Molecular characterization and functional study of a galectin-9 from a teleost fish, Boleophthalmus pectinirostris.

Galectin-9, a tandem-repeat galectin, plays an important role in the regulation of innate immune response against various microbial infections. Here, galectin-9 from mudskipper (Boleophthalmus pectinirostris) was identified and named as BpGal-9. Putative BpGal-9 contains two conserved carbohydrate recognition domains (CRDs), one CRD within N-terminal (N-CRD) and the other one within C-terminal (C-CRD). Multi-alignment analysis indicated that BpGal-9 shared the highest amino acid sequence identity of 64.3 % with that of Southern platyfish (Xiphophorus maculatus). Phylogenetic analysis showed that BpGal-9 grouped tightly with other teleosts galectin-9 and was most closely related to that of Southern platyfish. BpGal-9 transcripts were more abundant in the intestine, and its expression upregulated significantly in the intestine, kidney, spleen, gills, and skin after Edwardsiella tarda infection. Meanwhile, BpGal-9 expression significantly increased in hemocytes and serum of mudskipper infected by E. tarda. The recombinant BpGal-9 (rBpGal-9) and rBpGal-9C-CRD could agglutinate all tested bacteria, whereas rBpGal-9N-CRD could only agglutinate three kinds of bacteria. When targeting the same bacteria, rBpGal-9 showed stronger agglutinating activities than rBpGal-9C-CRD or rBpGal-9N-CRD. In addition, the induction effect of three recombinant proteins on the mRNA expression of anti-inflammatory cytokines (BpIL-10 and BpTGF-ß) was better than that on the pro-inflammatory cytokines (BpIL-1ß and BpTNF-α). Our result suggested that the N-CRD and C-CRD of galectin-9 contribute differently to its multiple functions in innate immunity in teleosts.

CCS-UNet: a cross-channel spatial attention model for accurate retinal vessel segmentation.

Precise segmentation of retinal vessels plays an important role in computer-assisted diagnosis. Deep learning models have been applied to retinal vessel segmentation, but the efficacy is limited by the significant scale variation of vascular structures and the intricate background of retinal images. This paper supposes a cross-channel spatial attention U-Net (CCS-UNet) for accurate retinal vessel segmentation. In comparison to other models based on U-Net, our model employes a ResNeSt block for the encoder-decoder architecture. The block has a multi-branch structure that enables the model to extract more diverse vascular features. It facilitates weight distribution across channels through the incorporation of soft attention, which effectively aggregates contextual information in vascular images. Furthermore, we suppose an attention mechanism within the skip connection. This mechanism serves to enhance feature integration across various layers, thereby mitigating the degradation of effective information. It helps acquire cross-channel information and enhance the localization of regions of interest, ultimately leading to improved recognition of vascular structures. In addition, the feature fusion module (FFM) module is used to provide semantic information for a more refined vascular segmentation map. We evaluated CCS-UNet based on five benchmark retinal image datasets, DRIVE, CHASEDB1, STARE, IOSTAR and HRF. Our proposed method exhibits superior segmentation efficacy compared to other state-of-the-art techniques with a global accuracy of 0.9617/0.9806/0.9766/0.9786/0.9834 and AUC of 0.9863/0.9894/0.9938/0.9902/0.9855 on DRIVE, CHASEDB1, STARE, IOSTAR and HRF respectively. Ablation studies are also performed to evaluate the the relative contributions of different architectural components. Our proposed model is potential for diagnostic aid of retinal diseases.

Sema 3A as a biomarker of the activated mTOR pathway during hexavalent chromium-induced acute kidney injury.

