Analysis of SARS-CoV-2 Mutations after Nirmatrelvir Treatment in a Lung Cancer Xenograft Mouse Model.

Paxlovid is the first approved oral treatment for coronavirus disease 2019 and includes nirmatrelvir, a protease inhibitor targeting the main protease (Mpro) of SARS-CoV-2, as one of the key components. While some specific mutations emerged in Mpro were revealed to significantly reduce viral susceptibility to nirmatrelvir in vitro, there is no report regarding resistance to nirmatrelvir in patients and animal models for SARS-CoV-2 infection yet. We recently developed xenograft tumors derived from Calu-3 cells in immunodeficient mice and demonstrated extended replication of SARS-CoV-2 in the tumors. In this study, we investigated the effect of nirmatrelvir administration on SARS-CoV-2 replication. Treatment with nirmatrelvir after virus infection significantly reduced the replication of the parental SARS-CoV-2 and SARS-CoV-2 Omicron at 5 days post-infection (dpi). However, the virus titers were completely recovered at the time points of 15 and 30 dpi. The virus genomes in the tumors at 30 dpi were analyzed to investigate whether nirmatrelvir-resistant mutant viruses had emerged during the extended replication of SARS-CoV-2. Various mutations in several genes including ORF1ab, ORF3a, ORF7a, ORF7b, ORF8, and N occurred in the SARS-CoV-2 genome; however, no mutations were induced in the Mpro sequence by a single round of nirmatrelvir treatment, and none were observed even after two rounds of treatment. The parental SARS-CoV-2 and its sublineage isolates showed similar IC50 values of nirmatrelvir in Vero E6 cells. Therefore, it is probable that inducing viral resistance to nirmatrelvir in vivo is challenging differently from in vitro passage.

Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.

A mouse xenograft long-term replication yields a SARS-CoV-2 Delta mutant with increased lethality.

We recently established a long-term SARS-CoV-2 infection model using lung-cancer xenograft mice and identified mutations that arose in the SARS-CoV-2 genome during long-term propagation. Here, we applied our model to the SARS-CoV-2 Delta variant, which has increased transmissibility and immune escape compared with ancestral SARS-CoV-2. We observed limited mutations in SARS-CoV-2 Delta during long-term propagation, including two predominant mutations: R682W in the spike protein and L330W in the nucleocapsid protein. We analyzed two representative isolates, Delta-10 and Delta-12, with both predominant mutations and some additional mutations. Delta-10 and Delta-12 showed lower replication capacity compared with SARS-CoV-2 Delta in cultured cells; however, Delta-12 was more lethal in K18-hACE2 mice compared with SARS-CoV-2 Delta and Delta-10. Mice infected with Delta-12 had higher viral titers, more severe histopathology in the lungs, higher chemokine expression, increased astrocyte and microglia activation, and extensive neutrophil infiltration in the brain. Brain tissue hemorrhage and mild vacuolation were also observed, suggesting that the high lethality of Delta-12 was associated with lung and brain pathology. Our long-term infection model can provide mutant viruses derived from SARS-CoV-2 Delta and knowledge about the possible contributions of emergent mutations to the properties of new variants.

Insights on interferon-independent induction of interferon-stimulated genes shaping the lung's response in early SARS-CoV-2 infection.

While mRNA vaccine efficacy against the 2019 coronavirus disease (COVID-19) outbreak remains high, research on antiviral innate immune responses in the early stages of infection is essential to develop strategies to prevent the dissemination of SARS-CoV-2. In this study, we investigated the induction of both interferon (IFN)-stimulated genes (ISGs) and IFN-independently upregulated ISGs following SARS-CoV-2 infection in Syrian golden hamsters. The viral titers were highest at 3 days post-infection (dpi). Over time, the viral titer gradually decreased while ISGs such as Mx1, Ifit2, Ifit3, Ifi44, and Rsad2 were markedly induced in the lung. The transcription of ISGs significantly increased from 2 dpi, and SARS-CoV-2-induced ISGs were maintained in the hamster lung until 7 dpi. The transcription of Ifnb and Ifng was minimally elevated, while Ifnl2/3 was significantly induced in the lung at 5 days after SARS-CoV-2 infection. RNA sequencing results also showed that at 3 dpi, SARS-CoV-2 initiated the activation of ISGs, with lesser increases of Ifnl2 and Ifnl3 transcription. In addition, Ddx58 and cGAS, which encode factors for virus sensing, Stat1, Stat2, and IFN regulatory factor 7 and 9 mRNA levels were also induced at the initial stage of infection. Our data demonstrate that ISGs might be upregulated in the lung in response to SARS-CoV-2 during the early stages of infection, and the rapid induction of ISGs was not associated with the activation of IFNs. Elucidation of IFN-independent induction of ISGs could further our understanding of alternative defense mechanisms employed by the lungs against SARS-CoV-2 and provide more effective antiviral strategies for patients with severe COVID-19.

