Effect of Chromium Propionate Substituting 25% Rumen-protected Choline on Production Performance and Blood Indicators of Perinatal Dairy Cows

The object of this study was to investigate the effects of chromium propionate replacing 25%rumen-protected choline(RPC)on production performance and blood indicators of perinatal dairy cows.According to the principle of ensuring that chromium propionate and RPC were fed 14 days prepartum,27 healthy Holstein cows(age,parity,lactation volume,body condition and expectancy were similar)were randomly divided into three groups(GroupsⅠ,ⅡandⅢ),with nine cows in each group.Cows in GroupⅠwere fed basal diets;cows in GroupⅡwere fed the same basal diets with 10 g•d-1 RPC per cow;and cows in GroupⅢwere fed the same basal diets,but 7.5 g RPC and 2.5 g chromium propionate(4 mg chromium)per cow.The results showed that dry matter intake(DMI)of prepartum in GroupsⅡandⅢwas significantly increased(P<0.05),compared with GroupⅠ;however,there was no significant difference between GroupsⅡandⅢ(P>0.05).The milk fat content in GroupⅢwas significantly higher than that in GroupⅠon the 7th day of postpartum(P<0.05).The urea nitrogen content in GroupsⅡandⅢsignificantly lower than that in GroupⅠon the 21st day of postpartum(P<0.05),but there were no significant difference between GroupsⅡandⅢ(P>0.05).The somatic cell counts in GroupⅢwere significantly lower than those in GroupsⅠandⅡon the 14th and 21st days of postpartum(P<0.05).The contents of the total cholesterol in GroupⅢdecreased significantly compared to GroupⅡon the 7th day of prepartum,on the day of calving and the 7th day of postpartum(P<0.05).The concentration of high-density lipoprotein(HDL)in GroupⅢwas significantly higher than that in GroupⅡon the 7th day of prepartum,on the day of calving and the 7th day of postpartum(P<0.05).On the 7th day of postpartum the concentration of insulin in GroupⅢwas obviously higher than that in GroupⅡ(P<0.05).In summary,replacing 25%of rumen-protected choline with chromium propionate reduced production costs and improved dairy farming benefits.

Tryptophan metabolism and piglet diarrhea:Where we stand and the challenges ahead

The intestinal architecture of piglets is vulnerable to disruption during weaning transition and leads to diarrhea,frequently accompanied by inflammation and metabolic disturbances(including amino acid metabolism).Tryptophan(Trp)plays an essential role in orchestrating intestinal immune tolerance through its metabolism via the kynurenine,5-hydroxytryptamine,or indole pathways,which could be dictated by the gut microbiota either directly or indirectly.Emerging evidence suggests a strong asso-ciation between piglet diarrhea and Trp metabolism.Here we aim to summarize the intricate balance of microbiota-host crosstalk by analyzing alterations in both the host and microbial pathways of Trp and discuss how Trp metabolism may affect piglet diarrhea.Overall,this review could provide valuable in-sights to explore effective strategies for managing piglet diarrhea and the related challenges.

Effects of Rumen-protected Methionine on Rumen Fermentation, lactation Performance and Plasma Characteristics in Dairy Cows

