The present study examined the effects of sustained anticipatory anxiety on the affective modulation of the eyeblink startle reflex. Towards this end, pleasant, neutral and unpleasant pictures were presented as a continuous stream during alternating threat-of-shock and safety periods, which were cued by colored picture frames. Orbicularis-EMG to auditory startle probes and electrodermal activity were recorded. Previous findings regarding affective picture valence and threat-of-shock modulation were replicated. Of main interest, anticipating aversive events and viewing affective pictures additively modulated defensive activation. Specifically, despite overall potentiated startle blink magnitude in threat-of-shock conditions, the startle reflex remained sensitive to hedonic picture valence. Finally, skin conductance level revealed sustained sympathetic activation throughout the entire experiment during threat- compared to safety-periods. Overall, defensive activation by physical threat appears to operate independently from reflex modulation by picture media. The present data confirms the importance of simultaneously manipulating phasic-fear and sustained-anxiety in studying both normal and abnormal anxiety. A large body of evidence supports the notion that the startle reflex is modulated by defensive system activation. When anticipating danger, the startle reflex is potentiated as compared to control conditions, for instance in aversive conditioning paradigms Learning about aversive events is critical in organizing defensive behavior. Accordingly, the mere verbal instruction about potential threats is sufficient to prime defensive response programs Building upon these findings, the present study examined coincident effects of sustained periods of anticipatory anxiety (72 s) and emotional pictures presented as a continuous stream (4 s), when both manipulations were unrelated. Measuring startle reflexes, the main purpose was to explore whether the concurrent activation of motivational systems by phasic picture cues and sustained periods of unpredictable threat-of-shock operate simultaneously but independent from each other, or whether they exert synergistic effects M = 22) recruited from University of Granada. Because of excessive noise in orbicularis oculi EMG, 3 participants  were excluded from startle data analyses.Participants were 36 healthy volunteers  between the ages of 18 to 27  M = 6.3 and 5.8, neutral M = 5.4 and 3.3, unpleasant M = 1.9 and 6.6), Fs = 271.35 and 154.49, ps <.001. All post hoc comparisons were significant, ps <.01.Highly arousing emotional picture contents were selected, as these materials elicit most pronounced modulations in defensive reflex, autonomic measures, and brain imaging studies The IAPS pictures (640×480 pixels) were fleetingly presented for 4 s without perceivable inter-stimulus interval see . IAPS piStartle responses were provoked by 105 dB, 50 ms white noise with instantaneous rise time, produced by Coulbourn V85–05 noise generator , gated through IMG Stage-Line® amplifier and presented over matched PPA-1 headphones. Probes were delivered 1, 1.5 or 2 s after picture onset in 72 trials, equally often for each picture category within each block (15.4 s mean distance between startle probes).Pictures were presented on a 22-inch monitor located 1 m in front of the participants. Electrical pulses  were generated by a Letica-shock-module , and administered to the left forearm during shock-workup procedure. Stimulus control and physiological data acquisition were accomplished using VPM Eyeblinks were recorded electromyographically from the orbicularis oculi muscle with Ag/AgCl-electrodes. Raw EMG signals were amplified (5K) and bandpass filtered (13–1000 Hz), using Coulbourn V75–04 bioamplifier, then rectified and integrated on-line using Coulbourn V76–23 module (time constant 20 ms). The sampling rate for the integrated signal was 1000 Hz, recorded from 50 ms prior to 300 ms after probe onset. A computer program scored startle blink magnitude peak and onset latency interactively controlled while blinded to the conditions Skin conductance activity was recorded through Ag/AgCl-electrodes, placed on the hypothenar eminence of the left palm, using Coulbourn V71–23 module with a sampling rate of 20 Hz. Averages were computed off-line for each 4 s picture period and tonic changes were determined by subtracting activity in 3-s before the first picture onset (within each block) from the means across periods. Logarithms of raw scores (log(change+10)) were computed for statistical analyses for non-startle trials only . To parallel a previous study utilizing ERP measures After sensors were attached, 12 practice trials were presented, including pictures, frames and two initial startle probes . Then the shock electrode was placed and a brief workup procedure was carried out to ensure credibility of the threat-of-shock instruction. In order to set the shock intensity individually at a level rated as “maximal unpleasant but not painful” participants received up to ten shocks with increasing intensity preceding the experiment t-tests for valence and arousal ratings were conducted on self-reports of threat and safety conditions.Separate To assess combined effects of threat-of-shock and picture valence on the startle reflex, repeated measures ANOVAs including the factors Picture Category  and Condition  were performed. Furthermore, to examine the time course of threat-of-shock and affective picture modulation, an additional factor (Time) was included by averaging the beginning, middle and last part of the experiment . The resulting statistical design was Picture Category (3)×Condition (2)×Time (3).Skin conductance changes were analyzed with repeated measures ANOVAs, including the factors Condition (2) and Time (3) summarizing the beginning, middle and last part of the experiment . Greenhouse-Geisser corrections were applied where relevant.M = 2.83, SD = 1.2) than safety periods , t(35) = −8.81, p<.001. In addition, threat-of-shock condition was rated as more arousing  than safety condition , t(35) = 7.44, p<.001.Participants perceived threat-of-shock periods as more unpleasant  = 65.76, p<.001. In addition, startle reflex was modulated by picture valence, F = 8.13, p<.001, ε = .98. Post-hoc tests revealed that blink magnitude for unpleasant pictures was potentiated as compared to pleasant pictures, F = 17.38, p<.001, and larger but not significantly different from neutral pictures, F = 2.08, p = .16. Moreover, blink magnitude for pleasant pictures was inhibited in contrast to neutral pictures, F = 6.33, p<.05.F <1  >31.92, ps <.001. Interestingly, threat-of-shock potentiated startle for both pleasant and neutral pictures exceeded significantly the startle potentiation for unpleasant pictures in safety condition, Fs >12.88, ps <.01. Additionally, startle modulation by picture valence was present in both safety and threat-of-shock conditions, Fs >3.37, ps <.05, ε = .91 and.99.Of main interest, the interaction of hedonic picture valence and instructed threat condition was not significant, F <1 see . ExploraFs<1.Including the additional factor Time revealed that neither the three-way interaction Category×Condition×Time nor the Category×Condition interaction (tested separately for each time window) approached significance, all F = 323.58, p<.001, ε = .95. Furthermore, threat-of-shock and picture valence effects varied across time, Time×Condition F = 13.24, p<.001, ε = .81, Time×Picture Category, F = 3.4, p<.05, ε = .67. Threat-of-shock effects were pronounced during the first, F = 45.58, p<.001, and second time period, F = 53.76, p<.001, while still significant in the third time period, F = 14.19, p<.001. Picture category effects were also reduced across time. Specifically, affective modulation of blink magnitude was significant at the first, F = 4.8, p<.05, ε = .91, second, F = 10.6, p<.001, ε = .98, but not the third time period, F = 1.28, p = .28, ε = .99.As expected, startle blink magnitude decreased along the experiment due to habituation, F = 14.45, p<.001  = 6.01, p<.05, second, F = 13.07, p<.001, and third time period of the experiment, F = 6.62, p<.05.Tonic electrodermal changes were enhanced during threat-of-shock compared to safety conditions, .001 see . This diF = 14.35, p<.001. Skin conductance decreased over Time, F = 6.74, p<.01, but there was no interaction of Condition×Time, F <1.A supplementary analysis of the electrodermal data for the excluded trials  revealed augmented skin conductance for threat as compared to safety periods, The present study examined the modulation of the eyeblink startle reflex as a function of sustained anticipatory anxiety and affective picture valence. Key findings regarding both manipulations were replicated. Specifically, the startle blink magnitude was potentiated when anticipating a rather unpredictable aversive event Investigating threat-of-shock and emotional picture processing, Bradley and colleagues The finding of an additive relationship between threat-of-shock and picture valence may be specific to motor output stages. Similar to the current design, a recent study examined the perceptual and evaluative processing of emotional pictures by measuring event-related potentials Both sustained threat-of-shock periods and phasic processing of unpleasant pictures elicited potentiated startle reflexes in the current study. Thus, the startle reflex remained sensitive to picture valence even in a threatening context. Several findings support the notion that threat-of-shock is more powerful in activating the human defense system compared to emotional picture media These findings may be interpreted from the perspective of the defense cascade model The possibility to assess both aversive anticipation and emotional picture effects simultaneously may be informative in the study of the anxiety disorder spectrum Several limitations of the present study need to be acknowledged and may be addressed in future research. First, the temporal design features  prevented the direct analysis of the interaction of threat-of-shock and picture valence by means of skin conductance data. Accordingly, accounting for the present hypothesis of concurrent but independent activation of motivational systems by fleeting picture cues and sustained threat signals, additional measures of phasic sympathetic activation  would be needed. Finally, to elucidate effects of habituation (picture repetition) and extinction (threat repetition) on autonomic and reflex activity the usage of block designs  and threat/safety periods varying in predictability would be highly informative In summary, anticipating an aversive event and passively viewing unpleasant pictures potentiated the startle reflex magnitude. When these two avenues that activate the human defense system – threat-of-shock and emotional picture media – coincide but have no inherent relationship, the startle reflex is sensitive to both manipulations. Despite similar and presumably shared neural structures and pathways


