Abstract:

Abstract:The growth, differentiation and proliferation of adipose cells run through the whole life process. Dysregulation of lipid metabolism in adipose cells affects adipose tissue immunity and systemic energy metabolism. Increasingly available data suggest that lipid metabolism is involved in regulating the occurrence and development of various diseases, such as hyperlipidemia, nonalcoholic fatty liver disease, diabetes and cancer, which pose a major threat to human and animal health. Hypoxia inducible factor (HIF) is a major transcription factor mediating oxygen receptors in tissues and organs. HIF can induce disease by regulating lipid synthesis, fatty acid metabolism and lipid droplet formation. However, due to the difference of hypoxia degree, time and mode of action, there is no conclusive conclusion whether it has harmful or beneficial effects on the development of adipocytes and lipid metabolism. This article summarizes the regulation of hypoxia stress mediated transcription regulators and regulation of adipocyte development and lipid metabolism, aiming to reveal the potential mechanism of hypoxia induced changes in adipocyte metabolism pathways.

Abstract:Traditional pig breeding has a long cycle and high cost, and there is an urgent need to use new technologies to revitalize the pig breeding industry. The recently emerged CRISPR/Cas9 genome editing technique shows great potential in pig genetic improvement, and has since become a research hotspot. Base editor is a new base editing technology developed based on the CRISPR/Cas9 system, which can achieve targeted mutation of a single base. CRISPR/Cas9 technology is easy to operate and simple to design, but it can lead to DNA double strand breaks, unstable gene structures, and random insertion and deletion of genes, which greatly restricts the application of this technique. Different from CRISPR/Cas9 technique, the single base editing technique does not produce double strand breaks. Therefore, it has higher accuracy and safety for genome editing, and is expected to advance the pig genetic breeding applications. This review summarized the working principle and shortcomings of CRISPR/Cas9 technique, the development and advantages of single base editing, the principles and application characteristics of different base editors and their applications in pig genetic improvement, with the aim to facilitate genome editing-assisted genetic breeding of pig.

Abstract:Endosomal sorting complex required for transport (ESCRT) system drives various cellular processes, including endosome sorting, organelle biogenesis, vesicle transport, maintenance of plasma membrane integrity, membrane fission during cytokinesis, nuclear membrane reformation after mitosis, closure of autophagic vacuoles, and enveloped virus budding. Increasing evidence suggests that the ESCRT system can be hijacked by different family viruses for their proliferation. At different stages of the virus life cycle, viruses can interfere with or exploit ESCRT-mediated physiological processes in various ways to maximize their chance of infecting the host. In addition, many retroviral and RNA viral proteins possess "late domain" motifs, which can recruit host ESCRT subunit proteins to assist in virus endocytosis, transport, replicate, budding and efflux. Therefore, the "late domain" motifs of viruses and ESCRT subunit proteins could serve as promising drug targets in antiviral therapy. This review focuses on the composition and functions of the ESCRT system, the effects of ESCRT subunits and virus "late domain" motifs on viral replication, and the antiviral effects mediated by the ESCRT system, aiming to provide a reference for the development and utilization of antiviral drugs.

Abstract:Messenger RNA (mRNA) vaccines emerge as promising vaccines to prevent infectious diseases. Compared with traditional vaccines, mRNA vaccines present numerous advantages, such as high potency, safe administration, rapid production potentials, and cost-effective manufacturing. In 2020, two COVID-19 vaccines (BNT162b2 and mRNA-1273) were approved by the Food and Drug Administration (FDA). The two vaccines showed high efficiency in combating COVID-19, which indicates the great advantages of mRNA technology in developing vaccines against emergent infectious diseases. Here, we summarize the type, immune mechanisms, modification methods of mRNA vaccines, and their applications in preventing infectious diseases. Current challenges and future perspectives in developing mRNA vaccines are also discussed.

Abstract:Listeria monocytogenes is recognized as a significant foodborne pathogen, capable of causing listeriosis in humans, which is a global public health concern. This pathogen is particularly dangerous for pregnant women, as it can lead to invasive listeriosis in fetuses and neonates, posing a significant threat to both maternal and fetal health. Therefore, establishing suitable in vitro and in vivo models for L. monocytogenes placenta infection, as well as analyzing and exploring the infection process and its pathogenic mechanism, are important approaches to prevent and control L. monocytogenes infection in mothers and infants. In this study, we reviewed the in vitro and in vivo placental models used for studying the infection of L. monocytogenes in maternal and infant, summarized and discussed the advantages and limitations of each model, and explored the potential of in vitro cell models and organoids for the study of L. monocytogenes infection. This paper aims to support the study of the infection pathway and pathogenesis of listeriosis and provide scientific references for the prevention and control of L. monocytogenes infection.

