Generation of a DMD loss-of-function mutant human embryonic stem cell lines by CRISPR base editing.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder, which is caused mostly by frame-disrupting, out-of-frame variation in the dystrophin (DMD) gene. Loss-of- function mutations are the most common type of mutation in DMD, accounting for approximately 60-90% of all DMD variations. In this study, we used adenine base editing to generate a human embryonic stem cell line with splice-site mutations to mimic exon deletion variants in clinical Duchenne muscular dystrophy patients. This cell line has differentiation potential and a normal karyotypic.

Proteomic and metabolomic analysis of the serum of patients with tick-borne encephalitis.

Tick-borne encephalitis virus (TBEV) is a common virus in Europe and Asia, causing around 10,000 to 10,500 infections annually. It affects the central nervous system and poses threats to public health. However, the exact molecular mechanisms of TBE pathogenesis are not yet fully understood due to the complex interactions between the virus and its host. In this study, a comprehensive analysis was conducted to characterize the serum metabolome and proteome of adult patients infected with TBEV, in comparison to a control group of healthy individuals. Liquid chromatography tandem mass spectrometry (LC-MS) was employed to monitor metabolic and proteomic alternations throughout the progression of the disease, significant physiological changes associated with different stages of the disease were identified. A total of 44 proteins and 115 metabolites exhibited significantly alternations in the sera of patients diagnosed with TBE. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of these metabolites and proteins revealed differential enrichment of genes associated with the extracellular matrix, complement binding, hemostasis, lipid metabolism, and amino acid metabolism between TBE patients and healthy controls. We gained valuable understanding of the specific metabolites implicated in the host's responses to TBE, establishing a basis for further research on TBE disease. SIGNIFICANCE: The current investigation revealed a comprehensive and systematic differences on TBE using LC-MS platform from human serum samples of TBE patients and healthy individuals providing the immune response to the invasion of TBE.

Deep learning-based radiomic nomogram to predict risk categorization of thymic epithelial tumors: A multicenter study.

PURPOSE: The study was aimed to develop and evaluate a deep learning-based radiomics to predict the histological risk categorization of thymic epithelial tumors (TETs), which can be highly informative for patient treatment planning and prognostic assessment. METHOD: A total of 681 patients with TETs from three independent hospitals were included and separated into derivation cohort and external test cohort. Handcrafted and deep learning features were extracted from preoperative contrast-enhanced CT images and selected to build three radiomics signatures (radiomics signature [Rad_Sig], deep learning signature [DL_Sig] and deep learning radiomics signature [DLR_Sig]) to predict risk categorization of TETs. A deep learning-based radiomic nomogram (DLRN) was then depicted to visualize the classification evaluation. The performance of predictive models was compared using the receiver operating characteristic and decision curve analysis (DCA). RESULTS: Among three radiomics signatures, DLR_Sig demonstrated optimum performance with an AUC of 0.883 for the derivation cohort and 0.749 for the external test cohort. Combining DLR_Sig with age and gender, DLRN was depict and exhibited optimum performance among all radiomics models with an AUC of 0.965, accuracy of 0.911, sensitivity of 0.921 and specificity of 0.902 in the derivation cohort, and an AUC of 0.786, accuracy of 0.774, sensitivity of 0.778 and specificity of 0.771 in the external test cohort. The DCA showed that DLRN had greater clinical benefit than other radiomics signatures. CONCLUSIONS: Our study developed and validated a DLRN to accurately predict the risk categorization of TETs, which has potential to facilitate individualized treatment and improve patient prognosis evaluation.

Bivalirudin plus a high-dose infusion versus heparin monotherapy in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention: a randomised trial.

