Advancing Cordyceps militaris Industry: Gene Manipulation and Sustainable Biotechnological Strategies.

Cordyceps militaris is considered to be of great medicinal potential due to its remarkable pharmacological effects, safety, and edible characteristics. With the completion of the genome sequence and the advancement of efficient gene-editing technologies, coupled with the identification of gene functions in Cordyceps militaris, this fungus is poised to emerge as an outstanding strain for medicinal engineering applications. This review focuses on the development and application of genomic editing techniques, including Agrobacterium tumefaciens-mediated transformation (ATMT), PEG-mediated protoplast transformation (PMT), and CRISPR/Cas9. Through the application of these techniques, researchers can engineer the biosynthetic pathways of valuable secondary metabolites to boost yields; such metabolites include cordycepin, polysaccharides, and ergothioneine. Furthermore, by identifying and modifying genes that influence the growth, disease resistance, and tolerance to environmental stress in Cordyceps militaris, it is possible to stimulate growth, enhance desirable traits, and increase resilience to unfavorable conditions. Finally, the green sustainable industrial development of C. militaris using agricultural waste to produce high-value-added products and the future research directions of C. militaris were discussed. This review will provide future directions for the large-scale production of bioactive ingredients, molecular breeding, and sustainable development of C. militaris.

Development and evaluation of a disulfidoptosis-related lncRNA index for prognostication in clear cell renal cell carcinoma.

Background: This study introduces a novel prognostic tool, the Disulfidoptosis-Related lncRNA Index (DRLI), integrating the molecular signatures of disulfidoptosis and long non-coding RNAs (lncRNAs) with the cellular heterogeneity of the tumor microenvironment, to predict clinical outcomes in patients with clear cell renal cell carcinoma (ccRCC). Methods: We analyzed 530 tumor and 72 normal samples from The Cancer Genome Atlas (TCGA), employing k-means clustering based on disulfidoptosis-associated gene expression to stratify ccRCC samples into prognostic groups. lncRNAs correlated with disulfidoptosis were identified and used to construct the DRLI, which was validated by Kaplan-Meier and receiver operating characteristic curves. We utilized single-cell deconvolution analysis to estimate the proportion of immune cell types within the tumor microenvironment, while the ESTIMATE and TIDE algorithms were employed to assess immune infiltration and potential response to immunotherapy. Results: The Disulfidoptosis-Related lncRNA Index (DRLI) effectively stratified ccRCC patients into high and low-risk groups, significantly impacting survival outcomes (P < 0.001). High-risk patients, marked by a unique lncRNA profile associated with disulfidoptosis, faced worse prognoses. Single-cell analysis revealed marked tumor microenvironment heterogeneity, especially in immune cell makeup, correlating with patient risk levels. In prognostic predictions, DRLI outperformed traditional clinical indicators, achieving AUC values of 0.779, 0.757, and 0.779 for 1-year, 3-year, and 5-year survival in the training set, and 0.746, 0.734, and 0.750 in the validation set. Notably, while the constructed nomogram showed exceptional predictive capability for short-term prognosis (AUC = 0.877), the DRLI displayed remarkable long-term predictive accuracy, with its AUC value reaching 0.823 for 10-year survival, closely approaching the nomogram's performance. Conclusions: The study introduces the DRLI as a groundbreaking molecular stratification tool for ccRCC, enhancing prognostic precision and potentially guiding personalized treatment strategies. This advancement is particularly significant in the context of long-term survival predictions. Our findings also elucidate the complex interplay between disulfidoptosis, lncRNAs, and the immune microenvironment in ccRCC, offering a comprehensive perspective on its pathogenesis and progression. The DRLI and the nomogram together represent significant strides in ccRCC research, highlighting the importance of molecular-based assessments in predicting patient outcomes.

Strategies for the Enhancement of Secondary Metabolite Production via Biosynthesis Gene Cluster Regulation in Aspergillus oryzae.

