Mandarin fish von Hippel-Lindau protein regulates the NF-κB signaling pathway via interaction with IκB to promote fish ranavirus replication.

The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( Siniperca chuatsi) VHL ( scVHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of scVHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα, scIKKß, scIκBα, or scp65. Co-immunoprecipitation (Co-IP) analysis identified scIκBα as a novel target protein of scVHL. Moreover, scVHL targeted scIκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.

Radiographic and clinical outcomes of minimally invasive surgery versus open osteotomies for the correction of hallux valgus.

PURPOSE: Hallux valgus is a common disease which causes pain and dysfunction of the foot. Although numerous methods of procedures have been introduced, a single procedure cannot correct all deformities of hallux valgus. The study aims to evaluate the radiographic and clinical effectiveness of a new minimally invasive surgery (MIS) versus open Chevron-Akin procedures. METHODS: This was a retrospective comparative study. Data were collected from May 2018 to January 2020. A total of 27 patients (31 feet) undergoing MIS for hallux valgus were included in this study. The average age of patients underwent MIS was 59.9 years. The mean follow-up was 25.1 months. Open osteotomies were performed in 30 patients (31 feet) during the same period. The mean age of these patients at the time of surgery was 59.1 years. The mean follow-up was 26.1 months. Pre-operative and post-operative radiographic outcome measures included HVA, IMA, DMAA, the Sgarlato's angle and the length of the first metatarsal, and distance between the dorsal cortex of first and second metatarsal necks. The AOFAS and VAS were used to assess foot function. RESULTS: The preoperative HVA in MIS group and open group were 34.8° and 33.1° respectively. The post-operative HVA were 20.4° and 13.7°. The pre-operative IMA in MIS group and open group were 13.0° and 12.1°. The post-operative IMA were 11.4° and 5.5° respectively. The pre-operative DMAA were 14.8° and 15.1° respectively. The post-operative DMAA were 6.3° and 8.7°. The AOFAS increased from 44.0 to 90.2 in MIS group and from 47.6 to 89.5 in open group. The VAS decreased from 7.3 to 1.3 in MIS group and from 7.1 to 1.2 in open group. CONCLUSION: Although open osteotomies were superior than MIS in HVA and IMA, MIS showed advantages in correcting DMAA. MIS provided equivalent functional outcomes compared to open surgery.

Hypoxia triggers the outbreak of infectious spleen and kidney necrosis virus disease through viral hypoxia response elements.

Hypoxia frequently occurs in aquatic environments, especially in aquaculture areas. However, research on the relationship between hypoxic aquatic environments with viral diseases outbreak is limited, and its underlying mechanisms remain elusive. Herein, we demonstrated that hypoxia directly triggers the outbreak of infectious spleen and kidney necrosis virus (ISKNV) disease. Hypoxia or activated hypoxia-inducible factor (HIF) pathway could remarkably increase the levels of viral genomic DNA, titers, and gene expression, indicating that ISKNV can response to hypoxia and HIF pathway. To reveal the mechanism of ISKNV respond to HIF pathway, we identified the viral hypoxia response elements (HREs) in ISKNV genome. Fifteen viral HREs were identified, and four related viral genes responded to the HIF pathway, in which the hre-orf077r promoter remarkably responded to the HIF pathway. The level of orf077r mRNA dramatically increased after the infected cells were treated with dimethyloxalylglycine (DMOG) or the infected cells/fish subjected to hypoxic conditions, and overexpressed orf077r could remarkably increase the ISKNV replication. These finding shows that hypoxic aquatic environments induce the expression of viral genes through the viral HREs to promote ISKNV replication, indicating that viral HREs might be important biomarkers for the evaluation of the sensitivity of aquatic animal viral response to hypoxia stress. Furthermore, the frequencies of viral HREs in 43 species aquatic viral genomes from 16 families were predicted and the results indicate that some aquatic animal viruses, such as Picornavirdea, Dicistronviridae, and Herpesviridae, may have a high risk to outbreak when the aquatic environment encounters hypoxic stress.

The Interaction of Mandarin Fish DDX41 with STING Evokes type I Interferon Responses Inhibiting Ranavirus Replication.

