Ribosomal S6 kinases 2 mediates potato resistance to late blight, through WRKY59 transcription factor.

Potato late blight is the most devastating pre- and post-harvest crop disease in the world, which is widespread and difficult to control, causing serious economic losses. Cultivating resistant varieties is a major way to prevent and control late blight in a green way. However, due to the rapid evolution of pathogens, the plant resistance is losing. Therefore, mining effective and durable genes involved in disease resistance is crucial for breeding resistant varieties against late blight. In this study, we took "potato-Phytophthora infestans" as the "host-pathogen" model system to discover the potential disease resistance-related genes and elucidate their molecular functional mechanism. Through yeast two-hybridization, bimolecular fluorescence complementation, Co-immunoprecipitation assays, and gene function validation etc., we found that ribosomal protein S6 kinase 2 (StS6K2) is a key resistant protein, which is interacted with StWRKY59 transcription factor. Overexpression of StS6K2 and StWRKY59 both enhanced the plants resistance to P. infestans, and promoted the host immune response, such as ROS burst and callose deposition. In OEStWRKY59 lines, DEGs involved in secondary metabolites synthesis, plant hormone signaling transduction and plant-pathogen interaction were significantly enriched. These findings provide novel genetic resources for the breeding of resistant varieties.

Computational identification of novel potential genetic pathogenesis and otherwise biomarkers in acute liver allograft rejection.

Acute cellular rejection (ACR) is a prevalent postoperative complication following liver transplantation (LT), exhibiting an increasing incidence of morbidity and mortality. However, the molecular mechanisms of ACR following LT remain unclear. To explore the genetic pathogenesis and identify biomarkers of ACR following LT, three relevant Gene Expression Omnibus (GEO) datasets consisting of data on ACR or non-ACR patients after LT were comprehensively investigated by computational analysis. A total of 349 upregulated and 260 downregulated differentially expressed genes (DEGs) and eight hub genes (ISG15, HELZ2, HNRNPK, TIAL1, SKIV2L2, PABPC1, SIRT1, and PPARA) were identified. Notably, HNRNPK, TIAL1, and PABPC1 exhibited the highest predictive potential for ACR with AUCs of 0.706, 0.798, and 0.801, respectively. KEGG analysis of hub genes revealed that ACR following LT was predominately associated with ferroptosis, protein processing in the endoplasmic reticulum, complement and coagulation pathways, and RIG-I/NOD/Toll-like receptor signaling pathway. According to the immune cell infiltration analysis, γδT cells, NK cells, Tregs, and M1/M2-like macrophages had the highest levels of infiltration. Compared to SIRT1, ISG15 was positively correlated with γδT cells and M1-like macrophages but negatively correlated with NK cells, CD4+ memory T cells, and Tregs. In conclusion, this study identified eight hub genes and their potential pathways, as well as the immune cells involved in ACR following LT with the greatest levels of infiltration. These findings provide a new direction for future research on the underlying mechanism of ACR following LT.

Lot-to-lot consistency, immunogenicity and safety of a quadrivalent split virion inactivated influenza vaccine in healthy population aged 9-59 years: A randomized, double-blind, controlled, phase IV clinical trial.

OBJECTIVES: This study was to assess the lot-to-lot consistency, immunogenicity and safety of three manufacturing lots of a quadrivalent inactivated influenza vaccine (IIV4). METHODS: A randomized, double-blind, phase IV clinical trial was conducted in healthy children, adolescents and adults aged 9-59 years in Guizhou Province, China. Eligible participants were enrolled and randomized into three groups in a ratio of 1:1:1 to receive a single dose of one of three manufacturing lots of IIV4. Serum samples were collected before and 28 days after vaccination for hemagglutination inhibition (HI) antibody testing. Safety data were collected for up to 28 days after vaccination. The primary objective was to evaluate the lot-to-lot consistency of immune response as assessed by the geometric mean titer (GMT) of HI antibody at 28 days after vaccination. RESULTS: Between November 27, 2022 and December 18, 2022, 1260 eligible participants were enrolled, with similar participant demographics among groups. Immune responses after vaccination were comparable across groups, with the 95% confidence intervals (CIs) of GMT ratios for all 4 strains falling into the equivalence criterion of (0.67, 1.5). The seroconversion rates (SCRs) and seroprotection rates (SPRs) met the US Center or Biologics Evaluation and Research (CBER) criteria for all strains for each lot (lower limit of 95% CI of SCR ≥ 40% and SPR ≥ 70%). The incidences of solicited and unsolicited adverse reactions were similar among three groups, most of which (91.9%) were mild or moderate in severity. A total of 11 serious adverse events were reported during the study, and all were considered unrelated to vaccination. CONCLUSION: The three manufacturing lots of IIV4 demonstrated consistent immunogenicity. IIV4 can elicit satisfactory immune responses for all four strains and no safety concerns were identified. CLINICAL TRIAL REGISTRATION: Identifier No. NCT05512494.