Abstract:T cells play central roles in anti-tumor immune responses. Immune checkpoint therapy, which is based on modulation of T cell reactivity, has achieved breakthrough in clinical treatment of multiple tumors. Moreover, adoptive T cell therapy, which includes mainly genetically engineered T cells, has shown substantial treatment efficacy in hematoma. Immune therapy has tremendously changed the scenario of clinical tumor treatment and become critical strategies for treating multiple tumors. T cell receptor (TCR) is the fundamental molecule responsible for the specificity of T cell recognition. TCRs could recognize peptides, which are derived from intracellular or extracellular tumor antigens, presented by major histocompatibility complex (MHC) and are therefore highly sensitive to low antigen level. Thereby, TCRs are broadly recognized as promising molecules for the development of anti-tumor drugs. The approval of the first TCR drug in 2022 has initiated a new era for TCR-based therapeutics and since then, multiple TCR drugs have shown substantial treatment efficacy in multiple tumors. This review summarizes the progress of TCR-based immune therapeutic strategies, including T cell receptor-engineered T cell (TCR-T), TCR-based protein drugs, and other cell therapies based on TCR signaling, providing useful information for future design of immune therapeutics based on TCR.

Abstract:Mechanosensitive channels (MSCs) are special membrane proteins that can convert mechanical stimulation into electrical or chemical signals. These channels have become potential targets for ultrasonic neuromodulation due to their properties. The good spatial resolution and focusing effect of ultrasound make it theoretically possible to achieve non-invasive whole-brain localization. Therefore, ultrasonic neuromodulation is a promising method for performing physical neuromodulation and treating neurological disorders. To date, only a few ion channels have been reported to be activated by ultrasound, while recent research has identified more channels with mechanosensitive properties. Moreover, the opening process and mechanism of MSCs under ultrasound excitation remain unknown. This review provides an overview on recent research advances and applications in MSCs, including large conductance mechanosensitive channels, transient receptor potential channels, degenerated protein/epithelial sodium channels, two-pore potassium channels, and piezo channels. These findings will facilitate future studies and applications of ultrasonic neuromodulation.

Abstract:3D bioprinting technology is a rapidly developing technique that employs bioinks containing biological materials and living cells to construct biomedical products. However, 3D-printed tissues are static, while human tissues are in real-time dynamic states that can change in morphology and performance. To improve the compatibility between in vitro and in vivo environments, an in vitro tissue engineering technique that simulates this dynamic process is required. The concept of 4D printing, which combines "3D printing + time" provides a new approach to achieving this complex technique. 4D printing involves applying one or more smart materials that respond to stimuli, enabling them to change their shape, performance, and function under the corresponding stimulus to meet various needs. This article focuses on the latest research progress and potential application areas of 4D printing technology in the cardiovascular system, providing a theoretical and practical reference for the development of this technology.

Abstract:Artificial nerve guidance conduits (NGCs) are synthetic nerve grafts that are capable of providing the structural and nutritional support for nerve regeneration. The ideal NGCs have plenty of requirements on biocompatibility, mechanical strength, topological structure, and conductivity. Therefore, it is necessary to continuously improve the design of NGCs and establish a better therapeutic strategy for peripheral nerve injury in order to meet clinical needs. Although current NGCs have made certain process in the treatment of peripheral nerve injury, their nerve regeneration and functional outcomes on repairing long-distance nerve injury remain unsatisfactory. Herein, we review the nerve conduit design from four aspects, namely raw material selection, structural design, therapeutic factor loading and self-powered component integration. Moreover, we summarize the research progress of NGCs in the treatment of peripheral nerve injury, in order to facilitate the iterative updating and clinical transformation of NGCs.

