Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BackgroundLawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate.ResultsBatch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers.ConclusionsConsidering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.How to cite: Salazar S, Gutiérrez N, Sánchez O, et al. Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis. Electron J Biotechnol 2021.https://doi.org/10.1016/j.ejbt.2021.01.002     

Discrimination between the human prostate normal cell and cancer cell by using a novel electrical impedance spectroscopy controlling the cross-sectional area of a microfluidic channel

The prostate biopsy method shows a high false negative result because the suspicious tissue considered as cancer is not confirmed during tissue sampling. Thus, repeated biopsy procedures and diagnostic errors in relation to prostate cancer frequently occur. The purpose of this research is to enhance the prostate cancer detection rate by using microfluidic electrical impedance spectroscopy (μEIS), which allows real-time measurement of the electrical impedance of a single human prostate normal cell and cancer cell. The μEIS was equipped with a movable flexible membrane, which is operated by pneumatic pressure to capture the single cell on the surface of sensing electrodes. The forced tight contact between the cell and electrodes makes it possible to measure the electrical characteristics of the cell with a high sensitivity. The μEIS discriminates well between normal human prostate cells (RWPE-1) and cancer cells (PC-3) at 8.7 kHz based on the electrical signal responses of the cells. The average difference rates of admittance magnitude and susceptance are 54.55% and 54.59%, respectively. The developed μEIS also shows high repeatability, which was verified by a deionized water test conducted before and after each cell assay; the maximum variance of both the impedance and admittance at 8.7 kHz was as small as 9.48%.     

Single cell rheometry with a microfluidic constriction: Quantitative control of friction and fluid leaks between cell and channel walls

We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data.     

Conversion of renewable substrates for biosurfactant production by Rhizopus arrhizus UCP 1607 and enhancing the removal of diesel oil from marine soil

BackgroundThe use of agro-industrial wastes to produce high value-added biomolecules such as biosurfactants is a promising approach for lowering the total costs of production. This study aimed to produce biosurfactants using Rhizopus arrhizus UCP 1607, with crude glycerol (CG) and corn steep liquor (CSL) as substrates. In addition, the biomolecule was characterized, and its efficiency in removing petroderivatives from marine soil was investigated.ResultsA 2² factorial design was applied, and the best condition for producing the biosurfactant was determined in assay 4 (3% CG and 5% CSL). The biosurfactant reduced the surface tension of water from 72 to 28.8 mN/m and produced a yield of 1.74 g/L. The preliminary biochemical characterization showed that the biosurfactant consisted of proteins (38.0%), carbohydrates (35.4%), and lipids (5.5%). The compounds presented an anionic character, nontoxicity, and great stability for all conditions tested. The biomolecule displayed great ability in dispersing hydrophobic substrates in water, thereby resulting in 53.4 cm² ODA. The best efficiency of the biosurfactant in removing the pollutant diesel oil from marine soil was 79.4%.ConclusionsThis study demonstrated the ability of R. arrhizus UCP1607 to produce a low-cost biosurfactant characterized as a glycoprotein and its potential use in the bioremediation of the hydrophobic diesel oil pollutant in marine soil.How to cite: Pele MA, Ribeaux DR, Vieira ER, et al. Conversion of renewable substrates for biosurfactant production by Rhizopus arrhizus UCP 1607 and enhancing the removal of diesel oil from marine soil. Electron J Biotechnol 2019;38. https://doi.org/10.1016/j.ejbt.2018.12.003.     

Enhanced production of glutaminase-free l-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines

BackgroundThe increasing rate of breast cancer globally requires extraordinary efforts to discover new effective sources of chemotherapy with fewer side effects. Glutaminase-free l-asparaginase is a vital chemotherapeutic agent for various tumor malignancies. Microorganisms from extreme sources, such as marine bacteria, might have high l-asparaginase productivity and efficiency with exceptional antitumor action toward breast cancer cell lines.Resultsl-Asparaginase-producing bacteria, Bacillus velezensis isolated from marine sediments, were identified by 16S rRNA sequencing. l-Asparaginase production by immobilized cells was 61.04% higher than that by free cells fermentation. The significant productivity of enzyme occurred at 72 h, pH 6.5, 37°C, 100 rpm. Optimum carbon and nitrogen sources for enzyme production were glucose and NH₄Cl, respectively. l-Asparaginase was free from glutaminase activity, which was crucial medically in terms of their severe side effects. The molecular weight of the purified enzyme is 39.7 KDa by SDS-PAGE analysis and was ideally active at pH 7.5 and 37°C. Notwithstanding, the highest stability of the enzyme was found at pH 8.5 and 70°C for 1 h. The enzyme kinetic parameters displayed V_max at 41.49 μmol/mL/min and a K_m of 3.6 × 10⁻⁵ M, which serve as a proof of the affinity to its substrate. The anticancer activity of the enzyme against breast adenocarcinoma cell lines demonstrated significant activity toward MDA-MB-231 cells when compared with MCF-7 cells with IC₅₀ values of 12.6 ± 1.2 μg/mL and 17.3 ± 2.8 μg/mL, respectively.ConclusionThis study provides the first potential of glutaminase-free l-asparaginase production from the marine bacterium Bacillus velezensis as a prospect anticancer pharmaceutical agent for two different breast cancer cell lines.How to cite: Mostafa Y, Alrumman S, Alamri S, et al. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.001.     

