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.

Hydroxyl-Directed Regio- and Diastereoselective Allylic Sulfone Reductions with [Sm(H2O)n]I2.

Allylic 1,2- and 1,3-hydroxy phenyl sulfones undergo regioselective and diastereoselective desulfonylation with double bond migration upon treatment with [Sm(H2O)n]I2. Selectivity in these reactions is thought to arise from the formation of a chelated organosamarium intermediate followed by intramolecular protonation by samarium-bound water, which is supported by observed diastereoselectivity and stereospecificity trends along with deuterium labeling experiments. The reaction was then featured in the synthesis of the phenolic fragment of the thailandamide natural products.

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.

The identification of exceptional skills in school-age autistic children: Prevalence, misconceptions and the alignment of informant perspectives.

BACKGROUND: Although autism is commonly described in terms of deficits, many autistic individuals have been found to demonstrate exceptional skills. The shift to a strengths-based approach in the field of autism necessitates increased understanding of these skills. AIMS: This study examined (1) rates of exceptional skills in autistic school-age children as reported by parents and teachers, (2) associations between exceptional skills, autism severity and intellectual disability and (3) correlations between parent and teacher reports of exceptional skills. METHOD: Parents and teachers of 76 children attending autism-specific schools in Australia completed online questionnaires. Thereafter, 35 parents and teachers who identified their child as having one or more exceptional skills were interviewed by a clinical psychologist. RESULTS: Forty parents (53%) and 16 (21%) teachers reported that their child had at least one exceptional skill (agreement between the parent and teacher reports was low; κ = .03, p = .74). In comparison, clinical psychologist assessments identified 22 children (29%) as having at least one such skill. No statistically significant relationships were identified between exceptional skills, autism severity and intellectual disability. CONCLUSION: While different exceptional skills were identified, regardless of children's intellectual functioning or autism severity, parents and teachers varied substantially in their evaluations of these skills. Furthermore, the identified prevalence rates of exceptional skills did not always align with the rates identified in previous studies. The study findings highlight the need for definitional consensus on different types of exceptional skills, and the importance of multiple criteria/multi-instrument approaches in the identification of exceptional skills in autistic children.

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.

Damned if you monitor, damned if you don't: medical malpractice and intraoperative neuromonitoring for spinal surgery.

OBJECTIVE: The aim of this study was to identify trends in medical malpractice litigation related to intraoperative neuromonitoring. METHODS: The Westlaw Edge legal research service was queried for malpractice litigation related to neuromonitoring in spine surgery. Cases were reviewed to determine if the plaintiff's assertion of negligence was due to either failure to use neuromonitoring or negligent monitoring. Comparative statistics and a detailed qualitative analysis of the resulting cases were performed. RESULTS: Twenty-six cases related to neuromonitoring were identified. Spinal fusion was the procedure in question in all cases, and defendants were nearly evenly divided between orthopedic surgeons and neurosurgeons. Defense verdicts were most common (54%), followed by settlements (27%) and plaintiff verdicts (19%). Settlements resulted in a mean $7,575,000 damage award, while plaintiff verdicts resulted in a mean $4,180,213 damage award. The basis for litigation was failure to monitor in 54% of the cases and negligent monitoring in 46%. There were no significant differences in case outcomes between the two allegations of negligence. CONCLUSIONS: The use and interpretation of intraoperative neuromonitoring findings can be the basis for a medical malpractice litigation. Spine surgeons can face malpractice risks by not monitoring when required by the standard of care and by interpreting or reacting to neuromonitoring findings inappropriately.

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.

Sports-related injuries in New Zealand: National Insurance (Accident Compensation Corporation) claims for five sporting codes from 2012 to 2016.

