Biophysical and structural studies of fibulin-2.

Fibulin-2 is a multidomain, disulfide-rich, homodimeric protein which belongs to a broader extracellular matrix family. It plays an important role in the development of elastic fiber structures. Malfunction of fibulin due to mutation or poor expression can result in a variety of diseases including synpolydactyly, limb abnormalities, eye disorders leading to blindness, cardiovascular diseases and cancer. Traditionally, fibulins have either been produced in mammalian cell systems or were isolated from the extracellular matrix, a procedure that results in poor availability for structural and functional studies. Here, we produced seven fibulin-2 constructs covering 62% of the mature protein (749 out of 1195 residues) using a prokaryotic expression system. Biophysical studies confirm that the purified constructs are folded and that the presence of disulfide bonds within the constructs makes them extremely thermostable. In addition, we solved the first crystal structure for any fibulin isoform, a structure corresponding to the previously suggested three motifs related to anaphylatoxin. The structure reveals that the three anaphylatoxins moieties form a single-domain structure.

A Novel UHPLC-MS/MS Based Method for Isomeric Separation and Quantitative Determination of Cyanogenic Glycosides in American Elderberry.

LC-MS/MS analyses have been reported as challenging for the reliable separation and quantification of cyanogenic glycosides (CNGs), especially (R)-prunasin and sambunigrin isomers found in American elderberry (Sambucus nigra L. subsp. canadensis (L.) Bolli). Hence, a novel multiple reaction monitoring (MRM)-based ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated in the present study for simultaneous separation and quantification of five CNGs, including amygdalin, dhurrin, linamarin, (R)-prunasin, and (S)-prunasin (commonly referred to as sambunigrin). Initially, the role of ammonium formate was investigated as an aqueous mobile-phase additive in developing MRM-based UHPLC-MS/MS. Later, chromatographic conditions for the resolved separation of (R)-prunasin and sambunigrin were identified. Validation studies confirmed that the developed method has good linearity and acceptable precision and accuracy. A noticeable matrix effect (mainly signal enhancement) was observed in leaf samples only. This method was used to detect and quantify CNGs, including (R)-prunasin and sambunigrin, in leaf and fruit samples of American elderberry. Among the studied CNGs, only (R)-prunasin was detected in the leaf samples. Interestingly, (S)-prunasin (sambunigrin) was not detected in the samples analyzed, even though it has been previously reported in elderberry species.

Assessing Quality in Interventional Practice: Identifying Meaningful and Actionable Metrics.

Interventional cardiologists should insist on quality assessment techniques that indisputably reflect the merit of care delivered. Only measurable outcomes and metrics that are modifiable should be identified and collected. An evaluation process should be adopted that genuinely appraises clinical practice, incorporating appropriate benchmarks for comparison.

Escherichia coli Cytoplasmic Expression of Disulfide-Bonded Proteins: Side-by-Side Comparison between Two Competing Strategies.

The production of disulfide bond-containing recombinant proteins in Escherichia coli has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of E. coli: i) engineered strains with deletions in the disulfide reduction pathways, e.g. SHuffle, and ii) the co-expression of oxidative folding catalysts, e.g. CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.

Perspective: use and reuse of NMR-based metabolomics data: what works and what remains challenging.

BACKGROUND: The National Cancer Institute issued a Request for Information (RFI; NOT-CA-23-007) in October 2022, soliciting input on using and reusing metabolomics data. This RFI aimed to gather input on best practices for metabolomics data storage, management, and use/reuse. AIM OF REVIEW: The nuclear magnetic resonance (NMR) Interest Group within the Metabolomics Association of North America (MANA) prepared a set of recommendations regarding the deposition, archiving, use, and reuse of NMR-based and, to a lesser extent, mass spectrometry (MS)-based metabolomics datasets. These recommendations were built on the collective experiences of metabolomics researchers within MANA who are generating, handling, and analyzing diverse metabolomics datasets spanning experimental (sample handling and preparation, NMR/MS metabolomics data acquisition, processing, and spectral analyses) to computational (automation of spectral processing, univariate and multivariate statistical analysis, metabolite prediction and identification, multi-omics data integration, etc.) studies. KEY SCIENTIFIC CONCEPTS OF REVIEW: We provide a synopsis of our collective view regarding the use and reuse of metabolomics data and articulate several recommendations regarding best practices, which are aimed at encouraging researchers to strengthen efforts toward maximizing the utility of metabolomics data, multi-omics data integration, and enhancing the overall scientific impact of metabolomics studies.

