Genetic fusion of CCL11 to antigens enhances antigenicity in nucleic acid vaccines and eradicates tumor mass through optimizing T-cell response.

Nucleic acid vaccines have shown promising potency and efficacy for cancer treatment with robust and specific T-cell responses. Improving the immunogenicity of delivered antigens helps to extend therapeutic efficacy and reduce dose-dependent toxicity. Here, we systematically evaluated chemokine-fused HPV16 E6/E7 antigen to improve the cellular and humoral immune responses induced by nucleotide vaccines in vivo. We found that fusion with different chemokines shifted the nature of the immune response against the antigens. Although a number of chemokines were able to amplify specific CD8 + T-cell or humoral response alone or simultaneously. CCL11 was identified as the most potent chemokine in improving immunogenicity, promoting specific CD8 + T-cell stemness and generating tumor rejection. Fusing CCL11 with E6/E7 antigen as a therapeutic DNA vaccine significantly improved treatment effectiveness and caused eradication of established large tumors in 92% tumor-bearing mice (n = 25). Fusion antigens with CCL11 expanded the TCR diversity of specific T cells and induced the infiltration of activated specific T cells, neutrophils, macrophages and dendritic cells (DCs) into the tumor, which created a comprehensive immune microenvironment lethal to tumor. Combination of the DNA vaccine with anti-CTLA4 treatment further enhanced the therapeutic effect. In addition, CCL11 could also be used for mRNA vaccine design. To summarize, CCL11 might be a potent T cell enhancer against cancer.

Characteristics of phenotypic antibiotic resistance of Helicobacter pylori and its correlation with genotypic antibiotic resistance: A retrospective study in Ningxia.

BACKGROUND: Geographic differences exist in the antibiotic resistance patterns of Helicobacter pylori. Personalized treatment regimens based on local or individual resistance data are essential. We evaluated the current status of H. pylori resistance in Ningxia, analyzed resistance-related factors, and assessed the concordance of phenotypic and genotypic resistance. METHODS: Strains were isolated from the gastric mucosa of patients infected with H. pylori in Ningxia and relevant clinical information was collected. Phenotypic antibiotic susceptibility assays (Kirby-Bauer disk diffusion) and antibiotic resistance gene detection (Sanger sequencing) were performed. RESULTS: We isolated 1955 H. pylori strains. The resistance rates of H. pylori to amoxicillin, levofloxacin, clarithromycin, and metronidazole were 0.9%, 42.4%, 40.4%, and 94.2%, respectively. Only five tetracycline-resistant and one furazolidone-resistant strain were identified. Overall, 3.3% of the strains were sensitive to all six antibiotics. Multidrug-resistant strains accounted for 22.9%, of which less than 20% were from Wuzhong. Strains isolated from women and patients with nonulcerative disease had higher rates of resistance to levofloxacin and clarithromycin. Higher rates of resistance to metronidazole, levofloxacin, and clarithromycin were observed in the older age group than in the younger age group. The kappa coefficients of phenotypic resistance and genotypic resistance for levofloxacin and clarithromycin were 0.830 and 0.809, respectively, whereas the remaining antibiotics showed poor agreement. CONCLUSION: H. pylori antibiotic resistance is severe in Ningxia. Therefore, furazolidone, amoxicillin, and tetracycline are better choices for the empirical therapy of H. pylori infection in this region. Host sex, age, and the presence of ulcerative diseases may affect antibiotic resistance of the bacteria. Personalized therapy based on genetic testing for levofloxacin and clarithromycin resistance may be a future direction for the eradication therapy of H. pylori infection in Ningxia.

Synthesis and antitumor activity of novel 2, 3-didithiocarbamate substituted naphthoquinones as inhibitors of pyruvate kinase M2 isoform.

The M2 isoform of pyruvate kinase (PKM2) is a potential antitumor therapeutic target. In this study, we designed and synthesised a series of 2, 3-didithiocarbamate substituted naphthoquinones as PKM2 inhibitors based on the lead compound 3k that we previously reported. Among them, compound 3f (IC50 = 1.05 ± 0.17 µM) and 3h (IC50 = 0.96 ± 0.18 µM) exhibited potent inhibition of PKM2, and their inhibitory activities are superior to compound 3k (IC50 = 2.95 ± 0.53 µM) and the known PKM2 inhibitor shikonin (IC50 = 8.82 ± 2.62 µM). In addition, we evaluated in vitro antiproliferative effects of target compounds using MTS assay. Most target compounds exhibited dose-dependent cytotoxicity with IC50 values in nanomolar concentrations against HCT116, MCF7, Hela, H1299 and B16 cells. These small molecule PKM2 inhibitors not only provide candidate compounds for cancer therapy, but also offer a tool to probe the biological effects of PKM2 inhibition on cancer cells.

Endoscopic Ruler for varix size measurement: A multicenter pilot study.

BACKGROUND: We invented Endoscopic Ruler, a new endoscopic device to measure the size of varices in patients with cirrhosis and portal hypertension. AIM: To assess the feasibility and safety of Endoscopic Ruler, and evaluate the agreement on identifying large oesophageal varices (OV) between Endoscopic Ruler and the endoscopists, as well as the interobserver agreement on diagnosing large OV using Endoscopic Ruler. METHODS: We prospectively and consecutively enrolled patients with cirrhosis from 11 hospitals, all of whom got esophagogastroduodenoscopy (EGD) with Endoscopic Ruler. The primary study outcome was a successful measurement of the size of varices using Endoscopic Ruler. The secondary outcomes included adverse events, operation time, the agreement of identifying large OV between the objective measurement of Endoscopic Ruler and the empirical reading of endoscopists, together with the interobserver agreement on diagnosing large OV by Endoscopic Ruler. RESULTS: From November 2020 to April 2022, a total of 120 eligible patients with cirrhosis were recruited and all of them underwent EGD examinations with Endoscopic Ruler successfully without any adverse event. The median operation time of Endoscopic Ruler was 3.00 min [interquartile range (IQR): 3.00 min]. The kappa value between Endoscopic Ruler and the endoscopists while detecting large OV was 0.52, demonstrating a moderate agreement. The kappa value for diagnosing large OV using Endoscopic Ruler among the six independent observers was 0.77, demonstrating a substantial agreement. CONCLUSION: The data demonstrates that Endoscopic Ruler is feasible and safe for measuring the size of varices in patients with cirrhosis and portal hypertension. Endoscopic Ruler is potential to promote the clinical practice of the two-grade classification system of OV.

Immunogenicity of the Xcl1-SARS-CoV-2 Spike Fusion DNA Vaccine for COVID-19.

SARS-CoV-2 spike (S) variants that may evade antibody-mediated immunity are emerging. Evidence shows that vaccines with a stronger immune response are still effective against mutant strains. Here, we report a targeted type 1 conventional dendritic (cDC1) cell strategy for improved COVID-19 vaccine design. cDC1 cells specifically express X-C motif chemokine receptor 1 (Xcr1), the only receptor for chemokine Xcl1. We fused the S gene sequence with the Xcl1 gene to deliver the expressed S protein to cDC1 cells. Immunization with a plasmid encoding the S protein fused to Xcl1 showed stronger induction of antibody and antigen-specific T cell immune responses than immunization with the S plasmid alone in mice. The fusion gene-induced antibody also displayed more powerful SARS-CoV-2 wild-type virus and pseudovirus neutralizing activity. Xcl1 also increased long-lived antibody-secreting plasma cells in bone marrow. These preliminary results indicate that Xcl1 serves as a molecular adjuvant for the SARS-CoV-2 vaccine and that our Xcl1-S fusion DNA vaccine is a potential COVID-19 vaccine candidate for use in further translational studies.

Identification of a new small molecule that initiates ferroptosis in cancer cells by inhibiting the system Xc- to deplete GSH.

Ferroptosis is a non-apoptotic cell death characterized by iron-mediated ROS accumulation and increasing lipid peroxidation. The activation of ferroptosis results in the destruction of cancer cells and overcoming the drug resistance associated with existing chemotherapeutic agents. It is essential to develop new ferroptosis inducers to provide new opportunities for cancer therapy. In this study, we found a small molecule Compound 8 which we had demonstrated to inhibit tumor growth in vivo initiated ferroptosis. Compound 8 treatment elevated the ferroptosis-related genes PTGS2 and CHAC1 mRNA levels in tumor cells. Ferroptosis inhibitors but not the necroptosis inhibitor or the apoptosis inhibitor can suppress the cell death induced by Compound 8. Compound 8 causes overall greater quantity of lipid peroxidation than the classic ferroptosis inducer Erastin through Flow cytometry analysis. The non-targeted lipidomic analysis also showed Compound 8 treatment resulted in oxidized lipid metabolites, similar to Erastin. The mechanism research showed that Compound 8 initiated ferroptosis by inhibiting the system Xc- to deplete GSH. Based on our previous study that Compound 8 blocked the interaction of PKM2 and VDAC3 (a regulator of ferroptosis) to inhibit tumor growth in vivo, Compound 8 may also trigger ferroptosis by regulating VADC3. Thus, Compound 8 not only will offer a potential tumor therapeutic alternative, but also provide an entrance to explore the new mechanism of ferroptosis.

