Objective To investigate the role and mechanism of RNA-Specific adenosine deaminase 3 (Adar3) in regulating macrophage polarization during Coxsackievirus B3(CVB3)-induced viral myocarditis (VM). Methods Bone marrow-derived macrophages (BMDM) from mice were cultured in vitro and induced into M1/M2 macrophages using interferon-gamma (IFN-γ)/lipopolysaccharide (LPS) or interleukin 4 (IL-4), respectively. The mRNA expression levels of Adar1, Adar2, and Adar3 in each group of cells were assessed by real-time quantitative PCR (qRT-PCR). Specific siRNAs targeting the Adar3 gene were designed, synthesized, and transiently transfected into M2 macrophages. The mRNA levels of M2 polarization-related marker genes-including arginase 1 (Arg1), chitinase 3-like molecule 3 (YM1/Chi3l3), and resistin-like molecule alpha (RELMα/FIZZ1)-were detected by qRT-PCR. RNA sequencing was performed to analyze the signaling pathways affected by Adar3. The expression levels of Wnt/β-catenin signaling pathway were further validated using qRT-PCR and Western blot. The adeno-associated virus overexpressing Adar3 was designed, synthesized, and injected into mice via tail vein. Three weeks later, a myocarditis mouse model was established. After an additional week, the phenotype and function of cardiac macrophages, as well as multiple indicators of VM (including echocardiography, body weight, histopathology and serology) were examined. Additionally, the protein levels of the Wnt/β-catenin signaling pathway were assessed. Results Compared to M0-type macrophages, the expression level of Adar3 was significantly increased in M2-type macrophages. After transfection of Adar3 siRNA, the mRNA levels of Arg1, YM1 and FIZZ1 in M2 macrophages were downregulated. RNA sequencing revealed 149 upregulated genes and 349 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and subsequent validation experiments indicated that Adar3 modulated the Wnt/β-catenin signaling pathway. In vivo experiments demonstrated that Adar3 overexpression alleviated the cardiac dysfunction of VM mice. The proportion of M1 macrophages in the heart decreased, while the proportion of M2 macrophages increased. At the same time, the Adar3 overexpression activated the Wnt/β-catenin signaling pathway. Conclusion Adar3 promotes macrophage polarization toward the M2 phenotype by activating the Wnt/β-catenin signaling pathway, thereby alleviating VM.
Animals ; Adenosine Deaminase/metabolism* ; Macrophages/immunology* ; Wnt Signaling Pathway/genetics* ; Myocarditis/immunology* ; Mice ; Coxsackievirus Infections/metabolism* ; Male ; Mice, Inbred BALB C ; Enterovirus B, Human/physiology* ; beta Catenin/genetics*

Metastasis remains the primary cause of cancer-related mortality worldwide. Circulating tumor cells (CTCs) represent critical targets for metastasis prevention and treatment. Traditional Chinese medicine may prevent lung cancer metastasis through long-term intervention in CTC activity. Tiao-Shen-Zhi-Ai Formular (TSZAF) represents a Chinese medicine compound prescription utilized clinically for lung cancer treatment. This study combined three principal active ingredients from TSZAF into a novel TSZAF monomer combination (TSZAF mc) to investigate its anti-metastatic effects and mechanisms. TSZAF mc demonstrated significant inhibition of proliferation, migration, and invasion in CTC-TJH-01 and LLC cells, while inducing cellular apoptosis in vitro. Moreover, TSZAF mc substantially inhibited LLC cell growth and metastasis in vivo. Mechanistically, TAZSF mc significantly suppressed the Wnt/β-catenin signaling pathway and CXCL5 expression in lung cancer cells and tissues. Additionally, TAZSF mc notably reduced neutrophil infiltration in metastatic lesions. These findings indicate that TSZAF mc inhibits lung cancer growth and metastasis by suppressing the Wnt/β-catenin signaling pathway and reducing CXCL5 secretion, thereby decreasing neutrophil recruitment and infiltration. TSZAF mc demonstrates potential as an effective therapeutic agent for lung cancer metastasis.
Lung Neoplasms/genetics* ; Wnt Signaling Pathway/drug effects* ; Animals ; Humans ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Neoplasm Metastasis/prevention & control* ; Cell Proliferation/drug effects* ; Cell Line, Tumor ; Neutrophil Infiltration/drug effects* ; Down-Regulation/drug effects* ; Cell Movement/drug effects* ; beta Catenin/genetics* ; Apoptosis/drug effects* ; Mice, Inbred C57BL ; Male ; Neoplastic Cells, Circulating/drug effects*

