A novel protein encoded by circFOXP1 enhances ferroptosis and inhibits tumor recurrence in intrahepatic cholangiocarcinoma.

CircRNAs participates in the development and occurrence of multiple tumor types. However, the specific effects and underlying mechanisms of circRNA in intrahepatic cholangiocarcinoma (ICC) progression and recurrence remain poorly understood. CircRNA sequencing was performed to screen circRNAs related to ICC recurrence after surgery using 53 ICC frozen tumor specimens. We found that compared with patients who experienced postsurgical recurrence, circFOXP1 had high expression in tumor tissues from patients with no postoperative recurrence. Functional experiments revealed that circFOXP1 inhibited ICC progression in vitro and in vivo. We then found that circFOXP1 inhibited ICC progression via encoding a novel protein, circFOXP1-231aa. Mechanistically, circFOXP1-231aa directly interacted with OTUD4, which regulates NCOA4 protein stability via deubiquitination modification, and thereby enhances ferroptosis of ICC cells. Examination of clinical ICC samples found positive correlations between circFOXP1 expression levels and levels of OTUD4 and NCOA4. These three factors are predictors of prognosis in patients with ICC. Collectively, we identified circFOXP1 encoded circFOXP1-231aa, which interacted with OTUD4 to suppress ubiquitination of NCOA4 and, thereby, promoted ferroptosis and inhibited ICC recurrence.

Comparison of intranasal dexmedetomidine alone and dexmedetomidine-chloral hydrate combination sedation for electroencephalography in children: A large retrospective cohort study and propensity score-matched analysis.

Aim: To compare the safety and efficacy of intranasal high-dose dexmedetomidine (DEX) versus a combination of intranasal low-dose dexmedetomidine and oral chloral hydrate (DEX-CH) sedation during electroencephalography (EEG) in children. Methods: Unadjusted analysis, 1:1 propensity score matching (PSM), and inverse probability of treatment weighting (IPTW) were used to compare the sedation success rate, adverse effects, onset time, and recovery time of these two sedation methods for 6967 children who underwent EEG. Results: A total of 6967 children were enrolled in this study, of whom 846 (12.1 %) underwent DEX intranasal sedation while 6121 (87.9 %) received DEX-CH sedation. No significant differences were observed in the sedation success rate with the first dose between the two groups [824 (97.4 %) for DEX vs. 5971 (97.6 %) for DEX-CH; RR 0.99; 95 % CI, 0.98-1.01; P = 0.79]. Similarly, there were no notable disparities in the incidence of adverse events [16 (1.9 %) for DEX vs. 101 (1.7 %) for DEX-CH; RR 1.15; 95 % CI, 0.68-1.93; P = 0.32]. However, intranasal DEX sedation compared with DEX-CH sedation was associated with lower vomiting [0 vs. 95(1.6 %); RR 0.04; 95 % CI, 0.02-0.6; P = 0.02] or more bradycardia [13(1.5 %) vs. 2(0.03 %); RR 47.03; 95 % CI, 10.63-208.04; P < 0.001]. Multivariate analysis using PSM and IPTW analysis yielded similar results. Conclusion: Both methods for EEG had high sedation success rate and low incidence of adverse events. High-dose intranasal DEX was more likely to induce bradycardia and had a shorter recovery time than the DEX-CH sedation, which was more likely to induce vomiting.

Tumor-associated macrophage-induced circMRCKα encodes a peptide to promote glycolysis and progression in hepatocellular carcinoma.

The tumor-associated macrophages (TAMs) play multifaceted roles in the progression of hepatocellular carcinoma (HCC). However, the involvement of circular RNAs in the interplay between TAMs and HCC remains unclear. Based on Transwell co-culturing and circular RNA sequencing, this study revealed that TAMs enhanced tumor glycolysis and progression by upregulating circMRCKα in HCC cells. Patients with HCC who exhibited elevated circMRCKα levels presented significantly reduced overall survival and greater cumulative recurrence. Notably, we identified a novel functional peptide of 227 amino acids named circMRCKα-227aa, encoded by circMRCKα. Mechanistically, circMRCKα-227aa bound to USP22 and enhanced its protein level to obstruct HIF-1α degradation via the ubiquitin-proteasome pathway, thereby augmenting HCC glycolysis and progression. In clinical HCC samples, a positive correlation was observed between the expression of circMRCKα and the number of infiltrating CD68+ TAMs and expression of USP22. Furthermore, circMRCKα emerged as an independent prognostic risk factor both individually and in conjunction with CD68+ TAMs and USP22. This study illustrated that circMRCKα-227aa, a novel TAM-induced peptide, promotes tumor glycolysis and progression via USP22 binding and HIF-1α upregulation, suggesting that circMRCKα and TAMs could be combined as therapeutic targets in HCC.

