The neuronal PAS domain 3 (NPAS3) is a member of the basic helix-loop-helix (bHLH) PAS family of transcription factors and is implicated in psychiatric and neurodevelopmental disorders. NPAS3 is robustly expressed in the cortical ventricle zone (VZ), a transient proliferative zone containing progenitor cells, mainly radial glial cells, destined to give rise to cortical excitatory neurons. However, the role of NPAS3 in corticogenesis remains largely unknown. In this study, we knocked down Npas3 expression in the neural progenitor cells residing in the cortical VZ to investigate the role of Npas3 in cerebral cortical development in mice. We demonstrated that Npas3 knockdown profoundly impaired neuronal radial migration and changed the laminar cell fate of the cells detained in the deep cortical layers. Furthermore, the downregulation of Npas3 led to the stemness maintenance of radial glial cells and increased the proliferation rate of neural progenitor cells residing in the VZ/subventricular zone (SVZ). These findings underline the function of Npas3 in the development of the cerebral cortex and may shed light on the etiology of NPAS3-related disorders.

Background Neuroinflammation is involved in the mechanisms of levodopa-induced dyskinesia (LID). The canonical NF-κB activation signaling pathway plays a critical role in the neuroinflammation development and BET protein-induced NF-κB-mediated neuroinflammation. The inhibition of the BET protein function has been reported to alleviate LID; however, its association with the canonical NF-κB signaling pathway in the 6-OHDA-lesioned striatum of the LID rat model remains unknown. Accordingly, we identified the status of the canonical NF-κB signaling pathway in the 6-OHDA-lesioned striatum of the LID rat model and whether the anti-dyskinetic effect of the BET inhibitor JQ1 was associated with its suppression on NF-κB-mediated neuroinflammation. Methods 6-OHDA PD rat models were treated with either L-dopa plus JQ1 or L-dopa alone. L-dopa treatment was given for 2 weeks, and the JQ1 treatment was given for 3 weeks and was initiated a week prior to L-dopa treatment. As a control, the sham rats were treated with JQ1 or Veh for 3 weeks. The ALO AIM assessment and cylinder test were performed during the treatment. Glial activation markers, pro-inflammatory substances, and critical proteins in the canonical NF-κB signaling pathway were tested in the lesioned striatum after the final treatment. Results JQ1 effectively alleviated LID without influencing motor improvement. In the lesioned striatum, L-dopa triggered an overactivation of the canonical NF-κB signaling pathway, with an increase in the phospho-IKKα/β, phospho-IκBα, and NF-κB nuclear translocation and its phosphorylation at Ser 536 and Ser 276 sites ( p < 0.01 vs. sham group). L-dopa induced an overexpression of the pro-inflammatory substances of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase (iNOS), and the glial activation markers CD68 and GFAP. All the molecular changes were greatly inhibited by JQ1. Conclusion L-dopa triggered an overactivation of the canonical NF-κB signaling pathway, leading to an enhanced neuroinflammation response in the 6-OHDA-lesioned striatum of LID rat models. The inhibition of the BET protein function significantly suppressed the activation of the canonical NF-κB signaling pathway in the striatum, alleviating the neuroinflammation response and the severity of LID.

Immunotherapy targeting checkpoint blockade to rescue T cells from exhaustion has become an essential therapeutic strategy in treating cancers. Till now, little is known about the PD-L1 graphic pattern and characteristics in CD8(+) T cells. We combined cytometry by time-of-flight (CyTOF) and imaging mass cytometry (IMC) approaches to analyze CD8(+) T cells from primary lung cancers and discovered that PD-L1(+)CD8(+) T cells were enriched in tumor lesions, spatially localized with PD-1+CD8(+) T cells. Furthermore, PD-L1(+)CD8(+) T cells exerted regulatory functions that inhibited CD8(+) T cells proliferation and cytotoxic abilities through the PD-L1/PD-1 axis. Moreover, tumor-derived IL-27 promotes PD-L1(+)CD8(+) T cells development through STAT1/STAT3 signaling. Single-cell RNA sequencing data analysis further clarified PD-L1(+)CD8(+) T cells elevated in the components related to downregulation of adaptive immune response. Collectively, our data demonstrated that PD-L1(+)CD8(+) T cells enriched in lung cancer engaged in tolerogenic effects and may become a therapeutic target in lung cancer.

