Human ACE2 protein is a molecular switch controlling the mode of SARS-CoV-2 transmission.

BACKGROUND: Human angiotensin-converting enzyme 2 (hACE2) is the receptor mediating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. hACE2 expression is low in the lungs and is upregulated after SARS-CoV-2 infection. How such a hACE2-limited pulmonary environment supports efficient virus transmission and how dynamic hACE2 expression affects SARS-CoV-2 infection are unclear. METHODS: We generated stable cell lines with different expression levels of hACE2 to evaluate how the hACE2 expression level can affect SARS-CoV-2 transmission. RESULTS: We demonstrated that the hACE2 expression level controls the mode of SARS-CoV-2 transmission. The hACE2-limited cells have an advantage for SARS-CoV-2 shedding, which leads to cell-free transmission. By contrast, enhanced hACE2 expression facilitates the SARS-CoV-2 cell-to-cell transmission. Furthermore, this cell-to-cell transmission is likely facilitated by hACE2-containing vesicles, which accommodate numerous SARS-CoV-2 virions and transport them to neighboring cells through intercellular extensions. CONCLUSIONS: This hACE2-mediated switch between cell-free and cell-to-cell transmission routes provides SARS-CoV-2 with advantages for either viral spread or evasion of humoral immunity, thereby contributing to the COVID-19 pandemic and pathogenesis.

Effects of a ground-glass opacity component on the recurrence and survival of pathological stage IA3 lung adenocarcinoma: a multi-institutional retrospective study.

Background: This study aimed to evaluate the effect of the presence of a radiographically manifested ground-glass opacity (GGO) component on the prognosis of patients with pathological stage IA3 lung adenocarcinoma. Methods: Patients diagnosed with pathological stage IA3 lung adenocarcinoma who underwent radical surgery at two medical institutions in China between July 2012 and July 2020 were enrolled. The cumulative incidence of recurrence (CIR) and cumulative incidence of death (CID) in patients with and without a GGO component were compared. Risk curves for the recurrence and tumor-related death overtime were analyzed between the two groups according to life table. In order to validate the prognostic value of GGO components, the recurrence-free survival (RFS) and cancer-specific survival (CSS) were estimated. Decision curve analysis (DCA) was performed to evaluate the clinical benefit rate of different models. Results: Among the 352 included patients, the presence of a GGO component was radiographically shown in 166 (47.2%) patients, while 186 (52.8%) displayed solid nodules. Patients exhibiting the absence of a GGO component had higher incidences of total recurrence (17.2% vs. 3.0%, P<0.001), local-regional recurrence (LRR) (5.4% vs. 0.6%, P=0.010), distant metastasis (DM) (8.1% vs. 1.8%, P=0.008), and multiple recurrences (4.3% vs. 0.6%, P=0.028) than the presence-GGO component group. The 5-year CIR and CID were 7.5% and 7.4% in the presence-GGO component group, and 24.5% and 17.0% in the absence-GGO component group, respectively, with statistically significant differences between the two groups (P<0.05). The risk of recurrence in patients with the presence of GGO components showed a single peak at 3 years postoperatively, while patients with the absence of GGO components showed a double peak at 1 and 5 years after surgery, respectively. However, the risk of tumor-related death peaked in both groups at 3 and 6 years postoperatively. Multivariate Cox analysis showed that the presence of a GGO component was a favorable independent risk factor for pathological stage IA3 lung adenocarcinoma patients (P<0.05). Conclusions: Pathological stage IA3 lung adenocarcinoma with or without GGO components are two types of tumors with different invasive abilities. In clinical practice, we should develop different treatment and follow-up strategies.

Anti-influenza virus activity of the REV-ERBα agonist SR9009 and related analogues.

