The effects of starter microbiota and the early life feeding of medium chain triglycerides on the gastric transcriptome profile of 2-or 3-week-old cesarean delivered piglets

Background

The stomach is a key point in the interface between the ingesta and the digestive system,affecting post-gastric efficiency of digestion,playing a role in several endocrine functions and being involved in several diseases of domestic animals and humans.Nevertheless the main molecular-cellular processes that govern its development and homeostasis have not yet been well defined.Current evidence indicates that the stomach can actively interact with the microbiota resident in or in transit to the lower digestive tracts.In pigs,the stomach shows an array of important tools related to immunity and barrier defense,also　developmentally　controlled:toll-like　receptors(TLRs)2,3 and 4[1]among the receptors specialized in detecting　microorganismsand　activating　an　innate immune response,the polymeric immunoglobulin receptor responsible for the transport of secretory immunoglobulins[1],submucosal and mucosal lymphoid follicles with and without compartmental organization of T and B lymphocytes[2]and plasma cells[3].This implies the relevance of adequate stimulation of the local microbiota as has been demonstrated in the intestine[4].This would also have practical relevance for managing the piglets,namely stimulating the diffusion of management solutions favoring the acquisition by neonate piglets of microbiota also out of their litter.Conversely,the acquisition of the dominant importance of specific core microbiota would indicate that certain bacterial pools can be used for the early orientation or acceleration of gut maturation.However,surprisingly,studies regarding the impact of different microbiota on the development of the stomach of mammals are lacking.It has generally been recognized that the availability of a single ‘standard’microbiome could circumvent several problems in studies focusing on the interaction of the host and its microbiota[5].For this purpose,Laycock et al.[6]selected a few bacteria strains as a standardized microbiota owing to their positive influence on a new early microbial colonization in the porcine gut.

Two studies were designed to elucidate the molecular networks in the oxyntic mucosa of the young pig which characterize the long-term individual response after various associations with a simplified or complex starter microbiota during their first days of life.Furthermore,an additional aim in the second study was to evidence the possible action of a dietary source of medium chain triglycerides on the transcriptome of the oxyntic mucosa.

Methods

Two different trials(Fig.1)were carried out on 12 and 24 piglets obtained from from 4 and 6 sows respectively[(Great York × Pietrain) × ‘Dalland’cross]by cesarean delivery(CD)(d 0);they were divided over two microbiota association treatments,and were then slaughtered and sampled at wk 2(Trial 1)or wk 3(Trial 2)of age,respectively.Piglets from different litters were balanced over the various groups based on litter,body weight and gender and in each pen/room 3 piglets per group were housed.

The piglets were housed in two pens per clean,nonsterile room having an automatic feeding system for supplying a moist diet,and were balanced for birth weight(BW)and litter of origin.At 1 h and 5 h after birth,each piglet received 110 mL sow serum(Trial 1)or 90 mL pasteurized(30 min at 60°C)sow colostrum by oral gavage[13,14](Trial 2).All the piglets received a simple starter microbiota consisting of Lactobacillus amylovorus(3.6×107CFU),Clostridium glycolicum(5.7×107CFU)and Parabacteroides spp.(4.8×107CFU)by oral inoculation(2 mL)on d1,d2 and d3 after birth.These bacteria　are　the　most　frequently　identified　of　the phylogenetic groups in the intestines of piglets as have previously　been　proposed　as　minimal　intestinal colonization microbiota for gnotobiotic pigs[6].

1.3 统计学处理 采用SPSS19.0统计软件进行资料分析。计数资料部分如两组MDRO感染终末消毒流程缺陷率比较使用χ2检验。部分计量资料如年龄的比较，使用两独立样本t检验。P＜0.05为差异有统计学意义。

Fig.1　General summary of the factors in the two trials

On d 3(at 15:30 and 21:00 h)and on d 4(at 8:00 h),the piglets of treatment CA received a fecal-oral inoculant immediately after feeding in the form of a 2-mL suspension of feces(feces in 0.1 mol/L sterile potassium phosphate buffer(pH 7.2),diluted 1:10).The SA-treated piglets received a placebo treatment(2 mL of a sterile 0.1 mol/L potassium phosphate buffer,pH 7.2).

Average birth weight was 1213±144 g and 1228±164(Trial 1),and 1273±138 g and 1275±153 g(Trial 2)for the CA and the SA piglets,respectively.The piglets were fed a milk replacer diet ad libitum during a period of 5 d(d0-d4).It consisted of bovine skimmed milk powder,whey powder,vegetable oil,hydrolyzed wheat protein,wheat starch,sucrose,and a vitamin and mineral premix,and contained 230 g crude protein per kg milk replacer(Table 1).All the piglets in Trial 1 and half of the piglets in Trial 2 were then fed a moist diet(CTRL)based on whey powder,maize,wheat,toasted full fat soybeans,oat flakes,sucrose,soybean meal,vegetable oil,coconut oil,wheat gluten,potato protein,rice protein,and brewer’s yeast during the remainder of the study(Table 2).The other half of the piglets in Trial 2 were fed the same diet but containing 7%of coconut oil in place of 4.68%soy oil and of 2.32%palm oil in order to generate a diet fortified in medium chain triglycerides(MCTs)(Table 2).In other words,the 24 piglets of the 2ndtrial were allocated by BW to 4 treatments arranged in a 2×2 factorial design,with 2 different early associations with microbiota(CA and SA)and two diets(CTRL and MCT).On d 16(Trial 1),and on d 21 and d 22(Trial 2),the piglets were euthanized,and the stomach wassampled always in the center of the area corresponding to the oxyntic mucosa.

