Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies

Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5 M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins.     

Tallgrass prairie restoration: implications of increased atmospheric nitrogen deposition when site preparation minimizes adventive grasses

Site preparation designed to exhaust the soil seedbank of adventive species can improve the success of tallgrass prairie restoration. Despite these efforts, increased rates of atmospheric nitrogen (N) deposition over the next century could potentially promote the growth of nitrophilic, adventive species in tallgrass restoration projects. We used a field experiment to examine how N addition affected species composition and plant productivity over the first 3 years of a tallgrass prairie restoration that was preceded by the planting of glyphosate‐resistant crops and multiple applications of glyphosate to exhaust the pre‐existing seedbank. We predicted that N addition would increase the percent cover of adventive plant species not included in the original seeding. Contrary to our prediction, only the cover of native species increased with N addition; native non‐leguminous forbs increased substantially, with Conyza canadensis (a weedy native species not part of the restoration seed mix) exploiting the combination of high N and bare ground in the first year, and non‐leguminous forbs (in particular Monarda fistulosa) and native C₃ grasses, all of which were seeded, increasing with N addition by the third year. Native legumes was the only functional group that exhibited lower cover in N addition plots than in control plots. There was no significant response by native C₄ grasses to N addition, and adventive grasses remained mostly absent from the plots. Overall, our results suggest that site pre‐treatment with herbicide may continue to be effective in minimizing adventive grasses in restored tallgrass prairie, despite future increases in atmospheric N deposition.     

Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa

Xylooligosaccharides (XOS) with DP 2-4 are important synbiotics used as food ingredients based on its prebiotic characteristics. In this work, the production of XOS from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylan was prepared from triploid Populas tomentosa, and bioconverted into XOS by crude xylanase solution obtained from Pichia stipitis. The effects of reaction time, temperature, enzyme dosage, and pH value on the production of XOS were fully evaluated. Under the optimal condition (25 U g⁻¹ substrate, pH 5.4 and 50 °C), 36.8% of the xylan preparation was converted to XOS, equivalent to 3.95 mg/mL of the hydrolyzate. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzymatic hydrolyzate, which together accounted for over 95% of the released oligosaccharides. Meanwhile, the effect of sonication pretreatment on the conversion efficiency of the xylan preparation was also investigated.     

小麦种子过氧化物酶WP1 基因的原核表达、纯化及多克隆抗体制备

WP1是小麦种子中最主要的阳离子过氧化物酶,该酶不仅参与种子的发育过程,而且影响面粉的加工品质。首先构建了WP1基因原核表达载体pET28a-WP1,并将其转化到T7 Expression大肠杆菌菌株中诱导表达。His-tag融合的WP1主要以包涵体形式存在,使用Ni-NTA亲和层析柱在变性条件下进行纯化,获得纯度大于98％的重组蛋白。重组WP1经尿素梯度透析复性溶解后免疫新西兰大白兔,最终获得WP1多克隆抗体。ELISA分析结果显示制备的WP1兔抗血清的效价大于1∶625 000;Western blotting结果证明制备的多克隆抗体对WP1具有很好的专一性。     

中华蚊母树染色体制片及核型分析

以中华蚊母树根尖为材料,采用常规压片法制片,比较材料的不同采集时间、预处理方法、固定剂、解离方法、解离时间及染色方法对中华蚊母树根尖染色体制片的影响.结果表明最佳的制片技术为:取材时间为上午9:00~11:00或下午13:00~15:00,以饱和对二氯苯预处理3 h,用1 mol?L-1盐酸60℃下解离8 min,卡诺固定剂固定,以改良石碳酸品红染色10 min.以该最佳制片方案对中华蚊母树进行体细胞染色体核型分析,首次揭示了中国特有植物———中华蚊母树体细胞染色体数目为2n=2x=24,染色体基数x=12,染色体核型公式为2n=2x=24=12m(2SAT)+10sm+2st,主要由中部和近中部着丝点染色体组成;染色体相对长度组成为2n=24=2L+8M2+12M1+2S,核型不对称指数为64.29%,属于2A型.结果显示中华蚊母树核型对称程度较高,在进化中属于比较原始的类型.     

两种榕属植物的染色体核型分析

采用根尖压片法对菩提树(Ficus religiosa Linn.)和小叶榕(Ficus microcarpa L.f.)进行染色体核型分析.结果显示两种植物的染色体数目均为2n=30,核型类型均为1B,核型公式分别为菩提树2n=2x=30=24m(2SAT)+6sm,小叶榕2n=2x=30=28m(2SAT)+2sm.比较发现,两种植物的核型相似,亲缘关系相近,二者中小叶榕的不对称系数较小(58.16%),为较原始的类型.两者的核型均为首次报道.     

低温电子显微技术在膜蛋白结构研究中的应用和展望

介绍了蛋白质电子晶体学和单颗粒分析技术这两种低温电子显微技术在膜蛋白和膜蛋白复合体结构研究中的具体方法和近10~20年来的实际应用,并分别分析了这两种方法的优势和瓶颈。此外,还介绍了Amphipol替代、Streptavidin二维晶体锚定脂质体和纳米球包被脂质体等近两年来出现的新的用于低温电镜成像的膜蛋白样品制备方法。最后对膜蛋白的低温电子显微研究的未来发展做了展望。     

家蚕核型多角体病毒BmNPV囊膜蛋白P74膜外区克隆和原核表达

通过PCR扩增家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)囊膜蛋白p74基因膜外区片段,对切胶纯化得到的DNA目的片段与原核表达载体pET28a进行连接,通过不同浓度的IPTG对含有pET28a-p74重组质粒的大肠杆菌BL21(DE3)进行诱导,对诱导产物进行SDS-PAGE电泳.结果表明p74基因膜外区获得了表达;通过His单抗对原核表达产物进行Western blotting分析,其结果证实诱导蛋白带为融合有组氧酸的目的蛋白.对割胶获得的P74蛋白和免疫佐剂进行克分研磨,以研磨后的匀浆液对昆明小鼠进行皮下多点注射,通过收获的抗血清对BmNPV ODV病毒粒子进行Westem blotting分析,检测到一条分子量大小为74 kD的特异杂交带,表明获得的多抗可用于P74蛋白功能的进一步研究.     

后磨牙模拟根管模型制备与研究

目的：制备后磨牙模拟根管模型。方法：依照预实验中测试的牙本质和树脂根管模型材料的布氏硬度值（牙本质HB72,树脂块HB54）选取纯铜质铜管为材料（纯铜HB58）同时利用预实验中确认的后磨牙模拟根管模型弯曲角度的范围（70°-60°）并参照树脂根管模型相关数据,设计不同弯曲角度（70°,64°,60°）的纯铜质根管模型,将20套镍钛根管锉随机分为4组,其中3组根管锉分别预备相应角度的10个纯铜质后磨牙根管模型,另一组在临床预备10个后磨牙根管作为对照组。所有镍钛根管锉均在疲劳寿命测试装置中测试疲劳寿命。结果：选用根管长度为16mm±1mm,弯曲半径为5mm,内径为0.70mm的纯铜质铜管制作后磨牙根管模型。其中在弯曲角度为70°的后磨牙根管模型中有33%（2/6）近似临床;在弯曲角度为64°的后磨牙根管模型中有67%（4/6）近似临床;在弯曲角度为60°的后磨牙根管模型中有33%（2/6）近似临床。结论：弯曲角度为64°的纯铜制后磨牙模拟根管模型更接近于临床实际,可为体外研究镍钛根管锉各项指标提供一个较为理想的模型。