Clinical effects of physiologic lesion testing in influencing treatment strategy for multi-vessel coronary artery disease.

Background: The application of fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR) in multivessel coronary artery disease (CAD) patients has not been definitively explored. We herein assessed how treatment strategies were decided based on FFR/iFR values in vessels selected clinically. Specifically, we sought to determine whether treatment selection was based on whether the vessel tested was the clinical target stenosis. Methods: 270 consecutive patients with angiographically determined multivessel disease who underwent FFR/iFR testing were included. Patients were classified initially based on their angiographic findings, then re-evaluated from FFR/iFR results (normal or abnormal). Tested lesions were classified into target or non-target lesions based on clinical and non-invasive evaluations. Results: Abnormal FFR/iFR values were demonstrated in 51.9 % of patients, in whom 51.4 % received coronary stenting (PCI) and 44.3 % had bypass surgery (CABG). With two-vessel CAD patients, medical therapy was preferred when the target lesion was normal (72.6 %), while PCI was preferred when it was abnormal (78.4 %). In non-target lesions, PCI was preferred regardless of FFR/iFR results (78.0 %). With three-vessel CAD patients, CABG was preferred when the target lesion was abnormal (68.5 %), and there was no difference in the selected modality when it was normal. Furthermore, the incidence of tested lesions was higher in the left anterior descending (LAD) compared to other coronary arteries, and two-vessel CAD patients with LAD stenoses were more frequently treated by PCI. Conclusion: The use of invasive physiologic testing in multivessel CAD patients may alter the preferred treatment strategy, leading to an overall increase in PCI selection.

Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.

In the methylotrophic yeast Komagataella phaffii, we identified an endoplasmic reticulum-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG. This combination of active site motifs has so far been unreported in PDI-family members. Homology searches revealed ERp41 is present in the genome of some plants, fungal parasites, and a few nonconventional yeasts, among which are Komagataella spp. and Yarrowia lipolytica. These yeasts are both used for the production of secreted recombinant proteins. Here, we analyzed the activity of K. phaffii Erp41. We report that it is nonessential in K. phaffii, and that it can catalyze disulfide bond formation in partnership with the sulfhydryl oxidase Ero1 in vitro with higher turnover rates than the canonical PDI from K. phaffii, Pdi1, but slower activation times. We show how Erp41 has unusually fast glutathione-coupled oxidation activity and relate it to its unusual combination of active sites in its thioredoxin-like domains. We further describe how this determines its unusually efficient catalysis of dithiol oxidation in peptide and protein substrates.

Cytoplasmic production of Fabs in chemically defined media in fed-batch fermentation.

Fragment of antigen-binding region (Fab) of antibodies are important biomolecules, with a broad spectrum of functionality in the biomedical field. While full length antibodies are usually produced in mammalian cells, the smaller size, lack of N-glycosylation and less complex structure of Fabs make production in microbial cell factories feasible. Since Fabs contain disulfide bonds, such production is often done in the periplasm, but there the formation of the inter-molecular disulfide bond between light and heavy chains can be problematic. Here we studied the use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) to express two Fabs (Herceptin and Maa48) in the cytoplasm of E. coli in fed-batch fermentation using a generic chemically defined media. We were able to solubly express both Fabs with purified yields of 565 mg/L (Maa48) and 660 mg/L (Herceptin) from low density fermentation. Both proteins exhibited CD spectra consistent with natively folded protein and both were biologically active. To our knowledge this is the first demonstration of high-level production of biological active Fabs in the cytoplasm of E. coli in industrially relevant fermentation conditions.

Quantification and characterization of biological activities of glansreginin A in black walnuts (Juglans nigra).