Cremastranone-Derived Homoisoflavanes Suppress the Growth of Breast Cancer Cells via Cell Cycle Arrest and Caspase-Independent Cell Death.

Breast cancer is the most common cancer and a frequent cause of cancer-related deaths among women wordlwide. As therapeutic strategies for breast cancer have limitations, novel chemotherapeutic reagents and treatment strategies are needed. In this study, we investigated the anti-cancer effect of synthetic homoisoflavane derivatives of cremastranone on breast cancer cells. Homoisoflavane derivatives, SH-17059 and SH-19021, reduced cell proliferation through G2/M cell cycle arrest and induced caspase-independent cell death. These compounds increased heme oxygenase-1 (HO-1) and 5-aminolevulinic acid synthase 1 (ALAS1), suggesting downregulation of heme. They also induced reactive oxygen species (ROS) generation and lipid peroxidation. Furthermore, they reduced expression of glutathione peroxidase 4 (GPX4). Therefore, we suggest that the SH-17059 and SH-19021 induced the caspase-independent cell death through the accumulation of iron from heme degradation, and the ferroptosis might be one of the potential candidates for caspase-independent cell death.

Turning Carbon Dioxide into Sustainable Food and Chemicals: How Electrosynthesized Acetate Is Paving the Way for Fermentation Innovation.

The agricultural and chemical industries are major contributors to climate change. To address this issue, hybrid electrocatalytic-biocatalytic systems have emerged as a promising solution for reducing the environmental impact of these key sectors while providing economic onboarding for carbon capture technology. Recent advancements in the production of acetate via CO2/CO electrolysis as well as advances in precision fermentation technology have prompted electrochemical acetate to be explored as an alternative carbon source for synthetic biology. Tandem CO2 electrolysis coupled with improved reactor design has accelerated the commercial viability of electrosynthesized acetate in recent years. Simultaneously, innovations in metabolic engineering have helped leverage pathways that facilitate acetate upgrading to higher carbons for sustainable food and chemical production via precision fermentation. Current precision fermentation technology has received much criticism for reliance upon food crop-derived sugars and starches as feedstock which compete with the human food chain. A shift toward electrosynthesized acetate feedstocks could help preserve arable land for a rapidly growing population.Technoeconomic analysis shows that using electrochemical acetate instead of glucose as a fermentation feedstock reduces the production costs of food and chemicals by 16% and offers improved market price stability. Moreover, given the rapid decline in utility-scale renewable electricity prices, electro-synthesized acetate may become more affordable than conventional production methods at scale. This work provides an outlook on strategies to further advance and scale-up electrochemical acetate production. Additional perspective is offered to help ensure the successful integration of electrosynthesized acetate and precision fermentation technologies. In the electrocatalytic step, it is critical that relatively high purity acetate can be produced in low-concentration electrolyte to help ensure that minimal treatment of the electrosynthesized acetate stream is needed prior to fermentation. In the biocatalytic step, it is critical that microbes with increased tolerances to elevated acetate concentrations are engineered to help promote acetate uptake and accelerate product formation. Additionally, tighter regulation of acetate metabolism via strain engineering is essential to improving cellular efficiency. The implementation of these strategies would allow the coupling of electrosynthesized acetate with precision fermentation to offer a promising approach to sustainably produce chemicals and food. Reducing the environmental impact of the chemical and agricultural sectors is necessary to avoid climate catastrophe and preserve the habitability of the planet for future generations.

Intranasal inoculation of IFN-λ resolves SARS-CoV-2 lung infection via the rapid reduction of viral burden and improvement of tissue damage.

Introduction: The innate immune responses of upper airway could further our understanding toward antiviral strategies against SARS-CoV-2. We characterize the potential of interferon (IFN)-λ as an innate immune inducer for the rapid clearance of SARS-CoV-2 in the lung and the therapeutic efficacy of intranasal inoculation of IFN-λ to resolve acute lung infection. Methods: Syrian golden hamsters were infected with SARS-CoV-2 and the dynamics of SARS-CoV-2 infection depending on IFN-λ inoculation were tested. Results: SARS-CoV-2-infected Syrian golden hamsters exhibited a significant decrease in body weight and high viral mRNA level at 3 days post-infection (dpi). Although viral replication was reduced completely from 7 dpi, the pathologic findings remained prominent until 14 dpi in the lung of hamsters. The transcription of IFN-λ was significantly induced in response to SARS-CoV-2 infection with the increase of IFN-stimulated genes. Intranasal inoculation of IFN-λ restricted SARS-CoV-2 replication in the lungs of infected completely from 3 dpi with markedly reduction of inflammatory cytokines. The transcriptional phenotypes were altered to the direction of damage repair and tissue remodeling in the lungs of SARS-CoV-2-infected hamsters following intranasal inoculation of IFN-λ, which improved SARS-CoV-2-caused lung damage. Conclusion: Collectively, our findings suggest that IFN-λ might be a potent innate immune inducer in the lung and intranasal inoculation of IFN-λ resolves SARS-CoV-2 infection with rapid viral clearance and improvement of lung damage.

Synthetic Homoisoflavane Derivatives of Cremastranone Suppress Growth of Colorectal Cancer Cells through Cell Cycle Arrest and Induction of Apoptosis.

Colorectal cancer is diagnosed as the third most prevalent cancer; thus, effective therapeutic agents are urgently required. In this study, we synthesized six homoisoflavane derivatives of cremastranone and investigated their cytotoxic effects on the human colorectal cancer cell lines HCT116 and LoVo. We further examined the related mechanisms of action using two of the potent compounds, SH-19027 and SHA-035. They substantially reduced the cell viability and proliferation in a dose-dependent manner. Treatment with SH-19027 and SHA-035 induced cell cycle arrest at the G2/M phase and increased expression of p21 both of which are implicated in cell cycle control. In addition, the apoptotic cell population and apoptosis-associated marker expression were accordingly increased. These results suggest that the synthesized cremastranone derivatives have anticancer effects through the suppression of cell proliferation and induction of apoptosis. Therefore, the synthesized cremastranone derivatives could be applied as novel therapeutic agents against colorectal cancer.

Inhaled delivery of recombinant interferon-lambda restores allergic inflammation after development of asthma by controlling Th2- and Th17-cell-mediated immune responses.

Remarkable progress has recently been achieved to identify the biological function and potential value of novel therapeutic targets for the effective control of allergic asthma. Interferon (IFN)-λ has been suggested to restrict chronic inflammation in the lungs of asthmatic mice and we sought to determine the contribution of IFN-λ as an asthma therapeutic. We show that inhaled IFN-λ can restrict Th2 and Th17 inflammation in the lungs of asthmatic mice, accompanied with alteration of IL-10 secretion. BALB/C mice were used for an asthmatic mouse model with OVA. Recombinant IFN-λs (IFN-λ2: 2 µg, IFN-λ3: 2 µg) were inoculated into asthmatic mice after OVA challenge by intranasal delivery. Lungs of asthmatic mice were severely inflamed, with extensive inflammatory cell infiltration and increased goblet cell metaplasia with higher total lung resistance. Transcription of IL-4, IL-5, IL-13, and IL-17A was significantly higher until five days after the final OVA challenge. Asthmatic mice were administered recombinant IFN-λ via inhalation three times after the last challenge and the asthmatic mice showed improvement in lung histopathologic findings, and total lung resistance was maintained under normal range. IFN-λ inhalation exhibited significant decreases in Th2 and Th17 cytokine levels, and the populations of Th2 and Th17 cells were recovered from the lungs of asthmatic mice. Additionally, increase in IL-10 secretion from CD4 + Th cells population was observed in response to inhaled delivery of IFN-λ along with alterations in Th2 and Th17 cell-derived inflammation. Our findings show that inhaled delivery of IFN-λ can restrict airway inflammation in the lungs of asthmatic mice by controlling Th2- and Th17-mediated responses accompanied by regulation of IL-10 secretion even after asthma development.

