Emerging and potential use of CRISPR in human liver disease.

CRISPR is a gene editing tool adapted from naturally occurring defense systems from bacteria. It is a technology that is revolutionizing the interrogation of gene functions in driving liver disease, especially through genetic screens and by facilitating animal knockout and knockin models. It is being used in models of liver disease to identify which genes are critical for liver pathology, especially in genetic liver disease, hepatitis, and in cancer initiation and progression. It holds tremendous promise in treating human diseases directly by editing DNA. It could disable gene function in the case of expression of a maladaptive protein, such as blocking transthyretin as a therapy for amyloidosis, or to correct gene defects, such as restoring the normal functions of liver enzymes fumarylacetoacetate hydrolase or alpha-1 antitrypsin. It is also being studied for treatment of hepatitis B infection. CRISPR is an exciting, evolving technology that is facilitating gene characterization and discovery in liver disease and holds the potential to treat liver diseases safely and permanently.

Regulation of adipose tissue development and energy metabolism by VEGFB isoforms.

Obesity is caused by imbalanced energy intake and expenditure. Excessive energy intake and storage in adipose tissues are associated with many diseases. Several studies have demonstrated that vascular growth endothelial factor B (VEGFB) deficiency induces obese phenotypes. However, the roles of VEGFB isoforms VEGFB167 and VEGFB186 in adipose tissue development and function are still not clear. In this study, genetic mouse models of adipose-specific VEGFB167 and VEGFB186 overexpression (aP2-Vegfb167 tg/+and aP2-Vegfb186tg/+) were generated and their biologic roles were investigated. On regular chow, adipose-specific VEGFB186 is negatively associated with white adipose tissues (WATs) and positively regulates brown adipose tissues (BATs). VEGFB186 upregulates energy metabolism and metabolism-associated genes. In contrast, VEGFB167 has a nominal role in adipose development and function. On high-fat diet, VEGFB186 expression can reverse the phenotypes of VEGFB deletion. VEGFB186 overexpression upregulates BAT-associated genes and downregulates WAT-associated genes. VEGFB186 and VEGFB167 have very distinct roles in the regulation of adipose development and energy metabolism. As a key regulator of adipose tissue development and energy metabolism, VEGFB186 may be a target for obesity prevention and treatment.

Protective Effect of Hydrogen Sulfide on Cerebral Ischemia-Reperfusion Injury.

The brain is the most sensitive organ to hypoxia in the human body. Hypoxia in the brain will lead to damage to local brain tissue. When the blood supply of ischemic brain tissue is restored, the damage will worsen, that is, cerebral ischemia-reperfusion injury. Hydrogen sulfide (H2S) is a gaseous signal molecule and a novel endogenous neuroregulator. Indeed, different concentrations of H2S have different effects on neurons. Low concentration of H2S can play an important protective role in cerebral ischemia-reperfusion injury by inducing anti-oxidative stress injury, inhibition of inflammatory response, inhibition of cell apoptosis, reduction of cerebrovascular endothelial cell injury, regulation of autophagy, and other ways, which provides a new idea for clinical diagnosis and treatment of related diseases. This review aims to report the recent research progress on the dual effect of H2S on brain tissue during cerebral ischemia/reperfusion injury.

Novel characterization discoveries of ferroptosis-associated molecules in COAD microenvironment based TCGA data.