OBJECTIVES: To provide epidemiological data and related costs for sport-related injuries of five sporting codes (cricket, netball, rugby league, rugby union and football) in New Zealand for moderate-to-serious and serious injury claims. METHODS: A retrospective analytical review using detailed descriptive epidemiological data obtained from the Accident Compensation Corporation (ACC) for 2012-2016. RESULTS: Over the 5 years of study data, rugby union recorded the most moderate-to-serious injury entitlement claims (25 226) and costs (New Zealand dollars (NZD$)267 359 440 (£139 084 749)) resulting in the highest mean cost (NZD$10 484 (£5454)) per moderate-to-serious injury entitlement claim. Rugby union recorded more serious injury entitlement claims (n=454) than cricket (t(4)=-66.6; P<0.0001); netball (t(4)=-45.1; P<0.0001); rugby league (t(4)=-61.4; P<0.0001) and football (t(4)=66.6; P<0.0001) for 2012-2016. There was a twofold increase in the number of female moderate-to-serious injury entitlement claims for football (RR 2.6 (95%CI 2.2 to 2.9); P<0.0001) compared with cricket, and a threefold increase when compared with rugby union (risk ratio (RR) 3.1 (95%CI 2.9 to 3.3); P<0.0001). Moderate-to-serious concussion claims increased between 2012 and 2016 for netball (RR 3.7 (95%CI 1.9 to 7.1); P<0.0001), rugby union (RR 2.0 (95% CI 1.6 to 2.4); P<0.0001) and football (RR 2.3 (95%CI 1.6 to 3.2); P<0.0001). Nearly a quarter of moderate-to-serious entitlement claims (23%) and costs (24%) were to participants aged 35 years or older. CONCLUSIONS: Rugby union and rugby league have the highest total number and costs associated with injury. Accurate sport exposure data are needed to enable injury risk calculations.

Theory and New Applications of Ex Situ Lift Out.

The ex situ lift out (EXLO) adhesion forces are reviewed and new applications of EXLO for focused ion beam (FIB)-prepared specimens are described. EXLO is used to manipulate electron transparent specimens on microelectromechanical systems carrier devices designed for in situ electron microscope analysis. A new patented grid design without a support film is described for EXLO. This new slotted grid design provides a surface for holding the specimen in place and also allows for post lift out processing. Specimens may be easily manipulated into a backside orientation to reduce FIB curtaining artifacts with this slotted grid. Large EXLO specimens can be manipulated from Xe+ plasma FIB prepared specimens. Finally, applications of EXLO and manipulation of FIB specimens using a vacuum probe lift out method are shown. The vacuum probe provides more control for placing specimens on the new slotted grids and also allows for easy manipulation into a backside configuration.

Use of the concussion check protocol for concussion assessment in a female soccer team over two consecutive seasons in New Zealand.

AIM: Address deficiencies in access to sports sideline medical care by using a Concussion Check Protocol (CCP) for non-medically-trained people. METHOD: A prospective observational cohort study was undertaken on a single amateur female club-based soccer team over two consecutive years in New Zealand utilising a non-medically trained support person termed a Safety officer. CCP is an extension of the King-Devick test with features such as warning signs and symptoms of concussion built into the application. All players suspected of having a potential concussive injury were tested on the match sideline. RESULTS: The study overall incidence of match-related concussions was 20.8 (95% CI: 11.8 to 36.6) per 1000 match-hrs, with mean missed-match duration of 31 (95% CI: 27.9 to 34.1) days. Twelve players over the study had a significantly slower post-injury KD (49.9 [44.3 to 64.1]s; χ2(1) = 11.0; p = 0.0009; z = -2.9; p = 0.0033; d = 0.30) and/or reported symptoms, compared with their own baseline (47.2 [44.3 to 64.1]s). CCP had an overall sensitivity of 100% (95% CI: 73.5% to 100.0%), specificity of 100% (95% CI: 69.2% to 100.0%) and positive predictive value (PPV) of 100% (84.6% to 100.0%). CONCLUSION: Sideline use of CCP was undertaken successfully by non-medically trained people and provided a reliable platform for concussion identification.

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.

Thermal and Radiation Stability in Nanocrystalline Cu.

Nanocrystalline metals have presented intriguing possibilities for use in radiation environments due to their high grain boundary volume, serving as enhanced irradiation-induced defect sinks. Their promise has been lessened due to the propensity for nanocrystalline metals to suffer deleterious grain growth from combinations of irradiation and/or elevated homologous temperature. While approaches for stabilizing such materials against grain growth are the subject of current research, there is still a lack of central knowledge on the irradiation-grain boundary interactions in pure metals despite many studies on the same. Due to the breadth of available reports, we have critically reviewed studies on irradiation and thermal stability in pure, nanocrystalline copper (Cu) as a model FCC material, and on a few dilute Cu-based alloys. Our study has shown that, viewed collectively, there are large differences in interpretation of irradiation-grain boundary interactions, primarily due to a wide range of irradiation environments and variability in materials processing. We discuss the sources of these differences and analyses herein. Then, with the goal of gaining a more overarching mechanistic understanding of grain size stability in pure materials under irradiation, we provide several key recommendations for making meaningful evaluations across materials with different processing and under variable irradiation conditions.

