Background

Betaine is a derivative of the amino acid glycine with three chemically reactive methyl groups.Betaine is distributed widely in animals,plants and microorganisms,and it is also a metabolite of choline oxidation in animals[1].The principal physiologic role of betaine is as a methyl group donor[2],which means betaine participates in many important biochemical pathways,including methionine-homocysteine cycle and the biosynthesis of many compounds such as carnitine,creatine and phospholipids.Since carnitine is required for transport of long chain fatty acids into mitochondria[3],scientists have paid much attention to effects of betaine on energy metabolism especially lipid metabolism in animals.Studies showed that dietary betaine supplementation affected energy partitioning in pigs[4,5]and it’s also widely reported that betaine promotes animal growth and decreases carcass fat percentage in finishing pigs[6-10].Further investigations found that betaine supplementation could decrease hepatic triglyceride accumulation[11,12]and prevent fatty liver in rats fed high-fat-diets[13,14].The intramuscular fat content in the longissimus muscle was increased when pigs were fed betaine[15,16].Madeira et al.[17]reported that betaine might be involved in the differential regulation of some key genes of lipid metabolism in muscle and subcutaneous adipose tissue.However,studies on the mechanism of betaine affecting lipid metabolism in muscle are lacking.Therefore,the objective of the present study was to investigate possible mechanisms of betaine in altering lipid metabolism in muscle tissue of finishing pigs.

Methods

Animals and treatments

The experiment protocol used in this study was approved by the Institutional Animal Care and Use Committee of Zhejiang University.A total of 120 crossbred gilts(Landrace×Yorkshire×Duroc)with an average initial body weight of 70.1 kg(SD 0.70 kg)were randomly allotted to three dietary treatments.Each treatment consisted of four pens replicates with 10 gilts per pen.The treatment diets included a corn-soybean meal basal(Table 1)supplemented with 0,1250 mg/kg(Low Betaine)or 2500 mg/kg(High Betaine)betaine(provided by Healthy Husbandry Sci-tech Co.,Ltd.Hangzhou,China)respectively at the expense of corn.The basal diet was formulated to meet or exceed the nutrient requirements of finishing pigs[18].Chemical analyses of the basal diet were carried out according to the methods of AOAC[19].The feeding experiment lasted 42 d after a 7-day adaptation period.All pigs were housed in a curtain-sided pig barn with concrete slotted floors.Feed and water were provided for ad libitum consumption throughout the experiment.

这样的场景真是无数次了，这时的周小羽背个书包，踢一脚小石头再一踢一脚小石头，其实他就是这样一脚一脚地踢着石头前进，前进到老樟树下。老樟树下是村里连接学校的唯一途径，路过是必须的。所以，在后来的周小羽看来，这几乎就是一种仪式，村里那些无所事事的人就每天守在老樟树下目接他回来，这是迎接他回家的仪式。

Table 1　Nutrition formulation of basic diet

aAll of the data were analyzed value except digestible energy which was calculated using swine NRC(2012)values bProvided the following amounts per kilogram of diet:Fe(FeSO4·7H2O),50 mg;Cu(CuSO4·5H2O),5 mg;Mn(MnSO4·H2O),5 mg;Zn(ZnSO4·7H2O),50 mg;I(KI),0.35 mg;Se(NaSe2O3),0.15 mg cProvided the following amounts per kilogram of diet:vitamin A,3000 IU;vitamin D3,610 IU;vitamin E,20 IU;vitamin B2,5 mg;vitamin B12,0.021 mg;biotin,0.1 mg;pantothenic acid,10 mg;nicotinic acid,15 mg

Ingredients　%　Nutrient　%Corn　67.83　Digestible energy,MJ/kga　13.42 Soybean meal　23　Dry matter　87.09 Rapeseed meal　3　Crude protein　17.02 Wheat midding　3　Crude fat　3.98 CaHPO4　1.5　Calcium　0.85 Limestone　1.0　Phosphorus　0.64 Salt　0.3　Lysine　0.92 Lysine　0.10　Met　0.27 Trace element premixb　0.25 Vintamin premixc　0.02

Sample collection

At the end of the trial,eighteen pigs(six from each dietary treatment)weighing about 111.8 kg(SD 2.08 kg)were selected to collect tissue samples.Following an overnight fast,pigs were stunned by electrical shock and bleeding.Individual blood samples were collected at slaughter during exsanguinations.After collection of blood,samples were kept at room temperature for 2 h and then centrifuged for 10 min at 3000×g at 4 °C.Serum was collected and frozen at-80°C until subsequent analyses.Samples of longissimus muscle between the 6thand 7thrib were obtained on the left side of the carcass within 5 min after slaughter,and then snap frozen in liquid nitrogen and stored at-80°C until subsequent analyses.

