Quercetin inhibits SARS-CoV-2 infection and prevents syncytium formation by cells co-expressing the viral spike protein and human ACE2.

BACKGROUND: Several in silico studies have determined that quercetin, a plant flavonol, could bind with strong affinity and low free energy to SARS-CoV-2 proteins involved in viral entry and replication, suggesting it could block infection of human cells by the virus. In the present study, we examined the ex vivo ability of quercetin to inhibit of SARS-CoV-2 replication and explored the mechanisms of this inhibition. METHODS: Green monkey kidney Vero E6 cells and in human colon carcinoma Caco-2 cells were infected with SARS-CoV-2 and incubated in presence of quercetin; the amount of replicated viral RNA was measured in spent media by RT-qPCR. Since the formation of syncytia is a mechanism of SARS-CoV-2 propagation, a syncytialization model was set up using human embryonic kidney HEK293 co-expressing SARS-CoV-2 Spike (S) protein and human angiotensin converting enzyme 2 (ACE2), [HEK293(S + ACE2) cells], to assess the effect of quercetin on this cytopathic event by microscopic imaging and protein immunoblotting. RESULTS: Quercetin inhibited SARS-CoV-2 replication in Vero E6 cells and Caco-2 cells in a concentration-dependent manner with a half inhibitory concentration (IC50) of 166.6 and 145.2 µM, respectively. It also inhibited syncytialization of HEK293(S + ACE2) cells with an IC50 of 156.7 µM. Spike and ACE2 co-expression was associated with decreased expression, increased proteolytic processing of the S protein, and diminished production of the fusogenic S2' fragment of S. Furin, a proposed protease for this processing, was inhibited by quercetin in vitro with an IC50 of 116 µM. CONCLUSION: These findings suggest that at low 3-digit micromolar concentrations of quercetin could impair SARS-CoV-2 infection of human cells partly by blocking the fusion process that promotes its propagation.

AAV-Vectored Expression of Marburg Virus-Neutralizing Antibody MR191 Provides Complete Protection From Challenge in a Guinea Pig Model.

Although there are no approved countermeasures available to prevent or treat disease caused by Marburg virus (MARV), potently neutralizing monoclonal antibodies (mAbs) derived from B cells of human survivors have been identified. One such mAb, MR191, has been shown to provide complete protection against MARV in nonhuman primates. We previously demonstrated that prophylactic administration of an adeno-associated virus (AAV) expressing MR191 protected mice from MARV. Here, we modified the AAV-MR191 coding sequence to enhance efficacy and reevaluated protection in a guinea pig model. Remarkably, 4 different variants of AAV-MR191 provided complete protection against MARV, despite administration 90 days prior to challenge. Based on superior expression kinetics, AAV-MR191-io2, was selected for evaluation in a dose-reduction experiment. The highest dose provided 100% protection, while a lower dose provided ∼88% protection. These data confirm the efficacy of AAV-mediated expression of MR191 and support the further development of this promising MARV countermeasure.

AAV-monoclonal antibody expression protects mice from Ebola virus without impeding the endogenous antibody response to heterologous challenge.

Filoviruses cause severe hemorrhagic fever with case fatality rates as high as 90%. Filovirus-specific monoclonal antibodies (mAbs) confer protection in nonhuman primates as late as 5 days after challenge, and FDA-approved mAbs REGN-EB3 and mAb114 have demonstrated efficacy against Ebola virus (EBOV) infection in humans. Vectorized antibody expression mediated by adeno-associated virus (AAV) can generate protective and sustained concentrations of therapeutic mAbs in animal models for a variety of infectious diseases, including EBOV. Here we demonstrate that AAV6.2FF-mediated expression of murine IgG2a EBOV mAbs, 2G4 and 5D2, protects from mouse-adapted (MA)-EBOV infection with none of the surviving mice developing anti-VP40 antibodies above background. Protective serum concentrations of AAV6.2FF-2G4/AAV6.2FF-5D2 did not alter endogenous antibody responses to heterologous virus infection. AAV-mediated expression of EBOV mAbs 100 and 114, and pan-ebolavirus mAbs, FVM04, ADI-15878, and CA45, as human IgG1 antibodies conferred protection against MA-EBOV at low serum concentrations, with minimum protective serum levels as low as 2 µg/mL. Vectorized expression of murine IgG2a or human IgG1 mAbs led to sustained expression in the serum of mice for >400 days or for the lifetime of the animal, respectively. AAV6.2FF-mediated mAb expression offers an alternative to recombinant antibody administration in scenarios where long-term protection is preferable to passive immunization.

