CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.-Mao, C.-Z., Zheng, L., Zhou, Y.-M., Wu, H.-Y., Xia, J.-B., Liang, C.-Q., Guo, X.-F., Peng, W.-T., Zhao, H., Cai, W.-B., Kim, S.-K., Park, K.-S., Cai, D.-Q., Qi, X.-F. CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.

CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells.

High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [(3)H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD.

Plasminogen kringle 5 induces endothelial cell apoptosis by triggering a voltage-dependent anion channel 1 (VDAC1) positive feedback loop.

Human plasminogen kringle 5 (K5) is known to display its potent anti-angiogenesis effect through inducing endothelial cell (EC) apoptosis, and the voltage-dependent anion channel 1 (VDAC1) has been identified as a receptor of K5. However, the exact role and underlying mechanisms of VDAC1 in K5-induced EC apoptosis remain elusive. In the current study, we showed that K5 increased the protein level of VDAC1, which initiated the mitochondrial apoptosis pathway of ECs. Our findings also showed that K5 inhibited the ubiquitin-dependent degradation of VDAC1 by promoting the phosphorylation of VDAC1, possibly at Ser-12 and Thr-107. The phosphorylated VDAC1 was attenuated by the AKT agonist, glycogen synthase kinase (GSK) 3ß inhibitor, and siRNA, suggesting that K5 increased VDAC1 phosphorylation via the AKT-GSK3ß pathway. Furthermore, K5 promoted cell surface translocation of VDAC1, and binding between K5 and VDAC1 was observed on the plasma membrane. HKI protein blocked the impact of K5 on the AKT-GSK3ß pathway by competitively inhibiting the interaction of K5 and cell surface VDAC1. Moreover, K5-induced EC apoptosis was suppressed by VDAC1 antibody. These data show for the first time that K5-induced EC apoptosis is mediated by the positive feedback loop of "VDAC1-AKT-GSK3ß-VDAC1," which may provide new perspectives on the mechanisms of K5-induced apoptosis.

Akt Activation and Inhibition of Cytochrome C Release: Mechanistic Insights into Leptin-promoted Survival of Type II Alveolar Epithelial Cells.

Fetal growth restriction (FGR) increases the risk of perinatal death, partly due to defects in lung development. Leptin, a polypeptide hormone, is involved in fetal lung development. We previously demonstrated that treatment with exogenous leptin during gestation significantly promotes fetal lung maturity in the rat model of FGR. In this study, to delineate the molecular pathways through which leptin may enhance fetal lung development, we investigated the impact of leptin treatment on the survival of type II alveolar epithelial cells (AECs), essential leptin-responsive cells involved in lung development, in a rat model of FGR. The rat model of FGR was induced in pregnant Sprague-Dawley rats by partial uterine artery and vein ligation. In vivo and in vitro analyses of fetal lung tissues and freshly-isolated cultured AECs, respectively, showed that leptin protects type II AECs from hypoxia-induced apoptosis. Further molecular studies revealed the role of Akt activation in the leptin-mediated promotion of survival of type II AECs. The data also showed that the anti-apoptotic effects of leptin are dependent on phosphoinositol 3-kinase (PI3K) activation, and involve the down-regulation of caspases 3 and 9, upregulation of pro-survival proteins Bcl-2, and p-Bad, and inhibition of the release of cytochrome c from mitochondria. Taken together, our data suggested that leptin enhances the maturity of fetal lungs by mediating the regulation of caspase-3 and -9 during hypoxia-induced apoptosis of type II AECs and provide support for the potential of leptin as a therapeutic agent for promoting lung development in FGR.

Short-term effects of cupping and scraping therapy for chronic nonspecific low-back pain: A prospective, multicenter randomized trial.

