Interaction Metabolomics to Discover Synergists in Natural Product Mixtures.

Mass spectrometry metabolomics has become increasingly popular as an integral aspect of studies to identify active compounds from natural product mixtures. Classical metabolomics data analysis approaches do not consider the possibility that interactions (such as synergy) could occur between mixture components. With this study, we developed "interaction metabolomics" to overcome this limitation. The innovation of interaction metabolomics is the inclusion of compound interaction terms (CITs), which are calculated as the product of the intensities of each pair of features (detected ions) in the data matrix. Herein, we tested the utility of interaction metabolomics by spiking known concentrations of an antimicrobial compound (berberine) and a synergist (piperine) into a set of inactive matrices. We measured the antimicrobial activity for each of the resulting mixtures against Staphylococcus aureus and analyzed the mixtures with liquid chromatography coupled to high-resolution mass spectrometry. When the data set was processed without CITs (classical metabolomics), statistical analysis yielded a pattern of false positives. However, interaction metabolomics correctly identified berberine and piperine as the compounds responsible for the synergistic activity. To further validate the interaction metabolomics approach, we prepared mixtures from extracts of goldenseal (Hydrastis canadensis) and habañero pepper (Capsicum chinense) and correctly correlated synergistic activity of these mixtures to the combined action of berberine and several capsaicinoids. Our results demonstrate the utility of a conceptually new approach for identifying synergists in mixtures that may be useful for applications in natural products research and other research areas that require comprehensive mixture analysis.

Modulation of in Vitro SARS-CoV-2 Infection by Stephania tetrandra and Its Alkaloid Constituents.

Botanical natural products have been widely consumed for their purported usefulness against COVID-19. Here, six botanical species from multiple sources and 173 isolated natural product compounds were screened for blockade of wild-type (WT) SARS-CoV-2 infection in human 293T epithelial cells overexpressing ACE-2 and TMPRSS2 protease (293TAT). Antiviral activity was demonstrated by an extract from Stephania tetrandra. Extract fractionation, liquid chromatography-mass spectrometry (LC-MS), antiviral assays, and computational analyses revealed that the alkaloid fraction and purified alkaloids tetrandrine, fangchinoline, and cepharanthine inhibited WT SARS-CoV-2 infection. The alkaloids and alkaloid fraction also inhibited the delta variant of concern but not WT SARS-CoV-2 in VeroAT cells. Membrane permeability assays demonstrate that the alkaloids are biologically available, although fangchinoline showed lower permeability than tetrandrine. At high concentrations, the extract, alkaloid fractions, and pure alkaloids induced phospholipidosis in 293TAT cells and less so in VeroAT cells. Gene expression profiling during virus infection suggested that alkaloid fraction and tetrandrine displayed similar effects on cellular gene expression and pathways, while fangchinoline showed distinct effects on cells. Our study demonstrates a multifaceted approach to systematically investigate the diverse activities conferred by complex botanical mixtures, their cell-context specificity, and their pleiotropic effects on biological systems.

How Low Can You Go? Selecting Intensity Thresholds for Untargeted Metabolomics Data Preprocessing.

Untargeted mass spectrometry (MS) metabolomics is an increasingly popular approach for characterizing complex mixtures. Recent studies have highlighted the impact of data preprocessing for determining the quality of metabolomics data analysis. The first step in data processing with untargeted metabolomics requires that signal thresholds be selected for which features (detected ions) are included in the dataset. Analysts face the challenge of knowing where to set these thresholds; setting them too high could mean missing relevant features, but setting them too low could result in a complex and unwieldy dataset. This study compared data interpretation for an example metabolomics dataset when intensity thresholds were set at a range of feature heights. The main observations were that low signal thresholds (1) improved the limit of detection, (2) increased the number of features detected with an associated isotope pattern and/or an MS-MS fragmentation spectrum, and (3) increased the number of in-source clusters and fragments detected for known analytes of interest. When the settings of parameters differing in intensities were applied on a set of 39 samples to discriminate the samples through principal component analyses (PCA), similar results were obtained with both low- and high-intensity thresholds. We conclude that the most information-rich datasets can be obtained by setting low-intensity thresholds. However, in the cases where only a qualitative comparison of samples with PCA is to be performed, it may be sufficient to set high thresholds and thereby reduce the complexity of the data processing and amount of computational time required.

