The death domain-associated protein suppresses porcine epidemic diarrhea virus replication by interacting with signal transducer and activator of transcription 1 and inducing downstream ISG15 expression.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute enteric disease in piglets and severely threatens the pig industry all over the world. Death domain-associated protein (DAXX) is a classical chaperone protein involved in multiple biological processes, such as cell apoptosis, transcriptional regulation, DNA damage repair, and host innate immunity. However, whether DAXX functions in the anti-PEDV innate immune responses remains unclear. In this study, we found that PEDV infection upregulated DAXX expression and induced its nucleocytoplasmic translocation in IPEC-J2 cells. Furthermore, we found that DAXX overexpression was inhibitory to PEDV replication, while downregulation of DAXX by RNA interference facilitated PEDV replication. The antiviral activity of DAXX was due to its positive effect on IFN-λ3-STAT1 signaling, as DAXX positively regulated STAT1 activation through their interaction in cytoplasm and enhancing the downstream ISG15 expression. Mutation of tryptophan at 621 to alanine in DAXX increased its abundance in the cytoplasm, leading to the upregulation of STAT1 phosphorylation and ISG15 expression. It indicated that cytoplasmic fraction of DAXX was advantageous for the STAT1-ISG15 signaling axis and PEDV inhibition. In summary, these results show that DAXX inhibits PEDV infection by increasing IFN-λ3-induced STAT1 phosphorylation and the downstream ISG15 expression.

Npro of classical swine fever virus enhances HMGB1 acetylation and its degradation by lysosomes to evade from HMGB1-mediated antiviral immunity.

Classical swine fever virus (CSFV) can dampen the host innate immunity by destabilizing IRF3 upon its binding with viral Npro. High mobility group box 1 (HMGB1), a non-histone nuclear protein, has diverse functions, including inflammation, innate immunity, etc., which are closely related to its cellular localization. We investigated potential mutual interactions between CSFV and HMGB1 and their effects on virus replication. We found that HMGB1 at the protein level, but not at mRNA level, was markedly reduced in CSFV-infected or Npro-expressing IPEC-J2 cells. HMGB1 in the nuclear compartment is anti-CSFV by promoting IFN-mediated innate immune response, as evidenced by overexpression of nuclear or cytoplasmic dominant HMGB1 mutant in IPEC-J2 cells stimulated with poly(I:C). However, CSFV Npro upregulates HMGB1 acetylation, a modification that promotes HMGB1 translocation into the cytoplasmic compartment where it is degraded by lysosomes. Ethyl pyruvate could downregulate HMGB1 acetylation and prevent Npro-mediated HMGB1 reduction. Inhibition of deacetylase HDAC1 with MS275 or by RNA silencing could promote Npro-mediated HMGB1 degradation. Taken together, our study elucidates the mechanism with which HMGB1 in the nuclei initiates antiviral innate immune response to suppress CSFV replication and elaborates the pathway by which CSFV uses its Npro to evade from HMGB1-mediated antiviral immunity through upregulating HMGB1 acetylation with subsequent translocation into cytoplasm for lysosomal degradation.

N-terminal domain of classical swine fever virus Npro induces proteasomal degradation of specificity protein 1 with reduced HDAC1 expression to evade from innate immune responses.

IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

Porcine Epidemic Diarrhea Virus Antagonizes Host IFN-λ-Mediated Responses by Tilting Transcription Factor STAT1 toward Acetylation over Phosphorylation To Block Its Activation.

Porcine epidemic diarrhea virus (PEDV) is the main etiologic agent causing acute swine epidemic diarrhea, leading to severe economic losses to the pig industry. PEDV has evolved to deploy complicated antagonistic strategies to escape from host antiviral innate immunity. Our previous study demonstrated that PEDV downregulates histone deacetylase 1 (HDAC1) expression by binding viral nucleocapsid (N) protein to the transcription factor Sp1, inducing enhanced protein acetylation. We hypothesized that PEDV inhibition of HDAC1 expression would enhance acetylation of the molecules critical in innate immune signaling. Signal transducer and activator of transcription 1 (STAT1) is a crucial transcription factor regulating expression of interferon (IFN)-stimulated genes (ISGs) and anti-PEDV immune responses, as shown by overexpression, chemical inhibition, and gene knockdown in IPEC-J2 cells. We further show that PEDV infection and its N protein overexpression, although they upregulated STAT1 transcription level, could significantly block poly(I·C) and IFN-λ3-induced STAT1 phosphorylation and nuclear localization. Western blotting revealed that PEDV and its N protein promote STAT1 acetylation via downregulation of HDAC1. Enhanced STAT1 acetylation due to HDAC1 inhibition by PEDV or MS-275 (an HDAC1 inhibitor) impaired STAT1 phosphorylation, indicating that STAT1 acetylation negatively regulated its activation. These results, together with our recent report on PEDV N-mediated inhibition of Sp1, clearly indicate that PEDV manipulates the Sp1-HDAC1-STAT1 signaling axis to inhibit transcription of OAS1 and ISG15 in favor of its replication. This novel immune evasion mechanism is realized by suppression of STAT1 activation through preferential modulation of STAT1 acetylation over phosphorylation as a result of HDAC1 expression inhibition. IMPORTANCE PEDV has developed sophisticated evasion mechanisms to escape host IFN signaling via its structural and nonstructural proteins. STAT1 is one of the key transcription factors in regulating expression of ISGs. We found that PEDV and its N protein inhibit STAT1 phosphorylation and nuclear localization via inducing STAT1 acetylation as a result of HDAC1 downregulation, which, in turn, dampens the host IFN signaling activation. Our study demonstrates a novel mechanism that PEDV evades host antiviral innate immunity through manipulating the reciprocal relationship of STAT1 acetylation and phosphorylation. This provides new insights into the pathogenetic mechanisms of PEDV and even other coronaviruses.

Development and Characterization of Novel Orthodontic Adhesive Containing PCL-Gelatin-AgNPs Fibers.

Enamel demineralization around brackets is a relatively common complication of fixed orthodontic treatment, which seriously affects the aesthetics of teeth. In this study, a novel orthodontic adhesive containing polycaprolactone−gelatin−silver nanoparticles (PCL−gelatin−AgNPs) composite fibers was prepared to prevent enamel demineralization of orthodontic treatment. First, PCL−gelatin−AgNPs fibers film prepared by electrospinning was made into short fibers and added to traditional orthodontic adhesives (Transbond XT, 3M Unitek) in three different ratios to design a series of composite adhesives containing antibacterial materials. The antimicrobial performance of the control product and the three samples were then evaluated by bacterial live/dead staining, colony-forming unit (CFU) counts, tensile bond strength (TBS), and adhesive residue index (ARI) scores. The composite adhesives' antimicrobial properties increased with the increasing content of PCL−gelatin−AgNPs short fibers. The addition of complex antimicrobial fibers to 3M Transbond XT adhesive can significantly reduce the CFU of bacterial biofilms (p < 0.05). The bacterial survival rate on the surface of the specimen decreased with the increase of PCL−gelatin−AgNPs short fibers (p < 0.05). The TBS and ARI values (n = 10) indicated that adding PCL−gelatin−AgNPs short fibers had no significant adverse effect on adhesion. Therefore, adding PCL−gelatin−AgNPs short fibers makes it possible to fabricate orthodontic adhesives with strong antibacterial properties without compromising the bonding ability, which is essential for preventing enamel demineralization around the brackets.

DNAJA3 Interacts with PEDV S1 Protein and Inhibits Virus Replication by Affecting Virus Adsorption to Host Cells.