BACKGROUND: As one of the most common musculoskeletal ailments, chronic nonspecific low-back pain (CNLBP) causes persistent disability and substantial medical expenses. Epidemiological evidence shows that the incidence rate of CNLBP in young and middle-aged people who are demanded rapidly recovery and social contribution is rising. Recent guidelines indicate a reduced role for medicines in the management of CNLBP. OBJECTIVE: The present study investigates the short-term effects of cupping and scraping therapy using a medicated balm, compared to nonsteroidal anti-inflammatory drug (NSAID) with a capsaicin plaster, in the treatment of CNLBP. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: We designed a prospective multicenter randomized clinical trial enrolling patients from January 1, 2022 to December 31, 2022. A total of 156 patients with CNLBP were randomized into two parallel groups. Diclofenac sodium-sustained release tablets were administered orally to participants in the control group for one week while a capsaicin plaster was applied externally. Patients in the test group were treated with cupping and scraping using a medical device and medicated balm. MAIN OUTCOME MEASURES: Primary outcome was pain recorded using the visual analogue scale (VAS). Two secondary outcomes were recorded using the Japanese Orthopedic Association low-back pain scale (JOA) and the traditional Chinese medicine (TCM) syndrome integral scale (TCMS) as assessment tools. RESULTS: Between baseline and postintervention, all changes in outcome metric scales were statistically significant (P < 0.001). Compared to the control group, patients in the test group had a significantly greater treatment effect in all outcome variables, as indicated by lower VAS and TCMS scores and higher JOA scores, after the one-week intervention period (P < 0.001). Further, according to the findings of multivariate linear regression analysis, the participants' pain (VAS score) was related to their marital status, age, smoking habits and body mass index. No adverse reactions were reported for any participants in this trial. CONCLUSION: The effectiveness of TCM combined with the new physiotherapy tool is superior to that of NSAID combined with topical plasters, regarding to pain intensity, TCM symptoms and quality of life. The TCM plus physiotherapy also showed more stable and long-lasting therapeutic effects. TRIAL REGISTRATION: This study was registered at Chinese Clinical Trial Registry (ChiCTR2200055655). Please cite this article as: He JY, Tu XY, Yin ZF, Mu H, Luo MJ, Chen XY, Cai WB, Zhao X, Peng C, Fang FF, Lü C, Li B. Short-term effects of cupping and scraping therapy for chronic nonspecific low-back pain: A prospective, multicenter randomized trial. J Integr Med. 2024; 22(1): 39-45.

Long-term statins administration exacerbates diabetic nephropathy via ectopic fat deposition in diabetic mice.

Statins play an important role in the treatment of diabetic nephropathy. Increasing attention has been given to the relationship between statins and insulin resistance, but many randomized controlled trials confirm that the therapeutic effects of statins on diabetic nephropathy are more beneficial than harmful. However, further confirmation of whether the beneficial effects of chronic statin administration on diabetic nephropathy outweigh the detrimental effects is urgently needed. Here, we find that long-term statin administration may increase insulin resistance, interfere with lipid metabolism, leads to inflammation and fibrosis, and ultimately fuel diabetic nephropathy progression in diabetic mice. Mechanistically, activation of insulin-regulated phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway leads to increased fatty acid synthesis. Furthermore, statins administration increases lipid uptake and inhibits fatty acid oxidation, leading to lipid deposition. Here we show that long-term statins administration exacerbates diabetic nephropathy via ectopic fat deposition in diabetic mice.

Loss of pigment epithelium-derived factor leads to ovarian oxidative damage accompanied by diminished ovarian reserve in mice.

