Frequent Occurrence of Mitochondrial DNA Mutations in Barrett’s Metaplasia without the Presence of Dysplasia

BACKGROUND: Barrett's esophagus (BE) is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA). The numerous molecular events may play a role in the neoplastic transformation of Barrett's mucosa such as the change of DNA ploidy, p53 mutation and alteration of adhesion molecules. However, the molecular mechanism of the progression of BE to EA remains unclear and most studies of mitochondrial DNA (mtDNA) mutations in BE have performed on BE with the presence of dysplasia. METHODS/FINDINGS: Thus, the current study is to investigate new molecular events (Barrett's esophageal tissue-specific-mtDNA alterations/instabilities) in mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissue (NT) and tissue (BT) from 34 patients having Barrett's metaplasia without the presence of dysplasia. Eighteen patients (53%) exhibited mtDNA mutations which were not found in adjacent NT. mtDNA copy number was about 3 times higher in BT than in adjacent NT. The activity of the mitochondrial respiratory chain enzyme complexes in tissues from Barrett's metaplasia without the presence of dysplasia was impaired. Reactive oxygen species (ROS) level in BT was significantly higher than those in corresponding samples. CONCLUSION/SIGNIFICANCE: High ROS level in BT may contribute to the development of mtDNA mutations, which may play a crucial role in disease progression and tumorigenesis in BE

Effects of Reynolds Number and Surface Roughness Magnitude and Location on Compressor Cascade Performance

The article of record as published may be located at http://dx.doi.org/10.1115/1.4003821An experimental investigation has been conducted to characterize the influence of Reynolds number and surface roughness magnitude and location on compressor cascade performance. Flow field surveys have been conducted in a low-speed, linear compressor cascade. Pressure, velocity, and loss have been measured via a five-hole probe, pitot probe, and pressure taps on the blades. Four different roughness magnitudes, Ra values of 0.38 lm (polished), 1.70 lm (baseline), 2.03 lm (rough 1), and 2.89 lm (rough 2), have been tested. Furthermore, various roughness locations have been examined. In addition to the as manufactured (baseline) and entirely rough blade cases, blades with roughness covering the leading edge, pressure side, and 5%, 20%, 35%, 50%, and 100% of suction side from the leading edge have been studied. All of the tests have been carried out for Reynolds numbers ranging from 300,000 to 640,000. For Reynolds numbers under 500,000, the tested roughnesses do not significantly degrade compressor blade loading or loss. However, loss and blade loading become sensitive to roughness at Reynolds numbers above 550,000. Cascade performance is more sensitive to roughness on the suction side than pressure side. Furthermore, roughness on the aft 2=3 of suction side surface has a greater influence on loss. For a given roughness location, there exists a Reynolds number at which loss begins to significantly increase. Finally, increasing the roughness area on the suction surface from the leading edge reduces the Reynolds number at which the loss begins to increase.The authors gratefully acknowledge financial support from the BK21 Program of the Korean Government, the Institute of Advanced Machinery and Design of Seoul National University, the Korea Institute of Energy Technology Evaluation and Planning, and the Naval Postgraduate School. Lastly, the authors thank William B. Roberts for his helpful comments and insight

Adipose Tissue-Derived Stem Cells Alleviate Cold Allodynia in a Rat Spinal Nerve Ligation Model of Neuropathic Pain

Neuropathic pain caused by lesions or nervous system dysfunction is a neuroimmune disease with limited therapeutic options. Adipose tissue-derived stem cells (ASCs) are multipotent mesenchymal stem cells with potent immunosuppressive properties, and their use as novel cell-based therapeutics have been proposed in many immune diseases. However, the analgesic effect and efficacy of ASCs to treat neuropathic pain remain unclear. This study, thus, investigated whether ASCs or ASC-derived culture medium can relieve neuropathic pain behaviors (i.e., mechanical and cold allodynia) in a rat model with L5 spinal nerve ligation. Intrathecal injection of ASCs significantly reduced cold allodynia, but not mechanical allodynia. Importantly, cold allodynia was completely reversed in rats with repeated injections of ASCs. In contrast, intrathecal injection of ASC-derived culture medium or retro-orbital injection of ASCs had no effect on neuropathic pain behaviors. These results suggest a novel and alternative therapeutic application of ASCs to target specific neuropathic pain behaviors

Orphan nuclear receptor Errγ induces C-reactive protein gene expression through induction of ER-bound Bzip transmembrane transcription factor CREBH.

