Metabolomic Analysis of Plasma in Huntington's Disease Transgenic Sheep (Ovis aries) Reveals Progressive Circadian Rhythm Dysregulation.

BACKGROUND: Metabolic abnormalities have long been predicted in Huntington's disease (HD) but remain poorly characterized. Chronobiological dysregulation has been described in HD and may include abnormalities in circadian-driven metabolism. OBJECTIVE: Here we investigated metabolite profiles in the transgenic sheep model of HD (OVT73) at presymptomatic ages. Our goal was to understand changes to the metabolome as well as potential metabolite rhythm changes associated with HD. METHODS: We used targeted liquid chromatography mass spectrometry (LC-MS) metabolomics to analyze metabolites in plasma samples taken from female HD transgenic and normal (control) sheep aged 5 and 7 years. Samples were taken hourly across a 27-h period. The resulting dataset was investigated by machine learning and chronobiological analysis. RESULTS: The metabolic profiles of HD and control sheep were separable by machine learning at both ages. We found both absolute and rhythmic differences in metabolites in HD compared to control sheep at 5 years of age. An increase in both the number of disturbed metabolites and the magnitude of change of acrophase (the time at which the rhythms peak) was seen in samples from 7-year-old HD compared to control sheep. There were striking similarities between the dysregulated metabolites identified in HD sheep and human patients (notably of phosphatidylcholines, amino acids, urea, and threonine). CONCLUSION: This work provides the first integrated analysis of changes in metabolism and circadian rhythmicity of metabolites in a large animal model of presymptomatic HD.

Highly efficient direct visible-light-mediated oxidative esterification of aldehydes.

A very efficient one-pot procedure for the direct oxidation of aldehydes to esters mediated by visible-light is presented. Utilizing a combination of indium triflate and N-bromosuccinimide, NBS, with either ortho-esters or alcohols provided rapid access to a variety of esters. Certain substrates convert fully within a few seconds, other more challenging tert-butyl esters were formed in good yield after no more than 4 h.

Tick-Tock Consider the Clock: The Influence of Circadian and External Cycles on Time of Day Variation in the Human Metabolome-A Review.

The past decade has seen a large influx of work investigating time of day variation in different human biofluid and tissue metabolomes. The driver of this daily variation can be endogenous circadian rhythms driven by the central and/or peripheral clocks, or exogenous diurnal rhythms driven by behavioural and environmental cycles, which manifest as regular 24 h cycles of metabolite concentrations. This review, of all published studies to date, establishes the extent of daily variation with regard to the number and identity of 'rhythmic' metabolites observed in blood, saliva, urine, breath, and skeletal muscle. The probable sources driving such variation, in addition to what metabolite classes are most susceptible in adhering to or uncoupling from such cycles is described in addition to a compiled list of common rhythmic metabolites. The reviewed studies show that the metabolome undergoes significant time of day variation, primarily observed for amino acids and multiple lipid classes. Such 24 h rhythms, driven by various factors discussed herein, are an additional source of intra/inter-individual variation and are thus highly pertinent to all studies applying untargeted and targeted metabolomics platforms, particularly for the construction of biomarker panels. The potential implications are discussed alongside proposed minimum reporting criteria suggested to acknowledge time of day variation as a potential influence of results and to facilitate improved reproducibility.

Truly unentangled photon pairs without spectral filtering.

We demonstrate that an integrated silicon microring resonator is capable of efficiently producing photon pairs that are completely unentangled; such pairs are a key component of heralded single-photon sources. A dual-channel interferometric coupling scheme can be used to independently tune the quality factors associated with the pump and signal and idler modes, yielding a biphoton wavefunction with a Schmidt number arbitrarily close to unity. This will permit the generation of heralded single-photon states with unit purity.

Proteoforms in Peripheral Blood Mononuclear Cells as Novel Rejection Biomarkers in Liver Transplant Recipients.

Biomarker profiles of acute rejection in liver transplant recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform signatures of acute rejection in circulating immune cells, using an emergent "top-down" proteomics methodology. We prepared differentially processed and cryopreserved cell lysates from 26 nonviral liver transplant recipients by molecular weight-based fractionation and analyzed them by mass spectrometry of whole proteins in three steps: (i) Nanocapillary liquid chromatography coupled with high-resolution tandem mass spectrometry; (ii) database searching to identify and characterize intact proteoforms; (iii) data processing through a hierarchical linear model matching the study design to quantify proteoform fold changes in patients with rejection versus normal liver function versus acute dysfunction without rejection. Differentially expressed proteoforms were seen in patients with rejection versus normal and nonspecific controls, most evidently in the cell preparations stored in traditional serum-rich media. Mapping analysis of these proteins back to genes through gene ontology and pathway analysis tools revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these novel rejection signatures and test their predictive value for use in clinical management.

Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.

BACKGROUND: The actual mechanisms underlying human hippocampal epileptogenicity, a process ultimately mediated by neurochemical events, remains to be fully elucidated. We submit early insight data regarding microdialysis (MD) recovery of the neuroactive amino acids glutamate, aspartate and gamma-aminobutyric acid (GABA) from the intraoperative and intact, spontaneously epileptiform human hippocampus. METHOD: Generally anaesthetised temporal lobe epilepsy (TLE) patients (N=7) undergoing therapeutic and anatomically standardised resective surgery were also subjected to ipsilateral anterior hippocampal MD with concomitant hippocampal electrocorticography (ECoG). Recovered 10-min dialysate samples were quantified for glutamate, aspartate and GABA using high-performance liquid chromatography; corresponding ECoG data was assessed for epileptiform activity (EA); mesial resection tissue was postoperatively examined and graded for hippocampal sclerosis. FINDINGS: Mean 'Sample 3' dialysate absolute recovery of glutamate, aspartate and GABA from hippocampi with minimal EA (N=5) was ( micro M+/-SEM): 6.406+/-2.143, 0.600+/-0.215, and 0.357+/-0.093, respectively. In contrast, 'Sample 3' dialysate absolute glutamate, aspartate and GABA levels ( micro M) from the hippocampi of two patients with vigorous EA were: 101.099 and 211.861, 21.860 and 14.482, and 4.241 and 4.817, respectively. Mesial resection tissue in all cases demonstrated hippocampal sclerosis, though the histopathological degree of sclerosis varied between patients. INTERPRETATION: These preliminary intraoperative findings suggest that dialysate glutamate, aspartate, and GABA levels from the sclerotic anterior hippocampus likely reflects the functional status of the sampled tissue - i.e., lower levels of these neuroactive amino acids are to be expected during quiescent or minimal EA versus considerably higher levels corresponding to vigorous EA.

Reconstruction of real world head injury accidents resulting from falls using multibody dynamics.

OBJECTIVE: To reconstruct real life head injury accidents resulting from falls using multibody modelling software, with the aim of comparing simulation output to injuries sustained. BACKGROUND: Much previous research on head injury biomechanics has focussed on animals and cadavers. However, focus is increasingly turning towards the examination of real life head injury. Falls are a major cause of head injury and, in general, are simpler to model than other accident types. DESIGN AND METHODS: Five cases of simple falling accidents resulting in focal head injury were examined, and reconstructions were performed using a multibody model of the human body. Each case was reconstructed a number of times, varying the initial conditions and using two different sets of properties for head contact. RESULTS: Results obtained included velocities, accelerations and forces on the head during impact. This output appeared more sensitive to changes in head contact characteristics than to changes in initial conditions. Depending on the contact characteristics used, results were consistent with proposed tolerance limits from the literature for various lesion types. CONCLUSIONS: Provided it is used with caution, this method could prove a useful source of biomechanical data for the investigation of head injury biomechanics. RELEVANCE: Biomechanical investigation of real-life cases of head injury is very important, yet not as prevalent as work with animals and cadavers. Reconstruction of real life accidents is a good method of obtaining data that will aid in the investigation of mechanisms of head injury and human tolerance to head injury.

Elevated extracellular levels of glutamate, aspartate and gamma-aminobutyric acid within the intraoperative, spontaneously epileptiform human hippocampus.

We report preliminary results from four patients subjected to hippocampal electrocorticography and microdialysis during temporal lobe epilepsy surgery. In two cases, spontaneously vigorous hippocampal epileptiform activity (EA) was identified; basal dialysate levels for hippocampal glutamate, aspartate, and gamma-aminobutyric acid ranged from approximately 23- to 84-fold, 19- to 33-fold and 10- to 34-fold higher, respectively, compared to the two cases of minimal hippocampal EA. These findings represent the first intraoperative evidence of elevated extracellular levels of neuroactive amino acids within the spontaneously epileptiform human hippocampus.

Structure of the mouse calcitonin/calcitonin gene-related peptide alpha and beta genes.