Background and Objective: One of the most recent forms of programmed cell death, ferroptosis, is crucial in tumorigenesis. Ferroptosis is characterized by iron-dependent oxidative destruction of cellular membranes following the antioxidant system's failure. However, it is unknown whether ferroptosis-related genes (FRGs) are associated with colon adenocarcinoma (COAD) metastasis, immune cell infiltration, and oxidative stress in COAD. The current study concentrated on FRGs expression in colon cancer metastasis, their relationship to immune cell infiltration (ICI), and potential pathological pathways in COAD. Methods and Results: Clinical information and mRNA expression patterns for patients with COAD metastasis were obtained from the public TCGA database. Patients with low mRNA levels showed good overall survival than patients with high mRNA levels. The genomic-clinicopathologic nomogram was subsequently created by combining risk score and clinicopathological features. Absolute Shrinkage and Selection Operator have shown a 4 gene signature that can stratify cancer patients into high-risk versus low-risk. These four FRGs were found to be significantly linked to the overall survival of COAD patients and predicted high risk score. Next, age, stage, and PTNM were combined in univariate and multivariate cox regression models to perform a filtering procedure. The receiver operating characteristic (ROC) and calibration curves indicated that constructed signature model exhibited high prediction accuracy and clinical relevance in COAD. ARID3A showed a strong negative correlation with a wide range of immune tumour-infiltrating cells in COAD microenvironment. According to the single sample gene set enrichment analysis (ssGSEA) results, FRGs are involved in variety of pathological pathways including PI3K-AKT-mTOR pathway, reactive oxygen species (ROS) pathway, response to hypoxia pathway, and other inflammation related pathways. Moreover, dysregulation of FRGs in COAD patients showed a significance correlation with wide range of miRNAs and transcription factors (TFs). Conclusion: We identified new diagnostic biomarkers and established prognostic models for ferroptosis related programmed cell death in COAD metastasis. FRGs may improve tumor cell survival by activating the TGFB pathway, which can stimulate ROS production, accelerates ECM breakdown, and promote tumor progression and invasion. Genes implicated in ferroptosis, as revealed by the Kaplan Meier and a genomic-clinicopathologic nomogram, are potential therapeutic targets and prognosis indications for metastasis COAD patients.

Immune checkpoint inhibitor (ICI) genes and aging in malignant melanoma patients: a clinicogenomic TCGA study.

BACKGROUND: Cancer diagnoses and deaths among the elderly (65 +) are expected to increase significantly over the next decade. Immune checkpoint inhibitors specifically target ICI genes and enhance immune system function. However, poor outcomes may be associated with aging. METHODS: We downloaded the Genomic Data Commons from the Cancer Genome Atlas (TCGA) and collected gene expression data from malignant melanoma (MM) tissues, the third level as the primary site. The CKTTD ICI genes database were applied and validated using the GEO database and lab experiments. RESULTS: In 414 patients, 13 ICI genes were obtained as risk gene signature by univariate and multivariate Cox hazard models and were associated with poor survival in the older group. At 1, 3, and 5 years (79%, 76%, and 76%, respectively), we investigate TNFRFS4 gene and age prediction using novel nomogram-associated aging (HR = 1.79, P 0.001, CI = 1.32-2.45) with higher sensitivity testing.TNFRSF4 gene expression was significantly high in younger (15 years interval) MM patients (P < 0.001). By correlation analysis, a significant negative association was determined (P < 0.001). The validation of gene correlation from GEO (GSE59455) and (GSE22153) was obtained as external validation. We tested the TNFRSF4 protein levels by IHC in 14 melanoma tissue samples. TNFRSF4 expression was observed to be lower expressed in the older of melanoma tissues, and higher in the younger age group (P = 0.02). Besides the connectivity of ICI gene proteins, the biological processes of cell aging, aging, and the immune system were found to be highly related. CONCLUSIONS: Along with the risk score evaluation, the ICI gene (TNFRSF4) was identified as a tumor suppressor gene related to inequalities in age survival and associated with immune cell infiltrations. The aging responses of melanoma patients and related gene expression need further investigation in order to identify potential therapeutic targets.

Heterozygous loss of Dip2B enhances tumor growth and metastasis by altering immune microenvironment.

Cancer is caused by abnormal cell growth and metastasis to other tissues. Development of cancers is complex and underlining mechanisms are mostly unknown. Disco-interacting protein 2 homolog B (DIP2B) is a member of Dip2. There have been reports suggesting that Dip2B may participate in tumor growth and development. However, direct link between DIP2B and cancer development is missing. In this study, Dip2btm1a/+ heterozygous knockout mouse model was used to investigate tumor growth and metastasis. Results show that one allele knockout of Dip2B significantly promoted tumor growth and metastasis, decreased tumor cell apoptosis and reduced immune cell infiltration in tumors, most likely by altering immune system that includes reduction of macrophage and cytotoxic T-cells infiltration into tumor microenvironment.

