TM9SF4 is an F-actin disassembly factor that promotes tumor progression and metastasis.

F-actin dynamics is crucial for many fundamental properties of cancer cells, from cell-substrate adhesion to migration, invasion and metastasis. However, the regulatory mechanisms of actin dynamics are still incompletely understood. In this study, we demonstrate the function of a protein named TM9SF4 in regulating actin dynamics and controlling cancer cell motility and metastasis. We show that an N-terminal fragment (NTF) cleaved from TM9SF4 can directly bind to F-actin to induce actin oxidation at Cys374, consequently enhancing cofilin-mediated F-actin disassembly. Knockdown of TM9SF4 reduces cell migration and invasion in ovarian cancer cells A2780, SKOV3 and several high grade serous ovarian cancer lines (HGSOCs). In vivo, knockdown of TM9SF4 completely abolishes the tumor growth and metastasis in athymic nude mice. These data provide mechanistic insights into TM9SF4-mediated regulation of actin dynamics in ovarian cancer cells.

Dietary supplementation of exopolysaccharides from Lactobacillus rhamnosus GCC-3 improved the resistance of zebrafish against spring viremia of carp virus infection.

Spring viremia of carp virus (SVCV) can cause high mortality of fish. The aim of this study was to investigate the effects of Lactobacillus rhamnosus GCC-3 exopolysaccharides (GCC-3 EPS) on zebrafish (Danio rerio) infected with SVCV and elucidate the underlying mechanisms. Zebrafish were fed with a control diet or diet supplemented with 0.5% and 1% of GCC-3 EPS for 2 weeks. The results showed that supplementation of GCC-3 EPS significantly improved the survival rate of zebrafish compared with the control group. In addition, dietary 0.5% and 1% GCC-3 EPS significantly up-regulated the expression of genes related to type I interferon (IFN) antiviral immunity. Consistent with in vivo results, GCC-3 EPS significantly inhibited SVCV replication in zebrafish embryonic fibroblast (ZF4) cells while significantly increased the expression of type I IFN signaling pathway related genes. Furthermore, knocking down TANK-binding kinase 1 significantly blocked the antiviral effect of GCC-3 EPS. Dietary GCC-3 EPS improved gut microbiota, and the culture supernatant of GCC-3 EPS-associated microbiota significantly inhibited SVCV replication in ZF4 cells compared with the control-microbiota counterpart. In conclusion, our results indicate that dietary GCC-3 EPS can improve the resistance of zebrafish against SVCV infection, and the mechanism may involve enhanced type I interferon signaling.

Nuclease-Treated Stabilized Fermentation Product of Cetobacterium somerae Improves Growth, Non-specific Immunity, and Liver Health of Zebrafish (Danio rerio).

High-fat diets (HFD) are harmful to fish health. Probiotics are commonly utilized to improve fish nutrition metabolism, immune response, and health. Nucleic acids of the probiotic bacterium can be hydrolyzed by nuclease to generate nucleotides. The present study aimed to evaluate the effects of stabilized fermentation product of nuclease-treated Cetobacterium somerae XMX-1 [XMX-1 (N)] on growth, non-specific immunity, and liver health of zebrafish (Danio rerio). Compared to the HFD group, 100 g/kg XMX-1 (N) significantly increased weight gain and decreased feed conversion ratio (FCR). However, 5 or 10 g/kg XMX-1 (N) had no influence on zebrafish growth. In addition, supplementation of 100 g/kg XMX-1 (N) significantly increased lysozyme activity and total antioxidant capacity in skin mucus, and the expression of inflammation related genes interleukin 1 beta (IL-1ß), interleukin 10 (IL-10), and interleukin 6 (IL-6) in the gut as well as fatty acid oxidation related genes uncoupling protein 2 (UCP2) and proliferator-activated receptor γ coactivator 1α (PGC1α) in the liver, while decreased the content of hepatic triacylglycerol (TAG) in zebrafish. The gene sequencing, 16S rRNA, showed that 100 g/kg XMX-1 (N) enhanced the relative abundance of Firmicutes while lowered Proteobacteria and Actinobacteria. 10 g/kg XMX-1 (N) significantly increased lysozyme activity and complement component 4 (C4) in skin mucus, and intestinal expression of inflammation-related genes. In the 5 g/kg XMX-1 (N) group, however, only an increase in C4 level in skin mucus was observed. Together, these results reveal that dietary supplementation with nuclease-treated C. somerae XMX-1 (N) has a dose-dependent beneficial effect on fish health.