Porcine epidemic diarrhea virus (PEDV) infection causes huge economic losses to the pig industry worldwide. DNAJA3, a member of the Hsp40 family proteins, is known to play an important role in the replication of several viruses. However, it remains unknown if it interacts with PEDV. We found that DNAJA3 interacted with PEDV S1, initially with yeast two-hybrid screening and later with Co-IP, GST pull-down, and confocal imaging. Further experiments showed the functional relationship between DNAJA3 and PEDV in the infected IPEC-J2 cells. DNAJA3 overexpression significantly inhibited PEDV replication while its knockdown had the opposite effect, suggesting that it is a negative regulator of PEDV replication. In addition, DNAJA3 expression could be downregulated by PEDV infection possibly as the viral strategy to evade the suppressive role of DNAJA3. By gene silencing and overexpression, we were able to show that DNAJA3 inhibited PEDV adsorption to IPEC-J2 cells but did not affect virus invasion. In conclusion, our study provides clear evidence that DNAJA3 mediates PEDV adsorption to host cells and plays an antiviral role in IPEC-J2 cells.

Porcine Circovirus 2 Activates the PERK-Reactive Oxygen Species Axis To Induce p53 Phosphorylation with Subsequent Cell Cycle Arrest at S Phase in Favor of Its Replication.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), is known to induce oxidative stress, activate p53 with induction of cell cycle arrest, and trigger the PERK (protein kinase R-like endoplasmic reticulum kinase) branch of the endoplasmic reticulum (ER) stress pathway. All these cellular responses could enhance PCV2 replication. However, it remains unknown whether PERK activation by PCV2 is involved in p53 signaling with subsequent changes of cell cycle. Here, we demonstrate that PCV2 infection induced cell cycle arrest at S phase to favor its replication via the PERK-reactive oxygen species (ROS)-p53 nexus. PCV2 infection promoted phosphorylation of p53 (p-p53) at Ser15 in porcine alveolar macrophages. Inhibition of PERK by RNA silencing downregulated total p53 (t-p53) and p-p53. Treatment with the MDM2 inhibitor nutlin-3 led to partial recovery of t-p53 in perk-silenced and PCV2-infected cells. perk silencing markedly downregulated ROS production. Scavenging of ROS with N-acetylcysteine (NAC) of PCV2-infected cells downregulated t-p53 and p-p53. Increased accumulation of p-p53 in the nuclei during PCV2 infection could be downregulated by silencing of perk or NAC treatment. Further studies showed that perk silencing or NAC treatment alleviated S phase accumulation and downregulated cyclins E1 and A2 in PCV2-infected cells. These findings indicate that the PCV2-activated PERK-ROS axis promotes p-p53 and contributes to cell cycle accumulation at S phase when more cellular enzymes are available to favor viral DNA synthesis. Overall, our study provides a novel insight into the mechanism how PCV2 manipulates the host PERK-ROS-p53 signaling nexus to benefit its own replication via cell cycle arrest. IMPORTANCE Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown. Recent studies have revealed that PERK-mediated ER stress, oxidative stress, and cell cycle arrest during PCV2 infection are conducive to viral replication. However, how PCV2 employs such host cell responses requires further research. Here, we provide a novel mechanism of PCV2-induced ER stress and enhanced viral replication: the PCV2-activated PERK-ROS-p53 nexus increases S phase cell population, a cell cycle period of DNA synthesis favorable for PCV2 replication. The fact that PCV2 deploys the simple ROS molecules to activate p53 to benefit its replication provides novel insights into the triggering factors, that is, certain stimuli or management measures that induce ER stress with subsequent generation of ROS would exacerbate PCVAD. Use of antioxidants is justified on farms where PCVAD is severe.

"Zero ischemia" laparoscopic partial nephrectomy by high-power GreenLight laser enucleation for renal carcinoma: A single-center experience.

BACKGROUND: Laparoscopic partial nephrectomy has been widely used in renal cell carcinoma treatment. The efficacy of GreenLight laser on Laparoscopic partial nephrectomy is still unknown. AIM: To present the first series of laparoscopic partial nephrectomy (LPN) by GreenLight laser enucleation without renal artery clamping. Due to the excellent coagulation and hemostatic properties of the laser, laser-assisted LPN (LLPN) makes it possible to perform a "zero ischemia" resection. METHODS: Fifteen patients with T1a exogenous renal tumors who received high-power GreenLight laser non-ischemic LPN in our hospital were retrospectively analyzed. All clinical information, surgical and post-operative data, complications, pathological and functional outcomes were analyzed. RESULTS: Surgery was successfully completed in all patients, and no open or radical nephrectomy was performed. The renal artery was not clamped, leading to no ischemic time. No blood transfusions were required, the average hemoglobin level ranged from 96.0 to 132.0 g/L and no postoperative complications occurred. The mean operation time was 104.3 ± 8.2 min. The postoperative removal of negative pressure drainage time ranged from 5.0 to 7.0 d, and the mean postoperative hospital stay was 6.5 ± 0.7 d. No serious complications occurred. Postoperative pathological results showed clear cell carcinoma in 12 patients, papillary renal cell carcinoma in 2 patients, and hamartoma in 1 patient. The mean creatinine level was 75.0 ± 0.8 µmol/L (range 61.0-90.4 µmol/L) at 1 mo after surgery, and there were no statistically significant differences compared with pre-operation (P > 0.05). The glomerular filtration rate ranged from 45.1 to 60.8 mL/min, with an average of 54.0 ± 5.0 mL/min, and these levels were not significantly different from those before surgery (P > 0.05). CONCLUSION: GreenLight laser has extraordinary cutting and sealing advantages when used for small renal tumors (exogenous tumors of stage T1a) during LPN. However, use of this technique can lead to the generation of excessive smoke.

Transcriptome analysis revealed inhibition of lipid metabolism in 2-D porcine enteroids by infection with porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is a swine enterovirus that causes huge economic losses to the swine industry. It is of great interest to understand the gene expression patterns of host responses to PEDV infection and the mechanistic insights. Here, we report the differences of gene expression profiles by RNA-seq in the porcine small intestinal 2-D enteroids cells infected with low-passage (16 passages, P16) and high-passage (120 passages, P120) PEDV strains for 12, 24 and 36 h. Of the 57 genes differentially expressed in P16 PEDV infected enteroids, 49 were upregulated and 7 downregulated at all time points. There were 247 genes with different patterns of expression in the enteroids infected with P120 PEDV: upregulation seen with 105 genes and downregulation with the remaining majority at all time points. Infection of both P16 and P120 PEDV strains led to significant upregulation of ISGs, such as ISG15, MX1 and RSAD2. In particular, P120 PEDV infection inhibited transcription of genes related to lipid metabolism, including those involved in lipid decomposition, absorption, bile secretion and cholesterol metabolism. Treatment of the infected enteroids with palmitic acid resulted in marked reduction of replication of both P16 and P120 PEDV strains. These results indicate that PEDV might manipulate lipid metabolism of the host to benefit its replication. Further research is warranted to study the mechanisms how palmitic acid inhibits PEDV replication.

Establishment of enzyme-linked immunosorbent assays based on recombinant S1 and its truncated proteins for detection of PEDV IgA antibody.

Porcine epidemic diarrhea virus (PEDV) can infect pigs of all ages, especially piglets. PEDV has spread across Asia since the 1980s. The highly virulent variant PEDV broke out on a large scale and caused huge economic losses to the pig industry in late 2010 in China. Rapid detection methods with high specificity and sensitivity are urgently needed for the diagnosis and control of the disease. In this study, we divided the PEDV S1 gene into three segments and constructed the recombinant plasmids pFastBac1-S1T1 (aa 21-279), pFastBac1-S1T2 (aa 280-539) and pFastBac1-S1T3 (aa 540-788), which carry the different antigenic regions of the S1 gene. Truncated S1 proteins PEDV-S1T1/S1T2/S1T3 were obtained by a Bac-to-Bac expression system, with protein sizes of 36 kDa, 38 kDa and 38 kDa, respectively. Recombinant proteins presented high reactivity with the monoclonal antibody against PEDV and positive pig serum. Based on full-length S1 protein and these truncated proteins, we established indirect ELISA methods for the detection of PEDV IgA antibody. A total of 213 clinical serum samples were tested by the above indirect ELISA methods, and IFA was used as the gold standard. ROC curves revealed a significant correlation between S1-ELISA and S1T2-ELISA with a 0.9134 correlation coefficient and favourable sensitivity and specificity of S1-ELISA (93.24%, 95.68%) and S1T2-ELISA (89.33%, 94.16%). Our results also indicated that serum with higher neutralizing activity (SNT ≥ 40) had a higher IgA antibody level based on S1-ELISA, S1T1-ELISA and S1T2-ELISA. In conclusion, both S1-ELISA and S1T2-ELISA can be used as candidate systems for detecting anti-PEDV IgA antibody titers in serum, which can reflect the level of neutralizing activity in pigs after natural infection or vaccination. The above research results provide a basis for the prevention and control of PEDV and can be used in the detection of host anti-infective immunity and evaluation of vaccine immune effects.

Coronaviruses Nsp5 Antagonizes Porcine Gasdermin D-Mediated Pyroptosis by Cleaving Pore-Forming p30 Fragment.

Coronaviruses (CoVs) are a family of RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans. Here, we use porcine epidemic diarrhea virus (PEDV) as a model of CoVs to illustrate the reciprocal regulation between CoV infection and pyroptosis. For the first time, we elucidate the molecular mechanism of porcine gasdermin D (pGSDMD)-mediated pyroptosis and demonstrate that amino acids R238, T239, and F240 within pGSDMD-p30 are critical for pyroptosis. Furthermore, 3C-like protease Nsp5 from SARS-CoV-2, MERS-CoV, PDCoV, and PEDV can cleave pGSDMD at the Q193-G194 junction to produce two fragments unable to trigger pyroptosis. The two cleaved fragments could not inhibit PEDV replication. In addition, Nsp5 from SARS-CoV-2 and MERS-CoV also cleave human GSDMD (hGSDMD). Therefore, we provide clear evidence that PEDV may utilize the Nsp5-GSDMD pathway to inhibit pyroptosis and, thus, facilitate viral replication during the initial period, suggesting an important strategy for the coronaviruses to sustain their infection. IMPORTANCE Recently, GSDMD has been reported as a key executioner for pyroptosis. This study first demonstrates the molecular mechanism of pGSDMD-mediated pyroptosis and that the pGSDMD-mediated pyroptosis protects host cells against PEDV infection. Notably, PEDV employs its Nsp5 to directly cleave pGSDMD in favor of its replication. We found that Nsp5 proteins from other coronaviruses, such as porcine deltacoronavirus, severe acute respiratory syndrome coronavirus 2, and Middle East respiratory syndrome coronavirus, also had the protease activity to cleave human and porcine GSDMD. Thus, we provide clear evidence that the coronaviruses might utilize Nsp5 to inhibit the host pyroptotic cell death and facilitate their replication during the initial period, an important strategy for their sustaining infection. We suppose that GSDMD is an appealing target for the design of anticoronavirus therapies.

Porcine Epidemic Diarrhea Virus Inhibits HDAC1 Expression To Facilitate Its Replication via Binding of Its Nucleocapsid Protein to Host Transcription Factor Sp1.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns toward severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1, in porcine IPEC-J2 cells. HDACs are considered important regulators of innate immunity. We hypothesized that PEDV interacts with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout, and overexpression. A GC-box (GCCCCACCCCC) within the HDAC1 promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor mithramycin A inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to HDAC1 transcription. The putative nuclear localization sequence 261PKKNKSR267 contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for HDAC1 expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. IMPORTANCE The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, also make use of their N proteins to intercept the host immune responses in favor of their infection.

Long non-coding RNA NORAD promotes the prostate cancer cell extracellular vesicle release via microRNA-541-3p-regulated PKM2 to induce bone metastasis of prostate cancer.

BACKGROUND: Bone metastasis is the leading cause of mortality and reduced quality of life in patients with metastatic prostate cancer (PCa). Long non-coding RNA activated by DNA damage (NORAD) has been observed to have an abnormal expression in various cancers. This article aimed to explore the molecular mechanism underlying the regulatory role of NORAD in bone metastasis of PCa. METHODS: NORAD expression in clinical PCa tissues and cell lines was detected with the application of qRT-PCR. Cancer cells were then transfected with plasmids expressing NORAD, after which Transwell assay and CCK-8 assay were carried out to detect proliferation, migration, and bone metastasis of PCa. NORAD downstream target molecules were screened through bioinformatics analysis, followed by further verification using dual luciferase assay. Extracellular vesicles (EVs) were labeled with PKH67 and interacted with bone marrow stromal cells. The gain- and loss-function method was applied to determine the internalization and secretion of PCa cells-derived EVs under the intervention of downstream target molecules or NORAD. RESULTS: PCa tissues and cell lines were observed to have a high expression of NORAD, particularly in tissues with bone metastasis. NORAD knockdown resulted in reduced secretion and internalization of EVs, and suppressed proliferation, migration, and bone metastasis of PCa cells. It was indicated that NORAD interacted with miR-541-3p, leading to the upregulation of PKM2. Forced expression of PKM2 promoted the transfer of PKH67-labeled EVs to bone marrow stromal cells. CONCLUSIONS: NORAD might serve as a ceRNA of miR-541-3p to promote PKM2 expression, thereby enhancing the development of bone metastasis in PCa by promoting internalization and transfer of EVs of cancer cells, providing an insight into a novel treatment for the disorder.

Utility Evaluation of Porcine Enteroids as PDCoV Infection Model in vitro.

Porcine deltacoronavirus (PDCoV) is a novel emerging enteric coronavirus found in pigs. Intestinal enteroids, which partially recreate the structure and function of intestinal villi-crypts, have many physiological similarities to the intestinal tissues in vivo. Enteroids exhibit advantages in studying the interactions between intestines and enteric pathogens. To create a novel infection model for PDCoV, we developed an in vitro system to generate porcine intestinal enteroids from crypts of duodenum, jejunum, and ileum of pigs. Enterocytes, enteroendocrine cells, Paneth cells, stem cells, proliferating cells, and goblet cells were found in the differentiated enteroids. Replication of PDCoV was detected in the cultured enteroids by immunofluorescence and quantitative RT-PCR. Double immunofluorescence labeling demonstrated that PDCoV was present in Sox9-positive intestinal cells and Villin1-positive enterocytes. There were multiple cellular responses shown as changes of transcription of genes related to mucosal immunity, antiviral genes, and marker genes of stem cells and other cells in the enteroids infected with PDCoV. We conclude that the 2-D enteroids derived from porcine jejunum can be used as an in vitro multicellular model for the investigation of pathogenesis and host immune responses to porcine enteric pathogens, such as PDCoV.

LRP11 activates ß-catenin to induce PD-L1 expression in prostate cancer.

Prostate cancer (PRAD) is associated with abnormal cholesterol metabolism and low-density lipoprotein (LDL) receptor-related protein (LRP) family is essential for the homeostasis of cholesterol. Immune check points like PD-L1 are vital for tumour cells to evade immune attack. However, the potential cross-talk between these two pathways has not been explored before in PRAD. Insight from the regulation mechanism of PD-L1 in PRAD may help to optimise PD-L1 based immunotherapy. In this study, we investigated a regulation network of LRP11/ß-catenin/PD-L1 in PRAD. We showed that the expression of LRP11 and PD-L1 was up-regulated in PRAD compared to paired normal tissues. LRP11 expression was positively correlated to PD-L1 expression in PRAD tissues. Further experiments in two PRAD cell lines with LRP11 over-expression and knockdown showed that LRP11 induced PD-L1 expression through ß-catenin signalling. In addition, LRP11 over-expression in PRAD cell line induced immunosuppression of Jurkat cell in in-vitro co-culture system. The effects of LRP11 could be blocked by neutralising LRP11 or PD-L1 antibody. Our results provide evidence for a novel regulation mechanism of PD-L1 expression in PRAD and LRP11 may be a potential therapeutic target in PRAD.

The polymeric immunoglobulin receptor-like protein from Marsupenaeus japonicus is a receptor for white spot syndrome virus infection.

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR, and blocking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.

