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Objectives: Physical inactivity in end-stage renal disease (ESRD) patients is associated with increased mortality, and might be related to abnormalities in body composition (BC) and physical performance. It is uncertain to what extent starting dialysis influences the effects of ESRD on physical activity (PA). This study aimed to compare PA and physical performance between stage 5 chronic kidney disease (CKD-5) non-dialysis and dialysis patients, and healthy controls, to assess alterations in PA during the transition from CKD-5 non-dialysis to dialysis, and to relate PA to BC. Methods: For the cross-sectional analyses 44 CKD-5 non-dialysis patients, 29 dialysis patients, and 20 healthy controls were included. PA was measured by the SenseWear™ pro3. Also, the walking speed and handgrip strength (HGS) were measured. BC was measured by the Body Composition Monitor©. Longitudinally, these parameters were assessed in 42 CKD-5 non-dialysis patients (who were also part of the cross-sectional analysis), before the start of dialysis and 6 months thereafter. Results: PA was significantly lower in CKD-5 non-dialysis patients as compared to that in healthy controls but not as compared to that in dialysis patients. HGS was significantly lower in dialysis patients as compared to that in healthy controls. Walking speed was significantly lower in CKD-5 non-dialysis patients as compared to that in healthy controls but not as compared to that in dialysis patients. Six months after starting dialysis, activity related energy expenditure (AEE) and walking speed significantly increased. Conclusions: PA is already lower in CKD-5 non-dialysis patients as compared to that in healthy controls and does not differ from that of dialysis patients. However, the transition phase from CKD-5 non-dialysis to dialysis is associated only with a modest improvement in AEE.

Background: Extended haemodialysis (EHD) has been associated with better outcomes compared to conventional (CHD) regimes. The cardiovascular (CV) profile of these patients has not been assessed in detail. Methods: We report baseline demographic and CV phenotype of 36 CHD and 36 EHD participants to a longitudinal multicentre study. We measured pulse wave velocity (PWV), 24-h ambulatory blood pressure, sublingual dark-field capillaroscopy and vascular biomarkers. Results: EHD patients were younger (p < 0.01), with less CV comorbidity (p = 0.04) and higher dialysis vintage (p < 0.01). Higher PWV in CHD (p = 0.02) was not independent of demographic differences in the 2 groups. Biomarker profiles were similar in EHD and CHD but abnormal compared to healthy controls. Conclusion: Although CV profiles in these 2 cohorts were similar, EHD patients were distinct from the CHD population in terms of age and dialysis vintage and appear to comprise a unique group. Direct comparison of outcomes in these groups is challenging due to clinical bias.

Pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation.

Background: The group of rare hereditary anemias includes a large variety of intrinsic defects of the red blood cell, as well as erythropoiesis. They include hemolytic anemias (e.g. enzyme deficiencies), hemoglobinopathies, hypoplastic anemias (e.g. Diamond-Blackfan Anemia, DBA), and dyserythropoietic anemias. As a result of the rapid developments in genetic testing and the subsequent increased knowledge of molecular defects underlying hereditary anemias, our understanding of the pathophysiology of rare anemias has increased during the last decade. However, in a substantial number of patients, the clinical phenotype does not fit classical criteria of a disease, response to therapy is less than expected, or a molecular defect cannot be found. In addition, in patients with well-described molecular defects, there is often no clear genotype-phenotype correlation. In order to better understand the underlying pathophysiological mechanisms driving ineffective erythropoiesis in patients and to improve their classification and clinical evaluation, novel functional tests are needed. Metabolomics is the large-scale, unbiased study of metabolites and their interactions within a biological system, directly reflecting the underlying biochemical activity and state of cells. Metabolomics can be used to identify novel disease biomarkers, study deregulated cellular pathways, and to determine the cellular responses to therapeutic interventions. In this study we demonstrate that dried blood spots (DBS) can be used as a minimal invasive and validated technical approach to perform large scale metabolomics in a variety of rare hereditary anemias. Methods: DBS samples from >100 patients suffering from a variety of rare anemiaswere collected during regular hospital visits. Quantification of metabolites was performed by direct infusion high resolution mass spectrometry (DI-HRMS) followed by an untargetedmetabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with DBS samples of 70 healthy adult controls and 35 pediatric patients negatively screened for metabolic diseases Results: For each patient sample, Z-scores were calculated for all mass peaks annotated with metabolites (HMDB, 3930). Mass peak, intensity and corresponding Z-scores were compared with two distinct groups of controls (∆Z-scores): pediatric patients who were screened for metabolic diseases but were found negative, and healthy adult controls. For data interpretation, two strategies were used. First, by untargeted statistical analysis in Metabo-analyst, we identified metabolites (and/or isomers) that showed either increased or decreased intensity. For the second strategy we specifically focused on red blood cell metabolic pathways, including glycolysis, the pentose phosphate pathway, ascorbate and glutathione metabolism, arginine and polyamine metabolism, and erythrocyte membrane turnover and transport. We corrected for a potential hematocrit effect and performed subgroup analyses correcting for reticulocyte counts. Our preliminary data indicate potential biomarkers for distinct disease entities, including altered polyamine metabolism (DBA, SCD), glycolysis (DBA, HS), and aberrant arginine metabolism (SCD) (Figure 1). Further in-depth pathway analyses, and targeted validation of biomarker profiles are currently being performed. Conclusion: Untargeted metabolomics using dried blood spots provides a novel functional tool to identify disease biomarkers and common and distinct deregulated cellular pathways. This will improve diagnostic evaluation and clinical management of patients with rare hereditary anemias, contribute to a better understanding of disease pathophysiology, and aid in the development of therapeutic strategies. Disclosures van Beers: Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:RR Mechatronics: Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding.

