CYLD mediates human pulmonary artery smooth muscle cell dysfunction in congenital heart disease-associated pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a common complication of congenital heart disease (CHD). Deubiquitinase cylindromatosis (CYLD) has been reported to significantly aggravate vascular smooth muscle cell (VSMC) phenotypic transformation, proliferation, and migration. Here, we aimed to further investigate its roles and underlying mechanisms in the CHD-PAH development. The expression of CYLD in the lung tissues from CHD-PAH patients and monocrotaline (MCT) plus aortocaval (AV)-induced PAH rats, pulmonary artery smooth muscle cells (PASMCs) from MCT-AV-induced PAH rats, and human PASMCs (HPASMCs) was evaluated. After infection with CYLD siRNA or pcNDA3.1-CYLD, the proliferation, migration, and apoptosis of HPASMCs were measured using an EdU assay, transwell and scratch wound healing assays, and flow cytometric assay, respectively. An adeno-associated virus (AAV) vector encoding CYLD was used to suppress CYLD expression by being intratracheally instilled in rats 7 days before MCT-AV treatment. The results showed that CYLD was increased in the lung tissues from CHD-PAH patients and MCT-AV-induced PAH rats, and in PASMCs from MCT-AV-induced PAH rats. The contractile-type HPASMCs expressed low levels of CYLD, while the proliferative synthetic-type HPASMCs expressed high levels of CYLD. In addition, CYLD could mediate HPASMC dysfunction, which regulated HPASMC phenotypic transformation and proliferation via the modulation of p38 and ERK activation, while CYLD regulated HPASMC migration via the modulation of p38 activation. In vivo results demonstrated that the local suppression of CYLD expression could attenuate the increased levels of PAH and its associated pulmonary vascular remodeling in MCT-AV-induced PAH rats. Collectively, these results indicated that CYLD might be a potential novel therapeutic target for the prevention of PAH and pulmonary vascular remodeling in CHD-PAH through the modulation of HPASMC dysfunction.

Balloon pulmonary angioplasty reverse right ventricular remodelling and dysfunction in patients with inoperable chronic thromboembolic pulmonary hypertension: a systematic review and meta-analysis.

OBJECTIVES: Right ventricular (RV) function is considered the major determinant of prognosis in patients with chronic thromboembolic pulmonary hypertension (CTEPH). The aim of this meta-analysis was to evaluate RV remodelling and function following balloon pulmonary angioplasty (BPA) in patients with inoperable CTEPH or persistent/recurrent pulmonary hypertension (PH) after pulmonary endarterectomy (PEA). METHODS: We reviewed all studies evaluating RV function by cardiac magnetic resonance (CMR) and/or echocardiography pre- and post-BPA from PubMed/Medline prior to 15 December 2019. Ten (299 patients) of the 29 studies retrieved met the inclusion criteria: 5 CMR and 5 echocardiography studies. The systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines. RESULTS: Pooled data from CMR studies revealed BPA resulted in a significantly decreased RV end-diastolic volume index (weighted mean difference (WMD) - 28.33 ml/m2, p < 0.00001) and RV end-systolic volume index (WMD - 29.06 ml/m2, p < 0.00001) accompanied by an increased RV ejection fraction (RVEF, WMD 8.97%, p < 0.00001). Data from the echocardiography studies showed BPA resulted in decreased RV basal diameter (WMD - 0.37 cm, p = 0.0009) and an increase of RV fractional area change (WMD 5.97 %, p = 0.003), but improvements of tricuspid annular plane systolic excursion (TAPSE) and S' were not significant. CONCLUSIONS: BPA improves RVEF and decreases RV volumes in patients with inoperable CTEPH or persistent/recurrent PH after PEA. KEY POINTS: • Balloon pulmonary angioplasty improves RVEF and decreases RV volumes in patients with inoperable CTEPH or persistent/recurrent PH after PEA.

Litopenaeus vannamei Sma and Mad related protein 5 gene is involved in stress response and white spot syndrome virus infection.