BACKGROUND: Previous randomised trials of bivalirudin versus heparin in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI) have reported conflicting results, in part because of treatment with different pharmacological regimens. We designed a large-scale trial examining bivalirudin with a post-PCI high-dose infusion compared with heparin alone, the regimens that previous studies have shown to have the best balance of safety and efficacy. METHODS: BRIGHT-4 was an investigator-initiated, open-label, randomised controlled trial conducted at 87 clinical centres in 63 cities in China. Patients with STEMI undergoing primary PCI with radial artery access within 48 h of symptom onset who had not received previous fibrinolytic therapy, anticoagulants, or glycoprotein IIb/IIIa inhibitors were randomly assigned (1:1) to receive bivalirudin with a post-PCI high-dose infusion for 2-4 h or unfractionated heparin monotherapy. There was no masking. Glycoprotein IIb/IIIa inhibitor use was reserved for procedural thrombotic complications in both groups. The primary endpoint was a composite of all-cause mortality or Bleeding Academic Research Consortium (BARC) types 3-5 bleeding at 30 days. This trial is registered with ClinicalTrials.gov (NCT03822975), and is ongoing. FINDINGS: Between Feb 14, 2019, and April 7, 2022, a total of 6016 patients with STEMI undergoing primary PCI were randomly assigned to receive either bivalirudin plus a high-dose infusion after PCI (n=3009) or unfractionated heparin monotherapy (n=3007). Radial artery access was used in 5593 (93·1%) of 6008 patients. Compared with heparin monotherapy, bivalirudin reduced the 30-day rate of the primary endpoint (132 events [4·39%] in the heparin group vs 92 events [3·06%] in the bivalirudin group; difference, 1·33%, 95% CI 0·38-2·29%; hazard ratio [HR] 0·69, 95% CI 0·53-0·91; p=0·0070). All-cause mortality within 30 days occurred in 118 (3·92%) heparin-assigned patients and in 89 (2·96%) bivalirudin-assigned patients (HR 0·75; 95% CI 0·57-0·99; p=0·0420), and BARC types 3-5 bleeding occurred in 24 (0·80%) heparin-assigned patients and five (0·17%) bivalirudin-assigned patients (HR 0·21; 95% CI 0·08-0·54; p=0·0014). There were no significant differences in the 30-day rates of reinfarction, stroke, or ischaemia-driven target vessel revascularisation between the groups. Within 30 days, stent thrombosis occurred in 11 (0·37%) of bivalirudin-assigned patients and 33 (1·10%) of heparin-assigned patients (p=0·0015). INTERPRETATION: In patients with STEMI undergoing primary PCI predominantly with radial artery access, anticoagulation with bivalirudin plus a post-PCI high-dose infusion for 2-4 h significantly reduced the 30-day composite rate of all-cause mortality or BARC types 3-5 major bleeding compared with heparin monotherapy. FUNDING: Chinese Society of Cardiology Foundation (CSCF2019A01), and a research grant from Jiangsu Hengrui Pharmaceuticals.

Comprehensive safety profile evaluation of bivalirudin in Chinese ST-segment elevation myocardial infarction patients receiving percutaneous coronary intervention: a prospective, multicenter, intensive monitoring study.

BACKGROUND: This prospective, multi-center, intensive monitoring study aimed to systematically assess the occurrence of adverse events (AEs) and adverse drug reactions (ADRs), especially thrombocytopenia and bleeding, as well as their risk factors in Chinese ST-segment elevation myocardial infraction (STEMI) patients receiving bivalirudin as anticoagulant for percutaneous coronary intervention (PCI). METHODS: In total, 1244 STEMI patients undergoing PCI and receiving bivalirudin as anticoagulant were enrolled in the present study. Safety data were collected from hospital admission to 72 h after bivalirudin administration; in addition, patients were further followed up at the 30th day with safety data collected at that time. RESULTS: AEs, severe AEs, ADRs and severe ADRs were reported in 224 (18.0%), 15 (1.2%), 49 (3.9%) and 5 (0.4%) patients, respectively. Importantly, 4 (0.3%) patients were submitted to hospitalization and 6 (0.5%) patients died due to AEs, while 1 (0.1%) patient was submitted to hospitalization but no (0.0%) patient died due to ADRs. Meanwhile, thrombocytopenia and bleeding occurred in 24 (1.9%) and 21 (1.7%) patients, respectively. Further multivariate logistic analysis identified several important independent factors related to AEs, ADRs, thrombocytopenia or bleeding, which included history of cardiac surgery and renal function impairment, high CRUSADE risk stratification, elective operation and combination with glycoprotein IIb/IIIa inhibitors. Moreover, 4 multivariate models were constructed based on the above-mentioned factors, which all showed acceptable predictive value for AEs, ADRs, thrombocytopenia and bleeding, respectively. CONCLUSION: Bivalirudin is a well-tolerant anticoagulant in Chinese STEMI patients undergoing PCI procedure.

