水稻耐冷性鉴定评价方法

水稻耐冷性是我国东北和西南高海拔地区、日本、韩国和朝鲜等国水稻育种的重要研究目标,至今许多学者对水稻耐冷性进行了广泛的研究,并且目前所采用的耐冷性鉴定评价方法已基本成熟,但国内还没有对其进行技术规范化.本文汇总了国内外至今所采用的水稻耐冷性鉴定方法和耐冷指标及分级标准等,将为水稻耐冷性种质资源鉴定和育种以及我国水稻耐冷性鉴定评价技术规范的制定提供参考依据.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Multiomics characterization of mesenchymal stromal cells cultured in monolayer and as aggregates

Mesenchymal stromal cells (MSCs) have failed to consistently demonstrate their therapeutic efficacy in clinical trials, due in part to variability in culture conditions used for their production. Of various culture conditions used for MSC production, aggregate culture has been shown to improve secretory capacity (a putative mechanism of action in vivo) compared with standard monolayer culture. The purpose of this study was to perform multiomics characterization of MSCs cultured in monolayer and as aggregates to identify aspects of cell physiology that differ between these culture conditions to begin to understand cellular-level changes that might be related to secretory capacity. Targeted secretome characterization was performed on multiple batches of MSC-conditioned media, while nontargeted proteome and metabolome characterization was performed and integrated to identify cellular processes differentially regulated between culture conditions. Secretome characterization revealed a reduction in MSC batch variability when cultured as aggregates. Proteome and metabolome characterization showed upregulation of multiple protein and lipid metabolic pathways, downregulation of several cytoskeletal processes, and differential regulation of extracellular matrix synthesis. Integration of proteome and metabolome characterization revealed individual lipid metabolites and vesicle-trafficking proteins as key features for discriminating between culture conditions. Overall, this study identifies several aspects of MSC physiology that are altered by aggregate culture. Further exploration of these processes and pathways is needed to determine their potential role in regulating cell secretory capacity.     

Antibody characterization using novel ERLIC-MS/MS-based peptide mapping

Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is a technique that is increasingly being used as a trapping/enrichment tool for glycopeptides/phosphorylated peptides or sample fractionation in proteomics research. Here, we describe a novel ERLIC-MS/MS-based peptide mapping method that was successfully used for the characterization of denosumab, in particular the analysis of sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation and glycopeptides. Compared to reversed phase liquid chromatography (RPLC)-MS/MS methods, ERLIC demonstrated unique advantages in the retention of small peptides, resulting in 100% sequence coverage for both the light and heavy chains. It also demonstrated superior performance in the separation and characterization of asparagine deamidated peptides, which is known to be challenging by RPLC-MS/MS. The developed method can be used alone for peptide mapping-based characterization of monoclonal antibodies, or as an orthogonal method to complement the RPLC-MS/MS method. This study extends the applications of ERLIC from that of a trapping/fractioning column to biologic therapeutics characterization. The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping-based characterization.     

Characterization of commercial yeast strains by flow microcalorimetry

A flow microcalorimetric procedure has been developed for the characterization of representative strains of Saccharomyces cerevisiae and Sacch. uvarum (carlsbergensis) used in the food industry. Growth in a chemically defined medium containing single and mixed sugars gave reproducible and characteristic power-time curves. The technique is proposed as a rapid alternative method for the characterization of yeast strains selected for commercial use.     

Potential of biosensor technology for the characterization of interactions by quantitative affinity chromatography

This review places the characterization of interactions by biosensor technology in the broader context of their study by quantitative affinity chromatography. The general reluctance to consider biosensor-based characterization as a form of quantitative affinity chromatography on the grounds of a difference in aims of the two techniques reflects a mistaken belief that BIAcore and IAsys studies characterize the kinetics of the chemical reaction responsible for biospecific adsorption of a soluble reactant to an immobilized form of its affinity partner. It now transpires that the association and dissociation rate constants thereby determined refer to thermodynamic characterization of biospecific adsorption in terms of a single-phase model in which affinity sites are distributed uniformly throughout the liquid-phase volume accessible to the partitioning reactant—the model used for characterization of biospecific adsorption by quantitative affinity chromatography. In that light the most important attribute of biosensor technology is its potential for thermodynamic characterization of biospecific adsorption by virtue of its ability to monitor complex formation directly; and hence its potential for the characterization of interactions with affinities that are too strong for study by forms of quantitative affinity chromatography that monitor complex formation on the basis of reactant depletion from the liquid phase. Kinetic as well as thermodynamic analyses of biosensor data are described for attainment of that potential.     