We previously identified common differentially expressed (DE) genes in bladder cancer (BC). In the present study we analyzed in depth, the expression of several groups of these DE genes.Samples from 30 human BCs and their adjacent normal tissues were analyzed by whole genome cDNA microarrays, qRT-PCR and Western blotting. Our attention was focused on cell-cycle control and DNA damage repair genes, genes related to apoptosis, signal transduction, angiogenesis, as well as cellular proliferation, invasion and metastasis. Four publicly available GEO Datasets were further analyzed, and the expression data of the genes of interest (GOIs) were compared to those of the present study. The relationship among the GOI was also investigated. GO and KEGG molecular pathway analysis was performed to identify possible enrichment of genes with specific biological themes.−31).Unsupervised cluster analysis of DNA microarray data revealed a clear distinction in BC vs. control samples and low vs. high grade tumors. Genes with at least 2-fold differential expression in BC vs. controls, as well as in non-muscle invasive vs. muscle invasive tumors and in low vs. high grade tumors, were identified and ranked. Specific attention was paid to the changes in osteopontin  expression, due to its multiple biological functions. Similarly, genes exhibiting equal or low expression in BC vs. the controls were scored. Significant pair-wise correlations in gene expression were scored. GO analysis revealed the multi-facet character of the GOIs, since they participate in a variety of mechanisms, including cell proliferation, cell death, metabolism, cell shape, and cytoskeletal re-organization. KEGG analysis revealed that the most significant pathway was that of Bladder Cancer (p = 1.5×10The present work adds to the current knowledge on molecular signature identification of BC. Such works should progress in order to gain more insight into disease molecular mechanisms. The remainder present with invasion of the muscular layer of the bladder wall or extend to perivesical tissue, adjacent organs and the pelvic wall. More than 60% of the superficial tumors will recur at least once and progress to less differentiated or invasive neoplasms with worse prognosis in a significant percentage of patients Urinary bladder cancer (BC) is the most common malignancy of the urinary tract, responsible for significant mortality and morbidity worldwide. In Europe, an estimated 105,000 new cases of bladder cancer are diagnosed annually, while approximately 30,000 patients die from bladder cancer each year We previously identified common differentially expressed (DE) genes in urinary BC Our results focused on genes which play a significant role in the most important cellular processes and demonstrated a large difference in their expression patterns between BC and control samples. Genes with at least 2-fold differential expression in BC vs. controls, as well as in non-muscle-invasive vs. muscle-invasive tumors, and in low vs. high grade tumors, were identified and ranked.Paired tumor and normal tissue samples from a consecutive series of 30 patients with newly diagnosed BCs undergoing transurethral bladder tumor resection at the Department of Urology, “Asklipieio” General Hospital, Athens, were acquired after the amount of tissue necessary for routine pathology examination had been removed Table 1.Tissue samples were obtained at surgery from the tumor and the following three grossly normal selected sites (cold cup biopsies): posterior wall, trigone, and the area adjacent to the tumor. Parts of the resected normal samples were sent for histopathological analysis. Tumor and normal tissues were frozen immediately in liquid nitrogen, transported and stored at −80°C until DNA extraction.Patients with non-muscle-invasive BCs were followed up with periodical cystoscopic examinations and intravesical treatment as indicated. Patients with invasive BCs were offered radical cystectomy with or without systemic chemotherapy. After a mean follow-up of 24±3 months, 8 (26.6%) patients had recurrent tumors. In Ta/T1 tumors the frequency of recurrence was 29.4% (5/17) compared with 23% (3/13) of T2-T3 tumors. In patients with non-muscle-invasive BCs, the progression rate was 11.1% and 22.2% for grade 2 and grade 3 tumors, respectively. All recurrences were proven by biopsy.2O2 at room temperature for 15 min. Antigen unmasking was performed by 30 min of incubation at 80°C in 10 mM trisodium citrate (pH 6.1). Immunostaining and revelation were performed on a Dako automate. Slides were incubated at room temperature with primary polyclonal goat antibodies against anti-ErbB2 , cyclin D1 , monoclonal antibody against anti-p53  and monoclonal antibody against anti-Ki-67 . Epitopes of the primary antibody were localized by the immunoperoxidase technique using the secondary antibody avidin-biotin complex and peroxidase substrate kit , according to the manufacturer's protocol. The sections were then treated with Chromagen 30-30 diaminobenzidene tetrahydrochloride to identify sites of immunoprecipitation by light microscopy. Finally, sections were washed, counterstained with hematoxylin, and mounted under coverslips. No specific staining was observed when the primary antibody was omitted from the protocol (negative control). Specificity of the immunostaining was additionally controlled by simultaneous staining of breast cancer samples with known ErbB2, cyclin D1, p53 and Ki67 expression patterns. An experienced pathologist scored the staining intensity at four levels , considering both color intensity and number of stained cells. In brief, the proportion score represented the estimated percentage of positive-stained tumor cells , and the intensity score represented the depths of the tumor cell staining .Sections, 3-mm thick, of formalin-fixed, paraffin-embedded tissue were cut and placed on slides coated with 3-aminopropyltriethoxysilone. Slides were dried at 56°C for 1 h before immunohistochemical staining. Tissue sections were deparaffinized in xylene before rehydration in graded alcohols, and endogenous peroxidase activity was blocked by treatment with 3% HWe isolated total RNA from crude tumor biopsy samples using a power homogenizer and the TRIzol® reagent , as previously described http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE27448). Furthermore, each gene was tested for its significance in differential expression using a z-test. Genes were considered to be significantly differentially expressed if they obtained a p-value <0.05. The False Discovery Rate was calculated as described previously Oligos microarray chips (∼57K genes) were obtained from GE HealthCare (IL) and AppliedMicroarrays (MA) (CodeLink 57K Human Whole Genome). Hybridization was performed with the CodeLink RNA amplification and Labeling kit, utilizing the Cy5 fluorescent dye. Slides were scanned with a microarray scanner (ScanArray 4000XL). Images were generated with ScanArray microarray acquisition software . cRNAs from three experimental setups were used in single experiments with internal spikes as controls. The experimental setups consisted of 10 urinary BC samples of different histologies  and 5 control samples. The scanned images were further processed with the CodeLink Expression Analysis Software v5.0 from Amersham Biosciences. The experimental setup was analyzed based on the reference design as described previously Table S1. GAPDH and ACTB genes were used as internal controls Transcribed products were subjected to real-time PCR assay with SYBR Green I in an Mx3000P programmable thermal controller apparatus . The primer pairs were designed to span at least one intron in order to avoid amplification of the contaminating genomic DNA along with cDNA. Their sequence and the corresponding PCR product sizes are listed in 2), 300 nM of each primer and 30 µM Rox passive reference dye in a final volume of 20 µl. After initial denaturation at 95°C for 10 min, samples were subjected to 40 amplification cycles comprising denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and elongation at 72°C for 30 sec. Amplification and elongation steps were followed by a melt curve analysis in which the temperature was increased from 55°C to 95°C at a linear rate of 0.2°C/sec. Data collection was performed during both annealing and extension, with two measurements at each step and at all times during melt curve analysis. To verify the results of the melt curve analysis, qPCR products were analyzed by electrophoresis on 2% agarose gel, stained with ethidium bromide and photographed on a UV light transilluminator . In each qPCR reaction two negative controls were included, one with no cDNA template and one with no reverse transcription treatment. All samples were treated in duplicate. Gene transcription levels were calculated using the ΔΔCt method, as previously described One microliter of cDNA from normal or TCC samples, respectively, was amplified in a PCR reaction with 2x Brilliant SYBR® Green QPCR Master Mix , the homogenized tissue samples were centrifuged at 14,000 x g for 15 min at 4°C, to remove insoluble material, and the supernatant was collected and stored at −80°C. Protein concentration of each sample was determined by the method of Bradford using a protein assay dye reagent . Samples were dissolved in LDS sample buffer (Invitrogen) and heated at 70°C for 7 min. Equal amounts of the samples (20–30 µg) were electrophoresed in NuPAGE 4–12% Bis-Tris gel (Invitrogen) and transferred to a 0.45- µm nitrocellulose membrane . The membrane was immersed in 5% nonfat milk or BSA, 0.1% Tween 20 and dissolved in Tris-buffered saline to block the nonspecific binding. The membranes were incubated overnight at 4°C with the following primary antibodies: mouse polyclonal anti-HRAS , mouse monoclonal anti-CDKN2A , mouse monoclonal anti-p53 , mouse monoclonal anti-VEGFA , rabbit polyclonal anti-TGFβ1  and mouse monoclonal anti-OPN . The membranes were then washed and incubated for 90 min. at RT with a secondary antibody that included horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG . Western blots were normalized using a monoclonal anti-β-actin antibody . The specific signals were visualized by ECL reagent  after exposition to ECL film. The relative density of the polypeptide bands detected on the ECL film was determined using the spot denso tool of the AlphaEaseFC software.After addition of 250 µl ice-cold GST-Fish lysis buffer , 100 mM NaCl, 1% (v/v) Nonidet P-40, 2 mM MgClARF, p16INK4A, p53, KRAS, HRAS, NRAS, ARAF, BRAF, RAF1, RKIP, MMP2, MMP9, TIMP1, TIMP2 and CyclinD1 transcript levels among different urinary bladder cancer types, its metastatic counterpart and the relative normal tissue. Four publicly available Gene Expression Omnibus (GEO) datasets were analyzed for this reason, with GEO series accession numbers GSE89, GSE7476, GSE3167 and GSE12630 2 intensity (Table S2).Computational analysis was performed to further investigate the differences in the OPN, VEGFA, TGFβ1, FGF2, EGFR, EGF, p14http://www.genome.jp/kegg/pathway.html).Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) molecular pathway analysis were performed to identify possible enrichment of genes with specific biological themes . The Mann-Whitney U test was used to examine the expression status of the genes with the various clinicopathological parameters after stratification. The Kaplan-Meier method was used to estimate overall survival as a function of time. Survival differences were assessed by Log-rank (Mantel Cox) and Gehan-Breslow-Wilcoxon tests. Numerical values are expressed as the mean±standard deviation (SD), and medians. Data extracted from the GEO databases were statistically compared by the Mann-Whitney U test. Statistical significance was set at the 95% level (p<0.05). All statistical analyses were performed with SPSS 11.5 .Tumor samples were stained with antibodies for ErbB2, cyclin D1, p53 and Ki-67 Figure 1ARF/p16INK4A), MMP9 and MKI67, in 10 BC samples vs. 5 controls. A detailed view of the sample cluster dendrogram is displayed in 2 transformed fold expression pattern of the GOI and partitioned the tumors into 3 main groups based on the differential expression of these 22 genes: The first principle branch contained T1-grade 3, T2-grade 3, T1-grade 2 and T3-grade 3 tumors. The second branch consisted of T1-grade 2 and T2-grade 3 tumors. The third branch consisted of tumors with in situ carcinoma, T2-grade 3 (CIS). The 22-gene set was divided into two main clusters. The first cluster was composed of genes that were over-expressed  and the second cluster contained genes that were equally or under-expressed  in TCC vs. normal tissue. The relative expression levels of the GOIs in each tumor group were expressed as a ratio of the normalized expression levels from the tumor group to the mean levels of the normal tissues, which was set to 1.0 (Table S2).We previously identified 831 genes that were differentially expressed in all 10 BC samples, simultaneously. Of these, 33 genes were up-regulated and 85 genes were down-regulated in all bladder cancer samples compared to the 5 normal tissues, simultaneously (data not shown). In the present study, we performed two-way average-linkage hierarchical clustering with Euclidian distance for a 22-gene set that included the following genes: VEGFA, ARAF, BRAF, OPN (SPP1), MMP2, KRAS, NRAS, TGFβ1, AKT1, HRAS, TIMP1, EGF, RKIP (PBP), FGF2, EGFR, RAF1, CDKN2D, TP53, CDKN2A .The fold expression (mean±SD) of the GOIs in each one of the three tumor groups compared to the normal tissue, as acquired by our microarray analysis, is depicted in (ARF/p16INK4A), MMP9 was determined by qPCR in both bladder cancer and normal tissue (Table S3). Scatter plots were constructed for better visualization of the genes that were up-regulated (>2-fold), down-regulated (<2-fold) or equally expressed (2-fold difference threshold) between BC and controls. Volcano plots graphing the log2 of the fold change in the expression of each gene between BC samples vs. its p-value from the t-test, were also constructed , MMP2, KRAS, NRAS, TGFβ1, AKT1, HRAS, EGF, RKIP (PBP), FGF2, EGFR, RAF1, TP53, CDKN2A  . The mean mRNA expression values between BC and normal tissue for each gene are depicted in The genes OPN, VEGFA, TGFβ1, p16ARF, AKT1, HRAS, ARAF, BRAF, RAF1, MMP9, EGFR and KRAS, did not differ significantly between BC and control tissue . The mean mRNA expression values between BC and normal tissue for each gene, were as follows: p14ARF, 0.0325±0.0488 vs. 0.0100±0.0118 (p = 0.1087); AKT1, 0.0321±0.0230 vs. 0.0262±0.0174 (p = 0.3632); HRAS, 0.0015±0.0021 vs. 0.0015±0.0021 (p = 0.9752); ARAF, 0.9931±0.0047 vs. 0.9942±0.0040 (p = 0.5201); BRAF, 0.8933±0.0657 vs. 0.9053±0.0486 (p = 0.8999); RAF1, 0.9348±0.0527 vs. 0.9419±0.0293 (p = 0.6681); MMP9, 0.0014±0.0015 vs. 0.0023±0.0054 (p = 0.1265); EGFR, 0.0078±0.0073 vs. 0.0049±0.0048 (p = 0.0948); KRAS, 0.0876±0.0997 vs. 0.0773±0.0900  .The mRNA expression levels of the genes p14Figure S3C).EGF, FGF2 and MMP2 exhibited significant under-expression in BC vs. normal tissue: EGF, 0.00004±0.000047 vs. 0.000151±0.000235 (p = 0.0017); FGF2, 0.0003±0.0004 vs. 0.0023±0.0019 (p<0.0001); MMP2, 0.0752±0.0858 vs. 0.1341±0.0834  , HRAS and TP53, was also verified at the protein level, by densitometric analysis of the Western blots (The expression of OPN (SPP1), TGFβ1, VEGFA, CDKN2A  test; p = 0.0041, χ2 = 8.260, Gehan-Breslow-Wilcoxon test]. Moreover, patients with grade 2 tumors showed a better overall survival vs. those of grade 3 tumors .A total of 30 urinary bladder cancer cases were investigated for overall survival rates. Patients with tumors of stage 1 presented better overall survival compared to those of stage 2 or 3 . Patients with high TGFβ1, FGF2, p142 transformed ratios of BC samples vs. controls are depicted in Table S2.We further compared our qPCR results to 4 publicly available BC microarray GEO datasets: GSE89 by Dyrskjot et al. (2003) 2 transformed ratios of BC vs. these extracted data, which we used as controls (Table S2).Dataset GSE12630 contained data only from transitional cell carcinoma of urinary bladder and metastatic urothelial carcinoma. Since no data from the normal tissue were available for this dataset, we extracted normal tissue data from dataset GSE89, and computed the mean logTable S4). In BC, MMP2 was significantly correlated with BRAF (p = 0.032), RAF1 (p = 0.039) and RKIP (p = 0.014). MMP9 correlated with VEGFA (p = 0.005) and FGF2 (p = 0.016). OPN correlated with NRAS (p = 0.027). VEGFA correlated with FGF2 (p = 0.019). TGFβ1 correlated with p53 (p = 0.033), EGFR (p = 0.048) and RKIP (p = 0.032). FGF2 correlated with EGFR (p = 0.015), HRAS (p = 0.005) and KRAS (p = 0.029). p14ARF correlated with p16INK4A (p<0.0001), KRAS (p = 0.016), NRAS (p = 0.009) and ARAF (p = 0.044). p16INK4A correlated with EGF (p = 0.017) and NRAS (p = 0.002). p53 correlated with AKT1 (p<0.0001), EGFR (p = 0.002) and RAF1 (p = 0.041). AKT1 correlated with EGFR (p<0.0001) and RKIP (p = 0.022). EGF correlated with NRAS (p = 0.027). HRAS correlated with NRAS (p = 0.007) and ARAF (p = 0.030). KRAS correlated with RKIP (p = 0.035). NRAS correlated with ARAF (p = 0.003) and BRAF (p = 0.004). ARAF correlated with BRAF (p = 0.001) and RAF1 (p = 0.001). BRAF correlated with RAF1 (p = 0.040). All positive and negative interactions among the genes, indicating their synergism in bladder tumorigenesis, can be deduced from the correlation coefficient values depicted in Table S4.We explored the correlation of the mRNA levels of the GOIs in BC as well as in normal tissue, performing the Spearman rank test . OPN, TP53, BRAF and EGFR exhibited the second highest number of significant interactions (18 of the GOIs), whereas VEGFA, RAF1, CDKN2A. ki67, ERBB2 and FGF2 were associated with 17 of the GOIs. NRAS was associated with 16 GOIs, KRAS, MMP2 and MMP9 with 15 GOI, TGFβ1 with 14 GOIs, ARAF with 13 GOIs, EGF with 6 GOIs and RKIP/PEBP1 with 5 of the GOIs, according to the literature .Moreover, we used the BioNetwork Tools from Figure S2 and ). HCL was performed on the microarray expression values, where the average of all samples in a tumor category was calculated and further processed.We performed hierarchical cluster analysis on this selected small gene dataset. HCL analysis classified the tumors based on their degree of aggressiveness, as expected .GO and KEGG molecular pathway annotation analysis in the selected genes is presented in As additional molecular determinants are added to a constantly growing list of prognostic indicators for BC, there is a growing need to combine such markers into logical groups and employ them for determination of outcome and therapeutic response in individual patients. It is clear that several molecular alterations can operate together to influence the pathogenesis of BC and its ultimate clinical behavior. Analyzing these alterations in combination may therefore provide deeper insight into the pathobiology of the disease, while also generating panels of markers that may be able to better predict patient outcome and treatment response.In the present study, we analyzed the expression profile of genes involved in cell cycle control, DNA damage repair, apoptosis, signal transduction, transcription factors, angiogenesis, cellular proliferation, invasion and metastasis. Our results showed an observable overlap between the tumor and normal urothelium samples with respect to the expression of the GOIs. Nevertheless, the detected changes in the expression of the GOIs were statistically significant in the tumor vs. paired non-tumor samples. The aforementioned overlap in the expression of the GOIs can be explained by the fact that overt carcinomas are frequently surrounded by histologically normal, but genetically aberrant urothelium. Increasing evidence suggests that TCCs and the surrounding urothelium share common genetic and epigenetic alterations. This genetic similarity may be the result either of the field cancerization effect or of intraepithelial migration. The two mechanisms, together with intraluminal seeding, appear to be strongly responsible for multi-focal disease as well as for localized and distant recurrences within the urinary bladder.Different studies have indicated that alteration in cell-cycle regulation is a key event in determining the biological behavior of bladder cancer INK4a over-expression has been previously detected in urothelial carcinomas INK4A has been statistically associated with disease stage but not with either tumor grade or disease progression INK4A expression has also been related to recurrence and survival INK4A were higher in BC vs. normal tissue, as assessed by qPCR. Western blot analysis verified the higher protein expression for CDKN2A in BC vs. normal tissue. Tumors of T2/T3-grade 3 exhibited higher p16INK4A mRNA levels vs. those of lower stage/grade. Finally, we could not relate p16INK4A expression to survival, probably due to the small sample number. p14ARF did not show any significant differences between BC and normal tissue, or among tumors of different histology. In contrast, high p14ARF expression showed a tendency to correlate with favorable prognosis.The most common genetic damage in BC is partial loss of chromosome 9. The area surrounding 9p21 where the CDKN2A/ARF gene is located is one of the major sites for deletion. p16Cyclin D1 plays an important role in the cell cycle, binds to cyclin-dependent kinases (CDK4/6), and promotes phosphorylation of RB1, orchestrating progression through the G1 restriction point. Recently, Del Rey et al. found that CCND1 amplification in homogeneously staining regions was present in 6 out of 14 CIN-positive tumors; 3 of them also showed amplification of this gene in double minutes Alterations in apoptotic pathways contribute to tumorigenesis and progression, as they allow cancerous cells to survive longer, resist normally harmful stresses, and become more invasive Recently, two research groups used immunohistochemistry and found that OPN was over-expressed in bladder cancer Signal transduction involves diverse pathways that modulate growth signals from cell-surface receptors to transcription factors in the nucleus. Alterations within these pathways can result in the deregulation of genes that control cellular homeostasis. One of the most important signaling pathways in BC is the Ras-mitogen-activated protein kinase (MAPK) signaling cascade.When RAS family members become activated, through physiological stimuli or through mutation Another oncogene implicated in BC is the epidermal growth factor receptor (EGFR). It's over-expression has been described in several tumors including bladder AKT1 is one of three family members of the serine-threonine kinases that are activated by phosphorylation of Thr308 by PDK1 and Ser473 by TORC2 The RAF  and MEK (MEK1/MAP2K1 and MEK2/MAP2K2) family of proteins are critical downstream mediators of the RAS and other oncogenic signaling pathways Angiogenesis is essential for tissue development, wound healing and reproduction Of the angiogenic factors, VEGF has been identified as a crucial