MicroRNA-146b-5p/EPHA7 axis regulates cell invasion, metastasis, proliferation, and temozolomide-induced chemoresistance via regulation of IRAK4/TRAF6/NF-κB signaling pathway in aggressive pituitary adenoma.

BACKGROUND: Aggressive pituitary adenoma (APA) is a huge challenge for neurosurgeons. Temozolomide (TMZ) is conventionally used in chemotherapy against APA, but acquired resistance developed during long-term therapy limits its benefits. MiRNA-146b-5p has been confirmed to inhibit tumor metastasis. This study aimed to explore the underlying biological functions of miRNA-146b-5p in APA. METHODS: Sixty confirmed APA tissues and corresponding adjacent normal tissues were collected. We established a TMZ-resistant cell line (GH3/TMZ) by exposing GH3 cells to gradually increasing doses of TMZ for 5 months. Cell Counting Kit-8 assay, flow cytometric analysis, RNA pull-down assay, 5-ethynyl-20-deoxyuridine assay, dual-luciferase reporter gene assay, wound healing assay, and invasion assay were used to explore the malignant biological characteristics of cells. Immunohistochemistry (IHC), western blotting analysis, and real-time quantitative PCR (qRT-PCR) were used to analyze the expression level of related proteins and nucleic acids. RESULTS: The expression of miRNA-146b-5p was down-regulated not only in APA tissues but also in PA cell lines compared with the matched adjacent non-tumor tissues or normal human astrocyte (NHA) cells. Low expression of miRNA-146b-5p was notably associated with poorer disease-free survival rate (P=0.032), overall survival rate (P=0.039), larger tumor size (P=0.028), poorer Knosp grade (P=0.020), and poorer Hardy grade (P=0.006) in APA patients. MiRNA-146b-5p negatively regulated cell proliferation, invasion, migration, and induced apoptosis in GH3 cells. Overexpression of miRNA-146b-5p suppressed IRAK4 and TRAF6 protein expression and negatively regulated NF-κB phosphorylation. The restoration of EPHA7 expression in GH3 cells notably reversed the inhibitory effects of miRNA-146b-5p. MiRNA-146b-5p expression was significantly down-regulated and EPHA7 gene expression was significantly up-regulated in GH3/TMZ cells, compared to the parental cell line. Similarly, EPHA7 was up-regulated, while the miRNA-146b-5p level was down-regulated in chemoresistance tissues more than in chemosensitive tissues. The autophagic activity was decreased markedly with increasing miRNA-146b-5p expression, while it was enhanced after Lv-EPHA7 treatment in GH3/TMZ cells. CONCLUSIONS: MiRNA-146b-5p can inhibit EPHA7 expression, suppress the IRAK4/TRAF6/NF-κB signaling pathway, and weaken PA cell invasion, metastasis, proliferation, and TMZ-induced chemoresistance in vitro.

Different Effects of Hematoma Expansion on Short-Term Functional Outcome in Basal Ganglia and Thalamic Hemorrhages.