Glansreginin A has been reported to be an indicator of the quality of walnuts (Juglans spp.). However, bioactive properties of glansreginin A have not been adequately explored. In the present study, we quantified concentrations of glansreginin A in black walnuts (Juglans nigra) using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed an array of in vitro bioassays to characterize biological activities (e.g., antibacterial, antioxidant, anticancer capacities) of this compound. Results from HPLC-MS/MS analysis indicated that glansreginin A was presented in all 12 black cultivars examined and its contents were variable among black walnut cultivars, ranged from 6.8 mg/kg (Jackson) to 47.0 mg/kg (Hay). Glansreginin A possessed moderate antibacterial activities against Gram-positive pathogens (Staphylococcus aureus and Bacillus anthracis). This compound exhibited no antioxidant activities, did not induce the activity of antioxidant response element signaling pathways, and exerted no antiproliferative effects on tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells.

A Data Deposition Platform for Sharing Nuclear Magnetic Resonance Data.

Nuclear magnetic resonance (NMR) data are rarely deposited in open databases, leading to loss of critical scientific knowledge. Existing data reporting methods (images, tables, lists of values) contain less information than raw data and are poorly standardized. Together, these issues limit FAIR (findable, accessible, interoperable, reusable) access to these data, which in turn creates barriers for compound dereplication and the development of new data-driven discovery tools. Existing NMR databases either are not designed for natural products data or employ complex deposition interfaces that disincentivize deposition. Journals, including the Journal of Natural Products (JNP), are now requiring data submission as part of the publication process, creating the need for a streamlined, user-friendly mechanism to deposit and distribute NMR data.

Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli.

With increased accessibility and tissue penetration, smaller antibody formats such as antibody fragments (Fab) and single chain variable fragments (scFv) show potential as effective and low-cost choices to full-length antibodies. These formats derived from the modular architecture of antibodies could prove to be game changers for certain therapeutic and diagnostic applications. Microbial hosts have shown tremendous promise as production hosts for antibody fragment formats. However, low target protein yields coupled with the complexity of protein folding result in production limitations. Here, we report an alternative antibody fragment format 'FabH3' designed to overcome some key bottlenecks associated with the folding and production of Fabs. The FabH3 molecule is based on the Fab format with the constant domains replaced by engineered immunoglobulin G1 (IgG1) CH3 domains capable of heterodimerization based on the electrostatic steering approach. We show that this alternative antibody fragment format can be efficiently produced in the cytoplasm of E. coli using the catalyzed disulfide-bond formation system (CyDisCo) in a natively folded state with higher soluble yields than its Fab counterpart and a comparable binding affinity against the target antigen.

Integrated multi-omic analyses provide insight into colon adenoma susceptibility modulation by the gut microbiota.

Colon cancer onset is strongly associated with the differences in microbial taxa in the gastrointestinal tract. Although recent studies highlight the role of individual taxa, the effect of a complex gut microbiome (GM) on the metabolome and host transcriptome is still unknown. We used a multi-omics approach to determine how differences in the GM affect the susceptibility to adenoma development in a rat model of human colon cancer. Ultra-high performance liquid chromatography mass spectrometry of feces collected prior to observable disease onset identified putative metabolite profiles that likely predict future disease severity. Transcriptome analyses performed after disease onset from normal colonic epithelium and tumor tissues show a correlation between GM and host gene expression. Integrated pathway analyses of the metabolome and transcriptome based on putatively identified metabolic features indicate that bile acid biosynthesis is enriched in rats with high tumors along with increased fatty acid metabolism and mucin biosynthesis. Targeted pyrosequencing of the Pirc allele indicates that the GM alters the mechanism of adenoma development and may drive an epigenetic pathway of tumor suppressor silencing. This study reveals how untargeted metabolomics identifies signatures of susceptibility and integrated analyses uncover pathways of differential mechanisms of loss of tumor suppressor gene function and for potential prevention and therapeutic intervention. IMPORTANCE The association between the gut microbiome and colon cancer is significant but difficult to test in model systems. This study highlights the association of differences in the pathogen-free gut microbiome to changes in the host transcriptome and metabolome that correlate with colon adenoma initiation and development in a rat genetic model of early colon cancer. The utilization of a multi-omics approach integrating metabolomics and transcriptomics reveals differences in pathways including bile acid biosynthesis and fatty acid metabolism. The study also shows that differences in gut microbiomes significantly alter the mechanism of adenoma formation, shifting from genetic changes to epigenetic changes that initiate the early loss of tumor suppressor function. These findings enhance our understanding of the gut microbiome's role in colon cancer susceptibility, offer insights into potential biomarkers and therapeutic targets, and may pave the way for future prevention and intervention strategies.

