The CTSC-RAB38 Fusion Transcript Is Associated With the Risk of Hemorrhage in Brain Arteriovenous Malformations.

Brain arteriovenous malformations (bAVMs) are congenital anomalies of blood vessels that cause intracranial hemorrhage in children and young adults. Chromosomal rearrangements and fusion genes play an important role in tumor pathogenesis, though the role of fusion genes in bAVM pathophysiological processes is unclear. The aim of this study was to identify fusion transcripts in bAVMs and analyze their effects. To identify fusion transcripts associated with bAVM, RNA sequencing was performed on 73 samples, including 66 bAVM and 7 normal cerebrovascular samples, followed by STAR-Fusion analysis. Reverse transcription polymerase chain reaction and Sanger sequencing were applied to verify fusion transcripts. Functional pathway analysis was performed to identify potential effects of different fusion types. A total of 21 fusion transcripts were detected. Cathepsin C (CTSC)-Ras-Related Protein Rab-38 (RAB38) was the most common fusion and was detected in 10 of 66 (15%) bAVM samples. In CTSC-RAB38 fusion-positive samples, CTSC and RAB38 expression was significantly increased and activated immune/inflammatory signaling. Clinically, CTSC-RAB38 fusion bAVM cases had a higher hemorrhage rate than non-CTSC-RAB38 bAVM cases (p < 0.05). Our study identified recurrent CTSC-RAB38 fusion transcripts in bAVMs, which may be associated with bAVM hemorrhage by promoting immune/inflammatory signaling.

Transthyretin Upregulates Long Non-Coding RNA MEG3 by Affecting PABPC1 in Diabetic Retinopathy.

The aim of the study was to demonstrate how transthyretin (TTR) could affect long non-coding RNA (lncRNA) of maternally expressed gene 3 (MEG3) and play important roles in diabetic retinopathy (DR). A DR model in C57BL/6 mice was established after intraperitoneal injection of streptozotocin (STZ). After intravitreal injection with TTR pAAV vector, MEG3 short hairpin RNA (shRNA), scrambled shRNA, or MEG3, retinal imaging, retinal trypsin digestion, and fundus vascular permeability tests were performed. Cell counting kit-8 (CCK8), transwell, and Matrigel assays were employed to detect the proliferation and migration of human retinal microvascular endothelial cells (hRECs). The binding between long non-coding RNA of maternally expressed gene 3 (lncRNA-MEG3) and microRNA-223-3p (miR-223-3p) was observed by using luciferase reporter assays, while co-immunoprecipitation (co-IP) was employed to confirm the interaction between TTR and the target. In the DR mice model, retinal vascular leakage and angiogenesis were repressed by overexpressing TTR. In vitro, the added TTR promoted the level of lncRNA-MEG3 by interacting with poly (A) binding protein cytoplasmic 1 (PABPC1), and then repressed proliferation and angiogenesis of hRECs. In vivo, silencing or overexpressing lncRNA-MEG3 significantly affected retinal vascular phenotypes. Additionally, the interaction between lncRNA-MEG3 and miR-223-3p was confirmed, and silencing of miR-223-3p revealed similar effects on hRECs as overexpression of lncRNA-MEG3. In summary, in the DR environment, TTR might affect the lncRNA MEG3/miR-223-3p axis by the direct binding with PABPC1, and finally repress retinal vessel proliferation.

Transcriptome analysis identified a novel 3-LncRNA regulatory network of transthyretin attenuating glucose induced hRECs dysfunction in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is the leading cause of blindness in the working age population. Transthyretin (TTR) showed a significantly decreased concentration in DR patients and exerted a visual protective effect by repressing neovascularization. This work intended to identify long non coding RNAs (lncRNAs) and explore their potential mechanism underlying the protective role of TTR. METHODS: Transcriptome of human retinal endothelial cells (hRECs) treated with low glucose (LG), high glucose (HG) or high glucose with 4 µM TTR (HG + TTR) was conducted. Differentially expressed lncRNAs, mRNAs and TTR related lncRNAs and mRNA were acquired. Functional annotation and Gene Set Enrichment Analysis were applied to analyse TTR affected pathways and processes. Weighted gene co-expression network analysis (WGCNA) was implemented to obtain hub modules and genes. LncRNA-mRNA regulatory networks were constructed based on cis, trans and competing endogenous RNAs acting mode. QRT-PCR was conducted to validate the expression of lncRNAs in aqueous humor and serum samples from 30 DR patients and 10 normal controls. RESULTS: RNA-sequencing of hRECs treated with low glucose (LG), high glucose (HG) or high glucose with 4 µM TTR (HG + TTR) was conducted. 146,783 protein-coding transcripts, 12,403 known lncRNA transcripts and 1184 novel non-coding transcripts were characterized. A total of 11,407 differentially expressed mRNAs (DE-mRNAs), 679 differentially expressed lncRNAs (DE-lncRNAs) in HG group versus LG group, 6206 DE-mRNAs and 194 DE-lncRNAs in HG + TTR versus HG group were obtained, respectively. 853 TTR-mRNAs and 48 TTR-lncRNAs were acquired, and functionally involved in cell cycle, apoptosis, inflammation signalling pathway, response to oxidative stress, neovascularization and autophagy. The WGCNA analysis identified a hub module of 133 genes, with the core function of oxidative stress response, angiogenesis, MAPK pathway, cell proliferation and apoptosis. After qRT-PCR validation, a 3-lncRNA regulatory network was proposed. At last, lncRNAs MSTRG.15047.3 and AC008403.3 showed significantly relative higher expression levels in both aqueous humor and serum samples, compared with normal controls, and FRMD6-AS2 was significantly down-regulated. CONCLUSIONS: TTR regulated mRNAs and biological processes including oxidative stress, inflammation signalling and autophagy. A 3-lncRNA regulatory network was characterized underlying TTR repressing neovascularization, and showed potential diagnostic performance in DR.