Table 1　Analyzed nutrient composition of the artificial milk provided to the piglets during the first 4-day after birth(in g/kg,unless stated otherwise)

Nutrient Content Dry matter　957 Ash　78 Crude protein　222 Crude fiber　1 Starch　27 Glucose　29 Sugar　435 Lactose　405 Phosphorus　7.2 Calcium　8.6 Crude fat　227 Total fatty acids(g/kg fat)　913 Saturated fatty acids(g/kg fat)　461 Monounsaturated fatty acids(g/kg fat)　360 Polyunsaturated fatty acids(g/kg fat)　86

Table 2　Ingredient composition of the experimental diets provided to the piglets from day 5 after birth until the end of the study(g/kg)

Ingredients　CTRL　MCT Maize　200.0　200.0 Whey powder　180.0　180.0 Wheat　150.0　150.0 Oat hulls　80.0　80.0 Barley　70.0　70.0 Wheat gluten meal　50.0　50.0 Soybeans,heat treated　40.0　40.0 Soycomill-P　33.0　33.0 Lactose　30.0　30.0 Protastar　30.0　30.0 Monocalciumphosphate　0.6　0.6 Rice meal　12.0　12.0 Limestone　0.9　0.9 Salt　3.6　3.6 Dicalciumphosphate　19.4　19.4 Sodium bicarbonate　4.5　4.5 Piglet premix 0-5%　5.0　5.0 L-Lysine HCl　5.9　5.9 DL-Methionine　2.0　2.0 L-Threonine　1.1　1.1 L-Tryptophan　0.9　0.9 L-Valine　0.5　0.5 Soy oil　50.6　3.8 Palm oil　30.0　6.8 Coconut oil　0　70.0

RNA isolation and gene array quantification

Total RNA was isolated from the gastric tissue samples using the FastPure RNA kit(TaKaRa Bio Inc.,Shiga,Japan).All other procedures were in agreement with the manufacturer’s protocol.The RNA purity and integrity were evaluated using Agilent Bioanalyzer 2100(Agilent Technologies,Palo Alto,CA)just before the transcriptome analysis.The total RNA was hybridized on Affymetrix Porcine Gene 1.1 ST array strips.The hybridized arrays were scanned on a GeneAtlas imaging station(Affymetrix,Santa Clara,CA,USA).Performance quality tests of the arrays including the labelling,hybridization,scanning and background signals using Robust Multichip Analysis were carried out on the CEL files using the Affymetrix Expression Console.

Statistics

The Affymetrix Trascript IDs,in general each characterized by several exonic sequences,were associated with 13,406 human gene names,based on Sus scrofa Ensembl(release 79,www.ensembl.org),where data regarding each porcine gene could be directly accessed using the names of genes which were adopted for the present text.Regarding the processed gene expression values,two exploratory functional analyses were applied with Gene Set Enrichment Analysis using the Hallmark(aggregating many gene sets to represent well-defined biological states or processes)and C5.BP(based on Gene Ontology(GO))catalogues of gene sets from Molecular Signatures Database　v5.1　(http://software.broadinstitute.org/gsea/msigdb/).The normalized enrichment score(NES)was calculated for each gene set,and statistical significance was defined when a False Discovery Rate(FDR)%＜25 and P-values of NES＜0.05.Enrichment score P-values were estimated using a gene set-based permutation test procedure.A new data set was created and added to the Hallmark catalogue which included 19 genes typically characterizing the stomach as compared with the different intestinal sections[15],and were more or equally expressed in the porcine oxyntic mucosa as compared with pyloric mucosa[16].Reference sequences for all these gene are available on Sus scrofa Ensembl except for the following genes that have the Ensembl gene accession number:ATP4A,ENSSSCT00000003207;EGF,ENSSSCT00000010002,or the NCBI Reference Sequence:LPL,NM_214286.1;TFF1,XM_003358973.3.

The overlap in enriched GO terms was visualized using the　Enrichment　Map　(http://baderlab.org/Software/EnrichmentMap20)plugin for Cytoscape 2.8.0(http://www.cytoscape.org/),including the gene sets with a P-value＜0.005 and an FDR q-value＜0.10.The nodes were joined if the overlap coefficient was≥0.5.

Results

As general information,body weight gain was similar among piglet groups both in trial 1 and trial 2.Average birth weight was approx.1 kg,increased to 2.5 kg at 2 wk of age and to 4 kg at 3 wk of age.Feed intake on a pen base did not differ.The general health score(including diarrhea)was similar among piglets,both for trial 1 and 2,and diarrhea was absent in trial 1 and in trial 2 diarrhea was only incidental and erratic among the 4 groups of piglets as judged on a daily basis from birth till 3 wk of age.

Figure 2a visualizes the nodes of the gene sets of the Hallmark catalogue,enriched(color red)or impoverished(color blue)in the porcine oxyntic mucosa of piglets early associated with complex microbiota(CA)as compared to simplex microbiota(SA)piglets,and sampled at two or three weeks of age(represented by the center and the ring of each node,respectively).The CA upregulated 9 and 10 gene sets in piglets of two and three weeks of age,respectively as compared to the SA.Of these sets,4 were upregulated at both ages.The CA downregulated 6 gene sets at both ages,all related to cycling activity,mitosis regulation steps and general protein synthesis.In addition,another 1 and 8 gene sets were downregulated in piglets of two and three weeks of age,respectively as compared to the SA.

In addition to the gene sets of the Hallmark catalogue,represented in Fig.2a,the new cured gene set oxyntic mucosa was upregulated by the CA at both two and three weeks of age(Table 3).Nevertheless,the upregulation involved more genes in two-week old piglets than in the older piglets.Potassium voltage-gated channel,Isk-related family,member 2(KCNE2),epidermal growth factor(EGF),Anion exchanger 2(SLC4A2),Lipoprotein lipase(LPL),potassium inwardly rectifying channel,subfamily J,member 15(KCNJ15)and solute carrier family 2(facilitated glucose transporter),member 4　(SLC2A4)genes were enriched at both ages of the piglets.

