High-throughput functional annotation of natural products by integrated activity profiling.

Determining mechanism of action (MOA) is one of the biggest challenges in natural products discovery. Here, we report a comprehensive platform that uses Similarity Network Fusion (SNF) to improve MOA predictions by integrating data from the cytological profiling high-content imaging platform and the gene expression platform Functional Signature Ontology, and pairs these data with untargeted metabolomics analysis for de novo bioactive compound discovery. The predictive value of the integrative approach was assessed using a library of target-annotated small molecules as benchmarks. Using Kolmogorov-Smirnov (KS) tests to compare in-class to out-of-class similarity, we found that SNF retains the ability to identify significant in-class similarity across a diverse set of target classes, and could find target classes not detectable in either platform alone. This confirmed that integration of expression-based and image-based phenotypes can accurately report on MOA. Furthermore, we integrated untargeted metabolomics of complex natural product fractions with the SNF network to map biological signatures to specific metabolites. Three examples are presented where SNF coupled with metabolomics was used to directly functionally characterize natural products and accelerate identification of bioactive metabolites, including the discovery of the azoxy-containing biaryl compounds parkamycins A and B. Our results support SNF integration of multiple phenotypic screening approaches along with untargeted metabolomics as a powerful approach for advancing natural products drug discovery.

The Natural Products Atlas 2.0: a database of microbially-derived natural products.

Within the natural products field there is an increasing emphasis on the study of compounds from microbial sources. This has been fuelled by interest in the central role that microorganisms play in mediating both interspecies interactions and host-microbe relationships. To support the study of natural products chemistry produced by microorganisms we released the Natural Products Atlas, a database of known microbial natural products structures, in 2019. This paper reports the release of a new version of the database which includes a full RESTful application programming interface (API), a new website framework, and an expanded database that includes 8128 new compounds, bringing the total to 32 552. In addition to these structural and content changes we have added full taxonomic descriptions for all microbial taxa and have added chemical ontology terms from both NP Classifier and ClassyFire. We have also performed manual curation to review all entries with incomplete configurational assignments and have integrated data from external resources, including CyanoMetDB. Finally, we have improved the user experience by updating the Overview dashboard and creating a dashboard for taxonomic origin. The database can be accessed via the new interactive website at https://www.npatlas.org.

Caryophyllene Sesquiterpenes from a Chaetomium globosum Endophyte of the Canadian Medicinal Plant Empetrum nigrum.

Punctaporonins T (1) and U (2), new caryophyllene sesquiterpenes, were isolated with three known punctaporonins, A (3), B (4), and C (5), from the endophytic fungus Chaetomium globosum (TC2-041). The structures and relative configurations of punctaporonins T and U were elucidated based on a combination of HRESIMS, 1D/2D NMR spectroscopic analysis, and X-ray diffraction analysis, while their absolute configuration is presumed to be consistent with the co-isolated 3-5 on biogenetic arguments. Compound 1 showed weak inhibitory activity against both Mycobacterium tuberculosis and Staphylococcus aureus.

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.

Evaluation of Ion Mobility Spectrometry for Improving Constitutional Assignment in Natural Product Mixtures.

The comprehensive chemical characterization of biological samples remains a central challenge in the field of natural products. Conventional workflows using liquid chromatography (LC)-coupled high-resolution tandem mass spectrometry (MS/MS or MS2) allow the detection of relevant small molecules while providing diagnostic fragment ions for their structural assignment. Still, many natural product extracts are of a molecular complexity that challenges the resolving power of modern LC-MS2 pipelines. In this study, we examined the effect of integrating ion mobility spectrometry (IMS) to our LC-MS2 platform for the characterization of natural product mixtures. IMS provides an additional axis of separation in the gas phase as well as experimental collision cross-sectional (CCS) values. We analyzed a mixture of 20 commercial standards at 2 concentration ranges, either solubilized in solvent or spiked into an actinobacterial extract. Data were acquired in positive ion mode using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) MS2 fragmentation approaches and assessed for both chemical coverage and spectral quality. IMS-DIA identified the largest number of standards in the spiked extract at the lower concentration of standards (17), followed by IMS-DDA (10), DDA (8), and DIA (6). In addition, we examined how these data sets performed in the Global Natural Products Social Molecular Networking (GNPS) platform. Overall, integrating IMS increased both metabolite detection and the quality of MS2 spectra, particularly for samples analyzed in DIA mode.

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.

Discovery of an Isothiazolinone-Containing Antitubercular Natural Product Levesquamide.

