eisai and UCB. Dr. Licchetta received support for travel to congresses from Eisai. Dr. Tinuper has participated in clinical trials for GW, UCB and LivaNova; received research grants from Eisai, UCB and Livanova; received support for travel to congresses from Cyberonics, Eisai, UCB and Bial; participated in advisory boards for Eisai, UCB, LivaNova and GW; has received speaker honoraria from Eisai, UCB and LivaNova. For the remaining authors none were declared., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Levodopa-carbidopa intestinal gel (LCIG) treatment has shown variable effect on dyskinesia in Parkinson's disease (PD)., Objective: To identify PD patients who are likely to have troublesome dyskinesia under LCIG treatment and describe the pharmacokinetic-dynamic profile and dyskinesia phenomenology of those patients., Methods: PD patients were assessed for clinical and therapeutic variables, before LCIG treatment ( T0 ) and at last outpatient visit ( T1 ). Sub-groups of patients with and without "troublesome dyskinesia" (UPDRS IV, item 33 ≥2), matched for disease and LCIG treatment duration, underwent a pharmacokinetic-dynamic assessment., Results: We included 53 PD patients. After a mean of 51.7 ± 34.1 months of LCIG treatment, "off-time" was significantly reduced, whereas, dyskinesia duration/disability did not change. The multivariate regression model, adjusted for LCIG treatment duration, showed that being female increases the risk of presenting troublesome dyskinesia at T1 (odds ratio [OR] = 9.2; 95% confidence interval [CI] = 2.4-37.4) that was also significantly associated to longer off periods at T1 (OR= 4.4; 95% CI = 1.1-14.3). Female patients showed a higher risk for a higher dyskinesia score at T1 (sum of the items 32 and 33: P = 0.001). Patients with troublesome dyskinesia showed a tendency for a lower motor benefit and the appearance of more severe dyskinesia despite similar levodopa plasma concentration., Conclusion: Dyskinesia should be carefully monitored in patients undergoing LCIG, with particular caution for female patients. Whether combined clinical and pharmacodynamic assessments could be helpful to manage patients with troublesome dyskinesia under LCIG treatment needs further evaluation in a larger group of patients., Competing Interests: The study had no specific funding. The authors report no conflict of interest., (© 2020 International Parkinson and Movement Disorder Society.)

The involvement of calcium-dependent cytosolic phospholipase A2α (cPLA2α) in aortic valve calcification is not exhaustively elucidated. Here, cPLA2α expression in aortic valve interstitial cell (AVIC) pro-calcific cultures simulating either metastatic or dystrophic calcification was estimated by qPCR, Western blotting, and counting of cPLA2α-immunoreactive cells, with parallel ultrastructural examination of AVIC calcific degeneration. These evaluations also involved pro-calcific AVIC cultures treated with cPLA2α inhibitor dexamethasone. cPLA2α over-expression resulted for both types of pro-calcific AVIC cultures. Compared to controls, enzyme content was found to increase by up to 300% and 186% in metastatic and dystrophic calcification-like cultures, respectively. Increases in mRNA amounts were also observed, although they were not as striking as those in enzyme content. Moreover, cPLA2α increases were time-dependent and strictly associated with mineralization progression. Conversely, drastically lower levels of enzyme content resulted for the pro-calcific AVIC cultures supplemented with dexamethasone. In particular, cPLA2α amounts were found to decrease by almost 88% and 48% in metastatic and dystrophic calcification-like cultures, respectively, with mRNA amounts showing a similar trend. Interestingly, these drastic decreases in cPLA2α amounts were paralleled by drastic decreases in mineralization degrees, as revealed ultrastructurally. In conclusion, cPLA2α may be regarded as a crucial co-factor contributing to AVIC mineralization in vitro, thus being an attractive potential target for designing novel therapeutic strategies aimed to counteract onset or progression of calcific aortic valve diseases.

Objective: The Bologna motor and non-motor prospective study on parkinsonism at onset (BoProPark) was designed to prospectively characterize motor and non-motor features in patients with a progressive neurodegenerative disease starting with parkinsonism since early disease stage and to investigate their diagnostic and prognostic role in the differential diagnosis of Parkinson's disease from atypical parkinsonisms. The aim of this paper is to describe the method and population of the BoProPark study., Methods: Patients referred to our Department with parkinsonism within 3 years from motor onset were recruited. Secondary causes of parkinsonism were excluded. Each patient underwent a comprehensive evaluation of motor and non-motor symptoms, assessed by means of quantitative, objective instrumental tests in addition to scales and questionnaires. The evaluations were performed at enrolment (T0), after 16 months (T1) and after 5 years (T2). Diagnoses were made according to consensus criteria., Results: We recruited 150 patients, with mean age 61.5 ± 9.8 years and mean disease duration 20 ± 9 months. H&Y stage was 1 in 47.3% and 2 in 46.7% of cases. Mean UPDRS-III was 17.7 ± 9.2. Fifty-four patients were on dopaminergic treatment with median levodopa equivalent daily dose (LEDD) of 200 mg., Conclusions: We expect that the prospective nature of the BoProPark study as well as the comprehensive, instrumental evaluation of motor and non-motor symptoms in patients with parkinsonism will provide important new insights for both clinical practice and research. Our data could be used for comparison with other cohorts and shared with national and international collaborators to develop new innovative projects.

Biallelic TYMP variants result in the mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a juvenile-onset disorder with progressive course and fatal outcome. Milder late-onset (>40 years) form has been rarely described. Gene panel sequencing in a cohort of 60 patients featuring muscle accumulation of mitochondrial DNA (mtDNA) deletions detected TYMP defects in three subjects (5%), two of them with symptom onset in the fifth decade. One of the patients only displayed ptosis and ophthalmoparesis. Biochemical and molecular studies supported the diagnosis. Screening of TYMP is recommended in adult patients with muscle mtDNA instability, even in the absence of cardinal MNGIE features., (Copyright © 2020 Ronchi, Caporali, Manenti, Meneri, Mohamed, Bordoni, Tagliavini, Contin, Piga, Sciacco, Saetti, Carelli and Comi.)

