Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.     

Does Serum 25-Hydroxyvitamin D Influence Muscle Development during Puberty in Girls? - A 7-Year Longitudinal Study

Vitamin D is well known for its regulatory role in calcium and phosphate homeostasis, but its role in muscle mass and strength during growth remains inconclusive. We explored the association of serum 25-hydroxyvitamin D (25(OH)D) with muscle development in girls from 11 to 18-years old. Whole body lean tissue mass (LM_WB), appendicular lean mass (aLM), muscle cross-sectional area at the lower leg (mCSA), maximal voluntary contraction of elbow flexors (MVC_elbow) and knee extensors (MVC_knee) were assessed in 217 girls aged 10–13 years (at baseline), 215 in 2-year and 226 in 7.5-year follow-up. Serum concentration of 25(OH)D and intact parathyroid hormone (PTH) were analyzed retrospectively and girls were categorized according to their 25(OH)D levels (consistently insufficient 25(OH)D G_LL <50 nmol/l and consistently sufficient G_HH >50 nmol/l from baseline to 7-year follow-up). We found that 25(OH)D level declined until menarche (p<0.05) while LM_WB, aLM, mCSA, MVC_elbow and MVC_knee continued to increase (p<0.001 for all) post menarche. At pre-menarche, the G_LL (n = 34) had higher LM_WB and aLM than the G_HH (n = 21, p<0.05), while post-menarche the G_HH (n = 15) had a greater catch-up gain in LM_WB (p = 0.004), aLM (p = 0.001) and mCSA (p = 0.027) compared to the G_LL (n = 65) over the first 2-year period. At the age of 18, no differences in muscle mass/strength between the low (n = 151) and high (n = 77) levels of 25(OH)D groups were found. This finding was independent of vitamin D receptor genotype and other confounders. In conclusion, our results showed that levels of 25(OH)D have no significant negative influence on the development of muscle mass and strength during pubertal growth both with longitudinal and cross-sectional comparison. On the contrary, our results suggest that the temporary negative association between 25(OH)D and muscle mass arises as a consequence of fast growth prior to menarche, and this negative association is diminished through catch-up growth after menarche.     

冬眠和非冬眠状态达乌尔黄鼠肾单位及相关功能因子的比较

冬眠是动物应对冬季低温和食物匮乏的一种生存策略。达乌尔黄鼠(Spermophilus dauricus)是典型的贮脂类冬眠动物。为研究冬眠动物肾脏的适应机制,本实验采用组织学、血液生化分析及酶联免疫方法检测了夏季活动期(7月)、冬眠期(12月)和早春出眠后(3月)达乌尔黄鼠肾单位形态学及血清肌酐、尿素和抗利尿激素(ADH)的变化,并用qPCR方法检测了肾脏水通道蛋白基因(AQP1、AQP2和AQP3)、ADH受体(V2R)及内皮型一氧化氮合酶基因(eNOS)的表达。结果发现,冬眠期和早春出眠期的达乌尔黄鼠肾小球密度、近曲小管和远曲小管的相对管径、皮质部近曲小管数与远曲小管数比值均低于夏季活动期;冬眠期血清肌酐和尿素浓度高于夏季活动期和早春出眠期,ADH浓度及其受体V2R基因表达低于夏季活动期;冬眠期AQP1基因表达高于早春出眠期,AQP3基因表达低于夏季活动期,AQP2基因表达无显著差异;冬眠期eNOS基因表达低于早春出眠期。这些结果表明冬眠的达乌尔黄鼠表现出较低的肾功能;不同时期的水通道蛋白,eNOS及ADH表现出适应性的功能调节。该实验结果丰富了对冬眠动物肾脏适应机制的认识。     

The Molecular Mechanisms of Protective Role of Se on the G<Subscript>2</Subscript>/M Phase Arrest of Jejunum Caused by AFB<Subscript>1</Subscript>

Aflatoxin B₁ (AFB₁) is the most toxic among the mycotoxins and causes detrimental health effects on human and animals. Selenium (Se) plays an important role in chemopreventive, antioxidant, anticarcinogen, and detoxification and involved in cell cycle regulation. The aim of this study was to explore the molecular mechanisms of selenium involved in inhibition of G₂/M cell cycle arrest of broiler’s jejunum. A total of 240 one-day-old healthy Cobb broilers were randomly divided into four groups and fed with basal diet (control group), 0.6 mg/kg AFB₁ (AFB₁ group), 0.4 mg/kg Se (+Se group), and 0.6 mg/kg AFB₁ + 0.4 mg/kg Se (AFB₁ + Se group) for 21 days, respectively. The histological observation and morphological analysis revealed that 0.4 mg/kg Se prevented the AFB₁-associated lesions of jejunum including the shedding of the apical region of villi, the decreased villus height, and villus height/crypt ratio. The cell cycle analysis by flow cytometry showed that 0.4 mg/kg Se ameliorated the AFB₁-induced G₂/M phase arrest in jejunal cells. Moreover, the expressions of ATM, Chk2, p53, Mdm2, p21, PCNA, Cdc25, cyclin B, and Cdc2 analyzed by immunohistochemistry and qRT-PCR demonstrated that 0.4 mg/kg Se restored these parameters to be close to those in the control group. In conclusion, Se promoted cell cycle recovery from the AFB₁-induced G₂/M phase arrest by the molecular regulation of ATM pathway in the jejunum of broilers. The outcomes from the present study may lead to a better understanding of the nature of selenium’s essentiality and its protective roles against AFB₁.     

聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶 *

聚乙二醇二缩水甘油醚(PEGDGE)作为双功能环氧试剂,在实验中被用于交联氨基载体LX-1000EA共价固定化海洋脂肪酶,经过处理后的载体共价固定化脂肪酶具有良好的效果。实验经过单因素初筛和正交试验,得到最佳的交联及固定化条件为0.75%交联剂浓度、交联温度35℃、交联时间3h、载体量1.25g、pH9.0、固定化温度55℃、固定化时间1h。对LX-1000EA-PEGDGE固定化酶与游离酶、戊二醛(GA)交联LX-1000HA-GA的固定化酶进行酶学性质的比较,发现LX-1000EA- PEGDGE固定化酶较游离酶最适反应温度未改变,与LX-1000HA-GA相同的是最适反应pH都由7.0提高为8.0。在最适条件中所测LX-1000EA-PEGDGE酶活达到78.84U/g,固定化改变了游离酶的酸碱耐受性,热稳定性和操作稳定性较游离酶和LX-1000HA-GA固定化酶均有提高。LX-1000EA-PEGDGE的热稳定表现优异,在60℃孵育3h后保留90%酶活;使用5次后仍能残余50%酶活;保存30天酶活仍保留60%。首次使用新型双环氧交联剂PEGDGE交联有机氨基载体共价结合固定化脂肪酶,为更有效的固定化方法提供了技术支持,同时也发现交联剂对固定化酶的性质存在较大影响。     

Skin aging caused by intrinsic or extrinsic processes characterized with functional proteomics

The skin provides protection against environmental stress. However, intrinsic and extrinsic aging causes significant alteration to skin structure and components, which subsequently impairs molecular characteristics and biochemical processes. Here, we have conducted an immunohistological investigation and established the proteome profiles on nude mice skin to verify the specific responses during aging caused by different factors. Our results showed that UVB‐elicited aging results in upregulation of proliferating cell nuclear antigen and strong oxidative damage in DNA, whereas chronological aging abolished epidermal cell growth and increased the expression of caspase‐14, as well as protein carbonylation. Network analysis indicated that the programmed skin aging activated the ubiquitin system and triggered obvious downregulation of 14‐3‐3 sigma, which might accelerate the loss of cell growth capacity. On the other hand, UVB stimulation enhanced inflammation and the risk of skin carcinogenesis. Collectively, functional proteomics could provide large‐scale investigation of the potent proteins and molecules that play important roles in skin subjected to both intrinsic and extrinsic aging.     

Sponge non-metastatic Group I Nme gene/protein - structure and function is conserved from sponges to humans

Background  

Nucleoside diphosphate kinases NDPK are evolutionarily conserved enzymes present in Bacteria, Archaea and Eukarya, with human Nme1 the most studied representative of the family and the first identified metastasis suppressor. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestor's genomic features. Recent studies show that sponges have a wide repertoire of genes many of which are involved in diseases in more complex metazoans. The original function of those genes and the way it has evolved in the animal lineage is largely unknown. Here we report new results on the metastasis suppressor gene/protein homolog from the marine sponge Suberites domuncula, NmeGp1Sd. The purpose of this study was to investigate the properties of the sponge Group I Nme gene and protein, and compare it to its human homolog in order to elucidate the evolution of the structure and function of Nme.     

用固定化弗劳地柠檬酸杆菌XP05从溶液中回收铂

比较了5种固定弗劳地柠檬酸杆菌XP05菌体的方法,其中明胶海藻酸钠包埋法为固定菌体的最佳方法。扫描电子显微镜观察表明,XP05菌体较均匀地分布于包埋基质中。固定化XP05菌体吸附Pt^4＋受吸附时间、固定化菌体浓度、溶液的pH值和Pt^4＋起始浓度的影响。吸附作用是一个快速的过程;吸附Pt^4＋的最适pH值为1.5;在50～250 mg P^4＋/L范围内,吸附量与Pt^4＋起始浓度成线性关系,吸附过程符合Langmuir和Freundlich吸附等温模型。在Pt^4＋起始浓度250 mg/L、固定化菌体2.0 g/L、pH 1.5和30℃条件下,振荡吸附60 min, 吸附量为35.3 mg/g。0.5 mol/L HCl能使吸附在固定化菌体上的Pt解吸98.7%。从废铂催化剂处理液回收铂的结果表明,在Pt^4＋起始浓度111.8 mg/L、固定化菌体4.0 g/L、pH 1.5和30℃条件下,振荡吸附60 min, 吸附量为20.9 mg/g。在填充床反应器中,在Pt^4＋起始浓度50 mg/L、流速1.2 ml/min、固定化菌体1.86 g的条件下,饱和吸附量达24.7 mg/g; 固定化XP05菌体经4次吸附解吸循环后吸附率仍达78％。