A natural product YSK-A blocks SARS-CoV-2 propagation by targeting multiple host genes.

Natural products and herbal medicine have been widely used in drug discovery for treating infectious diseases. Recent outbreak of COVID-19 requires various therapeutic strategies. Here, we used YSK-A, a mixture of three herbal components Boswellia serrata, Commiphora myrrha, and propolis, to evaluate potential antiviral activity against SARS-CoV-2. We showed that YSK-A inhibited SARS-CoV-2 propagation with an IC50 values of 12.5 µg/ml and 15.42 µg/ml in Vero E6 and Calu-3 cells, respectively. Using transcriptome analysis, we further demonstrated that YSK-A modulated various host gene expressions in Calu-3 cells. Among these, we selected 9 antiviral- or immune-related host genes for further study. By siRNA-mediated knockdown experiment, we verified that MUC5AC, LIF, CEACAM1, and GDF15 host genes were involved in antiviral activity of YSK-A. Therefore, silencing of these genes nullified YSK-A-mediated inhibition of SARS-CoV-2 propagation. These data indicate that YSK-A displays an anti-SARS-CoV-2 activity by targeting multiple antiviral genes. Although the exact antiviral mechanism of each constituent has not been verified yet, our data indicate that YSK-A has an immunomodulatory effect on SARS-CoV-2 and thus it may represent a novel natural product-derived therapeutic agent for treating COVID-19.

SARS-CoV-2 exploits cellular RAD51 to promote viral propagation: implication of RAD51 inhibitor as a potential drug candidate against COVID-19.

IMPORTANCE: Viruses are constantly evolving to promote propagation in the host. Here, we show that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes host RAD51 for replication. Silencing of RAD51 impaired SARS-CoV-2 propagation. Viral RNA colocalized with RAD51 in the cytoplasm of SARS-CoV-2-infected cells, suggesting that both viral RNA and RAD51 may form a replication complex. We, therefore, evaluated RAD51 inhibitors as possible therapeutic agents against SARS-CoV-2. Indeed, RAD51 inhibitors exerted antiviral activities against not only Wuhan but also variants of SARS-CoV-2. Molecular docking model shows that RAD51 inhibitors impede SARS-CoV-2 propagation by interfering with dimerization of RAD51. These data suggest that RAD51 may represent a novel host-based drug target for coronavirus disease 2019 treatment.

Polygalic acid inhibits african swine fever virus polymerase activity: findings from machine learning and in vitro testing.

African swine fever virus (ASFV), an extremely contagious virus with high mortality rates, causes severe hemorrhagic viral disease in both domestic and wild pigs. Fortunately, ASFV cannot be transmitted from pigs to humans. However, ongoing ASFV outbreaks could have severe economic consequences for global food security. Although ASFV was discovered several years ago, no vaccines or treatments are commercially available yet; therefore, the identification of new anti-ASFV drugs is urgently warranted. Using molecular docking and machine learning, we have previously identified pentagastrin, cangrelor, and fostamatinib as potential antiviral drugs against ASFV. Here, using machine learning combined with docking simulations, we identified natural products with a high affinity for AsfvPolX proteins. We selected five natural products (NPs) that are located close in chemical space to the six known natural flavonoids that possess anti-ASFV activity. Polygalic acid markedly reduced AsfvPolX polymerase activity in a dose-dependent manner. We propose an efficient protocol for identifying NPs as potential antiviral drugs by identifying chemical spaces containing high-affinity binders against ASFV in NP databases.

