Choice of Host Cell Line Is Essential for the Functional Glycosylation of the Fc Region of Human IgG1 Inhibitors of Influenza B Viruses.

Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG plays a major role in this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary-K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B-mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary-K1, but not in human endothelial kidney 293-F cells.

Modified recombinant human IgG1-Fc is superior to natural intravenous immunoglobulin at inhibiting immune-mediated demyelination.

Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Although Fc fragments derived from IVIG have shown efficacy in controlling immune thrombocytopenia in children, the mechanisms of action are unclear and controversial. The aim of this study was to dissect IVIG effector mechanisms using further adapted Fc fragments on demyelination in an ex vivo model of the central nervous system-immune interface. Using organotypic cerebellar slice cultures (OSCs) from transgenic mice, we induced extensive immune-mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective effects of adapted Fc fragments were assessed by live imaging of green fluorescent protein expression, immunohistochemistry and confocal microscopy. Cysteine- and glycan-adapted Fc fragments protected OSC from demyelination in a dose-dependent manner where equimolar concentrations of either IVIG or control Fc were ineffective. The protective effects of the adapted Fc fragments are partly attributed to interference with complement-mediated oligodendroglia damage. Transcriptome analysis ruled out signatures associated with inflammatory or innate immune responses. Taken together, our findings show that recombinant biomimetics can be made that are at least two hundred-fold more effective than IVIG in controlling demyelination by anti-MOG antibodies.

Prognostic Score-Based Stratification Analysis Reveals Universal Benefits of Radiotherapy on Lowering the Risk of Ipsilateral Breast Event for Ductal Carcinoma In Situ Patients with Different Risk Levels.

BACKGROUND: We aimed to analyze the effects of radiotherapy (RT) on the incidence rate of ipsilateral breast event (IBE) in ductal carcinoma in situ (DCIS) patients with lumpectomy after being stratified by prognostic score. METHODS: We identified DCIS patients who received lumpectomy, from the Surveillance, Epidemiology, and End Results (SEER) database from 1988 to 2015. Cumulative incidence functions for competing risk were used to evaluate the effects of RT on IBE risk over time. Three multivariate regression models (weighted, non-weighted, and Fine-Gray) were applied to compare the IBE risk between the RT and non-RT groups after stratifying patients by prognostic score. RESULTS: Overall, 72,623 DCIS patients were identified from the SEER database and 49,206 (66.8%) patients received RT. During the follow-up period (ranging from 7 to 347 months), the cumulative probability of invasive and in situ IBE was significantly lower in the RT group than in the non-RT group (p < 0.001). After being stratified by prognostic score, the weighted IBE incidence rate increased as the risk level increased (p < 0.050). In multivariate regression models, RT lowered the IBE incidence rate by at least 30% in low-, moderate-, and high-risk DCIS (p < 0.010). In particular, the in situ and invasive IBE incidence rate decreased by over 50% in low-risk DCIS with RT (p < 0.001). CONCLUSIONS: RT is associated with a lowered IBE incidence rate in DCIS patients, regardless of the assigned risk levels for patients. The significant reduction in the IBE incidence rate in low-risk DCIS patients also indicates the potential benefits for recommending RT to such a patient population in clinical practice.

Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus.

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein-Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A-mediated agglutination of human erythrocytes.

Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 µM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.

Inkjet-printed SnOx as an effective electron transport layer for planar perovskite solar cells and the effect of Cu doping.

Inkjet printing is a more sustainable and scalable fabrication method than spin coating for producing perovskite solar cells (PSCs). Although spin-coated SnO2 has been intensively studied as an effective electron transport layer (ETL) for PSCs, inkjet-printed SnO2 ETLs have not been widely reported. Here, we fabricated inkjet-printed, solution-processed SnOx ETLs for planar PSCs. A champion efficiency of 17.55% was achieved for the cell using a low-temperature processed SnOx ETL. The low-temperature SnOx exhibited an amorphous structure and outperformed high-temperature crystalline SnO2. The improved performance was attributed to enhanced charge extraction and transport and suppressed charge recombination at ETL/perovskite interfaces, which originated from enhanced electrical and optical properties of SnOx, improved perovskite film quality, and well-matched energy level alignment between the SnOx ETL and the perovskite layer. Furthermore, SnOx was doped with Cu. Cu doping increased surface oxygen defects and upshifted energy levels of SnOx, leading to reduced device performance. A tunable hysteresis was observed for PSCs with Cu-doped SnOx ETLs, decreasing at first and turning into inverted hysteresis afterwards with increasing Cu doping level. This tunable hysteresis was related to the interplay between charge/ion accumulation and recombination at ETL/perovskite interfaces in the case of electron extraction barriers.

