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球毛壳菌丝氨酸蛋白酶基因克隆及原核表达研究

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球毛壳菌丝氨酸蛋白酶基因克隆及原核表达研究

作者： olivia Sakhile Juba 哈尔滨工业大学

学位级别：硕士

球毛壳菌(Chaetomium globosum)能侵染多种植物病原真菌,通过其体内产生和释放不同的的水解酶类和抗生素物质来抑制真菌的生长。众多的研究结果显示:球毛壳菌(C. globosum)的各种酶能渗入到合适的宿主中并利用宿主的生物体作为营养来源... 详细信息

球毛壳菌(Chaetomium globosum)能侵染多种植物病原真菌,通过其体内产生和释放不同的的水解酶类和抗生素物质来抑制真菌的生长。众多的研究结果显示:球毛壳菌(C. globosum)的各种酶能渗入到合适的宿主中并利用宿主的生物体作为营养来源进行生长,由于这样的寄生作用,球毛壳菌(C. globosum)已被用于多种植物病原真菌的生物防治。多种证据表明球毛壳菌(C. globosum)能够减少许多重要农业病原菌的发病率和危害范围。球毛壳菌(C. globosum)中一些与生物防治相关的基因已经被克隆出来,如几丁质酶和木聚糖酶。然而,关于球毛壳菌(C. globosum)中在生物防治中起着重要作用的蛋白酶类的研究较少。本研究从球毛壳菌(C. globosum)中获得了与枯草杆菌蛋白酶相似的丝氨酸蛋白酶基因(ser)的DNA和cDNA序列。全长cDNA克隆的5’-和3’-端序列,已经从全菌的EST文库中采取分子生物学和生物信息学的方法获得。在这两个EST的基础上,球毛壳菌的类枯草杆菌丝氨酸蛋白酶已经通过PCR方法扩增出来,cDNA和mRNA的全序列已经呈递给NCBI,并得到序列号分别为FJ589720 and FJ589721。结合生物信息学和分子生物学方法,从球毛壳菌(C. globosum)的cDNA文库中成功克隆出丝氨酸蛋白酶基因(ser)的cDNA序列。cDNA的全长为1608-bp,编码535个氨基酸,预测的分子量为57.2 kDa;等电点为5.93。DNA序列长为1682-bp ,由2个外显子和1个内含子组成。两个外显子的长度分别为372和1236-bp,内含子的长度为74-bp。外显子和内含子之间的剪切位点符合GT/AG规则。SignalP预测分析显示:丝氨酸蛋白酶基因(ser)为分泌蛋白,在N端有一个由16个氨基酸残基组成的信号肽序列,信号肽的切割位点位于第15位和第16位的丙氨酸(Ala)之间。从此基因推导出的氨基酸序列与其它真菌来源的丝氨酸蛋白酶有超过80%的同源性,初步断定它属于S8超家族。在细菌启动子P7的作用下,用用大肠杆菌表达质粒pET-52b(+)构建了重组质粒pET-52b/Ser,转化大肠杆菌E. coli TOP10中,并将抽提出的经过复制的重组质粒转入大肠杆菌E. coli BL21后进行蛋白酶的表达研究。这两次转化过程,均经过菌落PCR和限制性内切酶双酶切检验。SDS-PAGE结果显示,目的基因表达不明显;酶活性检测为在诱导5h后丝氨酸蛋白酶活性最高为0.171U/10ml。丝氨酸蛋白酶的最适pH为4.5,在pH范围3.0–6.0内酶活稳定,在pH为4.6时能保持95%的酶活性。最适温度为40°C,当温度高于60°C,丝氨酸蛋白酶迅速失去活性。尽管丝氨酸蛋白酶基因(ser)在大肠杆菌(E. coli)的表达量不高,但对于研究该酶的最适反应条件还是可能的。在球毛壳菌(C. globosum)中成功的克隆到丝氨酸蛋白酶基因(ser)并在大肠杆菌(E. coli)表达为研究蛋白酶在生物防治中的作用和开发奠定基础。

关键词：球毛壳蛋白酶基因克隆大肠杆菌表达生物防治

Controlling self-assembling and tumor cell-targeting of protein-only nanoparticles through modular protein engineering

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Science China Materials 2020年第1期63卷 147-156页