DDX41 is an intracellular DNA sensor that evokes type I interferon (IFN-I) production via the adaptor stimulator of interferon gene (STING), triggering innate immune responses against viral infection. However, the regulatory mechanism of the DDX41-STING pathway in teleost fish remains unclear. The mandarin fish (Siniperca chuatsi) is a cultured freshwater fish species that is popular in China because of its high market value. With the development of a high-density cultural mode in mandarin fish, viral diseases have increased and seriously restricted the development of aquaculture, such as ranavirus and rhabdovirus. Herein, the role of mandarin fish DDX41 (scDDX41) and its DEAD and HELIC domains in the antiviral innate immune response were investigated. The level of scDDX41 expression was up-regulated following treatment with poly(dA:dT) or Mandarin fish ranavirus (MRV), suggesting that scDDX41 might be involved in fish innate immunity. The overexpression of scDDX41 significantly increased the expression levels of IFN-I, ISGs, and pro-inflammatory cytokine genes. Co-immunoprecipitation and pull-down assays showed that the DEAD domain of scDDX41 recognized the IFN stimulatory DNA and interacted with STING to activate IFN-I signaling pathway. Interestingly, the HELIC domain of scDDX41 could directly interact with the N-terminal of STING to induce the expression levels of IFN-I and ISGs genes. Furthermore, the scDDX41 could enhance the scSTING-induced IFN-I immune response and significantly inhibit MRV replication. Our work would be beneficial to understand the roles of teleost fish DDX41 in the antiviral innate immune response.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Hallux Valgus Correction Comparing Percutaneous Oblique Osteotomy and Open Chevron Osteotomy at a 2-year Follow-up.

OBJECTIVE: This study aimed to compare the percutaneous oblique osteotomy (POO) and the open chevron osteotomy technique for correction of hallux valgus deformity at a 2-year follow-up. METHODS: This is a retrospective study of consecutive patients undergoing operative correction of hallux valgus using one of two techniques (POO vs open chevron osteotomy) from 2014 to 2018. Forty eight feet (41 patients) that underwent the POO was compared with 64 feet (58 patients) that underwent open chevron osteotomy. The hallux valgus angle (HVA), intermetatarsal angle (IMA) and American Orthopedic Foot & Ankle Society Hallux Metatarsophalangeal-Interphalangeal scores (AOFAS-HMI) were assessed preoperatively and postoperatively at the 1, 2-year follow-up. The Manchester-Oxford Foot Questionnaire (MOXFQ) were assessed preoperatively and postoperatively at the 2-year follow-up. The VAS score was collected preoperatively and on 2 weeks,1 year and 2-year follow-up. RESULTS: Both groups achieved significant correction of the hallux deformity. The HVA in the POO group during the follow-up period were 12.5 ± 2.22 and 17.9 ± 9.31, respectively, and in the open chevron group were 14.1 ± 6.78 and 14.8 ± 7.83, respectively. The IMA in the POO group during the follow-up period were 7.61 ± 1.63 and 6.94 ± 1.53, respectively, and in the open chevron group were 6.89 ± 3.06 and 6.97 ± 2.95, respectively. Postoperative MOXFQ scores in all domains were significantly improved in both groups, however there was no significant difference in the improvement of any domain between POO and open groups at a 2-year follow-up. The AOFAS HMI scores in the POO group during the follow-up period were 86.5 ± 10.7 and 85.2 ± 13.8, respectively, and in the open chevron group were 88.2 ± 10.8 and 79.5 ± 23.7, respectively. The VAS scores in the POO group during the follow-up period were 2.00 ± 0.98, 2.00 ± 0.99 and 1.55 ± 1.11, respectively, and in the open chevron group were 5.51 ± 1.45, 2.56 ± 2.88 and 2.56 ± 2.88 respectively. The 1-year and 2-year follow-up outcomes between POO and open groups showed no significant difference regarding AOFAS HMI scores and VAS scores, however the POO group showed statistically significant improvement of VAS scores in the postoperative 2 weeks (P < 0.001). There was no statistical significance between the POO and open group in terms of complications rates (8.3% vs 12.5%, P = 0.480). CONCLUSION: The POO technique is reliable and shows a comparable outcome to the open chevron osteotomy. However, the POO technique shows significantly less pain in the first 2 weeks after surgery.

Intra-articular opening osteotomy combined with lateral ligament reconstruction for varus ankle arthritis.