Let G = (V(G),E(G)) is a connected simple graph. Let ec(v) is the eccentricity of vertex v, D(v) = Σ u∈V(G) d(u,v) is the sum of all distances from vertex v and deg(v) is the degree of vertex v in G. The eccentric distance sum index of G is defined as ξd (G) = Σ v∈V(G) ec(v)D(v) and the adjacent eccentric distance sum index of G is defined as ξ s v ( G ) = ∑ v ∈ V ( G ) e c ( v ) D ( v ) deg ( v ) . For positive integer m and m ≥ 3, let D 2m be dihedral group of order 2m and N is a normal subgroup of D 2m . The subgroup graph Γ N (D 2m ) of dihedral group D 2m is a simple graph with vertex set D 2m and two distinct vertices x and y are adjacent if and only if xy ∈ N. In the present paper, we compute eccentric distance sum and adjacent eccentric distance sum index of complement of subgroup graph of dihedral group D 2m . Total eccentricity, eccentric connectivity index, first Zagreb index, and second Zagreb index of these graphs are also determined.

Pyridox(am)ine 5’-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5’-phosphate (PLP), the biologically active form of VB6, and involved in the synthesis of neuro-transmitters including GABA, dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously we identified a c.95 C > A missense mutation insgll- theDrosophilahomolog of humanPNPO(hPNPO) and found mutant (sgll95) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here we report the establishment of bothsgll95and ubiquitoussgllknockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Bothsgll95andsgllKD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-typesgllorhPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specificsgllKD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, thePNPOgene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

Over the last decades, advances in clinical care for patients suffering from propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) have resulted in improved survival. These advances were possible thanks to new pathophysiological insights. However, patients may still suffer from devastating complications which largely determine the unsatisfying overall outcome. To optimize our treatment strategies, better insight in the pathophysiology of complications is needed. Here, we perform a systematic data-analysis of cohort studies and case-reports on PA and MMA. For each of the prevalent and rare complications, we summarize the current hypotheses and evidence for the underlying pathophysiology of that complication. A common hypothesis on pathophysiology of many of these complications is that mitochondrial impairment plays a major role. Assuming that complications in which mitochondrial impairment may play a role are overrepresented in monogenic mitochondrial diseases and, conversely, that complications in which mitochondrial impairment does not play a role are underrepresented in mitochondrial disease, we studied the occurrence of the complications in PA and MMA in mitochondrial and other monogenic diseases, using data provided by the Human Phenotype Ontology. Lastly, we combined this with evidence from literature to draw conclusions on the possible role of mitochondrial impairment in each complication. Altogether, this review provides a comprehensive overview on what we, to date, do and do not understand about pathophysiology of complications occurring in PA and MMA and about the role of mitochondrial impairment herein.

Contains fulltext : 202680.pdf (Publisher’s version ) (Closed access) Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

Upon exposure to platinum analogs, mesenchymal stem cells were recently found to excrete minute amounts of specific lipid mediators that induce chemotherapy resistance. One of these lipids is hexadeca-4,7,10,13-tetraenoic acid (FA(16:4)n-3). Importantly, FA(16:4)n-3 is present in high concentrations in certain fish oils and algae and oral intake of these products also potently induced chemotherapy resistance. These findings suggested that certain foods could negatively affect clinical chemotherapy treatment. In order to allow further study of the relation between FA(16:4)n-3 and clinical chemotherapy resistance, a method for the detection and quantification of FA(16:4)n-3 in plasma is required. Therefore, a quantification method for FA(16:4)n-3 in human and mouse plasma was developed consisting of a liquid–liquid extraction, solid phase clean-up and LC–MS/MS (MRM) analysis. The method was fully validated over a period of three weeks according to the standard protocols and requirements. The linearity range of the method is 1–100 nmol/L (r2 > 0.99) using deuterated FA(16:3)n-3 as an internal standard. The limits of quantification and detection are 1.0 nmol/L and 0.8 nmol/L, respectively. Recoveries for spiked concentrations range from 103 to 108%. The intra-day and inter-day mean precision amounts to 98–106% and 100–108%, respectively. No significant loss of FA(16:4)n-3 is observed upon storage at −80 °C. The developed assay for the detection and quantification of FA(16:4)n-3 in human plasma is robust and reproducible. The validation parameters are within limits of acceptance.