The objective was to evaluate effects of rumen-protected methionine （RP-Met） supplementation on rumen fermentation, lactation performance and plasma characteristics in dairy cows. Twenty-four multiparous （2.2 - 0.40 parity） Holstein dairy cows, averaged 620 ± 12.3 kg of BW, 68 ± 2.5 day in milk and daily milk production averaged 26.0 ±0.3 kg/cow were used in a replicated 4 × 4 Latin square experiment. The treatments were： control （without RP-Met）, LRP-Met, MRP-Met and HRP-Met with 20, 40 and 60 g RP-Met per cow per day, respectively. RP-Met was hand-mixed into the top one-third of the daily ration. Experimental periods were 30 days with 15 d of adaptation and 15 d of sampling. Dry matter （DM） intake and milk yields were not affected （P 〉 0.05） with increasing RP-Met supplementation. Yields of 4%FCM, ECM, milk fat and milk protein were higher （P 〈 0.03） for RP-Met supplementation than control and were quadratically （P 〈 0.05） changed due to the higher percentage of milk fat and protein for RP-Met supplementation than control （P 〈 0.03）. Ruminal pH tended to be lower （P = 0.071） for RP-Met supplementation than control, whereas total VFA concentration tended to be higher （P = 0.086） for RP-Met supplementation than control. Ratio of acetate to propionate decreased linearly （P = 0.001） from 4.01 to 3.57 as RP-Met supplementation increased due to the increase in propionate production. Digestibilities of DM, OM, CP, NDF and ADF in the total tract were higher （P 〈 0.05） for RP-Met supplementation than control and were quadratically （P 〈 0.05） increased with increasing RP-Met supplementation. Plasma concentrations of glucose not affected （P 〉 0.05） with RP-Met supplementation, Plasma concentrations of non-esterified fatty acids （NEFA） were significantly lower （P = 0.017） for RP-Met supplementation than control and were linearly （P = 0.011） changed. Plasma concentrations of beta-hydroxybutyrate tended to be lower （P = 0.068） for RP-Met supplementation than control and were linearly （P = 0.001） changed. The present results indicate that supplementation of diet with RP-Met improved the content of milk fat and protein, rumen fermentation and feed digestion, decreased plasma concentrations of NEFA. It was suggested that the RP-Met stimulated the digestive microorganisms or enzymes in a dose-dependent manner. In the experimental conditions of this trial, the optimum RP-Met dose was about 25 g RP-Met per cow per day.

Bidirectional effects of the tryptophan metabolite indole-3-acetaldehyde on colorectal cancer

BACKGROUND Colorectal cancer(CRC)has a high incidence and mortality.Recent studies have shown that indole derivatives involved in gut microbiota metabolism can impact the tumorigenesis,progression,and metastasis of CRC.AIM To investigate the effect of indole-3-acetaldehyde(IAAD)on CRC.METHODS The effect of IAAD was evaluated in a syngeneic mouse model of CRC and CRC cell lines(HCT116 and DLD-1).Cell proliferation was assessed by Ki-67 fluorescence staining and cytotoxicity tests.Cell apoptosis was analysed by flow cytometry after staining with Annexin V-fluorescein isothiocyanate and propidium iodide.Invasiveness was investigated using the transwell assay.Western blotting and real-time fluorescence quantitative polymerase chain reaction were performed to evaluate the expression of epithelial-mesenchymal transition related genes and aryl hydrocarbon receptor(AhR)downstream genes.The PharmMapper,SEA,and SWISS databases were used to screen for potential target proteins of IAAD,and the core proteins were identified through the String database.RESULTS IAAD reduced tumorigenesis in a syngeneic mouse model.In CRC cell lines HCT116 and DLD1,IAAD exhibited cytotoxicity starting at 24 h of treatment,while it reduced Ki67 expression in the nucleus.The results of flow cytometry showed that IAAD induced apoptosis in HCT116 cells but had no effect on DLD1 cells,which may be related to the activation of AhR.IAAD can also increase the invasiveness and epithelial-mesenchymal transition of HCT116 and DLD1 cells.At low concentrations(<12.5μmol/L),IAAD only exhibited cytotoxic effects without promoting cell invasion.In addition,predictions based on online databases,protein-protein interaction analysis,and molecular docking showed that IAAD can bind to matrix metalloproteinase-9(MMP9),angiotensin converting enzyme(ACE),poly(ADP-ribose)polymerase-1(PARP1),matrix metalloproteinase-2(MMP2),and myeloperoxidase(MPO).CONCLUSION Indole-3-aldehyde can induce cell apoptosis and inhibit cell proliferation to prevent the occurrence of CRC;however,at high concentrations(≥25μmol/L),it can also promote epithelial-mesenchymal transition and invasion in CRC cells.IAAD activates AhR and directly binds MMP9,ACE,PARP1,MMP2,and MPO,which partly reveals why it has a bidirectional effect.

Expression of Recombinant Tryptophan Decarboxylase in Different Subcellular Compartments in Tobacco Plant

The gene encoded for tryptophan decarboxylase (TDC), which is the key enzyme in terpenoil indole alkaloids pathway, was targeted to different subcellular compartments and stably expressed in transgenic tobacco (Nicotiana tabacum L.) plants at the levels detected by Western blot and tryptamine accumulation analysis. It was shown that the TDC was located in subcellular compartments, the chloroplasts and cytosol. The recombinant TDC targeted to chloroplasts and cytosol in tobacco plants was effectively expressed as soluble protein by Western blot analysis and enzymatic assay. The level of tryptamine accumulation in chloroplast was higher than that in cytosol and very low in vacuole and endoplasmic reticulum (ER) to be hardly detected by Western blot analysis. It was indicated that the highest amount of tryptamine was in chloroplasts, lower in endoplasmic reticula and the lowest in vacuoles as compared to those in wild type plants. The TDC targeted to different subcellular compartments of tobacco plants and its expression level were studied by different nucleotide sequences coding signal peptides at 5'-end of tdc gene in order to know the effects of the TDC in compartmentation on its functionality.