Establishment of a pheasant (Phasianus colchicus) spermatogonial stem cell line for the production of interspecies germ line chimeras

BackgroundSpermatogonial stem cells (SSCs) are important for the production of interspecies germ line chimeras. The interspecies germ cell transfer technique has been suggested as a way to conserve endangered birds. Our objective was to develop a technique for restoring endangered birds by developing interspecies germ line chimeras between pheasant (Phasianus colchicus) and chicken (Gallus gallus) with SSCs.ResultsSSCs were isolated from the surgically removed testis of a pheasant. Growth conditions for pheasant SSCs were established by co-culturing STO (SIM mouse embryo-derived thioguanine and ouabain resistant) cells and pheasant SSCs. The colony-forming cells divided and proliferated stably to yield an established SSC line. Pheasant SSCs showed strong reactivity for GDNF family receptor alpha1 (GFRα1) marker. Finally, production of germ line chimeras was attempted by transferring pheasant SSCs into recipient embryos. Although final embryo survival was 5.6% (20/354), the initial survival rate was 88% (312/354). To measure the percent transfer of donor SSC to gonads, the pheasant SSCs were labeled with PKH 26 fluorescent dye. We observed 30% donor cells and 9.48% c-kit/CD117-positive cells in the gonads of recipient chickens. Donor SSCs were thus stably engrafted in the recipient gonads.ConclusionsThis study showed that SSCs can be used as a tool for the conservation of endangered birds and the production of germ line chimeras. Our findings yield insights into how we may use the pheasant spermatogonial stem cell line for efficient production of interspecies germ line chimeras and ultimately, to the restoration of endangered birds.     

正确认识和处理科技期刊编校合一

编辑与校对是整个出版流程中的两个相对独立的工作,二者又是紧密相关、相互渗透的。编校合一具有可弥补编辑加的不足,发现校样中的“是”、“非”问题,有利于提高编辑加工水平,以及节省时间、人力财力,便于版式调整等优势。但也可能存在编辑对发排稿质量的要求降低,或编辑不遵循校对规律,不从校对人员的角度去校去等弊端。正确认识编校合一,妥善处理二者之间的关系,充分发挥编校合一的优势,严格按照校对步骤,采用切实有效的校对办法,使科技期刊的质量不断提高。     

Construction and application of a novel genetically engineered Aspergillus oryzae for expressing proteases

BackgroundWe aimed to test the possibility of improving polypeptide production from soybean meal fermentation by engineered Aspergillus oryzae strains. Four different protease genes were cloned and transformed into wild-type A. oryzae, and the engineered A. oryzae strains were then used for soybean meal fermentation.ResultsThe results showed different degrees of improvement in the protease activity of the four transformants when compared with wild-type A. oryzae. A major improvement in the polypeptide yield was achieved when these strains were used in soybean meal fermentation. The polypeptide conversion rate of one of the four transformants, A. oryzae pep, reached 35.9%, which was approximately twofold higher than that exhibited by wild-type A. oryzae. Amino acid content analysis showed that the essential amino acid content and amino acid composition of the fermentation product significantly improved when engineered A. oryzae strains were used for soybean meal fermentation.ConclusionsThese findings suggest that cloning of microbial protease genes with good physicochemical properties and expressing them in an ideal host such as A. oryzae is a novel strategy to enhance the value of soybean meal.     

创新驱动下企业创新能力提升路径及机制——基于单案例的探索性研究

在调查研究的基础上,通过对一家能源企业10多年的纵向案例研究,依据战略引领创新、创新促进能力发展、能力支撑战略的思想,分析和归纳其创新能力提升的路径和机制。研究表明:该企业创新能力的发展经历了二次创新到集成创新、再到各创新要素共同演进的路径,而创新机制中的积累机制、学习机制、协同机制在不同的阶段对创新能力的提升发挥了重要的作用。     

An assessment model for virtual communities of practice: a study in the oil and gas industry

This paper proposes a model to assess the level of maturity in Virtual Communities of Practice (VCoPs). The model is based on a theoretical construction attained from the analysis of previous frameworks proposed in literature. Using this construction, we have proposed eight main organizational, managerial and technological levers to improve the performance of VCoPs. Additionally, the model presents the analysis of four performance metrics obtained from these VCoP management levers. The model presented can be used as a benchmarking tool for analysing how companies perform in their management of VCoPs. After developing the model, we applied it to a benchmarking study of four global oil and gas companies. Our results include a performance comparison among these companies as well as the main practices and technologies they use to achieve success in the management of VCoP.     