Effect of dietary supplementation of Cetobacterium somerae XMX-1 fermentation product on gut and liver health and resistance against bacterial infection of the genetically improved farmed tilapia (GIFT, Oreochromis niloticus).

The purpose of this study was to evaluate the effects of Cetobacterium somerae XMX-1 fermentation product on gut and liver health and resistance against bacterial infection in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Fingerling GIFTs (n = 120; initial weight 1.33 ± 0.00 g) were randomly assigned to twelve 90-L tanks (four tanks per diet, 10 fish per tank) with three groups: control group (basal high fat diet), 1% XMX-1 group and 2% XMX-1 group (basal diet supplemented with 10 and 20 g XMX-1/kg feed respectively). After 49 days feeding trial, the growth performance and gut and liver health parameters of tilapia were evaluated. Also the gut microbiota and virome were detected by sequencing. 2% XMX-1 fermentation product had no effect on growth performance. For gut health, the expression of hypoxia-inducible factor-lα (Hif-1α) tend to increase in 1% XMX-1 group (P = 0.053). The expression of intestinal interleukin-6 (IL-6) and tumor growth factor ß (TGF-ß) was significantly down-regulated in 1% and 2% XMX-1 groups (P < 0.05), and the intestinal expression of interleukin-1ß (IL-1ß) had a trend to decrease (P = 0.08) in 1% XMX-1 group versus control. 1% and 2% XMX-1 groups also increased the intestinal expression of tight junction genes Claudin (P = 0.06 and 0.07, respectively). For liver health, XMX-1 fermentation product significantly decreased liver TAG (P < 0.05). Furthermore, the hepatic expression of lipid synthesis gene fatty acid synthase (FAS) was significantly decreased and the expression of lipid catabolism related-gene uncoupling protein 2 (UCP2) was significantly increased in 1% XMX-1 and 2% XMX-1 groups (P < 0.01). And the hepatic expression of IL-1ß and IL-6 significantly decreased in 1% XMX-1 and 2% XMX-1 groups (P < 0.05). XMX-1 fermentation product increased the abundance of Fusobacteria in the gut microbiota and 2% XMX-1 group led to alteration in the virome composition at family level. Lastly, the time of tilapia death post Aeromoans challenge was delayed in 1% XMX-1 and 2% XMX-1 groups compared with control. To sum up, our results show that the dietary supplementation of XMX-1 fermentation product can improve the gut and liver health as well as the resistance against pathogenic bacteria of tilapia.

TM9SF4 Is a Crucial Regulator of Inflammation and ER Stress in Inflammatory Bowel Disease.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a major intestinal disease. Excessive inflammation and increased endoplasmic reticulum (ER) stress are the key events in the development of IBD. Search of a genome-wide association study database identified a remarkable correlation between a TM9SF4 single-nucleotide polymorphism and IBD. Here, we aimed to resolve its underlying mechanism. METHODS: The role of TM9SF4 was determined with experimental mouse models of IBD. ER stress cascades, barrier functions, and macrophage polarization in colonic tissues and cells were assessed in vivo and in vitro. The expression of TM9SF4 was compared between inflamed regions of ulcerative colitis patients and normal colon samples. RESULTS: In mouse models of IBD, genetic knockout of the TM9SF4 gene aggravated the disease symptoms. In colonic epithelial cells, short hairpin RNA-mediated knockdown of TM9SF4 expression promoted inflammation and increased ER stress. In macrophages, TM9SF4 knockdown promoted M1 macrophage polarization but suppressed M2 macrophage polarization. Genetic knockout/knockdown of TM9SF4 also disrupted epithelial barrier function. Mechanistically, TM9SF4 deficiency may act through Ca2+ store depletion and cytosolic acidification to induce an ER stress increase. Furthermore, the expression level of TM9SF4 was found to be much lower in the inflamed colon regions of human ulcerative colitis patients than in normal colon samples. CONCLUSIONS: Our study identified a novel IBD-associated protein, TM9SF4, the reduced expression of which can aggravate intestinal inflammation. Deficiency of TM9SF4 increases ER stress, promotes inflammation, and impairs the intestinal epithelial barrier to aggravate IBD.

Stabilized fermentation product of Cetobacterium somerae improves gut and liver health and antiviral immunity of zebrafish.

