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Background: The heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition. Methods: Weighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria. Results: The CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice. Conclusions: The results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques. [ABSTRACT FROM AUTHOR]

Background: The internal limiting membrane (ILM) insertion technique was widely used to treat large macular hole (MH) for the high closure rate. However, the prognosis of closed MH after ILM insertion compared to ILM peeling remains controversial. This study aimed to compare foveal microstructure and microperimeter in large idiopathic MH surgically closed by ILM peeling and ILM insertion. Methods: This retrospective, non-randomized, comparative study included patients with idiopathic MH (minimum diameter ≥ 650 μm) who underwent primary pars plana vitrectomy (PPV) with ILM peeling or ILM insertion. The initial closure rate was recorded. Patients with initially closed MHs were divided into two groups according to the surgery methods. The best-corrected visual acuity (BCVA), optical coherence tomography (OCT) and microperimeter-3 (MP-3) outcomes of two groups were compared at baseline, 1 and 4 months postoperatively. Results: For idiopathic MH (minimum diameter ≥ 650 μm), ILM insertion had a significantly higher initial closure rate than ILM peeling (71.19% vs. 97.62%, P = 0.001). Among 39 patients with initially closed MHs who were on regular follow-up, twenty-one were assigned to the ILM peeling group and 18 to the ILM insertion group. Postoperative BCVA improved significantly in both groups. The final BCVA (logMAR) (0.40 vs. 0.88, P < 0.001), macular hole sensitivity (19.66 dB vs. 14.14 dB, P < 0.001), peripheral sensitivity of macular hole (24.63 dB vs. 21.95 dB, P = 0.005), and fixation stability (FS) within 2 degrees (82.42% vs. 70.57%, P = 0.031) were significantly better and external limiting membrane (ELM) defect (330.14 μm vs. 788.28 μm, P < 0.001) and ellipsoid zone (EZ) defect (746.95 μm vs. 1105.11 μm, P = 0.010) were significantly smaller in the ILM peeling group than in the ILM insertion group. Conclusion: For initially closed MHs (minimum diameter ≥ 650 μm), both ILM peeling and ILM insertion significantly improved the microstructure and microperimeter in the fovea. However, ILM insertion was less efficient at microstructural and functional recovery after surgery. [ABSTRACT FROM AUTHOR]

Background: The pathogenesis of age-related macular degeneration (AMD) is complex. It has been shown that vitreomacular traction (VMT) plays a role in the pathogenesis of AMD. We speculate that the continuous stretch induced by VMT might impair the function of retinal pigment epithelium (RPE) cells and it might also be involved in the progression of AMD.Methods: Cultured ARPE-19 cells were subjected to cyclic stretch on the Flexcell Strain system at a level of 25% increment on the surface area for 8 h, 14 h, 20 h, 24 h. In another group, the stretch was withdrawn at 14 h and the cell cultured for another 6 h. Then, we observed the changes in morphology, apoptosis and expression of interleukin 6 (IL6) and vascular endothelial growth factor (VEGF) in RPE cells under stretch.Results: We found that stretch induced the RPE cells to change from a spreading shape into a rounded shape, and that the morphological changes were positively correlated with the duration of the stretch. The expression of pFAK397 and pRac1/cdc42 were elevated in a time-dependent fashion. The stretch resulted in an increase in the apoptosis ratio, with Bcl2, Bax and p53 also showing time-dependent changes. In addition, up-regulation of IL6 and VEGF expression levels was also observed. After withdrawal of the stretch, all of these changes were significantly diminished.Conclusion: Stretch may induce morphological, cell apoptosis, and up-regulation of cytokines changes in RPE cells, indicating that cyclic stretching may participate in the progression of AMD by impeding the functions of the RPE. [ABSTRACT FROM AUTHOR]

Backgroud: Choroidal detachment (CD) following primary rhegmatogenous retinal detachment (RRD) is a special type of RRD. The purpose of this study is to investigate the potential risk factors of RRD with CD in a Chinese population.Methods: All of 201 consecutive RRD with CD patients and 210 RRD without CD patients were enrolled in this case-control retrospective study. The clinical data from these cases were reviewed here. Patients were undergone scleral buckling or encircling or both, or pars plana vitrectomy with or without scleral buckling or encircling or both according the patients' condition. The incidence of RRD with CD in this Chinese population was measured, and the potential risk factors for the development of RRD with CD were investigated by multivariate logistic regression analysis.Results: In this population, the incidence of RRD with CD was 8.6 %. The incidence of RRD with CD was significantly higher in patients with macular hole (P < 0.05), retinal breaks located posterior to the equator (P < 0.05), and total detachment (P < 0.05). Furthermore, the incidence of RRD with CD was significantly higher in patients with longer axial length (P < 0.05) only when ages, IOP, AL and duration time was set for categorical variables.Conclusions: Hypotony, retinal breaks located posteriorly especially macular hole, longer axial length, and the whole retinal detachment might be the potential risk factors for the development of CD in RRD patients. [ABSTRACT FROM AUTHOR]

