Antiviral treatments targeting the coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new agents having higher antiviral potentials than the drug candidates such as remdesivir and chroloquine: the anti-inflammatory drug Cepharanthine and HIV protease inhibitor Nelfinavir. Cepharanthine inhibited SARS-CoV-2 entry into cells, whilst Nelfinavir inhibited the catalytic activity of viral main protease to suppress viral replication. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will shorten the period until viral clearance by 5.5-days and the combining Cepharanthine/Nelfinavir enhanced their predicted efficacy to control viral proliferation. In summary, this study identifies a new multidrug combination treatment for COVID-19.Funding: This work was supported by The Agency for Medical Research and Development (AMED) emerging/re-emerging infectious diseases project (JP19fk0108111, JP19fk0108110, JP20fk0108104); the AMED Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS, JP19am0101114, JP19am0101069, JP19am0101111) program; The Japan Society for the Promotion of Science 260 KAKENHI (JP17H04085, JP20H03499, JP15H05707, 19H04839); The JST MIRAI program; and Wellcome Trust funded Investigator award (200838/Z/16/Z). Conflict of Interest: None.
Fractional flow reserve (FFR) measured after percutaneous coronary intervention (PCI) carries prognostic information. Yet, myocardial mass subtended by a stenosis influences FFR. We hypothesized that a smaller coronary lumen volume and a large myocardial mass might be associated with lower post-PCI FFR.We sought to assess the relationship between vessel volume, myocardial mass, and post-PCI FFR.This was a subanalysis with an international prospective study of patients with significant lesions (FFR ≤ 0.80) undergoing PCI. Territory-specific myocardial mass was calculated from coronary computed tomography angiography (CCTA) using the Voronoi's algorithm. Vessel volume was extracted from quantitative CCTA analysis. Resting full-cycle ratio (RFR) and FFR were measured before and after PCI. We assessed the association between coronary lumen volume (V) and its related myocardial mass (M), and the percent of total myocardial mass (%M) with post-PCI FFR.We studied 120 patients (123 vessels: 94 left anterior descending arteries, 13 left Circumflex arteries, 16 right coronary arteries). Mean vessel-specific mass was 61 ± 23.1 g (%M 39.6 ± 11.7%). The mean post-PCI FFR was 0.88 ± 0.06 FFR units. Post-PCI FFR values were lower in vessels subtending higher mass (0.87 ± 0.05 vs. 0.89 ± 0.07, p = 0.047), and with lower V/M ratio (0.87 ± 0.06 vs. 0.89 ± 0.07, p = 0.02). V/M ratio correlated significantly with post-PCI RFR and FFR (RFR r = 0.37, 95% CI: 0.21-0.52, p < 0.001 and FFR r = 0.41, 95% CI: 0.26-0.55, p < 0.001).Post-PCI RFR and FFR are associated with the subtended myocardial mass and the coronary volume to mass ratio. Vessels with higher mass and lower V/M ratio have lower post-PCI RFR and FFR.
ABSTRACT Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.