The diet containing an oil source of medium chain fatty acids in each group of piglets assigned to different early priming programs(3 wk of age,trial 2),in general,activated the inflammatory response,with the involvement of T cell cycling(indicated by the enrichment of IL2_STAT5_sSIGNALING together with TNFA_SIGNALING_VIA_NFKB)(Fig.3a).This was also particularly associated with the activation of CCL2(Chemokine(C-C Motif)Ligand 2)and CXCL10(Chemokine(C-X-C Motif)Ligand 10)involved in monocyte chemotaxis and T-cell migration[35].However,this was particularly evident when MCTs were given to the SA piglets,with a general enrichment of the nodes related to chemokine activity and cytokine binding(Fig.3b).Interestingly,feeding piglets a diet supplemented with coconut oil increased the intestinal translocation of the bacterial enterotoxin　ascompared　to　the　controldietora polyunsaturated oil[36].The Authors of this study explained this observation with the increased endocytosis of the enterotoxin favored by the innate immune receptor complex based on CD 14/Toll-like receptor 4(TLR4)/Lymphocyte Antigen 96.The Authors detected consistent expression of the TLR4 gene in our data set and a similar action regarding the MCT diet cannot be excluded,with the consequent induction of the inflammatory response by the enterotoxin eventually present in the stomach and derived from resident or transient bacteria.

A second gene enrichment study was generated to demonstrate the effect of early association with microbiota of different complexities regarding the enrichment of gene sets expressed in the porcine oxyntic mucosa fed each of the two diets.Figure 3a visualizes the nodes of the gene sets of the Hallmark catalogue,enriched(color red)or impoverished(color blue)in samples from the CA piglets as compared to the SA piglets,and fed the MCT or the CTRL diet(represented by the center and the ring of each node,respectively).As compared to the SA piglets,the CA enriched genes in the sets related to both the interferon α and γ response when the CTRL diet was given while the same sets were impoverished by the CA with the MCT diet.The CA downregulated 5 gene sets in both the diets,all related to controlling the cell cycling,cellular transformations and the related stress-induced homeostatic mechanisms of DNA control and replication.Another 3 gene sets were also downregulated by the CA,including mTORC-related protein synthesis and cellular stress response related to the endoplasmic reticulum.Conversely,3 gene sets were downgraded by the CA only when the CTRL diet was used,including the set related to protein secretion.The set of genes related to inflammatory response was enriched in the CA piglets fed only the CTRL diet while the TNFα and reactive oxygen species pathway sets were enriched in the SA piglets.

Fig.2　The nodes of the gene sets enriched in the oxyntic mucosa of piglets early associated with CA as compared to the SA piglets and sampled at two or three weeks of age.a The entire picture regarding the Hallmark gene set,summarizing the main biological states or processes;b Picture of nodes obtained with the more detailed gene ontology(GO)gene set after the removal of those related to cell mitosis;the nodes represent gene sets.The color on the center of the node represents the results of the trial ending at 2 wk;the color on the ring visualizes the trial ending at 3 wk.The edges represent the link of two or more gene sets sharing the same core group of genes explaining the enrichment of each of the gene sets.Enrichment significance(P-value)is conveyed as node color intensity where red stands for upregulation at 2 or 3 wk in the CA piglets,and blue stands for downregulation as compared with the SA piglets.The node size represents the number of genes in the gene set

Figure 3b shows the nodes of the gene sets of the C5 catalogue　(based　on　Gene　Ontology),differentially enriched in the CA or the SA piglets,supplemented with medium chain fatty acids(MCTs)or not(CTRL),following the same scheme as Fig.2a.Compared to the SA,the gene sets related to transmembrane receptor activity,including that of the neurotransmitters,were enriched in the CA piglets,particularly when fed the MCT diet(as demonstrated by the more intense red coloration).With the MCT diet,several gene sets related to anion channel activity were enriched by the CA as compared to the SA.

艺术创作是灵活而多变的。这些年，无论民族声乐演唱风格，还是情感表达，都在不断变化。除此之外，同一首民族声乐作品，演唱者不同，风格也存在差别，但不影响情感表达。民族声乐表演者倾向于通过肢体语言进行表演，并赋予欣赏者以想象空间。民族声乐表演过程中，表演者要注重自身情绪变化，情感随着歌词的改变而有所不同，使表演过程更加生动，更富想象力。以知名民族声乐作品《在希望的田野上》为例，优美的歌词和动听的旋律，会使听众的脑海中浮现歌曲中的画面和场景，以此拉近演唱者和受众的距离，帮助后者融入到声乐表演中，与表演者产生情感共鸣，真正将这首作品最真挚的情感表达出来[3]。

A third gene enrichment study was generated to demonstrate the effect of the different diets on the enrichment of the gene sets in the oxyntic mucosa from pigs whichhad an early association with microbiota of different complexities.Figure 4a shows the nodes of the gene sets of the Hallmark catalogue,enriched(color red)or impoverished(color blue)in samples from piglets fed the MCT diet compared to CTRL-fed piglets,and belonging to the CA or the SA group(represented by the ring and the center of each node,respectively).The MCT diet enriched gene sets of INFLAMMATORY_RESPONSE,TNFA_SIGNALING_VIA_NFKB,IL2_STAT5_SIGNALING,COMPLEMENT and also EPITHELIAL_MESENCHYMAL_TRANSITION,whatever the type of early association with the microbiota.As compared to the CTRL diet,feeding with the MCT diet impoverished genes in the sets related to both interferon α and γ response in the CA piglets while the same gene sets were enriched with the MCT diet in the SA piglets.Furthermore,ALLOGRAFT_REJECTION,IL6_JAK_STAT3_SIGNALING,APOPTOSIS,KRAS_SIGNALING_UP were also enriched by MCTs only in the SA piglets.