Background: Pre-diabetes and non-alcoholic fatty liver disease (NAFLD) are associated with an unhealthy lifestyle and pose extremely high costs to the healthcare system. In this study, we aim to explore whether individualized aerobic exercise (AEx) and low carbohydrate diet (LCh) intervention affect hepatic fat content (HFC) in pre-diabetes via modification of gut microbiota composition and other post-interventional effects. Methods/design A 6-month randomized intervention with 6-month follow-up is conducted from January 2013 to December 2015. The target sample size for intervention is 200 postmenopausal women and middle-aged men aged 50–65 year-old with pre-diabetes and NAFLD. The qualified subjects are randomized into 4 groups with 50 subjects in each group: 1 = AEx, 2 = LCh, 3 = AEx + LCh, and 4 = control. In addition, two age-matched reference groups (5 = pre-diabetes without NAFLD (n = 50) and 6 = Healthy without pre-diabetes or NAFLD (n = 50)) are included. The exercise program consists of progressive and variable aerobic exercise (intensity of 60 to 75% of initial fitness level, 3–5 times/week and 30–60 min/time). The diet program includes dietary consultation plus supplementation with a special lunch meal (40% of total energy intake/day) which aims to reduce the amount of carbohydrate consumption (30%). The control and reference groups are advised to maintain their habitual habits during the intervention. The primary outcome measures are HFC, serum metabolomics and gut microbiota composition. The secondary outcome measures include body composition and cytokines. In addition, socio-psychological aspects, social support, physical activity and diet will be performed by means of questionnaire and interview. Discussion Specific individualized exercise and diet intervention in this study offers a more efficient approach for liver fat reduction and diabetes prevention via modification of gut microbiota composition. Besides, the study explores the importance of incorporating fitness assessment and exercise in the management of patients with pre-diabetes and fatty liver disorders. If our program is shown to be effective, it will open new strategies to combat these chronic diseases. Trial registration Current Controlled Trials: ISRCTN42622771.

Background: To determine whether preoperative hole diameter ratio (HDR) is a predictive factor for postoperative anatomical outcome for stage III or IV idiopathic macular holes (IMHs).Methods: One-hundred and one eyes with stage III or IV IMH were included in this retrospective case series study. All cases were treated with vitrectomy combined with internal limiting membrane (ILM) peeling and room air tamponade. The macular hole (MH) minimum and maximum diameter was measured on preoperative optical coherence tomography (OCT) images. The HDR was defined as the minimum to maximum diameter ratio.Results: Eighty-one eyes (80.2%) got a Type I closure after surgery (group A). Postoperative unclosed MHs were found in 20 eyes (19.8%) (group B). The preoperative minimal diameter (703.6 ± 116.1 μm vs. 597.6 ± 120.1 μm, P < 0.01) and HDR (0.6 ± 0.1 vs. 0.5 ± 0.1, P = 0.01) were both significantly smaller in postoperative closed eyes. The closure rate of IMHs with HDR < 0.6 was significantly higher than those with HDR ≥ 0.6 (90.2% vs. 65.0%P = 0.002) .Conclusions: Preoperative HDR < 0.6 is predictive for a good postoperative anatomical outcome in stage III or IV IMHs. [ABSTRACT FROM AUTHOR]

