Enhanced NK cell activation via eEF2K-mediated potentiation of the cGAS-STING pathway in hepatocellular carcinoma.

BACKGROUND: Liver cancer, particularly hepatocellular carcinoma (HCC), is characterized by a high mortality rate, attributed primarily to the establishment of an immunosuppressive microenvironment. Within this context, we aimed to elucidate the pivotal role of eukaryotic elongation factor 2 kinase (eEF2K) in orchestrating the infiltration and activation of natural killer (NK) cells within the HCC tumor microenvironment. By shedding light on the immunomodulatory mechanisms at play, our findings should clarify HCC pathogenesis and help identify potential therapeutic intervention venues. METHODS: We performed a comprehensive bioinformatics analysis to determine the functions of eEF2K in the context of HCC. We initially used paired tumor and adjacent normal tissue samples from patients with HCC to measure eEF2K expression and its correlation with prognosis. Subsequently, we enrolled a cohort of patients with HCC undergoing immunotherapy to examine the ability of eEF2K to predict treatment efficacy. To delve deeper into the mechanistic aspects, we established an eEF2K-knockout cell line using CRISPR/Cas9 gene editing. This step was crucial for verifying activation of the cGAS-STING pathway and the subsequent secretion of cytokines. To further elucidate the role of eEF2K in NK cell function, we applied siRNA-based techniques to effectively suppress eEF2K expression in vitro. For in vivo validation, we developed a tumor-bearing mouse model that enabled us to compare the infiltration and activation of NK cells within the tumor microenvironment following various treatment strategies. RESULTS: We detected elevated eEF2K expression within HCC tissues, and this was correlated with an unfavorable prognosis (30.84 vs. 20.99 months, P = 0.033). In addition, co-culturing eEF2K-knockout HepG2 cells with dendritic cells led to activation of the cGAS-STING pathway and a subsequent increase in the secretion of IL-2 and CXCL9. Moreover, inhibiting eEF2K resulted in notable NK cell proliferation along with apoptosis reduction. Remarkably, after combining NH125 and PD-1 treatments, we found a significant increase in NK cell infiltration within HCC tumors in our murine model. Our flow cytometry analysis revealed reduced NKG2A expression and elevated NKG2D expression and secretion of granzyme B, TNF-α, and IFN-γ in NK cells. Immunohistochemical examination confirmed no evidence of damage to vital organs in the mice treated with the combination therapy. Additionally, we noted higher levels of glutathione peroxidase and lipid peroxidation in the peripheral blood serum of the treated mice. CONCLUSION: Targeted eEF2K blockade may result in cGAS-STING pathway activation, leading to enhanced infiltration and activity of NK cells within HCC tumors. The synergistic effect achieved by combining an eEF2K inhibitor with PD-1 antibody therapy represents a novel and promising approach for the treatment of HCC.

Effects of Phase â Cardiac Rehabilitation Combined with Cognitive Behavioural Therapy on Cardiac Function, Exercise Capacity and Mental Health in Patients after Aortic Valve Replacement: A Retrospective Study.