regulator of normal and pathological angiogenesis. VEGF produces a number of important biological effects such as endothelial mitogenesis and migration, extracellular matrix remodeling via induction of proteinases, increased vascular permeability and maintenance of newly formed vasculature At the early stage of tumor growth, FGF2 expression plays an important role in the regulation of angiogenesis, tumorigenicity and subsequent metastases of human bladder cancer TGFβ1 is regarded as a negative growth regulator because of its potent anti-proliferative effects on many cell types, such as endothelial and epithelial cells and various cell types of haematopoietic origin in vitro Despite the reported over-expression of EGFR in bladder cancer, leading to uncontrolled cell proliferation, increased angiogenesis and reduced apoptosis, to our knowledge, this is the first report regarding the expression of EGF in urinary bladder cancer. Our data imply significantly low levels of EGF, both by microarray and qPCR analysis (p = 0.0017).Angiogenesis-related molecular markers are commonly altered in urothelial carcinoma of the bladder, making them a target for therapy. Understanding the mechanisms of invasion, metastasis and angiogenesis in BC sets the stage for the development of novel targeted agents.The gene MKI67 encodes Ki-67, a nuclear protein that is associated with and may be necessary for cellular proliferation. In cancer cells, Ki-67 plays an important role as an index for the replication and the prognosis and is well associated with tumor grade, stage and recurrence The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes present in both normal and pathological tissues in which matrix remodelling is involved, including embryonic development, wound healing, arthritis, angiogenesis, tumor invasion and metastasis Most studies have previously determined MMP2 and MMP9 activity levels in urine or tissue from patients with BC, using gelatin zymography or ELISA Apart from MMPs, TIMP expression has also been previously reported in BC INK4A, p53, AKT1, HRAS, Cyclin D1, RAF1 and RKIP, exhibited significant over-expression in BC vs. normal tissue. OPN (SPP1), VEGFA, p14ARF and BRAF, exhibited equal expression levels between BC and normal tissue, whereas MMP2, MMP9, TIMP1, TIMP2, FGF2, EGFR, EGF, Ki-67, KRAS, NRAS, and ARAF showed significantly lower expression levels in BC vs. normal tissue.Regarding Dataset GSE89, TGFβ1, p16INK4A, p53, EGFR, EGF, Ki-67, KRAS, NRAS, and cyclin D1. The genes, MMP9, OPN (SPP1), TIMP1, TIMP2, TGFβ1, p14ARF, AKT1, HRAS, BRAF, and RKIP, presented equal expression levels between BC and normal tissue; whereas genes that presented significantly lower expression levels in BC vs. normal tissue were MMP2, FGF2, ARAF and RAF1.As for Dataset GSE7476, the genes that exhibited significant over-expression in BC vs. normal tissue were the following: VEGFA, p16ARF, p16INK4A, EGFR, EGF and BRAF showed significantly lower expression in BC vs. normal tissue (Table S2).Regarding Dataset GSE3167, OPN (SPP1), VEGFA, p53, AKT1, KRAS, HRAS, cyclin D1, RAF1, and RKIP exhibited significant over-expression in BC vs. normal tissue. The genes, MMP2, TIMP1, TIMP2, TGFβ1, Ki-67, NRAS, and ARAF exhibited identical expression between BC and normal tissue; whereas MMP9, FGF2, p142 transformation of the fold-expression rates minimized these differences and provided us with a more definite understanding of the expression profile of the GOIs in BC.Expression of the majority of the genes demonstrated good agreement among all GEO Datasets. Small differences detected among the 5 GEO Datasets were expected to occur due to divergence in the methodology used in the experimental procedures and in the analysis of each gene. However, logFurthermore, we performed GO and KEGG molecular pathway annotation analysis in the selected genes. Since the dataset was small, we expected a small number of significant functions to be represented in gene ontology. To our surprise, GO analysis manifested a large variety of functions for the GOIs. The variety of gene functions that were revealed implies the multi-facet character of the selected genes. It appears that they participate in a variety of mechanisms including cell proliferation, cell death (particularly negative regulation of cell death), metabolism and very importantly these same genes participate in cell shape and cytoskeletal re-organization.−31). When the certain group of genes was isolated for further processing from microarray experimentation, tumor types were successfully classified based only on these specific genes. This implies that the genes on which our interest was focused for further study, probably characterize bladder neoplasias. Looking further into the functions of the selected genes it appears that, in their majority, they participate in metabolic processes. This was on one hand expected, since tumors are known to be very active metabolically, and on the other hand it was interesting since it confirms the hypotheses stated in recent years that metabolic pathways could be used as therapeutic targets. The participation in many functions of the GOIs in the present study makes them good therapeutic targets. Since these specific genes appear to have key cellular functions and characterize tumor type and grade, future investigations as far as their potency as therapeutic targets are warranted.Importantly, when we performed an analysis for KEGG pathway participation of our selected dataset, the most significant pathway appeared to be the Bladder Cancer Pathway Click here for additional data file.Figure S2Differential fold expression of the genes of interest in 10 BC samples vs. 5 controls, as detected by microarray analysis. The Mann-Whitney test was performed to examine statistically different expression patterns between groups with p<0.05 considered statistically significant.(TIF)Click here for additional data file.Figure S3B. Scatterplot depicting the mRNA levels of the genes that were equally expressed among urinary bladder cancer and normal tissue. Groups pairs were statistically compared using the Mann-Whitney U test. Bars depict the median values. C. Scatterplot depicting the mRNA levels of the genes that were under-expressed in urinary bladder cancer versus normal tissue. Groups pairs were statistically compared using the Mann-Whitney U test. Bars depict the median values.Scatterplot depicting the mRNA levels of the genes that were over-expressed in urinary bladder cancer (BC) versus normal tissue. Groups pairs were statistically compared using the Mann-Whitney U test. Bars depict the median values. (TIF)Click here for additional data file.Figure S4www.pubgene.org, relationships between the GOIs were investigated. HRAS confirmed its role as a key molecule, since it presented interaction with all the GOIs (19 interactions). OPN, TP53, BRAF and EGFR exhibited the second highest number of significant interactions (18 of the GOIs), whereas VEGFA, RAF1, CDKN2A. ki67, ERBB2 and FGF2 were associated with 17 of the GOIs. NRAS was associated with 16 GOIs, KRAS, MMP2 and MMP9 with 15 GOIs, TGFβ1 with 14 GOIs, ARAF with 13 GOIs, EGF with 6 GOIs and RKIP/PEBP1 with 5 of the GOIs.Using the BioNetwork Tools from (TIF)Click here for additional data file.Figure S5A), PCA for experiments (B) and genes (C) as well as CA analyses (D) for the microarray dataset selected to be the same with the genes tested with qPCR. Colours of clusters in HCL correspond to the gene colours in all other analyses.HCL ((TIF)Click here for additional data file.Table S1Information of the primer sequences used for qPCR and the corresponding PCR product sizes.(DOC)Click here for additional data file.Table S22 intensity and statistically compared by the Mann-Whitney U test .Gene expression analysis in urinary bladder cancer was performed on data extracted from the following publicly available datasets: GSE89, GSE3167, GSE7476 and GSE12630. T, tumor; M, metastasis, N, normal tissue; sTCC with CIS, superficial transitional cell carcinoma (sTCC) with surrounding carcinoma in situ (CIS); sTCC without CIS, superficial transitional cell carcinoma (sTCC) without surrounding carcinoma in situ (CIS); mTCC, muscle-invasive carcinomas (mTCC). Results were expressed as mean levels of the log(DOC)Click here for additional data file.Table S3Fold differential change of each gene in BC vs. the control tissues. Red color indicates >2-fold expression; black color indicates equal expression with a threshold of 2; blue indicates <2-fold expression. T-test was performed between the expression levels of BC and control samples. The fold up- or down-regulation in BC vs. Control, was also explored using the mathematical formula: IF. If the fold change is positive or negative, it means up-or down-regulation, respectively.(DOC)Click here for additional data file.Table S4A) and control tissue (B).Spearman's bivariate correlation-coefficient test in order to detect correlations among the expression levels of all the genes studied in transitional cell carcinomas of the urinary bladder ((DOC)Click here for additional data file.Table S5Enriched KEGG pathways. This table lists the enriched KEGG pathways, number of Entrez IDs in the data set for the pathway, the corresponding Entrez IDs, and the statistics for the enrichment of the pathway. The statistics column lists the number of reference genes in the category (C), number of genes in the gene set and also in the category (O), expected number in the category (E), Ratio of enrichment (R), p value from hypergeometric test (rawP), and p value adjusted by the multiple test adjustment (adjP). The most significant pathway revealed, was the Bladder Cancer pathway. Interestingly, the most significant pathway revealed was Bladder Cancer pathway.(DOC)Click here for additional data file.


Hypothalamic hamartoma is a rare neoplastic heteropia caused by organic developmental failure. The most common clinical findings in hypothalamic hamartoma are pubertas precox with or without gelastic seizure, and behavioural disturbance. The aim of this case report to inform a rare case of hypothalamic hamartoma with pubertas precox and gelastic seizure in a boy.1P2G2-3, and neurological status was normal. Laboratory finding: FSH: 0,5 mlU/ml, LH: 0,85 mlU/ml, testosterone: 239,20 ng/dL, 17α-hydroxyprogesterone: 107 ng/dl. Cranial CT scan and MRI showed a space occupying lession in suprasellar region which was suspected as a tubercinerium hamartroma. Bone age result was appropriate for an 8 year and 6 month old boy. He was treated with leuprorelin 1x3,75mg intramuscular every 4 week and valproic acid 2x200mg. After 3 years evaluation, patient’s Tanner pubertal status was A1P2G2-3 and the bone age was appropriate for an 13 years old boy. No gelastic seizure.A 5 year and 7 month old boy, admitted to the hospital with a chief complain of premature pubic hair growth and frequent sudden laughing without apparent reason which seemed to be forced, uncontrolled, irregular without deteroratoin of consciousness. Physical examination found accelarate height growth with appropriate proportion. Tanner pubertal status was ALeuproleline effectivity as a therapy for pubertas precox with hypothalamic hamartoma is still a controversion, so management through surgical approach may be considered, and valproic acid has a good effectivity for gelastic seizure in this case.


GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty acids might have an important role in this pain control system. Inflammatory chronic pain, such as arthritis pain, joint pain and inflammatory bowel disease, is a significant health problem, and is initiated by tissue damage or inflammation n-3 series polyunsaturated fatty acids (PUFAs) or their derived lipid mediators have been found to have crucial roles in the local control and programming of acute inflammatory response and its resolution n-3 PUFAs results in a reduction of pain associated with rheumatoid and inflammatory joint pain Recently, endogenous 3) signaling pathway and leads to intracellular Ca2+ mobilization. GPR40 activation in pancreatic β-cells causes insulin secretion in response to increased blood glucose levels. In the central nervous system (CNS), GPR40 is predominantly expressed in the subventricular zone as well as in newborn and mature neurons Accumulating evidence clearly shows that free fatty acids (FFAs) can act as ligands for several G-protein-coupled receptors (GPCR), including GPR41 It has been reported that DHA produces an antinociceptive effect via β-endorphin release in response to various pain stimuli similar to those used in the present study In the present study, these issues were addressed using a complete Freund's adjuvant (CFA)-induced inflammatory model in mice, a well-characterized model of inflammatory pain. The functional role of hypothalamic GPR40 during inflammatory chronic pain was examined, and the involvement of astrocytes in the mechanisms of GPR40-induced antinociception was estimated.ad libitum.The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals adopted by the Japanese Pharmacological Society. All experiments were approved by the Ethical Committee for Animal Experimentation of Kobe Gakuin University . Male ddY mice  were obtained from Japan SLC . Mice were housed in cages at 23–24°C with a 12-h light–dark cycle (lights from 8 am to 8 pm) and food and water DHA , the selective GPR40-agonist GW9508  and the GPR40 antagonist GW1100  were dissolved in 1% dimethyl sulfoxide  and the solution was diluted with saline before von Frey testing . The doses of GW9508 were chosen based upon our previous publication post-mortem confirmation of dye placement within brain sections.The i.c.v. injection was performed by using a Hamilton microsyringe fitted with a 27-gauge i.c.v. needle This protocol was conducted as previously described Hyperplasia of paw tissue was measured by means of a digital caliper  before and at several times after i.pl. CFA injection during the 1- or 7-day period of study.Mechanical allodynia was evaluated using von Frey filaments  as previously described Thermal hyperalgesia of the hind paw was assessed using the plantar test , according to a previously described methodology The wet weight of hypothalamic tissue was measured, and that tissue was homogenized in methanol/acetone . The homogenate was centrifuged at 16,000 g for 5 min at 4°C. The resulting supernatant was moved into a glassy vial as the analysis sample of each FFA. To measure C12:0, C14:0, C15:0, C16:0, C16:1, C17:0, C18:0, C18:1, C18:2, C18:3, C20:0, C20:3, C20:4, C20:5 and C22:6, the supernatant was diluted by a factor of 10 or 50 FFAs comparative analysis was measured as previously described with some modifications Western blotting was done as previously described Mice were deeply anesthetized with sodium pentobarbital (65 mg/kg) and perfused transcardially with phosphate-buffered saline (PBS), pH 7.4, followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Brain sections were collected, post-fixed in 4% paraformaldehyde for 3 h, and dehydrated in 10% sucrose at 4°C for 3 h, and 20% sucrose at 4°C overnight. The following day, tissues were frozen in optimal cutting temperature compound  and held at −80°C until use. Sections were cut at 15 µm with a cryostat , and mounted on an MAS-coated glass slide .The sections were washed with PBS containing 0.1% Triton X-100 (PBST) three times at 5 min intervals and incubated with blocking buffer (3% BSA in PBST) for 2 h at room temperature. The sections were then incubated in specific antibodies against GPR40 , or GPR40, which was prelabeled with the Zenon Alexa Fluor 594 Rabbit IgG Labeling Kit . NeuN , GFAP , proopiomelanocortin (POMC) , c-Fos  and β-endorphin  were stored overnight at 4°C, at which time the antibody was diluted in reaction buffer (0.01% Triton X-100 and 1% BSA in PBS). The next day, sections were washed with PBST and incubated in secondary antibody conjugated with AlexaFluor 488 and/or 594  at room temperature for 2 h, at which time the secondary antibody was diluted with reaction buffer. Finally, sections were washed with PBST and coverslipped with Perma Fluor , and immunoreactivity was detected with a confocal fluorescence microscope . In the immunohistochemical control studies, no staining was detected when the corresponding primary or secondary antibody was omitted.t-test for comparison between two groups. A p value of <0.05 was regarded as significant.Data were expressed as mean ± S.E.M. Significance differences were evaluated by one-way analysis of variance followed by Dunnett's or Scheffe's multiple-comparison tests for comparisons between more than three groups or by Student's P<0.01) were elicited in CFA-treated mice, compared with saline-injected control mice, appearing on day 1 and continuing until day 14. No pain behavior was observed in saline-injected mice . However, there was no change in GPR40 expression in the hypothalamus at day 1, 3 or 14 after CFA injection, compared with saline groups  in the saline group .P<0.05). In addition, in immunohistochemical studies, increases in GFAP-positive cells were observed at day 1 after CFA injection . Behaviorally, CFA-treated mice with flavopiridol at day 1 showed significant recovery in mechanical allodynia and thermal hyperalgesia (P<0.001).At day 1 after CFA injection, hyperplasia had no effect in mice with the i.c.v. injection of flavopiridol . Concurrently, mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection were also attenuated to the levels of the saline group (P<0.01 or P<0.05).At day 7 after CFA injection, hyperplasia had no effect in mice with the i.c.v. injection of flavopiridol . GPR40 pAll long chain FFAs were detected in the hypothalamus of normal mice. Notably, saturated palmitate (C16:0) and stearate (C18:0), monosaturated oleinic acid (C18:1), polyunsaturated linoleic acid (C18:2), arachidonic acid (C20:4) and DHA (C22:6) were abundantly expressed in the hypothalamus, and the FFA contents (µg/g wet tissue) were 31965±3977, 24516±2408, 29711±4011, 2907±759, 18419±1654 and 13686±1154, respectively . The ratAt day 1 after CFA injection, mechanical allodynia  and therP<0.05). When 1% DMSO was given as a control or GW1100 was used alone, there was no effect on CFA-induced inflammatory pain.On the other hand, administration of DHA (50 µg) or GW9508 (1.0 µg) significantly suppressed mechanical allodynia  and therP<0.05) in the mice treated with GW9508, compared with the saline-treated group. In contrast, in saline-injected mice, there were almost no c-Fos or POMC-positive neurons  In conclusion, the important functional role of fatty acids, their receptors, and their metabolites in both the onset and suppression of pain has become increasingly apparent in recent years. The present findings support the idea that GPR40 signaling in the supraspinal area may contribute to regulation of the pain control system. In addition, GPR40 expressed on POMC neurons was shown to possibly control excitation signaling caused by inflammatory chronic pain. Taken together, application and study of a GPR40 agonist in this model might provide valuable information regarding a novel therapeutic approach for pain control in the future.


Researchers have provided evidence that telomere dysfunction play an important role in cancer development. MNS16A is a polymorphic tandem repeats minisatellite of human telomerase (hTERT) gene that influences promoter activity of hTERT and thus implicates to relate with risk of several malignancies. However, results on association between MNS16A and cancer risk remain controversial. We therefore conduct a meta-analysis to derive a more precise estimation of association between MNS16A and cancer risk.A systematic literature search was conducted by searching PubMed, ISI Web of Knowledge, Human Genome and Epidemiology Network Navigator and Google Scholar digital database for publications on associations between MNS16A and cancer risk. Variants with statistically significant associations by meta-analysis were assessed using Venice criteria.P for heterogeneity > 0.1). The cumulative analysis in chronologic order suggested a clear tendency towards a significant association with additional study samples.10 case-control articles enrolling 6101 cases and 10521 controls were brought into our meta-analysis. The relationships were strong epidemiological credibility in cerebral cancer and breast cancer population (The results provided a more accurate depiction of the role of MNS16A in cerebral cancer and breast cancer susceptibility. Additional larger studies were warranted to validate our findings. Telomeres  are crucial for genomic stability All methodology was based on guidelines proposed by the Human Genome Epidemiology Network (HuGENet) For statistical analysis, number of tandem repeats was classified as either short (S) or long (L) alleles (LS classification system): S alleles, 213bp, 240bp, 243bp, 271bp, 272bp, 274bp; L alleles, 299bp, 302bp, 331bp, 333bp, 364bp, frequently applied in literature. On basis of classification, MNS16A genotypes were assigned to SS, LS or LL genotype groups. ORs and 95% CIs were recalculated and assessed in gene models based on MNS16A length comparisons : a co dominant genetic model , a dominant genetic model (SS+LS versus LL) and a recessive model (SS versus LS + LL). To explore in depth of different lengths of MNS16A under S allele group, we classified the 271bp, 272bp and 274bp allele as middle alleles  and 213bp, 240bp and 243bp alleles still as S alleles (LMS classification system) described by Jin et al χ2-based Cochran’s Q statistic test and 2I metric P<0.1 for the Q statistic (to assess whether observed variance exceeds expected variance). And for the 2I metric (2I = 100% × (Q-df)/Q), the following cut-off points were used: 2I = 0–25%, no heterogeneity; 2I = 25–50%, moderate heterogeneity; 2I =  50–75%, large heterogeneity; 2I = 75–100%, extreme heterogeneity. The significance of the combined ORs was determined using the Z test . The DerSimonian and Laird random effect model P value of less than 0.05 was considered significant, except for Q test for heterogeneity, for which a less than 0.1 level of statistical significance was applied.Between-study heterogeneity was assessed by the minor): grade A, B, C requires nminor > 1000, 100 ≤ nminor ≤ 1000, nminor<100. Replication was graded by the heterogeneity statistic: grades A, B, and C were assigned for 2I less than 25%, 25–50%, and greater than 50%, respectively. Assessment of protection from bias was graded as grade A if there was no observable bias, grade B if bias could be present, or grade C if bias was evidence  Each variant with statistically significant associations by meta-analysis were assessed on the basis of the Human Genome Epidemiology Network Venice criteria. Credibility was defined as “strong,” “moderate,” or “weak” based on grades A, B, or C in three categories: 1) amount of evidence; 2) replication; and 3) protection from bias. Amount of evidence was assessed by size of test allele among case and controls in meta-analysis  for LS genotype versus LL genotype, and 1.17  for dominant model. In fixed-effects model, overall ORs were 1.32  for SS genotype versus LL genotype, and 1.23  for recessive model.As shown in Subsequently we categorized the data in LMS classification described by Jin et al. to explore in depth the effect of MNS16A S allele  and M allele  with cancer risk. As shown in P for heterogeneity > 0.1), and all genetic models presented a significantly increased cancer risk, with ORs of 1.16 (95%CI = 1.05–1.28), 1.33 (95%CI = 1.15–1.54), 1.19 (95%CI = 1.09–1.31), and 1.23 (95%CI = 1.07–1.42) for LS versus LL genotype, SS versus LL genotype, dominant model, and recessive model, respectively. However, all genetic models presented no statistical differences of cancer risk among Asian population . Patients with MNS16A-S allele had a significant statistically association with cerebral cancer risk: with ORs of 1.42 (95%CI = 1.19–1.70), 1.22 (95%CI = 1.09–1.37), 1.32 (95%CI = 1.11–1.56) for SS versus LL genotype, dominant and recessive model (P for heterogeneity > 0.1). For breast cancer, patients carried with LS genotype had higher risk than SS genotype, which ORs and 95%CI were 1.52 (1.19–1.94) and 1.46 (1.16–1.84) for LS versus LL genotype and dominant models. However, no statistically significant associations were observed with lung cancer patients  and ORs = 1.20  before and after removal, respectively. Omission of studies by Andersson et al. changed the pooled ORs fractionally , indicating the results were statistically robust.As reflected by either visualization of funnel plot or Egger’s and Begg’s test, there was no indication of publication bias in the genotypic models of LS versus LL, SS versus LL, dominant, and recessive model ; replication was assigned to category B, because the amount of between-study heterogeneity (2I =  40.5%); and protection from bias was graded as category B, due to the presence of summary ORs less than 1.15, which can easily be dissipated even by relatively small biases in a meta-analysis of published data. The overall assessment of association between MNS16A and cancer risk were moderate cumulative evidence (ABB). After stratification by ethnicity, the meta-analysis consistently showed a significant association cancer risk in Caucasian population and were assigned an overall strong epidemiological credibility (AAA). Asian population lacked of statistically significant findings and was placed to weak evidence. In addition, strong epidemiological credibility (AAA) was also observed for association between MNS16A with cerebral cancer and breast cancer . In comparison, our meta-analysis added more publications to consider association of MNS16A with all available type of cancer; analyzed data in different MNS16A classification system (LS and LMS classification system); stratified ethnicity and cancer types for further research.CTCCCAGTCTC) in putative promoter region of the antisense RNA transcript, was first reported to increase expression of hTERT mRNA in lung cancer tissues. In the current study, we conducted a meta-analysis of 10 previously published articles comprising 6101 cases and 10521 controls concerning association of MNS16A with cancer risk. Although all genetic models of MNS16A showed a moderate association with cancer risk, the effect could very well be driven by the effect on cerebral cancer. Thereupon, we stratified cancer types and found cerebral cancer and breast cancer patients showed strong cumulative evidence for associations, but lung cancer was not. Apart from this, ethnicity was also stratified in this work. Caucasian population presented a significantly increased relationship with cancer risk, whereas Asians not. The variance of effect between Caucasians and Asians might be contribute to that approximate 70% Caucasians were cerebral cancer, while similarly the absence of effect in Asians might well be due to the fact that only non-cerebral cancer were carried in this population. In addition, there was almost no obviously heterogeneity by stratified for cancer type, which suggested differential effects of MNS16A in diverse kinds of cancer. However, functional importance of the antisense transcript activity and exact molecular mechanisms of MNS16A with different cancer types were still unclear.A number of well designed genome wide association studies (GWAS) had implicated variants at hTERT locus to be significantly associated with almost all malignant tumors In this work, we analyzed data in different classification system: LS and LMS . The reason might due to lower frequencies of S allele in Asians. Additionally, omission article by Carpentier, the ORs were still presented increased risk, and 95%CI were nearby statistically significant , which not meaningfully changed the pooled ORs, as well as the article by Andersson.Some limitations needed serious consideration. First, our result was based on unadjusted estimates. Individual data were not available for an adjusted estimate by age and sex, which might potentially lead to false positive results. Another limitation was lacking original data to limit our further evaluation of gene-environment interaction such as smoking, alcohol use and other clinical characteristics. Finally, lacking of sufficient original studies limited our further evaluation of colorectal cancer, breast cancer and nasopharyngeal carcinoma risk with MNS16A.This work verified the important role of MNS16A minisatellites in cerebral and breast cancer predisposition. Additional larger studies were warranted to validate our findings.Checklist S1(DOC)Click here for additional data file.


T) ventilation during resuscitation of preterm lambs results in brain injury evident histologically within hours after birth. We aimed to investigate whether magnetic resonance spectroscopy (MRS) and/or diffusion tensor imaging (DTI) can be used for early in vivo detection of ventilation-induced brain injury in preterm lambs.High tidal volume  5 cmH2O) or an initial 15 minutes of “injurious ventilation”  followed by PROT ventilation for the remainder of the experiment. At 1 hour, lambs underwent structural magnetic resonance imaging . For measures of mean/axial/radial diffusivity  and fractional anisotropy (FA), 30 direction DTI was performed. Regions of interests encompassed the thalamus, internal capsule, periventricular white matter and the cerebellar vermis. MRS was performed using a localized single-voxel  encompassing suptratentorial deep nuclear grey matter and central white matter. Peak-area ratios for lactate (Lac) relative to N-acetylaspartate (NAA), choline (Cho) and creatine (Cr) were calculated. Groups were compared using 2-way RM-ANOVA, Mann-Whitney U-test and Spearman's correlations.Newborn lambs (0.85 gestation) were stabilized with a “protective ventilation” strategy (PROT, n = 7: prophylactic Curosurf, sustained inflation, VNo cerebral injury was seen on structural MR images. Lambs in the INJ group had higher mean FA and lower mean RD in the thalamus compared to PROT lambs, but not in the other regions of interest. Peak-area lactate ratios >1.0 was only seen in INJ lambs. A trend of higher mean peak-area ratios for Lac/Cr and Lac/Cho was seen, which correlated with lower pH in both groups.Acute changes in brain diffusion measures and metabolite peak-area ratios were observed after injurious ventilation. Early MRS/DTI is able to detect the initiation of ventilation-induced brain injury. T) during mask-ventilation, and the actual measured VT delivered to the baby, can be substantial, with many infants receiving excessive VT. High VT ventilation is harmful both for the lungs T ventilation causes cerebral blood flow instability, white matter injury, inflammation, vascular leakage and oxidative stress, evident as early as 90 minutes after birth. It is not possible to detect brain injury histologically in survivors of preterm birth; instead, magnetic resonance imaging (MRI) provides a potential biomarker for acute brain injury in preterm newborns in vivo MRI examinations in preterm animals are scarce, complicating translation of the findings to the human setting.Preterm infants are at high risk of brain injury and long-term neurodevelopmental impairments Diffusion tensor imaging (DTI) is an MRI method based on measures of the motion of water molecules in brain tissue and provides information on brain organization and microstructure in vivo information on brain metabolism. Similar to diffusion measures, the neurochemical profile changes during development Magnetic resonance spectroscopy (MRS) measures brain metabolites and provides in vivo detection of ventilation-induced brain injury in preterm lambs. We hypothesized that lambs that were stabilized with an “injurious” ventilation strategy at birth would display altered metabolite patterns on spectroscopy, such as increased lactate peak-area ratios, possibly in association with decreased creatine and NAA and increased choline ratios. Moreover, we hypothesized that high VT ventilation would have adverse effects on brain microstructure and that specific changes in MD/FA/AD/RD would provide information on particularly vulnerable regions and tissue components.The overall aim of the present study was to investigate whether structural MRI, DTI and MRS can be used for early The experimental protocol was performed in accordance with guidelines established by the National Health and Medical Research Council of Australia and was approved by the Monash Medical Centre animal ethics committee (MMCA) at Monash University (AEC number: MMCA-2012-26).2O delivered with a Neopuff . Ventilation  was commenced 1 minute prior to clamping of the umbilical cord, using volume guarantee (VG) mode with a set VT of 7 mL/kg and a positive end expiratory pressure (PEEP) of 5 cm H2O . The INJ strategy consisted of intubation and early cord clamping, followed by VG ventilation with a target VT of 12 mL/kg, no PEEP and administration of Curosurf at 15 minutes. Fractional inspired oxygen (FiO2) was initially set at 0.4 in both groups, and then adjusted to maintain arterial oxygen saturation (SaO2) between 88–95%. After the initial 15 minutes of intervention, both groups were managed using the PROT strategy for the remainder of the experiment.Pregnant anaesthetized ewes underwent caesarean section at 0.85 of gestation . Lambs were delivered and randomly assigned to receive stabilization with either a “protective ventilation” strategy  or an initial 15 minutes of “injurious ventilation”  as described previously 2) probes . Umbilical catheters were placed for intravenous administration of anesthesia, arterial blood gas sampling and invasive real-time monitoring of heart rate (HR) and blood pressure (BP) . Ventilator settings were noted regularly. The lambs were anaesthetized throughout the study using a continuous infusion of Alfaxane  10 mg/kg/h in 5% dextrose. Arterial blood samples were collected regularly and measured for partial pressure of oxygen (PaO2), partial pressure of carbon dioxide (PaCO2), pH, Base Excess and lactate . The ewes were humanely euthanized using sodium pentobarbitone (100 mg/kg i.v) after the caesarean section, and the lambs were euthanized after completion of the MRI.All lambs were dried, placed on a heated mattress, and fitted with rectal temperature and transcutaneous arterial oxygen saturation  in controlled mandatory ventilation mode. Lambs were imaged in supine position, swaddled and monitored using continuous HR and SpOAll scans were performed on a 3T MR scanner  at Monash University Biomedical Imaging , using a 15-channel RF coil for both RF transmission and receive. The MRI protocol consisted of structural imaging sequences , susceptibility weighted imaging (SWI) and a single voxel MRS. The total scanning time was approximately 40 minutes.2, slab thickness  = 102.5 mm, data matrix  = 256×256×128, resulting in a voxel size  = 0.78×0.78×0.8 mm3, number of averages  = 2. T2-weighted images were acquired with a 3 D SPACE sequence with parameters: TR/TE  = 1000/132 ms, flip angle  = 120°, FOV = 192×192 mm2, 44 mm slab thickness, acquisition matrix  = 384×384×88, voxel size  = 0.5×0.5×0.5 mm3, number of averages  = 2. SWI was acquired using a standard 3 D gradient echo sequence, with TR/TE  = 28/20 ms, flip angle  = 15°, FOV = 176×132 mm2 and 72 mm slab thickness, acquisition matrix  = 288×216×60, voxel size  = 0.6×0.6×1.2 mm3. The second phase encoding direction (in the 1.2 mm voxel direction) was orientated parallel to the main magnetic field to produce the best susceptibility effect.A 3D MPRAGE sequence was used to acquire T1-weighted images with parameters: repetition time (TR)/echo time (TE)/inversion time (TI) = 1440/3.92/900 ms, flip angle = 9°, field of view (FOV) = 200×200 mm2, data matrix  = 128×128, 50 continuous slices with 1.2 mm thickness, voxel size  = 1.2×1.2×1.2 mm3. Diffusion encoding gradients were applied in 30 directions with b value  = 1500 s/mm2. The diffusion-weighted acquisition was repeated twice and 5 b = 0 volumes were acquired.DTI was acquired in the axial plane using a spin-echo EPI sequence with parameters: TR/TE  = 11400/99 ms, FOV  = 154×154 mm3, encompassing supratentorial deep grey matter (GM) and central WM. A representative example of voxel placement and one sample spectra from a single subject are shown in MRS was performed using a localized single-voxel spin echo sequence with TR/TE  = 2000/270 ms and voxel size  = 15×15×20 mmThe structural MR images  and SWI were reviewed for malformations and cerebral injuries such as haemorrhages and infarcts.DTI data was processed using the FMRIB Software Library  and range. A p-value of <0.05 was accepted as significant.2), systolic, diastolic and mean BP were similar in both groups at all investigated times during and after the intervention (data not shown).The mean birth weight of the study sample was 3250 (±245) grams (range 2800–3800 g), with no significant difference between the groups: PROT 3329±250 g versus (vs) INJ 3189±237 g. The mean gestational age was 125.8±1 days (range 125–127 d), and similar between groups. There were 8 female and 9 male lambs, and these were similarly distributed between groups. Heart rate, peripheral oxygen saturation (SpOT/kg (p = 0.002) and lower PEEP (p<0.001) during the first 15 min compared to PROT lambs as expected. The PIP was higher throughout the study in INJ lambs  from 15 min.Injuriously ventilated lambs had higher V2 at 10 min , and lower PaCO2 at 15 min , compared to the PROT group (Lambs in the INJ group had lower PaOOT group . Consequ−4 mm2/sec vs (8.4±0.3)×10−4 mm2/sec, p = 0.002] compared to PROT lambs, whereas AD and MD were not significantly different. Two of the INJ lambs had significantly higher FA, lower MD, lower AD and lower RD  in the cerebellum compared to the other animals  and lower mean RD [(7.8±0.3)×10 animals , but no Mean arterial pH correlated negatively with FA  and AD  in the internal capsule but not in the other investigated ROIs.Compared to the PROT group, MRS peak-area ratios of lactate relative to other metabolites  were more scattered in INJ lambs . At a grT ventilation during the initial stabilization of preterm lambs causes white matter gliosis, inflammation and vascular leakage compared to lambs that received a protective ventilation strategy in vivo imaging, using advanced MRI techniques, could detect ventilation-induced brain injury. While no macroscopic injury was observed using conventional MRI, we observed acute changes in diffusion measures and metabolite peak-area ratios in injuriously ventilated lambs compared with protective ventilated lambs, which may reflect the initiation of brain injury.High VT ventilation. This was an expected finding supported by longitudinal studies where acute injury, although originating in the immediate newborn period, evolves over time and is not evident until many hours, or even days after birth In analogy with our histopathological study In a recent large study of very preterm neonates, Card et al We were unable to demonstrate differences in peak-area ratios for NAA/Cr, NAA/Cho or Cho/Cr between intervention groups. As these ratios are thought to indicate neuronal integrity and cell membrane turnover Several studies in neonates have used DTI to investigate neurodevelopment and injury in vivo imaging approach that to our knowledge has not been conducted previously in ventilated lambs.The current animal model provides several translational advantages. The sheep brain has many similarities with the human in terms of anatomy, physiology and development A limitation of the present study is that the lamb lungs at 0.85 of gestation are functionally equivalent to a 25–27 week preterm infant in vivo assessment of brain metabolites and diffusion parameters may provide useful indicators of effective neuroprotective strategies. Importantly, the present research contributes to bridging the gap between neonatal cardio-pulmonary and brain imaging research findings.In conclusion, injurious ventilation during stabilization of preterm lambs has early adverse effects on the brain that are detectable with MRS and DTI. The observed changes may precede morphological abnormalities that are not seen on conventional MRI and could reflect progression towards overt and/or permanent brain injury. Further


The antioxidant activity of these four bromophenols was examined using one biochemical and two cellular assays: Oxygen Radical Absorbance Capacity (ORAC), Cellular Antioxidant Activity (CAA) and Cellular Lipid Peroxidation Antioxidant Activity (CLPAA) assays. Compound 2 distinguished itself by showing potent activity, having a better antioxidant effect than luteolin in both the CAA and CLPAA assays and of quercetin in the CLPAA assay. Although several bromophenols are known to be potent antioxidants in biochemical assays, this is the first time their cellular antioxidant activity has been demonstrated.Three known bromophenols, 2,3-dibromo-4,5-dihydroxybenzylaldehyde ( Marine macroalgae are known to contain structurally diverse natural compounds with a range of different biological activities . One claStructurally, the BPs contain one or several benzene rings and a varying number of bromine and hydroxyl-substituents . The relPolysiphonia morrowii in 1955 +, calculated for C7H4Br2O3 292.8448. Compound 2: m/z 494.8067 [M − H]+, calculated for C14H11Br3O5 494.8078. Compound 3: m/z 572.7182 [M − H]+, calculated for C14H10Br4O5 571.7103. Compound 4: m/z 972.6160 [M − H]+, calculated for C28H20Br6O9 972.6129.Compound 1–3 and elucidation of compound 4. The NMR experiments were acquired on an Agilent (Varian) Inova spectrometer operating at 599.934 and 150.863 MHz for 1H and 13C, respectively, using a cryogenically cooled inverse detection HCN probe with enhanced proton channel (2nd generation). Typically, 16k data points were acquired for the 1D 1H experiments using 90° reading pulses, 5 seconds of relaxation delay and 64 transients. Data matrices of 1440 × 200 and 1440 × 256 complex points were acquired for the gradient selected edited HSQC and absolute value HMBC, respectively, using 32 transients. For carbon 1D spectra, 32k complex data points were acquired, using 45° reading pulses, 1-second relaxation delay and 20,000 transients, applying 1H decoupling during both relaxation and acquisition time. All spectra were acquired at 298 K in methanol-d4 or CDCl3 (Sigma-Aldrich) using sweep widths of 9600 and 30,200–37,800 Hz for 1H and 13C, respectively. All experiments were referenced to the residual solvent peak; 3.34 ppm for methanol-d4 and 7.26 ppm for CDCl3  (MTS) assay.Cell viability was determined by a colorimetric  and green [485 (excitation)/520 (emission)] fluorescence was recorded during ~1 h at 37 °C. Cells were washed with PBS between additions of new reagents. The total reaction volume was 100 μL (where 20 μL was the volume of the test compound). All incubations were carried out at 37 °C in a humidified atmosphere of 5% CO2.The method was carried out in the same plates as in the CAA assay. Fluorescence was recorded using the same plate reader, software and temperature as for the ORAC and CAA assays. HepG2 cells were seeded and incubated overnight, as described in the CAA assay. The cells were labelled with 5 µM C11-BODIPY was calculated based on the increase of green fluorescence as: activity (%) = (fluorescence treated well − fluorescence positive control)/(fluorescence negative control − fluore scence positive control) × 100. Inhibition (%) was calculated as: 100% − activation%. The results are presented as relative values compared to the positive and negative controls, with and without cumOOH, respectively.Cells in MEM Earle’s medium were used as negative control and cells treated with cumOOH as positive control. The oxidative degeneration of C2), have been reported for the first time. The fact that the compound was active in the CAA assay demonstrates that it enters cells, and as it was also active in the CLPAA assay, it shows the ability to decrease lipid peroxidation in cell membranes. Compound 2 was a better antioxidant than luteolin in both cellular assays and of quercetin in the CLPAA assay, and it was active well below the cytotoxic concentration in all assays. Cellular antioxidant effects for the bromophenol, 2,2′,3-tribromo-3′,4,4′,5-tetrahydroxy-6′-hydroxymethyl-diphenylmethane  for BPs, and the ones published are all from l assays . Hydroxyl assays ,8,30,31.ch other . The degy of BPs ,31. Haloy of BPs . Thus, ain vivo conditions, taking into account factors like bioactivity and membrane permeability. The cellular assays can be used to, at an early stage, exclude compounds lacking the ability of penetrating membranes and, hence, do not have an effect in vivo. This present identification of cellular antioxidant activity of structurally related BPs can be used as a starting point for SAR studies, which are more relevant to


