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The likelihood of experiencing social isolation increases later in life, particularly for females. It remains unknown how late-life social isolation impacts cognition and affective behavior in aged mice. We assessed the impact of late-life social isolation in 18-month old female mice. One month of single-housing did not lead to robust depressive-like symptomology, altered social interaction behavior, or sensitivity to context fear acquisition or memory. Rather, isolation increased hyperactivity and exploration, and reduced anxiety-like behavior in the open field and elevated plus maze, findings that have been similarly observed in young female and male mice following early-life isolation. These findings suggest that hyperactivity is a robust behavior following social isolation across the lifespan.

Enteroviruses frequently result in respiratory and gastrointestinal illness; however, multiple subtypes, including poliovirus, can cause severe neurologic disease. Recent biennial increases (i.e., 2014, 2016, and 2018) in cases of non-polio acute flaccid paralysis have led to speculations that other enteroviruses, specifically enterovirus D68 (EV-D68), are emerging to fill the niche that was left from poliovirus eradication. A cluster of 11 suspect cases of pediatric acute flaccid myelitis (AFM) was identified in 2016 in Phoenix, AZ. Multiple genomic analyses identified the presence of EV-D68 in the majority of clinical AFM cases. Beyond limited detection of herpesvirus, no other likely etiologies were found in the cluster. These findings strengthen the likelihood that EV-D68 is a cause of AFM and show that the rapid molecular assays developed for this study are useful for investigations of AFM and EV-D68., Enteroviruses are a common cause of respiratory and gastrointestinal illness, and multiple subtypes, including poliovirus, can cause neurologic disease. In recent years, enterovirus D68 (EV-D68) has been associated with serious neurologic illnesses, including acute flaccid myelitis (AFM), frequently preceded by respiratory disease. A cluster of 11 suspect cases of pediatric AFM was identified in September 2016 in Phoenix, AZ. To determine if these cases were associated with EV-D68, we performed multiple genomic analyses of nasopharyngeal (NP) swabs and cerebrospinal fluid (CSF) material from the patients, including real-time PCR and amplicon sequencing targeting the EV-D68 VP1 gene and unbiased microbiome and metagenomic sequencing. Four of the 11 patients were classified as confirmed cases of AFM, and an additional case was classified as probable AFM. Real-time PCR and amplicon sequencing detected EV-D68 virus RNA in the three AFM patients from which NP swabs were collected, as well as in a fourth patient diagnosed with acute disseminated encephalomyelitis, a disease that commonly follows bacterial or viral infections, including enterovirus. No other obvious etiological causes for AFM were identified by 16S or RNA and DNA metagenomic sequencing in these cases, strengthening the likelihood that EV-D68 is an etiological factor. Herpes simplex viral DNA was detected in the CSF of the fourth case of AFM and in one additional suspect case from the cluster. Multiple genomic techniques, such as those described here, can be used to diagnose patients with suspected EV-D68 respiratory illness, to aid in AFM diagnosis, and for future EV-D68 surveillance and epidemiology.

Approximately 90% of persons with amyotrophic lateral sclerosis (ALS) have the sporadic form, which may be caused by the interaction of multiple environmental factors and previously unknown genes.We performed a genomewide association analysis using 766,955 single-nucleotide polymorphisms (SNPs) found in 386 white patients with sporadic ALS and 542 neurologically normal white controls (the discovery series). Associations of SNPs with sporadic ALS were confirmed in two independent replication populations: replication series 1, with 766 case patients with the disease and 750 neurologically normal controls, and replication series 2, with 135 case patients and 275 controls.We identified 10 genetic loci that are significantly associated (P0.05) with sporadic ALS in three independent series of case patients and controls and an additional 41 loci that had significant associations in two of the three series. The most significant association with disease in white case patients as compared with controls was found for a SNP near an uncharacterized gene known as FLJ10986 (P=3.0x10(-4); odds ratio for having the genotype in patients vs. controls, 1.35; 95% confidence interval, 1.13 to 1.62). The FLJ10986 protein was found to be expressed in the spinal cord and cerebrospinal fluid of patients and of controls. Specific SNPs seem to be associated with sex, age at onset, and site of onset of sporadic ALS.Variants of FLJ10986 may confer susceptibility to sporadic ALS. FLJ10986 and 50 other candidate loci warrant further investigation for their potential role in conferring susceptibility to the disease.

Neurons adapt their electrophysiological properties to maintain stable levels of electrical excitability when faced with a constantly changing environment. We find that exposing freely swimming Xenopus tadpoles to 4–5 hr of persistent visual stimulation increases the intrinsic excitability of optic tectal neurons. This increase is correlated with enhanced voltage-gated Na+ currents. The same visual stimulation protocol also induces a polyamine synthesis-dependent reduction in Ca2+-permeable AMPAR-mediated synaptic drive, suggesting that the increased excitability may compensate for this reduction. Accordingly, the change in excitability was prevented by blocking polyamine synthesis during visual stimulation and was rescued when Ca2+-permeable AMPAR-mediated transmission was selectively reduced. The changes in excitability also rendered tectal cells more responsive to synaptic burst stimuli, improving visual stimulus detection. The synaptic and intrinsic adaptations function together to keep tectal neurons within a constant operating range, while making the intact visual system less responsive to background activity yet more sensitive to burst stimuli.