Cell survival is based on the stability of intracellular state. It was well known that biochemical reactions in cells require specific intracellular environments, such as pH and calcium concentration. While the mechanism of stabilizing the intracellular environment is complex and far from clear. In this study, a Sma and Mad related protein 5 gene (LvSmad5) of Litopenaeus vannamei was cloned. LvSmad5 was located to both cytoplasm and nucleus. And subcellular localization of LvSmad5 was responsed to the changing of cells internal and external environment. Besides, it was found that subcellular localization of LvSmad5 was also regulated by unfolded protein response. Moreover, it was proved that nucleic localization of LvSmad5 could significantly increase the white spot syndrome virus (WSSV) infection in shrimp, and knockdown expression of LvSmad5 decreased the cumulative mortality of WSSV infection shrimp. Further investigation revealed that cytoplasm LvSmad5 could interplay with shrimp hexokinase 1, and contribute to glycolysis. These results indicated that LvSmad5 played a role in L. vannamei environmental stress response, and was used by WSSV for its replication.

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.

Nestin represents a potential marker of pulmonary vascular remodeling in pulmonary arterial hypertension associated with congenital heart disease.

OBJECTIVE: Reportedly, nestin was re-expressed in proliferative synthetic-type pulmonary artery smooth muscle cells (PASMCs) and obligatory for PASMC proliferation in pulmonary arterial hypertension (PAH). Accordingly, nestin is increased in pulmonary vascular lesions of congenital heart disease (CHD)-associated PAH patients. We tested the hypothesis whether nestin was re-expressed in proliferative synthetic-type PASMCs and associated with pulmonary vascular remodeling in CHD-PAH. MATERIALS AND METHODS: Nestin expression was tested using lung tissues from CHD-PAH patients and monocrotaline (MCT) plus aortocaval (AV) shunt-induced PAH rats, human PASMCs (HPASMCs), and pulmonary artery endothelial cells (PAECs) and PASMCs from MCT-AV-induced PAH rats. The role and possible mechanism of nestin on HPASMC proliferation, apoptosis, cell cycle and migration were investigated by assays of CCK-8, EdU, TUNEL, flow cytometry, transwell chamber and immunoblotting assays. RESULTS: Nestin was solely expressed in proliferative synthetic-type PASMCs, but rarely detected in PAECs. Nestin was barely detected in normal pulmonary arterioles and occlusive pulmonary vascular lesions. Its expression was robustly increased in developing pulmonary vasculature, but returned to normal levels at the late stage of pulmonary vascular remodeling in lung tissues from CHD-PAH patients and MCT-AV-induced PAH rats. Besides, nestin peaks were consistent with the histological features in lung tissues of MCT-AV-induced PAH rats. Moreover, nestin overexpression effectively promoted HPASMC phenotypic transformation, proliferation, apoptosis resistance and migration via enhancing Wnt/ß-catenin activation. CONCLUSIONS: These data indicated that nestin was re-expressed in proliferative synthetic-type PASMCs and might represent a potential marker of pulmonary vascular remodeling in CHD-PAH.

Evaluation of Macitentan in Patients With Eisenmenger Syndrome.

BACKGROUND: Eisenmenger syndrome describes congenital heart disease-associated severe pulmonary hypertension accompanied by right-to-left shunting. The multicenter, double-blind, randomized, placebo-controlled, 16-week, phase III MAESTRO study (Macitentan in Eisenmenger Syndrome to Restore Exercise Capacity) evaluated the efficacy and safety of the endothelin receptor antagonist macitentan in patients with Eisenmenger syndrome. METHODS: Patients with Eisenmenger syndrome aged ≥12 years and in World Health Organization functional class II-III were randomized 1:1 to placebo or macitentan 10 mg once daily for 16 weeks. Patients with complex cardiac defects, Down syndrome and background PAH therapy were eligible. The primary end point was change from baseline to week 16 in 6-minute walk distance. Secondary end points included change from baseline to week 16 in World Health Organization functional class. Exploratory end points included NT-proBNP (N-terminal pro-B-type natriuretic peptide) at end of treatment expressed as a percentage of baseline. In a hemodynamic substudy, exploratory end points included pulmonary vascular resistance index (PVRi) at week 16 as a percentage of baseline. RESULTS: Two hundred twenty six patients (macitentan n=114; placebo n=112) were randomized. At baseline, 60% of patients were in World Health Organization functional class II and 27% were receiving phosphodiesterase type-5 inhibitors. At week 16, the mean change from baseline in 6-minute walk distance was 18.3 m and 19.7 m in the macitentan and placebo groups (least-squares mean difference, -4.7 m; 95% confidence limit (CL), -22.8, 13.5; P=0.612). World Health Organization functional class improved from baseline to week 16 in 8.8% and 14.3% of patients in the macitentan and placebo groups (odds ratio, 0.53; 95% CL, 0.23, 1.24). NT-proBNP levels decreased with macitentan versus placebo (ratio of geometric means, 0.80; 95% CL, 0.68, 0.94). In the hemodynamic substudy (n=39 patients), macitentan decreased PVRi compared with placebo (ratio of geometric means, 0.87; 95% CL, 0.73, 1.03). The most common adverse events with macitentan versus placebo were headache (11.4 versus 4.5%) and upper respiratory tract infection (9.6 versus 6.3%); a hemoglobin decrease from baseline of ≥2 g/dL occurred in 36.0% versus 8.9% of patients. Five patients (3 macitentan; 2 placebo) prematurely discontinued treatment and 1 patient died (macitentan group). CONCLUSIONS: Macitentan did not show superiority over placebo on the primary end point of change from baseline to week 16 in exercise capacity in patients with Eisenmenger syndrome. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT01743001.

