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Purification and identification of simian parvovirus protein Vp2 expressed in ***

Journal of Microbiology and Immunology 2005年第3期3卷 219-223页

作者： ZHeNG WeN LIU YONG LIe CHU kevin e.brown Department of Infectious Diseases First Hospital of Xi-an Jiaotong University Xi-an P. R. China Department of Microbiology Medical School Xi-an Jiaotong University Xi-an P. R. China Virus Discovery Group National Heart Lung and Blood Institute NIH Bethesda MD USA

To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in e. coli, fusion protein of SPV Vp2 was expressed in e. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacterial protein extraction reagent was used to extract the protein. Detergents with different characteristics were used to solubilize the fusion protein, and metal chelating resin （ProBond） with a continuous elusion polyacrylamide gel electrophoresis procedure was employed to purify the fusion protein. SDS-PAGe gel stained with coomassie blue and Western-blotting probed with anti-thio and anti-SPV Vp2 antibodies were used to identify the specificity of the expressed and purified fusion proteins. It was found that the SPV Vp2 protein expressed in e. coli was highly insoluble, and could not be solublized by the commonly used detergent. However, 6 M urea could solubilize the fusion proteins and was then employed for the further purification procedure, but metal chelating resin could not be used for this procedure, because of the loss of the tertiary structure of HP-thiaoredoxin and the metal-binding domain. The technique with continuous elusion polyacrylamide gel electrophoresis yielded a homogenous protein with a single band on the gel stained with coomassie blue and retained reactivity with anti-thio or anti-SPV Vp2 antibodies. It is evident that this technique with successful purification of SPV Vp2 protein has practical significance for the further investigation on the simian parvovirus infection.

关键词： Simian parvovirus Protein expression Protein purification

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2010-2015年全球实验室网络支持的消除麻疹和风疹工作的进展

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上海预防医学 2018年第2期30卷 156-159页

作者： mick n.mulders paul a.rota joseph p.icenogle kevin e.brown makoto takeda gloria j.rey myriam c.ben mamou annick r.g.a.dosseh charles r.byabamazima hinda j.ahmed sirima pattamadilok yan zhang marta gacic-dobo peter m.strebel james l.goodson 周惠嘉(翻译) department of immunization vaccinesand biologicalsworld health organization(who) division of viral diseases national center for immunization and respiratory diseasescdc virus reference department public health englandcolindale-london national institute of infectious diseases comprehensive family immunization who regional office for the americaspan american health organization vaccine-preventable diseases and immunization who regional office for europe accelerated immunisations initiatives program who regional office for africaintercountry support team west and central africa accelerated immunisations initiatives program who regional office for africaintercountry support team east and southern africa vaccine-preventable diseases and immunization who regional office for the eastern mediterranean region immunization and vaccine development who regional office for southeast asia expanded programme on immunization who regional office for western pacific global immunization division center for global healthcdc 不详

2012年,世界卫生大会（World Health Assembly）签署全球疫苗行动计划（Global Vaccine Action Plan,GVAP）,该计划旨在2020年前消除世界卫生组织（World Health Qrganization,WHO）5个区域的麻疹和风疹。2013年9月,WHO 6个区域的所有... 详细信息

关键词：风疹病毒基因型消除麻疹麻疹病毒实验室

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ezQTL:A Web Platform for Interactive Visualization and Colocalization of QTLs and GWAS Loci

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Genomics, Proteomics & Bioinformatics 2022年第3期20卷 541-548页

作者： Tongwu Zhang Alyssa Klein Jian Sang Jiyeon Choi kevin M.brown Division of Cancer epidemiology and Genetics National Cancer InstituteBethesdaMD 20892USA

Genome-wide association studies(GWAS)have identified thousands of genomic loci associated with complex diseases and traits,including *** vast majority of common traitassociated variants identified via GWAS fall in non-coding regions of the genome,posing a challenge in elucidating the causal variants,genes,and mechanisms *** quantitative trait locus(eQTL)and other molecular QTL studies have been valuable resources in identifying candidate causal genes from GWAS loci through statistical colocalization *** QTL colocalization is becoming a standard analysis in post-GWAS investigation,an easy web tool for users to perform formal colocalization analyses with either user-provided or public GWAS and eQTL datasets has been ***,we present ezQTL,a web-based bioinformatic application to interactively visualize and analyze genetic association data such as GWAS loci and molecular QTLs under different linkage disequilibrium(LD)patterns(1000 Genomes Project,UK Biobank,or user-provided data).This application allows users to perform data quality control for variants matched between different datasets,LD visualization,and two-trait colocalization analyses using two state-of-the-art methodologies(eCAVIAR and HyPrColoc),including batch *** is a free and publicly available cross-platform web tool,which can be accessed online at https://***/ezqtl.

关键词： Genome-wide association study expression quantitative trait locus Colocalization Linkage disequilibrium Visualization

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Influence of 3d transition-metal substitution on the oxygen reduction reaction electrocatalysis of ternary nitrides in acid

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Nano Research 2019年第9期12卷 2307-2312页

作者： kevin e. Fritz Yichen Yan Jin Suntivich Department of Material Science and engineering Cornell UniversityIthacaNew York 14850USA Kavli Institute at Cornell for Nanoscale Science IthacaNew York 14850USA

The development of non-precious,acid-stable,oxygen reduction reaction(ORR)electrocatalysts can significantly aid the commercialization of proton exchange membrane fuel cells(PeMFCs).We report a survey of the ORR electrocatalysis on 3d metal substituted(M=Mn,Fe,Co)molybdenum and tungsten nitrides in acidic *** find that molybdate catalysts are more active than tungstates,with the specific activity depending on the chemistry of the substituted 3d *** both families,more electronegative 3d metals led to higher ORR activity(i.e.,Co>Fe>Mn).We attribute this result to the ability of the more electro negative 3d metal to withdraw electro ns from the Mo-or W-based active sites,effectively oxidizing the metal centers of the *** on our observation,future nitride ORR electrocatalysts can be further optimized by oxidizing the Mo sites further by,for example,addi ng eve n more electro negative dopa nt metals or in corporati ng anion vaca ncies.

关键词： nitrides electrocatalysis fuel cells oxygen reduction

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Subcellular western blotting of single cells

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Microsystems & Nanoengineering 2017年第1期3卷 405-413页

作者： kevin A.Yamauchi Amy e.Herr Department of Bioengineering University of CaliforniaBerkeleyCA 94720USA The UC Berkeley—UCSF Graduate Program in Bioengineering University of CaliforniaBerkeleyCA 94720USA

Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells,antibody probe cross-reactivity and fixation artifacts remain confounding *** enhance selectivity while providing single-cell resolution,we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual *** confer precision fluidic control,we describe a passive multilayer microdevice that leverages the rapid transport times afforded by *** isolating single cells in microwells,we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate,whereas the nucleus remains intact in the ***,we lyse the intact nucleus and western blot the nuclear *** index each protein analysis to the originating subcellular compartment,we utilize bi-directional electrophoresis,a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation ***-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in *** subcellular,single-cell western blot is demonstrated for six targets per cell,and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes,even for closely sized proteins(a 7 kDa difference).Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with *** chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells,thus providing insight into protein signaling in heterogeneous cell populations.

关键词： cytometry microfluidic design proteomics single-cell analysis

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Digestive retention times for Allen’s swamp monkey and L’Hoest’s monkey: data with implications for the evolution of cercopithecine digestive strategy

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Integrative Zoology 2012年第2期7卷 183-191页

作者： kevin P.BLAINe Joanna e.LAMBeRT Department of Anesthesia Stanford University Medical CenterStanfordCaliforniaUSA Department of ecological Anthropology University of Texas at San AntonioSan AntonioTexasUSA

Primates access energy from plant fiber via bacterial fermentation in either a modified forestomach(‘foregut’),a caecocolic(‘hindgut’)chamber of the large intestine,or *** digestive retention times allow for more complete fermentation;as such,primates that consume an herbivorous diet high in fiber are expected to have both relatively and absolutely longer retention times than those mammals that rely on more readily digestible plant foods,such as *** used particulate markers to measure the digestive retention times of captive Allen’s swamp monkeys[Allenopithecus nigroviridis(Pocock,1907)](n=3)and L’Hoest’s monkey(Cercopithecus lhoesti ***,1899)(n=2).Results indicate mean retention times of 23.2-29.4 h and 23.2-24.0 h for *** and ***,*** from this study,in combination with previously published data on digestive retention times in other primate species,indicate that cercopithecines differ from other primate taxa by having lengthier retention times that can be predicted by body mass *** data are consistent with the hypothesis that relatively lengthy retention times are a primitive trait for Cercopithecinae.

关键词： Cercopithecinae digestive retention time evolution of diet fermentation plant fiber

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猴细小病毒Vp2的克隆、表达和鉴定

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西安交通大学学报（医学版） 2004年第2期25卷 111-113,117页

作者：刘正稳 kevin e.brown 楚雍烈西安交通大学第一医院传染科陕西西安710061 Virus Discovery Group 西安交通大学医学院微生物学教研室陕西西安710061

目的　克隆、表达和鉴定猴细小病毒 (simianparvovirus,SPV)Vp2蛋白。方法　用设计的SPVVp2区的特异性引物 ,PCR法扩增SPVVp2基因DNA片断 ;将获得的基因片断插入 pThioHisA载体中 ,转化大肠杆菌DH5α后挑选阳性克隆 ,利用限制性内切酶... 详细信息