Adeno-associated virus mediated expression of monoclonal antibody MR191 protects mice against Marburg virus and provides long-term expression in sheep.

Vectored monoclonal antibody (mAb) expression mediated by adeno-associated virus (AAV) gene delivery leads to sustained therapeutic mAb expression and protection against a wide range of infectious diseases in both small and large animal models, including nonhuman primates. Using our rationally engineered AAV6 triple mutant capsid, termed AAV6.2FF, we demonstrate rapid and robust expression of two potent human antibodies against Marburg virus, MR78 and MR191, following intramuscular (IM) administration. IM injection of mice with 1 × 1011 vector genomes (vg) of AAV6.2FF-MR78 and AAV6.2FF-MR191 resulted in serum concentrations of approximately 141 µg/mL and 195 µg/mL of human IgG, respectively, within the first four weeks. Mice receiving 1 × 1011 vg (high) and 1 × 1010 vg (medium) doses of AAV6.2FF-MR191 were completely protected against lethal Marburg virus challenge. No sex-based differences in serum human IgG concentrations were observed; however, administering the AAV-mAb over multiple injection sites significantly increased serum human IgG concentrations. IM administration of three two-week-old lambs with 5 × 1012 vg/kg of AAV6.2FF-MR191 resulted in serum human IgG expression that was sustained for more than 460 days, concomitant with low levels of anti-capsid and anti-drug antibodies. AAV-mAb expression is a viable method for prolonging the therapeutic effect of recombinant mAbs and represents a potential alternative "vaccine" strategy for those with compromised immune systems or in possible outbreak response scenarios.

Adult diffuse hepatic hemangiomatosis: A case report and review of the literature.

Diffuse hepatic hemangiomatosis (DHH) is an extremely rare disease, especially in adults. We present a case of DHH involving the entire liver in a 62-year-old male with a giant hemangioma next to the superior mesenteric vein. Based on what we could find in PubMed with pathological evidence, there are only seventeen cases of adult DHH reported in the literature. The female-to-male ratio is 2.4:1. Most patients consult for abdominal pain or distension. Radiographic examination shows multiple diffuse liver nodules. On MRI, these lesions show hypointense T1 and hyperintense T2. Some lesions may show peripheral rim enhancement in the arterial phase but no portovenous washout. In total, 47% of patients with DHH have one or more giant hemangioma(s). Pathology shows that the lesions are lined with flat endothelial cells without cellular atypia, which are stained positive for vascular endothelial markers. Liver failure is the main cause of death. Some patients can be improved by partial hepatectomy. However, there is no effective treatment for most patients. Liver transplantation should be considered in patients with liver failure or congestive heart failure caused by DHH. We attempt to classify DHH into two types based on the distribution of DHH and their treatment.

Development of an antigen detection assay for early point-of-care diagnosis of Zaire ebolavirus.

The 2013-2016 Ebola virus (EBOV) outbreak in West Africa and the ongoing cases in the Democratic Republic of the Congo have spurred development of a number of medical countermeasures, including rapid Ebola diagnostic tests. The likelihood of transmission increases as the disease progresses due to increasing viral load and potential for contact with others. Early diagnosis of EBOV is essential for halting spread of the disease. Polymerase chain reaction assays are the gold standard for diagnosing Ebola virus disease (EVD), however, they rely on infrastructure and trained personnel that are not available in most resource-limited settings. Rapid diagnostic tests that are capable of detecting virus with reliable sensitivity need to be made available for use in austere environments where laboratory testing is not feasible. The goal of this study was to produce candidate lateral flow immunoassay (LFI) prototypes specific to the EBOV glycoprotein and viral matrix protein, both targets known to be present during EVD. The LFI platform utilizes antibody-based technology to capture and detect targets and is well suited to the needs of EVD diagnosis as it can be performed at the point-of-care, requires no cold chain, provides results in less than twenty minutes and is low cost. Monoclonal antibodies were isolated, characterized and evaluated in the LFI platform. Top performing LFI prototypes were selected, further optimized and confirmed for sensitivity with cultured live EBOV and clinical samples from infected non-human primates. Comparison with a commercially available EBOV rapid diagnostic test that received emergency use approval demonstrates that the glycoprotein-specific LFI developed as a part of this study has improved sensitivity. The outcome of this work presents a diagnostic prototype with the potential to enable earlier diagnosis of EVD in clinical settings and provide healthcare workers with a vital tool for reducing the spread of disease during an outbreak.

Characteristics of acute ischemic stroke in hospitalized patients in Tibet: a retrospective comparative study.

BACKGROUND: Numerous studies on acute ischemic stroke (AIS) have been conducted at low-altitude regions, and the related findings have been used to guide clinical management. However, corresponding studies at high altitude are few. This study aimed to analyse the clinical characteristics of AIS patients at high-altitude regions through a hospital-based comparative study between Tibet and Beijing. METHODS: This study included the diagnoses of AIS patients from People's Hospital of Tibet Autonomous Region (PHOTAR) and Peking University First Hospital (PUFH) between 1 January 2014 and 31 December 2017, where data including patient demographics, treatment time, onset season, risk factors, infarction location, laboratory data, image examination results, treatments, and AIS subtype were collected and compared. Continuous and categorical variables were analysed with a two-sample t-test or Wilcoxon rank sum test and chi-square test, respectively. Significant risk factors were examined with binary logistic regression analysis. RESULTS: In total, 236 and 1021 inpatients from PHOTAR and PUFH were included, respectively. The PHOTAR patients were younger than the PUFH patients (P < 0.001). Young adult stroke, erythrocytosis, and hyperhomocysteinemia were more frequent in PHOTAR patients (all P < 0.001). Other vascular risk factors, including hypertension, diabetes mellitus, hyperlipidaemia, smoking and alcohol consumption history, were less prevalent in PHOTAR patients than in PUFH patients. The rate of intravenous thrombolysis and the rate of within intravenous thrombolysis window time were also lower in PHOTAR patients (both P < 0.001). The PHOTAR group also tended to have anterior circulation infarction. Erythrocytosis and hyperhomocysteinemia were independent risk factors in PHOTAR, and young adults accounted for a larger proportion of stroke cases. CONCLUSION: In Tibet, AIS patients were relatively younger, and anterior circulation infarctions were more common. Erythrocytosis and hyperhomocysteinemia may contribute to these differences. Here, young adult stroke also accounted for a higher proportion, and this may be associated with erythrocytosis. Our findings present the first hospital-based comparative study in Tibet and may contribute to policies for stroke prevention in this region.

Redox-Responsive Transcription Factor 1 (RRFT1) Is Involved in Extracellular ATP-Regulated Arabidopsis thaliana Seedling Growth.

Extracellular adenosine triphosphate (eATP) is an apoplastic signaling molecule that plays an essential role in the growth and development of plants. Arabidopsis seedlings have been reported to respond to eATP; however, the downstream signaling components are still not well understood. In this study, we report that an ethylene-responsive factor, Redox-Responsive Transcription Factor 1 (RRTF1), is involved in eATP-regulated Arabidopsis thaliana seedling growth. Exogenous adenosine triphosphate inhibited green seedling root growth and induced hypocotyl bending of etiolated seedlings. RRTF1 loss-of-function mutant (rrtf1) seedlings showed decreased responses to eATP, while its complementation or overexpression led to recovered or increased eATP responsiveness. RRTF1 was expressed rapidly after eATP stimulation and then migrated into the nuclei of root tip cells. eATP-induced auxin accumulation in root tip or hypocotyl cells was impaired in rrtf1. Chromatin immunoprecipitation and high-throughput sequencing results indicated that eATP induced some genes related to cell growth and development in wild type but not in rrtf1 cells. These results suggest that RRTF1 may be involved in eATP signaling by regulating functional gene expression and cell metabolism in Arabidopsis seedlings.