Antitubercular agent levesquamide is a new polyketide-nonribosomal peptide (PK-NRP) hybrid marine natural product isolated from Streptomyces sp. RKND-216. The structure contains a rare isothiazolinone moiety which has only been reported in collismycin SN. Structure elucidation by NMR spectroscopy was a significant challenge due to a deficiency of protons in this aromatic moiety. Therefore, the genome of Streptomyces sp. RKND-216 was sequenced to identify the levesquamide biosynthetic gene cluster (BGC). Analysis of the BGC provided structural insights and guided stable-isotope labeling experiments, which led to the assignment of the fused pyridine-isothiazolinone moiety. The BGC and the labeling experiments provide further insights into the biosynthetic origin of isothiazolinones. Levesquamide exhibited antimicrobial activity in the microplate alamarBlue assay (MABA) and low oxygen recovery assay (LORA) against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) values of 9.65 and 22.28 µM, respectively. Similar activity was exhibited against rifampicin- and isoniazid-resistant M. tuberculosis strains with MIC values of 9.46 and 9.90 µM, respectively. This result suggests levesquamide has a different mode of action against M. tuberculosis compared to the two first-line antitubercular drugs rifampicin and isoniazid. Furthermore, levesquamide shows no cytotoxicity against the Vero cell line, suggesting it may have a useful therapeutic window.

Physical Demands of Amateur Domestic and Representative Netball in One Season in New Zealand Assessed Using Heart Rate and Movement Analysis.

King, DA, Cummins, C, Hume, PA, and Clark, TN. Physical demands of amateur domestic and representative netball in one season in New Zealand assessed using heart rate and movement analysis. J Strength Cond Res 34(7): 2062-2070, 2020-The purpose of this descriptive cohort study was to describe physical demands of netball for positions and between playing levels using microtechnology. Data were collected from 34 female netball players across 3 teams at different levels (over 19 years representative [O19], under 19 years representative [U19], and open-age amateur club domestic) for 20 games using heart rate and microtechnology data. Total distance, maximal velocity, PlayerLoad ([PL] accumulated accelerometer-derived load), and individual PL vectors (PL forward [PLF], PL sideward [PLS] and PL vertical [PLV]) were examined. Analysis by playing level and netball position were conducted. The O19 players recorded a higher mean distance (3,365.7 ± 1,875.1 m) per match than U19 (p = 0.0095) players. The O19 players recorded a higher PL (p = 0.0003), PLF (p = 0.004), PLS (p = 0.0039), and PLV (p = 0.0352) than the domestic players. Domestic players recorded a higher maximal velocity than O19 players (p = 0.0003; d = 0.32) throughout the study. Domestic players recorded a higher average maximal heart rate (202.2 ± 28.2 b·min) than O19 (p < 0.0001) and U19 (p = 0.0002) players. Given the high physical demands of netball, individual player- and position-specific training programs are required to develop players for the specific demands of competition while also reducing the impact of excessive physical exertion to facilitate safer engagement within netball. The identification of the differing physical and physiological profiles of individual positional groups throughout match-play highlights the importance of integrating microtechnology into the routine monitoring of intermittent court-based sports, such as netball.

Isolation and bioassay screening of medicinal plant endophytes from eastern Canada.

Eighty-one distinct fungal endophytes were isolated from 12 traditionally used medicinal plants from New Brunswick, Canada. This is the first report of endophytes from 8 of the 12 host plants. One hundred and sixty-two crude extracts derived from the mycelia and spent fermentation broths of liquid cultures of each endophyte were screened for antibacterial and antifungal activity. Twenty-two extracts were active against Staphylococcus aureus while 30 were active against Pseudomonas aeruginosa. Twelve crude extracts were found to be active against Candida albicans.

Annotation of natural product compound families using molecular networking topology and structural similarity fingerprinting.

Spectral matching of MS2 fragmentation spectra has become a popular method for characterizing natural products libraries but identification remains challenging due to differences in MS2 fragmentation properties between instruments and the low coverage of current spectral reference libraries. To address this bottleneck we present Structural similarity Network Annotation Platform for Mass Spectrometry (SNAP-MS) which matches chemical similarity grouping in the Natural Products Atlas to grouping of mass spectrometry features from molecular networking. This approach assigns compound families to molecular networking subnetworks without the need for experimental or calculated reference spectra. We demonstrate SNAP-MS can accurately annotate subnetworks built from both reference spectra and an in-house microbial extract library, and correctly predict compound families from published molecular networks acquired on a range of MS instrumentation. Compound family annotations for the microbial extract library are validated by co-injection of standards or isolation and spectroscopic analysis. SNAP-MS is freely available at www.npatlas.org/discover/snapms .

