Correlation between RNA N6-methyladenosine and ferroptosis in cancer: current status and prospects.

N6-methyladenosine (m6A) is the most abundant chemical modification in eukaryotic cells. It is a post-transcriptional modification of mRNA, a dynamic reversible process catalyzed by methyltransferase, demethylase, and binding proteins. Ferroptosis, a unique iron-dependent cell death, is regulated by various cell metabolic events, including many disease-related signaling pathways. And different ferroptosis inducers or inhibitors have been identified that can induce or inhibit the onset of ferroptosis through various targets and mechanisms. They have potential clinical value in the treatment of diverse diseases. Until now, it has been shown that in several cancer diseases m6A can be involved in the regulation of ferroptosis, which can impact subsequent treatment. This paper focuses on the concept, function, and biological role of m6A methylation modification and the interaction between m6A and ferroptosis, to provide new therapeutic strategies for treating malignant diseases and protecting the organism by targeting m6A to regulate ferroptosis.

Solamargine improves the therapeutic efficacy of anti-PD-L1 in lung adenocarcinoma by inhibiting STAT1 activation.

OBJECTIVE: The effect of solamargine on lung adenocarcinoma and its effect on STAT1 signaling pathway mediated immune escape were studied through network pharmacology and in vitro and in vivo experiments. METHODS: The solamargine targets were screened using the TCMSP and the LUAD targets were screened using the GeneCard, OMIM, PharmGkb, TTD and DrugBank databases. PPI network analysis and target prediction were performed using GO and KEGG. Colony formation assay, EDU staining, wound healing, transwell assay, Hoechst and flow cytometry were used to detect the effects of solamargine on the proliferation, migration and apoptosis of LUAD. Western blotting (WB) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to detect P-STAT1 and PD-L1 expression. And immunofluorescence was used to detect P-STAT1 expression. In vivo experiments, C57BL/6 mice were divided into control group, low concentration group, high concentration group, positive control group and combination group. Every other day, following seven consecutive doses, the size of the tumor was assessed. Finally, the expressions of P-STAT1, STAT1, PD-L1 and apoptosis index proteins were detected by WB. RESULTS: The anti-LUAD effect of solamargine was found by wound healing, colony formation assay, transwell assay, hoechst and EdU staining. The results of network pharmacological analysis showed that solamargine could suppress STAT1 expression level. Further enrichment assay of STAT1 showed that STAT1 was associated with immune-related pathways. In addition, molecular signal analysis by WB and RT-qPCR indicated that solamargine could reduce the expression levels of P-STAT1 and PD-L1 in a concentration-dependent manner. According to the results of in vivo assays, combination of solamargine and immune checkpoint inhibitors (ICIs) durvalumab could significantly inhibit the growth of Lewis transplanted tumors in C57BL/6 mice, and no toxic side effect was recoded. CONCLUSION: These results indicated that solamargine could inhibit the proliferation and promote the apoptosis of LUAD. It also could reduce the expression level of P-STAT1 protein and inhibit the expression level of PD-L1. At the same time, the combination with the ICIs can better block the expression of PD-L1 in cells, thereby inhibiting the immune escape pathway of tumor cells and achieving anti-tumor effects. This study proposed a novel combined therapeutic approach, involving the inhibition of STAT1 by solamargine in conjunction with ICIs.

The ratio of alpha-calcitonin gene-related peptide to substance P is associated with the transition of bone metabolic states during aging and healing.

Alpha-calcitonin gene-related peptide (αCGRP) and substance P (SP) are functionally correlated sensory neuropeptides deeply involved in bone homeostasis. However, they are usually studied individually rather than as an organic whole. To figure out whether they are interdependent, we firstly recorded the real-time αCGRP and SP levels in aging bone and healing fracture, which revealed a moderate to high level of αCGRP coupled with a low αCGRP/SP ratio in an anabolic state, and a high level of αCGRP coupled with a high αCGRP/SP ratio in a catabolic state, suggesting the importance of αCGRP/SP ratio in driving aging and healing scenarios. During facture healing, increase in αCGRP/SP ratio by adding αCGRP led to better callus formation and faster callus remodeling, while simultaneous addition of αCGRP and SP resulted in hypertrophic callus and delayed remodeling. The characteristics in inflammation and osteoclast activation further confirmed the importance of high αCGRP/SP ratio during catabolic bone remodeling. In vitro assays using different mixtures of αCGRP-SP proved that the osteogenic potential of the mixtures depended mostly on αCGRP, while their effects on osteoclasts and neutrophils relied on both peptides. These results demonstrated that αCGRP and SP were spatiotemporally interdependent. The αCGRP/SP ratio may be more important than the dose of a single neuropeptide in managing age-related and trauma-related bone diseases.