RPS27, a sORF-Encoded Polypeptide, Functions Antivirally by Activating the NF-κB Pathway and Interacting With Viral Envelope Proteins in Shrimp.

A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of <100 amino acids in eukaryotes (50 amino acids in prokaryotes). Studies have shown that several sORF-encoded peptides (SEPs) have important physiological functions in different organisms. Many ribosomal proteins belonging to SEPs play important roles in several cellular processes, such as DNA damage repair and apoptosis. Several studies have implicated SEPs in response to infection and innate immunity, but the mechanisms have been unclear for most of them. In this study, we identified a sORF-encoded ribosomal protein S27 (RPS27) in Marsupenaeus japonicus. The expression of MjRPS27 was significantly upregulated in shrimp infected with white spot syndrome virus (WSSV). After knockdown of MjRPS27 by RNA interference, WSSV replication increased significantly. Conversely, after MjRPS27 overexpression, WSSV replication decreased in shrimp and the survival rate of the shrimp increased significantly. These results suggested that MjRPS27 inhibited viral replication. Further study showed that, after MjRPS27 knockdown, the mRNA expression level of MjDorsal, MjRelish, and antimicrobial peptides (AMPs) decreased, and the nuclear translocation of MjDorsal and MjRelish into the nucleus also decreased. These findings indicated that MjRPS27 might activate the NF-κB pathway and regulate the expression of AMPs in shrimp after WSSV challenge, thereby inhibiting viral replication. We also found that MjRPS27 interacted with WSSV's envelope proteins, including VP19, VP24, and VP28, suggesting that MjRPS27 may inhibit WSSV proliferation by preventing virion assembly in shrimp. This study was the first to elucidate the function of the ribosomal protein MjRPS27 in the antiviral immunity of shrimp.

Thermodynamic and Economic Analyses of Reformative Design for High Back-Pressure Heating in Coal-Fueled Cogeneration Units.

High back-pressure (HBP) heating technology has been identified as an effective approach to improve the efficiency of combined heat and power (CHP). In this study, the novel concept of a HBP heating system with energy cascade utilization is developed and its probability examined. In the reformative design, the extracted heating steam from the intermediate-pressure turbine (IPT) is first drawn to an additional turbine where its excess pressure can be converted into electricity, then steam with a lower pressure can be employed to heat the supply water. As a consequence, the exergy destruction in the supply water heating process can be reduced and the efficiency of the cogeneration unit raised. A detailed thermodynamic investigation was performed based on a typical coal-fired HBP-CHP unit incorporating the proposed configuration. The results show that the artificial thermal efficiency (ATE) promotion was as much as 2.01 percentage points, with an additional net power output of 8.4 MW compared to the reference unit. This was attributed to a 14.65 percentage-point increment in the exergy efficiency of the supply water heating process caused by the suggested retrofitting. The influences of the unit power output, unit heat output, supply water and return water temperatures and turbine back pressure on the thermal performance of the modified system are discussed as well. In addition, the economic performance of the new design is assessed, indicating that the proposed concept is financially feasible.

Protein Inhibitor of Activated STAT (PIAS) Negatively Regulates the JAK/STAT Pathway by Inhibiting STAT Phosphorylation and Translocation.

Protein inhibitor of activated STAT (PIAS) proteins are activation-suppressing proteins for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. The inhibitory mechanism of PIAS proteins in the Janus kinase (JAK)/STAT signaling pathway has been well studied in mammals and Drosophila. However, the roles of PIAS in crustaceans are unclear. In the present study, we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation. To explore the function of PIAS in shrimp infected with V. anguillarum, we performed an RNA interference assay. After knockdown of PIAS expression in shrimp subjected to V. anguillarum infection, bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp (dsGFP injection). Simultaneously, the expression levels of antimicrobial peptides (AMPs), including anti-lipopolysaccharide factor (ALF) A1, C1, C2, and CruI-1, increased. Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation. Pulldown assay indicated that PIAS interacts with activated STAT in shrimp. In conclusion, PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT, which leads to the reduction of AMP expression in shrimp. Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway.