Over-activation of microglia cells in the brain contributes to neurodegenerative processes promoted by the production of various neurotoxic factors including pro-inflammatory cytokines and nitric oxide. Recently, accumulating evidence has suggested that mitochondrial dynamics are an important constituent of cellular quality control and function. However, the role of mitochondrial dynamics in microglial activation is still largely unknown. In this study, we determined whether mitochondrial dynamics are associated with the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated immortalization of murine microglial cells (BV-2) by a v-raf/v-myc carrying retrovirus (J2). Excessive mitochondrial fission was observed in lentivirus-transfected BV-2 cells stably expressing DsRed2-mito following LPS stimulation. Furthermore, mitochondrial localization of dynamin-related protein 1 (Drp1) (a key regulator of mitochondrial fission) was increased and accompanied by de-phosphorylation of Ser637 in Drp1.Interestingly, inhibition of LPS-induced mitochondrial fission and reactive oxygen species (ROS) generation by Mdivi-1 and Drp1 knock-down attenuated the production of pro-inflammatory mediators via reduced nuclear factor kappa-light-chainenhancer of activated B cells (NF-jB) and mitogen-activated protein kinase (MAPK) signaling. Our results demonstrated for the first time that mitochondrial fission regulates mitochondrial ROS production in activated microglial cells and influences the expression of pro-inflammatory mediators through the activation of NF-jB and MAPK. We therefore suggest that mitochondrial dynamics may be essential for understanding pro-inflammatory mediator expression in activated microglial cells. This could represent a new therapeutic approach for preventing neurodegenerative diseases. Keywords: Drp1, lipopolysaccharide, Mdivi-1, microglia, mitochondrial dynamics, neuroinflammation. Address correspondence and reprint requests to Dong-Seok Lee, College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea. E-mail: lee1@knu.ac.kr 1 These authors contributed equally to this work. Abbreviations used: CMVie, cytomegalovirus immediate early; Cox-2, cyclooxygenase-2; COXIV, cytochrome c oxidase; cPPT, central polypurine tract; DMEM, Dulbecco's modified Eagle's medium; Drp1, dynamin-related protein 1; DsRed2, discosoma sp. red fluorescent; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; Fis1, mitochondrial fission 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; LTR, long terminal repeat; MAPK, mitogen-activated protein kinase; Mdivi-1, 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; Mfn1, mitofusin 1; Mfn2, mitofusin 2; MOI, multiplicity of infection; MTS, mitochondrial targeting sequence; NF-jB, nuclear factor kappa-light-chainenhancer of activated B cells...

Background/AimsEnvironmental factors such as food, lifestyle and prevalence of Helicobacter pylori infection are widely different in Asian countries compared to the West, and physiological functions and genetic factors of Asians may also be different from those of Westerners. Establishing an Asian consensus for functional dyspepsia is crucial in order to attract attention to such data from Asian countries, to articulate the experience and views of Asian experts, and to provide a relevant guide on management of functional dyspepsia for primary care physicians working in Asia.MethodsConsensus team members were selected from Asian experts and consensus development was carried out using a modified Delphi method. Consensus teams collected published papers on functional dyspepsia especially from Asia and developed candidate consensus statements based on the generated clinical questions. At the first face-to-face meeting, each statement was reviewed and e-mail voting was done twice. At the second face-to-face meeting, final voting on each statement was done using keypad voting system. A grade of evidence and a strength of recommendation were applied to each statement according to the method of the GRADE Working Group.ResultsTwenty-nine consensus statements were finalized, including 7 for definition and diagnosis, 5 for epidemiology, 9 for pathophysiology and 8 for management. Algorithms for diagnosis and management of functional dyspepsia were added.ConclusionsThis consensus developed by Asian experts shows distinctive features of functional dyspepsia in Asia and will provide a guide to the diagnosis and management of functional dyspepsia for Asian primary care physicians.

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Various in vivo mutagenesis methods have been developed to facilitate fast and efficient continuous evolution of proteins in cells. However, they either modify the DNA region that does not match the target gene, or suffer from low mutation rates. Here, we report a mutator, eMutaT7 (enhanced MutaT7), with very fast in vivo mutation rate and high gene-specificity in Escherichia coli. eMutaT7, a cytidine deaminase fused to an orthogonal RNA polymerase, can introduce up to ∼4 mutations per 1 kb per day, rivalling the rate in typical in vitro mutagenesis for directed evolution of proteins, and promotes rapid continuous evolution of model proteins for antibiotic resistance and allosteric activation. eMutaT7 provides a very simple and tunable method for continuous directed evolution of proteins, and suggests that the fusion of new DNA-modifying enzymes to the orthogonal RNA polymerase is a promising strategy to explore the expanded sequence space without compromising gene specificity.

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Under endoplasmic reticulum (ER)‐stress conditions, the unfolded protein response (UPR) generates a defense mechanism in mammalian cells. The regulation of UPR signaling is important in oocyte maturation, embryo development, and female reproduction of pigs. Recent studies have shown that melatonin plays an important role as an antioxidant to improve pig oocyte maturation. However, there is no report on the role of melatonin in the regulation of UPR signaling and ER‐stress during in vitro maturation (IVM) of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidative effects of melatonin on porcine oocyte maturation through the regulation of ER‐stress and UPR signaling. We investigated the changes in the mRNA/protein expression levels of three UPR signal genes (Bip/Grp78, ATF4, P90/50ATF6, sXbp1, and CHOP) on oocytes, cumulus cells, and cumulus‐oocyte complexes (COCs) during IVM (metaphase I; 22 hours and metaphase II; 44 hours) by Western blot and reverse transcription‐polymerase chain reaction analysis. Treatment with the ER‐stress inducer, tunicamycin (Tm), significantly increased expression of UPR markers. Additionally, cumulus cell expansion and meiotic maturation of oocytes were reduced in COCs of Tm‐treated groups (1, 5, and 10 μg/mL). We confirmed the reducing effects of melatonin (0.1 μmol/L) on ER‐stress after pretreatment with Tm (5 μg/mL; 22 hours) in maturing COCs. Addition of melatonin (0.1 μmol/L) to Tm‐pretreated COCs recovered meiotic maturation rates and expression of most UPR markers. In conclusion, we confirmed a role for melatonin in the modulation of UPR signal pathways and reducing ER‐stress during IVM of porcine oocytes.