Evaluation of nanoparticle albumin-bound paclitaxel loaded macrophages for glioblastoma treatment based on a microfluidic chip.

Introduction: Glioblastoma (GBM) is a primary brain malignancy with a dismal prognosis and remains incurable at present. In this study, macrophages (MΦ) were developed to carry nanoparticle albumin-bound paclitaxel (nab-PTX) to form nab-PTX/MΦ. The aim of this study is to use a GBM-on-a-chip to evaluate the anti-GBM effects of nab-PTX/MΦ. Methods: In this study, we constructed nab-PTX/MΦ by incubating live MΦ with nab-PTX. We developed a microfluidic chip to co-culture GBM cells and human umbilical vein endothelial cells, mimicking the simplified blood-brain barrier and GBM. Using a syringe pump, we perform sustainable perfusion of nutrient media. To evaluate the anti-GBM effects nab-PTX/MΦ, we treated the GBM-on-a-chip model with nab-PTX/MΦ and investigated GBM cell proliferation, migration, and spheroid formation. Results: At the chosen concentration, nab-PTX did not significantly affect the viability, chemotaxis and migration of MΦ. The uptake of nab-PTX by MΦ occurred within 1 h of incubation and almost reached saturation at 6 h. Additionally, nab-PTX/MΦ exhibited the M1 phenotype, which inhibits tumor progression. Following phagocytosis, MΦ were able to release nab-PTX, and the release of nab-PTX by MΦ had nearly reached its limit at 48 h. Compared with control group and blank MΦ group, individual nab-PTX group and nab-PTX/MΦ group could inhibit tumor proliferation, invasion and spheroid formation. Meanwhile, the anti-GBM effect of nab-PTX/MΦ was more significant than nab-PTX. Discussion: Our findings demonstrate that nab-PTX/MΦ has a significant anti-GBM effect compared to individual nab-PTX or MΦ administration, suggesting MΦ as potential drug delivery vectors for GBM therapy. Furthermore, the developed GBM-on-a-chip model provides a potential ex vivo platform for innovative cell-based therapies and tailored therapeutic strategies for GBM.

Protein profile analysis of Jilin white goose testicles at different stages of the laying cycle by DIA strategy.

BACKGROUND: Jilin white goose is an excellent local breed in China, with a high annual egg production and laying eggs mainly from February to July each year. The testis, as the only organ that can produce sperm, can affect the sexual maturity and fecundity of male animals. Its growth and development are affected and regulated by a variety of factors. Proteomics is generally applied to identify and quantify proteins in cells and tissues in order to understand the physiological or pathological changes that occur in tissues or cells under specific conditions. Currently, the female poultry reproductive system has been extensively studied, while few related studies focusing on the regulatory mechanism of the reproductive system of male poultry have been conducted. RESULTS: A total of 1753 differentially expressed proteins (DEPs) were generated in which there were 594, 391 and 768 different proteins showing differential expression in three stages, Initial of Laying Cycle (ILC), Peak of Laying Cycle (PLC) and End of Laying Cycle (ELC). Furthermore, bioinformatics was used to analyze the DEPs. Gene ontology (GO) enrichment, Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analysis were adopted. All DEPs were found to be implicated in multiple biological processes and pathways associated with testicular development, such as renin secretion, Lysosomes, SNARE interactions in vesicle trafficking, the p53 signaling pathway and pathways related to metabolism. Additionally, the reliability of transcriptome results was verified by real-time quantitative PCR by selecting the transcript abundance of 6 selected DEPs at the three stages of the laying cycle. CONCLUSIONS: The funding in this study will provide critical insight into the complex molecular mechanisms and breeding practices underlying the developmental characteristics of testicles in Jilin white goose.

An early warning indicator of mortality risk in patients with COVID-19: the neutrophil extracellular traps/neutrophilic segmented granulocyte ratio.

