4-Nitrophenol at environmentally relevant concentrations mediates reproductive toxicity in Caenorhabditis elegans via metabolic disorders-induced estrogen signaling pathway.

4-Nitrophenol (4-NP), as a toxic and refractory pollutant, has generated significant concern due to its adverse effects. However, the potential toxic effects and mechanism remained unclear. In this study, the reproduction, development, locomotion and reactive oxygen species (ROS) production of Caenorhabditis elegans were investigated to evaluate the 4-NP toxicity. We used metabolomics to assess the potential damage mechanisms. The role of metabolites in mediating the relationship between 4-NP and phenotypes was examined by correlation and mediation analysis. 4-NP (8 ng/L and 8 µg/L) caused significant reduction of brood size, ovulation rate, total germ cells numbers, head thrashes and body bends, and an increase in ROS. However, the oosperm numbers in uterus, body length and body width were decreased in 8 µg/L. Moreover, 36 differential metabolites were enriched in the significant metabolic pathways, including lysine biosynthesis, ß-alanine metabolism, tryptophan metabolism, pentose phosphate pathway, pentose and glucuronate interconversions, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, galactose metabolism, propanoate metabolism, glycerolipid metabolism, and estrogen signaling pathway. The mechanism of 4-NP toxicity was that oxidative stress caused by the perturbation of amino acid, which had effects on energy metabolism through disturbing carbohydrate and lipid metabolism, and finally affected the estrogen signaling pathway to exert toxic effects. Moreover, correlation and mediation analysis showed glycerol-3P, glucosamine-6P, glucosamine-1P, UDP-galactose, L-aspartic acid, and uracil were potential markers for the reproduction and glucose-1,6P2 for developmental toxicity. The results provided insight into the pathways involved in the toxic effects caused by 4-NP and developed potential biomarkers to evaluate 4-NP toxicity.

Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling.

Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked ß-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gαs:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.

The development of yellow lupin anthers depends on the relationship between jasmonic acid and indole-3-acetic acid.

The main purpose of this study was to demonstrate that the course of anther development, including post-meiotic maturation, dehiscence and senescence, is ensured by the interdependencies between jasmonic acid (JA) and indole-3-acetic acid (IAA) in yellow lupin (Lupinus luteus L.). The concentration of JA peaked during anther dehiscence when IAA level was low, whereas the inverse relationship was specific to anther senescence. Cellular and tissue localization of JA and IAA, in conjunction with broad expression profile for genes involved in biosynthesis, signalling, response, and homeostasis under different conditions, allowed to complete and define the role of studied phytohormones during late anther development, as well as predict events triggered by them. The development/degeneration of septum and anther wall cells, dehydration of epidermis, and rupture of stomium may involve JA signalling, while the formation of secondary thickening in endothecial cell walls is rather JA independent. The IAA is involved in programmed cell death (PCD)-associated processes during anther senescence but does not exclude its participation in the anther dehiscence processes, mainly related to cell disintegration and degeneration. A detailed understanding of these multistage processes, especially at the level of phytohormonal interplay, can contribute to the effective control of male fertility, potentially revolutionizing the breeding of L. luteus.

Preliminary evaluation of antiproliferative and apoptotic activities of novel indolin-2-one derivatives.

Indole-based agents are frequently used in targeted or supportive therapy of several cancers. In this study, we investigated the anticancer properties of originally synthesized novel indolin-2-one derivatives (6a-d) against Malignant Mesothelioma, Breast cancer, and Colon Cancer cells. Our results revealed that all derivatives were effectively delayed cell proliferation by inhibiting the ERK1/2, AKT, and STAT3 signaling pathways in a concentration-dependent manner. Additionally, these variants induced cell cycle arrest in the S phase, accompanied by elevated levels of p21 and p27 expressions. Derivatives also initiated mitochondrial apoptosis through the upregulation of Bax and downregulation of Bcl-2 proteins, leading to the activation of caspase 3 and PARP cleavage in exposed cells. Remarkably, three of the indolin-2-one derivatives displayed significant selectivity towards Breast and Colon Cancer cells, with compound 6d promising as the most potent and wide spectral one for all cancer cell lines.

