Several p-value combining methods have been developed and applied to genetic data; see Dai et al.  are often correlated due to linkage disequilibrium (LD). Other genetic data, including variants from next generation sequencing, gene expression levels measured by microarray, protein and DNA methylation data, etc. also contain complex correlation structures. Ignoring correlation structures among genetic variants may lead to severe inflation of Type I error rates for omnibus testing of p-values. In this work, we propose modifications to the Lancaster procedure by taking the correlation structure among p-values into account. The weight function in the Lancaster procedure allows meaningful biological information to be incorporated into the statistical analysis, which can increase the power of the statistical testing and/or remove the bias in the process. Extensive empirical assessments demonstrate that the modified Lancaster procedure largely reduces the Type I error rates due to correlation among p-values, and retains considerable power to detect signals among p-values. We applied our method to reassess published renal transplant data, and identified a novel association between B cell pathways and allograft tolerance.Rapid developments in molecular technology have yielded a large amount of high throughput genetic data to understand the mechanism for complex traits. The increase of genetic variants requires hundreds and thousands of statistical tests to be performed simultaneously in analysis, which poses a challenge to control the overall Type I error rate. Combining i et al.  for a co From a systems biology perspective, genes, cells, tissues, and organs function as a system through metabolic networks and cell signaling networks. In non-Mendelian inheritance patterns, such as complex disorders, a subset of genetic variants may jointly confer moderate effects in mediating molecular activities. As a result, signals may not be significant in single marker-single trait analysis, but many such values from related genes might provide valuable information on gene function and regulation. For instance, in pathway analysis  of p-values evaluates the pattern (distribution) of p-values instead of selecting p-values less than an arbitrary threshold. Therefore, this method has the potential to identify multiple genes with small effects. If we assume that all individual tests are independent and arise from genetic variants with no effects, then p-values are identically and independently distributed as Uniform . Taking this as a null hypothesis for the pattern of p-values in the global test, one can assess whether p-values, especially small p-values, are generated by chance. The global test of p-values is robust and can be applied to p-values from varying statistical models including t-tests, analysis of variance (ANOVA), linear mixed models, and so forth. Multiple simulation studies and case studies have demonstrated that this approach usually has sufficient power to detect signals of genetic association from a group of genes. For instance, Peng et al. , where p-values might first be transformed by a function H. So far, several statistical methods have been developed to combine p-values. Let ip be independent p-values obtained from n hypothesis tests. Under the null hypothesis (H0) that p-values follow a Uniform  distribution, Fisher (ip) follows a chi-square distribution with 2n degrees of freedom. For a one sided test with a nominal error rate of α, one can reject the null hypothesis when the test statistics exceeds the (1 − α)*100% percentile of χ2n2. Stouffer  for N. Under the null hypothesis, the z-test statistic follows N.One category of global tests of , Fisher  shows thp-values, Littell and Folks  under H0.Although there is no consensus regarding the most powerful method of combining nd Folks , 1973 dend Folks . Subsequancaster  generaliand Bush  proposedz-test. The Bahadur efficiency ratio gives the limiting ratio of sample sizes required by two statistics to attain an equally small significance level. Thus, Bahadur efficiency is an important method to compare test statistics. From the perspective of Bahadur efficiency, the Lancaster procedure asymptotically requires a relatively smaller sample size than other weighted p-value combining methods. This prompted us to focus on modification