Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

Leaf gas exchange and grain yield of common bean exposed to spermidine under water stress

Three prevalent aliphatic polyamines (PAs) include putrescine, spermidine, and spermine; they are low-molecular-mass polycations involved in many physiological processes in plants, especially, under stressful conditions. In this experiment, three bean (Phaseolus vulgaris L.) genotypes were subjected to well-watered conditions and two moderate and severe water-stressed conditions with and without spermidine foliar application. Water stress reduced leaf relative water content (RWC), chlorophyll contents, stomatal conductance (gs), intercellular CO₂ concentration (C_i), transpiration rate, maximal quantum yield of PSII (F_v/F_m), net photosynthetic rate (P_N), and finally grain yield of bean plants. However, spermidine application elevated RWC, gs, C_i, F_v/F_m, and P_N, which caused an increase in the grain yield and harvest index of bean plants under water stress. Overall, exogenous spermidine could be utilized to alleviate water stress through protection of photosynthetic pigments, increase of proline and carotenoid contents, and reduction of malondialdehyde content.

     

Germination and emergence of Astrodaucus orientalis (L.) Drude populations influenced by environmental factors and seed burial depth

Astrodaucus orientalis is a weed species in cropping systems and rangelands in Iran. The effects of temperature, light, NaCl concentration, water potential, seed burial depth and crop residue cover were assessed on seed germination and seedling emergence of two populations of A. orientalis from Ardabil (Meshginshahr population) and East Azarbayjan (Tabriz population) provinces of Iran. The A. orientalis populations indicated different responses to environmental factors and burial depth. In the Tabriz population the greatest germination (88.5%) was observed in 20/12°C day/night temperature but in the Meshginshahr population (83.2%) it was obtained in 24/16°C day/night temperature. Over a broad range of light period (10–24 hr light) germination was 74–83%, but it decreased (less than 37%) under 24 hr dark in both A. orientalis populations. With respect to water potential, the C₅₀ parameters were −0.62 and − 0.49 MPa for Tabriz and Meshginshahr populations, respectively. The D₅₀ parameters (the burial depth that caused 50% decrease in emergence) for Tabriz and Meshginshahr populations were 2.42 and 3.13 cm, respectively. Generally, the results showed that emergence of both populations of A. orientalis was delayed as depth of burial increased up to 4 cm and in cropping systems a shallow tillage that locates the seeds to >4 cm of depth in soil could be used in order to suppress seedling emergence. Our findings also could be useful in integrated management of A. orientalis in winter annual crops and rangelands.     

Accounting for activity respiration results in realistic trophic transfer efficiencies in allometric trophic network (ATN) models

Allometric trophic network (ATN) models offer high flexibility and scalability while minimizing the number of parameters and have been successfully applied to investigate complex food web dynamics and their influence on food web diversity and stability. However, the realism of ATN model energetics has never been assessed in detail, despite their critical influence on dynamic biomass and production patterns. Here, we compare the energetics of the currently established original ATN model, considering only biomass-dependent basal respiration, to an extended ATN model version, considering both basal and assimilation-dependent activity respiration. The latter is crucial in particular for unicellular and invertebrate organisms which dominate the metabolism of pelagic and soil food webs. Based on metabolic scaling laws, we show that the extended ATN version reflects the energy transfer through a chain of four trophic levels of unicellular and invertebrate organisms more realistically than the original ATN version. Depending on the strength of top-down control, the original ATN model yields trophic transfer efficiencies up to 71% at either the third or the fourth trophic level, which considerably exceeds any realistic values. In contrast, the extended ATN version yields realistic trophic transfer efficiencies ≤?30% at all trophic levels, in accordance with both physiological considerations and empirical evidence from pelagic systems. Our results imply that accounting for activity respiration is essential for consistently implementing the metabolic theory of ecology in ATN models and for improving their quantitative predictions, which makes them more powerful tools for investigating the dynamics of complex natural communities.     

Assembly of the RAG1/RAG2 Synaptic Complex

Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.