Background: Neutrophil extracellular traps (NETs) play a key role in thrombus formation in patients with coronavirus disease 2019 (COVID-19). However, the existing detection and observation methods for NETs are limited in their ability to provide quantitative, convenient, and accurate descriptions of in situ NETs. Therefore, establishing a quantitative description of the relationship between NETs and thrombosis remains a challenge. Objective: We employed morphological observations of blood cells and statistical analyses to investigate the correlation between the NETs/neutrophilic segmented granulocyte ratio and mortality risk in patients with COVID-19. Methods: Peripheral blood samples were collected from 117 hospitalized patients with COVID-19 between November 2022 and February 2023, and various blood cell parameters were measured. Two types of smudge cells were observed in the blood and counted: lymphatic and neutral smudge cells. Statistical data analysis was used to establish COVID-19 mortality risk assessment indicators. Results: Morphological observations of neutrophilic smudge cells revealed swelling, eruption, and NETs formation in the neutrophil nuclei. Subsequently, the NETs/neutrophilic segmented granulocyte ratio (NNSR) was calculated. A high concentration of NETs poses a fatal risk for thrombus formation in patients. Statistical analysis indicated that a high NNSR was more suitable for evaluating the risk of death in patients with COVID-19 compared to elevated fibrinogen (FIB) and D-dimer (DD) levels. Conclusion: Observing blood cell morphology is an effective method for the detection of NETs, NNSR are important markers for revealing the mortality risk of patients with COVID-19.

miR-140-y targets TCF4 to regulate the Wnt signaling pathway and promote embryonic feather follicle development in Hungarian white goose.

Goose down feather has become one of the most important economical products in the goose breeding industry and it provides several essential physiological roles in birds. Therefore, understanding and regulating the development of skin and feather follicles during embryogenesis is critical for avian biology and the poultry industry. MicroRNAs are known to play an important role in controlling gene expression during skin and feather follicle development. In this study, bioinformatics analysis was conducted to select miR-140-y as a potential miRNA involved in skin and feather follicle development and to predict TCF4 as its target gene. This gene was expressed at significant levels during embryonic feather follicle development, as identified by qPCR and Western blot. The targeting relationship was confirmed by a dual-luciferase assay in 293T cells. Then, the miR-140-y/TCF4 function in dermal fibroblast cells was explored. The results showed that miR-140-y could suppress the proliferation of goose embryonic dermal fibroblast cells (GEDFs) by suppressing the activity of some Wingless-types (Wnt) pathway related genes and proliferation marker genes, while miR-140-y inhibition led to the opposite effect. Similarly, the inhibition of the TCF4 gene results in blocking the proliferation of GEDFs by reducing the activity of some Wnt pathway-related genes. Finally, the co-transfection of miR-140-y inhibitor and siRNA-TCF4 results in a rescue of the TCF4 function and an increase of the Wnt signaling pathway and GEDFs proliferation. In conclusion, these results demonstrated that the miR-140-y-TCF4 axis influences the activity of the Wnt signaling pathway and works as a dynamic regulator during skin and feather follicle development.

Recent research advances on polysaccharide-, peptide-, and protein-based hemostatic materials: A review.

Hemorrhage is a potentially life-threatening emergency that can occur at any time or place. Whether traumatic, congenital, surgical, disease-related, or drug-induced, bleeding can lead to severe complications or death. Therefore, the development of efficient hemostatic materials is critical. However, the results and prognosis demonstrated by clinical means of hemostasis do not reach expectations. With the development of technology, novel hemostatic materials have been developed from polysaccharides (chitosan, hyaluronic acid, alginate, cellulose, cyclodextrins, starch, dextran, and carrageenan), peptides (self-assembling peptides), and proteins (silk fibroin, collagen, gelatin, keratin, and thrombin). These new materials exhibit high hemostatic efficacy due to the enhancement or interaction of various hemostatic mechanisms. The main forms include adhesives, sealants, bandages, hemostatic powders, and hemostatic sponges. This article introduces the clotting process and principles of hemostatic methods and reviews the research on polysaccharide-, peptide-, and protein-based hemostatic materials in the last five years. The design ideas and hemostatic principles of polysaccharide-, peptide-, and protein-based hemostatic materials are mainly introduced. Finally, we summarize material designs, advantages, disadvantages, and challenges regarding hemostatic materials.

Porcine delta coronavirus inhibits NHE3 activity of porcine intestinal epithelial cells through miR-361-3p/NHE3 regulatory axis.

