SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 ( 4 ) has an IC 50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC 50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC 50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC 50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Sublethal, Behavioral, and Developmental Effects of the Neonicotinoid Pesticide Imidacloprid on Larval Wood Frogs (Rana sylvatica).

Imidacloprid, a neonicotinoid pesticide, is used to prevent the spread of the hemlock woolly adelgid, currently affecting Eastern Hemlock trees across North America. When the pesticide is sprayed directly onto soil around infested trees (soil drenching), it can run off into aquatic systems, with potential negative effects on biota. Simultaneously, climate change may lead to faster pool drying, which acts as an additional stressor for sensitive species such as amphibians. We evaluated the sublethal effects of imidacloprid (10 ppb), and interaction with shorter hydroperiods on the larval behavior, growth, and survival of a model organism, the wood frog (Rana sylvatica). We performed 3 behavioral experiments evaluating swimming speed, time spent swimming, and distance the larvae swam. We found that larvae raised in 10 ppb imidacloprid or shorter hydroperiod did not differ in their swimming time, distance, and speed from nonexposed larvae. Naïve larvae exposed for 20 min to 10- to 500-ppb concentrations also showed similar performance to nonexposed larvae. However, when we applied a stimulus halfway through each experiment, we found that larvae exposed to 10 ppb imidacloprid (short and long term) swam shorter distances and spent less time swimming, suggesting that imidacloprid exposure may slow reaction time, potentially increasing the risk of predation. To minimize impacts on pool-breeding amphibians, imidacloprid application to combat the invasive hemlock woolly adelgid should use trunk injection and avoid soil drenching. Environ Toxicol Chem 2021;40:1840-1849. © 2021 SETAC.

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.

Complex hydroperiod induced carryover responses for survival, growth, and endurance of a pond-breeding amphibian.

Assessing carryover effects from the aquatic to the terrestrial stage of pond-breeding amphibians is critical as temperature and hydrologic regimes of temporary ponds continue to be altered as a result of climate change and other stressors. We evaluated carryover effects of hydroperiod length (50-62 days) on amphibian survival, developmental rates, and locomotor performance using a model organism, the wood frog (Rana sylvatica), through aquatic and terrestrial mesocosm experiments with individual tests of locomotor performance. We found that shorter hydroperiods (50 days) had low larval survival (0.44 ± 0.03) compared to the 62-day hydroperiod (0.91 ± 0.09) and increased developmental rates, resulting in smaller sizes at metamorphosis. We did not find evidence of carryover effects on terrestrial survival three months post-metamorphosis with all hydroperiod treatments showing high terrestrial survival (0.88 ± 0.07). However, post-metamorphic frogs from the longer hydroperiod treatments grew faster and larger compared to individuals from shortest hydroperiods and performed significantly better during endurance trials at 18 °C. Disentangling complex carryover effects across multiple life stages in species with high phenotypic plasticity can shed light on the physiological capacity of species to respond to changing environments and inform mechanistic predictions of persistence in the face of anthropogenic stressors.

RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements.

It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes, but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback and then barcoded for tracking via sequencing (i.e., random deletion library sequencing [RanDeL-seq]). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1's cis and trans elements. The resulting landscape recapitulated HIV-1's known cis-acting elements (i.e., long terminal repeat [LTR], Ψ, and Rev response element [RRE]) and, surprisingly, indicated that HIV-1's central DNA flap (i.e., central polypurine tract [cPPT] to central termination sequence [CTS]) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300-bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV's cis-acting regions are larger than the untranscribed (UTR) termini, encompassing a large fraction of the nonstructural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants, enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections.IMPORTANCE Recent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)-a subset of viral deletion mutants that conditionally replicate. Identifying and engineering DIPs require that viral cis- and trans-acting elements be accurately mapped. Here, we introduce a high-throughput method (random deletion library sequencing [RanDeL-seq]) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central polypurine tract (cPPT), and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.

Possible roles of epigenetics in stem cell therapy for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease with loss of dopaminergic neurons. PD has genetic and epigenetic influences that determine specific changes in the brain. Epigenetic changes result in defective methylation of genes leading to differential gene-expression causing PD. This review provides an overview of stem cell transplantations as potential therapies for PD, with a focus on the epigenetic changes, prior or following transplantation. To date, no reports have addressed epigenetic alterations following stem cell transplantation into the PD brain. Given the potential for affecting the efficacy of stem cell therapy, increased attention needs to be given to the epigenetic processes that occur during stem cell culture and transplantation to maximize the therapeutic potential of stem cells to PD.