More Than Just a Weed: An Exploration of the Antimicrobial Activity of Rumex crispus using a Multivariate Data Analysis Approach.

Plants have a long history of use for their medicinal properties. The complexity of botanical extracts presents unique challenges and necessitates the application of innovative approaches to correctly identify and quantify bioactive compounds. For this study, we used untargeted metabolomics to explore the antimicrobial activity of Rumex crispus (yellow dock), a member of the Polygonaceae family used as an herbal remedy for bacterial infections. Ultra-performance liquid chromatography coupled with high resolution mass-spectrometry (UPLC-MS) was used to identify and quantify the known antimicrobial compound emodin. In addition, we used biochemometric approaches to integrate data measuring antimicrobial activity from R. crispus root starting material and fractions against methicillin-resistant Staphylococcus aureus (MRSA) with UPLC-MS data. Our results support the hypothesis that multiple constituents, including the anthraquinone emodin, contribute to the antimicrobial activity of R. crispus against MRSA.

Kratom (Mitragyna speciosa) Validation: Quantitative Analysis of Indole and Oxindole Alkaloids Reveals Chemotypes of Plants and Products.

Many consumers are turning to kratom (Mitragyna speciosa) to self-manage pain and opioid addiction. In the United States, an array of capsules, powders, and loose-leaf kratom products are readily available. Additionally, several online sites supply live kratom plants. A prerequisite to establishing quality control and quality assurance standards for the kratom industry, or understanding how alkaloid levels effect clinical outcomes, is the identification and quantitation of major and minor alkaloid constituents within available products and preparations. To this end, an ultra-high performance liquid chromatography-high resolution mass spectrometry method was developed for the analysis of 8 indole alkaloids (7-hydroxymitragynine, ajmalicine, paynantheine, mitragynine, speciogynine, isopaynantheine, speciociliatine, and mitraciliatine) and 6 oxindole alkaloids (isomitraphylline, isospeciofoleine, speciofoline, corynoxine A, corynoxeine, and rhynchophylline) in US-grown kratom plants and commercial products. These commercial products shared a qualitatively similar alkaloid profile, with 12 - 13 detected alkaloids and high levels of the indole alkaloid mitragynine (13.9 ± 1.1 - 270 ± 24 mg/g). The levels of the other major alkaloids (paynantheine, speciociliatine, speciogynine, mitraciliatine, and isopaynantheine) and the minor alkaloids varied in concentration from product to product. The alkaloid profile of US-grown M. speciosa "Rifat" showed high levels of the indole alkaloid speciogynine (7.94 ± 0.83 - 11.55 ± 0.18 mg/g) and quantifiable levels of isomitraphylline (0.943 ± 0.033 - 1.47 ± 0.18 mg/g). Notably, the alkaloid profile of a US-grown M. speciosa seedling was comparable to the commercial products with a high level of mitragynine (15.01 ± 0.20 mg/g). This work suggests that there are several M. speciosa chemotypes.

Interlaboratory Comparison of Untargeted Mass Spectrometry Data Uncovers Underlying Causes for Variability.

Despite the value of mass spectrometry in modern natural products discovery workflows, it remains very difficult to compare data sets between laboratories. In this study we compared mass spectrometry data for the same sample set from two different laboratories (quadrupole time-of-flight and quadrupole-Orbitrap) and evaluated the similarity between these two data sets in terms of both mass spectrometry features and their ability to describe the chemical composition of the sample set. Somewhat surprisingly, the two data sets, collected with appropriate controls and replication, had very low feature overlap (25.7% of Laboratory A features overlapping 21.8% of Laboratory B features). Our data clearly demonstrate that differences in fragmentation, charge state, and adduct formation in the ionization source are a major underlying cause for these differences. Consistent with other recent literature, these findings challenge the conventional wisdom that electrospray ionization mass spectrometry (ESI-MS) yields a simple one-to-one correspondence between analytes in solution and features in the data set. Importantly, despite low overlap in feature lists, principal component analysis (PCA) generated qualitatively similar PCA plots. Overall, our findings demonstrate that comparing untargeted metabolomics data between laboratories is challenging, but that data sets with low feature overlap can yield the same qualitative description of a sample set using PCA.