Amuvatinib Blocks SARS-CoV-2 Infection at the Entry Step of the Viral Life Cycle.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2 propagation is mediated by the protein interaction between viral proteins and host cells. Tyrosine kinase has been implicated in viral replication, and hence, it has become a target for developing antiviral drugs. We have previously reported that receptor tyrosine kinase inhibitor blocks the replication of hepatitis C virus (HCV). In the present study, we investigated two receptor tyrosine kinase-specific inhibitors, amuvatinib and imatinib, for their potential antiviral efficacies against SARS-CoV-2. Treatment with either amuvatinib or imatinib displays an effective inhibitory activity against SARS-CoV-2 propagation without an obvious cytopathic effect in Vero E6 cells. Notably, amuvatinib exerts a stronger antiviral activity than imatinib against SARS-CoV-2 infection. Amuvatinib blocks SARS-CoV-2 infection with a 50% effective concentration (EC50) value ranging from ~0.36 to 0.45 µM in Vero E6 cells. We further demonstrate that amuvatinib inhibits SARS-CoV-2 propagation in human lung Calu-3 cells. Using pseudoparticle infection assay, we verify that amuvatinib blocks SARS-CoV-2 at the entry step of the viral life cycle. More specifically, amuvatinib inhibits SARS-CoV-2 infection at the binding-attachment step. Moreover, amuvatinib exhibits highly efficient antiviral activity against emerging SARS-CoV-2 variants. Importantly, we demonstrate that amuvatinib inhibits SARS-CoV-2 infection by blocking ACE2 cleavage. Taken together, our data suggest that amuvatinib may provide a potential therapeutic agent for the treatment of COVID-19. IMPORTANCE Tyrosine kinase has been implicated in viral replication and has become an antiviral drug target. Here, we chose two well-known receptor tyrosine kinase inhibitors, amuvatinib and imatinib, and evaluated their drug potencies against SARS-CoV-2. Surprisingly, amuvatinib displays a stronger antiviral activity than imatinib against SARS-CoV-2. Amuvatinib blocks SARS-CoV-2 infection by inhibiting ACE2 cleavage and the subsequent soluble ACE2 receptor. All these data suggest that amuvatinib may be a potential therapeutic agent in SARS-CoV-2 prevention for those experiencing vaccine breakthroughs.

Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.

Effects of Gender-Affirming Hormone Therapy on Progression Along the HIV Care Continuum in Transgender Women.

BACKGROUND: Progression along the HIV care continuum has been a key focus for improving outcomes for people with HIV (PWH). Transgender women with HIV (TGWWH) have not made the same progress as their cisgender counterparts. METHODS: All PWH identifying as transgender women receiving care at our clinic from 1/1/2015 to 12/31/2019 were identified from the electronic health records (EHRs) using International Classification of Diseases (ICD) codes. Demographics, laboratory data, prescription of gender-affirming hormone therapy (GAHT), and visit history were abstracted from the EHR. Retention in care and viral suppression were defined using Centers for Disease Control and Prevention definitions. The proportions of TGWWH who were consistently retained in care or virally suppressed over time were calculated using a binary response generalized mixed model including random effects and correlated errors. RESULTS: Of the 76 PWH identified by ICD codes, 2 were excluded for identifying as cisgender and 15 for insufficient records, leaving 59 TGWWH included for analysis. Patients were on average 35 years old and Black (86%), with a median CD4 count of 464 cells/µL. There were 13 patients on GAHT at study entry and 31 receiving GAHT at any point during the study period. Fifty-five percent were virally suppressed at study entry and 86% at GAHT initiation. The proportion of TGWWH who were consistently virally suppressed over time was greater among those receiving GAHT compared with those who were not (P = .04). CONCLUSIONS: Rates of viral suppression were significantly greater among TGWWH receiving GAHT when compared with those who were not. More research to evaluate the reasons behind this effect is needed.

Bond Strength and Fracture Resistance of Flowable Bulk Fill Composite Posts and Cores in Endodontically Treated Teeth.

OBJECTIVES: Smart dentin replacement (SDR) is a new flowable bulk fill composite with many useful characteristics such as low viscosity and higher depth of cure. This study aimed to evaluate the bond strength and fracture resistance of flowable bulk fill composite posts and cores versus that of fiber posts and cores. MATERIALS AND METHODS: Forty intact, extracted human maxillary central incisor roots were endodontically treated. Group A (n = 20) was prepared for the composite space and group B (n = 20) was prepared for the fiber post space. Group A and B were divided into two subgroups A1 and A2 and B1 and B2, respectively (10 roots for each subgroup). Root canal spaces of group A1 were filled with SDR composite, X-Post fiber post with Core X Flow composite was inserted into the root canal spaces of group B1. Group A2 was restored using SDR and group B2 was restored using post and core composite. Five hundred thermocycles were applied for the sample. Bond strength values were measured for segments in A1 and B1. Fracture force values were measured for specimens of A2 and B2. RESULTS: No significant difference was observed between the two paired groups (A1 and B1) and (A2 and B2) in bond strength and fracture force values. CONCLUSION: SDR could be used for restoring endodontically treated teeth.

