Tryptophan deficiency induced by indoleamine 2,3-dioxygenase 1 results in glucose transporter 1-dependent promotion of aerobic glycolysis in pancreatic cancer.

Indoleamine 2,3-dioxygenase 1 (IDO1), the key enzyme in the catabolism of the essential amino acid tryptophan (Trp) through kynurenine pathway, induces immune tolerance and is considered as a critical immune checkpoint, but its impacts as a metabolism enzyme on glucose and lipid metabolism are overlooked. We aim to clarify the potential role of IDO1 in aerobic glycolysis in pancreatic cancer (PC). Analysis of database revealed the positive correlation in PC between the expressions of and genes encoding important glycolytic enzyme , , and .

Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry.

Aggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as "Fab/c"), a commonly observed impurity during manufacturing and stability.

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS.

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment.

Preparation of astaxanthin-loaded composite micelles with coaxial electrospray technology for enhanced oral bioavailability and improved antioxidation capability.

Background: Astaxanthin (AST) is approved by the US Food and Drug Administration (FDA) as a safe dietary supplement for humans. As a potent lipid-soluble keto-carotenoid, it is widely used in food, cosmetics, and the pharmaceutical industry. However, its low solubility limits its powerful biological activity and its application in these fields.

Quantification of diaphragmatic dynamic dysfunction in septic patients by bedside ultrasound.

Although diaphragmatic dysfunction is an important indicator of severity of illness and poor prognosis in ICU patients, there is no convenient and practical method to monitor diaphragmatic function. This study was designed to analyze diaphragmatic dynamic dysfunction by bedside ultrasound in septic patients and provide quantitative evidence to assess diaphragm function systematically. This prospective observational study was conducted from October 2019 to January 2021 in the Department of Respiratory and Critical Care Medicine.

Predictive value of D-dimer and analysis of risk factors in pregnant women with suspected pulmonary embolism after cesarean section.

Background: Acute pulmonary embolism (PE) is one of the leading causes of maternal mortality, and cesarean section is an established independent risk factor for PE. The diagnostic utility of D-dimer for PE in non-pregnant women has been well-established, but its role in women with suspected PE after cesarean section is unclear. Furthermore, the optimal threshold level in this patient population is unknown.

A Highly Sensitive LC-MS/MS Method for Targeted Quantitation of Lipase Host Cell Proteins in Biotherapeutics.

Identification and accurate quantitation of host cell proteins (HCPs) in biotherapeutic drugs has become increasingly important due to the negative impact of certain HCPs on the safety, stability, and other product quality of biotherapeutics. Recently, several lipase HCPs have been identified to potentially cause the enzymatic degradation of polysorbate, a widely used excipient in the formulation of biotherapeutics, which can severely impact the stability and product quality of drug products. In this study, we identified three lipase HCPs that were frequently detected in Chinese hamster ovary (CHO) cell cultures using shotgun proteomics, including phospholipase B-like 2 (PLBL2), lipoprotein lipase (LPL), and lysosomal acid lipase (LIPA).

Development and Application of a Liquid Chromatography-Mass Spectrometry Method for Residual Iodixanol Quantification in AAV-Based Gene Therapy Product Development.

Adeno-associated viruses (AAVs) are nonenveloped viruses that have become popular gene transfer vectors to deliver DNA to target cells in clinical gene therapy. Iodixanol-based density gradient is one of the widely used purification methods for serotype-independent AAVs. However, residual iodixanol in AAV could be a safety concern, and further purification to remove this process-related impurity is typically needed.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications.

Astragaloside IV attenuates chronic intermittent hypoxia-induced myocardial injury by modulating Ca homeostasis.

Obstructive sleep apnea syndrome (OSAS) is an important consequence of chronic intermittent hypoxia (CIH). Astragaloside IV (AS-IV) exerts multiple protective effects in diverse diseases. However, whether AS-IV can attenuate CIH-induced myocardial injury is unclear.

Molecular cloning and characterization of a cathepsin L-like cysteine protease of Angiostrongylus cantonensis.