We report the cloning, genomic organization and sequence of the mouse alpha-CALC and beta-CALC genes. The two genes share extensive sequence homology. The transcription units of both genes contain 6 exons. Transcripts of the alpha-CALC gene were found to alternatively include exon 4 or exons 5 and 6. For the beta-CALC gene exon 4 was not detected in transcripts derived from this gene. The predicted mouse alpha-CGRP was found to be identical to rat alpha-CGRP, however, beta-CGRP predicted amino acid sequences revealed three amino acid differences suggesting these residues are not critical to CGRP function.

A novel sulfonylurea receptor family member expressed in the embryonic Drosophila dorsal vessel and tracheal system.

Sulfonylurea receptors (SURx) are required subunits of the ATP-sensitive potassium channel. SURx alone is electrophysiologically inert. However, when SURx is combined with an inward rectifier Kir6.2 subunit, ATP-sensitive potassium channel activity is generated. We report the identification, characterization, and localization of Dsur, a novel Drosophila gene that is highly related to the vertebrate SUR family. The Dsur coding sequence contains structural features characteristic of the ABC transporter family and, in addition, harbors 1.7 kilobases of a distinctive sequence that does not share homology with any known gene. When Dsur alone is expressed in Xenopus oocytes glibenclamide-sensitive potassium channel activity occurs. During Drosophila embryogenesis, the Dsur gene is specifically expressed in the developing tracheal system and dorsal vessel. Studies of the Drosophila genome support that only a single Dsur gene is present. Our data reveal conservation of glibenclamide-sensitive potassium channels in Drosophila and suggest that Dsur may play an important role during Drosophila embryogenesis. The lack of gene duplication in the Drosophila system provides a unique opportunity for functional studies of SUR using a genetic approach.

Genetic mutations as a cause of hyperinsulinemic hypoglycemia in children.

Hyperinsulinemic hypoglycemia in children is associated with unregulated secretion of insulin and hypoglycemia, a condition that is now known to be genetically diverse. This article reviews recent progress that has elucidated several beta-cell molecular defects responsible for the pathogenesis of this disorder.

A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland.

Mutations in genes encoding the ATP-regulated potassium (K(ATP)) channels of the pancreatic beta-cell (SUR1 and Kir6.2) are the major known cause of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We collected all cases of PHHI diagnosed in Finland between 1983 and 1997 (n = 24). The overall incidence was 1:40,400, but in one area of Central Finland it was as high as 1:3,200. Haplotype analysis using polymorphic markers spanning the SUR1/Kir6.2 gene cluster confirmed linkage to the 11p region. Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No K(ATP) channel activity was observed in beta-cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Thus, the mutation produces a nonfunctional channel and, thereby, continuous insulin secretion. This unique SUR1 mutation explains the majority of PHHI cases in Finland and is strongly associated with a severe form of the disease. These findings provide diagnostic and prognostic utility for suspected PHHI patients.

Complex orthodontic problems: the orthognathic patient with temporomandibular disorders.

The diagnosis and treatment of temporomandibular disorders (TMD) remain controversial despite considerable research and publication in this area. The relationship of these problems to dental and skeletal malocclusion is equally debatable. Recent studies suggest that although malocclusion may have a role, it is a small one. Accordingly, treatment of TMD with occlusion-altering therapy, such as orthodontics and orthognathic surgery, should be limited to specific situations. This report discusses the management of patients with coexisting TMD and skeletal malocclusion. Current concepts in clinical and radiographic diagnosis are discussed, as well as an overview of noninvasive therapy. A case report is used to illustrate an approach to diagnosis and treatment planning in an individual with active TMD and a skeletal malocclusion requiring orthognathic surgery for correction.

A human succinate-ubiquinone oxidoreductase CII-3 subunit gene ending in a polymorphic dinucleotide repeat is located within the sulfonylurea receptor (SUR) gene.

We report the cloning of two variant genes encoding the CII-3 subunit of succinate-ubiquinone oxidoreductase complex II. One gene is located within intron 10 of the human sulfonylurea receptor gene. The 3' boundary of this gene ends in a polymorphic dinucleotide repeat. The second gene CII-3b is expressed at a low level and contains a 102-bp internal deletion compared to CII-3 cDNA. These genes should prove valuable in the characterization of Complex II disorders.

Recovery of reproductive function in rats treated with the aromatase inhibitor fadrozole.

