Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody.

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

A Spike-destructing human antibody effectively neutralizes Omicron-included SARS-CoV-2 variants with therapeutic efficacy.

Neutralizing antibodies (nAbs) are important assets to fight COVID-19, but most existing nAbs lose the activities against Omicron subvariants. Here, we report a human monoclonal antibody (Ab08) isolated from a convalescent patient infected with the prototype strain (Wuhan-Hu-1). Ab08 binds to the receptor-binding domain (RBD) with pico-molar affinity (230 pM), effectively neutralizes SARS-CoV-2 and variants of concern (VOCs) including Alpha, Beta, Gamma, Mu, Omicron BA.1 and BA.2, and to a lesser extent for Delta and Omicron BA.4/BA.5 which bear the L452R mutation. Of medical importance, Ab08 shows therapeutic efficacy in SARS-CoV-2-infected hACE2 mice. X-ray crystallography of the Ab08-RBD complex reveals an antibody footprint largely in the ß-strand core and away from the ACE2-binding motif. Negative staining electron-microscopy suggests a neutralizing mechanism through which Ab08 destructs the Spike trimer. Together, our work identifies a nAb with therapeutic potential for COVID-19.

Construction and efficacy testing of DNA vaccines containing HLA-A*02:01-restricted SARS-CoV-2 T-cell epitopes predicted by immunoinformatics.

Vaccines play essential roles in the fight against the COVID-19 pandemic. The development and assessment of COVID-19 vaccines have generally focused on the induction and boosting of neutralizing antibodies targeting the SARS-CoV-2 spike (S) protein. Due to rapid and continuous variation in the S protein, such vaccines need to be regularly updated to match newly emerged dominant variants. T-cell vaccines that target MHC I- or II-restricted epitopes in both structural and non-structural viral proteins have the potential to induce broadly cross-protective and long-lasting responses. In this work, the entire proteome encoded by SARS-CoV-2 (Wuhan-hu-1) is subjected to immunoinformatics-based prediction of HLA-A*02:01-restricted epitopes. The immunogenicity of the predicted epitopes is evaluated using peripheral blood mononuclear cells from convalescent Wuhan-hu-1-infected patients. Furthermore, predicted epitopes that are conserved across major SARS-CoV-2 lineages and variants are used to construct DNA vaccines expressing multi-epitope polypeptides. Most importantly, two DNA vaccine constructs induce epitope-specific CD8 + T-cell responses in a mouse model of HLA-A*02:01 restriction and protect immunized mice from challenge with Wuhan-hu-1 virus after hACE2 transduction. These data provide candidate T-cell epitopes useful for the development of T-cell vaccines against SARS-CoV-2 and demonstrate a strategy for quick T-cell vaccine candidate development applicable to other emerging pathogens.

Functional and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential host range of SARS-CoV-2.

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs-including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria-could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

Suppression of Vanadium Oxide Dissolution via Cation Metathesis within a Coordination Supramolecular Network for Durable Aqueous Zn-V2 O5 Batteries.

Aqueous zinc metal batteries (ZMBs) are a promising sustainable technology for large-scale energy storage applications. However, the water is often associated with problematic parasitic reactions on both anode and cathode, leading to the low durability and reliability of ZMBs. Here, a multifunctional separator for the Zn-V2 O5 batteries by growing the coordination supramolecular network (CSN:Zn-MBA, MBA = 2-mercaptobenzoic acid) on the conventional non-woven fabrics (NWF) is developed. CSN tends to form a stronger coordination bond as a softer cation, enabling a thermodynamically preferred Zn2+ to VO2 + substitution in the network, leading to the formation of VO2 -MBA interface, that strongly obstructs the VO2 (OH)2 - penetration but simultaneously allows Zn2+ transfer. Moreover, Zn-MBA molecules can adsorb the OTF- and distribute the interfacial Zn2+ homogeneous, which facilitate a dendrite-free Zn deposition. The Zn-V2 O5 cells with Zn-MBA@NWF separator realize high capacity of 567 mAh g-1 at 0.2 A g-1 , and excellent cyclability over 2000 cycles with capacity retention of 82.2% at 5 A g-1 . This work combines the original advantages of the template and new function of metals via cation metathesis within a CSN, provides a new strategy for inhibiting vanadium oxide dissolution.

