A Frameshift Variant of GluN2A Identified in an Epilepsy Patient Results in NMDA Receptor Mistargeting.

N-methyl-D-aspartate receptors (NMDARs) are crucial for neuronal development and synaptic plasticity. Dysfunction of NMDARs is associated with multiple neurodevelopmental disorders, including epilepsy, autism spectrum disorder, and intellectual disability. Understanding the impact of genetic variants of NMDAR subunits can shed light on the mechanisms of disease. Here, we characterized the functional implications of a de novo mutation of the GluN2A subunit (P1199Rfs*32) resulting in the truncation of the C-terminal domain. The variant was identified in a male patient with epileptic encephalopathy, multiple seizure types, severe aphasia, and neurobehavioral changes. Given the known role of the CTD in NMDAR trafficking, we examined changes in receptor localization and abundance at the postsynaptic membrane using a combination of molecular assays in heterologous cells and rat primary neuronal cultures. We observed that the GluN2A P1199Rfs*32-containing receptors traffic efficiently to the postsynaptic membrane but have increased extra-synaptic expression relative to WT GluN2A-containing NMDARs. Using in silico predictions, we hypothesized that the mutant would lose all PDZ interactions, except for the recycling protein Scribble1. Indeed, we observed impaired binding to the scaffolding protein postsynaptic protein-95 (PSD-95); however, we found the mutant interacts with Scribble1, which facilitates the recycling of both the mutant and the WT GluN2A. Finally, we found that neurons expressing GluN2A P1199Rfs*32 have fewer synapses and decreased spine density, indicating compromised synaptic transmission in these neurons. Overall, our data show that GluN2A P1199Rfs*32 is a loss-of-function variant with altered membrane localization in neurons and provide mechanistic insight into disease etiology.

Plasma derived extracellular vesicle biomarkers of microglia activation in an experimental stroke model.

Chronic microglia activation post-stroke is associated with worse neurological and cognitive outcomes. However, measurement of microglia activation in vivo is currently limited. Plasma derived extracellular vesicles (EVs) are cell-specific indicators that may allow for non-invasive measurement of microglia phenotype. The aim of this study was to identify activation-state specific microglia EVs (MEVs) in vitro followed by validation in an experimental stroke model. Following pro-inflammatory activation, MEVs contain the microglia protein TMEM119 alongside increased expression of the Toll-like receptor 4 co-receptor CD14. Immunoprecipitation followed by fluorescent nanoparticle tracking analysis (ONI Nanoimager) was used to confirm the isolation of TMEM119+/CD14+ EVs from rat plasma. Electron microscopy confirmed that TMEM119 and CD14 localize to the MEV membrane. To model ischemia, plasma was collected from 3-month wildtype Fischer344 rats prior to, 7 and 28 days after endothelin-1 or saline injection into the dorsal right striatum. Fluorescently labelled MEVs were directly measured in the plasma using nanoflow cytometry (Apogee A60 Microplus). We report a significant increase in circulating TMEM119+/CD14+ EVs 28-days post-stroke in comparison to baseline levels and saline-injected rats, which correlated weakly with stroke volume. TMEM119+/MHC-II+ EVs were also increased post-stroke in comparison to baseline and saline-injected animals. This study is the first to describe an EV biomarker of activated microglia detected directly in plasma following stroke and represents a future tool for the measurement of microglia activity in vivo.

Differential effects of air conditioning type on residential endotoxin levels in a semi-arid climate.

Residential endotoxin exposure is associated with protective and pathogenic health outcomes. Evaporative coolers, an energy-efficient type of air conditioner used in dry climates, are a potential source of indoor endotoxins; however, this association is largely unstudied. We collected settled dust biannually from four locations in homes with evaporative coolers (n=18) and central air conditioners (n=22) in Utah County, Utah (USA), during winter (Jan-Apr) and summer (Aug-Sept), 2014. Dust samples (n=281) were analyzed by the Limulus amebocyte lysate test. Housing factors were measured by survey, and indoor temperature and relative humidity measures were collected during both seasons. Endotoxin concentrations (EU/mg) were significantly higher in homes with evaporative coolers from mattress and bedroom floor samples during both seasons. Endotoxin surface loads (EU/m2 ) were significantly higher in homes with evaporative coolers from mattress and bedroom floor samples during both seasons and in upholstered furniture during winter. For the nine significant season-by-location comparisons, EU/mg and EU/m2 were approximately three to six times greater in homes using evaporative coolers. A plausible explanation for these findings is that evaporative coolers serve as a reservoir and distribution system for Gram-negative bacteria or their cell wall components in homes.

