An ascidian Polycarpa aurata-derived pan-inhibitor against coronaviruses targeting Mpro.

Coronaviruses (CoVs) are responsible for a wide range of illnesses in both animals and human. The main protease (Mpro) of CoVs is an attractive drug target, owing its critical and highly conserved role in viral replication. Here, we developed and refined an enzymatic technique to identify putative Mpro inhibitors from 189 marine chemicals and 46 terrestrial natural products. The IC50 values of Polycarpine (1a), a marine natural substance we studied and synthesized, are 30.0 ± 2.5 nM for SARS-CoV-2 Mpro and 0.12 ± 0.05 µM for PEDV Mpro. Our research further demonstrated that pretreatment with Polycarpine (1a) inhibited the betacoronavirus SARS-CoV-2 and alphacoronavirus PEDV multiplication in Vero-E6 cells. As a result, Polycarpine (1a), a pan-inhibitor of Mpro, will function as an effective and promising antiviral option to combat CoVs infection and as a foundation for further therapeutic research.

Revealing the Mechanism of Ink Flaking from Surfaces of Palm Leaves (Corypha umbraculifera).

Palm leaves are the primary literary support in South and Southeast Asia before the widespread use of paper. However, palm leaf manuscripts face the threat of information loss due to the persistent issue of ink flaking during long-term preservation. Herein, we focus on studying the botanical structure, surface properties, and surface composition of palm leaves to gain an insightful understanding of the mechanism of ink flaking. According to the surface energy analysis, the surface of palm leaves is dominated by the dispersive component due to the presence of hydrophobic substances, resulting in the weak interaction between the handwriting and palm leaves. Moreover, the accumulation of silicon on palm leaves creates a "cuticle-silicon double layer", leading to a dense structure that hinders deep ink absorption. These two main reasons are considered to cause the ink flaking easily, which is further proven by the ink flaking test with the simulated palm leaf manuscripts. To the best of our knowledge, this is the first in-depth technical study on the adhesion performance of handwriting on plant leaves. This work also provides a theoretical basis for the study of the deterioration, adhesive repair, enhancement of flexibility, handwriting reinforcement, and beyond, which contributes to the conservation of precious palm leaf manuscripts.

C60- and CdS-Co-Modified Nano-Titanium Dioxide for Highly Efficient Photocatalysis and Hydrogen Production.

The inherent properties of TiO2, including a wide band gap and restricted spectral response range, hinder its commercial application and its ability to harness only 2-3% of solar energy. To address these challenges and unlock TiO2's full potential in photocatalysis, C60- and CdS-co-modified nano-titanium dioxide has been adopted in this work to reduce the band gap, extend the absorption wavelength, and control photogenerated carrier recombination, thereby enhancing TiO2's light-energy-harnessing capabilities and hydrogen evolution capacity. Using the sol-gel method, we successfully synthesized CdS-C60/TiO2 composite nanomaterials, harnessing the unique strengths of CdS and C60. The results showed a remarkable average yield of 34.025 µmol/h for TiO2 co-modified with CdS and C60, representing a substantial 17-fold increase compared to pure CdS. Simultaneously, the average hydrogen generation of C60-modified CdS surged to 5.648 µmol/h, a notable two-fold improvement over pure CdS. This work opens up a new avenue for the substantial improvement of both the photocatalytic degradation efficiency and hydrogen evolution capacity, offering promise of a brighter future in photocatalysis research.

Hybrid nanospheres of silica covalently containing yellow-emitting cationic iridium(III) complex: preparation and application in white light-emitting diodes.

A yellow-emitting cationic iridium(III) complex [(dfppy)2Ir(TBD)]PF6 (TBD: N4,N4'-bis(3-(triethoxysilyl)propyl)-[2,2'-bipyridine]-4,4'-dicarboxamide; dfppy: 2-(2,4-difluorophenyl)pyridine) containing hydrolysable alkoxysilanes was synthesized. Then, a series of silica-based hybrid nanospheres with diameters of around 400 nm was prepared via the hydrolysis of this complex together with tetraethyl orthosilicate (TEOS, a silica source). When the amount of the complex used was 5.0 wt%, hybrid nanospheres showed the best photoluminescence (PL) properties, relative to the PL quantum yield of pure solid [(dfppy)2Ir(TBD)]PF6 (12.7%), that of hybrid nanospheres increased to 26.2%. Moreover, the thermal decomposition temperature (Td) of pure solid [(dfppy)2Ir(TBD)]PF6 was 331 °C, the Td of the complex in hybrid nanospheres increased to 447 °C. However, the yellow light emission was almost unchanged and was still located at 500-750 nm with a maximum wavelength (λem,max) of 577 nm. Under the excitation of blue-emitting chips (λem,max ≈ 455 nm), cold/neutral/warm white light-emitting diodes (WLEDs) with good luminous quality can all be fabricated using these hybrid nanospheres as phosphors in epoxy resin at different blending concentrations. Compared with two or three iridium(III) complexes being contained in silica-based particles as phosphors as described in literatures, in this study, silica-based hybrid nanospheres covalently containing only one yellow-emitting cationic iridium(III) complex as phosphors provide a more effective and simpler method for preparation high-performance WLEDs.

