CELL INJURY,REPAIR,AGING,AND APOPTOSIS
Critical Role of FoxO3a in Alcohol-Induced Autophagy and Hepatotoxicity
Hong-Min Ni,*Kuo Du,*Min You,y and Wen-Xing Ding*
From the Department of Pharmacology,Toxicology,and Therapeutics,*University of Kansas Medical Center,Kansas City,Kansas;and the Department of Molecular Pharmacology and Physiology,y University of South Florida Health Sciences Center,Tampa,Florida
Accepted for publication August19,2013.
Address correspondence to Wen-Xing Ding,Ph.D., Department of Pharmacology, Toxicology,and Therapeutics, University of Kansas Medical Center,MS1018,3901 Rainbow Blvd.,Kansas City, KS66160.E-mail:wxding@ https://www.doczj.com/doc/784585657.html,.Autophagy is a lysosomal degradation process that degrades long-lived cellular proteins and damaged organelles as a critical cell survival mechanism in response to stress.We recently reported that acute ethanol induces autophagy,which then reduces ethanol-induced liver injury.However,the mechanisms by which ethanol induces autophagy are not known.In the present study,ethanol treatment signi?cantly increased both mRNA and protein levels of various essential autophagy-related genes in primary cultured mouse hepatocytes and in mouse liver.Both nuclear translocation of FoxO3a and expression of FoxO3a target genes were increased in ethanol-treated primary hepatocytes and mouse liver.Overexpression of a dominant negative form of FoxO3a inhibited ethanol-induced autophagy-related gene expression and enhanced ethanol-induced cell death in primary hepatocytes,which suggests that FoxO3a is a key factor in regulating ethanol-induced autophagy and cell survival.Resveratrol,a well-known SIRT1agonist, further enhanced ethanol-induced expression of autophagy-related genes,likely via increased deacety-lation of FoxO3a.Moreover,acute ethanol e treated Foxo3aà/àmice exhibited decreased autophagy-related gene expression,but enhanced steatosis and liver injury,compared with wild-type mice. FoxO3a thus plays a critical role in ethanol-induced autophagy in mouse liver.Modulating the FoxO3a autophagy pathway may offer novel therapeutic approaches for treating alcoholic liver pathogenesis. (Am J Pathol2013,183:1815e1825;https://www.doczj.com/doc/784585657.html,/10.1016/j.ajpath.2013.08.011)
Macroautophagy(hereafter referred to simply as autophagy) is a bulk intracellular degradation system that is mainly responsible for the degradation of long-lived proteins to provide nutrients for survival in response to starvation.1,2 Accumulating evidence indicates that autophagy can also selectively remove damaged or excess organelles,including mitochondria,endoplasmic reticulum,ribosome,and per-oxisome.3e5In the liver,autophagy can also help to remove excess lipid droplets to attenuate steatosis(in which case it is termed lipophagy).6
Alcoholic liver disease is a major disease of the liver in Western countries and worldwide.The clinical characteristic of this disease is the accumulation of excess fat in the liver in response to alcohol consumption.In humans,it is known that accumulation of excess fat can progress to more detrimental forms of liver injury,such as in?ammation,?brosis,and cirrhosis.However,it is also well known that only a small portion of alcohol drinkers can develop advanced liver in?ammation and?brosis,suggesting that a cellular protective mechanism or mechanisms could play a critical role in miti-gating alcohol-induced liver injury.7,8Our research group recently demonstrated that acute ethanol treatment induces autophagy in primary cultured mouse hepatocytes and in mouse liver;pharmacological induction of autophagy attenu-ated and pharmacological inhibition of autophagy exacerbated ethanol-induced steatosis and liver injury in mice.9However, the mechanisms by which acute ethanol induces autophagy in hepatocytes are not known.
FoxO3a is a member of the FoxO(forkhead box O) family of transcription factors.FoxO3a regulates expression of genes involved in multiple cellular functions,including oxidative stress,apoptosis,and cell-cycle transition,as well as DNA repair.10,11Recent evidence suggests that FoxO3a Supported in part by NIH grants R01-AA020518-01and P20-RR021940 (W.X.D),R01-AA015951and R01-AA013623(M.Y.),and P20-GM103549(COBRE grant to the University of Kansas Medical Center Cell Isolation Core laboratory).
Copyrighta2013American Society for Investigative Pathology. Published by Elsevier Inc.All rights reserved.
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also regulates expression of autophagy-related(Atg)genes in mouse skeletal muscle12,13and cardiomyocytes,which promotes cardiomyocyte survival on induction of oxidative stress.14
FoxO3a is regulated by multiple post-translational modi-?cations,including phosphorylation,acetylation,and ubiq-uitination.10,11FoxO3a is phosphorylated by the serine/ threonine protein kinase Akt and becomes sequestered in the cytoplasm,where it is unable to regulate gene expression.In contrast,SIRT1[sirtuin(silent mating type information regulation2homolog)1(S.cerevisiae)]deacetylates FoxO3a and modulates its gene transcription and speci?city,which increases antioxidative stress genes and suppresses apoptosis genes in response to oxidative stress.10,15However,it is not known whether acetylation of FoxO3a affects expression of autophagy-related genes.
In the present study,we demonstrated that acute ethanol treatment activates FoxO3a in mouse liver and in primary cultured mouse hepatocytes.The increased nuclear retention of FoxO3a by ethanol could be mediated by decreased Akt activity.Genetic inhibition of FoxO3a led to the suppression of ethanol-induced expression of autophagy-related genes and exacerbated ethanol-induced liver injury in mice.Moreover, we also found increased autophagy-related gene expression and autophagic?ux in ethanol-treated human hepatocytes.
Materials and Methods
Reagents
The following antibodies were used:Atg5(PM050)from MBL International(Woburn,MA);Beclin1(sc11427)and ULK1 (sc33182)from Santa Cruz Biotechnology(Santa Cruz,CA); Bnip3(Ab10433)and Nix(Ab8399)from Abcam(Cambridge, MA);p62(H00008878-M01)from Abnova(Walnut,CA); Atg7(2631),p-FoxO3a S253(9466),FoxO3a(2497),p-Akt S473 (4060),Akt(2966),Sirt1(2028),anti e acetylated-lysine (9441),and Lamin A/C(2032)from Cell Signaling Technol-ogy(Danvers,MA);and b-actin(A5441)and Atg14(A6358) from Sigma-Aldrich(St.Louis,MO).Horseradish perox-idase e conjugated or Cy3-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories(West Grove,PA).Rabbit polyclonal anti-LC3B antibody was generated as described previously.9Adenovirus GFP-LC3 (Ad-GFP-LC3),Ad-GFP,and adenovirus-dominant negative FoxO3a(Ad-DN-Foxo3a)were generated as described previ-ously.12,16Chloroquine(CQ),rapamycin,actinomycin D (ActD),and resveratrol were from Sigma-Aldrich.Ethanol was from Pharmco(Brook?eld,CT),and all other chemicals were from Sigma-Aldrich,Life Technologies(Carlsbad,CA),or Calbiochem(EMD Millipore,Billerica,MA).
Animals
Wild-type C57BL/6mice were purchased from the Jackson Laboratory(Bar Harbor,ME).Foxo3aà/àmice were generated as described previously.17Cryopreserved Foxo3at/àmouse embryos were purchased from the RIKEN BioResource Center (Ibaraki,Japan)and recovered at the Animal Transgenic Core facility at the University of Kansas Medical Center.The Foxo3at/àmice were maintained in a B6;129background. Female Foxo3aà/àmice are infertile;Foxo3aà/àmice were therefore generated by crossing male Foxo3at/àwith female Foxo3at/àmice.The generated Foxo3at/tmice were used as wild-type controls.
All animals received humane care according to the guidelines of the NIH and the University of Kansas Medical Center.
Mouse Ethanol Binge Treatment
Mouse ethanol treatment was modi?ed from the model of Carson and Pruett,18as we have described previously.9This model was designed to achieve blood alcohol levels,behavioral effects,and physiological effects comparable to those of human binge drinking.After6hours of fasting,male Foxo3aà/àmice and their wild-type littermates were administered33%(v/v) ethanol at a total cumulative dose of4.5g/kg body weight by four equally divided gavages at15-minute intervals.Control mice received the same volume of double-distilled water.After 6,12,and16hours of treatment,the mice were sacri?ced,and blood samples and liver tissues were collected.Liver injury was assessed by determination of serum alanine aminotransferase activity and H&E staining of liver sections,as we have de-scribed previously.19Total liver lysates were prepared using radioimmunoprecipitation assay buffer[1%NP40,0.5%so-dium deoxycholate,0.1%sodium dodecyl(lauryl)sulfate]. Primary Hepatocyte Culture
Mouse hepatocytes were isolated by a retrograde,nonre-circulating perfusion of livers with0.05%collagenase type IV (Sigma-Aldrich),as we have described previously.20Cells were cultured in Williams’medium E with10%fetal bovine serum,but no other supplements,for2hours to allow for attachment.Human hepatocytes were isolated and cultured according to methods described previously by Gramignoli et al.20All cells were maintained in a37 C incubator with5% CO2.All human liver specimens were obtained in accordance with the protocol(no.13513)approved by the University of Kansas Medical Center Human Subjects Committee. Immunoblot Assay
Cells were washed in PBS and lysed in radioimmunopre-cipitation assay buffer.From each sample,20m g of protein was separated by SDS-PAGE and transferred to polyvinylidene di?uoride membranes.The membranes were immunoblotted with primary antibodies,followed by secondary horseradish peroxidase-conjugated antibodies.The membranes were further developed with Pierce SuperSignal West Pico chemi-luminescent substrate(Thermo Fisher Scienti?c,Rockford,
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IL).Densitometry analysis was performed using Un-Scan-It software version6.1(Silk Scienti?c,Orem,UT).Densitometry data were further normalized with either b-actin or the respective total protein and expressed as means?SEM. qPCR
RNA was isolated from mouse livers using TRIzol reagent(Life Technologies)and was reverse-transcribed into cDNA with Fermentas RevertAid Reverse Transcriptase(Thermo Fisher Scienti?c,Waltham,MA).Quantitative real-time PCR(qPCR) to quantify gene expression of Atg5,Beclin1(Becn1;alias Atg6),Atg7,microtubule-associated protein1light chain3 beta(Map1lc3b;alias LC3b),Ulk1,BCL2/adenovirus E1B interacting protein3(Bnip3),and b-actin(Actb)was per-formed on an ABI Prism7900HT real-time PCR instrument (Life Technologies)using Fermentas Maxima SYBR Green/ ROX qPCR reagents(Thermo Fisher Scienti?c).The forward and reverse primer sequences were as follows:b-actin,50-TGTTACCAACTGGGACGACA-30and50-GGGGTGTT-GAAGGTCTCAAA-30;Atg5,50-GACCACAAGCAGCT-CTGGAT-30and50-GGTTTCCAGCATTGGCTCTA-30; Beclin1(Atg6),50-TGATCCAGGAGCTGGAAGAT-30and 50-CAAGCGACCCAGTCTGAAAT-30;Atg7,50-TCCGT-TGAAGTCCTCTGCTT-30and50-CCACTGAGGTTCAC-CATCCT-30;LC3B,50-CCGAGAAGACCTTCAAGCAG-30and50-ACACTTCGGAGATGGGAGTG-30;ULK1,50-GGTGGAGACCTGGCTGACTA-30and50-TCTTGACTC-GGATGTTGCTG-30;and Bnip3,50-AGCTTTGGCGAGA-AAAACAG-30and50-TCCAATGTAGATCCCCAAGC-30. Microscopy for Autophagy and Cell Death
To examine autophagy,primary mouse hepatocytes were seeded in a12-well plate(2?105cells per well)and infected overnight with Ad-GFP-LC3(100viral particles per cell).Cells were treated with80mmol/L ethanol in the presence or absence of0.1m g/mL ActD or with50m mol/L resveratrol for6hours. After treatment,cells were?xed with4%paraformaldehyde in PBS for2hours at room temperature or were kept at4 C for microscopy.Fluorescence images were acquired under an Eclipse200?uorescence microscope(Nikon,Tokyo,Japan) with MetaMorph software version6.1(Downingtown,PA). For the immunostaining assay,?xed primary hepatocytes or cryosections of liver tissues were immunostained with anti-FoxO3a antibody followed by Cy3-conjugated secondary antibody and Hoechst33342nuclear staining,as described previously.21Cell death and morphological changes were analyzed with1m g/mL Hoechst33342staining and?uores-cence imaging.All images were obtained using digital?uo-rescence microscopy as described above.
Nuclear Fractionation
Mouse liver nuclear proteins were extracted using Pierce NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scienti?c)according to the manufacturer’s instructions.In brief,liver tissues were cut into small pieces, homogenized in cytoplasmic extraction reagent I with sub-sequent addition of cytoplasmic extraction reagent II,and centrifuged.The supernatants resulting from centrifugation were cytoplasmic fractions.The insoluble pellets were suspended in nuclear extraction reagent and centrifuged;the supernatants resulting from this centrifugation contained nuclear proteins.
Hepatic Triglyceride Analysis
Triglyceride level was determined as described previously.9 Frozen liver tissues(50to100mg)were ground using a mortar and pestle.The crushed liver tissues were further incubated with1mL of a chloroform/methanol(2:1)mix with vigorous shaking for1hour at room temperature.Then, 200m L of double-distilled water was added into the mix and centrifuged for5minutes at3000?g.The lower lipid phase was collected and dried at room temperature,and the pellet was redissolved in a tert-butanol and Triton X-114/meth-anol(2:1)mix.Triglyceride analysis was performed with a colorimetric assay kit according to the manufacturer’s in-structions(Sigma-Aldrich).
Statistical Analysis
Experimental data were subjected to a Student’s t-test or one-way analysis of variance analysis with Scheffé’s post hoc test where appropriate.P<0.05was considered signi?cant. Results
Acute Ethanol Treatment Increases the Expression of Autophagy Genes in Mouse Liver and in Primary Cultured Mouse Hepatocytes
To investigate the expression of autophagy-related genes in mouse liver,male wild-type C57BL/6mice were adminis-tered4.5g/kg of water or ethanol for6,12,and16hours.This animal model,initially established by Carson and Pruett,18 has been widely used to mimic human binge drinking and acute alcohol abuse.22,23In this model,an ethanol dose of4to 6g/kg via gavage resulted in a peak blood ethanol concen-tration of43.4to86.8mmol/L,which can be commonly achieved in human binge drinkers.22Using this acute alcohol gavage model,we found that the mRNA levels of various autophagy-related genes,including Ulk1(yeast Atg1),Atg5, Becn1(yeast Atg6),Atg7,LC3b(yeast Atg8),Atg14,and Pik3c3(alias Vps34)were all increased in ethanol-treated mouse liver at all time points.Expression levels of most genes that we assessed(Ulk1,Becn1,LC3b,Atg14,and Vps34)peaked after6hours of treatment with ethanol (Figure1A).Expression levels of these autophagy-related genes started to decrease after12and16hours of treatment with ethanol,but were still signi?cantly higher than in
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controls (Figure 1A).Consistent with increased mRNA expression,ethanol treatment also increased Atg5,Beclin 1,Atg7,and LC3B-II protein levels;however,the extent of increase and the respective peak time varied.The expression levels of some proteins (such as Atg5)increased approxi-mately threefold,whereas expression levels of others (such as Atg7and Beclin 1)increased 60%to 80%,compared with control (Figure 1,B and C).It seems that Atg5,Beclin 1,and Atg14were maintained at higher levels during the treatment,whereas Atg7and LC3B-II increased at 6hours but then slightly declined at 12and 16hours of ethanol treatment.We have previously demonstrated an increase in autophagic ?ux induced by ethanol;in the presence of CQ,which blocks lysosomal degradation,ethanol-induced LC3B-II levels were further enhanced by CQ treatment.9Therefore,the decreased LC3B-II levels after prolonged ethanol treatment (12and 16hours)were likely due to autophagic degradation of LC3B-II.Consistent with in vivo ?ndings,we also found elevated mRNA levels of several autophagy-related genes in ethanol-treated primary mouse hepatocytes as early as 3hours and sustained to 16hours (Figure 1C).Moreover,ethanol treat-ment also increased Atg5,Beclin 1,Atg7,and LC3B-II protein levels in a time-dependent manner (Figure 1D).
ActD Suppresses Ethanol-Induced Expression of Autophagy-Related Proteins and GFP-LC3Puncta
To further investigate the role of gene transcription in ethanol-induced autophagy,we treated primary cultured mouse hepatocytes with ethanol in the presence or absence of ActD,a general transcription inhibitor.Beclin 1,Atg7,Bnip3,and LC3B-II protein levels induced by ethanol decreased in the presence of ActD (Figure 2A).Consistent
with our previous reports,9ethanol treatment increased the number of GFP-LC3puncta in primary cultured mouse hepatocytes,and these puncta were signi ?cantly diminished by ActD (Figure 2,B and C).More importantly,ActD also blocked ethanol-induced autophagic ?ux,as demonstrated by the decreased LC3B-II levels when cells were treated with ethanol together with ActD and CQ,compared with the cells treated with ethanol and CQ.Furthermore,ethanol treatment also decreased levels of p62,an autophagic sub-strate protein that is usually degraded by autophagy.Ethanol-induced decrease of p62was blocked by ActD,indicating that ActD inhibited ethanol-induced autophagic ?ux in hepatocytes (Figure 2D).These results suggest that ethanol-induced expression of autophagy-related genes could play an important role in ethanol-induced autopha-gosome formation and autophagic ?ux.
Ethanol Activates FoxO3a Both in Vivo and in Vitro
During mouse muscle atrophy,FoxO3a is activated to pro-mote autophagy by up-regulating autophagy-related genes,including LC3b ,Atg12,and Becn1.12In the present study,ethanol treatment increased Beclin 1,Atg7,Bnip3,and LC3B protein levels,and expression of these proteins was sup-pressed by ActD.We therefore investigated whether ethanol also activates FoxO3a and in turn increases the transcription of Atg genes in hepatocytes.FoxO3a signals were diffuse in both cytosol and nucleus in the liver of control mice,but the FoxO3a nuclear signal dramatically increased after ethanol treatment,indicating that ethanol increased nuclear FoxO3a retention in mouse liver (Figure 3A).Western blot analysis using nuclear fractions showed that ethanol treatment increased nuclear FoxO3a levels approximately
twofold
Figure 1Acute ethanol treatment increases the expression of autophagy-related genes in mouse liver and primary hepatocytes.A :Male C57BL/6mice were treated with 4.5g/kg water or ethanol by gavage for 6,12,or 16hours.Hepatic mRNA was isolated,and RT-qPCR was performed.B :Mice were treated as for A ,and total liver ly-sates were subjected to Western blot analysis,followed by densitometry analysis.C :Primary cultured mouse hepatocytes were treated with 80mmol/L ethanol for 3,6,9,and 16hours.mRNA was isolated from cultured hepatocytes,and RT-qPCR was performed.D :Primary cultured mouse hepatocytes were treated as for C ,and total cell lysates were subjected to Western blot anal-ysis,followed by densitometry analysis.Data are presented as means ?SEM (fold of control).n Z 4e 6(A );n Z 4(B and C );n Z 3(D ).*P <0.05,one-way analysis of variance with Scheffé’s post hoc test.
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(Figure 3B).Moreover,hepatic mRNA levels of three known FoxO3a target genes [Bnip3,Bim (reclassi ?ed as Bcl2l11),and manganese superoxide dismutase (Sod2;alias MnSOD )]also increased in ethanol-treated mouse liver (Figure 3C).Similar to the in vivo ?ndings,ethanol treatment also sig-ni ?cantly increased nuclear FoxO3a levels in primary cultured mouse hepatocytes (Figure 3,D and E).In addition,ethanol treatment also increased the expression of the known FoxO3a target genes Bnip3,Bnip3L ,and MnSOD in vitro (Figure 3F).These ?ndings indicate that ethanol activates FoxO3a in mouse liver and in primary cultured mouse hepatocytes.
Ethanol Induces Alterations of the Post-Translational Modi ?cations of FoxO3a in Mouse Liver
Because FoxO3a is regulated by multiple post-translational modi ?cations,including phosphorylation and acetylation,10we next investigated whether ethanol treatment affects phosphorylation and acetylation of https://www.doczj.com/doc/784585657.html,pared with control mice,ethanol treatment decreased the level of p-FoxO3a in mouse liver (Figure 4A).These ?ndings are consistent with our immunostaining data and nuclear Western blot analysis,showing that ethanol increased nu-clear FoxO3a (Figure 3A),because p-FoxO3a is liable to be exported to the cytosol from the nucleus.This decreased phosphorylation of FoxO3a could be due to decreased Akt phosphorylation,because Akt phosphorylates FoxOs to promote their nuclear exportation.11Indeed,the p-Akt levels in ethanol-treated mouse livers were decreased,compared with control (Figure 4A).In addition to phosphorylation,the transcriptional activity of FoxO3a can also be regulated by
Sirt1-mediated deacetylation.Ethanol treatment decreased hepatic Sirt1(Figure 4B),which was correlated with increased acetylated FoxO3a (Figure 4C).These data indi-cate that acute ethanol treatment decreases the level of phosphorylated FoxO3a,but increases acetylated FoxO3a;the latter is likely associated with a decrease in hepatic Sirt1induced by ethanol treatment.
Resveratrol Further Increases Ethanol-Induced Autophagy Gene Expression by Decreasing FoxO3a Acetylation
To investigate the in ?uence of acetylation of FoxO3a on ethanol-induced expression of autophagy-related genes,we treated primary mouse hepatocytes with ethanol in the presence or absence of resveratrol,a well-known Sirt1agonist 24that decreases ethanol-induced liver injury.25We found that resveratrol alone slightly increased the mRNA levels of Atg5,LC3b ,and Vps34,but the combined treat-ment of ethanol and resveratrol further increased ethanol-induced expression of these autophagy-related genes (Figure 5A).Ethanol-induced acetylation of FoxO3a was suppressed by resveratrol (Figure 5B),suggesting that deacetylated FoxO3a may further promote autophagy-related gene transcription.More importantly,in the pres-ence of resveratrol,the number of ethanol-induced GFP-LC3puncta was further increased,suggesting that resveratrol enhances ethanol-induced autophagosome formation (Figure 5,C and D).These ?ndings indicate that Sirt1-mediated deacetylation of FoxO3a may promote expres-sion of autophagy-related genes and autophagy induction in ethanol-treated
hepatocytes.
Figure 2
ActD inhibits ethanol-induced auto-phagy in primary mouse hepatocytes.A :Primary cultured hepatocytes were treated with ethanol in the presence or absence of ActD for 6hours.Total cell lysates were subjected to Western blot anal-ysis,followed by densitometry analysis.B :Ad-GFP-LC3e infected hepatocytes were treated as for A and were examined with ?uorescence microscopy.Original magni ?cation,?60.C :GFP-LC3puncta were quanti ?ed for each experiment,with at least 30cells counted in each experiment.D :Primary hepatocytes were treated as for A ,with or without 20m mol/L CQ for 6hours,followed by Western blotting and densitometry analysis.Data are expressed as means ?SEM.n Z 3(A and D );n Z 4(C ).**P <0.01,one-way analysis of variance with Scheffé’s post hoc test.
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Overexpression of a DN-FoxO3a Suppresses Ethanol-Induced Expression of Autophagy-Related Genes and Enhances Ethanol-Induced Apoptosis in Hepatocytes
To determine whether overexpression of DN-FoxO3a sup-presses ethanol-induced expression of autophagy-related
genes,we infected primary mouse hepatocytes with Ad-GFP control and Ad-DN-FoxO3a,which also contains GFP from a separate cytomegalovirus promoter.12More than 80%of cells showed very high ef ?ciency for the infection after 24hours of incubation (Figure 6A).After the cells were further treated with ethanol for another 6hours,ethanol-induced expression of Becn1,Atg7,LC3b ,and Atg12was inhibited by expression of DN-FoxO3a (Figure 6B).Consistent with the changes at the mRNA level,the ethanol-induced increases in Atg5(Atg12)and Atg6(Beclin 1)protein levels were also inhibited by DN-FoxO3a (Figure 6C).These results suggest that FoxO3a is respon-sible for ethanol-induced expression of autophagy-related genes.Furthermore,overexpression of DN-FoxO3a inhibited ethanol-induced autophagic ?ux in primary he-patocytes (Figure 6D).Interestingly,we also found that CQ treatment alone only mildly increased LC3B-II levels in DN-FoxO3a e infected cells (1.6versus 1),suggesting that DN-FoxO3a could also inhibit basal autophagy in hepato-cytes (Figure 6D).We previously found that inhibition of autophagy led to increased cell death and liver injury in ethanol-treated primary hepatocytes and mice.
9
Figure 3
Ethanol activates FoxO3a in mouse liver and primary mouse hepatocytes.A :Male C57BL/6mice were treated with 4.5g/kg water or ethanol by gavage for
16hours.Cryosectioned liver tissue was subjected to immunostaining for FoxO3a using a rabbit anti-FoxO3a antibody,followed by a Cy3-conjugated goat anti-rabbit antibody;nuclei were stained with Hoechst 33342.Representative ?uorescence microscopy images are shown.Boxed regions are shown at higher magni ?cation at the right .B :Mice were treated as for A ,and nuclear fractions were isolated from control and ethanol-treated mouse liver,followed by Western blotting and densitometry analysis.C :Hepatic mRNA was isolated,and RT-qPCR was performed.D :Primary cultured hepatocytes were treated with 80mmol/L ethanol for 6hours and ?xed in 4%paraformaldehyde,then immunostained for FoxO3a as for A ,and examined with ?uorescence microscopy.Representative images are shown.E :Cells with increased nuclear FoxO3a staining were quanti ?ed for each experiment,with at least 100cells counted in each experiment.F :mRNAs were isolated from cultured hepatocytes,and RT-qPCR was performed.Data are presented as means ?SEM.n Z 3(A ,B ,D ,E ,and F );n Z 4e 6(C ).*P <0.05,Student ’s t -test;**P <0.01,Z -test.
GAPDH FoxO3a p-FoxO3a
Control
EtOH
p-Akt S473
Akt
Sirt1
β-Actin
Control EtOH
Acetyl-FoxO3a
FoxO3a
Control
EtOH
1±0.4 0.6±0.3
1±0.2 0.5±0.2
1±0.5 13.2±0.5
1±0.2 0.4±0.1
Figure 4Ethanol induces post-translational modi ?cation of FoxO3a in mouse liver.A and B :C57BL/6mice were treated with 4.5g/kg water or ethanol by gavage for 16hours.Total liver lysates were subjected to Western blot analysis for phosphorylated and total FoxO3a and phosphorylated and total Akt (A )and Sirt1(B ),followed by densitometry analysis.C :Mice were treated as for A ,and total liver lysates were immunoprecipitated with an anti-FoxO3a anti-body,followed by Western blotting using an anti e acetylated lysine antibody and densitometry analysis.Data are expressed as means ?SEM.n Z 4.
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Interestingly,overexpression of DN-FoxO3a also enhanced ethanol-induced apoptosis in primary mouse hepatocytes (Figure 6E).Therefore,the increased cell death caused by expression of DN-FoxO3a could be due to the inhibition of
ethanol-induced expression of autophagy-related genes and subsequent autophagy.
Ethanol-Induced Expression of Autophagy-Related Genes Is Suppressed in Foxo3a à/àMice,Resulting in Increased Liver Injury and Steatosis
To further investigate the role of FoxO3a in ethanol-induced expression of autophagy-related genes and liver injury in vivo ,we treated Foxo3a à/àand age-matched wild-type mice with acute ethanol.Although the basal expression of autophagy genes in Foxo3a à/àmice did not differ sig-ni ?cantly from that of wild-type mice,ethanol-induced expression of autophagy-related genes was signi ?cantly suppressed in Foxo3a à/àmice (Figure 7A).The expression levels of several autophagy genes in ethanol-treated FoxO3a wild-type mice (B6;129background),such as Becn1and Atg7,were not induced as signi ?cantly as in ethanol-treated C57BL/6J wild-type mice (Figure 1A).These variations could be due to the difference in genetic background.Similar to ?ndings at the mRNA level,several of the ethanol-induced autophagy proteins were also suppressed in Foxo3a à/àmice (Figure 7D).More importantly,we further found that serum alanine aminotransferase and hepatic tri-glyceride levels were all signi ?cantly increased in ethanol-treated Foxo3a à/àmice,compared with wild-type mice (Figure 7,B and C).Notably,electron microscopy studies revealed that the number of lipid droplets in ethanol-treated Foxo3a à/àmouse liver was signi ?cantly higher than that of wild-type mice,whereas the number of autophagosomes was signi ?cantly decreased in ethanol-treated Foxo3a à/
à
Figure 5
Resveratrol enhances ethanol-induced expression of autophagy-related genes.A :Primary cultured mouse hepatocytes were treated with ethanol in the presence or absence of 50m mol/L resveratrol (Res)for 6hours.mRNA was isolated from cultured hepatocytes before RT-qPCR was performed.B :Total cell lysates were immunoprecipitated with an anti-FoxO3a antibody,followed by Western blotting using an anti e acetylated lysine antibody and densitometry analysis.C :Ad-GFP-LC3e infected hepatocytes were treated as for A and then were examined with ?uorescence microscopy.Representative GFP-LC3images are shown.Original magni ?cation,?60.D :The number of GFP-LC3puncta per cell was quanti ?ed and twenty cells were counted in each experi-ment.Data are expressed as means ?SEM.n Z 3(A and B );n Z 4(C and D ).*P <0.05,one-way analysis of variance with Scheffé’s post hoc
test.
Figure 6Overexpression of DN-FoxO3a suppresses ethanol-induced expression of autophagy-related genes,but enhances ethanol-induced apoptosis.A :Representative ?uorescence images of primary cultured mouse hepatocytes,infected with 10multiplicity of infection (MOI)Ad-GFP or Ad-DN-FoxO3a,which contain a GFP using separate cytomegalovirus promoters for 24hours.Original magni ?cation,?20.B :Primary hepatocytes,infected as in A ,were treated with 80mmol/L ethanol for 6hours.mRNA was isolated from cultured hepatocytes,and RT-qPCR was performed.C :Total cell lysates were subjected to Western blotting and densitometry analysis.D :Primary hepatocytes were treated as in B ,with or without 20m mol/L CQ,for 6hours.Total lysates were subjected to Western blotting and densitometry analysis.E :Primary hepatocytes,infected as in A ,were treated with 80mmol/L ethanol for 24hours.Cells were then stained with Hoechst 33342for nuclei,and fragmented apoptotic nuclei were quanti ?ed.Data are expressed as means ?SEM.n Z 3.*P <0.05,one-way analysis of variance with Scheffé’s post hoc test.
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mouse liver,compared with wild-type mice (Figure 8).These ?ndings suggest that the decreased expression of autophagy-related genes and autophagy in Foxo3a à/àmice may contribute to the increased steatosis and liver injury after ethanol treatment.
Ethanol Induces Expression of Autophagy-Related Genes and Autophagic Flux in Primary Human Hepatocytes
Given that we observed ethanol-induced expression of autophagy-related genes (Figure 1)and autophagic ?ux in primary mouse hepatocytes,9we next asked whether ethanol
induces similar effects in human hepatocytes.As with mouse hepatocytes,ethanol treatment increased the expression of several autophagy-related genes (Figure 9A),as well as autophagic ?ux,as demonstrated by further increased levels of LC3B-II in the presence of CQ,compared with either ethanol or CQ treatment alone (Figure 9B).
Discussion
The FoxO family of transcription factors regulates multiple cellular processes (including apoptosis,cell cycle,DNA damage repair,oxidative stress,and glucose metabolism)
and
Figure 7
Foxo3a à/àmice have decreased
expression of autophagy-related genes and increased liver injury after ethanol administration.A :Male Foxo3a à/àmice and their wild-type lit-termates were treated with 4.5g/kg water or ethanol by gavage for 6hours.Hepatic mRNA was isolated,and RT-qPCR was performed.B and C :Mice were treated as in A ,and serum alanine aminotransferase (ALT)(B )and hepatic tri-glycerides (TG)(C )were measured.D :Mice were treated as in A ,and total liver lysates were sub-jected to Western blotting and densitometry anal-ysis.Data are expressed as means ?SEM.n Z 4e 6(A e C );n Z 4(D ).*P <0.05,one-way analysis of variance with Scheffé’s post hoc
test.
Figure 8
Foxo3a à/àmice have decreased
number of hepatic lipid droplets and autophago-somes after ethanol treatment.Wild-type and Foxo3a à/àmice were treated with ethanol as re-ported for Figure 7.A :Representative electron microscopy images are shown.An autophagosome (boxed )is shown at higher magni ?cation to the right .The numbers of lipid droplets (B )and autophagosomes (C )per 100-m m 2cytosolic areas were quanti ?ed.Data are expressed as means ?SEM (>20cell sections).*P <0.05,one-way analysis of variance with Scheffé’s post hoc test.Scale bar Z 500nm.AV,autophagosome;LD,lipid droplet;M,mitochondria;N,nucleus.
Ni et al
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is highly conserved,from Caenorhabditis elegans to mam-mals.10,11The FoxO family has been shown to regulate autophagy in various systems,including skeletal muscle,12,13cardiomyocytes,14kidney,26liver,27and cancer cells.28Depending on the stress conditions,autophagy can be acti-vated as a cell-survival mechanism,either to provide necessary nutrients on nutrient deprivation or to remove cellular damaged or excess organelles and protein aggregates.1,2We have previously demonstrated that acute ethanol treatment induces autophagy as a compensatory mechanism to remove hepatic lipid droplets and damaged mitochondria to attenuate ethanol-induced liver steatosis and injury in mice.9,29How-ever,it was not known how acute ethanol modulates hepatic autophagy in mice.Here,we show that acute ethanol treatment promoted nuclear localization of FoxO3a in both primary cultured mouse hepatocytes and in mouse liver.In primary cultured hepatocytes,overexpression of DN-FoxO3a inhibited ethanol-induced up-regulation of autophagy genes and enhanced ethanol-induced cell death.Moreover,acute ethanol
treatment led to enhanced steatosis and liver injury in Foxo3a à/àmice,compared with wild-type mice.