The Legionella pneumophila effector WipA disrupts host F-actin polymerisation by hijacking phosphotyrosine signalling.

The major virulence determinant of Legionella pneumophila is the type IVB secretion system (T4BSS), which delivers approximately 330 effector proteins into the host cell to modulate various cellular processes. However, the functions of most effector proteins remain unclear. WipA, an effector, was the first phosphotyrosine phosphatase of Legionella with unknown function. In this study, we found that WipA induced relatively strong growth defects in yeast in a phosphatase activity-dependent manner. Phosphoproteomics data showed that WipA was likely involved into endocytosis, FcγR-mediated phagocytosis, tight junction, and regulation of actin cytoskeleton pathways. Western blotting further confirmed WipA dephosphorylates several proteins associated with actin polymerisation, such as p-N-WASP, p-ARP3, p-ACK1, and p-NCK1. Thus, we hypothesised that WipA targets N-WASP/ARP2/3 complex signalling pathway, leading to disturbance of actin polymerisation. Indeed, we demonstrated that WipA inhibits host F-actin polymerisation by reducing the G-actin to F-actin transition during L. penumophila infection. Furthermore, the intracellular proliferation of wipA/legK2 double mutant was significantly impaired at the late stage of infection, although the absence of WipA does not confer any further effect on actin polymerisation to the legK2 mutant. Collectively, this study provides unique insights into the WipA-mediated regulation of host actin polymerisation and assists us to elucidate the pathogenic mechanisms of L. pnuemophila infection.

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.

A child with household transmitted COVID-19.

BACKGROUND: Although people of all ages are susceptible to the novel coronavirus infection, which is presently named "Coronavirus Disease 2019" (COVID-19), there has been relatively few cases reported among children. Therefore, it is necessary to understand the clinical characteristics of COVID-19 in children and the differences from adults. CASE PRESENTATION: We report one pediatric case of COVID-19. A 14-month-old boy was admitted to the hospital with a symptom of fever, and was diagnosed with a mild form of COVID-19. The child's mother and grandmother also tested positive for SARS-CoV-2 RNA. However, the lymphocyte counts were normal. The chest computed tomography (CT) revealed scattered ground glass opacities in the right lower lobe close to the pleura and resorption after the treatment. The patient continued to test positive for SARS-CoV-2 RNA in the nasopharyngeal swabs and stool at 17 days after the disappearance of symptoms. CONCLUSION: The present pediatric case of COVID-19 was acquired through household transmission, and the symptoms were mild. Lymphocyte counts did not significantly decrease. The RNA of SARS-CoV-2 in stool and nasopharyngeal swabs remained positive for an extended period of time after the disappearance of symptoms. This suggests that attention should be given to the potential contagiousness of pediatric COVID-19 cases after clinical recovery.

Effects of environmental stress on shrimp innate immunity and white spot syndrome virus infection.

The shrimp aquaculture industry is plagued by disease. Due to the lack of deep understanding of the relationship between innate immune mechanism and environmental adaptation mechanism, it is difficult to prevent and control the diseases of shrimp. The shrimp innate immune system has received much recent attention, and the functions of the humoral immune response and the cellular immune response have been preliminarily characterized. The role of environmental stress in shrimp disease has also been investigated recently, attempting to clarify the interactions among the innate immune response, the environmental stress response, and disease. Both the innate immune response and the environmental stress response have a complex relationship with shrimp diseases. Although these systems are important safeguards, allowing shrimp to adapt to adverse environments and resist infection, some pathogens, such as white spot syndrome virus, hijack these host systems. As shrimp lack an adaptive immune system, immunization therapy cannot be used to prevent and control shrimp disease. However, shrimp diseases can be controlled using ecological techniques. These techniques, which are based on the innate immune response and the environmental stress response, significantly reduce the impact of shrimp diseases. The object of this review is to summarize the recent research on shrimp environmental adaptation mechanisms, innate immune response mechanisms, and the relationship between these systems. We also suggest some directions for future research.