Tunable rigidity of PLGA shell-lipid core nanoparticles for enhanced pulmonary siRNA delivery in 2D and 3D lung cancer cell models.

Faced with the threat of lung cancer-related deaths worldwide, small interfering RNA (siRNA) can silence tumor related messenger RNA (mRNA) to tackle the issue of drug resistance with enhanced anti-tumor effects. However, how to increase lung tumor targeting and penetration with enhanced gene silencing are the issues to be addressed. Thus, the objective of this study is to explore the feasibility of designing non-viral siRNA vectors for enhanced lung tumor therapy via inhalation. Here, shell-core based polymer-lipid hybrid nanoparticles (HNPs) were prepared via microfluidics by coating PLGA on siRNA-loaded cationic liposomes (Lipoplexes). Transmission electron microscopy and energy dispersive spectroscopy study demonstrated that HNP consists of a PLGA shell and a lipid core. Atomic force microscopy study indicated that the rigidity of HNPs could be well tuned by changing thickness of the PLGA shell. The designed HNPs were muco-inert with increased stability in mucus and BALF, good safety, enhanced mucus penetration and cellular uptake. Crucially, HNP1 with the thinnest PLGA shell exhibited superior transfection efficiency (84.83%) in A549 cells, which was comparable to that of lipoplexes and Lipofectamine 2000, and its tumor permeability was 1.88 times that of lipoplexes in A549-3T3 tumor spheroids. After internalization of the HNPs, not only endosomal escape but also lysosomal exocytosis was observed. The transfection efficiency of HNP1 (39.33%) was 2.26 times that of lipoplexes in A549-3T3 tumor spheroids. Moreover, HNPs exhibited excellent stability during nebulization via soft mist inhaler. In conclusion, our study reveals the great potential of HNP1 in siRNA delivery for lung cancer therapy via inhalation.

Challenges and Strategies to Enhance the Systemic Absorption of Inhaled Peptides and Proteins.

Proteins and peptides-based therapeutics are making substantial access to the market due to their obvious advantages of strong potency, high specificity and desirable safety profile. However, most clinical products are mainly delivered via parenteral route with inferior convenience. Lung is an attractive non-invasive alternative passage for systemic administration of biologics with numerous outstanding features, as examples of large absorptive surface area, extensive vascularization and mild local microenvironment. Even so, mucociliary clearance, alveolar macrophage phagocytosis, enzymatic metabolism, pulmonary surfactant adsorption and limited epithelium permeability constitute major obstacles affecting the systemic absorption of inhaled proteins and peptides. This article begins by giving a brief overview of challenges for the systemic absorption of inhaled proteins and peptides, and then goes on to a comprehensive review of possible strategies for enhanced pulmonary absorption, including chemical modification, addition of protease inhibitors, incorporation of absorption enhancers, modification with fusion proteins and development of particulate-based drug delivery systems. These strategies can provide enhanced transmembrane absorption capacity while avoiding pulmonary clearance, offering a valuable reference for designing pulmonary delivery systems of protein and peptide drugs.

Mechanisms and challenges of nanocarriers as non-viral vectors of therapeutic genes for enhanced pulmonary delivery.

With the rapid development of biopharmaceuticals and the outbreak of COVID-19, the world has ushered in a frenzy to develop gene therapy. Therefore, therapeutic genes have received enormous attention. However, due to the extreme instability and low intracellular gene expression of naked genes, specific vectors are required. Viral vectors are widely used attributed to their high transfection efficiency. However, due to the safety concerns of viral vectors, nanotechnology-based non-viral vectors have attracted extensive investigation. Still, issues of low transfection efficiency and poor tissue targeting of non-viral vectors need to be addressed. Especially, pulmonary gene delivery has obvious advantages for the treatment of inherited lung diseases, lung cancer, and viral pneumonia, which can not only enhance lung targeting and but also reduce enzymatic degradation. For systemic diseases therapy, pulmonary gene delivery can enhance vaccine efficacy via inducing not only cellular, humoral immunity but also mucosal immunity. This review provides a comprehensive overview of nanocarriers as non-viral vectors of therapeutic genes for enhanced pulmonary delivery. First of all, the characteristics and therapeutic mechanism of DNA, mRNA, and siRNA are provided. Thereafter, the advantages and challenges of pulmonary gene delivery in exerting local and systemic effects are discussed. Then, the inhalation dosage forms for nanoparticle-based drug delivery systems are introduced. Moreover, a series of materials used as nanocarriers for pulmonary gene delivery are presented, and the endosomal escape mechanisms of nanocarriers based on different materials are explored. The application of various non-viral vectors for pulmonary gene delivery are summarized in detail, with the perspectives of nano-vectors for pulmonary gene delivery.

Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like "off-target" toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment. METHODS: The peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups. RESULTS: The mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal "off-target" combination existed on CoG133-CAR T cells and major organs. CONCLUSION: Our study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.

Folate Receptor-Alpha Targeted 7x19 CAR-γδT Suppressed Triple-Negative Breast Cancer Xenograft Model in Mice.

Background: Triple-negative breast cancer (TNBC) is the worst prognosis subtype of breast cancer due to lack of specific targets. Recent studies have shown that immunotherapy may solve that problem by targeting folate receptor-alpha (FRα). Methods: Gene modified γδ T cells were manufactured to express FRa specific chimeric antigen receptor (FRa CAR) and secrete interleukin-7 (IL-7) and chemokine C-C motif ligand 19 (CCL19). CAR-γδT cells that secrete IL-7 and CCL19 (7 × 19 CAR-γδT) were evaluated for their antitumor activity both in vitro and in vivo. Results: 7 × 19 CAR-γδT showed remarkable antitumor activity in vitro. Combined with PBMC, 7 × 19 CAR-γδT inhibited TNBC xenograft model growth superiorly compared with single-application or conventional CAR-γδT cells. Histopathological analyses showed increased DC or T cells infiltration to tumor tissues. Conclusion: Taken together, our results showed that 7 × 19 CAR-γδT have remarkable anti-TNBC tumor activity and showed a broad application prospect in the treatment of incurable TNBC patients.

Minicircle DNA-Mediated CAR T Cells Targeting CD44 Suppressed Hepatocellular Carcinoma Both in vitro and in vivo.

PURPOSE: Based on the continuous exploration of solid tumor immunotherapy, we focused on hepatocellular carcinoma with a high level of morbidity and mortality. We confirm the stability of mcDNA-based CAR T cell generating platform, and investigate the antitumor activity of CD44-CAR T cells against hepatocellular carcinoma both in vitro and in vivo. MATERIALS AND METHODS: We fused anti-CD44 scFv structure with transmembrane domain and intracellular domain. Using a non-viral mcDNA vector to load CD44-CAR gene, then transfected the mcDNA-CD44-CAR into human T cells by electroporation. We exhibited the transfection efficacy of CAR T cells and the CD44 expression of tumor cell lines by flow cytometry. The antitumor efficacy of CD44-CAR T cells in vitro and in vivo was detected through CCK-8 and ELISA assays, and xenograft mouse models, respectively. RESULTS: We obtained mcDNA-CD44-CAR with a high level of density after repeated extraction and purification. The expression efficacy of CD44-CAR in T cells was more than 50% after seven days electroporation and the phenotype of CD44-CAR T cells was no difference compared with normal T cells. For CD44-positive hepatocellular carcinoma xenograft mice, CD44-CAR T cells had stronger tumor growth suppression compared to normal T and mock T cells. The same results occurred on the in vitro experiments including cytokine secretion and cytotoxicity assays. H&E staining graphs revealed that CD44-CAR T cells did not induce side effects in xenograft mice. CONCLUSION: The strategy for generating CAR T cells targeting cancer stem cell antigens was efficient and concise. The mcDNA had superior transgene ability without virus-related adverse effects. CD44-CAR T cells had strong suppression capacity against hepatocellular carcinoma.

Resveratrol Prevents Cognitive Impairment in Type 2 Diabetic Mice by Upregulating Nrf2 Expression and Transcriptional Level.