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 .

Nature of tackles that result in injury in professional rugby league.

A total of 31,655 tackles in 48 professional rugby league matches were coded from video for height and direction of tackle on the ball carrier. Injuries were recorded by team medical staff for injury date, time, site, type, mechanism, severity, and player position. Tackle-related injuries were most frequently the result of two tacklers being involved in tackling the ball carrier from the side at shoulder or midtorso body levels. The ball carrier had a higher injury rate when tackled from behind his visual field at shoulder height and in the fourth quarter of matches. Tacklers had a higher risk of injury when tackling from the side of the ball carrier, as the first tackler, and in the third quarter of matches. Coaches should focus on practicing correct technique during tackling with two or more tacklers and when tackling in the ball carrier's blind vision area.

Sample Preparation and Experimental Design for In Situ Multi-Beam Transmission Electron Microscopy Irradiation Experiments.

There is a need to understand materials exposed to overlapping extreme environments such as high temperature, radiation, or mechanical stress. When these stressors are combined there may be synergistic effects that enable unique microstructural evolution mechanisms to activate. Understanding of these mechanisms is necessary for the input and refinement of predictive models and critical for engineering of next generation materials. The basic physics and underlying mechanisms require advanced tools to be investigated. The in situ ion irradiation transmission electron microscope (I³TEM) is designed to explore these principles. To quantitatively probe the complex dynamic interactions in materials, careful preparation of samples and consideration of experimental design is required. Particular handling or preparation of samples can easily introduce damage or features that obfuscate the measurements. There is no one correct way to prepare a sample; however, many mistakes can be made. The most common errors and things to consider are highlighted within. The I³TEM has many adjustable variables and a large potential experimental space, therefore it is best to design experiments with a specific scientific question or questions in mind. Experiments have been performed on large number of sample geometries, material classes, and with many irradiation conditions. The following are a subset of examples that demonstrate unique in situ capabilities utilizing the I3TEM. Au nanoparticles prepared by drop casting have been used to investigate the effects of single ion strikes. Au thin films have been used in studies on the effects of multibeam irradiation on microstructure evolution. Zr films have been exposed to irradiation and mechanical tension to examine creep. Ag nanopillars were subjected to simultaneous high temperature, mechanical compression, and ion irradiation to study irradiation induced creep as well. These results impact fields including: structural materials, nuclear energy, energy storage, catalysis, and microelectronics in space environments.

Match and training injury incidence in rugby league: A systematic review, pooled analysis, and update on published studies.

In studies reporting rugby league injuries, match injuries varied depending upon participation level. To review and update pooled data estimates for rugby league injury epidemiology and add information for participation levels in match and training environments. A systematic review and pooled analysis for published studies reporting rugby league match and training injuries. Searches were performed in the PubMed, CINHAL, ScienceDirect, Scopus, SPORTDiscus, SpringerLink, and Wiley Online databases. Studies were considered if they reported on rugby league match or training injuries between Jan 1990 to June 2021. Two authors (DK, TC) extracted the study characteristics, numerical data and assessed the article quality, by adhering to the protocol for systematic review of observational studies (MOOSE) and the STrengthening and Reporting of OBservational studies in Epidemiology (STROBE) statement. The 46 studies included a combined exposure of 419,037 h and 18,783 injuries incorporating 158,003 match-hr and 15,706 match injuries (99.4 [95%CI: 97.9-101.0] per 1000 match-hr) and 264,033 training-hr and 3077 training injuries (11.8 [95%CI: 11.4-12.2] per 1000 training-hr). Of included studies, 47.9% utilised a medical attention/treatment injury definition. There was a five-fold difference in injuries for the semi-professional participation level (431.6 per 1000 match-hr) compared with professional (RR: 4.92; p < 0.001) and elite (RR: 3.77; p < 0.001) participation levels. The hooker recorded the highest pooled injury incidence (93.1 per 1000 match-hr). Compared to the 2014 analysis there was a 10-fold increase for head-neck region (RR: 10.7; p < 0.001) injury incidence, and more injuries for the ball carrier (RR: 1.1; p = 0.008) and tackler (RR: 1.2; p = 0.001). There was a three-fold decrease in injury incidence in the first half (RR: 2.9; p < 0.001) and a two-fold decrease in the second half (RR: 2.3; p < 0.001) of matches. While rugby league match and training injury incidence had decreased since 2014, the increase in head injuries, and greater injury rate at the semi-professional level, mean further injury prevention interventions are needed.