Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency

Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease.     

Revising the Concept of Pore Hierarchy for Ionic Transport in Carbon Materials for Supercapacitors

Rapid motion of electrolyte ions is a crucial requirement to ensure the fast charging/discharging and the high power densities of supercapacitor devices. This motion is primarily determined by the pore size and connectivity of the used porous carbon electrodes. Here, the diffusion characteristics of each individual electrolyte component, that is, anion, cation, and solvent confined to model carbons with uniform and well‐defined pore sizes are quantified. As a result, the contributions of micropores, mesopores, and hierarchical pore architectures to the overall transport of adsorbed mobile species are rationalized. Unexpectedly, it is observed that the presence of a network of mesopores, in addition to smaller micropores—the concept widely used in heterogeneous catalysis to promote diffusion of sorbates—does not necessarily enhance ionic transport in carbon materials. The observed phenomenon is explained by the stripping off the surrounding solvent shell from the electrolyte ions entering the micropores of the hierarchical material, and the resulting enrichment of solvent molecules preferably in the mesopores. It is believed that the presented findings serve to provide fundamental understanding of the mechanisms of electrolyte diffusion in carbon materials and depict a quantitative platform for the future designing of supercapacitor electrodes on a rational basis.     

Prey capture behavior and kinematics of the Atlantic cownose ray, Rhinoptera bonasus

The structurally reinforced jaws of the cownose ray, Rhinoptera bonasus testify to this species' durophagous diet of mollusks, but seem ill-suited to the behaviors necessary for excavating such prey. This study explores this discordance by investigating the prey excavation and capture kinematics of R. bonasus. Based on the basal suction feeding mechanism in this group of fishes, we hypothesized a hydraulic method of excavation. As expected, prey capture kinematics of R. bonasus show marked differences relative to other elasmobranchs, relating to prey excavation and use of the cephalic lobes (modified anterior pectoral fin extensions unique to derived myliobatiform rays). Prey are excavated by repeated opening and closing of the jaws to fluidize surrounding sand. The food item is then enclosed laterally by the depressed cephalic lobes, which transport it toward the mouth for ingestion by inertial suction. Unlike in most sharks, upper jaw protrusion and mandibular depression are simultaneous. During food capture, the ray's spiracle, mouth, and gill slit movements are timed such that water enters only the mouth (e.g., the spiracle closes prior to prey capture and reopens immediately following). Indigestible parts are then hydraulically winnowed from edible prey portions, by mouth movements similar to those used in excavation, and ejected through the mouth. The unique sensory/manipulatory capabilities of the cephalic lobes, as well as the cownose ray's hydraulic excavation/winnowing behaviors and suction feeding, make this species an effective benthic predator, despite its epibenthic lifestyle.     

Oxidative Imbalance,Nitrative Stress,and Inflammation in C6 Glial Cells Exposed to Hexacosanoic Acid: Protective Effect of <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-cysteine,Trolox, and Rosuvastatin

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder caused by disfunction of the ABCD1 gene, which encodes a peroxisomal protein responsible for the transport of the very long-chain fatty acids from the cytosol into the peroxisome, to undergo β-oxidation. The mainly accumulated saturated fatty acids are hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in tissues and body fluids. This peroxisomal disorder occurs in at least 1 out of 20,000 births. Considering that pathophysiology of this disease is not well characterized yet, and glial cells are widely used in studies of protective mechanisms against neuronal oxidative stress, we investigated oxidative damages and inflammatory effects of vesicles containing lecithin and C26:0, as well as the protection conferred by N-acetyl-l-cysteine (NAC), trolox (TRO), and rosuvastatin (RSV) was assessed. It was verified that glial cells exposed to C26:0 presented oxidative DNA damage (measured by comet assay and endonuclease III repair enzyme), enzymatic oxidative imbalance (high catalase activity), nitrative stress [increased nitric oxide (NO) levels], inflammation [high Interleukin-1beta (IL-1β) levels], and induced lipid peroxidation (increased isoprostane levels) compared to native glial cells without C26:0 exposure. Furthermore, NAC, TRO, and RSV were capable to mitigate some damages caused by the C26:0 in glial cells. The present work yields experimental evidence that inflammation, oxidative, and nitrative stress may be induced by hexacosanoic acid, the main accumulated metabolite in X-ALD, and that antioxidants might be considered as an adjuvant therapy for this severe neurometabolic disease.     