Identification of a clinical compound losmapimod that blocks Lassa virus entry.

Lassa virus (LASV) causes Lassa hemorrhagic fever in humans and poses a significant threat to public health in West Africa. Current therapeutic treatments for Lassa fever are limited, making the development of novel countermeasures an urgent priority. In this study, we identified losmapimod, a p38 mitogen-activated protein kinase (MAPK) inhibitor, from 102 screened compounds as an inhibitor of LASV infection. Losmapimod exerted its inhibitory effect against LASV after p38 MAPK down-regulation, and, interestingly, had no effect on other arenaviruses capable of causing viral hemorrhagic fever. Mechanistic studies showed that losmapimod inhibited LASV entry by affecting the stable signal peptide (SSP)-GP2 subunit interface of the LASV glycoprotein, thereby blocking pH-dependent viral fusion. As an aryl heteroaryl bis-carboxyamide derivative, losmapimod represents a novel chemical scaffold with anti-LASV activity, and it provides a new lead structure for the future development of LASV fusion inhibitors.

Generation and Characterization of Anti-Filovirus Nucleoprotein Monoclonal Antibodies.

Filoviruses cause lethal hemorrhagic fever in humans. The filovirus nucleoprotein (NP) is expressed in high abundance in infected cells and is essential for virus replication. To generate anti-filovirus monoclonal antibodies (mAbs) against the NP, mice were immunized with peptides known as B-cell epitopes corresponding to different filovirus NPs, and hybridomas were screened using FLAG-tagged filovirus NP constructs. Numerous mAbs were identified, isotyped, and characterized. The anti-NP mAbs demonstrated different ranges of binding affinities to various filovirus NPs. Most of the clones specifically detected both recombinant and wild-type NPs from different filoviruses, including Ebola (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Marburg (MARV), Tai Forest (TAFV), and Reston (RESTV) viruses in western blot analysis. The mAbs were also able to detect native NPs within the cytoplasm of infected cells by immunofluorescence confocal microscopy. Thus, this panel of mAbs represents an important set of tools that may be potentially useful for diagnosing filovirus infection, characterizing virus replication, and detecting NP⁻host protein interactions.

Complete protection of the BALB/c and C57BL/6J mice against Ebola and Marburg virus lethal challenges by pan-filovirus T-cell epigraph vaccine.

There are a number of vaccine candidates under development against a small number of the most common outbreak filoviruses all employing the virus glycoprotein (GP) as the vaccine immunogen. However, antibodies induced by such GP vaccines are typically autologous and limited to the other members of the same species. In contrast, T-cell vaccines offer a possibility to design a single pan-filovirus vaccine protecting against all known and even likely existing, but as yet unencountered members of the family. Here, we used a cross-filovirus immunogen based on conserved regions of the filovirus nucleoprotein, matrix and polymerase to construct simian adenovirus- and poxvirus MVA-vectored vaccines, and in a proof-of-concept study demonstrated a protection of the BALB/c and C57BL/6J mice against high, lethal challenges with Ebola and Marburg viruses, two distant members of the family, by vaccine-elicited T cells in the absence of GP antibodies.

Intramuscular Adeno-Associated Virus-Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice.

The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

Synergistic drug combination effectively blocks Ebola virus infection.

Although a group of FDA-approved drugs were previously identified with activity against Ebola virus (EBOV), most of them are not clinically useful because their human blood concentrations are not high enough to inhibit EBOV infection. We screened 795 unique three-drug combinations in an EBOV entry assay. Two sets of three-drug combinations, toremifene-mefloquine-posaconazole and toremifene-clarithromycin-posaconazole, were identified that effectively blocked EBOV entry and were further validated for inhibition of live EBOV infection. The individual drug concentrations in the combinations were reduced to clinically relevant levels. We identified mechanisms of action of these drugs: functional inhibitions of Niemann-Pick C1, acid sphingomyelinase, and lysosomal calcium release. Our findings identify the drug combinations with potential to treat EBOV infection.

