Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst: Application of the Enzyme-Labeled Antigen Method

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.     

Multi-analyte profiling of inflammatory mediators in COPD sputum – The effects of processing

Prior to using a new multi-analyte platform for the detection of markers in sputum it is advisable to assess whether sputum processing, especially mucus homogenization by dithiothreitol (DTT), affects the analysis. In this study we tested a novel Human Inflammation Multi Analyte Profiling® Kit (v1.0 Luminex platform; xMAP®).Induced sputum samples of 20 patients with stable COPD (mean FEV1, 59.2% pred.) were processed in parallel using standard processing (with DTT) and a more time consuming sputum dispersion method with phosphate buffered saline (PBS) only. A panel of 47 markers was analyzed in these sputum supernatants by the xMAP®.Twenty-five of 47 analytes have been detected in COPD sputum. Interestingly, 7 markers have been detected in sputum processed with DTT only, or significantly higher levels were observed following DTT treatment (VDBP, α-2-Macroglobulin, haptoglobin, α-1-antitrypsin, VCAM-1, and fibrinogen). However, standard DTT-processing resulted in lower detectable concentrations of ferritin, TIMP-1, MCP-1, MIP-1β, ICAM-1, and complement C3. The correlation between processing methods for the different markers indicates that DTT processing does not introduce a bias by affecting individual sputum samples differently.In conclusion, our data demonstrates that the Luminex-based xMAP® panel can be used for multi-analyte profiling of COPD sputum using the routinely applied method of sputum processing with DTT. However, researchers need to be aware that the absolute concentration of selected inflammatory markers can be affected by DTT.     

Comparative LCA studies of simulated HMF biorefineries from maize and miscanthus as an example of first- and second-generation biomass as a tool for process development

5-Hydroxymethylfurfural (HMF) is a versatile platform chemical for a fossil free, bio-based chemical industry. HMF can be produced by using fructose as a feedstock. Using edible, first-generation biomass to produce chemicals has been questioned in terms of potential competition with food supply. Second-generation biomass like miscanthus could be an alternative. However, there is a lack of information if second-generation lignocellulosic biomass is a more sustainable feedstock to produce HMF. Therefore, a life cycle assessment was performed in this study to determine the environmental impacts of HMF production from miscanthus and to compare it with HMF from high-fructose corn syrup (HFCS). HFCS from either Hungary or Baden-Württemberg (Germany) was considered. Compared to the HFCS biorefineries the miscanthus concept is producing less emissions in all impact categories studied, except land occupation. Overall, the production and usage of second-generation biomass could be especially beneficial in areas where the use of N fertilizers is restricted. Besides, conclusions for the further development of the on-farm biorefinery concept were elaborated. For this purpose, process simulations from a previous study were used. Results of the previous study in terms of TEA and the current LCA study in terms of environmental sustainability indicate that the lignin depolymerization unit in the miscanthus biorefinery has to be improved. The scenario without lignin depolymerization performs better in all impact categories. The authors recommend to not further convert the lignin to products like phenol and other aromatic compounds. The results of the contribution analyses show that the major impact in the HMF production is caused by the auxiliary materials in the separation units and the required heat. Further technical development should focus on efficient heat as well as solvent use and solvent recovery. At this point further optimizations will lead to reduced emissions and costs at the same time.     

Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon-densation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.     

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

Stratigraphy and sedimentology of Muschelkalk carbonates of the Southern Iberian Continental Palaeomargin (Siles and Cehegín Formations,Southern Spain)

The Triassic sediments of the External Zones of the Betic Cordillera were deposited on the Southern Iberian Continental Palaeomargin. Two coeval Ladinian formations, namely the Siles Formation and the Cehegín Formation, are described to illustrate the facies and lithostratigraphic variability in the Muschelkalk carbonates. There has been some dispute over the number of carbonate units present in the Siles Formation. Our studies assign a tectonic origin to these recurrent carbonate units. Both formations comprise only one carbonate unit, which is correlated to the Upper Muschelkalk of the Catalan and Germanic basins and some Iberian Range sections. To characterize the sedimentological features of these formations, 14 facies were defined. The most widespread sediment was originally lime mud, although bioclastic deposits are also common. In the facies succession, a main transgressive-regressive sequence could be identified. According to the facies model proposed here, a muddy coastal and shallow-water platform prograded over mid ramp deposits. There is no evidence for a seawards reefal or oolitic-bioclastic sandy barrier. The most significant feature of this sedimentary interpretation is that these carbonate facies show clear characteristics of an epicontinental platform.     

A platform to standardize,store, and visualize proteomics experimental data

With the development of functional genomics research, large-scale proteomics studies are now widespread, presenting significant challenges for data storage, exchange, and analysis. Here we present the Integrated Proteomics Exploring Database （IPED） as a platform for managing proteomics experimental data （both process and result data）. IPED is based on the schema of the Proteome Experimental Data Repository （PEDRo）, and complies with the General Proteomics Standard （GPS） drafted by the Proteomics Standards Committee of the Human Proteome Organization. In our work, we developed three components for the IPED platform： the IPED client editor, IPED server software, and IPED web interface. The client editor collects experimental data and generates an extensible markup language （XML） data file compliant with PEDRo and GPS; the server software parses the XML data file and loads information into a core database; and the web interface displays experimental results, to provide a convenient graphic representation of data. Given software convenience and data abundance, IPED is a powerful platform for data exchange and presents an important resource for the proteomics community. In its current release, IPED is available at http：//www. biosino.org/iped2.     

基于无人机和决策树算法的榆树疏林草原植被类型划分和覆盖度生长季动态估计

植被覆盖度是评估生态环境质量与植被生长的重要指标,也是全球众多陆面过程模型和生态系统模型中表达植被动态的重要参数。卫星遥感和地面测量是估算植被覆盖度的常见方法。然而,如何精确估计榆树疏林草原上木本、草本不同类型植被的覆盖度仍然具有挑战性。无人机飞行系统有效的补充了区域尺度低空间分辨率的卫星遥感影像与样地尺度实地调查之间的缺口,为精确的监测、评估疏林草原的植被动态提供了新途径。利用无人机监测平台和决策树算法构建了一套快速、准确、自动获取景观尺度植被类型和估算植被覆盖度的自动化工具,以浑善达克沙地榆树疏林草原为对象,应用无人机监测平台对榆树疏林草原长期定位监测大样地2017年生长季植被状况进行7次监测。结果表明:1)无人机植被监测平台数据飞行高度100 m,获取的样地数字正射影像空间分辨率为2.67 cm/像元,远高于高分卫星影像,利用决策树算法基于数字正射影像可以实现自动划分榆树疏林草原木本和草本植被类型和估算植被覆盖度; 2)生长季内榆树疏林草原木本植被覆盖度为(19±2)%,草本植被覆盖度为(50±8)%,植被总覆盖度为(69±9)%,相对于木本植被,草本植被生长季内盖度变幅较大; 3)在整个生长季中,木本植被和草本植被对植被总覆盖度的平均贡献率分别为27%和73%,草本植被对植被总盖度的贡献远大于木本植被,榆树疏林草原植被的盖度主要受草本植被的影响。本研究证明无人机监测平台是一种高效、准确的植被监测工具,结合机器学习算法,实现了景观尺度植被类型的自动划分和不同类型植被覆盖度快速获取;在浑善达克沙地榆树疏林草原地区首次获取了木本植被和草本植被覆盖度的生长季动态。该平台未来可应用于各种类型生态系统植被类型划分、监测和评估。     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.