Age-Based Disparities in Metastatic Melanoma Patients Treated in the Immune Checkpoint Inhibitors (ICI) Versus Non-ICI Era: A Population-Based Study.

Immune checkpoint inhibitors (ICIs) have revolutionized metastatic melanoma treatment, but our knowledge of ICI activity across age groups is insufficient. Patients in different age groups with advanced melanoma were selected based on the ICI approval time in this study. Patients with melanoma were identified in the Surveillance, Epidemiology, and End Result (SEER) database program 2004-2016. The results showed that 4,040 patients had advanced melanoma before the advent of ICI (referred to as the "non-ICI era"), whereas there were 6,188 cases after ICI approval (referred to as the "ICI era"). In all age groups, the cases were dominated by men. The differences between the first (20-59 years) and second (60-74 years) age groups in both eras were significant in terms of surgery performance and holding of insurance policies (p = 0.05). The first and second groups (20-59 and 60-70 years old, respectively) showed no difference in survival (median = 8 months) during the non-ICI era, but the difference was evident in the first, second, and third age groups in the ICI era, with the younger group (20-59 years) having significantly better survival (median = 18, 14, and 10 months, respectively, p = 0.0001). Multivariate analysis of the first group (the youngest) in the ICI era revealed that surgery was significantly associated with an increase in survival among patients compared with those who did not undergo surgery (p < 0.0001). Furthermore, having an insurance policy among all age groups in the ICI era was associated with favorable survival in the first (20-59 years) and second (60-74 years) age groups (p = 0.0001), while there were no survival differences in the older ICI group (>74 years). Although there were differences in survival between the ICI era and the non-ICI era, these results demonstrate that ICI positively affected the survival of younger patients with advanced melanoma (first age group) than it had beneficial effects on older patients. Moreover, having had cancer surgery and holding an insurance policy were positive predictors for patient survival. This study emphasizes that adequate clinical and preclinical studies are important to enhance ICI outcomes across age groups.

Analyzing differentially expressed genes and pathways of Bex2-deficient mouse lung via RNA-Seq.

Bex2 is well known for its role in the nervous system, and is associated with neurological disorders, but its role in the lung's physiology is still not reported. To elucidate the functional role of Bex2 in the lung, we generated a Bex2 knock-out (KO) mouse model using the CRISPR-Cas9 technology and performed transcriptomic analysis. A total of 652 genes were identified as differentially expressed between Bex2 -/- and Bex2 +/+ mice, out of which 500 were downregulated, while 152 were upregulated genes. Among these DEGs, Ucp1, Myh6, Coxa7a1, Myl3, Ryr2, RNaset2b, Npy, Enob1, Krt5, Myl2, Hba-a2, and Nrob2 are the most prominent genes. Myl2, was the most downregulated gene, followed by Npy, Hba-a2, Rnaset2b, nr0b2, Klra8, and Ucp1. Tcte3, Eno1b, Zfp990, and Pcdha9 were the most upregulated DEGs. According to gene enrichment analysis, PPAR pathway, cardiac muscle contraction, and cytokine-cytokine receptor interaction were the most enriched pathways. Besides, the nuclear factor-κB signaling pathway and hematopoietic cell linage pathways were also enriched. Chronic obstructive pulmonary disease (COPD) is enriched among KEGG disease pathways. RT-qPCR assays confirmed the RNA-Seq results. This study opens a new window toward the biological functions of Bex2 in different systems.

Atractylenolide II induces cell cycle arrest and apoptosis in breast cancer cells through ER pathway.

In this research, atractylenolide II (ATR II) on apoptosis, cell cycle cells via ER pathway in breast cancer (MDA-MB-231 and MCF-7) cells are assessed. The effect of ATR II on cell proliferation was detected by MTT assay. Additional flow cytometry, luciferase, the western blot were performed to detect the signaling pathway cytotoxicity of ATR II. We have also carried out autodock measurements to validate our results. Our findings showed ATR II could inhibit breast cancer cell growth by apoptosis mainly through G2/M-phase cell cycle arrest. Besides, the cytotoxicity of ATTR II on breast cancer was also correlated by the regulation of endrogen receptors and promising an anti-inflammatory activity via inhibiting NF-KB signaling pathways. Taking together, ATR II could be a potential anti-cancer drug for breast cancer.

