Mitochondrial dysfunction elevates cellular NADH/NAD+ ratios, inducing reductive stress that impairs biosynthesis and cell viability. However, the mechanisms by which cells buffer excess NADH to maintain redox homeostasis remain unclear. In this study, we identify a mitochondrial-cytosolic metabolic circuitry involving pyruvate carboxylase (PC), malate dehydrogenases (MDH1/2), and malic enzyme 1 (ME1) that mitigates NADH overload by coupling anaplerotic flux with NADH oxidation. Under reductive stress induced by electron transport chain (ETC) dysfunction, oxaloacetate derived from PC is converted to malate by MDH1/2, which is then oxidized by ME1, transforming both cytosolic and mitochondrial NADH into cytosolic NADPH. Although cellular NADPH is typically associated with antioxidant defense and biosynthesis, our experiments show that TPNOX, which can oxidase NADPH to NADP+, restores proliferation under ETC inhibition, and this rescue is entirely dependent on the mitochondrial-cytosolic metabolic circuitry. This finding highlights that the primary function of the circuit is to maintain NADH homeostasis rather than to generate NADPH. Notably, glucose-6-phosphate dehydrogenase (G6PD) enhances ME1 activity independently of its catalytic function by acting as a scaffold, thereby preventing net NADPH production while facilitating the conversion of NADH to NADPH. Disruption of the PC-MDH1/2-ME1 pathway through PC inhibition sensitizes cells to complex I inhibitors and/or glutaminase blockade, synergistically suppressing tumor growth both in vitro and in vivo. These findings uncover a redox-buffering strategy that redirects NADH into NADPH production, revealing a metabolic vulnerability in redox-adapted cancer cells.

Ethnopharmacological relevanceIxeris sonchifolia alias Kudiezi, it was named Ixeris sonchifolia (Bunge) Hance, a synonym for Crepidiastrum sonchifolium (Bunge) Pak & Kawano in the https://www.iplant.cn/. And it was first published in J. Linn. Soc., Bot. 13: 108 (1873), which was named Ixeris sonchifolia (Maxim.) Hance in the MPNS (http://mpns.kew.org). As a widely distributed medicinal and edible wild plant, it possesses unique bitter-cold characteristics and constituents with various pharmacological activities. Its main antitumor substances, same as artemisinin and paclitaxel, are classified as terpenoids and have become research foci in recent years. However, its specific biological activity and role in antitumor treatment remain largely unclear.Aim of the studyThis study aimed to elucidate the molecular targets and potential mechanisms of hepatocellular carcinoma apoptosis induced by Ixeris sonchifolia.Materials and methodsWe used network pharmacology methods to analyze and screen the active ingredients and possible underlying mechanisms of Ixeris sonchifolia in treating liver cancer and employed integrative time- and dose-dependent toxicity, transcriptomics, and molecular biology approaches to comprehensively verify the function of Ixeris sonchifolia extract (IsE) in human hepatoblastoma cell (HepG2) apoptosis and its potential mechanism.ResultsA total of 169 common targets were screened by network pharmacology, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that IsE inhibited HepG2 cell activity in a time- and dose-dependent manner. Western blot analysis confirmed that IsE promoted HepG2 cell apoptosis by inhibiting the PI3K/AKT signaling pathway and that the PI3K/AKT inhibitor LY294002 also substantially enhanced IsE-induced apoptosis. The PI3K/AKT signaling pathway exhibited significant differences compared to that in the control group.ConclusionCombining network pharmacology with experimental verification, IsE inhibited mitochondrial function and the PI3K/AKT pathway while inducing hepatoma cell apoptosis. IsE may have promising potential for liver cancer treatment and chemoprevention.