Abstract:Unique factors in the space environment can cause dysbiosis of astronauts' gut microbiota and its metabolites, which may exert systematic physiological effects on human body. Recent progress regarding the effect of space flight/simulated space environment (SF/SPE) on the composition of gut microbiota and its metabolites was reviewed in this paper. SF/SPE may cause the increase of invasive pathogenic bacteria and the decrease of beneficial bacteria, aggravating intestinal inflammation and increasing intestinal permeability. SF/SPE may also cause the decrease of beneficial metabolites or the increase of harmful metabolites of gut microbiota, leading to metabolism disorder in vivo, or inducing damage of other systems, thus not beneficial to the health and working efficiency of astronauts. Summarizing the effects of SF/SPE on gut microbiota may provide scientific basis for further researches in this field and the on-orbit health protection of astronauts.

Abstract:To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV:C:L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV:C:L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV:C:L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV:C immunization, EsxV:C:L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV:C:L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.

Abstract:Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

Abstract:Meiotic initiation is a critical step in gametogenesis. Recently, some genes required for meiotic initiation have been identified. However, meiosis-initiating factors and the underlying mechanisms are far from being fully understood. We have established a long-term culture system of spermatogonial stem cells (SSCs) and an in vitro model of meiotic initiation using mouse SSCs. Our previous study revealed that the RNA-binding protein RBFOX2 may regulate meiotic initiation, but the role and the mechanism need to be further elucidated. In this study, we constructed RBFOX2 knockdown SSC lines by using lentivirus-mediated gene delivery method, and found that the knockdown SSCs underwent normal self-renewal, mitosis and differentiation. However, they were unable to initiate meiosis when treated with retinoic acid, and they underwent apoptosis. These results indicate that RBFOX2 plays an essential role in meiotic initiation of spermatogonia. This work provides new clues for understanding the functions of RNA-binding proteins in meiotic initiation.

Abstract:Heterotypic cell-in-cell structures (heCICs) are closely related to tumor development and progression, and have become a new frontier in life science research. Ras-related C3 botulinum toxin substrate 1 (Rac1) belongs to the classic Rho GTPase, which plays a key role in regulating the cytoskeleton and cell movement. To investigate the role and mechanism of Rac1 in the formation of heCICs, tumor cells and immune killer cells were labeled with cell-tracker, respectively, to establish the heCICs model. Upon treatment with the Rac1 inhibitor NSC23766, the formation of heCICs between tumor and immune cells was significantly reduced. The plasmid pQCXIP-Rac1-EGFP constructed by gene cloning was packaged into pseudoviruses that subsequently infect tumor cells to make cell lines stably expressing Rac1. As a result, the formation of heCICs was significantly increased upon Rac1 overexpression. These results demonstrated a promotive role of Rac1 in heCICs formation, which may facilitate treating cell-in-cell related diseases, such as tumors, by targeting Rac1.

Abstract:The biofilms formed by pathogenic microorganisms seriously threaten human health and significantly enhance drug resistance, which urgently call for developing drugs specifically targeting on biofilms. Chitooligosaccharides extracted from shrimp and crab shells are natural alkaline oligosaccharides with excellent antibacterial effects. Nevertheless, their inhibition efficacy on biofilms still needs to be improved. Spirulina (SP) is a microalga with negatively charged surface, and its spiral structure facilitates colonization in the depth of the biofilm. Therefore, the complex of Spirulina and chitooligosaccharides may play a synergistic role in killing pathogens in the depth of biofilm. This research first screened chitooligosaccharides with significant bactericidal effects. Subsequently, Spirulina@Chitooligosaccharides (SP@COS complex was prepared by combining chitooligosaccharides with Spirulina through electrostatic adsorption. The binding of the complex was characterized by zeta potential, z-average size, and fluorescence labeling. Ultraviolet-visible spectroscopy (UV-Vis) showed the encapsulation efficiency and the drug loading efficiency reached up to 90% and 16%, respectively. The prepared SP@COS2 exhibited a profound synergistic inhibition effect on bacterial and fungal biofilms, which was mainly achieved by destroying the cell structure of the biofilm. These results demonstrate the potential of Spirulina-chitooligosaccharides complex as a biofilm inhibitor and provide a new idea for addressing the harm of pathogenic microorganisms.