Protein arginine methyltransferase 5 (PRMT5) participates in the symmetric dimethylation of arginine residues of proteins and contributes to a wide range of biological processes. However, how PRMT5 affects the transcriptional and epigenetic programs involved in the establishment and maintenance of T cell subset differentiation and roles in antitumor immunity is still incompletely understood. In this study, using single-cell RNA and chromatin immunoprecipitation sequencing, we found that mouse T cell-specific deletion of PRMT5 had greater effects on CD8(+) than CD4(+) T cell development, enforcing CD8(+) T cell differentiation into Klrg1(+) terminal effector cells. Mechanistically, T cell deficiency of PRMT5 activated Prdm1 by decreasing H4R3me2s and H3R8me2s deposition on its loci, which promoted the differentiation of Klrg1(+)CD8(+) T cells. Furthermore, effector CD8(+) T cells that transited to memory precursor cells were decreased in PRMT5-deficient T cells, thus causing dramatic CD8(+) T cell death. In addition, in a mouse lung cancer cell line-transplanted tumor mouse model, the percentage of CD8(+) T cells from T cell-specific deletion of PRMT5 mice was dramatically lost, but CD8(+)Foxp3(+) and CD8(+)PDL1(+) regulatory T cells were increased compared with the control group, thus accelerating tumor progression. We further verified these results in a mouse colon cancer cell line-transplanted tumor mouse model. Our study validated the importance of targeting PRMT5 in tumor treatment, because PRMT5 deficiency enforced Klrg1(+) terminal CD8(+) T cell development and eliminated antitumor activity.

Unlike intracellular chromosome, extracellular DNA (eDNA) may accelerate the spreading of antibiotic resistance genes (ARGs) through natural transformation, but one of the core issues regarding to the taxonomic characterization of eDNA in the complex water environments is largely unknown. Hence, Illumina Miseq sequencing was used to identify the genotype of eDNA from wastewater (WW), river water (RW) and stormwater (SW) runoff. High-throughput qPCR targeting 384 genes was implemented to detect extracellular ARGs (eARGs) and mobile genetic elements (eMGEs). We obtained 2,708,291 high quality sequences from 66 eDNA samples. The SW exhibited the significant higher Shannon Index. Subsequently, we identified 34 core bacteria sources of eDNA widely distributed in the three water compartments. Among which, Pseudomonas, Flavobacterium, Limnohabitans, Burkholderiaceae_unclassified, Methylotenera and Acinetobacter were the most prevalent. A total of 302 eARGs and eMGEs were detected, suggesting that eDNA is an important antibiotic resistance reservoir. Among the 127 shared genes of the three groups, 15 core resistance genes were filtered, including IS6100, sul1 NEW, intI1, ISPps1-pseud, aac3-Via, qacH_351 and ISSm2-Xanthob. The Procrustes analysis and Variance Partitioning Analysis (VPA) demonstrated that core bacteria and MGEs were significantly correlated with eARGs. These results suggested that the occurrence and changes of eARGs in the water compartments may be largely attributed to the core microbiota and eMGEs.

Untreated rural sewage seriously affects the universal access to clean water of rural residents. The lack of decision-support tools in rural sewage treatment (RuST) planning makes it difficult for RuST system to achieve the expected results and is not conducive to the optimal allocation of limited funds. Hence, there is an urgent need to develop a decision-support framework for large-scale RuST planning. For the first time, RuST planning decision-support framework was developed using divide-and-conquer strategy based on rural residents' spatial pattern (RESP) and the optimal pattern of RuST. This framework can be transferred to other countries/regions easily by correcting RESP dataset according to the spatial and environmental characteristics. We confirmed that the variation of RESP made the ideal RuST pattern varied significantly under different topography. And community-based pattern could be the optimal pattern for large-scale RuST planning, when spatial obstacle and RESP were fully considered. The price of onsite sewage treatment facility is the most significant factor for RuST planning. In our selected case, requited onsite facility accounted for 65.51%. For the total investment, the cost of sewer systems accounted for 56.01%, and the average investment in plains, hills, platforms and mountains was 1401, 1803, 1903 and 1859 USD/household, respectively. We expect this research could provide reference for RuST planning in other developing countries/regions all around the world.