HBV covalently closed circular DNA minichromosomes in distinct epigenetic transcriptional states differ in their vulnerability to damage.

BACKGROUND AND AIMS: HBV covalently closed circular DNA (cccDNA) is a major obstacle for a cure of chronic hepatitis B. Accumulating evidence suggests that epigenetic modifications regulate the transcriptional activity of cccDNA minichromosomes. However, it remains unclear how the epigenetic state of cccDNA affects its stability. APPROACHES AND RESULTS: By using HBV infection cell models and in vitro and in vivo recombinant cccDNA (rcccDNA) and HBVcircle models, the reduction rate of HBV cccDNA and the efficacy of apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A)-mediated and CRISPR/CRISPR-associated 9 (Cas9)-mediated cccDNA targeting were compared between cccDNAs with distinct transcriptional activities. Interferon-α treatment and hepatitis B x protein (HBx) deletion were applied as two strategies for cccDNA repression. Chromatin immunoprecipitation and micrococcal nuclease assays were performed to determine the epigenetic pattern of cccDNA. HBV cccDNA levels remained stable in nondividing hepatocytes; however, they were significantly reduced during cell division, and the reduction rate was similar between cccDNAs in transcriptionally active and transcriptionally repressed states. Strikingly, HBV rcccDNA without HBx expression exhibited a significantly longer persistence in mice. The cccDNA with low transcriptional activity exhibited an epigenetically inactive pattern and was more difficult to access by APOBEC3A and engineered CRISPR-Cas9. The epigenetic regulator activating cccDNA increased its vulnerability to APOBEC3A. CONCLUSIONS: HBV cccDNA minichromosomes in distinct epigenetic transcriptional states showed a similar reduction rate during cell division but significantly differed in their accessibility and vulnerability to targeted nucleases and antiviral agents. Epigenetic sensitization of cccDNA makes it more susceptible to damage and may potentially contribute to an HBV cure.

Forward genetic analysis using OCT screening identifies Sfxn3 mutations leading to progressive outer retinal degeneration in mice.

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N-ethyl-N-nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3-/- mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3-/- mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.

Constructing Hydrophobic Interface with Close-Packed Coordination Supramolecular Network for Long-Cycling and Dendrite-Free Zn-Metal Batteries.

Commercialization of aqueous zinc-metal batteries remains unrealistic due to the substantial dendrite growth and side reaction issues on the zinc anodes. It is highly demanded to develop easy-to-handle approaches for constructing stable, dense, as well as homogeneous solid anode/electrolyte interfaces. Herein, the authors construct the zinc anode interface with a close-packed Zn-TSA (TSA = thiosalicylate) coordination supramolecular network through the facile and up-scalable wet-chemical method. The hydrophobic Zn-TSA network can block solvated water and establish a solid-state diffusion barrier to well-distribute the interfacial Zn2+ , thus inhibiting hydrogen evolution and zinc dendrite growth on the anode. Meanwhile, the Zn-TSA network induces the formation of a uniform and stable solid electrolyte interphase composed of multiple inorganic-organic compounds. This denser structure can accommodate and self-heal the crack/degradation of the anode interphase associated with the repeated volume changes, and suppress the generation of detrimental by-product, Znx (OTF- )y (OH)2x-y ·nH2 O. Such a rationally fabricated anode/electrolyte interface further endows the assembled symmetric cells with superior plating/stripping stability for over 2000 h without dendrite formation (at 1 mA cm-2 and 1 mAh cm-2 ). Furthermore, this zinc anode has practical application in the Zn-MoS2 and Zn-V2 O5 full cells. This study provides a new train of thought for constructing the dense interface of zinc-metal anode.

The positive role of parental attachment and communication in Chinese adolescents' health behavior and mental health during COVID-19.

INTRODUCTION: Given that coronavirus disease 2019 (COVID-19) has largely influenced adolescents' physical and mental health around the globe, it is important to identify protective factors that may promote adolescents' positive adjustment during the pandemic. This study aimed to examine the role of parental attachment and COVID-19 communication in adolescents' health behavior and mental health during COVID-19. METHODS: A total of 442 Chinese parent-adolescent dyads (mean age of adolescents = 13.35 years; 50% girls) completed two-wave longitudinal surveys over the span of 2 months during the pandemic (Wave 1: July 2020; Wave 2: September 2020). At each wave, adolescents reported on their COVID-19-related health behavior, general health behavior, depressive symptoms, and anxiety symptoms. At Wave 1, parent-adolescent attachment security and COVID-19 communication were also assessed. RESULTS: Adolescents' attachment security to parents was associated with their increased COVID-19-related and general health behavior as well as decreased depression and anxiety over 2 months during COVID-19. Moreover, more frequent parent-adolescent COVID-19 communication was associated with adolescents' increased COVID-19-related and general health behavior over time. Notably, attachment security's and COVID-19 communication's associations with health behavior largely remained the same after taking into account both factors simultaneously. In addition, results from exploratory analyses suggest that more frequent COVID-19 communication mediates the link between attachment security and increased health behavior. CONCLUSIONS: These findings highlight the importance of promoting attachment security and COVID-19 communication between parents and adolescents during the pandemic, which may play a positive role in adolescents' health behavior and mental health.

Translatomic profiling reveals novel self-restricting virus-host interactions during HBV infection.

BACKGROUND & AIMS: HBV remains a global threat to human health. It remains incompletely understood how HBV self-restricts in the host during most adult infections. Thus, we performed multi-omics analyses to systematically interrogate HBV-host interactions and the life cycle of HBV. METHODS: RNA-sequencing and ribosome profiling were conducted with cell-based models for HBV replication and gene expression. The novel translational events or products hereby detected were then characterized, and functionally assessed in both cell and mouse models. Moreover, quasi-species analyses of HBV subpopulations were conducted with patients at immune tolerance or activation phases, using next- or third-generation sequencing. RESULTS: We identified EnhI-SL (Enhancer I-stem loop) as a new cis element in the HBV genome; mutations disrupting EnhI-SL were found to elevate viral polymerase expression. Furthermore, while re-discovering HpZ/P', a previously under-explored isoform of HBV polymerase, we also identified HBxZ, a novel short isoform of HBX. Having confirmed their existence, we functionally characterized them as potent suppressors of HBV gene expression and genome replication. Mechanistically, HpZ/P' was found to repress HBV gene expression partially by interacting with, and sequestering SUPV3L1. Activation of the host immune system seemed to reduce the abundance of HBV mutants deficient in HpZ/P' or with disruptions in EnhI-SL. Finally, SRSF2, a host RNA spliceosome protein that is downregulated by HBV, was found to promote the splicing of viral pre-genomic RNA and HpZ/P' biogenesis. CONCLUSION: This study has identified multiple self-restricting HBV-host interactions. In particular, SRSF2-HpZ/P' appeared to constitute another negative feedback mechanism in the HBV life cycle. Targeting host splicing machinery might thus represent a strategy to intervene in HBV-host interactions. LAY SUMMARY: There remain many unknowns about the natural history of HBV infection in adults. Herein, we identified new HBV-host mechanisms which could be responsible for self-restricting infections. Targeting these mechanisms could be a promising strategy for the treatment of HBV infections.

Impacts of Parental Burnout on Chinese Youth's Mental Health: The Role of Parents' Autonomy Support and Emotion Regulation.