REV-ERBα is a member of the nuclear receptor superfamily of transcription factors that aids in the regulation of many diseases. However, the prospect of using REV-ERBα for anti-influenza virus treatment remains poorly described, and there is an urgent need to develop effective anti-influenza agents due to the emergence of drug-resistant influenza viruses. In this study, eight SR9009 analogues were designed, synthesized, and evaluated for their biological activities against multiple influenza virus strains (H1N1, H3N2, adamantane- and oseltamivir-resistant H1N1 and influenza B virus), using ribavirin as the positive control. SR9009 and its analogues showed low micromolar or submicromolar EC50 values and exhibited modestly improved antiviral potency compared to that of ribavirin. In particular, compound 5a possessed the most potent inhibitory activity (EC50 = 0.471, 0.644, 1.644, 0.712 and 0.661 µM for A/PR/8/34, A/WSN/33, A/Wisconsin/67/2005, B/Yamagata/16/88 and Hebei/SWL1/2006, respectively). Cotransfection assays showed that all synthesized derivatives efficaciously suppressed transcription driven by the Bmal1 promoter. Mechanistic study results indicated that 5a efficiently inhibited IAV replication and interfered with the ealry stage of influenza virus life cycle. In addition, we found that 5a upregulated the key antiviral interferon-stimulated genes MxA, OAS2 and CH25H. Further in-depth transcriptome analysis revealed a series of upregulated genes that may contribute to the antiviral activities of 5a. These findings may provide an important direction for the development of new host-targeted broad-spectrum antiviral agents.

Design, synthesis, and insecticidal activities of propargyloxy-naphthalene-sulfonamide derivatives.

In our previous studies, a kind of novel benzenesulfonamides was found to be a candidate insecticidal compounds. It was shown that propargyloxy and sulfonamide groups are pharmacodynamic groups. One hundred and twenty-six (126) naphthalenesulfonamides derivatives with propargyloxy functionality were designed and synthesized, and their insecticidal activities were determined. Some of them showed outstanding activity, with LC50 values as low as 0.202 mg ml-1, much lower than that of the positive control celangulin V (23.9 mg ml-1). In addition, the structure-activity relationships were discussed, and molecular docking was used to verify the binding mode of the compound and the target receptor.

Synthesis and Insecticidal Activity Evaluation of Virtually Screened Phenylsulfonamides.

The fastest and most effective way to control pests is to use pesticides. However, with the accumulation of pesticide resistance and the difficulties of rapidly producing new pesticides, it is of great significance to create new pesticides through new synthetic methods. In this study, we report a computer-aided drug design (CADD)-assisted method to obtain two lead sulfonamides by homology modeling and virtual screening. On this basis, the lead compounds were synthesized from p-chlorocresol by four steps of esterification, sulfonation, sulfonamidation, and amidation. Further, 71 derivatives were synthesized by optimizing the lead compounds, and their insecticidal activities against Mythimna separata were evaluated by the leaf-dipping method. Notably, seven sulfonamides (5a, 5g, 5h, 5m, 6b, 6g, and 6m) with excellent insecticidal activity were obtained, and the possible binding modes between receptors and active groups in sulfonamides were verified by structure-activity relationship and docking simulation, which provided theoretical support for the subsequent development of these novel candidate insecticides.

Cell-to-Cell Spread of Dengue Viral RNA in Mosquito Cells.

Dengue virus (DENV) is an important mosquito-borne arbovirus that is particularly prevalent in tropical and subtropical areas of the world. The virus is generally ingested with a blood meal, replicates in host tissues, and disseminates into salivary glands for transmission to the next host. Membrane-bound vacuoles carrying DENV particles have been documented in mosquito cells and play a role in the cell-to-cell transmission of DENV2. C189 is one member of the tetraspanin family and generally increases its expression as one component of the vacuoles (C189-VCs) within C6/36 cells infected with DENV2. In the present study, we have further demonstrated via sucrose gradient centrifugation as well as magnetic immune isolation (MI) that the RNA of DENV2 was eventually carried by C189-VCs. In addition, viral RNA was shown to spread from donor to recipient cells in a coculture assay even when 20 mM NH4Cl was added to inhibit virus replication in the culture. In an alternate assay using the transwell system, viral RNA was only detected in recipient cells in the absence of 40 mM NH4Cl, suggesting that cell-cell contact is required for the intercellular spread of DENV2. In turn, the formation of viral synapse (VS) derived from aggregates of viral particles was frequently observed at sites of cell contact. Taken together, the formation of C189-VCs in C6/36 cells is induced by DENV2 infection, which may serve as a vehicle for transferring virions and also viral RNA to neighboring cells by cell-to-cell transmission after cell-cell contact. This finding provides insight into the understanding of viral spread between mosquito cells. It may also elucidate the benign persistent infection in mosquito cells and efficient dissemination of DENV infection within a mosquito vector.

