The CT10 regulator of kinase (CRK) family adaptor proteins are key mediators of intracellular signaling, linking multiple signaling molecules through their Src homology (SH) 2 and SH3 domains. They regulate essential cellular processes, including cell adhesion, migration, phagocytosis, and apoptosis, and are critically involved in host immune signaling during viral infection. However, the function of CRK in white spot syndrome virus (WSSV) infection remains largely unknown in crustaceans. In the present study, a CRK family adaptor protein from the crayfish Cherax quadricarinatus (CqCRK) was identified and characterized. The gene contains an 807 bp open reading frame encoding a 268 amino acid protein with one SH2 domain and two SH3 domains, consistent with conserved CRK family features. Analysis of tissue distribution revealed that CqCRK was broadly expressed, with the highest levels in hemocytes. Upon WSSV infection, CqCRK mRNA expression significantly increased at 24 hpi. Knockdown of CqCRK expression significantly increased viral VP28 expression, indicating that CqCRK suppresses WSSV replication in crayfish and contributes to antiviral immunity. Mechanistically, CqCRK interacts with phosphorylated CqSTAT; meanwhile, gene silencing of CqCRK promotes the accumulation of phosphorylated CqSTAT in the cytoplasm and impairs its nuclear translocation. In addition, CqCRK inhibited the expression of apoptosis related factors, including Cqp53 and CqCaspase1, as well as crayfish hemocyte apoptosis. Collectively, these findings suggest that CqCRK contributes to antiviral defense against WSSV by facilitating the nuclear translocation of CqSTAT and suppressing apoptosis in the late stages of viral infection, thereby shedding new light on the molecular mechanisms underlying antiviral immunity in crustaceans.

White spot syndrome virus (WSSV) is a devastating pathogen causing substantial economic losses in crustacean aquaculture. Successful viral replication depends on complex interactions with host cellular processes, including those that maintain redox homeostasis. However, the molecular mechanisms by which host antioxidant proteins influence WSSV replication remain poorly understood. In this study, we characterized a sestrin 2 homolog, CqSESN2, from red claw crayfish (Cherax quadricarinatus) and investigated its role in WSSV infection. The full-length cDNA of CqSESN2 contained an open reading frame of 1,242 bp encoding 413 amino acids. Phylogenetic analysis revealed that CqSESN2 is a conserved SESN2 family member. CqSESN2 was ubiquitously expressed in all examined tissues and significantly upregulated upon WSSV infection. Knockdown of CqSESN2 significantly enhanced WSSV replication, indicating an antiviral role for CqSESN2. Mechanistically, CqSESN2 functioned as a conserved negative regulator of intracellular reactive oxygen species (ROS), as its silencing led to elevated ROS levels. ROS scavenger NAC abolished WSSV-induced CqSESN2 upregulation, confirming ROS-dependent expression. Furthermore, CqSESN2 activated the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant pathway, promoting downstream antioxidant genes (CqGST, CqSrx, CqHO-1) and its own expression, forming a positive feedback loop. Pulldown assays revealed a specific interaction between CqSESN2 and Cqp62. Simultaneous knockdown of both CqSESN2 and CqNrf2 synergistically enhanced viral replication. Collectively, these findings reveal that CqSESN2 is an antiviral protein that inhibits WSSV replication by modulating ROS homeostasis and activating the Nrf2-mediated antioxidant response, providing new insights into host-virus interactions in crustaceans and potential targets for antiviral strategies in aquaculture.

