Toll-like receptor 3 (TLR3) induces host innate immune response on recognition of viral double-stranded RNA (dsRNA). Although several studies of avian TLR3 have been reported, none of these studies used a gene knockout (KO) model to directly assess its role in inducing the immune response and effect on other dsRNA receptors. In this study, we determined the coding sequence of quail TLR3, identified isoforms, and generated TLR3 KO quail fibroblast (QT-35) cells using a CRISPR/Cas9 system optimized for avian species. The TLR3-mediated immune response was studied by stimulating the wild-type (WT) and KO QT-35 cells with synthetic dsRNA or polyinosinic:polycytidylic acid [poly(I:C)] or infecting the cells with different RNA viruses such as influenza A virus, avian reovirus, and vesicular stomatitis virus. The direct poly(I:C) treatment significantly increased IFN-β and IL-8 gene expression along with the cytoplasmic dsRNA receptor, melanoma differentiation-associated gene 5 (MDA5), in WT cells, whereas no changes in all corresponding genes were observed in KO cells. We further confirmed the antiviral effects of poly(I:C)-induced TLR3-mediated immunity by demonstrating significant reduction of virus titer in poly(I:C)-treated WT cells, but not in TLR3 KO cells. On virus infection, varying levels of IFN-β, IL-8, TLR3, and MDA5 gene upregulation were observed depending on the viruses. No major differences in gene expression level were observed between WT and TLR3 KO cells, which suggests a relatively minor role of TLR3 in sensing and exerting immune response against the viruses tested in vitro. Our data show that quail TLR3 is an important endosomal dsRNA receptor responsible for regulation of type I interferon and proinflammatory cytokine, and affect the expression of MDA5, another dsRNA receptor, most likely through cytokine-mediated communication.

Organic fractions and extracts of willow () leaves, produced by sequential solvent extraction as well as infusion and decoction, exhibited anticancer potencies in four cancerous cell lines, including breast (MCF-7), colorectal (HCT-116), cervical (HeLa) and liver (HepG2). Results of the MTT assay revealed that chloroform (CHCl) and ethyl acetate (EtOAc)-soluble fractions exhibited specific anticancer activities as marginal toxicities were observed against two non-cancerous control cell lines (BJ-1 and MCF-12). Ultra-high-resolution mass spectrometry Q-Exactive™ HF Hybrid Quadrupole-Orbitrap™ coupled with liquid chromatography (UHPLC) indicated that both extracts are enriched in features belonging to major phenolic and purine derivatives. Fluorescence-activated cell sorter analysis (FACS), employing annexin V-FITC/PI double staining indicated that the observed cytotoxic potency was mediated via apoptosis. FACS analysis, monitoring the increase in fluorescence signal, associated with oxidation of DCFH to DCF, indicated that the mechanism of apoptosis is independent of reactive oxygen species (ROS). Results of immunoblotting and RT-qPCR assays showed that treatment with organic fractions under investigation resulted in significant up-regulation of pro-apoptotic protein and mRNA markers for Caspase-3, p53 and Bax, whereas it resulted in a significant reduction in amounts of both protein and mRNA of the anti-apoptotic marker Bcl-2. FACS analysis also indicated that pre-treatment and co-treatment of human amniotic epithelial (WISH) cells exposed to the ROS HO with EtOAc fraction provide a cytoprotective and antioxidant capacity against generated oxidative stress. In conclusion, our findings highlight the importance of natural phenolic and flavonoid compounds with unparalleled and unique antioxidant and anticancer properties.

Mitral valve prolapse (MVP) is the most common anomaly of the mitral valve. Several studies have shown prevalence of MVP in atrial septal defect (ASD) especially secundum types (II). The aims of this study is to show the potential role of 3D echocardiography in improving the diagnosis of MVP and to depict the relationship between reverse remodeling of the right and left ventricles (RV, LV) and MVP after transcatheter closure of ASD II. Sixty patients underwent transcatheter closure of ASD II and completed follow up by 2D and 3D echocardiography in Cairo University Children Hospital before the procedure and at 24 hours, 1 and 6 months after the procedure. 3D echocardiography was more accurate than 2D echocardiography in detecting MVP frequency in ASD II patients (75% vs. 50%). Maximum statistically significant remodeling was detected by 3D echocardiography 1 month after the procedure (RV: LV ratio by 3D echocardiography 1.9±0.03 24 hours after the procedure vs. 1.6±0.03 1 months after the procedure, <0.01) while 2D echocardiography was delayed in detecting biventricular reverse remodeling. 3D derived RV: LV ratio was accurate in detecting MVP status with a sensitivity of 88%. MVP in ASD II may be related to Biventricular remodeling; 3D echocardiography is accurate in the detection of reverse remodeling as well as MVP in ASD II patients before and after device closure.