Acute minocycline treatment inhibits microglia activation, reduces infarct volume, and has domain-specific effects on post-ischemic stroke cognition in rats.

Ischemic stroke affects millions of individuals worldwide and a high prevalence of survivors experience cognitive deficits. At present, the underlying mechanisms that drive post-stroke cognitive decline are not well understood. Microglia play a critical role in the post-stroke inflammatory response, but experimental studies show that an accumulation of chronically activated microglia can be harmful and associates with cognitive impairment. This study assessed the effect of acute post-stroke minocycline treatment on chronic microglia and astrocyte expression within the infarct and remote white matter regions, as well as its effect on various domains of cognitive function post-stroke. Nine-month-old male rats received an injection of endothelin-1 into the right dorsal striatum to induce transient focal ischemia, and then were treated with minocycline or saline for 4 days post-stroke. Rats were tested using a series of lever-pressing tasks and the Morris water maze to assess striatal-based learning, cognitive flexibility, and spatial learning and reference memory. We found that minocycline-treated rats had smaller stroke-induced infarcts and less microglia activation in the infarct area and remote white matter regions compared to saline-treated rats at 28 days post-stroke. The behavioural testing results differed according to the cognitive domain; whereas minocycline-treated rats trended towards improved striatal-based learning in a lever-pressing task, but cognitive flexibility was unaffected during the subsequent set-shifting task. Furthermore, minocycline treatment unexpectedly impaired spatial learning, yet it did not alter reference memory. Collectively, we show that post-stroke minocycline treatment can reduce chronic microglia activation even in remote brain regions, with domain-specific effects on cognitive function.

Transcriptional repression by REST: recruitment of Sin3A and histone deacetylase to neuronal genes.

Many genes whose expression is restricted to neurons in the brain contain a silencer element (RE1/NRSE) that limits transcription in nonneuronal cells by binding the transcription factor REST (also named NRSF or XBR). Although two independent domains of REST are known to confer repression, the mechanisms of transcriptional repression by REST remain obscure. We provide multiple lines of evidence that the N-terminal domain of REST represses transcription of the GluR2 and type II sodium-channel genes by recruiting the corepressor Sin3A and histone deacetylase (HDAC) to the promoter region in nonneuronal cells. These results identify a general mechanism for controlling the neuronal expression pattern of a specific set of genes via the RE1 silencer element.

Manipulation of spontaneous emission in a tapered photonic crystal fibre.

We characterize the spontaneous emission of dye that is introduced into the central core of a tapered photonic crystal fiber. Since the photonic crystal period in the fibre cladding varies along the taper, the transmission and spontaneous emission spectra over a wide range of relative frequencies can be observed. The spontaneous emission spectra of the fibre transverse to the fiber axis show suppression due to partial band-gaps of the structure, and also enhancement of spontaneous emission near the band edges. We associate these with van Hove features, as well as finite cluster size effects.

A correlational study of cardiovascular autonomic functioning and unipolar depression.

Cardiovascular autonomic functioning was assessed in 22 drug-free inpatients diagnosed by DSM-III criteria as having a unipolar depression. Sympathetic cholinergic, alpha- and beta-adrenergic activity were assessed via the measurement of forearm blood flow (FBF), digital blood flow (DBF), and the cardiac pre-ejection period (PEP), respectively. These parameters were correlated with total Hamilton score (HT) (using partial correlations to control for extraneous autonomic variables) to identify the specific autonomic correlates of unipolar depression. Significant negative correlations were found between HT and supine FBF and significant positive correlations between HT and PEP. Large effect-size, negative correlations (which approached significance) were found between HT and DBF. It is concluded that there is a specific autonomic profile of unipolar depression, characterized by a decrease in central sympathetic cholinergic outflow, coupled with increases in alpha-adrenergic and decreases in beta-adrenergic activity. Further, this profile is not merely a static hallmark of depression but covaries with the severity of the depression, independent of other autonomic activity.

Src family kinase activation in glycosphingolipid-rich membrane domains of endothelial cells treated with oxidised low density lipoprotein.