The filamentous fungus Aspergillus oryzae (A. oryzae) has been extensively used for the biosynthesis of numerous secondary metabolites with significant applications in agriculture and food and medical industries, among others. However, the identification and functional prediction of metabolites through genome mining in A. oryzae are hindered by the complex regulatory mechanisms of secondary metabolite biosynthesis and the inactivity of most of the biosynthetic gene clusters involved. The global regulatory factors, pathway-specific regulatory factors, epigenetics, and environmental signals significantly impact the production of secondary metabolites, indicating that appropriate gene-level modulations are expected to promote the biosynthesis of secondary metabolites in A. oryzae. This review mainly focuses on illuminating the molecular regulatory mechanisms for the activation of potentially unexpressed pathways, possibly revealing the effects of transcriptional, epigenetic, and environmental signal regulation. By gaining a comprehensive understanding of the regulatory mechanisms of secondary metabolite biosynthesis, strategies can be developed to enhance the production and utilization of these metabolites, and potential functions can be fully exploited.

Aspergillus oryzae as a Cell Factory: Research and Applications in Industrial Production.

Aspergillus oryzae, a biosafe strain widely utilized in bioproduction and fermentation technology, exhibits a robust hydrolytic enzyme secretion system. Therefore, it is frequently employed as a cell factory for industrial enzyme production. Moreover, A. oryzae has the ability to synthesize various secondary metabolites, such as kojic acid and L-malic acid. Nevertheless, the complex secretion system and protein expression regulation mechanism of A. oryzae pose challenges for expressing numerous heterologous products. By leveraging synthetic biology and novel genetic engineering techniques, A. oryzae has emerged as an ideal candidate for constructing cell factories. In this review, we provide an overview of the latest advancements in the application of A. oryzae-based cell factories in industrial production. These studies suggest that metabolic engineering and optimization of protein expression regulation are key elements in realizing the widespread industrial application of A. oryzae cell factories. It is anticipated that this review will pave the way for more effective approaches and research avenues in the future implementation of A. oryzae cell factories in industrial production.

Effects of Hericium erinaceus polysaccharide in porcine IPEC-J2 intestinal epithelial cells against apoptosis induced by oxidative stress.

This study was intended to investigate whether Hericium erinaceus polysaccharides (HEP) prevent oxidative stress and apoptosis of intestinal porcine epithelial cells from jejunum (IPEC-J2 cells) induced by hydrogen peroxide (H2O2). Crude HEP were extracted and purified by chromatography. The ultraviolet and infrared spectra and monosaccharide composition of HEP were analyzed. Reactive oxygen species (ROS) generation was quantified by flow cytometry method, and lactate dehydrogenase (LDH) and malondialdehyde (MDA) production were determined by TBARS. Also, apoptosis was analyzed by flow cytometry method and the apoptosis-related regulatory molecules were determined by microplate or western blotting method. Our results showed that pretreatment of IPEC-J2 cells with HEP significantly scavenged ROS and reduced LDH and MDA production. HEP also reduced apoptosis and kept polarity of the mitochondrial membrane potential. Moreover, HEP increased the content of caspase-3 and PARP, and protein expression of Bcl-2, while inhibited Bax and Bad and reduced the content of caspase-9 and release of CytC. Meanwhile, HEP inhibited the protein expression of TNFR1, FAS, and FASL, and decreased the content of caspase-8. The results indicated that HEP had a protective effect against oxidative stress in IPEC-J2 cells and the underlying mechanism was reducing apoptosis via mitochondrial and death receptor pathways.

Inflammatory responses and barrier disruption in the trachea of chicks following Mycoplasma gallisepticum infection: a focus on the TNF-α-NF-κB/MLCK pathway.