Analysis of lipid metabolites in serum

Serum concentration of high-density lipoprotein cholesterol(HDLC),total cholesterol(TC),free fatty acid(FFA)and triglyceride were measured with commercial assay kits(Nanjing Jiancheng Bio-Engineering Institute,Code No.A112-2,A111-2,A042-1 and A110-2,respectively,Nanjing,China)following the manufacturer’s instructions.

The level of FFA and TC were markedly higher in muscle when pigs were fed betaine(P＜0.05,Fig.2).Compared to the control group,the level of triglyceride in　muscle　was　notaffected　by　betaine　addition(P＞0.05).

Total RNA was extracted from frozen porcine muscle tissue using the Trizol reagent as described by the manufacturer(Invitrogen).The RNA concentration and purity were determined by the NanoDrop ND-2000 spectrophotometer(Thermofisher,USA)and its integrity was confirmed by agarose gel electrophoresis.The cDNA synthesis was performed in a 10-μL reaction volume containing 2 μg total RNA using the SYBR Prime-Script™RT-PCR kit with gDNA Eraser(Code No.RR047A,TaKaRa,Dalian,China).Genomic DNA is eliminated by treatment for 2 min at 42°C with gDNA Eraser,which has potent DNA degrading activity.Then a reverse-transcription reaction reagent is added that includes a component that completely inhibits DNA degradation activity,and the reverse-transcription reaction proceeds for 15 min at 37°C.The abundance of the target genes was measured by quantitative real-time PCR,performed with the ABI Stepone Plus™RT-PCR system(ABI Biotechnology,USA)using SYBR Premix Ex Taq™(Tli RNaseH Plus)RT-RCR kit(TaKaRa,Dalian,China).Primers for the selected genes were synthesized commercially by Invitrogen(Shanghai,China),shown in Table 2.The reaction protocol comprised a cycle of 95°C for 1 min,40 cycles of 95 °C for 10 s and 64 °C for 25 s.The expression of the target genes were normalized by the endogenous housekeeping gene(β-actin)[21,22].Each sample was analyzed in triplicate and the PCR amplification efficiency was close to 100%.The gene expression was calculated by using the comparative(2-ΔΔCt)method[23].

RT-PCR analysis

A 10%muscle homogenate was prepared with a mixture of chloroform and formaldehyde(a volume ratio of 2:1).Then extracted at room temperature for 24 h[20].The organic solvent layer was taken and the level of triglyceride in muscle was measured with commercial assay kit(Nanjing Jiancheng Bio-engineering Institute,A110-2,Nanjing,China).Before the levels of TC and FFA in muscle were measured by the kits(Nanjing Jiancheng Bio-engineering Institute,Code No.A112-2,A111-2,A042-1 and A110-2,respectively,Nanjing,China),muscle tissue was made homogenate with physiological saline.The concentrations of carnitine and malonyl-CoA were measured using ELISA kits(Biovol Technologies,Code　No.50R-E.3088P　&　50R-E.3035P,Shanghai,China)for porcine assay according to the instructions.

Western blot analysis

Protein form muscle samples was extracted by T-PER Tissue Protein Extraction Reagent containing protease inhibitorcocktail(Thermo　Pierce,CodeNo.78510,USA),and quantified with BCA protein assay kit(Beyotime,Code No.P0010,Shanghai,China)according to kit instructions.Proteins were separated on SDS-PAGE gels(12%),and then electrophoretically transferred onto immobilon-P polyvinylidene fluoride membranes(PVDF membrane,Millipore,Code No.IPVH00010,America).Membranes were blocked 1 h in Tris-buffered saline containing 5%nonfat-dried milk at room temperature.Membranes were then incubated overnight at 4°C in blocking buffer containing primary antibodies(as shown in Table 3).A goat anti-rabbit IgG(H+L)Secondary antibody(Thermo Pierce,Code NO.31210,USA)with 1/5000 dilution was used in the detection of specific proteins.For loading control,β-actin antibody was used as control.In addition,the relative expression of p-AMPK was normalized with AMPK.Finally,Super Signal West Dura Extended Duration Substrate(Thermo Pierce,Code No.34075,USA)was used to visualize the protein bands.Band　intensitiesweredetermined　byusing BandScan 5.0 software.

