5 - World Journal of Gastroenterology

ISSN 1949-8454 (online)World Journal ofBiological ChemistryWorld J Biol Chem 2012 May 26; 3(5): 93-109www.wjgnet.com

Jun-Ming Guo, NingboChang-Jiang Jin, HefeiDong-Yan Jin, Hong KongHui-Hua Li, BeijingChun Liang, Hong KongFeng Liu, NanjingShu-Wen Liu, GuangzhouPei-Yuan Qian, Hong KongLei Ren, XiamenHong-Bo Shao, YantaiTao Tao, XiamenKarl Tsim, Hong KongPaulus S Wang, TaipeiLing-Yun Wu, BeijingZhi-Heng Xu, BeijingYong-Bin Yan, BeijingTang-Bin Yang, BeijingZeng-Ming Yang, XiamenXue-Wu Zhang, GuangzhouYiguo Zhang, ChongqingHai-Meng Zhou, BeijingRong-Jia Zhou, WuhanXiao-Feng Zheng, BeijingWei-Guo Zhu, BeijingChao-Chun Zou, HangzhouShan Cen, ChinaPetr Draber, PragueCzech RepublicDenmarkRasmus Hartmann-Petersen, CopenhagenFinlandVille-Petteri Mäkinen, HelsinkiMikko Juhani Nikinmaa, TurkuMika Rämet, TampereFranceYannick Allanore, ParisOlivier Berteau, Jouy En JosasJean-Yves Bouet, ToulouseAnthony William Coleman, LyonCristine Alves da Costa, ValbonneYannick Goumon, StrasbourgHerve Hoste, ToulouseAnne Imberty, GrenobleEric J Kremer, MontpellierFlorian Lesage, Sophia-AntipolisJean-Louis Mergny, LyonSylvie Rebuffat, ParisNorbert Rolland, GrenobleSandrine Sagan, ParisGermanyMaik Behrens, NuthetalMatthias Eckhardt, BonnHarald Genth, HannoverMartin Gotte, MuensterChristian Hallermann, MuensterMichael Hecker, GreifswaldBernhard Lüscher, AachenWerner Müller, MainzJörg Nickelsen, Planegg-MartinsriedWolfgang Obermann, BochumMatthias Ocker, MarburgSatish Raina, BorstelMichael Ristow, JenaM Lienhard Schmitz, GiessenKlaus Schulze-Osthoff, TübingenGerhild van Echten-Deckert, BonnGreeceEvangelia Papadimitriou, PatrasMaria Papagianni, ThessalonikiGeorgia Sotiropoulou, Rion-PatrasNiki Chondrogianni, AthensIndiaSubrata Chattopadhyay, MumbaiVirendra S Gomase, LaturSiddhartha S Jana, KolkataSunil Kumar Manna, HyderabadVinay K Nandicoori, New DelhiMN Ponnuswamy, ChennaiManoj Raje, ChandigarhShio Kumar Singh, VaranasiTP Singh, New DelhiIranMehrdad Mohri, MashhadSeyed Nasser Ostad, TehranIsraelShoshana Bar-Nun, Tel AvivShaul Mordechai, Beer ShevaZvi Naor, Tel AvivEitan Shaulian, JerusalemVarda Shoshan-Barmatz, Beer ShevaItalyAndrea Battistoni, RomeAnnamaria Bevilacqua, MilanAntonio Brunetti, CatanzaroSantina Bruzzone, GenovaGaetano Cairo, MilanoGiovanna De Chiara, RomeRita De Santis, PomezaRosario Donato, PerugiaVittorio Gentile, NaplesFabio Grizzi, MilanMaria Luisa Mangoni, RomeLuca Munaron, TorinoAntonio Musarò, RomeSergio Papa, BariAlberto Passi, VareseRinaldo Pellicano, TurinLuca Rampoldi, MilanAndrea Rasola, PadovaGianfranco Risuleo, RomeVito Ruggiero, PomeziaRoberto Scatena, RomeMassimo Stefani, FlorenceAndrea Trabocchi, FlorenceCarlo Ventura, BolognaElena Zocchi, GenovaJapanNaohiko Anzai, TokyoNoriko Fujiwara, NishinomiyaYoshiaki Furukawa, YokohamaHiroshi Harada, KyotoMakoto Hashimoto, TokyoTadashi Hatanaka, Kaga-gunEiichi Hinoi, KanazawaSatoshi Inoue, TokyoTakaki Ishikawa, OsakaYoshizumi Ishino, FukuokaHiroaki Itamochi, YonagoHideaki Kaneto, OsakaKoichi Kato, OkazakiEiichi N Kodama, SendaiKenji Kuwasako, MiyazakiKatsumi Maenaka, FukuokaHisao Masai, TokyoShin-Ichiro Miura, FukuokaEiji Miyoshi, SuitaRyuichi Morishita, SuitaYasu S Morita, OsakaTatsuya Sakamoto, SetouchiToshiyasu Sasaoka, ToyamaHiroshi Shibuya, BunkyoToru Shimizu, SendaiHiroshi Takahashi, TottoriTakashi Takeuchi, YonagoTomohiro Tamura, SapporoKengo Tanabe, TokyoTakuji Tanaka, GifuIkuo Tooyama, OtsuHirokazu Tsukahara, FukuiToshimitsu Uede, SapporoNobutaka Wakamiya, AsahikawaJi-Yang Wang, YokohamaRichard W Wong, KanazawaSho-Ichi Yamagishi, KurumeMichiaki Yamashita, YokohamaKiyotsugu Yoshida, TokyoTsutomu Mikawa, YokohamaLithuaniaArunas Ramanavicius, VilniusMauritiusTheeshan Bahorun, ReduitMexicoAlejandra Bravo, MorelosGerardo Corzo, MorelosNetherlandsEgbert J Boekema, GroningenN Bovenschen, UtrechtBart Maarten Gadella, UtrechtLeo Nijtmans, NijmegenWJBC|www.wjgnet.comII May 26, 2012

W J B CWorld Journal ofBiological ChemistryOnline Submissions: http://www.wjgnet.com/1949-8454officewjbc@wjgnet.comdoi:10.4331/wjbc.v3.i5.93World J Biol Chem 2012 May 26; 3(5): 93-97ISSN 1949-8454 (online)© 2012 Baishideng. All rights reserved.Hui-Ling Chiang, PhD, Professor, Series EditorPeroxisomes, oxidative stress, and inflammationTOPIC HIGHLIGHTStanley R Terlecky, Laura J Terlecky, Courtney R GiordanoStanley R Terlecky, Laura J Terlecky, Courtney R Giordano,Department of Pharmacology, Wayne State University School ofMedicine, 540 E. Canfield Ave., Detroit, MI 48201, United StatesAuthor contributions: Terlecky SR wrote the majority of thereview; Terlecky LJ and Giordano CR contributed to the writing,designed and produced the figures, and edited the manuscript.Correspondence to: Stanley R Terlecky, PhD, Department ofPharmacology, Wayne State University School of Medicine, 540E. Canfield Ave., Detroit, MI 48201,United States. srterlecky@med.wayne.eduTelephone: +1-313-5773557 Fax: +1-313-5776739Received: July 27, 2011 Revised: May 10, 2012Accepted: May 17, 2012Published online: May 26, 2012AbstractPeroxisomes are intracellular organelles mediating awide variety of biosynthetic and biodegradative reactions.Included among these are the metabolism ofhydrogen peroxide and other reactive species, moleculeswhose levels help define the oxidative state ofcells. Loss of oxidative equilibrium in cells of tissuesand organs potentiates inflammatory responses whichcan ultimately trigger human disease. The goal of thisarticle is to review evidence for connections betweenperoxisome function, oxidative stress, and inflammationin the context of human health and degenerativedisease. Dysregulated points in this nexus are identifiedand potential remedial approaches are presented.© 2012 Baishideng. All rights reserved.Key words: Peroxisomes; Oxidative stress; InflammationPeer reviewers: Antonio Brunetti, MD, PhD, Professor, Cattedradi Endocrinologia, Università di Catanzaro “Magna Græcia”,88100 Catanzaro, Italy; Manuel Vazquez-Carrera, Dr., Universityof Barcelona, Unitat de Farmacologia. Facultat de Farmacia. Diagoanl643, E-08028 Barcelona, SpainTerlecky SR, Terlecky LJ, Giordano CR. Peroxisomes, oxidativestress, and inflammation. World J Biol Chem 2012; 3(5): 93-97Available from: URL: http://www.wjgnet.com/1949-8454/full/v3/i5/93.htm DOI: http://dx.doi.org/10.4331/wjbc.v3.i5.93INTRODUCTIONPeroxisomes are essential organelles of human cells. Inthis article, we review peroxisome biology; summarizinghow the organelle is formed, how it functions, andwhat happens when these processes are compromised.In addition, we describe an emergent link between theorganelle and cellular aging pathways. In the latter analysis,we connect peroxisome function with the generationand destruction of specific inflammatory mediators andspeculate on the organelle’s involvement in initiating andprogressing human disease.PEROXISOME FUNCTIONDegradationPeroxisomes synthesize and degrade a wide variety ofcellular compounds [1] . Through α- and β-oxidations, specificlong-chain, very-long-chain, and 3-methyl-branchedchainfatty acids are degraded. These processes may occurentirely within the organelle or may involve participationof other organelles - e.g., mitochondria. The notion thatperoxisomes shuttle metabolites for continued processingand/or anaplerotic metabolism is part of an emergenttheme for the organelle; specifically, that it is integratedinto a variously interacting endomembrane system responsiblefor a number of critical cellular processes [2] .The peroxisome’s handling of hydrogen peroxide, areactive oxygen species produced by oxidative reactionsoccurring within the organelle, also bears on this point.Under most conditions, hydrogen peroxide is producedand immediately processed by the organelle’s residentWJBC|www.wjgnet.com93 May 26, 2012|Volume 3|Issue 5|

Terlecky SR et al . Peroxisomes, oxidative stress, and inflammationOxidativestressPeroxisomesMitochondriaInflammationFigure 1 Emergent connections between peroxisomes and cellular metabolism.marker enzyme, catalase. However, conditions exist inwhich the balance of hydrogen peroxide production isupset, and the potentially toxic metabolite accumulates [3-7] .As discussed further below, such phenomena set cells on apro-aging program with potentially important health ramifications[2] .Other catabolic functions carried out by peroxisomesinclude degradation of polyamines, glyoxylate, certainamino acids, and several xenobiotics [1] . In addition, theorganelle breaks down the arachidonic acid derivativesknown as eicosanoids. Eicosanoids are critically importantsignaling molecules which exert tremendous controlover inflammatory reactions [8] . Among the arachidonicacid derivatives under consideration here are prostaglandins,thromboxanes, leukotrienes, and prostacyclins.These compounds elicit broad ranging inflammatory reactionsdepending on concentration and location.Included among the activities engendered by thesemolecules are modulating vasoconstriction/vasodilationand smooth muscle contraction/relaxation, controllingplatelet aggregation, regulating hormone release/metabolism,and initiating pyrogenic (i.e., febrile) responses [8] .And these examples only partially represent the broadphysiological effects elicited by eicosanoids. The importantpoint is that through their ability to be metabolized byperoxisomes - organelle function is linked to the inflammatoryresponse.Inflammation could not be a more popular topic incurrent medical research. There is a sense in the clinicalcommunity that inflammation plays a major role in agingand in chronic diseases. Indeed, the term “inflammaging”has been coined and is in use. Targeting inflammationis popularly seen as a major strategy to combat theseprocesses. Specific pathways involving the presence ofreactive oxygen species and chronic activation of inflammatorypathways are examined further below.SynthesisPeroxisomes contain enzymes which contribute to thesynthesis of critical cellular constituents including bileacids, ether phospholipids, and docosahexaenoic acids,among others [1] . Bile acids, derived from cholesterol, areimportant molecules involved in digestion through theirability to