Hypermethylated genome of a fish vertebrate iridovirus ISKNV plays important roles in viral infection.

Iridoviruses are nucleocytoplasmic large dsDNA viruses that infect invertebrates and ectothermic vertebrates. The hypermethylated genome of vertebrate iridoviruses is unique among animal viruses. However, the map and function of iridovirus genomic methylation remain unknown. Herein, the methylated genome of Infectious spleen and kidney necrosis virus (ISKNV, a fish iridovirus), and its role in viral infection, are investigated. The methylation level of ISKNV is 23.44%. The hypermethylated genome is essential for ISKNV amplification, but there is no correlation between hypermethylation and viral gene expression. The hypomethylated ISKNV (obtained via 5-Azacytidine) activates a strong immunoreaction in vitro and reduces its pathogenicity in vivo. The unmethylated viral DNA can induce a stronger immunoreaction in vitro, whereas inactivated hypomethylated ISKNV can induce a stronger immunoreaction in vivo, suggesting ISKNV may evade from immune system by increasing its genome methylation level. Our work provides new insights into the role of genome methylation in viral infection.

Low-temperature immunization attenuates the residual virulence of orf074r gene-deleted infectious spleen and kidney necrosis virus: a candidate immersion vaccine.

IMPORTANCE: Global aquaculture production yielded a record of 122.9 million tons in 2022. However, ~10% of farmed aquatic animal production is lost each year due to various infectious diseases, resulting in substantial economic waste. Therefore, the development of vaccines is important for the prevention and control of aquatic infectious diseases. Gene-deletion live attenuated vaccines are efficacious because they mimic natural pathogen infection and generate a strong antibody response, thus showing good potential for administration via immersion. However, most gene-deletion viruses still have residual virulence, and thus, gene-deletion immersion vaccines for aquatic viruses are rarely developed. In this study, an orf074r deletion strain (Δorf074r) of ISKNV with residual virulence was constructed, and an immunization process was developed to reduce its residual virulence at 22°C, thereby making it a potential immersion vaccine against ISKNV. Our work will aid in the development of an aquatic gene-deletion live-attenuated immersion vaccine.

Development and characterization of a spleen cell line from yellowfin seabream Acanthopagrus latus and its susceptibility to Mandarinfish ranavirus.

Yellowfin seabream (Acanthopagrus latus) is one of the most commercially important marine fish in China. In this study, a new continuous cell line, named ALS cells, was developed from the spleen tissue of A. latus. The cell line was maintained in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 Ham (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and successfully cultured up to 50 passages. The cell line was authenticated by amplifying and sequencing mitochondrial cytochrome C oxidase subunit-I (coi-I) gene. The ALS cell line had the maximum growth rate in DMEM/F-12 medium containing 20% FBS at 27°C. Chromosome number analysis showed that the ALS cells have a modal diploid chromosome number of 34. The ALS cell line was transfected with the pEGFP-N1 plasmid, and green fluorescence was observed. The ALS cell line was used for testing Mandarinfish ranavirus (MRV) susceptibility, and the cytopathic effects in the cell line were observed at 4 days post-infection (dpi). Furthermore, the susceptibility of the ALS cell line to MRV and the levels of MRV mRNA and viral loads were found to be significantly increased at 1-7 dpi. This study revealed that the ALS cell line could be useful for molecular, virological, and biotechnological studies on yellowfin seabream.

Interaction of Teleost Fish TRPV4 with DEAD Box RNA Helicase 1 Regulates Iridovirus Replication.