Table 3　List of genes enriched or not inside the new cured gene set oxyntic mucosa tested inside the Hallmark catalogue

aReflects the relative degree to which a gene is overrepresented at the top(positive sign)or bottom(negative sign)of the ordered dataset

Trial 1(2-week-old piglets)　Trial 2(3-week-old piglets)Gene product　Gene name　Rank enrichment scorea　Core enrichment　Rank enrichment scorea　Core enrichment Potassium voltage-gated channel,Isk-related family,member 2 KCNE2　0.729　Yes　0.330　Yes Epidermal growth factor　EGF　0.583　Yes　0.185　Yes Anion exchanger 2　SLC4A2　0.580　Yes　0.176　Yes Lipoprotein lipase　LPL　0.578　Yes　0.321　Yes Potassium inwardly rectifying channel,subfamily J,member 15 KCNJ15　0.576　Yes　0.406　Yes Chloride intracellular channel 6　CLIC6　0.495　Yes　0.070　No Solute carrier family 2(facilitated glucose transporter),member 4 SLC2A4　0.479　Yes　0.275　Yes ATPase,H+/K+exchanging,β polypeptide　ATP4B　0.417　Yes　0.058　No ATPase,H+/K+exchanging,α polypeptide　ATP4A　0.371　Yes　0.013　No Insulin-like growth factor binding protein 5　IGFBP5　0.266　Yes　0.055　No Pheromaxein,subunit C　PHEROC　0.248　Yes　-0.048　No Acquaporin 4　AQP4　0.236　Yes　0.006　No Potassium inwardly rectifying channel,subfamily J,member 13 KCNJ13　0.167　No　0.202　Yes Ghrelin　GHRL　0.094　No　-0.237　No Trefoil factor 1　TFF1　0.093　No　0.002　No Pepsinogen C　PGC　0.075　No　-0.072　No Insulin-like growth factor 1　IGF1　0.067　No　0.401　Yes Pepsinogen B　PGB　-0.023　No　-0.202　No Chitinase,acidic　CHIA　-0.089　No　-0.121　No

Several nodes were also observed with the more detailed GO catalogue(Fig.4b)with the MCT diet in the SA piglets as compared to the CTRL diet:a large network of gene sets related to chemokine and cytokine activity and binding,immune cell activation and,in general,the positive regulation of the immune system process;sparse gene sets including those related to tissue modelling and differentiation.

Discussion

ATP4A:ATPase,H+/K+exchanging,α polypeptide;ATP4B:ATPase,H+/K+exchanging,α polypeptide;CA:Treatment with association with complex microbiota inoculum;CCL2:Chemokine(C-C Motif)Ligand 2;CHRNA2:Cholinergic receptor,nicotinic,alpha 2(neuronal);CLCA1:For chloride channel accessory 1;CTRL:Control diet;CXCL10:Chemokine(C-X-C Motif)Ligand 10;CXCL10:Chemokine(C-X-C Motif)Ligand 10;EGF:Epidermal growth factor;FDR:False discovery rate;GABRG2:Gamma-aminobutyric acid(GABA)A receptor,gamma 2;GO:Gene Ontology;HTRIB:for 5-hydroxytryptamine(Serotonin)receptor 1B,G protein-coupled;JCHAIN:Joining chain of multimeric IgA and IgM;KCNE2:Potassium voltage-gated channel,Isk-related family,member 2;KCNJ15:Potassium inwardly rectifying channel,subfamily J,member 15;LPL:Lipoprotein lipase;MCT:Diet with medium chain tryglicerides;MMP3:Matrix metalloproteinase 3 matrix metalloproteinase 3;NES:Normalized enrichment score;SA:Treatment with simplified microbiota;SLC2A4:Solute carrier family 2(facilitated glucose transporter),member 4;SLC4A2:Anion exchanger 2;T1-T2:Trial 1 and Trial 2;TGFB:Transforming growth factor β;TLR4:Toll like receptor 4

Proliferative response in the oxyntic mucosa

Fig.3　The nodes of the gene sets enriched in the oxyntic mucosa of piglets early associated with the CA as compared to the SA piglets,and supplemented with medium chain fatty acids(MCTs)or not(CTRL).a The entire picture for the Hallmark gene set,summarizing the main biological states or processes;b Picture of nodes obtained with the more detailed GO gene set;Nodes represent gene sets.The color on the center of the node represents the MCT diet;the color on the ring represents the CTRL diet.The edges represent the link of two or more gene sets sharing the same core group of genes,thus explaining the enrichment of each of the gene sets.Enrichment significance(P-value)is conveyed as node color intensity where red stands for upregulation in the CA piglets fed the MCT or the CTRL diets,and blue for downregulationas compared with the SA piglets.The node size represents the number of genes in the gene set

The local action of pathogenic bacteria can overactivate a range of growth signaling pathways within the gastric mucosa,thus altering the physiological balance between growth and apoptosis[17,18].On the contrary,in rats,the probiotic bacteria can have a restorative action on an acetic-induced gastric ulcer,reestablishing correct cell proliferation[19].Bacteria can also produce putrescine and spermidine,the short-chain aliphatic amines involved in the regulation of cell proliferation and differentiation[20].In our study,the early provision of a complex starter microbiota to the piglets favorably prevented the activation of pathways related to cycling activity and mitosis regulation steps(Fig.1a)in the gastric mucosa at an age of two and three weeks as compared to the association with a simple microbiota.This may have been related to the observation that a short encounter with a complex microbiota in early life could be sufficient to influence the intestinal microbiota in the following weeks([21]which was the same animal experimental design of trial 1.Nevertheless,the reduction of proliferative activity in the CA piglets was not accompanied by an insufficient maturation of the oxyntic mucosa but,on the contrary,several major genes implicated in the activity of the parietal cells or typical of the gastric mucosa were upregulated(Table 1).