Novel Peptide from Commensal Staphylococcus simulans Blocks Methicillin-Resistant Staphylococcus aureus Quorum Sensing and Protects Host Skin from Damage.

Recent studies highlight the abundance of commensal coagulase-negative staphylococci (CoNS) on healthy skin. Evidence suggests that CoNS actively shape the skin immunological and microbial milieu to resist colonization or infection by opportunistic pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), in a variety of mechanisms collectively termed colonization resistance. One potential colonization resistance mechanism is the application of quorum sensing, also called the accessory gene regulator (agr) system, which is ubiquitous among staphylococci. Common and rare CoNS make autoinducing peptides (AIPs) that function as MRSA agr inhibitors, protecting the host from invasive infection. In a screen of CoNS spent media, we found that Staphylococcus simulans, a rare human skin colonizer and frequent livestock colonizer, released potent inhibitors of all classes of MRSA agr signaling. We identified three S. simulans agr classes and have shown intraspecies cross talk between noncognate S. simulans agr types for the first time. The S. simulans AIP-I structure was confirmed, and the novel AIP-II and AIP-III structures were solved via mass spectrometry. Synthetic S. simulans AIPs inhibited MRSA agr signaling with nanomolar potency. S. simulans in competition with MRSA reduced dermonecrotic and epicutaneous skin injury in murine models. The addition of synthetic AIP-I also effectively reduced MRSA dermonecrosis and epicutaneous skin injury in murine models. These results demonstrate potent anti-MRSA quorum sensing inhibition by a rare human skin commensal and suggest that cross talk between CoNS and MRSA may be important in maintaining healthy skin homeostasis and preventing MRSA skin damage during colonization or acute infection.

Identification of adulteration in botanical samples with untargeted metabolomics.

Adulteration remains an issue in the dietary supplement industry, including botanical supplements. While it is common to employ a targeted analysis to detect known adulterants, this is difficult when little is known about the sample set. With this study, untargeted metabolomics using liquid chromatography coupled to ultraviolet-visible spectroscopy (LC-UV) or high-resolution mass spectrometry (LC-MS) was employed to detect adulteration in botanical dietary supplements. A training set was prepared by combining Hydrastis canadensis L. with a known adulterant, Coptis chinensis Franch., in ratios ranging from 5 to 95% adulteration. The metabolomics datasets were analyzed using both unsupervised (principal component analysis and composite score) and supervised (SIMCA) techniques. Palmatine, a known H. canadensis metabolite, was quantified as a targeted analysis comparison. While the targeted analysis was the most sensitive method tested in detecting adulteration, statistical analyses of the untargeted metabolomics datasets detected adulteration of the goldenseal samples, with SIMCA providing the greatest discriminating potential. Graphical abstract.

The Chemistry of Kratom [Mitragyna speciosa]: Updated Characterization Data and Methods to Elucidate Indole and Oxindole Alkaloids.

Two separate commercial products of kratom [Mitragyna speciosa (Korth.) Havil. Rubiaceae] were used to generate reference standards of its indole and oxindole alkaloids. While kratom has been studied for over a century, the characterization data in the literature for many of the alkaloids are either incomplete or inconsistent with modern standards. As such, full 1H and 13C NMR spectra, along with HRESIMS and ECD data, are reported for alkaloids 1-19. Of these, four new alkaloids (7, 11, 17, and 18) were characterized using 2D NMR data, and the absolute configurations of 7, 17, and 18 were established by comparison of experimental and calculated ECD spectra. The absolute configuration for the N(4)-oxide (11) was established by comparison of NMR and ECD spectra of its reduced product with those for compound 7. In total, 19 alkaloids were characterized, including the indole alkaloid mitragynine (1) and its diastereoisomers speciociliatine (2), speciogynine (3), and mitraciliatine (4); the indole alkaloid paynantheine (5) and its diastereoisomers isopaynantheine (6) and epiallo-isopaynantheine (7); the N(4)-oxides mitragynine-N(4)-oxide (8), speciociliatine-N(4)-oxide (9), isopaynantheine-N(4)-oxide (10), and epiallo-isopaynantheine-N(4)-oxide (11); the 9-hydroxylated oxindole alkaloids speciofoline (12), isorotundifoleine (13), and isospeciofoleine (14); and the 9-unsubstituted oxindoles corynoxine A (15), corynoxine B (16), 3-epirhynchophylline (17), 3-epicorynoxine B (18), and corynoxeine (19). With the ability to analyze the spectroscopic data of all of these compounds concomitantly, a decision tree was developed to differentiate these kratom alkaloids based on a few key chemical shifts in the 1H and/or 13C NMR spectra.