It has been suggested that cytosolic FoxO1,particularly the acetylated form,can bind with Atg7to promote auto-phagy in certain cancer cells,independent of its transcrip-tional activity.28In contrast,it has been demonstrated that the transcriptional activities of FoxOs are required for the induction of autophagy in normal tissues,such as mouse skeletal muscle,12,13heart 14and liver.27In the present study,FoxO3a-mediated transcription of autophagy-related genes played a critical role in alcohol-induced autophagy in mouse liver and primary cultured hepatocytes.This notion is sup-ported by the following evidence:i)ethanol treatment increased the nuclear levels of FoxO3a;ii)suppression of ethanol-mediated expression of autophagy-related genes by ActD inhibited ethanol-induced autophagy;iii)over-expression of DN-FoxO3a inhibited ethanol-induced ex-pression of autophagy-related genes,resulting in reduced autophagy and increased cell death;and iv)Foxo3a à/àmice exhibited decreased expression of autophagy genes in ethanol-treated mouse liver,but increased steatosis and liver injury.Currently,it is not clear why FoxO-mediated tran-scription of autophagy genes is important for the autophagy process in normal tissues,but cytosolic FoxOs are important for autophagy induction in cancer cells.One possible ex-planation is that activation of Akt is one of the most frequent alterations observed in human cancer cells,and Akt acti-vation may lead to increased cytosolic FoxO levels because of increased Akt-mediated phosphorylation of FoxOs.
The activity of FoxO3a is largely regulated by its multiple post-translational modi ?cations,including phosphorylation,acetylation,ubiquitination,and methylation.10,11FoxOs are phosphorylated by the serine/threonine protein kinase Akt and become sequestered in the cytoplasm,where they are unable to regulate gene expression.11Acute ethanol treat-ment decreased the level of phosphorylated Akt and FoxO3a.These ?ndings suggest that acute ethanol-induced nuclear retention of FoxO3a could be mediated by the Akt signaling pathway.In contrast to negative regulation of FoxOs by phosphorylation,the acetylation of FoxOs seems to be more important for determination of speci ?city of FoxO target genes.15FoxO acetylation levels are regulated through acetylation by acetyltransferases (eg,p300/CBP)and through deacetylation by deacetylases (eg,SIRT1).10,30SIRT1deacetylates FoxO3a and modulates its gene tran-scription and speci ?city,which increases the expression of oxidative stress genes and suppresses the expression of apoptosis genes in response to oxidative stress.10,15Ethanol is reported to decrease SIRT1expression or activity by increasing the NADH/NAD tratio in hepatocytes.31In agreement with these reports,we also found that acute ethanol treatment reduced Sirt1proteins in mouse liver,but increased acetylation of FoxO3a.However,ethanol in-creased the expression of autophagy-related genes in primary hepatocytes and mouse liver,suggesting that ethanol-induced nuclear translocation of FoxO3a could be a
critical
Figure 9
Ethanol induces autophagy-related gene expression and
autophagic ?ux in primary human hepatocytes.A :Primary human he-patocytes were treated with 80mmol/L ethanol for 3or 6hours.mRNA was isolated,and RT-qPCR was performed.B :Primary human hepatocytes were treated with 80mmol/L ethanol in the presence or absence of 20m mol/L CQ for 6hours.Total cell lysates were subjected to Western blotting and densitometry analysis.C :A proposed model for the role of FoxO3a in ethanol-induced autophagy and liver injury.In ethanol-exposed hepatocytes,ethanol is metabolized by hepatic enzymes,which leads to an increase in the NADH/NAD tratio and to subsequent inhibition of Sirt1.Ethanol also leads to AKT inhibition,resulting in FoxO3a dephosphorylation and nuclear retention,which increases the expression of autophagy-related genes and autophagy.Resveratrol ac-tivates Sirt1,which leads to FoxO3a deacetylation and enhances expression of autophagy-related genes.FoxO3a-mediated autophagy pro-tects against ethanol-induced liver injury,likely by removal of ethanol-induced accumulation of lipid droplets and damaged mitochondria.Data are expressed as means ?SEM.n Z 4(A );n Z 3(B ).*P <0.05,one-way analysis of variance with Scheffé’s post hoc test.
FoxO3a in Alcohol-Induced Autophagy
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factor,and that this translocation is suf?cient to increase
FoxO3a-mediated expression of autophagy-related genes.
The?nding that resveratrol further enhanced ethanol-
induced up-regulation of autophagy genes suggests that
Sirt1-mediated deacetylation of FoxO3a may favor and
promote expression of autophagy-related genes.Further
studies are needed to determine whether the deacetylated
form of FoxO3a has increased binding af?nity for the pro-
moters of autophagy-related genes.Nevertheless,our present ?ndings collectively indicate that FoxO3a is a major target in the regulation of ethanol-induced autophagy-related gene
expression and autophagy.
The present results support a novel mechanism for FoxO-
mediated hepatoprotection against acute ethanol adminis-
tration in mice through activation of expression of
autophagy-related genes.This notion is supported by the
observation that overexpression of DN-FoxO3a enhanced
ethanol-induced apoptosis and that Foxo3aà/àmice
exhibited exacerbated liver injury and steatosis after acute
ethanol administration.Our results are also consistent with a