PURPOSE: To investigate the impact of hematoma expansion (HE) on short-term functional outcome of patients with thalamic and basal ganglia intracerebral hemorrhage. METHODS: Data of 420 patients with deep intracerebral hemorrhage (ICH) that received a baseline CT scan within 6 hours from symptom onset and a follow-up CT scan within 72 hours were retrospectively analyzed. The poor functional outcome was defined as modified Rankin score (mRS) > 3 at 30 days. Receiver operating characteristic (ROC) curves for relative and absolute growth of HE were generated and compared. Multivariable logistic regression models were used to analyze the impact of HE on the functional outcome in basal ganglia and thalamic hemorrhages. The predictive values for different thresholds of HE were calculated, and correlation coefficient matrices were used to explore the correlation between the covariables. RESULTS: Basal ganglia ICH showed a higher possibility of absolute hematoma growth than thalamic ICH. The area under the curve (AUC) for absolute and relative growth of thalamic hemorrhage was lower than that of basal ganglia hemorrhage (AUC 0.71 and 0.67, respectively) in discriminating short-term poor outcome with an AUC of 0.59 and 0.60, respectively. Each threshold of HE independently predicted poor outcome in basal ganglia ICH (P < 0.001), with HE > 3 ml and > 6 ml showing higher positive predictive values and accuracy compared to HE > 33%. In contrast, thalamic ICH had a smaller baseline volume (BV, 9.55 ± 6.85 ml) and was more likely to initially involve the posterior limb of internal capsule (PLIC) (85/153, 57.82%), and the risk of HE was lower without PLIC involvement (4.76%, P = 0.009). Therefore, in multivariate analysis, the effect of thalamic HE on poor prognosis was largely replaced by BV and the involvement of PLIC, and the adjusted odds ratios (ORs) of HE was not significant (P > 0.05). CONCLUSION: Though HE is a high-risk factor for short-term poor functional outcome, it is not an independent risk factor in thalamic ICH, and absolute growth is more predictive of poor outcome than relative growth for basal ganglia ICH.

Serum Metabolomics of Tick-Borne Encephalitis Based on Orbitrap-Mass Spectrometry.

BACKGROUND: Tick-borne encephalitis virus (TBEV), the most prevalent arbovirus, causes potentially fatal encephalitis in humans. Prevalent in northeast China, tick-borne encephalitis (TBE) poses a major threat to public health, local economies and tourism. There are no biomarkers for TBE, which is classified serologically and clinically. Due to sample heterogeneity of samples and different detection platforms, obtaining stable markers is a great challenge for metabolomics. Accurate annotation is vital for data mining and interpretation. OBJECTIVE: To identify reliable biomarkers of TBEV infection. METHODS: An untargeted metabolomics analysis of serum from 30 TBE patients and 30 healthy controls was carried out. Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics methods were used to characterize the subjects' serum metabolic profiles and to screen and validate TBE biomarkers. RESULTS: A total of 3370 molecular features were extracted from each sample, and the peak intensity of each feature was obtained. Pattern analysis, principal component analysis, partial least squares discriminant analysis were used to screen for potential metabolites. Bilirubin, LysoPC (18:1[9Z]), palmitic acid, and CL (8:0/8:0/8:0/8:0) were significantly different. Pathway enrichment analysis showed that these metabolites were in the fatty acid biosynthesis and glycerophospholipid metabolism pathways. The phospholipid family had a significant difference in both the difference ratio and the abundance. CONCLUSION: Phospholipids may be used to distinguish TBEV patients from healthy controls. TBEV infection affects the normal metabolic activity of host cells, providing insight into the pathogenesis of TBE.