Human cytomegalovirus (HCMV) poses a significant risk of neural damage during pregnancy. As the most prevalent intrauterine infectious agent in low- and middle-income countries, HCMV disrupts the development of neural stem cells, leading to fetal malformations and abnormal structural and physiological functions in the fetal brain. This review summarizes the current understanding of how HCMV infection dysregulates the Wnt signaling pathway to induce fetal malformations and discusses current management strategies.
Humans ; Cytomegalovirus Infections/virology* ; Wnt Signaling Pathway ; Pregnancy ; Female ; Cytomegalovirus/physiology* ; Pregnancy Complications, Infectious/virology* ; Congenital Abnormalities/virology* ; Animals

Formate is an important solar fuel, with large application potential in bioconversion. Especially, the win-win collaboration is achieved when formate is applied to the cultivation of microalgae, which combines the advantages from both artificial and natural photosynthesis. However, the inhibition of formate on the photosynthetic electron transport hinders the application of formate at high concentrations. The engineering or directed evolution of the regulation pathway is a case-by-case and time-consuming strategy. Here, we developed a new strategy by introducing a Stenotrophomonas sp. strain which was isolated and identified from the long-term self-evolution process of Chlamydomonas reinhardtii for adapting to high concentrations of formate. The co-culture with the strain or the fermentation broth relieved the inhibition of formate (50 mmol/L) on C. reinhardtii and promoted the growth of the microalga. Especially, the protein content increased significantly to nearly 50% of the dried weight. In addition, the co-culture also benefited the growth of both Chlorella pyrenoidesa and Synechocystis sp. PCC 6803 exposed to formate, which indicated broader applicability of this strategy. This strategy provides the opportunity to overcome the bottleneck in the formate-mediated artificial-natural hybrid photosynthesis and to aid the development of technologies for solar energy-driven production of bulk biomass, including proteins, by carbon dioxide reduction.
Photosynthesis/physiology* ; Formates/pharmacology* ; Stenotrophomonas/growth & development* ; Microalgae/metabolism* ; Chlamydomonas reinhardtii/growth & development*

Prunus mume is an ecologically and economically valuable plant with both medicinal and edible values. However, drought severely limits the promotion and cultivation of P. mume in the arid and semi-arid areas in northern China. In this study, we treated P. mume 'Meiren' with natural drought and then assessed photosynthetic and physiological indexes such as osmoregulatory substances, photosynthetic parameters, and antioxidant enzyme activities. Furthermore, we employed transcriptome sequencing to explore the internal regulatory mechanism of P. mume under drought stress. As the drought stress aggravated, the levels of chlorophyll a (Chla), chlorophyll b (Chlb), chlorophyll (a+b)[Chl(a+b)], and soluble protein (SP) in P. mume first elevated and then declined. The net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), maximum photochemical efficiency (Fv/Fm), effective photochemical quantum yield [Y(Ⅱ)], photochemical quenching (qP), and relative electron transport rate (ETR) all kept decreasing, while the levels of malondialdehyde, superoxide dismutase (SOD), peroxidase (POD), and osmoregulatory substances rose. Transcriptome sequencing revealed a total of 24 853 high-quality genes. Gene ontology (GO) enrichment showed that differentially expressed genes (DEGs) were the most under severe drought. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that the DEGs during the four drought periods were mainly involved in the biosynthesis of secondary metabolites, plant-pathogen interaction, plant hormone signal transduction, starch and sucrose metabolism, and mitogen-activated protein kinase signaling pathways. Furthermore, we identified 16 key genes associated with the drought tolerance of P. mume 'Meiren'. This study discovered that P. mume might up-regulate or down-regulate the expression of drought tolerance-related genes such as SUS, P5CS, LEA, SOD, POD, SOD1, TPPD, and TPPA via transcription factors like MYB, ERF, bHLH, NAC, and WRKY to promote the accumulation of osmoregulatory substances like sucrose and enhance the activities of antioxidant enzymes such as SOD and POD, thus reducing the harm of reactive oxygen species and protecting the structure and function of the membrane system under drought stress. The findings provide theoretical references for further exploration of candidate genes of P. mume in response to drought stress and breeding of drought-tolerant varieties.
Droughts ; Photosynthesis/physiology* ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics* ; Prunus/genetics* ; Chlorophyll/metabolism* ; Plant Proteins/genetics*