recent report that overexpression of Atg14,a target gene of
FoxOs,decreases hepatic triglyceride contents.27Another
signi?cant?nding from our present study was that ethanol
also increased autophagy-related gene expression and
autophagic?ux in primary human hepatocytes.However,in
addition to regulating autophagy-related genes,FoxO3a also
regulates expression of genes that have multiple functions,
such as antioxidant and DNA repair genes,which could also
attenuate ethanol-induced liver injury.30
Controversial evidence suggests that autophagy status
could differ between chronic and acute ethanol treatment in
mice.32,33Although it has been suggested that autophagy
could be decreased in the chronic ethanol feeding model,the
evidence to support this argument is still not clear,because
autophagic?ux data in these models are lacking(probably
because the dynamic nature of the autophagic process and of
the constitutive synthesis and turnover of LC3makes it
dif?cult to assess autophagic?ux in this model).In a recent
study by Lin et al,34autophagic?ux analysis was performed
after the mice were fed a Lieber e DeCarli liquid diet;the
results indicated increased autophagic?ux after chronic
ethanol feeding,at least at the4-week time point.These re-
sults were generally consistent with the acute alcohol binge
model.Although more careful autophagic?ux assays should
be performed(and with multiple time points)to further
dissect the autophagy process in the chronic ethanol feeding
model,the Lin et al34study also demonstrated that pharma-
cological promotion of autophagy by rapamycin and carba-
mazepine attenuated ethanol-induced liver injury in this
4-week feeding model.Regardless of the acute or chronic
ethanol treatment,therefore,our?ndings,taken together with
those of others,suggest that autophagy modulation could be
one of the ideal approaches to treat alcoholic liver diseases.
Although modulation of Akt or Sirt1may indirectly enhance
FoxO3a activity,the speci?c pharmacological activators for
FoxOs remain to be identi?ed.Developing speci?c FoxO activators will be a challenge,but could lead to new ap-proaches for treatment of alcoholic diseases.
In summary,our data suggest that FoxO3a-mediated auto-phagy-related gene expression plays a critical role in acute ethanol-induced autophagy.Genetic loss of FoxO3a enhanced acute ethanol-induced liver injury and steatosis in mice.The cellular and molecular events regulating ethanol-induced FoxO3a-mediated autophagy are summarized in Figure9C. Acknowledgments
We thank Drs.Jinghui Zhao and Alfred L.Goldberg (Harvard Medical School)for the reagents,Jessica Williams for critical reading of this manuscript,Dr.Melissa Larson [University of Kansas Medical Center(KUMC)Transgenic Core Facility]for the recovery of FoxO3a knockout mice from cryopreserved embryos,and Ken Dorko for providing the human hepatocytes.The hepatocytes used in this study were derived from samples collected and provided by the KUMC Department of Pharmacology,Toxicology and Therapeutics Cell Isolation Core lab and the University of Kansas Liver Center.
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FoxO3a in Alcohol-Induced Autophagy
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引言 本次主要是简要的介绍年产10万吨10度淡色啤酒厂的工厂设计。它主要包括啤酒厂的规划，啤酒工艺计算，啤酒厂资金的估算等方面的内容。一个年产量10万吨啤酒厂主要车间平面图及项目工艺方案的设计原则、方法、程序、设备等等。本次设计一共画三张图：全厂平面布置图、工艺流程图、车间工艺布置图。 1 厂址的选择 根据我国的具体情况，食品工厂一般建在距原产地附近大中城市的郊区。由于啤酒属于消费性强的休闲饮品，为了有利于销售，所以选择建于市区比较合适。这样不但可以获得足够的原料，而且利于产品的销售，同时还可以减少运输费用。 厂址选择的原则 （1）厂址的位置要符合城市规划（供气、供电、给排水、交通运输等）和工厂 对环境的要求。 （2）厂址地区要接近原料基地和产品销售市场，还要接近水源和能源。（3）具有良好的交通运输条件。 （4）场地有效利用系数高，并有远景规划的最终总体布局。 （5）有一定的施工条件和投产后的协作条件。
（6）厂址选择要有利于三废处理，保证环境卫生[1]。 1．2自然条件及能源 根据食品工厂厂址选择的要求，将啤酒厂建于淮安市郊区内。厂址地势平坦，周围无污染源，符合标准。场地面积有利于合理布置，符合工厂发展需要，并有一定扩建余地。该地自来水使用方便，且水质良好，可不用地下水，减少处理费用。接近排水系统，有利废水排放。供电系统也配备良好，可以满足生产需要。附近有居民区和学校，方便销售。 1．3政治经济和交通 该地区在城市规划区内，经规划部门批准，符合规划布局。并且接近销售渠道，有良好的经济开发前景。附近有发达的交通运输条件，接近高速公路，使原料入厂和啤酒出厂顺利进行。 2 总平面设计 2．1 总品面设计原则 （1）符合生产工艺要求。 （2）布置紧凑合理，节约用地，同时为长期发展留有余地。 （3）必须满足食品工厂卫生要求和食品卫生要求。 （4）优化建筑物间距，按有关规划进行设计。 （5）适合运输要求。
有时造句大全 [标签:栏目] ，有时造句大全 1、人们有时候是在做正确的决定，有时候是在证明决定正确。 2、家不只是房子，更重要的是人。有时候你会想方设法的离开他，有时候你却要不顾一切地回归他的怀抱。 3、再高的人有时也需踮足，再矮的人有时也需屈身。 4、人生啊，是这样不可预测，没有永恒的痛苦，也没有永恒的幸福，生活象流水一般，有时是那么平展，有时又是那么曲折。 5、使你惨败的，有时是你的朋友；使你成功的，有时是你的敌手。我们应学会感恩；更应该学会感怨。 6、这雨让我懂得了，父母是爱我么的，虽然有时很罗嗦，虽然有时脾气会很大，但是他们是爱我们的。 7、爱情是缘，就像瞌睡碰到了枕头，久旱遇到了雨天；爱情是思念，有时想着会失眠，有时梦里笑得甜。愿你爱情甜蜜！ 8、没有比时间更容易浪费的，同时没有比时间更珍贵的了，因为没有时间我们几乎无法做任何事。 9、一阵春风吹来，小草跳起欢快的舞蹈，有时舒展双臂，有时弯腰触地，有时左右摇晃，有时拥抱春风，真是姿态万千。 10、天鹅们同临一乱湖水，有时心怀幽情，咯守规行，有时也会意会神，雌雄彼此呵护。 11、月亮有时候象圆盘，有时候像西瓜，有时候象镰刀，有时候又像个光环。 12、炒股和赛车一样，有时候要停，有时候要冲，不要永远想着冲那样会撞死的。 13、最高明的骗子，可能在某个时刻欺骗所有人，也可能在所有时刻欺骗某些人，但不可能在所有时刻欺骗所有的人。 14、天空由于多变化，又是晴朗的天空是蓝蓝的，有时天空是黑沉沉的，有时天空像披上了雪白的衣服。 15、世界上没有原则，只有世故，没有法律，只有时势，高明的人同世故跟
时事打成一片，任意支配。 16、身体偶尔的背叛可以原谅，心灵的长期背叛不能容忍。有时要难得糊涂，有时要当机立断，这是婚姻的大智慧。 17、天空中的白云，有时像飞腾的巨龙，有时如威武的雄狮，有时又似奔腾的骏马。 18、小明正在做回家作业，只见他有时抓耳挠腮，有时写写画画，正急得满头大汗。 19、含羞草的**力是不可抵。有时，它的魅力甚至比牡丹还强，它的风格是典雅的，它的性格是独一无二的！含羞草，是真正值得我去爱的。 20、天上的云彩变化多端，有时候像一匹骏马在奔驰，有时候像一条狗在摆头摇尾，有时候像一只鸡在找食，有时候又像一头大象在喝水。 21、**常常走向自己的反面：吝啬有时导致挥霍，挥霍有时导致吝啬；我们常常经由软弱而达到坚强，经由怯懦而达到勇敢。 22、春雨，有时像飘渺的云烟，有时像如丝的针线，有时像密织的罗莎。 23、我的爸爸长着乌黑的头发，浓浓的眉毛下长着一双大大的眼睛，唱闪烁着智慧和机敏的神采，有时还使人感到几分诙谐和幽默。 24、对小钱不要过分去计较。金钱是生着羽翼的东西，有时它会自行飞去，有时必须将它放出去，才能带更多回来。 25、鸟们有时在天空中展翅高飞，有时停在树枝上婉转啼叫，有时在林间欢蹦乱跳。 26、我课余生活很丰富，有时看书，有时跳绳，有的画画。 27、春雨有时像牛毛，有时像花针，有时像细丝，像细小的珍珠，有时像细小的尘埃。 28、我很好奇，有时候我仿佛尝到了爱的味道，有时候爱很甜。 29、伤心有时是一种动力，失望有时是一种解脱，执迷不悟有时是一种磨练。 30、有时候相爱是一种无奈，有时候离开是另一种安排。为了爱你和你爱的人，请不要吸烟。 31、有时可能别人不在乎你，但你不能不在乎自己。 32、金翅雀唱着、跳跃着，有时也扑打着，像一群不知疲倦的孩子，给这幽