Recombinant adeno-associated virus (rAAV) has emerged as one of the best gene delivery vectors for human gene therapy in vivo. However, the clinical efficacy of rAAV gene therapy is often hindered by the host immune response against its transgene products. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is specialised to process peptides presented by class I molecules of major histocompatibility complex. Therefore, we hypothesise that modulation of the ERAP1 activity in rAAV transduced cells may be favoured to evade immune response against transgene products. In this study, we incorporated either miRNA-UL112-5p or ERAP1 shRNA into rAAV vectors expressing full-length ovalbumin (OVA) as a model antigen, and evaluated their effects for antigen presentation, cellular and humour immune response induced by OVA expression. The results indicated that silencing ERAP1 using miR-UL112-5p or ERAP1 shRNA did not affect the expression of OVA in cells, but inhibited the processing and presentation of OVA antigen peptide SIINFEKL in antigen presenting cells (APCs). Moreover, the rAAV vector co-expressing ERAP1 shRNA maintains stable and high expression of OVA in vivo, while simultaneously suppressing the humoral immunity of OVA. In addition, experimental results demonstrated that rAAV vectors incorporated ERAP1 shRNA efficiently repress costimulatory signals in dendritic cells (DCs), significantly attenuated the cytotoxic T-cell response, allowed for sustained transgene expression and reduced clearance of transduced muscle cells in mice. Moreover, our study suggested that the incorporation of miRNA-UL112-5p or ERAP1 shRNA into rAAV vectors effectively reduced transgene products induced immune response. The proposed method may potentially be applied in clinics to deliver therapeutic proteins safely and efficiently.

Protein acetylation, a crucial post-translational modification, plays a significant role in antiviral immunity of host and viral replication processes after infection. Our previous research showed that an acetyltransferase KAT2A significantly enhanced white spot syndrome virus (WSSV) replication in hematopoietic tissue (Hpt) cells from Cherax quadricarinatus, suggesting that alterations in intracellular acetylation states affect viral infection. Nevertheless, the mechanisms by which WSSV invasion regulates intracellular protein acetylation remain unexplored. To explore the connection between protein acetylation and WSSV infection, differentially acetylated proteins (DAcPs) were identified in Hpt cells during WSSV infection by proteomic analysis. The results revealed that 25 DAcPs were identified with primarily involving in transporter activity and proton ATPase activity complex during the early stage of WSSV infection, i.e., the intracellular transport of the virions. Furthermore, that 36 DAcPs associated with chromatin assembly and acetyltransferase complex were identified in the replication stage of WSSV infection, i.e., the process of extensive viral replication. Additionally, acetylation at the K27 and K36 sites of histone H3 was strongly activated during WSSV replication; similarly, the inhibition of histone acetylation by acetyltransferase inhibiter C646 significantly suppressed WSSV replication, implying that WSSV replication requires high levels of histone acetylation, while the acetylation at the K27 and K36 sites of histone H3 probably serves as critical viral regulatory targets. This study deepens the understanding of WSSV-host interaction, and lays the groundwork for future research into the relationship between protein acetylation and viral infection in crustaceans.

White spot syndrome virus (WSSV) is a large nuclear-replicating DNA virus of crustaceans such as shrimp and crayfish; however, the molecular mechanisms facilitating its transport from the invasion site to the cell nucleus have not yet been well elucidated. In this study, a CqProfilin (CqPFN) with a conserved PROF domain was identified from the red claw crayfish Cherax quadricarinatus. CqPFN was ubiquitously expressed in all examined tissues and hemocyte, with the highest levels in the hemocyte, followed by hematopoietic tissue (Hpt) from which the hemocyte were derived in crayfish. The transcript of WSSV genes such as IE1 and VP28 was obviously decreased both in vivo in hemocyte and Hpt, as well as in vitro in cultured Hpt cells, after CqPFN gene silencing; in contrast, the expression of viral genes was significantly increased by the introduction of a recombinant CqPFN protein in Hpt cells in vitro. Moreover, CqPFN was clearly colocalized with the main viral nucleocapsid protein VP664 and F-actin cytoskeleton, respectively, during the early stage of WSSV infection in Hpt cells. In addition, CqPFN was confirmed to interact with a truncated VP6642,405-2,535 and another viral nucleocapsid protein VP15 of WSSV and Cqβ-Actin from Hpt by co-immunoprecipitation assays. Further studies found that VP664 also colocalized with F-actin in the Hpt cell cytoplasm after WSSV infection, suggesting that the actin cytoskeleton was involved in the intracellular transport of incoming viral nucleocapsid. Taken together, CqPFN might combine with the actin cytoskeleton to promote WSSV infection through binding with viral nucleocapsid proteins VP664 and VP15, promoting intracellular transport of viral incoming nucleocapsid for further releasing genome into the nucleus for transcription. Collectively, these results provided an understanding of the WSSV pathogenesis, which will contribute to the development of an antiviral strategy against WSSV disease.

