Neuroinflammation is a complex immune response of the central nervous system. While it has a positive protective effect on nerves，continuous activation can lead to nerve damage and degenerative diseases. Its mechanism mainly involves the activation of microglia and astrocytes，as well as the complex regulation of multiple signaling pathways，such as NF-κB，PI3K/Akt，and MAPK. Numerous factors，including environmental stimuli such as lipopolysaccharide （LPS），can trigger neuroinflammation by activating receptors and inflammatory pathways. 3'-Sialyllactose （3'-SL） and 6'-Sialyllactose （6'-SL），as important sialic acid oligosaccharides found in human milk，play a key role in the neurodevelopment and immune system regulation of infants and young children. Their unique chemical structure gives them the potential to promote intestinal probiotic growth，enhance intestinal barrier function，and support nerve protection in the brain. This research aims to explore the protective effects and mechanisms of the human milk oligosaccharides 3'-SL and 6'-SL on neuroinflammation. The intervention effects of 3'-SL and 6'-SL on neuroinflammation were evaluated using two models：an in vitro BV-2 microglia model and an in vivo LPS-induced mouse neuroinflammation model. The experimental results show that 3'-SL and 6'-SL can effectively inhibit the upregulation of inflammatory factor expression in LPS-induced BV-2 cells and reduce excessive microglia activation in the hippocampus of mice. The mechanism of action may be achieved by down-regulating the expression of proteins related to the TLR4/Myd88/NF-κB/p65 signaling pathway. This research not only expands our understanding of the mechanisms of action of 3'-SL and 6'-SL，but also provides valuable references for developing new anti-neuroinflammatory drugs.
Objective：To explore the function of the long non-coding RNA （lncRNA） DGUOK-AS1 in kidney renal clear cell carcinoma （KIRC） and its impact on lipid metabolism. Methods：（1） The ENCORI database was used to analyze the expression levels of the lncRNA DGUOK-AS1 and SIX1 in KIRC tissues，as well as their relationship with the survival and prognosis of KIRC patients. （2） Real-time fluorescence quantitative polymerase chain reaction （PCR） was performed to detect the relative expression levels of DGUOK-AS1 and the mRNAs of lipid synthesis-related genes. （3） The effects of DGUOK-AS1，and the inhibitors of miR-145-5p and fatty acid synthase on KIRC cell proliferation were detected using a CCK8 assay. （4） The effect of DGUOK-AS1 on KIRC cell clonogenicity was assessed using a colony formation assay. （5） The impact of DGUOK-AS1 on KIRC cell migration and invasion was evaluated through wound healing and Transwell invasion assays. （6） The mRNA and protein levels of SIX1，which is regulated by DGUOK-AS1 were measured using quantitative real-time PCR （qRT-PCR） and Western blotting. （7）The effects of DGUOK-AS1 and SIX1 on lipid metabolism in renal cancer cells were determined by measuring total cholesterol and triglyceride levels. Results：（1） Analysis of the ENCORI database revealed that both DGUOK-AS1 and SIX1 were highly expressed in KIRC tissues，and patients with high expression of either DGUOK-AS1 or SIX1 had lower overall survival rates than those with low expression. （2） Compared with renal tubular epithelial cells，DGUOK-AS1 was highly expressed in KIRC cell lines. （3） Overexpression of DGUOK-AS1 promoted the proliferation，migration，and invasion of KIRC cells，while the knockdown of DGUOK-AS1 inhibited these processes. （4） Overexpression of DGUOK-AS1 upregulated the mRNA and protein levels of SIX1 and increased the mRNA expression of lipid synthesis-related genes. Conversely，knockdown of DGUOK-AS1 downregulated the mRNA and protein levels of SIX1 and the mRNA expression of lipid synthesis-related genes. （5） Overexpression of DGUOK-AS1 elevated total cholesterol and triglyceride levels，while its knockdown decreased these levels. （6） Overexpression of SIX1 increased total cholesterol and triglyceride levels，whereas knockdown of SIX1 reduced these levels. （7） DGUOK-AS1 promoted the expression of SIX1，as well as the proliferation and invasion of KIRC cells，by interacting with miR-145-5p. （8） C75 impaired the ability of DGUOK-AS1 to promote KIRC progression. Conclusion：The lncRNA DGUOK-AS1 is highly expressed in KIRC and acts as a potential oncogenic molecule. The DGUOK-AS1 gene affects the expression of SIX1 and lipid levels by interacting with miR-145-5p，thereby further modulating the proliferation and invasion of KIRC.