A novel transthyretin/STAT4/miR-223-3p/FBXW7 signaling pathway affects neovascularization in diabetic retinopathy.

MicroRNAs (miRNAs) are small RNAs without protein-coding functions that negatively regulate target genes and play important roles in physiological and pathological processes. The aim of this work was to reveal a novel miRNA/gene pathway in diabetic retinopathy (DR). A microarray was used to screen miRNAs in samples from nondiabetic controls and patients with DR, and miR-223-3p was screened as a potential candidate. Quantitative real-time PCR (qRT-PCR) revealed that the level of miR-223-3p was frequently overexpressed in DR samples and human retinal endothelial cells (hRECs) in hyperglycemia, but it was decreased in hyperglycemia after the addition of transthyretin (TTR). In addition, according to cell proliferation, tube formation, and wound healing assays, the downregulation of miR-223-3p suppressed cell migration and proliferation, whereas miR-223-3p upregulation showed the opposite effects. Furthermore, luciferase assays identified F-box and WD repeat domain-containing 7 (FBXW7) as a target mRNA of miR-223-3p. High glucose conditions facilitated the recruitment of signal transducer and activator of transcription 4 (STAT4) and promoted the transcription of miR-223-3p. In hRECs, in a hyperglycemic environment, TTR inhibited STAT4 expression, downregulated the level of miR-223-3p, and finally promoted FBXW7 expression. This study found a novel mechanism whereby TTR might affect neovascularization through a newly identified STAT4/miR-223-3p/FBXW7 cascade in DR.

KCNQ1OT1 affects the progression of diabetic retinopathy by regulating miR-1470 and epidermal growth factor receptor.

Long noncoding RNAs have been reported to play important roles in the pathogenesis of diabetic retinopathy (DR), which has been considered as the most common disease leading to vision loss. However, it is still unclear whether KCNQ1 overlapping transcript 1 (KCNQ1OT1) could affect DR. In this study, regarding quantitative reverse transcription polymerase chain reaction assay, KCNQ1OT1 level was upregulated while microRNA-1470 (miR-1470) was decreased in DR patients and human retinal endothelial cells. High KCNQ1OT1 expression was correlated with DR stage and low visual function. Using miR-1470 mimic or knockdown of KCNQ1OT1 could lead to the similar phenomenon; phospho-AKT, Bax, B-cell lymphoma 2, and cleaved poly-ADP ribose polymerase (PARP) were regulated, while vascularization was inhibited and apoptosis was promoted. Regarding bioinformatics analysis and in vitro dual luciferase reporter assay, there should be a negative correlation between KCNQ1OT1 and miR-1470. Additionally, mRNA of epidermal growth factor receptor (EGFR) was proved as the target of miR-1470 and EGFR targeting by miR-1470 initiated KCNQ1OT1 deficiency-induced apoptosis and promoted proliferation. KCNQ1OT1 and miR-1470 were proved to be the promoter and repressor of EGFR, respectively. The results suggested that KCNQ1OT1 could sponge miR-1470 and further regulate EGFR in DR.

Preparation and characterization of copper-Brevibacterium cholesterol oxidase hybrid nanoflowers.

Brevibacterium cholesterol oxidase (COD)-Cu hybrid nanoflowers were prepared, optimized and characterized for structural and catalytic properties. Regarding scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FTIR) and X-ray diffraction (XRD) assays, COD molecules were successfully encapsulated with Cu3(PO4)2·3H2O based hybrid nanoflowers. After immobilization in hybrid nanoflowers, the interaction between COD and flavo-cofactor (FAD) was enhanced; and regarding to differential scanning calorimetry (DSC) assay, the Tm value of immobilized COD was increased from 60.5 °C (free enzyme) to 138.49 °C (nanoflowers). Additionally, in activity assay, Cu-COD nanoflowers revealed improved resistance to temperature and pH. After 10 times of recycling, approximately 70% of initial activity of Cu-COD nanoflowers was maintained, while the free COD was inactivated after 3 times of recycling. Furthermore, using cholesterol as substrate, in n-octane/water biphasic reaction system, the stability of Cu-COD nanoflowers was significantly promoted, and the initial conversion ration could be over two times as that of free enzyme. In brief, the hybrid nanoflowers dramatically enhanced the structural and thermo stability, the tolerance to biphasic mixture, and the catalytic efficiency of COD; and Cu-COD nanoflowers should be of great potential in the bioconversion of sterol derivatives.

