Explore the vast range of titles available.

Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.

Poor corrosion resistance of magnesium alloys remains a major obstacle to their extensive application. Phosphate conversion coating (PCC) is one of the most direct and effective strategies to enhance the corrosion resistance of magnesium alloys. To overcome the environmental damage of traditional phosphating technology, a PCC free of fluorine, chromium and nitrite was prepared in the present work. The effects of surface pre-activation process and preparation temperature on the surface morphology and corrosion resistance of PCC on ZK60 magnesium alloy were investigated. Surface pre-activation could significantly reduce the ultimate grain size of phosphate. At 90 °C, the prepared PCC showed perfect morphology and corrosion resistance. To better understand the phosphating nucleation and growth process, the surface and cross-sectional morphologies of PCC prepared for different times were observed. The phase composition of the prepared PCC was detected to be Hureaulite (Mn 5 (PO 4 ) 2 (PO 3 OH) 2 ·4H 2 O) and the coating growth mechanism was suggested in the end.

Mammalian cell-based dual display and secretion (dualDS) systems enable both surface display of antibodies for binding analysis and simultaneous secretion for functional testing within the same cells. While powerful, their efficiency depends heavily on how light (LC) and heavy chains are co-expressed. In this study, we compared three vector designs in Chinese Hamster Ovary cells: (1) multiple promoters (MP), (2) internal ribosomal entry sites (IRES), and (3) a minimal furin cleavage sequence linked to the 2A peptide (Fm-2A). Vectors were stably integrated into a genomic landing pad using recombinase-mediated cassette exchange. Our results show MP constructs produced heterogeneous expression due to integration errors and transcriptional variability. IRES constructs yielded more homogenous expression but suffered from low secretion due to translational inefficiency. In contrast, the Fm-2A design achieved most uniform antibody display and highest secretion, though residual 2A fragments indicated incomplete furin cleavage. Sequence engineering of Fm-2A improved cleavage efficiency and enhanced antibody quality. These findings underscore the importance of vector design in optimizing dualDS systems and provide a framework for improving library screening, engineering, and other mammalian cell-based applications for therapeutic development.

Cell metabolism plays a pivotal role in regulating the effector functions of immune cells. Stimulatory cytokines, such as interleukin (IL)-2 or IL-12 and IL-15, activate glycolysis and oxidative phosphorylation in natural killer (NK) cells to support their enhanced effector functions. IL-10, a pleiotropic cytokine, is known to suppress macrophage activation but stimulate NK cells. However, it remains unclear if IL-10 has an effect on the metabolism of human NK cells and if so, what metabolic mechanisms are affected, and how these metabolic changes are regulated and contribute to the effector functions of NK cells. In this study, we demonstrate that IL-10 upregulates both glycolysis and oxidative phosphorylation in human NK cells, and these metabolic changes are crucial for the enhanced effector functions of NK cells. Mechanistically, we unravel that IL-10 activates the mammalian target of rapamycin complex 1 (mTORC1) to regulate metabolic reprogramming in human NK cells.

A new Frisch-grid ionization chamber has been built to explore the appropriate choice of Frisch-grid. Detailed studies of the relationship between grid geometries and detector performance have been performed with an 241Am source. This paper describes and compares the energy resolution of ionization chambers with parallel-wire and mesh grids of different grid parameters. Some specific recommendations for grid selection are provided based on the data currently available. To obtain optimal energy resolution, the operating voltage of the chamber must satisfy the condition of minimum electron collection on the grid with distinct geometries and parameters, respectively. Since there is no established theory applicable to both types of grids, we have devised a careful simulation procedure incorporating the COMSOL and Garfield++ codes to search for the conditions of the minimum electron collection on the grid. The simulation results fit the experimental data well, suggesting that this simulation method successfully predicts the suitable voltage setting when using a mesh grid or parallel wires grid as the shielding electrode.

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

The charge exchange (CE) reaction is an effective probe to study the structure of atomic nuclei in the isospin dimension, which has been studied for decades. To expand the range of nuclei studied by CE reactions to a wider range and research the structure characteristics of unstable nuclei, including the isospin symmetry, spin-isospin excitation, and nuclear symmetry energy, a semi-cylindrical time projection chamber (scTPC) prototype was designed and constructed to probe ( 3 He, t ) CE reactions in inverse kinematics. The 266 nm UV laser was used to achieve electron-drift-velocity calibration. The scTPC has an energy resolution (FWHM) of 5.6% for α particles emitted by 241 Am radioactive source. The position resolution of scTPC is described by the residual method. The spatial resolution on the pad plane is 409 μ m. And the position resolution in the drift direction is 326 μ m, equivalent to an angular resolution of 0.4 ∘ . These performances suggest that the scTPC can measure Δ E and particle tracks precisely. The successful development of the scTPC prototype provides better conditions for the next step of experimental data analysis and processing.