MroQ Is a Novel Abi-Domain Protein That Influences Virulence Gene Expression in Staphylococcus aureus via Modulation of Agr Activity.

Numerous factors have, to date, been identified as playing a role in the regulation of Agr activity in Staphylococcus aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigated the product of SAUSA300_1984 (termed MroQ), a transmembrane Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a USA300 mroQ mutant, we observed a drastic reduction in proteolysis, hemolysis, and pigmentation that was fully complementable. This appears to result from diminished agr activity, as transcriptional analysis revealed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA sequencing data sets for both mroQ and agr mutants revealed a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr-controlling effect; however, this was not wielded at the level of AgrD processing. Virulence assessment demonstrated that both mroQ and agr mutants exhibited increased formation of renal abscesses but decreased skin abscess formation alongside diminished dermonecrosis. Collectively, we present the characterization of a novel agr effector in S. aureus which would appear to be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase.

Orthogonal Method for Double-Bond Placement via Ozone-Induced Dissociation Mass Spectrometry (OzID-MS).

Most often, the structures of secondary metabolites are solved using a suite of NMR techniques. However, there are times when it can be challenging to position double bonds, particularly those that are fully substituted or when there are multiple double bonds in similar chemical environments. Ozone-induced dissociation mass spectrometry (OzID-MS) serves as an orthogonal structure elucidation tool, using predictable fragmentation patterns that are generated after ozonolysis across a carbon-carbon double bond. This technique is finding growing use in the lipidomics community, suggestive of its potential value for secondary metabolites. This methodology was evaluated by confirming the double-bond positions in five fungal secondary metabolites, specifically, ent-sartorypyrone E (1), sartorypyrone A (2), sorbicillin (3), trichodermic acid A (4), and AA03390 (5). This demonstrated its potential with a variety of chemotypes, ranging from polyketides to terpenoids and including those in both conjugated and nonconjugated polyenes. In addition, the potential of using this methodology in the context of a mixture was piloted by studying Aspergillus fischeri, first examining a traditional extract and then sampling a live fungal culture in situ. While the intensity of signals varied from pure compound to extract to in situ, the utility of the technique was preserved.

Prenylated Diresorcinols Inhibit Bacterial Quorum Sensing.

Current treatment options for bacterial infections are dependent on antibiotics that inhibit microbial growth and viability. These approaches result in the evolution of drug-resistant strains of bacteria. An anti-infective strategy that is less likely to lead to the development of resistance is the disruption of quorum sensing mechanisms, which are involved in promoting virulence. The goal of this study was to identify fungal metabolites effective as quorum sensing inhibitors. Three new prenylated diresorcinols (1-3), along with two known compounds, (4 R) -regiolone and decarboxycitrinone, were isolated from a freshwater fungus (Helotiales sp.) from North Carolina. Their structures were assigned on the basis of HRESIMS and NMR experiments. The structure of compound 1 was confirmed via X-ray diffraction analysis, and its absolute configuration was established by TDDFT-ECD and optical rotation calculations. Compounds 1-3 suppressed quorum sensing in a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), with IC50 values ranging from 0.3 to 12.5 µM. These compounds represent potential leads in the development of antivirulence therapeutics.

Signal Biosynthesis Inhibition with Ambuic Acid as a Strategy To Target Antibiotic-Resistant Infections.