Comparative study of antifungal susceptibility testing methods for clinical Candida albicans isolates.

PURPOSE: Candida albicans is the second most common cause of candidemia in Malaysia. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution method is the gold standard for determining its minimum inhibitory concentration (MIC); however, it is laborious and time-consuming. This study was conducted to evaluate the usefulness of alternative methods, namely Sensititre YeastOne (SYO), VITEK 2 system, and E-test for determining the MIC of clinical C. albicans isolates. MATERIALS AND METHODS: The susceptibilities of 95 C. albicans isolates were compared between SYO, VITEK 2 system, and E-test with CLSI broth microdilution method. The categorical agreement (CA), essential agreement (EA), very major errors (VME), major errors (ME) and minor errors (MiE) were calculated. RESULTS: Our finding showed the CA varied for SYO from 96.8% to 100%, while the EA ranged from 91.6% to 100%. The SYO method showed 1.1% of VME and ME, and up to 3.2% of MiE. Next, the CA and EA ranges for the VITEK 2 system were 97.8%-100% and 23.2%-100%, respectively. In the VITEK 2 technique, 1.1% of VME were found. For the E-test, the CA varied from 83.2% to 100% while the EA ranged from 64.2% to 98.9%. The E-test method showed 1.1% of VME and up to 16.8% of MiE. CONCLUSIONS: In conclusion, SYO and VITEK 2 (except flucytosine) could be potential alternatives to the CLSI broth microdilution method in determining the MIC of C. albicans.

Structural and functional insights into the 2'-O-methyltransferase of SARS-CoV-2.

A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16 (nsp16), 2'-O-methyltransferase (2'-O-MTase), to cap their RNAs through ribose 2'-O-methylation modification. This process is crucial for maintaining viral genome stability, facilitating efficient translation, and enabling immune escape. Despite considerable advances in the ultrastructure of SARS-CoV-2 nsp16/nsp10, insights into its molecular mechanism have so far been limited. In this study, we systematically characterized the 2'-O-MTase activity of nsp16 in SARS-CoV-2, focusing on its dependence on nsp10 stimulation. We observed cross-reactivity between nsp16 and nsp10 in various coronaviruses due to a conserved interaction interface. However, a single residue substitution (K58T) in SARS-CoV-2 nsp10 restricted the functional activation of MERS-CoV nsp16. Furthermore, the cofactor nsp10 effectively enhanced the binding of nsp16 to the substrate RNA and the methyl donor S-adenosyl-l-methionine (SAM). Mechanistically, His-80, Lys-93, and Gly-94 of nsp10 interacted with Asp-102, Ser-105, and Asp-106 of nsp16, respectively, thereby effectively stabilizing the SAM binding pocket. Lys-43 of nsp10 interacted with Lys-38 and Gly-39 of nsp16 to dynamically regulate the RNA binding pocket and facilitate precise binding of RNA to the nsp16/nsp10 complex. By assessing the conformational epitopes of nsp16/nsp10 complex, we further determined the critical residues involved in 2'-O-MTase activity. Additionally, we utilized an in vitro biochemical platform to screen potential inhibitors targeting 2'-O-MTase activity. Overall, our results significantly enhance the understanding of viral 2'-O methylation process and mechanism, providing valuable targets for antiviral drug development.