This study is designed to address the development, synthesis, and screening of non-animal-derived nanoantibody libraries. Furthermore, it seeks to elucidate the impact of framework region selection and complementarity-determining region (CDR) design on the characteristics of synthesized nanoantibody libraries. These investigations aim to establish a robust theoretical and technical foundation for enhancing the efficacy, diversity, and practical applicability of synthetic nanoantibody libraries. In this study, a new framework (IGHV3S65*01-IGHJ4*01) was identified based on the high-throughput sequencing results of natural nanobodies, and degenerate primers were designed based on the frequency of amino acids at each position in the complementarity-determining region (CDR) region to synthesize the coding fragments of nanobodies by overlap PCR. After 40 times of electro-transformation, a single-frame synthesized nanobody library (SS-Library) containing 6×10⁹ clones was obtained, and the titer of the library was demonstrated to be 10¹³ PFU/mL after rescue by the helper phage M13K07. Random 48 single colonies were picked for PCR, which revealed an insertion rate of 95.8%. Sanger sequencing results showed that 38 clones had complete sequences, none of which showed cysteines or stop codons, and no identical sequences appeared, suggesting that the library had higher diversity. The library was screened and validated with three antigens, including bovine serum albumin (BSA), acetylcholinesterase (AchE), and immunoglobulin G (IgG). Finally, 2 nanobodies against BSA, 10 against AchE, and 15 against IgG were obtained. One positive clone of each antigen was singled out for recombinant expression, and the results showed that all the three nanobodies were expressed in a soluble form. The binding activity of recombinantly expressed nanobodies was evaluated using indirect enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI). The results demonstrated that the anti-AChE and anti-IgG nanobodies exhibited specific binding to their respective antigens, with affinity constants (KD) of 294 nmol/L and 250 nmol/L, respectively. The nanobody synthetic library preparation method proposed in this study is simple and easy to use with low preference, and it is expected to be a universal nanobody discovery platform for the preparation and development of lead specific nanobodies.
Single-Domain Antibodies/biosynthesis* ; Peptide Library ; Complementarity Determining Regions/immunology* ; Animals

Microalgae possess high photosynthetic efficiency, robust adaptability, and substantial biomass, serving as excellent biological resources for large-scale cultivation. Malic enzyme (ME), a ubiquitous metabolic enzyme in living organisms, catalyzes the decarboxylation of malate to produce pyruvate, CO₂, and NAD(P)H, playing a role in multiple metabolic pathways including energy metabolism, photosynthesis, respiration, and biosynthesis. In this study, we identified the Scenedesmus quadricauda malic enzyme gene (SqME) and its biological functions, aiming to provide excellent target genes for the genetic improvement of higher plants. Based on the RNA-seq data from S. quadricauda under the biofilm cultivation mode with high CO₂ and light energy transfer efficiency and small water use, a highly expressed gene (SqME) functionally annotated as ME was cloned. The physicochemical properties of the SqME-encoded protein were systematically analyzed by bioinformatics tools. The subcellular localization of SqME was determined via transient transformation in Nicotiana benthamiana leaves. The biological functions of SqME were identified via genetic transformation in Nicotiana tabacum, and the potential of SqME in the genetic improvement of higher plants was evaluated. The ORF of SqME was 1 770 bp, encoding 590 amino acid residues, and the encoded protein was located in chloroplasts. SqME was a NADP-ME, with the typical structural characteristics of ME. The ME activity in the transgenic N. tabacum plant was 1.8 folds of that in the wild-type control. Heterologous expression of SqME increased the content of chlorophyll a, chlorophyll b, and total chlorophyll by 20.9%, 26.9%, and 25.2%, respectively, compared with the control. The transgenic tobacco leaves showed an increase of 54.0% in the fluorescence parameter NPQ and a decrease of 30.1% in Fo compared with the control. Moreover, the biomass, total lipids, and soluble sugars in the transgenic tobacco leaves enhanced by 20.5%, 25.7%, and 9.5%, respectively. On the contrary, the starch and protein content in the transgenic tobacco leaves decreased by 22.4% and 12.2%, respectively. Collectively, the SqME-encoded protein exhibited a strong enzymatic activity. Heterologous expressing of SqME could significantly enhance photosynthetic protection, photosynthesis, and biomass accumulation in the host. Additionally, SqME can facilitate carbon metabolism remodeling in the host, driving more carbon flux towards lipid synthesis. Therefore, SqME can be applied in the genetic improvement of higher plants for enhancing photosynthetic carbon fixation and lipid accumulation. These findings provide scientific references for mining of functional genes from S. quadricauda and application of these genes in the genetic engineering of higher plants.
Nicotiana/genetics* ; Photosynthesis/physiology* ; Malate Dehydrogenase/biosynthesis* ; Plant Leaves/genetics* ; Scenedesmus/enzymology* ; Carbon Cycle/genetics* ; Lipid Metabolism/genetics* ; Plants, Genetically Modified/metabolism*

