AZD7762 induces CRBN dependent BAG3 degradation through ubiquitin-proteasome pathway.

Protein degraders are currently under rapid development as a promising modality for drug discovery. They are compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting intracellular protein degradation through proteasomal pathway. More protein degraders identification will greatly promote the development of this field. BAG3 is widely recognized as an excellent therapeutic target in cancer treatments. Exploring protein degraders that target BAG3 degradation has profound implications. Herein, molecular docking was applied to assess binding energy between 81 clinical phase I kinase inhibitors and BAG3. BAG3 protein and mRNA level were detected by western blot and quantitative real-time PCR. CCK8 assay and colony formation assay were applied to detect the cell viability and proliferation rate. Cell death was accessed using flow cytometry combined with PI and Annexin V double staining. AZD7762, a Chk1 kinase inhibitor, was identified to induce BAG3 degradation in a ubiquitin-proteasome pathway. AZD7762-induced BAG3 degradation was not dependent on Chk1 expression or activity. CRBN, an E3 ligase, was identified to bind to BAG3 and mediated BAG3 ubiquitination in the presence of AZD7762. By targeting Chk1 and BAG3, two ideal therapeutic targets in cancer treatment, AZD7762 would be a powerful chemotherapy agent in the future.

Triptolide inhibits the progression of Glioblastoma U251 cells via targeting PROX1.

Background: Glioblastoma multiforme (GBM) is the most lethal brain cancer in adults, characterized by rapid growth, extensive invasiveness, and poor prognosis, and there is still a lack of effective treatments. Here, we aimed to explore the role of triptolide (TPL), purified from Tripterygium wilfordii Hook F, on glioblastoma cell growth, apoptosis, proliferation, migration and invasion, as well as potential underlying mechanisms. Methods: The publicly available clinical data of Brain Lower Grade Glioma (LGG) from The Cancer Genome Atlas (TCGA) had been screened to observe PROX1 expression. The Kaplan-Meier analysis was used to analyze the relationship between PROX1 expression and GBM prognosis. CCK8, cell cycle, EDU, apoptosis, wound healing, and transwell assays were performed to detect the effects of TPL on glioblastoma U251 cell viability, cell cycle, proliferation, apoptosis, migration and invasion, respectively. Further, a soft agar colony assay was used to calculate the growth of glioblastoma cells. The qRT-PCR and western blot were conducted to quantify PROX1 mRNA and protein levels. The transcriptional regulation of TPL was detected by Dual luciferase reporter assay. Results: We found that TPL inhibited glioblastoma cell viability, proliferation, cell cycle, migration and invasion, but enhanced apoptosis in a dose-dependent manner. The expression of cell cycle inhibitor, P21, and pro-apoptosis factor, Bax was increased, while invasion-related factors MMP2 and MMP9 were silenced after TPL treatments. Mechanistically, TPL showed transcriptional inhibition of PROX1 appearance. Moreover, ectopic expression of PROX1 partially rescued the effects of TPL on glioblastoma cell viability, proliferation, apoptosis, migration and invasion, and on the expression of cell function-related genes. Conclusion: This study verified that TPL inhibited the progression of glioblastoma cells by transcriptionally depressing the expression of PROX1.

Total Thoracoscopic Surgery for Late Mitral Paravalvular Leakage Repair in A Beating Heart

Abstract Paravalvular leakage (PVL) after mitral valve replacement is a troublesome complication that may lead to severe symptoms and reoperation. Previous case reports on total thoracoscopic cardiac surgery without aortic cross-clamping for repairing late PVL are rare. We describe a 64-year-old man who had undergone aortic and mitral valve replacement via median sternotomy eight years earlier, and who recently developed cardiac failure due to severe tricuspid regurgitation (TR) and PVL in the posterior mitral annulus. During total thoracoscopic surgery with using the beating heart technique, direct closure of the PVL was achieved via pledgeted mattress sutures, and tricuspid valvuloplasty was routinely performed to treat TR. This case indicated that total thoracoscopic surgery on a beating heart may be an excellent option for treating PVL concomitant with TR.

HMOX1 Promotes Ferroptosis Induced by Erastin in Lens Epithelial Cell through Modulates Fe2+ Production.

