Identification and validation of a CCL18-related signature for prediction of overall survival in patients with uveal melanoma.

Uveal melanoma (UM), the most frequent primary intraocular tumor in adults, has poor prognosis. High C-C motif chemokine ligand 18 (CCL18) has been detected in various tumors and is closely correlated with patients' clinicopathological characteristics. However, the essential role of CCL18 in UM remains unclear. Therefore, this study aimed to explore the prognostic value of CCL18 in UM. Uveal melanoma cells (M17) were transfected with pcDNA3.1-CCL18 si-RNA using Lipofectamine™ 2000. Cell growth and invasion abilities were measured through Cell Counting Kit-8 assay and invasion assay. RNA expression data and clinical and histopathological details were downloaded from the UM in The Cancer Genome Atlas (TCGA-UM) and GSE22138 datasets, which were defined as the training and validation cohorts, respectively. Univariate and multivariate Cox regression analyses were performed to identify significant prognostic biomarkers. The coefficients of these significant biomarkers generated by multivariate Cox proportional hazard regression analysis were used to establish a risk score formula. Functional enrichment analyses were also carried out. We found that downregulated CCL18 inhibits M17 cell growth and invasion in vitro. CCL18 may affect UM progression by altering C-C motif receptor 8 related pathways. Higher CCL18 expression was associated with worse clinical outcomes and tumor-specific death in the TCGA-UM dataset. Based on the coefficients obtained from the Cox proportional hazard regression analysis, a CCL18-related prognostic signature formula was constructed as follows: risk score = 0.05590 × age +2.43437 × chromosome 3 status +0.39496 × ExpressionCCL18. Notably, in this formula, the normal chromosome 3 was coded as 0, whereas the chromosome 3 loss was coded as 1. Each patient was assigned to either low-risk or high-risk groups using the median cut-off in the training cohort. High-risk patients survived for a shorter time than low-risk patients. The time-dependent and multivariate receiver operating characteristic curves showed promising diagnostic efficacy. Multivariate Cox regression analysis demonstrated the potential of this CCL18-related signature as an independent prognostic indicator. These results were validated using the GSE22138 dataset. In addition, in both TCGA-UM and GSE22138 datasets, stratification of clinical correlations and survival analyses based on this signature indicated the involvement of clinical progression and survival outcome in UM. In the high-risk group, Gene Ontology analyses mainly indicated the enrichment of immune response pathways, such as the T cell activation, response to interferon-gamma, antigen processing and presentation, interferon-gamma-mediated signaling pathway, MHC protein complex, MHC class II protein complex, antigen binding, and cytokine binding. Meanwhile, Kyoto Encyclopedia of Genes and Genomes analyses showed enrichments of pathways in cancer, cell adhesion, cytokine-cytokine receptor interaction, chemokine signaling pathway, Th1 and Th2 cell differentiation, and chemokine signaling pathway. Moreover, single-sample gene set enrichment analysis demonstrated the enrichment of almost all immune cells and immune functions in the high-risk group. In summary, a new prognostic CCL18-related signature was successfully established using the TCGA-UM dataset and validated using the GSE22138 dataset with meaningful predictive and diagnostic efficacies. This signature could serve as an independent and promising prognostic biomarker for patients with UM.

Identification of a prognostic six-immune-gene signature and a nomogram model for uveal melanoma.

BACKGROUND: To identify an immune-related prognostic signature and find potential therapeutic targets for uveal melanoma. METHODS: The RNA-sequencing data obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. The prognostic six-immune-gene signature was constructed through least absolute shrinkage and selection operator and multi-variate Cox regression analyses. Functional enrichment analysis and single sample GSEA were carried out. In addition, a nomogram model established by integrating clinical variables and this signature risk score was also constructed and evaluated. RESULTS: We obtained 130 prognostic immune genes, and six of them were selected to construct a prognostic signature in the TCGA uveal melanoma dataset. Patients were classified into high-risk and low-risk groups according to a median risk score of this signature. High-risk group patients had poorer overall survival in comparison to the patients in the low-risk group (p < 0.001). These findings were further validated in two external GEO datasets. A nomogram model proved to be a good classifier for uveal melanoma by combining this signature. Both functional enrichment analysis and single sample GSEA analysis verified that this signature was truly correlated with immune system. In addition, in vitro cell experiments results demonstrated the consistent trend of our computational findings. CONCLUSION: Our newly identified six-immune-gene signature and a nomogram model could be used as meaningful prognostic biomarkers, which might provide uveal melanoma patients with individualized clinical prognosis prediction and potential novel treatment targets.