The purpose of this study is to develop an inorganic nanoparticle-emulsion complex system for vaccine adjuvant research and development. This study employed inorganic calcium oxide nanoparticles （CaO NPs） and zinc oxide nanoparticles （ZnO NPs） to evaluate their antigen adsorption capacity by loading the model antigen ovalbumin （OVA）. Subsequently，the inorganic nanoparticles were incorporated into the internal aqueous phase of a water-in-oil-in-water （W/O/W） emulsion. A particle-emulsion complex system （WNP/O/W） was prepared using a two-step emulsification method，and the optimal formulation was determined by adjusting the surfactant ratio in the oil phase and the volume of the external aqueous phase. The antigen loading capacity of the complex system was determined using the BCA protein assay. In an experimental animal model，Cy7-labeled OVA （Cy7-OVA） was used as a fluorescent tracer to evaluate the antigen depot effect of the WNP/O/W via in vivo fluorescence imaging. Furthermore，the system’s specific immune enhancement on OVA was comprehensively evaluated by measuring humoral and cellular immune responses. The results demonstrated that both positively charged CaO NPs and ZnO NPs strongly adsorbed the negatively charged model antigen OVA，with an antigen adsorption rate of approximately 60%. The nanoparticle-emulsion complex system with a particle size of 1-2 μm was successfully prepared through formulation optimization，and the antigen loading efficiency of OVA was greater than 90%. In vivo fluorescence imaging revealed that both WNP/O/W systems notably extended the retention time of antigens at the injection site，surpassing the commercial aluminum adjuvant （Al NPs）. Fluorescence signals were detectable for up to 168 h，indicating a substantial antigen depot effect. Further immunological evaluation confirmed that the WNP/O/W formulation has excellent adjuvant activity. It synergistically elicits robust humoral and cellular immune responses and has great potential as a novel vaccine adjuvant platform.
Objective：The nematode-trapping fungus Arthrobotrys oligospora can produce a series of sesquiterpenyl epoxy-cyclohexenoids （SECs）. As fungal signal regulatory molecules，these compounds play a crucial role in the fungus’s predation of nematodes. However，the biosynthetic process of SECs has yet to be fully elucidated. This study aimed to preliminarily investigate the biosynthetic process of SECs by expressing key genes involved in SEC biosynthesis heterologously and analyzing the differences in secondary metabolites. Methods：Key biosynthetic target genes were extracted from the AOL s00215g gene cluster of Arthrobotrys oligospora，and recombinant plasmid expression vectors were constructed. The recombinant plasmids were introduced into the host strain of Aspergillus oryzae for expression using the protoplast transformation method. Following the liquid fermentation of the expression strain，high-performance liquid chromatography （HPLC） and ultra-high-performance liquid chromatography-mass spectrometry （UHPLC-MS） were employed to identify differential secondary metabolites in the fermentation broth. Results：Three key target genes were successfully extracted，and seven recombinant plasmid vectors were constructed. After the recombinant plasmid vectors were introduced into Aspergillus oryzae protoplasts for transformation，seven expression strains were obtained through screening. Analysis of the fermentation broth from the expression strains via HPLC and UHPLC-MS revealed that，compared with the blank control strain，all seven expression strains consumed the precursor toluquinol and produced differential compounds. Among them，it was inferred from the combination of secondary mass spectrometry that the Asor-276-277-278 expression strain contained SECs. Conclusion：Seven expression strains containing differential SECs were identified，thereby establishing a foundation for in-depth exploration of the biosynthetic process of SECs.
