Description: Background— Recent studies suggest that the oxygen-sensing pathway consisting of transcription factor hypoxia-inducible factor and prolyl hydroxylase domain proteins (PHDs) plays a critical role in glucose metabolism. However, the role of adipocyte PHD in the development of obesity has not been clarified. We examined whether deletion of PHD2 , the main oxygen sensor, in adipocytes affects diet-induced obesity and associated metabolic abnormalities. Methods and Results— To delete PHD2 in adipocyte, PHD2 -floxed mice were crossed with aP2-Cre transgenic mice ( Phd2 f/f /aP2-Cre) . Phd2 f/f /aP2-Cre mice were resistant to high-fat diet–induced obesity (36.7±1.7 versus 44.3±2.0 g in control; P 〈0.01) and showed better glucose tolerance and homeostasis model assessment–insulin resistance index than control mice (3.6±1.0 versus 11.1±2.1; P 〈0.01). The weight of white adipose tissue was lighter (epididymal fat, 758±35 versus 1208±507 mg in control; P 〈0.01) with a reduction in adipocyte size. Macrophage infiltration into white adipose tissue was also alleviated in Phd2 f/f /aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2 f/f /aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2 f/f /aP2-Cre mice. Knockdown of PHD2 in 3T3L1 adipocytes induced a decrease in the glucose level and an increase in the lactate level in the supernatant with upregulation of glycolytic enzymes and reduced lipid accumulation. Conclusions— PHD2 in adipose tissue plays a critical role in the development of diet-induced obesity and glucose intolerance. PHD2 might be a novel target molecule for the treatment of obesity and associated metabolic abnormalities.

Description: Rationale: Both fusion and fission contribute to mitochondrial quality control. How unopposed fusion affects survival of cardiomyocytes and left ventricular function in the heart is poorly understood. Objective: We investigated the role of dynamin-related protein 1 (Drp1), a GTPase that mediates mitochondrial fission, in mediating mitochondrial autophagy, ventricular function, and stress resistance in the heart. Methods and Results: Drp1 downregulation induced mitochondrial elongation, accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes at baseline. Drp1 downregulation also suppressed autophagosome formation and autophagic flux at baseline and in response to glucose deprivation in cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy. Mitochondrial elongation and accumulation of damaged mitochondria were also observed in tamoxifen-inducible cardiac-specific Drp1 knockout mice. After Drp1 downregulation, cardiac-specific Drp1 knockout mice developed left ventricular dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks. Autophagic flux is significantly suppressed in cardiac-specific Drp1 knockout mice. Although left ventricular function in cardiac-specific Drp1 heterozygous knockout mice was normal at 12 weeks of age, left ventricular function decreased more severely after 48 hours of fasting, and the infarct size/area at risk after ischemia/reperfusion was significantly greater in cardiac-specific Drp1 heterozygous knockout than in control mice. Conclusions: Disruption of Drp1 induces mitochondrial elongation, inhibits mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting cardiac dysfunction and increased susceptibility to ischemia/reperfusion.

Description: Background Oral mucositis is one of the most common side-effects of 5-fluorouracil (5-FU)-based chemotherapy. The objective of this study was to evaluate the effects of irsogladine maleate (IM) on fluorouracil-induced oral mucositis through a double-blind, placebo controlled trial. Patients and methods Patients ( N = 66) were randomly assigned to receive either placebo or IM (4 mg/day for 14 consecutive days). The incidence and maximum severity of fluorouracil-induced oral mucositis and safety of the irsogladine dosing regimen were evaluated. Results A cohort of 33 patients received placebo and 33 patients received IM. The incidence of oral mucositis was significantly lower for IM than for placebo (27% versus 73%; P 〈 0.001 by chi-square test). Specific adverse events considered related to IM were not found. Conclusion IM significantly reduced the incidence and maximum severity of oral mucositis in patients treated with 5-FU-chemotherapy.