Granulocytes play important roles in cancer, and their apoptotic status is often changed by the influence of tumor environment. However, the changes and the function on granulocyte apoptosis in cancer are unclear. In this study, we used tumor-bearing mouse model and tumor patients to analyzed the apoptosis of granulocytes in different tissues by flow analysis and TUNEL fluorescence staining, and found that the percentage of apoptosis cells in granulocytes was significantly decreased in late-stage tumor-bearing mouse and patients. The in vitro co-culture experiment showed that these antiapoptotic granulocytes could significantly inhibit T cell proliferation, and RNA-seq proved that there was obvious difference on the transcriptome between these cells and control cells, particularly immune-related genes. What is important, adoptive transfer of these antiapoptotic granulocytes promoted tumor progress in mouse model. Conclusively, we found that granulocytes in late-stage tumor could delay the process of apoptosis, inhibit T cell proliferation, and acquire pro-tumor activity, which provides a new therapeutic target for tumor immunity.

Proinflammatory cytokine interleukin 32 (IL-32) is involved in infectious diseases and cancer, but what subtypes of immune cells express IL-32 and its roles in tumor microenvironment (TME) have not been well discussed. In this study, we applied bioinformatics to analyze single-cell RNA sequencing data about tumor-infiltrating immune cells from esophageal squamous cell carcinoma (ESCC) TME and analyzed IL-32 expression in different immune cell types. We found CD4 + regulatory T cells (Treg cells) express the highest level of IL-32, while proliferating T and natural killer cells expressed relatively lower levels. Knocking down of IL-32 reduced Foxp3 and interferon gamma (IFNγ) expressions in CD4 + and CD8 + T cells, respectively. IL-32 was positively correlated with Foxp3, IFNG, and GZMB expression but was negatively correlated with proliferation score. IL-32 may have a contradictory role in the TME such as it promotes IFNγ expression in CD8 + T cells, which enhances the antitumor activity, but at the same time induces Foxp3 expression in CD4 + T cells, which suppresses the tumor immune response. Our results demonstrate different roles of IL-32 in Treg cells and CD8 + T cells and suggest that it can potentially be a target for ESCC cancer immunosuppressive therapy.

Liver fibrosis is characterized by the transdifferentiation of hepatic stellate cells (HSCs) to myofibroblasts and poor response to treatment. This can be attributed to the myofibroblast-specific resistance to phenotype reversal. In this study, we complemented miR-16 into miR-16-deficient myofibroblasts and analyzed the global role of miR-16 using transcriptome profiling and generating a pathway-based action model underlying transcriptomic regulation. Phenotypic analysis of myofibroblasts and fibrogenic characterization were used to understand the effect of miR-16 on phenotypic remodeling of myofibroblasts. miR-16 expression altered the transcriptome of myofibroblasts to resemble that of HSCs. Simultaneous targeting of Smad2 and Wnt3a, etc. by miR-16 integrated signaling pathways of TGF-β and Wnt, etc., which underlay the comprehensive regulation of transcriptome. The synergistic effect of miR-16 on the signaling pathways abolished the phenotypic characteristics of myofibroblasts, including collagen production and inhibition of adipogenesis. In vivo, myofibroblast-specific expression of miR-16 not only eliminated mesenchymal cells with myofibroblast characteristics but also restored the phenotype of HSCs in perisinusoidal space. This phenotypic remodeling resolved liver fibrosis induced by chronic wound healing. Therefore, miR-16 may integrate signaling pathways crucial for the fate determination of myofibroblasts. Its global effect induces the reversal of HSC-to-myofibroblast transdifferentiation and, subsequently, the resolution of fibrogenesis. Taken together, these findings highlight the potential of miR-16 as a promising therapeutic target for liver fibrosis.

Although CD4(+)CD45RA(-)Foxp3(l)degrees cytokine-secreting T cells (Fr.III cells) have been reported to be increased in systemic lupus erythematosus (SLE), their function and effects on response of B cells are still unclear. Here, we dissect how BACH2 regulates Fr.III cells function and promotes B-cell response in active SLE patients. We measured cytokines and BACH2 expression, and found that Fr.III cells from SLE patients produce much more inflammatory cytokines and were more able to promote B- cell proliferation, IgG, IgA, and TNF-alpha production than controls in a co-culture system. Fr.III cells expressed high levels of ICOS and CD154, but a low level of Tfr and BACH2, BACH2 expression was negatively correlated with SLE Disease Activity Index. Overexpressed of BACH2 in Fr.III cells, decreased cytokines expression and reduced B-cell response. Furthermore, we identified a reduction of H3K27ac level binding at the BACH2 locus in the SLE Fr.III cells and SLE serum stimulation decreased H3K27ac binding at the BACH2 locus, which could be restored using trichostatin A (TSA). In conclusion, BACH2 was associated with SLE disease activity, regulated the function of Fr.III cells, and promoted B-cells response. Targeting BACH2 may be a new immune intervention therapy of SLE.