Abstract:The neurotrophin-tyrosine receptor kinase B (TrkB) signaling pathway plays an important role in regulating the balance of excitation and inhibition in the primary visual cortex (V1). Previous studies have revealed its mechanism of regulating the level of cortical excitability by increasing the efficiency of excitatory transmission, but it has not been elucidated how TrkB receptors regulate the balance of excitation and inhibition through the inhibitory system, which in turn affects visual cortex function. Therefore, the objective of this study was to investigate how the TrkB signaling pathway specifically regulates the most important inhibitory neuron-PV neurons affects the visual cortex function of mice. The expression of TrkB receptor on PV neurons in the V1 region was specifically reduced by the virus, the functional changes of inhibitory and excitatory neurons in the primary visual cortex were recorded by multi-channel electrophysiological in vivo. The orientation discrimination ability of mice was tested by behavioral experiments, and altered orientation discrimination ability of mice was tested by behavioral experiments. The results showed that reduced expression of TrkB receptors on PV inhibitory neurons in primary visual cortex significantly increased the response intensity of excitatory neurons, reduced the orientation discrimination ability of inhibitory and excitatory neurons, and increased the signal-to-noise ratio, but the orientation discrimination ability at the individual level in mice showed a decrease. These results suggest that the TrkB signaling pathway does not modulate the function of PV neurons solely by increasing excitatory transmission targeting PV neurons, and its effect on neuronal signal-to-noise ratio is not due to enhancement of the inhibitory system.

Abstract:Cluster of differentiation 36 (CD36) is a membrane glycoprotein receptor capable of binding and transporting fatty acid. Nogo-B regulates the metabolism of fatty acids in the liver and affects the development of liver cancer. To date, it remains unclear whether the interaction between CD36 and Nogo-B affects the proliferation and migration of breast cancer cells. In the current study, we aimed to determine whether the interference of CD36 and Nogo-B affects the proliferation and migration of triple-negative breast cancer (TNBC) cells. The results showed that inhibition of CD36 or Nogo-B alone can inhibit the proliferation and migration of TNBC cells, and the inhibitory effect was more pronounced when CD36 and Nogo-B were inhibited simultaneously. Meanwhile, it was found that inhibition of CD36 and Nogo-B expression can inhibit the expression of Vimentin, B-cell lympoma-2 (BCL2) and proliferating cell nuclear antigen (PCNA). In vivo, knockdown of CD36 or Nogo-B in E0771 cells reduced its tumorigenic ability, which was further enhanced by knockdown of CD36 and Nogo-B simultaneously. Mechanistically, inhibition of CD36 and Nogo-B expression can decrease fatty acid binding protein 4 (FABP4) and fatty acid transport protein 4 (FATP4) expression. Moreover, overexpression of CD36 and Nogo-B-induced cell proliferation was attenuated by FABP4 siRNA, indicating that inhibition of CD36 and Nogo-B expression could inhibit the absorption and transport of fatty acids, thereby inhibiting the proliferation and migration of TNBC. Furthermore, inhibition of CD36 and Nogo-B expression activated the P53-P21-Rb signaling pathway which contributed to the CD36 and Nogo-B-inhibited proliferation and migration of TNBC. Taken together, the results suggest that inhibition of CD36 and Nogo-B can reduce the proliferation and migration of TNBC, which provides new targets for the development of drugs against TNBC.

Abstract:Silver nanoparticles (AgNPs) is known as one of the most valuable metal nanoparticles in antibacterial and anticancer application. AgNPs-resistant bacteria has been documented, but it is unclear whether cancer cells can also escape the anti-cancer effect of AgNPs. In this study, we aimed to investigate this phenomenon and its underlying mechanism. The antibacterial activity and cytotoxicity of AgNPs were measured in the presence of HeLa cell metabolites. The status of AgNPs in the system associated with metabolites were characterized by UV-Vis, Zetasizer Nano ZS, and transmission electron microscopy. Non-targeted metabolomics was used to reveal the metabolites components that bind with AgNPs. HeLa cells were injected intraperitoneally to establish the tumor-bearing mice model, and the stability of AgNPs in mice serum was analyzed. The results manifested that HeLa cell metabolites inhibited the anticancer and antibacterial effects of AgNPs in a dose-dependent manner by causing AgNPs aggregation. Effective metabolites that inhibited the biological activity of AgNPs were stable in 100℃, insoluble in chloroform, containing sulfur elements, and had a molecular weight less than 1 kDa in molecular weight. There were 115 compounds bound with AgNPs. In vitro experiments showed that AgNPs aggregation occurred only when the concentration of α-ketoglutarate (AKG) and glutathione (GSH) together reached a certain threshold. Interestingly, the concentration of AKG and GSH in HeLa cellular metabolites was 10 and 6 times higher than that in normal cervical epithelial cells, respectively, which explained why the threshold was reached. Furthermore, the stability of AgNPs in the serum of tumor-bearing mice decreased by 20% (P<0.05) compared with the healthy mice. In conclusion, our study demonstrates that HeLa cells escaped the anti-cancer effect of AgNPs through the synergistic effect of AKG and GSH, suggesting the need to develop strategies to overcome this limitation.