Iron is an essential dietary micronutrient for maintaining physiological homeostasis. However, disruption of cerebral iron regulation with the accumulation of iron in different brain structures appears to have a role in the pathogenesis of various neurodegenerative disorders. Studies have reported that autophagy induction could potentially mitigate progression in neurodegenerative diseases with iron deposition, but the relationship between autophagy and iron remains poorly understood. Meanwhile, abnormal autophagy in microglia is closely related to the occurrence of neurodegenerative diseases. Therefore, the effect of iron on microglia autophagy needs to be elaborated. In the present study, we found that iron induces autophagosome accumulation but inhibits its initiation in an Akt-mTOR pathway independent manner. Meanwhile, it caused autophagy flux defects and dysfunction of lysosomes. We also found that iron overload reduced the expression of Rab7, which is an essential protein for the fusion of autophagosomes and lysosomes. These results suggest that iron induces the accumulation of autophagosome in microglia and disrupts the autophagic flux in late stage of autophagy. Therefore, our work provides new insights into the molecular mechanisms of iron neurotoxicity.

Recombinant adeno-associated virus (rAAV) is an extremely attractive vector in the in vivo delivery of gene therapy as it is safe and its genome is simple. However, challenges including low permissiveness to specific cells and restricted tissue specificity have hindered its clinical application. Based on the previous studies, epidermal growth factor receptor-protein tyrosine kinase (EGFR-PTK) negatively regulated rAAV transduction, and EGFR-positive cells were hardly permissive to rAAV transduction. We constructed a novel rAAV-miRNA133b vector, which co-expressed miRNA133b and transgene, and investigated its in vivo and in vitro transduction efficiency. Confocal microscopy, live-cell imaging, pharmacological reagents and labelled virion tracking were used to analyse the effect of miRNA133b on rAAV2 transduction and the underlying mechanisms. The results demonstrated that miRNA133b could promote rAAV2 transduction and the effects were limited to EGFR-positive cells. The increased transduction was found to be a direct result of decreased rAAV particles degradation in the cytoplasm and enhanced second-strand synthesis. ss-rAAV2-miRNA133b vector specifically increased rAAV2 transduction in EGFR-positive cells or tissues, while ss-rAAV2-Fluc-miRNA133b exerted an antitumor effect. rAAV-miRNA133b vector might emerge as a promising platform for delivering various transgene to treat EGFR-positive cell-related diseases, such as non-small-cell lung cancer.

Iron deposition and chronic inflammation are associated with chronic liver diseases, such as alcoholic liver disease, nonalcoholic fatty liver disease, and chronic hepatitis B and C. However, the relationship between iron deposition and chronic inflammation in these diseases is still unclear. In the current study, we aimed to investigate the effect of iron on chronic inflammation in HepG2 cells and mice liver. We demonstrated that iron treatment enhanced the expression of cGAS, STING, and their downstream targets, including TBK1, IRF-3, and NF-κB in HepG2 cells and mice liver. We also found that treatment of HepG2 cells and mice with ferric ammonium citrate increased the expression of inflammatory cytokines, such as IFN-β. Finally, we found that genes involved in iron metabolism and the STING signaling pathway were up-regulated in liver cancer tissues, and the survival time of patients with high expression of these genes in tumor tissues was significantly shortened. These results suggest that iron overload may promote the progress of the chronic liver disease by activating cGAS-STING-mediated chronic inflammation, which provides a new idea for the development of drugs for the treatment of the chronic liver disease.

