Large-scale N-glycoproteome map of rat brain tissue: simultaneous characterization of insoluble and soluble protein fractions

The large-scale N-glycosylation analysis is critical for biomedical research, since a variety of diseases are found to be associated with glycoproteins. By a combination of glycoprotein analysis in insoluble protein fraction solubilized with 1% v/v 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM BF(4)) and those in soluble fraction, a total number of 462 non-redundant N-glycoprotein groups, including 316 transmembrane glycoproteins, were successfully identified. Correspondingly, 849 unique N-glycosites were confidently recognized. The data set could provide a support for the further in-depth research of brain N-glycosylation, such as for the discovery of candidate drug targets and biomarkers.     

Genetic, proteomic and metabolic analysis of the regulation of energy storage in rice seedlings in response to drought

We used proteomic analysis to determine the response of rice plant seedlings to drought-induced stress. The expression of 71 protein spots was significantly altered, and 60 spots were successfully identified. The greatest down-regulated protein functional category was translation. Up-regulated proteins were mainly related to protein folding and assembly. Additionally, many proteins involved in metabolism (e.g. carbohydrate metabolism) also showed differences in expression. cDNA microarray and GC-MS analysis showed 4756 differentially expressed mRNAs and 37 differentially expressed metabolites. Once these data were integrated with the proteomic analysis, we were able to elucidate the metabolic pathways affected by drought-induced stress. These results suggest that increased energy consumption from storage substances occurred during drought. In addition, increased expression of the enzymes involved in anabolic pathways corresponded with an increase in the content of six amino acids. We speculated that energy conversion from carbohydrates and/or fatty acids to amino acids was increased. Analysis of basic metabolism networks allowed us to understand how rice plants adjust to drought conditions.     

Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith

A metal‐ion chelate immobilized enzyme reactor (IMER) supported on organic–inorganic hybrid silica monolith was developed for rapid digestion of proteins. The monolithic support was in situ prepared in a fused silica capillary via the polycondensation between tetraethoxysilane hydrolytic sol and iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane. After activated by Cu²⁺, trypsin was immobilized onto the monolithic support via metal chelation. Proteolytic capability of such an IMER was evaluated by the digestion of myoglobin and BSA, and the digests were further analyzed by microflow reversed‐phase liquid chromatography with ESI‐MS/MS. Similar sequence coverages of myoglobin and BSA were obtained by IMER, in comparison to those obtained by in‐solution digestion (91 versus 92% for 200 ng myoglobin, and 26 versus 26% for 200 ng BSA). However, the digestion time was shortened from 12 h to 50 s. When the enzymatic activity was decreased after seven runs, the IMER could be easily regenerated by removing Cu²⁺ via EDTA followed by trypsin immobilization with fresh Cu²⁺ introduced, yielding the equal sequence coverage (26% for 200 ng BSA). For ～5 μg rat liver extract, even more proteins were identified with the immobilized trypsin digestion within 150 s in comparison to the in‐solution digestion for 24 h (541 versus 483), demonstrating that the IMER could be a promising tool for efficient and high‐throughput proteome profiling.     

功能矫形前伸青春期大鼠下颌后浅层嚼肌细胞凋亡的研究

目的：研究功能矫形前伸大鼠下颌后浅层嚼肌细胞凋亡的变化规律,探讨功能矫形的肌肉改建机理。方法：选用50只5周龄Sprague-Dawley（SD）雄性大白鼠,随机分为实验组和对照组各25只。实验组大鼠戴自制上颌功能矫治嚣,引导下颌前伸,并打开咬合。利用RT-PCR方法检测两组大鼠浅层嚼肌Bcl-2和Bax基因表达情况,利用TUNEL方法检测浅层嚼肌细胞凋亡情况。结果：①Bcl-2和Bax基因表达随大鼠戴用矫治器时间的延长而升高,至第3周开始下降但仍高于对照组,但Bax的表达高于Bcl-2。Bax/Bcl-2比值随大鼠戴用矫治器时间的延长而升高,至第4周开始下降。②TUNEL实验结果显示浅层嚼肌细胞在戴用矫治器1天后,开始出现凋亡,随着时间延长而增加,至第3周达到顶峰,第4周开始下降。结论：①Bax/Bcl-2比值升高促进浅层嚼肌细胞凋亡。②功能矫形可引起浅层嚼肌细胞凋亡,导致肌肉的结构和功能发生适应性改建。     