[Temporal and Spatial Evolution and Prediction of Ecosystem Carbon Storage in Jiangxi Province Based on PLUS-InVEST Model].

Land-use changes are an important factor affecting the change in carbon storage in terrestrial ecosystems. Exploring the relationship between land-use changes and carbon storage provides reliable data support for optimizing regional land-use structure and maintaining regional carbon balance. Taking Jiangxi Province as an example, we first analyzed the land-use changes; then simulated the land-use pattern under three scenarios (i.e., natural development, ecological priority, and economic development scenarios) in 2030 based on the PLUS model; and finally estimated the carbon storage change in the past (i.e., 1990-2020) and future periods (i.e., three scenarios in 2030) using the InVEST model, analyzed the spatial-temporal characteristics, and proposed the corresponding suggestions. The results showed:① The carbon storage in Jiangxi Province showed a downward trend from 1990 to 2020, with a total reduction of 4.58×107 t. The increase in the water bodies and construction land and the decrease in cultivated land, woodland, grassland, and unused land was the major cause. ② The carbon storage under natural development, ecological priority, and economic development scenarios in Jiangxi Province in 2030 were 2.20×109, 2.24×109 and 2.19×109 t, respectively. ③ The carbon storage under the three scenarios showed similar spatial characteristics, wherein the high carbon storage was distributed in northern, northwest, and western regions, and the low carbon storage was distributed near the central region. These results can provide data support for future land spatial planning and improving the carbon storage of terrestrial ecosystems in Jiangxi Province.

A Case of Refractory Posterior Scleritis with Marked Retinochoroidal Detachment Associated with Panuveitis.

An 84-year-old man presented with decreased right-eye visual acuity. Upon initial examination, the rightand left-eye visual acuities were 0.03 and 1.2, respectively; moreover, the right- and left-eye intraocular pressure was 12 mmHg and 13 mmHg, respectively. Examination revealed a shallow anterior chamber of the right eye, anterior chamber inflammation, vitreous opacity, and marked retinochoroidal detachment. Optical coherence tomography (OCT) revealed retinal detachment (RD) and choroidal folds; moreover, B-scan ultrasonography (B-scan) showed RD as well as thickened sclera with fluid in Tenon's space. Fluorescent fundus angiography revealed hyperfluorescence in the optic disc and vascular hyperpermeability in the right eye. The left eye lacked extra-ocular symptoms or abnormalities. The right ocular axis measured 23.4 mm with no apparent subretinal fluid migration due to positional changes. Accordingly, the patient was diagnosed with panuveitis associated with posterior scleritis and immediately started on 40 mg prednisolone, which improved his symptoms. However, at 3 post-treatment months, choroidal folds were observed and was restarted on 20 mg prednisolone. The choroidal folds subsequently disappeared, with a current visual acuity of 0.3 in the right eye and no recurrence. Our findings indicated the utility of accurate diagnosis of posterior scleritis by B-scan and prompt systemic steroid administration.

Antifungal Susceptibility and Genotypic Analysis of cyp51A Mutations in Aspergillus fumigatus Isolates in Malaysia.

Purpose: Azole resistance in Aspergillus fumigatus poses a significant challenge in the management of invasive aspergillosis. This study aimed to investigate the antifungal susceptibility and cyp51A mutation profiles of A. fumigatus isolates in Malaysia. Patients and Methods: Sixty clinical A. fumigatus isolates were collected and subjected to antifungal susceptibility testing (AFST) and molecular analysis. The antifungal susceptibility testing was performed according to CLSI M38 guideline. The geometric mean (GM) minimum inhibitory concentration (MIC), MIC50/MIC90 for voriconazole, itraconazole, posaconazole, amphotericin B, and isavuconazole against A. fumigatus in non-invasive cases and invasive cases were calculated. In addition, the presence of cyp51A mutations was also identified. Results: The present study revealed an overall resistance rate of 6.7% among the isolates. In non-invasive cases, isavuconazole and posaconazole demonstrated the lowest GM MIC of 0.08 µg/mL. Following them were itraconazole, voriconazole, and amphotericin B with concentrations of 0.15µg/mL, 0.16µg/mL and 0.90µg/mL, respectively. Similarly, in invasive cases, isavuconazole and posaconazole exhibited the lowest GM MIC of 0.09µg/mL. Following them were itraconazole, voriconazole, and amphotericin B with concentrations of 0.14µg/mL, 0.17µg/mL and 0.80µg/mL, respectively. Genotypic analysis revealed various cyp51A mutations, including F46Y, M172V, N248K, R34L, V244A, V244S, and E427K. However, not all mutations corresponded to antifungal resistance. Conclusion: The majority of clinical Aspergillus fumigatus isolates demonstrated susceptibility to the antifungal agents tested, with isavuconazole and posaconazole demonstrating the lowest MIC values. However, cyp51A mutations were discovered without a consistent correlation to antifungal resistance, emphasising the need for additional research.

