COVID-19 National Football League (NFL) Injury Analysis: Follow-Up Study.

Background: In 2020, COVID-19 spread across the world and brought normal daily life to a halt, causing the shutdown of nearly everything in order to prevent its spread. The National Football League (NFL) similarly experienced shutdowns and the resulting effects, leaving athletes unable to train in some of the most advanced facilities with many of the best trainers in the world. A previous study, titled COVID-19 Return to Sport: NFL Injury Prevalence Analysis, determined that there was increased injury prevalence during the 2020 season, likely due to decreased physiological adaptations within athletes' bodies as a result of facility shutdowns. Understanding injury epidemiology is vital to the prevention of injuries and the development of return-to-play protocols. Objective: The objective of this study is to perform a follow-up study to COVID-19 Return to Sport: NFL Injury Prevalence Analysis in order to examine the longitudinal effects of the COVID-19 pandemic on injury epidemiology. This study examines if there was a recovery to baseline levels of injuries or if there are still lingering effects from the COVID-19 pandemic-induced spike in injuries. Methods: To determine if there was change in the number of injuries for each season, injury tallies collected from the 17-week-long 2018, 2019, and 2020 NFL regular seasons were compared with those from the 18-week-long 2021 and 2022 NFL regular seasons. A Kruskall-Wallis test with post hoc Dunn analysis was conducted to compare the rate of injuries per team per week between each of the 2018, 2019, 2020, 2021, and 2022 regular seasons. Results: The Kruskall-Wallis test revealed an H statistic of 32.61 (P<.001) for the comparison of the injury rates across the 5 seasons. The post hoc Dunn analysis showed that 2020 had a statistically significant difference when compared with each of the 2018 (P<.001), 2019 (P=.04), 2021 (P=.02), and 2022 (P=.048) seasons. The 2019 season showed no statistical significance when compared with the 2021 (P=.23) and 2022 (P=.13) seasons. Conclusions: The results of this follow-up study, combined with the previous study, show that extended training interruptions stemming from COVID-19 in 2020 induced detraining and led to increased injuries. Additionally, the results of this study show that retraining can occur, resulting in the development of injury protective factors, as injury rates returned to baseline levels after 2020. This is the first large-scale and long-term opportunity to demonstrate the effects of these principles and how they are important to understanding injury epidemiology.

COVID-19 Return to Sport: NFL Injury Prevalence Analysis.

Background: Sport injuries have been common among athletes across the globe for decades and have the potential to disrupt athletic careers, performance, and psyche. Many health professionals and organizations have undertaken injury mitigation strategies to prevent sport injuries through protective equipment, training protocols, and a host of other evidence-based practices. Many of these specialized training methods were disrupted due to protocols to mitigate the spread of COVID-19. This research examines the effects of the COVID-19 pandemic in relation to the prevalence of athletic injuries in the National Football League (NFL). Objective: During the COVID-19 pandemic, NFL teams and athletes across all levels of sport were reported to have reduced training in preparation for their seasons due to protocols to mitigate the spread of COVID-19. This study compares the prevalence of injury during the 2018, 2019, and 2020 NFL seasons, with the aim to determine the potential causes of the differences in injury prevalence. Methods: Official injury reports from each team were counted during the 17-week regular season of each year (2018, 2019, and 2020). The data were analyzed using an unpaired t test to compare the injury prevalence between each of the three seasons. Results: The 2018 season produced a total of 1561 injuries and a mean of 48.8 injuries per team. The 2019 season produced a total of 1897 injuries and a mean of 59.3 injuries per team, while the 2020 season produced a total of 2484 injuries and a mean of 77.6 injuries per team. An unpaired t test was performed using the data to compare the mean number of injuries per team during each of the seasons. Comparison of the 2020 season against the 2019 season showed a statistically significant difference (P<.001); comparison of the 2020 season to the 2018 season found a statistically significant difference (P<.001); and comparison between the 2019 and the 2018 seasons found a statistically significant difference (P=.03). Conclusions: Although the 2019 and 2018 seasons showed a statistically significant difference (P=.03), this difference is not as large when we compare the 2020 seasons versus the 2019 (P<.001) and 2018 (P<.001) seasons. The astronomical increase in injury prevalence during the 2020 season over the previous years raises the possibility that there was a reduced physiological adaptation to stress, due to the limited amount of training as a result of the closure of practice facilities in order to slow the spread of COVID-19.

