Enhanced Membrane Incorporation of H289Y Mutant GluK1 Receptors from the Audiogenic Seizure-Prone GASH/Sal Model: Functional and Morphological Impacts on Xenopus Oocytes.

Epilepsy is a neurological disorder characterized by abnormal neuronal excitability, with glutamate playing a key role as the predominant excitatory neurotransmitter involved in seizures. Animal models of epilepsy are crucial in advancing epilepsy research by faithfully replicating the diverse symptoms of this disorder. In particular, the GASH/Sal (genetically audiogenic seizure-prone hamster from Salamanca) model exhibits seizures resembling human generalized tonic-clonic convulsions. A single nucleotide polymorphism (SNP; C9586732T, p.His289Tyr) in the Grik1 gene (which encodes the kainate receptor GluK1) has been previously identified in this strain. The H289Y mutation affects the amino-terminal domain of GluK1, which is related to the subunit assembly and trafficking. We used confocal microscopy in Xenopus oocytes to investigate how the H289Y mutation, compared to the wild type (WT), affects the expression and cell-surface trafficking of GluK1 receptors. Additionally, we employed the two-electrode voltage-clamp technique to examine the functional effects of the H289Y mutation. Our results indicate that this mutation increases the expression and incorporation of GluK1 receptors into an oocyte's membrane, enhancing kainate-evoked currents, without affecting their functional properties. Although further research is needed to fully understand the molecular mechanisms responsible for this epilepsy, the H289Y mutation in GluK1 may be part of the molecular basis underlying the seizure-prone circuitry in the GASH/Sal model.

Delving into the significance of the His289Tyr single-nucleotide polymorphism in the glutamate ionotropic receptor kainate-1 (Grik1) gene of a genetically audiogenic seizure model.

Genetic abnormalities affecting glutamate receptors are central to excitatory overload-driven neuronal mechanisms that culminate in seizures, making them pivotal targets in epilepsy research. Increasingly used to advance this field, the genetically audiogenic seizure hamster from Salamanca (GASH/Sal) exhibits generalized seizures triggered by high-intensity acoustic stimulation and harbors significant genetic variants recently identified through whole-exome sequencing. Here, we addressed the influence of the missense single-nucleotide polymorphism (C9586732T, p.His289Tyr) in the glutamate receptor ionotropic kainate-1 (Grik1) gene and its implications for the GASH/Sal seizure susceptibility. Using a protein 3D structure prediction, we showed a potential effect of this sequence variation, located in the amino-terminal domain, on the stability and/or conformation of the kainate receptor subunit-1 protein (GluK1). We further employed a multi-technique approach, encompassing gene expression analysis (RT-qPCR), Western blotting, and immunohistochemistry in bright-field and confocal fluorescence microscopy, to investigate critical seizure-associated brain regions in GASH/Sal animals under seizure-free conditions compared to matched wild-type controls. We detected disruptions in the transcriptional profile of the Grik1 gene within the audiogenic seizure-associated neuronal network. Alterations in GluK1 protein levels were also observed in various brain structures, accompanied by an unexpected lower molecular weight band in the inferior and superior colliculi. This correlated with substantial disparities in GluK1-immunolabeling distribution across multiple brain regions, including the cerebellum, hippocampus, subdivisions of the inferior and superior colliculi, and the prefrontal cortex. Notably, the diffuse immunolabeling accumulated within perikarya, axonal fibers and terminals, exhibiting a prominent concentration in proximity to the cell nucleus. This suggests potential disturbances in the GluK1-trafficking mechanism, which could subsequently affect glutamate synaptic transmission. Overall, our study sheds light on the genetic underpinnings of seizures and underscores the importance of investigating the molecular mechanisms behind synaptic dysfunction in epileptic neural networks, laying a crucial foundation for future research and therapeutic strategies targeting GluK1-containing kainate receptors.

Whole genome sequencing identifies candidate genes for familial essential tremor and reveals biological pathways implicated in essential tremor aetiology.