Probiotics are widely used in aquafeeds and exhibited beneficial effects on fish by improving host health and resisting pathogens. However, probiotics applied to aquaculture are mainly from terrestrial sources instead of the host animal. The purpose of the work was to evaluate the effects of stabilized fermentation product of commensal Cetobacterium somerae XMX-1 on gut, liver health and antiviral immunity of zebrafish. A total of 240 zebrafish were assigned to the control (fed a basal diet) and XMX-1 group (fed a basal diet with 10 g XMX-1/kg diet). After four weeks feeding, growth performance, feed utilization, hepatic steatosis score, TAG, lipid metabolism related genes and serum ALT were evaluated. Furthermore, serum LPS, the expression of Hif-1α, intestinal inflammation score, antioxidant capability and gut microbiota were tested. The survival rate and the expression of antiviral genes were analyzed after challenge by spring viremia of carp virus (SVCV). Results showed that dietary XMX-1 did not affect growth of zebrafish. However, dietary XMX-1 significantly decreased the level of serum LPS, intestinal inflammation score and intestinal MDA, as well as increased T-AOC and the expression of Hif-1α in zebrafish intestine (p < 0.05). Furthermore, XMX-1 supplementation decreased the relative abundance of Proteobacteria and increased Firmicutes and Actinobacteria. Additionally, XMX-1 supplementation significantly decreased hepatic steatosis score, hepatic TAG, serum ALT and increased the expression of lipolysis genes versus control (p < 0.05). Zebrafish fed XMX-1 diet exhibited higher survival rate after SVCV challenge. Consistently, dietary XMX-1 fermentation product increased the expression of IFNφ2 and IFNφ3 after 2 days of SVCV challenge and the expression of IFNφ1, IFNφ2 and MxC after 4 days of SVCV challenge in the spleen in zebrafish versus control (p < 0.05). In conclusion, our results indicate that dietary XMX-1 can improve liver and gut health, while enhancing antiviral immunity of zebrafish.

TM9SF4 is a novel regulator in lineage commitment of bone marrow mesenchymal stem cells to either osteoblasts or adipocytes.

BACKGROUND: Osteoporosis is a common bone disease in elderly population caused by imbalanced bone formation and bone resorption. Mesenchymal stem cells (MSCs) are responsible for maintaining this bone homeostasis. The phenotype of transmembrane 9 superfamily 4 (TM9SF4) knockout mice suggests a relationship between TM9SF4 proteins and bone homeostasis. But the effect of TM9SF4 in osteology has never been reported. In the present study, we investigated the function of TM9SF4 in MSC differentiation commitment, as well as its role in osteoporosis. METHODS: Primary bone marrow MSCs, isolated from TM9SF4 wildtype (TM9SF4+/+) and knockout (TM9SF4-/-) mice, were induced to differentiate into osteoblasts or adipocytes, respectively. The osteogenesis was examined by qRT-PCR detection of osteogenic markers, ALP staining and Alizarin Red S staining. The adipogenesis was tested by qRT-PCR quantification of adipogenic markers and Oil Red O staining. The cytoskeletal organization of MSCs was observed under confocal microscope. The osteoporotic model was induced by ovariectomy in TM9SF4+/+ and TM9SF4-/- mice, followed by Toluidine blue and H&E staining to assess lipid accumulation in trabecular bones, as well as micro-computed tomography scanning and immunohistochemistry staining for bone mass density assessment. The experiments on signaling pathways were conducted using qRT-PCR, Western blot and Alizarin Red S staining. RESULTS: We determined the role of TM9SF4 in MSC differentiation and found that TM9SF4-/- MSCs had higher potential to differentiate into osteoblasts and lower capability into adipocytes, without affecting osteoclastogenesis in vitro. In ovariectomy-induced osteoporotic model, TM9SF4-/- mice retained higher bone mass and less lipid accumulation in trabecular bones, indicating an important role of TM9SF4 in the regulation of osteoporosis. Mechanistically, TM9SF4-depleted cells showed elongated actin fibers, which may act through mTORC2/Akt/ß-catenin pathway to promote their commitment into osteoblasts. Furthermore, TM9SF4-depleted cells showed higher activity of canonical Wnt pathway, suggesting the participation of Wnt/ß-catenin during TM9SF4-regulated osteogenesis. CONCLUSIONS: Our study demonstrates TM9SF4 as a novel regulator for MSC lineage commitment. Depletion of TM9SF4 preferentially drives MSCs into osteoblasts instead of adipocytes. Furthermore, TM9SF4-/- mice show delayed bone loss and reduced lipid accumulation during ovariectomy-induced osteoporosis. Our results indicate TM9SF4 as a promising target for the future clinical osteoporotic treatment.