OBJECTIVE: To explore the application effect of phase Ⅰ cardiac rehabilitation (CR-Ⅰ) combined with cognitive behavioural therapy (CBT) on patients after aortic valve replacement (AVR). METHODS: This study retrospectively analysed the data of 441 patients after AVR in our hospital from January 2020 to May 2023. A total of 38 patients who did not meet the inclusion criteria were excluded. A total of 403 patients were included. In accordance with different postoperative management schemes, the included patients were divided into the reference group (n = 202, received CR-Ⅰ) and the observation group (n = 201, received CR-Ⅰ+CBT). The cardiac function, exercise capacity and mental health of the two groups were compared. RESULTS: Before management, both groups had no significant differences in left ventricular end diastolic diameter (LVEDD), left ventricular end systolic dimension (LVESD), left ventricular ejection fraction (LVEF) and six-minute walking test (6MWT) scores (p > 0.05). At discharge and 3 months after discharge, the observation group had significantly lower LVEDD and LVESD and remarkably higher LVEF and 6MWT scores than the reference group (p < 0.001). The proportions of autonomous activity in bed within 3-4 days after surgery, autonomous out-of-bed activity within 8-10 days after surgery and autonomous walking 200 m within 12-15 days after surgery were distinctly higher (p < 0.001) and the incidence of adverse reactions was overtly lower (p < 0.001) in the observation group than in the reference group. Before management, both groups had no significant difference in their scores on the State-Trait Anxiety Inventory (STAI) (p > 0.05). At discharge and 3 months after discharge, the observation group had lower STAI scores than the reference group (p < 0.001). CONCLUSION: CR-Ⅰ combined with CBT effectively improves the cardiac function, independent exercise capacity and mental health level of patients after AVR and provides a new direction for the formulation and selection of follow-up clinical management.

Protective efficacy of a novel recombinant influenza virus carrying partial human metapneumovirus F protein epitopes.

Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infections in infants and children. Currently, no approved HMPV vaccine is available. We developed a novel recombinant influenza virus, which carried partial HMPV F protein (HMPV-F) epitopes, utilizing reverse genetics. The novel single-stranded RNA virus, termed rFLU-HMPV/F-NA, was synthesized in the neuraminidase (NA) fragment of influenza virus A/PuertoRico/8/34 (PR8). The morphological characteristics of rFLU-HMPV/F-NA were consistent with the wild-type flu virus. The virus could passage in specific pathogen-free (SPF) chicken embryos for at least five consecutive generations with haemagglutinin (HA) titres of 28-9 or 8-9LogTCID50/mL. BALB/c mice were intranasally immunized at 21-day intervals with 104 TCID50 (low-dose group) or 106 TCID50 (high-dose group) rFLU-HMPV/F-NA, and PBS or PR8 vaccine was used for the control group. rFLU-HMPV/F-NA induced robust humoral, mucosal, and cellular immune responses in vivo in a dose-dependent manner. More importantly, wt clinical HMPV isolate challenge studies showed that rFLU-HMPV/F-NA provided significant immune protection against HMPV infection compared to the PBS or PR8 vaccine control group, as shown by improved histopathological changes and reduced viral titres in the lungs of immunized mice post-challenge. These findings demonstrate that rFLU-HMPV/F-NA has potential as a promising HMPV candidate vaccine and warrants further investigation into its control of HMPV infection.

Effects of Cells Self-aggregation in the Treatment of Neurogenic Erectile Dysfunction With Traditional Single Cell Suspension of Adipose-derived Stem Cells.

OBJECTIVE: To clarify the effects of cellular self-aggregation of adipose-derived stem cells (ADSCs) on erectile function (EF). METHODS: A model of neurogenic erectile dysfunction was performed using bilateral cavernous nerve crush injury in rats. ADSCs suspensions (1 × 106/0.2 ml), were administered via intracavernous injection (ICI) after being allowed to shelve for 0 minute (ICI 0) or 60 minutes (ICI 60) in vitro, as well as cell aggregates isolated from ICI 60 (ICI A). The caudal vein injection group (CVI 60) was used to evaluate whether cell self-aggregation was beneficial to EF when introduced into the peripheral circulation. One day after the transplantation, the distribution of cells was observed. EF and histopathological changes were evaluated after 4 weeks. RESULTS: Approximately 85% of ADSCs self-aggregated into cell clusters at 60 minutes. The ICI 60 had more significant improvements in EF and more visualized ADSCs retained in the corpus cavernosum (CC) than ICI 0 and CVI 60 (P <.05), but no significant difference between ICI 60 and ICI A. In the CVI 60 group, the cell clusters formed by self-aggregation could hardly reach the CC and were mostly found in lung tissue. Immunofluorescence staining showed increased the content of expressing biomarkers of smooth muscle, nerve within the CC tissue in the ICI groups when compared to the CVI group. CONCLUSION: ADSCs self-aggregation before ICI may be an influential factor in the treatment of neurogenic erectile dysfunction. Its potential mechanism may be through improving cell retention in the CC.