BACKGROUND: Takakura 3B ankle arthritis is featured as obliteration of ankle space with subchondral bone contact. Among these patients, some have medial distal tibial platform erosion. It is hard to treat this kind of patients. The purpose of this study was to evaluate the therapeutic outcomes of intra-articular opening osteotomy combined with lateral ligament reconstruction for Takakura 3B ankle arthritis with medial distal tibial platform erosion. METHODS: From September 2009 to May 2016, 17 patients with Takakura 3B ankle arthritis were reviewed, including 3 male and 14 female patients. All underwent the operation of intra-articular opening osteotomy combined with lateral ligament reconstruction. All patients were available for analysis. The main outcome measurements included TT angle, AOFAS score, VAS score, SF-36 scale, and AOS scale. RESULTS: All patients were followed for a mean follow-up of 87.2 months (range, 49 to 129 months). The VAS scale improved from 5.5 ± 1.6 to 2.3 ± 1.9. The mean AOFAS score improved from 47.7 ± 15.7 to 75.8 ± 12.0. The SF-36 scale improved from 41.6 ± 14.0 to 67.7 ± 14.6. The AOS improved from 60.9 ± 13.9 to 28.2 ± 17.7. The TT angle improved from 14.3 ± 5.0° to 5.3 ± 4.0°. The TAS and TLS changed from 83.4 ± 2.6° and 77.5 ± 2.3° to 90.7 ± 2.3° and 78.6 ± 2.2°. However, the LTAS was not corrected significantly. CONCLUSION: Intra-articular opening osteotomy combined with lateral ligament reconstruction is an effective method to treat varus ankle arthritis with medial distal tibial platform erosion.

Roles of extracellular matrix components in Tiger frog virus attachment to fathead minnow (Pimephales promelas) cells.

Tiger frog virus (TFV) belongs to the genus Ranavirus (family Iridoviridae) and causes significant harm in cultured frogs, resulting in substantial losses in ecological and economic field in Southern China. Attachment is the first step in viral life cycle, which is dependent on the interactions of virions with extracellular matrix (ECM) components. Studying this process will help in understanding virus infection and controlling viral diseases. In this study, the roles of primary ECM components in TFV attachment were investigated. The results on the kinetics of virus attachment showed TFV successful attachment to the cell surface as a relatively rapid process after TFV was used to inoculate cells for 10 min at 4 °C. Western blot and quantitative PCR analyses results showed that soluble fibronectin, collagen IV, laminin, or hyaluronic acid treatment with TFV caused no significant effect on virus attachment. Soluble heparin, heparan sulfate and chondroitin sulfate A/B could inhibit TFV attachment in a dose-dependent manner. Enzymic digestion by cell surface heparin/heparan sulfate using heparinase I, II, and III could significantly prevent TFV attachment, suggesting that heparan sulfate plays an important role in TFV attachment. Furthermore, the binding assays of heparin-agarose beads and virion showed that TFV virions specifically bound with heparin in a dose-dependent manner. Given that heparin is a structural analogue of heparan sulfate, the above results suggest that heparan sulfate might serve as an attachment factor of TFV infection. Our work would be beneficial to understand the mechanisms of TFV attachment and the interactions of TFV with cellular receptor(s).

The roles of mandarin fish STING in innate immune defense against Infectious spleen and kidney necrosis virus infections.

The mandarin fish Siniperca chuatsi is a cultured freshwater fish species that is popular in China because of its high market value. With the development of high-density cultural mode in mandarin fish, viral diseases such as Infectious spleen and kidney necrosis virus (ISKNV) are becoming increasingly serious. Stimulator of interferon genes (STING) is a central component in the innate immune response to cytosolic DNA and RNA derived from different pathogens. However, the roles of STING in innate immune response of mandarin fish remain unknown. In the present study, S. chuatsi STING (scSTING)-mediated host immune response against ISKNV infection was investigated. ScSTING transcription level increased remarkably in response to ISKNV infection, LPS, PMA, or poly (I:C) stimulation in mandarin fish fry (MFF-1) cells. Immunofluorescence results showed that scSTING localized majorly in the endoplasmic reticulum. scSTING overexpression remarkably increased the expression levels of scIFN-h, scMx, scISG15, scPKR, scViperin, scIL-1ß, scIL-18, and scTNF-α genes. IFN-ß-luciferase report assay results showed that the relative expressions of luciferin were remarkably increased in MFF-1 cells. Site mutation of serine (S) on C-terminus of scSTING showed that both S388 and S396 were important for mediated signaling. Furthermore, scSTING overexpression inhibited ISKNV infection, and knockdown of scSTING promoted ISKNV infection, indicating that scSTING could suppress ISKNV infection in MFF-1 cells. These observations suggested that the scSTING played an important role in innate immune against ISKNV infection. Our work would help elucidate the roles of teleost fish STING in innate immunity.