Epstein-Barr Virus-Positive Plasma Cell Neoplasms in Immunocompetent Patients: A Clinicopathological Study of 15 Cases from South China and Literature Review.

Epstein-Barr virus (EBV)-positive plasma cell neoplasms (PCNs) in immunocompetent patients are a rare entity, the clinicopathological and prognostic features of which have not been well characterized. Fifteen cases of EBV-positive PCN arising in immunocompetent patients from south China were retrospectively analyzed, and an additional 44 cases from the literature were reviewed. The overall EBV-positive rate defined by EBV-encoded small RNAs (EBERs) in-situ hybridization of PCNs was 12.3% (15/122), and it was significantly higher in plasmacytoma (17.1%, 13/76) than in plasma cell myeloma/multiple myeloma (4.3%, 2/46; P=0.031). The age of the patients ranged from 17 to 79 years, with a median age of 56 years. There was a large preponderance of men, with a male-to-female ratio of 4:1. Solitary plasmacytoma of bone (23.8%, 5/21) had comparable EBV-encoded small RNAs-positive rates with extramedullary plasmacytoma arising in the upper respiratory tract (19.5%, 8/41; P=0.949). Anaplastic and classic cytologic appearance was observed in 61.5% (8/13) and 38.5% (5/13) of EBV-positive plasmacytomas, respectively. Cases with an anaplastic cytologic appearance had a significantly higher Ki-67 proliferation index than those with a classic cytologic appearance (median: 55% vs. 10%, P=0.001). In the combined cohorts, anaplastic/plasmablastic cytologic appearance was significantly more common in extramedullary plasmacytoma arising in the upper respiratory tract (72.0%, 18/25) than outside the upper respiratory tract (11.1%, 1/9; P=0.006). Among the 59 cases of EBV-positive PCN, survival data of 34 cases were available for analysis, including 30 cases of plasmacytoma and 4 cases of plasma cell myeloma/multiple myeloma. There was no statistically significant difference in overall survival between patients with EBV-positive plasmacytomas in the combined cohorts and EBV-negative plasmacytomas in the present cohort. The prevalence of EBV in PCN in immunocompetent patients varies according to histologic subtype and tumor location. Compared with EBV-negative cases, EBV-positive plasmacytomas tend to have an anaplastic/plasmablastic cytologic appearance. No significant impact of EBV infection on clinical outcomes is observed in the limited number of reported cases.

Computational redesign of taxane-10ß-hydroxylase for de novo biosynthesis of a key paclitaxel intermediate.

Paclitaxel (Taxol®) is the most popular anticancer diterpenoid predominantly present in Taxus. The core skeleton of paclitaxel is highly modified, but researches on the cytochrome P450s involved in post-modification process remain exceedingly limited. Herein, the taxane-10ß-hydroxylase (T10ßH) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5α-yl-acetate (T5OAc) to taxadiene-5α-yl-acetoxy-10ß-ol (T10OH), was investigated in Escherichia coli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10ßH, M3 (I75F/L226K/S345V), exhibited a remarkable 9.5-fold increase in protein expression, accompanied by respective 1.3-fold and 2.1-fold improvements in turnover frequency (TOF) and total turnover number (TTN). Upon integration into the engineered strain, the variant M3 resulted in a substantial enhancement in T10OH production from 0.97 to 2.23 mg/L. Ultimately, the titer of T10OH reached 3.89 mg/L by fed-batch culture in a 5-L bioreactor, representing the highest level reported so far for the microbial de novo synthesis of this key paclitaxel intermediate. This study can serve as a valuable reference for further investigation of other P450s associated with the artificial biosynthesis of paclitaxel and other terpenoids. KEY POINTS: • The T10ßH from T. cuspidata was expressed and engineered in E. coli unprecedentedly. • The expression and activity of T10ßH were improved through protein engineering. • De novo biosynthesis of T10OH was achieved in E. coli with a titer of 3.89 mg/L.