图2中，每次预修后设备会变“年轻”，役龄回退到维修前(1-αi-1)τi-1时刻，此时设备起始故障率较维修前有所减小，但不会降为零，随后以速率bi-1加速增长，使设备加快老化衰退，因此称αi为役龄回退因子，称bi为故障率递增因子[11]。其中：

The relative expressions of target proteins=(The optical density of target proteins/The optical density of βactin).

Statistical analyses

论商标评审程序中“一事不再理”原则的适用............................................................................................周丽婷 06.38

As shown in Fig.3,the gene expression of FAT/CD36,FATP1 and PPARγ(P＜0.05)were higher in betaine-fed groups than control group.The addition of 2500 mg/kg betaine markedly up-regulated the gene expression of FABP3 and LPL(P＜0.05).In addition,the abundance of fatty acid transport protein and fatty acid binding protein were significantly increased by betaine supplementation(P＜0.05,Fig.4).

Results

Betaine on serum lipid metabolites

As shown in Fig.1,there was no significant difference in the levels of serum FFA and triglyceride in the pigs fedbetaine compared with control group.Additionally,the concentration of HDLC and TC were significantly lower in the betaine treated pigs(P＜0.05).

Table 2　Primers of target genes for RT-PCR

Genes　GenBank accession　Primers sequences(5′to 3′)　Product size,bp　Annealing temperature,°C β-actin　XM_003124280.3　CCTGCGGCATCCACGAAAC　123　63 TGTCGGCGATGCCTGGGTA AMPKα2　AY159788.1　GGTCTGGTTCCTCAACACCTCA　90　63 GGCTCTCCGCAGTGACAGAAT PPARγ　NM_214379　GTGGAGACCGCCCAGGTTTG　108　64 GGGAGGACTCTGGGTGGTTCA LPL　NM_214286.1　CCCTATACAAGAGGGAACCGGAT　138　63 CCGCCATCCAGTCGATAAACGT CPT1　NM_001007191.1　GGACGAGGAGTCTCACCACTATGAC　128　63 TCTTGAACGCGATGAGGGTGA FATP1　NM_001083931.1　CCCTCTGCGTCGCTTTGATG　151　63 GCTGCGGTCCCGGAAATACA FAT/CD36　NM_001044622.1　CTGGTGCTGTCATTGGAGCAGT　160　63 CTGTCTGTAAACTTCCGTGCCTGTT FABP3　NM_001099931.1　CCAACATGACCAAGCCTACCACA　176　63 ACAAGTTTGCCTCCATCCAGTGT PPARα　NM_001044526.1　GGCTTACGGCAATGGCTTCA CGGTCTCCGCACCAAATGA　168　64

Table 3　The primary antibodies for Western blot

Primary antibody　Order numbers　Dilution　Size,kDa Anti-Cardiac FABP　abca ab45966　1:1500　15 Anti-FATP1　abcam ab81875　1:2000　65 Anti-CPT1B　abcam ab104662　1:2000　88 Anti-Phospho-AMPK 25351:1000　62Anti-AMPKα 58321:1000　62β-actin(C4)　Santa Cruz SC-47778　1:1500　43

Betaine on muscle lipid metabolites

Muscle lipid metabolites analysis

Availability of data and materials

Results were presented as means and standard deviations.Statistical analysis was performed by one-way analysis of variance(ANOVA)and the Duncan method was used to put up multiple comparison with the statistical software SPSS 19.0.In all analyses,the level of significant difference was set at P＜0.05.

Key factors involved in muscle FA oxidation

In present study,betaine supplementation increased the level of free fatty acids in muscle,which may have resulted due to a change in the balance of fatty acid uptake and oxidation.Betaine may promote fatty acid uptake via increasing the expression of fatty acid transporters including FAT/CD36,FATP1 and FABP3 in muscle.Additionally,betaine activated AMPK and up-regulated PPARα and CPT1,and may enhance fatty acid oxidation in muscle.Fatty acid accretion in muscle represents a balance between uptake and oxidation,and it seems that the effect of betaine on uptake was stronger than oxidation.

The gene expression of AMPKα2,PPARα and CPT1 were significantly higher in pigs fed with betaine than the control group.(P＜0.05,Fig.6).Furthermore,betaine supplementation markedly increased the abundance of　phosphorylated-AMPK　and　CPT1　in　muscle(P＜0.05,Fig.7).