ADCA-DN and HSN-IE are rare neurodegenerative syndromes caused by dominant mutations in the replication foci targeting sequence (RFTS) of the DNA methyltransferase 1 (DNMT1) gene. Both phenotypes resemble mitochondrial disorders, and mitochondrial dysfunction was first observed in ADCA-DN. To explore mitochondrial involvement, we studied the effects of DNMT1 mutations in fibroblasts from four ADCA-DN and two HSN-IE patients. We documented impaired activity of purified DNMT1 mutant proteins, which in fibroblasts results in increased DNMT1 amount. We demonstrated that DNMT1 is not localized within mitochondria, but it is associated with the mitochondrial outer membrane. Concordantly, mitochondrial DNA failed to show meaningful CpG methylation. Strikingly, we found activated mitobiogenesis and OXPHOS with significant increase of H2O2, sharply contrasting with a reduced ATP content. Metabolomics profiling of mutant cells highlighted purine, arginine/urea cycle and glutamate metabolisms as the most consistently altered pathways, similar to primary mitochondrial diseases. The most severe mutations showed activation of energy shortage AMPK-dependent sensing, leading to mTORC1 inhibition. We propose that DNMT1 RFTS mutations deregulate metabolism lowering ATP levels, as a result of increased purine catabolism and urea cycle pathways. This is associated with a paradoxical mitochondrial hyper-function and increased oxidative stress, possibly resulting in neurodegeneration in non-dividing cells., (© The Author(s) 2020. Published by Oxford University Press.)

Background: Brivaracetam is an antiepileptic drug used as an add-on therapy for partial-onset seizures in subjects aged 4 years and older. Owing to potential drug interactions and intersubject variability in plasma concentrations, therapeutic monitoring for brivaracetam may be useful. The aim of this study was to develop a simplified method for measuring brivaracetam plasma concentrations applicable to therapeutic drug monitoring in epilepsy., Methods: An ultra high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated according to current guidelines for bioanalytical methods. Sample preparation (100 µL) involved only a simple precipitation step by acetonitrile. Brivaracetam-d7 was used as internal standard. The chromatographic analysis was performed by a Synergi Fusion column using 0.1% formic acid in water/acetonitrile as a binary gradient mobile phase, at a flow rate of 0.3 mL/min. Both brivaracetam and the internal standard eluted at 1.01 minutes. This method was applied to measure trough and 1-hour postmorning dose brivaracetam plasma concentrations of 11 patients with epilepsy., Results: The method was validated over a concentration range of 0.10-10 mcg/mL. The mean recovery was 95%. Both intra- and inter-assay imprecision and inaccuracy were <15% for all quality control samples. The lower limit of quantitation and detection was 0.10 and 0.05 mcg/mL, respectively. No interferences or carry-over was observed. Median (25%-75% quartiles) trough and 1-hour postdosing brivaracetam plasma concentrations were 0.61 mcg/mL (0.47-0.83 mcg/mL) and 1.55 mcg/mL (1.24-2.12 mcg/mL), respectively, at a median dose of 80 mg/d (50-150 mg/d). Large, up to 8-fold, intrasubject fluctuations of brivaracetam concentrations between trough and 1-hour postdosing were observed., Conclusions: The present assay is faster and simpler than previously published analytical reports for brivaracetam in human plasma and is suitable for therapeutic drug monitoring.

Coenzyme Q10 (CoQ 10 ) is essential in mitochondrial bioenergetics and is a potent endogenous antioxidant. Low CoQ 10 levels are associated with neurodegenerative, metabolic, muscular and cardiovascular disorders. Early treatment with high doses (5-50 mg/kg/day) demonstrated to limit the onset and progression of neuropathology. Recently, we developed an oleogel matrix able to support a high dose of oil-dissolved CoQ 10 , easy to swallow by CoQ 10 -deficient patients who suffer from secondary dysphagia. In the present study, we evaluated the bioavailability of oleogel-dissolved CoQ 10 and plasma antioxidant status in healthy adults in single-dose and repeated-dose studies. The single-dose study demonstrated that, in terms of CoQ 10 bioavailability, 1 g CoQ 10 /5g oleogel-disk was equivalent to the solid form (1 g CoQ 10 /three 00-size-capsules), whereas the repeated-dose study (14-days-administration) demonstrated a significantly higher increase in plasma CoQ 10 when administered through the oleogel, which could be compatible with the levels necessary to achieve an adequate therapeutic response. Also, a trend to a higher plasma apparent half-life (greater than24 h) was observed for the oleogel-loaded-CoQ 10 . In conclusion, the oleogel matrix does not compromise the oil-dissolved CoQ 10 bioavailability and can prevent the non-adherence to this vital supplementation in patients with high CoQ 10 requirements. No significant variation in the plasma antioxidant status (vitamins A, E and C, glutathione and TBARs) was observed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Parkinson disease (PD) is characterized by a clear beneficial motor response to levodopa (LD) treatment. However, with disease progression and longer LD exposure, drug-related motor fluctuations usually occur. Recognition of the individual relationship between LD concentration and its effect may be difficult, due to the complexity and variability of the mechanisms involved. This work proposes an innovative procedure for the automatic estimation of LD pharmacokinetics and pharmacodynamics parameters, by a biologically-inspired mathematical model. An original issue, compared with previous similar studies, is that the model comprises not only a compartmental description of LD pharmacokinetics in plasma and its effect on the striatal neurons, but also a neurocomputational model of basal ganglia action selection. Parameter estimation was achieved on 26 patients (13 with stable and 13 with fluctuating LD response) to mimic plasma LD concentration and alternate finger tapping frequency along four hours after LD administration, automatically minimizing a cost function of the difference between simulated and clinical data points. Results show that individual data can be satisfactorily simulated in all patients and that significant differences exist in the estimated parameters between the two groups. Specifically, the drug removal rate from the effect compartment, and the Hill coefficient of the concentration-effect relationship were significantly higher in the fluctuating than in the stable group. The model, with individualized parameters, may be used to reach a deeper comprehension of the PD mechanisms, mimic the effect of medication, and, based on the predicted neural responses, plan the correct management and design innovative therapeutic procedures., Competing Interests: The authors have declared that no competing interests exist.