HDAC inhibitor and proteasome inhibitor induce cleavage and exosome-mediated secretion of HSP90 in mouse pluripotent stem cells.

Heat shock protein 90 (HSP90), one of the molecular chaperones, stabilizes several proteins necessary to maintain pluripotency of embryonic stem (ES) cells. Recently, we reported that HDAC inhibitors and proteasome inhibitors down-regulate HSP90 activity through HSP90 cleavage induced by reactive oxygen species (ROS) generation and caspase 10 activation in various cancer cells. In this study, we investigated HSP90 cleavage in mouse ES cells. HDAC inhibitors and proteasome inhibitors induced HSP90 cleavage in the mouse ES cell line R1, and the cleaved HSP90 was barely found in the cells and instead secreted out of the cells through the exosome. The HSP90 cleavage was associated with ROS generation and caspase 10 activation. In addition, HDAC inhibitor and proteasome inhibitor induced Fas expression, and the inhibition of caspase 8, a downstream molecule of Fas, blocked HSP90 cleavage. Therefore, HDAC inhibitor- and proteasome inhibitor-mediated HSP90 cleavage was induced by ROS generation and Fas expression. We observed similar results in mouse induced pluripotent stem (iPS) cells. Taken together, HSP90 cleavage was induced in mouse pluripotent cells similarly to cancer cells but differently regulated through Fas expression and exosomal secretion. These findings will be helpful in elucidating the regulation of HSP90 upon stress in pluripotent stem cells.

Scalable Gas Diffusion Electrode Fabrication for Electrochemical CO2 Reduction Using Physical Vapor Deposition Methods.

Electrochemical CO2 reduction (ECR) promises the replacement of fossil fuels as the source of feedstock chemicals and seasonal storage of renewable energy. While much progress has been made in catalyst development and electrochemical reactor design, few studies have addressed the effect of catalyst integration on device performance. Using a microfluidic gas diffusion electrolyzer, we systematically studied the effect of thickness and the morphology of electron beam (EB) and magnetron-sputtered (MS) Cu catalyst coatings on ECR performance. We observed that EB-Cu outperforms MS-Cu in current density, selectivity, and energy efficiency, with 400 nm thick catalyst coatings performing the best. The superior performance of EB-Cu catalysts is assigned to their faceted surface morphology and sharper Cu/gas diffusion layer interface, which increases their hydrophobicity. Tests in a large-scale zero-gap electrolyzer yielded similar product selectivity distributions with an ethylene Faradaic efficiency of 39% at 200 mA/cm2, demonstrating the scalability for industrial ECR applications.

Electrochemical Reduction of Gaseous Nitrogen Oxides on Transition Metals at Ambient Conditions.

Mitigating nitrogen oxide (NOx) emissions is critical to tackle global warming and improve air quality. Conventional NOx abatement technologies for emission control suffer from a low efficiency at near ambient temperatures. Herein, we show an electrochemical pathway to reduce gaseous NOx that can be conducted at high reaction rates (400 mA cm-2) under ambient conditions. Various transition metals are evaluated for electrochemical reduction of NO and N2O to reveal the role of electrocatalyst in determining the product selectivity. Specifically, Cu is highly selective toward NH3 formation with >80% Faradaic efficiency in NO electroreduction. Furthermore, the partial pressure study of NO electroreduction revealed that a high NO coverage facilitates the N-N coupling reaction. In acidic electrolytes, the formation of NH3 is greatly favored, whereas the N2 production is suppressed. Additional mechanistic studies were conducted by using flow electrochemical mass spectrometry to gain further insights into reaction pathways. This work provides a promising avenue toward abating gaseous NOx emissions at ambient conditions by using renewable electricity.

The nasal symbiont Staphylococcus species restricts the transcription of SARS-CoV-2 entry factors in human nasal epithelium.