Not applicable.

(See figure on previous page.) Fig.4 The effect of supplemention with a fortified diet with(MCT)or not(CTRL)on the nodes of the gene sets enriched in the oxyntic mucosa of piglets early associated with the CA)or the SA.a The entire picture for Hallmark gene set,summarizing the main biological states or processes;b Picture of nodes obtained with the more detailed GO gene set;the nodes represent the gene sets.The color on the ring represents the CA piglets while the color at the center of the node represents the SA piglets.The edges represent the link of two or more gene sets sharing the same core group of genes,thus explaining the enrichment of each of the gene sets.Enrichment significance(P-value)is conveyed as node color intensity where red stands for upregulation in the CA or the SA piglets fed the MCT diet,and blue for downregulation as compared with the CTRL-fed piglets.The node size represents the number of genes in the genen set

Acidic secretion function

Inside the stomach,the oxyntic mucosa is characterized by a very large and highly expressed array of genes for ion and water protein channels[16]as compared to the pyloric mucosa(and also to the intestinal mucosa).Thus,the observation that the new cured set of genes typical of the oxyntic mucosa was enriched in the CA piglets as compared to the SA piglets was important.The enrichment involved fewer genes in piglets sacrificed at an older age;especially at 3 wk of age no different expression was seen for the two genes,ATP4A and ATP4B,involved in the synthesis of H+/K+-ATPase(Table 1).This may indicate that the potential of the proton pump to exchange luminal potassium with cytoplasmic hydronium was no different at this age between the CA and the SA piglets.Nevertheless,epidermal growth factor(EGF)is a growth factor important for gastric maturation and the constant upregulation of EGF may indicate that the local paracrine control was more active in the CA,including the stimulation of gastrin from the pyloric area[22].Other potassium channels implicated in the K+balance of parietal cells were also upregulated.In particular,several overlapping nodes of genes related to anion channel activity were upregulated in piglets fed MCTs which were early associated with a complex microbiota as compared to SA-associated piglets fed the same diet(Fig.2b).This indirectly confirmed again that the oxyntic mucosa from the CA piglets was already more able to adapt to different dietary conditions.Notably,the most representative gene of these nodes for differential expression was the gene coding for chloride channel accessory 1(CLCA1)which ranked 5th in the list of upregulated genes.The CLCA1 and CLCA2 products in the stomach were characteristic of the luminal membranes of the murine gastric parietal cells[23]and also of surface mucosa.

(1) 傅里叶红外光谱 (FT-IR)：测试仪器为20SXB型傅里叶红外光谱，上海精密仪器仪表公司，样品与KBr粉末混合压片测试。

Maturation of gastric signaling,enteroendocrine control and neuronal controls

2) 高压油泵等高压燃油系统泄漏引起燃油喷射压力降低，喷入缸内燃油量减少，前期燃烧放热量下降，燃烧爆压降低，膨胀冲程压力曲线下移，影响柴油机的做功性能和工作平稳性。

In the piglets that had been associated with a complex microbiota,particularly when fed the MCT diet,the connected nodes G_PROTEIN_COUPLED_RECEPTOR(GPCR)_ACTIVITY,RHODOPSIN_LIKE_RECEPTOR_ACTIVITY and NEUROTRANSMITTER__RECEPTOR_ACTIVITY were enriched of activated genes(Fig.2b).Several GPCRs are important for the maturation of the gastric mucosa[24]and for the ghrelin-secreting cells[25].In the list of upregulated genes of these nodes,the highest rank was for the gene for 5-hydroxytryptamine(Serotonin)receptor 1B,G protein-coupled(HTR1B).Serotonin is produced in the gastric mucosa by the enterochromaffin cells and the variation of this gene demonstrates the interacting role of early priming by the microbiota and the diet which controls the serotonergic signaling in the oxyntic mucosa.Some metabolites derived from the gut microbiota can modulate serotonin expression in the colon[26].Furthermore,the gene for another serotonin receptor(number 4)was co-associated with the serotonin transporter gene across different states of microbial association colonization stages in the mouse colon[27].

3)课后，教师通过收集评价反馈，开展教学反思，以便修正教学设计，亦可以在平台上教师系统中查看学生的学习情况并对学生的完成情况做出评价，另外在收集平台讨论区上，将学生反馈的疑难问题收集整理出来，作为设计课中学习活动的依据；在学生方面，学生通过平台不断地复习巩固、在课后进行实践练习，检测学习情况，反思学习的收获与不足，反思学习中出现的问题并寻找解决问题的办法，促进知识的升华。

The CA treatment enriched the complex of nodes related to neurotransmitter binding,to neurotransmitter receptor activity and to transmembrane receptor activity.The gastric parietal cells are innervated by secretomotor neurons and stimulated to release acid,and the same is also true for the chief cells which release pepsinogen.The gastric acid secretomotor neurons are cholinergic[28].Thus,the CA could have better activated neuronal control of the gastric secretions,as indicated by its first rank in the list of the genes of the cholinergic receptor,nicotinic,alpha 2(neuronal)(CHRNA2).Another highranking gene was the gamma-aminobutyric acid(GABA)A receptor,gamma 2(GABRG2).In the stomach,the cholinergic enteric neurons present GABAergic neuron immunoreactivity;GABA may also be sensed as hormonal and paracrine signaling[29].Interestingly,GABA is produced by several common gut commensals,including some lactic acid bacteria[30].In fact,our observation could be associated with the presence of a more developed and complex gastric microbiota.