A novel approach to label bone marrow-derived mesenchymal stem cells with mixed-surface PAMAM dendrimers.

BACKGROUND: Transplantation of mesenchymal stem cells has created enormous opportunities as a potential treatment for various diseases including neurodegenerative diseases. Given current techniques, such as Hoechst labeling, have safety and leakage issues, our study focused, as a proof-of-concept, on a new dendrimer-based technique for labeling these stem cells to ensure their efficacy and safety following transplantation into the brain of a healthy mice. METHODS AND RESULTS: The bone marrow-derived mesenchymal stem cells (BM-MSCs) were labeled using polyaminoamine (PAMAM) dendrimers following which their stemness based on their proliferation and differentiation ability were analyzed by gold standard methods. These labeled BM-MSCs were transplanted into the striatum of C57BL/6J mice and were tracked using in vivo imaging system (IVIS) and analyzed using tissue imaging, 2 weeks after transplantation. Our results showed that the dendrimer-labeled BM-MSCs were able to successfully maintain their stemness and were tracked in vivo following transplantation. Unlike Hoechst, we did not find the dendrimers to be leaking out of the cells and were very specific to the cells that up took the dendrimers. Moreover, no adverse events were found in the transplanted animals proving that this is a safer method. CONCLUSIONS: Labeling BM-MSCs using fluorescently tagged PAMAM dendrimers can be used as a potentially safe and efficient method for labeling cells, particularly stem cells, in vitro and in vivo following transplantation in rodents.

Altered spinal-level sensorimotor control related to pain and perceived instability in people with chronic ankle instability.

OBJECTIVES: To compare soleus spinal reflex excitability, presynaptic inhibition and recurrent inhibition between chronic ankle instability (CAI), acute Lateral Ankle Sprain coper (LAS-coper) and healthy populations. The relationship between spinal reflex excitability and pain and perceived instability in people with CAI was also examined. DESIGN: Cross-sectional laboratory experiment. METHODS: Twelve individuals with CAI, twelve 'copers' and twelve healthy age, limb and gender-matched controls participated. Soleus H-reflex recruitment curves, pre-synaptic excitability and recurrent inhibition of the spinal-reflex pathway were examined during static double- and single-leg stance. Reporting of pain and perceived instability were used to perform a regression analysis on measures of soleus spinal excitability in people with CAI, LAS-coper and healthy controls. RESULTS: Soleus spinal reflex excitability was greater during single-leg stance in CAI compared to healthy and coper individuals (p=<0.001). Pre-synaptic inhibition was three-times less in CAI participants compared to both healthy controls and copers (p=<0.001). There were no differences between healthy and coper participants in spinal-level measures of sensorimotor control. Reports of pain explained 15-16% of the variance in soleus spinal reflex excitability and presynaptic inhibition during single and double-leg stance, while perceived instability explained 20% of the variance in spinal reflex during single leg stance only. CONCLUSIONS: CAI participants presented with an inability to suppress soleus spinal reflexes during tasks with increased postural threat; likely due to disinhibition of pre-synaptic mechanisms. Pain and perceived instability may contribute to changes in spinal-level sensorimotor control in CAI.

A Post-Transcriptional Feedback Mechanism for Noise Suppression and Fate Stabilization.

Diverse biological systems utilize fluctuations ("noise") in gene expression to drive lineage-commitment decisions. However, once a commitment is made, noise becomes detrimental to reliable function, and the mechanisms enabling post-commitment noise suppression are unclear. Here, we find that architectural constraints on noise suppression are overcome to stabilize fate commitment. Using single-molecule and time-lapse imaging, we find that-after a noise-driven event-human immunodeficiency virus (HIV) strongly attenuates expression noise through a non-transcriptional negative-feedback circuit. Feedback is established through a serial cascade of post-transcriptional splicing, whereby proteins generated from spliced mRNAs auto-deplete their own precursor unspliced mRNAs. Strikingly, this auto-depletion circuitry minimizes noise to stabilize HIV's commitment decision, and a noise-suppression molecule promotes stabilization. This feedback mechanism for noise suppression suggests a functional role for delayed splicing in other systems and may represent a generalizable architecture of diverse homeostatic signaling circuits.

Factors Contributing to Chronic Ankle Instability: A Systematic Review and Meta-Analysis of Systematic Reviews.

