Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.     

The relevance of tissue thiol histochemistry to diagnostic hematopathology

Expression analyses suggest that alterations of the antioxidant state of some diffuse large B-cell lymphomas can assist prognosis; reversibly oxidized thiols may serve as a surrogate marker for identifying such cases. Little is known about the distribution of free thiols and reversibly oxidized thiols in human tissues. We developed a staining technique that enables visualization of tissue thiols in situ using bright field microscopy and validated it using gastrointestinal tissue specimens. We used our thiol staining technique to assess benign tonsillectomy and diffuse large B-cell lymphoma specimens. The gastrointestinal series revealed the presence of free thiols within epithelial cells and cells of the lamina propria. Staining for reversibly oxidized thiols was robust in gastric foveolar cells, intestinal goblet cells and the mucus they produce. Tonsillectomy specimens exhibited diffuse presence of free thiols. Staining for reversibly oxidized thiols was confined to germinal center macrophages and sinus histiocytes. Among the diffuse large B-cell lymphoma specimens, we observed strong staining for free thiols within malignant cells. By contrast to benign B-cells, the malignant cells demonstrated pronounced and diffuse staining for reversibly oxidized thiols. We demonstrated intrinsic differences between benign and malignant cells.     

Effects of spaceflight, simulated spaceflight and countermeasures on single muscle fiber physiology

Since the first human flew in space in 1961, there has been extensive scientific interest in the responses of the human body and how it adapts to this unique environment. From the available data, it appears that all major systems in the human body undergo an adaptive change while in a microgravity environment. In particular the human muscle system appears to undergo loss of muscle mass and strength which greatly influences the maximal work capacity of the muscle. Recently, our research group has been involved in a series of whole muscle and cellular studies during periods of short duration space flight, bed rest, and unilateral lower limb suspension (ULLS) in an attempt to elucidate the changes that are occurring in the whole muscle and single muscle fiber contractile properties with unloading. In addition, various countermeasure activities for skeletal muscle have been part of the space flights and ground-based studies. The intention of this paper will be to briefly review our findings in whole muscle, cellular, and countermeasure effectiveness with human muscle.     

C5a initiates the inflammatory cascade in immune complex peritonitis

Immune complex (IC)-induced inflammation is integral to the pathogenesis of several autoimmune diseases. ICs activate the complement system and interact with IgG FcgammaR. In this study, we demonstrate that activation of the complement system, specifically generation of C5a, initiates the neutrophilic inflammation in IC peritonitis. We show that ablation of C5a receptor signaling abrogates neutrophil recruitment in wild-type mice and prevents the enhancement of neutrophil migration seen in FcgammaRIIB(-/-) mice, suggesting that C5aR signaling is the crucial initial event upstream of FcgammaR signaling. We also provide evidence that C5a initiates the inflammatory cascade both directly, through C5aR-mediated effector functions on infiltrating and resident peritoneal cells, and indirectly, through shifting the balance between activating and inhibitory FcgammaRs on resident cells toward an inflammatory phenotype. We conclude that complement activation and C5a generation are prerequisites for IC-induced inflammation through activating FcgammaR, which amplifies complement-induced inflammation in autoimmunity.     

The human H2A and H2B histone gene complement

Sequences and expression patterns of newly isolated human histone H2A and H2B genes and the respective proteins were compared with previously isolated human H2A and H2B genes and proteins. Altogether, 15 human H2A genes and 17 human H2B genes have been identified. 14 of these are organized as H2A/H2B gene pairs, while one H2A gene and three H2B genes are solitary genes. Two H2A genes and two H2B genes turned outto be pseudogenes. The 13 H2A genes code for at least 6 different amino acid sequences, and the 15 H2B genes encode 11 different H2B isoforms. Each H2A/H2B gene pair is controlled by a divergent promoter spanning 300 to 330 nucleotides between the coding regions of the two genes. The highly conserved divergent H2A/H2B promoters can be classified in two groups based on the patterns of consensus sequence elements. Group I promoters contain a TATA box for each gene, two Oct-1 factor binding sites, and three CCAAT boxes. Group II promoters contain the same elements as group I promoters and an additional CCAAT box, a binding motif for E2F and adjacent a highly conserved octanucleotide (CACAGCTT) that has not been described so far. Five of the 6 gene pairs and 4 solitary genes with group I promoters are localized in the large histone gene cluster at 6p21.3-6p22, and one gene pair is located at 1q21. All group II promoter associated genes are contained within the histone gene subcluster at D6S105, which is located at a distance of about 2 Mb from the major subcluster at 6p21.3-6p22 containing histone genes with group I promoters. Almost all group II H2A genes encode identical amino acid sequences, whereas group I H2A gene products vary at several positions. Using human cell lines, we have analyzed the expression patterns of functional human H2A/H2B gene pairs organized within the two histone gene clusters on the short arm of chromosome 6. The genes show varying expression patterns in different tumor cell lines.     

Mycophagy by small mammals in the coniferous forests of North America: nutritional value of sporocarps of Rhizopogon vinicolor, a common hypogeous fungus

We evaluated the nutritional value of sporocarps of Rhizopogon vinicolor, a common hypogeous fungus in the coniferous forests of North America, for two small mammal species: the Californian red-backed vole (Clethrionomys californicus) and the northern flying squirrel (Glaucomys sabrinus). Although the nitrogen concentration of sporocarps was high, much of it was in non-protein form or associated with cell walls, suggesting that it may be of low nutritional value or protected from mammalian digestive enzymes. Sporocarps also had high concentrations of cell wall constituents, indicating low availability of digestible energy. When fed a diet of this fungus alone in a controlled feeding experiment both mammal species lost a small amount of body mass. Digestibilities of dry matter, nitrogen, cell wall constituents and energy from sporocarps by both species were lower than the digestibilities of other food types by other similarly sized small mammals. Red-backed voles digested the various components of sporocarps at least as well as the flying squirrels, even though they were almost six-fold smaller in body mass. This observation supports the notion that red-backed voles, like other microtine rodents, have morphological and physiological adaptations of the digestive system that are postulated to permit greater digestion of fibrous diets than predicted on the basis of body size. Despite this, our results re-affirm previous conclusions that hypogeous fungi are only of moderate nutritional value for most small, hindgut-fermenting mammals. Future studies should focus on the importance of mixed-species of fungi in the diet of small mammalian mycophagists. Accepted: 4 December 1998     

微机在牙形刺研究中的应用

该文对微机在牙形刺研究中的应用方法上进行了探索,在大量原始资料和数据的基础上,采用了模糊聚类分析和CAI计算机程序系统的研究,在地层划分,化石组合,沉积环境分析及生油气评价等方面都取得了一定的成果。     

Characterization of 'safe sites' for pioneers in primary succession on recently deglaciated terrain

1 We characterized safe sites for individuals of five early colonizers ( Abies lasiocarpa , Juncus drummondii , J. mertensianus , Saxifraga ferruginea , S. tolmiei ) that had survived at least one growing season on the recently deglaciated forefront of the Lyman Glacier in the North Cascade Mountains, Washington, USA.
2 Sites with concave surfaces, coarse surface substrate and in the vicinity of large rocks were more likely to be colonized by pioneering plant species.
3 We speculate that the distribution of plants is determined by the presence of sites that facilitate seed trapping and protect seeds and seedlings from desiccation.
4 The data identify the abiotic factors that determine initial recruitment and spatial distribution of plants. Such controls precede biotic interactions in this primary successional sere.