DNA polymerase δ (Polδ) plays a crucial and versatile role in DNA replication and DNA repair processes. Vent shrimp Rimicaris exoculata is the primary megafaunal community living in hydrothermal vents. In this study, the Polδ from shrimp Rimicaris exoculata was cloned, expressed and characterized. The results showed that the Polδ catalytic subunit (POLD1), 852 amino acids in length, shared high homology with crayfish Procambarus clarkii and shrimp Oratosquilla oratoria. The recombinant POLD1 expressed in Escherichia coli showed that the enzyme was active in a range of 20°C to 40°C with an optimum temperature at 25°C and in a wide range of pH with an optimum at pH 6.0. The activities of POLD1 were significantly enhanced in the presence of Triton-X 100, Tween 20 and Mn2+. The Km (dNTP) value of POLD1 was 4.7 μmol/L. The present study would be helpful to reveal the characterization of Polδ of deep-sea vent animals.

为构建应用型本科院校,提高细胞生物学实验教学水平,以细胞生物学实验课程改革实践为例,探析新建应用型本科高校细胞生物学实验教学存在的问题及改革方法,不断优化教学内容,丰富教学方式与手段,完善教学条件与考核.促进应用型本科高校细胞生物学实验教学示范中心建设,保障研究性教学的实施,提高学生自主学习和独立研究的能力.

Background: With the emergence of more and more cyclodextrin derivatives, cyclodextrin becomes an effective adjuvant for improving the prescription of drugs. Its application in pharmacy, especially in the sustained and controlled release, targeting, transdermal and mucosal drug delivery systems, is also being expanded and deepened. In this study, novel cyclodextrin derivatives were developed to investigate the impact of the charge on antitumor efficiency by introducing different groups (carboxymethyl or quaternary ammonium group) to poly-beta-cyclodextrin (beta-CD). Methods: These novel beta-CD derivatives were prepared by the nucleophilic substitution reaction and characterized by IR and H-1 NMR. Fluorouracil (5-FU) was adopted as a model drug to form inclusion compounds. The content of 5-FU in inclusion compounds was evaluated using fluorine element analysis. Also, the cytotoxicity of poly-beta-CD derivatives was studied. Finally, the effect of negative and positive charges on the antitumor activity of poly-beta-CD derivatives-5-FU inclusion compounds on HepG2 cancer cells was evaluated. Human liver cancer HepG2 cells (CYP3A4G/7R clone 87, RRID: CVCL_1x10) were purchased from Cell Bank, Shanghai Institutes for Biological Sciences (China). Results: The results of IR and H-1 NMR indicated consistently that both carboxymethyl poly-beta-CD (poly-CM-beta-CD) and glycidyl trimethyl ammonium chloride (GTMAC) poly-beta-CD (poly-GTAC-beta-CD) were successfully prepared. Fluorouracil was successfully loaded into poly-beta-CD derivatives. The results of fluorine analysis indicated that the content of 5-FU in 1 g poly-beta-CD, poly-GTAC-beta-CD and poly-CM-beta-CD was 1,214, 921 and 1,187 pg, respectively. No cytotoxicity of poly-beta-CD derivatives on HepG2 cells was observed. The killing effect of poly-beta-CD-5-FU on HepG2 cells was similar to that of poly-GTAC-beta-CD-5-FU. Poly-CM-beta-CD-5-FU had the worst killing effect on HepG2 cells. Conclusions: Charge had impact on antitumor efficiency. These novel poly-beta-CD derivatives have potential applications in tumor sustained-release targeted therapy.

