Functional analysis of the ube3a response in Japanese flounder (Paralichthys olivaceus) to CSBV infection.

Ube3a is a member of the E3 ubiquitin ligase HECTc family, and its role has been established in neurodevelopmental disorders. However, studies on its role in Japanese flounder are scarce. Thus, in this study, the ube3a of Japanese flounder was cloned, and its role in conferring resistance against Chinook salmon bafnivirus (CSBV) was analyzed. Japanese flounder ube3a encoded a protein containing 834 amino acids. Interestingly, its homology with the Atlantic halibut was determined to be 94%. In addition, there were differential expressions of ube3a in different tissues of Japanese flounder, with the highest expression level observed in the fin, followed by the gills and skin (P ≤ 0.05). Subcellular localization analysis revealed that Ube3a is a cytoplasmic protein. We established an in vitro CSBV infection model using Japanese flounder gill cell line (FG). After ube3a overexpression, the viral load was significantly lower than that of the control group (P ≤ 0.05). Contrastingly, after incubation of FG cells with an E3 ubiquitin ligase inhibitor, the viral load was significantly higher than in the control group (P ≤ 0.01). Then, the expression levels of nf-κb, traf3, and tnf-α after incubation with an E3 ubiquitin ligase inhibitor were examined. The results demonstrated that ube3a may exerted a significant antiviral effect in Japanese flounder via the ubiquitination pathway.

Characterization and function of Japanese flounder (Paralichthys olivaceus) slc2a6 in response to lymphocystis disease virus infection.

Slc2a6 is a member of the slc2 family (solute carrier 2 family) and previous reports have indicated its involvement in the inflammatory response. Slc2a6 is regulated by the NF-ĸB signaling pathway. This study investigated the differential expression of slc2a6 in the early embryonic development of Japanese flounder, revealing that the early gastrula stage had the highest level of slc2a6 expression. Moreover, slc2a6 expression was increased in vitro after stimulation by lymphocystis disease virus (LCDV), and in vivo experiments also showed significantly elevated levels in the spleen and muscle tissues following LCDV stimulation. Subcellular localization revealed that Slc2a6 was expressed in both the nucleus and cytoplasm of cells. The pcDNA3.1-slc2a6 overexpression plasmid was successfully constructed; the si-slc2a6 interfering strand was screened and samples were collected. The expression of NF-ĸB signaling pathway-related genes il-1ß, il-6, nf-ĸb, and tnf-α was evaluated in overexpressed, silenced, and LCDV-stimulated samples. The results showed that slc2a6 is involved in viral regulation in Japanese flounder by regulating innate immune responses.

Enhanced chiral sensing in achiral nanostructures with linearly polarized light.

Chiral plasmonic nanostructures can generate large superchiral near fields owing to their intrinsic chirality, leveraging applications for molecule chirality sensing. However, the large structural chirality of chiral nanostructures poses the risk of overshadowing molecular chiral signals, hampering the practical application of chiral nanostructures. Herein, we propose an achiral nanorod that shows no structural chirality and presents strong superchiral near-fields with linearly polarized incidence. The mechanism of the strong superchiral near-field originates from the coupling between the evanescent fields of the localized surface plasmon resonance and incident light. The enhanced near-field optical chirality at the corners of the nanorods reached 25 at a wavelength of 790 nm. Meanwhile, the sign of optical chirality can be tuned by the polarization of the incident light, which provides a convenient way to control the handedness of the light. Furthermore, the enantiomers of D- and L-phenylalanine molecules were experimentally characterized using an achiral platform, which demonstrated a promising nanophotonic platform for chiral biomedical sensing.

Down-regulation of lncRNA NEAT1 regulated by miR-194-5p/DNMT3A facilitates acute myeloid leukemia.