Extraction of ECV304 endothelial cells in 1% Triton X-100 at 4 degrees C resulted in a detergent-insoluble pellet that contained 90% of the caveolin, 78% of the src family kinases and 99% of the annexin II. When detergent-treated cells were loaded beneath a 10-30% sucrose gradient the caveolin and a large proportion of the cellular cholesterol floated at a density of 1.09 g/cm3, characteristic of caveolae and glycosphingolipid-rich membranes. With extended centrifugation the src family kinases, which were initially associated with this floating material, sedimented to the bottom of the gradient. Annexin II remained on the bottom of the gradient under both centrifugation conditions. After 24-h incubation with oxidised low density lipoprotein (oxLDL) about 7.5% of the total sterol in the cells was replaced by 7-ketocholesterol, the major oxysterol found in oxLDL. The majority of this 7-ketocholesterol was found in the light membrane fraction on sucrose gradients. Under these conditions src kinase activity more than doubled in the Triton-resistant fraction, without changes in the concentration of src kinase protein. Introducing oxysterols directly into the medium bathing ECV304 cells for 1 h also modulated the activity of src family kinases in the detergent-resistant membranes. An elevation in activity was observed for 7-ketocholesterol while 7alpha-hydroxycholesterol, 7alpha-hydroxycholesterol and cholesterol epoxide all produced decreases in the background level of src kinase activity. We conclude that 7-ketocholesterol and possibly other components of oxLDL can equilibrate into glycosphingolipid-rich membranes and increase the activity of src kinases, possibly by interaction with caveolin.

Effect of electronic cooking on fatty acids in meats.

This study was conducted to investigate the effects of microwave energy and conventional heating on the fatty acid composition of meats and poultry. Data on weight loss and cooking time with both heat treatments are reported. Gas-liquid chromatographic data of the methyl esters of myristic, palmitic, stearic, oleic, linoleic, and linolenic acids revealed no significant difference between fatty acid composition as a result of heat treatment. Decreases in fatty acids appeared to be mainly due to a change in oleic acid. Poultry appeared to be the species most affected by heat treatment. Cooking times were much shorter for the electronic method and varied among species. No significant difference was found in weight loss as a result of treatment of beef or pork. Poultry weight losses were greater for the conventional method.

Spinal cord arteriovenous malformation in a person with congenital lymphatic abnormalities.

Spinal cord arteriovenous malformations have been described in association with a variety of congenital diseases affecting the vasculature, including Klippel-Trenaunay-Weber syndrome, Rendu-Osler-Weber syndrome and others, but rarely in association with lymphatic abnormalities. We report the case of a young man with congenital lymphedema and arteriovenous malformations of one lower extremity and a spinal cord arteriovenous malformation. Awareness of the possible presence of a central nervous system arteriovenous malformation in individuals with pre-existing arteriovenous and lymphatic abnormalities may be helpful in their diagnosis and management.

Dural spinal cord arteriovenous malformation.

After multiple hospital admissions and an inpatient rehabilitation stay, a 68-year-old woman was transferred to our rehabilitation facility with a paraparesis of unknown etiology. Previous studies included four CT scans and three MRIs which did not demonstrate the lesion. A myelogram was noncharacteristic. The correct diagnosis, confirmed by selective angiography, was ultimately contingent upon recognition of the clinical features and natural history of dural spinal cord arteriovenous malformations (SCAVM). The unusual combination of this multitude of nondiagnostic imaging studies in the uncommon dural SCAVM served to delay diagnosis and treatment. Such delay may have great functional consequences. This report illustrates the importance of suspecting SCAVM and recognizing its features. Emphasis is placed on the physiatrist's role in assuring proper diagnosis to expedite a timely treatment and to obtain the best functional outcome. A brief review of the classification, clinical features, pathophysiology, diagnosis, and prognosis of SCAVM is presented.

Paraplegia following trauma in a patient with kyphosis.

The reported incidence of paraplegia following acute spinal trauma in patients with kyphosis is very low. One such case is presented here and discussed. Although deterioration of spinal cord function in patients with kyphosis who have not sustained trauma is treated by surgical decompression and stabilization of the spine, a more conservative approach may be beneficial in those patients with a history of trauma. Thus, the treatment appears to follow that now recommended for acute spinal injuries in patients with a premorbidly normal spine.