PURPOSE: Ferroptosis is defined by the iron-dependent cell death caused by the accumulation of lipid peroxidation. As a major intracellular Fe pools, heme could be metabolized into ferrous iron, carbon monoxide, and biliverdin by Heme oxygenase-1 (HMOX1). Aged human lens epithelium was reported to highly susceptible to ferroptosis, the functional molecular involved in this progress is not explored. Here, we have demonstrated the function of HMOX1 in human lens epithelium during ferroptotic cell death. METHODS: HMOX1 stably expressed cell line was constructed by lentivirus transfection. HMOX1 knock-out cell line was constructed by Crispr-cas9 technology. Protein expression was detected by western blot. Inverted microscope was applied to record the morphological changes among different treatments. CCK8 assay and colony formation assay were applied to detect the cell proliferation rate. Cell death was detected by PI staining. Lipid Peroxidation was detected by Cell malondialdehyde (MDA) assay. Intracellular Ferrous and Ferric ions were determined using an iron assay kit. RESULTS: HMOX1 expression was induced significantly in HLECs under erastin treatment in a time-dependent and dosage-dependent manner. Forced expression of HMOX1 increase the sensitivity of HLECs to erastin treatment. However, knock-out or knock-down of HMOX1 improved the cell viability of HLECs significantly under erastin treatment. Iron liberated from heme by HMOX1 might play pivotal role to improve the sensitivity of HLECs in response to erastin. CONCLUSION: HMOX1 is an essential pro-ferroptosis enzyme which increase the susceptibility of human lens epithelium to erastin. Ferrous iron, a byproduct of heme, might accelerate erastin triggered ferrotosis cell death in human lens epithelium cells.

Total Thoracoscopic Surgery for Late Mitral Paravalvular Leakage Repair in A Beating Heart.

Paravalvular leakage (PVL) after mitral valve replacement is a troublesome complication that may lead to severe symptoms and reoperation. Previous case reports on total thoracoscopic cardiac surgery without aortic cross-clamping for repairing late PVL are rare. We describe a 64-year-old man who had undergone aortic and mitral valve replacement via median sternotomy eight years earlier, and who recently developed cardiac failure due to severe tricuspid regurgitation (TR) and PVL in the posterior mitral annulus. During total thoracoscopic surgery with using the beating heart technique, direct closure of the PVL was achieved via pledgeted mattress sutures, and tricuspid valvuloplasty was routinely performed to treat TR. This case indicated that total thoracoscopic surgery on a beating heart may be an excellent option for treating PVL concomitant with TR.

A Modified Ascending Aortic Cannulation Technique in Minimally Invasive Totally Thoracoscopic Cardiac Surgery

ABSTRACT Cannulation through the femoral artery is the preferred method of establishing peripheral cardiopulmonary bypass in minimally invasive totally thoracoscopic cardiac surgery. However, faced with the contraindication of femoral artery cannulation, modified ascending aortic cannulation is an alternative approach to minimally invasive totally thoracoscopic cardiac surgery.

Identification of a Novel 15q21.1 Microdeletion in a Family with Marfan Syndrome.

Background: Marfan syndrome (MFS) is a connective tissue disease involving multiple systems, with thoracic aortic aneurysm (TAA) as the most common life-threatening manifestation. Method: A pedigree with TAA was investigated, and peripheral venous blood was extracted from six family members. After whole exome sequencing (WES) and chromosomal microarray analysis (CMA) in these individuals, bioinformatics and inheritance analyses were performed. Result: WES revealed a novel, small, 0.76 Mb microdeletion in 15q21.1, which cosegregated with the disease phenotype in the family and led to the haploinsufficiency of the fibrillin 1 (FBN1) gene, which is associated with MFS. This small copy number variant (CNV) was confirmed by CMA. Conclusion: Our study expands the phenotypic spectrum of the pathogenic CNV associated with MFS, thereby facilitating clinical genetic diagnosis and future genetic counseling for this family.

A Modified Ascending Aortic Cannulation Technique in Minimally Invasive Totally Thoracoscopic Cardiac Surgery.

Cannulation through the femoral artery is the preferred method of establishing peripheral cardiopulmonary bypass in minimally invasive totally thoracoscopic cardiac surgery. However, faced with the contraindication of femoral artery cannulation, modified ascending aortic cannulation is an alternative approach to minimally invasive totally thoracoscopic cardiac surgery.

Global identification of phospho-dependent SCF substrates reveals a FBXO22 phosphodegron and an ERK-FBXO22-BAG3 axis in tumorigenesis.