An Adenovirus Vaccine Expressing Ebola Virus Variant Makona Glycoprotein Is Efficacious in Guinea Pigs and Nonhuman Primates.

A licensed vaccine against Ebola virus (EBOV) remains unavailable, despite >11 000 deaths from the 2014-2016 outbreak of EBOV disease in West Africa. Past studies have shown that recombinant vaccine viruses expressing EBOV glycoprotein (GP) are able to protect nonhuman primates (NHPs) from a lethal EBOV challenge. However, these vaccines express the viral GP-based EBOV variants found in Central Africa, which has 97.3% amino acid homology to the Makona variant found in West Africa. Our previous study showed that a recombinant adenovirus serotype 5 (Ad5)-vectored vaccine expressing the Makona EBOV GP (MakGP) was safe and immunogenic during clinical trials in China, but it is unknown whether the vaccine protects against EBOV infection. Here, we demonstrate that guinea pigs immunized with Ad5-MakGP developed robust humoral responses and were protected against exposure to guinea pig-adapted EBOV. Ad5-MakGP also elicited specific B- and T-cell immunity in NHPs and conferred 100% protection when animals were challenged 4 weeks after immunization. These results support further clinical development of this candidate and highlight the utility of Ad5-MakGP as a prophylactic measure in future outbreaks of EBOV disease.

Mitogen- and stress-activated protein kinases 1 and 2 are required for maximal trefoil factor 1 induction.

Mitogen- and stress-activated protein kinases 1 and 2 (MSK1 and MSK2), activated downstream of the ERK- and p38-mitogen-activated protein kinase pathways are involved in cell survival, proliferation and differentiation. Following mitogenic or stress stimuli, they mediate the nucleosomal response, which includes phosphorylation of histone H3 at serine 10 (H3S10ph) coupled with transcriptional activation of immediate-early genes. While MSK1 and MSK2 are closely related, their relative roles may vary with cellular context and/or stimuli. However, our knowledge of MSK2 recruitment to immediate-early genes is limited, as research has primarily focused on MSK1. Here, we demonstrate that both MSK1 and MSK2, regulate the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced expression of the breast cancer marker gene, trefoil factor 1 (TFF1), by phosphorylating H3S10 at its 5' regulatory regions. The MSK-mediated phosphorylation of H3S10 promotes the recruitment of 14-3-3 isoforms and BRG1, the ATPase subunit of the BAF/PBAF remodeling complex, to the enhancer and upstream promoter elements of TFF1. The recruited chromatin remodeling activity leads to the RNA polymerase II carboxy-terminal domain phosphorylation at the TFF1 promoter, initiating TFF1 expression in MCF-7 breast cancer cells. Moreover, we show that MSK1 or MSK2 is recruited to TFF1 regulatory regions, but as components of different multiprotein complexes.

Dynamic distribution of HDAC1 and HDAC2 during mitosis: association with F-actin.

During mitosis, histone deacetylase 2 (HDAC2) becomes highly phosphorylated through the action of CK2, and HDAC1 and 2 are displaced from mitotic chromosomes. HDAC1 and 2 are components of corepressor complexes, which function with lysine acetyltransferases to catalyze dynamic protein acetylation and regulate gene expression. In this study, we show that HDAC1 and 2 associate with F-actin in mitotic cells. Inhibition of Aurora B or protein kinase CK2 did not prevent the displacement of HDAC1 and 2 from mitotic chromosomes in HeLa cells. Further, proteins of the HDAC1 and 2 corepressor complexes and transcription factors recruiting these corepressors to chromatin were dissociated from mitotic chromosomes independent of Aurora B activity. HDAC1 and 2 returned to the nuclei of daughter cells during lamin A/C reassembly and before Sp1, Sp3, and RNA polymerase II. Our results show that HDAC1 and 2 corepressor complexes are removed from the mitotic chromosomes and are available early in the events leading to the re-establishment of the gene expression program in daughter cells.