Mesenchymal stem cell-derived exosome microRNA as therapy for cardiac ischemic injury.

Cardiovascular diseases (CVD) are a significant cause of human health harm. In the past, stem cell therapy was reported to have functional defects, such as immune rejection, tumorigenicity, and infusion toxicity. Exosomes are extracellular vesicles with lipid bilayer membrane structure, containing proteins, lipids, mRNA, miRNA, DNA, and other molecules, which can mediate various biological functions such as immune response, inflammatory response, cell migration, and differentiation intercellular communication. Exosomal miRNAs have outstanding advantages in disease diagnosis and curative effect prediction. Likewise, paracrine factors could also mediate the main therapeutic effect of mesenchymal stem cells. Research has shown that mesenchymal stem cell-derived micro-exosomes, which may come from stem cells, accumulate in the ischemic tissue and regulate cell proliferation, apoptosis, inflammation, and angiogenesis sites of myocardial injury after being transplanted. This review reviewed the molecular mechanisms of exosomes and internal microRNAs derived from mesenchymal stem cells in cardiac ischemic injury repair.

Analysis of Dip2B Expression in Adult Mouse Tissues Using the LacZ Reporter Gene.

Disconnected (disco)-interacting protein 2 homolog B (Dip2B) is a member of the Dip2 superfamily and plays an essential role in axonal outgrowth during embryogenesis. In adults, Dip2B is highly expressed in different brain regions, as shown by in situ analysis, and may have a role in axon guidance. However, the expression and biological role of Dip2B in other somatic tissues remain unknown. To better visualize Dip2B expression and to provide insight into the roles of Dip2B during postnatal development, we used a Dip2btm1a(wtsi)komp knock-in mouse model, in which a LacZ-Neo fusion protein is expressed under Dip2b promoter and allowed Dip2B expression to be analyzed by X-gal staining. qPCR analyses showed that Dip2b mRNA was expressed in a variety of somatic tissues, including lung and kidney, in addition to brain. LacZ staining indicated that Dip2B is broadly expressed in neuronal, reproductive, and vascular tissues as well as in the kidneys, heart, liver, and lungs. Moreover, neurons and epithelial cells showed rich staining. The broad and intense patterns of Dip2B expression in adult mice provide evidence of the distribution of Dip2B in multiple locations and, thereby, its implication in numerous physiological roles.

CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

Surgery Treatment Improved the Overall Survival Rate in Locoregional Myxoid Leiomyosarcoma than Other Myxosarcomas in the United States.

Myxosarcomas are rare malignant tumors of soft connective tissues, classified into various subtypes, including myxoid liposarcoma, myxoid chondrosarcoma, and myxoid leiomyosarcoma. In this study, we proposed to study the demographic, tumor characteristics, and overall survival rate and compared the treatment modalities between these cancers. Patient data collected based on locoregional metastasis presentation of the abovementioned tumors with a cutoff study of survival duration up to 10 years were obtained from the SEER database during 1975-2016. Our results indicated that elderly patients and females were more in locoregional myxoid leiomyosarcoma than myxoid liposarcoma and myxoid chondrosarcoma with locoregional metastasis. The white race represented the most patients who suffered from these cancers than other races. The heart is the primary site for the abovementioned cancers, in addition to the female genitals to the myxoid leiomyosarcoma. Myxoid liposarcoma and myxoid chondrosarcoma patients with locoregional metastasis were suffering from grade II, while locoregional myxoid leiomyosarcoma patients with blank grading were due to missed data. Surgery was the most common treatment modality in this study compared with radiotherapy and chemotherapy. Kaplan-Meier analysis showed a significant difference in survival time between the three subtypes by using histology, and myxoid leiomyosarcoma showed prolonged survival than others. Elderly, female, white, unknown grade, surgery, no radiation, and no chemotherapy variables were independent factors associated with overall survival among these cancers. Multivariate analysis also showed significant differences in overall survival between the three tumors by histology, and myxoid leiomyosarcoma was with a better prognosis than others. Multivariate analysis of locoregional myxoid leiomyosarcoma showed the statistical significance of black race, grade, and radiotherapy, indicating them as independent prognostic factors of locoregional myxoid leiomyosarcoma. We conclude that surgery was the primary treatment modality against these cancers than radiotherapy and chemotherapy. And the locoregional myxoid leiomyosarcomas showed a better prognosis and higher survival rate than locoregional myxoid liposarcoma and locoregional myxoid chondrosarcoma.