Objective：The α-amylase gene L9A and the maltogenic amylase gene L9M from Bacillus licheniformis isolated from Daqu were cloned and then heterologously expressed in Escherichia coli. The enzymatic properties of the purified recombinant enzymes were characterized to establish a theoretical foundation for developing industrial amylase preparations. Methods：The L9A and L9M genes were cloned individually into the plasmid pET30a（+） to construct recombinant vectors，which were then transformed into E. coli BL21（DE3） for expression. Following expression and condition optimization，the recombinant enzymes were purified using nickel affinity chromatography. Enzyme activity was determined using the 3，5-dinitrosalicylic acid （DNS） method with soluble starch as the substrate. We characterized their enzymatic properties，including their optimal temperature and pH，thermostability and pH stability，the effects of metal ions，and their enzyme kinetic parameters. Results：Both L9A and L9M were successfully expressed in E. coli，with respective molecular weights of approximately 59.6 kDa and 68.4 kDa. L9A remained active at 100℃，with an optimal pH of 6.0. It retained over 85% of its activity after incubation at 80℃ for 1 h and showed good stability within the pH range of 6.0 to 10.6. Its activity increased with Ca2+. The Km and Kcat values were determined to be 3.0 mg/mL and 12 017.2 s-1，respectively. L9M exhibited optimal activity at a temperature of 40℃ and a pH of 6.0. It retained over 94% of its activity after incubation at 45℃ for 1 h，and showed good stability within the pH range of 6.0 to 10.0. Its activity was enhanced by Fe3+ and Mn2+. The Km and Kcat values were 26.3 mg/mL and 156.0 s-1，respectively. Conclusion：L9A is a thermostable α-amylase with high catalytic efficiency，while L9M is a mesophilic maltogenic amylase with distinct catalytic properties. Due to their significant differences in functional characteristics，they have the potential for synergistic applications in starch liquefaction and saccharification processes.
Metallothioneins （MTs） demonstrate significant potential for heavy metal adsorption. However，their practical application is limited by low expression levels，complex purification processes，and difficult recovery methods. Fusing MTs with a carbohydrate-binding module （CBM） and immobilizing them onto cellulose offers a promising strategy for overcoming these challenges. However，sources of CBM for MT fusion are currently limited. In this study，four CBMs of different origins were fused with MT and super-folder green fluorescent protein （sfGFP） to create recombinant MT-CBM-sfGFP proteins. By leveraging the fluorescent properties of sfGFP，the binding behavior of these recombinant proteins was evaluated on microcrystalline cellulose （MC），Sigmacell 101 （SC101），and two types of regenerated amorphous cellulose （RAC1 and RAC2）. Quantitative analysis focused on the loading capacity of the recombinant protein that incorporates the CBM from Caldicellulosiruptor bescii （CBM-Cb） on these cellulose substrates. The morphology of the four celluloses and the immobilization status of the protein were characterized using scanning electron microscopy （SEM） and transmission electron microscopy （TEM）. The optimal immobilization conditions were determined by systematically optimizing the pH and temperature. Additionally，the Langmuir adsorption isotherm model was employed to evaluate the adsorption capacity of the resulting protein-cellulose adsorbent for Pb2+ and Cd2+. The results indicated that the recombinant fusion protein containing CBM-Cb exhibited the highest immobilization efficiency on all types of cellulose，achieving a loading capacity of 18.90 mg/g on RAC2-more than nine times greater than that of previously reported systems. Structural characterization revealed that RAC2 possesses a porous network architecture with abundant binding sites，facilitating enhanced protein immobilization. The optimal immobilization conditions were determined to be a pH of 8.0 and a temperature of 40℃. This study demonstrates that combining CBM from Caldicellulosiruptor bescii and RAC2 significantly enhances the efficiency of MT immobilization providing a novel strategy and theoretical basis for developing high-performance bio-adsorbents for removing heavy metals.