N-Quinary heterocycle-4-sulphamoylbenzamides exert anti-hypoxic effects as dual inhibitors of carbonic anhydrases I/II.

Acute mountain sickness (AMS) affects approximately 25-50% of newcomers to high altitudes. Two human carbonic anhydrase isoforms, hCA I and II, play key roles in developing high altitude illnesses. However, the only FDA-approved drug for AMS is acetazolamide (AAZ), which has a nearly 100 times weaker inhibitory activity against hCA I (Ki = 1237.10 nM) than hCA II (Ki = 13.22 nM). Hence, developing potent dual hCA I/II inhibitors for AMS prevention and treatment is a critical medical need. Here we identified N-quinary heterocycle-4-sulphamoylbenzamides as potent hCA I/II inhibitors. The newly designed compounds 2b, 5b, 5f, 6d, and 6f possessed the desired inhibitory activities (hCA I: Ki = 16.95-52.71 nM; hCA II: Ki = 8.61-18.64 nM). Their hCA I inhibitory capacity was 22- to 76-fold stronger than that of AAZ. Relative to the control group for survival in a mouse model of hypoxia, 2b and 6d prolonged the survival time of mice by 21.7% and 29.3%, respectively, which was longer than those of AAZ (6.5%). These compounds did not display any apparent toxicity in vitro and in vivo. In addition, docking simulations suggested that the quinary aromatic heterocycle groups stabilised the interaction between hCA I/II and the inhibitors, which could be further exploited in structure optimization studies. Hence, future functional studies may confirm 2b and 6d as potential clinical candidate compounds with anti-hypoxic activity against AMS.

Antifungal, anti-inflamatory and neuritogenic activity of newly-isolated compounds from Disporopsis aspersa.

A new ester (1) and a terpenoid (2) were isolated from the dried whole plant of Disporopsis aspersa (HUA) ENGL. ex DIELS for the first time and their structures were elucidated, as well as their biological activities are described. The two compounds all showed good antifungal activities, especially furanone (2) exhibited better antifungal activity against Pseudoperonospora cubensis and Phytophthora infestans with EC50 value of 22.82, 18.90 µg/mL, respectively. Compound 1 exhibited a significant promotion on the neurite outgrowth in NGF-induced PC-12 cells, and moderate inhibition on the NO production induced by lipopolysaccharide (LPS) in BV-2 microglial cells.

Synthesis and evaluation of 4-(1,3,4-oxadiazol-2-yl)-benzenesulfonamides as potent carbonic anhydrase inhibitors.

Human Carbonic anhydrase (hCA) I and II are crucial targets for anti-acute mountain sickness. Twenty-one 4-(1,3,4-oxadiazol-2-yl) benzenesulfonamides were synthesized and screened against these two isoforms. The results illustrated that 5c, 5g, 5h, 5k were more potent against both hCA I and II than clinical drug AAZ. In particular, the value of compound 5c with hCA I (18.08 nM) was over 84-fold more than of AAZ with hCA I. The data of docking simulations were also in accord with the tendency of inhibitive activities. Furthermore, compound 6h, the methanesulfonate of 5h, showed better anti-hypoxia activity than AAZ in vivo, making it interesting lead compound.

Novel AR-12 derivatives, P12-23 and P12-34, inhibit flavivirus replication by blocking host de novo pyrimidine biosynthesis.