Molecular biology of mammalian amino acid receptors.

The amino acid receptor proteins are ubiquitous transducers of most excitatory and inhibitory synaptic transmission in the brain. In July 1987 two reports appeared describing the molecular cloning of a pair of subunits of the GABAA receptor (7) and one subunit of the glycine receptor (13). These papers sparked wide interest and led quickly to the concept of a ligand-gated receptor-ion channel superfamily that includes nicotinic acetylcholine receptors as well as certain amino acid receptors. The identification of additional subunits of each receptor followed; with the recent cloning of a kainate receptor subunit (14), only the NMDA receptor remains elusive. Several disciplines have been brought to bear on these receptor clones, including in situ hybridization and functional expression in Xenopus laevis oocytes and mammalian cell lines. In this review we compare cloning strategies that have been used for amino acid receptors and discuss structural similarities among the receptor subunits. Two findings that have arisen from molecular cloning and expression of these receptors receive special attention. First, the molecular heterogeneity of GABAA receptors is larger than expected from pharmacological studies of native receptors. Second, although the native receptors are thought to be heterooligomers, much like the model proposed for the nicotinic receptors, some individual amino acid receptor subunits can form functional receptor channels, presumably in a homomeric configuration. This review focuses, therefore, on what we have learned from cloning efforts about amino acid receptors and what might lie ahead in this field.

Licensed midwife-attended, out-of-hospital births in Washington state: are they safe?

The safety of out-of-hospital births attended by midwives who are licensed according to international standards has not been established in the United States. To address this issue, outcomes of births attended out of hospital by licensed midwives in Washington state were compared with those attended by physicians and certified nurse-midwives in hospital and certified nurse-midwives out of hospital between 1981 and 1990. Outcomes measured included low birthweight, low five-minute Apgar scores, and neonatal and postneonatal mortality. Associations between attendant and outcomes were measured using odds ratios to estimate relative risks. Multivariate analysis using logistic regression controlled for confounding variables. Overall, births attended by licensed midwives out of hospital had a significantly lower risk for low birthweight than those attended in hospital by certified nurse-midwives, but no significant differences were found between licensed midwives and any of the comparison groups on any other outcomes measured. When the analysis was limited to low-risk women, certified nurse-midwives were no more likely to deliver low-birthweight infants than were licensed midwives, but births attended by physicians had a higher risk of low birthweight. The results of this study indicate that in Washington state the practice of licensed nonnurse-midwives, whose training meets standards set by international professional organizations, may be as safe as that of physicians in hospital and certified nurse-midwives in and out of hospital.

Sensory conduction in peroneal and posterior tibial nerves using averaging techniques.

A method of obtaining pure sensory nerve conduction velocities in the lower extremities is described. This involves the use of electronic summation (signal averaging). Potentials were obtained and velocities calculated from all normal subjects examined. In patients with peripheral neuropathies it was often possible to obtain nerve velocities with signal amplitudes as low as 0·1 µV and these were often slower than those obtained from the normal subjects. The advantages and disadvantages of this method are discussed. It is of significant clinical value in that pure sensory nerve conduction velocities can be measured in the legs when this may be the only valuable parameter in the absence of motor involvement. In addition, investigation of neuropathies at an earlier stage of development and recovery may be facilitated. It is hoped that in the future this technique of obtaining low amplitude responses with an analogue averager can be incorporated with the more routine aspects of nerve conduction testing when clinically indicated.

Signal transduction and ligand specificity of the human monocyte chemoattractant protein-1 receptor in transfected embryonic kidney cells.

We have examined the ligand specificity and signal transduction pathways of a recently cloned receptor for monocyte chemoattractant protein-1 (MCP-1). In human 293 cells stably transfected with the MCP-1 receptor, MCP-1 bound specifically with high affinity (Kd = 260 pM) and induced a rapid mobilization of calcium from intracellular stores. The closely related chemokines MIP-1 alpha, MIP-1 beta, RANTES, interleukin 8 (IL-8), and Gro-alpha were inactive at concentrations as high as 300 nM. Activation of the MCP-1 receptor potently inhibited adenylyl cyclase with an IC50 = 90 pM. Activation of the MIP-1 alpha/RANTES receptor also mediated inhibition of adenylyl cyclase activity but with a different pharmacological profile: MIP-1 alpha (110 pM, IC50), RANTES (140 pM), MIP-1 beta (10 nM), and MCP-1 (820 nM). Mobilization of intracellular calcium and inhibition of adenylyl cyclase were blocked by pertussis toxin, suggesting that the MCP-1 receptor coupled to G alpha i. These results demonstrate that the MCP-1 receptor binds and signals in response to picomolar concentrations of MCP-1 in a highly specific manner. Signaling was manifested as mobilization of intracellular calcium and inhibition of adenylyl cyclase and was mediated by a pertussis toxin-sensitive G-protein(s).