Comprehensive analysis of cuproptosis-related genes in immune infiltration and prognosis in lung adenocarcinoma.

Copper-dependent cell death, called cuproptosis, is connected to tumor development, prognosis, and the immune response. Nevertheless, the function of cuproptosis-related genes (CRGs) in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD) remains unknown. This work used R software packages to classify the raw data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases of LUAD patients. Afterward, the connections of the various subgroups, clinical pathological traits, and immune infiltration (IMIF) features with the TME mutation status were explored. Ultimately, a nomogram and calibration curve were developed, aiming at enhancing the clinical application of CRG scores and estimating the survival probability of patients. Moreover, the relationships between cuproptosis and the molecular traits, immune cell infiltration of tumor tissue, prognosis, and clinical treatment of patients were investigated in this work. Subsequently, the CRG score was established to predict overall survival (OS), and its credible predictive ability in LUAD patients was identified. Afterward, a highly credible nomogram was created to contribute to the clinical viability of the CRG score. Furthermore, as demonstrated, gene signatures could be applied in assessing tumor immune cell infiltration, clinical traits, and prognosis. In addition, high tumor mutation burden, immunological activity, and significant survival probability were characterized by low CRG scores, and high CRG scores were related to immunosuppression and stromal pathway activation. The current work also discovered a predictive CRG-related signature for LUAD patients, probably contributing to TME trait clarification and more potent immunotherapy strategy exploration.

GPR37 promotes cancer growth by binding to CDK6 and represents a new theranostic target in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is associated with poor prognosis. Identifying novel cancer targets and helpful therapeutic strategies remains a serious clinical challenge. This study detected differentially expressed genes in The Cancer Genome Atlas (TCGA) LUAD data collection. We also identified a predictive DNA biomarker, G protein-coupled receptor 37 (GPR37), which was verified as a prognostic biomarker with a critical role in tumor progression. In human LUAD specimens and microarray analyses, we determined that GPR37 was significantly upregulated and associated with a poor prognosis. GPR37 downregulation markedly inhibited the proliferation and migration of LUAD both in vitro and in vivo. Mechanistically, GPR37 could bind to CDK6, thereby facilitating tumor progression in LUAD by inducing cell cycle arrest at the G1 phase. GPR37 also facilitates tumorigenesis in xenograft tumors in vivo. High-throughput screening for GPR37-targeted drugs was performed using the Natural Products Library, which revealed the potential of Hypocrellin B to inhibit GPR37 and cell growth in LUAD. We demonstrated that Hypocrellin B suppressed LUAD cell proliferation and migration both in vitro and in vivo via GPR37 inhibition. Collectively, our findings reveal the role of GPR37 in LUAD progression and migration and the potential of GPR37 as a target for the treatment of LUAD. Thus, the specific inhibition of GPR37 by the natural product Hypocrellin B may possess the potential for the treatment of LUAD.

Low-Intensity Pulsed Ultrasound Promotes Osteogenic Potential of iPSC-Derived MSCs but Fails to Simplify the iPSC-EB-MSC Differentiation Process.