SKP1-CUL1-F-box (SCF) ubiquitin ligases play fundamental roles in cellular functions. Typically, substrate phosphorylation is required for SCF recognition and subsequent degradation. However, phospho-dependent substrates remain largely unidentified. Here, using quantitative phoshoproteome approach, we performed a system-wide investigation of phospho-dependent SCF substrates. This strategy identified diverse phospho-dependent candidates. Biochemical verification revealed a mechanism by which SCFFBXO22 recognizes the motif XXPpSPXPXX as a conserved phosphodegron to target substrates for destruction. We further demonstrated BAG3, a HSP70 co-chaperone, is a bona fide substrate of SCFFBXO22. FBXO22 mediates BAG3 ubiquitination and degradation that requires ERK-dependent BAG3 phosphorylation at S377. FBXO22 depletion or expression of a stable BAG3 S377A mutant promotes tumor growth via defects in apoptosis and cell cycle progression in vitro and in vivo. In conclusion, our study identified broad phosphorylation-dependent SCF substrates and demonstrated a phosphodegron recognized by FBXO22 and a novel ERK-FBXO22-BAG3 axis involved in tumorigenesis.

miR-198 inhibits the progression of renal cell carcinoma by targeting BIRC5.

BACKGROUND: miR-198 is involved in the formation, migration, invasion, and metastasis of various malignant cancers. However, the function and mechanism of action of miR-198 in the tumorigenesis of renal cell carcinoma (RCC) remain elusive. Here, we aimed to explore the role of miR198 in RCC. METHODS: Immunohistochemistry was performed to estimate the level of survivin in RCC sections. Quantitative real-time polymerase chain reaction was performed to determine the expression level of miR-198 in fresh RCC tissues. Furthermore, the target relationship between miR-198 and BIRC5 was predicted using the TargetScanHuman 7.2 database and verified via dual-luciferase reporter assay and western blotting. The effects of miR-198 on the viability, apoptosis, invasion, and migration of A498 and ACHN cells were studied using Cell Counting Kit-8, flow cytometry, transwell migration assay, and wound healing assay, respectively. Additionally, a xenograft nude mouse model was established to evaluate the effect of miR-198 on RCC tumorigenesis. RESULTS: The expression levels of BIRC5 and miR-198 were respectively higher and lower in RCC tissues than those in normal adjacent tissues. Furthermore, miR-198 could inhibit luciferase activity and reduce the protein level of survivin without affecting the BIRC5 mRNA levels. miR-198 inhibited cell viability, migration, and invasion and promoted cell apoptosis; co-transfection with BIRC5 could rescue these effects. Moreover, miR-198 could repress tumor growth in the xenograft nude mouse model of RCC. CONCLUSIONS: Our study demonstrates that miR-198 suppresses RCC progression by targeting BIRC5.

The risk factors of the early postoperative residual tricuspid regurgitation after concomitant thoracoscopic tricuspid valve repair.

OBJECTIVE: Secondary tricuspid regurgitation will be aggravated if left uncorrected during the initial surgery. Recently, an aggressive strategy of routine concomitant tricuspid valve repair has been warranted. Follow this strategy, routine concomitant thoracoscopic tricuspid valve repair was performed and the surgical effect and postoperative residual TR were reviewed. METHODS: A two-center, retrospective, observational study was conducted. Patients who underwent concomitant thoracoscopic tricuspid valve repair performed by the same surgeon between May 2012 to April 2020 were recruited into the study. The data were collected from the hospital database and outpatient records from the most recent follow-up to analysis. RESULTS: There were 504 patients recruited in this study. No death occurred and all patients were discharged. The average follow-up time was 7.4 ± 7.5 months. After the surgery, the dimension of right ventricle and pulmonary artery systolic pressure were reduced significantly. There were 11 cases (2.2%) of postoperative residual tricuspid regurgitation. Multiple logistic regression analysis revealed left atrial dimension (p = .002) and tricuspid regurgitation (p = .002) positively associated with the residual tricuspid regurgitation occurrence rate significantly. Kaplan-Meier analysis indicated the more severe the tricuspid regurgitation, the higher the residual tricuspid regurgitation occurrence rate (p < .05). CONCLUSIONS: The tricuspid valve repair surgery may improve the patients' prognosis effectively if it was performed at the appropriate timing. The larger the left atrial dimension is, or the more severe the tricuspid regurgitation is, the higher the residual tricuspid regurgitation occurrence rate after concomitant thoracoscopic tricuspid valve repair. Our experience has shown that concomitant thoracoscopic tricuspid valve repair is reliable, effective, and safe, which may be beneficial to right heart remodeling in the short to midterm.

One-stage minimally surgical treatment for myocardial bridge with other cardiac malformations.

The myocardial bridge (MB) refers to the epicardial coronary artery being abnormally covered by the superficial myocardium and running within the cardiac muscle. Surgery should be considered as an optimal choice for severe symptomatic MBs or simultaneous surgery for other heart diseases with severe MBs. Median sternotomy is the most commonly used surgical method. In this case, the authors successfully treated a patient diagnosed with symptomatic MB combined with other cardiac malformations by one-stage minimally invasive surgery guided by three-dimensional reconstruction of cardiac computed tomography.

Transcriptome-wide study of the response of human trabecular meshwork cells to the substrate stiffness increase.

Elevated intraocular pressure, a major risk factor of glaucoma, is caused by the abnormal function of trabecular outflow pathways. Human trabecular meshwork (HTM) tissue plays an important role in the outflow pathways. However, the molecular mechanisms that how TM cells respond to the elevated IOP are largely unknown. We cultured primary HTM cells on polyacrylamide gels with tunable stiffness corresponding to Young's moduli ranging from 1.1 to 50 kPa. Then next-generation RNA sequencing (RNA-seq) was performed to obtain the transcriptomic profiles of HTM cells. Bioinformatics analysis revealed that genes related to glaucoma including DCN, SPARC, and CTGF, were significantly increased with elevated substrate stiffness, as well as the global alteration of HTM transcriptome. Extracellular matrix (ECM)-related genes were selectively activated in response to the elevated substrate stiffness, consistent with the known molecular alteration in glaucoma. Human normal and glaucomatous TM tissues were also obtained to perform RNA-seq experiments and supported the substrate stiffness-altered transcriptome profiles from the in vitro cell model. The current study profiled the transcriptomic changes in human TM cells upon increasing substrate stiffness. Global change of ECM-related genes indicates that the in vitro substrate stiffness could greatly affect the biological processes of HTM cells. The in vitro HTM cell model could efficiently capture the main pathogenetic process in glaucoma patients, and provide a powerful method to investigate the underlying molecular mechanisms.

Combined one-stage minimally invasive surgery for primary pulmonary carcinoma and mitral regurgitation.

BACKGROUND: We report the first successful short-term outcome of one-stage minimally invasive surgery (MIS) mitral valve repair and video-assisted thoracoscopic surgery (VATS) lobectomy. CASE PRESENTATION: We report the first successful short-term outcome of combined one-stage video-assisted thoracoscopic surgery lobectomy and minimally invasive surgery in a patient with operable primary right upper lobe adenocarcinoma and mitral regurgitation. Post- operative recovery was uneventful, and follow-up at 6 weeks confirmed an excellent surgical and oncologic outcome. CONCLUSIONS: We think one-stage minimally invasive surgery (MIS) cardiac surgery and video-assisted thoracoscopic surgery (VATS) lobectomy would benefit patients with satisfactory cardiac and pulmonary function.

Sustained ER stress promotes hyperglycemia by increasing glucagon action through the deubiquitinating enzyme USP14.

Endoplasmic reticulum (ER) stress plays an important role in metabolic diseases like obesity and type 2 diabetes mellitus (T2DM), although the underlying mechanisms and regulatory pathways remain to be elucidated. Here, we induced chronic low-grade ER stress in lean mice to levels similar to those in high-fat diet (HFD)-fed obese mice and found that it promoted hyperglycemia due to enhanced hepatic gluconeogenesis. Mechanistically, sustained ER stress up-regulated the deubiquitinating enzyme ubiquitin-specific peptidase 14 (USP14), which increased the stability and levels of 3',5'-cyclic monophosphate-responsive element binding (CREB) protein (CBP) to enhance glucagon action and hepatic gluconeogenesis. Exogenous overexpression of USP14 in the liver significantly increased hepatic glucose output. Consistent with this, liver-specific knockdown of USP14 abrogated the effects of ER stress on glucose metabolism, and also improved hyperglycemia and glucose intolerance in obese mice. In conclusion, our findings show a mechanism underlying ER stress-induced disruption of glucose homeostasis, and present USP14 as a potential therapeutic target against T2DM.