Genetic regulation of glutamate receptor ion channels.

Transcriptional and translational regulation of glutamate receptor expression determines one of the key phenotypic features of neurons in the brain--the properties of their excitatory synaptic receptors. Up- and down-regulation of various glutamate receptor subunits occur throughout development, following ischemia, seizures, repetitive activation of afferents, or chronic administration of a variety of drugs. The promoters of the genes that encode the NR1, NR2B, NR2C, GluR1, GluR2, and KA2 subunits share several characteristics that include multiple transcriptional start sites within a CpG island, lack of TATA and CAAT boxes, and neuronal-selective expression. In most cases, the promoter regions include overlapping Sp1 and GSG motifs near the major initiation sites, and a silencer element, to guide expression in neurons. Manipulating the levels of glutamate receptors in vivo by generating transgenic and knockout mice has enhanced understanding of the role of specific glutamate receptor subunits in long-term potentiation and depression, learning, seizures, neural pattern formation, and survival. Neuron-specific glutamate receptor promoter fragments may be employed in the design of novel gene-targeting constructs to deliver future experimental transgene and therapeutic agents to selected neurons in the brain.

Past history and degree of depression in paraplegic individuals.

The purpose of this study was to attempt to collect sufficient data to substantiate the clinical impression that the degree of depression in the paraplegic and his past history are correlated. It was hypothesised that the poorer the past history, the greater the degree of depression in paraplegic individuals. This study was conducted on ten paraplegic subjects from the Spinal Cord Clinic at Columbia Presbyterian Medical Center, Department of Rehabilitation Medicine, based on data obtained from the administration and scoring of the Depression Scale (D-scale) of the Minnesota Multiphasic Personality Inventory (MMPI) and by the clinical observation by the consulting psychiatrist with the Spinal Injury Clinic. Past history was assumed measurable as 'favourable' to 'poor' by the use of an original questionnaire. The results of this investigation suggested a relationship between a paraplegic individual's past history and the degree of depression. The study was deemed significant in presentation of an original past history questionnaire and in the prediction of future difficulties in the rehabilitation of paraplegics so that preventive measures could be instituted.

Differential induction of glyoxylate cycle enzymes by stress as a marker for seedling vigor in sugar beet (Beta vulgaris).

Significant differences in seedling vigor exist among sugar beet (Beta vulgaris) hybrids; however, traditional approaches to breeding enhanced vigor have not proven effective. Seedling vigor is a complex character, but presumably includes efficient mobilization of seed storage reserves during germination and efficient seedling growth in diverse environments. The involvement of lipid metabolism during germination of sugar beet under stress conditions was suggested by the isolation at high frequency of Expressed Sequence Tags (ESTs) with similarity to isocitrate lyase (EC 4.1.3.1). High-level expression of this glyoxylate cycle enzyme during germination and seedling emergence was also suggested by nucleotide sequencing of cDNA libraries obtained from a well emerging sugar beet hybrid during germination under stress. Genes involved in carbohydrate and lipid catabolism were differentially expressed in a strongly emerging hybrid, relative to a weakly emerging hybrid, during stress germination. Stress markedly reduced the levels of alpha-amylase transcripts in the weakly emerging hybrid. In contrast, the strongly emerging hybrid exhibited only a moderate reduction in alpha-amylase transcript levels under the same conditions, and showed large increases in the expression of genes involved in lipid metabolism, suggesting compensation by lipid for carbohydrate metabolism in the better emerging hybrid. Differential activity of the glyoxylate cycle thus appears to be a physiological marker that distinguishes between high- and low-vigor sugar beet cultivars. This finding suggests, for the first time, a biochemical target for selection for enhanced germination and improved emergence in sugar beet.