Viral diseases are a significant risk to the aquaculture industry. Transient receptor potential vanilloid 4 (TRPV4) has been reported to be involved in regulating viral activity in mammals, but its regulatory effect on viruses in teleost fish remains unknown. Here, the role of the TRPV4-DEAD box RNA helicase 1 (DDX1) axis in viral infection was investigated in mandarin fish (Siniperca chuatsi). Our results showed that TRPV4 activation mediates Ca2+ influx and facilitates infectious spleen and kidney necrosis virus (ISKNV) replication, whereas this promotion was nearly eliminated by an M709D mutation in TRPV4, a channel Ca2+ permeability mutant. The concentration of cellular Ca2+ increased during ISKNV infection, and Ca2+ was critical for viral replication. TRPV4 interacted with DDX1, and the interaction was mediated primarily by the N-terminal domain (NTD) of TRPV4 and the C-terminal domain (CTD) of DDX1. This interaction was attenuated by TRPV4 activation, thereby enhancing ISKNV replication. DDX1 could bind to viral mRNAs and facilitate ISKNV replication, which required the ATPase/helicase activity of DDX1. Furthermore, the TRPV4-DDX1 axis was verified to regulate herpes simplex virus 1 replication in mammalian cells. These results suggested that the TRPV4-DDX1 axis plays an important role in viral replication. Our work provides a novel molecular mechanism for host involvement in viral regulation, which would be of benefit for new insights into the prevention and control of aquaculture diseases. IMPORTANCE In 2020, global aquaculture production reached a record of 122.6 million tons, with a total value of $281.5 billion. Meanwhile, frequent outbreaks of viral diseases have occurred in aquaculture, and about 10% of farmed aquatic animal production has been lost to infectious diseases, resulting in more than $10 billion in economic losses every year. Therefore, an understanding of the potential molecular mechanism of how aquatic organisms respond to and regulate viral replication is of great significance. Our study suggested that TRPV4 enables Ca2+ influx and interactions with DDX1 to collectively promote ISKNV replication, providing novel insights into the roles of the TRPV4-DDX1 axis in regulating the proviral effect of DDX1. This advances our understanding of viral disease outbreaks and would be of benefit for studies on preventing aquatic viral diseases.

Hybrid Blood Purification in the Treatment of Multiple Organ Dysfunction Syndrome Following a Wasp Attack.

Severe wasp sting symptoms can progress rapidly, often causing multiple organ dysfunction syndrome (MODS) and, in some cases, even death. Early and comprehensive treatment is needed to avoid these outcomes. Here, we report the case of a patient with MODS due to severe wasp stings. The patient received conventional treatment combined with glucocorticoids, plasma exchange, hemoperfusion, and continuous renal replacement therapy and had a successful recovery. MODS is a serious potential complication of wasp stings. Early local wound treatment, antiallergy interventions, antishock therapy, fluid replacement, glucocorticoid administration, and blood purification treatments are required to treat MODS secondary to wasp stings. Our results suggest that a hybrid blood purification method involving plasma exchange combined with hemoperfusion and continuous renal replacement therapy is more clinically effective than the single blood purification method. Early use of high-dose glucocorticoids combined with a hybrid blood purification treatment method had a positive effect in managing our patient and may improve the prognosis of other patients with MODS.

Predictive markers for severe hypocalcemia in dialysis patients with secondary hyperparathyroidism after near-total parathyroidectomy.

BACKGROUND: Secondary hyperparathyroidism (SHPT) is common in dialysis patients with end-stage renal disease (ESRD). Parathyroidectomy (PTX) is an effective treatment for SHPT. Postoperative severe hypocalcemia (SH) is a common and severe complication after PTX. This study aimed to investigate the potential predictive markers of SH in dialysis ESRD patients with SHPT after near-total PTX (near-tPTX) without autotransplantation (AT). METHODS: A retrospective analysis involving 131 dialysis patients with SHPT who were treated with near-tPTX without AT between January and August 2018 was performed. Demographic characteristics (age, gender, type of dialysis modality, etc.) and perioperative laboratory parameters [serum calcium, phosphorus, alkaline phosphatase (ALP), intact parathyroid hormone (iPTH), and bone metabolism markers] were collected and analyzed. Postoperative serum calcium level <1.875 mmol/L (7.5 mg/dL) was defined as postoperative SH. RESULTS: Among the 131 patients, 73 (55.7%) had postoperative hypocalcemia and 43 (32.8%) had postoperative SH. Univariate analysis showed that values of preoperative serum iPTH, calcium, ALP, bone-specific alkaline phosphatase (BAP), and osteocalcin (OC) were significantly different between the SH and non-SH groups. In the multivariate logistic regression model, preoperative serum ALP was an independent risk predictor of postoperative SH. The receiver operating characteristic (ROC) curve for preoperative serum ALP was 277 U/L. The sensitivity of preoperative serum ALP was 73.8% and the specificity was 63.2%. CONCLUSIONS: The incidence rates of postoperative hypocalcemia and SH in dialysis patients with SHPT after near-tPTX without AT were 55.7% and 32.8%, respectively. Preoperative serum ALP was an independent predictor for the occurrence of postoperative SH, and dialysis patients with SHPT were susceptible to postoperative SH when preoperative serum ALP level was >277 U/L. Hence, we recommend that preoperative serum ALP be utilized to complement clinical protocols for postoperative SH management of dialysis ESRD patients with SHPT after near-tPTX without AT.

