A rapid and simple uhplc-ms/ms method for quantification of plasma globotriaosylsphingosine (Lyso-gb3)

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 ex-cellent 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

Case report: De novo mutation of a-galactosidase A in a female patient with end-stage renal disease: report of a case of late diagnosis of Anderson–Fabry disease

Background: Anderson-Fabry disease (AFD) is an X-linked disease that results from reduced activity of the enzyme galactosidase alpha (GLA). When the GLA gene sequence is altered by mutations that alter the normal DNA sequence, variants of the alpha-galactosidase A enzyme are produced, which may or may not function. These mutations are responsible for Fabry disease, and to date, over 800 different mutations of the gene have been described in patients with Anderson-Fabry disease. In this case, we report the case of a woman who is the sole family member with this type of mutation.Case presentation: We report a case of a 52-year-old woman with end-stage chronic kidney disease in dialysis treatment. The patient's alpha-galactosidase activity was 6.6 nmol/ml/h in whole blood, and lyso-GB3 levels were 11.45 nmol/L (normal range < 2.3 nmol/L). Alpha-galactosidase A gene sequence analysis revealed a pathogenic variant of c.947dupT in exon 6, leading to the p. I317NfsTer16 amino acid substitution. The genetic analysis did not detect the same mutation in any of the other screened family members.Conclusion: The international Fabry disease genotype-phenotype database (dbFGP) reports a pathogenic variant c.947dupT in exon 6 that is probably associated with a classical phenotype of Fabry disease. In this case report, we report the case of a woman who is the sole family member with this type of pathogenic variant. Similar situations have not been described in the literature for this pathogenic variant, and it represents an important case of inter- and intrafamilial variability in patients with Fabry disease. The literature shows that de novo pathogenic variants are frequently found in the context of Fabry disease

A second case with the V374A KCND3 pathogenic variant in an Italian patient with early-onset spinocerebellar ataxia

Background and Objectives To date, approximately 20 heterozygous mainly loss-of-function variants in KCND3 have been associated with spinocerebellar ataxia (SCA) type 19 and 22, a clinically heterogeneous group of neurodegenerative disorders. We aimed at reporting the second patients with the V374A KCND3 mutation from an independent family, confirming its pathogenic role. Methods We describe the clinical history of a patient with SCA and conducted genetic investigations including mitochondrial DNA analysis and exome sequencing. Results This male patient was reported to have unstable gait with tremors at the lower limbs and dysarthric speech since childhood. A neurologic examination also showed dysarthria, nystagmus, action tremor, dysmetria, and weak deep tendon reflexes. He had marked cerebellar atrophy at brain MRI, more evident at vermis. Molecular analysis, including exome sequencing and an in silico panel analysis of genes associated with SCA, revealed the c.1121T>C [p.V374A] mutation in KCND3. Discussion This report consolidates the pathogenicity of the V374A KCND3 mutation and suggests that the ataxic paroxysmal exacerbations are not a key phenotypic feature of this mutation

Rhanteriol, a New Rhanterium suaveolens Desf. Lignan with Pharmacological Potential as an Inhibitor of Enzymes Involved in Neurodegeneration and Type 2 Diabetes

Several specialized plant metabolites are reported to be enzyme inhibitors. In this investigation, the phytochemical composition and the biological activity of Rhanterium suaveolens Desf. were studied. One new lignan (rhanteriol 1) and seven known secondary metabolites were isolated from the aerial parts of R. suaveolens by using different chromatographic procedures. The biological properties of the R. suaveolens extracts and the new compound were evaluated by measuring their ability to inhibit the cholinesterase and carbohydrate-hydrolyzing enzymes, using cell-free in vitro methods. The new lignan, rhanteriol, was shown to inhibit alpha-amylase and alpha-glucosidase (IC50 = 46.42 +/- 3.25 mu M and 26.76 +/- 3.29 mu M, respectively), as well as butyrylcholinesterase (IC50 = 10.41 +/- 0.03 mu M), with an effect comparable to that of the respective standards, acarbose and galantamine. Furthermore, docking studies were performed suggesting the interaction mode of rhanteriol with the active sites of the investigated enzymes. The obtained data demonstrated that the aerial part of R. suaveolens could represent a source of active molecules, such as rhanteriol, usable in the development of treatments for preventing or treating type 2 diabetes mellitus and neurodegeneration