The GABA system is pivotal for an adequate response to a stressful environment but has remained largely unexplored in this context. The present study investigated the relationship of prospectively measured plasma GABA levels with psychopathology symptoms in military deployed to Afghanistan at risk for developing psychopathology following trauma exposure during deployment, including posttraumatic stress disorder (PTSD) and major depressive disorder (MDD). Plasma GABA levels were measured in military personnel (N=731) one month prior to deployment (T0), and one (T1) and six months (T2) after deployment using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Mental health problems and depressive symptoms were measured with the Dutch revised Symptom Checklist (SCL-90) and PTSD symptoms with the Dutch Self-Rating Inventory for PTSD (SRIP). Six months after deployment increases in GABA concentrations were present in individuals who had developed mental health problems (T2: β=0.06, p=1.6×10(-2), T1: β=4.7×10(-2), p=0.13), depressive symptoms (T2: β=0.29, p=7.9×10(-3), T1: β=0.23, p=0.072) and PTSD symptoms at T2 (T2: β=0.12, p=4.3×10(-2), T1: β=0.11, p=0.13). Plasma GABA levels prior to and one month after deployment poorly predicted a high level of psychopathology symptoms either one or six months after deployment. The number of previous deployments, trauma experienced during deployment, childhood trauma, age and sex were not significantly associated with plasma GABA levels over time. Exclusion of subjects who either started or stopped smoking, alcohol or medication use between the three time points rendered the association of increasing GABA levels with the emergence of psychopathology symptoms more pronounced (mental health problems at T2: β=0.09, p=4.2×10(-3); depressive symptoms at T2: β=0.35, p=3.5×10(-3), PTSD symptoms at T2: β=0.17, p=1.7×10(-2)). To our knowledge, this is the first study to provide prospective evidence that the development of psychopathology after military deployment is associated with increasing plasma GABA levels. Our finding that plasma GABA rises after the emergence of psychopathology symptoms suggests that GABA increase may constitute a compensatory mechanism and warrants further exploration of the GABA system as a potential target for treatment.

Biodegradable food packaging promises a more sustainable future. Among the many different biopolymers used, poly(lactic acid) (PLA) possesses the good mechanical property and cost-effectiveness necessary of a biodegradable food packaging. However, PLA food packaging suffers from poor water vapor and oxygen barrier properties compared to many petroleum-derived ones. A key challenge is, therefore, to simultaneously enhance both the water vapor and oxygen barrier properties of the PLA food packaging. To address this issue, we design a sandwich-architectured PLA-graphene composite film, which utilizes an impermeable reduced graphene oxide (rGO) as the core barrier and commercial PLA films as the outer protective encapsulation. The synergy between the barrier and the protective encapsulation results in a significant 87.6% reduction in the water vapor permeability. At the same time, the oxygen permeability is reduced by two orders of magnitude when evaluated under both dry and humid conditions. The excellent barrier properties can be attributed to the compact lamellar microstructure and the hydrophobicity of the rGO core barrier. Mechanistic analysis shows that the large rGO lateral dimension and the small interlayer spacing between the rGO sheets have created an extensive and tortuous diffusion pathway, which is up to 1450-times the thickness of the rGO barrier. In addition, the sandwiched architecture has imbued the PLA-rGO composite film with good processability, which increases the manageability of the film and its competency to be tailored. Simulations using the PLA-rGO composite food packaging film for edible oil and potato chips also exhibit at least eight-fold extension in the shelf life of these oxygen and moisture sensitive food products. Overall, these qualities have demonstrated the high potential of a sandwich-architectured PLA-graphene composite film for food packaging applications. (Figure Presented).

Background: Leptospiral sphingomyelinases are candidate virulence factors present only in pathogenic Leptospira spp. Leptospira interrogans serovar Lai encodes Sph1, Sph2, Sph3, Sph4 and SphH. Except for Sph4, they all possess the exo-endo-phosphatase domain that groups them under the DNase I superfamily. Methods, Results and Conclusions: Modeling of exo-endo-phosphatase domains reveals high-level structural similarity of Sph2 with the crystal structure of SmcL and BC SMase sphingomyelinases from Listeria ivanovii and Bacillus cereus, respectively. A β-hairpin loop, essential for host cell membrane interaction, is absent in leptospiral sphingomyelinases. Instead, several aromatic amino acids were oriented outward from the surface of these molecules and formed clusters of hydrophobic regions that possibly enables the anchoring of these molecules into the host cell membrane, as demonstrated in Sph2 and Sph3. Sph2 is unique and possesses the Mg++-binding Glu53 residue in the metal-binding site and two His residues (His151 and His286) in the catalytic site. We demonstrate experimentally the Mg++-dependent hemolysis of erythrocytes by rSph2 and its ability to cleave sphingomyelin to ceramide. Anti-Sph2 antibodies neutralized the hemolytic activity of Sph2. In conclusion, we provide evidence showing that Sph2 is a Mg++-dependent hemolysin with both sphingomyelinase and hemolytic activities.