In this paper, the theoretical model proposed in Zhang et al. (2019) is extended to compute the natural frequencies and modal shapes for two-dimensional moonpools with one or two recesses in finite ...water depth. In the framework of linear potential flow theory, the boundary value problem is solved by using a domain decomposition method, assuming the velocity potential at the outer boundaries to be nil. In particular, a new and efficient approximation method, double-mode approximations (DMA), is derived to estimate the natural frequencies and modal shapes for both piston-mode and sloshing-mode resonances. The present results using the derived DMA formulas are compared with the experimental results by Ravinthrakumar et al. (2019) and the solutions using the frozen-mode approximation (FMA). The comparisons show satisfactory agreement with the experimental data. In particular, it is shown that, in contrast to FMA, DMA can well predict the non-flat modal shape of the free surface at piston-mode resonance. Moreover, extensive parametric studies are performed to examine the effects of the moonpool geometry on natural frequencies and free-surface modal shapes.
•The present paper studies how the moonpool configurations affect the natural frequencies and modal shapes of two-dimensional moonpools with one or two recesses.•Double-mode approximation (DMA) is derived to predict the natural frequencies and modal shapes of the piston as well as the sloshing modes.•The effects of moonpool geometry on the piston-mode frequency is revealed by analyzing the variations of the stiffness term and inertial terms in the frozen mode approximation (FMA).•The different parametric sensitivities of the piston mode and sloshing modes for the moonpool with recesses are discussed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The porcine pseudorabies virus (PRV) is one of the most devastating pathogens and brings great economic losses to the swine industry worldwide. Viruses are intracellular parasites that have evolved ...numerous strategies to subvert and utilize different host processes for their life cycle. Among the different systems of the host cell, the cytoskeleton is one of the most important which not only facilitate viral invasion and spread into neighboring cells, but also help viruses to evade the host immune system. RhoA is a key regulator of cytoskeleton system that may participate in virus infection. In this study, we characterized the function of RhoA in the PRV replication by chemical drugs treatment, gene knockdown and gene over-expression strategy. Inhibition of RhoA by specific inhibitor and gene knockdown promoted PRV proliferation. On the contrary, overexpression of RhoA or activation of RhoA by chemical drug inhibited PRV infection. Besides, our data demonstrated that PRV infection induced the disruption of actin stress fiber, which was consistent with previous report. In turn, the actin specific inhibitor cytochalasin D markedly disrupted the normal fibrous structure of intracellular actin cytoskeleton and decreased the PRV replication, suggesting that actin cytoskeleton polymerization contributed to PRV replication in vitro. In summary, our data displayed that RhoA was a host restriction factor that inhibited PRV replication, which may deepen our understanding the pathogenesis of PRV and provide further insight into the prevention of PRV infection and the development of anti-viral drugs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pseudorabies virus (PRV) is a double-stranded DNA virus that causes Aujeszky's disease and is responsible for economic loss worldwide. Transmembrane protein 41B (TMEM41B) is a novel endoplasmic ...reticulum (ER)-localized regulator of autophagosome biogenesis and lipid mobilization; however, the role of TMEM41B in regulating PRV replication remains undocumented. In this study, PRV infection was found to upregulate TMEM41B mRNA and protein levels both
and
. For the first time, we found that TMEM41B could be induced by interferon (IFN), suggesting that TMEM41B is an IFN-stimulated gene (ISG). While TMEM41B knockdown suppressed PRV proliferation, TMEM41B overexpression promoted PRV proliferation. We next studied the specific stages of the virus life cycle and found that TMEM41B knockdown affected PRV entry. Mechanistically, we demonstrated that the knockdown of TMEM41B blocked PRV-stimulated expression of the key enzymes involved in lipid synthesis. Additionally, TMEM41B knockdown played a role in the dynamics of lipid-regulated PRV entry-dependent clathrin-coated pits (CCPs). Lipid replenishment restored the CCP dynamic and PRV entry in TMEM41B knockdown cells. Together, our results indicate that TMEM41B plays a role in PRV infection via regulating lipid homeostasis.
PRV belongs to the alphaherpesvirus subfamily and can establish and maintain a lifelong latent infection in pigs. As such, an intermittent active cycle presents great challenges to the prevention and control of PRV disease and is responsible for serious economic losses to the pig breeding industry. Studies have shown that lipids play a crucial role in PRV proliferation. Thus, the manipulation of lipid metabolism may represent a new perspective for the prevention and treatment of PRV. In this study, we report that the ER transmembrane protein TMEM41B is a novel ISG involved in PRV infection by regulating lipid synthesis. Therefore, our findings indicate that targeting TMEM41B may be a promising approach for the development of PRV vaccines and therapeutics.
Alphaherpesvirus infection results in severe health consequences in a wide range of hosts. USPs are the largest subfamily of deubiquitinating enzymes that play critical roles in immunity and other ...cellular functions. To investigate the role of USPs in alphaherpesvirus replication, we assessed 13 USP inhibitors for PRV replication. Our data showed that all the tested compounds inhibited PRV replication, with the USP14 inhibitor b-AP15 exhibiting the most dramatic effect. Ablation of USP14 also influenced PRV replication, whereas replenishment of USP14 in USP14 null cells restored viral replication. Although inhibition of USP14 induced the K63-linked ubiquitination of PRV VP16 protein, its degradation was not dependent on the proteasome. USP14 directly bound to ubiquitin chains on VP16 through its UBL domain during the early stage of viral infection. Moreover, USP14 inactivation stimulated EIF2AK3/PERK- and ERN1/IRE1-mediated signaling pathways, which were responsible for VP16 degradation through SQSTM1/p62-mediated selective macroautophagy/autophagy. Ectopic expression of non-ubiquitinated VP16 fully rescued PRV replication. Challenge of mice with b-AP15 activated ER stress and autophagy and inhibited PRV infection in vivo. Our results suggested that USP14 was a potential therapeutic target to treat alphaherpesvirus-induced infectious diseases.
Abbreviations ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; ATG5: autophagy related 5; ATG12: autophagy related 12; CCK-8: cell counting kit-8; Co-IP: co-immunoprecipitation; CRISPR: clustered regulatory interspaced short palindromic repeat; Cas9: CRISPR associated system 9; DDIT3/CHOP: DNA-damage inducible transcript 3; DNAJB9/ERdj4: DnaJ heat shock protein family (Hsp40) member B9; DUBs: deubiquitinases; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EP0: ubiquitin E3 ligase ICP0; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; FOXO1: forkhead box O1; FRET: Förster resonance energy transfer; HSPA5/BiP: heat shock protein 5; HSV: herpes simplex virus; IE180: transcriptional regulator ICP4; MAP1LC3/LC3: microtube-associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; PRV: pseudorabies virus; PRV gB: PRV glycoprotein B; PRV gE: PRV glycoprotein E; qRT-PCR: quantitative real-time polymerase chain reaction; sgRNA: single guide RNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID
50
: tissue culture infective dose; UB: ubiquitin; UBA: ubiquitin-associated domain; UBL: ubiquitin-like domain; UL9: DNA replication origin-binding helicase; UPR: unfolded protein response; USPs: ubiquitin-specific proteases; VHS: virion host shutoff; VP16: viral protein 16; XBP1: X-box binding protein 1; XBP1s: small XBP1; XBP1(t): XBP1-total.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Emerging viral pathogens cause substantial morbidity and pose a severe threat to health worldwide. However, a universal antiviral strategy for producing safe and immunogenic inactivated vaccines is ...lacking. Here, we report an antiviral strategy using the novel singlet oxygen (1O2)-generating agent LJ002 to inactivate enveloped viruses and provide effective protection against viral infection. Our results demonstrated that LJ002 efficiently generated 1O2 in solution and living cells. Nevertheless, LJ002 exhibited no signs of acute toxicity in vitro or in vivo. The 1O2 produced by LJ002 oxidized lipids in the viral envelope and consequently destroyed the viral membrane structure, thus inhibiting the viral and cell membrane fusion necessary for infection. Moreover, the 1O2-based inactivated pseudorabies virus (PRV) vaccine had no effect on the content of the viral surface proteins. Immunization of mice with LJ002-inactiviated PRV vaccine harboring comparable antigen induced more neutralizing antibody responses and efficient protection against PRV infection than conventional formalin-inactivated vaccine. Additionally, LJ002 inactivated a broad spectrum of enveloped viruses. Together, our results may provide a new paradigm of using broad-spectrum, highly effective inactivants functioning through 1O2-mediated lipid oxidation for developing antivirals that target the viral membrane fusion process.
•LJ002 efficiently generates 1O2 in solution and living cells.•LJ002 oxidizes lipids in the viral envelope, thus inhibiting fusion between the virus and cell membrane.•LJ002-inactivated PRV vaccine has no effect on the content of antigens on the viral surface.•LJ002-inactivated PRV vaccine elicits a strong neutralizing antibody response.•LJ002 can inactivate a broad spectrum of enveloped viruses.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since ...late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.
Abstract
Background: Anti-PD1/PDL1 therapy has been established as standard care in multiple tumor types, however, many patients will not benefit from this treatment as lack of infiltrating immune ...cells. Our aim was to explore the pan-cancer immune microenvironment and whether they impacted by ICI biomarkers.
Method: 7-plex mIHC was performed to assessed immune cell infiltrations were assessed. Positive PD-L1 expression was defined as ≥1% of tumor cells with membranous staining. Statistical analyses were performed using R version 3.4.4.
Result: Among 520 samples, CD8+T cell infiltration in tumor was assessed in Esophagus, Liver, Biliary Tract, Lung, Colorectum, Stomach tumors, ordered by descending median density at the center of the tumor. The median density at the periphery was almost double than the median density at the center. In addition, we have observed high TAM.M1 density in Bladder urinary Tract, Biliary Tract, Liver, with a higher or equivalent level of infiltration in stroma than in tumor. While in contrast, the TAM.M2 cell density was higher in stroma than in tumor.