Insights into the molecular basis of tick-borne encephalitis from multiplatform metabolomics.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is the most prevalent arbovirus, with a tentative estimate of 10,000 to 10,500 infections occurring in Europe and Asia every year. Endemic in Northeast China, tick-borne encephalitis (TBE) is emerging as a major threat to public health, local economies and tourism. The complicated array of host physiological changes has hampered elucidation of the molecular mechanisms underlying the pathogenesis of this disease. METHODOLOGY/PRINCIPLE FINDINGS: System-level characterization of the serum metabolome and lipidome of adult TBEV patients and a healthy control group was performed using liquid chromatography tandem mass spectrometry. By tracking metabolic and lipid changes during disease progression, crucial physiological changes that coincided with disease stages could be identified. Twenty-eight metabolites were significantly altered in the sera of TBE patients in our metabolomic analysis, and 14 lipids were significantly altered in our lipidomics study. Among these metabolites, alpha-linolenic acid, azelaic acid, D-glutamine, glucose-1-phosphate, L-glutamic acid, and mannose-6-phosphate were altered compared to the control group, and PC(38:7), PC(28:3;1), TAG(52:6), etc. were altered based on lipidomics. Major perturbed metabolic pathways included amino acid metabolism, lipid and oxidative stress metabolism (lipoprotein biosynthesis, arachidonic acid biosynthesis, leukotriene biosynthesis and sphingolipid metabolism), phospholipid metabolism and triglyceride metabolism. These metabolites were significantly perturbed during disease progression, implying their latent utility as prognostic markers. CONCLUSIONS/SIGNIFICANCE: TBEV infection causes distinct temporal changes in the serum metabolome and lipidome, and many metabolites are potentially involved in the acute inflammatory response and immune regulation. Our global analysis revealed anti- and pro-inflammatory processes in the host and changes to the entire metabolic profile. Relationships between metabolites and pathologies were established. This study provides important insight into the pathology of TBE, including its pathology, and lays the foundation for further research into putative markers of TBE disease.

Serum netrin-1 concentrations are associated with clinical outcome in acute intracerebral hemorrhage.

BACKGROUND: Netrin-1 is an axon guidance protein, which can inhibit inflammatory reaction and stabilize the blood-brain barrier to protect against experimental brain injury. We evaluated the concentration of netrin-1 in acute intracerebral hemorrhage (ICH) patients and explored whether netrin-1 is a potential prognostic biomarker for ICH. METHODS: This study recruited a total of 126 ICH patients and 126 healthy controls. Netrin-1 concentration was determined using a commercially available human enzyme-linked immune sorbent assay kit. National Institutes of Health Stroke Scale (NIHSS) score and hematoma volume were used to assess hemorrhagic severity. An unfavorable outcome was defined as modified Rankin Scale >2 at 90 days. RESULTS: ICH patients showed significantly lower serum netrin-1 concentrations than controls. Serum netrin-1 concentrations were strongly and inversely correlated with serum C-reactive protein concentrations, NIHSS score and hematoma volume. Multivariate analyses revealed that low netrin-1 concentration was associated with 90-day death, unfavorable outcome and overall survival after adjustment for other confounding variables. Under the receiver operating characteristic curve, serum netrin-1 remarkably discriminated patients at risk of 90-day death and unfavorable outcome. CONCLUSIONS: Serum netrin-1 concentrations are decreased in patients with ICH, and the concentrations of netrin-1 were intimately associated with inflammation, hemorrhagic severity and clinical outcome of ICH.

[Resveratrol reduces apoptosis of mouse RAW264.7 macrophage via down-regulating HIF-1α].

Objective To investigate the protective effect of resveratrol (Res) against mouse RAW264.7 macrophage injury induced by cobalt chloride (CoCl2) and its mechanism. Methods Macrophages were divided into control group, CoCl2 group and Res pretreatment group. CoCl2 group was treated with 500 µmol/L CoCl2 for 8 hours, and Res pretreatment group was pretreated with 40 µmol/L Res for 2 hours followed by 500 µmol/L CoCl2 treatment for 8 hours. The cell vitality and apoptotic rate in every group were detected by CCK-8 assay and flow cytometry. The distributions of caspase-3 and hypoxia-inducible factor 1 alpha (HIF-1α), as well as the influences of CoCl2 and Res on their expression were detected by immunofluorescence cytochemistry. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in every group were measured by the corresponding kits. The expression levels of Bcl2, BAX, c-caspase-3 and HIF-1α in every group were observed by Western blot analysis. Results Compared with the CoCl2 group, pretreatment with Res increased cell vitality, decreased apoptosis, enhanced the activity of SOD, and reduced the level of MDA. The caspase-3 was mainly distributed in the cytoplasm, and HIF-1α was mainly distributed on the nucleus. Compared with the CoCl2 group, Res up-regulated the expression of Bcl2 and down-regulated the expression of BAX, cleaved caspase-3 and HIF-1α. Conclusion Res can decrease apoptosis in macrophages, which may occur via reducing the accumulation of intracellular reactive oxygen species, enhancing cell antioxidant capacity, and down-regulating the expression of HIF-1α on cells.