A volumetric microsampling (VAMS) device (20 μl) was evaluated and validated for the analysis of γ-hydroxybutyric acid (GHB) in venous blood using a simple ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. GHB was extracted from VAMS device by acetonitrile, after a re-hydration step in a temperature-controlled ultrasonic bath at 60°C for 10 min. Chromatographic analysis was carried out on a Kinetex C 18 column using 0.1% formic acid in water and acetonitrile as binary gradient mobile phase (from 5 to 95% of acetonitrile from 1 to 2.5 min) at a flow rate of 0.3 ml/min. The VAMS method was fully validated according to current guidelines with satisfactory results in terms of linearity, selectivity, precision, absolute recovery, matrix effect and stability. The linearity was determined from 0.5 to 200 μg/ml and the lower limit of quantitation was 0.5 μg/ml. The novel VAMS-UHPLC-MS/MS method was successfully compared with plasma-based method in a GHB-treated patient as a proof of concept., (© 2019 John Wiley & Sons, Ltd.)

Conversion of conventional farming (CF) to organic farming (OF) is claimed to allow a sustainable management of soil resources, but information on changes induced on dissolved organic matter (DOM) are scarce. Among DOM components, dissolved humic substances (DHS) were shown to possess stimulatory effects on plant growth. DHS were isolated from CF and OF soil leacheates collected from soil monolith columns: first in November (bare soils) and then in April and June (bare and planted soils). DHS caused an enhancement of nitrate uptake rates in maize roots and modulated several genes involved in nitrogen acquisition. The DHS from OF soil exerted a stronger biostimulant action on the nitrate uptake system, but the first assimilatory step of nitrate was mainly activated by DHS derived from CF soil. To validate the physiological response of plants to DHS exposure, real-time RT-PCR analyses were performed on those genes most involved in nitrate acquisition, such as ZmNRT2.1 , ZmNRT2.2 , ZmMHA2 (coding for two high-affinity nitrate transporters and a PM H + -proton pump), ZmNADH:NR , ZmNADPH:NR , and ZmNiR (coding for nitrate reductases and nitrite reductase). All tested DHS fractions induced the upregulation of nitrate reductase (NR), and in particular the OF2 DHS stimulated the expression of both tested transcripts encoding for two NR isoforms. Characteristics of DHS varied during the experiment in both OF and CF soils: a decrease of high molecular weight fractions in the OF soil, a general increase in the carboxylic groups content, as well as diverse structural modifications in OF vs. CF soils were observed. These changes were accelerated in planted soils. Similarity of chemical properties of DHS with the more easily obtainable water-soluble humic substance extracted from peat (WEHS) and the correspondence of their biostimulant actions confirm the validity of studies which employ WEHS as an easily available source of DHS to investigate biostimulant actions on agricultural crops., (Copyright © 2020 Vujinović, Zanin, Venuti, Contin, Ceccon, Tomasi, Pinton, Cesco and De Nobili.)

The ability of the simultaneously extracted metals/acid volatile sulfides (∑SEM/AVS) index to ascertain environmental risk from potentially toxic elements in calcareous saltmarsh soils was tested using structural equation modeling. This technique allows the detection of both direct and indirect relationships among AVS, SEM, and other soil variables, representing results in a graphical view. The dataset included 90 soil samples from 21 different sites belonging to 6 different saltmarshes and featured a wide range of soil chemicophysical properties. Variables included in the a priori model were hydroperiod, pH, soil redox potential, labile organic carbon, carbonates, total iron, and total amount of potentially toxic elements (PTEs). The best optimized model pointed out the main soil properties that affect AVS accumulation and SEM speciation in these soils. Effect plots of AVS and SEM calculated with the partial linear mixed-effects models included in the piecewise structural equation modeling showed a significant and positive influence of pH and carbonates on AVS and a highly significant effect of carbonates and labile organic carbon on SEM. Single SEM components were also considered separately, to define the potential contributions of labile organic carbon or carbonates as alternative binding phases. Simultaneously extracted Cu, Ni, and Zn were preferentially bound to carbonates, followed by labile organic carbon, whereas Pb and Cd were easily bound to labile organic carbon. Environ Toxicol Chem 2019;38:2688-2697. © 2019 SETAC., (© 2019 SETAC.)

Sildenafil is a phosphodiesterase type 5 inhibitor which confers cardioprotection against myocardial ischaemia/reperfusion (I/R) injury. The aim of this study was to determine if Trx1 participates in cardioprotection exerted by sildenafil in an acute model of I/R, and to evaluate mitochondrial bioenergetics and cellular redox status. Langendorff-perfused hearts from wild type (WT) mice and a dominant negative (DN-Trx1) mutant of Trx1 were assigned to placebo or sildenafil (0.7 mg/kg i.p.) and subjected to 30 min of ischaemia followed by 120 min of reperfusion. WT + S showed a significant reduction of infarct size (51.2 ± 3.0% vs. 30 ± 3.0%, p < .001), an effect not observed in DN-Trx. After I/R, sildenafil preserved state 3 oxygen consumption from WT, but had a milder effect in DN-Trx1 only partially protecting state 3 values. Treatment restored respiratory control (RC) after I/R, which resulted 8% (WT) and 24% (DN-Trx1) lower than in basal conditions. After I/R, a significant increase in H 2 O 2 production was observed both for WT and DN-Trx (WT: 1.17 ± 0.13 nmol/mg protein and DN-Trx: 1.38 ± 0.12 nmol/min mg protein). With sildenafil, values were 21% lower only in WT I/R. Treatment decreased GSSG levels both in WT and DN-Trx1. In addition, GSSG/GSH 2 ratio was partially restored by sildenafil. Also, an increase in p-eNOS/eNOS even before the myocardial ischaemia was observed with sildenafil, both in WT (14%, p > .05) and in DN-Trx (35%, p < .05). Active Trx1 is required for the onset of the cardioprotective effects of sildenafil on I/R injury, together with the preservation of cellular redox balance and mitochondrial function.