emulsify fats. Ether phospholipids, includingplasmalogens, represent a vital class of membrane protectivemolecules, found throughout cells of the body.Myelin, the insulating layer of nerve sheaths, containsplasmalogens; absence of the ether phospholipid is associatedwith progressive neurological impairment [9] .Docosahexaenoic acids, peroxisomally produced omega-3fatty acids, are the pivotal precursors of resolvins(“resolution-phase interaction products”), maresins(“macrophage mediator in resolving inflammation”),and protectins (formerly called “neuroprotectins”) [10-12] .These molecules possess potent anti-inflammatory, inflammatoryresolving, and immunoregulatory activities.Importantly, conversion of docosahexaenoic acids tothese biologically active mediators is accelerated by nonsteroidalanti-inflammatory drugs, including aspirin,which inhibits the cyclooxygenase-2 enzyme [8] .PEROXISOME FORMATIONBiogenesisFrom a functional perspective, the peroxisome is clearly amajor player in cellular metabolism and a key componentof organismal physiology. As to how it acquires these capacities,the answer lies in a magnificently choreographedseries of biochemical processes which bring about itsbiogenesis [13] .Defining the origins of the peroxisome membranehas taken some time - many years in fact. Growth anddivision of existing organelles gained considerable earlysupport until evidence was obtained that the endoplasmicreticulum was also providing membrane [14] . Thecurrent consensus is that both processes contribute toperoxisome membrane growth and proliferation [13] .Once assembled, the peroxisome membrane acquiresadditional membrane proteins including those constitutingthe import machinery. Although still not describedin complete detail, this apparatus is known to consistof the following components: soluble receptors whichrecognize peroxisomal targeting signals on nascent proteins/enzymes;docking proteins which serve to concentrateand direct the receptor-ligand complex at theorganelle membrane; and several molecules involved infacilitating the translocation process and recycling essentialcomponents for additional rounds of import [13] .Mechanisms are in place to recycle unneeded, damaged,or aged import factors, as well as to degrade the entireorganelle when appropriate.WJBC|www.wjgnet.com94 May 26, 2012|Volume 3|Issue 5|

Terlecky SR et al . Peroxisomes, oxidative stress, and inflammationAB, Moser HW. Peroxisome biogenesis disorders. BiochimBiophys Acta 2006; 1763: 1733-174820 Wanders RJ, Waterham HR. Peroxisomal disorders: thesingle peroxisomal enzyme deficiencies. Biochim BiophysActa 2006; 1763: 1707-172021 Ivashchenko O, Van Veldhoven PP, Brees C, Ho YS, TerleckySR, Fransen M. Intraperoxisomal redox balance inmammalian cells: oxidative stress and interorganellar crosstalk.Mol Biol Cell 2011; 22: 1440-145122 Young CN, Koepke JI, Terlecky LJ, Borkin MS, Boyd SavoyL, Terlecky SR. Reactive oxygen species in tumor necrosisfactor-alpha-activated primary human keratinocytes: implicationsfor psoriasis and inflammatory skin disease. J InvestDermatol 2008; 128: 2606-261423 Undyala V, Terlecky SR, Vander Heide RS. Targeted intracellularcatalase delivery protects neonatal rat myocytesfrom hypoxia-reoxygenation and ischemia-reperfusion injury.Cardiovasc Pathol 2011; 20: 272-28024 Góth L, Eaton JW. Hereditary catalase deficiencies and increasedrisk of diabetes. Lancet 2000; 356: 1820-1821S- Editor Cheng JX L- Editor A E- Editor Zheng XMWJBC|www.wjgnet.com97 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK5signals were captured before and after 50% photobleachingYFP. FRET is indicated as the relative increase inCFP emission following YFP photobleaching. The imagingsystem was controlled by the Leica Application SuiteAdvanced Fluorescence software (http://www.leicamicrosystems.com).Western blottingFor detection of coimmunoprecipitates and SEPT8, sampleswere analyzed by SDS-PAGE on NuPAGE 4%-12%BisTris SDS-PAGE gels from Invitrogen according tothe manufacturer’s protocol and blotted onto a 0.45-μmpolyvinylidene difluoride membrane (Millipore). Immunoblottingwas performed by first blocking the membranewith phosphate buffered solution (PBS)-T [PBSwith 0.1% Tween 20 (Sigma) containing 10% (w/v) driedskimmed milk for 1 h and probed either by anti-PRAKor anti-SEPT8]. After three washes, the membrane wasincubated with the appropriate secondary antibody for 1 h.Visualization of proteins was achieved by using CDP Starsubstrate (Tropix, Bedford, MA, United States) and Lumi-Imager F1 from Roche Applied Sciences. MagicMarkwestern standard was from Invitrogen Life Technologies.GST pull downCell lysate was precleared with 25 mg GST and 50 mL50% slurry of glutathione-agarose beads for 2 h at 4 ℃.An input control was taken, and the cell lysate was dividedinto two tubes and 10 mg GST or GST-SEPT8 wereadded. The GST proteins were recovered after additionof glutathione beads and washed extensively in PBS-T.The proteins were eluted from the beads by addition of20 mL loading buffer (LDS-buffer with 100 mmol/LDTT) followed by 10 min incubation at 70 ℃. The GSTproteins were subsequently subjected to immunoblotanalysis.DensitometryDensitometry was performed using a BioRad ModelGS-700 Imaging Densitometer (Oslo, Norway) and theMulti-Analyst version 1.1 Software.RESULTSScreening of proteins that interact with MK5To elaborate the biological functions of MK5, we decidedto identify novel substrates for MK5. A yeast two-hybridscreen with MK5 as bait and a brain cDNA library wasdesigned. After selecting yeast colonies on uracil-deficientmedium, the activity of β-galactosidase was monitored.The URA + and lacZ + colonies were also tested for whetherthey could grow on adenosine-deficient medium. Tomaximize the specificity of the screening, protein-proteininteraction was tested using three independent reporterswith different types of GAL4-binding sites. This procedureeliminates interactions that result from nonspecificpromoter activation. In order to confirm the interaction,the prey plasmids from 29 candidates that expressed atleast two reporters were selected by E. coli transformation,and then the candidate prey plasmids were reintroducedinto yeast with MK5 bait plasmid or with a negative controlplasmid expressing GAL4-binding domain but lackingthe bait part. Twenty real positives were identified thatencoded three different proteins. Among 20 real positives,17 preys contained the same cDNA encoding SEPT8_v2(AF440762). Two other positive clones encoded the previouslyreported MK5 interaction partner ERK3 [10,11] .SEPT8 binds to MK5 in vitro and in vivoTo verify the interaction observed in the yeast two-hybridsystem, GST pull-down was performed. Lysates ofHEK293 cells transfected with the expression plasmid forGFP-MK5 were incubated with glutathione-sepharosebeads accompanied by either GST alone or GST-SEPT8.After immunoprecipitation, MK5 was detected by westernblotting using anti-PRAK antibody. The results showedthat MK5 could bind GST-SEPT8 (Figure 1A) but notGST alone.To establish whether complexes of MK5 and SEPT8existed within mammalian cells, HEK293 cells were cotransfectedwith MK5 and SEPT8 expression plasmidsbecause these cells give high transfection efficiency. Reciprocalcoimmunoprecipitation studies were conductedwith anti-PRAK antibody followed by western blottingwith anti-SEPT8 antibody, or immunoprecipitation withanti-SEPT8 antibody followed by western blotting withanti-PRAK antibody. Specific interaction was monitoredbetween the two proteins (Figure 1B and C). The signalin the last lane in Figure 1C (lysate of cells expressingectopically expressed SEPT8 but not MK5) was due tointeraction of SEPT8 with endogenous MK5. No interactionswere visualized in the control precipitation usingvehicles. To detect interaction between endogenous MK5and SEPT8, PC12 cells were used because both MK5 andSEPT8 are relatively highly expressed in neural cells [1,2,36] .MK5 could be detected when endogenous SEPT8 wasimmunoprecipitated (Figure 1D). Combined, our resultsclearly demonstrate that MK5 and SEPT8 specificallyinteract in vitro and in vivo. SEPT8 was originally identifiedas an interaction partner for SEPT5 [29] , therefore, wetested whether MK5 and SEPT5 complexes were foundin cells. Coimmunprecipitation studies failed to detectMK5:SEPT5 complexes (results not shown).We performed FRET analysis to monitor interactionbetween MK5 and SEPT8 in cells. HeLa cells were transfectedwith expression plasmids for MK5 and SEPT8.As a negative control, we included CREB because it doesnot interact with MK5 [4] . After photobleaching, interactionbetween MK5 and SEPT, but not MK5 and CREB,was observed as indicated by a relative increase in CFPemission following YFP photobleaching (Figure 2).MK5 phosphorylates SEPT8 in vitroIn previous studies, several septins have been shownto be phosphorylated in vitro and/or in vivo, includingSEPT1, SEPT2, SEPT3 and SEPT5 [40-44] . After describingthe interaction between two proteins, we wanted toknow whether MK5 possesses catalytic activity towardsWJBC|www.wjgnet.com101 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK5A8080MarkerInputGSTGST-SEPT8MK5GST-SEPT8BInputIP: a-SEPT8pcDNA3-HA-MK5 + + + +pRK5-Flag-SEPT8 + + + +pcDNA3-HA + +pRK5 + +WB: a-MK560501 2 3 4SEPT81 2 3 4 5 6WB: a-SEPT8CInputIP: a-MK5pcDNA3-HA-MK5 + + + +pRK5-Flag-SEPT8 + + + +pcDNA3-HA + +pRK5 + +D6050InputIP: a-SEPT8MK5WB: a-SEPT840WB: a-MK51 2 3 4 5 6WB: a-MK560501 2 3SEPT8WB: a-SEPT8Figure 1 Mitogen-activated protein kinase-activated protein kinase 5 and SEPT8 interact in vitro and in vivo. A: Glutathione S-transferase (GST) pull-downassay. GST-SEPT8 or GST alone purified from Escherichia coli and immobilized on glutathione-Sepharose beads were incubated for 60 min with lysate of HEK293cells transfected with GFP-mitogen-activated protein kinase-activated protein kinase 5 (MK5). After washing the beads five times, bound proteins were eluted byboiling, subjected to SDS-PAGE, and immunoblotted with anti-PRAK (upper panel) or anti-SEPT8 (lower panel) antibody. Lane 1: Protein molecular mass marker (inkDa); lane 2: Cell lysate; lane 3: Cell lysate after