Hepatocellular carcinoma （HCC） is a primary liver tumor characterized by high incidence and mortality，with a complex pathogenesis involving dysregulation of multiple signaling pathways. The Hippo signaling pathway，as one of the key regulatory pathways，primarily transmits signals through a cascade of core kinases，thereby inhibiting the nuclear translocation of the YAP/TAZ，transcriptional coactivators and reducing the transcription of downstream pro-proliferative and anti-apoptotic genes. Extensive evidence indicates that YAP/TAZ，as core effectors of the Hippo pathway，exhibit aberrant stability and activation，which represents a critical molecular event in HCC development and progression. Therefore，a thorough investigation of the function of YAP/TAZ proteins in the Hippo signaling pathway is of great significance for identifying novel therapeutic targets. This review systematically summarizes the regulation of hepatoma cell proliferation，apoptosis，metastasis，stemness，tumor microenvironment，and therapy resistance. It also discusses the classical ubiquitin-proteasome pathway governing YAP/TAZ stability，as well as emerging regulatory mechanisms such as phase separation and O-GlcNAcylation. Finally，therapeutic strategies centered on targeted degradation of YAP/TAZ proteins-particularly proteolysis-targeting chimeras （PROTACs） and molecular glues-are discussed in terms of their principles，advantages，and current challenges，aiming to provide a theoretical foundation and research direction for developing novel targeted therapies against HCC.
Viral vectors facilitate the highly efficient delivery of exogenous genes. In recent years，viral vectors have achieved significant breakthroughs in gene therapy for monogenic genetic diseases and anti-tumor immunotherapy，while also providing new therapeutic avenues for chronic diseases such as cardiovascular diseases and HIV. However，the challenges to the clinical application of viral vectors are manifold，including their targeting，immunogenicity，vector capacity，preparation/purification efficiency，and neutralizing antibodies. On the other hand，various vectors possess distinctive disadvantages. For instance，both LV and AdV have higher risks of gene mutation，off-target effect，and genome integration，while HSV has stronger viral protein toxicity. Therefore，the engineering modification of viral vectors is the current focus of gene therapyresearch.This review summarizes the advances and major challenges of gene delivery vectors’ engineering modification and application，including recombinant adeno-associated virus （AAV），lentivirus（LV），herpes simplex virus （HSV），and adenovirus （AD）.
In the fields of life science research and translational medicine，nanobodies （e.g.，variable domain of heavy-chain antibodies，or VHHs） have emerged as crucial tools due to their small molecular weight，structural stability，and ease of engineering. However，their clinical translation remains constrained by the maturity of engineering technologies. Currently，nanobody development is undergoing a profound transformation from “empirical screening”to“rational design”，achieving significant breakthroughs in key stages such as screening，optimization，and functional engineering. In high-efficiency screening，microfluidic single B-cell sorting technology bypasses traditional library construction processes，enabling direct and ultra-high-throughput mining of natural immune libraries，thereby significantly shortening the discovery cycle for high-affinity nanobodies. For performance optimization，the application of artificial intelligence and computational biology tools （e.g.，AlphaFold2 and Rosetta） enables precise prediction of binding interfaces and structural stability. This，in turn，facilitates virtual directed evolution and propels affinity maturation research into a data-driven paradigm. In functional engineering，protein engineering strategies，such as multispecific design and site-specific conjugation，have been employed to give nanobodies complex capabilities such as synergistic targeting，drug delivery，and molecular imaging，significantly broadening their biomedical applications. Based on the study of the structure and function of nanoantibodies，a systematic review of the engineering progress of the whole chain of nanoantibodies from efficient screening to functional optimization is presented in this paper. The objective is to provide a prospective perspective for overcoming the existing research and development bottlenecks and promoting clinical transformation，and to predict the future development trend.