Deletion of the Infectious spleen and kidney necrosis virus ORF069L reduces virulence to mandarin fish Siniperca chuatsi.

Mandarin fish (Siniperca chuatsi) is a significant cultured species with high added value in China. With the expansion of farming, diseases of mandarin fish such as Infectious spleen and kidney necrosis virus (ISKNV) diseases are becoming more and more serious. Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 (HHLA2) is a type 1 transmembrane molecule with three extracellular Ig domains (IgV-IgC-IgV) and plays important roles in the T cell proliferation and tumorigenesis. The HHLA2-homologues have not been found in virus. In this study, a viral HHLA2 protein encoded by ISKNV ORF069L was identified and the virulence of the deleted ORF069L reconstruction ISKNV strain (ΔORF069L) was investigated. ISKNV ORF069L gene was predicted to encode a 222-amino acids peptide. The bioinformation analysis revealed that ISKNV ORF069L contained an Ig HHLA2 domain and was homologous to vertebrate B7-CD28 family proteins. The recombinant virus strain of ΔORF069L was constructed by homologous recombination technology. The virus titer and growth curves between ISKNV wild type (WT) and ΔORF069L on cellular level showed no significant differences indicating that the ORF069L did not influence the ISKNV replication. The expression levels of immune-related genes (Mx1, IL-1ß, IL-8, TNF-a and IgM) were increased in fish infected with ΔORF069L, compared to those in fish infected with ISKNV WT. Furthermore, the lethality caused by ΔORF069L declined by 40% compared with ISKNV WT, indicating that ORF069L was a virulence gene of ISKNV. Most importantly, the protection rate was nearly 100% for fish immunized with ΔORF069L strain. Those results suggested that ΔORF069L could be developed as a potential attenuated vaccine against ISKNV. Our work will be beneficial to promote the development of gene deletion attenuated vaccines for ISKNV disease.

Intestinal Microbiota of Grass Carp Fed Faba Beans: A Comparative Study.

Many reports of the intestinal microbiota of grass carp have addressed the microbial response to diet or starvation or the effect of microbes on metabolism; however, the intestinal microbiota of crisp grass carp has yet to be elucidated. Moreover, the specific bacteria that play a role in the crispiness of grass carp fed faba beans have not been elucidated. In the present study, 16S sequencing was carried out to compare the intestinal microbiota in the fore-, mid- and hind-intestine segments of grass carp following feeding with either faba beans or formula feed. Our results showed that (1) the hind-intestine presented significant differences in diversity relative to the fore- or midintestine and (2) faba beans significantly increased the diversity of intestinal microbiota, changed the intestinal microbiota structure (Fusobacteria was reduced from 64.26% to 18.24%, while Proteobacteria was significantly increased from 17.75% to 51.99%), and decreased the metabolism of energy, cofactors and vitamins in grass carp. Furthermore, at the genus and species levels, Acinetobacter accounted for 15.09% of the microbiota, and Acinetobacter johnsonii and Acinetobacter radioresistens constituted 3.41% and 2.99%, respectively, which indicated that Acinetobacter of the family Moraxellaceae contributed to changes in the intestinal microbiota structure and could be used as a potential biomarker. These results may provide clues at the intestinal microbiota level to understanding the mechanism underlying the crispiness of grass carp fed faba beans.

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.

Tiger frog virus ORF104R interacts with cellular VDAC2 to inhibit cell apoptosis.