乙肝治疗耐药基因突变的焦磷酸测序技术诊断

目的：建立焦磷酸测序技术检测拉米夫定和阿德福韦酯治疗乙肝所致乙肝病毒基因耐药突变的定量检测方法,为临床乙肝耐药诊断和治疗提供依据。方法：针对乙肝病毒DNA聚合酶基因序列上4个常见基因突变位点的6种突变形式,分别克隆构建野生型和突变型质粒作为标准品,应用生物信息学手段设计目标基因通用PCR引物和各突变点的焦磷酸测序引物,建立焦磷酸测序的突变检测方法。对接受拉米夫定、阿德福韦酯治疗的慢性乙型肝炎患者血清标本进行检测。结果：构建了乙肝病毒四种常见耐药性突变的标准株和变异株克隆,建立了分别或同时检测拉米夫定、阿德福韦酯耐药突变的焦磷酸测序方法,对68例临床耐药或疑似耐药的患者血清标本进行检测,双脱氧测序验证,检出拉米夫定耐药突变32例,阿德福韦酯耐药突变5例,其中焦磷酸测序检出20例为混合突变,而双脱氧测序显示为6例。结论：成功建立了焦磷酸测序定量检测拉米夫定、阿德福韦酯耐药基因突变的方法,构建了乙肝病毒耐药基因突变的标准质粒,为临床动态监测乙肝病毒变异病毒株、指导合理用药奠定了基础。     

功能矫形前伸青春期大鼠下颌后翼外肌MyoD、myogenin的表达

目的：探讨＂应力-生长（改建）＂在细胞水平上的体现,为功能矫形治疗和矫治效果的保持提供新思路和实验依据。方法：本实验选用20只4周龄,雄性SD大鼠随机分为8组。其中实验组大鼠经戊巴比妥麻醉后佩戴上颌斜面导板,对照组未佩用。依据时间不同又分为四组：1d,7d,14d,21d。采用RT-PCR技术分析各组大鼠翼外肌组织中肌分化相关基因MyoD、myogenin mRNA的表达变化。结果：未施加功能矫形力的大鼠翼外肌组织MyoD表达伴随其生长发育呈现递减趋势,实验组在第7 d出现表达上调。同时,力学刺激后实验组动物myogenin的表达与对照组相比较在14 d组出现明显上调。结论：功能矫形力作用于翼外肌组织可以诱导MyoD和myogenin的表达上调进而诱导成肌细胞的分化。     

特发性腹膜后纤维化一例并文献复习

目的：分析腹膜后纤维化（RPF）的诊断以及治疗情况,以提高对RPF的认识。方法：回顾性分析我科18F-FDGPET/CT诊断的1例RPF患者的临床资料,并对相关文献进行复习。结果：本例患者以腹胀及右下腹部隐痛不适就诊,腹部CT表现为腹主动脉周围肿块,18F-FDGPET/CT显示腹膜后间隙中线大血管周围糖代谢增高肿块,经CT引导下穿刺及手术病理确诊为特发性腹膜后纤维化。结论：腹膜后纤维化属罕见病,CT、MRI在诊断中有较重要作用,PET/CT在IRPF的诊断及治疗随访中有比较重要的价值,在治疗方面,糖皮质激素治疗效果较好,晚期常需要手术治疗。     

我军某部赴利比里亚维和某部疾病构成及防治

目的：调查我维和部队官兵的疾病构成,从而探讨防治对策。方法：军医实行24小时值班制度,1周轮换。就诊官兵均按症状、体征进行临床诊断并实时登记。结果：感染性疾病、皮肤病、腰腿痛、胃炎、痔等5类疾病所占比例已经超过95%,而原来以为与军事行动关系较大的外伤、与热带环境工作相关的中暑倒处于一个较为次要的位置。结论：自我国派成建制的部队参加国际维和任务以来,所赴地区主要为热带或亚热带,各部队所处环境相似,各自的疾病防治经验可资借鉴。     

Intranasal immunization with recombinant HA and mast cell activator C48/80 elicits protective immunity against 2009 pandemic H1N1 influenza in mice

Background

Pandemic influenza represents a major threat to global health. Vaccination is the most economic and effective strategy to control influenza pandemic. Conventional vaccine approach, despite being effective, has a number of major deficiencies including limited range of protection, total dependence on embryonated eggs for production, and time consuming for vaccine production. There is an urgent need to develop novel vaccine strategies to overcome these deficiencies.

Methodology/Principal Findings

The major objective of this work was to develop a novel vaccine strategy combining recombinant haemagglutinin (HA) protein and a master cell (MC) activator C48/80 for intranasal immunization. We demonstrated in BALB/c mice that MC activator C48/80 had strong adjuvant activity when co-administered with recombinant HA protein intranasally. Vaccination with C48/80 significantly increased the serum IgG and mucosal surface IgA antibody responses against HA protein. Such increases correlated with stronger and durable neutralizing antibody activities, offering protection to vaccinated animals from disease progression after challenge with lethal dose of A/California/04/2009 live virus. Furthermore, protected animals demonstrated significant reduction in lung virus titers, minimal structural alteration in lung tissues as well as higher and balanced production of Th1 and Th2 cytokines in the stimulated splenocytes when compared to those without C48/80.

Conclusions/Significance

The present study demonstrates that the novel vaccine approach of combining recombinant HA and mucosal adjuvant C48/80 is safe and effective in eliciting protective immunity in mice. Future studies on the mechanism of action of C48/80 and potential combination with other vaccine strategies such as prime and boost approach may help to induce even more potent and broad immune responses against viruses from various clades.