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Abstract Background Ochratoxin A (OTA) is a toxin widely found in aquafeed ingredients, and hypoxia is a common problem in fish farming. In practice, aquatic animals tend to be more sensitive to hypoxia while feeds are contaminated with OTA, but no studies exist in this area. This research investigated the multiple biotoxicities of OTA and hypoxia combined on the liver of grass carp and explored the mitigating effect of curcumin (CUR). Methods A total of 720 healthy juvenile grass carp (11.06 ± 0.05 g) were selected and assigned randomly to 4 experimental groups: control group (without OTA and CUR), 1.2 mg/kg OTA group, 400 mg/kg CUR group, and 1.2 mg/kg OTA + 400 mg/kg CUR group with three replicates each for 60 d. Subsequently, 32 fish were selected, divided into normoxia (18 fish) and hypoxia (18 fish) groups, and subjected to hypoxia stress for 96 h. Results CUR can attenuate histopathological damage caused by coming to OTA and hypoxia by reducing vacuolation and nuclear excursion. The alleviation of this damage was associated with the attenuation of apoptosis in the mitochondrial pathway by decreasing the expression of the pro-apoptotic proteins Caspase 3, 8, 9, Bax, and Apaf1 while increasing the expression of the anti-apoptotic protein Bcl-2, and attenuation of endoplasmic reticulum stress (ERS) by reducing Grp78 expression and chop levels. This may be attributed to the fact that the addition of CUR increased the levels of catalase (CAT) and glutathione reductase (GSH), increased antioxidant capacity, and ensured the proper functioning of respiratory chain complexes I and II, which in turn reduced the high production of reactive oxygen species (ROS), thus alleviating apoptosis and ERS. Conclusions In conclusion, our data demonstrate the effectiveness of CUR in attenuating liver injury caused by the combination of OTA and hypoxia. This study confirms the feasibility and efficacy of adding natural products to mitigate toxic damage to aquatic animals.

D-mannose, essential for protein glycosylation, has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis. In addition to evaluating growth performance, we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine. All 1800 grass carp (16.20 ± 0.01 g) were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose (0.52, 1.75, 3.02, 4.28, 5.50 and 6.78 g/kg diet) for 70 d. The study revealed that D-mannose increased feed intake (P 0.05). D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value (P

Abstract Background Methionine (Met) is the only sulfur-containing amino acid among animal essential amino acids, and methionine deficiency (MD) causes tissue damage and cell death in animals. The common modes of cell death include apoptosis, autophagy, pyroptosis, necroptosis. However, the studies about the major modes of cell death caused by MD have not been reported, which worth further study. Methods Primary hepatocytes from grass carp were isolated and treated with different doses of Met (0, 0.5, 1, 1.5, 2, 2.5 mmol/L) to examine the expression of apoptosis, pyroptosis, autophagy and necroptosis-related proteins. Based on this, we subsequently modeled pyroptosis using lipopolysaccharides and nigericin sodium salt, then autophagy inhibitors chloroquine (CQ), AMP-activated protein kinase (AMPK) inhibitors compound C (CC) and reactive oxygen species (ROS) scavengers N-acetyl-L-cysteine (NAC) were further used to examine the expression of proteins related to pyroptosis, autophagy and AMPK pathway in MD-treated cells respectively. Results MD up-regulated B-cell lymphoma protein 2 (Bax), microtubule-associated protein 1 light chain 3 II (LC3 II), and down-regulated the protein expression levels of B-cell lymphoma-2 (Bcl-2), sequestosome 1 (p62), cleaved-caspase-1, cleaved-interleukin (IL)-1β, and receptor-interacting protein kinase (RIP) 1 in hepatocytes, while it did not significantly affect RIP3. In addition, MD significantly increased the protein expression of liver kinase B1 (LKB1), p-AMPK, and Unc-51-like kinase 1 (ULK1) without significant effect on p-target of rapamycin. Subsequently, the use of CQ increased the protein expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved-caspase-1, and cleaved-IL-1β inhibited by MD; the use of CC significantly decreased the protein expression of MD-induced LC3 II and increased the protein expression of MD-suppressed p62; then the use of NAC decreased the MD-induced p-AMPK protein expression. Conclusion MD promoted autophagy and apoptosis, but inhibited pyroptosis and necroptosis. MD inhibited pyroptosis may be related regarding the promotion of autophagy. MD activated AMPK by inducing ROS production which in turn promoted autophagy. These results could provide partial theoretical basis for the possible mechanisms of Met in ensuring the normal structure and function of animal organs. Furthermore, ferroptosis is closely related to redox states, it is worth investigating whether MD affects ferroptosis in hepatocytes.

Introduction: Osteoarthritis (OA) is a devastating whole-joint disease affecting a large population worldwide with no cure; its mechanism remains poorly defined. Abnormal mechanical stress is the main pathological factor of OA. Objectives: To investigate the effects of Piezo1 activation on OA development and progression and to explore Piezo1-targeting OA treatment. Methods: The expression levels of Piezo1 were determined in human OA cartilage and experimental OA mice. Mice with genetic Piezo1 deletion in chondrocytes or intra-articular injection of the Piezo1 activator Yoda1 were utilized to determine the effects on DMM-induced OA progression. Effects of artemisinin (ART), a potent antimalarial drug, on Piezo1 activation, chondrocyte metabolism and OA lesions were determined. Results: Piezo1 expression was elevated in articular chondrocytes in human OA and DMM-induced mouse OA cartilage. Piezo1 deletion in chondrocytes largely attenuates DMM-induced OA-like phenotypes. In contrast, intra-articular injection of Yoda1 aggravates the knee joint OA lesions in mice. PIEZO1 activation increases, while PIEZO1 siRNA knockdown decreases, expression of RUNX2 and catabolic enzymes MMP13 and ADAMTS5 in primary human articular chondrocytes in a PI3K-AKT dependent manner. We have provided strong evidence supporting that ART is a novel and potent inhibitor of Piezo1 activation in primary OA-HACs and all cell lines examined, including human endothelial HUVEC cells, ATDC5 chondrocyte-like cells and MLO-Y4 osteocytes-like cells. Results from in vitro experiments confirmed that ART decreases the Yoda1-induced increases in the levels of OA-related genes and p-PI3K and p-AKT proteins in OA-HACs and alleviates DMM-induced OA lesions in mice. Conclusions: We establish a critical role of Piezo1 in promoting OA development and progression and define ART as a potential OA treatment.

Abstract Background Ochratoxin A (OTA), a globally abundant and extremely hazardous pollutant, is a significant source of contamination in aquafeeds and is responsible for severe food pollution. The developmental toxicity of OTA and the potential relieving strategy of natural products remain unclear. This study screened the substance curcumin (Cur), which had the best effect in alleviating OTA inhibition of myoblast proliferation, from 96 natural products and investigated its effect and mechanism in reducing OTA myotoxicity in vivo and in vitro. Methods A total of 720 healthy juvenile grass carp, with an initial average body weight of 11.06 ± 0.05 g, were randomly assigned into 4 groups: the control group (without OTA and Cur), 1.2 mg/kg OTA group, 400 mg/kg Cur group, and 1.2 mg/kg OTA + 400 mg/kg Cur group. Each treatment consisted of 3 replicates (180 fish) for 60 d. Results Firstly, we cultured, purified, and identified myoblasts using the tissue block culture method. Through preliminary screening and re-screening of 96 substances, we examined cell proliferation-related indicators such as cell viability and ultimately found that Cur had the best effect. Secondly, Cur could alleviate OTA-inhibited myoblast differentiation and myofibrillar development-related proteins (MyoG and MYHC) in vivo and in vitro and improve the growth performance of grass carp. Then, Cur could also promote the expression of OTA-inhibited protein synthesis-related proteins (S6K1 and TOR), which was related to the activation of the AKT/TOR signaling pathway. Finally, Cur could downregulate the expression of OTA-enhanced protein degradation-related genes (murf1, foxo3a, and ub), which was related to the inhibition of the FoxO3a signaling pathway. Conclusions In summary, our data demonstrated the effectiveness of Cur in alleviating OTA myotoxicity in vivo and in vitro. This study confirms the rapidity, feasibility, and effectiveness of establishing a natural product screening method targeting myoblasts to alleviate fungal toxin toxicity. Graphical Abstract

Abstract Objective While joint immobilization is a useful repair method for intra-articular ligament injury and periarticular fracture, prolonged joint immobilization can cause multiple complications. A better understanding how joint immobilization and remobilization impact joint function and homeostasis will help clinicians develop novel strategies to reduce complications. Design We first determined the effects of long-term immobilization on joint pain and osteophyte formation in patients after an extraarticular fracture or ligament injury. We then developed a mouse model of joint immobilization and harvested the knee joint samples at 2, 4, and 8 weeks. We further determined the effects of remobilization on recovery of the osteoarthritis (OA) lesions induced by immobilization in mice. Results We found that the long-term (6 weeks) joint immobilization caused significant joint pain and osteophytes in patients. In mice, 2-week immobilization already induced moderate sensory innervation and increased pain sensitivity and infiltration in synovium without inducing marked osteophyte formation and cartilage loss. Long-term immobilization (4 and 8 weeks) induced more severe sensory innervation and inflammatory infiltration in synovium, massive osteophyte formation on both sides of the femoral condyle, and the edge of the tibial plateau and significant loss of the articular cartilage in mice. Remobilization, which ameliorates normal joint load and activity, restored to certain extent some of the OA lesions and joint function in mice. Conclusions Joint immobilization caused multiple OA-like lesions in both mice and humans. Joint immobilization induced progressive sensory innervation, synovitis, osteophyte formation, and cartilage loss in mice, which can be partially ameliorated by remobilization.

IntroductionHeavy metal-associated isoprenylated plant proteins (HIPPs) play vital roles in metal absorption, transport and accumulation in plants. However, so far, only several plant HIPPs have been functionally analyzed. In this study, a novel HIPP member OsHIPP17, which was involved in the tolerance to copper (Cu) was functionally characterized.MethodsIn this study, qRT-PCR, Yeast transgenic technology, Plant transgenic technology, ICP-MS and so on were used for research.ResultsOsHIPP17 protein was targeted to the nucleus. The Cu concentration reached 0.45 mg/g dry weight due to the overexpression of OsHIPP17 in yeast cells. Meanwhile, the overexpression of OsHIPP17 resulted in the compromised growth of Arabidopsis thaliana (Arabidopsis) under Cu stress. The root length of Oshipp17 mutant lines was also significantly reduced by 16.74- 24.36% under 25 mM Cu stress. The roots of Oshipp17 rice mutant showed increased Cu concentration by 7.25%-23.32%. Meanwhile, knockout of OsHIPP17 decreased the expression levels of OsATX1, OsZIP1, OsCOPT5 or OsHMA5, and increased the expression levels of OsCOPT1 or OsHMA4. Antioxidant enzyme activity was also reduced in rice due to the knockout of OsHIPP17. Moreover, the expression levels of cytokinin-related genes in plants under Cu stress were also affected by overexpression or knockout of OsHIPP17.DiscussionThese results implied that OsHIPP17 might play a role in plant Cu toxic response by affecting the expression of Cu transport genes or cytokinin-related genes. Simultaneously, our work may shed light on the underlying mechanism of how heavy metals affect the plant growth and provide a novel rice genetic source for phytoremediation of heavy metal-contaminated soil.

Abstract Background The Spo0B-associated GTP-binding protein (Obg) GTPase, has diverse and important functions in bacteria, including morphological development, DNA replication and ribosome maturation. Homologs of the Bacillus subtilis Obg have been also found in chloroplast of Oryza sativa, but their primary roles remain unknown. Results We clarify that OsObgC1 is a functional homolog of AtObgC. The mutant obgc1-d1 exhibited hypersensitivity to the DNA replication inhibitor hydroxyurea. Quantitative PCR results showed that the ratio of chloroplast DNA to nuclear DNA in the mutants was higher than that of the wild-type plants. After DAPI staining, OsObgC1 mutants showed abnormal nucleoid architectures. The specific punctate staining pattern of OsObgC1-GFP signal suggests that this protein localizes to the chloroplast nucleoids. Furthermore, loss-of-function mutation in OsObgC1 led to a severe suppression of protein biosynthesis by affecting plastid rRNA processing. It was also demonstrated through rRNA profiling that plastid rRNA processing was decreased in obgc1-d mutants, which resulted in impaired ribosome biogenesis. The sucrose density gradient profiles revealed a defective chloroplast ribosome maturation of obgc1-d1 mutants. Conclusion Our findings here indicate that the OsObgC1 retains the evolutionarily biological conserved roles of prokaryotic Obg, which acts as a signaling hub that regulates DNA replication and ribosome biogenesis in chloroplast nucleoids.

Uridine diphosphate glucose pyrophosphorylase (UDP-glucose pyrophosphorylase, UGPase), as one of the key enzymes in polysaccharide synthesis, plays important roles in the growth and development of plants. In this study, the DoUGP gene of Dendrobium officinale was overexpressed. The expression of DoUGP and genes playing roles in the same and other saccharide synthesis pathways was determined, and the total soluble polysaccharide was also tested in wild-type and transgenic seedlings. We also performed freezing and osmotic stress treatments to determine whether overexpression of DoUGP could influence stress resistance in transgenic seedlings. Results showed that mRNA expression levels of DoUGP and its metabolic upstream and downstream genes in the transgenic seedlings were increased compared to the expression of these genes in wild-type seedlings. Additionally, most CSLA genes involved in the biosynthesis of mannan polysaccharides were significantly upregulated. The total polysaccharide and mannose content of transgenic seedlings were increased compared to the content of wild type, and enhanced stress tolerance was found in the overexpressed seedlings compared to the wild type.