Endolysosomal ion channel MCOLN2 (Mucolipin-2) promotes prostate cancer progression via IL-1ß/NF-κB pathway.

BACKGROUND: Prostate cancer (Pca) is the most common cancer type among males worldwide. Dysregulation of Ca2+ signaling plays important roles during Pca progression. However, there is lack of information about the role of endolysosomal Ca2+ -permeable channels in Pca progression. METHODS: The expression pattern of MCOLN2 was studied by immunohistochemistry and western blot. Cell viability assay, transwell assay and in vivo tumorigenesis were performed to evaluate the functional role of MCOLN2. Downstream targets of MCOLN2 were investigated by cytokine array, enzyme-linked immunosorbent assay, Ca2+ release experiments and luciferase reporter assays. RESULTS: We report that MCOLN2 expression is significantly elevated in Pca tissues, and associated with poor prognosis. Overexpression of MCOLN2 promoted Pca cells proliferation, migration and invasion. Importantly, knockdown of MCOLN2 inhibited Pca xenograft tumor growth and bone lesion development in vivo. In addition, MCOLN2 promoted the production and release of IL-1ß. Moreover, luciferase reporter assay and western blot revealed that MCOLN2 promoted Pca development by regulating the IL-1ß/NF-κB pathway. CONCLUSION: In summary, MCOLN2 is crucially involved in Pca progression. Mechanistically, MCOLN2 regulates Pca progression via IL-1ß/NF-κB pathway. Our study highlights an intriguing possibility of targeting MCOLN2 as potential therapeutic strategy in Pca treatment.

Dietary Nucleotides Alleviate Hepatic Lipid Deposition via Exogenous AMP-Mediated AMPK Activation in Zebrafish.

BACKGROUND: Dietary nucleotides (NTs) have been reported to affect hepatic function and composition. However, the effects on hepatic lipid deposition are less studied. OBJECTIVES: We aimed to identify the regulatory role of dietary NTs in hepatic lipid deposition of zebrafish and elucidate the underlying mechanisms. METHODS: Zebrafish (60 ± 1.69 mg; 1 mo old) were fed control diet (16.2% energy as fat) or diet supplemented with 0.1% NTs or 0.02% AMP in feeding experiments 1 and 2. Experiment 3 was conducted with zebrafish larvae. In experiment 4, 1-mo-old zebrafish were fed a high-fat diet (HFD, 38.2% energy as fat) or an HFD supplemented with 0.1% NTs or 0.02% AMP. Hepatic lipid deposition was evaluated by triglyceride (TG) content and staining. Phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) was assayed by immunoblotting. Zebrafish liver (ZFL) cells were treated with exogenous adenosine. Small interfering RNA was used to knock down AMPK or nucleoside transporter SLC28a1 in ZFL cells. Vivo-morpholino was used to knock down AMPK in zebrafish larvae. RESULTS: Dietary 0.1% NTs or 0.02% AMP reduced hepatic TGs by 62% and 32%, respectively, compared with control (P < 0.05). Dietary AMP enhanced hepatic AMPK and ACC phosphorylation. Consistently, exogenous adenosine enhanced AMPK and ACC phosphorylation by 111% and 53%, respectively, in ZFL cells (P < 0.01) and reduced TG content by 56% (P < 0.05). Knockdown of AMPK and SLC28a1 abolished the effect of adenosine on lipid deposition in ZFL cells, and AMPK morpholino blocked the hepatic lipid-lowering effect of dietary AMP in vivo. Finally, dietary NTs and AMP activated AMPK and attenuated hepatic lipid deposition (28% and 30%, P < 0.05) in fish fed an HFD. CONCLUSIONS: Dietary NTs and AMP reduce hepatic lipid deposition in zebrafish, which involves exogenous AMP-mediated AMPK activation. Our results suggest that dietary NTs can contribute to alleviation of hepatic steatosis.

Ang II Promotes Cardiac Autophagy and Hypertrophy via Orai1/STIM1.

