Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.     

Pairwise and Bipartite Entanglements of Three Non-Equally Separated Quantum Dots in Plasmonic Nanowaveguide System

Plasmonics - We theoretically investigate properties of the pairwise and bipartite entanglements of three non-equally separated quantum dots (QDs) coupled to one-dimensional plasmonic nanowaveguide...     

WDNM1 is Associated with Differentiation and Apoptosis of Mammary Epithelial Cells

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFα treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFα induced WDNM1 expression showed the NFκB-dependent mechanism since it's expression was abrogated in IκBαM (super-repressor of NFκB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFα-NFκB signal pathway, is associated with HC11 cell apoptosis.     

The molecular mechanism of NELL2 movement and secretion in hippocampal progenitor HiB5 cells

Neural epidermal growth factor-like protein-like 2 (NELL2) is a secreted glycoprotein that is predominantly expressed in the nervous system, but little is known about the intracellular movement and secretion mechanism of this protein. By monitoring the localization and movements of enhanced green fluorescent protein (EGFP)-labeled NELL2 in living cultured hippocampal neuroprogenitor HiB5 cells, we determined the subcellular localization of NELL2 and its intracellular movement and secretion mechanism. Cterminal EGFP-fused NELL2 showed a typical expression pattern of secreted proteins, especially with respect to its localization in the endoplasmic reticulum, Golgi apparatus, and punctate structures. Vesicles containing NELL2 exhibited bidirectional movement in HiB5 cells. The majority of the vesicles (70.1%) moved in an anterograde direction with an average velocity of 0.454 μm/s, whereas some vesicles (28.7%) showed retrograde movement with an average velocity of 0.302 μm/s. The movement patterns of NELL2 vesicles were dependent upon the presence of microtubules in HiB5 cells. Anterograde movement of NELL2 did not lead to a detectable accumulation of NELL2 in the peripheral region of the cell, indicating that it was secreted into the culture medium. We also showed that the N-terminal 29 amino acids of NELL2 were important for secretion of this protein. Taken together, these results strongly suggest that the N-terminal region of NELL2 determines both the pattern of its intracellular expression and transport of NELL2 vesicles by high-velocity movement. Therefore, NELL2 may affect the cellular activity of cells in a paracrine or autocrine manner.     

Sphingosine and FTY720 modulate pacemaking activity in interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca²⁺ activated Cl⁻ channels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720- P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility.     

Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

Reprofiling of pyrimidine-based DAPK1/CSF1R dual inhibitors: identification of 2,5-diamino-4-pyrimidinol derivatives as novel potential anticancer lead compounds

Abstract

Hybridization of reported weakly active antiproliferative hit 5-amino-4-pyrimidinol derivative with 2-anilino-4-phenoxypyrimidines suggests a series of 2,5-diamino-4-pyrimidinol derivatives as potential antiproliferative agents. Few compounds belonging to the proposed series were reported as CSF1R/DAPK1 inhibitors as anti-tauopathies. However, the correlation between CSF1R/DAPK1 signalling pathways and cancer progression provides motives to reprofile them against cancer therapy. The compounds were synthesised, characterized, and evaluated against M-NFS-60 cells and a kinase panel which bolstered predictions of their antiproliferative activity and suggested the involvement of diverse molecular targets. Compound 6e, the most potent in the series, showed prominent broad-spectrum antiproliferative activity inhibiting the growth of hematological, NSCLC, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers by 84.1, 52.79, 72.15, 66.34, 66.48, 51.55, 55.95, 61.85, and 60.87%, respectively. Additionally, it elicited an IC₅₀ value of 1.97?µM against M-NFS-60 cells and good GIT absorption with P_e value of 19.0?±?1.1?×?10^?6?cm/s (PAMPA-GIT). Molecular docking study for 6e with CSF1R and DAPK1 was done to help to understand the binding mode with both kinases. Collectively, compound 6e could be a potential lead compound for further development of anticancer therapies.     

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At low dosage rates, it was more effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.