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Problem Statement: Object-oriented design has become a dominant method in software industry and many design metrics of object-oriented programs have been proposed for quality prediction, but there is no well-accepted statement on how significant those metrics are. In this study, empirical analysis is carried out to validate object-oriented design metrics for defects estimation. Approach: The Chidamber and Kemerer metrics suite is adopted to estimate the number of defects in the programs, which are extracted from a public NASA data set. The techniques involved are statistical analysis and neuro-fuzzy approach. Results: The results indicate that SLOC, WMC, CBO and RFC are reliable metrics for defect estimation. Overall, SLOC imposes most significant impact on the number of defects. Conclusions/Recommendations: The design metrics are closely related to the number of defects in OO classes, but we can not jump to a conclusion by using one analysis technique. We recommend using neuro-fuzzy approach together with statistical techniques to reveal the relationship between metrics and dependent variables, and the correlations among those metrics also have to be considered.

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.

White spot syndrome virus (WSSV) mainly infects crustaceans through the digestive tract. Whether C-type lectins (CLs), which are important receptors for many viruses, participate in WSSV infection in the shrimp stomach remains unknown. In this study, we orally infected kuruma shrimp Marsupenaeus japonicus to model the natural transmission of WSSV and identified a CL (designated as M. japonicus stomach virus–associated CL [MjsvCL]) that was significantly induced by virus infection in the stomach. Knockdown of MjsvCL expression by RNA interference suppressed the virus replication, whereas exogenous MjsvCL enhanced it. Further analysis by GST pull-down and coimmunoprecipitation showed that MjsvCL could bind to viral protein 28, the most abundant and functionally relevant envelope protein of WSSV. Furthermore, cell-surface calreticulin was identified as a receptor of MjsvCL, and the interaction between these proteins was a determinant for the viral infection–promoting activity of MjsvCL. The MjsvCL–calreticulin pathway facilitated virus entry likely in a cholesterol-dependent manner. This study provides insights into a mechanism by which soluble CLs capture and present virions to the cell-surface receptor to facilitate viral infection.

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.

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 ( Mj pIgR for short). Mj pIgR 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 Mj pIgR with its antibody inhibited WSSV infection in shrimp and overexpression of Mj pIgR facilitated the invasion of WSSV. Further analyses indicated that Mj pIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of Mj pIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin ( Mj CaM). Mj pIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of Mj pIgR 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 Mj pIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that Mj pIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.

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.

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