随着普惠金融工作统筹推动，2016年邯郸银监局制定了《2016年推进普惠金融发展工作方案》和《关于金融去杠杆支持供给侧结构性改革的意见》等，将发展普惠金融作为改革的重点领域，并以小微企业金融服务、农村金融服务及精准扶贫为抓手，明确发展普惠金融目标责任与任务，在实施过程中虽然取得了显著成效，但在提高金融服务覆盖率和渗透率方面还存在着许多问题和制约因素。

Induction and regulation of the gastric immune system and local inflammation by both the microbiota and the diet

In general,it has been well demonstrated that,in mammals,the first week of life are extremely relevant for the induction of proper maturation of all the key actors of innate and acquired defenses in the gut,in functional dependence on the host antigenic experience.Specifically,in piglets,in the passage from the 2ndto the 4th wk of age,the intestinal mucosa becomes colonized by CD4+T cells and the number of intraepithelial lymphocytes,and IgM+B cells start to appear in small numbers[31].

The degree of activation of different pathways related to the induction and the regulation of the gastric immune system and local inflammation by the microbiota changed with the age of the animals according to the type of previous association with microbiota and diet.With the control diet,the piglets sampled at three weeks of age which had been associated with a complex microbiota had overlapping enriched nodes related to interferon α and γ,cellular defense response and inflammatory response as compared to the SA piglets(Fig.1a).This activation was also linked to more activity and the receptor binding activation of the cytokines(Fig.1b).Of the genes in these groups,there was gene coding for chemokine(CX-C motif)ligand 11(CXCL11)which ranked 2ndin the list of all genes.The chemotactic protein CXCL11 is involved in mature T-cell recruitment and is induced by interferon γ[32].All the piglets were apparently healthy and,thus,this could be interpreted in the general situation of the higher functional maturity of the CA piglets.The presence of a remodeling phase in the older CA piglets versus the SA piglets could also be supported by the enrichment of gene sets COAGULATION and EPITHELIAL_MESENCHIMAL_TRANSITION where the first ranking gene was matrix metalloproteinase 3(MMP3)responsible for tissue remodeling[33].The differential maturation of the immune cells associated with the gastric tissue of the CA piglets as compared to the SA piglets is also indicated by the higher expression of a gene not considered in the two GSEA catalogues,JCHAIN(Joining chain of multimeric IgA and IgM).The product of the JCHAIN is essential for the fusion of two monomer units of either IgM or IgA,also cooperating with their binding to the secretory component.This　gene　is　induced　by　long-term　bacterial colonization[34].

对现有公路桥梁进行定期的检测维修与养护，不仅可以保证公路桥梁的完整性还可以有效延长公路桥梁的使用寿命，进一步提高公共资源的利用效率避免不必要浪费现象的发生。综上所述，对现有公路桥梁进行定期检测维修与养护，是促进国民经济又好又快发展的重要举措。

With MCTs,the interferon α and γ gene sets were stimulated in the SA piglets(Fig.2a),but not in the CA piglets,indicating that the presence of MCTs in the diet could condition these piglets to a maturation such as that of the CTRL-fed CA piglets.These observations relating to the CTRL-fed piglets are,in some ways,in contrast with those obtained in the 1sttrial with the 2-weekold piglets.In this case,in addition to the already mentioned general mitotic stimulation,the sets of genes for cytokine/chemokine activity and chemokine receptor binding were enriched with the SA diet.Several chemokines characterized by the CXC motif were represented at the top of the core genes characterizing the enriched gene sets,including CXCL11.This led us to conclude that an early association of newborn piglets with a complex microbiota favorably prevents the activation of pathways related to cell division and inflammatory development in the gastric mucosa at an age of two weeks as compared to the association with a simple microbiota.

改革时期是华语解禁与复苏的时期。印尼2009年第24号法律颁布以后，印尼语、地方语和外语有了更明确的定位。目前印尼华语的法律地位是外语，政府维护华人以华语为家庭用语的权利，但在教育体系中必须作为外语进行教学。因此，华校只能以三语学校的形式办学，华语只是学校的办学特色之一。与旧秩序末期一样，印尼语作为教学用语的规定限制了华语在学校的使用。华语只是一门课程，华语的使用范围也仅限于该课程。

Figure 2b visualizes the nodes of the gene sets of the C5 catalogue(based on Gene Ontology),differentially enriched in the CA or the SA piglets at 2 or 3 wk of age following the same scheme as Fig.2a.From the figure,all the gene sets related to cycling activity and mitosis regulation,already shown in Fig.2a,were depicted.The figure primarily shows that two groups of gene sets,related respectively to response to nutrient stimulus and to transforming the growth factor β(TGFB)pathway were upregulated by the CA in 2-week-old piglets,but not in older pigs;a group of gene sets related to chemokine activity was down-and upregulated with CA in piglets of two and three weeks of age respectively as compared to the SA.

Conclusions

The quality of early microbial colonization is important in promoting the functional maturation of the oxyntic mucosa in an age-dependent manner,as indicated by the variation of several genes typical of the oxyntic mucosa and of the gastric tissue in general.Furthermore,this was relevant for establishing a strong balance between the regulation of the immune system and the inflammatory response.The partial dietary substitution of unsaturated fatty acids with a medium chain fatty acid source may have affected the activation of several pathways addressed to the recruitment and the functional maturation of immune cells,especially when the piglets were early associated with a simplified starter microbiota.On the whole,these observations are important for the feed industry when designing strategies to stimulate the gut and immune system maturation of the piglet.Furthermore,the presence of an interaction of the complexity of early microbial colonization with the diet can explain the frequent variable response to early supplemented probiotics to the piglets and be relevant also as a general model for young human studies.

Abbreviations

The joint visualization of the results of the two experiments allowed demonstrating that some patterns of stimulation of gene groups are constantly related to the type of early association to microbiota,at least for the age range of our piglets.Conversely,other gene groups were regulated by the early treatment of pigs in a more age dependent manner.In general,early association with a complex starter microbiota tended to anticipate functional maturation while association with a simplified microbiota stimulated the cell cycles in the oxyntic mucosa.

