Correction: Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil.

[This corrects the article DOI: 10.1371/journal.pgen.1000621.].

Cortical gene expression: prognostic value for seizure outcome following temporal lobectomy and amygdalohippocampectomy.

Whole genome analyses were performed to test the hypothesis that temporal cortical gene expression differs between epilepsy patients rendered seizure-free versus non-seizure-free following anterior temporal lobectomy with amygdalohippocampectomy (ATL/AH). Twenty four patients underwent ATL/AH to treat medically intractable seizures of temporal lobe origin (mean age 35.5 years, mean follow-up 42.2 months); they were then dichotomized into seizure-free and non-seizure-free groups. Tissue RNA was isolated from the lateral temporal cortex and gene expression analysis was performed. Whole genome data were analyzed for prognostic value for seizure-free outcome following ATL/AH by logistic regression. Genes that could distinguish seizure outcome groups were identified based on providing an accuracy of >0.90 judging by area under the receiver operating characteristic curve, AUC, with a P value of the slope coefficient of <0.05. Four genes and seven RNA probes were with prognostic value for post-operative seizure-free outcome. Gene expression associated with seizure-free outcome included relative down-regulation of zinc finger protein 852 (ZNF852), CUB domain-containing protein 2 (CDCP2), proline-rich transmembrane protein 1 (PRRT1), hypothetical LOC440200 (FLJ41170), RNA probe 8047763, RNA probe 8126238, RNA probe 8113489, RNA probe 8092883, RNA probe 7935228, RNA probe 806293, and RNA probe 8104131. This study describes the predictive value of temporal cortical gene expression for seizure-free outcome after ATL/AH. Four genes and seven RNA probes were found to predict post-operative seizure-free outcome. Future prospective investigation of these genes and probes in human brain tissue and blood could establish new biomarkers predictive of seizure outcome following ATL/AH.

Cortical gene expression correlates of temporal lobe epileptogenicity.

INTRODUCTION: Despite being one of the most common neurological diseases, it is unknown whether there may be a genetic basis to temporal lobe epilepsy (TLE). Whole genome analyses were performed to test the hypothesis that temporal cortical gene expression differs between TLE patients with high vs. low baseline seizure frequency. METHODS: Baseline seizure frequency was used as a clinical measure of epileptogenicity. Twenty-four patients in high or low seizure frequency groups (median seizures/month) underwent anterior temporal lobectomy with amygdalohippocampectomy for intractable TLE. RNA was isolated from the lateral temporal cortex and submitted for expression analysis. Genes significantly associated with baseline seizure frequency on likelihood ratio test were identified based on >0.90 area under the ROC curve, P value of <0.05. RESULTS: Expression levels of forty genes were significantly associated with baseline seizure frequency. Of the seven most significant, four have been linked to other neurologic diseases. Expression levels associated with high seizure frequency included low expression of Homeobox A10, Forkhead box A2, Lymphoblastic leukemia derived sequence 1, HGF activator, Kelch repeat and BTB (POZ) domain containing 11, Thanatos-associated protein domain containing 8 and Heparin sulfate (glucosamine) 3-O-sulfotransferase 3A1. CONCLUSIONS: This study describes novel associations between forty known genes and a clinical marker of epileptogenicity, baseline seizure frequency. Four of the seven discussed have been previously related to other neurologic diseases. Future investigation of these genes could establish new biomarkers for predicting epileptogenicity, and could have significant implications for diagnosis and management of temporal lobe epilepsy, as well as epilepsy pathogenesis.

Alisertib added to rituximab and vincristine is synthetic lethal and potentially curative in mice with aggressive DLBCL co-overexpressing MYC and BCL2.

Pearson correlation coefficient for expression analysis of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) demonstrated Aurora A and B are highly correlated with MYC in DLBCL and mantle cell lymphoma (MCL), while both Auroras correlate with BCL2 only in DLBCL. Auroras are up-regulated by MYC dysregulation with associated aneuploidy and resistance to microtubule targeted agents such as vincristine. Myc and Bcl2 are differentially expressed in U-2932, TMD-8, OCI-Ly10 and Granta-519, but only U-2932 cells over-express mutated p53. Alisertib [MLN8237 or M], a highly selective small molecule inhibitor of Aurora A kinase, was synergistic with vincristine [VCR] and rituximab [R] for inhibition of cell proliferation, abrogation of cell cycle checkpoints and enhanced apoptosis versus single agent or doublet therapy. A DLBCL (U-2932) mouse model showed tumor growth inhibition (TGI) of ∼ 10-20% (p = 0.001) for M, VCR and M-VCR respectively, while R alone showed ∼ 50% TGI (p = 0.001). M-R and VCR-R led to tumor regression [TR], but relapsed 10 days after discontinuing therapy. In contrast, M-VCR-R demonstrated TR with no relapse >40 days after stopping therapy with a Kaplan-Meier survival of 100%. Genes that are modulated by M-VCR-R (CENP-C, Auroras) play a role in centromere-kinetochore function in an attempt to maintain mitosis in the presence of synthetic lethality. Together, our data suggest that the interaction between alisertib plus VCR plus rituximab is synergistic and synthetic lethal in Myc and Bcl-2 co-expressing DLBCL. Alisertib plus vincristine plus rituximab [M-VCR-R] may represent a new strategy for DLBCL therapy.

Using logistic regression to improve the prognostic value of microarray gene expression data sets: application to early-stage squamous cell carcinoma of the lung and triple negative breast carcinoma.