The Role of Cellular Immunity in the Protective Efficacy of the SARS-CoV-2 Vaccines.

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have been approved for clinical use. SARS-CoV-2 neutralizing antibody titers after immunization are widely used as an evaluation indicator, and the roles of cellular immune responses in the protective efficacy of vaccines are rarely mentioned. However, therapeutic monoclonal neutralizing antibodies have shown limited efficacy in improving the outcomes of hospitalized patients with coronavirus disease 2019 (COVID-19), suggesting a passive role of cellular immunity in SARS-CoV-2 vaccines. The synergistic effect of virus-specific humoral and cellular immune responses helps the host to fight against viral infection. In fact, it has been observed that the early appearance of specific T-cell responses is strongly correlated with mild symptoms of COVID-19 patients and that individuals with pre-existing SARS-CoV-2 nonstructural-protein-specific T cells are more resistant to SARS-CoV-2 infection. These findings suggest the important contribution of the cellular immune response to the fight against SARS-CoV-2 infection and severe COVID-19. Nowadays, new SARS-CoV-2 variants that can escape from the neutralization of antibodies are rapidly increasing. However, the epitopes of these variants recognized by T cells are largely preserved. Paying more attention to cellular immune responses may provide new instructions for designing effective vaccines for the prevention of severe disease induced by the break-through infection of new variants and the sequelae caused by virus latency. In this review, we deliberate on the role of cellular immunity against COVID-19 and summarize recent advances in the development of SARS-CoV-2 vaccines and the immune responses induced by vaccines to improve the design of new vaccines and immunization strategies.

Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment.

The widespread clinical application of cord blood (CB) for hematopoietic stem cell (HSC) transplantation is limited mainly by the inadequate number of hematopoietic stem and progenitor cells (HSPCs) in single CB units, which results in unsuccessful or delayed engraftment in recipients. The identification of agents to promote CB HSPC engraftment has significant therapeutic value. Here, we found that transient inhibition of the JNK pathway increased the HSC frequency in CB CD34+ cells to 13.46-fold. Mechanistic studies showed that inhibition of the JNK pathway upregulated the expression of quiescence-associated and stemness genes in HSCs, preventing HSCs from entering the cell cycle, increasing glucose uptake and accumulating reactive oxygen species (ROS). Importantly, transient inhibition of the JNK pathway during CB CD34+ cell collection also enhanced long-term HSC (LT-HSC) recovery and engraftment efficiency. Collectively, these findings suggest that transient inhibition of the JNK pathway could promote a quiescent state in HSCs by preventing cell cycle entry and metabolic activation, thus enhancing the HSC number and engraftment potential. Together, these findings improve the understanding of the regulatory mechanisms governing HSC quiescence and stemness and have the potential to improve HSC collection and transplantation.

Deubiquitinase OTUD3 regulates metabolism homeostasis in response to nutritional stresses.

The ovarian-tumor-domain-containing deubiquitinases (OTUDs) block ubiquitin-dependent protein degradation and are involved in diverse signaling pathways. We discovered a rare OTUD3 c.863G>A mutation in a family with an early age of onset of diabetes. This mutation reduces the stability and catalytic activity of OTUD3. We next constructed an experiment with Otud3-/- mice and found that they developed worse obesity, dyslipidemia, and insulin resistance than wild-type mice when challenged with a high-fat diet (HFD). We further found that glucose and fatty acids stimulate CREB-binding-protein-dependent OTUD3 acetylation, promoting its nuclear translocation, where OTUD3 regulates various genes involved in glucose and lipid metabolism and oxidative phosphorylation by stabilizing peroxisome-proliferator-activated receptor delta (PPARδ). Moreover, targeting PPARδ using a specific agonist can partially rescue the phenotype of HFD-fed Otud3-/- mice. We propose that OTUD3 is an important regulator of energy metabolism and that the OTUD3 c.863G>A is associated with obesity and a higher risk of diabetes.

Prevalence and risk factors of Helicobacter pylori infection in Ningxia, China: comparison of two cross-sectional studies from 2017 and 2022.

