SARS-CoV-2 Delta and Omicron variants resist spike cleavage by human airway trypsin-like protease.

Soluble host factors in the upper respiratory tract can serve as the first line of defense against SARS-CoV-2 infection. In this study, we described the identification and function of a human airway trypsin-like protease (HAT), capable of reducing the infectivity of ancestral SARS-CoV-2. Further, in mouse models, HAT analogue expression was upregulated by SARS-CoV-2 infection. The antiviral activity of HAT functioned through the cleavage of the SARS-CoV-2 spike glycoprotein at R682. This cleavage resulted in inhibition of the attachment of ancestral spike proteins to host cells, which inhibited the cell-cell membrane fusion process. Importantly, exogenous addition of HAT notably reduced the infectivity of ancestral SARS-CoV-2 in vivo. However, HAT was ineffective against the Delta variant and most circulating Omicron variants, including the BQ.1.1 and XBB.1.5 subvariants. We demonstrate that the P681R mutation in Delta and P681H mutation in the Omicron variants, adjacent to the R682 cleavage site, contributed to HAT resistance. Our study reports what we believe to be a novel soluble defense factor against SARS-CoV-2 and resistance of its actions in the Delta and Omicron variants.

Nanoplatform Based Intranasal Vaccines: Current Progress and Clinical Challenges.

Multiple vaccine platforms have been employed to develop the nasal SARS-CoV-2 vaccines in preclinical studies, and the dominating pipelines are viral vectored as protein-based vaccines. Among them, several viral vectored-based vaccines have entered clinical development. Nevertheless, some unsatisfactory results were reported in these clinical studies. In the face of such urgent situations, it is imperative to rapidly develop the next-generation intranasal COVID-19 vaccine utilizing other technologies. Nanobased intranasal vaccines have emerged as an approach against respiratory infectious diseases. Harnessing the power of nanotechnology, these vaccines offer a noninvasive yet potent defense against pathogens, including the threat of COVID-19. The improvements made in vaccine mucosal delivery technologies based on nanoparticles, such as lipid nanoparticles, polymeric nanoparticles, inorganic nanoparticles etc., not only provide stability and controlled release but also enhance mucosal adhesion, effectively overcoming the limitations of conventional vaccines. Hence, in this review, we overview the evaluation of intranasal vaccine and highlight the current barriers. Next, the modern delivery systems based on nanoplatforms are summarized. The challenges in clinical application of nanoplatform based intranasal vaccine are finally discussed.

Similarities and differences in dynamic properties of brain networks between internet gaming disorder and tobacco use disorder.

BACKGROUND: Internet gaming disorder (IGD) and tobacco use disorder (TUD) are two major addiction disorders that result in substantial financial loss. Identifying the similarities and differences between these two disorders is important to understand substance addiction and behavioral addiction. The current study was designed to compare these two disorders utilizing dynamic analysis. METHOD: Resting-state data were collected from 35 individuals with IGD, 35 individuals with TUD and 35 healthy controls (HCs). Dynamic coactivation pattern analysis was employed to decipher their dynamic patterns. RESULTS: IGD participants showed decreased coactivation patterns within the default mode network (DMN) and between the DMN and the salience network (SN). The SN showed reduced coactivation patterns with the executive control network (ECN) and DMN, and the ECN showed decreased coactivation patterns with the DMN. In the TUD group, the DMN exhibited decreased coactivation patterns with the SN, the SN exhibited reduced coactivation patterns with the DMN and ECN, and the ECN showed decreased coactivation patterns with the DMN and within the ECN. Furthermore, the triple network model was fitted to the dynamic properties of the two addiction disorders. Decoding analysis results indicated that addiction-related memory and memory retrieval displayed similar dysfunctions in both addictions. CONCLUSION: The dynamic characteristics of IGD and TUD suggest that there are similarities in the dynamic features between the SN and DMN and differences in the dynamic features between the DMN and ECN. Our results revealed that the two addiction disorders have dissociable brain mechanisms, indicating that future studies should consider these two addiction disorders as having two separate mechanisms to achieve precise treatment for their individualized targets.