目的　克隆、表达和鉴定猴细小病毒 (simianparvovirus,SPV)Vp2蛋白。方法　用设计的SPVVp2区的特异性引物 ,PCR法扩增SPVVp2基因DNA片断 ;将获得的基因片断插入 pThioHisA载体中 ,转化大肠杆菌DH5α后挑选阳性克隆 ,利用限制性内切酶酶切和核酸序列分析技术鉴定重组质粒 ;以LB培养液培养转化有插入SPVVp2基因的pThioHisA载体的大肠杆菌 ,以终浓度 1mmol·L -1的IPTG诱导蛋白的表达 ;用SDA PAGe和Westernblot分析和鉴定表达的蛋白。结果　经限制性内切酶酶切和核酸序列分析 ,获得了插入pThioHisA载体框架的SPVVp2基因的表达质粒 ;阳性转化大肠杆菌在IPTG诱导下获得了SPVVp2蛋白的表达 ;SDA PAGe和用抗 Thio及抗 SPVVp2的特异性抗体的Westernblot分析证实了表达蛋白的特异性。结论　获得了SPVVp2基因的表达质粒并在大肠杆菌中得了高效表达 ,为进一步建立SPV感染的检测方法和研究SPV的血清流行病学等奠定了基础。

关键词：猴细小病毒 Vp2 基因克隆基因表达基因鉴定检测基因扩增

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Solution pH jump during antibody and Fc-fusion protein thaw leads to increased aggregation

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Journal of Pharmaceutical Analysis 2018年第5期8卷 302-306页

作者： kevin P.Kent Chad e.Schroeder Chandana Sharma Upstream R&D MilliporeSigma

Freeze-thaw cycles impact the amount of aggregation observed in antibodies and Fc-fusion proteins. Various formulation strategies are used to mitigate the amount of aggregation that occurs upon putting a protein solution through a freeze-thaw cycle. Additionally, low pH solutions cause native antibodies to unfold, which are prone to aggregate upon pH neutralization, There is great interest in the mechanism that causes therapeutic proteins to aggregate since aggregate species can cause unwanted immunogenicity in patients, Herein, an increase in aggregation is reported when the pH is adjusted from pH 3 up to a pH ranging from pH 4 to pH 7 during the thaw process of a frozen antibody solution, Raising the pH during the thaw process caused a significant increase in the percent aggregation observed. Two antibodies and one Fc-fusion protein were evaluated during the pH jump thaw process and similar effects were observed. The results provide a new tool to study the kinetics of therapeutic protein ag- gregation upon pH increase,

关键词： Monoclonal antibodies Freeze-thaw Protein aggregation Protein stability

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Softening of phonon spectra in metallic glasses

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npj Computational Materials 2016年第1期2卷 31-35页

作者： Reza Mahjoub Nicholas e Hamilton kevin J Laws Michael Ferry School of Materials Science and engineering The University of New South WalesSydneyNSWAustralia

The vibrational spectra of a series of MgZnCa amorphous alloys were computed using density functional theory and implementing the small displacement *** atomic structures of the alloys were obtained by ab initio molecular dynamics *** vibrational thermodynamic properties were calculated as a function of temperature and,in particular,the specific heat at low temperature was approximated by temperature cubed based on the Debye *** computed the contribution of Mg vibrations to the specific heat and investigated the softening of Mg phonon spectra,where the maximum allowed vibrational frequency is lowered and highly collective diffusion processes are *** statistical correlation between the reported critical casting thickness of the alloys and softening of Mg phonons was *** calculations were performed for two distinctively different amorphous ZrTiCuAl alloys with large and small reported critical casting thickness,*** findings were consistent with those of the MgZnCa alloys.

关键词： alloys softening casting

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Sex-specific relationships between urbanization,parasitism,and plumage coloration in house finches

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Current Zoology 2021年第3期67卷 237-244页

作者： Brooke e.Sykes Pierce Hutton kevin J.McGraw School of Life Sciences Arizona State UniversityTempeAZ85287USA

Historically,studies of condition-dependent signals in animals have been male-centric,but recent work suggests that female ornaments can also communicate individual quality(e.g.,disease state,fecundity).There also has been a surge of interest in how urbanization alters signaling traits,but we know little about if and how cities affect signal expression in female *** measured carotenoid-based plumage coloration and coccidian(Isospora spp.)parasite burden in desert and city populations of house finches Haemorhous mexicanus to examine links between urbanization,health state,and feather pigmentation in males and *** earlier work,we showed that male house finches are less colorful and more parasitized in the city,and we again detected such patterns in this study for males;however,urban females were less colorful,but not more parasitized,than rural ***,contrary to rural populations,we found that urban birds(regardless of sex)with larger patches of carotenoid coloration were also more heavily infected with *** results show that urban environments can disrupt condition-dependent color expression and highlight the need for more studies on how cities affect disease and signaling traits in both male and female animals.

关键词： carotenoid pigmentation female ornaments Haemorhous mexicanus parasites urban ecology

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