The Incidence, Cost, and Burden of Concussion in Women's Rugby League and Rugby Union: A Systematic Review and Pooled Analysis.

BACKGROUND: The extent of concussion injury in the rugby codes for women is unclear. OBJECTIVE: Our aim was to review all published studies reporting concussion injuries from match and training participation in rugby codes and report the pooled data estimates for rugby league and union concussion injury epidemiology. METHODS: We conducted a systematic literature analysis of concussion in rugby league and rugby union for published studies from January 1990 to July 2021. Data from 16 studies meeting the inclusion criteria were extracted for women's concussion injuries and were subsequently pooled. Costs from Accident Compensation Corporation (ACC) data were attributed to the results to provide cost estimates. RESULTS: The pooled analysis match injury incidence of women's concussion was higher for rugby league (10.3 per 1000 match hours) than rugby 15 s (2.8 per 1000 match hours) or rugby 7 s (8.9 per 1000 match hours). There was a fourfold difference in the pooled incidence of concussion in women's rugby league (risk ratio [RR] 4.53, 95% confidence interval [CI] 1.8-11.3]; p = 0.0001) when compared with rugby 15 s. There was also a ninefold higher risk of a concussion during match participation compared with training participation for women's rugby 15 s (RR 9.3, 95% CI 1.29-66.78; p = 0.0070). The total estimated costs for the concussions reported were NZ$1,235,101. For rugby 7 s, the pooled concussive injury burden was 33.2 days. CONCLUSIONS: Our pooled analysis clarified the extent of concussion injury and the possible associated costs at several levels of the game for women's rugby codes. The pooled mean days lost because of concussions was 33 days. As this was considerably longer than the 7- to 10-day expected timeframe outlined in the Concussion in Sport Consensus statement, these guidelines need to be updated to include sex-specific differences.

The Natural Products Atlas: An Open Access Knowledge Base for Microbial Natural Products Discovery.

Despite rapid evolution in the area of microbial natural products chemistry, there is currently no open access database containing all microbially produced natural product structures. Lack of availability of these data is preventing the implementation of new technologies in natural products science. Specifically, development of new computational strategies for compound characterization and identification are being hampered by the lack of a comprehensive database of known compounds against which to compare experimental data. The creation of an open access, community-maintained database of microbial natural product structures would enable the development of new technologies in natural products discovery and improve the interoperability of existing natural products data resources. However, these data are spread unevenly throughout the historical scientific literature, including both journal articles and international patents. These documents have no standard format, are often not digitized as machine readable text, and are not publicly available. Further, none of these documents have associated structure files (e.g., MOL, InChI, or SMILES), instead containing images of structures. This makes extraction and formatting of relevant natural products data a formidable challenge. Using a combination of manual curation and automated data mining approaches we have created a database of microbial natural products (The Natural Products Atlas, www.npatlas.org) that includes 24 594 compounds and contains referenced data for structure, compound names, source organisms, isolation references, total syntheses, and instances of structural reassignment. This database is accompanied by an interactive web portal that permits searching by structure, substructure, and physical properties. The Web site also provides mechanisms for visualizing natural products chemical space and dashboards for displaying author and discovery timeline data. These interactive tools offer a powerful knowledge base for natural products discovery with a central interface for structure and property-based searching and presents new viewpoints on structural diversity in natural products. The Natural Products Atlas has been developed under FAIR principles (Findable, Accessible, Interoperable, and Reusable) and is integrated with other emerging natural product databases, including the Minimum Information About a Biosynthetic Gene Cluster (MIBiG) repository, and the Global Natural Products Social Molecular Networking (GNPS) platform. It is designed as a community-supported resource to provide a central repository for known natural product structures from microorganisms and is the first comprehensive, open access resource of this type. It is expected that the Natural Products Atlas will enable the development of new natural products discovery modalities and accelerate the process of structural characterization for complex natural products libraries.

Head impact exposure from match participation in women's rugby league over one season of domestic competition.