Mycoplasma gallisepticum (MG) can induce persistent inflammatory damage to the tracheal mucosa of poultry and cause chronic respiratory diseases in chickens. To further investigate the mechanism of MG-induced injury to the tracheal mucosa, we used chick embryo tracheal organ culture (TOC) as a model to study the invasion and reproduction of MG, the effect of MG on tracheal morphology, and the potential factors that promote MG tissue invasion. The results showed that MG infection significantly damaged the tracheal epithelial structure and weakened tracheal epithelial barrier function; MG also increased the occurrence of bacterial displacement, with a significant (p < 0.05) increase in the bacterial load of the infected TOCs at 5 and 7 days post-infection. In addition, MG significantly (p < 0.05) increased the expression levels of inflammatory cytokines, such as TNF-α, interleukin-1ß (IL-1ß), and IL-6, and activated the NF-κB signalling pathway, leading to increased nuclear translocation of NF-κB p65. Simultaneously, the map kinase pathway (MAPK) was activated. This activation might be associated with increased myosin light chain (MLC) phosphorylation, which could lead to actin-myosin contraction and disruption of tight junction (TJ) protein function, potentially compromising epithelial barrier integrity and further catalysing MG migration into tissues. Overall, our results contribute to a better understanding of the interaction between MG and the host, provide insight into the mechanisms of damage to the tracheal mucosa induced by MG infection, and provide new insights into the possible pathways involved in Mycoplasma gallisepticum infection in vivo.

Mycoplasma synoviae induce spleen tissue damage and inflammatory response of chicken through oxidative stress and apoptosis.

Mycoplasma synovium (MS) is a prominent avian pathogen known to elicit robust inflammatory responses in birds while evading immune detection, often leading to chronic infection and immune compromise. The mechanisms underpinning MS-mediated splenic tissue damage in chickens, however, remain undefined. In our investigation with 7-day-old SPF chickens, we administered an MS-Y bacterial solution (200 µl, 1 × 109 CCU/ml) through eye and nose droplets, collecting spleen samples on days 3, 6, and 12 post-infection. Comprehensive analyses utilizing histopathology, electron microscopy, TUNEL assay, qRT-PCR, and western blot were employed. Results demonstrated that MS-infection downregulated T-SOD, GSH-PX, and CAT, while concurrently elevating iNOS, NO, and MDA levels. Evidently, MS-induced oxidative stress compromised the spleen's antioxidant defences. Histological examinations pinpointed splenic damage characterized by lymphocyte reduction and increased inflammatory cell infiltration. Ultrastructural observations revealed clear apoptotic markers, including mitochondrial perturbations and nuclear anomalies. Importantly, MS induced significant spleen tissue apoptosis, as supported by TUNEL assay outputs and gene expression profiles associated with apoptosis. Concurrently, we observed upregulated expressions of mRNAs and proteins affiliated with the NF-κB/MAPK signalling cascade (p < 0.05). Collectively, our data elucidate that MS infection induces splenic apoptosis and oxidative disturbances, perturbs tissue integrity, and potentiates the NF-κB/MAPK-mediated inflammatory cascade.

A nomogram based on the preoperative neutrophil-to-lymphocyte ratio to distinguish sarcomatoid renal cell carcinoma from clear cell renal cell carcinoma.

Objective: Our study aimed to assess the predictive value of the preoperative neutrophil-to-lymphocyte ratio(NLR) in distinguishing sarcomatoid renal cell carcinoma (SRCC) from clear cell renal cell carcinoma(CCRCC) and to developing a nomogram based on the preoperative NLR and other factors to distinguish SRCC from CCRCC. Materials and methods: The database involved 280 patients, including 46 SRCC and 234 CCRCC. logistic analysis was conducted to select the variables associated with identifying SRCC preoperatively, and subgroup analysis was used to further validate the ability of NLR with preoperative identification of SRCC.In addition, The data were randomly separated into a training cohort(n=195) and a validation cohort(n=85). And an NLR-based nomogram was plotted based on the logistic analysis results. The nomogram was evaluated according to its discrimination, consistency, and clinical benefits. Results: Multivariate analysis indicated that NLR, flank pain, tumor size, and total cholesterol(TC) were independent risk factors for identifying SRCC. The results of subgroup analysis showed that higher NLR was associated with a higher probability of SRCC in most subgroups. The area under the curve(AUC) of the training and validation cohorts were 0.801 and 0.738, respectively. The results of the calibration curve show high consistency between predicted and actual results. Decision Curve Analysis(DCA) showed clinical intervention based on the model was beneficial over most of the threshold risk range. Conclusion: NLR is a potential indicator for preoperative differentiation of SRCC and CCRCC, and the predictive model constructed based on NLR has a good predictive ability. The new model could provide suggestions for the early identification of SRCC.