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.

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.

Functional characterization of an ER-stress responding Crustin gene in Litopenaeus vannamei.

Shrimp in culture ponds are challenged by various pathogens as well as harsh water environment. The innate immune system and environmental stress response system of shrimp paly an important role in shrimp survival and growth. For remission the endoplasmic reticulum (ER)-stress caused by environmental stress, unfolded protein response (UPR) may reduce the synthesis of most proteins, including great mass of immune factors, which could weaken the immune function of shrimp. Therefore, how cells keep appropriate amount of immune factor synthesis under such a situation is critical important for shrimp health and growth. In this study, we cloned a new Crustin gene (LvCruU) from Litopenaeus vannamei. We showed that LvCruU has antibacterial activity, and reducing its expression would increase the cumulative mortality of L. vannamei upon the Vibrio parahemolyticus infection. In addition, we found that promoter activity of LvCruU was enhanced not only by the deformed epidermal autoregulatory factor-1 (Deaf1), but also by activating transcription factor 3 (LvATF3) of shrimp UPR. Real-time RT-PCR showed that LvCruU and LvATF3 both were induced upon UPR activation. And moreover, in Thapsigargin plus dsLvCruU injection test, we showed that down-regulation of LvCruU increased the cumulative mortality of V. parahemolyticus-infected shrimp under ER-stress. These results suggest that LvCruU work as a downstream effector of UPR, and contribute to antimicrobic immune response upon ER-stress in L. vannamei.

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.

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.

Riociguat for the treatment of pulmonary arterial hypertension associated with connective tissue disease: results from PATENT-1 and PATENT-2.

BACKGROUND: The 12-week, phase III Pulmonary Arterial hyperTENsion sGC-stimulator Trial (PATENT)-1 study investigated riociguat in patients with pulmonary arterial hypertension (PAH). Here, we present a prospectively planned analysis of the safety and efficacy of riociguat in the subgroup of patients with PAH associated with connective tissue disease (PAH-CTD). METHODS: Patients with PAH-CTD were further classified post hoc as having PAH associated with systemic sclerosis or PAH-other defined CTD. In PATENT-1, patients received riociguat (maximum 2.5 or 1.5 mg three times daily) or placebo. Efficacy endpoints included change from baseline in 6-minute walking distance (6MWD; primary endpoint), haemodynamics and WHO functional class (WHO FC). In the long-term extension PATENT-2, patients received riociguat (maximum 2.5 mg three times daily); the primary endpoint was safety and tolerability. RESULTS: In patients with PAH-CTD, riociguat increased mean 6MWD, WHO FC, pulmonary vascular resistance and cardiac index. Improvements in 6MWD and WHO FC persisted at 2 years. Two-year survival of patients with PAH-CTD was the same as for idiopathic PAH (93%). Riociguat had a similar safety profile in patients with PAH-CTD to that of the overall population. CONCLUSIONS: Riociguat was well tolerated and associated with positive trends in 6MWD and other endpoints that were sustained at 2 years in patients with PAH-CTD. TRIAL REGISTRATION NUMBERS: PATENT-1 (NCT00810693), PATENT-2 (NCT00863681).

Litopenaeus vannamei activating transcription factor 6 alpha gene involvement in ER-stress response and white spot symptom virus infection.