Discussion

Fig.1　Effect of betaine supplementation on serum parameters of lipid metabolism.The levels of serum free fatty acid(FFA,a),triglyceride(b),total cholesterol(c)and high-density lipoprotein cholesterol(HDLC,d).a,bValues without common superscript letters differ significantly(P＜0.05).Low betaine and high betaine represent 1250 mg/kg and 2500 mg/kg betaine addition,respectively

Fig.2　Effect of betaine supplementation on total cholesterol,FFA and triglyceride in muscle.The levels of total cholesterol(a),free fatty acid(FFA,b)and triglyceride(c)in muscle.a,bValues without common superscript letters differ significantly(P＜0.05).Low betaine and high betaine represent 1250 mg/kg and 2500 mg/kg betaine addition,respectively

Fig.3　The relative gene expression of key factors involved fatty acid uptake in muscle.mRNA expression was performed by RT-PCR and β-actin was chosen as reference gene.aThe relative expression of FAT/CD36,FATP1,LPL and PPARγ in muscle,(b)The relative expression of FABP3 in muscle.a,bValues without common superscript letters differ significantly(P＜0.05).Low betaine and high betaine represent 1250 mg/kg and 2500 mg/kg betaine addition,respectively

Fig.4　The relative protein abundance of FATP1 and FABP3 in muscle.Western blot results were shown in a(The control group:1-1,1-2,1-3;Low betaine group:2-1,2-2,2-3;High betaine group:3-1,3-2,3-3).Data were normalized with β-actin as shown in b,c.a,bValues without common superscript letters differ significantly(P＜0.05).Low betaine and high betaine represent 1250 mg/kg and 2500 mg/kg betaine addition,respectively

Fatty acid metabolism in muscle includes uptake,synthesis and oxidation[24-26],but the synthesis is at a slow rate[27].The main source of fatty acid in muscle tissue includes transport from plasma and hydrolysis from chylomicron and very-low-density-lipoprotein(VLDL)with LPL.Our study found that the concentration of FFA was significantly increased in muscle when pigs were fed betaine,similarly to the studies carried out by Yang et al.[28]and Fernández-Fígares et al.[29].We speculated that the transport of FFA and/or the hydrolysis may be enhanced.More experiments were carried out regarding factors involved in fatty acid transport in muscle tissue.It is widely recognized that long chain fatty acid(LCFA)cross the plasma membrane via a protein-mediated mechanism.A number of fatty acid transporters have been identified,including fatty acid translocase/cluster of differentiation　(FAT/CD36)and fatty acid transport proteins(FATP1)[30].We found that betaine supplementation up-regulated gene expression for FATP1 and FAT/CD36.Experiments in vitro have shown that over expression of FATP1 increased the uptake of LCFA in cells[31]and studies in vivo documented that muscle-specific over-expression of FAT/CD36 enhanced cellular fatty acid uptake in mice[32].FABP3,another important protein in fatty acid transportation,plays a role in transporting fatty acid from the sarcolemma to their intracellular sites of metabolism[33].In muscle cells,the intracellular transport of LCFAs is facilitated to a great extent by FABP3[34]Additionally,FABP3 is confirmed to be associated with intramuscular fat in pigs[35].Our studies showed that feeding betaine up-regulated the protein abundance of FABP3.In addition,the gene expression of FABP3 was enhanced when pigs were fed with 2500 mg/kg betaine but no difference was found with 1250 mg/kgbetaine addition.The possible reason maybe that FABP3 expression is translationally rather than transcriptionally regulated[36].In summary,betaine may promote the uptake of fatty acids in muscle via regulating the expression of FAT/CD36,FATP1 and FABP3.As mentioned above,LPL is the principal enzyme that hydrolyzes circulating triglycerides and it also can increase lipid uptake[37].The results showed a significant increase in the gene expression of LPL with the addition of 2500 mg/kg betaine,which indicates betaine might enhance lipid uptake as well as chylomicron hydrolysis.The nuclear receptor PPARγ is a central regulator of adipose tissue development and an important modulator of expression in adipocytes[38].To date,only a limited number of genes are known to be direct targets of PPARγ in adipose tissue.The majority of these encode proteins with direct links to lipid metabolism including LPL,FATP and FAT/CD36[39,40].In present study,the gene expression of PPARγ was significantly higher in betaine-fed groups than the control group.We found that the effect of betaine on PPARγ was similar to its downstream target genes.All these results were similar to Albuquerque[41]and imply that betaine may facilitate fatty acids uptake in muscle via affecting key factors involved in FFA uptake,and the specific regulation mechanism needs more research.