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system.

Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

Preparation and Characterization of Polypropylene/Sepiolite Nanocomposites for Potential Application in Automotive Lightweight Materials.

Polypropylene (PP)/sepiolite nanocomposites were prepared using the melt blending technique. The effects of nano-sepiolite content on the mechanical property, thermal property, crystallinity, morphology and rheological property of PP/sepiolite nanocomposites were investigated. The organic modified sepiolites (OSep) were dispersed evenly in PP matrix after surface treatment. The addition of OSep improved the storage modulus and thermal stability, showing a strong interaction between OSep and PP matrix. With the increase of OSep content, the fluidity of PP/OSep composites first increased due to the lubrication of surface modifiers and then decreased due to the interaction between OSep and PP. The size of the toughening agent elastomer first increased and then decreased, and the impact notched strength of PP/Osep composites first decreased and then increased. The loading of OSep also reduced the crystallinity and shrinkage rate of PP. PP/OSep nanocomposites have potential applications in high-performance automotive lightweight materials.

Lipophagy: Molecular Mechanisms and Implications in Hepatic Lipid Metabolism.

The liver is the most significant metabolic organ in the body and plays an important role in lipid metabolism. Liver lipid metabolism disorders cause hepatic diseases such as hepatitis, hepatic cirrhosis, and hepatoma. Autophagy is a process of generating energy and building blocks by degrading redundant or damaged proteins and organelles. Thus, it helps in the maintenance of cellular homeostasis. Recent discoveries revealed that lipophagy plays a vital role in hepatic cellular homeostasis and lipid metabolism. Its imbalance is always associated with the perturbation of lipid metabolism in the liver. This article reviewed the molecular mechanisms involved in lipophagy and the interaction between lipophagy and hepatic lipid metabolism. Increasing evidence suggests that lipophagy is an effective method to resolve liver diseases.

Effects of earthworm hydrolysate in production performance, serum biochemical parameters, antioxidant capacity and intestinal function of Muscovy ducks.

Earthworm has a variety of molecular biological characteristic, for example, growth promotion, antioxidant, and anti-bacteria. Thus, we decomposed earthworm by earthworm's own protease for preparing of earthworm hydrolysate. Muscovy ducks were fed with basal diet that formulated to contain 1.5% and 2.5% earthworm hydrolysate. Then, we investigated the influences of earthworm hydrolysate on growth performance in Muscovy ducks by performance terminology and measurement for poultry (NY/T 823-2020). The morphology of duodenum and number of intraepithelial lymphocytes were tested by HE staining and immunohistochemical method. Serum biochemical parameters and antioxidant capacity were also determined. High-throughput sequencing technology can sequence 16S rDNA of cecal contents from experimental Muscovy ducks. Results showed that 1.5% earthworm hydrolysate increased ADG (16-70 days old), ALB, HDL-C, T-AOC, CAT, SOD, GSH-PX, villi length, intestine thickness and surface area of villi (P < 0.05 or P < 0.01), and reduced FCR (16-70 days old), UREA, CRE, LDL-C, MDA (P < 0.05 or P < 0.01). Meanwhile, 2.5% improved ADG (16-70 days old), abdominal fat yield, breast muscle yield, heart index, spleen index, ALP, UA, T-AOC, CAT, SOD, GSH-PX, villi length, crypt depth, intestine thickness, surface area of villi, the percentage of intraepithelial lymphocytes (P < 0.05 or P < 0.01), and decreased FCR (42-70 days old and 16-70 days old), UREA, UA, MDA (P < 0.05 or P < 0.01). The sequencing results of gut flora demonstrated that earthworm hydrolysate improved variety of the gut flora in the V4 area of ducks immensely. In a word, our results provide the foundation for preliminary researching the potential principles of earthworm hydrolysate in promoting production performance, adjusting antioxidant function and intestinal functions in the Muscovy duck industry.