Semaphorin 3A (sema 3A) is one of a class of secretory proteins belonging to a family of axon-directed factors found in podocytes, distal tubules, and collecting tubes of the kidney. It is considered to be a potential target molecule involved in the mammalian target of the rapamycin (mTOR) pathway in renal injury or renal diseases, but it has an unknown role in the course of hexavalent chromium-Cr(VI) induced nephrotoxicity. In the present study, an acute kidney injury (AKI) model in rats or cultured tubular epithelial HK-2 cells was employed for Cr(VI) exposure alone or in combination with rapamycin (Rap) or N-acetyl-l-cysteine (NAC) or recombinant sema 3A. The methods of histopathology, biochemics, and western blotting were applied to evaluate tubular injury and the role of sema 3A. The results showed that a significant increase of urinary sema 3A indicates an early occurrence of AKI exposed to Cr(VI), accompanied with a significant increase of tubular injury score and phosphorylated mTOR proteins. Further, Cr(VI) treatment, in combination with pretreatment of the mTOR pathway inhibitor, Rap, showed a considerably stronger protective effect of Rap in protecting against Cr(VI)-induced nephrotoxicity than that seen with the free radical scavenger NAC, highlighting the dominant renal protective role of the mTOR pathway in inhibiting toxicity by downregulating the expressed levels of sema 3A in renal tissue. This study has demonstrated that an increased expression of sema 3A occurs in Cr(VI)-induced AKI resulting from activation of the mTOR pathway, and that inhibition of this pathway has been shown to decrease the severity of the toxicity. In conclusion, this study has shown that increased urinary sema 3A is indicative of an activated mTOR pathway and is a valuable biomarker of the early AKI induced by Cr(VI) exposure.

Effects of alpha-tocopheryl acetate supplementation in preslaughter diet on antioxidant enzyme activities and fillet quality of commercial-size Sparus macrocephalus.

This study examined the effects of dietary alpha-tocopheryl acetate supplementation on antioxidant enzyme activities and fillet quality in commercial-size Sparus macrocephalus. Three hundred fish [main initial weight (350+/-12) g] were divided into three groups (E250, E500 and E1000) and reared in 9 cages. The fish were fed for 8 weeks with three diets containing different levels of dietary alpha-tocopheryl acetate (289, 553, 1 069 mg/kg). Over the experimental period, fish were fed to satiation and reached a final mean weight of (465+/-28) g without significant body weight difference and proximate composition difference. Fillet alpha-tocopherol was significantly (P<0.05) different between groups, reaching levels of 14.2, 22.1, 30.9 microg/mg fillet for groups E250, E500 and E1000, respectively. Total serum superoxide dismutase (SOD) activity increased significantly (P<0.05) in fish fed the diets high in alpha-tocopheryl acetate, but serum glutathione peroxidase (GPX) activity was unaffected. In storage on ice, fillets of fish fed the diets high in alpha-tocopheryl acetate exhibited significantly lower (P<0.05) levels of oxidation. These results suggested that increased dietary alpha-tocopheryl acetate could increase its flesh deposition, increase the activity of SOD and prevent lipid peroxidation of Sparus macrocephalus fillets in retail storage on ice.

Influence of fasting on muscle composition and antioxidant defenses of market-size Sparus macrocephalus.

The study was conducted to investigate fasting effects on flesh composition and antioxidant defenses of market-size Sparus macrocephalus. Two hundred fish (main initial weight 580 g) were divided into two groups (control and fasted) and reared in 6 cages. After two weeks of adaptation, group I fasted for 28 d; group II was fed normally as a control. In 3, 7, 14, 21 and 28 d, 6 fish per group were sampled for proximate flesh composition, liver antioxidant enzyme activities and malondialdehyde flesh content analyses. In fasted fish, the reduction of lipid content in muscle occurred after day 3, and, compared to controls, the content of protein decreased from day 14, the activities of liver antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) increased from day 3, and flesh malondialdehyde levels increased from day 21. Flesh fat reduction shows that fasting may be used as a technique to reduce flesh lipid content in Sparus macrocephalus. However, considering flesh protein loss and the subsequent oxidative stress, the fasting technique should be used with precautions.

[Detection of estrogenic effects of nonylphenol and bisphenol A in vitro reporter gene-based assays].

OBJECTIVE: To explore the estrogenic effects and disruptive mechanism of NP and BPA by reporter gene-based assays we developed. METHODS: pERE-Luc plamid was generated by inserting estrogen response element (ERE) fragment into MCS of pGL3-promoter vector. MCF7 cells were cotransfected with pERE-Luc and phRL-SV40 using Sofast transfection reagent. The cells then treated with 17beta-estradiol (E2), tamoxifen (Tam), nonylphenol(NP) and bisphenol A (BPA) and expression of the repoter gene in the cell lysates was assayed using Dual-Lucferase reporter assay system. RESULTS: The pERE-Luc plasmid was constructed. Luciferase activities of MCF7 cells transfected pERE-Luc showed dose-responed realitionship with E2. 1 x 10(-11) mol/L E2 could induce the expression of reporter gene and 1 x 10(-9) mol/L E2 resulted in the largest luciferase activity. E2 couldn't induce the luciferase activity without pERE-Luc. Tam is a complete antagonist, inhibited the E2-induced luciferase expression. NP induced the luciferase activity at concertrations > 1 x 10(-6) mol/L, BPA induced the luciferase activity at concertrations > 1 x 10(-6) mol/L. The estrogenic activity of NP was more than BPA. CONCLUSION: The assay we established is usful, NP and BPA showed estrogenic activities.

[Establishment of reporter gene-based assays for the identification of (anti) androgenic effects of chemicals in CHO cells].

OBJECTIVE: Develop reporter gene-based assays for the identification of (anti) androgenic effects of chemicals in CHO cells, explore the antiandrogenic effects of 2,4-DDT and methoxychlor. METHODS: Chinese Ovary cells were cotransfected with the human androgen receptor expression vector, mouse mammary tumour virus MMTV-luciferase vector and phRL-SV40 using the transfection reagent Sofast, test the luciferase activity. After treatment of the cells for 24 h with dihydrotestosterone (DHT), flutamide (FLU), validate the effectiveness and specificity, also test the effect of 2,4-DDT and methoxychlor (METH). RESULTS: DHT is a potent agonist, 1 x 10(-10) mol/L DHT could result in the enhancement of luciferase activity, FLU is a complete antagonist, that confirmed the effectiveness and specificity of test system. 2,4-DDT and METH are partial antagonists. 3 x 10(-7) mol/L and above 2,4-DDT and METH could inhibit the androgenic activity of DHT. CONCLUSION: The assay we developed is doful, 2,4-DDT and METH could antagonize the androgenic effects.

[Expression of sHsps of normal palate and cleft palate during mouse embryogenesis].

OBJECTIVE: To study the expression of sHsps in normal palate and cleft palate during mouse embryogenesis. METHODS: At GD10, gestational mice of the treatment and the control were administered with 80 mg/kg retinoic acid and the same volume vegetable oil separately, and the normal palate and cleft palate of embryos were harvested in GD15-GD17. The relative abundance of sHsps of all samples was measured by reverse transcript polymerase chain reaction (RT-PCR). RESULTS: In the normal limbs, except that there is no expression of Hspb10 at GD17, all the other Hsps expressed obviously in GD15-GD17. To the normal palates, the expressional abundance of Hsp20, Hsp25, Hsp27, Hsp32, Hspb2, Hspb3, Hspb7 was stable, that of Hsp30, Hspb5 was increased following the embryos aging, and the expressional peak of Hsp10, Hsp22, Hspb4 occurred at GD16. In GD15-GD17, the expressional abundance of Hsp30, Hsp32, Hspb4, Hspb10 of the cleft palates was higher than that of the normal palates, but the expressional abundance of Hsp60, Hspb5, Hspb9 of the cleft palates was lower than that of the normal palates. The expressional models of Hspb9, Hspb 10 of the normal palates were different from those of the cleft palates obviously. CONCLUSION: Except that there is no expression of HspblO at GD17, all the other Hsps expressed obviously in GD15-GD17 during normal clefts' development. To different Hsps, there is different expressional characteristic. Hsp30, Hsp32, Hspb4, Hspb10 maybe play a protective role in the stress action of cleft palate, and Hsp10, Hsp60, Hspb5, Hspb9, Hspb10 were maybe relative to cleft palate.