Genetic switch selectively kills hepatocellular carcinoma cell based on microRNA and tissue-specific promoter.

The clinical treatment of hepatocellular carcinoma (HCC) is still a heavy burden worldwide. Intracellular microRNAs (miRNAs) commonly express abnormally in cancers, thus they are potential therapeutic targets for cancer treatment. miR-21 is upregulated in HCC whereas miR-122 is enriched in normal hepatocyte but downregulated in HCC. In our study, we first generated a reporter genetic switch compromising of miR-21 and miR-122 sponges as sensor, green fluorescent protein (GFP) as reporter gene and L7Ae:K-turn as regulatory element. The reporter expression was turned up in miR-21 enriched environment while turned down in miR-122 enriched environment, indicating that the reporter switch is able to respond distinctly to different miRNA environment. Furthermore, an AAT promoter, which is hepatocyte-specific, is applied to increase the specificity to hepatocyte. A killing switch with AAT promoter and an apoptosis-inducing element, Bax, in addition to miR-21 and miR-122 significantly inhibited cell viability in Huh-7 by 70 % and in HepG2 by 60 %. By contrast, cell viability was not affected in five non-HCC cells. Thus, we provide a novel feasible strategy to improve the safety of miRNA-based therapeutic agent to cancer.

C-atrial natriuretic peptide (ANP)4-23 attenuates renal fibrosis in deoxycorticosterone-acetate-salt hypertensive mice.

Epithelial-mesenchymal transition (EMT) plays a critical role in hypertension-induced renal fibrosis, a final pathway that leads to end-stage renal failure. C-Atrial natriuretic peptide (ANP)4-23, a specific agonist of natriuretic peptide receptor-C (NPR-C), has been reported to have protective effects against hypertension. However, the role of C-ANP4-23 in hypertension-associated renal fibrosis has not yet been elucidated. In this study, mice were randomly divided into SHAM group, DOCA-salt group and DOCA-salt + C-ANP4-23 group. Renal morphology changes, renal function and fibrosis were detected. Human proximal tubular epithelial cells (HK2) stimulated by aldosterone were used for cell function and mechanism study. The DOCA-salt treated mice exhibited hypertension, kidney fibrosis and renal dysfunction, which were attenuated by C-ANP4-23. Moreover, C-ANP4-23 inhibited DOCA-salt treatment-induced renal EMT as evidenced by decrease of the mesenchymal marker alpha-smooth muscle actin (ACTA2) and vimentin and increase of epithelial cell marker E-cadherin. In HK2 cells, aldosterone induced EMT response, which was also suppressed by C-ANP4-23. The key transcription factors (twist, snail, slug and ZEB1) involved in EMT were increased in the kidney of DOCA-salt-treated mice, which were also suppressed by C-ANP4-23. Mechanistically, C-ANP4-23 inhibited the aldosterone-induced translocation of MR from cytosol to nucleus without change of MR expression. Furthermore, C-ANP4-23 rescued the enhanced expression of NADPH oxidase (NOX) 4 and oxidative stress after aldosterone stimulation. Aldosterone-induced Akt and Erk1/2 activation was also suppressed by C-ANP4-23. Our data suggest that C-ANP4-23 attenuates renal fibrosis, likely through inhibition of MR activation, enhanced oxidative stress and Akt and Erk1/2 signaling pathway.

Biotransformation and metabolite activity analysis of flavonoids from propolis in vivo.

Propolis is a natural resinous compound produced by bees, mixed with their saliva and wax, and has a range of biological benefits, including antioxidant and anti-inflammatory effects. This article reviews the in vivo transformation of propolis flavonoids and their potential influence on drug efficacy. Despite propolis is widely used, there is little research on how the active ingredients of propolis change in the body and how they interact with drugs. Future research will focus on these interactions and the metabolic fate of propolis in vivo.

Lymphatic Contrast-enhanced US to Improve the Diagnosis of Cervical Lymph Node Metastasis from Thyroid Cancer.

Background The presence of cervical lymph node (LN) metastases (LNMs) affects clinical staging and prognosis of thyroid cancer, but the role of conventional B-mode US is limited for preoperative diagnosis of LNMs. The diagnostic value of lymphatic contrast-enhanced US (LCEUS) in thyroid cancer is still being explored. Purpose To explore the diagnostic performance of LCEUS by means of thyroidal injection of contrast agent in comparison with US in detecting LNMs of suspected thyroid cancer. Materials and Methods In this single-center prospective study conducted from November 2020 to January 2021, consecutive participants with suspected thyroid cancer underwent B-mode US and LCEUS of cervical LNs before biopsy. LNMs were confirmed with fine-needle aspiration cytologic examination, thyroglobulin washout assessment, or histopathologic examination after surgery. The diagnostic performance of LCEUS for cervical LNs was compared with that of conventional B-mode US, and its association with LN size and location was evaluated. Results The final data set included 64 participants (mean age, 45 years ± 12 [SD]; 52 women) with 76 LNs. The sensitivity, specificity, and accuracy of LCEUS for LNM were 97%, 90%, and 93%, respectively, whereas they were 81%, 80%, and 80%, respectively, for LNM at conventional B-mode US. Compared with US, LCEUS had better diagnostic accuracy for the LNs smaller than 1 cm (82% vs 95%; P = .03) and for central neck LNs (level VI) (83% vs 96%; P = .04). Conclusion Lymphatic contrast-enhanced US had better diagnostic performance than conventional B-mode US for detecting cervical LN metastases in suspected thyroid cancer before surgery, especially for LNs smaller than 1 cm and central neck LNs. © RSNA, 2023 See also the editorial by Grant and Kwon in this issue.

Mitogen-Activated Protein Kinases Mediate Adventitial Fibroblast Activation and Neointima Formation via GATA4/Cyclin D1 Axis.

PURPOSE: Activation of mitogen-activated protein kinases (MAPKs) by pathological stimuli participates in cardiovascular diseases. Dysfunction of adventitial fibroblast has emerged as a critical regulator in vascular remodeling, while the potential mechanism remains unclear. In this study, we sought to determine the effect of different activation of MAPKs in adventitial fibroblast contributing to neointima formation. METHODS: Balloon injury procedure was performed in male 12-week-old Sprague-Dawley rats. After injury, MAPK inhibitors were applied to the adventitia of injured arteries to suppress MAPK activation. Adventitial fibroblasts were stimulated by platelet-derived growth factor-BB (PDGF-BB) with or without MAPK inhibitors. RNA sequencing was performed to investigate the change of pathway and cell function. Wound healing, transwell assay, and flow cytometry were used to analyze adventitial fibroblast function. RESULTS: Phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular regulated kinases 1/2 (ERK1/2) was increased in injured arteries after balloon injury. In primary culture of adventitial fibroblasts, PDGF-BB increased phosphorylation of p38, JNK, ERK1/2, and extracellular regulated kinase 5 (ERK5) in a short time, which was normalized by their inhibitors respectively. Compared with the injury group, perivascular administration of four MAPK inhibitors significantly attenuated neointima formation by quantitative analysis of neointimal area, intima to media (I/M) ratio, and lumen area. RNA sequencing of adventitial fibroblasts treated with PDGF-BB with or without four inhibitors demonstrated differentially expressed genes involved in multiple biological processes, including cell adhesion, proliferation, migration, and inflammatory response. Wound healing and transwell assays showed that four inhibitors suppressed PDGF-BB-induced adventitial fibroblast migration. Cell cycle analysis by flow cytometry demonstrated that JNK, ERK1/2, and ERK5 but not p38 inhibitor blocked PDGF-BB-induced G1 phase release associated with decrease expression of cell cycle protein Cyclin D1 and transcription factor GATA4. Moreover, four inhibitors decreased macrophage infiltration into adventitia and monocyte chemoattractant protein-1 (MCP-1) expression. CONCLUSION: These results suggest that MAPKs differentially regulate activation of adventitial fibroblast through GATA4/Cyclin D1 axis that participates in neointima formation.

[Oculo-facio-cardio-dental syndrome caused by BCOR gene mutations: a case report]. / BCORåŸºå› çªå˜è‡´æ–°ç”Ÿå„¿çœ¼-面-心-牙综合征1例.

A full-term female infant was admitted at 5 hours after birth due to heart malformations found during the fetal period and cyanosis once after birth. Mmultiple malformations of eyes, face, limbs, and heart were noted. The whole-exome sequencing revealed a pathogenic heterozygous mutation, c.2428C>T(p.Arg810*), in the BCOR gene. The infant was then diagnosed with oculo-facio-cardio-dental syndrome. He received assisted ventilation to improve oxygenation and nutritional support during hospitalization. Right ventricular double outlet correction was performed 1 month after birth. Ocular lesions were followed up and scheduled for elective surgery. The possibility of oculo-facio-cardio-dental syndrome should be considered for neonates with multiple malformations of eyes, face, and heart, and genetic testing should be performed as early as possible to confirm the diagnosis; meanwhile, active ophthalmic and cardiovascular symptomatic treatment should be given to improve the prognosis.

Inhibition of autophagy by 3-methyladenine restricts murine cytomegalovirus replication.

Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis. The eyecup isolated from adult C57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine, or rapamycin to block or stimulate autophagy. In cultured MEFs, the ratio of LC3I/II was reduced at 24 hours post infection (hpi), but was increased at 48 hpi In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7 days post infection (dpi). In addition, caspase-3 cleavage was increased at 48 hpi in MEFs and also elevated in MCMV infected eyecups at 4, 7, 10, and 14 dpi. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups. Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, the suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication.

Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Krüppel-like factor (KLF) 15 has emerged as a critical regulator of fibrosis in cardiovascular diseases. However, the precise role that KLF15 and its functional domain played in adventitial inflammation and fibrosis remains unclear. This study aims to investigate the role of the transactivation domain (TAD) of KLF15 in angiotensin II (Ang II)-induced adventitial pathologic changes. KLF15 expression was decreased in the vascular adventitia of Ang II-infused mice (1000 ng/kg/min, 14 d) and in adventitial fibroblasts (AFs) stimulated by Ang II (10-7 M). Adenovirus-mediated KLF15 overexpression normalized Ang II-induced vascular hypertrophy, increased collagen deposition, macrophage infiltration, and CCL2 and VCAM-1 expression. Interestingly, KLF15-ΔTAD (KLF15 with deletion of TAD at amino acids 132-152) overexpression showed no effect on the above pathologic changes. Similarly, perivascularly overexpression of KLF15 but not KLF15-ΔTAD in carotid arteries also attenuated Ang II-induced vascular inflammation and fibrosis. Furthermore, KLF15 overexpression after Ang II infusion rescued Ang II-induced vascular remodeling. CCL2 or VCAM-1-mediated monocyte and macrophage migration or adhesion to AFs in response to Ang II was negatively regulated by KLF15 through TAD. Ang II-enhanced Smad2/3 activation and adventitial migration, proliferation, and differentiation of AFs were suppressed by KLF15 but not KLF15-ΔTAD overexpression. Conversely, small interfering RNA knockdown of KLF15 aggravated Ang II-induced Smad2/3 activation and dysfunction of AFs. Luciferase, coimmunoprecipitation, and chromatin immunoprecipitation assay were used to demonstrate that interaction of KLF15 with Smad2/3 suppressed CCL2 expression through TAD. Mechanistically, activation of Ang II type 1 receptor/phospholipase Cγ 1/ERK1/2 signaling resulted in a decrease of KLF15 expression. In conclusion, these results demonstrate that KLF15 negatively regulates activation of AFs through TAD, which plays an important role in Ang II-induced adventitial inflammation and fibrosis.-Lu, Y.-Y., Li, X.-D., Zhou, H.-D., Shao, S., He, S., Hong, M.-N., Liu, J.-C., Xu, Y.-L., Wu, Y.-J., Zhu, D.-L., Wang, J.-G., Gao, P.-J. Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Complement 5a-mediated trophoblasts dysfunction is involved in the development of pre-eclampsia.

Pre-eclampsia (PE) is a life-threatening multisystem disorder leading to maternal and neonatal mortality and morbidity. Emerging evidence showed that activation of the complement system is implicated in the pathological processes of PE. However, little is known about the detailed cellular and molecular mechanism of complement activation in the development of PE. In this study, we reported that complement 5a (C5a) plays a pivotal role in aberrant placentation, which is essential for the onset of PE. We detected an elevated C5a deposition in macrophages and C5a receptor (C5aR) expression in trophoblasts of pre-eclamptic placentas. Further study showed that C5a stimulated trophoblasts towards an anti-angiogenic phenotype by mediating the imbalance of angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1) and placental growth factor (PIGF). Additionally, C5a inhibited the migration and tube formation of trophoblasts, while, C5aR knockdown with siRNA rescued migration and tube formation abilities. We also found that maternal C5a serum level was increased in women with PE and was positively correlated with maternal blood pressure and arterial stiffness. These results demonstrated that the placental C5a/C5aR pathway contributed to the development of PE by regulating placental trophoblasts dysfunctions, suggesting that C5a may be a novel therapeutic possibility for the disease.

The effect of labor epidural analgesia on maternal-fetal outcomes: a retrospective cohort study.

PURPOSE: To evaluate the impact of labor epidural analgesia on maternal-fetal safety outcomes in a signal Chinese academic medical center. METHODS: A single-intervention impact study was conducted at The Second Affiliated Hospital, Wenzhou Medical University. The study period was divided into three phases: (1) baseline phase: from January 1 and June 30, 2009 when no analgesic method was routinely employed during labor; (2) phase-in period: the epidural analgesia was implemented 8 a.m.-5 p.m. during weekdays; and (3) the post-No Pain Labor N'Delivery phase when the labor epidural was applied 24 h a day, 7 days a week, from June 1, 2010 and June 30, 2011. The maternal-fetal safety outcomes of delivery were compared between the different periods. RESULTS: There were 15,415 deliveries with 42.3% of nulliparous parturients in the 31-month study period. As the primary outcomes, the labor epidural analgesia rate increased from 0 to 57%, the vaginal delivery rate increased, and cesarean delivery rate decreased by 3.5% after full implementation. As the secondary outcomes, the rate of episiotomy and severe perineal injury after the implementation periods were significant decreased. The rate of postpartum oxytocin administration was decreased by 17.8%. No significant difference between the baseline and implementation periods was found in the rate of postpartum hemorrhage, Apgar scores less than 7 at both 1 and 5 min, 7-day mortality, and the overall neonatal intensive care unit admission rate. CONCLUSION: Implementation of labor epidural analgesia increased the vaginal delivery rate and use of labor epidural analgesia is safe to parturients and fetus.