吲哚胺2,3-双加氧酶1介导色氨酸代谢增强促进食管鳞癌放射抵抗

目的 探究食管鳞状细胞癌(esophageal squamous cell carcinoma, ESCC)发生放射抵抗的生物学机制,寻找有效增敏靶点。方法 从MSigDB数据库获取186条信号通路及通路相关基因信息。利用癌症基因图谱计划(the Cancer Genome Atlas, TCGA)和基因表达综合数据库(Gene Expression Omnibus, GEO)获得ESCC患者的RNA转录组数据。收集2013—2020年西安交通大学第一附属医院97例ESCC患者的临床病理特征及组织样本。使用基因集变异分析(gene set variation analysis, GSVA)计算KEGG信号通路评分,通过随机森林算法筛选放疗抵抗相关信号通路,利用DESeq2筛选通路中关键基因,基于支持向量机-递归特征消除(support vector machine-recursive feature elimination, SVM-RFE)构建放疗疗效预测模型;并通过蛋白印迹、克隆形成等实验进行验证。结果 基于KEGG信号通路的GSVA富集评分,随机森林分析显示,在TCGA队列及GSE45670队列中,色氨酸代谢通路富集值对ESCC放射抵抗的贡献程度显著优于其他通路。DESeq2分析发现,色氨酸代谢通路中关键分子吲哚胺2,3-双加氧酶1(IDO1)、ALDH1B1、AOC1、INMT、AFMID和ALDH7A1在ESCC抵抗组及敏感组中表达存在显著差异,利用上述差异基因基于SVM-RFE算法构建预测模型的曲线下面积为0.77,可较准确预测ESCC放疗疗效。蛋白印迹实验表明,IDO1在ESCC细胞中高表达,且IDO1抑制剂处理显著抑制KYSE-410细胞的存活及放射敏感性。入组患者中,免疫组化结果显示,IDO1在ESCC放疗抵抗组中高表达,且与ESCC患者的放疗不良预后相关;此外,进一步检测发现,IDO1在患者样本中的表达与其PD-L1表达正相关,且与CD3/CD8免疫细胞浸润比例负相关。结论 色氨酸分解代谢与ESCC放射抵抗相关,色氨酸代谢关键酶IDO1可作为ESCC放射增敏的治疗靶点。

高效分泌色氨酸羟化酶实现5-羟基色氨酸的生物转化

5-羟基色氨酸(5-hydroxytryptophan,5-HTP)是哺乳动物中重要的神经递质前体,已被证明在治疗多种神经疾病方面具有重要的医用价值,市场前景广阔。酶催化法具有高转化率和可控的反应条件等优点,是一种有前景的生产方法。然而,酶法催化也存在蛋白纯化过程复杂且成本高昂等问题。该研究旨在提升色氨酸羟化酶的表达、催化和分泌效率,无需复杂蛋白纯化过程,利用分泌的酶蛋白粗酶液催化实现5-HTP的高效生物转化。通过基因工程对表达宿主进行遗传改造,有效阻断了底物和产物的降解途径。通过蛋白截短和引入促融标签,获得了表达效率最佳且热稳定性较高的酶突变体M11。进一步通过优化催化体系参数,确立了最佳催化条件,提高了酶的催化效率,催化2 h获得了0.17 g/L的5-HTP,转化率达85%。结合绿色荧光蛋白实变体与促释放化学试剂,建立了一种高效胞外分泌酶蛋白的策略,蛋白分泌效率高达90%,节省了传统纯酶催化的操作成本,为5-HTP的工业化生产提供了潜在的替代方案。