Effect of Olyset Net on Mosquito Larvae and Their Heteroptera Predators in Water Jars in Southern Vietnam.

Residents of Vietnam living in areas with water shortages collect and store tap water, when it is available, in containers, such as jars, which is then used for their daily activities. Although these water jars are important breeding sites for mosquitoes, Vietnam legislation prohibits the use of chemical larvicides in such containers. The effect of Olyset Net on the abundance of mosquito larvae and their Heteroptera predators (Micronecta spp. and Veliidae) in jars was evaluated via field experiments conducted in Tan Chanh, Long An, Vietnam. The number of Aedes larvae decreased when the jars were covered with the Olyset Net, while the numbers of non-Aedes mosquito larvae (Culex and Anopheles), and those of Micronecta and Veliidae, did not vary until the end of the experiment. However, Micronecta population increased after removing the Olyset Net from the jars, and consequently Aedes larvae decreased due to these changes in the Micronecta, 26 days after the Olyset Net was removed. Incomplete use of Olyset Net failed to control mosquitoes in water jars based on the invasion of adult mosquitoes, and in water transferred by residents among the jars, which might contain larvae. These results suggest that Micronecta, as well as Olyset Net, can be considered as control agents for mosquito larvae.

Field trial on a novel control method for the dengue vector, Aedes aegypti by the systematic use of Olyset® Net and pyriproxyfen in Southern Vietnam.

BACKGROUND: Jars, tanks, and drums provide favorable rearing/breeding sites for Aedes aegypti in Vietnam. However, the use of insecticides to control mosquitoes at such breeding sites has not been approved in Vietnam since they are also often sources of drinking water, making larval vector control difficult. Mosquito nets pre-treated with long-lasting insecticide treated nets (LLITNs) form an effective measure for malaria control. We examined changes in the abundance of immature Aedes aegypti to evaluate the efficacy of covering ceramic jars with lids comprising one type of LLITN, Olyset® Net, in inhibiting oviposition by adult females, and to evaluate the effect of treating other breeding containers, such as flower vases, inside and around the outside of houses with a slow-release pyriproxyfen formulation to kill pupae. METHODS: We selected 313 households for the trial and 363 households for the control in Tan Chanh, Long An province, Vietnam. In the trial area, Olyset® Net lids were used to cover five major types of water container (ceramic jars, cylindrical concrete tanks, other concrete tanks, plastic drums, and plastic buckets), while pyriproxyfen was used to treat flower vases and ant traps. We also monitored dengue virus transmission by measuring anti-dengue IgM and IgG levels in healthy residents in both control and trial areas to estimate the effectiveness of Olyset® Net at controlling the dengue vector, Aedes aegypti. RESULTS: The container-index and house-index for immature Ae. aegypti fell steeply one month after treatment in the trial area. Lids with Olyset® Net that fit container openings clearly seemed to reduce the presence of immature Ae. aegypti as the density of pupae decreased 1 month after treatment in the trial area. Pyriproxyfen was also effective at killing pupae in the water containers in the trial area. Although the dengue seroconversion rate was not influenced by Olyset® Net, it was lower in two-five year old children when compared to older children and adults in both control and trial areas. CONCLUSIONS: Our study showed that the treatment by Olyset® Net and pyriproxyfen had a strong negative effect on the prevalence of immature Ae. aegypti, which persisted for at least 5 months after treatment.

Effect of plants on the bioavailability of metals and other chemical properties of biosolids in a column study.