There has been major interest by the scientific community in antivirulence approaches against bacterial infections. However, partly due to a lack of viable lead compounds, antivirulence therapeutics have yet to reach the clinic. Here we investigate the development of an antivirulence lead targeting quorum sensing signal biosynthesis, a process that is conserved in Gram-positive bacterial pathogens. Some preliminary studies suggest that the small molecule ambuic acid is a signal biosynthesis inhibitor. To confirm this, we constructed a methicillin-resistant Staphylococcus aureus (MRSA) strain that decouples autoinducing peptide (AIP) production from regulation and demonstrate that AIP production is inhibited in this mutant. Quantitative mass spectrometric measurements show that ambuic acid inhibits signal biosynthesis (50% inhibitory concentration [IC50] of 2.5 ± 0.1 µM) against a clinically relevant USA300 MRSA strain. Quantitative real-time PCR confirms that this compound selectively targets the quorum sensing regulon. We show that a 5-µg dose of ambuic acid reduces MRSA-induced abscess formation in a mouse model and verify its quorum sensing inhibitory activity in vivo Finally, we employed mass spectrometry to identify or confirm the structure of quorum sensing signaling peptides in three strains each of S. aureus and Staphylococcus epidermidis and single strains of Enterococcus faecalis, Listeria monocytogenes, Staphylococcus saprophyticus, and Staphylococcus lugdunensis By measuring AIP production by these strains, we show that ambuic acid possesses broad-spectrum efficacy against multiple Gram-positive bacterial pathogens but does not inhibit quorum sensing in some commensal bacteria. Collectively, these findings demonstrate the promise of ambuic acid as a lead for the development of antivirulence therapeutics.

Biochemometrics for Natural Products Research: Comparison of Data Analysis Approaches and Application to Identification of Bioactive Compounds.

A central challenge of natural products research is assigning bioactive compounds from complex mixtures. The gold standard approach to address this challenge, bioassay-guided fractionation, is often biased toward abundant, rather than bioactive, mixture components. This study evaluated the combination of bioassay-guided fractionation with untargeted metabolite profiling to improve active component identification early in the fractionation process. Key to this methodology was statistical modeling of the integrated biological and chemical data sets (biochemometric analysis). Three data analysis approaches for biochemometric analysis were compared, namely, partial least-squares loading vectors, S-plots, and the selectivity ratio. Extracts from the endophytic fungi Alternaria sp. and Pyrenochaeta sp. with antimicrobial activity against Staphylococcus aureus served as test cases. Biochemometric analysis incorporating the selectivity ratio performed best in identifying bioactive ions from these extracts early in the fractionation process, yielding altersetin (3, MIC 0.23 µg/mL) and macrosphelide A (4, MIC 75 µg/mL) as antibacterial constituents from Alternaria sp. and Pyrenochaeta sp., respectively. This study demonstrates the potential of biochemometrics coupled with bioassay-guided fractionation to identify bioactive mixture components. A benefit of this approach is the ability to integrate multiple stages of fractionation and bioassay data into a single analysis.

CodY-mediated regulation of the Staphylococcus aureus Agr system integrates nutritional and population density signals.

The Staphylococcus aureus Agr system regulates virulence gene expression by responding to cell population density (quorum sensing). When an extracellular peptide signal (AIP-III in strain UAMS-1, used for these experiments) reaches a concentration threshold, the AgrC-AgrA two-component regulatory system is activated through a cascade of phosphorylation events, leading to induction of the divergently transcribed agrBDCA operon and the RNAIII gene. RNAIII is a posttranscriptional regulator of numerous metabolic and pathogenesis genes. CodY, a global regulatory protein, is known to repress agrBDCA and RNAIII transcription during exponential growth in rich medium, but the mechanism of this regulation has remained elusive. Here we report that phosphorylation of AgrA by the AgrC protein kinase is required for the overexpression of the agrBDCA operon and the RNAIII gene in a codY mutant during the exponential-growth phase, suggesting that the quorum-sensing system, which normally controls AgrC activation, is active even in exponential-phase cells in the absence of CodY. In part, such premature expression of RNAIII was attributable to higher-than-normal accumulation of AIP-III in a codY mutant strain, as determined using ultrahigh-performance liquid chromatography coupled to mass spectrometry. Although CodY is a strong repressor of the agr locus, CodY bound only weakly to the agrBDCA-RNAIII promoter region, suggesting that direct regulation by CodY is unlikely to be the principal mechanism by which CodY regulates agr and RNAIII expression. Taken together, these results strongly suggest that cell population density signals inducing virulence gene expression can be overridden by nutrient availability, a condition monitored by CodY.