The genus Flavivirus contains many important pathogens, including dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV). AR-12 is a celecoxib-derived anticancer agent that possesses antiviral activity against a broad range of viruses. We pharmacologically exploited this unique activity to develop additional antiviral agents, resulting in the production of the AR-12 derivatives P12-23 and P12-34. At nanomolar concentrations, these compounds were effective in suppressing DENV, ZIKV and JEV replication, exhibiting 10-fold improvements in the efficacy and selectivity indices as compared to AR-12. Regarding the mode of antiviral action, P12-23 and P12-34 inhibited viral RNA replication but had no effect on viral binding, entry or translation. Moreover, these AR-12 derivatives co-localized with mitochondrial markers, and their antiviral activity was lost in mitochondria-depleted cells. Interestingly, exogenous uridine or orotate, the latter being a metabolite of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), abolished the antiviral activity of AR-12 and its derivatives. As DHODH is a key enzyme in the de novo pyrimidine biosynthesis pathway, these AR-12 derivatives may act by targeting pyrimidine biosynthesis in host cells to inhibit viral replication. Importantly, treatment with P12-34 significantly improved the survival of mice that were subcutaneously challenged with DENV. Thus, P12-34 may warrant further evaluation as a therapeutic to control flaviviral outbreaks.

Synthesis and insecticidal activity in vitro and vivo of novel benzenesulfonyl derivatives based on potent target subunit H of V-ATPase.

Two lead compounds with benzenesulfonamide were found through virtual screening based on the 3D structure of the subunit H of V-ATPase in previous study. 74 benzenesulfonyl derivatives were synthesized and their insecticidal activities were evaluated. The derivatives with propargyl substituents exhibit excellent insecticidal activities against Mythimna separata Walker. The LD50 values of compounds A5.7 (28.0 µg·g-1) and B5.7 (36.4 µg·g-1) were significantly less than that of Celangulin V (344.0 µg·g-1). Furthermore, Isothermal Titration Calorimetry (ITC) data indicate there is a strong binding affinity between A5.7 and V-ATPase Subunit H. These results demonstrate that it is a practical way to develop pesticides targeting at H subunit of V-ATPase.

Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein.

CCCH-type zinc-finger antiviral protein (ZAP) is a host factor that restricts the infection of many viruses mainly through RNA degradation, translation inhibition and innate immune responses. So far, only one flavivirus, yellow fever virus, has been reported to be ZAP-resistant. Here, we investigated the antiviral potential of human ZAP (isoform ZAP-L and ZAP-S) against three flaviviruses, Japanese encephalitis virus (JEV), dengue virus (DENV) and Zika virus (ZIKV). Infection of JEV but not DENV or ZIKV was blocked by ZAP overexpression, and depletion of endogenous ZAP enhanced JEV replication. ZAP hampered JEV translation and targeted viral RNA for 3'-5' RNA exosome-mediated degradation. The zinc-finger motifs of ZAP were essential for RNA targeting and anti-JEV activity. JEV 3'-UTR, especially in the region with dumbbell structures and high content of CG dinucleotide, was mapped to bind ZAP and confer sensitivity to ZAP. In summary, we identified JEV as the first ZAP-sensitive flavivirus. ZAP may act as an intrinsic antiviral factor through specific RNA binding to fight against JEV infection.

Bioassay-Guided Isolation of Antifungal Compounds from Disporopsis aspersa (Hua) Engl. ex Diels against Pseudoperonospora cubensis and Phytophthora infestans.

Oomycetes are one type of the most highly destructive of the diseases that cause damage to some important crop plants, such as potato late blight, cucumber downy mildew, and grape downy mildew. As main approach of the ongoing search for new botanical fungicide from plant, the secondary metabolites of D. aspersa were investigated. Through efficient bioassay-guided isolation, two new (1 and 2) and 12 known compounds (3 - 14) were isolated, and their structures were determined via extensive NMR, HR-ESI-MS, and IR. They were isolated from this genus for the first time except for compounds 11 and 12. The biological properties of 1 - 14 were evaluated against Pseudoperonospora cubensis and Phytophthora infestans. Compounds 1 - 8 showed potent antifungal activity in vitro. Additionally, compound 3 has preferable control effect on cucumber downy mildew, showing dual effect of protection and treatment in vivo.