ARTN-GFRA3 axis induces epithelial-mesenchymal transition phenotypes, migration, and invasion of gastric cancer cells via KRAS signaling.

Neural invasion underlies the local spread of gastric cancer and is associated with poor prognosis. This process has been receiving increasing attention in recent years. However, the relationship between neural invasion and the malignant phenotypes of gastric cancer cells, as well as the molecular mechanism involved in this process, remain unclear. In this study, bioinformatics analysis was performed using a dataset obtained from The Cancer Genome Atlas-Stomach Adenocarcinoma. The results revealed that high expression of GDNF family receptor alpha 3 (GFRA3) was associated with a poor prognosis of patients with gastric cancer. GFRA3 is a receptor for artemin (ARTN), a glial cell line-derived neurotrophic factor (GDNF). This association was indicated by short overall/disease-free survival, as well as the presence of high-stage and high-grade disease. Gene set enrichment analysis showed that two cancer-associated pathways, namely KRAS signaling and epithelial-mesenchymal transition (EMT), were activated when GFRA3 was highly expressed in gastric cancer. Further studies confirmed that GFRA3 activated KRAS downstream signaling phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) or extracellular signal-regulated kinase (ERK) and induced EMT markers, as well as promoted the migration and invasion of gastric cancer cells. As a ligand of GFRA3, ARTN induced the EMT, migration, and invasion of gastric cancer cells via GFRA3. Notably, the effects of the ARTN-GFRA3 axis were attenuated by treatment with a KRAS inhibitor. The present findings indicated that, during the neural invasion of gastric cancer, ARTN-mediated activation of GFRA3 induces EMT phenotypes, migration, and invasion of gastric cancer cells via KRAS signaling.

Antitumor activity of anlotinib in malignant melanoma: modulation of angiogenesis and vasculogenic mimicry.

Malignant melanoma presents a formidable challenge due to its aggressive metastatic behavior and limited response to current treatments. To address this, our study delves into the impact of anlotinib on angiogenesis and vasculogenic mimicry using malignant melanoma cells and human umbilical vein endothelial cells. Evaluating tubular structure formation, cell proliferation, migration, invasion, and key signaling molecules in angiogenesis, we demonstrated that anlotinib exerts a dose-dependent inhibition on tubular structures and effectively suppresses cell growth and invasion in both cell types. Furthermore, in a mouse xenograft model, anlotinib treatment resulted in reduced tumor growth and vascular density. Notably, the downregulation of VEGFR-2, FGFR-1, PDGFR-ß, and PI3K underscored the multitargeted antitumor activity of anlotinib. Our findings emphasize the therapeutic potential of anlotinib in targeting angiogenesis and vasculogenic mimicry, contributing to the development of novel strategies for combating malignant melanoma.

The correlation between TRIM28 expression and immune checkpoints in CRPC.

This study delves into the unexplored realm of castration-resistant prostate cancer (CRPC) by investigating the role of TRIM28 and its intricate molecular mechanisms using high-throughput single-cell transcriptome sequencing and advanced bioinformatics analysis. Our comprehensive examination unveiled dynamic TRIM28 expression changes, particularly in immune cells such as macrophages and CD8+ T cells within CRPC. Correlation analyses with TCGA data highlighted the connection between TRIM28 and immune checkpoint expression and emphasized its pivotal influence on the quantity and functionality of immune cells. Using TRIM28 knockout mouse models, we identified differentially expressed genes and enriched pathways, unraveling the potential regulatory involvement of TRIM28 in the cGAS-STING pathway. In vitro, experiments further illuminated that TRIM28 knockout in prostate cancer cells induced a notable anti-tumor immune effect by inhibiting M2 macrophage polarization and enhancing CD8+ T cell activity. This impactful discovery was validated in an in situ transplant tumor model, where TRIM28 knockout exhibited a deceleration in tumor growth, reduced proportions of M2 macrophages, and enhanced infiltration of CD8+ T cells. In summary, this study elucidates the hitherto unknown anti-tumor immune role of TRIM28 in CRPC and unravels its potential regulatory mechanism via the cGAS-STING signaling pathway. These findings provide novel insights into the immune landscape of CRPC, offering promising directions for developing innovative therapeutic strategies.