Dip2a regulates stress susceptibility in the basolateral amygdala

Dysregulation of neurotransmitter metabolism in the central nervous system contributes to mood disorders such as depression, anxiety, and post–traumatic stress disorder. Monoamines and amino acids are important types of neurotransmitters. Our previous results have shown that disco-interacting protein 2 homolog A(Dip2a) knockout mice exhibit brain development disorders and abnormal amino acid metabolism in serum. This suggests that DIP2A is involved in the metabolism of amino acid–associated neurotransmitters. Therefore, we performed targeted neurotransmitter metabolomics analysis and found that Dip2a deficiency caused abnormal metabolism of tryptophan and thyroxine in the basolateral amygdala and medial prefrontal cortex. In addition, acute restraint stress induced a decrease in 5-hydroxytryptamine in the basolateral amygdala. Additionally, Dip2a was abundantly expressed in excitatory neurons of the basolateral amygdala, and deletion of Dip2a in these neurons resulted in hopelessness-like behavior in the tail suspension test. Altogether, these findings demonstrate that DIP2A in the basolateral amygdala may be involved in the regulation of stress susceptibility. This provides critical evidence implicating a role of DIP2A in affective disorders.

基于核磁共振代谢组学揭示黄精通过调控苯丙氨酸、酪氨酸及色氨酸的生物合成发挥抗疲劳作用机制

目的:运用核磁共振(1H NMR)代谢组学方法,研究黄精的抗疲劳作用及可能的作用机制。方法:30只雄性ICR小鼠随机分为对照组、黄精低、高剂量组,每组10只。低、高剂量组分别灌胃给予剂量为1.125 g/kg、2.5 g/kg的黄精水提物,对照组给予相同体积蒸馏水,灌胃1次/d,连续3周。灌胃结束后进行负重力竭游泳实验,小鼠达到疲劳状态后,测试体质量变化肝脏肝糖原含量、血清乳酸及尿素氮(BUN)水平。进一步采用1H NMR代谢组学技术寻找小鼠腓肠肌内源性代谢物的变化,并进行接收者操作特征曲线(ROC)分析,筛选ROC曲线下面积>0.7的差异代谢物输入MetaboAnalyst网站中进行通路富集分析。结果:3组小鼠体质量均增加,但差异无统计学意义(P>0.05);与对照组比较,黄精高、低剂量组小鼠力竭游泳时间提高,肝糖原含量明显增高,血乳酸含量明显下降,差异均有统计学意义(P<0.01,P<0.001);黄精高、低剂量组小鼠血清BUN水平均下降,但差异无统计学意义(P>0.05)。在小鼠腓肠肌组织提取物中共指认34种代谢物,其中21个差异代谢物在黄精干预后发生显著变化,结合ROC曲线,筛选出17个差异代谢物。通路分析发现,黄精主要通过调控苯丙氨酸、酪氨酸及色氨酸的生物合成途径来发挥抗疲劳作用。结论:本研究采用1H NMR代谢组学技术结合多元统计分析阐明黄精的抗疲劳作用机制,为黄精抗疲劳应用以及相关产品研发提供理论依据。

疏肝方治疗肝癌并发抑郁症的生物信息学分析

目的:利用生物信息学方法,探讨疏肝方治疗肝癌并发抑郁症的作用机制。方法:通过TCMSP、BATMAN-TCM等获取疏肝方核心成分和靶蛋白,构建PPI和核心蛋白网络,进行KEGG和GO富集分析、分子对接、基因组学验证。结果:得到疏肝方的多种核心成分作用于TNF、IL-6、TP53等多种核心靶点,并通过调控细胞凋亡、衰老,抗氧化应激、调节免疫、抗炎症反应等信号通路抑制肝癌细胞增殖、促进凋亡并改善抑郁症状。分子对接显示,有效成分与靶蛋白间具有良好的亲和力。基因组学提示,TP53对于缩短肝癌患者总生存周期及色氨酸代谢通路的相关性均具有显著的统计学意义。结论:疏肝方可通过多成分、多靶点、多通路治疗肝癌并发抑郁症的症状,并初步推断疏肝方可能通过TP53靶向色氨酸代谢通路抑制肝癌的发生发展,并改善抑郁症状。