Characterization of bioreactors by in-situ fluorometry

A new microfluorometer with a special fiberoptic for the simultaneous detection of two different wavelengths was developed. Several tracers were tested for reactor characterization at different wavelengths, and the influence of the pH value and culture medium components on fluorescence was studied. In CST bioreactors, the effect of aeration rate and stirrer speed on the fluorescence can be used for reactor characterization. Mixing time experiments were performed in two different bubble columns.     

Electrophoresis and electroblotting of native collagens

Electrophoretic and immunoblotting techniques, while now used routinely for the biochemical characterization of many proteins, have not been used for the identification of native collagens. We present here an acidic electrophoresis system using very low percentage acrylamide gels which maintains collagen solubility and allows migration of native dermal collagens. The method gives uniform gels which can be made mechanically stable for subsequent electroblotting. The resulting nitrocellulose transfer allows immunological detection of collagens using either polyclonal or monoclonal antibodies and can be used to screen antibody specificities. The majority of murine monoclonal antibodies directed against collagen bind only to conformational epitopes on the native triple-helical collagen, and thus cannot be screened by Western blotting. This method therefore enables the electrophoretic screening of these monoclonal antibodies and provides an alternative approach for their characterization.     

The Use of the A.P.I. Lactobacillus System for the Characterization of Pediococci

A commercially produced system for the identification of Lactobacillus , by which the reactions of concentrated cell suspensions towards 50 substrates may be determined, is useful for the characterization of strains of Pediococcus . Suitable conditions for inoculum preparation vary for slow and fast growing strains; the influence of cell concentration on the results is described. The reactions are classified in terms of their value for species characterization and the results are shown to agree well with those in other studies where classical procedures have been used.     

De novo mass spectrometry sequencing and characterization of species-specific peptides from nucleoside diphosphate kinase B for the classification of commercial fish species belonging to the family Merlucciidae

The characterization by de novo peptide sequencing of the different protein nucleoside diphosphate kinase B (NDK B) from all the commercial hakes and grenadiers belonging to the family Merlucciidae is reported. A classical proteomics approach, consisting of two-dimmensional gel electrophoresis, tryptic in-gel digestion of the excised spots, MALDI-TOF MS, LC-MS/MS, and nanoESI-MS/MS analyses, was followed for the purification and characterization of the different isoforms of the NDK B. Fragmentation spectra were used for de novo peptide sequence. A high degree of homology was found between the sequences of all the species studied and the NDK B sequence from Gillichthys mirabilis, which is accessible in the protein databases. Particular attention was paid to the differential characterization of species-specific peptides that could be used for fish authentication purposes. These findings allowed us to propose a rapid and effective classification method, based in the detection of these biomarker peptides using the selective ion reaction monitoring (SIRM) scan mode in mass spectrometry.     

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology.     

Analytical methods for physicochemical characterization of antibody drug conjugates

Antibody-drug conjugates (ADCs), produced through the chemical linkage of a potent small molecule cytotoxin (drug) to a monoclonal antibody, have more complex and heterogeneous structures than the corresponding antibodies. This review describes the analytical methods that have been used in their physicochemical characterization. The selection of the most appropriate methods for a specific ADC is heavily dependent on the properties of the linker, the drug and the choice of attachment sites (lysines, inter-chain cysteines, Fc glycans). Improvements in analytical techniques such as protein mass spectrometry and capillary electrophoresis have significantly increased the quality of information that can be obtained for use in product and process characterization and for routine lot release and stability testing.Key words: antibody drug conjugates, physicochemical characterization, analytical methods, auristatins, maytansines, biophysical characterization, drug distribution, drug loading, drug to antibody ratio     

