Publications by authors named "Yize Li"

94 Publications

Adenovirus prevents dsRNA formation by promoting efficient splicing of viral RNA.

Nucleic Acids Res 2021 Oct 21. Epub 2021 Oct 21.

Division of Protective Immunity, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has long been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants defective for viral RNA processing. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively limit dsRNA formation by promoting efficient splicing and mRNA processing, thus avoiding detection and restriction by host innate immune sensors of pathogenic nucleic acids.
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http://dx.doi.org/10.1093/nar/gkab896DOI Listing
October 2021

Histone Deacetylase Inhibitor Trichostatin A Reduces Endothelial Cell Proliferation by Suppressing STAT5A-Related Gene Transcription.

Front Oncol 2021 23;11:746266. Epub 2021 Sep 23.

Department of Clinical Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.

Inhibitors of histone deacetylases (HDACi) have shown promising effects in preclinical applications for the treatment of many diseases. Confusedly though, the effects of the HDACi trichostatin A (TSA) on angiogenesis are variable among different diseases. This study investigated the direct effects of TSA on endothelial cells, which plays essential roles in angiogenesis and the underlying molecular events. TSA reduced the viability of human umbilical vein endothelial cells (HUVECs), in which proliferation-related genes including , , and were found to be involved. Furthermore, signal transducer and activator of transcription 5 A (STAT5A) was demonstrated to be reduced by TSA and to mediate TSA-induced downregulation of , , and and HUVEC proliferation. Mechanistically, data showed that STAT5A directly bound to the promoters of , , and and activated their transcription through special DNA sequence sites. Finally, the TSA-STAT5A-, , and axis also worked in a cancerous endothelial cell angiogenesis model. The results of this study revealed novel mechanisms underlying the effects of TSA on endothelial cells and provided insights for angiogenesis-associated diseases.
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http://dx.doi.org/10.3389/fonc.2021.746266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8506210PMC
September 2021

Genomic Profiling of Lung Adenocarcinoma in Never-Smokers.

J Clin Oncol 2021 Nov 30;39(33):3747-3758. Epub 2021 Sep 30.

Division of Oncology, Washington University School of Medicine, St Louis, MO.

Purpose: Approximately 10%-40% of patients with lung cancer report no history of tobacco smoking (never-smokers). We analyzed whole-exome and RNA-sequencing data of 160 tumor and normal lung adenocarcinoma (LUAD) samples from never-smokers to identify clinically actionable alterations and gain insight into the environmental and hereditary risk factors for LUAD among never-smokers.

Methods: We performed whole-exome and RNA-sequencing of 88 and 69 never-smoker LUADs. We analyzed these data in conjunction with data from 76 never-smoker and 299 smoker LUAD samples sequenced by The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium.

Results: We observed a high prevalence of clinically actionable driver alterations in never-smoker LUADs compared with smoker LUADs (78%-92% 49.5%; < .0001). Although a subset of never-smoker samples demonstrated germline alterations in DNA repair genes, the frequency of samples showing germline variants in cancer predisposing genes was comparable between smokers and never-smokers (6.4% 6.9%; = .82). A subset of never-smoker samples (5.9%) showed mutation signatures that were suggestive of passive exposure to cigarette smoke. Finally, analysis of RNA-sequencing data showed distinct immune transcriptional subtypes of never-smoker LUADs that varied in their expression of clinically relevant immune checkpoint molecules and immune cell composition.

Conclusion: In this comprehensive genomic and transcriptome analysis of never-smoker LUADs, we observed a potential role for germline variants in DNA repair genes and passive exposure to cigarette smoke in the pathogenesis of a subset of never-smoker LUADs. Our findings also show that clinically actionable driver alterations are highly prevalent in never-smoker LUADs, highlighting the need for obtaining biopsies with adequate cellularity for clinical genomic testing in these patients.
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http://dx.doi.org/10.1200/JCO.21.01691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601276PMC
November 2021

Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2.

Anal Chem 2021 09 20;93(38):13063-13071. Epub 2021 Sep 20.

Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.
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http://dx.doi.org/10.1021/acs.analchem.1c03016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8482318PMC
September 2021

Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Cell 2021 Sep;184(19):5031-5052.e26

Department of Oncology, Johns Hopkins University, Baltimore, MD 21205, USA.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.
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http://dx.doi.org/10.1016/j.cell.2021.08.023DOI Listing
September 2021

Comprehensive characterization of 536 patient-derived xenograft models prioritizes candidatesfor targeted treatment.

Nat Commun 2021 08 24;12(1):5086. Epub 2021 Aug 24.

Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA.

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs' recapitulation of human tumors.
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http://dx.doi.org/10.1038/s41467-021-25177-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384880PMC
August 2021

A proteogenomic portrait of lung squamous cell carcinoma.

Cell 2021 Aug;184(16):4348-4371.e40

Department of Genetics and Genomic Sciences, Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.
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http://dx.doi.org/10.1016/j.cell.2021.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8475722PMC
August 2021

P2Y1 Purinergic Receptor Contributes to Remifentanil-Induced Cold Hyperalgesia via Transient Receptor Potential Melastatin 8-Dependent Regulation of N-methyl-d-aspartate Receptor Phosphorylation in Dorsal Root Ganglion.

Anesth Analg 2021 09;133(3):794-810

From the Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin, People's Republic of China.

Background: Remifentanil can induce postinfusion cold hyperalgesia. N-methyl-d-aspartate receptor (NMDAR) activation and upregulation of transient receptor potential melastatin 8 (TRPM8) membrane trafficking in dorsal root ganglion (DRG) are critical to cold hyperalgesia derived from neuropathic pain, and TRPM8 activation causes NMDAR-dependent cold response. Contribution of P2Y1 purinergic receptor (P2Y1R) activation in DRG to cold pain hypersensitivity and NMDAR activation induced by P2Y1R upregulation in neurons are also unraveled. This study explores whether P2Y1R contributes to remifentanil-induced cold hyperalgesia via TRPM8-dependent regulation of NMDAR phosphorylation in DRG.

Methods: Rats with remifentanil-induced cold hyperalgesia were injected with TRPM8 antagonist or P2Y1R antagonist at 10 minutes before remifentanil infusion. Cold hyperalgesia (paw lift number and withdrawal duration on cold plate) was measured at -24, 2, 6, 24, and 48 hours following remifentanil infusion. After the last behavioral test, P2Y1R expression, TRPM8 expression and membrane trafficking, and NMDAR subunit (NR1 and NR2B) expression and phosphorylation in DRG were detected by western blot, and colocalization of P2Y1R with TRPM8 was determined by double-labeling immunofluorescence. Two-way repeated measures analysis of variance (ANOVA) or 2 × 2 factorial design ANOVA with repeated measures was used to analyze behavioral data of cold hyperalgesia. One-way ANOVA followed by Bonferroni post hoc comparisons was used to analyze the data in western blot and immunofluorescence.