OBJECTIVE: miR-194-5p and NEAT1 have been reported to be associated with multiple malignancies, but their roles in acute myeloid leukemia (AML) remains not fully understood. METHODS: Bone marrow samples were collected for monocyte separation. qRT-PCR assay was performed to investigate the expression patterns of NEAT1 and miR-194-5p in AML. CCK-8, soft agar colony formation, flow cytometry and transwell assays were employed to explore the biological functions of NEAT1 or miR-194-5p. Methylation PCR was performed to monitor the methylation of NEAT1. Luciferase reporter assay was subjected to verify the relationship between miR-194-5p and DNMT3A. Immunofluorescence and western blotting were performed to detect the alterations of protein expression. RESULTS: NEAT1 and miR-194-5p were both down-regulated in AML. Overexpression of either NEAT1 or miR-194-5p repressed proliferation, induced apoptosis and restrained migration and invasion of AML cells. There was a negative correlation between NEAT1 and DNMT3A in AML. Knockdown of DNMT3A dramatically decreased the methylation of NEAT1. Moreover, DNMT3A was identified as a downstream target of miR-194-5p. Furthermore, down-regulation of DNMT3A rescued the impacts on the malignant phenotypes of NEAT1 inhibition by miR-194-5p inhibitor. CONCLUSION: Altogether, down-regulation of NEAT1 mediated by miR-194-5p/DNMT3A axis promotes AML progression, which might provide therapeutic targets in AML treatment.

Applying copulas to predict the multivariate reduction effect of best management practices.

Best management practices (BMPs) have been widely applied to mitigate non-point source (NPS) pollution in agricultural watersheds. However, a prediction of the multivariate reduction effect of NPS pollutants by BMPs considering its stochastic nature has not been conducted. A new modeling approach combining a hydrological model and copulas was proposed to predict the multivariate effect of BMPs fully considering the stochastic characteristics of BMPs effects and the dependence structure between them. Two levels of reduction effect, i.e., the multi-indicator effect of a single BMP and the combined effect of multiple BMPs, were simulated. The approach was demonstrated in Zhangjiachong watershed, a typical small watershed in the Three Gorges Reservoir area, China. Results show that copulas can effectively simulate the dependence between the univariate effects of BMPs. The approach can accurately predict the probability to achieve the reduction objective for multiple pollutants and multiple BMPs in a watershed. It provides a stochastic way to predict the multivariate effect of BMPs and has great potential to be widely applied in BMPs related decision making.

Direct competitive chemiluminescence immunoassays based on gold-coated magnetic particles for detection of chloramphenicol.

Direct competitive chemiluminescence immunoassays (CLIA) based on gold-coated magnetic nanospheres (Au-MNPs) were developed for rapid analysis of chloramphenicol (CAP). The Au-MNPs were modified with carboxyl groups and amino groups by 11-mercaptoundecanoic acid (MUA) and cysteamine respectively, and then were respectively conjugated with CAP base and CAP succinate via an activating reaction using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). NSP-DMAE-NHS, a new and effective luminescence reagent, was employed to label anti-CAP antibody (mAb) as a tracer in direct CLIA for CAP detection using a 'homemade' luminescent measurement system that was set up with a photomultiplier tube (PMT) and a photon counting unit linked to a computer. The sensitivities and limits of detection (LODs) of the two methods were obtained and compared according to the inhibition curves. The 50% inhibition concentration (IC50 ) values of the two methods were about 0.044 ng/mL and 0.072 ng/mL respectively and LODs were approximately 0.001 ng/mL and 0.006 ng/mL respectively. To our knowledge, they were much more sensitive than any traditional enzyme-linked immunosorbent assay (ELISA) ever reported. Moreover, the new luminescence reagent NSP-DMAE-NHS is much more sensitive and stable than luminol and its derivatives, contributing to the sensitivity enhancement.

Spectral surface plasmon resonance imaging for the detection of clenbuterol via three-dimensional immobilization of bioprobes.

A method of immobilizing clenbuterol (CLEN) on the sensor chip for spectral surface plasmon resonance imaging (SPRi) was experimentally investigated. The bioprobes on the sensor chip were prepared by immobilizing bovine serum albumin (BSA) protein and conjugating CLEN molecules to BSA, which provides more active points and free orientations for specific binding. The calibration curve showed that the wavelength resonance shift decreased as the concentration of CLEN analyte increased, consistent with the inhibition principle. The limit of detection (LOD) was estimated to be 6.32 µg/ml. This method proved to be highly specific, high throughput, label free, and operationally convenient.

A New Cell Line Derived from the Spleen of the Japanese Flounder (Paralichthys olivaceus) and Its Application in Viral Study.

A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder (Paralichthys olivaceus) was established and characterized in this study. The JFSP cells grew rapidly at 29 °C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1ß, TNF-α, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases.