Transcriptome analysis reveals downregulation of virulence-associated genes expression in a low virulence Verticillium dahliae strain.

Verticillium dahliae causes wilt diseases and early senescence in numerous plants, including agricultural crops such as cotton. In this study, we studied two closely related V. dahliae strains, and found that V991w showed significantly reduced virulence on cotton than V991b. Comprehensive transcriptome analysis revealed various differentially expressed genes between the two strains, with more genes repressed in V991w. The downregulated genes in V991w were involved in production of hydrophobins, melanin, predicted aflatoxin, and membrane proteins, most of which are related to pathogenesis and multidrug resistance. Consistently, melanin production in V991w in vitro was compromised. We next obtained genomic variations between the two strains, demonstrating that transcription factor genes containing fungi specific transcription factor domain and fungal Zn2-Cys6 binuclear cluster domain were enriched in V991w, which might be related to pathogenicity-related genes downregulation. Thus, this study supports a model in which some virulence factors involved in V. dahliae pathogenicity were pre-expressed during in vitro growth before host interaction.

Applications of 3D printing technology in the treatment of mitral valve disease / 中国胸心血管外科临床杂志

@#Mitral valve disease is the most common cardiac valve disease. The main treatment of mitral valve disease is surgery or interventional therapy. However, as the anatomy of mitral valve is complicated, the operation is particularly difficult. As a result, it requires sophisticated experiences for surgeons. Three-dimensional (3D) printing technology can transform two-dimensional medical images into 3D solid models. So it can provide clear spatial anatomical information and offer safe and personalized treatment for the patients by simulating surgery process. This article reviews the applications of 3D printing technology in the treatment of mitral valve disease.

MicroRNA-transcription factor network analysis reveals miRNAs cooperatively suppress RORA in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) represents over 90% of oral cancer incidence, while its mechanisms of tumorigenesis remain poorly characterized. In this study, we applied RNA-seq and microRNA-seq methodologies in four pairs of cancer and adjacent normal tissues to profile the contribution of miRNAs to tumorigenesis-altered functional pathways by constructing a comprehensive miRNA-mediated mRNA regulatory network. There were 213 differentially expressed (DE) miRNAs and 2172 DE mRNAs with the involvement of negative miRNA-mRNA interactions identified by at least two pairs of cancerous tissues. GO analysis revealed that the upregulated microRNAs significantly contributed to a global down-regulation of a number of transcription factors (TFs) in OSCC. Among the negative regulatory networks between the selected miRNAs (133) and TFs (167), circadian rhythm genes (RORA, RORB, RORC, and CLOCK) simultaneously regulated by multiple microRNAs were of particular interest. For instance, RORA transcript was predicted to be targeted by 25 co-upregulated miRNAs, of which, miR-503-5p, miR-450b-5p, miR-27a-3p, miR-181a-5p and miR-183-5p were further validated to directly target RORA, resulting in a stronger effect on RORA suppression together. In addition, we showed that the mRNA and protein expression levels of RORα were significantly decreased in most OSCC samples, associated with advanced clinical stage and poor prognosis. RORα significantly suppressed the proliferation of OSCC cells in vitro and in vivo. Attenuated RORα decreased p53 protein expression and suppressed p53 phosphorylation activity. Altogether, our results strongly suggest the importance of the role of miRNAs in regulating the activity of circadian rhythm-related TFs network during OSCC tumorigenesis, and provide further clues to understand the clinical link between circadian rhythm and cancer therapy.

SRY-Box Containing Gene 4 Promotes Liver Steatosis by Upregulation of SREBP-1c.

Obesity is usually associated with an increased risk of nonalcoholic fatty liver disease that is characterized by accumulation of excessive triglyceride (TG) in hepatocytes. However, the factors involved in the obesity-induced hepatosteatosis are poorly defined. Here, we report that SRY-box containing gene 4 (Sox4), a transcription factor that regulates cell proliferation and differentiation, plays an important role in hepatic TG metabolism. Sox4 expression levels are markedly upregulated in livers of obese rodents and humans. Adenovirus-medicated overexpression of Sox4 in the livers of lean mice promotes liver steatosis, whereas liver-specific knockdown of Sox4 ameliorates TG accumulation and improves insulin resistance in obese mice. At the molecular level, we show that Sox4 could directly control the transcription of SREBP-1c gene through binding to its proximal promoter region. Thus, we have identified Sox4 as an important component of hepatic TG metabolism.