Angiostrongylus cantonensis is a parasitic nematode dwelling in the heart and pulmonary arteries of rats, which can cause angiostrongyliasis in human by accidental infections, manifested as eosinophilic meningitis or meningoencephalitis. Cysteine proteases are the major class of endopeptidases that are expressed at a high level in A. cantonensis, which suggests it may play key roles in pathogenesis of the disease.

Application of a label-free and domain-specific free thiol method in monoclonal antibody characterization.

Characterization of free thiol variants in antibody therapeutics is important for biopharmaceutical development, as the presence of free thiols may have an impact on aggregate formation, structural and thermal stability, as well as antigen-binding potency of monoclonal antibodies. Most current methods for free thiol quantification involve labeling of free thiol groups by different tagging molecules followed by UV, fluorescence or mass spectrometry (MS) detection. Here, we optimized a label-free liquid chromatography (LC)-UV/MS method for free thiol quantification at a subunit level and compared this method with two orthogonal and conventional approaches, Ellman's assay and peptide mapping with differential alkylation.

Discovery and Characterization of Histidine Oxidation Initiated Cross-links in an IgG1 Monoclonal Antibody.

Novel cross-links between an oxidized histidine and intact histidine, lysine, or cysteine residues were discovered and characterized from high-molecular weight (HMW) fractions of an IgG1 monoclonal antibody (mAb). The mAb HMW fractions were collected using preparative size-exclusion chromatography (SEC) and extensively characterized to understand the mechanism of formation of the nonreducible and covalently linked portion of the HMWs. The HMW fractions were IdeS digested, reduced, and analyzed by size-exclusion chromatography coupled with mass spectrometry (SEC-MS).

Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics.

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis.

Mechanism of Inactivation of Neuronal Nitric Oxide Synthase by (S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic Acid.

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and the second messenger nitric oxide. Three mechanistic pathways are proposed for the inactivation of neuronal NOS (nNOS) by (S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1): sulfide oxidation, oxidative dethiolation, and oxidative demethylation. Four possible intermediates were synthesized.

Proteomics guided discovery of flavopeptins: anti-proliferative aldehydes synthesized by a reductase domain-containing non-ribosomal peptide synthetase.

Due to the importance of proteases in regulating cellular processes, the development of protease inhibitors has garnered great attention. Peptide-based aldehydes are a class of compounds that exhibit inhibitory activities against various proteases and proteasomes in the context of anti-proliferative treatments for cancer and other diseases. More than a dozen peptide-based natural products containing aldehydes have been discovered such as chymostatin, leupeptin, and fellutamide; however, the biosynthetic origin of the aldehyde functionality has yet to be elucidated.

Gobichelin A and B: Mixed-Ligand Siderophores Discovered Using Proteomics.

"Omic" strategies have been increasingly applied to natural product discovery processes, with (meta-)genome sequencing and mining implemented in many laboratories to date. Using the proteomics-based discovery platform called PrISM (Proteomic Investigation of Secondary Metabolism), we discovered two new siderophores gobichelin A and B from Streptomyces sp. NRRL F-4415, a strain without a sequenced genome.

A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria.

Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases.

Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo.

Many lead compounds in the search for new drugs derive from peptides and polyketides whose similar biosynthetic enzymes have been difficult to engineer for production of new derivatives. Problems with generating multiple analogs in a single experiment along with lack of high-throughput methods for structure-based screening have slowed progress in this area. Here, we use directed evolution and a multiplexed assay to screen a library of >14,000 members to generate three derivatives of the antibacterial compound, andrimid.

Proteomics-based discovery of koranimine, a cyclic imine natural product.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzymes responsible for the biosynthesis of medically and ecologically important secondary metabolites. In a previous report, we described a proteomics approach to screen for expressed NRPSs or PKSs from bacteria with or without sequenced genomes. Here we used this proteome mining approach to discover a novel natural product arising from rare adenylation (A) and reductase (Red) domains in its biosynthetic machinery.