Parental burnout is a state that parents experience overwhelming exhaustion in their parental role. Given the detrimental impacts of parents' stress on adolescent development, youth may suffer from undesirable emotional adjustment due to parental burnout. Therefore, it is key to understand the underlying mechanisms through which parental burnout may play a role in youth's mental health and identify protective factors that may reduce the potential negative impacts. Using a sample of 442 Chinese parent-adolescent dyads (Mean age of youth = 13.35 years; 50% girls), this two-wave longitudinal study examined how parental burnout contributes to youth's mental health over the span of two months. Moreover, the current research investigated the potential mediating role of autonomy support and the potential moderating role of emotion regulation in the links between parental burnout and youth's mental health. The results showed that greater parental burnout was predictive of youth's greater depressive and anxiety symptoms two months later, and such effects were partially mediated by less autonomy-supportive parenting. Notably, the negative effects of parental burnout on autonomy-supportive parenting and youth's mental health were not significant when parents used more cognitive reappraisal to regulate their emotions. These findings demonstrate the underlying mechanisms of how parental burnout affects youth's mental health over time and highlight the protective role of healthy emotion regulation against parental burnout.

Effects of Pulsatile Frequency of Left Ventricular Assist Device (LVAD) on Coronary Perfusion: A Numerical Simulation Study.

BACKGROUND Left ventricular assist devices (LVADs) with counter-pulsation mode have been widely used to support left ventricular function and improve coronary circulation. However, the frequency characteristics of the coronary system have not been considered. The aim of this study was to investigate the effects of pulsatile frequency of LVADs on coronary perfusion. MATERIAL AND METHODS First, a lumped parameter (LP) model incorporating coronary circulation, systemic circulation, left heart, and LVAD was established to simulate the cardiovascular system. Then, the frequency characteristics of the coronary system were analyzed and the calculation results showed that the pulsatile frequency of the LVAD has a substantial effect on coronary blood flow. To verify the accuracy of the theoretical analysis, the hemodynamic effects of the LVAD on the coronary artery were compared under 4 support modes: co-pulsation mode, and counter-pulsation modes in synchronization ratios of 1: 1, 2: 1, and 3: 1. RESULTS We found that the coronary flow increased by 5% when the working mode changed from co-pulsation to counter-pulsation in a synchronization ratio of 1: 1, and by an additional 6% when the working mode changed from counter-pulsation in a synchronization ratio of 1: 1 to counter-pulsation in a synchronization ratio of 3: 1. CONCLUSIONS This work provides a useful method to increase coronary perfusion and may be beneficial for improving myocardial function in patients with end-stage heart failure, especially those with ischemic cardiomyopathy (ICM).

A Driving and Control Scheme of High Power Piezoelectric Systems over a Wide Operating Range.

Significant variation in impedance under a wide range of loads increases the difficulty of frequency tracking and vibration control in high-power piezoelectric systems (HPPSs). This paper proposed a wide operating range driving and control scheme for HPPSs. We systematically analyzed the impedance characteristics and deduced the load optimization frequency. In order to provide sufficient drive capability, the inverter combined with an LC matching circuit is configured. With the aid of a transformer ratio arm bridge (TRAB) combined with a proposed pulse-based phase detector (PBPD), the proposed scheme can control the vibration amplitude and keep parallel resonance status under a wide range of loads. Experiments conducted under actual operating conditions verify the feasibility of the proposed scheme under the modal resistance range from 7.40 to 500 Ω and the vibration range from 20% to 100%. Moreover, with the aid of a laser displacement sensor, our scheme is verified to have a vibration amplitude control accuracy better than 2% over a tenfold load variation. This research could be helpful for the driving and control of HPPSs operating in a wide range.

Recombinant covalently closed circular DNA of hepatitis B virus induces long-term viral persistence with chronic hepatitis in a mouse model.