第八章 毒品成瘾的教育矫治 第一节 毒品成瘾教育矫治的基础理论教育是毒品成瘾矫治的重要手段，也是毒品成瘾矫治的核心内容之一。毒品成瘾教育矫治是从社会稳定与健康发展的需要出发，对戒毒人员进行教育、矫治和挽救的过程。 一、毒品成瘾教育矫治的功能 （一）促进戒毒人员个体发展变化的功能 毒品成瘾教育矫治的目的在于通过对戒毒人员的身心施加影响，增长戒毒人员的知识与技能，转变思想观念，矫正吸毒等不良行为，塑造其社会所期待的人格，使之顺利回归社会。 毒品成瘾教育矫治对戒毒人员个体发展的影响主要表现在以下几个方面： 第一，增长知识与技能； 第二，转变错误的思想观念； 第三，矫正吸毒等不良行为； 第四，完善戒毒人员的人格。 （二）促进社会发展的功能。主要有以下两点： 第一，维护社会秩序的安全稳定； 第二，变消极因素为积极因素，促进生产力的发展。 二、毒品成瘾教育矫治的意义 （一）通过法制和道德教育转变戒毒人员的思想认识，树立其法律和道德观念。 对戒毒人员进行全面、系统的法律和道德教育，使其系统地掌握一定的法律和道德规范的要求，让法律知识和道德知识内化在戒毒人员的思想意识之中，会在不同程度上提高戒毒人员的法律认识和道德水平，指引戒毒人员的行为，做一个遵纪守法的好公民。 （二）通过文化知识教育提高戒毒人员的认识水平，提高其参与社会活动的能力。 在对戒毒人员的教育矫治过程中，文化知识教育与其他教育相比，所起的作用较为突出，在教育矫治中起着基础性的作用。对戒毒人员进行文化知识教育可以提高其认识水平，正视过去的违法行为，正确选择未来的生活道路。 （三）通过生活技能和职业教育增强戒毒人员重新生活的信心，提高其就业能力。 生活技能教育能够使戒毒人员正确认识自己、他人和环境，调整自
中油石化有限公司年产10万吨二甲醚生产装置项目环境影响报告书（简本） 浙江中油石化有限公司 10万t/a二甲醚生产线项目 环境影响报告书 （简? 本） 浙江大学环境影响评价研究室 Institute of Environmental Impact Assessment Zhejiang University 国环评证：甲字第2002号 二OO六年十月 目??? 录 附? 图 1? 项目概况 公司概况和项目由来 浙江中油石化有限公司系上海中油能源控股有限公司、江苏中油长江石化有限公司、上海华油有限公司等单位共同投资，公司主要经营石化产品的生产、储运和销售。公司拟在平湖独山港建设浙江中油石化有限公司10万t/a二甲醚生产线项目，以甲醇为原料气相制备二甲醚，生产规模10万t/a。平湖市经济贸易局以平经贸投资备[2006]244号《平湖市企业投资项目备案通知书（技术改造）》予以备案。 项目名称和性质 （1）项目名称：浙江中油石化有限公司10万t/a二甲醚生产线建设项目。 （2）项目性质：新建。 立项情况 平湖市经济贸易局以平经贸投资备[2006]244号《平湖市企业投资项目备案通知书（技术改造）》 建设规模 项目总用地面积19096.5m2，装置占地面积9567m2，建筑面积1788 m2，主要建设主装置区、甲醇储罐区、锅炉房、分析控制室、循环水站和空压站等。预计在2008年初可建成投产。 项目建设地点 浙江中油石化有限公司“综合型石化产品储运加工基地”位于平湖市独山港区临港工业发展区块内，基地的东侧为上海金山石化库区（属浙江境内），南侧为杭州湾海域，西侧为闲置工业用地，北侧为金桥村。 本项目位于“综合型石化产品储运加工基地”的东北侧，项目北侧为金桥村，其中金桥村居民距离本项目厂界最近距离为100m；东侧是上海金山石化库区；南侧是中油石化化学品库区（属于石化储运加工同期建设项目）；西侧是中油石化液态烃库区（属于石化储运加工同期建设项目）。据现场踏勘，目前，项目所在地现状为陆地、水塘（废弃蟹塘）和滩涂，无任何建构筑物，自然地势为北高南低。 2? 工程内容及污染因素分析 公用工程内容 供水。本项目总用水量约301449m3/a，其中职工生活用水1449m3/a，冷却水总用量100万m3/a，采用冷却塔降温后循环使用，其中冷却补充水300000 m3/a（37.5 m3/h），由平湖自来水公司供给。 供电。本项目年用电量300万kWh，由平湖市供电局供给。 供热。本项目近期临时设有1400万大卡燃煤导热油锅炉1台，以工艺废气和煤为燃料，用于各设备装置加热。远期待荣成纸业正式投产运行后由该企业统一集中供热。 排水。雨污分流，雨水排入厂区南侧杭州湾独山港，生产废水经厂内污水预处理后和生活污水一起纳入平湖市东片污水处理厂。 消防。包括火灾消防系统和水消防系统 生产工艺流程
项目策划书 鲁东大学设计题目：年产10万吨啤酒工厂设计 2010年06月05日
目录 一.可行性研究报告 (3) 1.1 总论 (3) 1.2 项目建设的目的和意义 (3) 1.3 产品方案及需求预测 (4) 1.4 建厂条件及厂址选择 (4) 1.5 项目实施预规划及资金支付 (6) 1.6 经济效益及社会效益的初步估算 (6) 二.总平面布局 (7) 三.淡色啤酒生产的工艺设计 (7) 3.1 原料 (7) 3.2 生产工艺 (8) 四．工艺计算 (10) 4.1 100000t/a啤酒厂糖化车间的物料衡算 (10) 4.2 100000t/a啤酒厂糖化车间的热量衡算 (12) 4.3 100000t/a啤酒厂发酵车间的耗冷量衡算 (15) 4.4 年产10万吨12度啤酒的用水量计算 (18) 4.5 总容积200立方米啤酒锥底发酵罐计算 (19) 五.设备计算及选型 (20) 5.1 主要设备的计算 (20) 5.2 设备清单 (21) 六.工厂布局 (22) 七.啤酒工厂卫生 (22) 7.1 工厂设计规范 (22) 7.2 厂库环境卫生 (22) 7.3 厂区设施卫生 (22) 7.4 车间卫生 (22) 7.5 厂区公共卫生 (22) 八.环境保护与综合利用 (23) 8.1 环保治理工艺的设计原则： (23) 8.2 三废处理 (23) 九. 经济技术及概算 (23) 9.1人力资源配置 (23) 9.2产品成本及利润估算 (24) 十．总结 (25) 参考文献 (25)
一.可行性研究报告 1.1 总论 1.1.1 项目名称：年产100000吨啤酒工厂设计 1.1.2 承办单位：青岛三德工艺品有限公司 昌邑得益工艺品有限公司 1.1.3 项目地址：潍坊市昌邑饮马工业园区 1.1.4 项目经理：杨玉琨 1.2项目建设的目的和意义 1.2.1 提出背景和依据 啤酒是夏秋季防暑降温解渴止汗的清凉饮料。 据医学和食品专家们研究，啤酒含有4%的酒精，能促进血液循环;含二氧化碳，饮用时有清凉舒适感;还能帮助消化，促进食欲。 啤酒花含有蛋白质、维生素、挥发油、苦味素、树脂等，具有强心、健胃、利尿，镇痛等医疗效能，对高血压病、心脏病及结核病等均有较好的辅助疗效。产妇喝啤酒，以增加母体乳汁，使婴儿得到更充分的营养。适量适用啤酒对心脏和高血压患者亦有一定疗效。啤酒生产是采用发芽的谷物作原料,经磨碎,糖化,发酵等工序制得.。在古代中国,也有类似于啤酒的酒精饮料,古人称之为醴.大约在汉代后,醴被酒曲酿造的黄酒所淘汰.清代末期开始,国外的啤酒生产技术引入我国,新中国成立后,尤其是80年代以来,啤酒工业得到了突飞猛进的发展,到现在中国已成为世界第二啤酒生产大国. 如今可说是中国的啤酒工业进入了旺盛的成熟期,一方面, 啤酒工业继续以高速度发展,在高速发展的同时,开始对啤酒的质量, 啤酒工业的经济效益更加重视,啤酒工业的规模按照国际上的惯例,开始向大型化,集团化方向发展.一些中小型啤酒厂被大型啤酒厂兼并. 1.2.2 投资的必要性和经济意义 现在我国啤酒产量方面跃居世界第二位，而且在质量、技术、装备水平等方 面也都有了较大幅度的提高，充分显示了我国啤酒工业强劲的发展势头。但是，我 国啤酒与世界发达国家相比，仍有很大差距。我国啤酒厂不合理企业规模偏多，达不到啤酒生产应有的经济规模。通过对国内外技术经济指标的数据分析得出，10万吨／年规模以上 的啤酒厂才有较好的技术经济指标水平。而现在这样的酒厂还较少，多数是设备陈旧、老化，生产能力不足，设备的自动化程度不高，工艺落后的小酒厂。所以建设一个现代化的大规模的啤酒厂势在必行。 1.2.3 产品优势 经过10年有价值的健康研究，专家们发现，经常性、中度啤酒摄入量——即每天1—2杯12盎司(350毫升)啤酒——对于男性和女性都有益，特别是如果你正面临衰老或受到最常见疾病的困扰。而以下7个你梦寐以求的好处，啤酒都可以带给你。 1护心脏健康： 大量的研究表明，适度饮酒，包括啤酒，可降低患心脏病的危险。 2护血管： 适度喝啤酒也有助于防止血栓形成，预防缺血性脑中风。 3低糖尿病风险： 研究显示，糖尿病人中度饮酒也能减少最大的杀手——冠心病发作的风险。这可能是因为，
但是造句大全 1、你可以选择坚持，也可以选择放弃。没有对错。我是说对爱情来说，但是重要的是坚持你的选择。 2、我站在窗前，静静地欣赏着，天空中下着一群可爱的小精灵，虽然都是雪花，但是，你只要仔细观察，就会发现有许多形状、图案不同的雪花。 3、以和蔼亲切的态度说话并不会伤到舌头，但是，以愤怒和不悦的态度说话，则可能招致不幸。 4、有自己的人生观和价值观。出现问题可以忍让并寻求解决，但是触及原则，要保持自己原则。丧失原则会让你失去生活的目的。 5、秋天，柳树的叶儿慢慢变黄，继而掉落，让人感到无限凄凉。这时的风也变得恶了，是不是柳树姑娘和风姐姐吵架了呢？我不知道。但是，我见到，秋天到处是落叶缤纷，仿佛一场自然界的葬礼。 6、虽然他不聪明，但是他学习十分的用功。 7、想要获得成功，不像数学题一样，没有一个绝对的公式。但是如果知道一些原则的话，可以离成功更近一步。 8、生活过，而不会宽容别人的人，是不配受到别人的宽容的。
但是谁能说是不需要宽容的呢？ 9、虽然这是你亲手做的巧克力蛋糕，但是我不喜欢。 10、虽然万物好像逝去了，但是，你瞧！那火红的枫叶在树枝上摇摆着，就像是一大群顽皮的孩子在手拉手一起跳着欢快的舞蹈呢。有些枫叶因跳舞不慎，从树枝上跌了下来，瞬间化作了一只只飞舞的蝴蝶，在空中飞来飞去。 11、对于18岁的我们来说，有些事情的确会影响我们的一生，但是没有一件事能决定我们的一生！高考试卷是一把刻度不均匀的尺子：对于你自己来说，难题的分值不一定高。 12、她虽然态度冷淡，但是还算客气。 13、是人都有惰性，这是与生俱来的，但是我们后天可以改变这种惰性，谁改变的越多，谁就越成功。 14、我们前进的脚步虽然会让挫折绊住，但是我们要做生活的主人，不要坐在绊脚石的面前唉声叹气而耗尽了自己。让我们学会微笑着用有限的生命来超越无限的自己！我坚信，挫折让我更自信，更成功！ 15、夏天是个多姿多彩的世界。在这个季节，是最充满快乐的。也许，尽管很枯燥；也许，令人热得无法忍受；也许，植物们懒洋洋
太原理工大学化学化工学院 《化工设计》课程设计说明书 年产20万吨甲醇制二甲醚生产工艺初步设计 学生学号： 学生姓名： 专业班级：化工工艺0904 指导教师： 起止日期： 2012.11.26～2012.12.21
化工设计课程设计任务书 一、化工课程设计题目 年产20万吨甲醇制二甲醚生产工艺的初步设计 二、化工课程设计要求及原始数据（资料）： 操作方式：连续操作 产品品种：二甲醚 拟建规模：20万吨/年 年操作日：365天 汽化塔：原料粗甲醇纯度90%（质量分数，下同），塔顶甲醇气体纯度≥99%，釜液甲醇含量≤0.5%； 合成塔：选择 -Al2O3做催化剂，转化率≥80%，选择性≥99.9%，脱水温度选择300摄氏度。 精馏塔：塔顶二甲醚纯度≥“99.9%”釜液二甲醚含量≤0.5%； 回收塔：塔顶回收甲醇纯度≥98%，废水中甲醇含量≤0.5%。 三、化工课程设计主要内容： 1、绪论 2、生产流程或方法的确定 3、物料衡算和热量衡算 4、主要工艺设备的计算及选型（包括设备一览表） 5、原材料、动力消耗定额及消耗量 6、参考文献 7、致谢 8、附图（带控制点的工艺流程图和关键设备的结构图） 四、时间安排： 共设计四周，前2周收集资料，进行工艺流程的设计、物料和热量衡算，后两周进行设计说明书的撰写、工艺流程图和设备图的绘制。 五、学生应交出的设计文件： 课程设计说明书一本 带控制点的工艺流程图一套（要求手工绘制2#图纸） 主要设备结构图一套（要求CAD绘制，2#图纸）
六、主要参考文献（资料）： 1、《化工设计》王静康主编 1995年版化学工业出版社出版 2、《化工原理》（上、下） 2001年版天津大学化工原理教研室编天津科学技术出版社出版 3．………… 专业班级化工工艺0904 学生武晓佩 要求设计工作起止日期 2012年11月25日至2012年12月21日 指导教师签字日期 教研室主任签字日期 系主任批准签字日期
本科生毕业设计年产10万吨啤酒厂设计 姓名 学号 专业食品科学与工程班级 指导教师 学部食品与环境学部答辩日期