BACKGROUND: Essential tremor (ET), one of the most common neurological disorders, has a phenotypically heterogeneous presentation characterized by bilateral kinetic tremor of the arms and, in some patients, tremor involving other body regions (e.g., head, voice). Genetic studies suggest that ET is genetically heterogeneous. METHODS: We analyzed whole genome sequence data (WGS) generated on 104 multi-generational white families with European ancestry affected by ET. Genome-wide parametric linkage and association scans were analyzed using adjusted logistic regression models through the application of the Pseudomarker software. To investigate the additional contribution of rare variants in familial ET, we also performed an aggregate variant non-parametric linkage (NPL) analysis using the collapsed haplotype method implemented in CHP-NPL software. FINDINGS: Parametric linkage analysis of common variants identified several loci with significant evidence of linkage (HLOD ≥3.6). Among the gene regions within the strongest ET linkage peaks were BTC (4q13.3, HLOD=4.53), N6AMT1 (21q21.3, HLOD=4.31), PCDH9 (13q21.32, HLOD=4.21), EYA1 (8q13.3, HLOD=4.04), RBFOX1 (16p13.3, HLOD=4.02), MAPT (17q21.31, HLOD=3.99) and SCARB2 (4q21.1, HLOD=3.65). CHP-NPL analysis identified fifteen additional genes with evidence of significant linkage (LOD ≥3.8). These genes include TUBB2A, VPS33B, STEAP1B, SPINK5, ZRANB1, TBC1D3C, PDPR, NPY4R, ETS2, ZNF736, SPATA21, ARL17A, PZP, BLK and CCDC94. In one ET family contributing to the linkage peak on chromosome 16p13.3, we identified a likely pathogenic heterozygous canonical splice acceptor variant in exon 2 of RBFOX1 (ENST00000547372; c.4-2A>G), that co-segregated with the ET phenotype in the family. INTERPRETATION: Linkage and association analyses of WGS identified several novel ET candidate genes, which are implicated in four major pathways that include 1) the epidermal growth factor receptor-phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha-AKT serine/threonine kinase 1 (EGFR-PI3K-AKT) and Mitogen-activated protein Kinase 1 (ERK) pathways, 2) Reactive oxygen species (ROS) and DNA repair, 3) gamma-aminobutyric acid-ergic (GABAergic) system and 4) RNA binding and regulation of RNA processes. Our study provides evidence for a possible overlap in the genetic architecture of ET, neurological disease, cancer and aging. The genes and pathways identified can be prioritized in future genetic and functional studies. FUNDING: National Institutes of Health, NINDS, NS073872 (USA) and NIA AG058131(USA).

A quantitative trait rare variant nonparametric linkage method with application to age-at-onset of Alzheimer's disease.

To analyze pedigrees with quantitative trait (QT) and sequence data, we developed a rare variant (RV) quantitative nonparametric linkage (QNPL) method, which evaluates sharing of minor alleles. RV-QNPL has greater power than the traditional QNPL that tests for excess sharing of minor and major alleles. RV-QNPL is robust to population substructure and admixture, locus heterogeneity, and inclusion of nonpathogenic variants and can be readily applied outside of coding regions. When QNPL was used to analyze common variants, it often led to loci mapping to large intervals, e.g., >40 Mb. In contrast, when RVs are analyzed, regions are well defined, e.g., a gene. Using simulation studies, we demonstrate that RV-QNPL is substantially more powerful than applying traditional QNPL methods to analyze RVs. RV-QNPL was also applied to analyze age-at-onset (AAO) data for 107 late-onset Alzheimer's disease (LOAD) pedigrees of Caribbean Hispanic and European ancestry with whole-genome sequence data. When AAO of AD was analyzed regardless of APOE ε4 status, suggestive linkage (LOD = 2.4) was observed with RVs in KNDC1 and nominally significant linkage (p < 0.05) was observed with RVs in LOAD genes ABCA7 and IQCK. When AAO of AD was analyzed for APOE ε4 positive family members, nominally significant linkage was observed with RVs in APOE, while when AAO of AD was analyzed for APOE ε4 negative family members, nominal significance was observed for IQCK and ADAMTS1. RV-QNPL provides a powerful resource to analyze QTs in families to elucidate their genetic etiology.

Inferior Colliculus Transcriptome After Status Epilepticus in the Genetically Audiogenic Seizure-Prone Hamster GASH/Sal.