Vitamin K2 inhibits rat vascular smooth muscle cell calcification by restoring the Gas6/Axl/Akt anti-apoptotic pathway.

Vascular calcification is associated with cardiovascular disease as a complication of hypertension, hyperlipidemia, diabetes mellitus, and chronic kidney disease. Vitamin K2 (VK2) delays vascular calcification by an unclear mechanism. Moreover, apoptosis modulates vascular smooth muscle cell (VSMC) calcification. This paper aimed to study VK2-modified VSMC calcification and survival cell signaling mediated by growth arrest-specific gene 6 (Gas6) and its tyrosine kinase receptor Axl. Primary-cultured VSMCs were dose-dependently treated with VK2 in the presence of calcification medium for 8 days, or pre-treated for 1 h with/without the Axl inhibitor R428 (2 µmol/L) or the caspase inhibitor Z-VAD-fmk (20 µmol/L) followed by treatment with VK2 (10 µmol/L) or rmGas6 (200 nmol/L) in calcification medium for 8 days. Calcium deposition was determined by the o-cresolphthalein complexone assay and Alizarin Red S staining. Apoptosis was determined by TUNEL and flow cytometry using Annexin V-FITC and propidium iodide staining. Western blotting detected the expressions of Axl, Gas6, p-Akt, Akt, and Bcl2. VK2 significantly inhibited CaCl2- and ß-sodium glycerophosphate (ß-GP)-induced VSMC calcification and apoptosis, which was dependent on restored Gas6 expression and activated downstream signaling by Axl, p-Akt, and Bcl2. Z-VAD-fmk significantly inhibited CaCl2- and ß-GP-induced VSMC calcification and apoptosis. Augmented recombinant mouse Gas6 protein (rmGas6) expression significantly reduced VSMC calcification and apoptosis. Furthermore, the Gas6/Axl interaction was inhibited by R428, which abolished the preventive effect of VK2 on CaCl2- and ß-GP-induced apoptosis and calcification. These results suggest that Gas6 is critical in VK2-mediated functions that attenuate CaCl2- and ß-GP-induced VSMC calcification by blocking apoptosis.

[Clinical study of pro 16-slice spiral CT perfusion imaging for high myopia].

PURPOSE: To study the hemodynamics of the eye-ring's microcirculation in patients with high myopia through 16-slice spiral CT perfusion image. METHODS: Twenty-eight patients (53 eyes) with high myopia and 32 cases with emmetropia (64 eyes) in control group were examined by GE lightspeed pro 16-slice spiral CT. The perfusion image and the blood volume (BV) of the posterior equatorial eye-ring were obtained and analyzed by SPSS 10.0 software. RESULTS: The BV of high myopia (4.61 +/- 1.48)ml/ 100 g is significantly less than that of the control group (7.72 +/- 1.92)ml/ 100 g (P < 0.01). It implies that the quantity of the small vessel and capillary in the eye-ring of the patient with high myopia is less than that of the control group. The diopter of high myopia has a significantly positive correlation with the BV of the posterior equatorial eye-ring (r = 0.793, P < 0.01). CONCLUSION: The perfusion image of 16-slice spiral CT is a quantitive method to evaluate the hemodynamics of the high myopia eye-ring's microcirculation.