Exploration of the miR-187-3p/CNR2 pathway in modulating osteoblast differentiation and treating postmenopausal osteoporosis through mechanical stress.

This study aimed to explore how mechanical stress affects osteogenic differentiation via the miR-187-3p/CNR2 pathway. To conduct this study, 24 female C57BL/6 mice, aged 8 weeks, were used and divided into four groups. The Sham and OVX groups did not undergo treadmill exercise, while the Sham + EX and OVX + EX groups received a 8-week treadmill exercise. Post-training, bone marrow and fresh femur samples were collected for further analysis. Molecular biology analysis, histomorphology analysis, and micro-CT analysis were conducted on these samples. Moreover, primary osteoblasts were cultured under osteogenic conditions and divided into GM group and CTS group. The cells in the CTS group underwent a sinusoidal stretching regimen for either 3 or 7 days. The expression of early osteoblast markers (Runx2, OPN, and ALP) was measured to assess differentiation. The study findings revealed that mechanical stress has a regulatory impact on osteoblast differentiation. The expression of miR-187-3p was observed to decrease, facilitating osteogenic differentiation, while the expression of CNR2 increased significantly. These observations suggest that mechanical stress, miR-187-3p, and CNR2 play crucial roles in regulating osteogenic differentiation. Both in vivo and in vitro experiments have confirmed that mechanical stress downregulates miR-187-3p and upregulates CNR2, which leads to the restoration of distal femoral bone mass and enhancement of osteoblast differentiation. Therefore, mechanical stress promotes osteoblasts, resulting in improved osteoporosis through the miR-187-3p/CNR2 signaling pathway. These findings have broad prospect and provide molecular biology guidance for the basic research and clinical application of exercise in the prevention and treatment of PMOP.

[Effect of oxymatrine on Cryptosporidium parvum infection in mice based on the HMGB1-TLR2/TLR4-NF-κB pathway].