Viral immediate early (IE) genes encode regulatory proteins that are critical for viral replication. WSV056 is an IE protein of white spot syndrome virus (WSSV), an important pathogen of farmed shrimp. It targets the host Rb protein(s) and, according to a previous study, may enhance the replication of the viral genome. However, the ectopic expression of WSV056 in transgenic Drosophila melanogaster exerted an inhibitory effect on the replication of Drosophila C virus (DCV). Transcriptome study using Affymetrix GeneChip suggested that the enrichment of serine proteases (SPs) likely accounts for DCV inhibition in WSV056-overexpressing Drosophila. Injection of recombinant WSV056 to the WSSV natural host Litopenaeus vannamei enhanced the expression of the SP family member prophenoloxidase-activating enzyme 2 (LvPPAE2) and conferred shrimp with more resistance to WSSV infection. LvPPAE2 knockdown contributed to decreased expression of antimicrobial peptides LvAlf1 and LvLyz1, reduced hemolymph phenoloxidase activity, and increased virus load, suggesting that LvPPAE2 is involved in the host defense against WSSV infection. Taken together, these results suggest that wsv056 plays a role in restricting viral replication by inducing the SP-mediated immune responses in the host.

Abstract Background Melanoma is a type of aggressive skin cancer with a poor survival rate. The resistance to conventional therapy of this disease is, at least in part, attributed to its cancer stem cell population. However, the mechanism of survival and stemness maintenance of cancer stem cells remains to be investigated. Methods Tumorsphere formation assay was used to study the stem-like property of melanoma stem-like cells (MSLC). Chromatin immunoprecipitation (ChIP), promoter luciferase reporter assay were included for exploring the role of MCL-1 in MSLC and electrophoretic mobility shift assay were used to evaluate the interaction between shrimp miR-965 and human Ago2 protein. Melanoma xenograft nude mice were used to study the inhibition of tumor development. Results In the present study, our results showed that myeloid cell leukemia sequence 1 (MCL-1) knocking down induced ER stress and apoptosis, and the expression reduction of stemness associated genes in MSLC, which implied a significant role of MCL-1 in MSLC. Further study indicated that ER stress agonist (tunicamycin) treatment in MSLC results in the translocation of XBP1, an ER stress sensor, into the nucleus to induce MCL-1 expression through direct binding to the − 313- to − 308-bp region of MCL-1 promoter. In addition, we found that a shrimp-derived miRNA (shrimp miR-965) could interact with the human Ago2 protein and suppressed the human MCL-1 expression by binding to the 3′ UTR of MCL-1 mRNA, thereby inhibiting the MSLC proliferation and stemness in vitro and in vivo in a cross-species manner. Conclusion In conclusion, we identified an important role of MCL-1-ER stress-XBP1 feedback loop in the stemness and survival maintenance of MSLC, and shrimp miR-965, a natural food derived miRNA, could regulate MSLC stemness and survival by targeting MCL-1 and disrupting the balance of MCL-1-ER stress-XBP1 feedback loop. In conclusion, this study indicated an important mechanism of the regulation of MSLC stemness and survival, otherwise it also demonstrated the significance of cross-species-derived miRNA as promising natural drugs in melanoma therapy.

Autophagy plays a vital role in innate and adaptive immunity against invading microorganisms, such as virus and bacteria. However, the mechanism underlying autophagy in shrimp is still limited. In our study, we challenged white shrimp L. vannamei with rapamycin to induce autophagy and employed Solexa/Illumina high-throughput RNA-seq method to examine the differences of transcriptome from gills of shrimps treated with or without rapamycin. More than 22.64 Gb raw data were produced, which were assembled into 62, 503 unigenes, with 14,126 unigenes over 1 kb in length. We then performed differential expression analysis and identified a total of 3050 differentially expressed genes (DEGs). Among them, 1456 were upregulated and 1594 were downregulated. We further annotated DEGs by matching against non-redundant protein sequence (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups of proteins (COG), euKaryotic Orthologous Groups (KOG), Gene ontology (GO), and Pfam databases. The assembled and annotated DEGs will facilitate our understanding of the molecular mechanism underlying autophagy and promote the studies on the role of autophagy in innate immunity of L. vannamei and other crustaceans.