Objectives: The aim was to evaluate plasma and cerebrospinal fluid (CSF) nimodipine concentrations in patients with aneurysmal subarachnoid hemorrhage and their correlation with clinical outcome., Methods: Nimodipine infusion was started at 1 mg/h and increased up to 2 mg/h and continued up to 21 days in surviving patients. Arterial and CSF samples were collected at least after 24 hours of stable nimodipine dosing. Delayed cerebral ischemia and vasospasm were documented by new neurological deficits and neuroimaging. The clinical outcome was assessed at 9 months by the modified Rankin scale., Results: Twenty-three patients were enrolled. Nimodipine dose was 13 to 38 μg/kg per hour. Nimodipine arterial and CSF concentrations were 24.9 to 71.8 ng/mL and 37 to 530 pg/mL, respectively. Dose did not correlate with arterial or CSF concentrations. Arterial concentrations did not correlate with corresponding CSF concentrations. Doses and arterial concentrations did not correlate with the clinical outcome and were not associated with the occurrence of delayed cerebral ischemia. However, patients with no significant disability after 9 months of hemorrhage showed significantly higher CSF nimodipine concentrations (P = 0.015) and CSF-to-plasma ratios (P = 0.011) compared with patients who showed some degree of disability or who died., Conclusions: Cerebrospinal fluid nimodipine concentrations measured during hospital drug infusion showed a correlation with long-term clinical outcome in patients with aneurysmal subarachnoid hemorrhage. These very preliminary data suggest that CSF concentrations monitoring may have some value in managing these patients.

Dendrimers are synthetic polymers that grow in three dimensions into well-defined structures. Their morphological appearance resembles a number of trees connected by a common point. Dendritic nanoparticles have been studied for a large number of pharmaceutical and biomedical applications including gene and drug delivery, clinical diagnosis and MRI. Despite the application of dendrimers, research is still in its childhood in comparison with liposomes and other nanomaterials. They are now playing a key role in several therapeutic strategies, with dendrimer-based products in clinical trials. The aim of this review is to describe the state-of-the-art of biomedical applications of dendrimers - and dendrimer conjugates - such as drug and gene delivery and antiviral activity.

Introduction: Pramipexole (PRA), ropinirole (ROP) and rotigotine (ROT) are non-ergoline dopaminergic agonists (DAs) used to treat Parkinson's disease (PD). Clinical pharmacokinetics of DAs is poorly characterized in PD. The main purpose of our study was to investigate the effect of dose, age and sex on steady-state plasma concentrations of DAs in real life PD patients on chronic DAs therapy., Methods: The study was single center, open and prospective. Blood samples for measurement of DAs plasma concentrations were drawn in the morning, at a median 18-h distance from the last DA dose., Results: Ninety-one patients treated with PRA, 50 with ROP and 37 with ROT were enrolled in the study. Plasma concentration of DAs significantly correlated with weight-adjusted daily dose in all subgroups, although at a given dose, matched plasma concentrations highly varied among patients. Median PRA plasma concentration-to-daily dose ratio (C/D) [(ng/mL)/(mg/kg/d)] was 68% higher in patients >65 years than ≤65 years (158 vs 94, p < 0.001), while was not affected by age in ROP and ROT subgroups. No sex-mediated differences in C/D ratios were observed in any group., Conclusion: These are the first observations on DAs pharmacokinetics in PD patients' everyday clinical practice. Of relevance, patients over 65yrs may require about one third of PRA dose compared to under 65yrs to achieve the same plasma concentration. Due to the high intersubject variability in plasma concentrations at the same dosage, we speculate that monitoring of plasma DAs might be helpful in the individualization of treatment in selected patients., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this work, several ribavirin analogues were synthesized and incorporated into a multivalent arrangement. Both were subsequently modified by the addition of polyhydroxylated residues. Their antiviral activity was tested against Junín virus, etiological agent responsible of Argentine hemorrhagic fever. Some compounds inhibited Junín virus in the range of 13.2-389.1 µM. Two modified ribavirin analogues presented an effective concentration comparable to ribavirin but with a higher selectivity index., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Metabolism and carbon, oxygen, and nutrient fluxes (DIC, DOC, DO 2 , NO 2 - , NO 3 - , NH 4 ) were studied during three surveys at two sites (VN1 and VN3) located at a fish farm at the Marano and Grado Lagoon (northern Adriatic Sea), using an in situ benthic chamber. Field experiments were conducted in July and October 2015 and March 2016 at a depth of approximately 2 m along the main channels of the fish farm. Water samples were collected by a scuba diver every 2 h in order to investigate daily fluxes of solutes across the sediment-water interface (SWI). Regarding the solid phase, C + , PO 4 3- and SiO 4 4- ) were studied during three surveys at two sites (VN1 and VN3) located at a fish farm at the Marano and Grado Lagoon (northern Adriatic Sea), using an in situ benthic chamber. Field experiments were conducted in July and October 2015 and March 2016 at a depth of approximately 2 m along the main channels of the fish farm. Water samples were collected by a scuba diver every 2 h in order to investigate daily fluxes of solutes across the sediment-water interface (SWI). Regarding the solid phase, C org /N tot and C org /P org molar ratios suggested an autochthonous marine origin of the organic matter and a minor preservation of P in the sediments, respectively; high values of sulphur (S tot ) were also encountered (0.8-2%). The conditions at VN3 were mostly anoxic with high NH 4 + levels (30-1027 μM) and the absence of NO 3 - . Substantial daily patterns of all solutes occurred especially in autumn and winter. On the contrary, fluxes at VN1 were less pronounced. Usually, inverse correlations appeared between dissolved O 2 and DIC trends, but in our system this was observed only at VN3 in autumn and accomplished by a parallel increase in NH 4 + , PO 4 3- and SiO 4 4- during intense nutrient regeneration. These results are significantly different than those reported for open lagoon environments, where nutrient regeneration at the SWI and in surface sediments is the primary source of nutrients available for assimilation processes, especially during the warmer period of the year when the natural nutrient input by fresh water inflows is limited. Due to the importance of this site for aquaculture, biodiversity and ecosystem services, useful suggestions have been provided from this study in order to improve the quality of this unique aquatic system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Monitoring of biogenic sulfide is important because acid volatile sulfides (AVS) represent a reactive pool responsible for immobilization of toxic metals. We propose a new sulfide paper sensor method for semiquantitative determination of AVS in which developed color is compared to a reference chart. The method was validated against the ion-selective microelectrode and the purge-and-trap methods. For fieldwork, readings should fall within 1 to 10 μmoles S 2- . Considering that the volume of soil used ranged between 1 and 16 cm 3 , the corresponding soil sulfides concentration range spans from 0.06 to 10 mmoles S 2- cm -3 . The sulfide paper sensor method is highly suitable for field screening and has sensitivity levels comparable to laboratory methods. Environ Toxicol Chem 2018;37:3025-3031. © 2018 SETAC., (© 2018 SETAC.)