Sarcomatoid-associated gene risk index for clear cell renal cell carcinoma.

Sarcomatoid renal cell carcinoma is a de-differentiated form of kidney cancer with an extremely poor prognosis. Genes associated with sarcomatoid differentiation may be closely related to the prognosis of renal cell carcinoma. The prognosis of renal cell carcinoma itself is extremely variable, and a new prognostic model is needed to stratify patients and guide treatment. Data on clear cell renal cell carcinoma with or without sarcomatoid differentiation were obtained from TCGA database, and a sarcomatoid-associated gene risk index (SAGRI) and column line graphs were constructed using sarcomatoid-associated genes. The predictive power of the SAGRI and column line graphs was validated using an internal validation set and an independent validation set (E-MTAB-1980). The SAGRI was constructed using four sarcoma-like differentiation-related genes, COL7A1, LCTL, NPR3, ZFHX4, and had a 1-year AUC value of 0.725 in the training set, 0.712 in the internal validation set, and 0.770 in the independent validation set for TCGA training cohort, with high model reliability. The molecular characteristics among the SAGRI subgroups were analyzed by multiple methods, and results suggested that the SAGRI-HIGH subgroup may benefit more from immunotherapy to improve prognosis. SAGRI satisfactorily predicted the prognosis of patients with clear cell renal cell carcinoma with or without sarcomatoid differentiation.

Study on the mechanism of Mycoplasma gallisepticum infection on chicken tracheal mucosa injury.

RESEARCH HIGHLIGHTSMG infection causes a persistent inflammatory response by increasing the expression of immune response genes.The ERK-MLCK signalling pathway activated by MG infection regulates tight junction proteins in the tracheal mucosa.These data provide a basis for future prevention and treatment studies to control MG infection.

Interactions of Muscovy duck reovirus, gut microbiota, and host innate immunity: Transcriptome and gut microbiota analysis.

It has been shown that Muscovy duck reovirus (MDRV) infection causes severe intestinal barrier damage and intestinal mucosal immune suppression. The health and balance of gut microbes is essential for the progression of intestinal infectious diseases. To investigate the interaction of MDRV, intestinal bacteria with host intestinal innate immunity, an MDRV contact-infection model was established in this study. High-throughput sequencing technology was used to sequence 16S rDNA and transcripts in ileal samples from experimental Muscovy ducklings. Our results suggest that intestinal opportunistic pathogens such as Streptococcus and Corynebacterium proliferated massively in MDRV-infected Muscovy ducklings. The body initiates antiviral and antibacterial immunity and actively fights the infection of gut microbes. The synthesis of peptidoglycan, lipopolysaccharide, and flagellin by intestinal bacteria activates the Toll-like receptor signaling pathway resulting in increased secretion of IFN-ß, IL-1ß, and IL-8. The RIG-I-like receptor signaling pathway is an important signaling pathway for the interaction between MDRV and the host. At the same time, we also observed that multiple genes in the JAK-STAT signaling pathway were significantly different. These genes are important targets for studying the immunosuppression caused by MDRV. In conclusion, we analyzed the interaction of MDRV, intestinal flora and host immune system during MDRV infection, which provides a basis for the further study on the mechanism of intestinal immunosuppression caused by MDRV.

