EphA4 expression promotes network activity and spine maturation in cortical neuronal cultures

<p>Abstract</p> <p>Background</p> <p>Neurons form specific connections with targets via synapses and patterns of synaptic connectivity dictate neural function. During development, intrinsic neuronal specification and environmental factors guide both initial formation of synapses and strength of resulting connections. Once synapses form, non-evoked, spontaneous activity serves to modulate connections, strengthening some and eliminating others. Molecules that mediate intercellular communication are particularly important in synaptic refinement. Here, we characterize the influences of EphA4, a transmembrane signaling molecule, on neural connectivity.</p> <p>Results</p> <p>Using multi-electrode array analysis on <it>in vitro </it>cultures, we confirmed that cortical neurons mature and generate spontaneous circuit activity as cells differentiate, with activity growing both stronger and more patterned over time. When EphA4 was over-expressed in a subset of neurons in these cultures, network activity was enhanced: bursts were longer and were composed of more spikes than in control-transfected cultures. To characterize the cellular basis of this effect, dendritic spines, the major excitatory input site on neurons, were examined on transfected neurons <it>in vitro</it>. Strikingly, while spine number and density were similar between conditions, cortical neurons with elevated levels of EphA4 had significantly more mature spines, fewer immature spines, and elevated colocalization with a mature synaptic marker.</p> <p>Conclusions</p> <p>These results demonstrate that experimental elevation of EphA4 promotes network activity <it>in vitro</it>, supporting spine maturation, producing more functional synaptic pairings, and promoting more active circuitry.</p

Genetic analysis of 7 medieval skeletons from the Aragonese Pyrenees

Aim To perform a genetic characterization of 7 skeletons from medieval age found in a burial site in the Aragonese Pyrenees. Methods Allele frequencies of autosomal short tandem repeats (STR) loci were determined by 3 different STR systems. Mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were determined by sequencing of the hypervariable segment 1 of mtDNA and typing of phylogenetic Y chromosome single nucleotide polymorphisms (YSNP) markers, respectively. Possible familial relationships were also investigated. Results Complete or partial STR profiles were obtained in 3 of the 7 samples. Mitochondrial DNA haplogroup was determined in 6 samples, with 5 of them corresponding to the haplogroup H and 1 to the haplogroup U5a. Ychromosome haplogroup was determined in 2 samples, corresponding to the haplogroup R. In one of them, the sub-branch R1b1b2 was determined. mtDNA sequences indicated that some of the individuals could be maternally related, while STR profiles indicated no direct family relationships. Conclusions Despite the antiquity of the samples and great difficulty that genetic analyses entail, the combined use of autosomal STR markers, Y-chromosome informative SNPs, and mtDNA sequences allowed us to genotype a group of skeletons from the medieval age

High incidence of Noonan syndrome features including short stature and pulmonic stenosis in patients carrying NF1 missense mutations affecting p.Arg1809: genotype-phenotype correlation

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple cafe-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P<0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844–848

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000–3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons—Leu844, Cys845, Ala846, Leu847, and Gly848—located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844–848 exists and will be valuable in the management and genetic counseling of a significant number of individuals

Lymphocyte-predominant Esophagitis: A Distinct and Likely Immune-mediated Disorder Encompassing Lymphocytic and Lichenoid Esophagitis

Lymphocytic esophagitis is a well-known manifestation of Crohn disease among children but is not considered to be an immune-mediated mucositis in adults. We hypothesize that adult-onset lymphocyte-predominant esophagitis is also an immune-mediated inflammatory pattern, the nature of which has been masked by other conditions that feature esophageal lymphocytosis and occur in older patients. We performed this study to consolidate diagnostic criteria for lymphocyte-predominant esophagitis and determine its clinical significance. We identified 61 patients with lymphocyte-rich inflammation in the mid or proximal esophagus, none of whom had another explanation for esophageal lymphocytosis. Affected patients were usually older adults and 72% were women. Most (56%) presented with dysphagia and 34% had eosinophilic esophagitis-like changes with rings, exudates, and/or edematous mucosa and linear furrows. Intraepithelial lymphocytosis was accompanied by mucosal injury featuring edema, basal zone hyperplasia, and scattered dyskeratotic cells. Some cases displayed occasional neutrophils or even superficial microabscesses; eosinophils were consistently infrequent. Most (67%) patients had at least 1 systemic immune-mediated disorder, particularly Crohn disease (30%) and connective tissue diseases (23%); only 1 had mucocutaneous lichen planus. We conclude that mild mucosal lymphocytosis (ie, ≥20 lymphocytes/HPF) alone is a frequent and nonspecific finding; criteria for lymphocyte-predominant esophagitis should include evidence of mucosal injury and allow for more than the occasional neutrophil. When this diagnosis is limited to cases that feature lymphocytosis unattributed to acid reflux, motility disorders, or infection, lymphocyte-predominant esophagitis may represent an immune-mediated disorder with characteristic clinical manifestations and a predilection for middle-aged women

Secondary analysis of merged American Hospital Association data and U.S. Census data: Beginning to understand the supply-demand chain in pediatric inpatient care