Source identification and impact of landscape pattern on riverine nitrogen pollution in a typical urbanized watershed, Beijing, China.

This study explored the sources of nitrate and the impact of landscape pattern on nitrogen pollution in the highly urbanized Beiyun River Watershed, China during 2016 by applying a dual stable isotope approach (δ15N-NO3-and δ18O-NO3-) combined with multiple statistical analyses. The sources of riverine nitrate principally originated from manure and sewage, nitrification of soil nitrogen, fertilizer nitrification, and atmospheric deposition. A Bayesian model was used to estimate the source contributions and results showed that manure and sewage were the major contributors to river nitrate with combined proportions of 77.59% and 89.57% in the rainy season and the dry season, respectively. Results from multiple stepwise regression indicated that the typical artificial land use types and landscape configuration metrics reflecting landscape fragmentation related well with riverine nitrogen variables for different seasons (R2>0.6). Industrial land, unused land and patch density of built-up land and road were positively correlated with riverine nitrogen over seasons, whereas the interspersion and juxtaposition index of forest land was negatively related with nitrate. Regulating built-up land and unused land, connecting forest land with other land use types and diminishing discharges of industrial and domestic wastewater would be effective ways to improve urban river water quality.

Natural Killer Group 2A Expressed on Both Peripheral CD3-CD56+NK Cells and CD3+CD8+T Cells Plays a Pivotal Negative Regulatory Role in the Progression of Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

To explore the role of surface receptors natural killer group 2A (NKG2A) and natural killer group 2D (NKG2D) on CD3+CD8+T cells and CD3-CD56+NK cells in the progression of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF), we measured the expression of NKG2A and NKG2D on the surface of these 2 types of circulating cells by flow cytometry in 3 groups. One group consists of 36 patients with chronic hepatitis B (CHB), another one consists of 22 patients with HBV-related ACLF, and the last one has 12 normal controls (NC). The experimental result indicated that there was no significant difference in the proportion of CD3+CD8+T cells in total lymphocytes between the 3 groups. However, the percentage of CD3-CD56+NK cells in ACLF group was evidently higher than that in the CHB group (P < 0.05). In addition, the expression of NKG2D on CD3+CD8+T cells in the ACLF group was significantly lower than that in the CHB group (P < 0.05), but there were no statistically significant differences in its percentages on CD3-CD56+NK cells between the 3 groups. The expression of NKG2A on CD3+CD8+T cells in the ACLF group was significantly higher than that in the NC group (P < 0.05), and on NK cells was significantly higher than that in the CHB group (P < 0.05) and NC group (P < 0.01). The increase in ratios of NKG2A to NKG2D on CD3+CD8+T cells and CD3-CD56+NK cells in the ACLF group was significantly more than that in the CHB group and NC group. The results indicate that the imbalance between NKG2A and NKG2D may contribute to the progression of HBV-related ACLF mediated by CD3-CD56+NK cells and CD3+CD8+T cells. Compared with NKG2D, NKG2A expressed on both peripheral CD3-CD56+NK cells and CD3+CD8+T cells plays a more pivotal negative regulatory role in the progression of HBV-related ACLF.

[Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells].

OBJECTIVE: To construct an eukaryotic expression vector containing human CD40 gene for its efficient, continuous and stable expression in human umbilical vein endothelial ECV-304 cells. METHODS: The recombinant plasmid pUCD40 was digested with endonucleases to obtain human CD40 gene fragment, which was cloned into pCDNA3.1 vector to construct recombinant eukaryotic expression vector pCDNA3.1(+)/CD40. The recombinant vector was identified by enzyme digestion before introduced into ECV-304 cells via liposome, with the positive cell clones selected with G418. The stable transfection and expression of CD40 in ECV-304 cells were identified by reverse transcription (RT)-PCR, Western blotting and flow cytometry, respectively. RESULTS: Enzyme digestion analysis showed that target gene had been cloned into the recombinant vector. The transfected ECV-304 cells successfully expressed human CD40 as determined by RT-PCR and Western-blotting, and 95% of the cells were CD40-positive as shown by flow cytometry. CONCLUSION: The recombinant eukaryotic expression vector pCDNA3.1(+)/CD40 has been successfully constructed, which is capable of stable transfection and expression of CD40 in ECV-304 cells to facilitate further investigation of the roles of CD40 molecule in antiatherosclerotic drug development.