Results: Remifentanil infusion (1 μg·kg-1·min-1 for 60 minutes) induced cold hyperalgesia (hyperalgesia versus control, paw lift number and withdrawal duration on cold plate at 2-48 hours, P < .0001) with upregulated NR1 (hyperalgesia versus naive, 48 hours, mean ± standard deviation [SD], 114.00% ± 12.48% vs 41.75% ± 5.20%, P < .005) and NR2B subunits expression (104.13% ± 8.37% vs 24.63% ± 4.87%, P < .005), NR1 phosphorylation at Ser896 (91.88% ± 7.08% vs 52.00% ± 7.31%, P < .005) and NR2B phosphorylation at Tyr1472 (115.75% ± 8.68% vs 59.75% ± 7.78%, P < .005), TRPM8 expression (115.38% ± 9.27% vs 40.50% ± 4.07%, P < .005) and membrane trafficking (112.88% ± 5.62% vs 48.88% ± 6.49%, P < .005), and P2Y1R expression (128.25% ± 14.86% vs 45.13% ± 7.97%, P < .005) in DRG. Both TRPM8 and P2Y1R antagonists attenuated remifentanil-induced cold hyperalgesia and downregulated increased NR1 and NR2B expression and phosphorylation induced by remifentanil (remifentanil + RQ-00203078 versus remifentanil + saline, NR1 phosphorylation, 69.38% ± 3.66% vs 92.13% ± 4.85%; NR2B phosphorylation, 72.25% ± 6.43% vs 111.75% ± 11.00%, P < .0001). NMDAR activation abolished inhibition of TRPM8 and P2Y1R antagonists on remifentanil-induced cold hyperalgesia. P2Y1R antagonist inhibited remifentanil-evoked elevations in TRPM8 expression and membrane trafficking and P2Y1R-TRPM8 coexpression (remifentanil + 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate [MRS2179] versus remifentanil + saline, coexpression, 8.33% ± 1.33% vs 22.19% ± 2.15%, P < .0001).

Conclusions: Attenuation of remifentanil-induced cold hyperalgesia by P2Y1R inhibition is attributed to downregulations in NMDAR expression and phosphorylation via diminishing TRPM8 expression and membrane trafficking in DRG.
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http://dx.doi.org/10.1213/ANE.0000000000005617DOI Listing
September 2021

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

Genome Biol 2021 06 3;22(1):169. Epub 2021 Jun 3.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Background: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.

Results: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.

Conclusions: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
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http://dx.doi.org/10.1186/s13059-021-02387-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173101PMC
June 2021

Zika virus employs the host antiviral RNase L protein to support replication factory assembly.

Proc Natl Acad Sci U S A 2021 06;118(22)

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.
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http://dx.doi.org/10.1073/pnas.2101713118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179202PMC
June 2021

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes.

Proc Natl Acad Sci U S A 2021 04;118(16)

Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104;

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in knockout A549 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in knockout A549 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.
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http://dx.doi.org/10.1073/pnas.2022643118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072330PMC
April 2021

Activation of STING Signaling Pathway Effectively Blocks Human Coronavirus Infection.

J Virol 2021 05 24;95(12). Epub 2021 May 24.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

The COVID-19 pandemic poses a serious global health threat. The rapid global spread of SARS-CoV-2 highlights an urgent need to develop effective therapeutics for blocking SARS-CoV-2 infection and spread. imulator of terferon enes (STING) is a chief element in host antiviral defense pathways. In this study, we examined the impact of the STING signaling pathway on coronavirus infection using the human coronavirus OC43 (HCoV-OC43) model. We found that HCoV-OC43 infection did not stimulate the STING signaling pathway, but the activation of STING signaling effectively inhibits HCoV-OC43 infection to a much greater extent than that of type I interferons (IFNs). We also discovered that IRF3, the key STING downstream innate immune effector, is essential for this anticoronavirus activity. In addition, we found that the amidobenzimidazole (ABZI)-based human STING agonist diABZI robustly blocks the infection of not only HCoV-OC43 but also SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as a potential therapeutic target that could be exploited for developing broad-spectrum antiviral therapeutics against multiple coronavirus strains in order to face the challenge of future coronavirus outbreaks. The highly infectious and lethal SARS-CoV-2 is posing an unprecedented threat to public health. Other coronaviruses are likely to jump from a nonhuman animal to humans in the future. Novel broad-spectrum antiviral therapeutics are therefore needed to control known pathogenic coronaviruses such as SARS-CoV-2 and its newly mutated variants, as well as future coronavirus outbreaks. STING signaling is a well-established host defense pathway, but its role in coronavirus infection remains unclear. In the present study, we found that activation of the STING signaling pathway robustly inhibits infection of HCoV-OC43 and SARS-CoV-2. These results identified the STING pathway as a novel target for controlling the spread of known pathogenic coronaviruses, as well as emerging coronavirus outbreaks.
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http://dx.doi.org/10.1128/JVI.00490-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316077PMC
May 2021

A Role for Transmembrane Protein 16C/Slack Impairment in Excitatory Nociceptive Synaptic Plasticity in the Pathogenesis of Remifentanil-induced Hyperalgesia in Rats.