Effect of low VEGF on lung development and function.

Vascular endothelial growth factor (VEGF) is important for lung development and function but ideal mouse models are limited to decipher the quantitative relationship between VEGF expression levels and its proper development and pathogenesis. Human SPC promoter has been used to faithfully express genes or cDNAs in the pulmonary epithelium in many transgenic mouse models. In the study, a mouse model of lung-specific and reversible VEGF repression (hspc-rtTRtg/+/VegftetO/tetO) was generated. Human SPC promoter was used to drive lung-specific rtTR expression, a cDNA coding for doxycycline-regulated transcription repression protein. By crossing with VegftetO/tetO mice, that has tetracycline operator sequences insertion in 5'-UTR region, it allows us to reversibly inhibit lung VEGF transcription from its endogenous level through doxycycline food, water or injection. The tissue-specific inhibition of VEGF is used to mimic abnormal expression levels of VEGF in lung. Reduced VEGF expression in lung is confirmed by quantitative real time PCR and immunoblotting. Lung development and structure was analyzed by histology analysis and found significantly affected under low VEGF. The pulmonary epithelium and alveolarization are found abnormal with swelling alveolar septum and enlargement of air space. Genome-wide gene expression analysis identified that immune activities are involved in the VEGF-regulated lung functions. The transgenic mouse model can be used to mimic human pulmonary diseases. The mouse model confirms the important regulatory roles of epithelial expressed VEGF in lung development and function. This mouse model is valuable for studying VEGF-regulated lung development, pathogenesis and drug screening under low VEGF expression.

TL1A/TNFR2 Axis Enhances Immunoregulatory Effects of Bone Marrow Derived Mesenchymal Stem Cell by Indian Hedgehog Signaling Pathway.

BACKGROUND AND OBJECTIVES: The immunomodulatory potential of mesenchymal stem cells (MSCs) can be regulated by a variety of molecules, especially cytokines. The inflammatory cytokine, TNF-like ligand 1A (TL1A), has been reported as an inflammation stimulator in-multiple autoimmune diseases. Here, we studied the effects of TL1A/TNF-receptor 2 (TNFR2) pathway on the therapeutic potency of bone marrow-derived MSCs (BMSCs). METHODS AND RESULTS: BMSCs, fibroblast-like synoviocytes (FLSs), and H9 and jurkat human T lymphocytes were used in this study. BMSCs paracrine activities, differentiation, proliferation, and migration were investigated after stimulation with TL1A, and intervened with anti-TNFR2. Additionally, the effects of TL1A on BMSCs therapeutic potency were evaluated by treating RA-FLSs, and H9 and jurkat T cells with TL1A-stimulated BMSCs conditioned medium (CM). Indian hedgehog (IHH) involvement was determined by gene silencing and treatment by recombinant IHH (rIHH). TL1A induced BMSCs stemness-related genes, COX-2, IL-6, IDO, TGF-ß and HGF through TNFR2. Also, TL1A corrected biased differentiation and increased proliferation, and migration through TNFR2. Meanwhile, CM of TL1A-stimulated BMSCs decreased the inflammatory markers of RA-FLSs and T cells. Moreover, TL1A-stimulated BMSCs experienced IHH up-regulation coupled with NF-κB and STAT3 signaling up-regulation, while p53 and oxidative stress were down-regulated. Furthermore, treatment of BMSCs by rIHH increased their anti-inflammatory effects. More importantly, knockdown of IHH decreased the ability of TL1A-stimulated BMSCs to alleviating the inflammation in RA-FLSs and T cells. CONCLUSIONS: This study reports the effects of TL1A/TNFR2 pathway on the biological behaviors and therapeutic potency of BMSCs through IHH. These findings could introduce novel procedures to increase the stemness of MSCs in cellular therapy.

