A novel Rac1-GSPT1 signaling pathway controls astrogliosis following central nervous system injury

Astrogliosis (i.e. glial scar), which is comprised primarily of proliferated astrocytes at the lesion site and migrated astrocytes from neighboring regions, is one of the key reactions in determining outcomes after CNS injury. In an effort to identify potential molecules/pathways that regulate astrogliosis, we sought to determine whether Rac/Rac-mediated signaling in astrocytes represents a novel candidate for therapeutic intervention following CNS injury. For these studies, we generated mice with Rac1 deletion under the control of the GFAP (glial fibrillary acidic protein) promoter (GFAP-Cre;Rac1(flox/flox)). GFAP-Cre;Rac1(flox/flox) (Rac1-KO) mice exhibited better recovery after spinal cord injury and exhibited reduced astrogliosis at the lesion site relative to control. Reduced astrogliosis was also observed in Rac1-KO mice following microbeam irradiation-induced injury. Moreover, knockdown (KD) or KO of Rac1 in astrocytes (LN229 cells, primary astrocytes, or primary astrocytes from Rac1-KO mice) led to delayed cell cycle progression and reduced cell migration. Rac1-KD or Rac1-KO astrocytes additionally had decreased levels of GSPT1 (G(1) to S phase transition 1) expression and reduced responses of IL-1β and GSPT1 to LPS treatment, indicating that IL-1β and GSPT1 are downstream molecules of Rac1 associated with inflammatory condition. Furthermore, GSPT1-KD astrocytes had cell cycle delay, with no effect on cell migration. The cell cycle delay induced by Rac1-KD was rescued by overexpression of GSPT1. Based on these results, we propose that Rac1-GSPT1 represents a novel signaling axis in astrocytes that accelerates proliferation in response to inflammation, which is one important factor in the development of astrogliosis/glial scar following CNS injury

Systematic analysis of mitochondrial genes associated with hearing loss in the Japanese population: dHPLC reveals a new candidate mutation

<p>Abstract</p> <p>Background</p> <p>Variants of mitochondrial DNA (mtDNA) have been evaluated for their association with hearing loss. Although ethnic background affects the spectrum of mtDNA variants, systematic mutational analysis of mtDNA in Japanese patients with hearing loss has not been reported.</p> <p>Methods</p> <p>Using denaturing high-performance liquid chromatography combined with direct sequencing and cloning-sequencing, Japanese patients with prelingual (N = 54) or postlingual (N = 80) sensorineural hearing loss not having pathogenic mutations of m.1555A > G and m.3243A > G nor <it>GJB2 </it>were subjected to mutational analysis of mtDNA genes (<it>12S rRNA</it>, <it>tRNA</it>^{<it>Leu(UUR)</it>}, <it>tRNA</it>^{<it>Ser(UCN)</it>}, <it>tRNA</it>^<it>Lys</it>, <it>tRNA</it>^<it>His</it>, <it>tRNA</it>^{<it>Ser(AGY)</it>}, and <it>tRNA</it>^<it>Glu</it>).</p> <p>Results</p> <p>We discovered 15 variants in <it>12S rRNA </it>and one homoplasmic m.7501A > G variant in <it>tRNA</it>^{<it>Ser(UCN)</it>}; no variants were detected in the other genes. Two criteria, namely the low frequency in the controls and the high conservation among animals, selected the m.904C > T and the m.1105T > C variants in <it>12S rRNA </it>as candidate pathogenic mutations. Alterations in the secondary structures of the two variant transcripts as well as that of m.7501A > G in <it>tRNA</it>^{<it>Ser(UCN) </it>}were predicted.</p> <p>Conclusions</p> <p>The m.904C > T variant was found to be a new candidate mutation associated with hearing loss. The m.1105T > C variant is unlikely to be pathogenic. The pathogenicity of the homoplasmic m.7501T > A variant awaits further study.</p