Natural evidence of coronaviral 2'-O-methyltransferase activity affecting viral pathogenesis via improved substrate RNA binding.

Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2'-O-MTase activity is essential for efficient viral replication and immune evasion. However, the K-D-K-E catalytic motif of 2'-O-MTase is highly conserved across numerous viruses, including flaviviruses, vaccinia viruses, coronaviruses, and extends even to mammals. Here, we observed a stronger 2'-O-MTase activity in SARS-CoV-2 compared to SARS-CoV, despite the presence of a consistently active catalytic center. We further identified critical residues (Leu-36, Asn-138 and Ile-153) which served as determinants of discrepancy in 2'-O-MTase activity between SARS-CoV-2 and SARS-CoV. These residues significantly enhanced the RNA binding affinity of 2'-O-MTase and boosted its versatility toward RNA substrates. Of interest, a triple substitution (Leu36 → Ile36, Asn138 → His138, Ile153 → Leu153, from SARS-CoV-2 to SARS-CoV) within nsp16 resulted in a proportional reduction in viral 2'-O-methylation and impaired viral replication. Furthermore, it led to a significant upregulation of type I interferon (IFN-I) and proinflammatory cytokines both in vitro and vivo, relying on the cooperative sensing of melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). In conclusion, our findings demonstrated that alterations in residues other than K-D-K-E of 2'-O-MTase may affect viral replication and subsequently influence pathogenesis. Monitoring changes in nsp16 residues is crucial as it may aid in identifying and assessing future alteration in viral pathogenicity resulting from natural mutations occurring in nsp16.

The lethal K18-hACE2 knock-in mouse model mimicking the severe pneumonia of COVID-19 is practicable for antiviral development.

Animal models of COVID-19 facilitate the development of vaccines and antivirals against SARS-CoV-2. The efficacy of antivirals or vaccines may differ in different animal models with varied degrees of disease. Here, we introduce a mouse model expressing human angiotensin-converting enzyme 2 (ACE2). In this model, ACE2 with the human cytokeratin 18 promoter was knocked into the Hipp11 locus of C57BL/6J mouse by CRISPR - Cas9 (K18-hACE2 KI). Upon intranasal inoculation with high (3 × 105 PFU) or low (2.5 × 102 PFU) dose of SARS-CoV-2 wildtype (WT), Delta, Omicron BA.1, or Omicron BA.2 variants, all mice showed obvious infection symptoms, including weight loss, high viral loads in the lung, and interstitial pneumonia. 100% lethality was observed in K18-hACE2 KI mice infected by variants with a delay of endpoint for Delta and BA.1, and a significantly attenuated pathogenicity was observed for BA.2. The pneumonia of infected mice was accompanied by the infiltration of neutrophils and pulmonary fibrosis in the lung. Compared with K18-hACE2 Tg mice and HFH4-hACE2 Tg mice, K18-hACE2 KI mice are more susceptible to SARS-CoV-2. In the antivirals test, REGN10933 and Remdesivir had limited antiviral efficacies in K18-hACE2 KI mice upon the challenge of SARS-CoV-2 infections, while Nirmatrelvir, monoclonal antibody 4G4, and mRNA vaccines potently protected the mice from death. Our results suggest that the K18-hACE2 KI mouse model is lethal and stable for SARS-CoV-2 infection, and is practicable and stringent to antiviral development.

