In the testicular cancer cell line, NT2, we previously demonstrated that differentially methylated regions were located in introns or intergenic regions, and postulated these might regulate non-coding RNAs. Three micro-RNAs and three small nucleolar RNAs were differentially methylated; one, miR-199a, was associated with the progression and prognosis of gastric and ovarian cancers. In this report we document, by epigenomic profiling of testicular tissue, that miR-199a is transcribed as antisense of dynamin 3 (chromosome 1q24.3), and hypermethylation of this region is correlated with miR-199a-5p/3p repression and tumor malignancy. Re-expression of miR-199a in testicular cancer cells led to suppression of cell growth, cancer migration, invasion and metastasis. The miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with tumor malignancy. We further identified the embryonal carcinoma antigen podocalyxin-like protein 1 (PODXL), an anti-adhesive protein expressed in aggressive tumors, as a target of miR-199a-5p. We demonstrated PODXL is overexpressed in malignant testicular tumor, and cellular depletion of PODXL resulted in suppression of cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests PODXL is a downstream effector mediating the action of miR199a-5p. This report identifies DNA methylation, miR-199a dysregulation and PODXL as critical factors in tumor malignancy.

Circadian rhythms are fundamental biological phenomena generated by molecular genetic mechanisms known as circadian clocks. There is increasing evidence that circadian synchronization of physiological and cellular processes contribute to the wellness of organisms, curbing pathologies such as cancer and premature aging. Therefore, there is a need to understand how circadian clocks orchestrate interactions between the organism's internal processes and the environment. Here, we explore the nexus between the clock and oxidative stress susceptibility in Drosophila melanogaster. We exposed flies to acute oxidative stress induced by hydrogen peroxide (H2O2), and determined that mortality rates were dependent on time at which exposure occurred during the day /night cycle. The daily susceptibility rhythm was abolished in flies with a null mutation in the core clock gene period (per) abrogating clock function. Furthermore, lack of per increased susceptibility to H2O2 compared to wild-type flies, coinciding with enhanced generation of mitochondrial H2O2 and decreased catalase activity due to oxidative damage. Taken together, our data suggest that the circadian clock gene period is essential for maintaining a robust anti-oxidative defense.

BACKGROUND: Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples. METHODS: In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs). RESULTS: We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system. CONCLUSION: This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.

Warfarin, an oral vitamin K antagonist, is used to prevent arterial and venous thromboembolism in patients suffering from a multitude of diseases. In 2004, 31 million warfarin prescriptions were dispensed in the United States. Warfarin inhibits the activation of the vitamin K–dependent clotting factors (Factors II, VII, IX, and X) and regulatory proteins (proteins C, S, and Z). It is one of the leading drugs implicated in emergency room visits for adverse drug reactions. Annually the frequency of bleeding complications associated with overanticoagulation is 15% to 20%, with fatal bleeds measuring as high as 1% to 3%. The most effective method of warfarin reversal involves the use of Four Factor Prothrombin Complex Concentrate (PCC), which is widely used throughout Europe but is unavailable in the United States. The current therapies available to emergency room physicians in the United States are fresh frozen plasma, recombinant Factor VIIa (rFVIIa), Factor Eight Inhibitory Bypassing Activity, or Three Factor PCC concomitantly administered with vitamin K. We review the advantages and disadvantages of these therapies and recommend Three Factor PCC with small doses of rFVIIa and with vitamin K in life-threatening situations if Four Factor PCC is unavailable.

Currently available prosthetic hands are capable of actuating anywhere from five to 30 degrees of freedom (DOF). However, grasp control of these devices remains unintuitive and cumbersome. To address this issue, we propose directly extracting finger commands from the neuromuscular system via electrodes implanted in residual innervated muscles and regenerative peripheral nerve interfaces (RPNIs). Two persons with transradial amputations had RPNIs created by suturing autologous free muscle grafts to their transected median, ulnar, and dorsal radial sensory nerves. Bipolar electrodes were surgically implanted into their ulnar and median RPNIs and into their residual innervated muscles. The implanted electrodes recorded local electromyography (EMG) with Signal-to-Noise Ratios ranging from 23 to 350 measured across various movements. In a series of single-day experiments, participants used a high speed pattern recognition system to control a virtual prosthetic hand in real-time. Both participants were able to transition between 10 pseudo-randomly cued individual finger and wrist postures in the virtual environment with an average online accuracy of 86.5% and latency of 255 ms. When the set was reduced to five grasp postures, average metrics improved to 97.9% online accuracy and 135 ms latency. Virtual task performance remained stable across untrained static arm positions while supporting the weight of the prosthesis. Participants also used the high speed classifier to switch between robotic prosthetic grips and complete a functional performance assessment. These results demonstrate that pattern recognition systems can use the high-quality EMG afforded by intramuscular electrodes and RPNIs to provide users with fast and accurate grasp control.

We present an analysis of a chromosomal walk in the region of the euchromatin-heterochromatin transition at the base of the X chromosome of Drosophila melanogaster. This region is difficult to analyse because of the presence of repeated sequences, and we have used cosmids to walk from the last euchromatic gene, suppressor of forked, towards the pericentric heterochromatin. The proximal 30-kb sequence we have isolated consists of repetitive DNA, including four tandem copies of a 5.9-kb sequence. This tandem repeat is itself a mosaic of other, mostly repeated, sequences, including part of a retrotransposon without long terminal repeats, a simple-sequence region of TAA repeats and part of a retrotransposon with long terminal repeats that has not been previously described. Although sequences homologous to these components are found elsewhere in the genome, this arrangement of repeated sequences is only found at the base of the X chromosome. It is conserved in D. melanogaster strains of different geographic origin, but is not conserved in even closely related species.

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