Porcine deltacoronavirus (PDCoV) infection in piglets can cause small intestinal epithelial necrosis and atrophic enteritis, which leads to severe damages to host cells, and result in diarrhea. In this study, we investigated the relationship between miR-361, SLC9A3(Solute carrier family 9, subfamily A, member 3), and NHE3(sodium-hydrogen exchanger member 3) in in porcine intestinal epithelial cells (IPI-2I) cells after PDCoV infection. Our results showed that the ssc-miR-361-3p expression inhibits the mRNA level of SLC9A3 gene which lead to the descending of NHE3 protein expression, and the NHE3 activity was suppressed. NHE3 activity was suppressed via down-regulation expression of SLC9A3 mRNA by transfection with siRNA. Ssc-miR-361-3p mimics and inhibitors were used to change the expression of ssc-miR-361-3p in IPI-2I cells. Ssc-miR-361-3p overexpression reduced the mRNA level of SLC9A3 gene, the level of NHE3 protein expression and NHE3 activity in IPI-2I cells, while ssc-miR-361-3p inhibits NHE3. Furthermore, luciferase reporter assay showed that SLC9A3 gene was a direct target of ssc-miR-361-3p. Ssc-miR-361-3p inhibition restored NHE3 activity in PDCoV infected IPI-2I cells by up-regulating SLC9A3 mRNA expression and NHE3 protein expression. These results demonstrate that the PDCoV infection can inhibit NHE3 activity through miR-361-3p/SLC9A3 regulatory axis. The relevant research is reported for the first time in PDCoV, which has significance in exploring the pathogenic mechanism of PDCoV and can provide a theoretical basis for its prevention and control. suggesting that NHE3 and ssc-miR-361-3p may be potential therapeutic targets for diarrhea in infected piglets.

Circ_103809 Aggravates the Malignant Phenotype of Pancreatic Cancer Through Modulating miR-197-3p/TSPAN3 Axis.

Pancreatic cancer (PC) is a malignant tumor with insidious clinical manifestations and dismal prognosis. Emerging reports have demonstrated that circRNAs exert pivotal biological function in PC. Here, we investigated the crucial biological role and underlying regulatory mechanisms of differentially expressed circ_103809 in PC. In this study, hsa_circ_103809 (hsa_circ_0072088) was identified as the research object via analyzing and screening the aberrantly expressed circRNAs in PC by GSE69362 dataset. The levels of circ_103809 in PC tissues and cells were assessed via qRT-PCR. Functional assays were conducted to monitor the impacts of circ_103809 on PC cells. Additionally, the downstream molecular targets and regulatory networks of circ_103809 were predicted by bioinformatics and validated using luciferase assays and rescue experiments. We found that circ_103809 was substantially upregulated in PC tissues and cells. Silencing circ_103809 restrained the growth viability, clonogenic rate, migration, and invasion capabilities of PC cells. Further mechanistic exploration disclosed that miR-197-3p was the downstream gene of circ_103809, while Tetraspanin-3 (TSPAN3) was a direct target of miR-197-3p. The suppressive effect of circ_103809 knockdown on malignant processes of PC cells was eliminated by miR-197-3p downregulation or TSPAN3 upregulation. Our study demonstrated that circ_103809 served as an innovative positive regulator in the growth and metastasis of PC cells. Furthermore, circ_103809 mediated the miR-197-3p/TSPAN3 axis to modulate the malignant progression of PC cells, which was prospected to be a probable biomarker and an efficient therapeutic target for PC.

Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification bacterium Acinetobacter ZQ-A1 and community characteristics analysis of its application in pig farm wastewater.

A heterotrophic nitrifying aerobic denitrifying (HN-AD) strain ZQ-A1 with excellent denitrification performance, identified as Acinetobacter, was isolated from simultaneous nitrification and denitrification (SND) craft. ZQ-A1 was capable of removing NH4+, NO2-, and NO3-; the 21-hour removal rates were 84.84%, 87.13%, and 92.63%. ZQ-A1 has the ability to treat mixed nitrogen sources. In addition, ZQ-A1 can be well applied to actual sewage. According to the analysis of microbial community characteristics, the relative abundance of Acinetobacter in the experimental group increased from 0.06% to 2.38%, which is an important reason for the removal rate of NH4+ exceeding 99% within 30 days. The results of KEGG function prediction showed that with the addition of ZQ-A1, the relative abundance of pathways related to bacterial metabolism, such as tricarboxylic acid cycle metabolism, was higher. The research expanded the thinking of HN-AD bacteria in actual production and laid a foundation for its application in sewage treatment.

Glioblastoma-on-a-chip construction and therapeutic applications.