Inflammatory profile in X‐linked adrenoleukodystrophy patients: Understanding disease progression

X‐linked adrenoleukodystrophy (X‐ALD) is an inherited disease characterized by progressive inflammatory demyelization in the brain, adrenal insufficiency, and an abnormal accumulation of very long chain fatty acids (VLCFA) in tissue and body fluids. Considering that inflammation might be involved in pathophysiology of X‐ALD, we aimed to investigate pro‐ and anti‐inflammatory cytokines in plasma from three different male phenotypes (CCER, AMN, and asymptomatic individuals). Our results showed that asymptomatic patients presented increased levels of pro‐inflammatory cytokines IL‐1β, IL‐2, IL‐8, and TNF‐α and the last one was also higher in AMN phenotype. Besides, asymptomatic patients presented higher levels of anti‐inflammatory cytokines IL‐4 and IL‐10. AMN patients presented higher levels of IL‐2, IL‐5, and IL‐4. We might hypothesize that inflammation in X‐ALD is related to plasmatic VLCFA concentration, since there were positive correlations between C26:0 plasmatic levels and pro‐inflammatory cytokines in asymptomatic and AMN patients and negative correlation between anti‐inflammatory cytokine and C24:0/C22:0 ratio in AMN patients. The present work yields experimental evidence that there is an inflammatory imbalance associated Th1, (IL‐2, IL‐6, and IFN‐γ), Th2 (IL‐4 and IL‐10), and macrophages response (TNF‐α and IL‐1β) in the periphery of asymptomatic and AMN patients, and there is correlation between VLCFA plasmatic levels and inflammatory mediators in X‐ALD. Furthermore, we might also speculate that the increase of plasmatic cytokines in asymptomatic patients could be considered an early biomarker of brain damage and maybe also a predictor of disease progression.     

Non-redundant function of dystroglycan and β1 integrins in radial sorting of axons

Radial sorting allows the segregation of axons by a single Schwann cell (SC) and is a prerequisite for myelination during peripheral nerve development. Radial sorting is impaired in models of human diseases, congenital muscular dystrophy (MDC) 1A, MDC1D and Fukuyama, owing to loss-of-function mutations in the genes coding for laminin α2, Large or fukutin glycosyltransferases, respectively. It is not clear which receptor(s) are activated by laminin 211, or glycosylated by Large and fukutin during sorting. Candidates are αβ1 integrins, because their absence phenocopies laminin and glycosyltransferase deficiency, but the topography of the phenotypes is different and β1 integrins are not substrates for Large and fukutin. By contrast, deletion of the Large and fukutin substrate dystroglycan does not result in radial sorting defects. Here, we show that absence of dystroglycan in a specific genetic background causes sorting defects with topography identical to that of laminin 211 mutants, and recapitulating the MDC1A, MDC1D and Fukuyama phenotypes. By epistasis studies in mice lacking one or both receptors in SCs, we show that only absence of β1 integrins impairs proliferation and survival, and arrests radial sorting at early stages, that β1 integrins and dystroglycan activate different pathways, and that the absence of both molecules is synergistic. Thus, the function of dystroglycan and β1 integrins is not redundant, but is sequential. These data identify dystroglycan as a functional laminin 211 receptor during axonal sorting and the key substrate relevant to the pathogenesis of glycosyltransferase congenital muscular dystrophies.     

Importance of phosphorus supply through endophytic <Emphasis Type="Italic">Metarhizium brunneum</Emphasis> for root:shoot allocation and root architecture in potato plants

Background and aims

Identification of organic P species is important to understand their origin, turnover in soils and their effects on soil fertility. Attention has been recently devoted to microbial inocula, referred to as Bioeffectors, that are capable to increase P bioavailability and plant uptake. Nevertheless, little is known on the effect of Bioeffectors on soil P forms and their dynamics in agricultural soils upon different P fertilization.

Methods

We investigated the effects of the application of different commercial inocula strains (Trichoderma harzianum T 22, Pseudomonas sp., and Bacillus amyloliquefaciens) alone or in combination with different P fertilizers (triple superphosphate, rock phosphate, and both composted cow- and horse-manure) on soil organic P forms. P forms were characterized by liquid-state ³¹P–NMR spectroscopy, while plant P uptake from P-treated soil was followed in a greenhouse pot experiment under maize cultivation.

Results

NMR spectra showed that the type of P fertilizer and bioeffectors inoculation, affected the abundance and the composition of organic P forms. The specific capacity of all bioeffectors, and especially Pseudomonas, was related to an increased content of diesters P forms. Pseudomonas, and, to a lesser extent, B. amyloliquefaciens showed the largest increase in combination with organic P amendments, which also provided the largest plant P uptake. This suggests a key role of Diester-P forms in determining P availability in agroecosystems.

Conclusions

Microbial inoculation plays an important role in the dynamics of soil P, inducing a rapid P cycling that prevents P fixation and losses from soils, thus enhancing the P fertilizer use efficiency in agricultural soils.

     

Identification of a Receptor for Extracellular Renalase

Background

An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI) and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI) in wild type (WT) mice. Therefore, we sought to identity the receptor for extracellular renalase.

Methods and Results

RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase.

Conclusions

PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.     

