Tonic extracellular glutamate and ischaemia: glutamate antiporter system xc - regulates anoxic depolarization in hippocampus.

In stroke, the sudden deprivation of oxygen to neurons triggers a profuse release of glutamate that induces anoxic depolarization (AD) and leads to rapid cell death. Importantly, the latency of the glutamate-driven AD event largely dictates subsequent tissue damage. Although the contribution of synaptic glutamate during ischaemia is well-studied, the role of tonic (ambient) glutamate has received far less scrutiny. The majority of tonic, non-synaptic glutamate in the brain is governed by the cystine/glutamate antiporter, system xc - . Employing hippocampal slice electrophysiology, we showed that transgenic mice lacking a functional system xc - display longer latencies to AD and altered depolarizing waves compared to wild-type mice after total oxygen deprivation. Experiments which pharmacologically inhibited system xc - , as well as those manipulating tonic glutamate levels and those antagonizing glutamate receptors, revealed that the antiporter's putative effect on ambient glutamate precipitates the ischaemic cascade. As such, the current study yields novel insight into the pathogenesis of acute stroke and may direct future therapeutic interventions. KEY POINTS: Ischaemic stroke remains the leading cause of adult disability in the world, but efforts to reduce stroke severity have been plagued by failed translational attempts to mitigate glutamate excitotoxicity. Elucidating the ischaemic cascade, which within minutes leads to irreversible tissue damage induced by anoxic depolarization, must be a principal focus. Data presented here show that tonic, extrasynaptic glutamate supplied by system xc - synergizes with ischaemia-induced synaptic glutamate release to propagate AD and exacerbate depolarizing waves. Exploiting the role of system xc - and its obligate release of ambient glutamate could, therefore, be a novel therapeutic direction to attenuate the deleterious effects of acute stroke.

Serum anti-tetanus and measles antibody titres in Ugandan children aged 4 months to 6 years: implications for vaccine programme.

To study the antibody response to tetanus toxoid and measles by age following vaccination in children aged 4 months to 6 years in Entebbe, Uganda. Serum samples were obtained from 113 children aged 4-15 months, at the Mother-Child Health Clinic (MCHC), Entebbe Hospital and from 203 of the 206 children aged between 12 and 75 months recruited through the Outpatients Department (OPD). Antibodies to measles were quantified by plaque reduction neutralisation test (PRNT) and with Siemens IgG EIA. VaccZyme IgG EIA was used to quantify anti-tetanus antibodies. Sera from 96 of 113 (85.0%) children attending the MCHC contained Measles PRNT titres below the protective level (120 mIU/ml). Sera from 24 of 203 (11.8%) children attending the OPD contained PRNT titres 0.15 IU/ml by EIA, a level considered protective. The overall concentration of anti-tetanus antibody was sixfold higher in children under 12 months compared with the older children, with geometric mean concentrations of 3.15 IU/ml and 0.49 IU/ml, respectively. For each doubling in age between 4 and 64 months, the anti-tetanus antibody concentration declined by 50%. As time since the administration of the third DTP vaccination doubled, anti-tetanus antibody concentration declined by 39%. The low measles antibody prevalence in the children presenting at the MCHC is consistent with the current measles epidemiology in Uganda, where a significant number of measles cases occur in children under 1 year of age and earlier vaccination may be indicated. The consistent fall in anti-tetanus antibody titre over time following vaccination supports the need for further vaccine boosters at age 4-5 years as recommended by the WHO.

Regulation of hippocampal synaptic strength by glial xCT.

Most extracellular glutamate in the brain is released by xCT, a glial antiporter that exports glutamate and imports cystine. The function of xCT, and extracellular glutamate in general, remains unclear. Several lines of evidence suggest that glutamate from xCT could act in a paracrine fashion to suppress glutamatergic synapse strength by triggering removal of postsynaptic glutamate receptors. To test this idea, we used whole-cell patch-clamp electrophysiology and immunohistochemistry to quantify receptor number and synapse function in xCT knock-out mouse hippocampal CA3-CA1 synapses. Consistent with the hypothesis that xCT suppresses glutamate receptor number and synapse strength, xCT knock-out synapses showed increased AMPA receptor abundance with concomitant large enhancements of spontaneous and evoked synaptic transmission. We saw no evidence for changes in GABA receptor abundance or the overall number of glutamatergic synapses. The xCT knock-out phenotype was replicated by incubating slices in the xCT inhibitor (S)-4-carboxyphenylglycine, and consistent with the idea that xCT works by regulating extracellular glutamate, the xCT knock-out phenotype could be reproduced in controls by incubating the slices in glutamate-free aCSF. We conclude that glutamate secreted via xCT suppresses glutamatergic synapse strength by triggering removal of postsynaptic AMPA receptors.

Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory.

Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. We tested this hypothesis in Drosophila, in which the role of the PKA pathway in associative learning has been well established. We first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. We next assayed memory performance 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that tomosyn is involved in a specific component of late associative memory.

A measles IgM rapid diagnostic test to address challenges with national measles surveillance and response in Malaysia.

INTRODUCTION: A lateral flow rapid diagnostic test (RDT) enables detection of measles specific immunoglobulin M (IgM) antibody in serum, capillary blood, and oral fluid with accuracy consistent with enzyme immunoassay (EIA). The objectives of the study were: 1) to assess measles RDT inter-reader agreement between two clinic staff; 2) to assess the sensitivity and specificity of the measles RDT relative to standard surveillance testing in a low transmission setting; 3) to evaluate the knowledge, attitudes, and practices of staff in clinics using the RDT; and 4) to assess the impact of RDT testing on the measles public health response in Malaysia. MATERIALS AND METHODS: The clinic-based prospective evaluation included all suspected measles cases captured by routine measles surveillance at 34 purposely selected clinics in 15 health districts in Malaysia between September 2019 and June 2020, following day-long regional trainings on RDT use. Following informed consent, four specimens were collected from each suspected case, including those routinely collected for standard surveillance [serum for EIA and throat swabs for quantitative reverse transcriptase polymerase chain reaction (RT-qPCR)] together with capillary blood and oral fluid tested with RDTs during the study. RDT impact was evaluated by comparing the rapidity of measles public health response between the pre-RDT implementation (December 2018 to August 2019) and RDT implementation periods (September 2019 to June 2020). To assess knowledge, attitudes, and practices of RDT use, staff involved in the public health management of measles at the selected sites were surveyed. RESULTS: Among the 436 suspect cases, agreement of direct visual readings of measles RDT devices between two health clinic staff was 99% for capillary blood (k = 0.94) and 97% for oral fluid (k = 0.90) specimens. Of the total, 45 (10%) were positive by measles IgM EIA (n = 44, including five also positive by RT-qPCR) or RT-qPCR only (n = 1), and 38 were positive by RDT (using either capillary blood or oral fluid). Using measles IgM EIA or RT-qPCR as reference, RDT sensitivity using capillary blood was 43% (95% CI: 30%-58%) and specificity was 98% (95% CI: 96%-99%); using oral fluid, sensitivity (26%, 95% CI: 15%-40%) and specificity (97%, 95% CI: 94%-98%) were lower. Nine months after training, RDT knowledge was high among staff involved with the public health management of measles (average quiz score of 80%) and was highest among those who received formal training (88%), followed by those trained during supervisory visits (83%). During the RDT implementation period, the number of days from case confirmation until initiation of public response decreased by about 5 days. CONCLUSION: The measles IgM RDT shows >95% inter-reader agreement, high retention of RDT knowledge, and a more rapid public health response. However, despite ≥95% RDT specificity using capillary blood or oral fluid, RDT sensitivity was <45%. Higher-powered studies using highly specific IgM assays and systematic RT-qPCR for case confirmation are needed to establish the role of RDT in measles elimination settings.

Membrane penetration by synaptotagmin is required for coupling calcium binding to vesicle fusion in vivo.

The vesicle protein synaptotagmin I is the Ca(2+) sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca(2+) binding by the C(2)B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca(2+) binding results in vesicle fusion remains controversial. Ca(2+)-dependent interactions between synaptotagmin and SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C(2)B Ca(2+)-binding motif, suggesting that these interactions are facilitatory rather than essential. Here we use Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca(2+)-binding pockets of synaptotagmin. Mutation of this residue in the C(2)A domain (F286) resulted in a ∼50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C(2)B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in syt(null) mutants, which is more severe than the phenotype of C(2)B Ca(2+)-binding mutants. Thus, mutation of a single, C(2)B hydrophobic residue required for Ca(2+)-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C(2)B domain of synaptotagmin, is the critical effector interaction for coupling Ca(2+) binding with vesicle fusion.

Nanoliter hemolymph sampling and analysis of individual adult Drosophila melanogaster.

The fruit fly (Drosophila melanogaster) is an extensively used and powerful, genetic model organism. However, chemical studies using individual flies have been limited by the animal's small size. Introduced here is a method to sample nanoliter hemolymph volumes from individual adult fruit-flies for chemical analysis. The technique results in an ability to distinguish hemolymph chemical variations with developmental stage, fly sex, and sampling conditions. Also presented is the means for two-point monitoring of hemolymph composition for individual flies.

Pre and postsynaptic roles for Drosophila CASK.

CASK ('calcium/calmodulin-dependent serine protein kinase'), also known in Drosophila as 'Caki' or 'Camguk/CMG', and in C. elegans as 'Lin-2', is thought to play an important role in cell-cell junction formation and at synapses in particular. To understand the role of CASK in synapse formation and function, we functionally and morphologically analyzed Drosophila embryonic and larval glutamatergic neuromuscular junctions (NMJs) after pan-cellular and tissue-specific manipulation of CASK expression. Our results show that Drosophila CASK is associated with both pre and postsynaptic membranes. Loss of presynaptic CASK led to less evoked synaptic transmission, fewer spontaneous synaptic events, and reduced synaptic vesicle cycling. These changes were accompanied by a reduction in the number of synapses but no change in overall NMJ size. Loss of postsynaptic CASK, on the other hand, caused reduced spontaneous synaptic current amplitudes and smaller glutamate-gated currents. These changes were accompanied by loss of postsynaptic glutamate receptors, but the receptor loss was subtype-specific: Only receptors containing GluRIIA subunits were lost in CASK mutants. Receptors containing GluRIIB were unaffected.

A glial amino-acid transporter controls synapse strength and courtship in Drosophila.

Mate choice is an evolutionarily critical decision that requires the detection of multiple sex-specific signals followed by central integration of these signals to direct appropriate behavior. The mechanisms controlling mate choice remain poorly understood. Here, we show that the glial amino-acid transporter genderblind controls whether Drosophila melanogaster males will attempt to mate with other males. Genderblind (gb) mutant males showed no alteration in heterosexual courtship or copulation, but were attracted to normally unappealing male species-specific chemosensory cues. As a result, genderblind mutant males courted and attempted to copulate with other Drosophila males. This homosexual behavior could be induced within hours using inducible RNAi, suggesting that genderblind controls nervous system function rather than its development. Consistent with this, and indicating that glial genderblind regulates ambient extracellular glutamate to suppress glutamatergic synapse strength in vivo, homosexual behavior could be turned on and off by altering glutamatergic transmission pharmacologically and/or genetically.

Risk analysis for measles reintroduction after global certification of eradication.

BACKGROUND: Measles virus will continue to exist after certification of global eradication as virus stocks and infectious materials held in laboratories, in persistently and chronically infected individuals, and possibly in undetected foci of transmission. A literature search was undertaken to identify and evaluate the main risks for reintroduction of measles transmission in the absence of universal measles immunization. METHODS: A qualitative risk assessment was conducted following a series of literature searches using the PubMed database. RESULTS: If the criteria for global certification of eradication are stringent and require rigorous validation, then the risk of undetected measles transmission after certification is very low. Risk of unintentional reintroduction from any source, including persistent infections and laboratory materials is low to very low but depends on the extent of measles vaccine use. If immunization levels decrease, measles will become a credible agent for bioterrorism through intentional release. CONCLUSIONS: Posteradication risks are low and should not deter any attempt at measles eradication. More information on measles transmission dynamics and the role of atypical infections is required to determine requirements for global certification of eradication. Posteradication risks would be minimized through development and implementation of an international risk management strategy, including requirements for a posteradication vaccine stockpile.

Evaluation of the World Health Organization global measles and rubella quality assurance program, 2001-2008.