pull down with GST; lane 4: Cell lysate after pull down with GST-SEPT8; B: Coimmunoprecipitation of FLAG-SEPT8and hemagglutinin (HA)-MK5. HA-MK5- and/or FLAG-SEPT8-encoding plasmids were transiently expressed in HEK293 cells. Total cellular lysates (input; lanes 1-3)and SEPT8 immunoprecipitates (IP: α-SEPT8; lanes 4-6) were probed with anti-PRAK (upper panel) and anti-SEPT8 (lower panel) antibody. Lane 1: Cell lysate ofcells cotransfected with expression plasmids for HA-tagged MK5 and FLAG-tagged SEPT8; lane 2: Cell lysate of cells cotransfected with expression plasmids for HAtaggedMK5 and empty vector for SEPT8; lane 3: Cell lysate of cells cotransfected with expression plasmids for FLAG-tagged SEPT8 and empty vector for MK5; lane4: Immunoprecipitated lysate of cells cotransfected with expression plasmids for HA-tagged MK5 and FLAG-tagged SEPT8; lane 5: Immunoprecipitated lysate of cellscotransfected with expression plasmids for HA-tagged MK5 and empty vector for SEPT8; lane 6: Immunoprecipitated lysate of cells cotransfected with expressionplasmids for FLAG-tagged SEPT8 and empty vector for MK5; C: Coimmunoprecipitation of HA-MK5 and FLAG-SEPT8. HEK293 cells were transiently transfectedwith expression plasmids for HA-MK5 and/or FLAG-SEPT8. Total cellular lysates (input) and HA-MK5 immunoprecipitates (IP: α-MK5) were probed with anti-SEPT8(upper panel) and anti-PRAK (lower panel) antibody. Lanes 1-3: Lysates from transfected cells; lanes 4-6: Immunoprecipitation of cell lysates. See (B) for details; D:Coimmunoprecipitation of endogenous MK5 and SEPT8. Endogenous SEPT8 was immunoprecipitated from PC12 cells with anti-SEPT8 antibodies and the precipitatewas analyzed for the presence of MK5 by anti-PRAK antibodies. Lane 1: Protein molecular mass marker (in kDa); lane 2: Lysate of PC12 cells (= input); lane 3:Immunoprecipitation with SEPT8 antibodies. The bottom panel shows control western blot with SEPT8 antibodies. IP: Immunoprecipitation; WB: Western blotting.SEPT8. To test this, GST-SEPT8 fusion protein was purifiedfrom bacteria and the GST moiety was enzymaticallycleaved. SEPT8 was then incubated with commerciallyavailable active MK5 in an in vitro kinase assay. Phosphoproteinswere fractionated by SDS-PAGE on a 4%-20%acrylamide gradient gel and visualized by autoradiography.As shown in Figure 3A, SEPT8 was phosphorylated byMK5. As previously reported, MK5 autophosphorylationactivity of MK5 was observed [38] . An additional band,corresponding to phosphorylated SEPT8 was detected.These results demonstrate that MK5 can phosphorylateof SEPT8 in vitro.Identification of phosphorylation sites on SEPT8To identify MK5 phosphoacceptor sites on SEPT8, weperformed an in vitro kinase assay using peptide arrays. Oneseparate peptide, YFIT 237 PT 239 GHS 242 LKS 245 LDLVT 250 MKK and two consecutive peptides IPIIAKADT 269 IS 271 KS 273ELHKFKI and IAKADT 269 IS 271 KS 273 ELHKFKIKIMappeared phosphorylated (Figure 3B). Putative phosphoacceptorsites for the serine/threonine MK5 kinase aremarked. We analyzed the SEPT8 sequence with NetPhos2.0 (accessible on the internet: http://www.cbs.dtu.dk/services/NetPhos/). This software algorithm predicts thepotential phosphorylation sites in proteins. According tothe NetPhos description, scores close to 1 are the mostlikely to represent an actual phosphorylation site. Amongall predicted sites, two serine residues in the sequences ofpeptides that were phosphorylated on peptide array had ahigh score, namely 0.994 for Ser-242 and 0.944 for Ser-271.We generated plasmids encoding GST-SEPTS242A, GST-SEPTS271A and GST-SEPTS242A/S271A in whichthese Ser residues were replaced by alanine and expressedthese fusion proteins in bacteria. GST was enzymaticallyremoved from the purified proteins and in vitro kinase assaywith active MK5 was performed. Mutation of Ser-242 orSer-271 into Ala reduced MK5-mediated phosphorylationof SEPT8, but the former substitution had less effect thanWJBC|www.wjgnet.com102 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK5AECFP-MK5EYFP-CREBPrebleach5.00 mm 5.00 mmPostbleachFRET signal (%)335.00 mm 5.00 mm0B ECFP-MK5 EYFP-SEPT8Prebleach5.00 mm 5.00 mmPostbleachFRET signal (%)335.00 mm 5.00 mm0Figure 2 Fluorescence resonance energy transfer analysis shows interaction between mitogen-activated protein kinase-activated protein kinase 5 andSEPT8. (A) HeLa cells were cotransfected with expression plasmids for heat shock protein (CFP)-tagged mitogen-activated protein kinase-activated protein kinase 5(MK5) and yellow fluorescent protein (YFP)-tagged CREB or (B) with expression vectors for CFP-tagged MK5 and YFP-tagged SEPT8. Twenty-four hours after transfection,cells were fixed and fluorescence resonance energy transfer (FRET) analysis was carried out. CFP and YFP were excited with separate laser channels of458 and 514 nm, respectively. Emission fluorescence intensity data were obtained at 465-500 nm (CFP) and 525-600 nm (YFP). CFP and YFP emission signals werecaptured before and after 50% photobleaching YFP. FRET is indicated as the relative increase in CFP emission following YFP photobleaching.WJBC|www.wjgnet.com103 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK5AMK5 SEPT8 MK5 + SEPT8BMK5SEPT8PonceauWB: anti-MK51 2 3SEPT8In vitrokinase12CSEPT8SEPT8S242AMK5SEPT8S271ASEPT8S242AS271A1 YFITPTGHS 242 LKSLDLVTMKK2 IPIIAKADTIS 271 KSELHKFKIIAKADTIS 271 KSELHKFKIKIMP-MK5P-SEPT81.0 0.7 0.4 0.4 RDUFigure 3 Mitogen-activated protein kinase-activated protein kinase 5 phosphorylates septin 8 in vitro. A: In vitro kinase assay on recombinant SEPT8. GlutathioneS-transferase (GST)-SEPT8 fusion protein was purified from Escherichia coli and the GST moiety was removed by thrombin. SEPT8 was incubated withactivated mitogen-activated protein kinase-activated protein kinase 5 (MK5) for 30 min at 30 ℃ in the presence of [γ- 32 P] ATP. Proteins were separated by SDS-PAGEand phosphorylation was visualized by autoradiography (upper panel). Loading control for MK5 (middle panel) and SEPT8 (lower panel) was performed by westernblotting with anti-PRAK and anti-SEPT8 antibodies, respectively. Lane 1: Recombinant activated MK5; lane 2: Purified SEPT8 protein; lane 3: recombinant MK5 andpurified SEPT8; B: SEPT8 peptide array was subjected to in vitro phosphorylation by activated MK5. Each spot represents a 20-mer peptide fragment of SEPT8. Thepeptide fragments constitute the complete SEPT8 protein. The sequential peptide has a 17-amino acid overlap with the previous peptide. Several spots with peptideswere detected by autoradiography (lower panel). Ser-242 and Ser-271 with highest prediction score 0.994 and 0.944, respectively by Netphos software, were chosenas possible phosphorylation sites in the peptide sequences. The upper panel represents Ponceau staining of the peptide array membrane used. The sequences ofthe peptides representing the positive spots are shown; C: In vitro phosphorylation of wild-type SEPT8, and SEPT8 mutants carrying a single amino acid substitution(Ser-242 into Ala or Ser-271 into Ala, respectively) or the double amino acid substitution Ser-242 and Ser-271 into Ala. The upper band represents autophosphorylatedMK5, while the lower band is phosphorylated SEPT8. Relative densitometry units (RDU) of the bands representing phosphorylated SEPT8 are shown. The valueobtain for wild-type SEPT8 was arbitrary set as 1.0 and the other values were related to this.the latter on phosphoSEPT8 levels. The double SEPT8S242A/S271A substitution did not further reduce thephosphorylation levels of SEPT8 (Figure 3C). These findingsindicate that Ser-271 and to a lesser extend Ser-242represent in vitro targets for MK5, but that additional aminoacid residues can be phosphorylated by MK5 in vitro.MK5 colocalizes with SEPT8 in HEK293 and PC12 cellsTo examine cellular localization of MK5 with SEPT8,confocal microscopy was performed on HEK293 cellsexpressing GFP-MK5 and AsRed-SEPT8 fusion proteinsseparately (Figure 4A) or simultaneously (Figure 4B). Aspreviously reported, GFP-MK5 protein resides predominantlyin the nucleus, but can also be detected in the cytoplasm[38] , while The RFP-SEPT8 protein is exclusivelylocated in the cytoplasm (Figure 4A). Cotransfection withexpression plasmid encoding tagged MK5 and SEPT8demonstrated colocalization of these two proteins in theperinuclear area (Figure 4B). Several studies have shownthe involvement of septins in neuronal transmission andexocytosis. Therefore, we wanted to test if a similar colocalizationpattern of proteins could be seen in neuroendocrinePC12 cells. Colocalization was observed not onlyin cell bodies but also in neurite terminals of PC12 cells(Figure 4C). Interestingly, a previous study showed thatSEPT5 was concentrated near the plasma membrane andin growth cones of PC12 cells [45] .MK5 and SEPT8 colocalize with synaptophysin in theneuroblastoma cell line SK-N-DZIn a previous study, SEPT8 colocalized with the synapticmarker synaptophysin [36] . To examine the cellular localizationof SEPT8 with synaptophysin in the neuroblastomacell line SK-N-DZ, cells were transfected with AsRed-SEPT8. Protein was visualized directly, and anti-synaptophysinFITC conjugate antibodies were used to monitorsynaptophysin localization. SEPT8 and synaptophysinshowed strong colocalization (Figure 5A), confirmingthe findings by Ito and colleagues [36] . We then investigatedwhether MK5 could colocalize with synaptophysinbecause we had observed colocalization with SEPT8 inseveral cell lines. Indeed, MK5 colocalized with synaptophysinin a similar fashion as SEPT8 (Figure 5B).DISCUSSIONSeveral genuine substrates have been identified for MK5 includingERK3, ERK4, 14-3-3ε, p53, Rheb and Hsp27 [6,8,10-15] .However, the functions of MK5 remain elusive, andMK5-deficient mice appear normal or die at embryonicstage E11.5, depending on the genetic background of themice [6,10,46] . In our search for other possible MK5 interactionpartners that may provide a clue to the biologicalfunctions of MK5, we have identified SEPT8 as an in vitroMK5 substrate. The interaction was originally revealed byWJBC|www.wjgnet.com104 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK5A