of the Lancaster procedure for correlated genetic data in this work.In a separate paper, we have proved that the Lancaster procedure achieves the optimal Bahadur efficiency. We further demonstrated that the Lancaster procedure yields higher Bahadur efficiency than the weighted p-value combining methods as compared to smaller genes  to a chi-square distribution, i.e., γ−1iw /2, 2)( (1−ip)~ χ2iw where χ2iw is a chi-square distribution with iw >0 degree(s) of freedom. The parameter iw serves as a weight function to adjust the individual p-values. When p-values are independent, T has an exact chi-square distribution with In this section, we allow p-values, T does not have an explicit analytical form. To address this issue, we consider a Satterthwaite approximation by approximating a scaled T statistic with a new chi-square distribution , we use the Satterthwaite method to match the mean and variance of cT and χ2v, and then solve the equations to derive c and v. Koziol  = α. It follows that the corresponding percentage point for Approximate ET approximation.cT by χ2v then perform saddle point approximation  = Φ (ya) − ϕ(yb−1 − ya−1) for y ≠ 1 and K(t) = −0.5log (1 − 2t), and yt = (y − 1)/2y.Approximate ij is unknown, one can use the permutation approach to estimate ρij by shuffling the phenotype variable among subjects. For the kth permutation , we keep the genetic variants within the subject to preserve the correlation structure, then randomly assign the phenotype variable to subjects. Individual hypothesis testing can be done on all n genetic variants separately to generate the p-value vector kp = t. The permutation is repeated m = 1000 times, and ρij is estimated from .When the covariance ρp-values with varying correlation structures. We consider six different types of correlation structures, including fixed and random compound symmetric as well as random positive definite variance-covariance structures for Σ. Let I be an identity matrix, t be the transposition. In Cases I–V, let Σ = Block ⊗ I20 be compound symmetric variance matrices with 20 blocks of size 5 where The accuracy of the five approximate distributions to the correlated Lancaster procedure is then assessed using T that combines n p-values is renamed as LancasternT in Figure y = x, indicating the limiting distribution for the test statistic in the original Lancaster procedure is less dispersed than the distribution of LancasternT under correlation structures. As a result, the original Lancaster procedure will have severely inflated Type I errors. In contrast, the five approximations  match the underlying distribution of LancasternT. For data with stronger internal correlation, AT, DT, and ET better approximate LancasternT. The Q-Q plots under other correlation structures are similar to Figure The quantile-quantile (Q-Q) plot assessing the accuracy of the proposed methods when the correlation coefficient ρ = 0.3 is shown in Figure q1 and q2 be the minor allele frequencies (MAFs) at loci 1 and 2. Assuming Hardy–Weinberg equilibrium, the genotype at locus 1 can be randomly generated using a binomial distribution. Given the distribution of SNP at locus 1, one can simulate the genotype at locus 2. To do so, let D be a measure of LD. Then the conditional probability for the genotype at locus 2 given the genotype at locus 1 can be expressed as P(A|B) = [A qBq + D]/Bq, P(a|B) = [(1−Aq)Bq −D]/Bq, P(A|b) = [Aq (1−Bq)−D]/(1−Bq), and P(a|b) = [(1−Aq)(1−Bq)+D]/(1−Bq) where A and B represent the minor alleles at the two loci. For a diploid genome, similar idea can be applied and the simulation details can be found at Cui et al. . The variable D = 0, 1.5, and 2 suggests no LD, moderate LD, and very strong LD among SNPs with the corresponding correlation R = 0, 0.71, and 0.95. Six scenarios for disease susceptibility (p) are simulatedWe assess the Type I error rates and power for the proposed correlated Lancaster procedures and compare them to the independent Lancaster procedure ) = β1G1, 2 +β2G1, 5 +β3G1, 7 +β4G1, 8 +β5G1, 12.Case I: ln (p/(1−p)) = β1G2, 2 +β2G2, 4 +β3G2, 6 +β4G3, 2 +β5G3, 3.Case II: ln (p/(1−p)) = β1G3, 2 +β2G3, 4 +β3G4, 1 +β4G4, 3 +β5G5, 1.Case III: ln (p/(1−p)) = β1G1, 1 +β2G1, 3 +β3G1, 7 +β4G1, 8G1, 10G1, 11 +β5G1, 12.Case IV: ln (p/(1−p)) = β1G3, 1 +β2G3, 3 +β3G4, 2 +β4G3, 2G3,4 +β5G4, 3G5, 1.Case V: ln (p/(1−p)) = β1G1, 2 +β2G2, 2 +β3G3, 3 +β4G5, 2 +β5G1, 5G1,7 +β6G3, 3G5, 1.Case VI: ln ( (1−pi) transforms p-value into a variable with χ22 distribution.Weight functions can be used to remove potential bias when combining g et al.  and othen = 200  have well controlled Type I error rates (<0.05).Due to LD, SNPs from the same gene are correlated. We first assess the Type I error rate of the test statistics by testing n = 200 to n = 400.The power of all test statistics was compared for regression coefficient values set at β = 0.4 and β = 0.6, respectively. The results in Tables D = 0, 1.5, 2, and uniform. To save the space, we only show the results for D = 1.5 (Table D = 2 (Tables AT − ET) have well-controlled Type I error rates and power for both moderate and strong LD (Tables We simulate different levels of LD for SNPs with .5 Table  and D = D Tables .AT − ET), the power is increased when a weighting function is used. This confirms that an appropriate weight function is beneficial to the Lancaster procedure.In Table http://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS4266). A group of tolerant renal transplant recipients , as defined by stable graft function in the absence of immunosuppression for more than 1 year, were compared to subjects with stable graft function who were receiving standard immunotherapy  as well as to a group of healthy controls . Gene expression profiles of whole-blood total RNA from all subjects were measured by microarray. The goal of the study was to identify genetic variants associated with long-term allograft survival without the requirement for continuous immunosuppression, a condition known as allograft tolerance. Newell et al.  preprocessing methodology, which included background subtraction, quantile normalization, and summarization via median-polish.F-test. This is equivalent to a One-Way ANOVA for each gene except that the residual mean squares have been moderated across genes. P-values from multiple hypothesis testing were adjusted by FDR  as described in Section Correlated Lancaster Procedures to combine p-values from pathways. Combining p-values allows us to integrate small effects in pathway and gain the power of statistical testing. A total of 1454 Gene Ontology human pathway gene sets were analyzed. The size of pathways ranged from 9 genes to 2131 genes, with a median of 27 genes per pathway. Also, the number of probes per gene was highly variable. In order to map genes to pathways, we removed genes without gene symbols from the analysis. Among 21,049 genes with gene symbols, approximately 48% (n = 10161) of genes were interrogated with a single probe, 26% (n = 5389) of genes were queried using 2 probes, 14% (n = 2842) of genes were assessed by 3 probes. There were 3 or more probes for each on the remaining genes (range: 4–17). This finding indicates that larger genes would have more p-values and a stronger impact to pathway analysis. To prevent this bias, we set the weight function as in is the number of probes for the ith gene.To address this issue, we performed the modified Lancaster procedure . The increase in number of significant pathways identified by the traditional approach is primarily due to false positive discovery, and is consistent with the inflation of Type I error rate as presented in Section Empirical Assessments. Thus, by accounting for correlation structures (LD) within pathways and the number of probes per gene, our proposed method minimized identification of larger, non-specific cellular processes pathways, and instead revealed more focused and functionally relevant biological pathways implicating a role for a humoral immune response in immunotolerance to renal transplants , the Satterthwaite approximation (AT) is the most computationally efficient. Other approximation methods  are based on the Satterthwaite approximation. Therefore, we recommend using the Satterthwaite approximation (AT) as the standard procedure to modify the Lancaster procedure. Among other approximation methods, simulation results in Section Correlated Lancaster Procedures show that, for data with stronger internal correlation, DT and ET have better approximation than BT and CT. Our simulation study and the case study further provide evidence that DT tends to have slightly higher power than the Satterthwaite approximation AT. The R code for five approximation is posted at http://d.web.umkc.edu/daih/.Among five proposed approximation methods (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our results suggest that Hv1 may be used as a potential biomarker for diagnosis and prognosis of colorectal carcinoma, and a potential target for anticancer drugs in colorectal cancer therapy.Solid tumors exist in a hypoxic microenvironment, and possess high-glycolytic metabolites. To avoid the acidosis, tumor cells must exhibit a dynamic cytosolic pH regulation mechanism(s). The voltage-gated proton channel Hv1 mediates NADPH oxidase function by compensating cellular loss of electrons with protons. Here, we showed for the first time, that Hv1 expression is increased in colorectal tumor tissues and cell lines, associated with poor prognosis. Immunohistochemistry showed that Hv1 is strongly expressed in adenocarcinomas but not or lowly expressed in normal colorectal or hyperplastic polyps. Hv1 expression in colorectal cancer is significantly associated with the tumor size, tumor classification, lymph node status, clinical stage and p53 status. High Hv1 expression is associated significantly with shorter overall and recurrence-free survival. Furthermore, real-time RT-PCR and immunocytochemistry showed that Hv1 is highly expressed in colorectal cancer cell lines, SW620, HT29, LS174T and Colo205, but not in SW480. Inhibitions of Hv1 expression and activity in the highly metastatic SW620 cells by small interfering RNA (siRNA) and Zn Increasing pHo or lowering pHi promotes H+ channel opening by shifting the activation threshold to more negative potentials 2+ and Cd2+ and other divalent cations The voltage-gated proton channel Hv1 was identified using bioinformatics searches based on known cation channels, which is mainly expressed in immune cells such as macrophages, neutrophils, and eosinophils + channels are comprised of four subunits, each of which has a pore domain and a VSD. The four pore domains come together to form one single central pore, and four peripheral VSDs control the gate of the pore + channels, the Hv1 contains a VSD but lacks the pore domain. Recent studies showed that Hv1 functions as a dimer in which the intracellular C-terminal domain is responsible for the dimeric architecture of the protein, and each subunit contains its own proton-transporting pathway α-helical coiled-coil and is essential for the protein localization Hv1 contains three predicted domains: N-terminal acid and proline-rich domain, transmembrane voltage-sensor domain (VSD), and C-terminal domain. Voltage-gated KTumor cells often exist in a hypoxic microenvironment, and possess high-glycolytic activity and produce acidic metabolites All of the procedures were done in accordance with the Declaration of Helsinki and relevant policies in China. We obtained the written informed consent from all participants involved in our study. The study obtained ethics approval for our study from the ethics committee of Tonghua Center Hospital.Colorectal cancer tissue samples were obtained from patients who underwent routine curative surgery at the Department of Surgery, Tonghua Center Hospital between 2001 and 2007. The patients were not pretreated with radiotherapy or chemotherapy prior to surgery. 139 colorectal cancer tissues and paired adjacent non-tumor colorectal tissues were fixed in 10% formalin and embedded in paraffin for immunohistochemical analysis. The clinicopathological features of these patients were shown in Escherichia coliAn anti-Hv1 polyclonal antibody was generated against the carboxyl terminal domain of Hv1 . The protein was purified to homogeneity after expression in 2 incubator at 37°C. Cells grown on glass coverslips at 50–70% confluence in a six-well plate were transiently transfected with pHv1-EGFP plasmid by using lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Cells were used for experiments 36–48 h after transfection.Hv1 cDNA was cloned into pEGFP-N1 (Clontech) to create Hv1 expression plasmid pHv1-EGFP, fused with the enhanced green fluorescent protein (EGFP) moiety attached to the C-terminus of Hv1 Western blotting was performed with an anti–Hv1 polyclonal antibody (1 mg/ml) as described above with a final dilution of 1 1000. The denatured proteins were separated by 12.5% SDS-PAGE and then transferred to a PVDF membrane  