Background and Purpose Though peripheral blood mRNA and microRNA change following ischemic stroke, any role for long noncoding RNA (lncRNA), which comprise most of the genome and have been implicated in various diseases, is unknown. Thus, we hypothesized that lncRNA expression also changes following stroke. Methods lncRNA expression was assessed in 266 whole-blood RNA samples drawn once per individual from ischemic stroke patients and matched vascular risk factor controls. Differential lncRNA expression was assessed by Analysis of Covariance (ANCOVA, p-value < 0.005; fold change > |1.2|), principal components analysis and hierarchical clustering on a derivation set (n=176) and confirmed on a validation set (n=90). Post-stroke temporal lncRNA expression changes were assessed using ANCOVA with confounding factor correction (p<0.005; partial correlation with time since event >|0.4|). Because sexual dimorphism exists in stroke, analyses were performed for each sex separately. Results 299 lncRNAs were differentially expressed between stroke and control males, whereas 97 lncRNAs were differentially expressed between stroke and control females. Significant changes of lncRNA expression with time after stroke were detected for 49 lncRNAs in males and 31 lncRNAs in females. Some differentially expressed lncRNAs mapped close to genomic locations of previously identified putative stroke-risk genes, including Lipoprotein, Lp(A)-Like 2, ABO blood group, Prostaglandin 12 Synthase, and α-Adducins. Conclusions This study provides evidence of altered and sexually dimorphic lncRNA expression in peripheral blood of stroke patients compared to controls and suggests lncRNAs have potential for stroke biomarker development. Some regulated lncRNA could regulate some previously identified putative stroke-risk genes.

Because our recent studies have demonstrated that miR-122 decreased in whole blood of patients and in whole blood of rats following ischemic stroke, we tested whether elevating blood miR-122 would improve stroke outcomes in rats. Young adult rats were subjected to a temporary middle cerebral artery occlusion (MCAO) or sham operation. A polyethylene glycol-liposome-based transfection system was used to administer a miR-122 mimic after MCAO. Neurological deficits, brain infarction, brain vessel integrity, adhesion molecule expression and expression of miR-122 target and indirect-target genes were examined in blood at 24 h after MCAO with or without miR-122 treatment. miR-122 decreased in blood after MCAO, whereas miR-122 mimic elevated miR-122 in blood 24 h after MCAO. Intravenous but not intracerebroventricular injection of miR-122 mimic decreased neurological deficits and brain infarction, attenuated ICAM-1 expression, and maintained vessel integrity after MCAO. The miR-122 mimic also down-regulated direct target genes (e.g. Vcam1, Nos2, Pla2g2a) and indirect target genes (e.g. Alox5, Itga2b, Timp3, Il1b, Il2, Mmp8) in blood after MCAO which are predicted to affect cell adhesion, diapedesis, leukocyte extravasation, eicosanoid and atherosclerosis signaling. The data show that elevating miR-122 improves stroke outcomes and we postulate this occurs via downregulating miR-122 target genes in blood leukocytes.

Understanding how the blood transcriptome of human intracerebral hemorrhage (ICH) differs from ischemic stroke (IS) and matched controls (CTRL) will improve understanding of immune and coagulation pathways in both disorders. This study examined RNA from 99 human whole-blood samples using GeneChip® HTA 2.0 arrays to assess differentially expressed transcripts of alternatively spliced genes between ICH, IS and CTRL. We used a mixed regression model with FDR-corrected p(Dx) < 0.2 and p < 0.005 and |FC| > 1.2 for individual comparisons. For time-dependent analyses, subjects were divided into four time-points: 0(CTRL), <24 h, 24-48 h, >48 h; 489 transcripts were differentially expressed between ICH and CTRL, and 63 between IS and CTRL. ICH had differentially expressed T-cell receptor and CD36 genes, and iNOS, TLR, macrophage, and T-helper pathways. IS had more non-coding RNA. ICH and IS both had angiogenesis, CTLA4 in T lymphocytes, CD28 in T helper cells, NFAT regulation of immune response, and glucocorticoid receptor signaling pathways. Self-organizing maps revealed 4357 transcripts changing expression over time in ICH, and 1136 in IS. Understanding ICH and IS transcriptomes will be useful for biomarker development, treatment and prevention strategies, and for evaluating how well animal models recapitulate human ICH and IS.

Objective: To evaluate microRNA let7i in ischemic stroke and its regulation of leukocytes.Methods: A total of 212 patients were studied: 106 with acute ischemic stroke and 106 controls matched for risk factors. RNA from circulating leukocytes was isolated from blood collected in PAXgene tubes. Let7i microRNA expression was assessed using TaqMan quantitative reverse transcription PCR. To assess let7i regulation of gene expression in stroke, messenger RNA (mRNA) from leukocytes was measured by whole-genome Human Transcriptome Array Affymetrix microarray. Given microRNAs act to destabilize and degrade their target mRNA, mRNAs that inversely correlated with let7i were identified. To demonstrate let7i posttranscriptional regulation of target genes, a 39 untranslated region luciferase assay was performed. Target protein expression was assessed using ELISA.Results: Let7i was decreased in patients with acute ischemic stroke (fold change 21.70, p , 0.00001). A modest inverse correlation between let7i and NIH Stroke Scale score at admission (r 5 20.32, p 5 0.02), infarct volume (r 5 20.21, p 5 0.04), and plasma MMP9 (r 5 20.46, p 5 0.01) was identified. The decrease in let7i was associated with increased expression of several of its mRNA targets, including CD86, CXCL8, and HMGB1. In vitro studies confirm let7i posttranscriptional regulation of target genes CD86, CXCL8, and HMGB1. Functional analysis predicted let7i regulates pathways involved in leukocyte activation, recruitment, and proliferation including canonical pathways of CD86 signaling in T helper cells, HMGB1 signaling, and CXCL8 signaling.Conclusions: Let7i is decreased in circulating leukocytes of patients with acute ischemic stroke.Mechanisms by which let7i regulates inflammatory response post stroke include targeting CD86, CXCL8, and HMGB1. Neurology ® 2016;87:2198-2205 GLOSSARY cDNA 5 complementary DNA; HTA 5 Human Transcriptome Array; IL 5 interleukin; mRNA 5 messenger RNA; NIHSS 5 NIH Stroke Scale; 39UTR 5 39 untranslated region; TNF-a 5 tumor necrosis factor a.Ischemic stroke remains a leading cause of disability. Although early reperfusion therapy can improve outcomes, it is available to a minority of stroke patients. Specific therapies are needed to reduce brain injury and improve outcomes after stroke. The immune system responds rapidly following cerebral ischemia. A range of damage-associated molecular patterns, cytokines, and chemokines are released to activate circulating leukocytes, vasculature, and brain cells.

The histone deacetylase 9 (HDAC9) polymorphism rs2107595 is associated with an increased risk for large vessel atherosclerotic stroke (LVAS). In humans, there remains a need to better understand this HDAC9 polymorphism's contribution to large vessel stroke. In this pilot study, we evaluated whether the HDAC9 polymorphism rs2107595 is associated with differences in leukocyte gene expression in patients with LVAS. HDAC9 SNP rs2107595 was genotyped in 155 patients (43 LVAS and 112 vascular risk factor controls). RNA isolated from blood was processed on whole genome microarrays. Gene expression was compared between HDAC9 risk allele-positive and risk allele-negative LVAS patients and controls. Functional analysis identified canonical pathways and molecular functions associated with rs2107595 in LVAS. In HDAC9 SNP rs2107595 risk allele-positive LVAS patients, there were 155 genes differentially expressed compared to risk allele-negative patients (fold change > |1.2|, p < 0.05). The 155 genes separated the risk allele-positive and risk allele-negative LVAS patients on a principal component analysis. Pathways associated with HDAC9 risk allele-positive status involved IL-6 signaling, cholesterol efflux, and platelet aggregation. These preliminary data suggest an association with the HDAC9 rs2107595 risk allele and peripheral immune, lipid, and clotting systems in LVAS. Further study is required to evaluate whether these differences are related to large vessel atherosclerosis and stroke risk.

Coccidioidomycosis is a fungal disease acquired through the inhalation of spores of Coccidioides spp., which afflicts primarily humans and other mammals. It is endemic to areas in the southwestern United States, including the San Joaquin Valley portion of Kern County, California, our region of interest (ROI). Recently, incidence of coccidioidomycosis, also known as valley fever, has increased significantly, and several factors including climate change have been suggested as possible drivers for this observation. Up to date details about the ecological niche of C. immitis have escaped full characterization. In our project, we chose a three-step approach to investigate this niche: 1) We examined Landsat-5-Thematic-Mapper multispectral images of our ROI by using training pixels at a 750 m×750 m section of Sharktooth Hill, a site confirmed to be a C. immitis growth site, to implement a Maximum Likelihood Classification scheme to map out the locations that could be suitable to support the growth of the pathogen; 2) We used the websoilsurvey database of the US Department of Agriculture to obtain soil parameter data; and 3) We investigated soil samples from 23 sites around Bakersfield, California using a multiplex Polymerase Chain Reaction (PCR) based method to detect the pathogen. Our results indicated that a combination of satellite imagery, soil type information, and multiplex PCR are powerful tools to predict and identify growth sites of C. immitis. This approach can be used as a basis for systematic sampling and investigation of soils to detect Coccidioides spp.

Objective Single nucleotide polymorphisms (SNPs) contribute to complex disorders such as ischemic stroke (IS). Since SNPs could affect IS by altering gene expression, we studied the association of common SNPs with changes in mRNA expression (i.e. expression quantitative trait loci; eQTL) in blood after IS. Methods RNA and DNA were isolated from 137 patients with acute IS and 138 vascular risk factor controls (VRFC). Gene expression was measured using Affymetrix HTA 2.0 microarrays and SNP variants were assessed with Axiom Biobank Genotyping microarrays. A linear model with a genotype (SNP) × diagnosis (IS and VRFC) interaction term was fit for each SNP‐gene pair. Results The eQTL interaction analysis revealed significant genotype × diagnosis interaction for four SNP‐gene pairs as cis‐eQTL and 70 SNP‐gene pairs as trans‐eQTL. Cis‐eQTL involved in the inflammatory response to IS included rs56348411 which correlated with neurogranin expression (NRGN), rs78046578 which correlated with CXCL10 expression, rs975903 which correlated with SMAD4 expression, and rs62299879 which correlated with CD38 expression. These four genes are important in regulating inflammatory response and BBB stabilization. SNP rs148791848 was a strong trans‐eQTL for anosmin‐1 (ANOS1) which is involved in neural cell adhesion and axonal migration and may be important after stroke. Interpretation This study highlights the contribution of genetic variation to regulating gene expression following IS. Specific inflammatory response to stroke is at least partially influenced by genetic variation. This has implications for progressing toward personalized treatment strategies. Additional research is required to investigate these genes as therapeutic targets.

The effect of spironolactone in the alleviation of the symptoms of the premenstrual syndrome was compared with placebo in a double-blind, parallel group controlled study. One tablet daily of 100 mg spironolactone or placebo was given to 63 women from Day 12 of the menstrual cycle until the first day of the next menstrual bleed. This regimen was repeated for two consecutive cycles. Spironolactone was statistically significantly superior in providing relief from bloatedness (p less than 0.001). No statistically significant changes were observed in blood biochemistry of plasma hormone levels of oestradiol, progesterone or prolactin, though an increase in serum aldosterone levels was seen in the spironolactone-treated group. No differences were detected in weight, blood pressure or the incidence and severity of complaints following treatment.