YouTube as a source of information for cryptococcal infection: A cross-sectional study.

Objective: Immunocompromised individuals, particularly HIV patients, worldwide are at risk from cryptococcal infection. There are a number of videos of cryptococcal infection and more and more individuals may search these videos, but the quality of videos on YouTube is unclear. This study set out to assess the content and quality of YouTube videos regarding cryptococcal infection. Methods: The keywords "Cryptococcus," "Cryptococcosis" and "Cryptococcal infection" were searched on YouTube. The videos were evaluated and graded by two impartial raters. A 14-point content score was used to categorize videos as bad, good or exceptional. The reliability and quality were evaluated utilizing the DISCERN instrument and a 5-point global quality score. Videos were then divided into groups based on uploading sources and content types. Results: A total of 46 videos were located, and the ratings provided by the two raters were identical. Our scoring algorithm determined that 54.3% (n = 25), 32.6% (n = 15) and 13.0% (n = 6) of the videos were poor, decent and exceptional, respectively. Regarding quality, no difference was identified between the various video categories. The global quality scale, number of views, days posted, content score and DISCERN showed a significant positive relationship. Conclusions: Professional individuals or healthcare organizations should be encouraged to submit high-quality videos for the expanding internet population, as only a small proportion of available videos had exceptional quality.

Effect of folic acid supplementation on diminished ovarian reserve: study protocol of a single-centre, open-label, randomised, placebo-controlled clinical trial.

INTRODUCTION: The prevalence of diminished ovarian reserve (DOR), a common gynaecological disorder, is approximately 10% across the world. Failure in early diagnosis and treatment may result in continuous decreases in ovarian function and the resultant loss in an opportunity of pregnancy, which greatly affects the happiness of the women's family and women's physical and mental health. Nevertheless, there has been no effective treatment for such a disorder until now. Folic acid, a member of the vitamin B family, is involved in one-carbon cycle and methylation regulation. It has been found that folic acid affects the whole period of pregnancy, and folic acid supplementation has shown effective to remarkably reduce the incidence of fetal neural tube defects and decrease plasma homocysteic acid levels, thereby resulting in a decline in the incidence of abortion. In addition, folic acid is reported to mediate ovarian functions. It is therefore hypothesised that folic acid may improve DOR. METHODS AND ANALYSIS: A single-centre, open-label, randomised, placebo-controlled clinical trial is designed. We plan to recruit 140 women with DOR at ages of 30-35 years. All participants will be randomised into the folic acid group and placebo group, and each subject will be given a tablet with the same appearance daily for 6 months. The primary outcome is antral follicle count, and the secondary outcomes are ovarian reserve markers, ovarian low-dose stimulation responses and safety. ETHICS AND DISSEMINATION: This study was approved by the Ethics Review Committee of Nanping First Hospital Affiliated to Fujian Medical University on 10 February 2021 (approval number: NPSY202002042). Written informed consent was obtained from all participants prior to randomisation, following a detailed description of the purpose of the study. The results of this clinical trial will be presented at scientific conferences and submitted to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry, ChiCTR2100047410.

Sialylated and sulfated N-Glycans in MDCK and engineered MDCK cells for influenza virus studies.