The effects of metal-accumulating plants (Salix x reichardtii and Populus balsamifera) on the chemical properties and dynamics of metals in biosolids were investigated using different techniques including diffusive gradients in thin films (DGT), sequential extraction procedures and partitioning coefficient (K(d)). Plants could effectively extract Cd, Ni, and Zn and decreased dissolved organic carbon (DOC). The presence of plants increased the potential bioavailability of these metals, as assessed by an increase in the ratio of metal measured by DGT and metals in the solution. The plants affected the Cd, Ni, and Zn pools (soluble/exchangeable; Fe/Mn oxide and organic matter bound) characterised by sequential extraction and K(d) but did not reduce the total metals in either substrate. However, plants had no effect on Cu, presumably because of the effective buffering of available Cu by organic matter in both solution and solid phases. A high density of plant roots was associated with increased leaching of metals.

Low entomological impact of new water supply infrastructure in southern Vietnam, with reference to dengue vectors.

We did a prospective study in southern Vietnam where new water infrastructure was added. New 1,200-L tanks may present potential breeding grounds for Aedes aegypti, particularly when sealed lids were not always supplied. Some householders in these communes received a piped water supply, however there was no reduction in water storage practices. The prevalence of Aedes aegypti immatures in tank and tap households reached 73%, but were non-significantly different from each other and from control households that received no infrastructure. In all three communes, standard jars comprised from 48% to 71% of containers but were associated with > 90% of III-IV instars and pupae on occasions. In contrast, project tanks contributed from 0-21% of the total population. Non-functional or no lids were apparent 4 months after installation in 45-76% of new tanks, but there was no difference between communes with lids and without lids.

Comparison of the histology, gene expression profile, and phenotype of cultured human limbal epithelial cells from different limbal regions.

PURPOSE: To investigate whether human limbal epithelial cells (HLECs) derived from various regions of the limbus exhibit differences in gene expression and epithelial characteristics. METHODS: HLECs were derived from explants taken from the superior, nasal, inferior, and temporal limbus and cultured for 21 days. Whole genome transcript profiling was performed with a gene microarray. The microarray results were validated by using RT-PCR. Epithelial morphology was studied with light microscopy and transmission electron microscopy, and phenotype was evaluated by immunohistochemistry. RESULTS: Epithelial outgrowth was present in most cultures of superior origin (88%) in contrast to cultures of temporal origin (38%). The epithelial thickness and number of cell layers were significantly greater in cultures of superior origin than in cultures from inferior and temporal areas. TRIM36, OSR2, and RHOU, which are involved in morphogenesis, were significantly differentially expressed in the superior region, compared with the other regions. Proposed limbal stem cell, progenitor, and differentiation markers were not differentially expressed. The uniform gene expression of ocular surface markers correlated with homogeneous immunostaining of corresponding protein markers in HLEC cultures from all regions, demonstrating an undifferentiated phenotype (p63(+), DeltaNp63alpha(+), ABCG2(+), K19(+), vimentin(+), integrin beta1(+), nestin(-), K3(-), K5(+), and E-cadherin(+)). CONCLUSIONS: No major transcriptional or phenotypic differences were observed in cultured HLECs derived from different regions of the limbus. However, explants of superior origin demonstrated the highest outgrowth success rate and generated epithelia with greater epithelial thickness and number of cell layers, which may prove useful for transplantation purposes.

Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children. It is thought to result from the transformation of granule cell precursors (GCPs) in the developing cerebellum, but little is known about the early stages of the disease. Here, we identify a pre-neoplastic stage of medulloblastoma in patched heterozygous mice, a model of the human disease. We show that pre-neoplastic cells are present in the majority of patched mutants, although only 16% of these mice develop tumors. Pre-neoplastic cells, like tumor cells, exhibit activation of the Sonic hedgehog pathway and constitutive proliferation. Importantly, they also lack expression of the wild-type patched allele, suggesting that loss of patched is an early event in tumorigenesis. Although pre-neoplastic cells resemble GCPs and tumor cells in many respects, they have a distinct molecular signature. Genes that mark the pre-neoplastic stage include regulators of migration, apoptosis and differentiation, processes crucial for normal development but previously unrecognized for their role in medulloblastoma. The identification and molecular characterization of pre-neoplastic cells provides insight into the early steps in medulloblastoma formation, and may yield important markers for early detection and therapy of this disease.