The aromatase inhibitor, fadrozole hydrochloride (CGS 16949A), was developed for the treatment of breast cancer, and has not been available for pediatric use because of the lack of information about potential reproductive toxicology. To determine the effect of fadrozole on subsequent fertility and reproductive performance in rats, peripubertal male and female Sprague-Dawley rats (10/group) were given fadrozole by oral gavage once a day for 60 consecutive days (age 21 through 80 d) at a dose of 0, 1200, or 6000 micrograms/kg/d (dose range in women with breast cancer: 60 to 240 micrograms/kg/d). Following a 30-d recovery period (days 81 through 111 of age), cohabitation with untreated rats of the opposite sex was accomplished for 30 d or until positive evidence of mating was obtained (daily vaginal smears). The nonfadrozole-treated males used for cohabitation were proven fertile breeders; the females were virgin with proven 4-d estrous cycles. The duration of pregnancy, number, sex, condition, and body weight of pups were determined. Pregnant rats were weighed on gestational days 7, 14, and 20. There was a profound decrease in the number of estrous cycles at both dose levels of fadrozole compared to the control (P < 0.001). During the 30-d recovery period, estrous cycles were reestablished within a few days in the treated rats and the number and length of estrous cycles were not statistically different between fadrozole-treated and control rats. The gestational body weights of fadrozole-treated and untreated females did not differ significantly. There were no statistically significant differences in the number of matings/number of pairings, gestational length, mean live pups/litter, % pups born alive/litter, and % male pups/litter in the three groups (vehicle-, low-, and high-dose fadrozole-treated females, cohabited with untreated males and fadrozole-treated males, cohabited with untreated females). Thus, young male and female rats treated for 60 d with large doses of fadrozole had no detectable adverse effect on subsequent reproductive function.

Inactivation of the first nucleotide-binding fold of the sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia of infancy.

Familial persistent hyperinsulinemic hypoglycemia of infancy is a disorder of glucose homeostasis and is characterized by unregulated insulin secretion and profound hypoglycemia. Loss-of-function mutations in the second nucleotide-binding fold of the sulfonylurea receptor, a subunit of the pancreatic-islet beta-cell ATP-dependent potassium channel, has been demonstrated to be causative for persistent hyperinsulinemic hypoglycemia of infancy. We now describe three additional mutations in the first nucleotide-binding fold of the sulfonylurea-receptor gene. One point mutation disrupts the highly conserved Walker A motif of the first nucleotide-binding-fold region. The other two mutations occur in noncoding sequences required for RNA processing and are predicted to disrupt the normal splicing pathway of the sulfonylurea-receptor mRNA precursor. These data suggest that both nucleotide-binding-fold regions of the sulfonylurea receptor are required for normal regulation of beta-cell ATP-dependent potassium channel activity and insulin secretion.

The molecular basis for familial persistent hyperinsulinemic hypoglycemia of infancy.

Familial persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a glucose metabolism disorder in neonates characterized by inappropriate insulin secretion in the presence of profound hypoglycemia. Loss of function mutations in the sulfonylurea receptor (SUR) gene recently have been implicated as a cause for familial PHHI in nine independent families. This review will describe the combined positional cloning and candidate gene strategy used to identify the SUR gene as the one responsible for PHHI. Potential roles for SUR in other disorders of insulin secretion remains to be determined.

Cloning of nod gene regions from mesquite rhizobia and bradyrhizobia and nucleotide sequence of the nodD gene from mesquite rhizobia.

Nitrogen-fixing symbiosis between bacteria and the tree legume mesquite (Prosopis glandulosa) is important for the maintenance of many desert ecosystems. Genes essential for nodulation and for extending the host range to mesquite were isolated from cosmid libraries of Rhizobium (mesquite) sp. strain HW17b and Bradyrhizobium (mesquite) sp. strain HW10h and were shown to be closely linked. All of the cosmid clones of rhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite also supported nodulation of a Sym- mesquite strain. The cosmid clones of bradyrhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite were only able to confer nodulation ability in the Sym- mesquite strain if they also contained a nodD-hybridizing region. Subclones containing just the nodD genes of either genus did not extend the host range of Rhizobium (Parasponia) sp. to mesquite, indicating that the nodD gene is insufficient for mesquite nodulation. The nodD gene region is conserved among mesquite-nodulating rhizobia regardless of the soil depth from which they were collected, indicating descent from a common ancestor. In a tree of distance relationships, the NodD amino acid sequence from mesquite rhizobia clusters with homologs from symbionts that can infect both herbaceous and tree legumes, including Rhizobium tropici, Rhizobium leguminosarum bv; phaseoli, Rhizobium loti, and Bradyrhizobium japonicum.