Objectives: We aimed to assess the potential relationship between intrasubject 9-tetrahydrocannabinol/cannabidiol (THC/CBD) oromucosal spray plasma profiles and clinical effects elicited by subacute dosing in chronically treated patients with multiple sclerosis (MS)., Methods: The study design was pilot, single center, open, and prospective. The patients were challenged with a morning test dose of 2 THC/CBD sprays at a 15-minute interval. Venous blood samples were collected before the first spray administration and every 30 minutes after the second spray, until 240 minutes postdosing. Patients rated their spasticity by the Numerical Rating Scale (NRS) simultaneously with blood drawings. Postural and motor tests were performed before the first spray and 90 and 180 minutes thereafter., Results: Twelve patients were recruited. Peak plasma concentrations of THC/CBD largely varied among patients, from 0.60 to 13.29 ng/mL for THC and 0.55 to 11.93 ng/mL for CBD. Time to peak plasma concentrations ranged from 150 to 240 minutes for THC and 90 to 240 minutes for CBD. Patients' NRS serial scores decreased after dosing, from a median value of 6 to 3.5 (P < 0.001). A significant inverse correlation was observed between median intrasubject repeated NRS scores and corresponding median values of both THC (P < 0.01) and CBD (P < 0.002) plasma concentrations. No significant effect of cannabinoids dosing could be appreciated according to posturographic and motor tests., Conclusions: Our kinetic dynamic findings from THC/CBD oromucosal spray are the first obtained in real MS patients. Although preliminary, they suggest that subacute dosing might elicit a subjective clinically significant effect on MS-related spasticity, paralleling cannabinoids measurable plasma concentrations.

We prospectively examined the effect of antiepileptic (AED) cotherapy on steady state plasma concentrations of perampanel (PMP) in epileptic patients. We classified AEDs as strong enzyme inducers (carbamazepine, phenobarbital, phenytoin, oxcarbazepine), not strong enzyme inducers/not inhibitors (levetiracetam, lamotrigine, topiramate, rufinamide, lacosamide, zonisamide, clobazam), and enzyme inhibitors (valproic acid [VPA]). The main outcome was the comparison of PMP plasma concentration to weight-adjusted dose ratio (C/D; [μg/mL]/mg kg -1  d -1 ) among comedication subgroups. From 79 patients (42 females, 37 males) aged (mean ± standard deviation) 33 ± 13 years (range = 12-66 years), 114 plasma samples were collected. Twenty-eight patients (44 samples) were cotreated with enzyme inducers (group A), 21 (27 samples) with not strong enzyme inducers/not inhibitors (group B), 21 (31 samples) with not strong enzyme inducers/not inhibitors + VPA (group C), and 9 (12 samples) with enzyme inducers + VPA (group D). PMP C/D was reduced (-56%, P < .001) in group A (1.79 ± 0.80) versus group B (4.05 ± 2.16) and increased (P < .001) in group C (6.72 ± 4.04) compared with groups A (+275%), B (+66%), and D (2.76 ± 2.00, +143%). Our study documents the unpublished higher PMP C/D in patients cotreated with VPA. These findings have both theoretical relevance, suggesting better characterization of PMP metabolic pathways with ad hoc studies, and clinical usefulness in managing patients on AED polytherapy., (Wiley Periodicals, Inc. © 2018 International League Against Epilepsy.)

We aimed to assess the intrasubject reproducibility of a technology-based levodopa (LD) therapeutic monitoring protocol administered in supervised versus unsupervised conditions in patients with Parkinson's disease (PD). The study design was pilot, intrasubject, single center, open and prospective. Twenty patients were recruited. Patients performed a standardized monitoring protocol instrumented by an ad hoc embedded platform after their usual first morning LD dose in two different randomized ambulatory sessions: one under a physician's supervision, the other self-administered. The protocol is made up of serial motor and non-motor tests, including alternate finger tapping, Timed Up and Go test, and measurement of blood pressure. Primary motor outcomes included comparisons of intrasubject LD subacute motor response patterns over the 3-h test in the two experimental conditions. Secondary outcomes were the number of intrasession serial test repetitions due to technical or handling errors and patients' satisfaction with the unsupervised LD monitoring protocol. Intrasubject LD motor response patterns were concordant between the two study sessions in all patients but one. Platform handling problems averaged 4% of total planned serial tests for both sessions. Ninety-five percent of patients were satisfied with the self-administered LD monitoring protocol. To our knowledge, this study is the first to explore the potential of unsupervised technology-based objective motor and non-motor tasks to monitor subacute LD dosing effects in PD patients. The results are promising for future telemedicine applications.

The Marano and Grado Lagoon is well known for being contaminated by mercury (Hg) from the Idrija mine (Slovenia) and the decommissioned chlor-alkali plant of Torviscosa (Italy). Experimental activities were conducted in a local fish farm to understand Hg cycling at the sediment-water interface. Both diffusive and benthic fluxes were estimated in terms of chemical and physical features. Mercury concentration in sediments (up to 6.81μg/g) showed a slight variability with depth, whereas the highest methylmercury (MeHg) values (up to 10ng/g) were detected in the first centimetres. MeHg seems to be produced and stored in the 2-3cm below the sediment-water interface, where sulphate reducing bacteria activity occurs and hypoxic-anoxic conditions become persistent for days. DMeHg in porewaters varied seasonally (from 0.1 and 17% of dissolved Hg (DHg)) with the highest concentrations in summer. DHg diffusive effluxes higher (up to 444ng/m 2 /day) than those reported in the open lagoon (~95ng/m 2 /day), whereas DMeHg showed influxes in the fish farm (up to -156ng/m 2 /day). The diurnal DHg and DMeHg benthic fluxes were found to be higher than the highest summer values previously reported for the natural lagoon environment. Bottom sediments, especially in anoxic conditions, seem to be a significant source of MeHg in the water column where it eventually accumulates. However, net fluxes considering the daily trend of DHg and DMeHg, indicated possible DMeHg degradation processes. Enhancing water dynamics in the fish farm could mitigate environmental conditions suitable for Hg methylation., (Copyright © 2017. Published by Elsevier B.V.)