XBB.1.16-RBD-based trimeric protein vaccine can effectively inhibit XBB.1.16-included XBB subvariant infection.

The newly identified XBB.1.16-containing sublineages, including XBB.1.5, have become the prevailing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant in circulation. Unlike previous Omicron XBB variants (e.g., XBB.1.5 and XBB.1.9) harboring the F486P substitution, XBB.1.16 also carries a T478R substitution in the receptor-binding domain (RBD). Numerous researchers have delved into the high transmissibility and immune evasion of XBB.1.16 subvariant. Therefore, developing a new vaccine targeting XBB.1.16, including variants of concern (VOCs), is paramount. In our study, we engineered a recombinant protein by directly linking the S-RBD sequence of the XBB.1.16 strain of SARS-CoV-2 to the sequences of two heptad repeat sequences (HR1 and HR2) from the SARS-CoV-2 S2 subunit. Named the recombinant RBDXBB.1.16-HR/trimeric protein, this fusion protein autonomously assembles into a trimer. Combined with an MF59-like adjuvant, the RBDXBB.1.16-HR vaccine induces a robust humoral immune response characterized by high titers of neutralizing antibodies against variant pseudovirus and authentic VOCs and cellular immune responses. Additionally, a fourth heterologous RBDXBB.1.16-HR vaccine enhances both humoral and cellular immune response elicited by three-dose mRNA vaccines. These findings demonstrate that the recombinant RBDXBB.1.16-HR protein, featuring the new T478R mutation, effectively induces solid neutralizing antibodies to combat newly emerged XBB variants.

Dopaminylation of endothelial TPI1 suppresses ferroptotic angiocrine signals to promote lung regeneration over fibrosis.

Lungs can undergo facultative regeneration, but handicapped regeneration often leads to fibrosis. How microenvironmental cues coordinate lung regeneration via modulating cell death remains unknown. Here, we reveal that the neurotransmitter dopamine modifies the endothelial niche to suppress ferroptosis, promoting lung regeneration over fibrosis. A chemoproteomic approach shows that dopamine blocks ferroptosis in endothelial cells (ECs) via dopaminylating triosephosphate isomerase 1 (TPI1). Suppressing TPI1 dopaminylation in ECs triggers ferroptotic angiocrine signaling to aberrantly activate fibroblasts, leading to a transition from lung regeneration to fibrosis. Mechanistically, dopaminylation of glutamine (Q) 65 residue in TPI1 directionally enhances TPI1's activity to convert dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP), directing ether phospholipid synthesis to glucose metabolism in regenerating lung ECs. This metabolic shift attenuates lipid peroxidation and blocks ferroptosis. Restoring TPI1 Q65 dopaminylation in an injured endothelial niche overturns ferroptosis to normalize pro-regenerative angiocrine function and alleviate lung fibrosis. Overall, dopaminylation of TPI1 balances lipid/glucose metabolism and suppresses pro-fibrotic ferroptosis in regenerating lungs.

A Fabric-Based Strain Sensor with a Microbridge Structure and the Supercapacitor-Powered Integrated Sensing System.

Developing fabric-based strain sensors with high sensitivity and stability is in high demand for wearable electronics. Herein, carbon nanotubes (CNTs) and polypyrrole (PPy) are coated on a thermoplastic polyurethane (TPU) fabric as strain sensors. A microbridge structure, in which CNT bridges the stretching-induced cracks, has been designed for the TPU-CNT-PPy strain sensor. The microbridge structure can significantly enhance the electrical resilience, ensuring the improved sensitivity and stability of strain sensors. As a result, our TPU-CNT-PPy strain sensors deliver high sensitivity (GF = 231.5) with a broad working range (150%) and fast response and recovery time (166/195 ms). In addition, our TPU-CNT-PPy could also be used as flexible electrodes of the microsupercapacitors (MSCs) as a power supplier for the integrated sensing system. The TPU-CNT-PPy-based MSCs exhibit a high specific capacitance (460.3 mF cm-2 at 0.5 mA cm-2) and excellent cycling stability (96.69% capacitance retention for 10,000 charge/discharge cycles). Finally, we demonstrated an integrated sensing system using TPU-CNT-PPy as both MSCs and strain sensors, where the current signals of the sensors could be well detected via Bluetooth. This study offers a microbridge strategy to fabricate strain sensors with high sensitivity and stability and develops an integrated sensing system for the actual applications of wearable electronics.

Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT.

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

Structure and activity of the septal peptidoglycan hydrolysis machinery crucial for bacterial cell division.

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.

A chimeric adenovirus-vectored vaccine based on Beta spike and Delta RBD confers a broad-spectrum neutralization against Omicron-included SARS-CoV-2 variants.

Urgent research into innovative severe acute respiratory coronavirus-2 (SARS-CoV-2) vaccines that may successfully prevent various emerging emerged variants, particularly the Omicron variant and its subvariants, is necessary. Here, we designed a chimeric adenovirus-vectored vaccine named Ad5-Beta/Delta. This vaccine was created by incorporating the receptor-binding domain from the Delta variant, which has the L452R and T478K mutations, into the complete spike protein of the Beta variant. Both intramuscular (IM) and intranasal (IN) vaccination with Ad5-Beta/Deta vaccine induced robust broad-spectrum neutralization against Omicron BA.5-included variants. IN immunization with Ad5-Beta/Delta vaccine exhibited superior mucosal immunity, manifested by higher secretory IgA antibodies and more tissue-resident memory T cells (TRM) in respiratory tract. The combination of IM and IN delivery of the Ad5-Beta/Delta vaccine was capable of synergically eliciting stronger systemic and mucosal immune responses. Furthermore, the Ad5-Beta/Delta vaccination demonstrated more effective boosting implications after two dosages of mRNA or subunit recombinant protein vaccine, indicating its capacity for utilization as a booster shot in the heterologous vaccination. These outcomes quantified Ad5-Beta/Delta vaccine as a favorable vaccine can provide protective immunity versus SARS-CoV-2 pre-Omicron variants of concern and BA.5-included Omicron subvariants.

The severity of addiction mediates loneliness and cortical volume in internet gaming disorder.

Internet gaming disorder (IGD) subjects reported higher loneliness scores than healthy controls. However, the neural correlates underlying the association between loneliness and IGD remain unclear. Thus, the aim of this study was to explore the relationship between loneliness, online gaming addiction and brain structure. In the current study, structural MRI data were acquired from 84 IGD subjects and 103 matched recreational game users (RGUs). We assessed and compared their addiction severity, loneliness scores, and cortical volumes and analyzed the correlations among these values. Significant correlations were found between loneliness scores and brain volumes in the postcentral cortex, the medial orbitofrontal cortex, the rostral anterior cingulate cortex, and the temporal cortex. In addition, the addiction severity scores partly mediated the relationship between loneliness score and cortical volume in IGD. The results showed that participants with extreme loneliness had significant correlations with brain regions responsible for executive control, social threat surveillance and avoidance. More importantly, the severity of addiction mediates loneliness and cortical volume. The findings shed new insight into the neural mechanisms of loneliness and IGD and have implications for potential treatment.

Development of a bispecific nanobody conjugate broadly neutralizes diverse SARS-CoV-2 variants and structural basis for its broad neutralization.

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and profound immune-escape capacity makes it an urgent need to develop broad-spectrum therapeutics. Nanobodies have recently attracted extensive attentions due to their excellent biochemical and binding properties. Here, we report two high-affinity nanobodies (Nb-015 and Nb-021) that target non-overlapping epitopes in SARS-CoV-2 S-RBD. Both nanobodies could efficiently neutralize diverse viruses of SARS-CoV-2. The neutralizing mechanisms for the two nanobodies are further delineated by high-resolution nanobody/S-RBD complex structures. In addition, an Fc-based tetravalent nanobody format is constructed by combining Nb-015 and Nb-021. The resultant nanobody conjugate, designated as Nb-X2-Fc, exhibits significantly enhanced breadth and potency against all-tested SARS-CoV-2 variants, including Omicron sub-lineages. These data demonstrate that Nb-X2-Fc could serve as an effective drug candidate for the treatment of SARS-CoV-2 infection, deserving further in-vivo evaluations in the future.