Regarding to PDL1 status in colorectum, there was little difference in the degree of CD8+ T cell infiltration between stroma and tumor. In the term of MSI status, CD8+T cell infiltration was significantly higher in MSIH colorectum tumor.
We compared the proportion of above immune cells. In stroma, except lung tumor, CD8+T cells made up the largest share of immune cells, following by NK cells. In tumor, except for lung cancer, NK cells accounted for the highest proportion. Cancer with the highest proportion of NK cells is bladder/urinary tract cancer. Although the proportion of NK cells in lung cancer is also high, macrophages proportion is the highest, especially TAM.M2.
Conclusions: Together, these data outlined the immune infiltration inter-, extra-tumor, and associations with TMB, MSI status. Tumor immune microenvironment might serve as promising predictive factors for immunotherapy.
Citation Format: Tianqing Chu, Zhang Bei. Pan-cancer characterization of tumor immune microenvironment abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2796.
β-Glucosidase belongs to the glycoside hydrolase I family, which is widely present in multiple species and responds to various biotic and abiotic stresses. In rice, whether β-glucosidase is involved ...in the interaction between plants and microorganisms is not clear. In this study, we found that the expression of several genes encoding β-glucosidases, including OsBGLU19 and OsBGLU23, were upregulated after inoculation with Xanthomonas oryzae pv. oryzicola (Xoc) and downregulated after inoculation with X. oryzae pv. oryzae ( Xoo). The respective insertion mutants of OsBGLU19 and OsBGLU23, bglu19 and bglu23 , were more susceptible to Xoc infection. The expression of OsAOS2, a key gene in the jasmonic acid signal pathway, was dramatically downregulated after inoculation with Xoc in the bglu19 and bglu23 mutants. Simultaneously, the expression of downstream disease resistance-related genes, such as OsPR1a, OsPR5 and a key transcription factors OsWRKY72 were obviously downregulated. The resistance mediated by OsBGLU19 and OsBGLU23 to bacterial leaf streak is related to disease resistance-related genes above mentioned.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Heat shock response in eukaryotes is transcriptionally regulated by conserved heat shock transcription factors (Hsfs). Hsf genes are represented by a large multigene family in plants and ...investigation of the Hsf gene family will serve to elucidate the mechanisms by which plants respond to stress. In recent years, reports of genome-wide structural and evolutionary analysis of the entire Hsf gene family have been generated in two model plant systems, Arabidopsis and rice. Maize, an important cereal crop, has represented a model plant for genetics and evolutionary research. Although some Hsf genes have been characterized in maize, analysis of the entire Hsf gene family were not completed following Maize (B73) Genome Sequencing Project.
A genome-wide analysis was carried out in the present study to identify all Hsfs maize genes. Due to the availability of complete maize genome sequences, 25 nonredundant Hsf genes, named ZmHsfs were identified. Chromosomal location, protein domain and motif organization of ZmHsfs were analyzed in maize genome. The phylogenetic relationships, gene duplications and expression profiles of ZmHsf genes were also presented in this study. Twenty-five ZmHsfs were classified into three major classes (class A, B, and C) according to their structural characteristics and phylogenetic comparisons, and class A was further subdivided into 10 subclasses. Moreover, phylogenetic analysis indicated that the orthologs from the three species (maize, Arabidopsis and rice) were distributed in all three classes, it also revealed diverse Hsf gene family expression patterns in classes and subclasses. Chromosomal/segmental duplications played a key role in Hsf gene family expansion in maize by investigation of gene duplication events. Furthermore, the transcripts of 25 ZmHsf genes were detected in the leaves by heat shock using quantitative real-time PCR. The result demonstrated that ZmHsf genes exhibit different expression levels in heat stress treatment.
Overall, data obtained from our investigation contributes to a better understanding of the complexity of the maize Hsf gene family and provides the first step towards directing future experimentation designed to perform systematic analysis of the functions of the Hsf gene family.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pseudorabies virus (PRV) is a contagious herpesvirus that causes Aujeszky's disease and economic losses worldwide. Liver X receptors (LXRs) belong to the nuclear receptor superfamily and are critical ...for the control of lipid homeostasis. However, the role of LXR in PRV infection has not been fully established. In this study, we found that PRV infection downregulated the mRNA and protein levels of LXRα and LXRβ in vitro and in vivo. Furthermore, we discovered that LXR activation suppressed PRV proliferation, while LXR inhibition promoted PRV proliferation. We demonstrated that LXR activation-mediated reduction of cellular cholesterol was critical for the dynamics of PRV entry-dependent clathrin-coated pits. Replenishment of cholesterol restored the dynamics of clathrin-coated pits and PRV entry under LXR activation conditions. Interestingly, T0901317, an LXR agonist, prevented PRV infection in mice. Our results support a model that PRV modulates LXR-regulated cholesterol metabolism to facilitate viral proliferation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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