OBJECTIVE: To investigate the involvement of the high mobility group box protein B1 (HMGB1)-Toll-like receptor 2 (TLR2)/TLR4-nuclear factor κB (NF-κB) pathway in the intestinal mucosal injury induced by Cryptosporidium parvum infection, and to examine the effect of oxymatrine (OMT) on C. parvum infection in mice. METHODS: Forty SPF 4-week-old BALB/c mice were randomly divided into four groups, including the control group, infection group, glycyrrhizin (GA) group and OMT group. Each mouse was orally administered with 1 × 105 C. parvum oocysts one week in the infection, GA and OMT groups following dexamethasone-induced immunosuppression to model C. parvum intestinal infections in mice. Upon successful modeling, mice in the GA group were intraperitoneally injected with GA at a daily dose of 25.9 mL/kg for successive two weeks, and animals in the OMT group were orally administered OMT at a daily dose of 50 mg/kg for successive two weeks, while mice in the control group were given normal food and water. All mice were sacrificed two weeks post-treatment, and proximal jejunal tissues were sampled. The pathological changes of mouse intestinal mucosal specimens were observed using hematoxylin-eosin (HE) staining, and the mouse intestinal villous height, intestinal crypt depth and the ratio of intestinal villous height to intestinal crypt depth were measured. The occludin and zonula occludens protein 1 (ZO1) expression was determined in mouse intestinal epithelial cells using immunohistochemistry, and the relative expression of HMGB1, TLR2, TLR4, myeloid differentiation primary response gene 88 (MyD88) and NF-κB p65 mRNA was quantified in mouse jejunal tissues using quantitative real-time PCR (qPCR) assay. RESULTS: HE staining showed that the mouse intestinal villi were obviously atrophic, shortened, and detached, and the submucosal layer of the mouse intestine was edematous in the infection group as compared with the control group, while the mouse intestinal villi tended to be structurally intact and neatly arranged in the GA and OMT groups. There were significant differences among the four groups in terms of the mouse intestinal villous height (F = 6.207, P = 0.000 5), intestinal crypt depth (F = 6.903, P = 0.000 3) and the ratio of intestinal villous height to intestinal crypt depth (F = 37.190, P < 0.000 1). The mouse intestinal villous height was lower in the infection group than in the control group [(321.9 ± 41.1) µm vs. (399.5 ± 30.9) µm; t = 4.178, P < 0.01] and the GA group [(321.9 ± 41.1) µm vs. (383.7 ± 42.7) µm; t = 3.130, P < 0.01], and the mouse intestinal crypt depth was greater in the infection group [(185.0 ± 35.9) µm] than in the control group [(128.4 ± 23.6) µm] (t = 3.877, P < 0.01) and GA group [(143.3 ± 24.7) µm] (t = 2.710, P < 0.05). The mouse intestinal villous height was greater in the OMT group [(375.3 ± 22.9) µm] than in the infection group (t = 3.888, P < 0.01), and there was no significant difference in mouse intestinal villous height between the OMT group and the control group (t = 1.989, P > 0.05). The mouse intestinal crypt depth was significantly lower in the OMT group [(121.5 ± 27.3) µm] than in the infection group (t = 4.133, P < 0.01), and there was no significant difference in mouse intestinal crypt depth between the OMT group and the control group (t = 0.575, P > 0.05). The ratio of the mouse intestinal villous height to intestinal crypt depth was significantly lower in the infection group (1.8 ± 0.2) than in the control group (3.1 ± 0.3) (t = 10.540, P < 0.01) and the GA group (2.7 ± 0.3) (t = 7.370, P < 0.01), and the ratio of the mouse intestinal villous height to intestinal crypt depth was significantly higher in the OMT group (3.1 ± 0.2) than in the infection group (t = 15.020, P < 0.01); however, there was no significant difference in the ratio of the mouse intestinal villous height to intestinal crypt depth between the OMT group and the control group (t = 0.404, P > 0.05). Immunohistochemical staining showed significant differences among the four groups in terms of occludin (F = 28.031, P < 0.000 1) and ZO1 expression (F = 14.122, P < 0.000 1) in mouse intestinal epithelial cells. The proportion of positive occluding expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.3 ± 4.5)% vs. (28.3 ± 0.5)%; t = 3.810, P < 0.01], and the proportions of positive occluding expression were significantly higher in mouse intestinal epithelial cells in the GA group [(30.3 ± 1.3)%] and OMT group [(25.8 ± 1.5)%] than in the infection group (t = 7.620 and 5.391, both P values < 0.01); however, there was no significant differences in the proportion of positive occluding expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 1.791 and 2.033, both P values > 0.05). The proportion of positive ZO1 expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.4 ± 1.8)% vs. (24.2 ± 2.8)%; t = 4.485, P < 0.01], and the proportions of positive ZO1 expression were significantly higher in mouse intestinal epithelial cells in the GA group [(24.1 ± 2.3)%] (t = 5.159, P < 0.01) and OMT group than in the infection group [(22.5 ± 1.9)%] (t = 4.441, P < 0.05); however, there were no significant differences in the proportion of positive ZO1 expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 0.037 and 0.742, both P values > 0.05). qPCR assay showed significant differences among the four groups in terms of HMGB1 (F = 21.980, P < 0.000 1), TLR2 (F = 20.630, P < 0.000 1), TLR4 (F = 17.000, P = 0.000 6), MyD88 (F = 8.907, P = 0.000 5) and NF-κB p65 mRNA expression in mouse jejunal tissues (F = 8.889, P = 0.000 7). The relative expression of HMGB1 [(5.97 ± 1.07) vs. (1.05 ± 0.07); t = 6.482, P < 0.05] 、TLR2 [(5.92 ± 1.29) vs. (1.10 ± 0.14); t = 5.272, P < 0.05] 、TLR4 [(5.96 ± 1.50) vs. (1.02 ± 0.03); t = 4.644, P < 0.05] 、MyD88 [(3.00 ± 1.26) vs. (1.02 ± 0.05); t = 2.734, P < 0.05] and NF-κB p65 mRNA [(2.33 ± 0.72) vs. (1.04 ± 0.06); t = 2.665, P < 0.05] was all significantly higher in mouse jejunal tissues in the infection group than in the control group. A significant reduction was detected in the relative expression of HMGB1 (0.63 ± 0.01), TLR2 (0.42 ± 0.10), TLR4 (0.35 ± 0.07), MyD88 (0.70 ± 0.11) and NF-κB p65 mRNA (0.75 ± 0.01) in mouse jejunal tissues in the GA group relative to the control group (t = 8.629, 5.830, 11.500, 4.729 and 6.898, all P values < 0.05), and the relative expression of HMGB1, TLR2, TLR4, MyD88 and NF-κB p65 mRNA significantly reduced in mouse jejunal tissues in the GA group as compared to the infection group (t = 7.052, 6.035, 4.084, 3.165 and 3.274, all P values < 0.05). In addition, the relative expression of HMGB1 (1.14 ± 0.60), TLR2 (1.00 ± 0.24), TLR4 (1.14 ± 0.07), MyD88 (0.96 ± 0.25) and NF-κ B p65 mRNA (1.12 ± 0.17) was significantly lower in mouse jejunal tissues in the OMT group than in the infection group (t = 7.059, 5.320, 3.510, 3.466 and 3.273, all P values < 0.05); however, there were no significant differences between the OMT and control groups in terms of relative expression of HMGB1, TLR2, TLR4, MyD88 or NF-κB p65 mRNA in mouse jejunal tissues (t = 0.239, 0.518, 1.887, 0.427 and 0.641, all P values > 0.05). CONCLUSIONS: C. parvum infection causes intestinal inflammatory responses and destruction of intestinal mucosal barrier through up-regulating of the HMGB1-TLR2/TLR4-NF-κB pathway. OMT may suppress the intestinal inflammation and repair the intestinal mucosal barrier through inhibiting the activity of the HMGB1-TLR2/TLR4-NF-κB pathway.