Ranaviruses belong to the family Iridoviridae, and have become a serious threat to both farmed and natural populations of fish and amphibians. Previous reports showed that ranaviruses could encode viral Bcl-2 family-like proteins (vBcl-2), which play a critical role in the regulation of cell apoptosis. However, the mechanism of ranaviruses vBcl-2 interactions with host protein in mediating apoptosis remains unknown. Tiger frog virus (TFV) belonging to the genus Ranavirus has been isolated from infected tadpoles of Rana tigrina rugulosa, and it causes a high mortality rate among tiger frog tadpoles cultured in southern China. This study elucidated the molecular mechanism underlying the interaction of TFV ORF104R with the VDAC2 protein to regulate cell apoptosis. TFV ORF104R is highly similar to other ranaviruses vBcl-2 and host Mcl-1 proteins, indicating that TFV ORF104R is a postulate vBcl-2 protein. Transcription and protein expression levels showed that TFV orf104r was a late viral gene. Western blot results suggested that TFV ORF104R was a viral structural protein. Subcellular localization analysis indicated that TFV ORF104R was predominantly colocalized with the mitochondria. Overexpressed TFV ORF104R could suppress the release of cytochrome C and the activities of caspase-9 and caspase-3. These results indicated that TFV ORF104R might play an important role in anti-apoptosis. Furthermore, the interaction between TFV ORF104R and VDAC2 was detected by co-immunoprecipitation in vitro. The above observations suggest that the molecular mechanism of TFV-regulated anti-apoptosis is through the interaction of TFV ORF104R with the VDAC2 protein. Our study provided a mechanistic basis for the ranaviruses vBcl-2-mediated inhibition of apoptosis and improved the understanding on how TFV subverts host defense mechanisms in vivo.

Identification and functional analysis of the Mandarin fish (Siniperca chuatsi) hypoxia-inducible factor-1α involved in the immune response.

Mandarin fish (Siniperca chuatsi) is a popular cultured freshwater fish species due to its high market value in China. With increasing density of breeding, mandarin fish is often cultured under low environmental oxygen concentrations (hypoxia). In this study, the relative expression levels of hypoxia response element (HRE)-luciferase reporter and the HIF signaling pathway downstream genes (scldha, scvegf, and scglut-1) were significantly increased by hypoxic stress, thereby indicating that mandarin fish has an HIF signaling pathway. The mandarin fish HIF-1α (scHIF-1α) was also characterized. Multiple sequence alignments showed that scHIF-1α presented similar architectures to other known vertebrates. Subcellular localization analysis showed that scHIF-1α was mainly located in the nucleus of the mandarin fish fry-1 (MFF-1) cells. The role of scHIF-1α in the regulation of the HIF signaling pathway was confirmed. Overexpression of scHIF-1α could induce the HIF signaling pathway, whereas knockdown of scHIF-1α inhibited the activity of the HIF-1 signaling pathway. Tissue distribution analysis showed that schif-1α was significantly highly expressed in the blood, heart, and liver, which indicated that the main function of scHIF-1α was closely related to the circulatory system. Furthermore, scHIF-1α expression was significantly induced by poly I:C, poly dG:dC or PMA, thereby indicating that scHIF-1α was involved in the immune response. HIF-1α plays an important role in pathogen infections in mammals, but its role in fish is rarely investigated. Overexpression of scHIF-1α could inhibit MRV and SCRV infections, whereas knockdown of scHIF-1α could promote such infections. Those results suggested that scHIF-1α played an important role in fish virus infection. Our study will help understand the hypoxia associated with the outbreaks of aquatic viral disease.

Puerarin alleviates streptozotocin (STZ)-induced osteoporosis in rats through suppressing inflammation and apoptosis via HDAC1/HDAC3 signaling.