The orphan nuclear receptor estrogen-related receptor-γ (ERRγ) is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER) stress response. cAMP responsive element-binding protein H (CREBH) is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERRγ directly regulates CREBH gene expression in response to ER stress. ERRγ bound to the ERRγ response element (ERRE) in the CREBH promoter. Overexpression of ERRγ by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP), whereas either knockdown of ERRγ or inhibition of ERRγ by ERRγ specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1α was required for ERRγ mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP) assay showing binding of both ERRγ and PGC1α on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERRγ and PGC1α binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERRγ directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions

ERRγ induces CREBH gene.

<p>(A,B), AML12 cells were infected with Ad-GFP or Ad-ERRγ for 24 hr. (A),Total RNA was isolated for qRT-PCR analyses to quantify CREBH mRNA levels using CREBH primers. (B), Western blot analyses shows CREBH full length (CREBH-F), and CREBH active form (CREBH-N) expression. (C), AML12 cells were infected with Ad-GFP or Ad-ERRα for 24 hr. Western blot analyses shows CREBH-N expression. (D,E), Ad-GFP or Ad-ERRγ were injected via tail vein into male C57BL/6J mice (n = 5 per group). Following completion of the experiments, mice were sacrificed, and liver tissues were obtained for (D), qRT-PCR analyses to quantify CREBH mRNA levels, and (E), western blot analyses of CREBH-N expression. (F,G), HepG2 cells were infected with Ad-GFP or Ad-ERRγ for 24 hr. (F),Total RNA was isolated for qRT-PCR analyses to quantify CREBH mRNA levels using CREBH primers. (G), Western blot analyses shows CREBH-N expression. Data are representative of three independently performed experiments and shown as mean±SD; *, P<0.05 using Student’s t-test.</p

ERRγ inverse agonist inhibits CREBH and CRP <i>in vivo</i>.

<p>(A), GSK5182 was administered prior to (40 mg/kg, p.o. in 30% PEG400/DW) and at 30 minutes after Tm (1 mg/kg, i.p. in 1% DMSO/DW) was administered into C57BL/6J mice (n = 5 per group). Following completion of the experiments, mice were sacrificed, and liver tissues were obtained for western blot analyses of CRP, CREBH-N, and ERRγ. (B), GSK5182 was administered (40 mg/kg, p.o. in 30% PEG400/DW) into C57BL/6J mice (n = 5 per group) or EtOH was administered into C57BL/6J mice (n = 5 per group) or first GSK5182 was administered and then EtOH was administered (n = 5 per group). After completion of the treatment, mice were sacrificed. Western blot analyses shows CRP, CREBH-N and ERRγ expression in liver tissue. (C), GSK5182 was administered (40 mg/kg, p.o. in 30% PEG400/DW) into db/db mice (n = 5 per group) for 30 days. After completion of the treatment, mice were sacrificed. Western blot analyses shows CRP, CREBH-N and ERRγ expression in liver tissue. (D), Schematic representation of the proposed model in which ER stress mediated induction of CREBH is mediated through ERRγ.</p

ERRγ regulates activation of CREBH gene promoter.

<p>(A), PGC1α-dependent activation of the mouse CREBH promoter by ERRγ. Transient transfection was performed in 293T cells with the indicated plasmid DNAs. (B), Deletion constructs of the CREBH promoter demonstrate the ERRγ binding site in 293T cells. Transient transfection was performed in 293T cells with the indicated plasmid DNAs. (C), ERRE-dependent activation of the CREBH promoter in 293T cells. 293T cells were transfected with the wild-type or ERRE-mutant CREBH promoter along with ERRγ plasmid DNAs. (D), ChIP assay shows the binding of ERRγ and PGC1α to the endogenous CREBH promoter by semiquantitative PCR. AML12 cells were treated with DMSO or Tm (5µg/mL) for 12 hr. After completion of the treatment, chromatin fragments were prepared and immunoprecipitated with ERRγ, PGC1α, or IgG control antibodies. DNA fragments covering –766 to –642 and –195 to –87 elements on the CREBH promoter were PCR amplified. 10% of the soluble chromatin was used as input. (E), ChIP assay for detection of histone acetylation at the ERRγ/PGC1α binding site under the indicated conditions in AML12 cells. Chromatin fragments were prepared and immunoprecipitated with Acetyl-Histone 3 and Acetyl-Histone 4 antibodies. DNA fragments covering –195 to –87 element on the CREBH promoter were qPCR-amplified as described in the ‘Materials and Methods’ section. Data are representative of three independently performed experiments and shown as mean±SD; *P<0.05, **P<0.005, and ***, p<0.0005 using Student’s t-test.</p