Background Allergic asthma is a common respiratory disease that significantly impacts human health. Through in silico analysis of human lung RNASeq, we found that asthmatic lungs display lower levels of Isthmin-1 (ISM1) expression than healthy lungs. ISM1 is an endogenous anti-inflammatory protein that is highly expressed in mouse lungs and bronchial epithelial cells, playing a crucial role in maintaining lung homeostasis. However, how ISM1 influences asthma remains unclear. This study aims to investigate the potential involvement of ISM1 in allergic airway inflammation and uncover the underlying mechanisms. Methods We investigated the pivotal role of ISM1 in airway inflammation using an ISM1 knockout mouse line ( ISM1 −/− ) and challenged them with house dust mite (HDM) extract to induce allergic-like airway/lung inflammation. To examine the impact of ISM1 deficiency, we analyzed the infiltration of immune cells into the lungs and cytokine levels in bronchoalveolar lavage fluid (BALF) using flow cytometry and multiplex ELISA, respectively. Furthermore, we examined the therapeutic potential of ISM1 by administering recombinant ISM1 (rISM1) via the intratracheal route to rescue the effects of ISM1 reduction in HDM-challenged mice. RNA-Seq, western blot, and fluorescence microscopy techniques were subsequently used to elucidate the underlying mechanisms. Results ISM1 −/− mice showed a pronounced worsening of allergic airway inflammation and hyperresponsiveness upon HDM challenge. The heightened inflammation in ISM1 −/− mice correlated with enhanced lung cell necroptosis, as indicated by higher pMLKL expression. Intratracheal delivery of rISM1 significantly reduced the number of eosinophils in BALF and goblet cell hyperplasia. Mechanistically, ISM1 stimulates adiponectin secretion by type 2 alveolar epithelial cells partially through the GRP78 receptor and enhances adiponectin-facilitated apoptotic cell clearance via alveolar macrophage efferocytosis. Reduced adiponectin expression under ISM1 deficiency also contributed to intensified necroptosis, prolonged inflammation, and heightened severity of airway hyperresponsiveness. Conclusions This study revealed for the first time that ISM1 functions to restrain airway hyperresponsiveness to HDM-triggered allergic-like airway/lung inflammation in mice, consistent with its persistent downregulation in human asthma. Direct administration of rISM1 into the airway alleviates airway inflammation and promotes immune cell clearance, likely by stimulating airway adiponectin production. These findings suggest that ISM1 has therapeutic potential for allergic asthma. Graphical abstract

Over recent decades, bispecific antibodies (bsAbs) have garnered significant attention for their superior therapeutic efficacy compared to progenitor monoclonal antibodies, enabling innovative treatment strategies. Despite their potential, the development of bsAbs presents significant challenges, with structural stability playing a pivotal role in manufacturability, therapeutic performance, and safety. Among the factors influencing stability, the design and incorporation of molecular linkers are particularly critical. In this study, we investigated the structural stability and fragmentation profiles of a symmetric bispecific antibody (Sym-bsAb), targeting HER2 and CD3, under forced degradation conditions. The Sym-bsAb exhibited pronounced fragmentation under prolonged thermal stress, particularly when combined with high pH and salt conditions. Intact mass analysis identified key degradation events, including sequential clipping along G4S and G4 linkers, fragmentations at interchain cystinyl residues and cleavage at the C-terminal of asparagine residues. The identification of G4S and G4 linkers as vulnerable regions prone to clipping in Sym-bsAb provided valuable insights into the stability and manufacturability of bsAbs incorporating linker sequences, underscoring critical considerations for their development.

Monoclonal antibodies (mAbs) eliminate cancer cells via various effector mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are influenced by the N-glycan structures on the Fc region of mAbs. Manipulating these glycan structures on mAbs allows for optimization of therapeutic benefits associated with effector functions. Traditional approaches such as gene deletion or overexpression often lead to only all-or-nothing changes in gene expression and fail to modulate the expression of multiple genes at defined ratios and levels. In this work, we have developed a CHO cell engineering platform enabling modulation of multiple gene expression to tailor the N-glycan profiles of mAbs for enhanced effector functions. Our platform involves a CHO targeted integration platform with two independent landing pads, allowing expression of multiple genes at two pre-determined genomic sites. By combining with internal ribosome entry site (IRES)-based polycistronic vectors, we simultaneously modulated the expression of α-mannosidase II (MANII) and chimeric β-1,4- N -acetylglucosaminyl-transferase III (cGNTIII) genes in CHO cells. This strategy enabled the production of mAbs carrying N-glycans with various levels of bisecting and non-fucosylated structures. Importantly, these engineered mAbs exhibited different degrees of effector cell activation and CDC, facilitating the identification of mAbs with optimal effector functions. This platform was demonstrated as a powerful tool for producing antibody therapeutics with tailored effector functions via precise engineering of N-glycan profiles. It holds promise for advancing the field of metabolic engineering in mammalian cells.