Detection of prions in skin punch biopsies of Creutzfeldt–Jakob disease patients

Prion real-time quaking-induced conversion (RT-QuIC) is an ultrasensitive assay detecting pathological aggregates of misfolded prion protein in biospecimens. We studied 71 punch biopsy skin samples of 35 patients with Creutzfeldt–Jakob disease (CJD), including five assessed in vitam. The results confirmed the high value of skin prion RT-QuIC for CJD diagnosis (89% sensitivity and 100% specificity) and support its use in clinical practice. Preliminary data based on a limited number of cases suggest that prion-seeding activity in the skin varies according to the prion strain, being higher in sporadic CJD subtypes linked to the V2 strain (VV2 and MV2K) than in typical CJDMM1

A comparative blind study between skin biopsy and seed amplification assay to disclose pathological α-synuclein in RBD

To compare the diagnostic accuracy of the immunofluorescence (IF) technique and aSyn-seed amplification assay (aSyn-SAA) of skin and cerebrospinal fluid (CSF) in disclosing pathological α-syn in idiopathic idiopathic REM sleep behavior disorder (iRBD) as early phase of a synucleinopathy. We prospectively recruited 41 patients with iRBD and 40 matched clinical controls including RBD associated with type 1 Narcolepsy (RBD-NT1, 21 patients), iatrogenic causes (2 pt) or OSAS (6 pt) and 11 patients with peripheral neuropathies. IF from samples taken by skin biopsy and aSyn-SAA from skin and CSF samples were analysed blinded to the clinical diagnosis. IF showed a good diagnostic accuracy (89%) that was lower in the case of skin and CSF-based aSyn-SAA (70% and 69%, respectively) because of a lower sensitivity and specificity. However, IF showed a significant agreement with CSF aSyn-SAA. In conclusion, our data may favor the use of skin biopsy and aSyn-SAA as diagnostic tools for a synucleinopathy in iRBD

The Association of Field Test Outcomes with Peak Oxygen Uptake in Patients with Cystic Fibrosis: A Systematic Review

International Journal of Exercise Science 15(3): 1381-1394, 2022. The purpose of the study was to evaluate the association of field test outcomes with peak oxygen uptake (VO2peak) in patients with cystic fibrosis (CF) and to describe the main prediction equations available. Data searches were performed in five databases (Pubmed, Embase, LILACs, Scopus and Web of Science) and also in the reference lists of articles included. The following inclusion criteria were used: studies including individuals with CF, presenting both a field test and a cardiopulmonary exercise testing (CPET), and describing a predictive equation or coefficient of correlation/determination. Case studies, abstracts, letters of reply, editorials and duplicate publications were excluded. The methodological quality analysis was performed using the JBI Critical Appraisal Checklist for Analytical Cross-Sectional Studies scale. Protocol registration number: CRD42020148363. Ten studies were eligible. Five equations were found to predict VO2peak. Equations derived from the shuttle tests (ST) showed strong correlations with VO2peak (r = 0.79 to 0.95). The six-minute walk test (6MWT) showed moderate associations with VO2peak in participants with moderate disease severity (r = 0.53 to 0.65). Furthermore, patients with lower maximum heart rate on the three-minute step test tended to have a higher percent predicted VO2peak (r = -0.40), and the one-minute sit-to-stand test demonstrated moderate correlations between VO2peak and the number of repetitions (r = 0.52 to 0.66). In conclusion, field test outcomes correlate with oxygen consumption assessed through CPET, although only the ST seems to be valid as a predictor of VO2peak in patients with CF