Genetic evolution of fowl adenovirus serotype 4 and its pathogenicity to Cherry Valley ducks in China.

Hydropericardium hepatitis syndrome (HHS), a novel poultry disease, is caused by fowl adenovirus 4 (FAdV-4). It mainly infects 3-5-week-old broilers. In July 2015, the first outbreak of HHS occurred in the broilers in east China, which caused great economic losses to the poultry industry. In June 2019, infectious disease was detected with suspected HHS symptoms on a duck farm in Linyi City, Shandong Province. The main necropsy lesions included pericardial effusion and hepatitis. In this study, we isolated a strain of FAdV-4 from naturally infected ducks and named it SDLY190604, and the hexon gene sequence was amplified and analyzed using polymerase chain reaction (PCR). In order to study the effect of FAdV-4 on Cherry Valley ducks, we inoculated three-week-old ducks with 0.2 ml of FAdV-4 virus fluid (TCID50 of 10-6.3/0.1 ml) by orally, subcutaneously and intramuscularly. Clinical signs, gross lesions and histopathological changes, cytokines and viral load were detected and recorded within 15 days after infection. The results showed that ducks in the experimental groups exhibited typical symptoms of hydropericardium and hepatitis. The histopathological sections showed multiple-organ damage, including serious liver and kidney damage with elevated levels of inflammatory cytokines, probably due to the infection and innate immune response. Later, immunosuppression occurred, resulting in decreased levels of cytokines. The viral load indicated that the virus could be present in several organs of the ducks, with the highest viral DNA found in the liver, followed by the kidney. Compared to the subcutaneous and oral groups, the intramuscular group exhibited the highest viral load. In summary, this study can increase our understanding of the pathogenicity of FAdV-4 in ducks and provide a basis for further understanding of the virus, imparting new insights into disease research.

High EIF4E2 expression is an independent prognostic risk factor for poor overall survival and recurrence-free survival in uveal melanoma.

Uveal melanoma (UM), as the most common primary intraocular carcinoma, is a relatively rare but lethal tumor. Upregulated eukaryotic translation initiation factor 4E family member 2 (EIF4E2) promotes the progression of multiple human carcinomas. However, its role remains unclear in UM. To identify the prognostic value of EIF4E2 in UM, we downloaded RNA-sequencing data along with clinical information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. EIF4E2 mRNA was significantly increased in three different subgroups in the TCGA-UM dataset. High mRNA expression was correlated with shorter overall survival (OS) and shorter recurrence-free survival (RFS). Moreover, we constructed a prognostic signature using Cox regression analyses in our training cohort TCGA-UM dataset as follows: risk score = 0.04335 × Age +0.49639 × expression of EIF4E2. Based on the risk score, each patient was classified as high-risk or low-risk. Additional survival analyses suggested that patients in the high-risk score group had an unfavorable OS compared with patients in the low-risk score group, which was validated in two external GEO datasets, including GSE84976 and GSE22138. Functional enrichment analysis demonstrated that UM was correlated with hypoxia-related functions. Gene set enrichment analysis (GSEA) indicated significant enrichments of the p53 and Notch pathways. In addition, EIF4E2 was genetically altered in 12.5% (10/80) of UM patients. Epigenetically, higher expression of cg03852847 was correlated with longer OS and longer RFS. In conclusion, our findings demonstrated that high EIF4E2 expression is an independent prognostic risk factor for UM patients. EIF4E2 might play an important role in hypoxia-related signaling pathways during UM progression. Both genetic and epigenetic alterations may contribute to UM pathogenesis. These findings could offer individualized clinical prognostication and potential novel treatment targets for UM patients.

[Prediction of postoperative visual acuity in cataract patients with idiopathic macular epiretinal membrane by hand-held retinal optometer and optical coherence tomography].