Involvement of Tetraspanin C189 in Cell-to-Cell Spreading of the Dengue Virus in C6/36 Cells.

Dengue virus (DENV) is naturally transmitted by mosquitoes to humans, infecting cells of both hosts. Unlike in mammalian cells, DENV usually does not cause extremely deleterious effects on cells of mosquitoes. Despite this, clustered progeny virions were found to form infection foci in a high density cell culture. It is thus interesting to know how the virus spreads among cells in tissues such as the midgut within live mosquitoes. This report demonstrates that cell-to-cell spread is one way for DENV to infect neighboring cells without depending on the "release and entry" mode. In the meantime, a membrane-bound vacuole incorporating tetraspanin C189 was formed in response to DENV infection in the C6/36 cell and was subsequently transported along with the contained virus from one cell to another. Knockdown of C189 in DENV-infected C6/36 cells is shown herein to reduce cell-to-cell transmission of the virus, which may be recovered by co-transfection with a C189-expressing vector in DENV-infected C6/36 cells. Moreover, cell-to-cell transmission usually occurred at the site where the donor cell directly contacts the recipient cell. It suggested that C189 is crucially involved in the intercellular spread of progeny viral particles between mosquito cells. This novel finding presumably accounts for the rapid and efficient infection of DENV after its initial replication within tissues of the mosquito.

Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells.

Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the 70 kDa heat-shock protein (Hsp70). Heat-shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is constitutively expressed; thus, it may be expressed under physiological conditions. In C6/36 cells, Hsc70 is upregulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at the late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C and D; of these, isoform D helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.

Discriminable roles of Aedes aegypti and Aedes albopictus in establishment of dengue outbreaks in Taiwan.

Aedes aegypti and Aedes albopictus were reported to be significant as vectors of dengue fever. In Taiwan, the latter is distributed throughout the island while the former appears only south of the Tropic of Cancer; i.e., 23.5°N. In the past decade, there were five outbreaks with over 1000 cases of dengue fever in Taiwan. Without exception, these outbreaks all occurred in the south where the two Aedes mosquitoes are sympartic. According to the Center for Disease Control of Taiwan, imported cases are thought to provide the seeds of dengue outbreaks every year. Mostly, the number of imported cases is greater in northern island, probably due to a larger population of travelers and imported workers from endemic countries. Looking at the example in 2002, northern, central, and southern parts of Taiwan reported 28, 11, and 13 imported cases, respectively. However, 54, 21, and 5309 total cases were confirmed in the corresponding regions over the entire year, indicating a significant skew of case distributions. A hypothesis is thus inspired that the existence of Ae. aegypti is a prerequisite to initiate a dengue outbreak, while participation of Ae. albopictus expands or maintains the scale until the de novo herd immunity reaches high level.

Additive protection by antioxidant and apoptosis-inhibiting effects on mosquito cells with dengue 2 virus infection.

Cytopathic effects (CPEs) in mosquito cells are generally trivial compared to those that occur in mammalian cells, which usually end up undergoing apoptosis during dengue virus (DENV) infection. However, oxidative stress was detected in both types of infected cells. Despite this, the survival of mosquito cells benefits from the upregulation of genes related to antioxidant defense, such as glutathione S transferase (GST). A second defense system, i.e., consisting of antiapoptotic effects, was also shown to play a role in protecting mosquito cells against DENV infection. This system is regulated by an inhibitor of apoptosis (IAP) that is an upstream regulator of caspases-9 and -3. DENV-infected C6/36 cells with double knockdown of GST and the IAP showed a synergistic effect on activation of these two caspases, causing a higher rate of apoptosis (> 20%) than those with knockdown of each single gene (-10%). It seems that the IAP acts as a second line of defense with an additional effect on the survival of mosquito cells with DENV infection. Compared to mammalian cells, residual hydrogen peroxide in DENV-infected C6/36 cells may signal for upregulation of the IAP. This novel finding sheds light on virus/cell interactions and their coevolution that may elucidate how mosquitoes can be a vector of DENV and probably most other arboviruses in nature.