by wetting electroblotting devices. Nonspecific protein absorption was prevented using 5% (w/v) skim milk in phosphate-buffered saline containing 0.1% Tween 20 (PBS-T) for 1 h. Primary antibody incubation in PBS-T was performed for 1 h at room temperature. The HRP-coupled anti-mouse secondary antibody was used at a final dilution of 1 15,000 in PBS-T, and HRP was revealed with a chemiluminescent detection system (Millipore).2O2) for 10 min to inhibit endogenous peroxidase activity. Subsequently, the sections were incubated in PBST containing 5% fetal bovine serum and 2% BSA for 30 min to reduce nonspecific binding. Incubation with primary antibody was performed overnight at 4°C in a humidified chamber. HRP-coupled anti-mouse secondary antibody was used at a final dilution of 1 400 for 1 h. Finally, the visualization signal was developed with diaminobenzidine (DAB) and the slides were counterstained in hematoxylin. Negative control was performed to treat with non-immune mouse serum as the primary antibody instead of anti-Hv1 antibody.Histological diagnoses of tumourous and non-tumourous formalin-fixed and paraffin-embedded tissues were confirmed in haematoxylin and eosin-stained sections. Immunohistochemistry was performed with an anti-Hv1 polyclonal antibody. The anti-Hv1 antibody (1.0 mg/ml) was diluted 100-fold with PBST  containing 1% (w/v) BSA. The paraffin-embedded sections filled with 10 mM ethylenediaminetetracetic acid (EDTA) buffer (pH 8.0) were heated in a microwave oven for 12 min. After cooling, the sections were treated with 0.5% Triton X-100 in PBS for 10 min, exposed to 3% (v/v) hydrogen peroxide . Briefly, the IRS assigns sub-scores for immunoreactive distribution (0–4) and intensity (0-3), then multiplies them to yield the IRS score. The percent positivity was scored as “0” (<5%), “1” (5–25%), “2” (25–50%), “3” (50–75%), “4” (>75%). The staining intensity was score according to the area of Hv1-positive staining cells as “0, −” , “1, +” , “2, ++” , and “3, +++” . The final Hv1 expression score was calculated from the values of percent positivity score and staining intensity score, which was ranged from 0 to 12. We estimated IRS by averaging the values in eight fields at ×500 magnification for each specimen. Hv1 expression levels were defined as follows: low expression (score ≤3) and high expression (score >3). Immunohistochemical analysis and scoring were performed by two independent experienced pathologists.2 with DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 mM L-glutamine.The human colorectal cancer cell lines SW620, HT29, LS174T, Colo205 and SW480 were cultivated at 37°C in an atmosphere of 95% air and 5% CO2O2) for 15 min, and blocked with 5% fetal bovine serum and 2% BSA in PBST for 20 min at room temperature. The blocked coverslips were incubated with the anti-Hv1 antibody (1.0 mg/ml) at a dilution of 1 200 with PBST containing 1% (w/v) BSA at 37°C for 1 h. After washing in PBS for three times, the coverslips were further incubated with HRP-coupled anti-mouse secondary antibody at a final dilution of 1 400 for 1 h. Signals were visualized by the HRP/DAB system. The cell nuclei were stained with hematoxylin.SW620, HT29, LS174T, Colo205 and SW480 cells grown on glass coverslips at confluence in six-well tissue culture plates were fixed with 4% (w/v) paraformaldehyde in PBS at room temperature for 30 min, washed in PBS, treated with 0.5% Triton X-100 in PBS for 20 min, exposed to 3% (v/v) hydrogen peroxide (H5′-CAGGTCATCATCATCTGCTTG-3′ (forward) and 5′-CCGTTCTGAACGTGTCTTAAC-3′ (Reverse); GAPDH, 5′-CCAAGGTCATCCATGACAAC-3′ (forward) and 5′-AGAGGCAGGGATGATGTTCT-3′ (reverse). PCR thermal condition used was: 94°C for 30 s; annealing, 52°C for 30 s; extension, 72°C for 30 s. Relative mRNA expression levels of proteins were calculated according to the equation: 2−ΔΔCT, in which ΔΔCT = (CTHv1 - CTGAPDH) - (CTHv1 - CTGAPDH)SW620. All experiments were performed in triplicate.The mRNA expression levels of Hv1 in SW620, HT29, LS174T, Colo205 and SW480 cells, were evaluated by quantitative real-time PCR using ABI PRISM 7000 Sequence Detection System . Total RNAs were extracted using RNAiso Reagent (Takara), and reverse-transcribed by MMLV super transcriptase (Takara). Real-time quantitative reverse transcription PCR was performed with SYBR PrimeScriptTM RT-PCR