BACKGROUND: Numerous microarray-based prognostic gene expression signatures of primary neoplasms have been published but often with little concurrence between studies, thus limiting their clinical utility. We describe a methodology using logistic regression, which circumvents limitations of conventional Kaplan Meier analysis. We applied this approach to a thrice-analyzed and published squamous cell carcinoma (SQCC) of the lung data set, with the objective of identifying gene expressions predictive of early death versus long survival in early-stage disease. A similar analysis was applied to a data set of triple negative breast carcinoma cases, which present similar clinical challenges. METHODS: Important to our approach is the selection of homogenous patient groups for comparison. In the lung study, we selected two groups (including only stages I and II), equal in size, of earliest deaths and longest survivors. Genes varying at least four-fold were tested by logistic regression for accuracy of prediction (area under a ROC plot). The gene list was refined by applying two sliding-window analyses and by validations using a leave-one-out approach and model building with validation subsets. In the breast study, a similar logistic regression analysis was used after selecting appropriate cases for comparison. RESULTS: A total of 8594 variable genes were tested for accuracy in predicting earliest deaths versus longest survivors in SQCC. After applying the two sliding window and the leave-one-out analyses, 24 prognostic genes were identified; most of them were B-cell related. When the same data set of stage I and II cases was analyzed using a conventional Kaplan Meier (KM) approach, we identified fewer immune-related genes among the most statistically significant hits; when stage III cases were included, most of the prognostic genes were missed. Interestingly, logistic regression analysis of the breast cancer data set identified many immune-related genes predictive of clinical outcome. CONCLUSIONS: Stratification of cases based on clinical data, careful selection of two groups for comparison, and the application of logistic regression analysis substantially improved predictive accuracy in comparison to conventional KM approaches. B cell-related genes dominated the list of prognostic genes in early stage SQCC of the lung and triple negative breast cancer.

Aurora A inhibitor (MLN8237) plus vincristine plus rituximab is synthetic lethal and a potential curative therapy in aggressive B-cell non-Hodgkin lymphoma.

PURPOSE: Aurora A and B are oncogenic serine/threonine kinases that regulate mitosis. Overexpression of Auroras promotes resistance to microtubule-targeted agents. We investigated mechanistic synergy by inhibiting the mitotic spindle apparatus in the presence of MLN8237 [M], an Aurora A inhibitor with either vincristine [MV] or docetaxel [MD] in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The addition of rituximab [R] to MV or MD was evaluated for synthetic lethality. EXPERIMENTAL DESIGN: Aggressive B-NHL cell subtypes were evaluated in vitro and in vivo for target modulation and anti-NHL activity with single agents, doublets, and triplets by analyzing cell proliferation, apoptosis, tumor growth, survival, and mechanisms of response/relapse by gene expression profiling with protein validation. RESULTS: MV is synergistic whereas MD is additive for cell proliferation inhibition in B-NHL cell culture models. Addition of rituximab to MV is superior to MD, but both significantly induce apoptosis compared with doublet therapy. Mouse xenograft models of mantle cell lymphoma showed modest single-agent activity for MLN8237, rituximab, docetaxel, and vincristine with tumor growth inhibition (TGI) of approximately 10% to 15%. Of the doublets, MV caused tumor regression, whereas TGI was observed with MD (approximately 55%-60%) and MR (approximately 25%-50%), respectively. Although MV caused tumor regression, mice relapsed 20 days after stopping therapy. In contrast, MVR was curative, whereas MDR led to TGI of approximately 85%. Proliferation cell nuclear antigen, Aurora B, cyclin B1, cyclin D1, and Bcl-2 proteins of harvested tumors confirmed response and resistance to therapy. CONCLUSIONS: Addition of rituximab to MV is a novel therapeutic strategy for aggressive B-NHL and warrants clinical trial evaluation.

Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors.

The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT-PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5-fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up- or down-regulated by at least 1.5-fold after spaceflight (P < or = 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT-PCR were as follows: Rbm3 (up-regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down-regulated). QRT-PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla-4, IFN-alpha2a (up-regulated) and CD44 (down-regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space.

Gene expression profiling in the fetal cardiac tissue after folate and low-dose trichloroethylene exposure.

BACKGROUND: Previous studies show gene expression alterations in rat embryo hearts and cell lines that correspond to the cardio-teratogenic effects of trichloroethylene (TCE) in animal models. One potential mechanism of TCE teratogenicity may be through altered regulation of calcium homeostatic genes with a corresponding inhibition of cardiac function. It has been suggested that TCE may interfere with the folic acid/methylation pathway in liver and kidney and alter gene regulation by epigenetic mechanisms. According to this hypothesis, folate supplementation in the maternal diet should counteract TCE effects on gene expression in the embryonic heart. APPROACH: To identify transcriptional targets altered in the embryonic heart after exposure to TCE, and possible protective effects of folate, we used DNA microarray technology to profile gene expression in embryonic mouse hearts with maternal TCE exposure and dietary changes in maternal folate. RESULTS: Exposure to low doses of TCE (10 ppb) caused extensive alterations in transcripts encoding proteins involved in transport, ion channel, transcription, differentiation, cytoskeleton, cell cycle, and apoptosis. Exogenous folate did not offset the effects of TCE exposure on normal gene expression, and both high and low levels of folate produced additional significant changes in gene expression. CONCLUSIONS: A mechanism by which TCE induces a folate deficiency does not explain altered gene expression patterns in the embryonic mouse heart. The data further suggest that use of folate supplementation, in the presence of this toxin, may be detrimental and not protective of the developing embryo.

Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil.

Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell-cell interaction.