作者： Eric Volta-Duran olivia Cano-Garrido Naroa Serna Hector Lopez-Laguna Laura Sanchez-Garcia Mireia Pesarrodona Alejandro Sanchez-Chardi Ramon Mangues Antonio Villaverde Esther Vazquez Ugutz Unzueta Institut de Biotecnologia i de Biomedicina Universitat Autònoma de BarcelonaBellaterra08193 BarcelonaSpain Departament de Genetica i de Microbiologia Universitat Autònoma de BarcelonaBellaterra08193 BarcelonaSpain CIBER de Bioingeniería Biomateriales y Nanomedicina(CIBER-BBN)C/Monforte de Lemos 3-528029 MadridSpain Nanoligent SL Edifici EUREKAUniversitat Autonoma de BarcelonaBellaterra08193 BarcelonaSpain Institut dInvestigacions Biomèdiques Sant Pau and Josep Carreras Research Institute Hospital de la Santa Creu i Sant Pau08041 BarcelonaSpain Departament de Biologia Evolutiva Ecologia i Ciencies AmbientalsFacultat de BiologiaUniversitat de BarcelonaAv.Diagonal 64308028 BarcelonaSpain Institute for Research in Biomedicine(IRB Barcelona) The Barcelona Institute of Science and Technology08028 BarcelonaSpain

Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional domains would impact and refine these activities, including the ability to organize as supramolecular entities and to generate multifunctional protein materials. To explore this concept, we have repositioned functional segments in the modular protein T22-GFP-H6 and characterized the resulting alternative fusions. In T22-GFP-H6, the combination of T22 and H6 promotes selfassembling as regular nanoparticles and selective binding and internalization of this material in CXCR4-overexpressing tumor cells, making them appealing as vehicles for selective drug delivery. The results show that the pleiotropic activities are dramatically affected in module-swapped constructs, proving the need of a carboxy terminal positioning of H6 for protein self-assembling, and the accommodation of T22 at the amino terminus as a requisite for CXCR4^+ cell binding and internalization. Furthermore, the failure of self-assembling as regular oligomers reduces cellular penetrability of the fusions while keeping the specificity of the T22-CXCR4 *** these data instruct how multifunctional nanoscale protein carriers can be designed for smart, protein-driven drug delivery, not only for the treatment of CXCR4^+ human neoplasias, but also for the development of anti-HIV drugs and other pathologies in which CXCR4 is a relevant homing marker.

关键词： nanoparticles protein materials recombinant proteins drug delivery self-assembling cancer cell targeting

Assessment of localization to sound in disorder of consciousness:own name versus bell ring

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Assessment of localization to sound in disorder of conscious...

Modeling the optical properties of twisted bilayer photonic crystals

中国神经科学学会第九届全国学术会议暨第五次会员代表大会

作者： olivia Gosseries Caroline Schnakers International vegetative state and consciousness science institution Hangzhou Normal University

＜正＞Objective The assessment of orientation to sound is an item in the behavioral evaluation of consciousness in patients with disorders of consciousness（DOC）.For the commonly used Coma Recovery Scale-Revised（CRS-R）, it is not specified what stimulus to use,so this study uses 2 different stimuli（with one auto-referential） to assess their sensitivity for auditory *** CRS-R scale was used for clinical measurement of consciousness in randomly selected 50 DOC patients diagnosed with vegetative state（VS） or minimally conscious state（MCS）. Patients’ location response to bell and own name were recorded and the sensitivity of stimuli localization was *** Difference was found between localization responses to own name and bell in 50 DOC patients. In MCS patients（n=27）,presentation of the subject’s own name elicited more location response than bell ring （P＜0.05）.In VS patients,there was no significant *** In this study,we compared the sensitivity to elicit localization response between subject’s own name and bell ring in DOC *** found that the subject’s own name was more sensitive to elicit location response than bell ring（P＜0.05） in MCS *** VS patients,no difference was *** result indicates that the subject’s own name may be more suitable to elicit a location response than a simple auditory stimulus such as bell ring and should be used as a standard stimulation when assessing auditory functions in DOC.