BACKGROUND: Many factors are thought to contribute to chronic ankle instability (CAI). Multiple systematic reviews have synthesised the available evidence to identify the primary contributing factors. However, readers are now faced with several systematic reviews that present conflicting findings. OBJECTIVE: The aim of this systematic review and meta-analysis was to establish the statistical significance and effect size of primary factors contributing to CAI and to identify likely reasons for inconsistencies in the literature. METHODS: Relevant health databases were searched: CINAHL, MEDLINE, PubMed, Scopus and SPORTDiscus. Systematic reviews were included if they answered a focused research question, clearly defined the search strategy criteria and study selection/inclusion and completed a comprehensive search of the literature. Included reviews needed to be published in a peer-reviewed journal and needed to review observational studies of factors and/or characteristics of persons with CAI, with or without meta-analysis. There was no language restriction. Studies using a non-systematic review methodology (e.g. primary studies and narrative reviews) were excluded. Methodological quality of systematic reviews was assessed using the modified R-AMSTAR tool. Meta-analysis on included primary studies was performed. RESULTS: Only 17% of primary studies measured a clearly defined CAI population. There is strong evidence to support the contribution of dynamic balance, peroneal reaction time and eversion strength deficits and moderate evidence for proprioception and static balance deficits to non-specific ankle instability. CONCLUSIONS: Evidence from previous systematic reviews does not accurately reflect the CAI population. For treatment of non-specific ankle instability, clinicians should focus on dynamic balance, reaction time and strength deficits; however, these findings may not be translated to the CAI population. Research should be updated with an adequately controlled CAI population. SYSTEMATIC REVIEW REGISTRATION: PROSPERO 2016, CRD42016032592.

Factors contributing to chronic ankle instability: a protocol for a systematic review of systematic reviews.

BACKGROUND: Ankle sprains are a significant clinical problem. Researchers have identified a multitude of factors contributing to the presence of recurrent ankle sprains including deficits in balance, postural control, kinematics, muscle activity, strength, range of motion, ligament laxity and bone/joint characteristics. Unfortunately, the literature examining the presence of these factors in chronic ankle instability (CAI) is conflicting. As a result, researchers have attempted to integrate this evidence using systematic reviews to reach conclusions; however, readers are now faced with an increasing number of systematic review findings that are also conflicting. The overall aim of this review is to critically appraise the methodological quality of previous systematic reviews and pool this evidence to identify contributing factors to CAI. METHODS: A systematic review will be conducted on systematic reviews that investigate the presence of various deficits identified in CAI. Databases will be searched using pre-determined search terms. Reviews will then be assessed for inclusion based on the set eligibility criteria. Two independent reviewers will assess the articles for inclusion before evaluating the methodological quality and presence of bias of the included studies; any disagreements will be resolved by discussion between reviewers to reach consensus or by a third reviewer. Data concerning the specific research question, search strategy, inclusion/exclusion criteria, population, method and outcomes will be extracted. Findings will be analysed with respect to the methodological quality of the included reviews. DISCUSSION: It is expected that this review will clarify the cause of contradicting findings in the literature and facilitate future research directions. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42016032592 .

H-reflex excitability is inhibited in soleus, but not gastrocnemius, at the short-latency response of a horizontal jump-landing task.

Impaired spinal-level neuromuscular control is suggested to contribute to instability and injury during dynamic landing tasks. Despite this suggestion, spinal-level neuromuscular control is yet to be examined during a horizontal jump-landing task. The aim of the current study was to assess changes in H-reflexes and its reliability at the short-latency response of landings from short and long distances. Eight healthy individuals (five male, three female; age, 22±1.2yrs; height, 178±8.1cm; weight, 72±15.7kg) participated in the study. H-reflexes were evoked at the SLR in the soleus and medial gastrocnemius muscles, during two landing conditions: 25% and 50% of maximal broad jump distance. H-reflexes were expressed relative to the background electromyography (EMG) and maximal M-wave responses (M-max). Soleus H-reflexes were inhibited when landing from shorter distance (25%, 13.9±7.6%; 50%, 8.3±6.5%; p<0.01). No change in H-reflex excitability was observed in medial gastrocnemius. Background EMG was unaltered across landing conditions. Inhibition of soleus H-reflex excitability from 25% to 50% landing condition indicates a reduced contribution of Ia-afferent feedback to the alpha-motor neuron during landings from greater distances, which may contribute to stiffness regulation at the ankle joint. Unaltered H-reflex excitability of medial gastrocnemius is most likely attributed to its functional role during the landing task.