BackgroundRadiofrequency ablation (RFA) is the primary curative treatment for hepatocellular carcinoma (HCC) patients who are not eligible for surgery. However, the effects of RFA on the global tumor immune response remain unclear.MethodIn this study, we examined the phenotypic and functional changes in peripheral blood mononuclear cells (PBMCs) from recurrent HCC patients who had undergone two RFA treatments using mass cytometry and high-throughput mRNA assays. ResultsWe observed significant increase in monocytes and decrease in T cell subpopulations three days after the first RFA treatment and three days after the second RFA treatment. The down-regulation of GZMB, GZMH, GZMK, and CD8A, which are involved in the cytotoxic function of T cells, was observed following RFA. Furthermore, the population of CD8 effector and memory T cells (CD8 Teff and CD8 Tem) significantly decreased after RFA. The expression of CD5 and CD161 in various T cell subpopulations also showed significant reductions. Additionally, elevated secretion of VEGF was observed in monocytes, B cells, regulatory T cells (Tregs), and CD4 naive T cells. ConclusionIn recurrent HCC patients, serum components derived from radiofrequency therapy can enhance the antigen-presenting capacity of monocytes. However, they also inhibit the anti-cancer immune response by reducing the population of CD8 effector and memory T cells and suppressing the activation of T cells, as well as down-regulating the expression of CD161 and CD5 in various T cell subpopulations. These tumor-derived components also contribute to an immunosuppressive microenvironment by promoting the secretion of VEGF in monocytes, Tregs, B cells, and CD4 naive T cells.

Hepatocellular carcinoma（HCC） is the most prevalent form of primary liver cancer, accounting for 75%-85% of cases. Although treatments are given to cure early-stage HCC, up to 50%-70% of individuals may experience a relapse of the illness in the liver after 5 years. Research on the fundamental treatment modalities for recurrent HCC is moving significantly further. The precise selection of individuals for therapy strategies with established survival advantages is crucial to ensuring better outcomes. These strategies aim to minimize substantial morbidity,support good life quality, and enhance survival for patients with recurrent HCC.For individuals with recurring HCC after curative treatment, no approved therapeutic regimen is currently available. A recent study presented novel approaches, like immunotherapy and antiviral medication, to improve the prognosis of patients with recurring HCC with the apparent lack of data to guide the clinical treatment. The data supporting several neoadjuvant and adjuvant therapies for patients with recurring HCC are outlined in this review. We also discuss the potential for future clinical and translational investigations.

Background: The host immune response to pneumocystis pneumonia (PCP) involves interactions among immune cell subsets and cytokines. However, the definite pathogenesis and immunological influences of P. jirovecii have not been fully elucidated.Methods: Mass cytometry and high-throughput sequencing of the T cell receptor (TCR) were used to profile the host immune response to human immunodeficiency virus-1 (HIV-1) and P. jirovecii infection. Results: Our findings demonstrated that patients with PCP showed different immune cell proportions when compared to healthy controls. Changes in cytokines were found after anti-PCP treatment, suggested that cytokines may play an important role in controlling the pathogen. Furthermore, PCP patients showed marked reduction of TCR repertoire diversity. The diversity of TCR repertoire was restored by the anti-PCP treatment.Conclusion: In summary, we profiled the composition and characteristics of immune environment in response to HIV-1 and P. jirovecii infection, which may contribute to elucidating host immunity.

[This corrects the article DOI: 10.1155/2019/8727935.].

Mitochondria, as the only metabolic organelles which exist independently of the cell nucleus with DNA, play an important role in the development and progression of colorectal cancer. There are many researches about the relationship between colorectal cancer and mitochondrial DNA (mtDNA) emerge in endlessly. As the starting site of transcription and replication, displacement loop region (D-loop) region becomes one of the hot spots among these researches. In this paper, we reviewed the progress research about the correlation between mutations, polymorphisms, methylation, microsatellite instability and copy number changes of D-loop region and colorectal cancer. Some studies have shown that the presence of tumor D-loop mutations are associated with poor prognosis and can be used as a predictor of postoperative adjuvant chemotherapy in patients with stage Ⅲ colorectal cancer. The frequent haplotypes of secondary haplotypes of nucleotide 16290T and the frequent haplotypes of nucleotide 16298T in hypervariable region 1 (HV1) are associated with the high survival rates of colorectal cancer, and the polymorphism at position 16 290 is identified as independent predictors of colorectal cancer prognosis. The demethylation of the D-loop region may be involved in the regulation of mtDNA copy number and nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND-2) expression, and its microsatellite instability is associated with drug resistance and poor prognosis of the fluorouracil-based adjuvant chemotherapy. Due to D-loop region as an important transcriptional initiation region of mtDNA, the in-depth and comprehensive study will help us understand its role in the pathogenesis and progression of colorectal cancer, and further provide guidance for clinical diagnosis and treatment.

CD8(+) CD45RA(+)/CD4(-) CD45RO(+)/CD4(-) CD16(-)/CD107A(+) CD3(+)/CD4(2+) CD3(+)/CD8(+) CD45RA(3+)/CD4(2+) CD45RO(+)/CD4(2+) CD16(2+)/CD107A(2+) CD3(+)/CD4(+) CD3(+)/CD8(+) CD45RA(4+)/CD4(+) CD45RO(+)/CD4(+) CD16(+)/

目的 构建HBx表达载体,筛选可稳定表达HBx的人肝细胞(HL)-7702细胞系.方法 采用RT-PCR法扩增HBx基因片段,并将其连接至pIRES载体,经酶切和测序鉴定pIRES-HBx重组质粒序列.然后,分别采用Real-timePCR和Western blot技术检测验证重组质粒的正确性.最后,应用G418抗生素筛选稳定表达HBx的HL-7702细胞系.结果 经PCR扩增得到正确的HBx片段,成功构建pIRES-HBx质粒,将重组质粒转染HEK 293细胞和HL-7702细胞均实现了HBx过量表达;经免疫荧光法鉴定,我们获得了稳定大量表达HBx的HL-7702细胞系.结论 成功构建的pIRES-HBx表达载体能在HL-7702细胞稳定表达HBx,为后续研究提供了基础实验工具.

Title: Down-regulated Summary : Suppressed T cell immune response and decreased T cells in COVID-19 patients related to down-regulated gene expression involved in T cell activation and differentiation Abstract Background: WHO characterizes novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a pandemic. Here, we investigated the clinical, cytokine levels, T cell proportion and related gene expression occurring in COVID-19 patients on admission and after intial treatment. Methods: 11 patients diagnosed as COVID-19 with similar initial treatment regimen were enrolled in the hospital. Plasma cytokines, CyTOF and microfluidic qPCR for gene expression were conducted. Results: 5 mild and 6 severe patients were included. Cough and fever were the top symptoms in the 11 COVID-2019 cases. The elder age, more neutrophils numbers and higher C-reactive protein level were found in severe cases. IL-10 level was significantly varied with disease progression and treatment. The decreased T cell proportions were observed in COVID-19 patients especially in severe cases, and all elevated to normal in mild patiens after initial treatment but only CD4+T cells return to normal in severe cases. The number of DEGs increased with the disease progress, and decreased after initial treatment. All down-regulated DEGs in severe cases mainly involved in Th17 cell differentiation, cytokine-mediated signaling pathway and T cell activation. After initial treatmen in severe cases, MAP2K7 and SOS1 were upregulated relative to that on admission. Conclusions: Our findings show a decreased T cell proportion with down-regulated gene expression related to T cell activation and differentiation were occurred in COVID-19 severe patients, which may help to provide effective treatment strategies for COVID-19 . reported clinical and plasma cytokines features of COVID-19, only limited information is available on the host innate immune status of SARS-CoV-2 infected patients. The changes of immune-related gene expression levels during SARS-CoV-2 infection were still unclear. Here, we comprehensively evaluated the characteristics of 11 patients with COVID-19 admitted to Beijing Youan Hospital. We aimed to compare the clinical, cytokine levels and immune related gene expression characteristics between different clinical stages. Our data provide basic information to understand the role of immune responses on the disease process of COVID-19. Results of microfluidic qPCR and following bioinformatics analysis demonstrated that SARS-CoV-2 infection could affect the mRNA expression level related with immune functions in COVID-19 cases. Our study revealed the number of misregulated genes increased as the disease progressed when compared to normal. A more DEGs were found in severe cases. Both DEGs of severe and mild patients are participated in such as Th17 cell differentiation, cytokine-mediated signaling pathway, sa mmp cytokine connection, natural killer cell mediated cytotoxicity and T cell activation. While DEGs specific to severe cases were mainly responsible for CD8 TCR pathway, EPO pathway, signaling by Interleukins, TNF signaling pathway, entry into host cell and regulation of T cell proliferation enrichment terms. These results indicated SARS-CoV-2 infection may resulting inspecific Th1/Th17 inactivation and impaired inflammatory responses, which may related to the decreased proportion of T cells expecially in COVID-19 severe patients.. 14 key genes related with immune functions in COVID-19 cases were screened, which revealed FC down-regulated more in group A1 than that in group B1 both relative to the NC group. Like influenza viruses, CoVs such as SARS-CoV and MERS-CoV also use a combination of ways to achieve host shut-off both at the transcriptional and the translational levels [7]. Innate and adaptive immune responses to viral infection during infection. Coronaviruses (CoVs) infects macrophages and presents CoV antigens to T cells, which process could results in T cell activation and differentiation that including the production of cytokines associated with different T cell subpopulations, and subsequent large release of cytokines for immune response expansion in the severe COVID-19 patients before treatment we demonstrated the changed immune response in COVID-19 patients through down-regulated at the transcriptional levels involved in T cell activation and differentiation especially in severe patients. Although clinical blood laboratory findings did not show significantly differences after intinal treatment, IL-10 level, T cells proportions and related mRNA expression level changed with the disease progression. These findings will help us extend our understanding of the SARS-CoV-2 infection mechanism, may help elucidate the COVID-19 infection and provide a basis for future novel immune therapeutic strategies.