AsRed-SEPT8 GFP-MK510 mm 10 mmBAsRed-SEPT8GFP-MK5Merged10 mm 10 mm10 mmCAsRed-SEPT8GFP-MK5Merged20 mm 20 mm20 mmDFigure 4 Mitogen-activated protein kinase-activated protein kinase 5 colocalizes with SEPT8. A: HeLa cells were transfected separately with an expressionplasmid for GFP-mitogen-activated protein kinase-activated protein kinase 5 (MK5) (left panel) or AsRed-SEPT8 (right panel); B: HeLa cells cotransfected with bothexpression plasmids. The subcellular localization of RFP-tagged SEPT8 (left panel), GFP-tagged MK5 (middle panel), or both proteins (merged; right panel) is shown; C:PC12 cells cotransfected with expression vectors for AsRed-SEPT8 (left panel) and GFP-MK5 (middle panel). Colocalization is shown in the right panel; D: Enlargementof the areas marked by arrows in figures (C) shows clear colocalization of MK5 and SEPT8. After 24 h, cells were fixed and MK5 (green channel) and SEPT8 (redchannel) were visualized directly.yeast-two hybrid assay and confirmed in mammalian cellcultures by coimmunoprecipitation, GST pull-down andFRET assays. Moreover, colocalization of both proteinswas observed in different cell lines transfected with expressionplasmids for GFP-MK5 and AsRed-SEPT8 fusion.We went on to investigate whether MK5 had kinaseWJBC|www.wjgnet.com105 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK5A Synaptophysin AsRed-SEPT8 Merged10 mm 10 mm10 mm20 mm 20 mm 20 mmBSynaptophysin MK5 Merged10 mm10 mm10 mmFigure 5 Mitogen-activated protein kinase-activated protein kinase 5 and SEPT8 colocalize with synaptophysin. A: SK-N-DZ cells were transfected with expressionvector encoding AsRed-SEPT8 and after 24 h, cells were fixed. Synaptophysin was visualized by staining with anti-synaptophysin fluorescein isothiocyanate(FITC) conjugate antibody (left panels), while AsRed-SEPT8 was visualized directly (red channel; middle panels). A merged image of red and green channels is shownin the right panel; B: SK-N-DZ cells were fixed and stained with anti-synaptophysin FITC conjugate antibody (left panel) and with anti-PRAK antibody followed by AlexaFluor 568 anti-rabbit antibody (middle panel). Merged image of red and green channels is shown in the right panel.activity towards SEPT8 and it appeared that SEPT8 wasindeed phosphorylated by MK5 in vitro. To the best of ourknowledge, this is the first report linking MAPK-activatedprotein kinases to the group of septin proteins. However,previous studies have identified several mammalian septinsas substrates of several kinases. For instance, SEPT1 hasbeen shown to be phosphorylated in vitro by aurora-Bkinase [41] . SEPT2 is a substrate of casein kinase 2, and itsphosphorylation on Ser-218 is crucial for the proliferationof hepatoma carcinoma cells [44] . Moreover, SEPT2 is alsoan in vitro substrate for protein kinase (PK)C and cAMPdependentPKA [47] . SEPT3 phosphorylation on Ser-91 bycGMP-dependent PKG may contribute to the regulationof its subcellular localization in neurons [40] . SEPT5 phosphorylationby cyclin-dependent kinase 5 at Ser-17 andSer-327 reduces binding of septin to syntaxin, thereforeplaying a role in modulating exocytic secretion [42,43] . MK5phosphorylates SEPT8 on Ser-271 and with lower stoichiometryon Ser-242 in an in vitro assay. The double SEPT8S242A/S271A mutant is still phosphorylated in vitro,indicating that other sites may act as MK5 phosphoacceptorsites in vitro. Both sites are conserved in SEPT8 fromdifferent species ranging from fish to humans (Figure 6).This indicates that the sites are crucial for normal functionof SEPT8. It remains to be tested whether MK5can phosphorylate these sites in vivo. Phosphoproteomicstudies have demonstrated that SEPT8 is phosphorylatedon Ser-10 and Ser-141 in mouse brain, mouse skin melanoma,and nocodazole-arrested HeLa cells [48-50] . Both sitesare conserved in fish, amphibians, and several mammalssuch as apes, primates, rodents and cattle. The proteinkinases responsible for these phosphorylations have notWJBC|www.wjgnet.com106 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK5Human (NP_00109228.1)TRIHVCLYFITPTGHSLKSLDLVTMKKLDSKVNIIPIIAKADTISKSELHKFKIKIMGELArmadillo (ACQ63032.1) ************************************************************Baboon (NP_001162506.1) ************************************************************Cattle (AAI20075.1) ************************************************************Chicken (XP_414648.3)*********************************************************S**Chimpanzee (XP_517927.3) ************************************************************Dog (XP_5319034) ************************************************************Dusky titi (B1MTN8.1) ************************************************************Dwarf hamster (EGW03012.1) ************************************************************Elephant (XP_003404794.1) ************************************************************Ferret (AER93422.1) ************************************************************Gibbon (XP_003259994.1) ************************************************************Guinea pig (XP_003464551.1) ************************************************************Horse (XP_001504480.3)**V*********************************************************Horseshoe bat (B2KIE9.1)**V*********************************************************Lancelet (XP_002588303.1)**V*A*****************************************N**********S**Lizard (XP_003217450.1)**************************N******************************S**Macaque (BAE87336.1) ************************************************************Marmoset (XP_002744650.1) ************************************************************Mouse (CAP19147.1) ************************************************************Naked mole rat (EHB07406.1) *************************************T**********************Nile tilapia (XP_003447038)****I*****A**********************************************S**Opossum (XP_003339562.1) *****************************************************S***S**Orangutan (XP_002815929.1) ************************************************************Panda (XP_002913002.1)**V****************************************V****************Pig (XP_003123974.1) ************************************************************Platypus (XP_001510590.2) ************************************************************Puffer fish (CAG11702.1)****I*****S*************************************************Rabbit (NP_001164820.1) ************************************************************Rat (NP_001100472.1) ************************************************************Salmon (NP_001167234.1)G*V*I*****A**********************************************S**Turkey (XP_003210544.1)*********************************************************S**Xenopus (NP_001087375.1)S********************************************************S**Zebra finch (XP_002188929) *********************************************************S**Zebrafish (NP_001108589)**********S**********************************************S**Figure 6 Mitogen-activated protein kinase-activated protein kinase 5 phosphoacceptor sites in human SEPT8 are conserved in SEPT8 from other species.Alignment of proven and putative SEPT8 proteins in different species of the region encompassing mitogen-activated protein kinase-activated protein kinase 5 phosphoacceptorsites Ser-242 and Ser-271 (numbering for human SEPT8). The code in parenthesis refers to the accession number in the Protein database (http://www.ncbi.nlm.nih.gov/protein/). Asterisk indicates identical amino acid residues. The non-conserved residues are shown in red. The one-letter amino acid code is used. Theresidues corresponding to human SEPT8 Ser-242 and Ser-271 are shown.been identified. Our in vitro kinase assay with the peptidearray did not indicate that Ser-10 and Ser-141 are in vitrophosphorylation sites for MK5.The biological implications of MK5-mediated phosphorylationof SEPT8 are not known but it may affectstructural properties by interfering with di- and trimericcomplex formation with other septins. It has been shownfor SEPT7 that substitution of tyrosine-318 into nonphosphorylatableAla prevents interaction with SEPT8 [30] .Phosphorylation of SEPT8 may have a similar effect oncomplex formation with septins. Phosphorylation mayalso affect the role of SEPT8 in cellular functions. Theexact functions of SEPT8 remain to be elucidated. In accordancewith SEPT5, SEPT8 is implicated in vesiculartransport and exocytosis processes of neurotransmitterrelease and platelet secretion. In neurons, SEPT5 bindssyntaxin, one of the SNARE proteins, and copurifies withsynaptic vesicles, suggesting a role in exocytosis and synapticvesicle dynamics [33,45,51] . Platelets from SEPT5 -/- micepossess altered serotonin secretion and aggregation [25] .SEPT8 expression is high in neurons and platelets, and arecent study has suggested the importance of SEPT8 inthe process of SNARE complex formation and subsequentneurotransmitter release, but actual experimentalproof is lacking [32,34,36,45,52] . Moreover, SEPT8 colocalizeswith the synaptic marker synaptophysin in primary culturedrat hippocampal neurons [36] . We also observed colocalizationof MK5 and synaptophysin in neuroblastomaSK-N-DZ cells. MAPK p38, which can act as an upstreamactivator of MK5 [53] , has been shown to be involved inSNARE-dependent exocytotic release of brain-derivedneurotrophic factor from microglia [54] . Finally, transgenicmice overexpressing an active variant of MK5 display differentanxiety-related traits as compared to wild-type littermates[5] . All these observations point to a possible roleof MK5 in exocytosis that may comprise SEPT8.ACKNOWLEDGMENTSThe authors thank Dr. Koh-Ichi Nagata and Dr. MasakiInagaki for the kind gift of SEPT8 expression plasmid, Dr.Terje Johansen for the generous gift of the Gateway destinationvectors pDest-EYFP and pDest-ECFP, Kenneth Larsen(Bioimaging platform, University of Tromsø) for helpwith the confocal microscope, Marte Singsås Dragset forassistance with cloning, and Dr. Ole Rumohr Blingsmo andDr. Elisa Bjørgo (Biotechnology Centre, Oslo, Norway) forsynthesis and valuable suggestions with peptide arrays.WJBC|www.wjgnet.com107 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK5COMMENTSBackgroundThe mitogen-activated protein kinase (MAPK) pathways transmit signalsthrough a cascade of phosphorylation events, thereby controlling cellular processessuch as proliferation, differentiation, migration, survival/apoptosis, andgene regulation. The precise function of one of the MAPKs, MAPK-activatedprotein kinase 5 (MK5), remains elusive. The aim of this study was to identifynew substrates for MK5 in an effort to better understand the function of thisprotein kinase.Research frontiersPhosphorylation of proteins at serine, threonine and tyrosine residues byprotein kinases is an important post-translational modification that controls theactivity of the substrates. The human genome encodes more than 500 differentprotein kinases, including a large group known as MAPKs. MAPKs controlthrough phosphorylation of their substrates crucial cellular processes. Perturbedaction of these kinases can lead to malignant and nonmalignant diseases. Itis therefore important to divulge the exact functions and genuine substrates ofthese kinases. This may allow designing therapeutic strategies for pathologicalconditions in which MAPKs play a causal role.Innovations and breakthroughsThe genuine function of MK5 remains incompletely understood because fewgenuine substrates have been characterized. This study identified septin 8 asa novel interaction partner for MK5 and is the first to demonstrate that MAPKscan phosphorylate a member of the septin family. Septins are small GTPbindingproteins that participate in processes ranging from cytoskeletal architecture,scaffolding, cytokinesis, ciliogenesis, and neurogenesis. The functionof septin 8 remains enigmatic, but it is enriched at presynapses and interactswith several proteins of synaptic vesicles. Hence, septin 8 may be involved inneurotransmitter release and the role of septin 8 may be modulated by MK5-mediated phosphorylation. Interestingly, transgenic mice overexpressing aconstitutive active variant of MK5 showed changes in anxiety-related behavior.ApplicationsCompounds that specifically modify the phosphorylation of septin 8 by modulatingthe activity of MK5 or that interfere with the interaction between MK5 andseptin 8 may have an impact on neuronal secretion and as such be used inpatients suffering from perturbed neurosecretion.Peer reviewOverall, this is a potentially interesting paper; however, the data presented arepreliminary.REFERENCES1 New L, Jiang Y, Zhao M, Liu K, Zhu W, Flood LJ, Kato Y,Parry GC, Han J. PRAK, a novel protein kinase regulated bythe p38 MAP kinase. EMBO J 1998; 17: 3372-33842 Ni H, Wang XS, Diener K, Yao Z. MAPKAPK5, a novelmitogen-activated protein kinase (MAPK)-activated proteinkinase, is a substrate of the extracellular-regulated kinase(ERK) and p38 kinase. Biochem Biophys Res Commun 1998;243: 492-4963 Gaestel M. MAPKAP kinases - MKs - two’s company, three’s a crowd. 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Genetic control of the cell division cycle inyeast. IV. Genes controlling bud emergence and cytokinesis.Exp Cell Res 1971; 69: 265-27618 Cao L, Yu W, Wu Y, Yu L. The evolution, complex structuresand function of septin proteins. Cell Mol Life Sci 2009; 66:3309-332319 Nishihama R, Onishi M, Pringle JR. New insights into thephylogenetic distribution and evolutionary origins of theseptins. Biol Chem 2011; 392: 681-68720 Hall PA, Russell SE. Mammalian septins: dynamic heteromerswith roles in cellular morphogenesis and compartmentalization.J Pathol 2012; 226: 287-29921 Oh Y, Bi E. Septin structure and function in yeast and beyond.Trends Cell Biol 2011; 21: 141-14822 Kinoshita M. Insight into septin functions from mouse models.In: Hall PA, Russell SEH, Pringle JR. The septins. WestSussex: Wiley-Blackwell, 2008: 319-33623 Ono R, Ihara M, Nakajima H, Ozaki K, Kataoka-FujiwaraY, Taki T, Nagata K, Inagaki M, Yoshida N, Kitamura T,Hayashi Y, Kinoshita M, Nosaka T. Disruption of Sept6, afusion partner gene of MLL, does not affect ontogeny, leukemogenesisinduced by MLL-SEPT6, or phenotype inducedby the loss of Sept4. Mol Cell Biol 2005; 25: 10965-1097824 Fujishima K, Kiyonari H, Kurisu J, Hirano T, Kengaku M.Targeted disruption of Sept3, a heteromeric assembly partnerof Sept5 and Sept7 in axons, has no effect on developingCNS neurons. J Neurochem 2007; 102: 77-9225 Dent J, Kato K, Peng XR, Martinez C, Cattaneo M, PoujolC, Nurden P, Nurden A, Trimble WS, Ware J. A prototypicplatelet septin and its participation in secretion. Proc NatlAcad Sci USA 2002; 99: 3064-3069WJBC|www.wjgnet.com108 May 26, 2012|Volume 3|Issue 5|

Shiryaev A et al . SEPT8 and MK526 Ihara M, Kinoshita A, Yamada S, Tanaka H, Tanigaki A,Kitano A, Goto M, Okubo K, Nishiyama H, Ogawa O, TakahashiC, Itohara S, Nishimune Y, Noda M, Kinoshita M.Cortical organization by the septin cytoskeleton is essentialfor structural and mechanical integrity of mammalian spermatozoa.Dev Cell 2005; 8: 343-35227 Ihara M, Yamasaki N, Hagiwara A, Tanigaki A, Kitano A,Hikawa R, Tomimoto H, Noda M, Takanashi M, Mori H,Hattori N, Miyakawa T, Kinoshita M. Sept4, a componentof presynaptic scaffold and Lewy bodies, is required for thesuppression of alpha-synuclein neurotoxicity. Neuron 2007;53: 519-53328 Liu M, Shen S, Chen F, Yu W, Yu L. Linking the septin expressionwith carcinogenesis. Mol Biol Rep 2010; 37: 3601-360829 Bläser S, Jersch K, Hainmann I, Zieger W, Wunderle D,Busse A, Zieger B. Isolation of new splice isoforms, characterizationand expression analysis of the human septinSEPT8 (KIAA0202). Gene 2003; 312: 313-32030 Sandrock K, Bartsch I, Bläser S, Busse A, Busse E, ZiegerB. Characterization of human septin interactions. Biol Chem2011; 392: 751-76131 Bläser S, Jersch K, Hainmann I, Wunderle D, Zgaga-GrieszA, Busse A, Zieger B. Human septin-septin interaction: CD-Crel-1 partners with KIAA0202. FEBS Lett 2002; 519: 169-17232 Bläser S, Horn J, Würmell P, Bauer H, Strümpell S, Nurden P,Pagenstecher A, Busse A, Wunderle D, Hainmann I, ZiegerB. The novel human platelet septin SEPT8 is an interactionpartner of SEPT4. Thromb Haemost 2004; 91: 959-96633 Beites CL, Campbell KA, Trimble WS. The septin Sept5/CD-Crel-1 competes with alpha-SNAP for binding to the SNAREcomplex. Biochem J 2005; 385: 347-35334 Zhang Y, Gao J, Chung KK, Huang H, Dawson VL, DawsonTM. Parkin functions as an E2-dependent ubiquitin- proteinligase and promotes the degradation of the synaptic vesicleassociatedprotein, CDCrel-1. 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J Biol Chem 2000; 275: 10047-1005648 Beausoleil SA, Villén J, Gerber SA, Rush J, Gygi SP. Aprobability-based approach for high-throughput proteinphosphorylation analysis and site localization. Nat Biotechnol2006; 24: 1285-129249 Trinidad JC, Thalhammer A, Specht CG, Lynn AJ, BakerPR, Schoepfer R, Burlingame AL. Quantitative analysis ofsynaptic phosphorylation and protein expression. Mol CellProteomics 2008; 7: 684-69650 Zanivan S, Gnad F, Wickström SA, Geiger T, Macek B, CoxJ, Fässler R, Mann M. Solid tumor proteome and phosphoproteomeanalysis by high resolution mass spectrometry. JProteome Res 2008; 7: 5314-532651 Beites CL, Peng XR, Trimble WS. Expression and analysis ofproperties of septin CDCrel-1 in exocytosis. Methods Enzymol2001; 329: 499-51052 Hsu SC, Hazuka CD, Roth R, Foletti DL, Heuser J, SchellerRH. Subunit composition, protein interactions, and structuresof the mammalian brain sec6/8 complex and septinfilaments. Neuron 1998; 20: 1111-112253 Shiryaev A, Moens U. Mitogen-activated protein kinase p38and MK2, MK3 and MK5: ménage à trois or ménage à quatre?Cell Signal 2010; 22: 1185-119254 Trang T, Beggs S, Wan X, Salter MW. P2X4-receptor-mediatedsynthesis and release of brain-derived neurotrophic factorin microglia is dependent on calcium and p38-mitogenactivatedprotein kinase activation. J Neurosci 2009; 29:3518-3528S- Editor Cheng JX L- Editor Kerr C E- Editor Zheng XMWJBC|www.wjgnet.com109 May 26, 2012|Volume 3|Issue 5|

W J B CWorld Journal ofBiological ChemistryOnline Submissions: http://www.wjgnet.com/1949-8454officewjbc@wjgnet.comwww.wjgnet.comWorld J Biol Chem 2012 May 26; 3(5): IISSN 1949-8454 (online)© 2012 Baishideng. All rights reserved.ACKNOWLEDGMENTSAcknowledgments to reviewers of World Journal ofBiological ChemistryMany reviewers have contributed their expertise andtime to the peer review, a critical process to ensure thequality of World Journal of Biological Chemistry. The editorsand authors of the articles submitted to the journal aregrateful to the following reviewers for evaluating thearticles (including those published in this issue and thoserejected for this issue) during the last editing time period.Antonio Brunetti, MD, PhD, Professor, Cattedra di Endocrinologia,Università di Catanzaro “Magna Græcia”, 88100 Catanzaro, ItalyWayne Grant Carter, PhD, School of Biomedical Sciences,University of Nottingham, Queen’s Medical Centre, Nottingham,NG7 2UH, United KingdomManuel Vazquez-Carrera, Dr., University of Barcelona, Unitatde Farmacologia. Facultat de Farmacia. Diagoanl 643,Barcelona E-08028, SpainHong-Gang Wang, PhD, Professor, Lois High BerstlerProfessor of Pharmacology, Penn State College of Medicine,Penn State Hershey Cancer Institute, CH74, 500 UniversityDrive, PO Box 850, Hershey, PA 17033-0850, United StatesWJBC|www.wjgnet.com May 26, 2012|Volume 3|Issue 5|

W J B CWorld Journal ofBiological ChemistryOnline Submissions: http://www.wjgnet.com/1949-8454officewjbc@wjgnet.comwww.wjgnet.comWorld J Biol Chem 2012 May 26; 3(5): IISSN 1949-8454 (online)© 2012 Baishideng. All rights reserved.MEETINGSEvents Calendar 2012January 10, 2012Annual Symposium-Frontiers inBiological CatalysisCambridge, United KingdomFebruary 1-2, 2012World Cancer Metabolism SummitWashington DC, WA 33601,United StatesFebruary 10-11, 20122012-Indo-Korean Conference onIntegrative Bioscience Research-Opportunities and ChallengesCoimbatore, IndiaFebruary 12, 20124th International Conference onDrug Discovery and TherapyDubai, United Arab EmiratesFebruary 19, 2012Applied PharmaceuticalAnalysis-IndiaAhmedabad, IndiaFebruary 20, 2012International Conference andExhibition on Metabolomics andSystems BiologySan Francisco, CA 95101,United StatesFebruary 20, 2012Healthcare India 2012New Delhi, IndiaFebruary 20, 2012Metabolomics2012Burlingama, CA 95101, United StatesFebruary 24, 201219th Annual Southeastern RegionalYeast Meeting 2012Atlanta, GA 30314, United StatesMarch 2-5, 2012Medicinal Chemistry Conference2012Lanzarote, SpainMarch 12, 2012Vaccine World SummitHyderabad, IndiaMarch 13, 2012ADME and Predictive ToxicologyMunich, GermanyMarch 19-22, 2012Society for Endocrinology: BES 2012Harrogate, United KingdomMarch 26-27, 2012Intrinsically disordered proteinsYork, United KingdomMarch 27, 2012RNAi2012: Gene Regulation bySmall RNAsOxford, United KingdomMarch 28, 2012LRRK2: Function and dysfunctionLondon, United KingdomMarch 28, 2012Advances in Microarray TechnologyConference and ExhibitionRiccarton, United KingdomApril 16, 2012Biologics World KoreaSeoul, South KoreaApril 23, 2012Flow Chemistry Congress andExhibitionBoston, MA 02110, United StatesApril 25, 2012European Algae BiomassLondon, United KingdomApril 30-May 03, 2012Association for Clinical Biochemistry2012Liverpool, United KingdomMay 5-9, 201215th International and14th European Congress ofEndocrinologyFlorence, ItalyMay 7-8, 2012LIPID MAPS Annual Meeting2012: Impact on Cell Biology,Metabolomics and TranslationalMedicineLa Jolla, CA 92093, United StatesMay 16, 201218th Annual International Stressand Behavior Neuroscience andBiopsychiatry Conference (NorthAmerica)Petersburg, FL 33063,United StatesJune 11, 2012Rab GTPases and their interactingproteins in health and diseaseCork, IrelandJuly 8-13, 2012BiocatalysisSmithfield, RI 02896, United StatesJuly 15-19, 20122012 AACC Annual MeetingLos Angeles, CA 90015,United StatesAugust 5-10, 2012Medicinal ChemistryNew London, NH 03257,United StatesAugust 18, 2012The 30th World Congress ofBiomedical Laboratory ScienceBerlin, GermanyAugust 18-22, 2012The 30th World Congress ofBiomedical Laboratory ScienceBerlin, GermanyAugust 25-29, 20129th International Symposium onBiomolecular ChemistryBeijing, ChinaSeptember 2-6, 201222nd International Symposium onMedicinal ChemistryBerlin, GermanySeptember 11-13, 2012Lipids and Membrane BiophysicsLondon, United KingdomSeptember 16, 201215th International BiotechnologySymposiumDaegu, South KoreaSeptember 25, 2012Molecular Diagnostics WorldCongress and ExhibitionSan Diego, CA 09963, United StatesNovember 5-9, 20127th International IUPAC Symposiumon Mycotoxins and PhycotoxinsRotterdam, NetherlandsWJBC|www.wjgnet.com May 26, 2012|Volume 3|Issue 5|

W J B CWorld Journal ofBiological ChemistryOnline Submissions: http://www.wjgnet.com/1949-8454officewjbc@wjgnet.comwww.wjgnet.comWorld J Biol Chem 2012 May 26; 3(5): I-VISSN 1949-8454 (online)© 2012 Baishideng. All rights reserved.INSTRUCTIONS TO AUTHORSGENERAL INFORMATIONWorld Journal of Biological Chemistry (World J Biol Chem, WJBC, onlineISSN 1949-8454, DOI: 10.4331), is a monthly, open-access (OA),peer-reviewed journal supported by an editorial board of 529 expertsin biochemistry and molecular biology from 40 countries.The biggest advantage of the OA model is that it provides free,full-text articles in PDF and other formats for experts and the publicwithout registration, which eliminates the obstacle that traditionaljournals possess and usually delays the speed of the propagation andcommunication of scientific research results. The open access modelhas been proven to be a true approach that may achieve the ultimategoal of the journals, i.e., the maximization of the value to the readers,authors and society.Maximization of personal benefitsThe role of academic journals is to exhibit the scientific levels ofa country, a university, a center, a department, and even a scientist,and build an important bridge for communication between scientistsand the public. As we all know, the significance of the publication ofscientific articles lies not only in disseminating and communicatinginnovative scientific achievements and academic views, as well as promotingthe application of scientific achievements, but also in formallyrecognizing the "priority" and "copyright" of innovative achievementspublished, as well as evaluating research performance and academiclevels. So, to realize these desired attributes of WJBC and create awell-recognized journal, the following four types of personal benefitsshould be maximized. The maximization of personal benefits refersto the pursuit of the maximum personal benefits in a well-consideredoptimal manner without violation of the laws, ethical rules and thebenefits of others. (1) Maximization of the benefits of editorial boardmembers: The primary task of editorial board members is to give apeer review of an unpublished scientific article via online office systemto evaluate its innovativeness, scientific and practical values anddetermine whether it should be published or not. During peer review,editorial board members can also obtain cutting-edge information inthat field at first hand. As leaders in their field, they have priority tobe invited to write articles and publish commentary articles. We willput peer reviewers’ names and affiliations along with the article theyreviewed in the journal to acknowledge their contribution; (2) Maximizationof the benefits of authors: Since WJBC is an open-accessjournal, readers around the world can immediately download andread, free of charge, high-quality, peer-reviewed articles from WJBCofficial website, thereby realizing the goals and significance of thecommunication between authors and peers as well as public reading;(3) Maximization of the benefits of readers: Readers can read or use,free of charge, high-quality peer-reviewed articles without any limits,and cite the arguments, viewpoints, concepts, theories, methods,results, conclusion or facts and data of pertinent literature so as tovalidate the innovativeness, scientific and practical values of their ownresearch achievements, thus ensuring that their articles have novelarguments or viewpoints, solid evidence and correct conclusion; and(4) Maximization of the benefits of employees: It is an iron law that afirst-class journal is unable to exist without first-class editors, and onlyfirst-class editors can create a first-class academic journal. We insiston strengthening our team cultivation and construction so that everyemployee, in an open, fair and transparent environment, could contributetheir wisdom to edit and publish high-quality articles, therebyrealizing the maximization of the personal benefits of editorial boardmembers, authors and readers, and yielding the greatest social andeconomic benefits.Aims and scopeThe major task of WJBC is to rapidly report the most recent developmentsin the research by the close collaboration of biologists andchemists in area of biochemistry and molecular biology, including:general biochemistry, pathobiochemistry, molecular and cellularbiology, molecular medicine, experimental methodologies and thediagnosis, therapy, and monitoring of human disease.ColumnsThe columns in the issues of WJBC will include: (1) Editorial: Tointroduce and comment on major advances and developments in thefield; (2) Frontier: To review representative achievements, commenton the state of current research, and propose directions for future research;(3) Topic Highlight: This column consists of three formats, including(A) 10 invited review articles on a hot topic, (B) a commentaryon common issues of this hot topic, and (C) a commentary on the 10individual articles; (4) Observation: To update the development of oldand new questions, highlight unsolved problems, and provide strategieson how to solve the questions; (5) Guidelines for Basic Research:To provide guidelines for basic research; (6) Guidelines for ClinicalPractice: To provide guidelines for clinical diagnosis and treatment;(7) Review: To review systemically progress and unresolved problemsin the field, comment on the state of current research, and make suggestionsfor future work; (8) Original Articles: To report innovativeand original findings in biochemistry and molecular biology; (9) BriefArticles: To briefly report the novel and innovative findings in biochemistryand molecular biology; (10) Case Report: To report a rareor typical case; (11) Letters to the Editor: To discuss and make reply tothe contributions published in WJBC, or to introduce and commenton a controversial issue of general interest; (12) Book Reviews: Tointroduce and comment on quality monographs of biochemistry andmolecular biology; and (13) Guidelines: To introduce Consensuses andGuidelines reached by international and national academic authoritiesworldwide on the research in biochemistry and molecular biology.Name of journalWorld Journal of Biological ChemistryISSNISSN 1949-8454 (online)Editor-in-chiefYin-Yuan Mo, PhD, Associate Professor, Medical Microbiology, Immunologyand Cell Biology, Southern Illinois University School ofMedicine, Springfield, IL 62702, United StatesEditorial officeWorld Journal of Biological ChemistryRoom 903, Building D, Ocean International Center,No. 62 Dongsihuan Zhonglu, Chaoyang District,Beijing 100025, ChinaTelephone: +86-10-85381892Fax: +86-10-85381893E-mail: wjbc@wjgnet.comhttp://www.wjgnet.comWJBC|www.wjgnet.com May 26, 2012|Volume 3|Issue 5|

Instructions to authorsIndexed and abstracted inPubMed Central, PubMed, Digital Object Identifier, and Directoryof Open Access Journals.Published byBaishideng Publishing Group Co., LimitedSPECIAL STATEMENTAll articles published in this journal represent the viewpoints of theauthors except where indicated otherwise.Biostatistical editingStatistical review is performed after peer review. We invite an expertin Biomedical Statistics from to evaluate the statistical method used inthe paper, including t-test (group or paired comparisons), chi-squaredtest, Ridit, probit, logit, regression (linear, curvilinear, or stepwise),correlation, analysis of variance, analysis of covariance, etc. The reviewingpoints include: (1) Statistical methods should be describedwhen they are used to verify the results; (2) Whether the statisticaltechniques are suitable or correct; (3) Only homogeneous data can beaveraged. Standard deviations are preferred to standard errors. Givethe number of observations and subjects (n). Losses in observations,such as drop-outs from the study should be reported; (4) Values suchas ED50, LD50, IC50 should have their 95% confidence limits calculatedand compared by weighted probit analysis (Bliss and Finney);and (5) The word ‘significantly’ should be replaced by its synonyms (ifit indicates extent) or the P value (if it indicates statistical significance).Conflict-of-interest statementIn the interests of transparency and to help reviewers assess any potentialbias, WJBC requires authors of all papers to declare any competingcommercial, personal, political, intellectual, or religious interestsin relation to the submitted work. Referees are also asked to indicateany potential conflict they might have reviewing a particularpaper. Before submitting, authors are suggested to read “UniformRequirements for Manuscripts Submitted to Biomedical Journals:Ethical Considerations in the Conduct and Reporting of Research:Conflicts of Interest” from International Committee of MedicalJournal Editors (ICMJE), which is available at: http://www.icmje.org/ethical_4conflicts.html.Sample wording: [Name of individual] has received fees for servingas a speaker, a consultant and an advisory board member for [namesof organizations], and has received research funding from [names oforganization]. [Name of individual] is an employee of [name of organization].[Name of individual] owns stocks and shares in [name oforganization]. [Name of individual] owns patent [patent identificationand brief description].Statement of informed consentManuscripts should contain a statement to the effect that all humanstudies have been reviewed by the appropriate ethics committeeor it should be stated clearly in the text that all persons gave theirinformed consent prior to their inclusion in the study. Details thatmight disclose the identity of the subjects under study should beomitted. Authors should also draw attention to the Code of Ethicsof the World Medical Association (Declaration of Helsinki, 1964,as revised in 2004).Statement of human and animal rightsWhen reporting the results from experiments, authors should followthe highest standards and the trial should conform to Good ClinicalPractice (for example, US Food and Drug Administration GoodClinical Practice in FDA-Regulated Clinical Trials; UK MedicinesResearch Council Guidelines for Good Clinical Practice in ClinicalTrials) and/or the World Medical Association Declaration of Helsinki.Generally, we suggest authors follow the lead investigator’s nationalstandard. If doubt exists whether the research was conductedin accordance with the above standards, the authors must explain therationale for their approach and demonstrate that the institutionalreview body explicitly approved the doubtful aspects of the study.Before submitting, authors should make their study approved bythe relevant research ethics committee or institutional review board.If human participants were involved, manuscripts must be accompaniedby a statement that the experiments were undertaken with theunderstanding and appropriate informed consent of each. Any personalitem or information will not be published without explicit consentsfrom the involved patients. If experimental animals were used,the materials and methods (experimental procedures) section mustclearly indicate that appropriate measures were taken to minimizepain or discomfort, and details of animal care should be provided.SUBMISSION OF MANUSCRIPTSManuscripts should be typed in 1.5 line spacing and 12 pt. BookAntiqua with ample margins. Number all pages consecutively, andstart each of the following sections on a new page: Title Page, Abstract,Introduction, Materials and Methods, Results, Discussion,Acknowledgements, References, Tables, Figures, and Figure Legends.Neither the editors nor the publisher are responsible for theopinions expressed by contributors. Manuscripts formally acceptedfor publication become the permanent property of BaishidengPublishing Group Co., Limited, and may not be reproduced by anymeans, in whole or in part, without the written permission of boththe authors and the publisher. We reserve the right to copy-edit andput onto our website accepted manuscripts. Authors should followthe relevant guidelines for the care and use of laboratory animalsof their institution or national animal welfare committee. For thesake of transparency in regard to the performance and reporting ofclinical trials, we endorse the policy of the ICMJE to refuse to publishpapers on clinical trial results if the trial was not recorded in apublicly-accessible registry at its outset. The only register now available,to our knowledge, is http://www.clinicaltrials.gov sponsoredby the United States National Library of Medicine and we encourageall potential contributors to register with it. However, in the casethat other registers become available you will be duly notified. Aletter of recommendation from each author’s organization shouldbe provided with the contributed article to ensure the privacy andsecrecy of research is protected.Authors should retain one copy of the text, tables, photographsand illustrations because rejected manuscripts will not be returnedto the author(s) and the editors will not be responsible for loss ordamage to photographs and illustrations sustained during mailing.Online submissionsManuscripts should be submitted through the Online SubmissionSystem at: http://www.wjgnet.com/1949-8454office. Authors arehighly recommended to consult the ONLINE INSTRUCTIONSTO AUTHORS (http://www.wjgnet.com/1949-8454/g_info_20100316155305.htm) before attempting to submit online. Forassistance, authors encountering problems with the Online SubmissionSystem may send an email describing the problem to wjbc@wjgnet.com, or by telephone: +86-10-85381892. If you submit yourmanuscript online, do not make a postal contribution. Repeatedonline submission for the same manuscript is strictly prohibited.MANUSCRIPT PREPARATIONAll contributions should be written in English. All articles must besubmitted using word-processing software. All submissions mustbe typed in 1.5 line spacing and 12 pt. Book Antiqua with amplemargins. Style should conform to our house format. Required informationfor each of the manuscript sections is as follows:Title pageTitle: Title should be less than 12 words.Running title: A short running title of less than 6 words shouldbe provided.Authorship: Authorship credit should be in accordance with theWJBC|www.wjgnet.comII May 26, 2012|Volume 3|Issue 5|

Instructions to authorsstandard proposed by ICMJE, based on (1) substantial contributionsto conception and design, acquisition of data, or analysis andinterpretation of data; (2) drafting the article or revising it criticallyfor important intellectual content; and (3) final approval of the versionto be published. Authors should meet conditions 1, 2, and 3.Institution: Author names should be given first, then the completename of institution, city, province and postcode. For example, Xu-Chen Zhang, Li-Xin Mei, Department of Pathology, ChengdeMedical College, Chengde 067000, Hebei Province, China. One authormay be represented from two institutions, for example, GeorgeSgourakis, Department of General, Visceral, and TransplantationSurgery, Essen 45122, Germany; George Sgourakis, 2nd SurgicalDepartment, Korgialenio-Benakio Red Cross Hospital, Athens15451, GreeceAuthor contributions: The format of this section should be:Author contributions: Wang CL and Liang L contributed equallyto this work; Wang CL, Liang L, Fu JF, Zou CC, Hong F and WuXM designed the research; Wang CL, Zou CC, Hong F and WuXM performed the research; Xue JZ and Lu JR contributed newreagents/analytic tools; Wang CL, Liang L and Fu JF analyzed thedata; and Wang CL, Liang L and Fu JF wrote the paper.Supportive foundations: The complete name and number ofsupportive foundations should be provided, e.g. Supported by NationalNatural Science Foundation of China, No. 30224801Correspondence to: Only one corresponding address should beprovided. Author names should be given first, then author title, affiliation,the complete name of institution, city, postcode, province,country, and email. All the letters in the email should be in lowercase. A space interval should be inserted between country name andemail address. For example, Montgomery Bissell, MD, Professor ofMedicine, Chief, Liver Center, Gastroenterology Division, Universityof California, Box 0538, San Francisco, CA 94143, United States.montgomery.bissell@ucsf.eduTelephone and fax: Telephone and fax should consist of +,country number, district number and telephone or fax number, e.g.Telephone: +86-10-85381892 Fax: +86-10-85381893Peer reviewers: All articles received are subject to peer review.Normally, three experts are invited for each article. Decision foracceptance is made only when at least two experts recommendan article for publication. Reviewers for accepted manuscripts areacknowledged in each manuscript, and reviewers of articles whichwere not accepted will be acknowledged at the end of each issue.To ensure the quality of the articles published in WJBC, reviewersof accepted manuscripts will be announced by publishing thename, title/position and institution of the reviewer in the footnoteaccompanying the printed article. For example, reviewers: ProfessorJing-Yuan Fang, Shanghai Institute of Digestive Disease, Shanghai,Affiliated Renji Hospital, Medical Faculty, Shanghai JiaotongUniversity, Shanghai, China; Professor Xin-Wei Han, Departmentof Radiology, The First Affiliated Hospital, Zhengzhou University,Zhengzhou, Henan Province, China; and Professor Anren Kuang,Department of Nuclear Medicine, Huaxi Hospital, Sichuan University,Chengdu, Sichuan Province, China.AbstractThere are unstructured abstracts (no less than 256 words) andstructured abstracts (no less than 480). The specific requirementsfor structured abstracts are as follows:An informative, structured abstracts of no less than 480 wordsshould accompany each manuscript. Abstracts for original contributionsshould be structured into the following sections. AIM (nomore than 20 words): Only the purpose should be included. Pleasewrite the aim as the form of “To investigate/study/…; MATERI-ALS AND METHODS (no less than 140 words); RESULTS (noless than 294 words): You should present P values where appropriateand must provide relevant data to illustrate how they were obtained,e.g. 6.92 ± 3.86 vs 3.61 ± 1.67, P < 0.001; CONCLUSION (no morethan 26 words).Key wordsPlease list 5-10 key words, selected mainly from Index Medicus,which reflect the content of the study.TextFor articles of these sections, original articles and brief articles, themain text should be structured into the following sections: INTRO-DUCTION, MATERIALS AND METHODS, RESULTS and DIS-CUSSION, and should include appropriate Figures and Tables. Datashould be presented in the main text or in Figures and Tables, but notin both. The main text format of these sections, editorial, topic highlight,case report, letters to the editors, can be found at: http://www.wjgnet.com/1949-8454/g_info_20100316160646.htm.IllustrationsFigures should be numbered as 1, 2, 3, etc., and mentioned clearlyin the main text. Provide a brief title for each figure on a separatepage. Detailed legends should not be provided under thefigures. This part should be added into the text where the figuresare applicable. Figures should be either Photoshop or Illustratorfiles (in tiff, eps, jpeg formats) at high-resolution. Examplescan be found at: http://www.wjgnet.com/1007-9327/13/4520.pdf; http://www.wjgnet.com/1007-9327/13/4554.pdf; http://www.wjgnet.com/1007-9327/13/4891.pdf; http://www.wjgnet.com/1007-9327/13/4986.pdf; http://www.wjgnet.com/1007-9327/13/4498.pdf. Keeping all elements compiled isnecessary in line-art image. Scale bars should be used rather thanmagnification factors, with the length of the bar defined in the legendrather than on the bar itself. File names should identify the figureand panel. Avoid layering type directly over shaded or texturedareas. Please use uniform legends for the same subjects. For example:Figure 1 Pathological changes in atrophic gastritis after treatment.A: ...; B: ...; C: ...; D: ...; E: ...; F: ...; G: …etc. It is our principleto publish high resolution-figures for the printed and E-versions.TablesThree-line tables should be numbered 1, 2, 3, etc., and mentionedclearly in the main text. Provide a brief title for each table. Detailedlegends should not be included under tables, but rather added intothe text where applicable. The information should complement,but not duplicate the text. Use one horizontal line under the title, asecond under column heads, and a third below the Table, above anyfootnotes. Vertical and italic lines should be omitted.Notes in tables and illustrationsData that are not statistically significant should not be noted. a P 0.05 should not be noted). Ifthere are other series of P values, c P < 0.05 and d P < 0.01 are used.A third series of P values can be expressed as e P < 0.05 and f P < 0.01.Other notes in tables or under illustrations should be expressed as1 F, 2 F, 3 F; or sometimes as other symbols with a superscript (Arabicnumerals) in the upper left corner. In a multi-curve illustration, eachcurve should be labeled with ●, ○, ■, □, ▲, △, etc., in a certain sequence.AcknowledgmentsBrief acknowledgments of persons who have made genuine contributionsto the manuscript and who endorse the data and conclusionsshould be included. Authors are responsible for obtainingwritten permission to use any copyrighted text and/or illustrations.REFERENCESCoding systemThe author should number the references in Arabic numerals ac-WJBC|www.wjgnet.comIII May 26, 2012|Volume 3|Issue 5|

Instructions to authorscording to the citation order in the text. Put reference numbers insquare brackets in superscript at the end of citation content or afterthe cited author’s name. For citation content which is part of thenarration, the coding number and square brackets should be typesetnormally. For example, “Crohn’s disease (CD) is associated withincreased intestinal permeability [1,2] ”. If references are cited directlyin the text, they should be put together within the text, for example,“From references [19,22-24] , we know that...”When the authors write the references, please ensure that theorder in text is the same as in the references section, and also ensurethe spelling accuracy of the first author’s name. Do not list the samecitation twice.PMID and DOIPleased provide PubMed citation numbers to the reference list, e.g.PMID and DOI, which can be found at http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed and http://www.crossref.org/SimpleTextQuery/,respectively. The numbers will be used in E-versionof this journal.Style for journal referencesAuthors: the name of the first author should be typed in bold-facedletters. The family name of all authors should be typed with the initialletter capitalized, followed by their abbreviated first and middleinitials. (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR). The title of the cited article and italicizedjournal title (journal title should be in its abbreviated form as shownin PubMed), publication date, volume number (in black), start page,and end page [PMID: 11819634 DOI: 10.3748/wjg.13.5396].Style for book referencesAuthors: the name of the first author should be typed in boldfacedletters. The surname of all authors should be typed with theinitial letter capitalized, followed by their abbreviated middle andfirst initials. (For example, Lian-Sheng Ma is abbreviated as Ma LS,Bo-Rong Pan as Pan BR) Book title. Publication number. Publicationplace: Publication press, Year: start page and end page.FormatJournalsEnglish journal article (list all authors and include the PMID where applicable)1 Jung EM, Clevert DA, Schreyer AG, Schmitt S, Rennert J,Kubale R, Feuerbach S, Jung F. Evaluation of quantitative contrastharmonic imaging to assess malignancy of liver tumors:A prospective controlled two-center study. World J Gastroenterol2007; 13: 6356-6364 [PMID: 18081224 DOI: 10.3748/wjg.13.6356]Chinese journal article (list all authors and include the PMID where applicable)2 Lin GZ, Wang XZ, Wang P, Lin J, Yang FD. Immunologiceffect of Jianpi Yishen decoction in treatment of Pixu-diarrhoea.Shijie Huaren Xiaohua Zazhi 1999; 7: 285-287In press3 Tian D, Araki H, Stahl E, Bergelson J, Kreitman M. Signatureof balancing selection in Arabidopsis. Proc Natl Acad Sci USA2006; In pressOrganization as author4 Diabetes Prevention Program Research Group. Hypertension,insulin, and proinsulin in participants with impaired glucosetolerance. Hypertension 2002; 40: 679-686 [PMID: 12411462PMCID:2516377 DOI:10.1161/01.HYP.0000035706.28494.09]Both personal authors and an organization as author5 Vallancien G, Emberton M, Harving N, van Moorselaar RJ;Alf-One Study Group. Sexual dysfunction in 1, 274 Europeanmen suffering from lower urinary tract symptoms. J Urol2003; 169: 2257-2261 [PMID: 12771764 DOI:10.1097/01.ju.0000067940.76090.73]No author given6 21st century heart solution may have a sting in the tail. BMJ2002; 325: 184 [PMID: 12142303 DOI:10.1136/bmj.325.7357.184]Volume with supplement7 Geraud G, Spierings EL, Keywood C. Tolerability and safetyof frovatriptan with short- and long-term use for treatmentof migraine and in comparison with sumatriptan. Headache2002; 42 Suppl 2: S93-99 [PMID: 12028325 DOI:10.1046/j.1526-4610.42.s2.7.x]Issue with no volume8 Banit DM, Kaufer H, Hartford JM. Intraoperative frozensection analysis in revision total joint arthroplasty. Clin OrthopRelat Res 2002; (401): 230-238 [PMID: 12151900 DOI:10.1097/00003086-200208000-00026]No volume or issue9 Outreach: Bringing HIV-positive individuals into care. HRSACareaction 2002; 1-6 [PMID: 12154804]BooksPersonal author(s)10 Sherlock S, Dooley J. Diseases of the liver and billiary system.9th ed. Oxford: Blackwell Sci Pub, 1993: 258-296Chapter in a book (list all authors)11 Lam SK. Academic investigator’s perspectives of medicaltreatment for peptic ulcer. In: Swabb EA, Azabo S. Ulcerdisease: investigation and basis for therapy. New York: MarcelDekker, 1991: 431-450Author(s) and editor(s)12 Breedlove GK, Schorfheide AM. Adolescent pregnancy.2nd ed. Wieczorek RR, editor. White Plains (NY): March ofDimes Education Services, 2001: 20-34Conference proceedings13 Harnden P, Joffe JK, Jones WG, editors. Germ cell tumours V.Proceedings of the 5th Germ cell tumours Conference; 2001Sep 13-15; Leeds, UK. New York: Springer, 2002: 30-56Conference paper14 Christensen S, Oppacher F. An analysis of Koza's computationaleffort statistic for genetic programming. In: Foster JA,Lutton E, Miller J, Ryan C, Tettamanzi AG, editors. Geneticprogramming. EuroGP 2002: Proceedings of the 5th EuropeanConference on Genetic Programming; 2002 Apr 3-5;Kinsdale, Ireland. Berlin: Springer, 2002: 182-191Electronic journal (list all authors)15 Morse SS. Factors in the emergence of infectious diseases.Emerg Infect Dis serial online, 1995-01-03, cited 1996-06-05;1(1): 24 screens. Available from: URL: http://www.cdc.gov/ncidod/eid/index.htmPatent (list all authors)16 Pagedas AC, inventor; Ancel Surgical R&D Inc., assignee.Flexible endoscopic grasping and cutting device and positioningtool assembly. United States patent US 20020103498. 2002 Aug1Statistical dataWrite as mean ± SD or mean ± SE.Statistical expressionExpress t test as t (in italics), F test as F (in italics), chi square test asχ 2 (in Greek), related coefficient as r (in italics), degree of freedomas υ (in Greek), sample number as n (in italics), and probability as P (initalics).UnitsUse SI units. For example: body mass, m (B) = 78 kg; blood pressure,p (B) = 16.2/12.3 kPa; incubation time, t (incubation) = 96 h,blood glucose concentration, c (glucose) 6.4 ± 2.1 mmol/L; bloodCEA mass concentration, p (CEA) = 8.6 24.5 mg/L; CO 2 volumefraction, 50 mL/L CO 2 , not 5% CO 2 ; likewise for 40 g/L formaldehyde,not 10% formalin; and mass fraction, 8 ng/g, etc. Arabicnumerals such as 23, 243, 641 should be read 23 243 641.WJBC|www.wjgnet.comIV May 26, 2012|Volume 3|Issue 5|

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