关键词： auditory localization/location vegetative state minimally conscious state own name bell

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Light(Science & Applications) 2021年第9期10卷 1672-1679页

作者： Haoning Tang Fan Du Stephen Carr Clayton DeVault olivia Mello Eric Mazur School of Engineering and Applied Sciences Harvard UniversityCambridgeMA02138USA Brown Theoretical Physics Center and Department of Physics Brown UniversityProvidenceRI02912USA

We demonstrate a photonic analog of twisted bilayer graphene that has ultra-flat photonic bands and exhibits extreme slow-light *** twisted bilayer photonic device,which has an operating wavelength in the C-band of the telecom window,uses two crystalline silicon photonic crystal slabs separated by a methyl methacrylate tunneling *** numerically determine the magic angle using a finite-element method and the corresponding photonic band structure,which exhibits a flat band over the entire Brillouin *** flat band causes the group velocity to approach zero and introduces light localization,which enhances the electromagnetic field at the expense of *** our original plane-wave continuum model,we find that the photonic system has a larger band *** band structure can easily be engineered by adjusting the device geometry,giving significant freedom in the design of *** work provides a fundamental understanding of the photonic properties of twisted bilayer photonic crystals and opens the door to the nanoscale-based enhancement of nonlinear effects.

关键词： twisted properties tunneling

Re-Testing in Batch Testing Model Based on Quality Control Process for Proportion Estimation

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Open Journal of Statistics 2021年第1期11卷 123-136页

作者： Ronald Waliaula Wanyonyi olivia Wanjeri Mwangi Charles Wambugu Mwangi Department of Mathematics Egerton University Njoro Kenya Data Science Institute University of Delaware Newark DE USA Department of Mathematics and Actuarial Kabarak University Kabarak Kenya

The quality of products manufactured or procured by organizations is an important aspect of their survival in the global market. The quality control processes put in place by organizations can be resource-intensive but substantial savings can be realized by using acceptance sampling in conjunction with batch testing. This paper considers the batch testing model based on the quality control process where batches that test positive are re-tested. The results show that re-testing greatly improves the efficiency over one stage batch testing based on quality control. This is observed using Asymptotic Relative Efficiency (ARE), where for values of p computed ARE > 1 implying that our estimator has a smaller variance than the one-stage batch testing. Also, it was found that the model is more efficient than the classical two-stage batch testing for relatively high values of proportion.

关键词： Quality Control Batch Testing Cut Off Value Proportion Re-Testing

FOLFIRI regimen in metastatic pancreatic adenocarcinoma resistant to gemcitabine and platinum-salts

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World Journal of Gastroenterology 2012年第33期18卷 4533-4541页

作者： Cindy Neuzillet olivia Hentic Benot Rousseau Vinciane Rebours Léla Bengrine-Lefèvre Franck Bonnetain Philippe Lévy Eric Raymond Philippe Ruszniewski Christophe Louvet Pascal Hammel Service de Gastroentérologie-Pancréatologie Hpital Beaujon 100 Boul-evard du Général Leclerc 92110 Clichy La Garenne AP-HP France Service d'Oncologie Médicale Hpital Saint Antoine 184 Rue du Faubourg Saint-Antoine 75012 Paris AP-HP France Unité de Biostatistique et d'Epidémiologie EA 4184 Centre Georges Franois Leclerc 1 Rue du Pr Marion 21079 Dijon France Service d'Oncologie Médicale Hpital Beau-jon 100 Boulevard du Général Leclerc 92110 Clichy La Garenne AP-HP France Département d'Oncologie Médicale Institut Mutualiste Montsouris 42 Boulevard Jourdan 75014 Paris France

AIM: To evaluate the efficacy and safety of the FOLFIRI regimen in patients with metastatic pancreatic adenocarcinoma (PAC) after the failure of gemcitabine and platinum salts. METHODS: All consecutive patients with histologically confirmed, metastatic PAC and World Health Organiza-tion performance status (PS) ≤ 2 received FOLFIRI-1 [irinotecan 180 mg/m2 on day 1 and leucovorin 400 mg/m2 followed by 5-fluorouracil (5-FU) 400 mg/m2 bolus, then 5-FU 2400 mg/m2 as a 46-h infusion, biweekly] or FOLFIRI-3 (irinotecan 100 mg/m2 on day 1 and leucovorin 400 mg/m2, then 5-FU 2400 mg/m2 as a 46-h infusion and irinotecan 100 mg/m2 repeated on day 3, biweekly) after failure of gemcitabine and platinum-based chemotherapies as a systematic policy in two institutions between January 2005 and May 2010. Tumor response, time to progression (TTP), overall survival rate (OS) and grade 3-4 toxicities were retrospectively studied. Subgroup analyses were performed to search for prognostic factors. RESULTS: Sixty-three patients (52.4% male, median age 59 years) were analyzed. Among them, 42.9% were PS 0, 38.1% were PS 1 and 19.0% were PS 2. Fifty one patients (81.0%) had liver metastases. Before the FOLFIRI regimen, patients had received 1 line (n = 19), 2 lines (n = 39) or 3 lines (n = 5) of chemotherapy. Median TTP obtained with the line before FOLFIRI was 3.9 mo (95% CI: 3.4-5.3 mo). A total of 480 cycles was completed (median: 6 cycles, range: 1-51 cycles). The main reason for discontinuing FOLFIRI was tumor progression (90.3%). Tumor control was achieved in 25 patients (39.7%) (partial response: n = 5, stable disease: n = 20) with FOLFIRI. Median TTP was 3.0 mo (95% CI: 2.1-3.9 mo) and median OS was 6.6 mo (95% CI: 5.3-8.1 mo). Dose adaptation was required in 36 patients (57.1%). Fifteen patients (23.8%) had grade 3-4 toxicities, mainly hematological (n = 11) or digestive (n = 4). Febrile neutropenia occurred in 3 patients. There was no toxic death. PS 2 was significantly associated w