A Comparative Study on ASIC, FPGAs, GPUs and General Purpose Processors in the O(N^2) Gravitational N-body Simulation

In this paper, we describe the implementation of gravitational force calculation for N-body simulations in the context of Astrophysics. It will describe high performance implementations on general purpose processors, GPUs, and FPGAs, and compare them using a number of criteria including speed performance, power efficiency and cost of development. These results show that, for gravitational force calculation and many-body simulations in general, GPUs are very competitive in terms of performance and performance per dollar figures, whereas FPGAs are competitive in terms of performance per Watt figures.2009 NASA/ESA Conference on Adaptive Hardware and Systems (AHS) : San Francisco, CA, USA, 2009.07.29-2009.08.

Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing <i>PDGFRA</i> Variant in Cultured Human Glioblastoma Cell Lines

Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions

Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis.

In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 103 or 2 × 104 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 103 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine

Protective Effects of Peroxiredoxin 4 (PRDX4) on Cholestatic Liver Injury

Accumulating evidence indicates that oxidative stress plays a critical role in initiating the progression of inflammatory and fibrotic liver diseases, including cholestatic hepatitis. Peroxiredoxin 4 (PRDX4) is a secretory antioxidase that protects against oxidative damage by scavenging reactive oxygen species (ROS) in both the intracellular compartments and extracellular space. In this study, we examined the in vivo net effects of PRDX4 overexpression in a murine model of cholestasis. To induce cholestatic liver injury, we subjected C57BL/6J wild-type (WT) or human PRDX4 (hPRDX4) transgenic (Tg) mice to sham or bile duct ligation (BDL) surgery for seven days. Our results showed that the liver necrosis area was significantly suppressed in Tg BDL mice with a reduction in the severity of liver injuries. Furthermore, PRDX4 overexpression markedly reduced local and systemic oxidative stress generated by BDL. In addition, suppression of inflammatory cell infiltration, reduced proliferation of hepatocytes and intrahepatic bile ducts, and less fibrosis were also found in the liver of Tg BDL mice, along with a reduced mortality rate after BDL surgery. Interestingly, the composition of the hepatic bile acids (BAs) was more beneficial for Tg BDL mice than for WT BDL mice, suggesting that PRDX4 overexpression may affect BA metabolism during cholestasis. These features indicate that PRDX4 plays an important role in protecting against liver injury following BDL and might be a promising therapeutic modality for cholestatic diseases

Distribution and favorable prognostic implication of genomic EGFR alterations in IDH‐wildtype glioblastoma

Abstract Background We aimed to evaluate the mutation profile, transcriptional variants, and prognostic impact of the epidermal growth factor receptor (EGFR) gene in isocitrate dehydrogenase (IDH)‐wildtype glioblastomas (GBMs). Methods We sequenced EGFR, evaluated the EGFR splicing profile using a next‐generation sequencing oncopanel, and analyzed the outcomes in 138 grade IV IDH‐wildtype GBM cases. Results EGFR mutations were observed in 10% of GBMs. A total of 23.9% of the GBMs showed EGFR amplification. Moreover, 25% of the EGFR mutations occurred in the kinase domain. Notably, EGFR alterations were a predictor of good prognosis (p = 0.035). GBM with EGFR alterations was associated with higher Karnofsky Performance Scale scores (p = 0.014) and lower Ki‐67 scores (p = 0.005) than GBM without EGFR alterations. EGFRvIII positivity was detected in 21% of EGFR‐amplified GBMs. We identified two other EGFR variants in GBM cases with deletions of exons 6–7 (Δe 6–7) and exons 2–14 (Δe 2–14). In one case, the initial EGFRvIII mutation transformed into an EGFR Δe 2–14 mutation during recurrence. Conclusions We found that the EGFR gene profiles of GBM differ among cohorts and that EGFR alterations are good prognostic markers of overall survival in patients with IDH‐wildtype GBM. Additionally, we identified rare EGFR variants with longitudinal and temporal transformations of EGFRvIII