Objective To investigate the effects of folate deficiency on changes in histone H3 lysine 4 (H3K4) mono-methylation (me1)-marked enhancers and the molecular mechanism underpinning the folate deficiency-induced neural tube defects (NTD). Methods Mouse embryonic stem cells (mESCs) were cultured in the folate-free DMEM medium (folate-deficient group) and the DMEM medium containing 4 mg/L folate (normal control group),respectively.Chromatin immunoprecipitation sequencing (ChIP-seq) was performed for H3K4me1. The mouse model of folate-induced NTD was established,and transcriptome sequencing (RNA-seq) was performed for the brain tissue of fetal mice to reveal the differential expression profiles.The results were validated through real-time quantitative polymerase chain reaction (RT-qPCR).The activity of the differential peak regions of H3K4me1 was verified through the luciferase reporter assay. Results The folate content in the mESCs cultured in the folate-free medium reduced compared with that in the normal control group (P=0.008).The H3K4me1-maked enhancers in the mESCs cultured in the folate-free medium induced significant changes in intronic regions,and these changes were concentrated in metabolic and energy metabolism processes (q=9.56×10^-48,P=1.28×10^-47).The differentially expressed genes harboring H3K4me1-marked enhancers in mESCs were mainly enriched in the Wnt signaling pathway (q=0.004,P=0.004 7).ChIP-qPCR results confirmed that H3K4me1 binding decreased in the differential peak regions of the Ldlrap1 gene (P=0.008),Camta1 gene (P=0.002),and Apc2 gene (P=0.012).The H3K4 demethylase inhibitor T-448 effectively reversed the H3K4me1 binding in the differential peak regions of the aforementioned genes (P=0.01).The results of RNA-seq for the brain tissue of NTD fetal mice showed significant enrichment of the differentially expressed genes in the Wnt signaling pathway (P=1.52×10^-5).The enrichment of differential peak regions of H3K4me1-marked enhancers in Apc2,Ldlrap1,and Camta1 genes in the brain tissue also showed significant changes.The differential peak region in Apc2 exhibited transcription factor activity (P=0.020). Conclusion Folate deficiency may affect changes in H3K4me1-marked enhancers to participate in the regulation of neural tube closure genes,thereby inducing the occurrence of NTD.
Neural Tube Defects/genetics* ; Animals ; Mice ; Folic Acid Deficiency/complications* ; Histones/metabolism* ; Folic Acid/metabolism* ; Methylation ; Mouse Embryonic Stem Cells/metabolism* ; Wnt Signaling Pathway ; Lysine/metabolism* ; Chromatin Immunoprecipitation Sequencing