Ergot Alkaloids (Re)generate New Leads as Antiparasitics.

Praziquantel (PZQ) is a key therapy for treatment of parasitic flatworm infections of humans and livestock, but the mechanism of action of this drug is unresolved. Resolving PZQ-engaged targets and effectors is important for identifying new druggable pathways that may yield novel antiparasitic agents. Here we use functional, genetic and pharmacological approaches to reveal that serotonergic signals antagonize PZQ action in vivo. Exogenous 5-hydroxytryptamine (5-HT) rescued PZQ-evoked polarity and mobility defects in free-living planarian flatworms. In contrast, knockdown of a prevalently expressed planarian 5-HT receptor potentiated or phenocopied PZQ action in different functional assays. Subsequent screening of serotonergic ligands revealed that several ergot alkaloids possessed broad efficacy at modulating regenerative outcomes and the mobility of both free living and parasitic flatworms. Ergot alkaloids that phenocopied PZQ in regenerative assays to cause bipolar regeneration exhibited structural modifications consistent with serotonergic blockade. These data suggest that serotonergic activation blocks PZQ action in vivo, while serotonergic antagonists phenocopy PZQ action. Importantly these studies identify the ergot alkaloid scaffold as a promising structural framework for designing potent agents targeting parasitic bioaminergic G protein coupled receptors.

Doxycycline as an inhibitor of p-glycoprotein in the alpaca for the purpose of maintaining avermectins in the CNS during treatment for parelaphostrongylosis.

Meningeal worms (Parelaphostrongylus tenuis) are a common malady of alpacas, often refractory to conventional treatments. Ivermectin is a very effective anthelmintic used against a variety of parasites but this drug is not consistently effective against alpaca meningeal worms once the parasite has gained access to the CNS, even if used in a protracted treatment protocol. Ivermectin is not effective against clinical cases of P. tenuis, raising the possibility that the drug is not sustained at therapeutic concentrations in the central nervous system (CNS). A specific protein (designated as p-glycoprotein (PGP)) effluxes ivermectin from the brain at the blood-brain barrier, thus hampering the maintenance of therapeutic concentrations of the drug in the CNS. Minocycline is a synthetic tetracycline antibiotic with an excellent safety profile in all animals tested to date. Minocycline has three unique characteristics that could be useful for treating meningeal worms in conjunction with ivermectin. First, minocycline is an inhibitor of PGP at the blood-brain barrier and this inhibition could maintain effective concentrations of ivermectin in the brain and meninges. Second, minocycline protects neurons in vivo through a number of different mechanisms and this neuroprotection could alleviate the potential untoward neurologic effects of meningeal worms. Third, minocycline is a highly lipid-soluble drug, thus facilitating efficient brain penetration. We thus hypothesized that minocycline will maintain ivermectin, or a related avermectin approved in ruminants (abamectin, doramectin, or eprinomectin), in the alpaca CNS. To test this hypothesis, we cloned the gene encoding the alpaca PGP, expressed the alpaca PGP in a heterologous expression system involving MDCK cells, and measured the ability of minocycline to inhibit the efflux of avermectins from the MDCK cells; doxycycline was used as a putative negative control (based on studies in other species). Our in vitro studies surprisingly revealed that doxycycline was effective at inhibiting the efflux of ivermectin and doramectin (minocycline had no effect). These two avermectins, in combination with doxycycline, should be considered when treating meningeal worms in alpacas.

Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi.

Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30-120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed new therapeutic strategies and unearth stage-specific diagnostic biomarkers.

Functional analysis of Girardia tigrina transcriptome seeds pipeline for anthelmintic target discovery.