血清色氨酸用于乙肝相关慢加急性肝衰竭90 d死亡风险分层管理的潜在价值:一项多中心回顾性研究

目的探索血清色氨酸与乙肝相关慢加急性肝衰竭(HBV-ACLF)死亡风险之间的相关性。方法回顾性纳入180例HBVACLF患者,根据确诊后90 d结局为患者分为存活组和死亡组;采用高效液相色谱法测定两组患者外周血中的血清色氨酸含量;比较两组患者基线血清色氨酸水平的差异;通过单因素及多因素回归分析基线血清色氨酸水平对于HBV-ACLF患者确诊后90 d病死率的影响;通过受试者曲线下面积探讨血清色氨酸对HBV-ACLF 90 d死亡风险的潜在预测价值。结果90 d随访期内,53例(29.4%)HBV-ACLF患者因疾病死亡,127例(70.6%)存活。死亡患者的基线色氨酸水平低于存活患者(P<0.001);经多因素回归分析校正混杂因素显示,色氨酸水平是HBV-ACLF死亡风险的独立预测因素。HBV-ACLF患者入组时低水平色氨酸(<10.14 pg/mL)与高水平色氨酸(≥10.14 pg/mL)相比具有更高的90 d死亡风险(43.3%vs 15.6%,HR:3.157,95%CI:1.713-5.817);同时合并肾功能不全和低水平色氨酸的90 d死亡风险更高(73.3%vs 15.0%,HR:7.558,95%CI:3.369-16.960);血清色氨酸水平用于预测HBV-ACLF患者的90 d死亡风险具有一定潜在价值(ROC=0.771,95%CI:0.699-0.844)。结论血清色氨酸与HBV-ACLF预后密切相关;低水平的血清色氨酸预示90 d高死亡风险;尤其是同时合并肾功能不全的患者90 d死亡风险更高。

Helicobacter pylori and serum kynurenine-tryptophan ratio in patients with colorectal cancer

AIM: To evaluate how Helicobacter pylori(H. pylori) is able to evade the immune response and whether it enhances systemic immune tolerance against colorectal cancer.METHODS: This prospective randomized study involved 97 consecutive colorectal cancer patients and 108 cancer-free patients with extra-digestive diseases. Colorectal cancer and cancer-free patients were assigned into subgroups according to H. pylori Ig G seropositivity. Exposure to H. pylori was determined by Ig G seropositivity which was detected by enzyme linked immunoassay(ELISA). Serum neopterin levels were measured by ELISA. Serum tryptophan, kynurenine, and urinary biopterin concentrations were measured by high performance liquid chromatography. Serum nitrite levels were detected spectrophotometrically. Serum indoleamine 2,3-dioxygenase activity was estimated by the kynurenine to tryptophan ratio and by assessing the correlation between serum neopterin concentrations and the kynurenine to tryptophan ratio. The frequencies of increased serum kynurenine to tryptophan ratio of H. pylori seronegative and seropositive colorectal cancer subgroups were estimated by comparing them with the average kynurenine to tryptophan ratio of H. pylori seronegative tumor-free patients.RESULTS: Compared with respective controls, in both H. pylori seronegative and seropositive colorectal cancer patients, while serum tryptophan levels were decreased(controls vs patients; seronegative: 20.37 ± 0.89 μmol/L vs 15.71 ± 1.16 μmol/L, P < 0.05; seropositive: 20.71 ± 0.81 μmol/L vs 14.97 ± 0.79 μmol/L, P < 0.01) the kynurenine to tryptophan ratio was significantly increased(controls vs patients; seronegative: 52.85± 11.85 μmol/mmol vs 78.91 ± 8.68 μmol/mmol, P < 0.01, seropositive: 47.31 ± 5.93 μmol/mmol vs 109.65 ± 11.50 μmol/mmol, P < 0.01). Neopterin concentrations in cancer patients were significantly elevated compared with controls(P < 0.05). There was a significant correlation between serum neopterin levels and kynurenine/tryptophan in control and colorectal cancer patients groups(r s = 0.494, P = 0.0001 and r s= 0.293, P = 0.004, respectively). Serum nitrite levels of H. pylori seropositive cancer cases were significantly decreased compared with seropositive controls(controls vs patients; 26.04 ± 2.39 μmol/L vs 20.41 ± 1.48 μmol/L, P < 0.05) The decrease in the nitrite levels of H. pylori seropositive cancer patients may be attributed to excessive formation of peroxynitrite and other reactive nitrogen species.CONCLUSION: A significantly high kynurenine/tryptophan suggested that H. pylori may support the immune tolerance leading to cancer development, even without an apparent upper gastrointestinal tract disease.