The pathophysiology of cardiac hypertrophy is complex and multifactorial. Both the store-operated Ca2+ entry (SOCE) and excessive autophagy are the major causative factors for pathological cardiac hypertrophy. However, it is unclear whether these two causative factors are interdependent. In this study, we examined the functional role of SOCE and Orai1 in angiotensin II (Ang II)-induced autophagy and hypertrophy using in vitro neonatal rat cardiomyocytes (NRCMs) and in vivo mouse model, respectively. We show that YM-58483 or SKF-96365 mediated pharmacological inhibition of SOCE, or silencing of Orai1 with Orail-siRNA inhibited Ang II-induced cardiomyocyte autophagy both in vitro and in vivo. Also, the knockdown of Orai1 attenuated Ang II-induced pathological cardiac hypertrophy. Together, these data suggest that Ang II promotes excessive cardiomyocyte autophagy through SOCE/Orai1 which can be the prime contributing factors in cardiac hypertrophy.

Research progress on the regulation of nutrition and immunity by microRNAs in fish.

MicroRNAs (miRNAs) are a class of highly conserved, endogenous non-coding single-stranded small RNA molecules with a length of 18-25 nucleotides. MiRNAs can negatively regulate the target gene through complementary pairing with the mRNA. It has been more than 20 years since the discovery of miRNA molecules, and many achievements have been made in fish research. This paper reviews the research progress in the regulation of fish nutrition and immunity by miRNAs in recent years. MiRNAs regulate the synthesis of long-chain polyunsaturated fatty acids, and are involved in the metabolism of glucose, lipids, as well as cholesterol in fish. Moreover, miRNAs play various roles in antibacterial and antiviral immunity of fish. They can promote the immune response of fish, but may also participate in the immune escape mechanism of bacteria or viruses. One important aspect of miRNAs regulation on fish immunity is mediated by targeting pattern recognition receptors and downstream signaling factors. Together, current results indicate that miRNAs are widely involved in the complex regulatory network of fish. Further studies on fish miRNAs may deepen our understanding of the regulatory network of fish nutrition and immunity, and have the potential to promote the development of microRNA-based products and detection reagents that can be applied in aquaculture industry.

Interactions between commensal bacteria and viral infection: insights for viral disease control in farmed animals.

Viral diseases cause serious economic loss in farmed animals industry. However, the efficacy of remedies for viral infection in farmed animals is limited, and treatment strategies are generally lacking for aquatic animals. Interactions of commensal microbiota and viral infection have been studied in recent years, demonstrating a third player in the interaction between hosts and viruses. Here, we discuss recent developments in the research of interactions between commensal bacteria and viral infection, including both promotion and inhibition effect of commensal bacteria on viral pathogenesis, as well as the impact of viral infection on commensal microbiota. The antiviral effect of commensal bacteria is mostly achieved through priming or regulation of the host immune responses, involving differential microbial components and host signaling pathways, and gives rise to various antiviral probiotics. Moreover, we summarize studies related to the interaction between commensal bacteria and viral infection in farmed animals, including pigs, chickens, fish and invertebrate species. Further studies in this area will deepen our understanding of antiviral immunity of farmed animals in the context of commensal microbiota, and promote the development of novel strategies for treatment of viral diseases in farmed animals.

Paraprobiotics and Postbiotics of Probiotic Lactobacilli, Their Positive Effects on the Host and Action Mechanisms: A Review.

Lactobacilli comprise an important group of probiotics for both human and animals. The emerging concern regarding safety problems associated with live microbial cells is enhancing the interest in using cell components and metabolites derived from probiotic strains. Here, we define cell structural components and metabolites of probiotic bacteria as paraprobiotics and postbiotics, respectively. Paraprobiotics and postbiotics produced from Lactobacilli consist of a wide range of molecules including peptidoglycans, surface proteins, cell wall polysaccharides, secreted proteins, bacteriocins, and organic acids, which mediate positive effect on the host, such as immunomodulatory, anti-tumor, antimicrobial, and barrier-preservation effects. In this review, we systematically summarize the paraprobiotics and postbiotics derived from Lactobacilli and their beneficial functions. We also discuss the mechanisms underlying their beneficial effects on the host, and their interaction with the host cells. This review may boost our understanding on the benefits and molecular mechanisms associated with paraprobiotics and probiotics from Lactobacilli, which may promote their applications in humans and animals.

Ability of prebiotic polysaccharides to activate a HIF1α-antimicrobial peptide axis determines liver injury risk in zebrafish.