Acknowledgements

编目员的综合素质包括人生经验、专业背景、思考和看待问题的视角、人文学素养等等，不同的职业视角对资料价值的敏感度不一样。价值甄别首先要解决视角问题，编目员要有明确的认识，不是从观众欣赏的角度，而是要解析、寻找有价值的信息内容。一般来说用户是个人特定情感和体验来理解音视频所传递的信息；而编目员必须从内容价值的角度审视视频资源，揭示有价值的部分。

Funding

This study was financially supported by the European Union(contract No.227549)through the Interplay project.The authors are solely responsible for the study described in this paper,and their opinions are not necessarily those of the European Union.

Availability of data and materials

The datasets supporting the conclusions of this article are available in the National Center for Biotechnology Information’s Gene Expression Omnibus(NCBI GEO)with GEO accession number GSE87124 and GSE87125(https://www.ncbi.nlm.nih.gov/geo/).

Authors’contributions

AJMJ,SJK and PB conceived and designed the experiments;DP and PT performed the experiments;PB and VM analyzed the data;SJK,PT,DP and DL contributed reagents/materials/analysis tools;PT,DP,AJMJ and PB wrote the manuscript.All authors read and approved the final manuscript.

Ethics approval

The protocol of the study was approved by the Committee on the Ethics of Animal Experiments of Wageningen University and Research Centre in Lelystad,The Netherlands(Permission Number:2,011,081.b and 2,012,083.e).

Competing interests

张乾乃鄂东怪杰，江湖人称铁头大哥，其父是油铺首富，在蕲州、汉口开有店铺，财纳四海，名满三江。张乾少时，家父曾以重金聘请本地名儒教授，均无果而终。张乾厌文喜武，力猛过人，好结英豪，素以关张自居。年方十六，即上武当学艺，三年出师，闯荡江湖，下汉口，走河南，年至三十始归，娶妻陈氏。不料陈氏因难产亡故，幸留一婴。张乾抱着婴儿讨百家奶，始知为父之艰。

The authors declare that they have no competing interests.

图12表示圆柱域约束下aF和bF的协调关系。图12中，左侧点集表示aF的变动，aF={aF0,aF1,aF2,aF3,,aFi,,aFu}，u=∞；右侧点集表示bF的变动，bF={bF0,bF1,bF2,,bFj,,bFw}，w=∞。aF和bF的协调关系可表示为aF×bF={〈aFi,bFj〉|aFi∈aF,bFj∈bF}。

Author details

10月25日，福布斯发布了最新的2018福布斯中国400富豪榜。马云自2014年以来首次回归榜首，但财富比一年前缩水40亿美元。

1DISTAL,University of Bologna,Viale Fanin 46,40127 Bologna,Italy.

冯先生先列举惠施的“历物十事”，认为惠施的特点是认为一切事物皆是变动的、有限的、相对的。世俗所谓同异是具体物之间的同异，这是“小同异”。而从“至大无外”的观点看事物，自其同处看，则万物莫不同；自其异处看，则万物莫不异。故万物可谓毕同毕异，这是“大同异”。惠施倡导从大处着眼，看出“天地与我并生，而万物与我为一”(《庄子·齐物论》)，由此“泛爱万物”。冯先生指出，庄子的齐物论是在惠施的基础上转进一步，惠施只说出了知识上的结论，庄子则又有无言、无知、心斋、坐忘等体会万物一体的修养方法。

2Wageningen University and Research,Wageningen,The Netherlands.

References

1.　Trevisi P,Gandolfi G,Priori D,Messori S,Colombo M,Mazzoni M,et al.Agerelated expression of the polymeric immunoglobulin receptor(pIgR)in the gastric mucosa of young pigs.PLoS One.2013;8:e81473.

2.　Mazzoni M,Bosi P,De Sordi N,Lalatta-Costerbosa G.Distribution,organization and innervation of gastric MALT in conventional piglet.J Anat.2011;219:611-21.

3.　De Bruyne E,Flahou B,Chiers K,Meyns T,Kumar S,Vermoote M,et al.An experimental Helicobacter suis infection causes gastritis and reduced daily weight gain in pigs.Vet Microbiol.2012;160:449-54.

4.　Hooper LV.Bacterial contributions to mammalian gut development.Trends Microbiol.2000;12:129-34.

5.　Stappenbeck TS.Accounting for reciprocal host-microbiome interactions in experimental science.Nature.2016;534:191-9.

6.　Laycock G,Sait L,Inman C,Lewis M,Smidt H,van Diemen P,et al.A defined intestinal colonization microbiota for gnotobiotic pigs.Vet Immunol Immunopathol.2012;49:216-24.

7.　Dierick NA,Decuypere JA,Molly K,Van Beek E,Vanderbeke E.The combined use of triacylglycerols(TAGs)containing medium chain fatty acids(MCFAs)and exogenous lipolytic enzymes as an alternative to nutritional antibiotics in piglet nutrition:II.In vivo release of MCFAs in gastric cannulated and slaughtered piglets by endogenous and exogenous lipases;effects on the luminal gut flora and growth performance.Liv Prod Sci.2002;76:1-16.

8.　Zentek J,Ferrara F,Pieper R,Tedin L,Meyer W,Vahjen W.Effects of dietary combinations of organic acids and medium chain fatty acids on the gastrointestinal microbial ecology and bacterial metabolites in the digestive tract of weaning piglets.J Anim Sci.2013;91:3200-10.

9.　Odle J.New insights into the utilization of medium-chain triglycerides by the neonate:observations from a piglet model.J Nutr.1997;127:1061-7.

