Model acetylcholinesterase-Fc fusion glycoprotein biotechnology system for the manufacture of an organophosphorus toxicant bioscavenging countermeasure.

Organophosphate (OP) toxicants remain an active threat to public health and to warfighters in the military. Current countermeasures require near immediate administration following OP exposure and are reported to have controversial efficacies. Acetylcholinesterase (AChE) fused to the human immunoglobulin 1 (IgG1) Fc domain (AChE-Fc) is a potential bioscavenger for OP toxicants, but a reproducible AChE-Fc biomanufacturing strategy remains elusive.

A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format.

Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS).

Impact of sunitinib resistance on clear cell renal cell carcinoma therapeutic sensitivity .

Sunitinib resistance creates a major clinical challenge for the treatment of advanced clear cell renal cell carcinoma (ccRCC) and functional and metabolic changes linked to sunitinib resistance are not fully understood. We sought to characterize the molecular and metabolic changes induced by the development of sunitinib resistance in ccRCC by developing and characterizing two human ccRCC cell lines resistant to sunitinib. Consistent with the literature, sunitinib-resistant ccRCC cell lines presented an aberrant overexpression of Axl and PD-L1, as well as a metabolic rewiring characterized by enhanced OXPHOS and glutamine metabolism.

SARS-CoV-2 infection establishes a stable and age-independent CD8 T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8 T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8 T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8 T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8 T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors.

Extended Opioid Exposure Modulates the Molecular Metabolism of Clear Cell Renal Cell Carcinoma.

Opioids are commonly prescribed for extended periods of time to patients with advanced clear cell renal cell carcinoma to assist with pain management. Because extended opioid exposure has been shown to affect the vasculature and to be immunosuppressive, we investigated how it may affect the metabolism and physiology of clear cell renal cell carcinoma. RNA sequencing of a limited number of archived patients' specimens with extended opioid exposure or non-opioid exposure was performed.

Comprehensive N- and O-Glycoproteomic Analysis of Multiple Chinese Hamster Ovary Host Cell Lines.

Glycoproteomic analysis of three Chinese hamster ovary (CHO) suspension host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in biopharmaceutical settings for recombinant protein production is reported. Intracellular and secreted glycoproteins were examined. We utilized an immobilization and chemoenzymatic strategy in our analysis.

Longitudinal analysis reveals age-related changes in the T cell receptor repertoire of human T cell subsets.

A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens, and TCR repertoire size is believed to decline with age. However, the precise size of human TCR repertoires, in both total and subsets of T cells, as well as their changes with age, are not fully characterized. We conducted a longitudinal analysis of the human blood TCRα and TCRβ repertoire of CD4+ and CD8+ T cell subsets using a unique molecular identifier-based (UMI-based) RNA-seq method.

PD-L1 Mediates IFNγ-Regulation of Glucose but Not of Tryptophan Metabolism in Clear Cell Renal Cell Carcinoma.

The immune checkpoint programmed death-ligand 1 (PD-L1) is expressed on the cell surface of tumor cells and is key for maintaining an immunosuppressive microenvironment through its interaction with the programmed death 1 (PD-1). Clear cell renal cell carcinoma (ccRCC) is a highly immunogenic cancer characterized by an aberrant aerobic glycolytic metabolism and is known to overexpress PD-L1. Multiple immunotherapies have been approved for the treatment of ccRCC, including cytokines and immune checkpoint inhibitors.

Differential T cell immune responses to deamidated adeno-associated virus vector.

Despite the high safety profile demonstrated in clinical trials, the immunogenicity of adeno-associated virus (AAV)-mediated gene therapy remains a major hurdle. Specifically, T-cell-mediated immune responses to AAV vectors are related to loss of efficacy and potential liver toxicities. As post-translational modifications in T cell epitopes have the potential to affect immune reactions, the cellular immune responses to peptides derived from spontaneously deamidated AAV were investigated.

In search of autophagy biomarkers in breast cancer: Receptor status and drug agnostic transcriptional changes during autophagy flux in cell lines.

Autophagy drives drug resistance and drug-induced cancer cell cytotoxicity. Targeting the autophagy process could greatly improve chemotherapy outcomes. The discovery of specific inhibitors or activators has been hindered by challenges with reliably measuring autophagy levels in a clinical setting.

SARS-CoV-2 B.1.1.7 (alpha) and B.1.351 (beta) variants induce pathogenic patterns in K18-hACE2 transgenic mice distinct from early strains.

SARS-CoV-2 variants of concern (VOC) B.1.1.

Anthracycline-Induced Cardiotoxicity: Molecular Insights Obtained from Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs).