OBJECTIVE: To investigate the value of hand-held retinal optometer and optical coherence tomography (OCT) in predicting postoperative visual acuity in patients with age-related cataract and idiopathic macular epiretinal membrane. METHODS: We retrospectively analyzed the data of patients undergoing phacoemulsification combined with intraocular lens implantation for age-related cataract in our hospital from January, 2019 to April, 2020.Preoperative examination detected idiopathic macular epiretinal membrane in 45 of the patients (52 eyes) with lens opacity grade C2N2P1 according to LOCSⅡ lens opacity classification criteria.Based on the thickness of the macular fovea, the eyes were divided into group A (9 eyes) with macular thickness < 300 µm by OCT examination, group B (25 eyes) with macular thickness of 300 to 400 µm, and group C (18 eyes) with macular thickness >400 µm.The best corrected visual acuity and retinal visual acuity before operation and the best corrected visual acuity on the first day and at 3 months after the surgery were compared among the 3 groups.The consistency between the preoperative retinal vision and the best corrected vision at 3 months after the surgery was analyzed. RESULTS: The best corrected visual acuity at one day and 3 months after the surgery differed significantly from that before the surgery in all the 3 groups (P < 0.05).The best corrected visual acuity recovered to 0.3-0.5 in most of the patients in the 3 groups. Thirty-one (91.18%) of the patients with a macular thickness less than 400 µm had a visual acuity≥0.3 after cataract surgery, as compared with only 14 patients (77.78%) among those with a macular thickness >400 µm.There was a positive linear correlation between preoperative retinal visual acuity and best corrected visual acuity at 3 months after the surgery (r=0.830, P < 0.05). CONCLUSIONS: For patients with cataract and idiopathic macular epiretinal membrane, phacoemulsification combined with intraocular lens implantation can improve postoperative vision.Hand-held retinal optometer can accurately assess postoperative vision in patients with stage C2N2P1 cataract.Patients with a macular thickness >400 µm caused by idiopathic macular epiretinal membrane are likely to have poor postoperative visual outcomes.

Dynamic Change of Red Cell Distribution Width Levels in Prediction of Hospital Mortality in Chinese Elderly Patients with Septic Shock.

BACKGROUND: The normal range of red cell distribution width (RDW) level is <15%. Several studies have indicated that a high RDW level was associated with mortality in critically ill patients, and the patients with a high RDW level need increased focus in clinical practice. In view of the difficulty in defining the specific value of high RDW level, the key is to focus on the patient with the level beyond the normal upper limit. This study aimed to determine whether dynamic change of RDW levels, rather than the level itself, is predictive of death in elderly patients with septic shock when RDW level is beyond 15%. METHODS: Between September 2013 and September 2015, the elderly septic shock patients with RDW level beyond 15% were enrolled in this study. The RDW levels were measured at enrollment (day 1), and days 4 and 7 after enrollment. Sequential Organ Failure Assessment (SOFA) scores were recorded simultaneously. RESULTS: A total of 45 patients, including 32 males and 13 females, were included in the final analysis. Based on their hospital outcomes, these patients were divided into the survivor group (n = 26) and the nonsurvivor group (n = 19). There were no significant differences in age, gender, body mass index, initial level of RDW, Acute Physiology and Chronic Health Evaluation II scores, and SOFA scores between survivors and nonsurvivors. At days 4 and 7 measurement, both RDW level (median [interquartile range]: day 4: 15.8 [2.0]% vs. 16.7 [2.0]%, P= 0.011; and day 7: 15.6 [1.8]% vs. 17.7 [2.5]%, P= 0.001) and SOFA scores (day 4: 7.0 [4.0] vs. 16.0 [5.0], P< 0.001, day 7: 5.5 [4.0] vs. 17.0 [5.0], P< 0.001) were significantly lower in survivors than those in nonsurvivors. Dynamic changes of RDW and SOFA scores in survivor group were significantly different from those in nonsurvivor group (all P< 0.05). Continuous increase in RDW level was observed in 10 of the 13 nonsurvivors, but only in 3 of the 26 survivors. The level of RDW7 and dynamic changes significantly correlated with their counterparts of SOFA scores (all P< 0.05), whereas the levels of RDW1 and RDW4 had no significant correlation with their counterparts of SOFA scores (all P> 0.05). CONCLUSIONS: Continuous increase in RDW level, rather than the level of RDW itself, was more useful in predicting hospital death in elderly patients with septic shock when the level of RDW was >15%. The dynamic changes of RDW were highly correlated with the SOFA score in the patients.

[Establishment of Double Antibody Sandwich ELISA for the Determination of Human Soluble VE-Cadherin in Human Plasma and Its Application].