BACKGROUND: During 2001-2008, the Victorian Infectious Diseases Reference Laboratory (VIDRL) prepared and provided a measles and rubella proficiency test panel for distribution to the World Health Organization (WHO) measles and rubella network laboratories as part of their annual laboratory accreditation assessment. METHODS: Panel test results were forwarded to VIDRL, and results from 8 consecutive years were analyzed. We assessed the type of assays used and results achieved on the basis of the positive and negative interpretation of submitted results, by year and WHO region, for measles and rubella. RESULTS: Over time, there has been a noticeable increase in laboratory and WHO regional participation. For all panels, the proportion of laboratories in all WHO regions using the WHO-validated Dade Behring assay for measles and rubella-specific IgM antibodies ranged from 35% to 100% and 59% to 100%, respectively. For all regions and years, the proportion of laboratories obtaining a pass score ranged from 87% to 100% for measles and 93% to 100% for rubella. CONCLUSIONS: During 2001-2008, a large proportion of laboratories worldwide achieved and maintained a pass score for both measles and rubella. Measles and rubella proficiency testing is regarded as a major achievement for the WHO measles and rubella laboratory program.

Improving global virologic surveillance for measles and rubella.

An important aspect of laboratory surveillance for measles and rubella is the genetic characterization of circulating wild-type viruses to support molecular epidemiologic studies and to track transmission pathways. Virologic surveillance that is sufficient to document the interruption of transmission of measles and rubella viruses will be an essential criterion for verification of elimination. Laboratories in the World Health Organization (WHO) Measles and Rubella Laboratory Network have worked to improve and expand virologic surveillance as many regions move toward elimination of measles and rubella/congenital rubella syndrome. As countries approach elimination, it will be necessary to obtain genetic information from as many chains of transmission as possible. In addition, baseline virologic surveillance, especially for rubella, needs to be improved in many countries. This report contains a summary of recent improvements to the methods used for virologic surveillance.

Expansion of the global measles and rubella laboratory network 2005-09.

Enhancing measles surveillance with integration of epidemiologic and laboratory information is one of the key strategies for accelerated measles control and elimination. The World Health Organization (WHO) Global Measles and Rubella Laboratory Network (LabNet) has been developed since 2000 to currently include 690 laboratories serving 183 countries. The LabNet testing strategy follows well-validated, standardized procedures for confirming suspected cases and for monitoring measles and rubella virus transmission patterns. The strength of the LabNet is a strong quality assurance program that monitors the performance of all laboratories through annual proficiency testing and continuous assessment. In the 5-year period 2005-2009, the results of >1 million measles immunoglobulin M (IgM) tests have been reported by the LabNet and, in addition, sequence information on >7000 measles and 600 rubella viruses has been shared. Progress with the development of the LabNet during 2005-2009 is discussed.

Global distribution of measles genotypes and measles molecular epidemiology.

A critical component of laboratory surveillance for measles is the genetic characterization of circulating wild-type viruses. The World Health Organization (WHO) Measles and Rubella Laboratory Network (LabNet), provides for standardized testing in 183 countries and supports genetic characterization of currently circulating strains of measles viruses. The goal of this report is to describe the lessons learned from nearly 20 years of virologic surveillance for measles, to describe the global databases for measles sequences, and to provide regional updates about measles genotypes detected by recent surveillance activities. Virologic surveillance for measles is now well established in all of the WHO regions, and most countries have conducted at least some baseline surveillance. The WHO Global Genotype Database contains >7000 genotype reports, and the Measles Nucleotide Surveillance (MeaNS) contains >4000 entries. This sequence information has proven to be extremely useful for tracking global transmission patterns and for documenting the interruption of transmission in some countries. The future challenges will be to develop quality control programs for molecular methods and to continue to expand virologic surveillance activities in all regions.

Status of global virologic surveillance for rubella viruses.

The suspected measles case definition captures rubella cases. Therefore, measles surveillance will be improved in the course of the control and eventual elimination of rubella transmission. One aspect of rubella control, virologic surveillance, is reviewed here. A systematic nomenclature for rubella viruses (RVs) based on 13 genotypes has been established and is updated when warranted by increases in information about RVs. From 2005 through 2010, the genotypes of RVs most frequently reported were 1E, 1G, and 2B, and genotypes 1a, 1B, 1C, 1h, 1j, and 2C were less frequently reported. Virologic surveillance can support rubella control and elimination. Synopses of rubella virologic surveillance in various countries, regions, and globally are given, including characterization of viruses from imported cases in a country that has eliminated rubella and studies of endemic viruses circulating in countries without rubella control objectives. Current challenges are discussed.