Structural and functional basis of low-affinity SAM/SAH-binding in the conserved MTase of the multi-segmented Alongshan virus distantly related to canonical unsegmented flaviviruses.

Alongshan virus (ALSV), a newly discovered member of unclassified Flaviviridae family, is able to infect humans. ALSV has a multi-segmented genome organization and is evolutionarily distant from canonical mono-segmented flaviviruses. The virus-encoded methyltransferase (MTase) plays an important role in viral replication. Here we show that ALSV MTase readily binds S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) but exhibits significantly lower affinities than canonical flaviviral MTases. Structures of ALSV MTase in the free and SAM/SAH-bound forms reveal that the viral enzyme possesses a unique loop-element lining side-wall of the SAM/SAH-binding pocket. While the equivalent loop in flaviviral MTases half-covers SAM/SAH, contributing multiple hydrogen-bond interactions; the pocket-lining loop of ALSV MTase is of short-length and high-flexibility, devoid of any physical contacts with SAM/SAH. Subsequent mutagenesis data further corroborate such structural difference affecting SAM/SAH-binding. Finally, we also report the structure of ALSV MTase bound with sinefungin, an SAM-analogue MTase inhibitor. These data have delineated the basis for the low-affinity interaction between ALSV MTase and SAM/SAH and should inform on antiviral drug design.

Cryo-EM structures of human GPR34 enable the identification of selective antagonists.

GPR34 is a functional G-protein-coupled receptor of Lysophosphatidylserine (LysoPS), and has pathogenic roles in numerous diseases, yet remains poorly targeted. We herein report a cryo-electron microscopy (cryo-EM) structure of GPR34 bound with LysoPS (18:1) and Gi protein, revealing a unique ligand recognition mode with the negatively charged head group of LysoPS occupying a polar cavity formed by TM3, 6 and 7, and the hydrophobic tail of LysoPS residing in a lateral open hydrophobic groove formed by TM3-5. Virtual screening and subsequent structural optimization led to the identification of a highly potent and selective antagonist (YL-365). Design of fusion proteins allowed successful determination of the challenging cryo-EM structure of the inactive GPR34 complexed with YL-365, which revealed the competitive binding of YL-365 in a portion of the orthosteric binding pocket of GPR34 and the antagonist-binding-induced allostery in the receptor, implicating the inhibition mechanism of YL-365. Moreover, YL-365 displayed excellent activity in a neuropathic pain model without obvious toxicity. Collectively, this study offers mechanistic insights into the endogenous agonist recognition and antagonist inhibition of GPR34, and provides proof of concept that targeting GPR34 represents a promising strategy for disease treatment.

Cryo-EM structure of the nucleocapsid-like assembly of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a nonsegmented, negative strand RNA virus that has caused severe lower respiratory tract infections of high mortality rates in infants and the elderly, yet no effective vaccine or antiviral therapy is available. The RSV genome encodes the nucleoprotein (N) that forms helical assembly to encapsulate and protect the RNA genome from degradation, and to serve as a template for transcription and replication. Previous crystal structure revealed a decameric ring architecture of N in complex with the cellular RNA (N-RNA) of 70 nucleotides (70-nt), whereas cryo-ET reconstruction revealed a low-resolution left-handed filament, in which the crystal monomer structure was docked with the helical symmetry applied to simulate a nucleocapsid-like assembly of RSV. However, the molecular details of RSV nucleocapsid assembly remain unknown, which continue to limit our complete understanding of the critical interactions involved in the nucleocapsid and antiviral development that may target this essential process during the viral life cycle. Here we resolve the near-atomic cryo-EM structure of RSV N-RNA that represents roughly one turn of the helical assembly that unveils critical interaction interfaces of RSV nucleocapsid and may facilitate development of RSV antiviral therapy.

Lipid-Polymer Hybrid Nanoparticles with Both PD-L1 Knockdown and Mild Photothermal Effect for Tumor Photothermal Immunotherapy.