Diabetic osteoporosis is a severe public health concern in the world. Puerarin (PU) is extensively attractive due to its superior bioactivities. In the study, we found that PU protected against streptozotocin (STZ)-induced osteoporotic changes in rats. PU treatment improved STZ-induced diabetes in rats, as evidenced by the reduced serum glucose and insulin levels. PU administration markedly attenuated bone loss and tartarate-resistant acid phosphatase (TRAP) activity in STZ-induced rats. Bone mineral density (BMD) was significantly decreased in diabetic rats, while being prevented by PU. STZ-induced impairments in microarchitecture of femoral tissues were markedly alleviated by PU treatment. In addition, bone-specific alkaline phosphatase (BALP), osteoprotegerin (OPG) and Runt-related transcription factor 2 (Runx2) levels in serum or tibia were improved by PU in STZ-injected rats; however, TRACP isoform 5b (TRACP-5b), carboxy-terminal collagen cross-links (ß-CTX) and receptor activator of nuclear factor-κB ligand (RANKL) levels were decreased. Further, PU treatment inhibited inflammation and apoptosis in STZ-treated rats. Additionally, STZ injection increased histone deacetylase (HDAC)-1 and -3 expressions in femoral heads of rats, which were relieved by PU treatment. Notably, both HDAC1 and HDAC3 could enhance osteoporosis in vitro, as proved by the decreased ALP and Runx2 levels and the increased TRAP expression. Inflammation and apoptosis were exacerbated by HDAC1/3 over-expression, which were markedly diminished by PU treatment. In contrast, suppressing HDAC1/3 significantly abrogated fructose (Fru)-elicited inflammation and apoptosis in cells. Collectively, our data illustrated that PU is a potential therapeutic option to prevent diabetic osteoporosis by inhibiting HDAC1/HDAC3 signaling.

Realignment Surgery for Malunited Ankle Fracture.

OBJECTIVE: To investigate the characteristics and the results of realignment surgery for the treatment of malunited ankle fracture. METHODS: Thirty-three patients with malunited fractures of the ankle who underwent reconstructive surgery at our hospital from January 2010 to January 2014 were reviewed. The tibial anterior surface angle (TAS), the tibiotalar tilt angle (TTA), the malleolar angle (MA), and the tibial lateral surface angle (TLS) were measured. Clinical assessment was performed with use of the American Orthopaedic Foot and Ankle Society (AOFAS) scale and visual analogue scale (VAS) scores, and the osteoarthritis stage was determined radiographically with the modified Takakura classification system. The Wilcoxon matched-pairs test was used to analyze the difference between the preoperative and the postoperative data. RESULTS: The mean follow-up was 36 months (range, 20-60 months). The mean age at the time of realignment surgery was 37.1 years (range, 18-62 years). Compared with preoperation, the TAS at the last follow-up showed a significant increase (88.50° ± 4.47° vs. 90.80° ± 3.49°, P = 0.0035); similar results were observed in TTA (1.62° ± 1.66° vs. 0.83° ± 0.90°, P < 0.01) and MA (82.30° ± 8.03° vs. 78.70° ± 4.76°, P = 0.005). At the last follow-up, the mean AOFAS score was significantly increased compared with the score at preoperation (44.5 ± 13.7 vs. 78.0 ± 8.9, P < 0.01). Significant differences in VAS scores were found at the last follow-up (6.76 ± 1.03 vs. 2.03 ± 1.21, P < 0.01). There was no significant difference in the Takakura grade between the preoperation and the last follow-up. One patient had increased talar tilt postsurgery; the postoperative talar tilt angle of this patient was 20°. One patient had progressive ankle osteoarthritis, and was treated by ankle joint distraction. CONCLUSIONS: Realignment surgery for a malunited ankle fracture can reduce pain, improve function, and delay ankle arthrodesis or total ankle replacement. Postoperative large talar tilt and advanced stages of ankle arthritis are the risk factors for the surgery.

Identification and differential expression analysis of MicroRNAs encoded by Tiger Frog Virus in cross-species infection in vitro.

BACKGROUND: Tiger frog virus (TFV), dsDNA virus of the genus Ranavirus and family Iridoviridae, causes a high mortality of tiger frog tadpoles cultured in Southern China. MicroRNAs (miRNAs) have been identified in many viruses especially DNA viruses such as Singapore Grouper Iridoviruses (SGIV). MicroRNAs play important roles in regulating gene expression for virus subsistence in host. Considering that TFV infects cells of different species under laboratory conditions, we aim to identify the specific and essential miRNAs expressed in ZF4 and HepG2 cells. METHODS: We identified and predicted novel viral miRNAs in TFV-infected ZF4 and HepG2 cells by deep sequencing and software prediction. Then, we verified and described the expression patterns of TFV-encoded miRNAs by using qRT-PCR and Northern blot. RESULTS: Deep sequencing predicted 24 novel TFV-encoded miRNAs, and qRT-PCR verified 19 and 23 miRNAs in TFV-infected ZF4 (Group Z) and HepG2 (Group H) cells, respectively. Northern blot was performed to validate eight and five TFV-encoded miRNAs in Groups H and Z, respectively. We compared the expression of TFV-encoded miRNAs from two groups and defined TFV-miR-11 as the essential viral miRNA and TFV-miR-13 and TFV-miR-14 as the specific miRNAs that contribute to HepG2 cell infection. CONCLUSIONS: We identified novel viral miRNAs and compared their expression in two host cells. The results of this study provide novel insights into the role of viral miRNAs in cross-species infection in vitro.