Glial solute carrier transporters in Drosophila and mice.

Glia regulate brain physiology primarily by regulating the movement and concentration of substances in the extracellular fluid. Therefore, one approach to understanding the role of glia in brain physiology is to study what happens when glial transporters are removed or modified. The largest and most highly conserved class of transporter is solute carrier (SLC) proteins. SLC proteins are highly expressed in brain, and many are found in glia. The function of many SLC proteins in the brain--particularly in glia--is very poorly understood. SLC proteins can be relatively easily knocked out or modified in genetic model organisms to better understand glial function. Drosophila are popular genetic model organisms that offer a nice balance between genetic malleability and brain complexity. They are ideal for such an endeavor. This article lists and discusses SLC transporter family members that are expressed in both mouse and Drosophila glia, in an effort to provide a foundation for studies of glial SLC transporters using Drosophila as a model.

Drosophila glutamate receptor mRNA expression and mRNP particles.

The processes controlling glutamate receptor expression early in synaptogenesis are poorly understood. Here, we examine glutamate receptor (GluR) subunit mRNA expression and localization in Drosophila embryonic/larval neuromuscular junctions (NMJs). We show that postsynaptic GluR subunit gene expression is triggered by contact from the presynaptic nerve, approximately halfway through embryogenesis. After contact, GluRIIA and GluRIIB mRNA abundance rises quickly approximately 20-fold, then falls within a few hours back to very low levels. Protein abundance, however, gradually increases throughout development. At the same time that mRNA levels decrease following their initial spike, GluRIIA, GluRIIB, and GluRIIC subunit mRNA aggregates become visible in the cytoplasm of postsynaptic muscle cells. These mRNA aggregates do not colocalize with eIF4E, but nevertheless presumably represent mRNP particles of unknown function. Multiplex FISH shows that different GluR subunit mRNAs are found in different mRNPs. GluRIIC mRNPs are most common, followed by GluRIIA and then GluRIIB mRNPs. GluR mRNP density is not increased near NMJs, for any subunit; if anything, GluR mRNP density is highest away from NMJs and near nuclei. These results reveal some of the earliest events in postsynaptic development and provide a foundation for future studies of GluR mRNA biology.

A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle.

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.

New measles virus genotype associated with outbreak, China.

To determine the origin of the virus associated with a measles outbreak in Menglian County, Yunnan Province, People's Republic of China, in 2009, we conducted genetic analyses. Phylogenetic analyses based on nucleoprotein (N) and hemagglutinin (H) gene sequences showed that these Menglian viruses were not closely related to sequences of any World Health Organization (WHO) reference strains representing the 23 currently recognized genotypes. The minimum nucleotide divergence between the Menglian viruses and the most closely related reference strain, genotype D7, was 3.3% for the N gene and 3.0% for the H gene. A search of the databases of GenBank, WHO, and the Health Protection Agency Measles Nucleotide Surveillance showed that the Menglian viruses, together with the 2 older non-Menglian viruses, could be members of a new proposed measles genotype, d11. The new genotype designation will allow for better description of measles transmission patterns, especially in the Southeast Asian and Western Pacific regions.

Genetic characterization of wild-type measles viruses isolated in China, 2006-2007.

Molecular characterization of wild-type measles viruses in China during 1995-2004 demonstrated that genotype H1 was endemic and widely distributed throughout the country. H1-associated cases and outbreaks caused a resurgence of measles beginning in 2005. A total of 210,094 measles cases and 101 deaths were reported by National Notifiable Diseases Reporting System (NNDRS) and Chinese Measles Laboratory Network (LabNet) from 2006 to 2007, and the incidences of measles were 6.8/100,000 population and 7.2/100,000 population in 2006 and 2007, respectively. Five hundred and sixty-five wild-type measles viruses were isolated from 24 of 31 provinces in mainland China during 2006 and 2007, and all of the wild type virus isolates belonged to cluster 1 of genotype H1. These results indicated that H1-cluster 1 viruses were the predominant viruses circulating in China from 2006 to 2007. This study contributes to previous efforts to generate critical baseline data about circulating wild-type measles viruses in China that will allow molecular epidemiologic studies to help measure the progress made toward China's goal of measles elimination by 2012.