The effect of dietary tryptophan levels on oxidative stress of liver induced by diquat in weaned piglets

Oxidative stress can induce abnormal tryptophan metabolism. The present study was mainly conducted to determine the effect of dietary tryptophan levels on oxidative stress in the liver of weaned pigs challenged by diquat. A total of 36 PIC piglets weaned at 21 days of age were randomly allotted to 1 of 3 diets containing dietary tryptophan levels of 0.18, 0.30, and 0A5% for 14 d. On day 8, the piglets were injected intraperitoneally with sterile 0.9% NaCI solution or diquat （10 mg/kg body weight）. During the first 7 d of trial, increasing dietary tryptophan levels enhanced average daily gain （P = 0.09） and average daily feed intake （P = 0.08）, and decreased the feed efficiency （P 〈 0.05） of piglets. The growth performance was decreased by diquat injection （P 〈 0.05）. Diquat injection also decreased the activities of the superoxide dismutase （SOD） and glutathione peroxidase （GPx） in the plasma and liver （P 〈 0.05）, increased plasma malondialdehyde （MDA） （P 〈 0.05） and urea nitrogen （P 〈 0.05） concentrations, and enhanced MDA concentration （P = 0.09） and tryptophan 2,3-dioxygenase （TDO） activity （P = 0.07） in liver of piglets. Increasing dietary tryptophan levels could attenuate the effects of diquat injection on the MDA （P = 0.06） concentration and the activities of SOD （P = 0.09） and GPx （P = 0.05） of the liver, and plasma urea nitrogen （P = 0.06） concentration in the piglet. There was a synergistic role for increasing TDO activity in the liver between dietary tryptophan levels and diquat injection （P 〈 0.05）. These results suggest that increasing dietary tryptophan levels could attenuate the oxidative stress of the liver in weaned piglets intraperitoneally injected with diquat via enhancing the antioxidant capacity.

Effects of dietary tryptophan and stocking density on the performance,meat quality,and metabolic status of broilers

Background： Highly automated cage-rearing systems are becoming increasingly popular in China. However, a high stocking density can cause oxidative stress and decrease broiler performance. The tryptophan （TRP） derivative 5-hydroxytryptophan （5-HT） has been shown to preserve membrane fluidity in birds suffering from oxidative stress Therefore, this experiment was conducted to determine the effects of dietan/TRP supplementation on performance, breast meat quality and oxidative stress in broilers reared in cages with a high or low stocking density. Methods： Female Arbor Acres broilers （25-d-old, n = 144） were randomly allocated to 1 of 4 treatments. The birds were fed a diet based on corn, soybean meal, cottonseed meal and corn gluten meal containing either 0.18 or 0.27% TRP and were housed with stocking densities of 11 or 15.4 birds/m2 in a 2 x 2 factorial experiment. Broiler performance was evaluated from d 25 to 42. Eight birds from each treatment were slaughtered on d 42 and plasma and breast muscle samples were collected to measure biochemical indices. Results： A higher stocking density tended to be associated with reduced weight gain （P 〈 0.10）, and significantly increased plasma glutamic-pyruvic transaminase （GPT） activity （P 〈 0.001）. Increased dietary TRP significantly reduced the activities of lactic dehydrogenase and GPT while increasing total cholesterol in the plasma （P 〈 0.01）, reducing drip loss of breast muscle （P 〈 0.10） and improving feed efficiency （P 〈 0.10）. Conclusions： An increase in dietary TRP, ].S-fold higher than the standard supplementation level, can alleviate oxidative stress as well as improve welfare and feed efficiency in broilers reared in cages with a high stocking density.

Lysine, Methionine and Tryptophan Requirements of Beijing Ducklings of 0-2 Weeks of Age

A feed trial was conducted with a total of 1 134 Beijing ducklings to study the optimum level of dietary lysine （Lys） （0.95, 1.10, 1.25%）, methionine （Met） （0.26, 0.46, 0.66%） and tryptophan （Trp） （0.20, 0.30, 0.40%） for those ducklings during a phase of 0-2 weeks. Ducklings were randomly allotted to 27 groups according to a 3 × 3× 3 factorial arrangement and fed a basal corn-soybean-peanut meal diet containing 20.26% CP, 12.45 MJ kg^-1 ME. The results from this study indicate that Lys affected body weight （P〈0.01）, feed intake （0-14 d） （P〈0.01）, but had no effect on feed/gain （0-14 d） （P〉0.05）, uric acid concentration （P 〉 0.05）. Methionine influenced body weight （P 〈 0.01）, feed/gain （P 〈 0.05）, and feed intake （P 〈 0.01）. Tryphtophan had no effect on indices measured. The requirement of the Lys and Met for Beijing ducklings of 0-2 weeks of age were 1.10 and 0.46%. The requirement of Trp for Beijing ducklings of 0-2 weeks of age was not more than 0.20%.

Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting

Unbalanced brain serotonin(5-HT) levels have implications in various behavioral abnormalities and neuropsychiatric disorders. The biosynthesis of neuronal 5-HT is regulated by the rate-limiting enzyme, tryptophan hydroxylase-2(TPH2). In the present study, the clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas) system was used to target the Tph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at38.5%. The biallelic modified colonies were used as donors for somatic cell nuclear transfer(SCNT) and 10 Tph2 targeted piglets were successfully generated. These Tph2 KO piglets were viable and appeared normal at the birth.However, their central 5-HT levels were dramatically reduced, and their survival and growth rates were impaired before weaning. These Tph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.

Changes of 5-hydroxytryptamine and tryptophan hydroxylase expression in the ventral horn of spinal cord

Objective To investigate changes of 5-hydroxytryptamine （5-HT） and its synthesis rate-limiting enzyme tryp-tophan hydroxylase （TPH） in the ventral horn of spinal cord after exercise-induced fatigue, and to further discuss the mecha- nism of exercise-induced central fatigue at spinal level. Methods Sixteen healthy adult Wistar rats were randomly divided into 2 groups： exercise-induced fatigue group and control group. Immunohistochemical staining for 5-HT and TPH in the ventral horn were performed and analysized quantitatively. The mean optic densities of 5-HT and TPH positive fibers or terminals were measured by computerized image analyzer. Results Both 5-HT and TPH positive fibers/terminals decreased in the exercise-induced fatigue group. The immunohistochemical staining was weaker and the mean optic densities decreased obviously in the fatigue group compared with those in the control group （P 〈 0.05）. Conclusion 5-HT and TPH in the ventral horn of spinal cord might be involved in exercise-induced fatigue.

Chemical Modification and Fluorescence Spectrum of Tryptophan Residues in Pullulanase

Tryptophan（Trp） residues in a pullulanase were modified by N-bromosuccinimide（NBS）. The results of the Spande method indicate that there are 18 Trp residues in the pullulanase and nine of them are located on the surface af the enzyme. Three of these Trp residues are nonessential residues which show the fastest reaction rate according to the Zou＇s plot. Two of the seven relative faster reacting residues are essential for the activity of the enzyme. The other eight are the slowest in the reaction rate or non-reactive residues for the reaction. The fluorescence and circular dichroism（CD） spectra of the pullulanase have been changed after the reaction with NBS. Potassium iodide（KI） and acrylamide also have remarkable influences on the fluorescence spectra of the pullulanase.

Flow-injection chemiluminescence determination of tryptophan using galangin-potassium permanganate-polyphosphoric acid system

A high sensitive flow-injection chemiluminescence （FI-CL） method for the determination of tryptophan has been developed. The method is based on the chemiluminescence reaction of galangin-potassium permanganate-tryptophan in polyphosphoric acid （PPA） media. Under the optimized conditions, tryptophan was determined in the range 0.05-10 μg/mL with the detection limit （3tr） of 5.0 × 10^-3 μg/mL. The relative standard deviation （RSD） was 1.0% for 11 replicate determinations of 1.0 μg/mL tryptophan. Three synthetic samples were determined selectively with recoveries in the range from 99.6% to 102.0% in the presence of other amino acids.

Determination of Enantiomeric Compositions of Tryptophan by Chemometric Analysis of the Fluorescence Spectra of Bovine Serum Albumin Receptor-ligand Mixtures

In this work, a novel method was constructed to determine the enantiomeric composition of tryptophan （Trp） by bovine serum albumin （BSA） based on the fluorescence spectra of the receptor-ligand mixtures coupled with partial least squares （PLS-1） analysis. As a result the enantiomeric composition of Trp was accurately determined.