Much attention has been focused on how the nursing shortage will impact the growing number of aging Americans. This study was conducted as a first step in understanding nursing supply relative to potential pediatric demand using merged data from the American Hospital Association\u27s annual survey and Census data by state from the year 2000. Findings indicate that there is tremendous variability among reporting states related to estimated pediatric nurses (registered nurse full-time equivalents), potential pediatric demand (persons from birth to 18 years), and allocated pediatric beds. Future research will examine how this supply-demand chain impacts clinical and cost outcomes for pediatric patients

Portal Cavernoma Cholangiopathy

Abstract Objectives Portal cavernoma cholangiopathy (formerly portal biliopathy) is a type of biliary injury that occurs in association with a portal vein thrombus or cavernoma. Although the radiographic features of portal cavernoma cholangiopathy have been enumerated in the literature, its histologic features have not been described in detail. Methods We describe the histologic findings in liver specimens from three patients with radiologically confirmed portal cavernoma cholangiopathy. Results Of the three patients, one underwent surgical resection due to a clinical suspicion for cholangiocarcinoma, one had a liver biopsy sample obtained for evaluation of possible cirrhosis, and one had a clinically suspicious “hilar mass” at the time of orthotopic liver transplant. Histologic features common among the three liver specimens included portal venous abnormalities, where the portal veins were obliterated or small relative to the portal tract size, and obstructive biliary changes, such as ductular reaction and reactive epithelial atypia accompanied by a mixed inflammatory cell infiltrate with neutrophils. Conclusions This case series provides clinicopathologic characteristics of portal cavernoma cholangiopathy. Histologic changes are reminiscent of hepatoportal sclerosis and/or bile duct obstruction. Attention to portal veins can provide helpful diagnostic clues, especially when biopsy samples are obtained from patients with a known portal vein thrombus or cavernoma

Simultaneous Detection of Single Nucleotide Variants and Expression of RNA and Protein in Multiple Myeloma Bone Marrow Aspirates

Introduction: Multiple myeloma (MM) is generally an incurable plasma cell neoplasm that evolves from a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS). Primary genomic events leading to the development of MGUS include hyperdiploidy, translocations involving the immunoglobulin heavy chain genes, and copy number variations. Single-nucleotide variants (SNVs) as well as insertions and deletions are secondary events that trigger progression to active MM. Identifying nucleotide variants that lead to changes in RNA and protein expression is critically important to understanding the biology of the cancer and ultimately identifying therapeutic targets. Analysis of DNA, RNA, and protein has traditionally involved integration of three separate assays and is frequently limited by the availability of sufficient material from MM patient specimens. We used a more efficient approach that simultaneously detects DNA variants, RNA and protein expression in a single sample from as little as 5 ng DNA, 25 ng RNA, and 250 ng protein. We evaluated the NanoString nCounter® Vantage 3D™ DNA:RNA:Protein Heme Assay performed on 1 x 106cells from bone marrow biopsies obtained from patients with MM. Methods: Bone marrow aspirates from 15 patients diagnosed with MM were obtained through an Institutional Review Board approved protocol. Seven patients had newly diagnosed MM and 8 had relapsed disease. DNA, RNA, and protein were extracted from unsorted, cryopreserved, ficoll-hypaque separated bone marrow mononuclear cells and hybridized with optical barcodes designed to detect 124 DNA somatic variants common to hematologic malignancies as well as 180 mRNA transcripts and 38 total and phospho-proteins. We also used the nCounter PanCancer Pathways Panel Assay to measure gene expression of 770 additional genes from 13 cancer-associated canonical pathways. The hybridized probes in each sample were counted using the nCounter Digital Analyzer and statistical analysis was performed using nSolver™ v4.0 alpha software. Results: The percentage of CD138+ bone marrow plasma cells measured by immunohistochemistry at the time of biopsy varied from 44 to 100% in each sample. One or more DNA variants were detected in 3 pre-treatment and 4 relapse samples. Mutations in KRAS, NRAS, and BRAF were most common and 2 patients were found to have variants in both KRAS and NRAS or KRAS and BRAF . Additionally, variants in DNMT3A and IDH2 were detected in 1 patient each. Since KRAS, NRAS, and BRAF are all members of the mitogen-activated protein kinase (MAPK) signaling pathway, a differential gene expression analysis of both RNA and protein was performed comparing samples with and without mutations in these genes. Findings confirmed that a higher level of expression was detectable in MAPK pathway genes, although this result did not reach statistical significance following correction for replicate testing, potentially the consequence of a small sample size. Conclusions: The Vantage 3D DNA:RNA:Protein Heme Assay is a rapid, sensitive and resource sparing platform for the simultaneous detection of DNA variants, RNA transcripts, and protein expression. We show that this method can feasibly be performed on as few as 1 x 106 cryopreserved bone marrow cells in patients with MM and our findings confirm detection of 3 of the most common MM variants. This technology should have broad application in other hematological malignancies and further evaluation of blood and lymph node biopsies from fresh, frozen and formalin-fixed paraffin-embedded (FFPE) samples is warranted. Demirkan:NanoString Technologies: Employment. Meredith:NanoString Technologies: Employment. Meredith:NanoString Technologies: Employment