AIMS: This study aims to investigate the pathophysiological role and mechanism of pigment epithelium-derived factor (PEDF) deletion in ovarian damage. METHODS: Female PEDF-knockout mice and their wild-type littermates were used in this study. Relevant tests were performed at 8-10 weeks or 32 weeks of age. KEY FINDINGS: Compared to the wild-type mice, the PEDF-knockout mice showed diminished ovarian reserve (DOR), worse ovum quality after injection to induce controlled ovarian stimulation, increased serum follicle stimulating hormone (FSH) level and an follicle stimulating hormone/luteinizing hormone (FSH/LH) ratio. Moreover, severe ovarian oxidative damage was found in ovaries of PEDF-knockout mice that mainly manifested as an accumulation of reactive oxygen species (ROS), NF­E2-related factor 2 (Nrf2) pathway activation, significantly upregulated expression of ROS-generating genes. Correspondingly, the PEDF-knockout mice exhibited lipid metabolism disorder and insulin resistance, which mainly manifested as obesity, abdominal fat accumulation, adipocyte enlargement, severe ectopic fat deposition, dyslipidemia, changes in adipokine levels, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired insulin tolerance and significantly declined protein kinase B (Akt) phosphorylation levels. SIGNIFICANCE: Loss of PEDF leads to ovarian oxidative damage accompanied by DOR in mice, this is related to PEDF deficiency induced severe insulin resistance and lipid metabolism disorder. Therefore, PEDF may be a potential target for the treatment of diseases related to ovarian oxidative damage.

Long-term follow-up of anterior approach preperitoneal hernia repair using the Kugel patch.

BACKGROUND: Despite many advantages of original Kugel hernia repair over other procedures, there exist certain disadvantages of technical difficulty, long learning curve, and high early recurrence. The aim of this study was to explore the outcomes of long-term follow-up using anterior approach preperitoneal hernia repair with the Kugel patch and determine its safety and efficacy. METHODS: Five hundred eighty-one inguinal hernias were performed in 560 patients, using anterior approach preperitoneal repair. Patients' age and sex, type of hernias, operative time, hospital stay, complications, and recurrence were evaluated. RESULTS: We included 581 hernias, with 354 on right side, 162 on left side, and 65 bilateral sides. All hernias were primary. There were 443 indirect hernias, 115 direct hernias, and 23 femoral hernias. Mean operative time was 50 minutes; local anesthesia was applied in 530 cases (91.2%). Postoperative complications affected 50 patients (8.9%). The patients were discharged from 4 to 8 days (with average of 6 days). The averaged follow-up time was 70 months (12 to 120 months). There were 3 recurrences in the period (.5%). CONCLUSIONS: The results of long-term follow-up with this procedure are safe and effective, easy to learn. We believe that this procedure should be adopted as an alternative method for Chinese patients with inguinal hernias.

[High-dose glucose induces human retinal endothelial cell apoptosis].

OBJECTIVE: To examine the direct effect of high glucose levels on primary cultured human retinal capillary endothelial cells (HRCEC). METHODS: HRCECs were isolated from donated eyes and cultured for 6 days in the media containing 5 or 25 mmol/L glucose. The cell viability was determined by trypan blue exclusion assay and cell cycle analyzed by flow cytometry, with the cell apoptosis assayed by TUNEL method. RESULTS: The cell viability was significantly decreased after exposure to 25 mmol/L glucose, and the number of apoptotic cells determined by flow cytometry and TUNEL was significantly increased in response to high-dose glucose treatment. CONCLUSION: High-dose glucose induces apoptosis in HRCEC, which may contribute to the development of diabetic retinopathy.

[A deletion mutant of plasminogen kringle 5 inhibits retinal capillary endothelial cell proliferation].

OBJECTIVE: To obtain purified deletion mutant of plasminogen kringle 5 (K5) using gene mutation and genetic recombination methods and assess its anti-angiogenic activity in vitro. METHODS: A deletion mutant of K5 was obtained by deleting 15 amino acids from K5 while retaining all the 3 disulfide bonds. This K5 mutant (Mut1) was expressed in E. coli and affinity purified. The inhibition effect of K5 Mut1 on primary retinal capillary endothelial cells and pericytes from the same origin was assessed by MTT assay. RESULTS: The K5 Mut1 inhibited the proliferation of primary retinal capillary endothelial cells in a concentration-dependent manner, with an apparent half-inhibition concentration (EC(50)) of approximately 35 nmol/L, which was 2-fold more potent than intact K5. In the same concentration range, this peptide did not inhibit pericytes from the same origin, suggesting an endothelial cell-specific inhibition. CONCLUSION: This K5 deletion mutant is a more potent angiogenic inhibitor than K5 and may have therapeutic potential in the treatment of such disorders with abnormal neovascularization as diabetic retinopathy, age-related macular degeneration and solid tumor.