Staphylococcus epidermidis agr quorum-sensing system: signal identification, cross talk, and importance in colonization.

Staphylococcus epidermidis is an opportunistic pathogen that is one of the leading causes of medical device infections. Global regulators like the agr quorum-sensing system in this pathogen have received a limited amount of attention, leaving important questions unanswered. There are three agr types in S. epidermidis strains, but only one of the autoinducing peptide (AIP) signals has been identified (AIP-I), and cross talk between agr systems has not been tested. We structurally characterized all three AIP types using mass spectrometry and discovered that the AIP-II and AIP-III signals are 12 residues in length, making them the largest staphylococcal AIPs identified to date. S. epidermidis agr reporter strains were developed for each system, and we determined that cross-inhibitory interactions occur between the agr type I and II systems and between the agr type I and III systems. In contrast, no cross talk was observed between the type II and III systems. To further understand the outputs of the S. epidermidis agr system, an RNAIII mutant was constructed, and microarray studies revealed that exoenzymes (Ecp protease and Geh lipase) and low-molecular-weight toxins were downregulated in the mutant. Follow-up analysis of Ecp confirmed the RNAIII is required to induce protease activity and that agr cross talk modulates Ecp activity in a manner that mirrors the agr reporter results. Finally, we demonstrated that the agr system enhances skin colonization by S. epidermidis using a porcine model. This work expands our knowledge of S. epidermidis agr system function and will aid future studies on cell-cell communication in this important opportunistic pathogen.

Investigations of analyte-specific response saturation and dynamic range limitations in atmospheric pressure ionization mass spectrometry.

With this study, we investigated why some small molecules demonstrate narrow dynamic ranges in electrospray ionization-mass spectrometry (ESI-MS) and sought to establish conditions under which the dynamic range could be extended. Working curves were compared for eight flavonoids and two alkaloids using ESI, atmospheric pressure chemical ionization (APCI), and heated electrospray ionization (HESI) sources. Relative to reserpine, the flavonoids exhibited narrower linear dynamic ranges with ESI-MS, primarily due to saturation in response at relatively low concentrations. Saturation was overcome by switching from ESI to APCI, and our experiments utilizing a combination HESI/APCI source suggest that this is due in part to the ability of APCI to protonate neutral quercetin molecules in the gas phase. Thermodynamic equilibrium calculations indicate that quercetin should be fully protonated in solution, and thus, it appears that some factor inherent in the ESI process favors the formation of neutral quercetin at high concentration. The flavonoid saturation concentration was increased with HESI as compared to ESI, suggesting that inefficient transfer of ions to the gas phase can also contribute to saturation in ESI-MS response. In support of this conclusion, increasing auxiliary gas pressure or switching to a more volatile spray solvent also increased the ESI dynamic range. Among the sources investigated herein, the HESI source achieved the best analytical performance (widest linear dynamic range, lowest LOD), but the APCI source was less subject to saturation in response at high concentration.

SARS-CoV-2 Main Protease Targets Host Selenoproteins and Glutathione Biosynthesis for Knockdown via Proteolysis, Potentially Disrupting the Thioredoxin and Glutaredoxin Redox Cycles.