A long-term cohort study: the immune evasion and decreasing neutralization dominated the SARS-CoV-2 breakthrough infection.

Most of vaccinees and COVID-19 convalescents can build effective anti-SARS-CoV-2 humoral immunity, which helps preventing infection and alleviating symptoms. However, breakthrough viral infections caused by emerging SARS-CoV-2 variants, especially Omicron subvariants, still pose a serious threat to global health. By monitoring the viral infections and the sera neutralization ability of a long-tracked cohort, we found out that the immune evasion of emerging Omicron subvariants and the decreasing neutralization led to the mini-wave of SARS-CoV-2 breakthrough infections. Meanwhile, no significant difference had been found in the infectivity of tested SARS-CoV-2 variants, even though the affinity between human angiotensin-converting enzyme 2 (hACE2) and receptor-binding domain (RBDs) of tested variants showed an increasing trend. Notably, the immune imprinting of inactivated COVID-19 vaccine can be relieved by infections of BA.5.2 and XBB.1.5 variants sequentially. Our data reveal the rising reinfection risk of immune evasion variants like Omicron JN.1 in China, suggesting the importance of booster with updated vaccines.

An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still epidemic around the world. The manipulation of SARS-CoV-2 is restricted to biosafety level 3 laboratories (BSL-3). In this study, we developed a SARS-CoV-2 ΔN-GFP-HiBiT replicon delivery particles (RDPs) encoding a dual reporter gene, GFP-HiBiT, capable of producing both GFP signal and luciferase activities. Through optimal selection of the reporter gene, GFP-HiBiT demonstrated superior stability and convenience for antiviral evaluation. Additionally, we established a RDP infection mouse model by delivering the N gene into K18-hACE2 KI mouse through lentivirus. This mouse model supports RDP replication and can be utilized for in vivo antiviral evaluations. In summary, the RDP system serves as a valuable tool for efficient antiviral screening and studying the gene function of SARS-CoV-2. Importantly, this system can be manipulated in BSL-2 laboratories, decreasing the threshold of experimental requirements.

CD39hi identifies an exhausted tumor-reactive CD8+ T cell population associated with tumor progression in human gastric cancer.

The ectonucleotidase CD39 has been regarded as a promising immune checkpoint in solid tumors. However, the expression of CD39 by tumor-infiltrating CD8+ T cells as well as their potential roles and clinical implications in human gastric cancer (GC) remain largely unknown. Here, we found that GC-infiltrating CD8+ T cells contained a fraction of CD39hi cells that constituted about 6.6% of total CD8+ T cells in tumors. These CD39hi cells enriched for GC-infiltrating CD8+ T cells with features of exhaustion in transcriptional, phenotypic, metabolic and functional profiles. Additionally, GC-infiltrating CD39hiCD8+ T cells were also identified for tumor-reactive T cells, as these cells expanded in vitro were able to recognize autologous tumor organoids and induced more tumor cell apoptosis than those of expanded their CD39int and CD39-CD8+ counterparts. Furthermore, CD39 enzymatic activity controlled GC-infiltrating CD39hiCD8+ T cell effector function, and blockade of CD39 efficiently enhanced their production of cytokines IFN-γ and TNF-α. Finally, high percentages of GC-infiltrating CD39hiCD8+ T cells correlated with tumor progression and independently predicted patients' poor overall survival. These findings provide novel insights into the association of CD39 expression level on CD8+ T cells with their features and potential clinical implications in GC, and empowering those exhausted tumor-reactive CD39hiCD8+ T cells through CD39 inhibition to circumvent the suppressor program may be an attractive therapeutic strategy against GC.

Knowledge, attitude, and practice of medication and its influencing factors among residents in western China: a large-scale cross-sectional study.

Background: This study aimed to understand the knowledge, attitude, and practice (KAP) of drug use among residents in western China and its influencing factors for accurately designing the knowledge, contents, and methods of popular science activities for safe drug use among residents to provide a reference for conducting rational drug use educational activities and improving residents' level of safe drug use. Methods: A cross-sectional questionnaire survey was conducted to investigate the KAP of medication among western China residents and its influencing factors from March to April 2023. Each question option was assigned a score according to logic, and the risk factors for resident medication safety KAP were explored through univariate and logistic regression analyses. Results: A total of 7,557 valid questionnaires were collected, with an effective recovery rate of 96.7%. The average scores of KAP were 72.77 ± 22.91, 32.89 ± 10.64, and 71.27 ± 19.09, respectively. In the evaluation criteria of the questionnaire, the score of medication knowledge reached "good," and the score of attitude and practice was "average." Multiple linear regression analysis indicated that male sex and low education level were significant factors affecting the lack of drug knowledge among residents. Old age and low education level were the factors of poor attitude toward medication. The low condition of medical security was a factor in residents' irregular drug use behavior. Conclusion: The overall level of rational drug use among residents in western China is good, but there are still some inconsistencies. Rational drug use education should be conducted according to the risk points of residents in drug safety KAP to further improve the level of rational drug use of residents.

Atypical Peripheral Retinal Necrosis Without Ocular Inflammation and Retinal Whitening in a Patient With Acute Myelogenous Leukemia: A Case Report.

Retinal necrosis is a severe condition that threatens visual function. It is caused by viruses that are known to cause acute retinal necrosis (ARN) and progressive outer retinal necrosis (PORN), which are called necrotizing herpetic retinopathies (NHR). ARN causes severe intraocular inflammation, including anterior chamber intravitreal cells, keratic precipitate, vitreous opacity, and retinal vasculitis, whereas intraocular inflammation in PORN is considered mild or virtually absent. In addition, PORN is a disease that manifests in immunosuppressive patients, such as those with acquired immunodeficiency syndrome. Here, we present a case of unilateral retinal necrosis after chemotherapy, allogeneic peripheral blood stem cell transplantation, and cord blood transplantation for acute myelogenous leukemia (AML) in a 31-year-old male patient. AML treatment resulted in metabolic remission, and oral steroids and tacrolimus were continued. After two days, the patient visited an ophthalmologist because he noticed a sudden onset of floaters and visual field disturbance in the left eye. The peripheral retina was already necrotic in all layers, causing total retinal detachment. Intraocular inflammation, retinal opacity, or hemorrhagic spots in the fundus were not observed. His previous CD4 count was 43 cells/µL. A polymerase chain reaction test of the anterior chamber fluid revealed varicella-zoster virus (VZV), and vitrectomy was performed four days after disease onset. The excised vitreous demonstrated minimal opacity. The peripheral necrotic retina was excised, photocoagulation was performed on the residual retinal limbus, and silicone oil was injected to maintain retinal attachment. The retinal restoration was maintained under silicone oil tamponade, and corrected visual acuity improved to 20/32 without strong inflammation after vitrectomy. However, two months postoperatively, he contracted coronavirus disease 2019 (COVID-19), his general condition rapidly deteriorated, and he died. This case of retinal necrosis without inflammatory results in an immunocompromised patient and VZV detection in an intraocular sample led us to suspect PORN. However, the patchy or spread retinal whitening characteristic of PORN was completely absent, whereas the well-defined, peripheral, full-layer retinal necrosis characteristic of ARN was present. Thus, this is a rare case of VZV-induced NHR with partial features of PORN and ARN that progressed very silently.

CD39 expression defines exhausted CD4+ T cells associated with poor survival and immune evasion in human gastric cancer.

Objectives: CD4+ T cell helper and regulatory function in human cancers has been well characterised. However, the definition of tumor-infiltrating CD4+ T cell exhaustion and how it contributes to the immune response and disease progression in human gastric cancer (GC) remain largely unknown. Methods: A total of 128 GC patients were enrolled in the study. The expression of CD39 and PD-1 on CD4+ T cells in the different samples was analysed by flow cytometry. GC-infiltrating CD4+ T cell subpopulations based on CD39 expression were phenotypically and functionally assessed. The role of CD39 in the immune response of GC-infiltrating T cells was investigated by inhibiting CD39 enzymatic activity. Results: In comparison with CD4+ T cells from the non-tumor tissues, significantly more GC-infiltrating CD4+ T cells expressed CD39. Most GC-infiltrating CD39+CD4+ T cells exhibited CD45RA-CCR7- effector-memory phenotype expressing more exhaustion-associated inhibitory molecules and transcription factors and produced less TNF-α, IFN-γ and cytolytic molecules than their CD39-CD4+ counterparts. Moreover, ex vivo inhibition of CD39 enzymatic activity enhanced their functional potential reflected by TNF-α and IFN-γ production. Finally, increased percentages of GC-infiltrating CD39+CD4+ T cells were positively associated with disease progression and patients' poorer overall survival. Conclusion: Our study demonstrates that CD39 expression defines GC-infiltrating CD4+ T cell exhaustion and their immunosuppressive function. Targeting CD39 may be a promising therapeutic strategy for treating GC patients.

Palladium-Catalyzed Sequential Cross-Coupling/Annulation of ortho-Vinyl Bromobenzene with Aryl Bromide: Bimetallic Pathway versus Pd(II)-Pd(IV) Pathway: A DFT Investigation.

The palladium-catalyzed sequential cross-coupling/annulation of ortho-vinyl bromobenzenes with aryl bromides generating phenanthrenes was characterized by density functional theory (DFT). The Pd(II)-Pd(IV) pathway (Path V) is shown to be less probable than the bimetallic pathway (Path I), the latter proceeding via the following six steps: oxidative addition, vinyl-C(sp2)-H activation, Pd(II)-Pd(II) transmetalation, C-C coupling, aryl-C(sp2)-H activation, and reductive elimination. The aryl-C(sp2)-H activation process acts as the rate-determining step (RDS) of the entire chemical transformation, with an activation free energy barrier of ca. 27.4-28.8 kcal·mol-1, in good agreement with the corresponding experimental data (phenanthrenes' yields of ca. 65-90% at 130 °C after 5 h of reaction). The K2CO3 additive effectively reduces the activation free energy barrier of the RDS through direct participation in the reaction while preferentially modulating the charge distributions and increasing the stability of corresponding intermediates and complexes along the reaction path. Furthermore, bonding and electronic structure analyses of the key structures indicate that the chemo- and regioselectivities of the reaction are strongly influenced by both electronic effects and steric hindrance.

Confirmation of pain-related neuromodulation mechanism of Bushen Zhuangjin Decoction on knee osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen Zhuangjin Decoction (BZD) are an herbal compound commonly used to treat osteoarthritis (OA) in China. AIM OF THE STUDY: This study aimed to verify the mechanism of Bushen Zhuangjin Decoction in relieving the pain of knee osteoarthritis. MATERIALS AND METHODS: Network pharmacology evaluation was used to discover the potential targets of BZD to relieve pain in KOA. The therapeutic effects of BZD treatment on KOA pain using histomorphology, behavioral assessments, suspension chip analysis, and ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) assays. The functional magnetic resonance imaging was used to explore the effects of BZD treatment on brain function associated to KOA. RESULTS: Network pharmacological analysis revealed the association between the analgesic effect of BZD on KOA and the pain signaling neurotransmitter 5-HT. Subsequently, we conducted experiments to verify the therapeutic effect of BZD on pain in KOA animal models. Behavioral tests demonstrated that the pain threshold of knee osteoarthritis rats decreased in PWT and PWL, but BZD was able to increase the pain threshold. Histopathological staining indicated thinning of the cartilage layer and sparse trabeculae in the subchondral bone. Suspension chip analysis revealed a significant increase in pro-inflammatory factors of IL-1α, IL-5, IL-12, IL-17A, RANTES, TNF-α and M-CSF in KOA, along with a significant decrease in anti-inflammatory factor of IL-13. However, BZD treatment decreased the expression of pro-inflammatory factors and increased the content of anti-inflammatory factor. UHPLC-MS/MS analysis showed a significant decrease in the serum levels of GABA, E, GSH, Kyn, Met, and VMA in KOA, which were significantly increased by BZD. Conversely, the serum levels of TrpA, TyrA, Spd, and BALa were significantly increased in KOA and significantly decreased by BZD. ELISA and Western blot analysis showed increased expression of subchondral bone pain-related neuropeptides SP, CGRP, TH, NPY, VEGFA, 5-HT3 in KOA, which were decreased in BZD. Functional magnetic resonance imaging demonstrated that BZD exerts its therapeutic effect on KOA by modulating the activity and functional connections of the cortex, hypothalamus, and hippocampus. CONCLUSIONS: This study confirmed the significant role of pain-related neuromodulation mechanisms in the analgesic therapy of BZD and provides a theoretical foundation for using BZD as a traditional Chinese medical treatment for KOA pain.

Effects of Chaihu Shugan San on Brain Functional Network Connectivity in the Hippocampus of a Perimenopausal Depression Rat Model.

In this study, we used Chaihu Shugan San (CSS), a traditional Chinese herbal formula, as a probe to investigate the involvement of brain functional network connectivity and hippocampus energy metabolism in perimenopausal depression. A network pharmacology approach was performed to discover the underlying mechanisms of CSS in improving perimenopausal depression, which were verified in perimenopausal depression rat models. Network pharmacology analysis indicated that complex mechanisms of energy metabolism, neurotransmitter metabolism, inflammation, and hormone metabolic processes were closely associated with the anti-depressive effects of CSS. Thus, the serum concentrations of estradiol (E2), glutamate (Glu), and 5-hydroxytryptamine (5-HT) were detected by ELISA. The brain functional network connectivity between the hippocampus and adjacent brain regions was evaluated using resting-state functional magnetic resonance imaging (fMRI). A targeted metabolomic analysis of the hippocampal tricarboxylic acid cycle was also performed to measure the changes in hippocampal energy metabolism using liquid chromatography-tandem mass spectrometry (LC-MS/MS). CSS treatment significantly improved the behavioral performance, decreased the serum Glu levels, and increased the serum 5-HT levels of PMS + CUMS rats. The brain functional connectivity between the hippocampus and other brain regions was significantly changed by PMS + CUMS processes but improved by CSS treatment. Moreover, among the metabolites in the hippocampal tricarboxylic acid cycle, the concentrations of citrate and the upregulation of isocitrate and downregulation of guanosine triphosphate (GTP) in PMS + CUMS rats could be significantly improved by CSS treatment. A brain functional network connectivity mechanism may be involved in perimenopausal depression, wherein the hippocampal tricarboxylic acid cycle plays a vital role.

Acteoside ameliorates learning and memory impairment in APP/PS1 transgenic mice by increasing Aß degradation and inhibiting tau hyperphosphorylation.

Alzheimer's disease (AD) is a neurodegenerative disease. Senile plaques and intracellular neurofibrillary tangles are pathological hallmarks of AD. Recent studies have described the improved cognitive and neuroprotective functions of acteoside (AS). This study aimed to investigate whether the improved cognition of AS was mediated by Aß degradation and tau phosphorylation in APP/PS1 mice. The open field, Y maze, and novel object recognition tests were used to assess cognitive behavioral changes. We evaluated the levels of Aß40 and Aß42 in serum, cortex, and hippocampus, and Aß-related scavenging enzymes, phosphorylated GSK3ß and hyperphosphorylated tau in the cortex and hippocampus of APP/PS1 mice by western blotting. Our results revealed that AS treatment ameliorated anxious behaviors, spatial learning, and memory impairment in APP/PS1 mice and significantly reduced Aß deposition in their serum, cortex, and hippocampus. AS significantly increased Aß degradation, inhibited the hyperphosphorylation of tau, and significantly decreased the activity of GSK3ß, which is involved in tau phosphorylation. Altogether, these findings indicated that the beneficial effects of AS on AD-associated anxious behaviors and cognitive impairments could be attributed to promoting Aß degradation and inhibiting tau hyperphosphorylation, which might be partly mediated by GSK3ß.