Background and Aims: Accumulating evidence suggests that a subset of cancer cells also known as the “cancer stem cells” (CSCs) influence various clinical outcomes in cancer, including tumor recurrence, metastasis and resistance to chemotherapy. Recently stemness has been recognized as a dynamic state governed by epigenetic modifiers including miRNAs. Despite identification of several self-renewal associated miRNAs, miRNA profile of CSCs remains unclear. Herein, we characterized miRNA expression of CSCs with high vs. low CD44v6 expression through RNA-Seq. Subsequently, we investigated the clinical significance of a novel miRNA identified from this systematic discovery approach. Methods: Colorectal CSCs from HCT116 and HT29 cells were grown as spheroid-derived cancer stem cells (SDCSCs). CD44v6+ and CD44v6- CSCs were subdivided by FACS and characterized by small RNA-Seq. Differentially expressed miRNAs were subsequently confirmed in CD44v6+ CSCs and chemoresistant cells. The expression of one such candidate, miR-1246, was assessed in a clinical cohort (n = 144) by qRT-PCR. Results: MiRNA profiling identified a unique overall pattern of CD44v6+ SDCSCs indicative of high self-renewal capacity. We noted that a panel of established self-renewal suppressive-miRNAs were downregulated (including miR-34a, 101 and 200 family) in CD44v6+ CSCs, and discovered upregulation of previously unreported miRNAs (miR-1246, 3605, 3182 and 4284). KEGG pathway analysis indicated that these miRNAs regulate Akt-MAPK and Wnt signaling pathways. Subsequently, we selected miR-1246 and validated its expression in CD44v6+ SDCSCs and chemoresistant cells. Clinically, the expression of miR-1246 was significantly elevated in CRC tissues compared to corresponding normal mucosa, and this occurred in a stage-dependent manner in primary CRCs. Furthermore, the expression of CD44v6 positively correlated with miR-1246 in CRC tissues. High miR-1246 expression resulted in poor disease free and overall survival. Conclusion: Using a systematic and comprehensive approach, we have identified a unique network of dysregulated miRNAs in CD44v6 CSCs indicative of high degree of stemness features in cancer. In particular, we identified miR-1246 to be frequently overexpressed in CSCs as well as chemoresistant cells and its expression was associated with poor prognosis in CRC patients. Collectively, we have identified a unique group of previously unreported miRNAs which appear to have important mechanistic roles in CSCs and could serve as a promising predictive biomarkers for recurrence and prognosis in patients with CRC. Citation Format: Shusuke Toden, Takatoshi Matsuyama, Elizabeth Hutchins, Kendall Jensen, Ajay Goel. Identification of a novel network of miRNAs that regulate stemness in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3355. doi:10.1158/1538-7445.AM2017-3355

INTRODUCTION: Biobanked brain tumor specimens share a critical shortcoming: the inability to efficiently screen tissue specimens prior to banking, potentially leading to storage of poor-quality samples. Standard histologic approaches to evaluate cellularity of tissue, such as frozen sectioning, may alter molecular characteristics and damage tissue for downstream analyses. Here, utilizing a novel ex vivo approach, we evaluate the feasibility of interrogating tissue cellularity and cytoarchitecture with label-free confocal reflectance microscopy (CRM) prior to biobanking. METHODS: Biopsies from patients with intracranial neoplasms (n = 3 for each; glioma, meningioma, pituitary adenoma, and schwannoma) were transported to the pathology department and placed in ice-cold saline. Samples were immediately positioned on the stage of our pathology-based Zeiss LSM710 confocal microscope and visualized with CRM. CRM images were evaluated for tumor cellularity, architecture and morphology, and then compared to corresponding permanent sections. RESULTS: CRM could contrast cellularity and stroma in all samples, as compared to corresponding permanent sections. Additionally, CRM contrasted vasculature and regions of necrosis in glioblastomas, tumor architecture in meningiomas and schwannomas, and sheets of neoplastic cells in pituitary ademonas. DISCUSSION: We found confocal reflectance microscopy to be a simple approach to screening biospecimens prior to biobanking. CRM quickly generated distinct ex vivo microscopic images from biopsies without tissue processing or application of exogenous dyes. CRM contrasted histopathological features present in our samples, and produced images that could be digitally stored and recalled with the matched specimen in the biobank. In our previous studies, we reported CRM did not alter the molecular integrity of fresh biopsies from brain tumor animal models. We hypothesize that wider use of this technique could improve the quality of banked tissue.