Thermal transistor: Heat flux switching and modulating

Thermal transistor is an efficient heat control device which can act as a heat switch as well as a heat modulator. In this paper, we study systematically one-dimensional and two-dimensional thermal transistors. In particular, we show how to improve significantly the efficiency of the one-dimensional thermal transistor. The study is also extended to the design of two-dimensional thermal transistor by coupling different anharmonic lattices such as the Frenkel-Kontorova and the Fermi-Pasta-Ulam lattices. Analogy between anharmonic lattices and single-walled carbon nanotube is drawn and possible experimental realization with multi-walled nanotube is suggested.Comment: To appear in J. Phys. Soc. Jp

Clinical and pathology characterization of small nerve fiber neuro(no)pathy in cerebellar ataxia with neuropathy and vestibular areflexia syndrome

Background and purpose: Biallelic mutation/expansion of the gene RFC1 has been described in association with a spectrum of manifestations ranging from isolated sensory neuro(no)pathy to a complex presentation as cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS). Our aim was to define the frequency and characteristics of small fiber neuropathy (SFN) in RFC1 disease at different stages. Methods: RFC1 cases were screened for SFN using the Neuropathic Pain Symptom Inventory and Composite Autonomic Symptom Score 31 questionnaires. Clinical data were retrospectively collected. If available, lower limb skin biopsy samples were evaluated for somatic epidermal and autonomic subepidermal structure innervation and compared to healthy controls (HCs). Results: Forty patients, median age at onset 54 years (interquartile range [IQR] 49–61) and disease duration 10 years (IQR 6–16), were enrolled. Mild-to-moderate positive symptoms (median Neuropathic Pain Symptom Inventory score 12.1/50, IQR 5.5–22.3) and relevant autonomic disturbances (median Composite Autonomic Symptom Score 31 37.0/100, IQR 17.7–44.3) were frequently reported and showed scarce correlation with disease duration. A non-length-dependent impairment in nociception was evident in both clinical and paraclinical investigations. An extreme somatic denervation was observed in all patients at both proximal (fibers/mm, RFC1 cases 0.0 vs. HCs 20.5, p < 0.0001) and distal sites (fibers/mm, RFC1 cases 0.0 vs. HCs 13.1, p < 0.0001); instead only a slight decrease was observed in cholinergic and adrenergic innervation of autonomic structures. Conclusions: RFC1 disease is characterized by a severe and widespread somatic SFN. Skin denervation may potentially represent the earliest feature and drive towards the suspicion of this disorder

Chromatic Pupillometry in Isolated Rapid Eye Movement Sleep Behavior Disorder

Background: Melanopsin retinal ganglion cell (mRGC)-mediated pupillary light reflex (PLR) abnormalities have been documented in several neurodegenerative disorders including Parkinson's disease. Overall, isolated rapid eye movement (REM) sleep behavior disorder (iRBD) represents the strongest prodromal risk factor for impending α-synucleinopathies. Objectives: To quantitatively compare PLR and mRGC-mediated contribution to PLR in 16 iRBD patients and 16 healthy controls. Methods: iRBD and controls underwent extensive neuro-ophthalmological evaluation and chromatic pupillometry. In iRBD, PLR metrics were correlated with clinical variables and with additional biomarkers including REM atonia index (RAI), DaTscan, and presence of phosphorylated-α-synuclein (p-α-syn) deposition in skin biopsy. Results: We documented higher baseline pupil diameter and decreased rod-transient PLR amplitude in iRBD patients compared to controls. PLR rod-contribution correlated with RAI. Moreover, only iRBD patients with evidence of p-α-syn deposition at skin biopsy showed reduced PLR amplitude compared to controls. Conclusion: The observed PLR abnormalities in iRBD might be considered as potential biomarkers for the risk stratification of phenoconversion of the disease. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society