Clenbuterol Assay by Spectral Imaging Surface Plasmon Resonance Biosensor System.

To prevent illegal use of clenbuterol and for quality control in the food industry, more efficient and reliable methods for clenbuterol detection are needed. In this study, clenbuterol was detected using a spectral imaging surface plasmon resonance sensor system via two inhibition methods: (1) the target site compensation method, in which anti-clenbuterol antibody was immobilized on the sensor chip as a bioprobe and (2) the solution competition method in which a clenbuterol-BSA conjugate was immobilized on the sensor chip as the bioprobe. The detectable clenbuterol concentration ranged between 6.25 and 100 µg/mL for both methods. The clenbuterol limit of detection for the target site compensation method and solution competition method are estimated to be 6.7 and 4.5 µg/mL, respectively. The proposed methods were successfully applied to the detection of clenbuterol molecules and were found to have high specificity and high-throughput and were label free and operationally convenient.

[Establishment and characterization of a new squamous cell carcinoma cell line CS1213 from the human uterine cervix].

OBJECTIVE: To establish a human cervical carcinoma cell line. METHODS: A primary culture was initiated from malignant tissue collected by dissection of cervical biopsy specimens. Characterizing cells in culture which included morphological observation, biological and karyotypic analysis, experimental tumorigenesis and the expression of p53, bcl-2 and Ki67 genes was carried out. RESULTS: The new established cervical carcinoma cell line (CS1213) had been maintained in culture for over 170 generations. The cells which were nonadherent had a common, rounded appearance with a cell cycle time of 25-hour and a 19 colony formation rate in soft agar. Electron micrographs demonstrated abundant tonofilaments in the cytoplasm. The karyotype showed a hyperdiploid feature with a main chromosome stem number ranged from 80 to 88. The culture was not contaminated by mycoplasma and had a distinct lactic acid dehydrogenase isozyme pattern. High expression level of p53 (31.9%), bcl-2 (89.3%) and Ki67 (33.7%) proteins was detected by flow cytometry. The xenogeneic tumors were grown in nude mice with the histological structure of the original one. CONCLUSIONS: The novel CS1213 cells have the characteristics of human cervical squamous cells and could be used as an appropriate cellular model system for studying tumor invasion and metastasis.

[Study on expression of cell cycle-related genes in subclonal cell lines of human cervical carcinoma].

OBJECTIVE: To explore the role of expression of cell cycle-related genes in cervical carcinoma cell lines. METHODS: A series of expression microarray analysis of two homologous cervical carcinoma subclonal cell lines were initiated by cDNA microarray which represent a set of 234 human cell cycle-related genes. RESULTS: In normal medium, the percent of G(1) phase in CS03 cells was higher than in CS07 cells dramatically, but the percent of S phase in CS07 cells was more than in CS03 cells. After cultured 48 h in serum-free medium, the percent of apoptosis cells (sub-G(1) phase) in CS03 cells was higher than in CS07 cells significantly and increased with time. By applying this cDNA microarray, we identified 3 differentially expressed genes in two homologous cell lines, which were BN51, hsp90 and Mcl-1 genes, further identified 3 upregulated genes in CS07 cell line, the ratio of Cy5/Cy3 was 0.480, 0.479 and 0.490 respectively. CONCLUSION: Differential expression of BN51, hsp90 and Mcl-1 genes is associated to various growth patterns of human cervical carcinoma cells.

Elevated serum IL-21 levels in hantavirus-infected patients correlate with the severity of the disease.

