Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer.

BACKGROUND: Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate. METHODS: Transcription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting. RESULTS: The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping. Finally, six live piglets and one stillborn piglet were collected from two recipients by caesarean section. Sequencing analyses of the target site confirmed the P53 biallelic knockout in all fetuses and piglets, consistent with the genotype of the donor cells. The qPCR analysis showed that the expression of the P53 mRNA had significant reduction in various tissues of the knockout piglets. Furthermore, confocal microscopy and western blotting analyses demonstrated that the fibroblast cells of Diannan miniature piglets with a P53 biallelic knockout were defective in mediating DNA damage when incubated with doxorubicin. CONCLUSION: TALENs combined with SCNT was successfully used to generate P53 KO Diannan miniature pigs. Although these genetically engineered Diannan miniature pigs had no tumorigenic signs, the P53 gene was dysfunctional. We believe that these pigs will provide powerful new resources for preclinical oncology and basic cancer research.

Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer.

BACKGROUND: α1,3-Galactosyltransferase (GGTA1) is essential for the biosynthesis of glycoproteins and therefore a simple and effective target for disrupting the expression of galactose α-1,3-galactose epitopes, which mediate hyperacute rejection (HAR) in xenotransplantation. Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation. METHODS: Transcription activator-like effector nucleases (TALENs) were designed to target exon 6 of porcine GGTA1 gene. The targeting activity was evaluated using a luciferase SSA recombination assay. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids. One cell line was selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. GTKO aborted fetuses, stillborn fetuses and live piglets were obtained. Genotyping of the collected cloned individuals was performed. The Gal expression in the fibroblasts and one piglet was analyzed by fluorescence activated cell sorting (FACS), confocal microscopy, immunohistochemical (IHC) staining and western blotting. RESULTS: The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 17.1-fold higher than those of the control. Three cell lines (3/126) showed GGTA1 biallelic knockout after modification by the TALENs. The GGTA1 biallelic modified C99# cell line enabled high-quality SCNT, as evidenced by the 22.3 % (458/2068) blastocyst developmental rate of the reconstructed embryos. The reconstructed GTKO embryos were subsequently transferred into 18 recipient gilts, of which 12 became pregnant, and six miscarried. Eight aborted fetuses were collected from the gilts that miscarried. One live fetus was obtained from one surrogate by caesarean after 33 d of gestation for genotyping. In total, 12 live and two stillborn piglets were collected from six surrogates by either caesarean or natural birth. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS, confocal microscopy, IHC and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts, kidneys and pancreas of one GTKO piglet. CONCLUSIONS: TALENs combined with SCNT were successfully used to generate GTKO Diannan miniature piglets.

A Discolorable Flexible Synaptic Transistor for Wearable Health Monitoring.

Multifunctional intelligent wearable electronics, providing integrated physiological signal analysis, storage, and display for real-time and on-site health status diagnosis, have great potential to revolutionize health monitoring technologies. Advanced wearable systems combine isolated digital processor, memory, and display modules for function integration; however, they suffer from compatibility and reliability issues. Here, we introduce a flexible multifunctional electrolyte-gated transistor (EGT) that integrates synaptic learning, memory, and autonomous discoloration functionalities for intelligent wearable application. This device exhibits synergistic light absorption coefficient changes during voltage-gated ion doping that modulate the electrical conductance changes for synaptic function implementation. By adaptively changing color, the EGT can differentiate voltage pulse inputs with different frequency, amplitude, and duration parameters, exhibiting excellent reversibility and reliability. We developed a smart wearable monitoring system that incorporates EGT devices and sensors for respiratory and electrocardiogram signal analysis, providing health warnings through real-time and on-site discoloration. This study represents a significant step toward smart wearable technologies for health management, offering health evaluation through intelligent displays.

Comparison of Third-Generation Sequencing Technology and Traditional Microbiological Detection in Pathogen Diagnosis of Lower Respiratory Tract Infection.