The Madin-Darby canine kidney (MDCK) cell line is an in vitro model for influenza A virus (IAV) infection and propagation. MDCK-SIAT1 (SIAT1) and humanized MDCK (hCK) cell lines are engineered MDCK cells that express N-glycans with elevated levels of sialic acid (Sia) in α2,6-linkage (α2,6-Sia) that are recognized by many human IAVs. To characterize the N-glycan structures in these cells and the potential changes compared to the parental MDCK cell line resulting from engineering, we analyzed the N-glycans from these cells at different passages, using both mass spectrometry and specific lectin and antibody binding. We observed significant differences between the three cell lines in overall complex N-glycans and terminal galactose modifications. MDCK cells express core fucosylated, bisected complex-type N-glycans at all passage stages, in addition to expressing α2,6-Sia on short N-glycans and α2,3-Sia on larger N-glycans. By contrast, SIAT1 cells predominantly express α2,6-Sia glycans and greatly reduced level of α2,3-Sia glycans. Additionally, they express bisected, sialylated N-glycans that are scant in MDCK cells. The hCK cells exclusively express α2,6-Sia glycans. Unexpectedly, hCK glycoproteins bound robustly to the plant lectin MAL-1, indicating α2,3-Sia glycans, but such binding was not Sia-dependent and closely mirrored that of an antibody that recognizes glycans with terminal 3-O-sulfate galactose (3-O-SGal). The 3-O-SGal epitope is highly expressed in N-glycans on multiple hCK glycoproteins. These results indicate vastly different N-glycomes between MDCK cells and the engineered clones that could relate to IAV infectivity.

Inkjet-Printed Electron Transport Layers for Perovskite Solar Cells.

Inkjet printing emerged as an alternative deposition method to spin coating in the field of perovskite solar cells (PSCs) with the potential of scalable, low-cost, and no-waste manufacturing. In this study, the materials TiO2, SrTiO3, and SnO2 were inkjet-printed as electron transport layers (ETLs), and the PSC performance based on these ETLs was optimized by adjusting the ink preparation methods and printing processes. For the mesoporous ETLs inkjet-printed from TiO2 and SrTiO3 nanoparticle inks, the selection of solvents for dispersing nanoparticles was found to be important and a cosolvent system is beneficial for the film formation. Meanwhile, to overcome the low current density and severe hysteresis in SrTiO3-based devices, mixed mesoporous SrTiO3/TiO2 ETLs were also investigated. In addition, inkjet-printed SnO2 thin films were fabricated by using a cosolvent system and the effect of the SnO2 ink concentrations on the device performance was investigated. In comparison with PSCs based on TiO2 and SrTiO3 ETLs, the SnO2-based devices offer an optimal power conversion efficiency (PCE) of 17.37% in combination with a low hysteresis. This work expands the range of suitable ETL materials for inkjet-printed PSCs and promotes the commercial applications of inkjet printing techniques in PSC manufacturing.

Predicting the target genes of miRNAs in preterm via targetscore algorithm.

Compared with normal neonates, preterm infants have an immature immune system which causes them to have a higher morbidity rate and even death. In order to reduce the mortality of newborns, we need to find the target genes which affect the preterm and understand their mechanism. It has been verified that microRNA (miRNA)-200 and miRNA-182 are closely related to the incidence of preterm. Therefore, it is significant to predict the target genes which are regulated by them for further understanding the mechanism of preterm. We chose the targetscore method for calculating the variational Bayesian-Gaussian mixture model (VB-GMM) as the target genes prediction method. It is designed for condition-specific target predictions and not limited to predict conserved genes, so the results are more accurate than previous sequence-based target prediction algorithms. In this study, our major contribution is to predict the target mRNAs of the chosen miRNAs with the gene expression profiles and a new method, which can effectively improve the accuracy of the prediction.

Jatrophane diterpenoid esters from Euphorbia sororia serving as multidrug resistance reversal agents.

Six (1-6) new jatrophane diterpenoid esters together with four known compounds (7-10) were isolated from the acetone extract of fructus Euphorbia sororia. Their structures were elucidated by the spectral technology, including the 2D NMR experiments (HMQC, HMBC and NOESY). The absolute configuration of compound 1 and compound 7 were first confirmed by X-ray crystallographic analysis. Compounds 1-7 were assayed for their antiproliferative activity in human cancer cell lines: human mammary adenocarcinoma (MCF-7) and human lung adenocarcinoma (A549). All the compounds were inactive for the cell lines. The multidrug-resistance reversal activity was also tested on KBv200 cells and compound 2 displayed strong multidrug resistance reversal activity, outperforming verapamil at 10 µM.