10.Bosi P,Mazzoni M,De Filippi S,Casini L,Trevisi P,Petrosino G,et al.Continuous dietary supply of free calcium formate negatively affects parietal cell population and gastric RNA expression for H+/K+-ATPase in weaning pigs.J Nutr.2006;136:1229-35.

11.Mazzoni M,Le Gall M,De Filippi S,Minieri L,Trevisi P,Wolinski J,et al.Supplemental sodium butyrate stimulates different gastric cells in weaned pigs.J Nutr.2008;138:1426-31.

12.Friis-Hansen L.Gastric functions in gastrin gene knock-out mice.Pharmacol Toxicol.2002;91:363-7.

13.Godden S,McMartin S,Feirtag J,Stabel J,Bey R,Goyal S,et al.Heat treatment of bovine colostrum.II.Effects of heating duration on pathogen viability and IgG.J Dairy Sci.2006;89:3476-83.

14.Elizondo-Salazar JA,Heinrichs AJ.Feeding heat-treated colostrum or unheated colostrum with two different bacterial concentrations to neonatal dairy calves.J Dairy Sci.2009;92:4565-71.

15.Goebel M,Stengel A,Lambrecht NW,Sachs G.Selective gene expression by rat gastric corpus epithelium.Physiol Genomics.2011;43:237-54.

16.Colombo M,Priori D,Trevisi P,Bosi P.Differential gene expression in the oxyntic and pyloric mucosa of the young pig.PLoS One.2014;9(10):e111447.

17.Varro A,Noble PJ,Pritchard DM,Kennedy S,Hart CA,Dimaline R,et al.Helicobacter pylori induces plasminogen activator inhibitor 2(PAI-2)in gastric epithelial cells through NF-κB and RhoA:implications for invasion and apoptosis.Cancer Res.2004;64:1695-702.

18.Romano M,Ricci V,Zarrilli R.Mechanisms of disease:Helicobacter pylorirelated gastric carcinogenesis-implications for chemoprevention.Nat Clin Pract Gastroenterol Hepatol.2006;3:622-32.

19.Lam EK,Yu L,Wong HP,WK W,Shin VY,Tai EK,et al.Probiotic Lactobacillus rhamnosus GG enhances gastric ulcer healing in rats.Eur J Pharmacol.2007;565:171-9.

20.Pegg AE,McCann PP.Polyamine metabolism and function.Am J Phys.1982;243:C212-21.

21.Jansman AJM,Zhang J,Koopmans SJ,Dekker RA,Smidt H.Effects of a simple or a complex starter microbiota on intestinal microbiota composition in caesarean derived piglets.J Anim Sci.2012;90(Supplement 4):433-5.

22.Merchant JL,Shiotani A,Mortensen ER,Shumaker DK,Abraczinskas DR.Epidermal growth factor stimulation of the human gastrin promoter requires Sp1.J Biol Chem.1995;270:6314-9.

23.Roussa E,Wittschen P,Wolff NA,Torchalski B,Gruber AD,Thévenod F.Cellular distribution and subcellular localization of mCLCA1/2 in murine gastrointestinal epithelia.J Histochem Cytochem.2010;58:653-8.

24.Nagata A,Ito M,Iwata N,Kuno J,Takano H,Minowa O,Chihara K,et al.G.protein-coupled cholecystokinin-B/gastrin receptors are responsible for physiological cell growth of the stomach mucosa in vivo.Proc Natl Acad Sci U S A.1996;93:11825-30.

25.Engelstoft MS,Park WM,Sakata I,Kristensen LV,Husted AS,Osborne-Lawrence S,et al.Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells.Mol Metab.2013;2:376-92.

26.Yano JM,Yu K,Donaldson GP,Shastri GG,Ann P,Ma L,et al.Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis.Cell.2015;161:264-76.

27.Reigstad CS,Linden DR,Szurszewski JH,Sonnenburg JL,Farrugia G,Kashyap PC.Correlated gene expression encoding serotonin(5-HT)receptor 4 and 5-HT transporter in proximal colonic segments of mice across different colonization states and sexes.Neurogastroenterol Motil.2016;28:1443-8.

28.Furness JB.Types of neurons in the enteric nervous system.J Auton Nerv Syst.2000;81:87-96.

29.Tsai LH.Function of GABAergic and glutamatergic neurons in the stomach.J Biomed Sci.2005;12:255-66.

30.Barrett E,Ross RP,O'Toole PW,Fitzgerald GF,Stanton C.γ-Aminobutyric acid production by culturable bacteria from the human intestine.J Appl Microb.2012;113:411-7.

31.Rothkötter HJ,Mollhoff S,Pabst R.The influence of age and breeding conditions on the number and proliferation of intraepithelial lymphocytes in pigs.Scand J Immunol.1999;50:31-8.

32.Cole KE,Strick CA,Paradis TJ,Ogborne KT,Loetscher M,Gladue RP,et al.Interferon-inducible T cell alpha chemoattractant(I-TAC):a novel Non-ELR CXC Chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.J Exp Med.1998;187:2009-21.

33.Page-McCaw A,Ewald AJ,Werb Z.Matrix metalloproteinases and the regulation of tissue remodelling.Nat Rev Mol Cell Bio.2007;8:221-33.

34.Johansson ME,Jakobsson HE,Holmén-Larsson J,Schütte A,Ermund A,Rodríguez-Piñeiro AM,et al.Normalization of host intestinal mucus layers requires long-term microbial colonization.Cell Host Microbe.2015;18:582-92.

35.Zlotnik A,Yoshie O.Chemokines:a new classification system and their role in immunity.Immunity.2000;12:121-7.

36.Mani V,Hollis JH,Gabler NK.Dietary oil composition differentially modulates intestinal endotoxin transport and postprandial endotoxemia.Nutri Metab.2013;10:6.