Combined analysis of single-cell sequencing and bulk transcriptome sequencing reveals new mechanisms for non-healing diabetic foot ulcers.

Diabetic foot ulcers (DFUs) pose a significant challenge in diabetes care. Yet, a comprehensive understanding of the underlying biological disparities between healing and non-healing DFUs remains elusive. We conducted bioinformatics analysis of publicly available transcriptome sequencing data in an attempt to elucidate these differences. Our analysis encompassed differential analysis to unveil shifts in cell composition and gene expression profiles between non-healing and healing DFUs. Cell communication alterations were explored employing the Cellchat R package. Pseudotime analysis and cytoTRACE allowed us to dissect the heterogeneity within fibroblast subpopulations. Our findings unveiled disruptions in various cell types, localized low-grade inflammation, compromised systemic antigen processing and presentation, and extensive extracellular matrix signaling disarray in non-healing DFU patients. Some of these anomalies partially reverted in healing DFUs, particularly within the abnormal ECM-receptor signaling pathway. Furthermore, we distinguished distinct fibroblast subpopulations in non-healing and healing DFUs, each with unique biological functions. Healing-associated fibroblasts exhibited heightened extracellular matrix (ECM) remodeling and a robust wound healing response, while non-healing-associated fibroblasts showed signs of cellular senescence and complement activation, among other characteristics. This analysis offers profound insights into the wound healing microenvironment, identifies pivotal cell types for DFU healing promotion, and reveals potential therapeutic targets for DFU management.

ULK2 suppresses ovarian cancer cell migration and invasion by elevating IGFBP3.

Background: Ovarian cancer is an aggressive malignancy with high mortality known for its considerable metastatic potential. This study aimed to explore the expression and functional role of Unc-51 like autophagy activating kinase 2 (ULK2) in the progression of ovarian cancer. Methods: ULK2 expression patterns in ovarian cancer tissues as well as benign tumor control samples obtained from our institution were evaluated using immunohistochemistry. Cell counting kit 8 and Transwell assays were applied to assess the effects of ULK2 overexpression on cell proliferation, migration and invasion, respectively. RNA sequencing was performed to explore potential mechanisms of action of ULK2 beyond its classical autophagy modulation. Results: Our experiments showed significant downregulation of ULK2 in ovarian cancer tissues. Importantly, low expression of ULK2 was markedly correlated with decreased overall survival. In vitro functional studies further demonstrated that overexpression of ULK2 significantly suppressed tumor cell proliferation, migration, and invasion. RNA sequencing analysis revealed a potential regulatory role of ULK2 in the insulin signaling pathway through upregulation of insulin-like growth factor binding protein-3 (IGFBP3) in ovarian cancer cells. Conclusions: In summary, the collective data indicated that ULK2 acted as a tumor suppressor in ovarian cancer by upregulating the expression of IGFBP3. Our study underscores the potential utility of ULK2 as a valuable prognostic marker for ovarian cancer.