Quantitative Analysis of the Mitochondrial and Plastid Proteomes of the Moss Physcomitrella patens Reveals Protein Macrocompartmentation and Microcompartmentation

Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as organelles, namely mitochondria and plastids in plants. During evolution, organellar proteomes are modified by gene gain and loss, by gene subfunctionalization and neofunctionalization, and by changes in protein targeting. To date, proteomics data for plastids and mitochondria are available for only a few plant model species, and evolutionary analyses of high-throughput data are scarce. We combined quantitative proteomics, cross-species comparative analysis of metabolic pathways, and localizations by fluorescent proteins in the model plant Physcomitrella patens in order to assess evolutionary changes in mitochondrial and plastid proteomes. This study implements data-mining methodology to classify and reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study the effects of postendosymbiotic evolution on organellar pathway partitioning. Our results indicate that, although plant morphologies changed substantially during plant evolution, metabolic integration of organelles is largely conserved, with exceptions in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization are common in the studied nucleus-encoded gene families of organelle-targeted proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions revealed novel proteins at organelle interfaces such as plastid stromules (stroma-filled tubules) and highlight microcompartments as well as intercellular and intracellular heterogeneity of mitochondria and plastids. Thus, we establish a comprehensive data set for mitochondrial and plastid proteomes in moss, present a novel multilevel approach to organelle biology in plants, and place our findings into an evolutionary context.Endosymbiosis has enabled and shaped eukaryotic evolution. The engulfment of an ancestral α-proteobacterium by a presumably archaebacterial host cell stands at the origin of mitochondrial and eukaryotic evolution over 1.5 billion years ago (Dyall et al., 2004). In plants, the subsequent uptake of a photosynthetic bacterium between 1.5 and 1.2 billion years ago led to the formation of chloroplasts (Dyall et al., 2004). Plants thereby evolved by the integration of three distinct genetic compartments. After the establishment of endosymbiosis, genes were transferred to a great extent, mainly from mitochondria and plastids to the nucleus (Bock and Timmis, 2008), necessitating an orchestrated flux of information in the form of proteins and metabolites between the compartments of eukaryotic cells to ensure homeostasis, growth, and development. This communication between organelles is facilitated by physical interactions (Kornmann et al., 2009), control of protein import (Ling et al., 2012), and retrograde signaling (Nargund et al., 2012). During radiation and diversification, especially of land plants, nuclear genomes substantially changed due to endosymbiotic and horizontal (Yue et al., 2012) gene transfer, genome duplication, and gene gain and loss (Duarte et al., 2006; Lang et al., 2010; Martin, 2010), obtruding the question of whether these phenomena are linked to alterations in metabolic pathway partitioning between organelles. Retained paralogs can either introduce a new function (neofunctionalization) or reconstitute existing functions (subfunctionalization; Duarte et al., 2006), for example by distinct spatiotemporal expression profiles or distinct subcellular localizations, resulting in the modulation or introduction of metabolic functions in the respective cellular compartments. Moreover, proteins can localize to several subcellular compartments, a phenomenon called dual or multiple targeting (Yogev and Pines, 2011; Xu et al., 2013). Consequently, many eukaroytic metabolic pathways, as well as the plastid and mitochondrial proteomes, are constituted of a mosaic of proteins of diverse evolutionary origins (Szklarczyk and Huynen, 2010), and evolution has shaped variable organellar functionalities across taxa. To date, the evolution and variability of postendosymbiotic metabolic partitioning is largely not characterized on a high-throughput level. So far, large-scale mitochondrial proteome data sets are only available for the green alga Chlamydomonas reinhardtii (Atteia et al., 2009), rice (Oryza sativa; Huang et al., 2009), and the model flowering plant Arabidopsis (Arabidopsis thaliana; Millar et al., 2001; Heazlewood et al., 2004), whereas plastid proteomics in plants is on an advanced level and covers more species (Polyakov et al., 2010; van Wijk and Baginsky, 2011).While higher plants diversified relatively recently but massively, simple moss plants can be traced back 330 million years (Hubers and Kerp, 2012), identifying them as prime candidates for an evolutionary view of organellar proteomes and organelle biology at a genome-wide scale. In contrast to specialized flowering plants, mosses are generalists with few tissues, high metabolic variability, and ancestral features such as high abiotic stress tolerance (Frank et al., 2007) and few plastid types (Cove, 2005).By integrating quantitative proteomics, multivariate analysis, metabolic pathway maps, phylogenomics, and localization with fluorescent proteins, we reliably characterize subcellular proteomes and gene family diversification. Key characteristics of postendosymbiotic organellar proteome evolution are identified by cross-species comparative analysis. In support of our high-throughput analyses, we conduct single-protein analyses and identify proteins that mark microcompartments within organelles and localize to dynamic contact sites between organelles. These proteins may facilitate the exchange of proteins and metabolites, while others influence the dynamics of individual chloroplasts and mitochondria. This study characterizes the mitochondrial and plastid proteomes of moss and reveals the heterogeneity of organelles within a single cell.