Analysis of the genetic changes in human tumors is often problematical because of the presence of normal stroma and the limited availability of pure tumor DNA. However, large amounts of highly reproducible “representations” of tumor and normal genomes can be made by PCR from nanogram amounts of restriction endonuclease cleaved DNA that has been ligated to oligonucleotide adaptors. We show here that representations are useful for many types of genetic analyses, including measuring relative gene copy number, loss of heterozygosity, and comparative genomic hybridization. Representations may be prepared even from sorted nuclei from fixed and archived tumor biopsies.

In a genome-wide association study (GWAS) of late-onset Alzheimer's disease (AD), we found an association between common haplotypes of the GAB2 gene and AD risk in carriers of the apolipoprotein E (APOE) e4 allele, the major late-onset AD susceptibility gene. We previously proposed the use of fluorodeoxyglucose positron emission tomography (FDG-PET) measurements as a quantitative pre-symptomatic endophenotype, more closely related to disease risk than the clinical syndrome itself, to help evaluate putative genetic and non-genetic modifiers of AD risk. In this study, we examined the relationship between the presence or absence of the relatively protective GAB2 haplotype and PET measurements of regional-to-whole brain FDG uptake in several AD-affected brain regions in 158 cognitively normal late-middle-aged APOEe4 homozygotes, heterozygotes, and non-carriers. GAB2 haplotypes were characterized using Affymetrix Genome-Wide Human SNP 6.0 Array data from each of these subjects. As predicted, the possibly protective GAB2 haplotype was associated with higher regional-to-whole brain FDG uptake in AD-affected brain regions in APOEe4 carriers. While additional studies are needed, this study supports the association between the possibly protective GAB2 haplotype and the risk of late-onset AD in APOEe4 carriers. It also supports the use of brain-imaging endophenotypes to help assess possible modifiers of AD risk.

Homer proteins are integral components of the postsynaptic density and are thought to function in synaptogenesis and plasticity. In addition, overexpression of Homer in the developing Xenopus retinotectal system results in axonal pathfinding errors. Here we report that Xenopus contains the homer1 gene, expressed as the isoform, xhomer1b, which is highly homologous to the mammalian homer1b. The mammalian homer1 gene is expressed as three isoforms, the truncated or short form homer1a and the long forms homer1b and -1c. For Xenopus, we cloned three very similar variants of homer1b, identified as Xenopus xhomer1b.1, xhomer1b.2, and xhomer1b.3, which display up to 98% homology with each other and 90% similarity to mammalian homer1b. Furthermore, we demonstrate that Xenopus also contains a truncated form of the Homer1 protein, which could be induced by kainic acid injection and is likely homologous to the mammalian Homer1a. xHomer1b expression was unaffected by neuronal activity levels but was developmentally regulated. Within the brain, the spatial and temporal distributions of both Homer isoforms were similar in the neuropil and cell body regions. Homer1 was detected in motor axons. Differential distribution of the two isoforms was apparent: Homer1b immunoreactivity was prominent at junctions between soma and the ventricular surface; in the retina, the Mueller radial glia were immunoreactive for Homer1, but not Homer1b, suggesting the retinal glia contain only the Homer1a isoform. Homer1b expression in muscle was prominent throughout development and was aligned with the actin striations in skeletal muscle. The high level of conservation of the xhomer1 gene and the protein expression in the developing nervous system suggest that Homer1 expression may be important for normal neuronal circuit development.

Electroporation is becoming more popular as a technique for transfecting neurons within intact tissues. One of the advantages of electroporation over other transfection techniques is the ability to precisely target an area for transfection. Here we highlight this advantage by describing methods to restrict transfection to either a single cell, clusters of cells, or to include large portions of the brain of the intact Xenopus tadpole. Electroporation is also an effective means of gene delivery in the retina. We have developed these techniques to examine the effects of regulated gene expression on various neuronal properties, including structural plasticity and synaptic transmission. Restriction of transfection to individual cells aids in imaging of neuronal morphology, while bulk cell transfection allows examination of the affects of gene expression on populations of cells by biochemical assays, imaging, and electrophysiological recording.

Corrigendum to “Association between GAB2 haplotype and higher glucose metabolism in Alzheimer's disease-affected brain regions in cognitively normal APOEe4 carriers” [NeuroImage 54/3 (2011) 1896–1902] Winnie S. Liang , Kewei Chen , Wendy Lee , Kunal Sidhar , Jason J. Corneveaux , April N. Allen , Amanda Myers , Stephen Villa , Bessie Meechoovet , Jeremy Pruzin , Daniel Bandy , Adam S. Fleisher , Jessica B.S. Langbaum , Matthew J. Huentelman , Kendall Jensen , Travis Dunckley , Richard J. Caselli , Susan Kaib , Eric M. Reiman a,b,c,j,⁎