Understanding the molecular crossroads in acute liver failure: A pathway to new therapies.

In this editorial we comment on the article published in a recent issue of the World Journal of Gastroenterology. Acute liver failure (ALF) is a critical condition characterized by rapid hepatocellular injury and organ dysfunction, and it often necessitates liver transplant to ensure patient survival. Recent research has elucidated the involvement of distinct cell death pathways, namely ferroptosis and pyroptosis, in the pathogenesis of ALF. Ferroptosis is driven by iron-dependent lipid peroxidation, whereas pyroptosis is an inflammatory form of cell death; both pathways contribute to hepatocyte death and exacerbate tissue damage. This comprehensive review explores the interplay between ferroptosis and pyroptosis in ALF, highlighting the role of key regulators such as silent information regulator sirtuin 1. Insights from clinical and preclinical studies provide valuable perspectives on the dysregulation of cell death pathways in ALF and the therapeutic potential of targeting these pathways. Collaboration across multiple disciplines is essential for translating the experimental insights into effective treatments for this life-threatening condition.

Suppressor of Cytokine Signaling Proteins 3 and 5 Potentially Delineate Polarization of Th cells in Chronic Rhinosinusitis.

Background: Chronic rhinosinusitis (CRS) is an inflammatory condition classified into chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). Th cells manage inflammatory cells in CRS. Suppressor of Cytokine Signaling (SOCS) proteins regulate Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in Th cells by polarizing toward Th1, Th2, and Th17 cells. This study evaluated the levels of SOCS1,3,5 in CRS patients to find associations with Th cells. Methods: In this cross-sectional study, 20 CRSwNP patients, 12 CRSsNP patients, and 12 controls participated. The infiltration of CD4+ T cells was determined using immunohistochemistry. The expression of specific transcription factors and SOCS proteins was assessed using real-time PCR. Cytokine levels were evaluated using ELISA. SOCS protein levels were investigated using western blot analysis. Results: The expression of SOCS3 increased in the CRSwNP group compared to CRSsNP and control groups (p <0.001). SOCS3 protein levels increased in the CRSwNP group compared to CRSsNP (p <0.05) and control (p <0.001) groups. Although there was a significant difference in SOCS5 expression between CRSsNP and control groups, SOCS5 protein levels were significantly different between CRSsNP and control (p <0.001) and CRSwNP (p <0.05) groups. Conclusions: Targeted therapies may be suggested for CRS by modulating SOCS3 and SOCS5 proteins that are responsible for polarization of Th cells toward Th2 or Th1 cells, respectively. JAK-STAT pathway targeting, which encompasses numerous cells, can be limited to SOCS proteins to more effectively orchestrate Th cell differentiation.

Interleukin-17 directly stimulates tumor infiltrating Tregs to prevent cancer development.

Background: Interleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance, but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses. Methods: We generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice (Il17ra ΔTreg mice). Subsequently, we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer (Cdx2-Cre +/Apc F/+), to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors, and compared to those of human tumor infiltrating Treg cells. Results: IL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA, specifically in Tregs, resulted in increased Th17 cells, and exacerbated tumor development. Mechanistically, tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation, maturation, and suppression function, and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming, we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing, and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function. Conclusion: IL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC.

SOCS1 is a critical checkpoint in immune homeostasis, inflammation and tumor immunity.