Background: This study explores the role of actin cytoskeleton depolymerization induced by Cytochalasin D and mechanical stretch on the interleukin-8 (IL-8) expression and c-jun N-terminal kinase (JNK) phosphorylation levels in human retinal pigment epithelial (RPE) cells.Methods: A Flexcell FX-5000 Tension system was used to apply cyclic stretch to cultured human RPE cells (ARPE-19) at 0.33 Hz with 20% elongation for 0 h, 6 h or 24 h. The cells were stretched alone or pre-treated with Cytochalasin D. The redistribution of the actin cytoskeleton was evaluated using phalloidin immunofluorescence staining. The protein expression levels of IL-8 and JNK in the RPE cells were determined via Western blotting.Results: The cells in the control groups displayed abundant and uniform phalloidin staining. After exposure to mechanical stretch for 24 h, phalloidin staining revealed an unclear and irregular actin cytoskeleton. In all Cytochalasin D-treated cells, the shrinkage and disruption of the cytoskeletal structure was observed regardless of mechanical stress. The stimulation of the RPE cells with cyclic stretch alone did not induce a significant increase in IL-8 expression and JNK phosphorylation levels, which were similar to those of the control groups. After pre-treatment with Cytochalasin D alone, IL-8 expression and JNK phosphorylation levels were not significantly different at 6 h but were significantly increased by approximately 1.2-fold (1.18 ± 0.05; P<0.01) and 3.0-fold (3.01 ± 0.02; P<0.01) at 24 h, respectively. After the pre-incubation of the RPE cells with Cytochalasin D followed by exposure to cyclic stretch, IL-8 expression and JNK phosphorylation levels increased by approximately 1.3-fold (1.31 ± 0.02; P<0.01) and 1.3-fold (1.31 ± 0.02; P<0.01) at 6 h, respectively, and by 1.7-fold (1.69 ± 0.06; P<0.01) and 3.2-fold (3.21 ± 0.12; P<0.01) at 24 h, respectively.Conclusions: This study demonstrates that disruption of actin polymerization by cytochalasin D and mechanical stretch upregulates interleukin-8 expression and JNK phosphorylation levels in human RPE cells, which indicates that the integrity of the actin cytoskeleton may play important roles in the pro-inflammatory processes in RPE cells. [ABSTRACT FROM AUTHOR]

Background: This study evaluates the macular function changes in patients with idiopathic macular epiretinal membrane (ERM) by multifocal electroretinography (mfERG) and their correlations with visual acuity and central macular thickness (CMT) by optical coherence tomography (OCT)., Methods: Twenty eyes of 20 patients with ERM underwent OCT and mfERG examinations. The response amplitude densities and implicit times of mfERG were compared to the control fellow eyes. Correlation analyses among visual acuity, central macular thickness and mfERG values in the central two concentric rings were performed., Results: The mfERG P1 response amplitude densities in ring 1-2 and P1 implicit time in ring1 were significantly changed in epiretinal membrane eyes compared with controls (P < 0.05). Multivariate stepwise linear regression analyses showed LogMAR visual acuity was significantly correlated with CMT (P = 0.004), and also with the P1 amplitude density in ring 1 (P = 0.002). CMT showed significant correlation with P1 implicit time in ring 2 (P = 0.013)., Conclusions: The mfERG abnormalities show macular function changes and correlate with visual acuity and central macular thickness in eyes with ERM. In first-order mfERG responses, P1 wave changes may be a sensitive functional measurement for ERM patients.

Background: The pathogenesis of age-related macular degeneration (AMD) is complex. It has been shown that vitreomacular traction (VMT) plays a role in the pathogenesis of AMD. We speculate that the continuous stretch induced by VMT might impair the function of retinal pigment epithelium (RPE) cells and it might also be involved in the progression of AMD., Methods: Cultured ARPE-19 cells were subjected to cyclic stretch on the Flexcell Strain system at a level of 25% increment on the surface area for 8 h, 14 h, 20 h, 24 h. In another group, the stretch was withdrawn at 14 h and the cell cultured for another 6 h. Then, we observed the changes in morphology, apoptosis and expression of interleukin 6 (IL6) and vascular endothelial growth factor (VEGF) in RPE cells under stretch., Results: We found that stretch induced the RPE cells to change from a spreading shape into a rounded shape, and that the morphological changes were positively correlated with the duration of the stretch. The expression of pFAK397 and pRac1/cdc42 were elevated in a time-dependent fashion. The stretch resulted in an increase in the apoptosis ratio, with Bcl2, Bax and p53 also showing time-dependent changes. In addition, up-regulation of IL6 and VEGF expression levels was also observed. After withdrawal of the stretch, all of these changes were significantly diminished., Conclusion: Stretch may induce morphological, cell apoptosis, and up-regulation of cytokines changes in RPE cells, indicating that cyclic stretching may participate in the progression of AMD by impeding the functions of the RPE.

Backgroud: Choroidal detachment (CD) following primary rhegmatogenous retinal detachment (RRD) is a special type of RRD. The purpose of this study is to investigate the potential risk factors of RRD with CD in a Chinese population., Methods: All of 201 consecutive RRD with CD patients and 210 RRD without CD patients were enrolled in this case-control retrospective study. The clinical data from these cases were reviewed here. Patients were undergone scleral buckling or encircling or both, or pars plana vitrectomy with or without scleral buckling or encircling or both according the patients' condition. The incidence of RRD with CD in this Chinese population was measured, and the potential risk factors for the development of RRD with CD were investigated by multivariate logistic regression analysis., Results: In this population, the incidence of RRD with CD was 8.6 %. The incidence of RRD with CD was significantly higher in patients with macular hole (P < 0.05), retinal breaks located posterior to the equator (P < 0.05), and total detachment (P < 0.05). Furthermore, the incidence of RRD with CD was significantly higher in patients with longer axial length (P < 0.05) only when ages, IOP, AL and duration time was set for categorical variables., Conclusions: Hypotony, retinal breaks located posteriorly especially macular hole, longer axial length, and the whole retinal detachment might be the potential risk factors for the development of CD in RRD patients.