Emerging evidence indicates that severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is transmitted through the human nasal mucosa via the principal entry factors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), which are highly expressed in the nasal epithelium. Therefore, the biologics targeting host entry factors on human nasal mucosa will be necessary for complete control of SARS-CoV-2. Our data reveal that ACE2 was more abundant in human nasal mucosa than lung tissue. Both ACE2 and TMPRSS2 transcriptions significantly decreased in nasal epithelium in response to S. epidermidis and were relatively lower in human nasal mucus with large numbers of S. epidermidis. ACE2 transcription was also reduced in nasal epithelium in response to nasal symbiont S. aureus. This study proposes that Staphylococcus species nasal commensals might potentially restrict SARS-CoV-2 entry to the nasal epithelium via down regulation of cellular receptors coupled with reduction of principal host protease.

Electrochemical reduction of acetonitrile to ethylamine.

Electrifying chemical manufacturing using renewable energy is an attractive approach to reduce the dependence on fossil energy sources in chemical industries. Primary amines are important organic building blocks; however, the synthesis is often hindered by the poor selectivity because of the formation of secondary and tertiary amine byproducts. Herein, we report an electrocatalytic route to produce ethylamine selectively through an electroreduction of acetonitrile at ambient temperature and pressure. Among all the electrocatalysts, Cu nanoparticles exhibit the highest ethylamine Faradaic efficiency (~96%) at -0.29 V versus reversible hydrogen electrode. Under optimal conditions, we achieve an ethylamine partial current density of 846 mA cm-2. A 20-hour stable performance is demonstrated on Cu at 100 mA cm-2 with an 86% ethylamine Faradaic efficiency. Moreover, the reaction mechanism is investigated by computational study, which suggests the high ethylamine selectivity on Cu is due to the moderate binding affinity for the reaction intermediates.

The impact of nitrogen oxides on electrochemical carbon dioxide reduction.

The electroreduction of carbon dioxide offers a promising avenue to produce valuable fuels and chemicals using greenhouse gas carbon dioxide as the carbon feedstock. Because industrial carbon dioxide point sources often contain numerous contaminants, such as nitrogen oxides, understanding the potential impact of contaminants on carbon dioxide electrolysis is crucial for practical applications. Herein, we investigate the impact of various nitrogen oxides, including nitric oxide, nitrogen dioxide, and nitrous oxide, on carbon dioxide electroreduction on three model electrocatalysts (i.e., copper, silver, and tin). We demonstrate that the presence of nitrogen oxides (up to 0.83%) in the carbon dioxide feed leads to a considerable Faradaic efficiency loss in carbon dioxide electroreduction, which is caused by the preferential electroreduction of nitrogen oxides over carbon dioxide. The primary products of nitrogen oxides electroreduction include nitrous oxide, nitrogen, hydroxylamine, and ammonia. Despite the loss in Faradaic efficiency, the electrocatalysts exhibit similar carbon dioxide reduction performances once a pure carbon dioxide feed is restored, indicating a negligible long-term impact of nitrogen oxides on the catalytic properties of the model catalysts.

Effects of calcination temperature for rate capability of triple-shelled ZnFe2O4 hollow microspheres for lithium ion battery anodes.

Triple-shelled ZnFe2O4 hollow microspheres (ZFO) as anode materials for lithium ion battery are prepared through a one-pot hydrothermal reaction using the composite solution consisting of sucrose in water and metal ions in ethylene glycol (EG), followed by different calcination processes. The architectures of ZFO micro spheres are differently synthesized through a mutual cooperation of inward and outward ripening with three different calcination temperatures. Thin triple-shelled ZnFe2O4 hollow microspheres calcined at 450 °C (ZFO-450) delivers a high reversible capacity of 932 mA h g-1 at a current density of 2 A g-1 even at the 200th cycle without obvious decay. Furthermore, ZFO-450 delivers 1235, 1005, 865, 834, and 845 mA h g-1 at high current densities of 0.5, 2, 5, 10, and 20 A g-1 after 100 cycles. Thin triple-shelled hollow microsphere prepared at an optimum calcination temperature provides exceptional rate capability and outstanding rate retention due to (i) the formation of nanoparticles leading to thin shell with morphological integrity, (ii) the facile mass transfer by thin shell with mesoporous structure, and (iii) the void space with macroporous structure alleviating volume change occurring during cycling.