关键词： Pancreatic cancer Pancreatic adenocarcinoma Metastases Chemotherapy 5-fluorouracil Irinotecan Camptothecin FOLFIRI regimen

Bacterial and archaeal communities in saline soils from a Los Negritos geothermal area in Mexico

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Pedosphere 2023年第2期33卷 312-320页

作者： Joseph GUEVARA-LUNA Mario HERNÁNDEZ-GUZMÁN Nina MONTOYA-CIRIACO Luc DENDOOVEN Marina olivia FRANCO-HERNÁNDEZ Paulina ESTRADA-DE LOS SANTOS María Soledad VÁSQUEZ-MURRIETA Escuela Nacional de Ciencias Biológicas Instituto Politécnico NacionalCiudad de México 11340(Mexico) Laboratory of Soil Ecology CinvestavMexico City 07360(Mexico) Centro Tlaxcala de Biología de la Conducta Universidad Autónoma de Tlaxcala.Carretera Federal Tlaxcala-Puebla km.1.5Tlaxcala 90070(Mexico) Unidad Profesional Interdisciplinaria de Biotecnología Instituto Politécnico NacionalCiudad de México 07340(Mexico)

In recent years,there has been a growing need to understand how salinity affects microbial communities in agricultural *** and bacterial community diversities and structures were investigated by high-throughput sequencing analysis of their 16S rRNA in two arable soils with low electrical conductivity(EC)(2.3 and 2.6 dS m^(-1))and a saline soil(EC=17.6 dS m^(-1)).The dominant bacterial phyla in the soils were Proteobacteria(relative abundance(RA)=46.2%),followed by Acidobacteria(RA=13.1%)and Actinobacteria(RA=10.0%),whereas Serratia(RA=6.0%)and Bacillus(RA=4.0%)were the dominant bacterial *** Nitrososphaera(53.5%)was the dominant archaeal phylotype in the arable soils,whereas Nitrosopumilus(RA=0.4%)dominated in the saline *** archaeal and bacterial community structures were different between the soils and significantly correlated with soil sand,arsenic,barium,and antimony contents,but not with soil salinity.

关键词： agricultural soil compositional approach microbial community mud volcano soil salinity soluble anion

Assessing the Effect of Global Travel and Contact Restrictions on Mitigating the COVID-19 Pandemic

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Engineering 2021年第7期7卷 914-923页

作者： Shengjie Lai Nick W.Ruktanonchai Alessandra Carioli Corrine W.Ruktanonchai Jessica R.Floyd olivia Prosper Chi Zhang Xiangjun Du Weizhong Yang Andrew J.Tatem WorldPop School of Geography and Environmental ScienceUniversity of SouthamptonSouthampton SO171BJUK Population Health Sciences Virginia TechBlacksburgVA 24061USA Department of Mathematics University of TennesseeKnoxvilleTN 37996USA School of Public Health(Shenzhen) Sun Yat-sen UniversityShenzhen 510275China School of Population Medicine and Public Health Chinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100730China

Travel restrictions and physical distancing have been implemented across the world to mitigate the coronavirus disease 2019(COVID-19)pandemic,but studies are needed to understand their effectiveness across regions and *** on the population mobility metrics derived from mobile phone geolocation data across 135 countries or territories during the first wave of the pandemic in 2020,we built a metapopulation epidemiological model to measure the effect of travel and contact restrictions on containing COVID-19 outbreaks across *** found that if these interventions had not been deployed,the cumulative number of cases could have shown a 97-fold(interquartile range 79–116)increase,as of May 31,***,their effectiveness depended upon the timing,duration,and intensity of the interventions,with variations in case severity seen across populations,regions,and ***,before effective vaccines are widely available and herd immunity is achieved,our results emphasize that a certain degree of physical distancing at the relaxation of the intervention stage will likely be needed to avoid rapid resurgences and subsequent lockdowns.