Description: Although transforming growth factor (TGF)-β1 is a well-known immunosuppressive cytokine, little is known about the role of its downstream transcription factors, Smad2 and Smad3, in the suppression of macrophage activation. Previous studies have demonstrated that Smad3 is critical for the suppression of LPS-mediated inducible nitric oxide (NO) synthase (iNOS) induction, although the role of Smad2 remains to be investigated. In this study, we found that iNOS induction was enhanced in Smad2-deficient bone marrow-derived macrophages (BMDMs) and peritoneal macrophages in vitro and tumor-associated macrophages in vivo , compared with wild-type (WT) macrophages. However, TGF-β1 still suppressed iNOS induction in Smad2-deficient macrophages. In Smad2/3 double knockout (KO) (Smad2/3 DKO) BMDMs, LPS-mediated NO/iNOS induction was more strongly elevated than in Smad2 or Smad3 single KO BMDMs, and its suppression by exogenous TGF-β1 was severely impaired. These data suggest that Smad2 and Smad3 redundantly regulate iNOS induction. Similarly, the production of IL-6 and TNFα, but not IL-10 was augmented in Smad2/3 DKO BMDMs, suggesting that Smad2 and Smad3 also redundantly suppressed some cytokines production. In Smad2/3 DKO macrophages, TLR3- as well as TLR4-mediated IRF3 activation and IFN-β production were strongly augmented, which resulted in hyper STAT1 phosphorylation. Furthermore, IFN-β- and IFN--induced iNOS induction in the absence of TLR signaling and STAT1 transcriptional activity were augmented in Smad2/3 DKO BMDMs. These results suggest that Smad2 and Smad3 negatively regulate iNOS induction in macrophages by suppressing multiple steps in the IRF3-IFN-β-STAT1 pathway.

Description: p32 is an evolutionarily conserved and ubiquitously expressed multifunctional protein. Although p32 exists at diverse intra and extracellular sites, it is predominantly localized to the mitochondrial matrix near the nucleoid associated with mitochondrial transcription factor A. Nonetheless, its function in the matrix is poorly understood. Here, we determined p32 function via generation of p32-knockout mice. p32-deficient mice exhibited mid-gestation lethality associated with a severe developmental defect of the embryo. Primary embryonic fibroblasts isolated from p32-knockout embryos showed severe dysfunction of the mitochondrial respiratory chain, because of severely impaired mitochondrial protein synthesis. Recombinant p32 binds RNA, not DNA, and endogenous p32 interacts with all mitochondrial messenger RNA species in vivo . The RNA-binding ability of p32 is well correlated with the mitochondrial translation. Co-immunoprecipitation revealed the close association of p32 with the mitoribosome. We propose that p32 is required for functional mitoribosome formation to synthesize proteins within mitochondria.

Description: Objective— BubR1, a cell cycle–related protein, is an essential component of the spindle checkpoint that regulates cell division. Mice with BubR1 expression reduced to 10% of the normal level display a phenotype characterized by progeria; however, the involvement of BubR1 in vascular diseases is still unknown. We generated mice in which BubR1 expression was reduced to 20% ( BubR1 L/L mice) of that in wild-type mice ( BubR1 +/+ ) to investigate the effects of BubR1 on arterial intimal hyperplasia. Approach and Results— Ten-week-old male BubR1 L/L and age-matched wild-type littermates ( BubR1 +/+ ) were used in this study. The left common carotid artery was ligated, and histopathologic examinations were conducted 4 weeks later. Bone marrow transplantation was also performed. Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aorta to examine cell proliferation, migration, and cell cycle progression. Severe neointimal hyperplasia was observed after artery ligation in BubR1 +/+ mice, whereas BubR1 L/L mice displayed nearly complete inhibition of neointimal hyperplasia. Bone marrow transplantation from all donors did not affect the reconstitution of 3 hematopoietic lineages, and neointimal hyperplasia was still suppressed after bone marrow transplantation from BubR1 +/+ mice to BubR1 L/L mice. VSMC proliferation was impaired in BubR1 L/L mice because of delayed entry into the S phase. VSMC migration was unaffected in these BubR1 L/L mice. p38 mitogen–activated protein kinase–inhibited VSMCs showed low expression of BubR1, and BubR1-inhibited VSMCs showed low expression of p38. Conclusions— BubR1 may represent a new target molecule for treating pathological states of vascular remodeling, such as restenosis after angioplasty.

Description: High-affinity memory B cells are preferentially selected during secondary responses and rapidly differentiate into antibody-producing cells. However, it remains unknown whether only high-affinity, mutated memory B cells simply expand to dominate the secondary response or if in fact memory B cells with a diverse V H repertoire, including those with no mutations, accumulate somatic mutations to create a new repertoire through the process of affinity maturation. In this report, we took a new approach to address this question by analyzing the V H gene repertoire of IgG1 + memory B cells before and after antigen re-exposure in a host unable to generate IgG + B cells. We show here that both mutated and unmutated IgG1 + memory B cells respond to secondary challenge and expand while accumulating somatic mutations in their V H genes in a stepwise manner. Both types of memory cells subsequently established a V H gene repertoire dominated by two major clonotypes, which are distinct from the original repertoire before antigen re-exposure. In addition, heavily mutated memory B cells were excluded from the secondary repertoire. Thus, both mutated and unmutated IgG1 + memory cells equally contribute to establish a new antibody repertoire through a dynamic process of mutation and selection, becoming optimally adapted to the recall challenge.

Description: Background— Mitochondrial autophagy is an important mediator of mitochondrial quality control in cardiomyocytes. The occurrence of mitochondrial autophagy and its significance during cardiac hypertrophy are not well understood. Methods and Results— Mice were subjected to transverse aortic constriction (TAC) and observed at multiple time points up to 30 days. Cardiac hypertrophy developed after 5 days, the ejection fraction was reduced after 14 days, and heart failure was observed 30 days after TAC. General autophagy was upregulated between 1 and 12 hours after TAC but was downregulated below physiological levels 5 days after TAC. Mitochondrial autophagy, evaluated by electron microscopy, mitochondrial content, and Keima with mitochondrial localization signal, was transiently activated at 3 to 7 days post-TAC, coinciding with mitochondrial translocation of Drp1. However, it was downregulated thereafter, followed by mitochondrial dysfunction. Haploinsufficiency of Drp1 abolished mitochondrial autophagy and exacerbated the development of both mitochondrial dysfunction and heart failure after TAC. Injection of Tat-Beclin 1, a potent inducer of autophagy, but not control peptide, on day 7 after TAC, partially rescued mitochondrial autophagy and attenuated mitochondrial dysfunction and heart failure induced by overload. Haploinsufficiency of either drp1 or beclin 1 prevented the rescue by Tat-Beclin 1, suggesting that its effect is mediated in part through autophagy, including mitochondrial autophagy. Conclusions— Mitochondrial autophagy is transiently activated and then downregulated in the mouse heart in response to pressure overload. Downregulation of mitochondrial autophagy plays an important role in mediating the development of mitochondrial dysfunction and heart failure, whereas restoration of mitochondrial autophagy attenuates dysfunction in the heart during pressure overload.

Description: In order to reveal the origin of the ultrafast outflows (UFOs) that are frequently observed in active galactic nuclei (AGNs), we perform two-dimensional radiation hydrodynamics simulations of the line-driven disc winds, which are accelerated by the radiation force due to the spectral lines. The line-driven winds are successfully launched for the range of M BH = 10 6–9 M and = 0.1–0.5, and the resulting mass outflow rate ( $\dot{M_{\rm w}}$ ), momentum flux ( $\dot{p_{\rm w}}$ ), and kinetic luminosity ( $\dot{E_{\rm w}}$ ) are in the region containing 90 per cent of the posterior probability distribution in the $\dot{M}_{\rm w}$ – L bol plane, $\dot{p}_{\rm w}$ – L bol plane, and $\dot{E}_{\rm w}$ – L bol plane shown in Gofford et al., where M BH is the black hole mass, is the Eddington ratio, and L bol is the bolometric luminosity. The best-fitting relations in Gofford et al., ${\rm d}\log \dot{M_{\rm w}}/{\rm d}\log {L_{\rm bol}}\sim 0.9$ , ${\rm d}\log \dot{p_{\rm w}}/{\rm d}\log {L_{\rm bol}}\sim 1.2$ , and ${\rm d}\log \dot{E_{\rm w}}/{\rm d}\log {L_{\rm bol}}\sim 1.5$ , are roughly consistent with our results, ${\rm d}\log \dot{M_{\rm w}}/{\rm d}\log {L_{\rm bol}}\sim 9/8$ , ${\rm d}\log \dot{p_{\rm w}}/{\rm d}\log {L_{\rm bol}}\sim 10/8$ , and ${\rm d}\log \dot{E_{\rm w}}/{\rm d}\log {L_{\rm bol}}\sim 11/8$ . In addition, our model predicts that no UFO features are detected for the AGNs with 0.01, since the winds do not appear. Also, only AGNs with M BH 10 8 M exhibit the UFOs when ~ 0.025. These predictions nicely agree with the X-ray observations. These results support that the line-driven disc wind is the origin of the UFOs.