Fig.5　Effect of betaine supplementation on the level of carnitine(a)and malonyl-CoA(b)in muscle.Low betaine and high betaine represent 1250 mg/kg and 2500 mg/kg betaine addition,respectively

Fig.6　The relative mRNA expression of factors involved in fatty acid oxidation in muscle.mRNA expression was performed by RT-PCR and β-actin was chosen as reference gene.a,bValues without common superscript letters differ significantly(P＜0.05).Low betaine and high betaine represent 1250 mg/kg and 2500 mg/kg betaine addition,respectively

Fig.7　The relative protein abundance of p-AMPK and M-CPT1.The results of western blot were showed a and b(The control group:1-1,1-2,1-3;Low betaine group:2-1,2-2,2-3;High betaine group:3-1,3-2,3-3).p-AMPK(the activated form of AMPK)was normalized with AMPK(shown in c)and MCPT1(the muscle type of CPT1)was normalized with β-actin(shown in d).a,bValues without common superscript letters differ significantly(P＜0.05).Low betaine and high betaine represent 1250 mg/kg and 2500 mg/kg betaine addition,respectively

The concentration of FFA in muscle tissue resultes from the balance of transport and oxidation.As a methyl donor,betaine participates in the biosynthesis of carnitine and because of this,betaine may be related to fatty acid β-oxidation.LCFAs are first transformed into acyl CoA,then transferred into mitochondria after combining with carnitine where it is oxidized.Carnitine palmitoyl transferase I(CPT1)is the rate-limiting enzyme that controls the step of combination and malony-CoA is an allosteric inhibitor of CPT1[42].Whereas the synthesis of malonyl-CoA is catalyzed by acetyl-CoA carboxylase(ACC),the activity of the ACC is regulated by phosphorylation of AMPK[43].Hence,AMPK-ACCCPT1 is an important signaling pathway to regulate fatty acid β-oxidation in mitochondria.Cai et al.[44]found that gestational dietary betaine supplementation downregulated expression of ACC in neonatal piglets and Pekkinen et al.[11]found betaine supplementation had an impact on carnitine metabolism in high-fat-fed mice.Our experiment didn’t find significant changes in muscle concentrations of malonyl-CoA or carnitine.The different results might be related to the different experiment condition and the mechanism needs to be further investigated.Increased gene expression and protein expression of CPT1 were up-regulated with betaine addition,which　implied　betainemayenhancefattyacid　　βoxidation in muscle tissue.However,others have shown betaine supplementation reduced the activity of CPT1 and mRNA abundance,and further increased IMF in finishing pigs[Duroc×(Seghers×Seghers)][15].We speculate that the effect of betaine addition on CPT1 might be influenced by breed and muscle type.In order to get a better understanding,we further analyzed effects of betaine on AMP-activated protein kinase(AMPK)and PPARα,which are both upstream regulatory factors of CPT1.AMPK is a crucial energy sensor for cells,which can promote the catabolism of fatty acids by enhancing their uptake into mitochondria and their consequent breakdown by beta-oxidation[45].It was reported that activated AMPK in muscle enhances the gene expression of PPARα and CPT1[46],and CPT1 also seems to be a target of PPARα[47].In the current experiment,the gene expression of both PPARα and AMPK were higher in betaine-fed groups as well as protein expression of p-AMPK(the activated form of AMPK).Similar to our previous report in rat liver[12],it can be inferred that betaine affected fatty acid oxidation in muscle via activating AMPK and up-regulated PPARα and CPT1 gene expression.

The effect of betaine supplementation on cholesterol metabolism was of interest.The present study showed that betaine supplementation decreased the concentration of serum cholesterol and HDLC and increased cholesterol level in muscle,which was consistent with the studies by Albuquerque et al.[41]and Yang et al.[20].However,Matthews et al.[48]and Martins et al.[49]reported that betaine supplemented pigs presented higher serum cholesterol.The efficacy of betaine in regulating the concentration of cholesterol in pigs shows variable results and seems to depend on both animal and dietary factors.Although the results were inconsistent,it seems to indicate that betaine might affect cholesterol partitioning or maybe enhances the transport of cholesterol in pigs,and more research is needed to clarify the specific mechanism.

Conclusions

Betaine supplementation did not affect carnitine or malonyl-CoA in muscle compared to the control group(P＞0.05,Fig.5).