Gastric cancer (GC) is the TOP3 leading cause of human mortality in malignant tumors. Notwithstanding, the association between GC and circRNAs is not clear. The purpose of this research was to determine the association between GC progression and circRNAs. The data of circRNAs was obtained from the Gene Expression Omnibus (GEO) database to identify gene, which differentially expressed circRNAs in GC tissues and paired normal tissues. The expression of circRNAs in cancer tissues and normal tissues were tested, and the target circRNA was verified before and after surgery in the plasma. A circRNA-micro(mi)RNA-mRNA competing endogenous RNAs (ceRNAs) network was established, and GO and KEGG analysis are performed. Five candidate circRNAs were identified through bioinformatics analysis. Hsa_circ_0021087 and hsa_circ_0005051 were both downregulated in GC tissues, cells and plasma by RTq-PCR. Additionally, there was a significant difference in the expression of plasma hsa_circ_0021087 in patients with GC at the preoperative and postoperative stages (P < 0.001). Hsa_circ_0021087 also promoted the proliferation of GC cells in vitro. Next, the circRNA-miRNA-mRNA network of hsa_circ_0021087 was predicted, which may be associated with the development of GC by bioinformatics analysis. In summary, the aforementioned dual-circular RNAs may have important implications on the potential, novel and non-invasive diagnostic method for patients with GC.

Increased expression of the kinesin family member 23 (KIF23) has been verified in gastric cancer (GC) and its upregulation contributes to cell proliferation. Even though, the role of KIF23 has not been fully elucidated in GC, and the mechanisms of KIF23 as an oncogene remain unknown. To further identify its potential role in GC, we analyzed gene expression data from GC patients in GEO and TCGA datasets. KIF23 was upregulated in GC, and increased expression of KIF23 correlated with poor prognosis. Importantly, KIF23 inhibition not only suppressed GC cell proliferation, tumorigenesis, but also migration and invasion, and arrested the cell cycle in the G2/M phase. Mechanistic investigations confirmed that KIF23 activated the Wnt/β-catenin signaling pathway by directly interacting with APC membrane recruitment 1 (Amer1). Furthermore, KIF23 exhibited competitive binding with Amer1 to block the association of Amer1 with adenomatous polyposis coli (APC), thus relocating Amer1 from the membrane and cytoplasm to the nucleus and attenuating the ability of Amer1 to negatively regulate Wnt/β-catenin signaling, resulting in activation of this signaling pathway. Collectively, our findings demonstrated that KIF23 promoted GC cell proliferation by directly interacting with Amer1 and activating the Wnt/β-catenin signaling pathway.

Cancer immunotherapy has revolutionized cancer treatment, and it relies heavily on the comprehensive understanding of the immune landscape of the tumor microenvironment (TME). Here, we obtain a detailed immune cell atlas of esophageal squamous cell carcinoma (ESCC) at single-cell resolution. Exhausted T and NK cells, regulatory T cells (Tregs), alternatively activated macrophages and tolerogenic dendritic cells are dominant in the TME. Transcriptional profiling coupled with T cell receptor (TCR) sequencing reveal lineage connections in T cell populations. CD8 T cells show continuous progression from pre-exhausted to exhausted T cells. While exhausted CD4, CD8 T and NK cells are major proliferative cell components in the TME, the crosstalk between macrophages and Tregs contributes to potential immunosuppression in the TME. Our results indicate several immunosuppressive mechanisms that may be simultaneously responsible for the failure of immuno-surveillance. Specific targeting of these immunosuppressive pathways may reactivate anti-tumor immune responses in ESCC.

Coagulation has been accepted as a cost-effective and environmental-friendly method to remove pollutants. In our recent work, two coagulants of polyaluminum chloride (PAC) and polyaluminum ferric chloride (PAFC) with dosage gradients, and one coagulant aid of anionic polyacrylamide (PAM) were used to investigate their potential to remove particle-associated (PA) and free-living (FL) ARGs and MGEs detected by high throughput qPCR (HT-qPCR) method. The results indicated that the maximum removal efficiencies of PA- and FL-ARGs (4.67- and 3.18-logs) were obtained at the PAFC dosage of 50.0 mg/L. Excessive PAFC dosage can hamper the removal of size-fractionated ARGs. As PAC aid, anionic PAM (1.0 mg/L) had limited effects to promote the removal of PA-ARG, while FL-ARG removal was enhanced by 0.34 log at the PAC dosage of 50.0 mg/L. The fitted curves suggested that the optimal chemical dosages of PAC, PAFC and PAC coupled with PAM in the removal of total ARGs and MGEs were 40.5, 64.7 and 50.0 mg/L, respectively. In addition, we found that much more coagulants were needed to remove FL-ARGs compared to that of PA-ARGs. The removal efficiencies of size-fractionated ARGs by flocculation can be affected by coagulant type, dosage, coagulant aid, Zeta potential and microorganism lifestyle (PA or FL).

Regulatory interleukin-10 (IL-10)-producing B cells (B10 cells) play a critical role in preventing and curing autoimmune diseases in experimental mouse models. However, the precise cellular and molecular mechanisms of action of B10 cells in humans, especially in patients with Crohn's disease (CD), remain to be determined. miR-155 regulates many physiological and pathological conditions, including inflammation such as that in CD. In this study, we aimed to explore the effect of miRNA-155 on IL-10 production by B cells in healthy controls (HCs) and CD patients. Interestingly, we found that CD24hiCD27+ B cells express high levels of miRNA-155 and IL-10, which are positively correlated. Additionally, CD24hiCD27+ B cells express higher levels of Toll-like receptor 9 than those found in other B cell subsets. Overexpression of miRNA-155 promotes IL-10 production, while inhibition of miRNA-155 decreases IL-10 production. We determined that miR-155 directly inhibits the expression of Jarid2, which reduces H3K27me3 binding to the IL10 promoter and increases IL-10 gene expression. In coculture systems, the CD24hiCD27+ B cells from HCs suppressed the secretion of TNFα and IFNγ by monocytes and T cells, respectively. However, the number and function of CD24hiCD27+ B cells from CD patients were decreased. Moreover, we found that miR-155 induces CD24hiCD27+ B cells to produce higher levels of TNFα instead of IL-10 in CD patients than in the controls and that the increased number of IL-10+TNFα+ B cells reduces the induction of Foxp3 expression and the inhibition of IFNγ production by CD4+CD25- T cells, as well as TNFα production by monocytes. Our study demonstrates the critical role of miRNA-155 in the regulation of IL-10 production by B cells and reveals the novel molecular mechanism underlying the functional impairment of B10 cells in CD patients. Our study has the potential to drive the development of B10 cell-based strategies to ameliorate disease progression in CD patients.

Morbidity from allergic diseases is increasing. Basophils play a critical role in systemic anaphylaxis and chronic allergic inflammation. The prenatal environment must be regarded as a possible early risk factor for allergic diseases in children. Our objective was to determine if basophils harvested from neonates genetically predisposed to atopic disease had different levels of CD63 expression and IL-4 release properties in response to various stimuli (peptidoglycan, Dermatophagoides farinae, hyperosmotic mannitol). Blood samples were collected from 16 asthmatic and 18 healthy women and their newborns. Peripheral blood basophil histamine was measured using the human basophil degranulation test (HBDT), whereas activation was assessed by flow cytometric measurement of CD63 expression on the cord blood basophil surface. IL-4 levels were quantified by ELISA following allergen stimulation. The basophil degranulation index (DI) in granulocytes harvested from the peripheral blood of asthmatic women was assessed following stimulation with peptidoglycan (PGN), Dermatophagoides farinae (Df ) extract, or hyperosmotic mannitol. The DI was significantly higher in atopic women than in healthy controls. Upregulation of CD63 on the cord blood basophil surface was also detected in response to these stimuli. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared to basophils purified from the controls. These data suggested that various stimuli play a role in augmenting allergic reactions via modulation of activated basophil cytokine secretion. It may require new methods to stabilize the basophils in allergic diseases.