Although most patients with COVID-19 pneumonia have a good prognosis, some patients develop to severe or critical illness, and the mortality of critical cases is up to 61.5%. However, specific molecular information about immune response in critical patients with COVID-19 is poorly understood. A total of 54 patients were enrolled and divided into three groups, among which 34 were common, 14 were severe, and 6 were critical. The constitution of peripheral blood mononuclear cells (PBMC) in patients was analyzed by CyTOF. The profile of cytokines was examined in plasma of patients using luminex. The IL-2 signaling pathway was investigated in the PBMC of patients by qRT-PCR. The count and percentage of lymphocytes were significantly decreased in critical patients compared to common and severe patients with COVID-19 pneumonia. The count of T cells, B cells, and NK cells was remarkably decreased in critical patients compared to normal controls. The percentage of CD8 + T cells was significantly lower in critical patients than that in common and severe patients with COVID-19 pneumonia. The expression of IL-2R, JAK1, and STAT5 decreased in PBMC of common, severe, and critical patients, but IL-2 level was elevated in severe patients and decreased in critical patients with COVID-19 pneumonia. The decrease of CD8 + T cells in critical patients with COVID-19 pneumonia may be related to the IL-2 signaling pathway. The inhibition of IL-2/IL-2R gives rise to CD8 + T cell and lymphocyte decrease through JAK1-STAT5 in critical patients with COVID-19 pneumonia.

BACKGROUND:The World Health Organization characterizes novel coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as a pandemic. Here, we investigated the clinical, cytokine levels; T-cell proportion; and related gene expression occurring in patients with COVID-19 on admission and after initial treatment.METHODS:Eleven patients diagnosed with COVID-19 with similar initial treatment regimens were enrolled in the hospital. Plasma cytokine, peripheral T cell proportions, and microfluidic quantitative polymerase chain reaction analyses for gene expression were conducted.RESULTS:Five patients with mild and 6 with severe disease were included. Cough and fever were the primary symptoms in the 11 COVID-19 cases. Older age, higher neutrophil count, and higher C-reactive protein levels were found in severe cases. IL-10 level significantly varied with disease progression and treatment. Decreased T-cell proportions were observed in patients with COVID-19, especially in severe cases, and all were returned to normal in patients with mild disease after initial treatment, but only CD4+ T cells returned to normal in severe cases. The number of differentially expressed genes (DEGs) increased with the disease progression, and decreased after initial treatment. All downregulated DEGs in severe cases mainly involved Th17-cell differentiation, cytokine-mediated signaling pathways, and T-cell activation. After initial treatment in severe cases, MAP2K7 and SOS1 were upregulated relative to that on admission.CONCLUSIONS:Our findings show that a decreased T-cell proportion with downregulated gene expression related to T-cell activation and differentiation occurred in patients with severe COVID-19, which may help to provide effective treatment strategies for COVID-19.

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.