OBJECTIVES: To quantify the magnitude, frequency, duration and distribution of head impact exposure in a women's rugby league competition. DESIGN: Prospective cohort study. METHODS: Twenty-one players had a wireless impact measuring device (X2Biosystems XPatch) behind their right ear during match participation. Head impact data were collected and downloaded for analysis. Median peak linear and rotational accelerations and impact locations between player positions were assessed using a Friedman repeated measures ANOVA on ranks with a Wilcoxon signed-rank test for post hoc analysis with a Bonferroni correction. RESULTS: A total of 1659 impacts to the head >10g were recorded (range 10g-91g) over the nine competition matches. There was a mean of 184±18 impacts per-match resulting in a mean of 14±12 impacts per-player per-match. The No. 8 prop recorded a mean of 29±27 impacts per-match, the No. 12 second-row forward recorded the highest median peak resultant linear acceleration (16g) per-match and the No. 11 second-row forward recorded the highest median peak resultant rotational acceleration (3696rad/s2). CONCLUSIONS: Our cohort of 21 female rugby league athletes were exposed to repetitive sub-concussive head impact exposure with an average of 14 impacts per-player per-match. Forwards were exposed to more impacts per-match than backs and these impacts were of higher magnitude. Most impacts occurred on the side of the head and were sustained during the second half of the game. Clinicians, coaches and players should be aware of the rates and magnitude of head impacts in female rugby league athletes.

Similar head impact acceleration measured using instrumented ear patches in a junior rugby union team during matches in comparison with other sports.

OBJECTIVE Direct impact with the head and the inertial loading of the head have been postulated as major mechanisms of head-related injuries, such as concussion. METHODS This descriptive observational study was conducted to quantify the head impact acceleration characteristics in under-9-year-old junior rugby union players in New Zealand. The impact magnitude, frequency, and location were collected with a wireless head impact sensor that was worn by 14 junior rugby players who participated in 4 matches. RESULTS A total of 721 impacts > 10g were recorded. The median (interquartile range [IQR]) number of impacts per player was 46 (IQR 37-58), resulting in 10 (IQR 4-18) impacts to the head per player per match. The median impact magnitudes recorded were 15g (IQR 12g-21g) for linear acceleration and 2296 rad/sec(2) (IQR 1352-4152 rad/sec(2)) for rotational acceleration. CONCLUSIONS There were 121 impacts (16.8%) above the rotational injury risk limit and 1 (0.1%) impact above the linear injury risk limit. The acceleration magnitude and number of head impacts in junior rugby union players were higher than those previously reported in similar age-group sports participants. The median linear acceleration for the under-9-year-old rugby players were similar to 7- to 8-year-old American football players, but lower than 9- to 12-year-old youth American football players. The median rotational accelerations measured were higher than the median and 95th percentiles in youth, high school, and collegiate American football players.

Isolation of Phomopsolide A and 6(E)-Phomopsolide A as Antimycobacterial Natural Products from an Unidentified Endophyte of the Canadian Medicinal Plant Heracleum maximum.

An extract of an unidentified endophyte from the Canadian medicinal plant Heracleum maximum exhibited a unique metabolomic profile and significant antimycobacterial activity against Mycobacterium tuberculosis H37Ra. Bioassay guided fractionation of the extract led to the isolation of phomopsolide A (1) and 6(E)-phomopsolide A (2). This is the first report of antimycobacterial activity for 1 and 2.

Synthesis and biological activity of arylspiroborate salts derived from caffeic Acid phenethyl ester.

Two novel boron compounds containing caffeic acid phenethyl ester (CAPE) derivatives have been prepared and characterized fully. These new compounds and CAPE have been investigated for potential antioxidant and antimicrobial properties and their ability to inhibit 5-lipoxygenase and whether chelation to boron improves their biological activity. Sodium salt 4 was generally more active than ammonium salt 5 in the biological assays and surpassed the radical scavenging ability of CAPE. Compounds 4 and 5 were more active than CAPE and Zileuton in human polymorphonuclear leukocytes. These results clearly show the effectiveness of the synthesized salts as transporter of CAPE.

Isolation of (-)-avenaciolide as the antifungal and antimycobacterial constituent of a Seimatosporium sp. Endophyte from the medicinal plant Hypericum perforatum .

An extract of Seimatosporium sp., an endophyte from the Canadian medicinal plant Hypericum perforatum, exhibited significant antifungal and antimycobacterial activity against Candida albicans and Mycobacterium tuberculosis H37Ra. Bioassay guided fractionation led to the isolation of (-)-avenaciolide as the only bioactive constituent of the extract. This is the first report of both the antimycobacterial activity of avenaciolide and its isolation from a Seimatosporium sp. fungus.

Isolation of the plant hormone (+)-abscisic acid as an antimycobacterial constituent of the medicinal plant endophyte Nigrospora sp.

An extract of the endophytic fungus Nigropsora sp. (isolate TC2-054) from the Canadian medicinal plant Fragaria virginiana exhibited significant antimycobacterial activity against Mycobacterium tuberculosis H37Ra. Bioassay guided fractionation revealed that linoleic acid derivatives and the plant hormone (+)-abscisic acid (ABA) were responsible for the observed antimycobacterial activity. This activity of ABA has not been previously reported.