Antioxidant defense is one of the mechanisms by which mosquito cells survive dengue 2 viral infection.

Dengue viruses (DENVs) generally induce apoptosis in mammalian cells but cause only minor damage in mosquito cells. To find genes involved in determining the cell fate, datasets derived from expressed sequence tags (ESTs) of C6/36 cells with and without infection were established. Of overexpressed genes found in infected dataset, chaperone proteins were validated significantly upregulated in C6/36 cells at 24 hpi. It suggests that DENV-2 in mosquito cells activates the unfolded protein response to cope with endoplasmic reticular stress. Changes in the mitochondrial membrane potential and generation of superoxide provided further evidence that DENV-2 induces oxidative stress in both C6/36 and BHK-21 cells. Significant elevation of glutathione S-transferase (GST) activity was shown in infected C6/36, but not BHK-21, cells, while suppression of GST produced superoxide at 36 hpi and increased the cell death rate at 48 hpi. This indicates that mosquito cells protect themselves against viral infection through antioxidant defenses.

Upregulation of a novel eukaryotic translation initiation factor 5A (eIF5A) in dengue 2 virus-infected mosquito cells.

BACKGROUND: Dengue virus, a mosquito-borne flavivirus, is the etiological agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. It generally induces apoptosis in mammalian cells, but frequently results in persistent infection in mosquito cells. That mechanism remains to be explored. In turn, a genomic survey through subtractive hybridization (PCR-select cDNA subtraction) was conducted in order to find gene(s) that may play a role in interactions between the virus and its host cells. RESULTS: Through this technique, we identified a novel eukaryotic translation initiation factor 5A (eIF5A) which is upregulated in Aedes albopictus-derived C6/36 cells infected by the type 2 dengue (Den-2) virus. The full-length of the identified eIF5A gene consisted of 1498 bp of nucleotides with a 41.39% G+C content, and it possessed a higher similarity and shorter evolutionary distance with insects than with other organisms. Upregulation of eIF5A in response to Den-2 virus infection was validated at both the RNA and protein levels. This phenomenon was also observed by confocal microscopy. In addition, cell death obviously occurred when eIF5A activity was inhibited in C6/36 cells even when they were infected by the virus. However, viral multiplication was not obviously affected in infected C6/36 cells when eIF5A activity was reduced. CONCLUSIONS: Taken together, we postulated that eIF5A plays a role in preventing mosquito cells from death in response to Den-2 viral infection, thus facilitating continued viral growth and potential persistent infection in mosquito cells. It would be worthwhile to further investigate how its downstream factors or cofactors contribute to this phenomenon of dengue infection.

Numerical simulation for meniscus shape and optical performance of a MEMS-based liquid micro-lens.

It is very difficult to fabricate tunable optical systems having an aperture below 1000 micrometers with the conventional means on macroscopic scale. Krogmann et al. (J. Opt. A 8, S330-S336, 2006) presented a MEMS-based tunable liquid micro-lens system with an aperture of 300 micrometers. The system exhibited a tuning range of back focal length between 2.3mm and infinity by using the electrowetting effect to change the contact angle of the meniscus shape on silicon with a voltage of 0-45 V. However, spherical aberration was found in their lens system. In the present study, a numerical simulation is performed for this same physical configuration by solving the Young-Laplace equation on the interface of the lens liquid and the surrounding liquid. The resulting meniscus shape produces a back focal length that agrees with the experimental observation excellently. To eliminate the spherical aberration, an electric field is applied on the lens. The electric field alters the Young-Laplace equation and thus changes the meniscus shape and the lens quality. The numerical result shows that the spherical aberration of the lens can be essentially eliminated when a proper electric field is applied.