Leptin promotes fetal lung maturity and upregulates SP-A expression in pulmonary alveoli type-II epithelial cells involving TTF-1 activation.

The placental hormone leptin has important functions in fetal and neonatal growth, and prevents depressed respiration in leptin-deficient mice. The effect of leptin on respiratory distress suffered by low birth weight and premature infants has been studied. However, it is unclear how leptin enhances lung maturity in the fetus and ameliorates neonatal respiratory distress. In the present study, we found that antenatal treatment with leptin for 2 d significantly enhanced the relative alveolus area and improved the maturity of fetal lungs in a rat model of fetal growth restriction (FGR). Mean birth weight and lung wet weight were higher in the leptin-treated group than in the PBS-treated group, indicating promotion of fetal growth. Leptin upregulated the intracellular expression and extracellular secretion of surfactant protein (SP) A in type-II alveolar epithelial cells (AECs) in vivo and in vitro. Dual positive effects of leptin were found on protein expression and transcriptional activity of thyroid transcription factor-1 (TTF-1), a nuclear transcription essential for branching morphogenesis of the lung and expression of SP-A in type-II AECs. Knockdown of TTF-1 by RNA interference indicated that TTF-1 may play a vital role in leptin-induced SP-A expression. These results suggest that leptin may have great therapeutic potential for the treatment of FGR, and leptin-mediated SP-A induction and lung maturity of the fetus are TTF-1 dependent.

The Prognostic Value of p16 Hypermethylation in Cancer: A Meta-Analysis.

BACKGROUND: The prognostic value of p16 promoter hypermethylation in cancers has been evaluated for several years while the results remain controversial. We thus performed a systematic review and meta-analysis of studies assessing the impact of p16 methylation on overall survival (OS) and disease-free survival (DFS) to clarify this issue. METHODS: We searched Pubmed, Embase and ISI web of knowledge to identify studies on the prognostic impact of p16 hypermethylation in cancers. A total of 6589 patients from 45 eligible studies were included in the analysis. Pooled hazard ratios (HRs) with 95% confidence interval (95% CI) were calculated to estimate the effect using random-effects model. RESULTS: The analysis indicated that p16 hypermethylation had significant association with poor OS of non-small cell lung cancer (NSCLC) (HR 1.74, 95% CI: 1.36-2.22) and colorectal cancer (CRC) (HR 1.80; 95% CI 1.27-2.55). Moreover, the significant correlation was present between p16 hypermethylation and DFS of NSCLC (HR 2.04, 95% CI: 1.19-3.50) and head and neck cancer (HR 2.24, 95% CI: 1.35-3.73). Additionally, in the analysis of the studies following REMARK guidelines more rigorously, p16 hypermethylation had unfavorable impact on OS of NSCLC (HR 1.79, 95% CI: 1.35-2.39) and CRC (HR 1.96, 1.16-3.34), and on DFS of NSCLC (HR 2.12, 95% CI: 1.21-3.72) and head and neck cancer (HR 2.24, 95% CI: 1.35-3.73). CONCLUSIONS: p16 hypermethylation might be a predictive factor of poor prognosis in some surgically treated cancers, particularly in NSCLC.

Dual inhibition of plasminogen kringle 5 on angiogenesis and chemotaxis suppresses tumor metastasis by targeting HIF-1α pathway.