The Genetic Audiogenic Seizure Hamster from Salamanca (GASH/Sal), an animal model of reflex epilepsy, exhibits generalized tonic-clonic seizures in response to loud sound with the epileptogenic focus localized in the inferior colliculus (IC). Ictal events in seizure-prone strains cause gene deregulation in the epileptogenic focus, which can provide insights into the epileptogenic mechanisms. Thus, the present study aimed to determine the expression profile of key genes in the IC of the GASH/Sal after the status epilepticus. For such purpose, we used RNA-Seq to perform a comparative study between the IC transcriptome of GASH/Sal and that of control hamsters both subjected to loud sound stimulation. After filtering for normalization and gene selection, a total of 36 genes were declared differentially expressed from the RNA-seq analysis in the IC. A set of differentially expressed genes were validated by RT-qPCR showing significant differentially expression between GASH/Sal hamsters and Syrian control hamsters. The confirmed differentially expressed genes were classified on ontological categories associated with epileptogenic events similar to those produced by generalized tonic seizures in humans. Subsequently, based on the result of metabolomics, we found the interleukin-4 and 13-signaling, and nucleoside transport as presumably altered routes in the GASH/Sal model. This research suggests that seizures in GASH/Sal hamsters are generated by multiple molecular substrates, which activate biological processes, molecular processes, cellular components and metabolic pathways associated with epileptogenic events similar to those produced by tonic seizures in humans. Therefore, our study supports the use of the GASH/Sal as a valuable animal model for epilepsy research, toward establishing correlations with human epilepsy and searching new biomarkers of epileptogenesis.

Triage and Ongoing Care for Critically Ill Patients in the Emergency Department: Results from a National Survey of Emergency Physicians.

INTRODUCTION: We conducted a cross-sectional study at the Icahn School of Medicine at Mount Sinai to elicit emergency physician (EP) perceptions regarding intensive care unit (ICU) triage decisions and ongoing management for boarding of ICU patients in the emergency department (ED). We assessed factors influencing the disposition decision for critically ill patients in the ED to characterize EPs' perceptions about ongoing critical care delivery in the ED while awaiting ICU admission. METHODS: Through content expert review and pilot testing, we iteratively developed a 25-item written survey targeted to EPs, eliciting current ICU triage structure, opinions on factors influencing ICU admission decisions, and views on caring for critically ill patients "boarding" in the ED for >4-6 hours. RESULTS: We approached 732 EPs at a large, national emergency medicine conference, achieving 93.6% response and completion rate, with 54% academic and 46% community participants. One-fifth reported having formal ICU admission criteria, although only 36.6% reported adherence. Common factors influencing EPs' ICU triage decisions were illness severity (91.1%), ICU interventions needed (87.6%), and diagnosis (68.2%), while ICU bed availability (13.5%) and presence of other critically ill patients in ED (10.2%) were less or not important. While 72.1% reported frequently caring for ICU boarders, respondents identified high patient volume (61.3%) and inadequate support staffing (48.6%) as the most common challenges in caring for boarding ICU patients. CONCLUSION: Patient factors (eg, diagnosis, illness severity) were seen as more important than system factors (eg, bed availability) in triaging ED patients to the ICU. Boarding ICU patients is a common challenge for more than two-thirds of EPs, exacerbated by ED volume and staffing constraints.

Behavioral and Molecular Effects Induced by Cannabidiol and Valproate Administration in the GASH/Sal Model of Acute Audiogenic Seizures.

Despite evidence that supports cannabidiol (CBD) as an anticonvulsant agent, there remains controversy over the antiseizure efficacy, possible adverse effects, and synergistic interactions with classic antiepileptics such as valproate (VPA). The genetic audiogenic seizure hamster from the University of Salamanca (GASH/Sal) is a reliable experimental model of generalized tonic-clonic seizures in response to intense sound stimulation. The present study examines the behavioral and molecular effects of acute and chronic intraperitoneal administrations of VPA (300 mg/kg) and CBD (100 mg/kg) on the GASH/Sal audiogenic seizures, as well as the coadministration of both drugs. The GASH/Sal animals were examined prior to and after the corresponding treatment at 45 min, 7 days, and 14 days for seizure severity and neuroethology, open-field behaviors, body weight variations, and various hematological and biochemical parameters. Furthermore, the brain tissue containing the inferior colliculus (so-called epileptogenic nucleus) was processed for reverse transcription-quantitative polymerase chain reaction analysis to determine the treatment effects on the gene expression of neuronal receptors associated with drug actions and ictogenesis. Our results indicated that single dose of VPA helps prevent the animals from getting convulsions, showing complete elimination of seizures, whereas 7 days of chronic VPA treatment had few effects in seizure behaviors. Acute CBD administration showed subtle attenuation of seizure behaviors, increasing seizure latency and decreasing the duration of the convulsion phase, but without entirely seizure abolition. Chronic CBD treatments had no significant effects on sound-induced seizures, although some animals slightly improved seizure severity. Acute and chronic CBD treatments have no significant adverse effects on body weight, hematological parameters, and liver function, although locomotor activity was reduced. The combination of VPA and CBD did not alter the therapeutic outcome of the VPA monotherapy, showing no apparent synergistic effects. As compared to sham animals, chronic treatments with CBD caused abnormal mRNA expression levels for Trpv1, Adora1, Slc29a1, and Cnr1 genes, whereas no differences in gene expression were found for Htr1a and Sigmar1. Our study shed light on the behavioral and molecular effects of CBD and VPA on the GASH/Sal model and constituted the basis to develop further studies on the pharmacological effects of CBD and its interactions with other anticonvulsants.

