Loss of heterozygosity (LOH) of the entire chromosome 10 is the most frequent genetic alteration in human glioblastoma (GBM). In addition to PTEN/MMAC1 on 10q23.3, clustering of partial deletion break-points on 10q25.3-26.1 points to a second suppressor locus. The proposed target gene DMBT1 was not confirmed. By somatic deletion mapping of this region, we identified the complementary DNA encoding the human homologue of rat orphan G protein-coupled receptor GPR26. GPR26 is highly expressed in fetal and adult brain, but frequently reduced or absent in glioma cells and biopsies, due to de novo methylation of its 5' CpG island. Silencing of GPR26 was reversed with 5-aza-deoxycytidine and the histone deacetylase inhibitor trichostatin A. Furthermore, overexpression of GPR26 in HEK and in U87 glioma cells increased intracellular cAMP concentration which is considered to induce astrocytic differentiation. Interestingly, we observed concomitant silencing of GPR26 with O6-methylguanine-DNA methyl transferase (MGMT), a DNA repair gene co-localized on 10q25.3-26.1 (p=0.0001). We conclude that epigenetic silencing is a common mechanism in malignant gliomas that simultaneously inactivates MGMT and GPR26. The 10q25.3-26.1 region may contain an important epigenetic pathway in brain tumorigenesis.

Background: The purpose of this study was to determine whether the application of high stimulation current thresholds (SCT) leads to a distant needle to nerve proximity (NNP) compared with low SCT during nerve localization for regional anaesthesia in pigs. Methods: A minimal motor response to the stimulation of femoral or brachial plexus nerves in 16 anaesthetized pigs was triggered either by a minimal SCT of a low (0.01-0.3 mA) or a high (0.8-1.0 mA) current in a random order. After eliciting a motor response with a predetermined SCT, synthetic resin was injected via the needle. After postmortem dissection of the injection site, the localization of the resin deposition was determined verifying the final position of the needle tip. Depending on the proximity of resin deposition to the nerve epineurium, the needle tip placement was considered either as a close or a distant NNP. Results: A total of 235 punctures were performed. Ninety-one punctures were carried out with low SCT and 92 with a high SCT. Fifty-two punctures served as a control (1.8-2.0 mA). All injectates following both high or low SCT were considered 'close needle tip to nerve placement', whereas 27 of 52 injectates of the control group appeared distant to nerve epineurium. Conclusion: Regardless of the applied SCT, i.e. high or low, all resin deposition was found adjacent to nerve epineurium. These findings suggest that high and low SCT result in equivalent needle tip localization in pigs.

This review highlights current information about the regulatory mechanisms that govern gene expression during mammalian hibernation, in particular the potential role of epigenetic controls in coordinating the global suppression of transcription. Hibernation is characterized by long periods of deep torpor (when core body temperature drops to near ambient) that are interspersed with brief arousal periods back to euthermia. Entry into torpor requires coordinated controls which strongly suppress and reprioritize all metabolic functions, including global controls on both transcription and translation. At the same time, however, selected hibernation-specific genes are up-regulated under the control of specific transcription factors to support the torpid state; this includes genes that encode proteins involved in lipid fuel catabolism and in long term cytoprotection (e.g. antioxidants, chaperones). We evaluate the currently available information on global transcriptional suppression in hibernation and propose that epigenetic mechanisms such as DNA methylation, histone modification, SUMOylation and the actions of sirtuins play crucial roles in transcriptional suppression during torpor. Global controls providing translational suppression also occur during hibernation including reversible phosphorylation control of ribosomal initiation and elongation factors as well as polysome dissociation. We also present initial data that mRNA transcripts are regulated via inhibitory interactions with microRNA species during torpor and provide the first evidence of differential expression of miRNAs in hibernators. When taken together, these mechanisms provide hibernators with multiple layers of regulatory controls that achieve both global repression of gene expression and selected enhancement of genes/proteins that achieve the hibernation phenotype.

In her 2006 book "My Stroke of Insight" Dr. Jill Bolte Taylor relates her experience of suffering from a left hemispheric stroke caused by a congenital arteriovenous malformation which led to a loss of inner speech. Her phenomenological account strongly suggests that this impairment produced a global self-awareness deficit as well as more specific dysfunctions related to corporeal awareness, sense of individuality, retrieval of autobiographical memories, and self-conscious emotions. These are examined in details and corroborated by numerous excerpts from Taylor's book.

The utilization of human embryonic stem cells (hESC) in regenerative medicine largely depends on the development of technologies that will allow efficient genetic manipulation of the cells in vitro. Although a few studies have described the transfection of hESC for generation of reporter lines stably expressing specific transgenes driven by different promoters, the optimal choice of promoter system for driving transgene in hESC has yet to be elucidated. We show for the first time that Chinese hamster elongation factor-1alpha (CHEF1) promoter robustly drove reporter gene expression higher than the human elongation factor 1alpha (hEF1alpha), other constitutive Chinese hamster promoters, human cytomegalovirus (CMV) immediate early enhancer/promoter and SV40 promoters in hESC by quantitative analysis. We also successfully generated stably transfected hESC lines using this CHEF1 promoter system and demonstrated that they continued to express enhanced green fluorescent protein (EGFP) during prolonged undifferentiated proliferation, in differentiated embryoid bodies (EBs), and in teratomas without transgene silencing. By immunofluorescence staining and D ow cytometry analysis, the pluripotent markers, OCT-4, SSEA-4, and TRA-1-60, continued to be expressed in undifferentiated CHEF1-EGFP expressing hESC lines. When the stably transfected hESC were directed to differentiate into neural precursors in vitro, high-level EGFP expression was maintained and co-expression of neural markers, Nestin, and beta-tubulin III was observed. The morphology, karyotype, and telomerase activity of CHEF1-EGFP expressing hESC were normal after >50 continuous passages, and the cells retained the ability to differentiate into derivatives of the three germ layers in vitro as confirmed by RT-PCR analysis and immunocytochemical staining and in vivo teratoma formation. Therefore, stable CHEF1-EGFP hESC lines retained the capability for self-renewal and pluripotency. The novel CHEF1 promoter system described here enables high-level transgene expression in the stably transfected hESC. It may have signi, cant implication for uses in bioprocess development and future development of gene-modified hESC in tissue regeneration and transplantation applications.

Mammalian hibernation includes long periods of profound torpor where the rates of all metabolic processes are strongly suppressed in a reversible manner. We hypothesized that microRNAs (miRNAs), small non-coding transcripts that bind to mRNA, could play a role in the global suppression of mRNA translation when animals enter torpor. Selected miRNA species (4-9 of the following: mir-1, mir-24, mir-15a, mir-16, mir-21, mir-122a, mir-143, mir-146 and mir-206) were evaluated in four organs of euthermic versus hibernating ground squirrels, Spermophilus tridecemlineatus using RT-PCR. Levels of mir-24 transcripts were significantly reduced in the heart and skeletal muscle of torpid animals as were mir-122a levels in the muscle. Mir-1 and mir-21 both increased significantly in the kidney during torpor by 2.0- and 1.3-fold, respectively. No changes were found for the four miRNA species analyzed in the liver. Protein levels of Dicer, an enzyme involved in miRNA processing were also quantified in the heart, kidney and liver. Dicer protein levels increased by 2.7-fold in the heart during hibernation but decreased by 60% in the kidney. These data are the first report that differential regulation of miRNA levels occurs during mammalian hibernation and they provide a mechanism for reversible gene silencing during torpor that can be rapidly reversed to allow renewed translation of mRNA when animals arouse back to euthermia.