BACKGROUND: It is common to obtain a low detection rate and unsatisfactory detection results in complex infection or rare pathogen detection. This retrospective study aimed to illustrate the application value and prospect of the third-generation sequencing technology in lower respiratory tract infection disease. METHODS: This study recruited 70 patients with lower respiratory tract infection (LRTI). Pathogen detection of bronchoalveolar lavage fluid (BALF) from all patients was performed using nanopore metagenomic sequencing technology and traditional culture. BALF culture combined with quantitiative PCR (qPCR) was used as a reference standard to analyze the sensitivity and specificity of nanopore sequencing technology. The current study also collected the examination results of enrolled samples using technical methods sputum culture, tuberculosis DNA (TB-DNA), and Xpert MTB/RIF and analyzed the detection efficiency of nanopore sequencing for Mycobacterium tuberculosis. RESULTS: The positive rates of pathogens in 70 BALF samples detected by conventional culture and nanopore sequencing were 25.71% and 84.29%, respectively. Among the 59 positive BALF cases using nanopore sequencing, a total of 31 pathogens were identified, of which the proportions of bacteria, fungi, viruses, and other pathogens were 50%, 17%, 32%, and 1%, respectively. Using the results combined with culture and qPCR detection methods as the standard, the pathogen detection of BALF using nanopore sequencing had a sensitivity of 70% and a specificity of 91.7%. Additionally, the positive rate of the detection of M. tuberculosis using nanopore sequencing was 33.3% (6/18). The clinical medication plans of 74.3% (52/70) of the patients were referred to the nanopore sequencing results, of which 31 cases changed their treatment strategy, 21 supported the previous treatment plans, and 90% (47/52) of the patients finally had clinical improvement. CONCLUSIONS: BALF detection using nanopore sequencing technology improves the process of detecting pathogens in patients with LRTI, especially for M. tuberculosis, fungi, and viruses, by reducing the report time from three days to six hours. The clinical application prospect of nanopore sequencing technology is promising in the pathogen diagnosis of LRTI.

System Analysis of Adaptor-Related Protein Complex 1 Subunit Mu 2 (AP1M2) on Malignant Tumors: A Pan-Cancer Analysis.

OBJECTIVE: To identify new tumor marker genes available for early tumor screening, differentially expressed gene profiles of multiple tumors were compared using Genotype-Tissue Expression (GTEx), Cancer Cell Line Encyclopedia (CCLE), and The Cancer Genome Atlas (TCGA) databases. As AP1M2 was highly and differentially expressed in invasive breast carcinoma, the purpose of this study was to explore the association of AP1M2 gene with the survival, immune invasion, and tumor neoantigens of patients on a pan-cancer basis. METHODS: The expression and distribution of AP1M2 gene in tumor tissues and the corresponding normal control tissues were analyzed using the pan-cancer databases GTEx, CCLE, and TCGA. Kaplan-Meyer survival plots and proportional hazards model (COX) were employed to evaluate actions of AP1M2 on the clinical prognosis of tumor patients. Subsequently, the association of AP1M2 expression with immune invasion in different tumor types was explored. Simultaneously, the investigation of the interrelationship of AP1M2 and tumor neoantigens of the immune system, unstable microsatellite, DNA repair genes, and DNA methyltransferases were explored, and the mutation frequency of AP1M2 gene in diverse tumors was studied. Several tumor types were analyzed using gene-set enrichment analysis (GSEA). RESULTS: AP1M2 was abundantly expressed in a wide range of cancers, and its expression level was positively correlated with the outcome of tumor victims. Through a study on AP1M2 action on clinical prognosis and immune infiltration in tumor patients, AP1M2 expression in breast-infiltrating carcinoma was found to be highly associated with patients' overall survival and infiltration levels of macrophages, dendritic cells, T cells (CD4+ and CD8+), and B cells. Also, AP1M2 expression was positively correlated with tumor immune neoantigens and microsatellite instability in breast invasive carcinoma. The effect of AP1M2 on tumors was analyzed by GSEA, and findings demonstrated that AP1M2 expression levels in most tumors influenced the activation of tumor-associated pathways and immune-associated pathways. CONCLUSIONS: These findings suggest that AP1M2 expression levels are significantly correlated to patients' outcomes and levels of immune infiltration in most cancer types, including T cells (CD8+ and CD4+), macrophages, neutrophils, and dendritic cells (DCs), particularly in breast cancer. The results indicate that AP1M2 may influence the tumor environment of invasive breast cancer patients and it may be a target contributing to early screening and treatment for breast cancer, helping improve the efficiency of early screening and overall survival rate in invasive breast cancer patients.

Screening of Key Prognosis Genes of Lung Adenocarcinoma Based on Expression Analysis on TCGA Database.