Targeted Disruption of Mouse Dip2B Leads to Abnormal Lung Development and Prenatal Lethality.

Molecular and anatomical functions of mammalian Dip2 family members (Dip2A, Dip2B and Dip2C) during organogenesis are largely unknown. Here, we explored the indispensable role of Dip2B in mouse lung development. Using a LacZ reporter, we explored Dip2B expression during embryogenesis. This study shows that Dip2B expression is widely distributed in various neuronal, myocardial, endothelial, and epithelial cell types during embryogenesis. Target disruption of Dip2b leads to intrauterine growth restriction, defective lung formation and perinatal mortality. Dip2B is crucial for late lung maturation rather than early-branching morphogenesis. The morphological analysis shows that Dip2b loss leads to disrupted air sac formation, interstitium septation and increased cellularity. In BrdU incorporation assay, it is shown that Dip2b loss results in increased cell proliferation at the saccular stage of lung development. RNA-seq analysis reveals that 1431 genes are affected in Dip2b deficient lungs at E18.5 gestation age. Gene ontology analysis indicates cell cycle-related genes are upregulated and immune system related genes are downregulated. KEGG analysis identifies oxidative phosphorylation as the most overrepresented pathways along with the G2/M phase transition pathway. Loss of Dip2b de-represses the expression of alveolar type I and type II molecular markers. Altogether, the study demonstrates an important role of Dip2B in lung maturation and survival.

Global transcriptome study of Dip2B-deficient mouse embryonic lung fibroblast reveals its important roles in cell proliferation and development.

Disco-interacting protein 2 homolog B (Dip2B) is a member of Dip2 family encoded by Dip2b gene. Dip2B has been reported to regulate murine epithelial KIT+ progenitor cell expansion and differentiation epigenetically via exosomal miRNA targeting during salivary gland organogenesis. However, its molecular functions, cellular activities and biological process remain unstudied. Here, we investigated the transcriptome of Dip2B-deficient mouse embryonic lung fibroblasts (MELFs) isolated from E14.5 embryos by RNA-Seq. Expression profiling identified 1369 and 1104 differentially expressed genes (DEGs) from Dip2b-/- and Dip2b+/- MELFs in comparisons to wild-type (Dip2b+/+ ). Functional clustering of DEGs revealed that many gene ontology terms belong to membrane activities such as 'integral component of plasma membrane', and 'ion channel activity', suggesting possible roles of Dip2B in membrane integrity and membrane function. KEGG pathway analysis revealed that multiple metabolic pathways are affected in Dip2b- / - and Dip2b +/ - when compared to Dip2b +/+ MELFs. These include 'protein digestion and absorption', 'pancreatic secretion' and 'steroid hormone synthesis pathway'. These results suggest that Dip2B may play important roles in metabolism. Molecular function analysis shows transcription factors including Hox-genes, bHLH-genes, and Forkhead-genes are significantly down-regulated in Dip2b- / - MELFs. These genes are critical in embryo development and cell differentiation. In addition, Dip2B-deficient MELFs demonstrated a reduction in cell proliferation and migration, and an increase in apoptosis. All results indicate that Dip2B plays multiple roles in cell proliferation, migration and apoptosis during embryogenesis and may participate in control of metabolism. This study provides valuable information for further understanding of the function and regulatory mechanisms of Dip2B.

Brain transcriptome study through CRISPR/Cas9 mediated mouse Dip2c gene knock-out.