Histone H3 phosphorylation, immediate-early gene expression, and the nucleosomal response: a historical perspective.

Histone H3 is modified at serines 10 and 28 in interphase cells following activation of the RAS-MAPK or p38-MAPK pathways by growth factors or stress. These modifications are involved in the regulation of immediate-early genes, including Jun and Fos, whose increased expression is a trademark of various cancers. This review outlines the series of discoveries that led to the characterization of these modifications, the kinase, MSK1/2, which is activated by both MAPK pathways and directs phosphorylation of H3, and the mechanistic function of these modifications in transcriptional activation. Research examining the effect of deregulated MSK1/2 in human disorders, namely cancer, is evaluated. Recently, a number of reports proposed novel, intervening pathways leading to enrichment of phosphorylated serine 10 and 28 and the activation of MSK1/2. These novel pathways predict an even more complicated signalling mechanism for cell growth, apoptosis, and the immune response, suggesting that MSK1/2 is intrinsically responsible for an even greater number of biological processes. This review proposes that MSK1/2 is an optimal target for cancer therapy, based on its fundamental role in transmitting external signals into varied responses involved in cancer development.

Role of MSK1 in the malignant phenotype of Ras-transformed mouse fibroblasts.

Activated by the RAS-MAPK signaling pathway, MSK1 is recruited to immediate-early gene (IEG) regulatory regions, where it phosphorylates histone H3 at Ser-10 or Ser-28. Chromatin remodelers and modifiers are then recruited by 14-3-3 proteins, readers of phosphoserine marks, leading to the occupancy of IEG promoters by the initiation-engaged form of RNA polymerase II and the onset of transcription. In this study, we show that this mechanism of IEG induction, initially elucidated in parental 10T1/2 murine fibroblast cells, applies to metastatic Hras1-transformed Ciras-3 cells. As the RAS-MAPK pathway is constitutively activated in Ciras-3 cells, MSK1 activity and phosphorylated H3 steady-state levels are elevated. We found that steady-state levels of the IEG products AP-1 and COX-2 were also elevated in Ciras-3 cells. When MSK1 activity was inhibited or MSK1 expression was knocked down in Ciras-3 cells, the induction of IEG expression and the steady-state levels of COX-2, FRA-1, and JUN were greatly reduced. Furthermore, MSK1 knockdown Ciras-3 cells lost their malignant phenotype, as reflected by the absence of anchorage-independent growth.

Selective association of peroxiredoxin 1 with genomic DNA and COX-2 upstream promoter elements in estrogen receptor negative breast cancer cells.

In a search for proteins differentially cross-linked to DNA by cisplatin or formaldehyde in normal breast epithelial and breast cancer cell lines, we identified peroxiredoxin 1 (PRDX1) as a protein preferentially cross-linked to DNA in estrogen receptor negative (ER-) MDA-MB-231 but not in estrogen receptor positive (ER+) MCF7 breast cancer cells. Indirect immunofluorescence microscopic analyses showed that PRDX1 was located in the cytoplasm and nucleus of normal and breast cancer cells, with nuclear PRDX1 associated with promyelocytic leukemia protein bodies. We demonstrated that PRDX1 association with the transcription factor nuclear factor-kappaB (NF-kappaB) in MDA-MB-231 but not in MCF7 cells contributed to PRDX1-selective recruitment to MDA-MB-231 genomic DNA. Furthermore, PRDX1 was associated with the cyclooxygenase (COX)-2 upstream promoter region at sites occupied by NF-kappaB in ER- but not in ER+ breast cancer cells. PRDX1 knockdown attenuated COX-2 expression by reducing NF-kappaB occupancy at its upstream promoter element in MDA-MB-231 but not in MCF7 cells. A phosphorylated form of PRDX1 was only present in ER- breast cancer cells. Because PRDX1 phosphorylation is known to inhibit its peroxidase activity and to promote PRDX1 oligomerization, we propose that PRDX1 acts as a chaperone to enhance the transactivation potential of NF-kappaB in ER- breast cancer cells.