Objective: The data of lung adenocarcinoma- (LUAD-) related gene expression profiles were mined from the Cancer Genome Atlas (TCGA) database using bioinformatics methods and potential biomarkers related to the occurrence, development, and prognosis of LUAD were screened out to explore the key prognostic genes and clinical significance. Methods: Following the LUAD gene expression profile data that were initially exported from the TCGA database, R software DESeq2 was employed to analyze the difference between the expression profiles of LUAD and normal tissues. The R package "clusterProfiler" was subsequently utilized to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the differential genes. A protein-protein interaction (PPI) network was constructed via the String database, and cytohubba, a plugin of Cytoscape, was applied to screen hub genes using the MCC algorithm. The Gene Expression Profile Data Interactive Analysis (GEPIA) was used to analyze expressions of 10 candidate genes in LUAD samples and healthy lung samples, and the selected genes were employed for survival analysis. Results: A total of 1,598 differential genes were identified through differential analyses and data mining, with 1,394 genes upregulated and 204 downregulated. A total of 10 hub genes CCNA2, CDC20, CCNB2, KIF11, TOP2A, BUB1, BUB1B, CENPF, TPX2, and KIF2C were obtained using the cytohubba plugin. The results of the GEPIA analysis indicated that compared with normal lung tissue, the mRNA expression level of the described hub genes in LUAD tissue was significantly increased (P < 0.05). Survival analysis revealed that these genes had a significant impact on the overall survival time of LUAD patients (P < 0.05). Conclusion: The previously described key genes related to LUAD identified in the TCGA database may be used as potential prognostic biomarkers, which will contribute to further comprehension of the occurrence and development of LUAD and provide references for its diagnosis and treatment.

MIP From Legionella pneumophila Influences the Phagocytosis and Chemotaxis of RAW264.7 Macrophages by Regulating the lncRNA GAS5/miR-21/SOCS6 Axis.

Background: The intracellular pathogen Legionella pneumophila (L. pneumophila) is a causative agent of pneumonia and does great harm to human health. These bacteria are phagocytosed by alveolar macrophages and survive to replicate within the macrophages. Despite macrophage infectivity potentiator (MIP) protein serving as an essential virulence factor during the invasion process of L. pneumophila, the regulatory mechanism of MIP protein in the process of bacterial infection to host cells is not yet completely understood. This research thus aims to explore the interaction between MIP and macrophage phagocytosis. Methods: Through the experiment of the co-culture of RAW264.7 macrophages with different concentrations of MIP, the chemotactic activity of macrophages was detected and the phagocytosis was determined by a neutral red uptake assay. The expression of long noncoding RNA (lncRNA) GAS5, microRNA-21 (miR-21), and suppressor of cytokine signaling (SOCS)6 was determined by qRT-PCR. Target genes were detected by dual luciferase assay. Results: MIP could reduce the phagocytosis and improve the chemotaxis of RAW264.7 macrophages. The expression of both lncRNA GAS5 and SOCS6 was increased whereas the expression of miR-21 was decreased when macrophages were treated with MIP. Dual luciferase assay revealed that lncRNA GAS5 could interact with miR-21, and SOCS6 served as the target of miR-21. After GAS5 overexpression, the phagocytosis of RAW264.7 treated with MIP was increased whereas the chemotaxis was decreased. In contrast, the opposite results were found in RAW264.7 following GAS5 interference. Conclusions: The present results revealed that MIP could influence RAW264.7 macrophages on phagocytic and chemotactic activities through the axis of lncRNA GAS5/miR-21/SOCS6.

Palmatine induces G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA.

Studies have shown that palmatine (PAL) has anti-cancer effects. However, the activity and potential mechanisms of PAL against colorectal cancer remain elusive. The results showed that PAL significantly inhibited the proliferation of colon cancer cells in vitro and in vivo without significant effect on non-tumorigenic colon cells. Target prediction and clinical sample database analysis suggested that PAL may contribute to colon cancer cells phase arrest and apoptosis by targeting aurora kinase A (AURKA). Inhibition and overexpression of AURKA proved that PAL induces G2/M phase arrest and apoptosis in colon cancer cells by targeting AURKA. Moreover, PAL promoted intracellular Reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (ΔΨm). PAL reduced the levels of AURKA, Bcl-xl and Bcl2 proteins, and promoted the expression of pro-apoptotic proteins P53, P73, Caspase3 and Caspase9, as well as the increase of cytochrome c (cyt. c) in cell lysates in vitro and in vivo. Together, our study confirmed that PAL induced G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA. PAL may provide a novel solution for the treatment of colon cancer by serving as a new AURKA inhibitor.