Wearable sensors for monitoring marine environments and their inhabitants.

Human societies depend on marine ecosystems, but their degradation continues. Toward mitigating this decline, new and more effective ways to precisely measure the status and condition of marine environments are needed alongside existing rebuilding strategies. Here, we provide an overview of how sensors and wearable technology developed for humans could be adapted to improve marine monitoring. We describe barriers that have slowed the transition of this technology from land to sea, update on the developments in sensors to advance ocean observation and advocate for more widespread use of wearables on marine organisms in the wild and in aquaculture. We propose that large-scale use of wearables could facilitate the concept of an 'internet of marine life' that might contribute to a more robust and effective observation system for the oceans and commercial aquaculture operations. These observations may aid in rationalizing strategies toward conservation and restoration of marine communities and habitats.

Highly efficient export of a disulfide-bonded protein to the periplasm and medium by the Tat pathway using CyDisCo in Escherichia coli.

High-value heterologous proteins produced in Escherichia coli that contain disulfide bonds are almost invariably targeted to the periplasm via the Sec pathway as it, among other advantages, enables disulfide bond formation and simplifies downstream processing. However, the Sec system cannot transport complex or rapidly folding proteins, as it only transports proteins in an unfolded state. The Tat system also transports proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Most of the studies related to Tat secretion have used the well-studied TorA signal peptide that is Tat-specific, but this signal peptide also tends to induce degradation of the protein of interest, resulting in lower yields. This makes it difficult to use Tat in the industry. In this study, we show that a model disulfide bond-containing protein, YebF, can be exported to the periplasm and media at a very high level by the Tat pathway in a manner almost completely dependent on cytoplasmic disulfide formation, by other two putative Tat SPs: those of MdoD and AmiC. In contrast, the TorA SP exports YebF at a low level.

Mechanistic insights into the ROS-mediated inactivation of human aldehyde oxidase.

Human aldehyde oxidase (hAOX1) is a molybdoenzyme that oxidizes aldehydes and N-heterocyclic compounds, thereby generating hydrogen peroxide (H2 O2 ) and superoxide during turnover. hAOX1 has been shown previously to be inactivated under turnover conditions by H2 O2 . Here, we investigated the effect of exogenously added H2 O2 on the activity of hAOX1. We show that exogenously added H2 O2 did not affect the enzyme activity under aerobic conditions, but completely inactivated the enzyme under anaerobic conditions. We propose that this effect is based on the reducing power of H2 O2 and the susceptibility of the reduced molybdenum cofactor (Moco) to lose the sulfido ligand. When oxygen is present, the enzyme is rapidly reoxidized. We believe that our study is significant in understanding the detailed effect of reactive oxygen species on the inactivation of hAOX1 and other molybdoenzymes.

Inactivation of Human Aldehyde Oxidase by Small Sulfhydryl-Containing Reducing Agents.

Human aldehyde oxidase (hAOX1) is a molybdoflavoenzyme that belongs to the xanthine oxidase (XO) family. hAOX1 is involved in phase I drug metabolism, but its physiologic role is not fully understood to date, and preclinical studies consistently underestimated hAOX1 clearance. In the present work, we report an unexpected effect of the common sulfhydryl-containing reducing agents, e.g., dithiothreitol (DTT), on the activity of hAOX1 and mouse aldehyde oxidases. We demonstrate that this effect is due to the reactivity of the sulfido ligand bound at the molybdenum cofactor with the sulfhydryl groups. The sulfido ligand coordinated to the Mo atom in the XO family of enzymes plays a crucial role in the catalytic cycle and its removal results in the total inactivation of these enzymes. Because liver cytosols, S9 fractions, and hepatocytes are commonly used to screen the drug candidates for hAOX1, our study suggests that DTT treatment of these samples should be avoided, otherwise false negative results by an inactivated hAOX1 might be obtained. SIGNIFICANCE STATEMENT: This work characterizes the inactivation of human aldehyde oxidase (hAOX1) by sulfhydryl-containing agents and identifies the site of inactivation. The role of dithiothreitol in the inhibition of hAOX1 should be considered for the preparation of hAOX1-containing fractions for pharmacological studies on drug metabolism and drug clearance.