Hemorrhagic fever with renal syndrome (HFRS) is an acute viral infection caused by Hantavirus (HTV). Capillary leakage is one of the hallmarks of HTV infection. The mechanisms underlying the pathogenesis of HFRS are not completely understood. However, it has been suggested that immune mechanisms, including cytokines, might have an important role in HFRS pathogenesis. In this study, we investigated the potential role of interleukin-21 (IL-21) which is a newly discovered cytokine that stimulates T-cell and B-cell responses in the pathogenesis of HFRS. Serum samples were collected from 58 patients hospitalized with laboratory-verified HTV infection and 20 healthy controls. Serum IL-21 concentration was measured using an enzyme-linked immunosorbent assay. Serum IL-21 levels began to increase in the fever phase when renal damage appeared. The highest serum IL-21 level was detected in oliguric phase along with the peak degree of urinary renal impairment. When entering the polyuric phase, with gradual increase in urine and recovered renal function, the serum IL-21 level was observed to fall, returning to normal level after the renal function recovered in the convalescent phase. The serum IL-21 level was positively correlated with blood urea nitrogen (BUN) and creatinine (Cr), suggesting that the serum IL-21 level is associated with the disease severity of HFRS. This study indicated that IL-21 may act as an important inflammatory mediator in the immunopathogenesis of HFRS. The strategy to control IL-21 may hamper the immune response in patients with HFRS.

Serum IL-21 levels associated with chronic hepatitis B and hepatitis B-related liver failure.

The aim of the present study was to investigate the role of interleukin (IL)-21 in chronic hepatitis B virus (HBV) infection. IL-21 stimulates T and B cell responses and plays a role in the control of chronic viral infections. Serum IL-21 levels were measured by enzyme immunoassay in 109 patients with chronic HBV infection at various clinical stages, as well as in 19 healthy controls (HCs). The proportion of T cells producing IL-21 in the peripheral blood was assessed by intracellular cytokine staining and flow cytometry. Mean serum IL-21 levels in patients with chronic hepatitis B (CHB) and the HCs were 303.54±152.77 pg/ml and 68.24±9.06 pg/ml, respectively (P=0.003). In addition, the mean serum IL-21 level in patients with hepatitis B-related acute-on-chronic liver failure (HB-ACLF) was 455.38±412.38 pg/ml, which exhibited a statistically significant difference when compared with the HCs (P=0.000). Serum IL-21 levels were highest in the patients with HB-ACLF (455.38±412.38 pg/ml) and exhibited a significant difference when compared with the CHB patients (P=0.04). The mean serum IL-21 levels in patients with cirrhosis also increased, but there was no statistically significant difference when compared with the HCs (P=0.82). The frequency of IL-21+CD4+ cells also increased compared with the HCs and correlated with the number and percentage of lymphocytes in the peripheral blood. Serum IL-21 levels increased in CHB and HB-ACLF patients. Relatively low serum IL-21 levels in CHB may have a causal role in the persistence of HBV infection. Higher serum levels in HB-ACLF may activate T and B cells to eliminate the virus or injure the liver via the release of inflammatory cytokines.

Detection of HLA-B*27 gene using a spectral plasmon resonance imaging system.

Detection of HLA-B*27 gene is clinically important due to its strong association with diseases, such as ankylosing spondylitis. Nucleic acid-based biosensors represent a promising clinical tool for gene diagnosis. Surface plasmon resonance (SPR) can be used to monitor DNA-DNA hybridization in real-time and without any prior labeling step. Here, a self-built HLA-B*27 positive PCR products spectral SPR imaging system was used for multichannel direct-detection of a specific sequence of the HLA-B*27 gene. Thiol-labeled single-stranded oligonucleotide probes, which were proved to be superior to amine-labeled probes in immobilization, were immobilized onto the surfaces of the gold spots on the sensor chip to target the specific sequence in the HLA-B*27 gene in blood. SPR measurements were performed with different concentrations of synthetic target DNA sequence. The calibration curve of synthetic target sequence showed a good linear relationship between the concentration of the synthetic target sequence and the shift of the SPR wavelength from 10 nM to 500 nM with a detection limit of 5 nM. The HLA-B*27 gene was isolated from human whole blood and amplified using polymerase chain reaction (PCR). PCR products were measured using the SPR imaging system. HLA-B*27 positive PCR products showed significant SPR wavelength shift, while synthetic non-complementary sequence and negative PCR products showed no significant SPR wavelength shift. The method is high-specific, high-throughput and label-free.

Restoring the Treg cell to Th17 cell ratio may alleviate HBV-related acute-on-chronic liver failure.

