Effects of three methionine sources in diets on temporal B0AT1 and ASCT2 mRNA expression in different intestinal segments of cobia,Rachycentron canadum

A 4‐week feeding trial was conducted using five groups of hybrid grouper (61.15 ± 0.15 g) to explore the potential effects of three methionine (Met) sources. Five isonitrogenous and isolipidic diets were formulated that included a fishmeal (FM) diet; nonsupplemented (NS) diet; or NS diets with the addition of the L‐methionine (L‐Met), DL‐methionine (DL‐Met), or coated Met (Co‐Met) to obtain the same Met level as the FM diet. Fish were randomly distributed into sea cages (30 fish per cage). Weight gain and specific growth rate in the DL‐Met group and FM group were significantly higher than those in all other groups (p < .05). In the proximal and distal intestines, of the 20 gene and time combinations (10 per gene), there were 14 combinations (70%) in which there were no significant differences in gene expression levels between the FM and DL‐Met groups (p > .05). The main reason for the same growth effect between the DL‐Met and FM groups may be attributed to the synchronized absorption at most time points after feeding, which had similar expression patterns of B⁰AT1 and ASCT2 of the proximal and distal intestines between two groups.     

绿色富硒生物猪饲料中AFB1达标生产工艺优化研究

饲料中黄曲霉毒素(AFB₁)容易超标,检出率达80%~100%,毒性大,具强致癌性,可抑制生猪免疫机能,降低动物生产性能,引起动物继发感染,还会在动物产品中残留而威胁人类健康,给生猪养殖带来经济损失,降低猪肉食品安全性。本文通过使用先进固体发酵系统设备和益生菌发酵技术,采用单因素试验和响应面中试优化,获得发酵降解猪饲料AFB₁的最佳工艺参数为硒浓度0.3 mg/kg,发酵时间12 h,量子波强度30 Hz,益生菌菌种组合CGMCC NO.17328混合CGMCC NO.15611。该工艺将猪饲料AFB₁量从63.41μg/kg降解到2.98μg/kg,降解率达到95.30%,AFB₁含量达到国家饲料安全标准。生产工艺适合养猪场低成本快速生产AFB₁达标猪饲料。     

稻瘟病菌CAAX蛋白酶MoRce1的功能研究

 蛋白质的翻译后异戊烯化修饰(CAAX修饰)能够介导真核生物中许多重要蛋白质的亚细胞定位以及蛋白与蛋白间的相互作用。稻瘟病菌(Magnaporthe oryzae)引致的稻瘟病是水稻最重要病害之一,造成全球水稻严重减产。为深入了解稻瘟病菌致病机理,更好地防控稻瘟病,我们研究了异戊烯修饰是否影响稻瘟病菌的生长发育和致病性。首先从稻瘟病菌基因组数据库中鉴定到一个稻瘟病菌的异戊烯蛋白酶MoRce1,同源比对发现MoRce1保守结构域在各物种之间变化较大,猜测在不同物种中该蛋白可能出现了功能分化。经同源重组方法敲除MoRCE1基因,发现MoRCE1缺失突变体在胁迫培养条件下细胞壁完整性明显缺陷,但是对营养生长、产孢、萌发以及致病性没有明显影响,说明MoRce1蛋白可能通过参与稻瘟病菌细胞壁合成相关蛋白的异戊烯化修饰进而影响该菌细胞壁的完整性,其具体机制还有待深入研究。     

Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.     

基于有限元的汽车扭臂热锻工艺研究

文章以汽车扭杆力臂为研究对象,通过DEFORM-3D有限元分析对汽车扭杆力臂热锻工艺过程进行模拟试验,分析了成形过程中的上模载荷情况、应力应变场分布情况和温度场的变化规律。以提高产品质量和降低载荷为主要目的,给出合理的热加工参数,分析结果能为类似锻件的热模锻工艺和模具设计提供依据。     

Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

过表达ZmIBH1-1提高玉米苗期抗旱性

【目的】干旱是严重影响玉米生长发育进程的一个重要因素。挖掘玉米抗旱相关基因,通过转基因功能验证和转录组分析,解析关键基因在响应干旱胁迫过程中的分子调控机制,为抗旱分子育种和遗传改良提供理论依据。【方法】以玉米自交系B104（WT）为背景材料,利用农杆菌介导方法构建过表达ZmIBH1-1转基因株系（ZmIBH1-1-OE）;通过对转基因植株进行草铵膦抗性筛选、标记基因和目的基因PCR检测,以及运用实时荧光定量PCR检测目的基因的表达情况,鉴定阳性植株和株系;以WT和ZmIBH1-1-OE转基因株系为材料,通过干旱处理（20% PEG6000）,进行表型鉴定和耐旱生理生化指标测定,验证ZmIBH1-1的抗旱功能;通过对干旱胁迫下玉米4叶期转录组的比较分析,鉴定出差异表达的基因（differentially expressed genes,DEGs）;结合DAP-seq（DNA affinity purification sequencing）分析,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的下游靶基因,利用基因组可视化软件IGV（integrative genomics viewer）分析ZmIBH1-1蛋白结合候选靶基因的位置,然后通过Dual-Luciferase试验验证ZmIBH1-1蛋白与靶基因的调控关系。【结果】通过玉米遗传转化获得12个转化事件;T₃代中,能同时检测到标记基因Bar和目的基因ZmIBH1-1的植株有458个,实时荧光定量PCR检测结果表明,ZmIBH1-1-OE中ZmIBH1-1的表达量显著高于WT,株系3和株系8表达量最高,将其自交获得T₄代转基因株系用于后续试验。在干旱胁迫条件下,ZmIBH1-1-OE株系存活率、叶片相对含水量、叶绿素含量、可溶性蛋白含量及其生理生化指标（超氧化物歧化酶、过氧化物酶、过氧化氢酶活性）均显著高于WT,说明玉米中过量表达ZmIBH1-1赋予玉米更高的耐旱性。转录组分析结果表明,WT与ZmIBH1-1-OE株系在干旱胁迫下有1 214个差异表达基因;Gene Ontology（GO）功能富集分析结果表明,差异表达基因主要涉及生物过程、细胞组分和分子功能,如在生物过程中主要涉及到光合作用、应激响应、脱水响应等;KEGG富集分析表明,差异表达基因主要参与植物激素信号传导、新陈代谢等过程。结合转录组显著差异表达基因和DAP-Seq分析所得到ZmIBH1-1蛋白的靶基因,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的11个候选靶基因,包括2个钙信号相关基因、3个半胱氨酸代谢相关基因、1个bHLH转录因子、1个应激响应蛋白、1个谷胱甘肽转移酶、1个氧化还原过程蛋白和2个乙烯响应因子;基因组可视化结果显示ZmIBH1-1蛋白可以结合靶基因启动子区;随后通过Dual-Luciferase试验进一步表明,ZmIBH1-1蛋白可以直接作用于11个候选靶基因,其中,ZmIBH1-1蛋白可以促进ZmCa-M、ZmSYCO、ZmbHLH54、ZmGlu-r1、ZmCLPB3和ZmP450-99A2的表达,抑制ZmAGD12、ZmCYS、ZmCYSB、ZmERF-107和ZmEIN3的表达。此外,在干旱胁迫下NAC、WRKY、MYB等转录因子在ZmIBH1-1-OE和WT株系中也存在差异表达。【结论】ZmIBH1-1的过表达可以增强玉米苗期的耐旱性;ZmIBH1-1蛋白通过直接调控乙烯信号通路中的ZmERF-107和ZmEIN3的表达提高玉米的耐旱性;ZmIBH1-1蛋白通过直接调控钙信号相关基因ZmCa-M和ZmAGD12增强玉米的耐旱性;ZmIBH1-1蛋白可能通过间接调控NAC、WRKY、MYB等转录因子响应干旱胁迫。     

Replication of neurovirulent equine herpesvirus type 1 (EHV-1) in CD172a+ monocytic cells

Equine herpesvirus type 1 (EHV-1) is responsible for respiratory disorders, abortion and myeloencephalopathy (EHM) in horses. Two pathotypes of EHV-1 strains are circulating in the field: neurovirulent (N) and non-neurovirulent (NN). For both strains, CD172a⁺ monocytic cells are one of the main carrier cells of EHV-1 during primary infection, allowing the virus to invade the horse’s body. Recently, we showed that EHV-1 NN strains showed a restricted and delayed replication in CD172a⁺ cells. Here we characterize the in vitro replication kinetics of two EHV-1 N strains in CD172a⁺ cells and investigate if the replication of these strains is similarly silenced as shown for EHV-1 NN strains. We found that EHV-1 N replication was restricted to 7–8% in CD172a⁺ cells compared to 100% in control RK-13 cells. EHV-1 N replication was not delayed in CD172a⁺ cells but virus production was significant lower (10^3.0 TCID₅₀/10⁵ inoculated cells) than in RK-13 cells (10^8.5 TCID₅₀/10⁵ inoculated cells). Approximately 0.04% of CD172a⁺ cells produced and transmitted infectious EHV-1 to neighbour cells compared to 65% of RK-13 cells. Unlike what we observed for the NN strain, pretreatment of CD172a⁺ cells with histone deacetylases inhibitors (HDACi) did not influence the replication of EHV-1 N strains in these cells. Overall, these results show that the EHV-1 replication of N strains in CD172a⁺ cells differs from that observed for NN strains, which may contribute to their different pathogeneses in vivo.     

Mapping the spatial distribution of shade provision of street trees in Boston using Google Street View panoramas

Street trees provide shade and increase human thermal comfort during hot summer. In this study, we investigated the spatial distribution of shade provision of street trees in Boston, Massachusetts. The sky view factor (SVF), which influences the solar radiation to the ground and affects human thermal comfort, was used to indicate the contribution of street trees on shade provision. Google Street View (GSV) panoramas were used to calculate the photographic method based SVF (SVF_P), with the consideration of all kinds of obstructions within street canyons. A building height model was used to calculate the simulation based SVF (SVFs), with consideration of obstruction of building blocks only. Considering the fact that street trees and building blocks are the two major obstructions of radiation within street canyons, therefore, the difference between the two SVF estimation results can be considered as the shade provision of street trees. The results show that street trees help to decrease the SVF by 24.61% in Boston, Massachusetts. The shading level varies spatially in the study area. Generally, the southwestern area has much higher shading level than the north and the east. We further explored the shading variation among different socioeconomic groups in the study area. Result shows that Hispanics tend to live in neighborhoods with lower shading level. This study can help to provide a reference for future urban greening projects for global climate change adaption.