Anthracyclines are a class of chemotherapy drugs that are highly effective for the treatment of human cancers, but their clinical use is limited by associated dose-dependent cardiotoxicity. The precise mechanisms by which individual anthracycline induces cardiotoxicity are not fully understood. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are emerging as a physiologically relevant model to assess drugs cardiotoxicity.

Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products.

Background: Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIII ) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIII can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated.

Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells.

In mammalian cells, N-glycans may include multiple N-acetyllactosamine (poly-LacNAc) units that can play roles in various cellular functions and properties of therapeutic recombinant proteins. Previous studies indicated that β-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and β-1,4-galactotransferase 1 (B4GALT1) are two of the primary glycosyltransferases involved in generating LacNAc units. In the current study, knocking out sialyltransferase genes slightly enhanced the LacNAc content (≥4 repeats per glycan) on recombinant EPO protein.

Optimization of GIG for -Glycopeptide Characterization with Sialic Acid Linkage Determination.

-Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in the release of -glycans. Moreover, chemical releasing methods, such as β-elimination/Michael addition, are not specific to -glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions.

NGS Evaluation of Colorectal Cancer Reveals Interferon Gamma Dependent Expression of Immune Checkpoint Genes and Identification of Novel IFNγ Induced Genes.

To evaluate the expression of immune checkpoint genes, their concordance with expression of IFNγ, and to identify potential novel ICP related genes (ICPRG) in colorectal cancer (CRC), the biological connectivity of six well documented ("classical") ICPs (CTLA4, PD1, PDL1, Tim3, IDO1, and LAG3) with IFNγ and its co-expressed genes was examined by NGS in 79 CRC/healthy colon tissue pairs. Identification of novel IFNγ- induced molecules with potential ICP activity was also sought. In our study, the six classical ICPs were statistically upregulated and correlated with IFNγ, CD8A, CD8B, CD4, and 180 additional immunologically related genes in IFNγ positive (FPKM > 1) tumors.

Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing.

DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression.

T cell-derived soluble glycoprotein GPIbα mediates PGE production in human monocytes activated with the vaccine adjuvant MDP.

Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors (PRRs) on antigen-presenting cells, including monocytes and macrophages, which can cause prostaglandin E (PGE) release and consequently undesired inflammatory responses and fever in vaccine recipients. Here, we studied the mechanism of PGE production by human monocytes activated with muramyl dipeptide (MDP) adjuvant, which activates cytosolic nucleotide-binding oligomerization domain 2 (NOD2). In rabbits, administration of MDP elicited an early increase in PGE followed by fever.

Novel reference genes in colorectal cancer identify a distinct subset of high stage tumors and their associated histologically normal colonic tissues.

Background: Reference genes are often interchangeably called housekeeping genes due to 1) the essential cellular functions their proteins provide and 2) their constitutive expression across a range of normal and pathophysiological conditions. However, given the proliferative drive of malignant cells, many reference genes such as beta-actin (ACTB) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) which play critical roles in cell membrane organization and glycolysis, may be dysregulated in tumors versus their corresponding normal controls METHODS: Because Next Generation Sequencing (NGS) technology has several advantages over hybridization-based technologies, such as independent detection and quantitation of transcription levels, greater sensitivity, and increased dynamic range, we evaluated colorectal cancers (CRC) and their histologically normal tissue counterparts by NGS to evaluate the expression of 21 "classical" reference genes used as normalization standards for PCR based methods. Seventy-nine paired tissue samples of CRC and their patient matched healthy colonic tissues were subjected to NGS analysis of their mRNAs.

The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain.

A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468.

Comprehensive analysis of -glycans in IgG purified from ferrets with or without influenza A virus infection.

Influenza viruses cause contagious respiratory infections, resulting in significant economic burdens to communities. Production of influenza-specific Igs, specifically IgGs, is one of the major protective immune mechanisms against influenza viruses. In humans, -glycosylation of IgGs plays a critical role in antigen binding and effector functions.

Trans-chalcone increases p53 activity via DNAJB1/HSP40 induction and CRM1 inhibition.

Naturally-occurring chalcones and synthetic chalcone analogues have been demonstrated to have many biological effects, including anti-inflammatory, anti-malarial, anti-fungal, and anti-oxidant/anti-cancerous activities. Compared to other chalcones, trans-chalcone exhibits superior inhibitory activity in cancer cell growth as shown via in vitro assays, and exerts anti-cancerous effects via the activation of the p53 tumor suppressor protein. Thus, characterization of the specific mechanisms, by which trans-chalcone activates p53, can aid development of new chemotherapeutic drugs that can be used individually or synergistically with other drugs.

Deciphering Protein O-Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment.

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. N-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release.

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing.

Background: Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported.