Molecular mechanism of bovine Gasdermin D-mediated pyroptosis.

Pyroptosis is a form of programmed cell death characterized by cell swelling, pore formation in the plasma membrane, lysis, and releases of cytoplasmic contents. To date, the molecular mechanism of human and murine Gasdermin D-mediated pyroptosis have been fully investigated. However, studies focusing on molecular mechanism of bovine Gasdermin D (bGSDMD)-mediated pyroptosis and its function against pathogenic infection were unclear. In the present study, we demonstrate that bovine caspase-1 (bCaspase-1) cleaves bGSDMD at amino acid residue D277 to produce an N-terminal fragment (bGSDMD-p30) which leads to pyroptosis. The amino acid residues T238 and F239 are critical for bGSDMD-p30-mediated pyroptosis. The loop aa 278-299, L293 and A380 are the key sites for autoinhibitory structure of the full length of bGSDMD. In addition, bCaspase-3 also cleaves bGSDMD at residue Asp86 without inducing cell death. Therefore, our study provides the first detailed elucidation of the mechanism of bovine GSDMD-mediated pyroptosis. The results will establish a significant foundation for future research on the role of pyroptosis in bovine infectious diseases.

Mycobacterial PE12 protein promotes bacterial survival through inhibiting cell apoptosis.

Mycobacterial PE_PGRS family proteins play key roles in pathogen-host interaction. However, the function of most PE_PGRS proteins remains unknown. In this study, we characterized the role of PE12 of Mycobacterium bovis (M. bovis) on bacterial growth, bacterial survival, and host cell apoptosis. Transcriptome sequencing of infected THP-1 cells was also performed. Compared to Ms_Vec, we found that M. bovis PE12 did not alter the colony morphology of M. smegmatis. The survival of Ms_PE12 was obviously higher than that of Ms_Vec. Furthermore, PE12 significantly suppressed the apoptosis of THP-1 induced by M. smegmatis infection. Transcriptome analysis results showed that there were 70 downregulated genes in the Ms_PE12 infection group in comparison with the Ms_Vec infection group, and these differentially expressed genes were enriched in 240 downregulated GO terms and 6 KEGG pathways. The downregulated expression genes are involved in cell adhesion, phagocytosis, apoptosis, inflammatory response, glycolysis and transmembrane transporter activity. Taken together, our study reveals that PE12 can suppress apoptosis and inhibit proinflammatory cytokine response. We propose that PE12 is related to macrophage phagocytosis and apoptosis, providing useful information to the pathogenic mechanisms of M. bovis.

Use of Non-Saccharomyces Yeast Co-Fermentation with Saccharomyces cerevisiae to Improve the Polyphenol and Volatile Aroma Compound Contents in Nanfeng Tangerine Wines.

This study attempted to improve the polyphenol and volatile aroma compound contents in Nanfeng tangerine wines using non-Saccharomyces yeast and Saccharomyces cerevisiae. The effects of fermentation with pure cultures of Candida ethanolica, Hanseniaspora guilliermondii and Hanseniaspora thailandica, as well as in sequential and mixed inoculations (1:1 or 1:100 ratio) with S. cerevisiae in Nanfeng tangerine wines were evaluated. C. ethanolica was found to produce the most polyphenols (138.78 mg/L) during pure fermentation, while H. guilliermondii produced the most volatile aroma compounds (442.34 mg/L). The polyphenol content produced during sequential fermentation with S. cerevisiae and H. guilliermondii (140.24 mg/L) or C. ethanolica (140.21 mg/L) was significantly higher than other co-fermentations. Meanwhile, the volatile aroma compounds were found to be more abundant in S. cerevisiae/H. guilliermondii mixed fermentation (1:1 ratio) (588.35 mg/L) or S. cerevisiae/H. guilliermondii sequential fermentation (549.31 mg/L). Thus, S. cerevisiae/H. guilliermondii sequential fermentation could considerably boost the polyphenol and volatile aroma component contents in Nanfeng tangerine wines. The findings of this study can be used to drive strategies to increase the polyphenol content and sensory quality of tangerine wines and provide a reference for selecting the co-fermentation styles for non-Saccharomyces yeast and S. cerevisiae in fruit wine fermentation.

Purification and Characterization of a Novel Fibrinolytic Enzyme from Cipangopaludina Cahayensis.

BACKGROUND: Cipangopaludina cahayensis contains active fibrinolytic proteins and has been considered a potential anti-cancer agent. However, its anti-cancer characteristics and functions have yet to be elucidated. OBJECTIVES: To study the fibrinolytic activity and anticancer activity of crude protein extracts from Cipangopaludina cahayensis. MATERIALS AND METHODS: Crude proteases were separated and extracted from the Cipangopaludina cahayensis through homogenization, desalting, ammonium sulfate fractionation, dialysis, and ion exchange chromatography. The fibrinolytic activity of extracted proteins was assessed using the fiber plate method. Total protein concentrations of the crude proteases were determined via BCA assay. Molecular weights (MWs) were determined through SDS-PAGE electrophoresis. RESULTS: The crude extract had a MW of ~ 50 kDa, and the highest protein concentration was 3.026 mg.mL-1. The optimum pH for fibrinolytic activity was 7.0. Cell culture assays demonstrated that the addition of the crude enzyme extracts to the human ovary cancer cell line Ovcar-3 resulted in significant growth defects. CONCLUSIONS: Our data showed that crude proteins purified from Cipangopaludina cahayensis are novel fibrinolytic proteases and have potential anti-cancer propertie.

Experimental co-infection of variant infectious bursal disease virus and fowl adenovirus serotype 4 increases mortality and reduces immune response in chickens.

Infectious bursal disease virus (IBDV) and fowl adenovirus serotype 4 (FAdV-4) cause infectious bursal disease (IBD) and hydropericardium-hepatitis syndrome, respectively. Recently, studies have reported co-infections of poultry with IBDV and FAdV-4, which is an important problem in the poultry industry. Here, the variant IBDV strain ZD-2018-1 and FAdV-4 isolate HB1501 were used to assess the pathogenicity of co-infection in 1-day-old specific pathogen-free (SPF) chickens. Compared with chickens infected with only FAdV-4, those coinfected with IBDV and FAdV-4 showed enhanced clinical symptoms, higher mortality, more severe tissue lesions, and higher biochemical index levels. Furthermore, the expression of interleukin (IL)-6, IL-1ß, and interferon-γ mRNAs in the IBDV-FAdV-4 coinfected chickens was delayed, and the antibody response levels were significantly lower in those birds compared with the FAdV-4-infected chickens. These results indicate that co-infection with variant IBDV ZD-2018-1 and FAdV-4 HB1501 could significantly promote the pathogenicity of FAdV-4 and reduce the immune response in chickens. This study provides the foundation for further investigation of the interaction mechanism in IBDV and FAdV-4 co-infection.

Isolation and pathogenicity testing of avian reticuloendotheliosis virus from layer chickens in China.

Reticuloendotheliosis virus (REV) can cause runting, immunosuppression, acute reticulum cell neoplasia, and chronic lymphoid tumors in a variety of domestic and wild birds. We diagnosed a case of reticuloendotheliosis with obvious tumors in liver and kidney. We isolated and sequenced the virus and performed pathogenicity testing of the REV strain. Immunohistochemistry and PCR confirmed that the diseased layer chickens were infected with REV. The strain, named BJ1503, was successfully isolated from the case by inoculation of tissue homogenates onto chicken embryo fibroblasts. The length of the proviral REV genome is 8,293 nucleotides. The isolate had 99.7% identity with REV-HA9901 (AY842951.1), which was isolated from Jiangsu, China, in 1999. The chickens infected with REV-BJ1503 had depressed weight gain and lymphoid atrophy. Our findings suggest that REV isolate BJ1503 was phylogenetically close to the earlier strain found in China, with minor variations, and the virus was associated with severe production problems.

The defective effect of starch branching enzyme IIb from weak to strong induces the formation of biphasic starch granules in amylose-extender maize endosperm.

KEYMESSAGE: Biphasic starch granules in maize ae mutant underwent the weak to strong SBEIIb-defective effect during endosperm development, leading to no birefringence in their exterior due to extended long branch-chains of amylopectin. Biphasic starch granules are usually detected regionally in cereal endosperm lacking starch branching enzyme (SBE). However, their molecular structure, formation mechanism, and regional distribution are unclear. In this research, biphasic starch granules were observed in the inner region of crown endosperm of maize ae mutant, and had poorly oriented structure with comb-like profiles in their exterior. The inner endosperm (IE) rich in biphasic starch granules and outer endosperm (OE) without biphasic starch granules were investigated. The starch had lower amylose content and higher proportion of long branch-chains of amylopectin in IE than in OE, and the exterior of biphasic starch granules had less amylose and more long branch-chains of amylopectin than the interior. Compared with OE, the expression pattern of starch synthesis related enzymes changed significantly in IE. The granule-bound starch synthase I activity within biphasic starch granules decreased slightly. The IE experienced more severe hypoxic stress than OE, and the up-regulated anaerobic respiration pathway indicated an increase in carbon consumption. The starch in IE underwent the SBEIIb-defective effect from weak to strong due to the lack of sufficient carbon inflow, leading to the formation of biphasic starch granules and their regional distribution in endosperm. The results provided information for understanding the biphasic starch granules.