OBJECTIVE: To establish double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human soluble VE-cadherin in human plasma and to investigate its value in clinical use. METHODS: The monoclonal antibodies against human VE-cadherin were prepared from BALB/c mice immunized with prokaryotic expression recombinant proteins, and the best combination of double antibodies was selected by checkerboard titration method. Double antibody sandwich ELISA for the determination of human VE-cadherin was established by using HRP-labeled McAb as a detection antibody and a capture antibody. The methodology performance was evaluated. The plasma concentrations of VE-cadherin in 28 healthy subjects and 60 patients with cancer were determined. RESULTS: The double antibody sandwich ELISA for the determination of human VE-cadherin was established by selecting the combination of double antibodies. The detection limit was 24.7 pg/ml, the coefficients of variation for inner-batch and inter-batch were 4.1%-7.7% and 8.7%-10.8% respectively. The average recovery was 96.7%. The plasma level of soluble VE-cadherin in normal controls was 262.1±11.75 pg/ml. The plasma level of soluble VE-cadherin was 173.9±17.98 pg/ml in 24 patients with leukemia, 311.7±25.24 pg/ml in 14 patients with stomach cancer, 206.8±25.01 pg/ml in 11 patients with lung cancer, and 310.7±11.82 pg/ml in others patients(9 patients with breast cancer, 1 patients with gliomas, 1 patients with liver cancer). CONCLUSION: The developed ELISA kit has better sensitivity and specificity, and can be used in detection of human soluble VE-cadherin in human plasma, therefore, it can provide a new mathod for diagnosis of cancer patients.

[Preparation and application of monoclonal antibody against human vascular endothelial cadherin 5].

OBJECTIVE: To prepare and identify monoclonal antibody (mAb) against human vascular endothelial cadherin 5 (VECDH5). METHODS: BALB/c mice were immunized with recombinant VECDH5 protein for preparing mAb using hybridoma technique. The positive clones were confirmed and selected by indirect ELISA for titer determination. Western blotting, flow cytometry, immunofluorescence staining, immunohistochemical staining were performed to identify the specificity and epitope. RESULTS: One hybridoma cell strain secreting specific mAb against VECDH5 was obtained (2C11). ELISA showed that the titer of the ascites was 1:10 000. Western blotting, flow cytometry, immunofluorescence, immunohistochemistry demonstrated that the mAb could specifically recognize and bind VECDH5. Epitope identification showed that the amino acid sequence recognized by 2C11 was LDREVVPWYNLTVEA. CONCLUSION: We have prepared mAb against human VECDH5, which has good binding ability and specificity.

Methylated +58CpG site decreases DCN mRNA expression and enhances TGF-ß/Smad signaling in NSCLC cells with high metastatic potential.

Decorin (DCN) has been suggested to display an anti-metastatic role by antagonizing bioactive TGF-ß in advanced human cancers. However, the epigenetic mechanisms by which defective expression of DCN causes cancer metastasis remain unclear. We focused on non-small cell lung cancer (NSCLC) cell lines with low metastatic potential (95C) and high metastatic potential (95D), which share a similar genetic background. Quantitative PCR and clonal bisulfite sequencing indicated that the methylation levels of the +58CpG site in the DCN 5'-UTR region was significantly higher in 95D cells with low expression of DCN mRNA compared to 95C cells with high expression of DCN mRNA. In silico prediction and ChIP assay showed a correlation between +58CpG site and AhR, which was reported as a transcriptional activator. Importantly, EMSA and luciferase reporter gene assays suggested that +58CpG methylation specifically diminished the recruitment of AhR to DCN 5'-UTR sequence and caused a reduction of approximately 50% in transcriptional activity. At baseline, 95D cells exhibited higher p-Smad3 levels and lower E-cadherin expression when compared with 95C cells. The demethylating agent 5-Aza significantly led to restoration of DCN expression, reduced level of p-Smad3, increased expression of E-cadherin in 95D cells. Taken together, we identified the methylated +58CpG in DCN 5'-UTR associated with reduced expression of DCN mRNA, and revealed that +58CpG methylation may be one of the mechanisms accounting for reduced recruitment of the transcriptional activator AhR to DCN 5'-UTR, and suggest that this mechanism promotes TGF-ß/Smad signaling by enhancing the phosphorylation of Smad3, thereby downregulating E-cadherin in NSCLC cells with high metastatic potential.

