In vivo and in vitro models of demyelinating diseases. V. Comparison of the assembly of mouse hepatitis virus, strain JHM, in two murine cell lines.

The developmental sequence of a neurotropic strain (JHM) of mouse hepatitis virus was examined by transmission electron microscopy and immunocytology. The nucleoprotein core of this coronavirus, which contains RNA of positive polarity and is helical in configuration, becomes incorporated into enveloped particles in the same manner as the nucleocapsids of the orthomyxo- and paramyxoviruses. However, JHM virus is assembled intracellularly by budding at surfaces of smooth membranous vacuoles. A comparison of JHM virus replication in L2 and 17Cl-1 cell lines revealed that L2 cells undergo more rapid cytopathology and cease virus production much sooner than 17Cl-l cells. In L2 cells the accumulation of core material appears to continue after the abrupt cessation of virus assembly. This is evident by the massive cytoplasmic accumulation of structure resembling nucleocapsids, which react with hybridoma antibody to the nucleocapsid antigen as demonstrated by the immunoperoxidase procedure. The current findings are consistent with our previously published demonstration, using cells of neural and other deviation, of the fundamental role of the host cell type in regulating the replication and expression of coronaviruses

Cystathionine beta-synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts

Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism

Tephrochronology and its application: A review

Tephrochronology (from tephra, Gk ‘ashes’) is a unique stratigraphic method for linking, dating, and synchronizing geological, palaeoenvironmental, or archaeological sequences or events. As well as utilising the Law of Superposition, tephrochronology in practise requires tephra deposits to be characterized (or ‘fingerprinted’) using physical properties evident in the field together with those obtained from laboratory analyses. Such analyses include mineralogical examination (petrography) or geochemical analysis of glass shards or crystals using an electron microprobe or other analytical tools including laser-ablation-based mass spectrometry or the ion microprobe. The palaeoenvironmental or archaeological context in which a tephra occurs may also be useful for correlational purposes. Tephrochronology provides greatest utility when a numerical age obtained for a tephra or cryptotephra is transferrable from one site to another using stratigraphy and by comparing and matching inherent compositional features of the deposits with a high degree of likelihood. Used this way, tephrochronology is an age-equivalent dating method that provides an exceptionally precise volcanic-event stratigraphy. Such age transfers are valid because the primary tephra deposits from an eruption essentially have the same short-lived age everywhere they occur, forming isochrons very soon after the eruption (normally within a year). As well as providing isochrons for palaeoenvironmental and archaeological reconstructions, tephras through their geochemical analysis allow insight into volcanic and magmatic processes, and provide a comprehensive record of explosive volcanism and recurrence rates in the Quaternary (or earlier) that can be used to establish time-space relationships of relevance to volcanic hazard analysis. The basis and application of tephrochronology as a central stratigraphic and geochronological tool for Quaternary studies are presented and discussed in this review. Topics covered include principles of tephrochronology, defining isochrons, tephra nomenclature, mapping and correlating tephras from proximal to distal locations at metre- through to sub-millimetre-scale, cryptotephras, mineralogical and geochemical fingerprinting methods, numerical and statistical correlation techniques, and developments and applications in dating including the use of flexible depositional age-modelling techniques based on Bayesian statistics. Along with reference to wide-ranging examples and the identification of important recent advances in tephrochronology, such as the development of new geoanalytical approaches that enable individual small glass shards to be analysed near-routinely for major, trace, and rare-earth elements, potential problems such as miscorrelation, erroneous-age transfer, and tephra reworking and taphonomy (especially relating to cryptotephras) are also examined. Some of the challenges for future tephrochronological studies include refining geochemical analytical methods further, improving understanding of cryptotephra distribution and preservation patterns, improving age modelling including via new or enhanced radiometric or incremental techniques and Bayesian-derived models, evaluating and quantifying uncertainty in tephrochronology to a greater degree than at present, constructing comprehensive regional databases, and integrating tephrochronology with spatially referenced environmental and archaeometric data into 3-D reconstructions using GIS and geostatistics

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Clonally expanded mitochondrial DNA deletions within the choroid plexus in multiple sclerosis

OBJECTIVE: Mitochondrial DNA deletions (Δ-mtDNA) are implicated in the pathogenesis of multiple sclerosis (MS), Parkinson’s disease (PD), Alzheimer’s disease (AD) and ageing. Given the diffuse nature of inflammation in MS, aim of this study was to determine whether Δ-mtDNA caused respiratory deficient cells in excess of age within choroid plexus (CP) and ongoing mutagenesis or clonal expansion accounted for the respiratory deficiency in MS. METHODS: Respiratory chain complex IV and complex II activity was determined sequentially using histochemistry. Δ-mtDNA were characterized using real time PCR, long range PCR, sequencing and single molecule PCR. Sources of reactive oxygen and nitrogen species (RONS) were explored using immunohistochemistry. RESULTS: Respiratory deficient cells (lacking complex IV and with intact complex II activity) within CP epithelium were in excess of age in MS, PD and AD. Subunit-I of complex IV was lacking to a greater extent in MS than controls. Percentage of respiratory deficient cells harboring >50% heteroplasmy level of Δ-mtDNA was significantly greater in MS than PD, AD and controls. Long range PCR and sequencing confirmed Δ-mtDNA. Single molecule PCR identified clonally expanded Δ-mtDNA in MS, despite an increase in sources of RONS. INTERPRETATION: Our findings establish clonal expansion of Δ-mtDNA causing respiratory deficiency in MS and the extraparenchymal intracranial location indicated the potential to involve multiple cell types. Understanding factors that influence clonal expansion of Δ-mtDNA, a molecular link between inflammation and delayed cellular energy failure, may identify potential therapeutic targets for progressive forms of MS as well as other neurodegenerative disorders

Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I

G41 is an interfacial residue located within the α-helix 34–42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (∼5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 → R mutation causes a partial unwinding of the 34–42 α-helix and a displacement of the first 44 N-terminal residues including the active site loop 24–32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations