MicroRNAs (miRNAs) regulate gene expression and have a critical role in many biologic and pathologic processes. We hypothesized that miRNA expression profiles in injured brain (hippocampus) would ...show common as well as unique profiles when compared with those of blood. Adult, untouched, control rats were compared with rats with sham surgeries, ischemic strokes, brain hemorrhage (lysed blood, fresh blood, or thrombin), and kainate-induced seizures. Brain and whole-blood miRNA expression profiles were assessed 24 h later using TaqMan rodent miRNA arrays. MicroRNA response profiles were different for each condition. Many miRNAs changed more than 1.5-fold in brain and blood after each experimental manipulation, and several miRNAs were upregulated or downregulated in both brain and blood after a given injury. A few miRNAs (e.g., miR-298, miR-155, and miR-362-3p) were upregulated or downregulated more than twofold in both brain and blood after several different injuries. The results show the possible use of blood miRNAs as biomarkers for brain injury; that selected blood miRNAs may correlate with miRNA changes in the brain; and that many of the mRNAs, previously shown to be regulated in brain and blood after brain injury, are likely accounted for by changes in miRNA expression.
Owing to their clinical success, there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia ...(AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform that consists of a universal effector module (EM) and individual target modules (TMs). Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (for example, the extracellular CD137 ligand domain) to the TM, the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector-to-target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.
Genome-wide association studies have identified putative ischemic stroke risk genes, yet, their expression after stroke is unexplored in spite of growing interest in elucidating their specific role ...and identifying candidate genes for stroke treatment. Thus, we took an exploratory approach to investigate sexual dimorphism, alternative splicing, and etiology in putative risk gene expression in blood following cardioembolic, atherosclerotic large vessel disease and small vessel disease/lacunar causes of ischemic stroke in each sex compared to controls. Whole transcriptome arrays assessed 71 putative stroke/vascular risk factor genes for blood RNA expression at gene-, exon-, and alternative splicing-levels. Male (
n
= 122) and female (
n
= 123) stroke and control volunteers from three university medical centers were matched for race, age, vascular risk factors, and blood draw time since stroke onset. Exclusion criteria included: previous stroke, drug abuse, subarachnoid or intracerebral hemorrhage, hemorrhagic transformation, infection, dialysis, cancer, hematological abnormalities, thrombolytics, anticoagulants or immunosuppressants. Significant differential gene expression (fold change > |1.2|,
p
< 0.05, partial correlation > |0.4|) and alternative splicing (false discovery rate
p
< 0.3) were assessed. At gene level, few were differentially expressed: ALDH2, ALOX5AP, F13A1, and IMPA2 (males, all stroke); ITGB3 (females, cardioembolic); ADD1 (males, atherosclerotic); F13A1, IMPA2 (males, lacunar); and WNK1 (females, lacunar). GP1BA and ITGA2B were alternatively spliced in both sexes (all patients vs. controls). Six genes in males, five in females, were alternatively spliced in all stroke compared to controls. Alternative splicing and exon-level analyses associated many genes with specific etiology in either sex. Of 71 genes, 70 had differential exon-level expression in stroke patients compared to control subjects. Among stroke patients, 24 genes represented by differentially expressed exons were male-specific, six were common between sexes, and two were female-specific. In lacunar stroke, expression of 19 differentially expressed exons representing six genes (ADD1, NINJ2, PCSK9, PEMT, SMARCA4, WNK1) decreased in males and increased in females. Results demonstrate alternative splicing and sexually dimorphic expression of most putative risk genes in stroke patients' blood. Since expression was also often cause-specific, sex, and etiology are factors to consider in stroke treatment trials and genetic association studies as society trends toward more personalized medicine.
Although peripheral blood mRNA and micro-RNA change after 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 after stroke.
lncRNA expression was assessed in 266 whole-blood RNA samples drawn once per individual from patients with ischemic stroke and matched with vascular risk factor controls. Differential lncRNA expression was assessed by ANCOVA (P<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). Poststroke 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.
A total of 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 men and 31 lncRNAs in women. Some differentially expressed lncRNAs mapped close to genomic locations of previously identified putative stroke-risk genes, including lipoprotein, lipoprotein(a)-like 2, ABO (transferase A, α1-3-N-acetylgalactosaminyltransferase; transferase B, α1-3-galactosyltransferase) blood group, prostaglandin 12 synthase, and α-adducins.
This study provides evidence of altered and sexually dimorphic lncRNA expression in peripheral blood of patients with stroke compared with that of controls and suggests that lncRNAs have potential for stroke biomarker development. Some regulated lncRNA could regulate some previously identified putative stroke-risk genes.
Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have ...identified an optimal set of the most stably expressed genes in human blood that can be used for normalization.
Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms.
Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder).
The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.
Cucumber mosaic virus (CMV) infects a wide variety of crop plants and in tomato (Lycopersicon esculentum Mill.) causes significant economic losses in many growing regions, particularly the ...Mediterranean. The objective of the present study was to identify the number and map locations of genes controlling resistance to CMV in breeding lines (BC^sub 1^-inbreds) derived from the related wild species L. chilense. These lines also carried the gene Tm-2 ^sup a^ for resistance to ToMV, which facilitated the interpretation of disease symptoms. The segregation for CMV resistance in the BC^sub 2^F^sub 1^ and BC^sub 2^F^sub 2^ generations, following mechanical inoculation with subgroup-I isolates, was consistent with expectations for a single dominant gene, for which the symbol Cmr (cucumber mosaic resistance) was given. Resistant and susceptible BC^sub 1^-inbreds were analyzed with RFLP and isozyme markers to identify genomic regions introgressed from L. chilense. The only L. chilense-specific markers found were on chromosome 12; some resistant lines contained a single introgression comprising the entire short arm and part of the long arm of this chromosome, while others contained a recombinant derivative of this introgression. The chromosome 12 markers were significantly associated with CMV resistance in both qualitative and quantitative models of inheritance. The qualitative analysis, however, demonstrated that CMV resistance was not expressed as a reliable monogenic character, suggesting a lack of penetrance, significant environmental effects, or the existence of additional (undetected) resistance factors. In the quantitative analysis, the marker interval TG68 - CT79 showed the most significant association with CMV resistance. No association between CMV resistance and the Tm-2 ^sup a^ gene was observed. These breeding lines are potentially useful sources of CMV resistance for tomato improvement, in which context knowledge of the map location of Cmr should accelerate introgression by marker-assisted selection.PUBLICATION ABSTRACT
Since RNA expression differences have been reported in autism spectrum disorder (ASD) for blood and brain, and differential alternative splicing (DAS) has been reported in ASD brains, we determined ...if there was DAS in blood mRNA of ASD subjects compared to typically developing (TD) controls, as well as in ASD subgroups related to cerebral volume.
RNA from blood was processed on whole genome exon arrays for 2-4-year-old ASD and TD boys. An ANCOVA with age and batch as covariates was used to predict DAS for ALL ASD (n=30), ASD with normal total cerebral volumes (NTCV), and ASD with large total cerebral volumes (LTCV) compared to TD controls (n=20).
A total of 53 genes were predicted to have DAS for ALL ASD versus TD, 169 genes for ASD_NTCV versus TD, 1 gene for ASD_LTCV versus TD, and 27 genes for ASD_LTCV versus ASD_NTCV. These differences were significant at P <0.05 after false discovery rate corrections for multiple comparisons (FDR <5% false positives). A number of the genes predicted to have DAS in ASD are known to regulate DAS (SFPQ, SRPK1, SRSF11, SRSF2IP, FUS, LSM14A). In addition, a number of genes with predicted DAS are involved in pathways implicated in previous ASD studies, such as ROS monocyte/macrophage, Natural Killer Cell, mTOR, and NGF signaling. The only pathways significant after multiple comparison corrections (FDR <0.05) were the Nrf2-mediated reactive oxygen species (ROS) oxidative response (superoxide dismutase 2, catalase, peroxiredoxin 1, PIK3C3, DNAJC17, microsomal glutathione S-transferase 3) and superoxide radical degradation (SOD2, CAT).
These data support differences in alternative splicing of mRNA in blood of ASD subjects compared to TD controls that differ related to head size. The findings are preliminary, need to be replicated in independent cohorts, and predicted alternative splicing differences need to be confirmed using direct analytical methods.
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