Covalently closed circular DNA of hepatitis B virus (HBV) is critical for viral persistence in vivo. We recently reported a technique involving recombinant covalently closed circular DNA (rcccDNA) of HBV by site-specific DNA recombination. Using hydrodynamic injection, rcccDNA induces a temporarily prolonged HBV antigenemia in immunocompetent mice, similar to acute resolving HBV infection. In this study, we simulated the pathophysiological impact of chronic hepatitis to reproduce rcccDNA persistence in mouse models. We showed that rcccDNA achieved long-lasting persistence in the presence of a compromised immune response or when transcriptional activity was repressed. To closely mimic chronic hepatitis, we used a replication-defective recombinant adenoviral vector to deliver rcccDNA to the liver, which led to prominent HBV persistence throughout the experiment duration (>62 weeks) in transgenic mice expressing Cre recombinase under the albumin promoter. A sustained necroinflammatory response and fibrosis were identified in mouse livers, with dysplastic lesions commonly seen during the late stage of viral persistence, analogous to the progressive pathology of clinical chronic hepatitis. CONCLUSION: rcccDNA was intrinsically stable in vivo, enabling long-term persistence in the context of chronic hepatitis, and viral persistence, in turn, may promote progression of chronic liver disease; our study also presented a surrogate model of HBV cccDNA persistence in mice that could advance our understanding of the pathogenesis of chronic hepatitis B. (Hepatology 2018;67:56-70).

Velocity Control of Traveling-Wave Ultrasonic Motors Based on Stator Vibration Amplitude.

Nonlinearity and resonance frequency shift make it difficult to control the operation of the traveling-wave ultrasonic motors (TWUSMs) in a wide velocity and load range. In this paper, a velocity control scheme based on the stator vibration amplitude and the parallel resonance frequency (VCBVF) of TWUSMs is proposed. Then, the stator vibration amplitude (SVA) and parallel resonance frequency ( f p ) are detected by a transformer ratio-arm bridge. Based on the linear relationship between the velocity and the SVA of TWUSMs, the proposed scheme achieves the control of the mechanical loop and the electrical loop. The linear relationship between the velocity and the SVA makes the mechanical loop achieve the target velocity efficiently, according to the SVA, and the electrical loop could provide the target SVA quickly. Experimental results show that the response time of velocity is 3-4 ms under different load torques and the overshoot is less than 22%. In addition, the proposed scheme improves the efficiency of TWUSMs due to f p tracking. Due to directing the SVA control, the proposed scheme can heighten the velocity response and the load adaptability of TWUSMs, and promote the application of TWUSMs under various conditions.

A wide drive frequency matching method for harmonic suppression and voltage stabilization of ultrasonic motors.

To reduce harmonic components, balance system impedance, and stabilize driving voltage, an additional matching circuit is required for ultrasonic motors (USMs) driver. However, the performance of inductor or capacitor matching can be seriously weakened with changes in driving frequency. Therefore, this paper presents a simple and effective LC matching method against driving frequency adjustment for USMs. First, the driving scheme of the USM is proposed and the electromechanical coupling model is analyzed. Subsequently, the output characteristics of the full-bridge inverter are derived theoretically when the driving frequency deviates from the mechanical resonant frequency. Then, the impedance circular transform method is proposed, which can intuitively analyze the effect of matching parameters on the voltage amplitude. A matching objective function is established that can consider both the voltage stabilization and harmonic suppression. The matching parameters are solved using random weight particle swarm optimization. Simulations and experiments demonstrate that within the operating frequency of the USM, the proposed matching method can effectively prevent overvoltage and suppress harmonic components. Furthermore, compared with the existing resonant matching method, the proposed matching method can realize more stable driving capability at different frequencies. The proposed method could be useful for USMs' variable-frequency driver design.

The Moderation Effect of Approach Motivation Between Schizotypy and Creative Ideational Behavior.

Introduction: The schizotypy-creativity link has been studied from different perspectives over the past few decades, yet the results of this relationship are inconsistent in the literature. Previous studies have suggested that two basic motivational systems-Behavioral Inhibition System (BIS, avoidance motivation) and Behavioral Activation System (BAS, approach motivation)-underlie the relationship between schizotypy and creativity. Few empirical studies, however, have examined how the relationship interacts with other variables. This study fills these gaps and explores the role of the approach and avoidance motivation assessed by trait behavioral activation and inhibition in the link between schizotypy as a dimensional personality trait and creative ideational behavior as a measure of creativity. Method: Undergraduate students (N = 388) completed questionnaires including the Schizotypal Personality Questionnaire (SPQ) measuring 3 dimensions of schizotypy, Runco Ideational Behavior Scale (RIBS) measuring creative ideational behavior, and BIS/BAS Scales measuring trait behavioral motivation. Bivariate Pearson correlation was computed, and hierarchical linear regression was performed to explore the effects of schizotypy, BIS/BAS, and their interaction on creative ideational behavior. The conditional effect of schizotypy based on different levels of the moderator was further tested. Results: The total score, positive dimension and disorganized dimension of SPQ were all positively correlated with RIBS, BAS, and BIS. Negative dimension of SPQ was not significantly correlated with the RIBS score but was positively correlated with BIS. Additionally, after controlling gender and age, BAS significantly moderated the relationship between the positive and disorganized dimensions of schizotypy and creative ideational behavior measured by RIBS. However, BIS was not a significant moderator. Discussion: The findings of this study regarding the relationships between different dimensions of schizotypy, two motivational systems, and creative ideational behavior were mostly consistent with previous findings. The significant moderated effect of BAS on the relationship between two dimensions of schizotypy and creative ideational behavior made significant contributions to the understanding of the relationship between schizotypy and creativity.

Development of a Cell Culture Model for Inducible SARS-CoV-2 Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/LoxP-mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/LoxP-mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics.

TGF-ß1/SMAD3-driven GLI2 isoform expression contributes to aggressive phenotypes of hepatocellular carcinoma.

Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-ß1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-ß1/SMAD3-mediated non-canonical activation was found in approximately half of GLI2-positive hepatocellular carcinoma (HCC), and two new GLI2 isoforms with transactivating activity were identified. Phospho-SMAD3 interacted with active GLI2 isoforms to transactivate downstream genes in modulation of stemness, epithelial-mesenchymal transition, chemo-resistance and metastasis in poorly-differentiated hepatoma cells. Non-canonical activation of hedgehog signaling was confirmed in a transgenic HBV-associated HCC mouse model. Inhibition of TGF-ß/SMAD3 signaling reduced lung metastasis in a mouse in situ hepatic xenograft model. In another cohort of 55 HCC patients, subjects with high GLI2 expression had a shorter disease-free survival than those with low expression. Moreover, co-positivity of GLI2 with SMAD3 was observed in 87.5% of relapsed HCC patients with high GLI2 expression, indicating an increased risk of post-resection recurrence of HCC. The findings underscore that suppressing the non-canonical hedgehog signaling pathway may confer a potential strategy in the treatment of HCC.

Hepatitis B virus core protein as a Rab-GAP suppressor driving liver disease progression.

Chronic hepatitis B virus (HBV) infection can lead to advanced liver pathology. Here, we establish a transgenic murine model expressing a basic core promoter (BCP)-mutated HBV genome. Unlike previous studies on the wild-type virus, the BCP-mutated HBV transgenic mice manifest chronic liver injury that culminates in cirrhosis and tumor development with age. Notably, agonistic anti-Fas treatment induces fulminant hepatitis in these mice even at a negligible dose. As the BCP mutant exhibits a striking increase in HBV core protein (HBc) expression, we posit that HBc is actively involved in hepatocellular injury. Accordingly, HBc interferes with Fis1-stimulated mitochondrial recruitment of Tre-2/Bub2/Cdc16 domain family member 15 (TBC1D15). HBc may also inhibit multiple Rab GTPase-activating proteins, including Rab7-specific TBC1D15 and TBC1D5, by binding to their conserved catalytic domain. In cells under mitochondrial stress, HBc thus perturbs mitochondrial dynamics and prevents the recycling of damaged mitochondria. Moreover, sustained HBc expression causes lysosomal consumption via Rab7 hyperactivation, which further hampers late-stage autophagy and substantially increases apoptotic cell death. Finally, we show that adenovirally expressed HBc in a mouse model is directly cytopathic and causes profound liver injury, independent of antigen-specific immune clearance. These findings reveal an unexpected cytopathic role of HBc, making it a pivotal target for HBV-associated liver disease treatment. The BCP-mutated HBV transgenic mice also provide a valuable model for understanding chronic hepatitis B progression and for the assessment of therapeutic strategies.