The Suppressor of Cytokine Signaling (SOCS) family proteins are important negative regulators of cytokine signaling. SOCS1 is the prototypical member of the SOCS family and functions in a classic negative-feedback loop to inhibit signaling in response to interferon, interleukin-12 and interleukin-2 family cytokines. These cytokines have a critical role in orchestrating our immune defence against viral pathogens and cancer. The ability of SOCS1 to limit cytokine signaling positions it as an important immune checkpoint, as evidenced by the detection of detrimental SOCS1 variants in patients with cytokine-driven inflammatory and autoimmune disease. SOCS1 has also emerged as a key checkpoint that restricts anti-tumor immunity, playing both a tumor intrinsic role and impacting the ability of various immune cells to mount an effective anti-tumor response. In this review, we describe the mechanism of SOCS1 action, focusing on the role of SOCS1 in autoimmunity and cancer, and discuss the potential for new SOCS1-directed cancer therapies that could be used to enhance adoptive immunotherapy and immune checkpoint blockade.

sUPRa is a dual-color reporter for unbiased quantification of the unfolded protein response with cellular resolution.

The unfolded protein response (UPR) maintains proteostasis upon endoplasmic reticulum (ER) stress, and is initiated by a range of physiological and pathological processes. While there have been advances in developing fluorescent reporters for monitoring individual signaling pathways of the UPR, this approach may not capture a cell's overall UPR activity. Here we describe a novel sensor of UPR activity, sUPRa, which is designed to report the global UPR. sUPRa displays excellent response characteristics, outperforms reporters of individual UPR pathways in terms of sensitivity and kinetics, and responds to a range of different ER stress stimuli. Furthermore, sUPRa's dual promoter and fluorescent protein design ensures that both UPR-active and inactive cells are detected, and controls for reporter copy number. Using sUPRa, we reveal UPR activation in layer 2/3 pyramidal neurons of mouse cerebral cortex following a period of sleep deprivation. sUPRa affords new opportunities for quantifying physiological UPR activity with cellular resolution.

Revisiting the evolution of bow-tie architecture in signaling networks.

Bow-tie architecture is a layered network structure that has a narrow middle layer with multiple inputs and outputs. Such structures are widely seen in the molecular networks in cells, suggesting that a universal evolutionary mechanism underlies the emergence of bow-tie architecture. The previous theoretical studies have implemented evolutionary simulations of the feedforward network to satisfy a given input-output goal and proposed that the bow-tie architecture emerges when the ideal input-output relation is given as a rank-deficient matrix with mutations in network link intensities in a multiplicative manner. Here, we report that the bow-tie network inevitably appears when the link intensities representing molecular interactions are small at the initial condition of the evolutionary simulation, regardless of the rank of the goal matrix. Our dynamical system analysis clarifies the mechanisms underlying the emergence of the bow-tie structure. Further, we demonstrate that the increase in the input-output matrix reduces the width of the middle layer, resulting in the emergence of bow-tie architecture, even when evolution starts from large link intensities. Our data suggest that bow-tie architecture emerges as a side effect of evolution rather than as a result of evolutionary adaptation.

Exosome-shuttled miR-150-5p from LPS-preconditioned mesenchymal stem cells down-regulate PI3K/Akt/mTOR pathway via Irs1 to enhance M2 macrophage polarization and confer protection against sepsis.

Rationale: Sepsis is a life-threatening organ dysfunction and lack of effective measures in the current. Exosomes from mesenchymal stem cells (MSCs) reported to alleviate inflammation during sepsis, and the preconditioning of MSCs could enhance their paracrine potential. Therefore, this study investigated whether exosomes secreted by lipopolysaccharide (LPS)-pretreated MSCs exert superior antiseptic effects, and explored the underlying molecular mechanisms. Methods: Exosomes were isolated and characterized from the supernatants of MSCs. The therapeutic efficacy of normal exosomes (Exo) and LPS-pretreated exosomes (LPS-Exo) were evaluated in terms of survival rates, inflammatory response, and organ damage in an LPS-induced sepsis model. Macrophages were stimulated with LPS and treated with Exo or LPS-Exo to confirm the results of the in vivo studies, and to explain the potential mechanisms. Results: LPS-Exo were shown to inhibit aberrant pro-inflammatory cytokines, prevent organ damages, and improve survival rates of the septic mice to a greater extent than Exo. In vitro, LPS-Exo significantly promoted the M2 polarization of macrophages exposed to inflammation. miRNA sequencing and qRT-PCR analysis identified the remarkable expression of miR-150-5p in LPS-Exo compared to that in Exo, and exosomal miR-150-5p was transferred into recipient macrophages and mediated macrophage polarization. Further investigation demonstrated that miR-150-5p targets Irs1 in recipient macrophages and subsequently modulates macrophage plasticity by down-regulating the PI3K/Akt/mTOR pathway. Conclusion: The current findings highly suggest that exosomes derived from LPS pre-conditioned MSCs represent a promising cell-free therapeutic method and highlight miR-150-5p as a novel molecular target for regulating immune hyperactivation during sepsis.