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) are a promising cell source for bone tissue engineering. However, iMSCs have less osteogenic potential than BMSCs, and the classical iPSC-EB-iMSC process to derive iMSCs from iPSCs is too laborious as it involves multiple in vitro steps. Low-intensity pulsed ultrasound (LIPUS) is a safe therapeutic modality used to promote osteogenic differentiation of stem cells. Whether LIPUS can facilitate osteogenic differentiation of iMSCs and simplify the iPSC-EB-iMSC process is unknown. We stimulated iMSCs with LIPUS at different output intensities (20, 40, and 60 mW/cm2) and duty cycles (20, 50, and 80%). Results of ALP activity assay, osteogenic gene expression, and mineralization quantification demonstrated that LIPUS was able to promote osteogenic differentiation of iMSCs, and it worked best at the intensity of 40 mW/cm2 and the duty cycle of 50% (LIPUS40/50). The Wnt/ß-catenin signaling pathway was involved in LIPUS40/50-mediated osteogenesis. When cranial bone defects were implanted with iMSCs, LIPUS40/50 stimulation resulted in a significant higher new bone filling rate (72.63 ± 17.04)% than the non-stimulated ones (34.85 ± 4.53)%. Daily exposure to LIPUS40/50 may accelerate embryoid body (EB)-MSC transition, but it failed to drive iPSCs or EB cells to an osteogenic lineage directly. This study is the first to demonstrate the pro-osteogenic effect of LIPUS on iMSCs. Although LIPUS40/50 failed to simplify the classical iPSC-EB-MSC differentiation process, our preliminary results suggest that LIPUS with a more suitable parameter set may achieve the goal. LIPUS is a promising method to establish an efficient model for iPSC application.

Neutrophil extracellular traps induce a hypercoagulable state in glioma.

BACKGROUND: Venous thromboembolism (VTE) is one of the leading complications in glioma patients. Neutrophil extracellular traps (NETs) have been reported to play a critical role in the physiopathology of cancer. We aimed to investigate the presence and potential role of NETs in the hypercoagulable state in glioma patients. Moreover, we evaluated the interaction between NETs and endothelial cells (ECs) in glioma patients. METHODS: The plasma levels of NETs were detected by enzyme-linked immunosorbent assay. The NET procoagulant activity was performed based on fibrin formation assays. The NET generation and NET-treated ECs in vitro were observed by confocal microscopy. Activated platelets (PLTs) and PLT-neutrophil aggregates were detected by flow cytometry. RESULTS: Plasma NET markers were significantly higher in stage III/IV glioma patients than in stage I/II glioma patients and healthy subjects. PLTs from glioma patients tended to induce NET formation than those from healthy subjects. NETs contributed to the hypercoagulable state in glioma patients. After ECs were incubated with NETs isolated from stage III/IV glioma patients, they lost their intercellular connections and were converted into procoagulant phenotypes. Combining DNase I and activated protein C markedly decreased endothelial dysfunction. CONCLUSIONS: Our results showed the interaction between NETs and hypercoagulability in glioma patients. Targeting NETs may be a potential therapeutic and prevention direction for thrombotic complications in glioma patients.

Comparative efficacy and acceptability of psychosocial treatments for disruptive behaviour disorders in children and adolescents: study protocol for a systematic review and network meta-analysis.

INTRODUCTION: Disruptive behaviour disorders are common among children and adolescents, with negative impacts on the youths, their families and society. Although multiple psychosocial treatments are effective in decreasing the symptoms of disruptive behaviour disorders, comprehensive evidence regarding the comparative efficacy and acceptability between these treatments is still lacking. Therefore, we propose a systematic review and network meta-analysis, integrating both direct and indirect comparisons to obtain a hierarchy of treatment efficacy and acceptability. METHODS AND ANALYSIS: The present protocol will be reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols. Ten databases, including Web of Science, PubMed, PsycINFO, MEDLINE, APA PsycArticles, Psychology and Behavioral Sciences Collection, OpenDissertations, The Cochrane Library, Embase and CINAHL, will be searched from inception for randomised controlled trials of psychosocial treatments for children and adolescents with disruptive behaviour disorders, without restrictions on language, publication year and status. The primary outcomes will be efficacy at post-treatment (severity of disruptive behaviour disorders at post-treatment) and acceptability (dropout rate for any reason) of psychosocial treatments. The secondary outcomes will involve efficacy at follow-up, severity of internalising problems and improvement of social functioning. Two authors will independently conduct the study selection and data extraction, assess the risk of bias using the revised Cochrane Collaboration's Risk of Bias tool and evaluate the quality of the evidence using the Grading of Recommendations Assessment, Development and Evaluation framework to network meta-analysis. We will perform Bayesian network meta-analyses with a random effects model. Subgroup and sensitivity analyses will be performed to evaluate the robustness of the findings. ETHICS AND DISSEMINATION: The research does not require ethical approval. Results are planned to be published in journals or presented at conferences. The network meta-analysis will provide information on a hierarchy of treatment efficacy and acceptability and help make a clinical treatment choice. PROSPERO REGISTRATION NUMBER: CRD42020197448.