Glioblastoma (GBM) is the most malignant type of primary intracranial tumor with a median overall survival of only 14 months, a very poor prognosis and a recurrence rate of 90%. It is difficult to reflect the complex structure and function of the GBM microenvironment in vivo using traditional in vitro models. GBM-on-a-chip platforms can integrate biological or chemical functional units of a tumor into a chip, mimicking in vivo functions of GBM cells. This technology has shown great potential for applications in personalized precision medicine and GBM immunotherapy. In recent years, there have been efforts to construct GBM-on-a-chip models based on microfluidics and bioprinting. A number of research teams have begun to use GBM-on-a-chip models for the investigation of GBM progression mechanisms, drug candidates, and therapeutic approaches. This review first briefly discusses the use of microfluidics and bioprinting technologies for GBM-on-a-chip construction. Second, we classify non-surgical treatments for GBM in pre-clinical research into three categories (chemotherapy, immunotherapy and other therapies) and focus on the use of GBM-on-a-chip in research for each category. Last, we demonstrate that organ-on-a-chip technology in therapeutic field is still in its initial stage and provide future perspectives for research directions in the field.

Identification of a receptor tyrosine kinase inhibitor CP-724714 inhibits SADS-CoV related swine diarrhea coronaviruses infection in vitro.

The outbreak of the COVID-19 epidemic in 2020 has caused unprecedented panic among all mankind, pointing the major importance of effective treatment. Since the emergence of the swine acute diarrhea syndrome coronavirus (SADS-CoV) at the end of 2017, multiple reports have indicated that the bat-related SADS-CoV possesses a potential threat for cross-species transmission. Vaccines and antiviral drugs development deserve more attention. In this study, we found that the HER2 phosphorylation inhibitor (CP-724714) inhibited SADS-CoV infection in a dose-dependent manner. Further validation demonstrated that CP-724714 affected at the post-entry stage of SADS-CoV infection cycle. Also, efficient SADS-CoV infection required the activation of HER2 and its cascade Ras-Raf-Mek-Erk signaling pathway. In addition, CP-724714 has a broad-spectrum anti-swine diarrhea coronaviruses activity, and can dose-dependently combat SADS-CoV, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) infection in vitro with a specificity index of greater than 21.98, 9.38, 95.23 and 31.62, respectively. These results highlight the potential utility of CP-724714 or antiviral drugs targeting with HER2 and its cascade Ras-Raf-Mek-Erk signaling pathway as host-targeted SADS-CoV and other related coronaviruses therapeutics.

Infectious Recombinant Senecavirus A Expressing p16INK4A Protein.

Senecavirus A (SVA) is an oncolytic RNA virus, and it is the ideal oncolytic virus that can be genetically engineered for editing. However, there has not been much exploration into creating SVA viruses that carry antitumor genes to increase their oncolytic potential. The construction of SVA viruses carrying antitumor genes that enhance oncolytic potential has not been fully explored. In this study, a recombinant SVA-CH-01-2015 virus (p15A-SVA-clone) expressing the human p16INK4A protein, also known as cell cycle-dependent protein kinase inhibitor 2A (CDKN2A), was successfully rescued and characterized. The recombinant virus, called SVA-p16, exhibited similar viral replication kinetics to the parent virus, was genetically stable, and demonstrated enhanced antitumor effects in Ishikawa cells. Additionally, another recombinant SVA virus carrying a reporter gene (iLOV), SVA-iLOV, was constructed and identified using the same construction method as an auxiliary validation. Collectively, this study successfully created a new recombinant virus, SVA-p16, that showed increased antitumor effects and could serve as a model for further exploring the antitumor potential of SVA as an oncolytic virus.

LncRNA 8244-ssc-miR-320-CCR7 Regulates IFN-ß during SVA Infecting PK-15 Cells.

Seneca Valley virus (SVV), a member of the Picornaviridae family, is an oncolytic RNA virus that can cause idiopathic vesicular disease and increase mortality in newborn piglets. Although research on the pathogenic characteristics, epidemiology, pathogenic mechanism, and clinical diagnosis of SVA has increased due to its emergence and prevalence, the interaction between SVA and its host lncRNA has not been fully studied. This study used qualcomm sequencing to analyze differentially expressed lncRNAs and found that during SVA infection, lncRNA 8244 was significantly down-regulated in both PK-15 cells and piglets. Further analysis through quantitative real-time PCR and dual luciferase experiments demonstrated that lncRNA8244 could compete with ssc-miR-320 to regulate the expression of CCR7. The lncRNA824-ssc-miR-320-CCR7 axis activated the TLR-mediated signaling pathway, which recognized viral molecules and induced the expression of IFN-ß. These findings provide new insight into the interaction between lncRNA and SVA infection, which could lead to a better understanding of SVA pathogenesis and contribute to the prevention and control of SVA disease.