This study was done to assess the variations of vaginal microbial flora according to the menstrual cycle. In order to be prepared for the study of correlativities of vaginal bacterial flora to the menstrual cycle, relations between vaginal bacterial flora vs. ovarian functions and between microbial flora vs. vaginal pH were studied.The subjects of the study were normal and healthy women who visited the Department of Gynecology at the Central Hospital of Japanese National Railways between October 1977 and September 1979.At first, by discharge samples obtained from the vaginal lateral wall, fornix and endocervix, pH and microbial isolation rates were studied in relation with the sites of sampling. PH was, in the higher to lower order, endocervix, fornix, vaginal lateral wall while the microbial isolation rates were, adversely, vaginal lateral wall, fornix, endocervix. As the types of bacteria were equal in three sites, the vaginal lateral wall was decided as the site of vaginal discharge sampling for the study, as follows.The study consisted of search for relations between the ovarian function and vaginal microbial flora variations at women of regular menstruation in sexually active phase of life, at climacteric women of irregular menstruation and also at postmenopausal women by determining their bacterial isolation rates and vaginal PH, as well as the predominant organism of vaginal microbial flora. The results shoved that the number of bacteria and the number of types of bacteria were both greatly reduced in climacteric and postmenopausal women. The vaginal PH of women in sexually active phase was low, and the predominant microorganism in them was lactobacillus, while in climacteric and postmenopausal women, the vaginal PH became higher and lactobacillus became less predominant. In many cases, no predominant organism could be determined in them.The variations of vaginal PH vs. menstrual cycle and PH vs. microbial flora were not found significantly correlative.The variation of vaginal microbial flora according to the menstrual cycle was studied in view of the following three facters.1) In view of the bacterial isolation rates: The 42 women of normal menstrual cycle were asked to visit the hospital in their first haif of follicular phase, second half of follicular phase, first half of luteal phase and second half of luteal phase for vaginal microbial study, and 168 samples obtained from them were studied. For anaerobic bacteria, the samples obtained were 120 from 30 of the women.The resulted isolation rates almost did not show any significant differences from phase to phase. But only Bacteroides increased in the luteal phase.2) In view of the bacterial amounts: The microbial amounts were studied by giving a score to each bacteria, and its found that Bacteroides increased in the luteal phase, but others did not show any significant differences from phase to phase.3) In view of the positive rates of Doderlein bacilli (Lactobacilli) by vaginal smear: The Doderlein bacilli on the smear were studied by vaginal cytological examination, but no significant variation of them in accordance with the menstrual cycles was recognized.The conclusion from the above study was that the increase or decrease of vaginal microbial flora is under influences of the ovarian function (estrogen) and vaginal PH variations. But there was no variation of them almost to follow the menstrual cycles, but only Bacteroides increase in the luteal phase.
Abstract Background Recently, non-hyperemic physiologic indices have become widespread for evaluating physiological lesion assessment. The resting full-cycle ratio (RFR) is a unique non-hyperemic index which is calculated as the point of absolutely lowest distal pressure to aortic pressure during entire cardiac cycle. It is unclear whether RFR may detect functionally significant coronary stenosis that cannot be detected with other resting indices due to differences in the cardiac cycle. The aim of this study is to compare the diagnostic performance of RFR based on cardiac cycle. Method This study was a prospectively enrolled observational study. A total of 156 consecutive patients with 220 intermediate lesions were enrolled in this study. The RFR was measured after adequately waiting for stable condition, while FFR was measured after intravenous administration of ATP (180mcg/kg/min). Lesions with FFR ≤0.80 were considered functionally significant coronary artery stenosis. Results In all lesions, reference diameter, diameter stenosis, lesion length, RFR, and FFR were 3.0±0.7mm, 45±13%, 13.0±8.8mm, 0.90±0.09, and 0.82±0.10, respectively. Functional significance was observed in 88 lesions (40%) of all lesions. RFR systole was observed in 24 lesions (10.9%). Regarding to the coronary lesions, RFR systole was more frequent in non-LAD (LAD; 4.2%, left circumflex artery (LCX); 9.8%, and right coronary artery (RCA); 30.4%, respectively, p<0.018). RFR showed a significant correlation with FFR in both systole and diastole (R = 0.918, p<0.001, R = 0.733, p<0.001, respectively). The ROC curve analysis showed similar agreement in both systole and diastole (AUC: 0.881, p<0.001, AUC: 0.864, p<0.001, respectively). RFR provided a good diagnostic accuracy and no difference in both systole and diastole (79.6% and 87.5%, respectively, p=0.58). Conclusion RFR is feasible and reliable non-hyperemic index regardless of the difference of cardiac cycle to evaluate physiological lesion severity in daily practice. Funding Acknowledgement Type of funding source: None
A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic of coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded to develop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP. These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able to detect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of SARS-CoV-2 and biological cell response against it.