BMP2 immune complexes promote new bone formation by facilitating the direct contact between osteoclasts and osteoblasts.

BMP2 antibody is proposed as a promising replacement for rhBMP2 in bone tissue engineering. Although studies have demonstrated its osteoinductive efficacy, the underlying osteogenic mechanism and adverse reactions of specific BMP2 antibody are not clarified yet, making it difficult to optimize the antibody for future application. By establishing BMP2 immune complexes (BMP2-ICs) ex vivo, we were able to introduce BMP2-ICs directly in vivo and found that BMP2-ICs promoted bone formation while suppressing osteoclastogenesis. However, ex vivo osteoclastogenic assays showed that BMP2-ICs promoted osteoclastogenesis by binding FcγR and activating PLCγ2 phosphorylation. Given that BMP2-ICs react with osteoblast and osteoclast lineage cells by the conjugated BMP2 domain and the Fc domain respectively, we introduced BMP2-ICs into coculture system of the two lineage cells and found that BMP2-ICs promoted osteogenesis while suppressing osteoclastogenesis by facilitating osteoblast-osteoclast contact and activating the EphrinB2-EphB4 signaling. This bidirectional function of BMP2-ICs was reproduced in the cranial bone resorption model, where osteoblast and osteoclast lineage cells co-localized. This study excluded the hidden problem of osteoclast overactivation that usually comes with rhBMP2 and clarified the first evidence of the mechanism of antibody-mediated bone regeneration, suggesting BMP2-ICs may present a promising therapy for bone diseases related with disrupted osteoclast-osteoblast interaction.

Neutrophil extracellular traps induce thrombogenicity in severe carotid stenosis.

BACKGROUND: Severe carotid stenosis is a common cause of stroke. In addition, previous clinical studies revealed that patients symptomatic of carotid stenosis suffer from increased episodes of stroke compared with their asymptomatic counterparts. However, the mechanism underlying these differences in the recurrence of stroke remains unclear. OBJECTIVE: The present study aimed to evaluate the levels of neutrophil extracellular traps (NETs) in the plasma of patients with severe carotid stenosis and investigate whether NETs induced procoagulant activity (PCA) in severe carotid stenosis. The study also sought to investigate the interactions between platelets or endothelial cells (ECs) and NETs. METHODS: The levels of NETs in plasma were quantified using enzyme-linked immunosorbent assay (ELISA). In addition, NETting neutrophils and neutrophil-platelet aggregates were detected through flow cytometry. On the other hand, the morphology of NETs formation and endothelial cells were analyzed through confocal microscopy. Finally, the procoagulant activity (PCA) of NETs and endothelial cells were assessed through ELISA and fibrin formation. RESULTS: Patients with symptomatic carotid stenosis patients had significantly higher levels of NETs markers compared with their asymptomatic counterparts and healthy subjects. In addition, increased levels of neutrophil-platelet aggregates induced the generation of NETs in patients with symptomatic carotid stenosis. Moreover, NETs contributed to PCA through tissue factor (TF), in patients with carotid stenosis. Furthermore, NETs disrupted the endothelial barrier and converted endothelial cells (ECs) into PCA to enhance the PCA in patients with carotid stenosis. CONCLUSIONS: The current study revealed differences in the levels of NETs in the plasma of symptomatic and asymptomatic patients suffering from carotid stenosis. The study also uncovered the interaction between NETs and thrombogenicity in carotid stenosis. Therefore, inhibiting NETs may be a potential biomarker and therapeutic target for recurring stroke in severe carotid stenosis.

The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis.

Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.