A previous study found that inositol-requiring enzyme-1-X-box binding protein 1 (IRE1-XBP1) pathway and the protein kinase RNA (PKR)-like ER kinase-eIF2α (PERK-eIF2α) pathway of shrimp play roles in the unfolded protein response (UPR). And they also be proved that was involved in white spot symptom virus (WSSV) infection. Yet the functions of the third branch in shrimp UPR are still unclear. In this study, we showed that upon UPR activation, activating transcription factor 6 alpha (LvATF6α) of Litopenaeus vannamei was cleaved and transferred from the cytoplasm to the nucleus in 293T cells, indicating that the ATF6 pathway in shrimp is also a branch of UPR. Furthermore, LvATF6α could reduce the apoptosis rate of Drosophila Schneider 2 (S2) cells treated with actinomycin, and knock-down expression of LvATF6α increased the apoptosis rate of shrimp hemocytes. In vivo testing revealed that the short from LvATF6α (LvATF6α-s) was obviously increased after UPR activation or WSSV infection, indicating that the ATF6 pathway was activated in L. vannamei gills under such circumstances. Moreover, knock-down expression of LvATF6α could reduce the cumulative mortality and WSSV copy number in WSSV-infected shrimp. Further study revealed that WSSV may profit from shrimp ATF6 pathway activation in two aspects. First, LvATF6α-s significantly upregulated the expression of the WSSV genes (wsv023, wsv045, wsv083, wsv129, wsv222, wsv249, and wsv343). Second, LvATF6α-s inhibited apoptosis by negatively regulating the apoptosis signal-regulating kinase 1 - (c-Jun N-terminal kinase) pathway. All of these evidences suggested that the ATF6 pathway is a member of the L. vannamei UPR, and it is also engaged in WSSV infection.

Functional characterization of a reactive oxygen species modulator 1 gene in Litopenaeus vannamei.

Reactive oxygen species (ROS) imparts a dual effect on multicellular organisms, wherein high levels are usually harmful, and low levels could facilitate in combating pathogenic microorganisms; therefore, the regulation of ROS production is critical. Previous studies have suggested that ROS contributes to resistance to the white spot syndrome virus (WSSV) or Vibrio alginolyticus in Litopenaeus vannamei. However, the regulation of ROS metabolism in L. vannamei remains elusive. In the present study, we proved that the overexpression of L. vannamei reactive oxygen species modulator 1 (LvROMO1) increases ROS production in Drosophila Schneider 2 (S2) cells. Real-time RT-PCR analysis indicated that LvROMO1 is induced by WSSV or V. alginolyticus infection and ß-glucan or microcystin (MC-LR) injection. Further investigation showed that LvROMO1 responding to MC-LR, thereby inducing hemocytes to undergo apoptosis, and ultimately resulting in hepatopancreatic damage. And LvROMO1 downregulation induced an increase in the cumulative mortality of WSSV-infected shrimp by reducing ROS production and suppressing the expression of antimicrobial peptides genes. The findings of present study suggest that LvROMO1 plays an important role in ROS production in L. vannamei and is involved in innate immunity.

Prognostic value of right ventricular ejection/filling parameters in IPAH using cardiac magnetic resonance: A prospective pilot study.

BACKGROUND AND OBJECTIVE: The potential prognostic value of cardiovascular magnetic resonance (CMR)-derived right ventricular (RV) ejection/filling parameters in the assessment of RV function remains to be fully established. The goal of this study was to explore the prognostic value of these parameters in idiopathic pulmonary arterial hypertension (IPAH) patients. METHODS: In this prospective investigation, newly diagnosed IPAH patients without targeted therapy were recruited. Patients underwent right heart catheterization (RHC), 6-min walk test and CMR imaging within 1 week. Cardiopulmonary haemodynamics derived from RHC, and RV ejection/filling parameters derived from CMR were collected. RESULTS: A total of 41 IPAH patients were recruited with the median follow-up time of 824 days. During this period, 13 patients significantly deteriorated and among them, 7 patients died. CMR-derived RV peak ejection rate (PER) correlated significantly with CMR-derived RV stroke volume index (r = 0.574, P < 0.001) and RV cardiac index (r = 0.611, P < 0.001), but had no significant correlation with RV end diastolic volume index (EDVI, r = 0.201, P = 0.208) and pulmonary vascular resistance (r = -0.134, P = 0.403). Multivariate Cox regression analysis showed that RV time to PER (TPER, hazard ratio (HR) = 1.010, 95% CI: 1.003-1.017, P = 0.005) and RV TPER/time of a cardiac cycle (TPER/TCC, HR = 1.085, 95% CI: 1.031-1.141, P = 0.002) were predictors of clinical deterioration after adjusting for N-terminal pro-brain natriuretic peptide (NT-proBNP) and RV EDVI. Kaplan-Meier survival analyses showed that patients with RV TPER ≥ 199.01 ms (log rank: P = 0.036) or RV TPER/TCC ≥ 20.02% (log rank: P = 0.007) had worse prognosis. CONCLUSION: CMR-derived RV TPER and TPER/TCC can reflect RV early systolic function, and may be promising predictors for long-term prognosis in IPAH patients.