ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

The end of late-generation European ethnicity in America?

Exploring late-generation ethnicity automatically raises a long-ignored question: whether it will end and how: through what terminal stages and processes? In America, this question currently makes sense only for late-generation descendants of the European immigration wave that lasted roughly from the 1870s to 1924. However, answering it prepares the ground for eventually asking it also about late-generation ethnics in newer waves of immigration.     

禽网状内皮组织增殖病病毒LTR序列的启动子功能

通过PCR方法,将禽网状内皮组织增殖病病毒(REV)的长末端重复序列(LTR)扩增并克隆进pUC-18质粒多克隆位点(MCS)的EcoR I和Sac I之间,并以BGH基因的多聚腺苷酸序列作为终止子克隆到SphI~HindIII之间,构建成重组质粒pUC-LTR。将GFP基因和REV囊膜糖蛋白gp90基因分别克隆到pUC-LTR载体中,获得质粒pUC-LTR-GFP和质粒pUC-LTR-gp90。重组质粒经转染48h,能够检测到外源基因的表达。本研究提示,REVLTR能够作为启动子构建表达质粒。     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

香丹注射液对急性冠脉综合征患者外周血C-反应蛋白、氨基末端脑钠肽及心肌酶水平的影响

目的:探讨香丹注射液治疗急性冠脉综合征血清C-反应蛋白、氨基末端脑钠肽以及心肌酶学的影响。方法:收集我院心内科收治的急性冠脉综合征患者66例,随机分为试验组和对照组,各33例。对照组予以阿司匹林片和硫酸氢氯吡格雷片治疗,试验组在对照组基础上予以香丹注射液治疗。观察并比较两组患者的临床疗效、血清肌钙蛋白T、C-反应蛋白、氨基末端脑钠肽、心肌酶学、不良反应以及心脏事件发生情况。结果:试验组临床总有效率高于对照组(P0.05)。治疗后两组肌钙蛋白T、C反应蛋白以及氨基末端脑钠肽水平降低,且试验组低于对照组(P0.05)。治疗后两组肌酸激酶同工酶、肌酸激酶、天门冬氨酸转移酶以及乳酸脱氢酶水平降低,且试验组低于对照组(P0.05)。试验组心绞痛发作次数、心肌缺血时间以及室性早搏次数较对照组相比显著降低(P0.05)。两组不良反应比较,差异无统计学意义(P0.05)。术后1个月内试验组心脏事件发生率显著低于对照组,差异有统计学意义(P0.05)。结论:香丹注射液治疗急性冠脉综合征的疗效显著,抑制炎症反应,安全性较高,降低心脏事件发生率,适宜临床应用推广。     

Influence of high inorganic selenium and manganese diets for fattening pigs on oxidative stability and pork quality parameters

Data are scarce regarding combined high Se and Mn supplementation in livestock diets, however, as Se and Mn are functionally related as cofactors of glutathione peroxidase (GPx) and Mn-superoxide dismutase (SOD), respectively, beneficial synergistic effects on oxidative stability of tissues may result. This experiment evaluated the effect of an oversupply of Se and Mn within European legal limits compared with recommendations on performance, oxidative stability of the organism and meat quality in a randomised complete block design. A total of 60 crossbred gilts were fed maize–barley–soya bean meal diets formulated in a 2×2 factorial approach with inorganic Se (0.2 v. 0.5 mg/kg Se dry matter (DM)) or inorganic Mn (20 v. 150 mg/kg Mn DM) from 31 to 116 kg BW. Se supplementation reduced feed intake, whereas high Mn diets impaired average daily gain (P<0.05). Qualitative carcass characteristics were impaired by Se and Mn predominantly in the semimembranosus muscle. Activity of GPx in liver was increased by high Se diets (P<0.05). Mn supplementation increased catalase (CAT) activity in liver, GPx in plasma and total antioxidative capacity (TAC) in muscle, whereas it decreased CAT activity in plasma (P<0.05). Cu/Zn-SOD in muscle showed higher activity in high-Se-low-Mn diets but lower activity when both high Se and Mn were combined (Se×Mn P<0.05). Mn supplementation increased Mn concentration in longissimus thoracis et lumborum, but simultaneously reduced Se concentration (P<0.05). Upon retail display, Mn increased lipid oxidation more pronouncedly (higher thiobarbituric acid reactive substances; P<0.05) than Se (P<0.10). Despite some positive effects (Mn increased TAC, Se increased GPx, Se and Mn increased tenderness), no synergistic effects of high Se and Mn diets or an overall beneficial impact on meat quality, especially during storage, could be observed. Including the negative effects on performance, feeding Se and Mn up to the maximum legal level cannot be recommended.     

Alternative operation models for using a feed‐in terminal as a part of the forest chip supply system for a CHP plant

The fuel supply of forest chips has to adapt to the annual fluctuations of power and heat generation. This creates inefficiency and unbalances the capacity utilization of the fuel supply fleet in the direct fuel supplies from roadside storages to power and heat generation. Terminals can offer an alternative approach for the fleet management of fuel supplies in terms of smoothing the unbalanced fleet use towards more even year‐round operations. The aim of the study was to compare the supply costs of a conventional direct forest chip supply to an alternative fuel supply with the use of a feed‐in terminal using the discrete‐event simulation method. The influences of the terminal location, terminal investment cost, outbound terminal transport method, terminal truck utilization and quality changes of terminal‐stored forest chips for the fuel supply cost were studied in the case environment. By introducing a feed‐in terminal and a shuttle truck for the transports of terminal‐stored forest chips, the total supply cost was 1.4% higher than the direct fuel supply scenario. In terminal scenarios, the supply costs increased 1–2% if the cost of the terminal investment increased 30%, the distance to the terminal increased from 5 to 30 km or the total annual use of a terminal truck decreased 1500 h. Moreover, a 1 per cent point per month increase in the dry matter loss of terminal‐stored chips increased the total supply cost 1%. The study revealed that with the relatively low additional cost, the feed‐in terminal can be introduced to the conventional forest chip supply. Cost compensation can be gained through the higher annual use of a fuel supply fleet and more secured fuel supply to power plants by decreasing the need for supplement fuel, which can be more expensive at a time of the highest fuel demand.     

Effect of copper variation in yeast hydrolysate on C‐terminal lysine levels of a monoclonal antibody

The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This article addresses the control of C‐terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large‐scale production of a murine cell line exhibited variation in the C‐terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture. Small‐scale studies demonstrated sensitivity of the cells to copper, where a single low dose of copper to the culture impacted cell lactate metabolism and C‐terminal lysine processing. Subsequent analytical tests indicated that the yeast hydrolysate powder, added to the basal media and nutrient feed in the process, contained varying levels of trace copper across lots. The measured copper concentrations in yeast hydrolysate lots correlated well with the variation in lactate and pH trends and C‐terminal lysine levels of the batches in manufacturing. Small‐scale studies further demonstrated that copper supplementation to yeast hydrolysate lots with low concentrations of copper can shift the metabolic performance and C‐terminal lysine levels of these cultures to match the control, high copper cultures. Hence, a strategy of monitoring, and if necessary supplementing, copper in yeast‐hydrolysate powders resulted in the ability to control and ensure product quality consistency. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:463–468, 2017