A high-resolution molecular-based panel of assays for identification and characterization of human embryonic stem cell lines

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies, particularly in view of the diverse methods for derivation and maintenance of these cell lines. However, characterization methods are generally not standardized and many currently used assays are subjective, making dependable and direct comparison of cell lines difficult. In order to address this problem, we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines, derived in two different laboratories using different derivation techniques, as pathogen free, genetically stable, and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control, a crucial element of successful hESC research and clinical translation.     

Dynamic analysis,with feedback characterization,of hybrid human musculoskeletal frameworks by the linegraph-flowgraph procedure

The dynamic response of human musculo-skeletal framework is treated by (i) idealization of the musculo-skeletal framework as hybrid structural networks possessing feedback characteristics and then (ii) employing linegraph-flowgraph procedures for the feedback characterization of the hybrid structural networks. Topological procedures are used in which a “tree” of a network furnishes the skeleton upon which the “linkage” (muscle representing) members provide interaction. Feedback characterization (representing the sensitivity of the skeletal members to the tensile forces) is defined, between the internal “linkage” and “tree” members, by means of the flowgraph. Mikusinski operational calculus is used to facilitate representation of inertia effects by dynamic feedback characterization, with inclusion of initial conditions.     

Recent advances in bioinorganic chemistry of bismuth

Bismuth has been used in medicine for over two centuries for the treatment of various diseases, in particular for gastrointestinal disorders, owing to its antimicrobial activity. Recent structural characterization of bismuth drugs provides an insight into assembly and pharmacokinetic pathway of the drugs. Mining potential protein targets inside the pathogen via metallomic/metalloproteomic approach and further characterization on the interactions of bismuth drugs with these targets laid foundation in understanding the mechanism of action of bismuth drugs. Such studies would be beneficial in rational design of new potential drugs.     

DNA typing of Pakistani cattle breeds Tharparkar and Red Sindhi by microsatellite markers

Microsatellite markers are used for any individual identity and breed characterization in animals that is an efficient and successful way of investigation. They are used for multiple purposes as genetic detectors including, rapid mutation rate, high level of polymorphism, and range of variety of microsatellite markers available. A panel of 19 microsatellite markers was developed for breed characterization in Tharparkar and Red Sindhi breeds of cattle in Pakistan. Forty four blood samples of cattle (each breed) were collected from Department of Livestock Management, Sindh Agriculture University, Tandojam, Tando Qaiser, Tharparkar Cattle Farm Nabi sar Road, Umer Kot, Sindh, and Govt. Red Sindhi Cattle Breeding Farm, Tando Muhammad Khan Pakistan. Breed characterization was 100% successful. Average PIC, He and Power of Exclusion values were found to be 0.91, 0.62 and 13.28, respectively. Pattern of allelic frequencies of most of the microsatellite markers were clearly distinct between two breeds. As a result of present study a reliable, efficient and very informative panel of microsatellite markers was successfully developed which was capable to interpret individual identity, forensic cases and breed characterization in cattle. This facility is ready to be provided to local cattle breeder at commercial level for DNA testing of cattle. This study will also be highly helpful for breed conservation of cattle. In addition this study can also become a basis to open up new disciplines of animal forensics in Pakistan.     

Application of high‐throughput mini‐bioreactor system for systematic scale‐down modeling,process characterization,and control strategy development

High‐throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high‐throughput mini‐bioreactor system viz. the Advanced Microscale Bioreactor (ambr15^TM), to perform process characterization in less than a month and develop an input control strategy. As a pre‐requisite to process characterization, a scale‐down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO₂ profiles as well as several other process and product quality parameters. The scale‐down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15^TM system is capable of replacing the bench scale bioreactor system for routine process development and process characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1623–1632, 2015