Sulforaphane enhances the cisplatin sensitivity through regulating DNA repair and accumulation of intracellular cisplatin in ovarian cancer cells.

OBJECTIVES: Cisplatin is commonly applied as anticancer agent for various cancers, including ovarian cancer. Unfortunately, the drug resistance frequently occurred which obstructing the effect of cisplatin on tumors. The goal of our research was to investigate the reversal actions and the potential mechanisms of sulforaphane (SFN) on cisplatin resistance in ovarian carcinoma. METHODS: The A2780 and IGROV1 cells and their cisplatin resistance cells A2780/CP70 and IGROV1-R10 were used in this study. Cell viability was detected by CCK-8. The DNA repair was measured by comet assay. The cisplatin transporter proteins were measured with western blotting. The concentration of intracellular cisplatin was detected by HPLC. The luciferase activity assay was applied to determine the target site of miR-30a-3p on the 3'UTR of ERCC1 and ATP7A. A2780/CP70 and IGROV1-R10 xenograft mouse model were established to confirm the antineoplastic action of SFN combined with cisplatin. RESULTS: SFN reversed the resistance of A2780/CP70 and IGROV1-R10 ovarian carcinoma cells to cisplatin through inducing DNA damage and accumulation of intracellular cisplatin. SFN treatment notably increased miR-30a-3p expression, which was decreased in cisplatin-resistant cells. Moreover, overexpressed miR-30a-3p enhanced the sensitivity of A2780/CP70 and IGROV1-R10 cells to cisplatin treatment, and inhibiting miR-30a-3p activity abated the reversal actions of SFN on cisplatin resistance. The luciferase assay findings showed that miR-30a-3p binds to ERCC1 and ATP7A which are the key regulators for DNA repair and cisplatin transportation. CONCLUSIONS: Our findings indicated that SFN could enhance cisplatin sensitivity of ovarian carcinoma cells through up-regulating miR-30a-3p to induce DNA damage and accumulation of intracellular cisplatin.

Cyclosporine attenuates Paraquat-induced mitophagy and pulmonary fibrosis.

Objectives: Paraquat (PQ) poisoning can induce mitophagy and pulmonary fibrosis. Cyclosporine A (CsA) is an inhibitor of mitophagy. This study aimed at investigating whether CsA could inhibit PQ-induced mitophagy and pulmonary fibrosis in rats.Materials and Methods: Male Sprague-Dawley (SD) rats were treated with vehicle saline (control), 50 mg/kg PQ by gavage alone, or together with different doses of CsA. At 14 days post-induction, the levels of pulmonary fibrosis and PTEN-induced putative kinase 1 (PINK1) and Parkin expression in individual rats and mitochondrial membrane potential (MMP) in lung cells were measured. Moreover, A549 cells were treated with PQ or PQ + CsA for 24 h and the levels of PINK1, Parkin, fibronectin, collagen I and LC3 I and II expression and MMP were examined. Finally, the impact of PINK1 overexpression on the PQ or PQ + CsA-modulated fibronectin and collagen I expression in A549 cells was tested.Results: PQ exposure significantly increased the levels of hydroxyproline and collagen I expression and collagen fiber accumulation in the lung of rats, which were mitigated by CsA treatment. Furthermore, treatment with CsA significantly improved the PQ-decreased MMP and abrogated PQ-upregulated PINK1 and Parkin expression in the lungs of rats. In addition, CsA treatment decreased the PQ-induced fibrosis and mitophagy and PQ-impaired MMP as well as PQ-upregulated PINK1 and Parkin expression in A549 cells. The later effect of CsA was abrogated by PINK1 overexpression in A549 cells.Conclusions: Therefore, CsA can inhibit the PQ-induced mitophagy and pulmonary fibrosis by attenuating the PINK1/Parkin signaling.

ß-Hydroxybutyrate Ameliorates Aß-Induced Downregulation of TrkA Expression by Inhibiting HDAC1/3 in SH-SY5Y Cells.

Tyrosine kinase receptor A (TrkA) plays an important role in the protection of cholinergic neurons in Alzheimer's disease (AD). This study was designed to investigate whether ß-hydroxybutyrate (BHB), an endogenous histone deacetylase (HDAC) inhibitor, upregulates the expression of TrkA by affecting histone acetylation in SH-SY5Y cells treated with amyloid ß-protein (Aß). The results showed that BHB ameliorated the reduction of cell vitality and downregulation of TrkA expression induced by Aß. Furthermore, BHB inhibited the upregulation of HDAC1/2/3 expression and downregulation of histone acetylation (Ace-H3K9 and Ace-H4K12) levels in Aß-treated cells. The expression of TrkA was upregulated in HDAC1- or 3-silenced SH-SY5Y cells. However, there was no significant difference in TrkA expression between the HDAC2 knockdown and control cells. In conclusion, this study demonstrates that BHB protects against Aß-induced neurotoxicity in SH-SY5Y cells. The underlying mechanism of the effect may be associated with the upregulation of TrkA expression by inhibiting HDAC1/3.

Molecular cloning of Y-Box binding protein-1 from mandarin fish and its roles in stress-response and antiviral immunity.

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.

Effect of extended hours dialysis on markers of chronic kidney disease-mineral and bone disorder in the ACTIVE Dialysis study.

BACKGROUND: Chronic Kidney Disease - Mineral and Bone Disorder (CKD-MBD) is a significant cause of morbidity among haemodialysis patients and is associated with pathological changes in phosphate, calcium and parathyroid hormone (PTH). In the ACTIVE Dialysis study, extended hours dialysis reduced serum phosphate but did not cause important changes in PTH or serum calcium. This secondary analysis aimed to determine if changes in associated therapies may have influenced these findings and to identify differences between patient subgroups. METHODS: The ACTIVE Dialysis study randomised 200 participants to extended hours haemodialysis (≥24 h/week) or conventional haemodialysis (≤18 h/week) for 12 months. Mean differences between treatment arms in serum phosphate, calcium and PTH; and among key subgroups (high vs. low baseline phosphate/PTH, region, time on dialysis, dialysis setting and frequency) were examined using mixed linear regression. RESULTS: Phosphate binder use was reduced with extended hours (- 0.83 tablets per day [95% CI -1.61, - 0.04; p = 0.04]), but no differences in type of phosphate binder, use of vitamin D, dose of cinacalcet or dialysate calcium were observed. In adjusted analysis, extended hours were associated with lower phosphate (- 0.219 mmol/L [- 0.314, - 0.124; P < 0.001]), higher calcium (0.046 mmol/L [0.007, 0.086; P = 0.021]) and no change in PTH (0.025 pmol/L [- 0.107, 0.157; P = 0.713]). The reduction in phosphate with extended hours was greater in those with higher baseline PTH and dialysing at home. CONCLUSION: Extended hours haemodialysis independently reduced serum phosphate levels with minimal change in serum calcium and PTH levels. With a few exceptions, these results were consistent across patient subgroups. TRIAL REGISTRATION: Clinicaltrials.gov NCT00649298 . Registered 1 April 2008.

Intermittent Fasting Protects against Alzheimer's Disease Possible through Restoring Aquaporin-4 Polarity.

The impairment of amyloid-ß (Aß) clearance in the brain plays a causative role in Alzheimer's disease (AD). Polarity distribution of aquaporin-4 (AQP4) is important to remove Aß from brain. AQP4 polarity can be influenced by the ratio of two AQP4 isoforms M1 and M23 (AQP4-M1/M23), however, it is unknown whether the ratio of AQP4-M1/M23 changes in AD. Histone deacetylase 3 has been reported to be significantly increased in AD brain. Moreover, evidence indicated that microRNA-130a (miR-130a) possibly mediates the regulation of histone deacetylase 3 on AQP4-M1/M23 ratio by repressing the transcriptional activity of AQP4-M1 in AD. This study aimed to investigate whether intermittent fasting (IF), increasing the level of an endogenous histone deacetylases inhibitor ß-hydroxybutyrate, restores AQP4 polarity via miR-130a mediated reduction of AQP4-M1/M23 ratio in protection against AD. The results showed that IF ameliorated cognitive dysfunction, prevented brain from Aß deposition, and restored the AQP4 polarity in a mouse model of AD (APP/PS1 double-transgenic mice). Additionally, IF down-regulated the expression of AQP4-M1 and histone deacetylase 3, reduced AQP4-M1/M23 ratio, and increased miR-130a expression in the cerebral cortex of APP/PS1 mice. In vitro, ß-hydroxybutyrate was found to down-regulate the expression of AQP4-M1 and histone deacetylase 3, reduce AQP4-M1/M23 ratio, and increase AQP4-M23 and miR-130a expression in 2 µM Aß-treated U251 cells. Interestingly, on the contrary to the result observed in 2 µM Aß-treated cells, AQP4 expression was obviously decreased in cells exposed to 10 µM Aß. miR-130a mimic decreased the expression of AQP4-M1 and the ratio of AQP4-M1/M23, as well as silencing histone deacetylase 3 caused the up-regulation of AQP4 and miR-130a, and the reduction of AQP4-M1/M23 ratio in U251 cells. In conclusion, IF exhibits beneficial effects against AD. The mechanism may be associated with recovery of AQP4 polarity, resulting from the reduction of AQP4-M1/M23 ratio. Furthermore, ß-hydroxybutyrate may partly mediate the effect of IF on the reduction of AQP4-M1/M23 ratio in AD, in which miR-130a and histone deacetylase 3 may be implicated.

Beneficial Effects of Sulforaphane Treatment in Alzheimer's Disease May Be Mediated through Reduced HDAC1/3 and Increased P75NTR Expression.

Alzheimer's disease is an irreversible, progressive neurodegenerative disorder. The accumulation of Aß in the brain is thought to play a causative role in the development of cognitive dysfunction in Alzheimer's disease. The p75 neurotrophin receptor is of great importance to protect against the Aß burden and its expression is regulated by histone acetylation. This study investigated whether the phytochemical sulforaphane, a pan-histone deacetylase inhibitor, up-regulates the p75 neurotrophin receptor expression via affecting histone acetylation in protection against Alzheimer's disease. We found that sulforaphane ameliorated behavioral cognitive impairments and attenuated brain Aß burden in Alzheimer's disease model mice. Additionally, sulforaphane reduced the expression of histone deacetylase1, 2, and 3, up-regulated p75 neurotrophin receptor, and increased levels of acetylated histone 3 lysine 9 and acetylated histone 4 lysine 12 in the cerebral cortex of Alzheimer's disease model mice as well as in Aß-exposed SH-SY5Y cells. Furthermore, silencing of histone deacetylase1 and 3, but not histone deacetylase2, gene expression with small interfering RNA caused up-regulation of p75 neurotrophin receptor in SH-SY5Y cells. In conclusion, this study demonstrates that sulforaphane can ameliorate neurobehavioral deficits and reduce the Aß burden in Alzheimer's disease model mice, and the mechanism underlying these effects may be associated with up-regulation of p75 neurotrophin receptor mediated, apparently at least in part, via reducing the expression of histone deacetylase1 and 3.

[Effects of resveratrol on lipid metabolism in C57BL/6J mice].

OBJECTIVE: To investigate the effects of resveratrol on serum and liver lipids in C57BL/6J mice. METHODS: 30 male C57BL/6J mice were randomly assigned to the following three groups: control group given a normal diet, high-fat diet group and resveratrol group (22.5 mg/kg BW) were given high fat diet. the resveratrol were dissolved in 0.5% sodium carboxymethyl cellulose and were given to the Resveratrol group by intragastric administration. The levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), the levels of TG and TC in liver, and the pathological changes of liver were measured after the treatment lasted for 8 weeks. RESULTS: The serum TC, LDL-C, HDL-C levels of high-fat diet and resveratrol groups were higher than those of control group (P < 0.05), and the serum TC and LDL-C levels of high-fat diet were also higher than those of resveratrol group (P < 0. 05). But the serum TG levels of high-fat diet and resveratrol groups were lower than those of control group (P < 0.05). The TC content of liver in high-fat diet group were higher than those of control and resveratrol groups (P < 0.05). CONCLUSION: The TC content in C57BL/6J mice can be decreased by resveratrol (22.5 mg/kg BW).