Associations between dietary selenium status and the clinical outcome of many viral infections, including SARS-CoV-2, are well established. Multiple independent studies have documented a significant inverse correlation between selenium status and the incidence and mortality of COVID-19. At the molecular level, SARS-CoV-2 infection has been shown to decrease the expression of certain selenoproteins, both in vitro and in COVID-19 patients. Using computational methods, our group previously identified a set of six host proteins that contain potential SARS-CoV-2 main protease (Mpro) cleavage sites. Here we show experimentally that Mpro can cleave four of the six predicted target sites, including those from three selenoproteins: thioredoxin reductase 1 (TXNRD1), selenoprotein F, and selenoprotein P, as well as the rate-limiting enzyme in glutathione synthesis, glutamate-cysteine ligase catalytic subunit (GCLC). Cleavage was assessed by incubating recombinant SARS-CoV-2 Mpro with synthetic peptides spanning the proposed cleavage sites, and analyzing the products via UPLC-MS. Furthermore, upon incubation of a recombinant Sec498Ser mutant of the full TXNRD1 protein with SARS-CoV-2 Mpro, the predicted cleavage was observed, destroying the TXNRD1 C-terminal redox center. Mechanistically, proteolytic knockdown of both TXNRD1 and GCLC is consistent with a viral strategy to inhibit DNA synthesis, conserving the pool of ribonucleotides for increased virion production. Viral infectivity could also be enhanced by GCLC knockdown, given the ability of glutathione to disrupt the structure of the viral spike protein via disulfide bond reduction. These findings shed new light on the importance of dietary factors like selenium and glutathione in COVID-19 prevention and treatment.

What was old is new again: Phenotypic screening of a unique fungal library yields pyridoxatin, a promising lead against extensively resistant Acinetobacter baumannii (AB5075).

Due to the emergence of resistance, the World Health Organization considers Gram-negative pathogen Acinetobacter baumannii a top priority for therapeutic development. Using this priority pathogen and a phenotypic, agar plate-based assay, a unique library of extracts from 2,500 diverse fungi was screened for antimicrobial activity against a highly virulent, drug-resistant strain of A. baumannii (AB5075). The most potent hit from this screen was an extract from the fungus Tolypocladium sp., which was found to produce pyridoxatin. Another active extract from the fungi Trichoderma deliquescens was characterized and yielded trichokonin VII and trichokonin VIII. Evaluation of pyridoxatin against A. baumannii (AB5075) in a broth microdilution assay revealed a minimum inhibitory concentration (MIC) of 38 µM, compared to the known antibiotic levofloxacin with MIC of 28 µM. Mass spectrometry, Marfey's analysis and nuclear magnetic resonance spectroscopy analyses confirmed the structures of trichokonins VII and VIII to be consistent with previous reports. In an in vivo Galleria mellonella model, pyridoxatin tested at 150 mg/kg exhibited minimal toxicity (90% survival) and promising antimicrobial efficacy (50% survival) after 5 days. Trichokonins VII and VIII tested at 150 mg/kg were toxic to G. mellonella, with 20% survival and 40% survival after 5 days, respectively. The findings of this project suggest that pyridoxatin may serve as a lead compound for the development of antimicrobials against A. baumannii. They also demonstrate the value of the phenotypic screening approach employed herein.

Metabolomics-Guided Comparison of Pollen and Microalgae-Based Artificial Diets in Honey Bees.

Managed honey bee colonies used for crop pollination are fed artificial diets to offset nutritional deficiencies related to land-use intensification and climate change. In this study, we formulated novel microalgae diets using Chlorella vulgaris and Arthrospira platensis (spirulina) biomass and fed them to young adult honey bee workers. Diet-induced changes in bee metabolite profiles were studied relative to a natural pollen diet using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) metabolomics. Untargeted analyses of pollen- and microalgae-fed bees revealed significant overlap, with 248 shared features determined by LC-MS and 87 shared features determined by GC-MS. Further metabolomic commonalities were evident upon subtraction of unique diet features. Twenty-five identified metabolites were influenced by diet, which included complex lipids, essential fatty acids, vitamins, and phytochemicals. The metabolomics results are useful to understand mechanisms underlying favorable growth performance as well as increased antioxidant and heat shock protein gene expression in bees fed the microalgae diets. We conclude that the tested microalgae have potential as sustainable feed additives and as a source of bee health-modulating natural products. Metabolomics-guided diet development could eventually help tailor feed interventions to achieve precision nutrition in honey bees and other livestock animals.