Abbreviations

CPT1:Carnitine palmitoyl transferase 1;FABP3:Fatty acid binding protein;FAT/CD36:Fatty acid translocase/cluster of differentiation;FATP1:Fatty acid transporter protein1;FFA:Free fatty acid;HDLC:High-density lipoprotein cholesterol;LPL:Lipoprotein lipase;TC:Total cholesterol

Acknowledgements

“雅颂”之声被古人视为“正声”“正体”[11]。《周颂》是人类理性精神开始自觉的产物，虽然杂糅着宗教情绪，但是非理性色彩并不浓郁。与《商颂》的“雅颂”思想拥有崇奉上帝的迷狂观念不同，《周颂》的“雅颂”思想具有伦理化特征。

We would like to thank Healthy Husbandry Sci-tech Co.,Ltd.(Hangzhou,China)for their offer with betaine.

作者简介： 卜琰，女，湖南常德人，常德市安乡县教师进修学校专任教师，讲师，本科学历，研究方向：农村中小学教师培训。

Key factors involved in muscle FFA intake

1.正确接产。当母猪有产仔征兆时，要有专人护理。当第一个仔猪出生后，要尽快接产。接产时，首先将仔猪嘴里的粘液清除干净（仔猪有叫声发出为标准），再把仔猪身上的粘液清除干净，在脐带距腹部1～1.5寸处，将脐带的血液向腹部推挤，然后用钝性断脐，用碘酒消毒断端，同时修剪仔猪的乳牙，然后放入保温箱内保温，等待第二个仔猪的出生。在第一个仔猪产出后1 h内，可以将已产出的仔猪喂奶，能够刺激母猪加快分娩。一般相邻两个仔猪产出时间间隔在5～10 min，整个产程在1～3 h。

The raw data for the current study are available from the corresponding author on reasonable request.

Funding

未来脊髓成像方法的发展需要MRI设备制造商和软件开发工程师进行不间断的更深层次沟通，以使方法和技术共享。未来发展的方向为促进方法和数据的广泛共享，如可以为脊髓成像软件和方法建立一个自由访问的方法库，可以用于共享脊髓成像数据。专门讨论脊髓成像方面的成果和新进展，使更多研究人员能参与，弥补相互不足而促进更快的发展。因此，在我们对脊髓损伤的病理生理学的详细了解和脊髓功能磁共振成像所利用的信号变化的生物物理机制之间架起桥梁是很重要的。放射科医生、神经学家和脊柱外科医生由于工作繁忙，无法亲自进行后期手动生成量化指标，所以数据分析需要完全自动化，方法耗时、不可靠或不准确都是不可接受的。

This work was funded and supported by National Basic Research Program of China(No.2012CB124705),Zhejiang Provincial Key Research and Development Program(2015C03006)and Provincial Key S&T Special Projects(2015C02022).

Authors’contributions

YW and JF designed the study.SL and HW were involved in performing the experiment and data interpretation.SL drafted the manuscript and XW revised the manuscript.All authors read and approved the final version of the manuscript.

Ethics approval

“所谓同声传译，是用一种语言把另一种语言所表达的思想和情感以与讲话人几乎同步的速度，用口头形式重新表述处理的一种翻译实践”(张维为)这种“同步性”意味着译员需要同时完成听力、理解、表达等多个任务。这个过程需要高强度的精力。在精力分配不佳的情况下，译者就会出现错误。然而译者在这一过程是主动的，可以对分配过程进行控制的。我们把这一过程称为译者的协调机制。

The experiment protocols used in this study was approved by the Institutional Animal Care and Use Committee of Zhejiang University.

Consent for publication

Not applicable.

其实王姐也没抱太大希望，来到哈尔滨市第一医院，仍不挂号，便直接领我们到住院部去主任办公室找蒋利学。很不凑巧，蒋利学刚好去查床了，王姐便不客气地掏手机，给蒋利学挂电话。

周内尿素连续小幅探涨为市场带来新的操作思路，业内谨慎心态渐感释怀，只是追涨的意愿有限，一旦价格继续探涨，下游需求或将被再度刺激。客观分析全国52%左右的开工率确为高价尿素的保障条件。目前，虽尿素外贸因瓶颈无从谈起，但内需方面却渐被业内认可。预计下周国内尿素主产区出厂报价1900-1950元/吨。

顶板结构特点及其运动规律决定了综放支架工作阻力显现方式，通过分析工作面顶板结构，确定导致压架事故的原因有3条[15-16]。

Competing interests

The authors declare that they have no completing interests.

考虑区域自治能力的主动配电网分层优化调度//孟繁星,孙英云,蒲天骄,陈乃仕,李晨,吴华华//(15):70

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