低碳农业与农作物碳足迹核算研究述评

持续提高农业产量、提高土壤生产力、降低环境污染的低碳农业是21世纪发展的主旋律,碳足迹评价方法是支撑低碳农业技术转型、碳交易、碳标签等管理措施的基础。本研究收集了水稻、小麦、玉米3种作物的22个研究结果,发现碳足迹仍有很大的不确定性,目前国内外尚没有建立统一的碳足迹核算方法体系,导致同一作物碳足迹相差达十几倍,例如水稻碳足迹从1895 kgce/hm²（ce：CO₂equal,即二氧化碳等当量）,到11 811kgce/hm²,而小麦和玉米的净排放却出现相反的两个结果,即固碳（净碳排放为负值）和排放（净碳排放为正值）。分析表明,主要问题在于模型边界、温室气体种类、数据获取途径、量化单位等方面不一致,因此需要重构作物生产碳足迹研究方法。本文提出了用生命周期方法量化农作物生产碳足迹的理论模型,提出以农业投入品上游排放、农田生产排放、以及土壤固碳为边界的生命循环为边界,量化全生命链条中的CO₂、CH₄、N₂O等直接排放因子以及NH₃和NO₃^-等间接排放因子,通过大样本数据建立关键排放因子的量化模型,实现区域性碳足迹的定量化分析。     

Measurement of expansin activity and plant cell wall creep by using a commercial texture analyzer

BackgroundExpansins play an important role in cell wall metabolism and fruit softening. Determination of expansin activity is a challenging problem since it depends on measuring cell wall properties by using ad hoc extensometers, a fact that has strongly restricted its study. Then, the objective of the work was to adapt a methodology to measure cell wall creep and expansin activity using a commercial texture meter, equipped with miniature tensile grips and an ad hoc cuvette of easy construction.ResultsIt was possible to measure hypocotyls acid growth and expansin activity in a reliable and reproducible way, using a commercial texture meter, common equipment found in laboratories of food science or postharvest technology. Expansin activity was detected in protein extracts from cucumber hypocotyls, tomato and strawberry fruits, and statistical differences in expansin activity were found in both fruit models at different ripening stages.ConclusionsThe possibility of measuring expansin activity following this adapted protocol with a commercial texture meter could contribute to ease and increase the analysis of expansin in different systems, leading to a better understanding of the properties of these proteins under different experimental conditions.     

Design and analysis of a novel cascade control algorithm for braking-by-wire system based on electromagnetic direct-drive valves

In order to improve the response speed and control precision of the braking system with parameters uncertainty and nonlinear friction, a braking-by-wire system based on the electromagnetic direct-drive valve and a novel cascade control algorithm was proposed in this paper. An electromagnetic linear actuator directly drives the valve spool and rapidly adjusts the pressure of braking wheel cylinders. A dynamic model of electromagnetic direct-drive valve considering improved LuGre dynamic friction is established. A novel cascade control algorithm with an outside loop pressure fuzzy controller and an inside loop electromagnetic direct-drive valve position controller was proposed. An adaptive integral robust inside loop controller is designed by combining friction compensation adaptive control law, linear feedback, and integral robust control. The uncertainty parameters and the friction state are estimated online. The stability of the cascade controller is proved by the Lyapunov method. Then a multi-objective opitimizemization design method of control parameters is proposed, which combines a multi-objective game theory and a technique for order preference by similarity to ideal solution (TOPSIS) based on entropy weight. The results show that the pressurization time of cascade control is less than 0.09 s under the 15 MPa step target signal. The control precision is improved effectively by the cascade controller under the ARTEMIS condition.     

新冠疫情下政府差异化复工复产路径研究

有序推进复工复产是降低新冠疫情影响的重要着力点,也是社会经济稳定发展的重要保障。然而,目前文献对复工复产背后多重因素间的复杂机理尚未开展深入研究。本文基于TOE框架,探究技术、组织和环境因素对地方政府复工复产的联动匹配效应。运用模糊集定性比较分析（fsQCA）方法,以我国29个省级政府为案例进行组态分析,研究结果表明：（1）技术、组织和环境条件“多重并发”,形成驱动复工复产的五种组态,即复工复产的驱动路径具有“殊途同归”的特点;（2）相对其他条件而言,数字化基础设施对政府复工复产起到更为重要的作用;（3）在一定情形下,技术、组织与环境条件之间具有替代关系;（4）数字化管理能力、注意力分配和财政资源支持等条件的缺失是造成政府非高水平复工复产率的主要原因。研究丰富了复工复产的研究视角,有助于政府根据各自的资源禀赋和受疫情影响等具体情况,选择合理的复工复产路径,制定精准的复工复产政策,从而避免政策覆盖面广、“一刀切”等问题,找到政策落实的“共赢点”。     

A microfluidic cell for studying the formation of regenerated silk by synchrotron radiation small- and wide-angle X-ray scattering

A tube-in-square-pipe microfluidic glass cell has been developed for studying the aggregation and fiber formation from regenerated silk solution by in-situ small-angle X-ray scattering using synchrotron radiation. Acidification-induced aggregation has been observed close to the mixing point of the fibroin and buffer solution. The fibrous, amorphous material is collected in a water bath. Micro-wide-angle X-ray scattering of the dried material confirms its β-sheet nature.