Dip2C is highly expressed in brain and many other tissues but its biological functions are still not clear. Genes regulated by Dip2C in brain have never been studied. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, adaptive immune systems of bacteria and archaea, have been recently developed and broadly used in genome editing. Here, we describe targeted gene deletions of Dip2c gene in mice via CRISPR/Cas9 system and study of brain transcriptome under Dip2C regulation. The CRISPR/Cas9 system effectively generated targeted deletions of Dip2c by pronuclei injection of plasmids that express Cas9 protein and two sgRNAs. We achieved targeted large fragment deletion with efficiencies at 14.3% (1/7), 66.7% (2/3) and 20% (1/5) respectively in 3 independent experiments, averaging 26.7%. The large deletion DNA segments are 160.4 kb (Dip2CΔ160kb), spanning from end of exon 4 to mid of exon 38. A mouse with two base pair deletion was generated from a single sgRNA targeting in exon 4 (Dip2cΔ2bp) by non-homologous end joining (NHEJ). Loss of gene expression for Dip2c mRNA was confirmed by quantitative real-time PCR (qPCR). Dip2C-regulated genes and pathways in brain were investigated through RNAseq of Dip2cΔ2bp. In total, 838 genes were found differentially regulated, with 252 up and 586 down. Gene ontology (GO) analysis indicated that DEGs in brain are enriched in neurological functions including 'memory', 'neuropeptide signaling pathway', and 'response to amphetamine' while KEGG analysis shows that 'neuroactive ligand-receptor interaction pathway' is the most significantly enriched. DEGs Grid2ip, Grin2a, Grin2c, Grm4, Gabbr2, Gabra5, Gabre, Gabrq, Gabra6 and Gabrr2 are among the highly regulated genes by Dip2C. Results confirm Dip2C may play important roles in brain development and function.

Indian Hedgehog regulates senescence in bone marrow-derived mesenchymal stem cell through modulation of ROS/mTOR/4EBP1, p70S6K1/2 pathway.

Premature senescence of bone marrow-derived mesenchymal stem cells (BMSC) remains a major concern for their application clinically. Hedgehog signaling has been reported to regulate aging-associated markers and MSC skewed differentiation. Indian Hedgehog (IHH) is a ligand of Hedgehog intracellular pathway considered as an inducer in chondrogenesis of human BMSC. However, the role of IHH in the aging of BMSC is still unclear. This study explored the role IHH in the senescence of BMSC obtained from human samples and senescent mice. Isolated BMSC were transfected with IHH siRNA or incubated with exogenous IHH protein and the mechanisms of aging and differentiation investigated. Moreover, the interactions between IHH, and mammalian target of rapamycin (mTOR) and reactive oxygen species (ROS) were evaluated using the corresponding inhibitors and antioxidants. BMSC transfected with IHH siRNA showed characteristics of senescence-associated features including increased senescence-associated ß-galactosidase activity (SA-ß-gal), induction of cell cycle inhibitors (p53/p16), development of senescence-associated secretory phenotype (SASP), activation of ROS and mTOR pathways as well as the promotion of skewed differentiation. Interestingly, BMSC treatment with IHH protein reversed the senescence markers and corrected biased differentiation. Moreover, IHH shortage-induced senescence signs were compromised after mTOR and ROS inhibition. Our findings presented anti-aging activity for IHH in BMSC through down-regulation of ROS/mTOR pathways. This discovery might contribute to increasing the therapeutic, immunomodulatory and regenerative potency of BMSC and introduce a novel remedy in the management of aging-related diseases.

Efficiency of Traditional Chinese medicine targeting the Nrf2/HO-1 signaling pathway.

Cardiovascular disease (CVD) is a significant cause of death worldwide. Because of its major individual differences in genetic background, pathogenesis, and disease progression pattern, the mortality risk rate remains high following conventional Western medicine diagnosis under current guidelines. Traditional Chinese medicine (TCM) has important multi-target, multi-pathway, and multi-layer benefits that can effectively address western medicine deficiencies. It was therefore commonly used in CVD diagnosis. Oxidative stress is also one of the main factors of CVD. Likewise, this main reaction regulator is the nuclear factor erythroid-2-related (Nrf2) factor. When activated, it can be transferred to the nucleus and initiated in the downstream pathway, thus playing an anti-oxidant stress role. As one of the most crucial endogenous protection systems in the body, Nrf2-related / heme oxygenase 1 (Nrf2/HO-1) signaling pathway is Nrf2's most classic approach to playing roles. Recently, various advances have been made to research and explain TCM by manipulating this pathway to treat CVD using modern molecular biology and other approaches. This analysis summarized the relationship between Nrf2/HO-1 signaling route, CVD and TCM. Further, Autodock calculation was also conducted to determine the binding amino acid on this TCM to Nrf2 and HO-1.