AIM: To investigate the role of T helper 17 cells (Th17) and regulatory T cells (Treg) in hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF). METHODS: We enrolled 79 patients with HBV infection into the study, 50 patients with HBV-related ACLF and 29 patients with chronic hepatitis B (CHB), from the First Affiliated Hospital of Medical College from January 2009 to June 2012. The ACLF patients were diagnosed according to the criteria recommended by The 19(th) Conference of the Asian Pacific Association for the Study of the Liver in 2009. Twenty healthy individuals with a similar gender and age structures to the two patient groups were also included as the normal controls (NC). Of the 50 ACLF patients, 28 were subsequently classified as non-survivors: 19 patients died from multi-organ failure, 3 underwent liver transplantation, and 6 discontinued therapy during follow-up because of financial reasons. The remaining 22 ACLF patients whose liver and anticoagulation function recovered to nearly normal levels within the next 6 mo were classified as survivors. The number of circulating Treg and Th17 cells was determined upon diagnosis and during the 8th week of follow-up through flow cytometry. RESULTS: The percentage of circulating Treg cells in the ACLF group was significantly higher than that in the CHB group (5.50% ± 1.15% vs 3.30% ± 1.13%, P < 0.01). The percentages of circulating Th17 cells in the ACLF and the CHB groups were significantly higher than that in the NC group (6.32% ± 2.22% vs 1.56% ± 0.44%, P < 0.01; 3.53% ± 1.65% vs 1.56% ± 0.44%, P < 0.01). No significant difference in Treg cell to Th17 cell ratio was observed between the ACLF group and the CHB group (0.98 ± 0.44 vs 1.12 ± 0.64, P = 0.991), whereas those in the two HBV infection groups were significantly lower than that in the NC group (1.85 ± 1.22; both P < 0.01). The percentage of Treg cells in the survivors during the 8(th) week of follow-up was significantly lower than that during peak ACLF severity [total bilirubin (TBIL) peak] (3.45% ± 0.97% vs 5.18% ± 1.02%, P < 0.01). The percentage of Th17 cells in survivors during the 8(th) week of follow-up was significantly lower than that during the peak TBIL (2.89% ± 0.60% vs 5.24% ± 1.46%; P < 0.01). The Treg cell to Th17 cell ratio during the 8(th) week of follow-up was significantly higher than that during the TBIL peak (1.22 ± 0.36 vs 1.10 ± 0.54; P < 0.05). CONCLUSION: Restoring the Treg cell to Th17 cell ratio during the follow-up phase of ACLF could maintain the immune system at a steady state, which favours good prognosis.

Identification of cyclophilin A as a potential prognostic factor for clear-cell renal cell carcinoma by comparative proteomic analysis.

The high recurrence and improved survival rate of clear-cell renal cell carcinoma (ccRCC) demand for continuous effort to search for novel prognostic factors. Herein a comparative proteomics approach based on two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to identify differentially expressed proteins between ccRCC cell line RLC-310 and normal renal cell line HK-2. Of the 31 proteins identified, Cyclophilin A (CypA) was a newly identified differentially expressed protein in ccRCC cell line. The overexpression of CypA in ccRCC tissues was confirmed using RT-PCR and Western blot analyses. Further immunohistochemistry revealed that overexpression of CypA was associated with poor differentiation and decreased survival (p < 0.05, p < 0.0001). These data suggest that CypA may serve as a novel prognostic factor for ccRCC.

[Differential expression of immune-associated genes in two subcloned cell lines from a same human bladder cancer].

AIM: To screen and identify differentially expressed genes in subcloned lines from a same human bladder transitional cell carcinoma (TCC). METHODS: Two bladder TCC cell lines (BLX and BLS-211) with different phenotypes but same origin were used to screen for differentially expressed genes by suppression subtractive hybridization (SSH). RESULTS: 9 over-expressed genes in BLX and 15 in BLS-211 cells were obtained, respectively. Some Bacillus Galmette-Guerin(BCG)- associated genes, such as BCG induced integral membrane protein(BIGM103), fibronectin(FN), complement factor B(BF), were over-expressed in BLX cells. And 8 new ESTs(Expressed Sequence Tag) in BLS-211 cells were collected by GenBank dbEST database with the accession number of DY505708-13, DY230447-8. CONCLUSION: SSH is a powerful method for the identification of differentially expressed genes in different cell lines or clones. Some BCG-associated genes, which are differentially expressed in different cells may contribute to the different response to clinical BCG therapy. The identified new ESTs can be cloned for full length to further study their functions.