OBJECTIVES: Helicobacter pylori (H. pylori) infection causes a variety of intragastric and extragastric diseases. Despite its decreasing global prevalence, it remains a major public health problem in many developing countries. This study aimed to understand the prevalence of H. pylori infection and its risk factors in five cities of the Ningxia Hui Autonomous Region, an area with high incidence of gastric cancer. METHODS: Cross-sectional studies were conducted in Ningxia from 2017 and 2022, to detect the prevalence of H. pylori using the 14C urea breath test. All participants completed a questionnaire that included demographics, personal habits, household economic characteristics, and previous health status. Multiple logistic regression analyses were used to identify independent factors for H. pylori infection. RESULTS: Our findings demonstrated that the prevalence of H. pylori infection in Ningxia decreased significantly from 60.3% in 2017 to 43.6% in 2022, with an increase in public awareness rate from 35.9% in 2017 to 68.5% in 2022. The lowest infection rate was found in Zhongwei and highest in Guyuan. The prevalence of H. pylori infection was higher among Hui ethnicity, farmers, individuals living in rural areas, individuals with lower income, low education, and those who consumed less fruit. Gallbladder, respiratory, cardiovascular and autoimmune diseases were not associated with H. pylori infection. CONCLUSIONS: The prevalence of H. pylori in Ningxia decreased in the past five years. Ethnicity, location, occupation, income, education, and consumption of fruits were independent risk factors for H. pylori infection in Ningxia. It was not associated with extra-gastric disease.

Efficacy and Safety of Central Memory T Cells Combined With Adjuvant Therapy to Prevent Recurrence of Hepatocellular Carcinoma With Microvascular Invasion: A Pilot Study.

BACKGROUND: Postoperative adjuvant transcatheter arterial chemoembolization (TACE) following curative hepatectomy has been reported to improve the clinical outcomes of hepatocellular carcinoma (HCC) patients with microvascular invasion (MVI), but more endeavors are required to achieve greater clinical benefit. Central memory T-cell (Tcm) self-transfusion has shown superior antitumor activity in several preclinical studies; however, clinical studies are rare. The aim of this study was to evaluate the clinical benefit and safety of combination treatment with Tcm self-transfusion and TACE as adjuvant treatment in HCC patients with MVI after curative hepatectomy. METHODS: From October 2016 to September 2018, primary HCC patients with histologically confirmed MVI who underwent curative hepatectomy at the Cancer Hospital of the Chinese Academy of Medical Sciences were recruited for this study. The patients were divided into a Tcm group (combined Tcm self-transfusion with TACE treatment) or a control group (TACE treatment alone) according to their willingness. The recurrence-free survival (RFS), quality-of-life (QOL) score, and adverse events of each patient were recorded within 2 years. RESULTS: A total of 52 patients were enrolled, and 48 were eligible for the final data analysis. The median follow-up time was 20.5 months (95% CI: 17.05-22.55 months). The median RFS time was 9.5 months in the control group; the cutoff date was not reached in the Tcm group (when the follow-up duration was 12 months, p = 0.049, HR = 0.40; 95% CI: 0.16-0.99). Compared with the control group, 1- and 2-year RFS rates were higher in the Tcm group (72.0% vs. 46.4% and 58.18% vs. 39.14%, respectively). Multivariate analysis did not indicate that Tcm treatment was an independent prognostic factor associated with HCC recurrence (p = 0.107, HR = 2.312; 95% CI: 0.835-6.400), which might be due to the small sample size of this study. Nevertheless, Tcm treatment effectively improved a reduced QOL due to HCC and liver function injury. Finally, the safety profile of Tcm treatment in this study was good, without any serious adverse events. CONCLUSIONS: This pilot study showed that Tcm self-transfusion combined with TACE treatment might be a beneficial adjuvant therapy with good safety for primary HCC patients with MVI after curative hepatectomy. TRIAL REGISTRATION NUMBER: NCT03575806.

Difference of physiological characters in dark green islands and yellow leaf tissue of Cucumber mosaic virus (CMV)-infected Nicotiana tabacum leaves.

Dark green islands (DGIs) are a common symptom of plants systemically infected with the mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow leaf tissue that is full of virus particles. In Cucumber mosaic virus (CMV)-infected Nicotiana tabacum leaves, the respiration and photosynthesis capabilities of DGIs and yellow leaf tissues were measured. The results showed that the cyanide-resistant respiration was enhanced in yellow leaf tissue and the photosynthesis was declined, while in DGIs they were less affected. The activities of the oxygen-scavenging enzymes catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in infected leaves were significantly higher than those in the healthy leaves, and the enzyme activities in DGIs were always lower than in the yellow leaf tissues. Reactive oxygen species (ROS) staining showed that the hydrogen peroxide content in yellow leaf tissues was apparently higher than that in DGIs, while the superoxide content was on the contrary. Formation of DGIs may be a strategy of the host plants resistance to the CMV infection.

Succinylation-dependent mitochondrial translocation of PKM2 promotes cell survival in response to nutritional stress.