Coronaviral Main Protease Induces LPCAT3 Cleavage and Endoplasmic Reticulum (ER) Stress.

Zoonotic coronaviruses infect mammals and birds, causing pulmonary and gastrointestinal infections. Some animal coronaviruses, such as the porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), lead to severe diarrhea and animal deaths. Gastrointestinal symptoms were also found in COVID-19 and SARS patients. However, the pathogenesis of gastrointestinal symptoms in coronavirus diseases remains elusive. In this study, the main protease-induced LPCAT3 cleavage was monitored by exogenous gene expression and protease inhibitors, and the related regulation of gene expression was confirmed by qRT-PCR and gene knockdown. Interestingly, LPCAT3 plays an important role in lipid absorption in the intestines. The Mpro of coronaviruses causing diarrhea, such as PEDV and MERS-CoV, but not the Mpro of HCoV-OC43 and HCoV-HKU1, which could induce LPCAT3 cleavage. Mutagenesis analysis and inhibitor experiments indicated that LPCAT3 cleavage was independent of the catalytic activity of Mpro. Moreover, LPCAT3 cleavage in cells boosted CHOP and GRP78 expression, which were biomarkers of ER stress. Since LPCAT3 is critical for lipid absorption in the intestines and malabsorption may lead to diarrhea in coronavirus diseases, Mpro-induced LPCAT3 cleavage might trigger gastrointestinal symptoms during coronavirus infection.

The Effect of Phonetic Similarity on Domain-General Executive Control in Color-Shape Task: Evidence from Cantonese-Mandarin and Beijing-Dialect-Mandarin Bidialectals.

The present study was carried out to investigate whether bidialectals have a similar advantage in domain-general executive function as bilinguals and if so whether the phonetic similarity between two different dialects can modulate the executive function performance in the conflicting-switching task. The results showed that the latencies for switching trials in mixed block (SMs) were longest, non-switching trials in mixed block (NMs) were medium, and non-switching trials in pure block (NPs) were the shortest in the conflict-switching task in all three groups of participants. Importantly, the difference between NPs and NMs varied as a function of phonetic similarity between two dialects with Cantonese-Mandarin bidialectal speakers being the minimum, Beijing-dialect-Mandarin bidialectals medium, and Mandarin native speakers maximum. These results provide strong evidence that there is an advantage in balanced bidialectals's executive function which is modulated by the phonetic similarity between two dialects suggesting that phonetic similarity plays an important role in domain-general executive function.

The effects of nurse-led family pulmonary rehabilitation intervention on quality of life and exercise capacity in rural patients with COPD.

AIM: To assess the effects of nurse-led family pulmonary rehabilitation intervention on quality of life and exercise capacity in rural patients with chronic obstructive pulmonary disease (COPD). DESIGN: A non-randomized clinical trial. METHODS: This study enrolled COPD patients at two hospitals from January 2019 to January 2020. The primary outcome was the quality of life. The secondary outcome was exercise capacity. RESULTS: Seventy-four rural COPD patients were enrolled, 38 in the comprehensive nursing group and 36 in the routine nursing group. After the intervention, the St. George's Respiratory Questionnaire (SGRQ) scores in the comprehensive nursing group were significantly lower than in the routine nursing group, especially in symptoms, activities and total scores. The SGRQ scores of the comprehensive nursing group were significantly lower than baseline, while the SGRQ scores of the routine nursing group were significantly higher than baseline. After the intervention, there was a significant difference in six-minute walking distance(6MWD) between the two groups.

Disrupting the phase separation of KAT8-IRF1 diminishes PD-L1 expression and promotes antitumor immunity.

Immunotherapies targeting the PD-1/PD-L1 axis have become first-line treatments in multiple cancers. However, only a limited subset of individuals achieves durable benefits because of the elusive mechanisms regulating PD-1/PD-L1. Here, we report that in cells exposed to interferon-γ (IFNγ), KAT8 undergoes phase separation with induced IRF1 and forms biomolecular condensates to upregulate PD-L1. Multivalency from both the specific and promiscuous interactions between IRF1 and KAT8 is required for condensate formation. KAT8-IRF1 condensation promotes IRF1 K78 acetylation and binding to the CD247 (PD-L1) promoter and further enriches the transcription apparatus to promote transcription of PD-L1 mRNA. Based on the mechanism of KAT8-IRF1 condensate formation, we identified the 2142-R8 blocking peptide, which disrupts KAT8-IRF1 condensate formation and consequently inhibits PD-L1 expression and enhances antitumor immunity in vitro and in vivo. Our findings reveal a key role of KAT8-IRF1 condensates in PD-L1 regulation and provide a competitive peptide to enhance antitumor immune responses.