Objective: To study the effect of apoptosis-stimulating protein 2 of p53 (ASPP2) on the activation and apoptosis of hepatic stellate cells induced by transforming growth factor-β1 (TGF - β1), and to explore the role of autophagy in this process. Methods: Mouse hepatic stellate cells were primarily isolated and cultured with green fluorescent protein (GFP) expressing empty vector adenovirus (Ad-GFP) and ASPP2 expressing adenovirus (Ad-ASPP2) for 12 h by transfection kit, and then treated with TGF-β1 (10ng/ml) for 24 h. The experiments were grouped as follows: control group: green fluorescent protein (GFP) expressing empty vector adeno (Ad-GFP); experimental group 1: transfected with Ad-GFP and added with TGF-β1; experimental group 2: transfected with Ad-ASPP2 and induced by TGF-β1. Western blot and quantitative fluorescence PCR were used to detect the expression of ASPP2, α-smooth muscle actin (SMA). At the same time, autophagy was determined by microtubule-associated protein 1 light chain 3-β (LC3). Autophagy and apoptosis of MHSc were observed by immunocytochemistry and RNA interference (RNAi). Multiple pairwise-comparisons between the mean of groups was performed by one-way ANOVA. Results: The relative expression of α-SMA mRNA in mHSC of TGF-β1 + Ad-GFP group (16.83 ± 2.41) was significantly higher than Ad-GFP group (3.62 ± 0.56) (P < 0.05), while the relative expression of α-SMA mRNA (4.22 ± 0.48) in TGF-β1 + Ad-GFP group was significantly lower than TGF-β1 + Ad-GFP group (P < 0.05). The expression of α-SMA protein in each group was consistent with mRNA expression. The proportion of mHSC autophagy in TGF-β1 + Ad-GFP group (80%) was significantly higher than Ad-GFP group (35%); however, there was no statistically significant difference between the two groups. The proportion of mHSC autophagy in TGF-β1 + Ad-ASPP2 group was 42%, which was significantly lower than TGF-β1 + Ad-GFP group, but the apoptotic rate was significantly increased. Cells were simultaneously treated with autophagy inhibitors 3-MA and TGF-β1. The level of autophagy was not statistically significantly different from that of TGF-β1 + Ad-ASPP2 group, but the apoptotic rate was increased. In addition, the RNAi group added with ASPP2 had increased autophagy (LC3-II/LC3-I) than control RNAi group, and the rate of apoptosis was significantly decreased. Conclusion: Overexpression of ASPP2 can alleviate the activation of mHSC and promote the apoptosis of HSC by inhibiting autophagy, so as to alleviate liver fibrosis.

Sorafenib is the standard first-line systemic chemotherapeutic drugs for advanced hepatocellular carcinoma (HCC), but acquired resistance to sorafenib is frequently observed in clinical practice. In this study, we first produced three sorafenib resistance (SR) HCC cell lines by using two human HCC cell lines (Hep3B and Huh7) and a human primary HCC cell line. We identified that epidermal growth factor receptor (EGFR) and Kruppel-like factor 4 (KLF4) are dramatically increased in the three SR HCC cell lines. Either inhibition of tyrosine kinase activity of EGFR with Erlotinib/Icotinib or inhibition of KLF4 expression with short hairpin RNA recovered the response of three SR HCC cell lines to sorafenib, suggesting the critical roles of EGFR tyrosine kinase and KLF4 on inducing SR. Luciferase activity and chromatin immunoprecipitation assays further determined that KLF4 promoted EGFR expression through inducing its transcription by directly binding to its promoter. EGFR, conversely, could also promote KLF4 expression through inducing its transcription by binding to its promoter in a tyrosine kinase-dependent manner, suggesting that a positive feedback loop formed by EGFR and KLF4 further amplifies their effects on inducing SR. Up to now, our findings that KLF4 induces the development of SR and it cooperates with EGFR to form a positive feedback loop to amplify their SR-inducing abilities have rarely been reported. Our findings bear possible implications for the improvement of the efficacy of sorafenib in HCC.

Purpose. Multidrug resistance (MDR) is a major obstacle in chemotherapy of leukemia treatments. In this paper, we investigated Usnea Acid (UA) as MDR reversal agent on hematologic K562/ADR cells via ROS dependent apoptosis. Methods. CCK8 assay was used to measure cell viability rate of K562/ADR. Intracellular reactive oxygen species (ROS) generation, cell cycle distribution, cell apoptosis were measured with flow cytometry, respectively. Proteins related to apoptosis were measured by Western blot. Intracellular Adriamycin accumulation was observed by confocal microscopy and measured by flow cytometry. Results. In vitro study showed intracellular Adriamycin accumulation was remarkably increased by UA. Cell viability treated with Adr (4 μM) was decreased from 89.8% ± 4.7 to 32% ± 8.9 by combined with UA (4 μM). Adr-induced apoptosis and G1/G0 phase cell cycle arrest were remarkably increased by UA, as well as, intracellular ROS level. However, MDR reversing activity of UA was inhibited by N-acetyl cysteine (NAC), a ROS scavenger. Conclusion. These data provide compelling evidence that UA is a promising agent against MDR in leukemia cell line and suggest a promising therapeutic approach for leukemia.