PURPOSE: This study aimed to determine whether the natural antioxidant resveratrol (RSV) prevents type 2 diabetes mellitus (T2DM)-induced cognitive impairment and to explore whether redox-associated factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in the neuroprotective effect of RSV. MATERIALS AND METHODS: We established a T2DM model with 8-week-old male ICR mice by administration of a high-fat diet for 2 months and low-dose streptozotocin for 3 days. Then, diabetic and age-matched control mice were treated with or without RSV for 4 months every other day and subjected to the Morris water maze test. After the mice were euthanized, whole brains were sectioned for Nissl staining and immunofluorescence labeling. Hippocampal sections were observed by transmission electron microscopy to evaluate the ultrastructure of synapses. Inflammatory factors, oxidative stress-related indexes, and Nrf2 and downstream target gene expression were analyzed in hippocampal tissues by quantitative real-time PCR, Western blotting, and associated quantitative kits. RESULTS: In the Morris water maze test, compared to control mice, T2DM mice showed learning and memory impairments, but RSV treatment prevented the learning and memory decline in T2DM mice. Similarly, RSV prevented T2DM-induced hippocampal neuron destruction and synaptic ultrastructural damage. The expression levels of inflammatory factors and oxidative stress-related indicators were increased in the T2DM group compared with the control group but were decreased significantly by RSV treatment in the T2DM group. Additionally, the expression of Nrf2 and its downstream target genes was decreased in the T2DM group compared with the control group and was significantly increased by RSV treatment in the T2DM group. CONCLUSION: RSV prevented T2DM-induced cognitive impairment through anti-inflammatory and antioxidant activities. This effect was accompanied by the upregulation of Nrf2 transcriptional activity and the increased expression of downstream antioxidant genes.

Serum Liver Fibrosis Markers in the Prognosis of Liver Cirrhosis: A Prospective Observational Study.

BACKGROUND The prognostic role of serum liver fibrosis markers in cirrhotic patients remains unclear. We performed a prospective observational study to evaluate the effect of amino-terminal pro-peptide of type III pro-collagen (PIIINP), collagen IV (CIV), laminin (LN), and hyaluronic acid (HA) on the prognosis of liver cirrhosis. MATERIAL AND METHODS All patients who were diagnosed with liver cirrhosis and admitted to our department were prospectively enrolled. PIIINP, CIV, LN, and HA levels were tested. RESULTS Overall, 108 cirrhotic patients were included. Correlation analysis demonstrated that CIV (coefficient r: 0.658, p<0.001; coefficient r: 0.368, p<0.001), LN (coefficient r: 0.450, p<0.001; coefficient r: 0.343, p<0.001), and HA (coefficient r: 0.325, p=0.001; coefficient r: 0.282, p=0.004) levels, but not PIIINP level (coefficient r: 0.081, p=0.414; coefficient r: 0.090, p=0.363), significantly correlated with Child-Pugh and MELD scores. Logistic regression analysis demonstrated that HA (odds ratio=1.00003, 95% confidence interval [CI]=1.000004-1.000056, p=0.022) was significantly associated with the 6-month mortality. Receiver operating characteristics analysis demonstrated that the area under the curve (AUC) of HA for predicting the 6-month mortality was 0.612 (95%CI=0.508-0.709, p=0.1531). CONCLUSIONS CIV, LN, and HA levels were significantly associated with the severity of liver dysfunction, but might be inappropriate for the prognostic assessment of liver cirrhosis.

[Compatible biomass models for main tree species with measurement error in Heilongjiang Province of Northeast China].

Based on the biomass data of 516 sampling trees, and by using non-linear error-in-variable modeling approach, the compatible models for the total biomass and the biomass of six components including aboveground part, underground part, stem, crown, branch, and foliage of 15 major tree species (or groups) in Heilongjiang Province were established, and the best models for the total biomass and components biomass were selected. The compatible models based on total biomass were developed by adopting the method of joint control different level ratio function. The heteroscedasticity of the models for total biomass was eliminated with log transformation, and the weighted regression was applied to the models for each individual component. Among the compatible biomass models established for the 15 major species (or groups) , the model for total biomass had the highest prediction precision (90% or more), followed by the models for aboveground part and stem biomass, with a precision of 87.5% or more. The prediction precision of the biomass models for other components was relatively low, but it was still greater than 80% for most test tree species. The modeling efficiency (EF) values of the total, aboveground part, and stem biomass models for all the tree species (or groups) were over 0.9, and the EF values of the underground part, crown, branch, and foliage biomass models were over 0.8.