Zinc oxide-coated zeolite (ZOCZ) and zinc oxide (ZnO) were compared in terms of their effectiveness in removing Staphylococcus aureus (S. aureus) from nutrient broth and phosphate-buffered saline (PBS) solution. ZOCZ was found to be extremely efficient in removing S. aureus. ZnO initially was much less effective. Photographs of removal S. aureus from PBS solution with ZOCZ confirmed that a multilayer of S. aureus cells forms on the surface of ZOCZ particles. The comparison of the images of confocal laser scanning microscope and inverted contrast fluorescence microscope further proved that a multilayer of S. aureus cells formed on the surface of ZnO-30N-zeolite. The FESEM images showed that the cell membranes of S. aureus attached to the surface of ZnO-30N-zeolite collapsed. Energy Dispersive X-Ray Spectrum and the atomic absorption spectroscopic analysis confirmed that zinc ions penetrate into S. aureus cells, causing their death. The dead cells were easily removed, allowing ZOCZ to be reused.

为了开发利用绿潮藻类条浒苔中含量丰富的蛋白质,采用木瓜蛋白酶酶解条浒苔蛋白,最佳酶解条件为:料液比1:25、加酶量1250 U/g pro、温度45.7℃、pH 7.2、震荡酶解时间120 min.在该条件下的酶解多肽经超滤分离获得不同分子量区间的多肽,并通过HUVEC、A549、H446、H460等细胞水平初步评价酶解条浒苔制备多肽抗肺癌的细胞生物学效果.结果表明,经上述条件获得的2-6 kD条浒苔多肽处理的HUVEC在抗氧化和小管形成等方面均受到抑制,其处理的NCI-H460、NCI-H446和A549的增殖、细胞周期、迁移也受到不同程度抑制,且能够促进细胞凋亡;2-6 kD浒苔多肽对H446、H460作用效果显著优于对A549的作用.这一细胞水平研究,不仅证实了酶解条浒苔获取新型天然抗肿瘤小肽的可行性,也为条浒苔蛋白质资源高值化利用探索提供了新的途径.

White shrimp Litopenaeus vannamei are widely cultured in the world and white spot syndrome virus (WSSV) led to huge economic losses in the shrimp industry every year. In the present study, miRNAs involved in the response of shrimp L. vannamei to WSSV infection were obtained through the Illumina HiSeq 2500 high-throughput next-generation sequencing technique. A total number of 7 known miRNAs and 54 putative novel miRNAs were obtained. Among them, 14 DEMs were identified in the shrimp infected with WSSV. The putative target genes of these DEMs were related to host immune response or signaling pathways, indicating the importance of miRNAs in shrimp against WSSV infection. The results will provide information for further research on shrimp response to virus infection and contribute to the development of new strategies for effective protection against WSSV infections.

Iron extensively exists in brain tissues, iron is needed for mitochondrial respiration and synthesis of myelin, neurotransmitters. Accumulation of iron in the brain increases with age. Excess iron in the brain is associated with diseases like Alzheimer's, Parkinson's disease, leading to the formation of reactive oxygen species and ER stress. Rabs are molecular switches controlling multiple steps in membrane traffic. Rab8A is founded to be a regulator of glucose and rhodopsin transport recently. In the present study, We found Rab8A is reduced in iron overloaded neural cells, indicating a mechanism of Alzheimer's and Parkinson's disease.

The removal of Escherichia coli (E. coli) from water by zinc oxide-coated zeolite (ZOCZ) and ZOCZ's antibacterial properties were examined in laboratory experiments using plate counting method and tests of cell apoptosis. Batch experiments showed that ZOCZ has a maximum removal capacity for E. coli of about 4.34 × 106 CFU g-1 at 25 °C. Element mappings confirm that zinc ions accumulate in the E. coli cells causing cell death. Pseudo-second-order kinetics and Freundlich isotherms were found to best describe the removal of E. coli, suggesting that a multilayer of E. coli cells forms on the surface of ZOCZ particles.

对虾是我国重要的海水养殖品种,但频繁爆发的病害威胁着对虾养殖的可持续发展.Toll通路是对虾重要的免疫应答通路,在对虾的抗病原体感染中起重要作用.文章综述了国内外关于对虾Toll通路的最新研究进展,总结了国内外的研究成果,指出对虾Toll通路研究的重要意义和未来的研究方向.