We present a simple and fast high-performance liquid chromatography method with fluorescence detection for the determination of the antiepileptic drug perampanel in human plasma. The chromatographic separation was performed on a Kinetex PFP (100 × 2.6 mm, 4.6 µm) column, using a mobile phase of sodium acetate 0.03 M pH 3.7 and acetonitrile (40/60, v/v), at a flow rate of 0.8 mL/min. Total chromatography time for each run was 5 min. Sample preparation (250 µL) involved only one simple precipitation step by acetonitrile spiked with mirtazapine as internal standard. The method was validated over a concentration range of 20-1000 ng/mL and successfully applied to measure perampanel concentrations in plasma samples obtained from patients with epilepsy. This assay combines the high specificity of fluorescence detection with a very simple and fast sample pretreatment and can offer real advantages over existing methods in terms of simplicity and transferability to a therapeutic drug monitoring setting.

A novel ultra-high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the dopamine receptor agonist rotigotine in human plasma. Following liquid-liquid extraction with tert-butyl methyl ether from 500 μL plasma, the chromatographic analysis was performed on a Gemini NX3 column using 5 mm pH 5.0 ammonium acetate-5 mm ammonium acetate in methanol as binary gradient mobile phase, at a flow rate of 0.3 mL/min. The MS/MS ion transitions were 316.00 → 147.00 for rotigotine and 256.10 → 211.00 for the internal standard (lamotrigine). The lower limit of quantitation was 50 pg/mL and the linearity was determined from 50 to 2500 pg/mL. The mean recovery was 96.9%. Both intra- and interassay imprecision and inaccuracy were ≤15% at all quality control concentrations. The method was successfully applied to measure morning trough plasma rotigotine concentrations in a series of patients with Parkinson's disease on chronic treatment. The present study describes the first fully validated method for rotigotine determination in human plasma., (Copyright © 2017 John Wiley & Sons, Ltd.)

Objective: To investigate whether Mucuna pruriens (MP), a levodopa-containing leguminous plant growing in all tropical areas worldwide, may be used as alternative source of levodopa for indigent individuals with Parkinson disease (PD) who cannot afford long-term therapy with marketed levodopa preparations., Methods: We investigated efficacy and safety of single-dose intake of MP powder from roasted seeds obtained without any pharmacologic processing. Eighteen patients with advanced PD received the following treatments, whose sequence was randomized: (1) dispersible levodopa at 3.5 mg/kg combined with the dopa-decarboxylase inhibitor benserazide (LD+DDCI; the reference treatment); (2) high-dose MP (MP-Hd; 17.5 mg/kg); (3) low-dose MP (MP-Ld; 12.5 mg/kg); (4) pharmaceutical preparation of LD without DDCI (LD-DDCI; 17.5 mg/kg); (5) MP plus benserazide (MP+DDCI; 3.5 mg/kg); (6) placebo. Efficacy outcomes were the change in motor response at 90 and 180 minutes and the duration of on state. Safety measures included any adverse event (AE), changes in blood pressure and heart rate, and the severity of dyskinesias., Results: When compared to LD+DDCI, MP-Ld showed similar motor response with fewer dyskinesias and AEs, while MP-Hd induced greater motor improvement at 90 and 180 minutes, longer ON duration, and fewer dyskinesias. MP-Hd induced less AEs than LD+DDCI and LD-DDCI. No differences in cardiovascular response were recorded., Conclusion: Single-dose MP intake met all noninferiority efficacy and safety outcome measures in comparison to dispersible levodopa/benserazide. Clinical effects of high-dose MP were similar to levodopa alone at the same dose, with a more favorable tolerability profile., Clinicaltrialsgov Identifier: NCT02680977., (Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Valve dystrophic calcification is a common disorder affecting normophosphatemic subjects. Here, cultured aortic valve interstitial cells (AVICs) were treated 3 to 28 days with phosphate (Pi) concentrations spanning the normal range in humans (0.8, 1.3, and 2.0 mM) alone or supplemented with proinflammatory stimuli to assess possible priming of dystrophic-like calcification. Compared with controls, spectrophotometric analyses revealed marked increases in calcium amounts and alkaline phosphatase activity for 2.0-mM-Pi-containing cultures, with enhancing by proinflammatory mediators. Ultrastructurally, AVICs treated with low/middle Pi concentrations showed an enormous endoplasmic reticulum (ER) enclosing organelle debris, so apparently executing a survival-related atypical macroautophagocytosis, consistently with ultracytochemical demonstration of ER-associated acid phosphatase activity and decreases in autophagosomes and immunodetectable MAP1LC3. In contrast, AVICs cultured at 2.0-mM Pi underwent mineralization due to intracellular release and peripheral layering of phospholipid-rich material acting as hydroxyapatite nucleator, as revealed by Cuprolinic Blue and von Kossa ultracytochemical reactions. Lack of immunoblotted caspase-3 cleaved form indicated apoptosis absence for all cultures. In conclusion, fates of cultured AVICs were crucially driven by Pi concentration, suggesting that serum Pi levels just below the upper limit of normophosphatemia in humans may represent a critical watershed between macroautophagy-associated cell restoring and procalcific cell death., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

A novel, simple and reliable method based on micellar electrokinetic chromatography with ultraviolet detection was developed to analyze idebenone in a pediatric formulation. Idebenone is a synthetic short chain benzoquinone that acts as an electron carrier in the mitochondrial electron transport chain facilitating the production of adenosine triphosphate. It can be found in two different redox states that differ in their physiological properties. Idebenone has been investigated as a treatment in several neurological disorders like Friedreich's ataxia, Leber's hereditary optic neuropathy, mitochondrial encephalomyopathies and senile dementia. Accordingly, a micellar electrokinetic chromatography was employed to discriminate both redox forms. The final optimized system was validated in terms of selectivity, linearity (r2 0.992), limit of detection (0.5 µg/mL), limit of quantification (1.8 µg/mL), intra- and inter-day precision (RSD ≤ 2) and accuracy in terms of recovery studies (99.3-100.5%). Robustness was studied following a Plackett-Burman design. Finally, the validated system was applied to the analysis of idebenone in a pediatric formulation., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a fatal, recessive disease caused by mutations in the gene encoding thymidine phosphorylase, leading to reduced enzymatic activity, toxic nucleoside accumulation, and secondary mitochondrial DNA damage. Thymidine phosphorylase replacement has been achieved by allogeneic hematopoietic stem cell transplantation, a procedure hampered by high mortality. Based on high thymidine phosphorylase expression in the liver, a 25-year-old severely affected patient underwent liver transplantation. Serum levels of toxic nucleosides rapidly normalized. At 400 days of follow-up, the patient's clinical conditions are stable. We propose liver transplantation as a new therapy for MNGIE. Ann Neurol 2016;80:448-455., (© 2016 American Neurological Association.)

We present a simple, fast and validated method for the determination of nimodipine in plasma and cerebrospinal fluid (CSF) of patients with subarachnoid haemorrhage using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Plasma or CSF 250μL aliquots were pretreated with acetonitrile spiked with lacosamide as internal standard. The chromatographic separation was performed on a Fusion (3μm) 50×2.0mm I.D. column with gradient elution of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at a flow rate of 0.35mL/min. The MS/MS ion transitions were 419.1→343 for nimodipine and 251.1→91 for the internal standard. The linearity was determined from 2.0 to 40.0ng/mL in plasma and 40.0-800.0pg/mL in CSF. The lower limit of quantitation (LLOQ) of nimodipine was 0.4ng/mL in plasma and 40pg/mL in CSF. The mean recovery for nimodipine was ≥75% in plasma and ≥90% in CSF at all three considered concentrations. Intra- and interassay precision and accuracy were ≤15% at all quality control concentrations in plasma and CSF. The method was applied to measure plasma and CSF concentrations of nimodipine in a series of patients with subarachnoid haemorrhage treated with intravenous nimodipine. The present procedure, omitting time-consuming liquid-liquid extraction and drying steps, is faster, simpler and cheaper than published LC-MS/MS analytical methods for nimodipine in plasma and the first validated one for nimodipine in CSF., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Molecularly imprinted polymer nanoparticles (MIPNPs) with the ability to recognize coenzyme Q10 (CoQ10) were synthesised in order to be employed as sorbent in a dispersive micro-solid phase extraction (DMSPE) for the determination of CoQ10 in a liver extract. CoQ10 is a redox-active, lipophilic substance integrated in the mitochondrial respiratory chain which acts as an electron carrier, shuttling electrons from complex I (NADH-ubiquinone oxidoreductase) and II (succinate-ubiquinone oxidoreductase) to complex III (ubiquinol-cytochrome c reductase), for the production of cellular energy. The MIPNPs were synthesised by precipitation polymerization using coenzyme Q0 as the dummy template, methacrylic acid as the functional monomer, an acetonitrile: water mixture as the porogen, ethylene glycol dimethacrylate as the crosslinker and potassium persulfate as initiator. The nanoparticles were characterized by microscopy, capillary electrophoresis, dynamic light scattering, N2 adsorption-desorption isotherms, and infrared spectroscopy. The MIPNPs demonstrated the presence of selective cavities complementary to the quinone nucleus of CoQ10, leading to a specific recognition of CoQ10 compared with related compounds. In the liver extract the relative CoQ10 peak area (CoQ10 area/total peak area) increased from 4.6% to 25.4% after the DMSPE procedure. The recovery percentage of CoQ10 from the liver matrix was between 70.5% and 83.7% quantified against CoQ10 standard processed under the same conditions. The DMSPE procedure allows the elution of almost all the CoQ10 retained (99.4%) in a small volume (200μL), allowing the sample to be concentrated 2.5 times (LOD: 1.1μgg(-1) and LOQ: 3.7μgg(-1) of tissue). The resulted clean up of the sample, the improvement in peak shape and baseline and the reduction of interferences, evidence that the MIPNPs could potentially be applied as sorbent in a DMSPE with satisfactory results and with a minimum amount of sorbent (1mg)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Malfunctions in the neural circuitry of the basal ganglia (BG), induced by alterations in the dopaminergic system, are responsible for an array of motor disorders and milder cognitive issues in Parkinson's disease (PD). Recently Baston and Ursino (2015a) presented a new neuroscience mathematical model aimed at exploring the role of basal ganglia in action selection. The model is biologically inspired and reproduces the main BG structures and pathways, modeling explicitly both the dopaminergic and the cholinergic system. The present work aims at interfacing this neurocomputational model with a compartmental model of levodopa, to propose a general model of medicated Parkinson's disease. Levodopa effect on the striatum was simulated with a two-compartment model of pharmacokinetics in plasma joined with a motor effect compartment. The latter is characterized by the levodopa removal rate and by a sigmoidal relationship (Hill law) between concentration and effect. The main parameters of this relationship are saturation, steepness, and the half-maximum concentration. The effect of levodopa is then summed to a term representing the endogenous dopamine effect, and is used as an external input for the neurocomputation model; this allows both the temporal aspects of medication and the individual patient characteristics to be simulated. The frequency of alternate tapping is then used as the outcome of the whole model, to simulate effective clinical scores. Pharmacokinetic-pharmacodynamic modeling was preliminary performed on data of six patients with Parkinson's disease (both "stable" and "wearing-off" responders) after levodopa standardized oral dosing over 4 h. Results show that the model is able to reproduce the temporal profiles of levodopa in plasma and the finger tapping frequency in all patients, discriminating between different patterns of levodopa motor response. The more influential parameters are the Hill coefficient, related with the slope of the effect sigmoidal relationship, the drug concentration at half-maximum effect, and the drug removal rate from the effect compartment. The model can be of value to gain a deeper understanding on the pharmacokinetics and pharmacodynamics of the medication, and on the way dopamine is exploited in the neural circuitry of the basal ganglia in patients at different stages of the disease progression.

Background: Parkinson's disease (PD) is a progressive neurological condition. Levodopa (LD) is the gold standard therapy for PD patients. Most PD patients in low-income areas cannot afford long-term daily Levodopa therapy. The aim of our study was to investigate if Mucuna pruriens (MP), a legume with high LD content that grows in tropical regions worldwide, might be potential alternative for poor PD patients., Methods: We analyzed 25 samples of MP from Africa, Latin America and Asia. We measured the content in LD in various MP preparations (dried, roasted, boiled). LD pharmacokinetics and motor response were recorded in four PD patients, comparing MP vs. LD+Dopa-Decarboxylase Inhibitor (DDCI) formulations., Results: Median LD concentration in dried MP seeds was 5.29%; similar results were obtained in roasted powder samples (5.3%), while boiling reduced LD content up to 70%. Compared to LD+DDCI, MP extract at similar LD dose provided less clinical benefit, with a 3.5-fold lower median AUC., Conclusion: Considering the lack of a DDCI, MP therapy may provide clinical benefit only when content of LD is at least 3.5-fold the standard LD+DDCI. If long-term MP proves to be safe and effective in controlled clinical trials, it may be a sustainable alternative therapy for PD in low-income countries., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Generic substitution of antiepileptic drugs (AEDs) is still a matter of controversy and concern among clinicians and patients. We aimed to assess intrasubject variation in plasma concentrations of lamotrigine (LTG), levetiracetam (LEV) and topiramate (TPM) after generic substitution compared with a stable brand-name drug regimen in a population of patients with epilepsy. A retrospective analysis was performed on prospectively collected and stored data from our therapeutic drug monitoring (TDM) database for the years 2009-2014. The main outcome variable was the proportion of patients who, after switching from branded to generic formulations, showed a greater than ±20% change in AED plasma concentrations compared to the proportion of control patients showing a change in AED plasma concentrations of the same extent while receiving stable branded formulations over repeated TDM tests. Fifty patients on LTG, 27 on LEV and 16 on TPM showing at least one TDM test while receiving generic products fulfilled the inclusion/exclusion criteria for the analysis and were compared with 200 control patients for LTG, 120 for LEV and 80 for TPM. The proportion of patients showing an intrasubject change greater than ±20% in AED plasma concentrations was similar in the brand name vs generic group compared with the control one for LTG (22% vs 33%) and LEV (44% vs 38%), while it was higher in the control group for TPM (41% vs 6%, p<0.01). These are the first data in the literature about the within-patient variation in steady-state plasma concentrations of a series of stable treatments with brand-name AEDs in a real clinical setting. In conclusion, a significant interday variability in intrapatient LTG, LEV and TPM plasma concentrations can be observed even in patients stabilized with the same brand name product over time. This suggests that any change in plasma AED concentration and possible related clinical effects after generic substitution may be not necessarily related to the switch. Our results should be confirmed by large, prospective, blinded, randomized controlled studies in people with epilepsy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A simple and validated ultra high pressure liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of the dopaminergic agents pramipexole and ropinirole in plasma of patients with Parkinson's disease. Following liquid-liquid extraction with tert-butyl methyl ether from 250μL plasma, the separation of the analytes was achieved on a Gemini NX3 column using 10mM pH 6.0 ammonium formate and 10mM ammonium formate in methanol as binary gradient mobile phase at a flow rate of 0.3mL/min. The MS/MS ion transitions were 212.10→153.03 for pramipexole, 261.2→114.2 for ropinirole and 256.1→211 for the internal standard (lamotrigine). The lower limit of quantitation (LLOQ) for both analytes was 80pg/mL and the linearity was determined from 80 to 4000pg/mL for pramipexole and from 200 to 10000pg/mL for ropinirole. Mean recoveries were 94% for PRA and 73% for ROP. Both intra- and inter-assay precision and accuracy were ≤20% at LLOQ concentration and ≤15% at other concentrations. The proposed validated method was successfully applied to measure plasma concentrations of pramipexole and ropinirole in a series of patients with Parkinson's disease on chronic treatment. By grouping the two dopaminergic agents in the same assay, the method allows a large series of patient samples to be processed in a single analytical session., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biochar has a charcoal polycyclic aromatic structure which allows its long half-life in soil, making it an ideal tool for C sequestration and for adsorption of organic pollutants, but at the same time raises concerns about possible adverse impacts on soil biota. Two biochars were tested under laboratory-controlled conditions on Eisenia andrei earthworms: a biochar produced at low temperature from wine tree cuttings (WTB) and a commercial low tar hardwood lump charcoal (HLB). The avoidance test (48-h exposure) showed that earthworms avoid biochar-treated soil with rates higher than 16 t ha(-1) for HLB and 64 t ha(-1) for WTB. After 42 days, toxic effects on earthworms were observed even at application rates (100 t ha(-1)) that are generally considered beneficial for most crops. The concentration of HLB and WTB required to kill half of earthworms' population (LC50; 95% confidence limits) in the synthetic OECD soil was 338 and 580 t ha(-1), respectively. Accumulation of polycyclic aromatic hydrocarbons (PAH) in earthworms exposed to the two biochar types at 100 t ha(-1) was tested in two soils of different texture. In biochar-treated soils, the average earthworm survival rates were about 64% in the sandy and 78% clay-loam soils. PAH accumulation was larger in the sandy soil and largest in soils amended with HLB. PAH with less than four rings were preferentially scavenged from the soil by biochars, and this behaviour may mask that of the more dangerous components (i.e. four to five rings), which are preferentially accumulated. Earthworms can accumulate PAH as a consequence of exposure to biochar-treated soils and transfer them along the food chain. Soil type and biochar quality are both relevant in determining PAH transfer.

Background and Purpose: Adult-onset autosomal dominant leukodystrophy (ADLD) is a rare progressive neurological disorder caused by Lamin B1 duplication (LMNB1). Our aim was to investigate longitudinally the pattern of the autonomic dysfunction and the degree of neuropsychological involvement., Methods: Three related ADLD patients and one asymptomatic carrier of LMNB1 duplication underwent a standardized evaluation of autonomic nervous system, including cardiovascular reflexes, pharmacological testing, microneurography, skin biopsy, Metaiodobenzylguanidine scintigraphy and a complete neuropsychological battery., Results: An early neurogenic orthostatic hypotension was detected in all patients and confirmed by a low rise in noradrenaline levels on Tilt Test. However infusion of noradrenaline resulted in normal blood pressure rise as well as the infusion of clonidine. At the insulin tolerance test the increase in adrenaline resulted pathological in two out three patients. Microneurography failed to detect muscle sympathetic nerve activity bursts. Skin biopsy revealed a poor adrenergic innervation, while cardiac sympathetic nerves were normal. None of ADLD patients showed a global cognitive deficit but a selective impairment in the executive functions., Conclusion: Autonomic disorder in ADLD involves selectively the postganglionic sympathetic system including the sympatho-adrenal response. Cognitive involvement consisting in an early impairment of executive tasks that might precede brain MR abnormalities., (Copyright © 2016 Elsevier B.V. All rights reserved.)