Determination of genotypic and clinical characteristics of Colombian patients with mucopolysaccharidosis IVA.

BACKGROUND: As mucopolysaccharidosis IVA (MPS IVA) is the most frequent MPS in Colombia, this paper aims to describe its clinical and mutational characteristics in 32 diagnosed patients included in this study. METHODS: Genotyping was completed by amplification and Sanger sequencing of the GALNS gene. The SWISS-model platform was used for bioinformatic analysis, and mutant proteins were generated by homology from the wild-type GALNS code 4FDI template from the Protein Data Bank (PDB) database. Docking was performed using the GalNAc6S ligand (PubChem CID: 193456) by AutoDock Vina 1.0 and visualized in PyMOL and LigPlot+. RESULTS: Eleven variants were identified, and one new pathogenic variant was described in the heterozygous state, which is consistent with genotype c. 319 G> T or p.Ala107Ser. The pathogenic variant c.901G>T or p.Gly301Cys was the most frequent mutation with 51.6% of alleles. Docking revealed affinity energy of -5.9 Kcal/mol between wild-type GALNS and the G6S ligand. Some changes were evidenced at the intermolecular interaction level, and affinity energy for each mutant decreased. CONCLUSION: Clinical variables and genotypic analysis were similar to those reported for other world populations. Genotypic data showed greater allelic heterogeneity than those previously reported. Bioinformatics tools showed differences in the binding interactions of mutant proteins with the G6S ligand, in regard the wild-type GALNS.

Bacteremia después de biopsias de próstata dirigidas por ultrasonido / Bacteremia after ultrasound guided transrectal prostate biopsy

Propósito: Determinar la incidencia de bacteremia después de biopsias por sextantes y octantes de próstata. Material y métodos. Se trata de un estudio prospectivo realizado en un lapso de tiempo de 5 meses, en el servicio de cografía urológica de la Clínica de Marly, en los pacientes que llegan remitidos para biopsias prostáticas por sospecha de cáncer de próstata. El examen es realizado por un único urólogo especialista, utilizando un ecógrafo Siemens Sonoline Prima, con un transductor biplanar de 5 a 7,5 MHz. Las biopsias ecodirigidas se obtienen con aguja de biopsia 18 gauge y pistola de biopsia automática. Las muestras se marcan y analizan por separado. Se obtiene una muestra de orina inmediatamente antes del examen, que se envía al laboratorio para urocultivo. Todos los pacientes se citan a las 24 horas del examen para toma del hemocultivo, el cual se procesa con antibiograma si se detecta crecimiento bacteriano. La preparación del paciente consiste en la toma de antibiótico (Ciprofloxacina) por 3 días, y la colocación de un enema evacuador previo al examen. Resultados: El estudio incluyó 69 pacientes. El promedio de edad del grupo de estudio fue de 62.9 años, con un volumen promedioprostático de 46.78 cc. La incidencia de bacteremia fue de 7.24 por cien, (n=5). No se observó diferencia en la incidencia de bacteremia en pacientes que toman antibiótico profiláctico genérico o comercial, ni en las biopsias por sextantes vs.octantes. El 32.69 por cien de los pacientes a quienes se le toman biopsias (n=104) tienen carcinoma de próstata. No existió diferencia significativa para la detección del cáncer prostático entre la toma de biopsias por octantes y sextantes. Conclusiones: En este estudio se encontró una incidencia de bacteremia y cuadro de sepsis, mas baja que la comparada con las estadísticas informadas en la literatura. Las técnicas actuales de biopsias transrectales ecodirigidas con la toma de múltiples muestras obtenidas con aguja fina, son seguras c...