Affinity adsorption of bovine hyaluronidase with ligands targeting to active site.

Four affinity ligands were designed from 6-chloromethyluracil and 2-aminobenzimidazole and simulated for the interaction with bovine hyaluronidase-1. Regarding sequence alignment, bovine hyaluronidase-1 precursor showed circa 83.6% similarity with human hyaluronidase-1. Regarding structural modeling and molecular docking, bovine hyaluronidase-1 interacted with ligands in the active site. Using epichlorohydrin, 1,3-propanediamine and cyanuric chloride as spacers, 6-chloromethyluracil and 2-aminobenzimidazole were composed to Sepharose beads. The modified Sepharose beads were then subjected to adsorption analysis with bovine hyaluronidase. After one step of affinity adsorption, the samples extracted from bovine testes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and activity assay. As calculated, the densities of four ligands on sorbents (entitled as L-1, L-2, L-3 and L-4) were 37.7 ±â€¯2.3, 36.4 ±â€¯3.2, 42.4 ±â€¯4.2 and 33.7 ±â€¯2.3 µmol/g wet gel; the theoretical maximum adsorption (Qmax) of bovine hyaluronidase on the four sorbents were 63.6 ±â€¯1.6, 72.0 ±â€¯0.7, 111.0 ±â€¯4.1 and 121.7 ±â€¯2.3 mg/g wet gel, respectively; the dissociation constants (Kd) of the four sorbents were 18.5 ±â€¯0.8, 48.1 ±â€¯4.3, 35.0 ±â€¯3.0, 40.6 ±â€¯2.7 µg/g wet gel, respectively. After optimization, the proteins captured by sorbents attaching 2-aminobenzimidazole based ligands (L-3 and L-4) revealed the main single band at approximately 50 kDa, and the purities were about 85.2 and 96.4%; the bioactivity recoveries were 83.5 and 89.4%. In addition, the bands on SDS-PAGE gel were also extracted and confirmed with linear trap quadropole mass spectrometry (LTQ-MS) analysis.

Investigation of toxin gene diversity and antimicrobial resistance of Clostridium difficile strains.

The incidence of Clostridium difficile infection (CDI) has been previously reported in a number of studies. However, data collected from the Chinese population is limited. In the present study, the diversity of the toxin genes, tcdA and tcdB, of 57 Clostridium difficile (C. difficile) isolates from a Chinese population were investigated by polymerase chain reaction (PCR) (38 A+B+, 14 A-B+ and 5 A-B-). Quantitative PCR was used to check the expression of these two genes and it was found that the genes were not expressed by all the strains. The absence of tcdA or tcdB expression in certain strains could be due to the lower expression of tcdD and the higher expression of tcdC, which are positive and negative regulators for these two toxin genes, respectively. In addition, the antimicrobial susceptibilities of 57 isolates were investigated. Therefore, these data would aid in the future prevention of CDI outbreaks and improve the understanding of the infection.

[Application of 3D-CT angiography in the diagnosis and post-operative evaluation of intracranial aneurysms].

OBJECTIVE: To assess the values of three-dimensional CT (3D-CT) angiography (3D-CTA) in the diagnosis and operative follow up of intracranial aneurysms after clip. METHODS: 3D-CTA and DSA were performed on 32 patients with clinical manifestations suggestive of harboring intracranial aneurysms. DSA and operation were regarded as gold standards. Five patients who had been treated with clip underwent CTA both preoperatively and postoperatively to evaluate the effects of aneurysm clipping. RESULTS: According to DSA results and surgical findings, the sensitivity, specificity, and the accuracy of 3D-CTA for the detection of aneurysms were 100%, 100%, and 93.9% respectively. The detection rate of aneurysm with a diameter<3 mm of CTA, with the smallest diameter of 2 mm, was higher than that of DSA, however, there was no significant difference in the detection rate of aneurysm with the diameter>3 mm between CTA and DSA. Postoperative CTA displayed a remnant of aneurysm body in one case. CONCLUSION: With satisfying sensitivity and specificity, 3D-CTA is a quick, reliable, and relatively noninvasive diagnostic tool for intracranial aneurysms. 3D-CTA combined with VR delineates the aneurysmal morphology in detail, and provides useful information for choosing and planning microsurgical or endovascular treatment.