Gene sequencing is an important method of genetic testing that mainly includes the following steps：nucleic acid extraction，library preparation，sequencing on an instrument，and bioinformatics analysis. Compared with traditional gene sequencing technologies，nanopore single-molecule sequencing has advantages such as ultra-long read length，real-time analysis，and easy integration. The instrument platform that combines nanopore single-molecule sequencing and microfluidic chip technology is compact，simple to operate，accurate and intelligent. To a certain extent，it solves the problems of the traditional laboratory sequencing process being complex，time-consuming，and requiring highly skilled technical personnel. This type of sequencing technology has broad application prospects in many fields such as rapid identification of pathogenic microorganisms，clinical diagnosis，analysis of drug resistance，and monitoring of epidemic prevention and control. This paper systematically reviews nanopore sequencing technology，sequencing processes，as well as its automated library preparation platforms and sequencing platforms，which can provide references for researchers to carry out the standardization of nanopore sequencing processes and formulate nanopore sequencing solutions based on specific needs.
γ-Polyglutamic acid （γ-PGA） is a polymer composed of glutamic acid monomers that are linked by γ-amide bonds，characterized by its abundance of carboxyl groups. γ-PGA’s unique molecular structure is responsible for its multiple functional properties，including exceptional water absorption and chelation capacity，as well as biodegradability，and biocompatibility. Due to these properties，γ-PGA exhibits broad application prospects in agriculture. It can be used to improve soil quality，enahnce fertilizer efficiency，and increase crop stress resistance，thereby promoting plant growth and increasing crop yield. The primary methods for γ-PGA production include extraction，chemical synthesis，enzymatic synthesis，and microbial fermentation. Among these methods，microbial fermentation has emerged as the dominant industrial-scale technique. However，large-scale production of γ-PGA faces critical challenges，including relatively low fermentation yields，high production costs，and difficulty in controlling the precise molecular weight，which constrain its widespread application. This review systematically summarizes the structure and properties of γ-PGA，its biosynthetic mechanisms，strategies to optimize fermentation production，and the latest advances in its agricultural applications. The goal is to provide insights into the eco-efficient and green production of γ-PGA，as well as to promote its integration into agricultural practices.
High-level biosafety laboratories are key infrastructure of the national biosafety defense. They carry out important tasks such as pathogen research，epidemic prevention and control，and emergency response. In recent years，China has made progress in constructing a high-level laboratory system and has initially formed a scientific research support system centered on national key laboratories，high-level biosafety experimental platforms，and special pathogen resource libraries. The development of high-level biosafety laboratories in China faces challenges such as an uneven regional layout，insufficient enforcement of laws and regulations，a lack of operational guarantees for facilities，a shortage of talent，and a need for stronger management mechanisms，all of which hinder its efficient operation. This article reviews the construction status of high-level biosafety laboratories in China，and thoroughly analyzes the difficulties and challenges encountered during their development. We proposed countermeasures and suggestions，such as optimizing the layout，improving laws and regulations，enahncing mechanisms，and strengthening guarantees. These measures aim to provide theoretical support and policy references for improving China’s biosafety governance capabilities and responding to major public health risks.
Superbug infections pose a global threat and challenge to public health，and phage therapy is a promising technology for preventing and controlling them. The European Union has promulgated the Guidelines on the quality，safety and efficacy of veterinary medicinal products specifically designed for phage therapy，which set out the scope of application and legal basis of the guidelines，the requirements for marketing authorization applications for veterinary phage products，and post-authorization modifications. The requirements for marketing authorization applications for veterinary phage products primarily include the following：product composition，expected product quality，research on product safety and efficacy，critical quality attributes of the active pharmaceutical ingredient and final product，production process，determination and control of raw material quality，product stability，commercial production records，and post-approval pharmacovigilance databases for the product or similar products，and general scientific knowledge. The guidelines offer general recommendations and do not delve into specifics. It is expected that China will establish regulatory provisions for phage-related veterinary drugs and technologies，enhance support for phage research and industrialization，expedite the legalization process of phages，and provide innovative solutions for drug-resistant bacteria in animal husbandry and human health.