Tumor growth and progression is characteristically associated with the synergistic effects of uncontrolled cellular proliferation and cell survival under stress. Pyruvate kinase M2 (PKM2) contributes to both of these effects. However, the specific mechanism by which PKM2 promotes uncontrolled proliferation or cell survival under stress in different nutritional environments is unclear. We show that succinylation mediated mitochondrial translocation of PKM2 under glucose starvation plays a role in switching the cellular machinery from proliferation to cell survival mode and vice versa. Mitochondrial PKM2 inhibits ubiquitination-mediated degradation of voltage-dependent anion channel 3 (VDAC3) and increases mitochondrial permeability to generate more ATP for cell survival under nutritional depletion. We found there is a positive correlation of upregulation of mitochondrial PKM2 and upregulation of VDAC3 in human colon cancer. This shows the mechanisms identified in this study in fact play a role in neoplastic biology. We therefore developed a small molecule designated compound 8 that blocks mitochondrial translocation of PKM2 and inhibits tumor development. Our data suggest that blocking PKM2 mitochondrial function with a small molecule inhibitor has potential for cancer treatment.

Anti-cancer effect of a novel 2,3-didithiocarbamate-substituted naphthoquinone as a tumor metabolic suppressor in vitro and in vivo.

Tumor cells reprogram their cellular metabolism by switching from oxidative phosphorylation to aerobic glycolysis to support aberrant cell proliferation. Suppressing tumor cell metabolism has become an attractive strategy for treating cancer patients. In this study, we identified a 2,3-didithiocarbamate-substituted naphthoquinone 3i that inhibited the proliferation of tumor cells by disturbing their metabolism. Compound 3i reduced cancer cell viability with IC50 values from 50 nM to 150 nM against HCT116, MCF7, MDA-MB231, HeLa, H1299 and B16 cells. Further, compound 3i was found to suppress ATP production in cultured cancer cells, inhibit the M2 isoform of pyruvate kinase (PKM2) which is a rate-limiting enzyme in the glycolytic pathway and block the subsequent transcription of the downstream genes GLUT1, LDH and CCND1. In addition, exposure to compound 3i significantly suppressed tumor growth in a B16 melanoma transplantation mouse model and a spontaneous breast carcinoma mouse model in vivo. The identification of compound 3i as a tumor metabolic suppressor not only offers a candidate compound for cancer therapy, but also provides a tool for an in-depth study of tumor metabolism.

Discovery of novel naphthoquinone derivatives as inhibitors of the tumor cell specific M2 isoform of pyruvate kinase.

Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme of the glycolytic pathway which is highly expressed in cancer cells. Cancer cells rely heavily on PKM2 for anabolic and energy requirements, and specific targeting of PKM2 therefore has potential as strategy for cancer therapy. Here, we report the synthesis and biologic evaluation of novel naphthoquinone derivatives as selective small molecule inhibitors of PKM2. Some target compounds, such as compound 3k, displayed more potent PKM2 inhibitory activity than the reported optimal PKM2 inhibitor shikonin. The well performing compound 3k also showed nanomolar antiproliferative activity toward a series of cancer cell lines with high expression of PKM2 including HCT116, Hela and H1299 with IC50 values ranging from 0.18 to 1.56 µM. Moreover, compound 3k exhibited more cytotoxicity on cancer cells than normal cells. The identification of novel potent small molecule inhibitors of PKM2 not only offers candidate compounds for cancer therapy, but also provides a tool with which to evaluate the function of PKM2 in depth.

PTENα Modulates CaMKII Signaling and Controls Contextual Fear Memory and Spatial Learning.

PTEN (phosphatase and tensin homology deleted on chromosome 10) has multiple functions, and recent studies have shown that the PTEN family has isoforms. The roles of these PTEN family members in biologic activities warrant specific evaluation. Here, we show that PTENα maintains CaMKII in a state that is competent to induce long-term potentiation (LTP) with resultant regulation of contextual fear memory and spatial learning. PTENα binds to CaMKII with its distinctive N terminus and resets CaMKII to an activatable state by dephosphorylating it at sites T305/306. Loss of PTENα impedes the interaction of CaMKII and NR2B, leading to defects in hippocampal LTP, fear-conditioned memory, and spatial learning. Restoration of PTENα in the hippocampus of PTENα-deficient mice rescues learning deficits through regulation of CaMKII. CaMKII mutations in dementia patients inhibit CaMKII activity and result in disruption of PTENα-CaMKII-NR2B signaling. We propose that CaMKII is a target of PTENα phosphatase and that PTENα is an essential element in the molecular regulation of neural activity.