Exclusive neuronal expression of SUCLA2 in the human brain

SUCLA2 encodes the ATP-forming subunit (A-SUCL-) of succinyl-CoA ligase, an enzyme of the citric acid cycle. Mutations in SUCLA2 lead to a mitochondrial disorder manifesting as encephalomyopathy with dystonia, deafness and lesions in the basal ganglia. Despite the distinct brain pathology associated with SUCLA2 mutations, the precise localization of SUCLA2 protein has never been investigated. Here we show that immunoreactivity of A-SUCL- in surgical human cortical tissue samples was present exclusively in neurons, identified by their morphology and visualized by double labeling with a fluorescent Nissl dye. A-SUCL- immunoreactivity co-localized >99% with that of the d subunit of the mitochondrial F0-F1 ATP synthase. Specificity of the anti-A-SUCL- antiserum was verified by the absence of labeling in fibroblasts from a patient with a complete deletion of SUCLA2. A-SUCL- immunoreactivity was absent in glial cells, identified by antibodies directed against the glial markers GFAP and S100. Furthermore, in situ hybridization histochemistry demonstrated that SUCLA2 mRNA was present in Nissl-labeled neurons but not glial cells labeled with S100. Immunoreactivity of the GTP-forming subunit (G-SUCL-) encoded by SUCLG2, or in situ hybridization histochemistry for SUCLG2 mRNA could not be demonstrated in either neurons or astrocytes. Western blotting of post mortem brain samples revealed minor G-SUCL- immunoreactivity that was however, not upregulated in samples obtained from diabetic versus non-diabetic patients, as has been described for murine brain. Our work establishes that SUCLA2 is expressed exclusively in neurons in the human cerebral cortex

Sampling the Solution Space in Genome-Scale Metabolic Networks Reveals Transcriptional Regulation in Key Enzymes

Genome-scale metabolic models are available for an increasing number of organisms and can be used to define the region of feasible metabolic flux distributions. In this work we use as constraints a small set of experimental metabolic fluxes, which reduces the region of feasible metabolic states. Once the region of feasible flux distributions has been defined, a set of possible flux distributions is obtained by random sampling and the averages and standard deviations for each of the metabolic fluxes in the genome-scale model are calculated. These values allow estimation of the significance of change for each reaction rate between different conditions and comparison of it with the significance of change in gene transcription for the corresponding enzymes. The comparison of flux change and gene expression allows identification of enzymes showing a significant correlation between flux change and expression change (transcriptional regulation) as well as reactions whose flux change is likely to be driven only by changes in the metabolite concentrations (metabolic regulation). The changes due to growth on four different carbon sources and as a consequence of five gene deletions were analyzed for Saccharomyces cerevisiae. The enzymes with transcriptional regulation showed enrichment in certain transcription factors. This has not been previously reported. The information provided by the presented method could guide the discovery of new metabolic engineering strategies or the identification of drug targets for treatment of metabolic diseases

Haematogenous Staphylococcus aureus meningitis. A 10-year nationwide study of 96 consecutive cases

BACKGROUND: Haematogenous Staphylococcus aureus meningitis is rare but associated with high mortality. Knowledge about the disease is still limited. The objective of this study was to evaluate demographic and clinical prognostic features of bacteraemic S. aureus meningitis. METHODS: Nationwide surveillance in Denmark from 1991 to 2000 with clinical and bacteriological data. Risks of death were estimated by Cox proportional hazards regression analysis. RESULTS: Among 12480 cases of S. aureus bacteraemia/sepsis, we identified 96 cases of non-surgical bacteraemic S. aureus meningitis (0.8%). Incidence rates were 0.24 (95% confidence interval [CI], 0.18 to 0.30)/100 000 population between 1991–1995 and 0.13 (CI, 0.08 to 0.17)/100 000 population between 1996–2000. Mortality was 56%. After adjustment, only co morbidity (hazard ratio [HR], 3.45; CI, 1.15 to 10.30) and critical illness (Pitt score ≥ 4) (HR, 2.14; CI, 1.09 to 4.19) remained independent predictors of mortality. CONCLUSION: The incidence, but not mortality of bacteraemic S. aureus meningitis decreased during the study period. Co morbidity and critical illness were independent predictors of a poor outcome

Molecular Cloning and Characterization of Two Genes Encoding Dihydroflavonol-4-Reductase from Populus trichocarpa

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars

Large-Scale Phenotyping of an Accurate Genetic Mouse Model of JNCL Identifies Novel Early Pathology Outside the Central Nervous System

Cln3Δex7/8 mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3Δex7/8 mice. Homozygous Cln3Δex7/8 mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10–14 weeks of age. Homozygous Cln3Δex7/8 mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12–13 week old homozygous Cln3Δex7/8mice, which were also seen to a lesser extent in heterozygous Cln3Δex7/8 mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15–16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3Δex7/8 mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3Δex7/8 neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3Δex7/8 mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3Δex7/8 mice that merit further study for JNCL biomarker development

A Novel Soluble Immune-Type Receptor (SITR) in Teleost Fish: Carp SITR Is Involved in the Nitric Oxide-Mediated Response to a Protozoan Parasite

Background- The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Methodology/Principal Findings - Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. Conclusion/Significance - We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite

Identification and Validation of Novel Cerebrospinal Fluid Biomarkers for Staging Early Alzheimer's Disease

Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the 'preclinical' stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome.CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85-0.94 95% confidence interval [CI]) and 0.88 (0.81-0.94 CI), respectively.Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions

Differential effects on gene transcription and hematopoietic differentiation correlate with GATA2 mutant disease phenotypes

Heterozygous GATA2 mutations underlie an array of complex hematopoietic and lymphatic diseases. Analysis of the literature reporting three recurrent GATA2 germline (g) mutations (gT354M, gR396Q and gR398W) revealed different phenotype tendencies. Although all three mutants differentially predispose to myeloid malignancies, there was no difference in leukemia-free survival for GATA2 patients. Despite intense interest, the molecular pathogenesis of GATA2 mutation is poorly understood. We functionally characterized a GATA2 mutant allelic series representing major disease phenotypes caused by germline and somatic (s) mutations in zinc finger 2 (ZF2). All GATA2 mutants, except for sL359V, displayed reduced DNA-binding affinity and transactivation compared with wild type (WT), which could be attributed to mutations of arginines critical for DNA binding or amino acids required for ZF2 domain structural integrity. Two GATA2 mutants (gT354M and gC373R) bound the key hematopoietic differentiation factor PU.1 more strongly than WT potentially perturbing differentiation via sequestration of PU.1. Unlike WT, all mutants failed to suppress colony formation and some mutants skewed cell fate to granulocytes, consistent with the monocytopenia phenotype seen in GATA2-related immunodeficiency disorders. These findings implicate perturbations of GATA2 function shaping the course of development of myeloid malignancy subtypes and strengthen complete or nearly complete haploinsufficiency for predisposition to lymphedema.C-E Chong, P Venugopal, PH Stokes, YK Lee, PJ Brautigan, DTO Yeung, M Babic, GA Engler, SW Lane, M Klingler-Hoffmann, JM Matthews, RJ D'Andrea, AL Brown, CN Hahn, and HS Scot