OBJECTIVES: To investigate the role and mechanism of Brahma-related gene 1 (Brg1) in regulating the Wnt/β-catenin signaling pathway in a bronchopulmonary dysplasia (BPD) model. METHODS: Wild-type C57BL/6 and Brg1^f1/f1 mice were randomly divided into four groups: wild-type control, wild-type BPD, Brg1^f1/f1 control, and Brg1^f1/f1 BPD (n=5 each). Immortalized mouse pulmonary alveolar type 2 cells (imPAC2) were cultured, and Brg1 gene was knocked down using lentivirus transfection technology. Cells were divided into three groups: control, empty vector, and Brg1 knockdown. Hematoxylin and eosin staining and immunofluorescence were used to detect pathological changes in mouse lung tissue. Western blot and real-time fluorescent quantitative PCR were used to measure Brg1 protein and mRNA expression levels in mouse lung tissue. Western blot and immunofluorescence were used to detect the expression of homeodomain-containing protein homeobox (HOPX), surfactant protein C (SPC), and Wnt/β-catenin signaling pathway proteins in mouse lung tissue and imPAC2 cells. The CCK8 assay was used to assess the proliferation of imPAC2 cells, and co-immunoprecipitation was performed to verify the interaction between Brg1 and β-catenin proteins in imPAC2 cells. RESULTS: Compared to the Brg1^f1/f1 control group and wild-type BPD group, the Brg1^f1/f1 BPD group showed increased alveolar diameter and SPC protein expression, and decreased relative density of pulmonary vasculature and HOPX protein expression (P<0.05). Compared to the control group, the Brg1 knockdown group showed increased cell proliferation ability, protein expression levels of SPC, Wnt5a and β-catenin, and β-catenin protein fluorescence intensity, along with decreased HOPX protein expression (P<0.05). An interaction between Brg1 and β-catenin proteins was confirmed. CONCLUSIONS The Brg1 gene may promote the proliferation of alveolar type 2 epithelial cells by regulating the Wnt/β-catenin signaling pathway, thus influencing the occurrence and development of BPD.
Animals ; DNA Helicases/genetics* ; Transcription Factors/genetics* ; Wnt Signaling Pathway/physiology* ; Nuclear Proteins/genetics* ; Mice ; Bronchopulmonary Dysplasia/etiology* ; Mice, Inbred C57BL ; beta Catenin/physiology* ; Disease Models, Animal ; Cell Proliferation ; Lung/pathology* ; Male

OBJECTIVE: To investigate the effect of Arsenic trioxide (ATO) combined with Norcantharidin (NCTD) on the proliferation and apoptosis of Jurkat cells, and to evaluate its effect on the proliferation and apoptosis of acute T-lymphoblastic leukemia (T-ALL) based on the Wnt/β-catenin signaling pathway. METHODS: Jurkat cell lines were used as the study subjects and treated with different concentrations of ATO (0, 2, 4, 8, 16 μmol/L) and NCTD (0, 10, 25, 50, 100 μmol/L) for 72 hours, and the cell proliferation was detected by CCK-8. Meanwhile, flow cytometry was used to detect the apoptosis rate, EdU staining to detect cell proliferation viability, cell clone formation assay to assess cell cloning ability, Transwell assay to assess cell invasion ability, and Western blot to detect apoptosis and the expression of Wnt/β-catenin signaling pathway-related proteins. RESULTS: Compared with the control group, both ATO and NCTD effectively inhibited Jurkat cell proliferation when used alone, and the inhibition effect was more significant when used in combination ( P < 0.05). The combination significantly increased the apoptosis rate of Jurkat cells ( P < 0.05). Meanwhile, the combination significantly decreased the proliferation vitality and clone formation ability of the cells ( P < 0.05), and inhibited the invasion ability of Jurkat cells ( P < 0.05). Western blot analysis showed that the combination of ATO and NCTD significantly up-regulated the expression of pro-apoptotic proteins Bax and E-cadherin, and down-regulated the expression of anti-apoptotic proteins Bcl-2, c-myc and Cyclin D1 ( P < 0.05). CONCLUSION The combination of ATO and NCTD had a synergistic effect in inhibiting proliferation and promoting apoptosis in Jurkat cells, which may be related to the inhibition of Wnt/β-catenin signaling pathway.
Humans ; Apoptosis/drug effects* ; Jurkat Cells ; Cell Proliferation/drug effects* ; Arsenic Trioxide ; Wnt Signaling Pathway/drug effects* ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; beta Catenin/metabolism* ; Arsenicals/pharmacology* ; Oxides/pharmacology*