BACKGROUND: Neglected diseases caused by helminth infections impose a massive hindrance to progress in the developing world. While basic research on parasitic flatworms (platyhelminths) continues to expand, researchers have yet to broadly adopt a free-living model to complement the study of these important parasites. METHODS: We report the high-coverage sequencing (RNA-Seq) and assembly of the transcriptome of the planarian Girardia tigrina across a set of dynamic conditions. The assembly was annotated and extensive orthology analysis was used to seed a pipeline for the rational prioritization and validation of putative anthelmintic targets. A small number of targets conserved between parasitic and free-living flatworms were comparatively interrogated. RESULTS: 240 million paired-end reads were assembled de novo to produce a strictly filtered predicted proteome consisting of over 22,000 proteins. Gene Ontology annotations were extended to 16,467 proteins. 2,693 sequences were identified in orthology groups spanning flukes, tapeworms and planaria, with 441 highlighted as belonging to druggable protein families. Chemical inhibitors were used on three targets in pharmacological screens using both planaria and schistosomula, revealing distinct motility phenotypes that were shown to correlate with planarian RNAi phenotypes. CONCLUSIONS: This work provides the first comprehensive and annotated sequence resource for the model planarian G. tigrina, alongside a prioritized list of candidate drug targets conserved among parasitic and free-living flatworms. As proof of principle, we show that a simple RNAi and pharmacology pipeline in the more convenient planarian model system can inform parasite biology and serve as an efficient screening tool for the identification of lucrative anthelmintic targets.

'Death and axes': unexpected Ca²âº entry phenologs predict new anti-schistosomal agents.

Schistosomiasis is a parasitic flatworm disease that infects 200 million people worldwide. The drug praziquantel (PZQ) is the mainstay therapy but the target of this drug remains ambiguous. While PZQ paralyses and kills parasitic schistosomes, in free-living planarians PZQ caused an unusual axis duplication during regeneration to yield two-headed animals. Here, we show that PZQ activation of a neuronal Ca²âº channel modulates opposing dopaminergic and serotonergic pathways to regulate 'head' structure formation. Surprisingly, compounds with efficacy for either bioaminergic network in planarians also displayed antischistosomal activity, and reciprocally, agents first identified as antischistocidal compounds caused bipolar regeneration in the planarian bioassay. These divergent outcomes (death versus axis duplication) result from the same Ca²âº entry mechanism, and comprise unexpected Ca²âº phenologs with meaningful predictive value. Surprisingly, basic research into axis patterning mechanisms provides an unexpected route for discovering novel antischistosomal agents.

Effects of intravenous administration of polymyxin B in neonatal foals with experimental endotoxemia.

OBJECTIVE: To evaluate the effect of IV administration of polymyxin B on clinical and serum biochemical variables in foals with experimental endotoxemia. DESIGN: Prospective experimental study. ANIMALS: 14 healthy neonatal foals. PROCEDURES: Foals were randomly assigned to a treatment or control group and were administered a single dose of lipopolysaccharide (0.5 µg/kg [0.23 µg/lb]) IV over 30 minutes. The treatment group received polymyxin B (6,000 U/kg [2,727 U/lb], IV) immediately after completion of lipopolysaccharide infusion; the control group was administered an equal volume of saline (0.9% NaCl) solution. Subsequent doses of polymyxin B or saline solution were administered IV at 8 and 16 hours. Blood was collected at various time points, and outcome variables, including heart rate, respiratory rate, rectal temperature, attitude score, WBC count, neutrophil count, lymphocyte count, monocyte count, platelet count, Hct, blood lactate concentration, blood glucose concentration, serum tumor necrosis factor-α concentration, and plasma thromboxane B2 concentration, were measured. Urine was collected prior to and after experimentation to determine whether nephrotoxicosis was associated with treatment. RESULTS: The treatment group had significantly lower blood lactate concentration and serum tumor necrosis factor-α and plasma thromboxane B2 concentrations and had higher blood glucose concentrations and better attitude scores, compared with the control group, at various time points during the study. No other significant differences and no evidence of overt nephrotoxicosis were detected. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of polymyxin B IV in healthy neonatal foals challenged with lipopolysaccharide attenuated some clinical and serum biochemical derangements associated with endotoxemia.

Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

Novel RNAi-mediated approach to G protein-coupled receptor deorphanization: proof of principle and characterization of a planarian 5-HT receptor.

G protein-coupled receptors (GPCRs) represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi) was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gα(i/o). The method was then extended to deorphanize a novel Gα(s)-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.