We had demonstrated that plasminogen kringle 5 (K5), a potent angiogenic inhibitor, inhibited retinal neovascularization and hepatocellular carcinoma growth by anti-angiogenesis. The current study investigated the effects and the underlying mechanisms of K5 on both tumor growth and spontaneous pulmonary metastasis in Lewis lung carcinoma (LLC) implanted mouse model. Similarly, K5 could decrease expression of VEGF in LLC cells and grafted tissues and suppress tumor angiogenesis and growth. K5 had no direct effect on proliferation and apoptosis of LLC. However, K5 could significantly inhibit SDF-1α-induced chemotaxis movement of LLC cells and resulted in a great reduction of surface metastatic nodules and micrometastases in the lungs of LLC tumor-bearing mice. K5 also decreased expression of chemokine (C-X-C motif) receptor 4 (CXCR4) in LLC cells and grafted tissues. Furthermore, K5 down-regulated SDF-1α expression in metastatic lung tissues of LLC-bearing mice. Therefore, K5 may suppress tumor pulmonary metastasis through inhibiting SDF-1α-CXCR4 chemotaxis movement and down-regulation of VEGF. Moreover, the role of hypoxia inducible factor-1α (HIF-1α), a crucial transcriptional factor for both VEGF and CXCR4 expression, was evaluated. The siRNA of HIF-1α attenuated expression of VEGF and CXCR4 and inhibited LLC migration. K5 decreased HIF-1α protein level and impaired nuclear HIF-1α accumulation. These results showed for the first time that K5 inhibits LLC growth and metastasis via the dual effects of anti-angiogenesis and suppression of tumor cell motility by targeting the pivotal molecule, HIF-1α.

Inducible nitric oxide synthase is involved in the oxidation stress induced by HIV-1 gp120 in human retina pigment epithelial cells.

BACKGROUND: The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 has been implicated in the development of AIDS-associated retinopathy. The present study tested the hypothesis that gp120 may induce oxidative stress including up regulation of inducible nitric oxide synthase (iNOS) and production of malondialdehyde (MDA) and nitric oxide (NO) to mediate retinopathy in retinal pigment epithelial (RPE) cells. METHODS: Human RPE cell line D407 was cultured and treated with gp120. HIV-1 gp120 protein induced lipid peroxidation product MDA. NO production and iNOS expression were examined in vitro by spectrophomtometry, real-time PCR, Western blotting, and confocal microscope. RESULTS: Addition of gp120 was able to induce RPE cells to produce NO and MDA in time- and dose-dependent manners (P < 0.05). Similarly, gp120 was also capable of up-regulating iNOS mRNA and protein in D407 cells in time- and dose-dependent manners. CONCLUSIONS: Gp120 induces oxidative stress in D407 cell by stimulating MDA and NO production, which is mediated by up-regulating iNOS expression. Gp120 may mediate oxidation stress in AIDS-associated retinopathy.

[Construction of carcinoembryonic antigen (CEA) gene vaccine and stable coexpression with an immune adjuvant].

BACKGROUND & OBJECTIVE: Carcinoembryonic antigen (CEA) positive cancers are poorly responded to different kinds of treatments. Gene vaccines are promising in research of gene immunotherapy for these tumors. In this study, a CEA gene vaccine with hIL-2 as an immune adjuvant was constructed into a pVAX1 vector for synchronous expression, so as to explore experimentally a new biotherapy strategy against tumors. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), CEA cDNA was obtained from a large intestine carcinoma tissue; its encoded protein was compared with the CEA presented in GenBank using the protein analysis software. The acquired CEA cDNA fragment was linked to hIL-2 cDNA via an IRES site and cloned into the pVAX1 vector. The recombinant plasmid was estimated by CEA luminometry assay and hIL-2 ELISA measurement respectively. RESULTS: The nucleotide sequences of the target gene fragments of the recombinant plasmid were verified. The acquired CEA sequence is highly homologous with M29540 and M17303 (99.8%) in GenBank; and the PCR sequence of hIL-2 is coincident with the original cDNA (100%) provided. The antigenicity,membrane-spanning segments, signal cleavage sites, secondary structure and 3D structure of the acquired CEA protein were similar to the original proteins of M29540 and M17303 predicted by the protein analysis software. Results showed the recombinant could steadily express CEA antigen and hIL-2 protein synchronously in CHO cells in vitro. CONCLUSION: The CEA cDNA was obtained from the tumor tissue and the CEA gene vaccine with hIL-2 coexpression was constructed successfully. It has provided a possible method for immunotherapy against CEA positive cancers in vivo.