Neurosci Bull 2021 May 29;37(5):669-683. Epub 2021 Mar 29.

Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Research Institute of Anesthesiology, Tianjin, 300052, China.

Remifentanil is widely used to control intraoperative pain. However, its analgesic effect is limited by the generation of postoperative hyperalgesia. In this study, we investigated whether the impairment of transmembrane protein 16C (TMEM16C)/Slack is required for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) activation in remifentanil-induced postoperative hyperalgesia. Remifentanil anesthesia reduced the paw withdrawal threshold from 2 h to 48 h postoperatively, with a decrease in the expression of TMEM16C and Slack in the dorsal root ganglia (DRG) and spinal cord. Knockdown of TMEM16C in the DRG reduced the expression of Slack and elevated the basal peripheral sensitivity and AMPAR expression and function. Overexpression of TMEM16C in the DRG impaired remifentanil-induced ERK1/2 phosphorylation and behavioral hyperalgesia. AMPAR-mediated current and neuronal excitability were downregulated by TMEM16C overexpression in the spinal cord. Taken together, these findings suggest that TMEM16C/Slack regulation of excitatory synaptic plasticity via GluA1-containing AMPARs is critical in the pathogenesis of remifentanil-induced postoperative hyperalgesia in rats.
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http://dx.doi.org/10.1007/s12264-021-00652-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099973PMC
May 2021

Hydrogen enriched saline alleviates morphine tolerance via inhibiting neuroinflammation, GLT-1, GS nitration and NMDA receptor trafficking and functioning in the spinal cord of rats.

Neurosci Lett 2021 06 24;755:135847. Epub 2021 Mar 24.

Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin, 300052, PR China; Tianjin Research Institute of Anesthesiology, Tianjin, 300052, PR China. Electronic address:

The development and maintenance of morphine tolerance showed association with neuroinflammation and dysfunction of central glutamatergic system (such as nitration of glutamate transporter). Recent evidence indicated that hydrogen could reduce the levels of neuroinflammation and oxidative stress, but its role in morphine tolerance has not been studied. The rats were intrathecally administered with morphine (10 μg/10 μL each time, twice/day for 5 days). Hydrogen enriched saline (HS) or saline was given intraperitoneally at 1, 3 and 10 mL/kg for 10 min before each dose of morphine administration. The tail-flick latency, mechanical threshold and thermal latency were assessed one day (baseline) before and daily for up to 5 days during morphine injection. The pro-inflammatory cytokine expressions [tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6)] (by western blotting), astrocyte activation (by immunofluorescence and western blotting), and nitration of glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) (by immunoprecipitation), membrane and total expression of N-methyl-d-aspartic acid (NMDA) receptor NR1 and NR2B subunits were carried out in the spinal dorsal horns. Chronic morphine administration induced antinociceptive tolerance, and together led to increased TNF-α, IL-1β and IL-6 expression, astrocyte activation, GLT-1 and GS nitration, increased membrane and total NR1, NR2B expression. Injection of HS attenuated morphine tolerance in a dose-dependent manner, decreased proinflammatory cytokine expression, inhibited astrocyte activation, decreased GLT-1 and GS nitration, and inhibited membrane trafficking of NMDA receptor. Our result showed that hydrogen pretreatment prevented morphine tolerance by reducing neuroinflammation, GLT-1, GS nitration, NMDA receptor trafficking in the spinal dorsal horn. Pretreatment with hydrogen might be considered as a novel therapeutic strategy for the prevention of morphine tolerance.
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http://dx.doi.org/10.1016/j.neulet.2021.135847DOI Listing
June 2021

P2X4 receptor in the dorsal horn contributes to BDNF/TrkB and AMPA receptor activation in the pathogenesis of remifentanil-induced postoperative hyperalgesia in rats.