[High-resolution melting analysis for the detection of EGFR mutations in circulating DNA of lung cancer patients].

OBJECTIVE: To discuss the practicability of detecting epidermal growth factor receptor (EGFR) mutations in plasma circulating DNA of lung cancer patients by high-resolution melting (HRM). METHODS: The sensitivity of HRM was analyzed by the detection of samples containing different proportions of EGFR-mutated plasmids. The mutations in exons 19 and 21 of EGFR were detected by HRM in 96 lung cancer patients from September 2009 to May 2010. And the results of HRM were compared with those of sequencing. RESULTS: The HRM detection could identify the EGFR mutations in a proportion of 5% of mutated plasmid DNA. And the EGFR mutations were detected in 17 (17.7%, 17/96) cases. Among which, the number of exons 19 and 21 mutations was 15 (88.2%, 15/17) and 2 (11.8%, 2/17) respectively. The results of sequencing were consistent. CONCLUSION: The HRM analysis may be an optimal method for clinical screening of EGFR mutation due to its simplicity and promptness with a high sensitivity.

Multivesicular liposome formulation for the sustained delivery of breviscapine.

Breviscapine, a well-known bioactive flavonoid ingredient extracted from the traditional Chinese medicine, has been extensively used in clinic to treat ischemic cerebrovascular and cardiovascular diseases in China. In order to prolong the duration of the drug in the circulation, reduce the frequency of injection administration and subsequently afford patient compliance, multivesicular liposome (MVL, namely DepoFoam) was utilized as a sustained-delivery system for breviscapine. In vitro release and in vivo pharmacokinetics of MVLs containing breviscapine (bre-MVLs) following intramuscular injection to rats were investigated compared with those of traditional liposomes containing breviscapine (bre-TLs). The drug durations both in vitro and in vivo were significantly prolonged for the bre-MVL, and that the drug release in vitro and the absorption in vivo showed a good linear correlation (R=0.9834), which provided an evidence for the suitability to select human plasma as the medium of drug release from MVLs in vitro. Drug release from bre-MVLs (triolein/tricaprylin, 10/0) in vitro extended a long period of 5-6 days, while the bre-TLs released 80% within only 4h. The mean residence time (MRT) obtained from the pharmacokinetics study of bre-MVL was about 16.6- and 5.04-fold longer than those of breviscapine solution (BS) and bre-TL, respectively. A duration in vivo for a period of 4-5 days was fulfilled for bre-MVL. In conclusion, MVL can be successfully used as a sustained delivery system of breviscapine.

[Studies on chiral inversion of dextropantoprazole in human].

AIM: To study the chiral inversion of dextropantoprazole in human. METHODS: Three healthy Chinese male volunteers after an oral dose of 40 mg dextropantoprazole. An HPLC method was developed and used to determine the total plasma concentrations of each enantiomer. The ratios of the enantiomers in plasma samples were measured on a Chiral-AGP column. The plasma concentration of each enantiomer was then calculated using the ratios of enantiomers and total concentrations of the two enantiomers previously measured. RESULTS: The AUC0-t of levopantoprazole was only 1.5% of the total AUC0-t of enantiomers. CONCLUSION: The chiral inversion from dextropantoprazole to levopantoprazole does not occur in the three healthy Chinese male volunteers after an oral dose of 40 mg dextropantoprazol.

Determination of scutellarin in rat plasma by high-performance liquid chromatography with ultraviolet detection.

A validated high-performance liquid chromatography method is described for the determination of scutellarin in rat plasma using a liquid-liquid extraction and ultraviolet (UV) absorbance detection. The separation used a Diamonsil ODS column (250 mm x 4.6mm i.d., 5 microm particle size) with an isocratic mobile phase consisting of methanol-acetonitrile-50mM dihydrogen ammonium phosphate buffer (22:15:63 (v/v/v), adjusted to pH 2.5 with 1M phosphoric acid). The ultraviolet detector operated at 335 nm. Plasma samples were extracted with ethyl acetate after acidification. The extraction recovery of scutellarin ranged from 68.1 to 80.5%. High selectivity and a low quantitation limit (0.050 microg/ml) were achieved. The linear range was 0.050-12.5 microg/ml, correlation coefficient r=0.9981. The method has a good reproducibility, R.S.D. values were below 7.9% for within-day and between-day precision. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.