Comprehensive comparison and applications of different sections in investigating the microstructure and histochemistry of cereal kernels.

This review summarizes the main applications of different sections and some improved sectioning methods in investigating the microstructure and histochemistry of cereal kernels. Thick sections of developing kernels prepared by free-hand and sliding microtome-aided sectioning method can be employed to elucidate tissue anatomy and histochemistry. The thin sections of mature kernels prepared by ultramicrotome-aided sectioning method can exhibit the micromorphology of starch granules when stained with iodine solution. The paraffin sections of developing kernels can exhibit the tissue anatomy of kernel, the accumulation of storage substances, and the location of protein and gene transcripts with immunohistochemistry and in situ hybridization techniques. The semithin resin sections can clearly exhibit the morphology of cells, starch granules, and protein bodies in kernel, but the sections prepared with different resins have various advantages and disadvantages for research investigating the morphology and histochemistry of cereal kernels. The improved methods of free-hand sectioning and ultramicrotome-aided sectioning of mature kernels are suitable for investigating the morphology of starch granules in a large number of samples in a short time. The modified method for preparing resin sections of whole kernels can be employed to determine the morphology and distribution of cells, starch granules, and storage protein in mature, developing, germinated, and cooked kernels in situ. This review could help researchers choose appropriate sections for investigating the microstructure and histochemistry of cereal kernels according to their study objectives.

Phylogenetic analyses and pathogenicity of a variant infectious bursal disease virus strain isolated in China.

Infectious bursal disease is an acute, highly contagious, immunosuppressive disease of young chickens caused by infectious bursal disease virus (IBDV). In recent years, there has been a notable increase in the isolation rates of variant IBDV strains in China; however, the pathogenicity of these variants is unclear. In the current study, we characterized variant IBDV strain ZD-2018-1 and assessed its pathogenicity in specific-pathogen-free chickens. Phylogenetic analysis revealed that ZD-2018-1 belonged to the variant IBDV strain, which showed several unique amino acid mutations compared with other previously-isolated variant IBDV strains. Pathogenicity assays showed that ZD-2018-1 was less virulent than very virulent IBDV strain SD-2013-1, and did not cause an obvious symptoms or death. In comparison, strain SD-2013-1 had a high mortality rate and caused severe lesions in various tissues. However, both of the strains induced obvious pathological lesions on the bursa of Fabricius, resulting in severe immunosuppression in the infected chickens. The results of this study present a systematic evaluation of the genetic characteristics, pathogenicity, and immunosuppressive properties of a new variant IBDV strain, and may help in the development of strategies for the prevention and control of IBDV in poultry.

Physicochemical properties of starches from vitreous and floury endosperms from the same maize kernels.

Starches were isolated from vitreous and floury endosperms from the same kernels, and their physicochemical properties were investigated to reveal their differences. Starch from vitreous endosperm had more surface indentations, larger granule size, and higher contents of protein, amylose and damaged starch than that from floury endosperm. Both starches from vitreous and floury endosperms exhibited A-type crystalline structure, but starch from vitreous endosperm had lower relative crystallinity and lamellar peak intensity than that from floury endosperm. Starch from floury endosperm had higher gelatinization temperature and enthalpy but lower gelatinization temperature range and percentage of retrogradation than that from vitreous endosperm. Native starch from floury endosperm was more resistant to enzyme hydrolysis than that from vitreous endosperm. Gelatinized and retrograded starches had similar digestion properties between starches from vitreous and floury endosperms. Principal component analysis indicated that starches from vitreous and floury endosperms had significant differences in their physicochemical properties.

Effects of Different Isolation Media on Structural and Functional Properties of Starches from Root Tubers of Purple, Yellow and White Sweet Potatoes.

Different-colored sweet potatoes have different contents of pigments and phenolic compounds in their root tubers, which influence the isolation of starch. It is important to justify the identification of the most suitable isolation medium of starch from different colored root tubers. In this study, starches were isolated from root tubers of purple, yellow and white sweet potatoes using four different extraction media, including H2O, 0.5% Na2S2O5, 0.2% NaOH, and both 0.5% Na2S2O5 and 0.2% NaOH. Their structural and functional properties were investigated and compared among different extraction media. The results showed that the granule size, apparent amylose content, lamellar peak intensity, thermal properties, and pasting properties were different among different-colored sweet potatoes due to their different genotype backgrounds. The four extraction media had no significant effects on starch structural properties, including apparent amylose content, crystalline structure, ordered degree, and lamellar peak intensity, except that the NaOH and Na2S2O5 treatment were able to increase the whiteness of purple and yellow sweet potato starches. The different extraction media had some effects on starch functional properties, including thermal properties, swelling power, water solubility, and pasting properties. The above results indicated that the H2O was the most suitable extraction medium to simply and fast isolate starch from root tubers of different-colored sweet potatoes.