Inhibition of Th17 cell differentiation by aerobic exercise improves vasodilatation in diabetic mice.

BACKGROUND: Aortic endothelial diastolic dysfunction is an early complication of diabetes and the abnormal differentiation of Th17 cells is involved in the development of diabetes. However, the exact role of exercise on regulating the Th17 cells differentiation and the underlying molecular mechanisms remain to be elucidated in diabetic mice. METHODS: db/db and db/m+ mice were randomly divided into exercise and sedentary groups. Mice in exercise group were exercised daily, 6 days/week, for 6 weeks and mice in sedentary groups were placed on a nonmoving treadmill for 6 weeks. Vascular endothelial function was measured via wire myograph and the frequencies of Th17 from peripheral blood in mice were assessed via flow cytometry. RESULTS: Our data showed that exercise improved insulin resistance and aortic endothelial diastolic function in db/db mice. In addition, the proportion of Th17 cells and IL-17A level in peripheral blood of db/db mice were significantly increased, and exercise could promote Th17 cell differentiation and reduce IL-17A level. More importantly, STAT3 or ROR-γt inhibitors could promote Th17 cell differentiation in db/db mice, while exercise significantly down-regulated p-STAT3/ROR-γt signaling in db/db mice, suggesting that exercise regulated Th17 differentiation through STAT3/ROR-γt signaling. CONCLUSIONS: This study demonstrated that exercise improved vascular endothelial function in diabetic mice via reducing Th17 cell differentiation through p-STAT3/ROR-γt pathway, suggesting exercise may be an important non-pharmacological intervention strategy for the treatment of diabetes-related vascular complications.

Expression of ISG60 is induced by TLR3 signaling in BEAS­2B bronchial epithelial cells: Possible involvement in CXCL10 expression.

Viral infections in the respiratory tract are common, and, in recent years, severe acute respiratory syndrome coronavirus 2 outbreaks have highlighted the effect of viral infections on antiviral innate immune and inflammatory reactions. Specific treatments for numerous viral respiratory infections have not yet been established and they are mainly treated symptomatically. Therefore, understanding the details of the innate immune system underlying the airway epithelium is crucial for the development of new therapies. The present study aimed to investigate the function and expression of interferon (IFN)­stimulated gene (ISG)60 in non­cancerous bronchial epithelial BEAS­2B cells exposed to a Toll­like receptor 3 agonist. BEAS­2B cells were treated with a synthetic TLR3 ligand, polyinosinic­polycytidylic acid (poly IC). The mRNA and protein expression levels of ISG60 were analyzed using reverse transcription­quantitative PCR and western blotting, respectively. The levels of C­X­C motif chemokine ligand 10 (CXCL10) were examined using an enzyme­linked immunosorbent assay, and the effects of knockdown of IFN­ß, ISG60 and ISG56 were examined using specific small interfering RNAs. Notably, ISG60 expression was increased in proportion to poly IC concentration, and recombinant human IFN­ß also induced ISG60 expression. By contrast, knockdown of IFN­ß and ISG56 decreased ISG60 expression, and ISG60 knockdown reduced CXCL10 and ISG56 expression. These findings suggested that ISG60 is partly implicated in CXCL10 expression and that ISG60 may serve a role in the innate immune response of bronchial epithelial cells. The present study highlights ISG60 as a potential target for new therapeutic strategies against viral infections in the airway.