Natural polysaccharides have received much attention for their ability to ameliorate hepatic steatosis induced by high-fat diet. However, the potential risks of their use have been less investigated. Here, we show that the exopolysaccharides (EPS) from Lactobacillus rhamnosus GG (LGG) and L. casei BL23 reduce hepatic steatosis in zebrafish fed a high-fat diet, while BL23 EPS, but not LGG EPS, induce liver inflammation and injury. This is due to the fact that BL23 EPS induces gut microbial dysbiosis, while LGG EPS promotes microbial homeostasis. We find that LGG EPS, but not BL23 EPS, can directly activate intestinal HIF1α, and increased HIF1α boosts local antimicrobial peptide expression to facilitate microbial homeostasis, explaining the distinct compositions of LGG EPS- and BL23 EPS-associated microbiota. Finally, we find that liver injury risk is not confined to Lactobacillus-derived EPS but extends to other types of commonly used natural polysaccharides, depending on their HIF1α activation efficiency.

Knockdown of TM9SF4 boosts ER stress to trigger cell death of chemoresistant breast cancer cells.

Drug resistance is one of the major obstacles to breast cancer therapy. However, the mechanisms of how cancer cells develop chemoresistance are still not fully understood. In the present study, we found that expression of TM9SF4 proteins was much higher in adriamycin (ADM)-resistant breast cancer cells MCF-7/ADM than in its parental line wild-type breast cancer cells MCF-7/WT. shRNA-mediated knockdown of TM9SF4 preferentially reduced cell growth and triggered cell death in chemoresistant MCF-7/ADM cells compared with MCF-7/WT cells. Knockdown of TM9SF4 also reduced cell growth and triggered cell death in chemoresistant MDA-MB-231/GEM cells. Mechanistic studies showed that TM9SF4 knockdown increased protein misfolding and elevated endoplasmic reticulum (ER) stress level in MCF-7/ADM cells, as indicated by aggresome formation and upregulated expression of ER stress markers, the effect of which was reversed by a small molecule chaperone 4-phenybutyric acid. In an athymic nude mouse model of ADM-resistant human breast xenograft tumor, knockdown of TM9SF4 decreased the growth of tumor xenografts. In chemoresistant breast cancer patients, chemotherapy increased the expression of TM9SF4 proteins in breast tumor samples. Taken together, these results uncovered a novel role of TM9SF4 proteins in alleviating ER stress and protecting chemoresistant breast cancer cells from apoptotic/necrotic cell death. These results highlight a possible strategy of targeting TM9SF4 to overcome breast cancer chemoresistance.

The TRPC5 channel regulates angiogenesis and promotes recovery from ischemic injury in mice.

Ischemia-related diseases are a leading cause of death worldwide, and promoting therapeutic angiogenesis is key for effective recovery from hypoxia-ischemia. Given the limited success of angiogenic factors, such as vascular endothelial growth factor, in clinical trials, it is important to find more promising angiogenic targets. Here, using both cell- and tissue-based assays and a mouse model of injury-induced ischemia, we investigated the involvement of the transient receptor potential canonical 5 (TRPC5) ion channel in angiogenesis and the effects of a TRPC5 activator, the Food and Drug Administration-approved drug riluzole, on recovery from ischemic injury. We demonstrate that TRPC5 is involved in endothelial cell sprouting, angiogenesis, and blood perfusion in an oxygen-induced retinopathy model and a hind limb ischemia model. We found a potential regulatory link between nuclear factor of activated T cell isoform c3 and angiopoietin-1 that could provide the mechanistic basis for the angiogenic function of TRPC5. Importantly, treatment with riluzole, which can activate TRPC5 in endothelial cells, improved recovery from ischemia in mice. Our study reveals TRPC5 as a potential angiogenic target and suggests riluzole as a promising drug for managing ischemic diseases.

Dietary nucleotides can directly stimulate the immunity of zebrafish independent of the intestinal microbiota.

In this study, we firstly tested the effects of dietary nucleotides on the disease resistance and innate immunity of zebrafish. Further, we investigated the role of intestinal microbiota in the nucleotides-induced immunostimulatory effect by using a germ-free zebrafish model and microbiota transfer technique. Fish were fed control or nucleotides (NT)-supplemented diets (at 0.05%,0.1%, 0.15% or 0.2%, m/m) for 4 weeks, followed by immersion challenge with Aeromonas hydrophila NJ-1. The results showed that 0.1% NT group enhanced the resistance of zebrafish against A. hydrophila infection. We further observed that the relative expressions of mucin, claudin16, occlusin1, hepcidin, defensin beta-like, myeloperoxidase (Mpo), and serum amyloid A (Saa) increased in the 0.1% NT group compared with control (P < 0.05), indicating that dietary nucleotides enhanced the physical barrier and mucosal immunity in the intestine of zebrafish. Moreover, ROS level in the head kidney was significantly increased in NT fed zebrafish versus control (P < 0.05), indicating enhanced systematic immunity. Furthermore, dietary NT significantly elevated the relative expressions of mpo, saa and the ROS activity in germ-free zebrafish, while germ-free zebrafish colonized with NT-altered microbiota had no significant difference in the relative expressions of mpo, saa and the ROS activity compared with the control microbiota-colonized fish, suggesting that the immunostimulatory effect of dietary NT is mediated by direct action of NT and does not involve the microbiota. Consistently, dietary NT can protect germ-free zebrafish from pathogenic infection, whereas germ-free zebrafish colonized with NT microbiota showed no difference in disease resistance compared with control microbiota colonized counterparts. Together, these results indicated that the immunostimulatory and disease protection effect of dietary nucleotides in zebrafish was mediated by direct action of the nucleotides, and does not involve the intestinal microbiota.

Aeromonas veronii and aerolysin are important for the pathogenesis of motile aeromonad septicemia in cyprinid fish.

Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ-free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono-infection, but also A. hydrophila NJ-1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii-targeted treatments in future efforts against MAS.

Treatment of hypertension by increasing impaired endothelial TRPV4-KCa2.3 interaction.

The currently available antihypertensive agents have undesirable adverse effects due to systemically altering target activity including receptors, channels, and enzymes. These effects, such as loss of potassium ions induced by diuretics, bronchospasm by beta-blockers, constipation by Ca2+ channel blockers, and dry cough by ACEI, lead to non-compliance with therapies (Moser, 1990). Here, based on new hypertension mechanisms, we explored a new antihypertensive approach. We report that transient receptor potential vanilloid 4 (TRPV4) interacts with Ca2+-activated potassium channel 3 (KCa2.3) in endothelial cells (ECs) from small resistance arteries of normotensive humans, while ECs from hypertensive patients show a reduced interaction between TRPV4 and KCa2.3. Murine hypertension models, induced by high-salt diet, N(G)-nitro-l-arginine intake, or angiotensin II delivery, showed decreased TRPV4-KCa2.3 interaction in ECs. Perturbation of the TRPV4-KCa2.3 interaction in mouse ECs by overexpressing full-length KCa2.3 or defective KCa2.3 had hypotensive or hypertensive effects, respectively. Next, we developed a small-molecule drug, JNc-440, which showed affinity for both TRPV4 and KCa2.3. JNc-440 significantly strengthened the TRPV4-KCa2.3 interaction in ECs, enhanced vasodilation, and exerted antihypertensive effects in mice. Importantly, JNc-440 specifically targeted the impaired TRPV4-KCa2.3 interaction in ECs but did not systemically activate TRPV4 and KCa2.3. Together, our data highlight the importance of impaired endothelial TRPV4-KCa2.3 coupling in the progression of hypertension and suggest a novel approach for antihypertensive drug development.

TrpC5 regulates differentiation through the Ca2+/Wnt5a signalling pathway in colorectal cancer.

Transient receptor potential channel 5 (TrpC5) is a member of the TrpC subgroup, and it forms a receptor-activated, non-selective Ca2+ channel. The architecture of the TrpC5 channel is poorly understood. In the present study, we report that TrpC5 is a key factor in regulating differentiation in colorectal cancer (CRC). Through a study of specimens from a large cohort of patients with CRC, we found that TrpC5 was highly expressed and its cellular level correlated with tumour grade. We showed further that up-regulated TrpC5 caused a robust rise in intracellular calcium concentration [Ca2+]i, increased Wnt5a expression and the nuclear translocation of ß-catenin, leading to a reduction in cancer differentiation and an increase in cancer cell stemness. Notably, patients with tumours that expressed high levels of TrpC5 showed significantly poorer disease-free and overall survival. Therefore, our findings suggest that TrpC5 is an independent adverse prognostic factor for death in CRC, reducing differentiation through the Ca2+/Wnt5a signalling pathway.