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Genetic characterization of four strains porcine circovirus-like viruses in pigs with diarrhea in Hunan Province of China.

In this study, we detected a circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus [named Po-Circo-like (PCL) virus] in intestinal tissue and fecal samples of pigs. PCL virus contains a single-stranded DNA genome, and ORF1 encodes the Rep and not the typical capsid protein encoded in PCV. The Rep protein may be responsible for viral genome replication. In addition, PCL virus may be one of the pathogens causing diarrhea symptoms in pigs. We identified four strains of PCL virus in two different pig farms with severe diarrhea outbreaks in Hunan Province, China. The strains in this study share 85.7-99.7% nucleic acid identity and 84.7-100% amino acid identity with Rep of the reference strains. A multiple sequence alignment of these PCL viruses and Bo-Circo-like CH showed a identity of 93.2% for the Rep protein, and the nucleotide identity was 86.7-89.3%. Moreover, Bo-Circo-like CH and HN75, HN39-01, HN39-02 had similar stem-loop sequences. In conclusion, the present study is the first detailed report of the PCL virus in Hunan provinces, which is a potential new virus in pigs that might be involved in cross-species transmission. Further investigation is needed to determine the pathogenesis of this virus and its epidemiologic impact.

Microfluidic-LAMP chip for the point-of-care detection of gene-deleted and wild-type African swine fever viruses and other four swine pathogens.

Introduction: Different pathogens causing mixed infection are now threatening the pig industry in the context of the African Swine Fever (ASF) circulating especially in China, and it is crucial to achieving the early diagnosis of these pathogens for disease control and prevention. Methods: Here we report the development of a rapid, portable, sensitive, high-throughput, and accurate microfluidic-LAMP chip detection system for simultaneous detection and differentiation of gene-deleted type and wild-type African swine fever virus (ASFV), pseudorabie virus (PRV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome (PRRSV). Results and discussion: The newly developed system was shown to be sensitive with detection limits of 101 copies/µl for ASFV-MGF505-2R/P72, PPV, and PCV2, 102 copies/µl for ASFV-CD2v, PRV, and PRRSV. The system was highly specific (100%) and stable (C.V.s < 5%) in its ability to detect different pathogens. A total 213 clinical samples and 15 ASFV nucleic acid samples were collected to assess the performance of the detection system, showing highly effective diagnosis. Altogether, the developed microfluidic-LAMP chip system provides a rapid, sensitive, high-throughput and portable diagnostic tool for the accurate detection of multiple swine pathogens.

Combined Analysis of the Whole Transcriptome of Piglets Infected with SADS-CoV Virulent and Avirulent Strains.

When piglets are infected by virulent and avirulent strains of swine acute diarrhea syndrome coronavirus (SADS-CoV), there are obvious differences in their clinical symptoms; however, the specific mechanisms of pathogenicity and the immune regulation of highly pathogenic and low pathogenic strains are unknown. We collected intestinal tissues from SADS-CoV-infected piglets, performed a whole transcriptome sequencing analysis, including mRNA, miRNA, lncRNA, cicrRNA, and TUCP, and performed functional and correlation analyses of differentially expressed RNAs. Our results showed that the differentially expressed RNAs in group A versus group B (AvsB), group A versus group C (AvsC), and group B versus group C (BvsC) were relevant to immune and disease-related signaling pathways that participate in the organisms' viral infection and immune regulation. Furthermore, data obtained from the HAllA analysis suggested that there was a strong correlation between the differentially expressed RNAs. Specifically, LNC_011487 in the P set was significantly negatively correlated with ssc-miR-215, and LNC_011487 was positively correlated with PI3. Moreover, we also constructed a differentially expressed RNA association network map. This study provides a valuable resource for studying the SADS-CoV transcriptome and pathogenic mechanism from the perspective of RNA to understand the differences in and consistency of the interaction between virulent and attenuated SADS-CoV strains and hosts.

A study of the growth mechanism of large-diameter double-wall TiO2 nanotube arrays fabricated by high voltage anodization.