Spatiotemporal accumulation and characteristics of starch in developing maize caryopses.

The accumulation and morphology of starch in the pericarp, embryo and endosperm of normal and waxy maize were investigated using whole sections of complete caryopses. Pericarp starch took the form of compound granules, was distributed in the bottom of caryopses, and degraded from the top to the bottom. Embryo starch mostly took the form of simple granules and accumulated in the scutellum beginning approximately 10 DAP. In the endosperm, starch accumulated longitudinally from the top to the bottom and transversely from the centre to the periphery with caryopsis development. The peripheral endosperm cells synthesized starch faster than did the inner ones. Simple and compound starches were both observed, but the compound starch granules were distributed in the central region of the endosperm. At a late stage of development, compound starch was only observed in the bottom central portion of the endosperm. The pericarp starch of normal maize showed higher amylose content than did the embryo and endosperm starch. The waxy maize pericarp and embryo starches had similar amylose contents, but amylose was hardly detected in the endosperm due to the granule-bound starch synthase I gene mutation. The starches from the endosperm, embryo and pericarp of normal and waxy maize all had A-type crystallinity.

Japanese encephalitis virus induces apoptosis by inhibiting Foxo signaling pathway.

Japanese encephalitis virus (JEV) infection induces brain tissue disease characterized by neuron death. however, little is known about the underlying mechanism. Using RNA sequencing, we profiled global mRNA expression changes in response to in vitro and in vivo JEV infection. Integration analysis of in vitro and in vivo mRNA transcriptome revealed that JEV infection regulated apoptosis-related Foxo signaling pathway. Foxo expression was reduced by JEV infection in vitro and in vivo. Knockdown of Foxo promoted apoptosis, while its overexpression reduced apoptosis in JEV-infected Neuro-2a cells. JEV infection in Neuro-2a cells decreased the expression of Foxo downstream genes including pro-apoptotic protein Bim, anti-apoptotic protein Bcl-6 and p21. Overexpression of anti-apoptotic proteins Bcl-6 and p21 repressed JEV-induced apoptosis. These findings suggest that Foxo primarily exerts an anti-apoptotic function via Bcl-6 and p21 in JEV-infected Neuro-2a cells. A STAT3 binding site was identified in the promoter region of Foxo by TFBIND software and confirmed by ChIP and reporter assays. JEV infection reduced STAT3 expression as well as its binding at the Foxo promoter compared to mock infection in Neuro-2a cells. Moreover, STAT3 knockdown reduced Foxo promoter activity and Foxo expression. Therefore, JEV reduced Foxo expression, at least in part, by downregulating STAT3. Taken together, we found that JEV induced cell apoptosis by inhibiting STAT3-Foxo-Bcl-6/p21 pathway, which provides a novel insight into JEV-caused encephalitis.

Hepatitis C virus and its protein NS4B activate the cancer-related STAT3 pathway via the endoplasmic reticulum overload response.

Oxidative stress induces the activation of signal transducer and activator of transcription 3 (STAT3), which plays an important role in hepatocellular carcinoma (HCC). We have previously reported that hepatitis C virus (HCV) and its protein NS4B induce the production of reactive oxygen species (ROS) via the endoplasmic reticulum overload response (EOR) in human hepatocytes. Here, we found that NS4B and HCV induce STAT3 activation and stimulate the expression of cancer-related STAT3 target genes, including VEGF, c-myc, MMP-9 and Mcl-1, by EOR in human hepatocytes. Moreover, the cancer-related STAT3 pathway activated by NS4B and HCV via EOR were found to promote human hepatocyte viability. Taken together, these findings revealed that HCV NS4B might contribute to HCC by activating the EOR-mediated cancer-related STAT3 pathway, and this could provide novel insights into HCV-induced HCC.

Japanese encephalitis virus induces apoptosis by the IRE1/JNK pathway of ER stress response in BHK-21 cells.

The role of endoplasmic reticulum (ER) stress in Japanese encephalitis is largely unknown. In this study, we found that Japanese encephalitis virus (JEV) strain SA14-14-2 regulates the expression of glucose-regulated protein 78 (GRP78), transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and splicing of X-box-binding protein 1 (XBP1) mRNA in BHK-21 cells. SA14-14-2-induced cytopathic effect and decrease in viability were also observed. Moreover, the inositol-requiring enzyme 1 (IRE1) inhibitor 3,5-dibromosalicylaldehyde and JNK inhibitor SP600125 increased cell viability and reduced cell apoptosis but did not alter virus replication in SA14-14-2-infected BHK-21 cells. These results, for the first time, demonstrate that JEV induces apoptosis by the IRE1/JNK pathway of ER stress response.