Structural characterization and epitope mapping of the AAVX affinity purification ligand.

The application of adeno-associated virus (AAV) vectors in human gene therapies requires reproducible and homogeneous preparations for clinical efficacy and safety. For the AAV production process, often scalable affinity chromatography columns are utilized, such as the POROS CaptureSelect AAVX affinity resin, during downstream processing to ensure highly purified AAV vectors. The AAVX ligand is based on a camelid single-domain antibody capturing a wide range of recombinant AAV capsids.

Rescue of mitochondrial dysfunction through alteration of extracellular matrix composition in barth syndrome cardiac fibroblasts.

Fibroblast-ECM (dys)regulation is associated with a plethora of diseases. The ECM acts as a reservoir of inflammatory factors and cytokines that mediate molecular mechanisms within cardiac cell populations. The role of ECM-mitochondria crosstalk in the development and progression of cardiac disorders remains uncertain.

Multilevel Proteomics Reveals Epigenetic Signatures in BCG-Mediated Macrophage Activation.

The bacillus Calmette-Guérin BCG vaccine (Mycobacterium bovis) is primarily used to prevent tuberculosis (TB) infections but has wide-ranging immunogenic effects. One of its most notable properties is its ability to induce trained immunity, a memory-like response in innate immune cells such as macrophages. Through targeted analyses of well-established histone marks, prior research has shown that these changes are generated through epigenetic modification.

An acyl-CoA thioesterase is essential for the biosynthesis of a key dauer pheromone in C. elegans.

Methyl ketone (MK)-ascarosides represent essential components of several pheromones in Caenorhabditis elegans, including the dauer pheromone, which triggers the stress-resistant dauer larval stage, and the male-attracting sex pheromone. Here, we identify an acyl-CoA thioesterase, ACOT-15, that is required for the biosynthesis of MK-ascarosides. We propose a model in which ACOT-15 hydrolyzes the β-keto acyl-CoA side chain of an ascaroside intermediate during β-oxidation, leading to decarboxylation and formation of the MK.

Investigation of Glycosylphosphatidylinositol (GPI)-Plasma Membrane Interaction in Live Cells and the Influence of GPI Glycan Structure on the Interaction.

Glycosylphosphatidylinositols (GPIs) need to interact with other components in the cell membrane to transduce transmembrane signals. A bifunctional GPI probe was employed for photoaffinity-based proximity labelling and identification of GPI-interacting proteins in the cell membrane. This probe contained the entire core structure of GPIs and was functionalized with photoreactive diazirine and clickable alkyne to facilitate its crosslinking with proteins and attachment of an affinity tag.

Phage predation, disease severity and pathogen genetic diversity in cholera patients.

Despite an increasingly detailed picture of the molecular mechanisms of phage-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. Here we report a year-long, nation-wide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified (prey) and its virulent phages (predators) using metagenomics and quantitative PCR, while accounting for antibiotic exposure using quantitative mass spectrometry.

Genome-wide association studies reveal distinct genetic correlates and increased heritability of antimicrobial resistance in under anaerobic conditions.

The antibiotic formulary is threatened by high rates of antimicrobial resistance (AMR) among enteropathogens. Enteric bacteria are exposed to anaerobic conditions within the gastrointestinal tract, yet little is known about how oxygen exposure influences AMR. The facultative anaerobe was chosen as a model to address this knowledge gap.

Nonpolar Lipids Contribute to Midday Fogging During Scleral Lens Wear.

Purpose: To determine correlations between lipids in the fluid reservoir (FR) and the severity of midday fogging (MDF) in scleral lens (SL) wear.

Methods: SL neophytes were recruited to wear custom SL for 4 days, examined after 8 hours on days 1 and 4. Lens vault and MDF were quantified from anterior segment optical coherence tomography (AS-OCT), and the FR was collected and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Optimization of the Sulfo-Phospho-Vanillin Assay for Total Lipid Normalization in Untargeted Quantitative Lipidomic LC-MS/MS Applications.

Liquid chromatography (LC)-mass spectrometry (MS)/MS lipidomic normalization is generally performed by equalizing pre-extraction sample materials or via DNA or protein pre-quantitation methods, which have known measurement inaccuracies. We propose the use of the sulfo-phospho-vanillin assay (SPVA), a total lipid colorimetric analysis, as a pre-quantitation method to normalize lipids in lipidomic LC-MS/MS applications. The assay has been applied to a 300 μL well volume in a 96-well plate and tested using Avanti total lipid standards of porcine brain and .

Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase.

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed.

Tear Proteomics of Children and Young Adult Soft Contact Lens, Orthokeratology and Spectacle Wearers - A Pilot Study.

Purpose: Contact lens complications occur more often in older teenagers and young adults compared to children. This study explored differences in tear proteomics between children and young adults wearing soft contact lens (SCL), orthokeratology or spectacles for >3 years.

Methods: Twelve children and 12 sex- and correction-matched young adults were enrolled.

Identification and characterization of proteins, lipids, and metabolites in two organic fertilizer products derived from different nutrient sources.

Unlabelled: The biochemical composition of organic fertilizers largely determines their nutrient supply characteristics following soil application as well as their potential impact on soil microbial communities. Yet, limited information is available regarding the biochemical composition of organic fertilizers derived from different nutrient sources. Here, we qualitatively analyzed the presence and abundance of proteins, lipids, and metabolites in a liquid fish fertilizer (LFF) product and a type of granular organic fertilizer (GOF) commonly used in organic vegetable production, using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide in a metazoan.

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis.

Combating Multidrug-Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling.

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy.

Characterization of Glycosphingolipids and Their Diverse Lipid Forms through Two-Stage Matching of LC-MS/MS Spectra.

Glycosphingolipids (GSLs) play a key role in various biological and pathological events. Thus, determination of the complete GSL compositions in human tissues is essential for comparative and functional studies of GSLs. In this work, a new strategy was developed for GSL characterization and glycolipidomics analysis based on two-stage matching of experimental and reference MS/MS spectra.

The Impacts of Sortase A and the 4'-Phosphopantetheinyl Transferase Homolog Sfp on Extracellular Membrane Vesicle Biogenesis.

Extracellular membrane vesicles (EMVs) are produced by many Gram-positive organisms, but information regarding vesiculogenesis is incomplete. We used single gene deletions to evaluate the impacts on EMV biogenesis of Sortase A (SrtA), which affects EMV composition, and Sfp, a 4'-phosphopantetheinyl transferase that affects EMV stability. Δ EMVs were notably larger than Δ and wild-type (WT) EMVs.

First Penicillin-Binding Protein Occupancy Patterns for 15 β-Lactams and β-Lactamase Inhibitors in Mycobacterium abscessus.

causes serious infections that often require over 18 months of antibiotic combination therapy. There is no standard regimen for the treatment of infections, and the multitude of combinations that have been used clinically have had low success rates and high rates of toxicities. With β-lactam antibiotics being safe, double β-lactam and β-lactam/β-lactamase inhibitor combinations are of interest for improving the treatment of infections and minimizing toxicity.

A Large Family of Enzymes Responsible for the Modular Architecture of Nematode Pheromones.

The nematode produces a broad family of pheromones, known as the ascarosides, that are modified with a variety of groups derived from primary metabolism. These modifications are essential for the diverse activities of the ascarosides in development and various behaviors, including attraction, aggregation, avoidance, and foraging. The mechanism by which these different groups are added to the ascarosides is poorly understood.

Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant and .

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against and , there are limited data on OM permeability especially in Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant and Both clinical isolates harbored a and several other β-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses.

The Proteome of Pancreatic Cancer-Derived Exosomes Reveals Signatures Rich in Key Signaling Pathways.

Exosomes are membrane-bound vesicles that traffic small molecular cargos. These cargos participate in cell-cell communication and contribute to the pathogenesis of many disease including cancer. How these mechanisms contribute to communication within the pancreatic adenocarcinoma (PDAC) microenvironment and how they contribute to PDAC biology are poorly understood.