Abstract:During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.

Abstract:This study aimed to explore the expression changes of VASA gene in sheep testis development and to construct VASA gene knock-in vector to prepare for the study on the differentiation of sheep germ cells in vitro. The testicular tissues of 3-month-old (3M) and 9-month-old (9M) sheep which represent immature and mature stages, respectively, were collected. The differential expression of VASA gene was analyzed by quantitative real-time PCR (qPCR) and Western blotting, and the location of VASA gene was detected by immunohistochemistry. The sgRNA targeting the VASA gene was designed and homologous recombination vectors were constructed by PCR. Subsequently, plasmids were transferred into sheep ear fibroblasts. The VASA gene was activated in combination with CRISPR/dCas9 technology to further verify the efficiency of the vector. The results showed that the expression level of VASA gene increased significantly with the development of sheep testis (P<0.01), and was mainly located in spermatocytes and round spermatids. The knock-in vector of VASA gene was constructed by CRISPR/Cas9 system, and the Cas9-gRNA vector and pEGFP-PGK puro-VASA vector were transfected into ear fibroblasts. After CRISPR/dCas9 system was activated, ear fibroblasts successfully expressed VASA gene. The results suggest that VASA gene plays a potential function in sheep testicular development and spermatogenesis, and the VASA gene knock-in vector can be constructed in vitro through the CRISPR/Cas9 system. Our results provided effective research tools for further research of germ cell development and differentiation.

Abstract:The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7% of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.

Abstract:Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.

Abstract:Anti-reflective nanocoatings that mimic the eyes of fruit flies are biodegradable materials with great market potential for a variety of optical devices that require anti-reflective properties. Microbial expression of retinin provides a new idea for the preparation of nanocoatings under mild conditions compared to physicochemical methods. However, the current expression level of retinin, the key to anti-reflective coating, is low and difficult to meet mass production. In this study, we analyzed and screened the best expression hosts for Drosophila-derived retinin protein, and optimized its expression. Chinese hamster ovary (CHO) cells were identified as the efficient expression host of retinin, and purified retinin protein was obtained. At the same time, the preparation method of lanolin nanoemulsion was explored, and the best anti-reflective ability of the nano-coating was determined when the ratio of specific concentration of retinin protein and wax emulsion was 16:4, the pH of the nano-coating formation system was 7.0, and the temperature was 30℃. The enhanced antireflective ability and reduced production cost of artificial antireflective nanocoatings by determining the composition of nanocoatings and optimizing the concentration, pH and temperature of system components may facilitate future application of artificial green degradable antireflective coatings.

Abstract:The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His₆-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His₆-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.

Abstract:We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R²=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation<5%, n=3.

Abstract:Gas vesicles (GVs) are gas-filled protein nanostructures that can regulate the buoyancy of microorganisms such as cyanobacteria and archaea. Recent studies have shown that GVs have the potential to be used as ultrasound molecular imaging probes in disease diagnosis and treatment. However, the mechanism of the inflation and deflation of GVs remains unclear, which hampers the preservation of GVs and gas replacement. In the present study, the environmental pH value was found to be an important factor in regulating the inflation and deflation of GVs. It can not only regulate the inflation and deflation of GVs in vivo to make Microcystis sp. cells present distinct levitation state, but also regulate the inflation and deflation of purified GVs in vitro, and the regulation process is reversible. Our results may provide a technical support for the large-scale production and preservation of biosynthetic ultrasound molecular imaging probes, especially for gas replacement to meet different diagnostic and therapeutic needs, and would facilitate the application of biosynthetic ultrasound molecular imaging probes.