Long noncoding RNA FAM66C promotes tumor progression and glycolysis in intrahepatic cholangiocarcinoma by regulating hsa-miR-23b-3p/KCND2 axis.

Long noncoding RNAs (lncRNAs) are known to be the important regulators in cancer progression. However, the role of lncRNA FAM66C (FAM66C) is yet to be investigated in intrahepatic cholangiocarcinoma (ICC). This study aimed to investigate the effects and related mechanisms of FAM66C in ICC. Human ICC tissues and cell lines were collected. The expression levels of FAM66C, hsa-miR-23b-3p (miR-23b-3p), and KCND2 were detected by qRT-RCR. The transfection experiments were employed to measure the effect of FAM66C on cell viabilities, migration, and invasion in ICC cells by CCK-8, transwell assays. Glycolysis was investigated by glucose consumption, lactate production and ATP levels. The dual-luciferase reporter and RNA pull down assays were conducted as a means of confirming the interactions between FAM66C, miR-23b-3p, and KCND2. Furthermore, the levels of the EMT-associated proteins (KCND2, GLUT1, PKM2, and LDHA) in ICC cells were detected by western blot. FAM66C was increased in ICC tissues and cells, increased cell viability, glycolysis, migration and invasion, and decreased apoptosis were shown in FAM66C overexpressing cells. Mechanistic analyses revealed that FAM66C regulated the downstream target gene KCND2 by sponging miR-23b-3p. FAM66C effect on ICC was further validated in murine xenograft assays. FAM66C knockdown cells gave rise to tumors that were smaller in size, consistent with the role of FAM66C as a promoter of in vivo tumor growth. These data revealed that FAM66C was able to drive ICC tumor progression and glycolytic activity via the miR-23b-3p/KCND2 axis, indicating FAM66C may be a viable target for treating ICC.

Upregulation of long noncoding RNA W42 promotes tumor development by binding with DBN1 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignancy found globally. Accumulating studies have shown that long noncoding RNAs (lncRNAs) play critical roles in HCC. However, the function of lncRNA in HCC remains poorly understood. AIM: To understand the effect of lncRNA W42 on HCC and dissect the underlying molecular mechanisms. METHODS: We measured the expression of lncRNA W42 in HCC tissues and cells (Huh7 and SMMC-7721) by quantitative reverse transcriptase polymerase chain reaction. Receiver operating characteristic curves were used to assess the sensitivity and specificity of lncRNA W42 expression. HCC cells were transfected with pcDNA3.1-lncRNA W42 or shRNA-lncRNA W42. Cell functions were detected by cell counting Kit-8 (CCK-8), colony formation, flow cytometry and Transwell assays. The interaction of lncRNA W42 and DBN1 was confirmed by RNA immunoprecipitation and RNA pull down assays. An HCC xenograft model was used to assess the role of lncRNA W42 on tumor growth in vivo. The Kaplan-Meier curve was used to evaluate the overall survival and recurrence-free survival after surgery in patients with HCC. RESULTS: In this study, we identified a novel lncRNA (lncRNA W42), and investigated its biological functions and clinical significance in HCC. LncRNA W42 expression was upregulated in HCC tissues and cells. Overexpression of lncRNA W42 notably promoted the proliferative and invasion of HCC, and inhibited cell apoptosis. LncRNA W42 directly bound to DBN1 and activated the downstream pathway. LncRNA W42 knockdown suppressed HCC xenograft tumor growth in vivo. The clinical investigation revealed that HCC patients with high lncRNA W42 expression exhibited shorter survival times. CONCLUSION: In vitro and in vivo results suggested that the novel lncRNA W42, which is upregulated in HCC, may serve as a potential candidate prognostic biomarker and therapeutic target in HCC patients.