Neurosci Lett 2021 04 24;750:135773. Epub 2021 Feb 24.

Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Research Institute of Anesthesiology, Tianjin, 300052, China. Electronic address:

The mechanism underlying the high incidence of remifentanil-induced postoperative hyperalgesia is unclear. Also, no effective prevention method exists. Inflammatory pain-related studies showed that P2X4 purinergic receptors (P2X4Rs) in the dorsal horn of the spinal cord and dorsal root ganglia are essential for maintaining allodynia caused by inflammation. However, little is known about its role in opioid-induced hyperalgesia. This study aimed to determine the role of P2X4R and related signaling pathways in the remifentanil-induced postoperative hyperalgesia (RIH) model. The study simulated the remifentanil infusion and surgical incision during general anesthesia. The mRNA and protein expression level of P2X4R in rats with RIH model increased from 2 h to 48 h after the surgery. The administration of P2X4R inhibitors prevented the occurrence of RIH, resulting in a reduction in mechanical and thermal pain. Moreover, P2X4R was involved in RIH in male and female rats, indicating no sex-specific difference. P2X4R also increased the expression of AMPA receptor subunit GluA1 in a brain-derived neurotrophic factor (BDNF) / tyrosine receptor kinase B (TrkB) dependent manner. The results from whole-cell patch-clamp recording suggested that P2X4R also regulated AMPA receptor-mediated miniature excitatory postsynaptic currents and participated in the synaptic plasticity of spinal dorsal horn neurons. In summary, P2X4R was involved in AMPAR expression, electrophysiological function, and synaptic plasticity of spinal dorsal horn neurons through BDNF/TrkB signaling. This might be the mechanism underlying RIH, and hence inhibition of P2X4R might be a potential treatment strategy.
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http://dx.doi.org/10.1016/j.neulet.2021.135773DOI Listing
April 2021

Glucocorticoid counteracts cellular mechanoresponses by LINC01569-dependent glucocorticoid receptor-mediated mRNA decay.

Sci Adv 2021 Feb 24;7(9). Epub 2021 Feb 24.

Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.

Mechanical stimuli on cells and mechanotransduction are essential in many biological and pathological processes. Glucocorticoid is an important hormone, roles, and mechanisms of which in cellular mechanotransduction remain unknown. Here, we report that glucocorticoid counteracted cellular mechanoresponses dependently on a novel long noncoding RNA (lncRNA), Further, mediated glucocorticoid effects on mechanotransduction by destabilizing messenger RNA (mRNA) of mechanosensors including early growth response protein 1 (), Cbp/P300-interacting transactivator 2 (), and bone morphogenic protein 7 () in glucocorticoid receptor-mediated mRNA decay (GMD) manner. Mechanistically, directly bound to the GMD factor Y-box-binding protein 1 (). Then, the complex was guided to the mRNAs of , , and through specific -mRNA interaction, thereby contributing to the successful assembly of GMD complex and triggering GMD. Our results uncovered roles of glucocorticoid in cellular mechanotransduction and novel lncRNA-dependent GMD machinery and provided potential strategy for early intervention in mechanical disorder-associated diseases.
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http://dx.doi.org/10.1126/sciadv.abd9923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904261PMC
February 2021

Proteogenomic and metabolomic characterization of human glioblastoma.

Cancer Cell 2021 04 11;39(4):509-528.e20. Epub 2021 Feb 11.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.

Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM is driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identify specific lipid distributions across subtypes and distinct global metabolic changes in IDH-mutated tumors. This work highlights biological relationships that could contribute to stratification of GBM patients for more effective treatment.
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http://dx.doi.org/10.1016/j.ccell.2021.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044053PMC
April 2021

NR2F1-AS1/miR-140/HK2 Axis Regulates Hypoxia-Induced Glycolysis and Migration in Hepatocellular Carcinoma.

Cancer Manag Res 2021 15;13:427-437. Epub 2021 Jan 15.

Department of Clinical Oncology, Xijing Hospital, Fourth Military Medical University, Xian 710032, People's Republic of China.

Background: Hypoxia is an important feature for the progression of hepatocellular carcinoma (HCC). Long noncoding RNA nuclear receptor subfamily 2 group F member 1 antisense RNA 1 (NR2F1-AS1) is dysregulated in HCC. However, the role and mechanism of N2RF1-AS1 in hypoxia-induced glycolysis and migration remain unclear.

Materials And Methods: Tumor tissues and adjacent samples were harvested from 40 HCC patients. HCC cells were treated by hypoxia. The levels of NR2F1-AS1, microRNA (miR)-140, and hexokinase 2 (HK2) were examined via quantitative reverse transcription polymerase chain reaction or Western blot. Glycolysis was analyzed via glucose uptake, lactate production, and adenosine triphosphate (ATP) levels. Cell migration was analyzed via transwell assay. The target association was analyzed via dual-luciferase reporter assay and RNA immunoprecipitation.

Results: NR2F1-AS1 level was enhanced in HCC tissues and cells. High expression of NR2F1-AS1 indicated poor overall survival. Silence of NR2F1-AS1 repressed hypoxia-induced glycolysis and migration in HCC cells. NR2F1-AS1 could regulate HK2 expression by modulating miR-140. miR-140 down-regulation or HK2 up-regulation mitigated the influence of NR2F1-AS1 silence on hypoxia-induced glycolysis and migration in HCC cells.

Conclusion: NR2F1-AS1 knockdown restrained hypoxia-induced glycolysis and migration in HCC cells via increasing miR-140 and decreasing HK2.
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http://dx.doi.org/10.2147/CMAR.S266797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815091PMC
January 2021

Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape.

Nat Commun 2021 01 20;12(1):469. Epub 2021 Jan 20.

Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, 77030, USA.

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.
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http://dx.doi.org/10.1038/s41467-020-20789-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817669PMC
January 2021

SARS-CoV-2 Genomic Variation in Space and Time in Hospitalized Patients in Philadelphia.

mBio 2021 01 19;12(1). Epub 2021 Jan 19.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients. Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.
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http://dx.doi.org/10.1128/mBio.03456-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829343PMC
January 2021

Spinal NR2B phosphorylation at Tyr1472 regulates IRE(-)DMT1-mediated iron accumulation and spine morphogenesis via kalirin-7 in tibial fracture-associated postoperative pain after orthopedic surgery in female mice.

Reg Anesth Pain Med 2021 04 18;46(4):363-373. Epub 2020 Dec 18.

Anesthesiology, Tianjin Medical University General Hospital, Tianjin, China

Background: Prolonged postoperative pain is a major concern and occurs more frequently in women, but mechanisms remain elusive. NR2B-containging N-methyl-d-aspartate (NMDA) receptor is a key component of nociception transduction. Divalent metal transporter 1 (DMT1)-mediated iron overload involves NMDA-induced neurotoxicity in males. Kalirin-7 is vital in synaptic plasticity underlying pathological pain in males. Herein, the requirement for kalirin-7 in NR2B phosphorylation-dependent iron accumulation and spine plasticity in postoperative pain after tibial fracture in female mice has been examined.

Methods: Pain-related behavior, spinal NR2B phosphorylation at Tyr1472, kalirin-7 expression, DMT1 with/without iron-responsive element (IRE (+) DMT1 and IRE (-) DMT1) level, iron concentration and spine morphology were assessed in females. NR2B antagonist Ro25-6981, iron chelator deferoxamine and kalirin-7 knockdown by short hairpin RNA were employed to assess the potential cascade.

Results: Tibial fracture initiates long-term allodynia lasting at least 21 days postoperatively, and upregulates spinal NR2B phosphorylation, kalirin-7 and IRE (-) DMT1 expression, iron overload and spine density. Ro25-6981 reduces postoperative mechanical and cold allodynia, spinal NR2B phosphorylation, kalirin-7 level and IRE (-) DMT1-mediated iron overload. Kalirin-7 knockdown impairs fracture-associated allodynia, IRE (-) DMT1-mediated iron overload and spine plasticity. Deferoxamine also attenuates behavioral allodynia and spine plasticity. Spinal NMDA application elicits NR2B-dependent mechanical allodynia and iron overload, which is reversed by kalirin-7 knockdown or coadministration of deferoxamine.

Conclusion: Spinal NR2B phosphorylation at Tyr1472 upregulates kalirin-7 expression to facilitate IRE (-) DMT1-mediated iron accumulation and spine morphogenesis in the development of fracture-associated postoperative pain in female mice.
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http://dx.doi.org/10.1136/rapm-2020-101883DOI Listing
April 2021

Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma.

Cancer Cell 2021 03 7;39(3):361-379.e16. Epub 2021 Jan 7.

Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Population Health Science and Policy, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

We present a proteogenomic study of 108 human papilloma virus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs). Proteomic analysis systematically catalogs HNSCC-associated proteins and phosphosites, prioritizes copy number drivers, and highlights an oncogenic role for RNA processing genes. Proteomic investigation of mutual exclusivity between FAT1 truncating mutations and 11q13.3 amplifications reveals dysregulated actin dynamics as a common functional consequence. Phosphoproteomics characterizes two modes of EGFR activation, suggesting a new strategy to stratify HNSCCs based on EGFR ligand abundance for effective treatment with inhibitory EGFR monoclonal antibodies. Widespread deletion of immune modulatory genes accounts for low immune infiltration in immune-cold tumors, whereas concordant upregulation of multiple immune checkpoint proteins may underlie resistance to anti-programmed cell death protein 1 monotherapy in immune-hot tumors. Multi-omic analysis identifies three molecular subtypes with high potential for treatment with CDK inhibitors, anti-EGFR antibody therapy, and immunotherapy, respectively. Altogether, proteogenomics provides a systematic framework to inform HNSCC biology and treatment.
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http://dx.doi.org/10.1016/j.ccell.2020.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946781PMC
March 2021

Spinal caspase-3 contributes to tibial fracture-associated postoperative allodynia via up-regulation of LRRTM1 expression in mice.

Neurosci Lett 2020 11 16;739:135429. Epub 2020 Oct 16.

Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300052, China; Tianjin Research Institute of Anesthesiology, Tianjin 300052, China. Electronic address:

Background: Bone fracture may subsequently cause chronic postoperative pain after orthopedic surgery, but mechanisms remain elusive. The necessity of caspase-3 in neuroinflammation and synaptic plasticity has been summarized in pathological pain. Leucine-rich repeat transmembrane protein 1 (LRRTM1) mediates synaptic delivery of AMPA receptor and synaptogenesis. This study evaluated whether caspase-3 and LRRTM1 are required for fracture-associated postoperative allodynia.

Methods: A model of tibial fracture with intramedullary pinning in mice was established for the induction of postoperative pain, verified by measurement of mechanical paw withdrawal threshold and cold scores response to acetone. The caspase-3 specific inhibitor, recombinant caspase-3 and LRRTM1 knockdown by shRNA were utilized for the investigation of pathogenesis as well as the prevention of allodynia. Also, the activity of caspase-3 and the expression of LRRTM1 in the spinal dorsal horn were examined by Western blot and RT-qPCR.