Background: Research on the growth mechanism of titanium dioxide (TiO2) nanotube arrays fabricated by anodic oxidation is essential to achieve artificial control of the microstructure and to expand their applications. In our previous work, we reported the preparation of highly ordered large-diameter double-wall TiO2 nanotube arrays prepared by high voltage anodization. Methods: In this paper, we observed and analyzed the initial growth process of large-diameter double-wall TiO2 nanotube arrays anodized at 120 V in ethylene glycol electrolyte containing aluminum fluoride (NH4F) and water (H2O), such as the evolution of surface and cross-sectional morphologies, the influence of current density on growth rate, the transition process from nanoholes to nanotubes, and the evolution of dimples on the remaining substrate. Results: On the basis of our observations and inspirations from the existing viewpoints, we established growth models of large-diameter double-wall TiO2 nanotube arrays corresponding to different growth stages to explain the growth process. The growth rate of anodic oxide film changes accordingly with the current density. The compact anodic oxide film formed initially actually contains outer layer and inner layer, with no obvious interface between them. Then, the bottom even levels of the inner layer and outer layer bulge towards the substrate and become individual hemisphere-like structures. The inner layer becomes the outer wall, and the outer layer becomes inner wall. Eventually, V-shaped large-diameter and double-wall TiO2 nanotube arrays form. Conclusions: The results presented in this work are significant and provide a better understanding of the growth mechanism of large-diameter double-wall TiO2 nanotube arrays anodized by high voltage.

Direct cloning of a herpesvirus genome for rapid generation of infectious BAC clones.

INTRODUCTION: The herpesviridae are DNA viruses with large and complicated genomes. The herpesvirus bacterial artificial chromosomes (BACs) have been useful for generating recombinant viruses to study the biology and pathogenesis. However, the conventional method using homologous recombination is not only time consuming but also prone to accumulate attenuating mutations during serial passage of the virus in cells. Elimination of the BAC vector from the recombinant viral genome requires additional step for phenotypically consistence with the original strain. OBJECTIVES: To generate a streamlined approach for generating infectious BAC clones of herpesvirus. METHODS: The 142-kb pseudorabies virus genome was directly cloned into a bacterial artificial chromosome (BAC) in Escherichia coli by Exonuclease Combined with RecET recombination (ExoCET). Placement of the BAC vector at the terminus of the linear virus genome enabled excision of the BAC backbone from the viral genome by restriction endonuclease for delivery into mammalian cells, with the subsequent rapid rescue of virus that was genetically identical to the original strain. RESULTS: This new approach for molecular cloning of the genome from a large DNA virus and isolation of pure virus lacking the BAC vector from transfected mammalian cells bypass the tedious and time-consuming method of multiple rounds of plaque purification. The viral BAC was stable in E. coli, allowing further mutagenesis mediated by the Red system or various site-specific recombination methods. CONCLUSION: An efficient method for construction of infectious clones of herpesvirus was established. It is expected to be potentially useful for other viruses with large double-stranded DNA genomes.

3D Microfluidic System for Evaluating Inhibitory Effect of Chinese Herbal Medicine Oldenlandia diffusa on Human Malignant Glioma Invasion Combined with Network Pharmacology Analysis.

OBJECTIVE: To investigate the anti-invasion efficacy of the ethanol extract of Oldenlandia diffusa Will. (EEOD) on a three-dimensional (3D) human malignant glioma (MG) cell invasion and perfusion model based on microfluidic chip culture and the possible mechanism of action of Oldenlandia diffusa Will. (OD). METHODS: The comprehensive pharmacodynamic analysis method in this study was based on microfluidic chip 3D cell perfusion culture technology, and the action mechanism of Chinese medicine (CM) on human MG cells was investigated through network pharmacology analysis. First, the components of EEOD were analyzed by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Then, cell viability and apoptosis were assessed to determine the optimum concentration of EEOD for invasion experiments, and two-dimensional (2D) migration and invasion abilities of U87 and U251 MG cells were evaluated using scratch wound and Transwell assays. The possible mechanism underlying the effects of EEOD on glioma was analyzed through a network pharmacology approach. RESULTS: Thirty-five compounds of EEOD were detected by UPLC-Q-TOF/MS. EEOD suppressed the viability of MG cells, promoted their apoptosis, and inhibited their migratory and invasive potentials (all P<0.05). Network pharmacology analysis showed that OD inhibited the invasion of MG cells by directly regulating MAPK and Wnt pathways through MAPK, EGFR, MYC, GSK3B, and other targets. The anti-invasion effect of OD was also found to be related to the indirect regulation of microtubule cytoskeleton organization. CONCLUSIONS: ]EEOD could inhibit the invasion of human MG cells, and the anti-invasion mechanism of OD might be regulating MAPK and Wnt signaling pathways and microtubule cytoskeleton organization.