Effects of Hericium erinaceus polysaccharide on immunity and apoptosis of the main immune organs in Muscovy duck reovirus-infected ducklings.

To investigate the effects of Hericium erinaceus polysaccharide (HEP) on immunity in Muscovy duck reovirus (MDRV)-infected ducklings and explore its mechanism of action, an MDRV contact-infection model was established. Then, we investigated the influence of HEP on morphology of main immune organs in MDRV-infected ducklings by HE staining, while antioxidant capacity (T-AOC, MDA), serum protein levels (TP, ALB, GLO), complement levels (C3, C4) and antibody levels (IgA, IgM, IgG) were detected. Apoptotic indexes (apoptosisi rate and FAS-L) were also quantified by TUNEL method and immunohistochemical staining. Meanwhile, FADD and CytC (apoptosis-related genes), were tested by quantitative RT-PCR. Results showed that HEP could reduce the injuries of immune organs caused by MDRV. Additionally, HEP markedly diminished MDA (p < 0.01), while significantly increased T-AOC, TP, ALB, GLO, C3, C4, IgA, IgM and IgG (p < 0.01 or p < 0.05). Then, HEP shifted apoptosis time to an early MDRV-infected stage and reduced apoptosis at later MDRV-infected stage. This was associated with changes of FADD and CytC. Collectively, our data suggested that HEP could reduce the immunesuppression by many ways, such as decreasing organs' injuries, improving antioxidant capacity, serum proteins levels, antibody levels and complement levels, while diminish the apoptosis by lowering the FADD and CytC.

Application of Microalgal Stress Responses in Industrial Microalgal Production Systems.

Adaptive laboratory evolution (ALE) has been widely utilized as a tool for developing new biological and phenotypic functions to explore strain improvement for microalgal production. Specifically, ALE has been utilized to evolve strains to better adapt to defined conditions. It has become a new solution to improve the performance of strains in microalgae biotechnology. This review mainly summarizes the key results from recent microalgal ALE studies in industrial production. ALE designed for improving cell growth rate, product yield, environmental tolerance and wastewater treatment is discussed to exploit microalgae in various applications. Further development of ALE is proposed, to provide theoretical support for producing the high value-added products from microalgal production.

Co-selection of antibiotic resistance genes, and mobile genetic elements in the presence of heavy metals in poultry farm environments.

Environmental selection of antibiotic resistance genes (ARGs) is considered to be caused by antibiotic or metal residues, frequently used in livestock. In this study we examined three commercial poultry farms to correlate the co-occurrence patterns of antibiotic and metal residues to the presence of ARGs. We quantified 283 ARGs, 12 mobile genetic elements (MGEs), 49 targeted antibiotics, 7 heavy metals and sequenced 16S rRNA genes. The abundance and type of ARG were significantly enriched in manure while soil harbored the most diverse bacterial community. Procrustes analysis displayed significant correlations between ARGs/MGEs and the microbiome. Cadmium (Cd), arsenic (As), zinc (Zn), copper (Cu) and lead (Pb) were responsible for a majority of positive correlations to ARGs when compared to antibiotics. Integrons and transposons co-occurred with ARGs corresponding to 9 classes of antibiotics, especially Class1 integrase intI-1LC. Redundancy analysis (RDA) and Variance partitioning analysis (VPA) showed that antibiotics, metals, MGEs and bacteria explain solely 0.7%, 5.7%, 12.4%, and 21.9% of variances of ARGs in the microbial community, respectively. These results suggested that bacterial composition and horizontal gene transfer were the major factors shaping the composition of ARGs; Metals had a bigger effect on ARG profile than detected antibiotics in this study.

Muscovy duck reovirus promotes virus replication by inhibiting autophagy-lysosomal degradation pathway.

Autophagy plays a momentous role in cellular responses against pathogens. However, the influence of the autophagy machinery on Muscovy duck reovirus (MDRV) infection is not yet confirmed. In this study, it was shown that MDRV infection significantly increased the number of autophagy-like vesicles in DF-1 cells under electron microscope and the LC3-I/LC3-II conversion, which was considered important indicators of autophagy. It was worth noting that the level of autophagy was positively correlated with MDRV replication. Further test results showed that MDRV-induced autophagy can promote virus replication in DF-1 cells, and both the envelope protein sigma A and non-structural protein sigma NS that play an important role in virus replication process can colocalize with the autophagosome marker molecule LC3-II by confocal immunofluorescence analysis. These results indicated that MDRV utilized the autophagosomes for replication. Through transfection of the dual fluorescent plasmid mcherry-EGFP-LC3 and fluorescence microscope observation, it was found that autophagosomes were more likely to fuse with lysosomes in MDRV-infected cells compared with the blank group. The phenomenon of pEGFP-LC3B fluorescent spot and LAMP1 co-localization appeared in MDRV infected cells, indicating that MDRV infection would promote the fusion of autophagosomes and the lysosomes. Conversely, accumulation of p62 was observed by immunoblotting, suggesting that autolysosomes does not exert effective degradation. MDRV infection triggered a incomplete autophagic response. Further studies found that the expression of LAMP1, a marker protein of late endosome/early lysosome, increased significantly in MDRV-infected cells, suggesting an increase in the number of immature lysosomes. In addition, the experiment detected the maturation of the lysosomal acid hydrolase Cathepsin D in the cells, and found that the expression of the 33 kDa mature form of Cathepsin D was significantly reduced after MDRV infection, indicating that MDRV inhibits the maturation of lysosomes. In general, MDRV infection induces autophagy of DF-1 cells, promotes the fusion of autophagosomes and lysosomes, inhibits autophagolysosome degradation, and promotes virus replication.

Effects of Astragalus polysaccharides on intestinal morphology and intestinal immune cells of Muscovy ducklings infected with Muscovy duck reovirus.

Muscovy duck reovirus (MDRV) infection induces serious immunosuppression and intestinal injury in Muscovy ducklings with a high morbidity and mortality, and Astragalus polysaccharide (APS) pretreatment could efficiently protect ducklings from MDRV infection, although the underlying immunoregulatory mechanisms remain unclear. Thus, the objective of this study was to investigate effects of APS on the intestinal mucosal immunity in MDRV-infected Muscovy ducklings. A total of 190 1-day-old healthy Muscovy ducklings were randomly assigned to 3 groups (n = 50): normal control group, APS pretreatment for MDRV-infected group, and cohabitation infection group, then pretreated with 0.6 g/L APS or only drinking water followed by MDRV cohabitation infection with the remaining 40 artificially infected ducklings, respectively. At the 2, 3, 4, 6, 9 and 15 d after cohabitation infection, the intestinal samples were prepared to measure intestinal parameters including villus length, villus length/crypt depth (V/C) ratio, and wall thickness, together with counts of intraepithelial lymphocyte (IEL) and goblet cell (GC) by hematoxylin-eosin staining. Meanwhile, ileal secretory IgA (sIgA) and duodenal cytokine levels of IL-4, IL-6, IL-15, tumor necrosis factor-alpha, and interferon gamma were detected by the ELISA and radioimmunoassay, respectively. The results showed that APS significantly improved intestinal injuries of villi length, V/C ratio, and wall thickness of the small intestine infected with MDRV, effectively inhibited the reduction of IEL and GC caused by MDRV infection, subsequently increased sIgA and all the cytokine secretions at most time points, suggesting that APS pretreatment can effectively stimulate mucosal immune function by improving intestinal morphology and repair MDRV caused injures of small intestinal mucosal immune barrier in infected ducklings. Our findings lay the foundation for further application of APS in prevention and treatment of MDRV infection.