黑龙江东方学院本科生毕业论文（设计）评语（一） 姓名学号专业 班级 05-D 总 成绩 毕业论文（设计）题目：年产10万吨啤酒厂设计 答 辩 委 员 会 评 语 答辩成绩 主任签字：年月日答辩委员会成员签字 学部 毕业 论文 （设 计）领 导小 组意 见 组长签字：年月日学部公章
黑龙江东方学院本科生毕业论文（设计）评语（二）姓名李季学号054131235 专业班级05-D 毕业论文（设计）题目：年产10万吨啤酒厂设计 指导教师成绩 指 导 教 师 评 语 指导教师签字：年月日
黑龙江东方学院本科生毕业论文（设计）评语（三）姓名李季学号054131235 专业班级05-D 毕业论文（设计）题目：年产10万吨啤酒厂设计 评阅教师成绩 评 阅 教 师 评 语 评阅教师签字：年月日
黑龙江东方学院本科生毕业论文（设计）任务书姓名李季学号054131235专业班级05-D 毕业论文（设计）题目：年产10万吨啤酒厂设计 毕业论文（设计）的立题依据 主要内容及要求 进度安排 学生签字： 指导教师签字： 年月日本表一式三份，学生本人、指导教师、学部各一份。
年产10万吨啤酒厂设计 摘要 本文主要是简要的介绍年产10万吨10度淡色啤酒厂的工厂设计。它主要包括啤酒发展，啤酒原料，啤酒厂建设的目的，啤酒厂的规划，啤酒工艺计算、啤酒厂设备的计算和重点设备的计算，啤酒厂的发展状况，啤酒厂资金的估算等方面的内容主要是糖化车间的工艺。本设计一共画二张图：全厂平面布置图、工艺流程图。 本文设计的工厂采用3班倒的工作制，每天工作时间24小时，除去设备清洗和升温时间4小时，实际生产时间按20小时计，本设计设计了一个年产量10万吨啤酒厂主车间平面图及项目工艺方案的设计原则、方法、程序、设备、等等。 关键词：啤酒厂；工厂设计；工艺流程
一会一会造句大全 导读：本文是关于一会一会造句大全，如果觉得很不错，欢迎点评和分享！ 1、天上的云，颜色一会儿红通通的，一会儿半紫半黄的，一会儿金灿灿的。 2、科学家们好勤奋，一会儿观察自然现象，一会儿观察天气，一会儿做实验，一会儿相互提问，忙得不可开交。 3、小明一会儿打扫房间一会儿拖地，忙着手忙脚乱。 4、灯下的影子一会儿变长，一会儿缩短，可真有趣。 5、她起身站着想，神经质地一会儿把两手绞在一起，一会儿又松开。 6、不知在远处的什么地方，一辆汽车一会儿起动，一会儿熄火，一会儿起动，一会儿熄火，终于无声无息了。 7、火烧云的变化形态万千，一会儿就变成了一只可爱的小狗，一会儿又变成了一匹飞奔的骏马。 8、他一边走，头一边不停地来回转动，一会儿看看桥，一会儿看看桥后面的树。 9、健身房里，人们一会儿跑步一会儿做仰卧起坐，快乐的挥洒着汗水。 10、妈妈一会儿在家织毛衣，一会儿在厨房里做饭，我回到家很快就可以吃饭了。
11、白云飘飘一会儿东一会儿西一会儿左一会儿右。 12、秋高气爽的时节，一群大雁正往南飞，一会儿排成一字形，一会儿排成人字形。 13、他像只调皮的猴子，一会儿挠腮一会儿跳上跳下。 14、我做作业老是不专心，一会儿做作业，一会儿又吃东西，一会儿又看电视。 15、上课时他一会儿东张西望，一会儿交头接耳，结果什么也没有学到。 16、美丽的小燕子一会儿蜻蜓点水般掠过湖面，一会儿像离弦的弓箭飞向远方。 17、小蜜蜂一会我采集花粉回来，一会儿采信蜂蜜回来。 18、我也不明白是怎么回事，一会儿觉得丧气，一会儿又觉得轻快。 19、动物园里的猴子一会儿爬上，一会儿窜下，可调皮了。 20、数学课上，小明一会儿跟同桌小刚聊天，一会儿和邻桌小红聊天，最后被老师批评了。 21、小猫钓鱼很不专心，一会儿捉蜻蜓，一会儿捉蝴蝶。 22、野鸭悠闲自在地浮着，一会儿跌入水底，一会儿又立在浪尖上，像孩子在打秋千。 23、小花狗跑到楼顶上，一会儿大声叫，一会儿趴在那不动。 24、我好喜欢我的语文老师，读课文的时候一会儿大声，一会儿小声，一会儿神色飞扬。
国内二甲醚发展现状及市场前景 摘要：文章重点介绍了近年来国内二甲醚产业发展状况，分析了二甲醚在我国发展存在的优势和问题，对其市场发展前景进行了展望。 关键词：二甲醚发展现状市场前景 二甲醚是一种新兴的煤化工产品，具有燃烧热值高、污染小等优点。在国际原油价格高企的背景下，二甲醚部分替代石油产品具有一定的经济优势，国内市场对于二甲醚的认同程度也渐渐提高。目前，国内二甲醚的主要用途是按一定比例（10%左右）添加到液化石油气中，作为民用燃气；其次，还可以替代柴油，作为汽车燃料。另外，二甲醚在医药、农药、金属焊接等领域也有一定的应用。近年来，由于国际原油价格持续上涨，液化气生产成本增加。二甲醚以其独特的优势逐步开始在市场上推广。 1国内二甲醚生产现状 1．1 2007年国内二甲醚生产情况 据统计，2007年我国共有二甲醚生产企业30家，产能合计261.15万吨/年，产量约130万吨。其中，外购甲醇生产二甲醚的企业共23家，产能合计170.65万吨/年；自配甲醇装置的企业7家，产能合计90.5万吨/年。我国主要二甲醚生产企业情况见表1 1．2 2008年产能扩张情况 2008年我国有8个二甲醚项目投产，产能合计147.5万吨/吨。其中自配甲醇装置的项目有2个，产能合计16万吨/年。需要外购甲醇的项目共6个，产能合计131.5万吨/年。我国二甲醚总产能达到408.65万吨/年，其中自配甲醇的产能为106.5万吨/年，外购甲醇的产能为302.15万吨/年。2008年投产的部分二甲醚项目统计见表2。
1．3 2009~2010年产能扩张情况 2009年~2010年投产的二甲醚项目共14个，产能合计395万吨/年（见表2）。其中，自配甲醇的项目共7个，产能合计125万吨/年，需要外购甲醇厚的项目也有7个，产能合计270万吨/年。预计到2010年底，国内二甲醚产能将至少达到803.65万吨/年，其中需要外购甲醇的生产能力为572.15万吨/年，若开工率按90%计算，则这部分二甲醚产量为514.9万吨，至少需要市场采购甲醇772.4万吨。 2 我国发展二甲醚产业的优势 2．1 资源优势 我国煤炭资源丰富，发展以煤为原料的化工产品原料充足，有利于保障行业的可持续发展，也符合我国“缺油富煤”的资源结构。国内拥有煤炭资源的企业发展二甲醚产业在保障原料来源的同时，也可以降低生产成本，提高产品竞争力，因此优势更加明显。从经济性考虑，建立在煤矿附近的甲醇生产企业可能有效降低甲醇生产成本，进而可以将二甲醚的生产成本相应控制在一定范围。 2.2市场优势 在两大应用领域——替代液化石油气领域和替代柴油领域，二甲醚都有广阔的市场前景。2007年我国液化石油气表现消费量为2300万吨，柴油表现消费量为1.25亿吨。随着国内经济的持续发展，市场对于液化气石油气和柴油的需求量都将保持稳定增长。预计到2010年，国内液化气石油气和柴油的市场需求量将分别达到2600万吨和1.4亿吨。但是，由于我国石油资源匮乏，原油和液化石油气的对外依存度不断上升。因此，发展替代产品有利于缓解我国石油供需矛盾，降低石油对外依存度。如果按照液化石油气替代10%，柴油替代3%计算，2010年二甲醚的市场需求量将会达到680万吨甚至更多。由此可见，只要二甲醚推广工作进展顺利、配套设施能够尽快完善，二甲醚的市场前景将会非常乐观。 2．3 政策优势 2007年8月，建设部发布了《城镇燃气用二甲醚》标准。该标准的实施表明，二甲醚作为液化气石油气的替代燃料已具有合法身份，可以正式进入城镇作为替代燃料。同时，该标准的实施也为二甲醚的大范围推广铺平了道路。除了在政策上给予支持，我国政府在二甲醚技术开发上也加大了投入。2006年12月，久泰化工获得了国家发改委总额730万元的财政扶持资金。此外，政府还直接推动中央企业参与二甲醚生产。由中煤、中石化等5家企业联合组建的中天合创420万吨/年甲醇、300万吨/年二甲醚项目已经在内蒙古鄂尔多斯签约，
年产12万吨啤酒厂糖化车间设计 本设计的内容 摘要：啤酒，但是酿造原理却是一样的。在整个酿造过程中，大体可以分为四大工序：麦芽制造；麦汁制备；啤酒发酵；啤酒包装与成品啤酒。其中麦汁制造是啤酒生产的重要环节，它包含了对原料的糊化、液化、糖化、麦醪过滤和麦汁煮沸等处理工艺。设计从实际生产出发，确定出生产10万吨啤酒所需要的物料量，热量和糖化车间内的常用设备如糊化锅、糖化锅、过滤槽、煮沸锅、沉淀槽及薄板冷却器的主要尺寸、选型以及其他辅助设备、管道的选型。设备均是现今国内常用的类型，具有一定的先进性。而且对整个车间的布局进行了设计，包括设备布置图，工艺流程图等。 关键词：糖化锅物料衡算热量衡算 一、前言： 啤酒是全世界分布最广，也是历史最悠久的酒精性饮料，它的酒精度低、营养丰富、有益于人的健康，因而有“液体面包”之美称，受到众人的喜爱。 我国最新的国家标准规定：啤酒是以大麦芽（包括特种麦芽）为主要原料，加酒花，经酵母发酵酿制而成的、含二氧化碳的、起泡的、低酒精度（2.5％～7.5％，V/V）的各类熟鲜啤酒。 目前，我国人均啤酒消费量虽然已接近22升，但中西部地区仅在10升左右，8亿多人口的农村人均连5 升不到。因此，我国啤酒市场还拥有很大的挖掘潜力，消费量仍将保持增长。 啤酒品种很多，一般可根据生产方式，按产品浓度、啤酒的色泽、啤酒的消费对象、啤酒的包装容器、啤酒发酵所用的酵母菌等种类来分类。 ◆根据原麦汁浓度分类 啤酒酒标上的度数与白酒上的度数不同，它并非指酒精度，它的含义为原麦汁浓度，即啤酒发酵进罐时麦汁的浓度。主要的度数有18、16、14、12、11、10、8度啤酒。日常生活中我们饮用的啤酒多为11、12度啤酒。 ◆根据啤酒色泽分类 淡色啤酒——色度在5-14EBC之间。淡色啤酒为啤酒产量最大的一种。浅色啤酒又分为浅黄色啤酒、金黄色啤酒。 浅黄色啤酒口味淡爽，酒花香味突出。金黄色啤酒口味清爽而醇和，酒花香味也突出。 浓色啤酒——色泽呈红棕色或红褐色，色度在14-40EBC之间。浓色啤酒麦芽香味突出、口味醇厚、酒花苦味较清。黑色啤酒——色泽呈深红褐色乃至黑褐色，产量较低。黑色啤酒麦芽香味突出、口味浓醇、泡沫细腻，苦味根据产品类型而有较大差异。 ◆根据杀菌方法分类 鲜啤酒——啤酒包装后，不经巴氏灭菌的啤酒。这种啤酒味道鲜美，但容易变质，保质期7天左右。 熟啤酒——经过巴氏灭菌的啤酒。可以存放较长时间，可用于外地销售，优级啤酒保质期为120天。 ◆根据包装容器分类 瓶装啤酒——国内主要为640ml和355ml两种包装。国际上还有500ml和330ml等其他规格。 易拉罐装啤酒——采用铝合金为材料，规格多为355ml。便于携带，但成本高。
用2个有时有时造句大全 用有时有时造句 1、天上的云彩变化多端，有时像一匹骏马在奔驰，有时像一条狗在摆头摇尾。 2、大白菜的价格有时高有时低。 3、小狗豆豆有时很温驯，有时又很顽皮。 4、在这个竞争激烈的社会里，我们有时会感到压力重重，而有时会感到生活很充实。 5、乐曲有时高昂，有时低沉。 6、鸟们有时在天空中展翅高飞，有时停在树枝上婉转啼叫，有时在林间欢蹦乱跳。 7、一分钟有时很长，有时又很短。 8、放学后，我有时直接回家，有时留在学校做作业，有时和同
学去打球。 9、课间休息时间，我有时去操场散步，有时和同学跳皮筋。 10、天上的月亮有时像一把镰刀，静静地挂在天空，懒得动一下；有时像一的大银盆，又圆又大的。 11、人生总是包含着五味杂陈，有时你会感到快乐，有时你会遭遇困难，有时你会期待梦想，有时也会充满感伤，必须每样都经历才算完整。 12、观看比赛的观众有时鼓掌为运动员加油，有时呐喊为选手鼓劲。 13、喷泉有时像百花怒放，有时像凤凰一飞冲天。 14、我的爸爸有时候严厉，有时候慈祥。 15、小猫咪有时淘气，有时可爱。 16、我有时可笑，有时悲伤。 17、在周末，小明有时在家里看电视，有时去爷爷奶奶家玩。 18、一群大雁往南飞，它们有时排成一字，有时排成人字。
19、天气千变万化，有时晴天，有时阴天。 20、人生际遇总是变幻莫测，有时如顺风行船，喜笑颜开；有时如雪中登山，凶险万分。 21、月亮有时像圆盘，有时像镰刀。 22、亮有时象圆盘，有是象镰刀，有时又象个光环。 23、我有时步行去学校，有时骑车去。 24、妈妈去外地出差了，我有时给妈妈打电话，有时我还给妈妈发短信。 25、这些天，她有时高兴，有时悲伤，有时又整天沉默无语，一定是发生了什么事。 26、生命如水，有时潺潺的流动，有时静静的盘桓。 27、仙女们在这片神奇的绿野上游戏，有时跳舞，有时歌唱，有时漫步，有时飞翔。 28、小鸟们有时在天空中展翅高飞，有时停在树枝上婉转啼叫。 29、他的课余生活丰富。有时打篮球，有时上网。