Design and syntheses of a bimolecular STING agonist based on the covalent STING antagonist.

Cyclic GMP-AMP synthase and stimulator of interferon genes (cGAS-STING) signaling stimulators, an essential innate immunity component, monitor invading pathogen DNA and damaged self-DNA, making them an appealing target for drug development. The natural STING agonist, 2'3'-cGAMP, mounts and stabilizes the STING homodimer to trigger an antiviral or antitumor immune responses. However, cyclic-dinucleotide-based STING agonists show limited clinical effects owing to their short half-lives. To explore whether STING-dimer stabilizers could trigger STING signaling instead of cyclic dinucleotide-based molecules, we analyzed the structural characteristics of STING to design and synthesize a series of compounds based on the covalent STING inhibitor C-170, three of which were 23, 26, and 27, exhibited STING-dependent immune activation, both in vitro and in vivo. Compound 23 could act synergistically with cGAMP and other STING agonists as a promising moderate STING agonist. This indicates that promoting STING dimerization is a promising strategy for designing next-generation STING agonists.

Discovery of a first-in-class orally available HBV cccDNA inhibitor.

BACKGROUND & AIMS: The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. METHODS: A phenotypic assay with HBV-infected primary human hepatocytes (PHHs) was employed to screen for novel cccDNA inhibitors. A HBVcircle mouse model and a uPA-SCID (urokinase-type plasminogen activator-severe combined immunodeficiency) humanized liver mouse model were used to evaluate the anti-HBV efficacy of the discovered cccDNA inhibitors. RESULTS: Potent and dose-dependent reductions in extracellular HBV DNA, HBsAg, and HBeAg levels were achieved upon the initiation of ccc_R08 treatment two days after the HBV infection of PHHs. More importantly, the level of cccDNA was specifically reduced by ccc_R08, while it did not obviously affect mitochondrial DNA. Additionally, ccc_R08 showed no significant cytotoxicity in PHHs or in multiple proliferating cell lines. The twice daily oral administration of ccc_R08 to HBVcircle model mice, which contained surrogate cccDNA molecules, significantly decreased the serum levels of HBV DNA and antigens, and these effects were sustained during the off-treatment follow-up period. Moreover, at the end of follow-up, the levels of surrogate cccDNA molecules in the livers of ccc_R08-treated HBVcircle mice were reduced to below the lower limit of quantification. CONCLUSIONS: We have discovered a small-molecule cccDNA inhibitor that reduces HBV cccDNA levels. cccDNA inhibitors potentially represent a new approach to completely cure patients chronically infected with HBV. IMPACT AND IMPLICATIONS: Covalently closed circular DNA (cccDNA) persistence in HBV-infected hepatocytes is the root cause of chronic hepatitis B. We discovered a novel small-molecule cccDNA inhibitor that can specifically reduce cccDNA levels in HBV-infected hepatocytes. This type of molecule could offer a new approach to completely cure patients chronically infected with HBV.

CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics.

MHA-Net: A Multibranch Hybrid Attention Network for Medical Image Segmentation.

The robust segmentation of organs from the medical image is the key technique in medical image analysis for disease diagnosis. U-Net is a robust structure for medical image segmentation. However, U-Net adopts consecutive downsampling encoders to capture multiscale features, resulting in the loss of contextual information and insufficient recovery of high-level semantic features. In this paper, we present a new multibranch hybrid attention network (MHA-Net) to capture more contextual information and high-level semantic features. The main idea of our proposed MHA-Net is to use the multibranch hybrid attention feature decoder to recover more high-level semantic features. The lightweight pyramid split attention (PSA) module is used to connect the encoder and decoder subnetwork to obtain a richer multiscale feature map. We compare the proposed MHA-Net to state-of-art approaches on the DRIVE dataset, the fluoroscopic roentgenographic stereophotogrammetric analysis X-ray dataset, and the polyp dataset. The experimental results on different modal images reveal that our proposed MHA-Net provides better segmentation results than other segmentation approaches.

Discovery of Novel cccDNA Reducers toward the Cure of Hepatitis B Virus Infection.

Chronic hepatitis B virus (HBV) infection is a worldwide disease that causes thousands of deaths per year. Currently, there is no therapeutic that can completely cure already infected HBV patients due to the inability of humans to eliminate covalently closed circular DNA (cccDNA), which serves as the template to (re)initiate an infection even after prolonged viral suppression. Through phenotypic screening, we discovered xanthone series hits as novel HBV cccDNA reducers, and subsequent structure optimization led to the identification of a lead compound with improved antiviral activity and pharmacokinetic profiles. A representative compound 59 demonstrated good potency and oral bioavailability with no cellular toxicity. In an HBVcircle mouse model, compound 59 showed excellent efficacy in significantly reducing HBV antigens, DNA, and intrahepatic cccDNA levels.

Cloning and functional analysis of SNX8 from grass carp (Ctenopharyngodon idellus).

Sorting nexin 8 (SNX8), a member of sorting nexin protein family, plays important roles in endocytosis, endosomal sorting, and innate immune response. To date, a few homologs of SNX8 have been found in fish except in mammals. In this study, a teleost SNX8 cDNA was identified from grass carp (Ctenopharyngodon idellus). CiSNX8 was up-regulated significantly after infection with poly I:C or GCRV. We found that SNX8 was mainly distributed in the endoplasmic reticulum (ER) in CIK cells. Further analysis indicated that CiSNX8 might negatively regulate RLR signaling pathway that is quite distinct from mammalian SNX8. In addition, CiSNX8 could interact with MAVS, STING, TBK1, IRF3 and IRF7. Either wild type CiSNX8 or mutants of N-terminal PX domain (aa 1-245) and C-terminal BAR domain (aa 256-519) could associate with STING. These results suggested that fish SNX8 participated in innate immune response through different molecular mechanisms.

The anti-inflammatory activity of specific-sized hyaluronic acid oligosaccharides.

Severe acute inflammatory conditions may cause tissue damage, sepsis, and death. As a critical component of the extracellular matrix, hyaluronic acid (HA) has been reported to possess pro- and anti-inflammatory properties via Toll-like receptors (TLRs). In this study, we prepared different sizes and structures of HA oligosaccharides and derivatives and investigated the effects on inflammation in vitro and in vivo. Our results showed that HA tetra-saccharide was the minimum fragment to enhance inflammation, whereas HA disaccharide competitively blocked TLR4-dependent inflammation. The enzymatic HA disaccharide (ΔHA2) inhibited lipopolysaccharide (LPS)-induced inflammation. Based on structure-activity relationship analysis, we observed that anti-inflammatory activity depended on HAs polymerization degree, acetyl group, and configuration. In addition, we demonstrated that ΔHA2 reduced LPS-induced pro-inflammatory cytokines production in vivo. ΔHA2, a native metabolite of HA polysaccharides, may have a potential role against LPS-mediated inflammatory diseases.

Erratum: Oncogenic roles of carbonic anhydrase IX in human nasopharyngeal carcinoma.

[This corrects the article on p. 2942 in vol. 7, PMID: 25031713.].

Prognostic Value of an Immunohistochemical Signature in Patients With Head and Neck Mucosal Melanoma.

Purpose: We aimed to develop a prognostic immunohistochemistry (IHC) signature for patients with head and neck mucosal melanoma (MMHN). Methods: In total, 190 patients with nonmetastatic MMHN with complete clinical and pathological data before treatment were included in our retrospective study. Results: We extracted five IHC markers associated with overall survival (OS) and then constructed a signature in the training set (n=116) with the least absolute shrinkage and selection operator (LASSO) regression model. The validation set (n=74) was further built to confirm the prognostic significance of this classifier. We then divided patients into high- and low-risk groups according to the IHC score. In the training set, the 5-year OS rate was 22.0% (95% confidence interval [CI]: 11.2%- 43.2%) for the high-risk group and 54.1% (95% CI: 41.8%-69.9%) for the low-risk group (P<0.001), and in the validation set, the 5-year OS rate was 38.1% (95% CI: 17.9%-81.1%) for the high-risk group and 43.1% (95% CI: 30.0%-61.9%) for the low-risk group (P=0.26). Multivariable analysis revealed that IHC score, T stage, and primary tumor site were independent variables for predicting OS (all P<0.05). We developed a nomogram incorporating clinicopathological risk factors (primary site and T stage) and the IHC score to predict 3-, 5-, and 10-year OS. Conclusions: A nomogram was generated and confirmed to be of clinical value. Our IHC classifier integrating five IHC markers could help clinicians make decisions and determine optimal treatments for patients with MMHN.