Budding of Tiger Frog Virus (an Iridovirus) from HepG2 Cells via Three Ways Recruits the ESCRT Pathway.

The cellular endosomal sorting complex required for transport (ESCRT) pathway is a multifunctional pathway involved in cell physiological activities. While the majority of RNA viruses bearing L-domains are known to hijack the ESCRT pathway to complete the budding process, the budding of large and complex enveloped DNA viruses, especially iridoviruses, has been rarely investigated. In the present study, we use the tiger frog virus (TFV) as a model to investigate whether iridoviruses are released from host cells through the ESCRT pathway. Inhibition of class E proteins and auxiliary proteins (VPS4A, VPS4B, Tsg101, Alix, and Nedd4.1) reduces extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in TFV release. The respective interactions of TFV VP031L, VP065L, VP093L with Alix, Tsg101, Nedd4 suggest the underlying molecular mechanism by which TFV gets access to the ESCRT pathway. Co-depletion of Alix, Tsg101, and Nedd4.1 induces a significant reduction in extracellular virion production, which implies the functional redundancy of host factors in TFV budding. Those results are first observation that iridovirus gains access to ESCRT pathway through three ways of interactions between viral proteins and host proteins. Our study provides a better understanding of the budding mechanism of enveloped DNA viruses.

Tiger frog virus ORF080L protein interacts with LITAF and impairs EGF-induced EGFR degradation.

Tiger frog virus (TFV) belongs to the genus Ranavirus, family Iridoviridae, and causes severe mortality in commercial cultures in China. TFV ORF080L is a gene homolog of lipopolysaccharide-induced TNF-α factor (LITAF), which is a regulator in endosome-to-lysosome trafficking through its function in the endosomal sorting complex required for transport machinery. The characteristics and biological roles of TFV ORF080L were identified. TFV ORF080L was predicted to encode an 84-amino acid peptide (VP080L). It had high-sequence identity with mammalian LITAF, but lacked the N-terminus of LITAF, which contains two PPXY motifs. Transcription and protein level analyses showed that TFV ORF080L was a late viral gene. Localization in the virons also showed that TFV VP080L was a viral structural protein. Immunofluorescence staining showed that TFV ORF080L was predominantly colocalized with plasma membrane and partly distributed with the late endosome in infected HepG2 cells. SiRNA-mediated TFV ORF080L silencing decreased viral reproduction. Moreover, TFV ORF080L interacted with human/zebrafish LITAF and impaired EGF-induced EGFR degradation, thereby indicating that TFV ORF080L played a role in endosome-to-lysosome trafficking. These findings suggested that TFV ORF080L might negate the function of cellular LITAF to impair endosomal sorting and trafficking. Results provide a clue to the link between the dysregulated endosomal trafficking and iridovirus pathogenesis.

Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles.

Caveolae are flask-shaped invaginations of the plasma membrane. Caveolae play important roles in the process of viruses entry into host cells, but the roles of caveolae at the late stage of virus infection were not completely understood. Tiger frog virus (TFV) has been isolated from the diseased tadpoles of the frog, Rana tigrina rugulosa, and causes high mortality of tiger frog tadpoles cultured in Southern China. In the present study, the roles of caveolae at the late stage of TFV infection were investigated. We showed that TFV virions were localized with the caveolae at the late stage of infection in HepG2 cells. Disruption of caveolae by methyl-ß-cyclodextrin/nystatin or knockdown of caveolin-1 significantly increase the release of TFV. Moreover, the interaction between caveolin-1 and TFV major capsid protein was detected by co-immunoprecipitation. Those results suggested that caveolae restricted TFV release from the HepG2 cells. Caveolae-associated proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into TFV virions. Different combinations of proteolytic and/or detergent treatments with virions showed that caveolae-associated proteins were located in viral capsid of TFV virons. Taken together, caveolae might be a restriction factor that affects virus release and caveolae-associated proteins were incorporated in TFV virions.