主题：吸食、注射毒品人员成瘾标准如何界定？回复：根据公安部《关于对吸食、注射毒品人员成瘾标准界定问题的批复》意见（1998年4月22日公复字[1998]3号）对吸食、注射毒品人员成瘾的界定标准是：有证据证明其吸毒，且查获时尿样毒品检测为阳性的，认定为成瘾；对曾经吸过毒，但有证据证明其没有继续吸毒；且查获时尿样毒品检测为阴性的，不认定为成瘾。 对尿样毒品检测呈阴性，但吸毒证据不足的，应进行尿样复检和进一步调查取证，有条件的可作药品催瘾、医学试验，然后作出确认。 根据国家《中国人民共和国禁毒法》 ——第三十一条国家采取各种措施帮助吸毒人员戒除毒瘾，教育和挽救吸毒人员。 吸毒成瘾人员应当进行戒毒治疗。 吸毒成瘾的认定办法，由国务院卫生行政部门、药品监督管理部门、公安部门规定。—— 中：吸毒成瘾的认定，——公安部《关于对吸食、注射毒品人员成瘾标准界定问题的批复》（公复字[1998]3号）规定：“有证据证明其吸毒，且查获时尿样毒品检测为阳性的，认定为成瘾；对曾经吸过毒，但有证据证明其没有继续吸毒，且查获时尿样毒品检测为阴性的，不认定为成瘾。对尿样毒品检测呈阳性，但吸毒证据不足的，应进行尿样复检和进一步调查取证，有条件的可作药品（纳屈酮）催瘾医学试验，然后作出确认。”结论：先进行认定，如果有证据证明达不到吸毒成瘾，就不违反38条规定，即不会被带到戒毒所！ 公安部关于对吸食、注射毒品人员成瘾标准界定问题的批复 发文单位：公安部 文号：公复字[1998]3号 发布日期：1998-4-22 执行日期：1998-4-22 浙江省公安厅： 你厅《关于对吸食。注射毒品人员成瘾标准如何的请示》（浙公刑[1998]9号）收悉。现批复如下： 鉴于目前和今后相当长的时间内，在基层执法部门推广使用药品催瘾医学试验的条件难以具备，以及国际社会和我国禁毒执法实践中普遍采取尿样毒品检测方法的实际情况，为保证强制戒毒工作的正常开展，同意你厅提出的对吸食。注射毒品人员成瘾标准的界定意见：有证据证明其吸毒，且查获时尿样毒品检测为阳性的，认定为成瘾；对曾今吸过毒，但有证据证明其没有继续吸毒，且查获时尿样毒品检测为阴性的，不认定为成瘾。 对尿样毒品检测呈阳性，但吸毒证据不足的，应进行尿样复检和进一步调查取证，有条件的可作药品（纳络酮）催瘾医学实验，然后作出确认。 一九九八年四月二十二日 公安部
2008“三井化学”杯大学生化工设计竞赛 广广西西大大学学 f f o o r r w w a a r r d d 团团队队
2008三井化学杯 大学生化工设计竞赛项目设计说明书 项目名称：以蔗渣（蔗髓）为原料年产10万吨二甲醚项目 参赛学校： 设计时间：2008.08-2008.09
目录 第一章总论 (1) 1.1 项目名称 (1) 1.2 企业和建设性质 (1) 1.3 编制依据 (1) 1.4 编制原则 (1) 1.5 项目背景 (1) 1.6 项目投资的必要性和经济意义 (2) 1.7 工程项目研究概述 (3) 1.8 本项目的特色与创新点 (4) 第二章市场预测分析 (5) 2.1 二甲醚特性 (5) 2.2 二甲醚产品用途 (5) 2.3 市场情况及预测 (7) 2.3.1 国内市场 (7) 2.3.2 国际市场 (8) 2.3.3 市场预测 (10) 2.3.4 二甲醚产品价格预测 (11) 第三章产品方案及生产规模 (12) 3.1 产品方案 (12) 3.1.1 产品方案构成 (12) 3.1.2 产品规格及质量指标 (12) 3.2 生产规模 (12) 第四章工艺技术方案 (13) 4.1 工艺技术方案的选择及技术来源 (13) 4.2 二甲醚合成工艺路线的现状及选择 (13) 4.2.1 二甲醚生产工艺对比 (13) 4.2.2 国内工艺发展路线 (17) 4.2.3 二甲醚合成工艺方案的确定 (18) 4.3 生物质超临界水气化氧化技术 (18) 4.3.1 生物质超临界水气化技术的选择及意义 (18) 4.3.2 超临界水中生物质气化原理及工艺选择 (19) 4.3.3 超临界水氧化技术的运用 (22) 4.4 合成二甲醚工艺条件的选取 (23)
工程策划书 鲁东大学 设计题目：年产10万吨啤酒工厂设计
目录 一.可行性研究报告 (3) 1.1 总论 (3) 1.2 工程建设地目地和意义 (3) 1.3 产品方案及需求预测 (4) 1.4 建厂条件及厂址选择 (4) 1.5 工程实施预规划及资金支付 (6) 1.6 经济效益及社会效益地初步估算 (6) 二.总平面布局 (7) 三.淡色啤酒生产地工艺设计 (7) 3.1 原料 (7) 3.2 生产工艺 (8) 四．工艺计算 (10) 4.1 100000t/a啤酒厂糖化车间地物料衡算 (10) 4.2 100000t/a啤酒厂糖化车间地热量衡算 (12) 4.3 100000t/a啤酒厂发酵车间地耗冷量衡算 (15) 4.4 年产10万吨12度啤酒地用水量计算 (18) 4.5 总容积200立方M啤酒锥底发酵罐计算 (19) 五.设备计算及选型 (20) 5.1 主要设备地计算 (20) 5.2 设备清单 (21) 六.工厂布局 (22) 七.啤酒工厂卫生 (22) 7.1 工厂设计规范 (22) 7.2 厂库环境卫生 (22) 7.3 厂区设施卫生 (22) 7.4 车间卫生 (22) 7.5 厂区公共卫生 (22) 八.环境保护与综合利用 (23) 8.1 环保治理工艺地设计原则： (23) 8.2 三废处理 (23) 九. 经济技术及概算 (23) 9.1人力资源配置 (23) 9.2产品成本及利润估算 (24) 十．总结 (25) 参考文献 (25) 一.可行性研究报告 1.1 总论 1.1.1 工程名称：年产100000吨啤酒工厂设计 1.1.2 承办单位：青岛三德工艺品有限公司 昌邑得益工艺品有限公司 1.1.3 工程地址：潍坊市昌邑饮马工业园区 1.1.4 工程经理：杨玉琨
出现造句大全 导读：本文是关于出现造句大全，如果觉得很不错，欢迎点评和分享！ 1、你们同学白天上课时的音量比晚自习时小多了，这是极不正常的，如果这种情况出现在野生动物身上，那就意味着大的自然灾害即将到来。 2、我不是带着将失败的情绪走进赛场的。我回来的时候满怀信心，我相信仍然能够有所作为。如果我只是坐在这里听别人告诉我不能复出，那我肯定不会出现在这里。 3、小贝，我去向琛哥要我的明天，全身上下最值钱的东西，我都留给你，谢谢你给了我一个值得活下来的理由，为了这个理由，我愿意用生命去赌我们的未来，如果没回来，不要来找我，就当我从来没出现在你的生命里。 4、夏天的晚上，太阳太劳累了，又钻回了云朵被窝去睡觉了，星星和月亮，养足了精神，出现在了天空，发出动人的光芒，蟋蟀和没有睡觉的青蛙知了，在草丛里池塘里大树上唱着歌。 5、曾经也有一个笑容出现在我的生命里，可是最后还是如雾般消散，而那个笑容，就成为我中心深深埋藏的一条湍急河流，无法泅渡，那河流的声音，就成为我每日每夜绝望的歌唱。 6、知道自己出现在别人的梦里是很让人开心的事，这能证明你的存在，而且在某种程度上，还可以证明你在别的地方也具有实体和
价值。 7、正当我高兴的流下泪水时，闹钟忽然响了，它把我从梦中拉出来，才知道原来是场梦，但我坚信，在不久的将来，我一定会出现在奥运会上，为中国队努力拼搏。 8、世界上没有谁选择谁，只有谁遇到谁，所以我不选ABCD，只看着时间刚刚好的时候，出现在你面前，然后，牵着手，去哪里那里，然后，就是一辈子。 9、只见月亮像一个害羞的姑娘，羞答答地从一片乌云背后伸出半个脑袋，偷偷地向下窥探，发现没有什么动静，一扭身，出现在天空中，天空就好像出现了一盏明亮的灯，周围的乌云被白色的月光照着。 10、情痴先生，多谢你的痴情。对于我在你的梦中出现这件事，令我十分震惊。你没有征求我的同意，便让我出现在你的梦里，侵犯了我的自由，我保留所有法律上追究的权利跟赔偿。还有，你没有告诉我在梦里你对我做了什么？ 11、到19世纪70年代，那些实际生活中的作奸犯科者、道德沦丧者，竟然已经堂而皇之地出现在歌舞表演中，这一幕与如今八卦电视节目何其相似，文化垃圾商品的真人版而已！ 12、以前只是一种经历与感觉，而不是证据，不需要为以前的喜欢付出现在或以后的责任。不要揪住以前的事情不放。现在的事实比以前的回忆更有实效性与说服力。 13、法律不等于正义，这是一种非常不完美的机制，如果你按
人是怎么上瘾的？ 看看我们现在的生活吧，各种短视频、娱乐节目、游戏等等，让大家沉浸在各种浅层的刺激里，这些浅层的刺激让我们上瘾，一个个碎片化的满足，把我们的生活填满了。 “上瘾”的逻辑是如何被设计出来的？人的大脑里有一种叫多巴胺的神经传导物质，当人被外界刺激的愉悦时，多巴胺会大量爆发出来，比如一个拥抱、一句赞扬的话，一个幻象，都会引起多巴胺的升高，当外界的刺激够强烈，人的身体就会进入到一种如痴如醉如梦如幻的感官体验，即涌起一阵阵“快感”，感到很“爽”。 “爽”的浅层次的体验有笑话、美食、挑逗、赞美、看热闹，甚至惊恐（不少女孩喜欢看恐怖片就是这个原因）；中等层次的体验有抽烟、游戏、整容；深层次体验有性爱、鸦片、豪赌、毒品，就是俗称的黄赌毒。对比这三者，如果说性最能让多巴胺含量提升100%，可卡因让多巴胺上升了350%，冰毒带来的是接近1200%，所以毒品的致瘾是很难戒掉的…... 现在的世界越来越开放和自由，人们时刻都在互联网上追求“爽”的各种体验，约会/娱乐/游戏/八卦秘闻/色情段子/性挑逗等等，于是人越来越沉迷于这种短暂的上瘾体验。更可怕的是：人是会对快感脱敏的，快感的阈值是会不断升高的。一个人要想一直获得快感，就得不断加强刺激的程度，你需要被更持续、更强烈的刺激，才能继续获得快感。比如有的人抽烟，从刚开始是两天一包，到一天一包，再到后来要一天两
包，最后甚至要两根烟一起抽才有感觉。鸦片、吸毒、色情、偷窥、赌博都遵循这个逻辑。 有这样一个实验：在小鼠脑中埋个电极，让小鼠踩踏板放电，每踩一次，电极就会刺激产生多巴胺的神经元兴奋。结果小鼠以每分钟几百次的速度踩踏，直到力竭而亡……因此，为什么现在很多人短视频可以刷一天不停，为什么现在很多人微信可以刷一天不停，因为一旦停下来就会感到空虚。最最可怕的是：这种“爽”的感觉，竟然可以被现代技术和算法设计出来。比如各种娱乐APP越来越多，大部分APP的本质是什么？每个APP背后都有一个强大的运营团队，他们用强大的运算和数据处理能力，通过声、光、交互、反馈等全方位途径，再在各种心理学、消费行为学、神经科学等理论指导下，不断的给你刺激，让你持续的“爽”，越来越离不开它们。 前段时间看过一篇文章，提到世界上有一个叫“多巴胺实验室”的公司，对外宣称“能运用神经科学理论，结合人工智能机器学习，用多巴胺让你的App 令人上瘾”。他们为各种App 定制这样的服务：在一些关键的地方和时间点设计“奖赏”，比如不断的惊喜和奖励，或物质或精神，从而提高用户的留存度、打开率和停留时间。这也就意味着增加APP的打开率，提高打开时间，这会让APP更值钱，赚更大钱。即便像谷歌这样的以“不作恶”来标榜自己的公司，其产品设计的核心逻辑依然是如何才能提升增加点击率，延长用户使用时间，然后见缝插针/算计的地去卖广告，满足这个逻辑的品才被定义成好产品。因为在资本的眼中，用户数量、日活、月活和平均在线长度等等数据决定了一个
第一章概述 1.1 概述 1.1.1项目名称、建设地点、建设单位 项目名称：年产120万吨甲醇、30万吨二甲醚项目 建设地点：内蒙古呼伦贝尔市鄂温克族自治旗浩勒堡 建设单位： 具体联系人： 编制时间： 1.1.2项目建设原因 石油、天然气和煤是目前世界能源的三大支柱，按现在石油消耗量和开采量计算，石油的开采年限约半个世纪，而煤的开采年限却超过200年。中国是一个煤炭资源极其丰富的国家，石油资源却相对较少，所有油田的出油率随开采年限的增长而下降，而石油的需求却正在逐年增加，加之天然气的价格比较高，工业经济效益难以得到保障。 我国煤炭资源丰富，2001年资源量为1万吨居世界第三位。估计到2010年消费量为18-19亿吨／年。坑口煤价较便宜，煤价为60-80元／吨。 内蒙古呼伦贝尔市有丰富的矿产资源，已探查到的矿产有4 0多种，主要有煤、石油、铁、铜、铅、锌、水泥灰岩、天然碱等。煤炭资源储量大、分布广，以海拉尔区为中心，
北有宝日希勒煤田，南有伊敏煤田，东有大雁煤田，西有扎赉诺尔煤田，四大煤田保有储量约280亿吨。内蒙古东部煤炭资源丰富保有储量大，占东北全区保有储量的58％，呼伦贝尔市煤炭储量占内蒙东部煤炭储量的67％，占东北煤炭储量的4O％左右，远景储量约1000亿吨，是黑龙江、吉林、辽宁三省总和的1.8倍，均是适于煤转化工用的优质褐煤。 1.1.3企业概况 大雁煤业集团公司为山东鲁能集团所属重点煤炭生产企业，属国有大型二档企业，是以煤炭生产为主，兼营电力、矿井建设、建筑安装、建材生产、绿色薯业以及生态旅游等多元化产业的经济实体，为全国煤炭百强企业。现有三对矿井，核定生产能力690万吨。 1.2项目建设的目的和意义 甲醇既是基本有机化工原料，又可以用来做汽车或民用的代用燃料。以甲醇为原料可以生产六十多个下游化工产品，经过再加工，又可以得到几百种化工产品，因此甲醇工业目前已成为碳一化学中的主要支柱。同时，我国对汽油掺烧甲醇已经进行了先期的试验，并取得了成功的经验。汽油掺烧甲醇被世界公认是可行的、经济的、安全的、环保的。为了在全国推广汽车用汽油掺烧甲醇，国家标准局在国家经贸委各有关司局的配合下正积极制订汽油中掺甲醇5％、10％和15％的各项标准规范和实施细则，并有望近期出台，