关键词： COVID-19 Pandemic Population mobility Travel restriction Physical distancing

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野生稻和栽培稻基因组抗性基因的鉴定与比较

野生稻和栽培稻基因组抗性基因的鉴定与比较

作者： Allasi Canales Nataly olivia 福建农林大学

学位级别：硕士

世界50%人口以水稻主食,保护水稻免受真菌、细菌、病毒、昆虫等病害是保障食物安全的重要议题。水稻以两种方式防御病害。本文主要研究了不同水稻品种中抗性蛋白(R蛋白),它们参与对病原的先天免疫。水稻中最常见的R蛋白由NBS-LRR基因编... 详细信息

世界50%人口以水稻主食,保护水稻免受真菌、细菌、病毒、昆虫等病害是保障食物安全的重要议题。水稻以两种方式防御病害。本文主要研究了不同水稻品种中抗性蛋白(R蛋白),它们参与对病原的先天免疫。水稻中最常见的R蛋白由NBS-LRR基因编码。首先,通过同源性分析和HMM方法鉴定2种栽培和7种野生稻品种中的NBS-LRR编码基因。我们在***,***,***,*** indica,***,*** japonica,***,*** 和 *** 中分别鉴定到 461,283,429,551,204,513,445,330和462个NBS-LRR编码基因。值得注意的是,除了 ***,所有的测试水稻品种都有TIR结构域。我们还发现所有9个品种的NBS-LRR编码基因在不同染色体上的共同分布趋势。1 1和12号染色体包含26-40%的R基因;染色体5和3具有较少数目的R基因。此外,第1 1号染色体的R基因最多,是12染色体R基因数量的两倍。其次,对R基因进行进化分析,考虑了从CC域跨到MHD结构域。结果表明,在9个品种中鉴定到了 405个直系同源R基因进化支。2个栽培稻品种总是存在于同一进化支中,提示可以通过R基因直系同源分子发现不同水稻品种之间的演化关系。与其他测试水稻品种相比,***和***在405个进化枝中的缺失最多。R基因的系统发育学分析结果证实了前述结果,显示***和***分别是日本晴和931 1的祖先。***与***密切相关,而***与其他品种很远。第三,以日本晴基因组为参照,检测了水稻基因组中的SNP。我们还确定了错义变体,以发现基因功能丢失或获得。***基因组具有最高数量的非同义突变,而***最少。同时我们发现R基因中CC和NBS结构域之间的SNP最多为5个。在NBS结构域中也检测到最多为5个SNP,而LRR结构域中SNP的最多只有4个。为了进一步表征R基因在水稻中的进化,我们计算了R基因直系同源分子中不同结构域的ka/ks值。结果表明直系同源组的LRR结构域受正向选择(ka/ks=1.22)。此外,其核心区域受较强的正选择(ka/ks=2.11)。该结果表明,R基因中的LRR结构域与水稻驯化密切相关,可能是水稻驯化过程中选择的关键位点。第四,我们从直系同源基因组中发现了具有非同义突变(nSNP)的3个R基因。两个是LOCs07g02590和LOCs07g02624,来自组 01(G01),另一个是组 03(G03)的 LOCs11g45090。LOCs07g02624中的位置144是一个组氨酸,它将突变为缬氨酸,因为在其密码子中检测到一个nSNP。在CC域中也发现了一个螺旋构象,从LOCs97g02624 的 77 到 86 位。3D 结构模型分析表明,LOCs97g02624位置144的突变将诱导失去并改变一个键连接。此外,α-螺旋结构倾向于破坏或甚至在突变蛋白中消失。这表明R基因的突变将导致栽培和野生稻品种对真菌的耐药能力不同。这些结果提供了对野生和栽培稻之间R基因进化的新见解,并且涉及水稻基因组中R基因中的几个先前的突变。

关键词：野生稻栽培稻抗性基因正交群错义变异系统发育学