Background: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses., Results: In this study, we mined the available expressed sequence tags (ESTs) of B. napus and identified a total of 25 CPK genes, among which cDNA sequences of 23 genes were successfully cloned from a double haploid cultivar of canola. Phylogenetic analysis demonstrated that they could be clustered into four subgroups. The subcellular localization of five selected BnaCPKs was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of 21 BnaCPK genes in response to salt, drought, cold, heat, abscisic acid (ABA), low potassium (LK) and oxidative stress were studied by quantitative RT-PCR and were found to respond to multiple stimuli, suggesting that canola CPKs may be convergence points of different signaling pathways. We also identified and cloned five and eight Clade A basic leucine zipper (bZIP) and protein phosphatase type 2C (PP2C) genes from canola and, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC), determined the interaction between individual BnaCPKs and BnabZIPs or BnaPP2Cs (Clade A). We identified novel, interesting interaction partners for some of the BnaCPK proteins., Conclusion: We present the sequences and characterization of CPK gene family members in canola for the first time. This work provides a foundation for further crop improvement and improved understanding of signal transduction in plants.

Background: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca2+ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola., Results: In the present study, we identified seven CBL and 23 CIPK genes from canola by database mining and cloning of cDNA sequences of six CBLs and 17 CIPKs. Phylogenetic analysis of CBL and CIPK gene families across a variety of species suggested genome duplication and diversification. The subcellular localization of three BnaCBLs and two BnaCIPKs were determined using green fluorescence protein (GFP) as the reporter. We also demonstrated interactions between six BnaCBLs and 17 BnaCIPKs using yeast two-hybrid assay, and a subset of interactions were further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore, the expression levels of six selected BnaCBL and 12 BnaCIPK genes in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and low potassium were examined by quantitative RT-PCR and these CBL or CIPK genes were found to respond to multiple stimuli, suggesting that the canola CBL-CIPK network may be a point of convergence for several different signaling pathways. We also performed a comparison of interaction patterns and expression profiles of CBL and CIPK in Arabidospsis, canola and rice, to examine the differences between orthologs, highlighting the importance of studying CBL-CIPK in canola as a prerequisite for improvement of this crop., Conclusions: Our findings indicate that CBL and CIPK family members may form a dynamic complex to respond to different abiotic or hormone signaling. Our comparative analyses of the CBL-CIPK network between canola, Arabidopsis and rice highlight functional differences and the necessity to study CBL-CIPK gene functions in canola. Our data constitute a valuable resource for CBL and CPK genomics.

Background: From October 2006 to February 2007, clinical specimens from 452 patients with symptoms related to respiratory tract infection in the northern region of Taiwan were collected. Real-time PCR and direct immunofluorescent antibody tests showed that 145 (32%) patients had influenza B virus infections. Subsequently, nucleotide sequence analyses of both hemagglutinin (HA) and neuraminidase (NA) genes of 39 isolates were performed. Isolated viruses were antigenically characterized using hemagglutinin inhibition (HI) test., Findings: Phylogenetic tree analysis showed that all the isolates belonged to the B reassortant lineage with HA gene belonged to the B/Victoria/2/87 lineage and the NA gene belonged to the B/Yamagata/16/88 lineage. In addition, a group of children aged between 6 to 8 years old resided in Yilan county were infected with a variant strain. Hemagglutinin inhibition (HI) tests confirmed that all the reassortant influenza B viruses were B/Malaysia/2506/04-like viruses. Pre- and post-immunized serum samples from 4 normal volunteers inoculated with 2007 influenza vaccine were evaluated for their HI activity on 6 reassortant B isolates including two variants that we found in the Yilan county. The results demonstrated that after vaccination, all four vaccinees had at least 4-fold increases of their HI titers., Conclusion: The results indicate that the 2006-2007 seasonal influenza vaccine was effective in stimulating protective immunity against the influenza B variants identified in Yilan county. Continuous surveillance of emerging influenza B variants in the northern region of Taiwan is important for the selection of proper vaccine candidate in the future.