In developing countries, the incidence of colorectal cancer (CRC) is on the rise. The combination of programmed cell death ligand-1 (PD-L1) siRNA (siPD-L1) and mild photothermal therapy (PTT) is a promising strategy for CRC treatment. In this study, dopamine-modified polyethylenimine (PEI) was prepared to fabricate an IR780 and siPD-L1 codelivery lipid-polymer hybrid nanoparticle (lip@PSD-siP) for the photothermal immunotherapy of CRC. The modification of dopamine can significantly reduce the cytotoxicity of PEI. lip@PSD-siP can be effectively taken up by CT26 cells and successfully escaped from lysosomes after entering the cells for 4 h. After CT26 cells were transfected with lip@PSD-siP, the PD-L1 positive cell rate decreased by 82.4%, and its PD-L1 knockdown effect was significantly stronger than the positive control Lipo3000-siP. In vivo studies showed that lip@PSD-siP-mediated mild PTT and efficient PD-L1 knockdown exhibited primary and distal tumor inhibition, metastasis delay, and rechallenged tumor inhibition. The treatment with lip@PSD-siP significantly promoted the maturation of dendritic cells in lymph nodes. The amount of T cell infiltration in the tumor tissues increased significantly, and the frequency of CD8+ T cells and CD4+ T cells was significantly higher than that of other groups. The percentage of immunosuppressive regulatory cells (Tregs) in the tumor tissue on the treatment side decreased by 88% compared to the PBS group, and the proportion of CD8+CD69+ T cells in the distal tumor tissue was 2.8 times that of the PBS group. The memory T cells of mice in the long-term antitumor model were analyzed. The results showed that after treatment with lip@PSD-siP, the frequency of effector memory T cells (Tem cells) significantly increased, suggesting the formation of immune memory.

Structural basis of BAM-mediated outer membrane ß-barrel protein assembly.

The outer membrane structure is common in Gram-negative bacteria, mitochondria and chloroplasts, and contains outer membrane ß-barrel proteins (OMPs) that are essential interchange portals of materials1-3. All known OMPs share the antiparallel ß-strand topology4, implicating a common evolutionary origin and conserved folding mechanism. Models have been proposed for bacterial ß-barrel assembly machinery (BAM) to initiate OMP folding5,6; however, mechanisms by which BAM proceeds to complete OMP assembly remain unclear. Here we report intermediate structures of BAM assembling an OMP substrate, EspP, demonstrating sequential conformational dynamics of BAM during the late stages of OMP assembly, which is further supported by molecular dynamics simulations. Mutagenic in vitro and in vivo assembly assays reveal functional residues of BamA and EspP for barrel hybridization, closure and release. Our work provides novel insights into the common mechanism of OMP assembly.

Deficient dynamics of prefrontal-striatal and striatal-default mode network (DMN) neural circuits in internet gaming disorder.

BACKGROUND: Studies have proven that individuals with internet gaming disorder (IGD) show impaired cognitive control over game craving; however, the neural mechanism underlying this process remains unclear. Accordingly, the present study aimed to investigate the dynamic features of brain functional networks of individuals with IGD during rest, which have barely been understood until now. METHODS: Resting-state fMRI data were collected from 333 subjects (123 subjects with IGD (males/females: 73/50) and 210 healthy controls (males/females: 135/75)). First, the data-driven methodology, named co-activation pattern analysis, was applied to investigate the dynamic features of nucleus accumbens (the core region involved in craving/reward processing and addiction)-centered brain networks in IGD. Further, machine learning analysis was conducted to investigate the prediction effect of the dynamic features on participants' addiction severity. RESULTS: Compared to controls, subjects in the IGD group showed decreased resilience, betweenness centrality and occurrence in the prefrontal-striatal neural circuit, and decreased in-degree in the striatal-default mode network (DMN) circuit. Moreover, these decreased dynamic features could significantly predict participants' addiction severity. LIMITATIONS: The causal relationship between IGD and the abnormal dynamic features cannot be identified in this study. All the subjects were university students. CONCLUSIONS: The present results revealed the underlying brain networks of uncontrollable craving and game-seeking behaviors in individuals with IGD during rest. The decreased dynamics of the prefrontal-striatal and striatal-DMN neural circuits might be potential biomarkers for predicting the addiction severity of IGD and potential targets for effective interventions to reduce game craving of this disorder.