Diagnostic values of FibroTest (FT) for hepatic fibrosis have rarely been assessed in Asian chronic hepatitis B (CHB) patients. We aimed to validate its diagnostic performances in comparison with liver stiffness (LS).From 2008 to 2010, 194 CHB patients who underwent liver biopsies along with FT and transient elastography were prospectively enrolled. Fibrosis stage was assessed according to the Batts and Ludwig system.To predict significant fibrosis (F≥2), advanced fibrosis (F≥3), and cirrhosis (F = 4), areas under receiver operating characteristic curves (AUROCs) of FT were 0.903, 0.907, and 0.866, comparable to those of LS . Optimized cutoffs of FT to maximize sum of sensitivity and specificity were 0.32, 0.52, and 0.68 for F≥2, F≥3, and F = 4, while those of LS were 8.8, 10.2, and 14.1 kPa, respectively. According to FT and LS cutoffs, 123 (63.4%) and 124 (63.9%) patients were correctly classified consistent with histological fibrosis , respectively. Overall concordance between each fibrosis stage estimated by FT and LS was observed in 111 patients, where 88 were correctly classified with histological results. A combination formula adding LS to FT (LS+FT) showed similar AUROC levels , while another multiplying LS by FT (LS×FT) showed the best AUROCs .FT provides good fibrosis prediction, with comparable outcomes to LS in Asian CHB patients. FT substantially reduces need for liver biopsy, especially when used in combination with LS. Accurate assessment of the severity of liver fibrosis in patients with chronic hepatitis B (CHB) is necessary not only for predicting the long-term clinical course, but also for determining whether and when to begin antiviral therapy. Most recent guidelines on the management of CHB have proposed that the presence of significant fibrosis with detection of serum hepatitis B virus (HBV) DNA is a clear indication to commence antiviral therapy, because maintenance of viral suppression can reduce liver-related complications in patients with CHB who have significant fibrosis or cirrhosis To date, liver biopsy has been the gold standard for assessing liver fibrosis. It is often limited, however, by its invasiveness, cost, risk of complications, poor acceptance, lack of availability of expert practitioners, and intra/inter-observer variability Recently, liver stiffness (LS) value assessed by transient elastography , which relies on calculating liver elasticity from the velocity of a low frequency elastic wave transmitted through the liver, has been introduced as a noninvasive surrogate for liver biopsy in the assessment of liver fibrosis et al.2-macroglobulin, apolipoproteinA1, haptoglobin, γ-glutamyl-transpeptidase (GGT), and total bilirubin for assessment of liver fibrosis, adjusted by age and gender. FT has been studied extensively as a surrogate marker for liver biopsy. It was initially validated primarily in Caucasian populations with CHC and the results showed a good correlation with liver fibrosis stage Meanwhile, Poynard The present study was performed to prospectively validate the diagnostic value of FT in Asian populations with CHB in comparison with TE, to define optimized thresholds for predicting liver fibrosis, and to investigate the performance of the combined use of FT and LS.Consecutive patients with CHB who underwent liver biopsy along with FT and TE on the same day at Severance Hospital, Yonsei University College of Medicine, Seoul, Korea, between July 2008 and June 2010, were considered eligible for this study. Liver biopsy was performed to assess the severity of fibrosis and inflammation prior to treatment.The exclusion criteria were as follows: previous history of antiviral therapy; history of hepatocellular carcinoma (HCC) treatment at the time of liver biopsy; diagnosis of malignancy other than HCC during follow-up; liver biopsy specimen shorter than 20 mm; coinfection with human immunodeficiency virus; invalid LS values with fewer than ten successful acquisitions, a success rate of less than 60%, or interquartile range (IQR)/median value ratio (IQR/M) greater than 0.3; alcohol ingestion in excess of 40 g/day for more than 5 years; or right-sided heart failure.The study was performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from each participant or responsible family member after possible complications of the diagnostic procedures had been fully explained. This study was approved by the Institutional Review Board of Severance Hospital.www.biopredictive.com) as follows: f = 4.467×log[α2-macroglobulin (g/L)]−1.357×log[haptoglobin (g/L)]+1.017×log[γ-GGT (IU/L)]+0.0281×[age (in years)]+1.737×log[bilirubin (µmol/L)]−1.184×[apolipoprotein A1 (g/L)]+0.301×sex −5.540.All laboratory data including specific parameters for calculating FT score including α2-macroglobulin level, apolipoprotein A1 level, haptoglobin level, γ-GGT level, and total bilirubin level were obtained on the same day as TE and liver biopsy. FT score was computed on the BioPredictive website  and Masson's trichrome. All liver tissue samples were evaluated by an experienced hepatopathologist (YN Park) who was blinded to the patients' clinical histories. Specimens that were shorter than 20 mm and considered by the pathologists to be unsuitable for fibrosis assessment were excluded from the analysis. Liver histology was evaluated semiquantitatively according to the Batts and Ludwig scoring system The major goals of this study were to prospectively validate the diagnostic performance of FT for detection of the presence of histological significant fibrosis, advanced fibrosis, and cirrhosis in comparison with LS and to suggest optimal cutoff values in patients with CHB. To assess the diagnostic performance of each noninvasive index, receiver operating characteristic (ROC) curves were constructed and the areas under the ROC curves (AUROCs) were calculated. Then, to evaluate the usefulness of the noninvasive method, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined from the ROC curves. Furthermore, we evaluated the usefulness of combined use of FT and LS as a surrogate marker for liver biopsy.p<0.05 was taken to indicate statistical significance.Statistical analyses were performed using SAS software version 9.1.3 . In all analyses, A total of 350 consecutive patients were screened for possible inclusion in the study. Based on the exclusion criteria, a total of 194 patients  were analyzed .The patients' characteristics are summarized in p<0.05). As the fibrosis stage increased from F1 to F4, the mean value of LS increased from 6.9±4.2 kPa in F1, 9.7±5.7 kPa in F2, 12.1±2.97 kPa in F3, and 21.1±10.1 kPa in F4, while that of FT also increased from 0.16±0.17 in F1, 0.34±0.18 in F2, 0.6±0.23 in F3, and 0.76±0.19 in F4. LS and FT were significantly different between F1 and F2 (p = 0.035 and p<0.001), F2 vs. F3 (p = 0.034 and p<0.001), and F3 vs. F4 (p<0.001 and p<0.001), respectively.As shown in p>0.05 by Hanley and McNeil test) With regard to the diagnostic performances of LS and FT in the prediction of histological liver fibrosis, the AUROCs of LS and FT were 0.873  and 0.903 (95% CI 0.838–0.968) for significant fibrosis (F≥2) , 0.897 , and 0.9The most discriminant cutoff values for LS and FT were determined from the ROC curves to maximize the sum of sensitivity and specificity LS agreed with liver biopsy on the diagnosis of F<2 vs. F≥2 in 154 patients (79.3%), F<3 vs. F≥3 in 172 patients (88.7%), and F<4 vs. F = 4 in 164 patients (84.5%), while FT agreed on the diagnosis of F<2 vs. F≥2 in 158 patients (81.4%), F<3 vs. F≥3 in 170 patients (87.6%), and F<4 vs. F = 4 in 160 patients (82.5%) . In addiIrrespective of matching with histological fibrosis stages, 152 patients (78.3%) showed agreement in fibrosis staging using LS and FT for noninvasive estimation of F<2 vs. F≥2, 158 patients (81.4%) for F<3 vs. F≥3, and 146 patients (75.3%) for F<4 vs. F = 4. Thus, when LS and FT agreed for noninvasive prediction of F<2 vs. F≥2, F<3 vs. F≥3, and F<4 vs. F = 4, these results agreed with those of liver biopsy examination in 88.8% (134 of 152 patients), 96.8% (153 of 158 patients), and 94.5% (138 of 146 patients), respectively.Overall agreement between each fibrosis stage  estimated by LS and FT was observed in 111 patients. Among them, 88 patients (79.3%) were correctly classified with reference to liver biopsy examinations, and they could avoid liver biopsy based upon concordant results between the two tests . Among these patients with discordance between LS and FT, 41 and 42 patients showed higher fibrosis stage by LS and by FT, respectively. The baseline characteristics were compared between these two groups, and none was identified as a significant factor capable of explaining this difference.Discordant results between fibrosis stages estimated by LS and FT were identified in 83 patients (42.8%). On multivariate analysis, only the presence of histological cirrhosis was identified as a single significant factor, which showed a negative association with discordance between LS and FT , which showed the best AUROC for prediction of F≥2 , F≥3 , and F = 4 , compared to LS, FT, and LS+FT. The optimized cutoff values of combination formula, LS+FT and LS×FT, are described in detail in Using the above cutoffs, LS+FT agreed with liver biopsy on the diagnosis of F<2 vs. F≥2 in 164 patients (84.5%), F<3 vs. F≥3 in 176 patients (90.7%), and F<4 vs. F = 4 in 169 patients (87.1%), while LS×FT agreed on the diagnosis of F<2 vs. F≥2 in 165 patients (85.1%), F<3 vs. F≥3 in 175 patients (90.2%), and F<4 vs. F = 4 in 170 patients (87.6%). Overall, 129 (66.5%) and 130 (67.0%) patients were correctly classified consistent with each histological fibrosis stage  using LS+FT and LS×FT, respectively.FT, a good surrogate marker for liver biopsy, has been widely studied regarding its usefulness for noninvasive prediction of fibrosis stage primarily in western populations with HCV infection Although the underlying mechanisms of fibrosis progression in chronic viral hepatitis are expected to be similar, several differences according to etiology may affect diagnostic accuracy This study has several strengths. First, we prospectively recruited patients who underwent the baseline blood tests and LS on the same day as liver biopsy, and the diagnostic performance of FT was compared to LS, which has already shown excellent diagnostic value in Asian populations with CHB In the present study, the diagnostic performance of FT was comparable to that of LS for diagnosing fibrosis stages: 0.903 vs. 0.873 for significant fibrosis (F≥2), 0.907 vs. 0.897 for advanced fibrosis (F≥3), and 0.866 vs. 0.910 for cirrhosis (F = 4), respectively. Using the Youden method Among subjects where LS and FT agreed with prediction of F≥2, F≥3, and F = 4 regardless of matching with histological examinations, concordance with liver biopsy examination was observed in 88.8%, 96.8%, and 94.5%, respectively. Furthermore, overall agreement between each fibrosis stage  estimated by LS and FT was observed in 111 patients (57.2%). Among them, 88 patients (79.3%) were correctly classified with reference to liver biopsy examinations, and they could avoid liver biopsy based on the concordant results between the two tests . In contp = 0.008). Because the higher ALT has been known as a well-known overestimating confounder of LS With regard to discordance between LS and FT, among various baseline factors, only the presence of histological cirrhosis was identified as a single significant factor with a negative association with such discrepancies. Consistent with a previous study Several investigators This study is limited in that its design was cross-sectional. It is not clear whether repeated determination of FT score may be useful for tracking the progression of fibrosis and related clinical outcomes, such as occurrence of hepatic decompensation and HCC in individual patients. Therefore, the diagnostic value for predicting the subsequent development of cirrhosis and its various complications with sequential FT measurements during long-term follow-up must be examined further in a longitudinal study. Second, our population did not include patients with F0. As our institute is a tertiary referral hospital and one of the largest medical centers in Korea, patients with relatively more advanced disease status are likely to be referred for close follow-up. It might have resulted in a selection bias and eventually a spectrum bias, since the diagnostic performance of a given noninvasive test tends to increase in general in a cohort with the high disease prevalence and thus the diagnostic performances of LS and FT might have been overestimated in our cohort accordingly. Therefore, another independent external validation study in a population with minimal fibrotic burden should be performed to provide more generalizable results in patients with CHB-related chronic liver disease.In summary, in a prospective study, we first assessed FT in Asian patients with CHB, demonstrating its comparable diagnostic accuracy to LS for predicting histological fibrosis stage. The optimal suggested cutoff values are expected to be useful as reference values for future studies in Asian patients with CHB. Our results suggest that combined use of LS and FT could avoid invasive liver biopsy in most patients with CHB. We hope that other researchers will evaluate the reproducibility of FT for the noninvasive diagnosis of fibrosis stage in independent populations.