Results: This study reported that tibial fracture and orthopedic surgery produced long-lasting mechanical allodynia and cold allodynia, along with the up-modulation of spinal caspase-3 activity (but not caspase-3 expression) and LRRTM1 expression. Spinal caspase-3 inhibition prevented fracture-associated behavioral allodynia in a dose-dependent manner. Caspase-3 inhibitor also reduced the spinal increased LRRTM1 level after tibial fracture with pinning. Spinal LRRTM1 deficiency impaired fracture-caused postoperative pain. Intrathecal recombinant caspase-3 facilitated acute pain hypersensitivity and spinal LRRTM1 expression in naïve mice, reversing by LRRTM1 knockdown.

Conclusion: Our current results demonstrate the spinal up-regulation of LRRTM1 by caspase-3 activation in the development of tibial fracture-associated postoperative pain in mice.
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http://dx.doi.org/10.1016/j.neulet.2020.135429DOI Listing
November 2020

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes.

bioRxiv 2020 Nov 2. Epub 2020 Nov 2.

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in knockout A549 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in knockout A549 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

Significance: SARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.09.24.312553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523129PMC
November 2020

Physiologic RNA targets and refined sequence specificity of coronavirus EndoU.

RNA 2020 12 28;26(12):1976-1999. Epub 2020 Sep 28.

Department of Immunology and Microbiology, Program in Molecular Biology, School of Medicine, University of Colorado, Aurora, Colorado 80045, USA.

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U A and C A sequences (endoY A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoU) or 2'-5' phosphodiesterase (PDE) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U A and C A sequences (endoY A) within viral (+) strand RNA to evade dsRNA-activated host responses.
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http://dx.doi.org/10.1261/rna.076604.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668261PMC
December 2020

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples.

Nat Commun 2020 09 21;11(1):4748. Epub 2020 Sep 21.

The McDonnell Genome Institute at Washington University, St. Louis, MO, 63108, USA.

The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts.
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http://dx.doi.org/10.1038/s41467-020-18151-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505971PMC
September 2020

A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency.

Proc Natl Acad Sci U S A 2020 10 21;117(40):24802-24812. Epub 2020 Sep 21.

Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada;

The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.
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http://dx.doi.org/10.1073/pnas.2006883117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547215PMC
October 2020

Broad Anti-coronavirus Activity of Food and Drug Administration-Approved Drugs against SARS-CoV-2 and SARS-CoV .

J Virol 2020 10 14;94(21). Epub 2020 Oct 14.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019 and has rapidly caused a pandemic, with over 20 million recorded COVID-19 cases in August 2020 (https://covid19.who.int/). There are no FDA-approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA-approved drugs. Rapid development and human testing of potential antivirals is urgently needed. Numerous drugs are already approved for human use, and subsequently, there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that 17 of these inhibit SARS-CoV-2 at non-cytotoxic concentrations. We directly followed up seven of these to demonstrate that all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we evaluated two of these, chloroquine and chlorpromazine, using a mouse-adapted SARS-CoV model and found that both drugs protect mice from clinical disease. There are no FDA-approved antivirals for any coronavirus, including SARS-CoV-2. Numerous drugs are already approved for human use that may have antiviral activity and therefore could potentially be rapidly repurposed as antivirals. Here, we present data assessing the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV We found that 17 of these inhibit SARS-CoV-2, suggesting that they may have pan-anti-coronaviral activity. We directly followed up seven of these and found that they all inhibit infectious-SARS-CoV-2 production. Moreover, we evaluated chloroquine and chlorpromazine using mouse-adapted SARS-CoV. We found that neither drug inhibited viral replication in the lungs, but both protected against clinical disease.
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http://dx.doi.org/10.1128/JVI.01218-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565640PMC
October 2020
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