Kit (Takara) according to the manufacturer's instructions. The primers were as follows: Hv1, SW620 and SW480 cells grown on glass coverslips were fixed with 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 30 min, washed in PBS, treated with 0.5% Triton X-100 in PBS for 20 min, and blocked with 5% fetal bovine serum and 2% BSA in PBST for 1 h. The blocked coverslips were incubated with anti-Hv1 antibody (1.0 mg/ml) at a dilution of 1 200 in 2% BSA at 4°C overnight. After washing with PBS for 5 min for four times, the coverslips were further incubated for 1 h at room temperature with a FITC-conjugated goat anti-mouse IgG at a dilution of 1 400 in 2% BSA, followed by another washing as described above. Confocal images of FITC fluorescence of SW620 and SW480 cells were recorded on a Leica TCS SP5 confocal microscopy  with the FITC-filter set for Hv1 and the DAPI filter set for the nuclear DAPI dye. The images were later processed by Adobe Photoshop software.5′-CTACAAGAAATGGGAGAAT-3′, and the random sense sequence was 5′-TTCTCCGAACGTGTCACGT-3′, both of which were obtained from Ribobio  The sequence of the siRNA targeting the Hv1 gene was 2+ respectively, were assessed in wounded monolayer model. To down-regulate Hv1 expression, SW620 cells were grown to confluence and transfected with siRNA and negative control for 24 h, respectively. To inhibit Hv1 activity, 1 M ZnCl solution was added into the DMEM medium to a final concentration of 100 µM 5 per well) and cultured for 24vh to confluence and subsequently wounded with a tip. Cell movement was observed under phase-contrast microscopy, and were captured with a digital camera every 12 h.To examine the effect of Hv1 on the migratory ability of colorectal cancer cells, migrations of SW620, SW480, and SW620 cells down-regulated Hv1 expression and inhibited Hv1 activity by siRNA and ZnIn vitro invasion and migration assays were performed to assess the effects of Hv1 on invasive and migratory abilities of SW620 and SW480 cells. SW620 and SW480 cells were cultured in six-well plate in DMEM medium with 10% FBS at confluence, transfected with the siRNA and negative control respectively for 24 h, and then trypsinized, washed and counted. For cell invasion, transwells with 8 µm pore size filters (Millipore) were covered with matrigel (Becton Dickinson) and inserted into 24-well plates. And for cell migration, the transwells were not coated with matrigel. DMEM medium (500 µl) containing 10% FBS was added to the lower chamber, and 200 µl of a cell suspension (5×104 cells) was placed in the upper chamber. The plates were incubated at 37°C in humidified atmosphere containing 5% CO2 for 24 h. To inhibit Hv1 activity, 1 M ZnCl solution was added into the DMEM medium to a final concentration of 100 µM SW620] ×100% and [invasion cell No. of test/invasion cell No. of control SW620] ×100%, respectively.i) in response to membrane depolarization by BCECF fluorescence 2, 1 mM MgCl2, 20 mM Tris, pH 7.5) for 3 times. Cells were incubated with NH4Cl/NMDG (N-methyl D-glucamine) solution  for 20 min and washed by ammonium free solution , rapidly inducing intracellular acidification + solution . Intracellular pH changes acid-loaded cells were detected by fluorescent probe BCECF at excitation wavelengths of 490 nm and 440 nm and an emission wavelength of 525 nm under membrane depolarizing condition using RF-5301PC Spectrofluorophotometer .The activity of Hv1 in colorectal cancer cells was assessed as a change in intracellular pH . Calibration of fluorescence vs pH was performed by equilibration of external and internal pH with nigericin (10 µM) in a high K+ buffer with a range of pH from 5.5 to 8.0. The high K+ buffer contained 145 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgCl2, and 20 mM HEPES (or MES). The relative fluorescence ratio values were plotted against corresponding pHi values, which allowed determination of the unknown pHi.Intracellular pH was measured using the pH sensitive fluorescent probe BCECF-AM. Cells cultured in the monolayers were incubated with 3.0 µM of BCECF-AM (Molecular Probe) in bicarbonate-free DMEM medium at 37°C for 30 min. After loading, the cells were washed three times with HEPES buffer to remove the extracellular dye, and made to remain in the identical buffer. The HEPES buffer contained 140 mM NaCl, 5 mM KCl, 1 mM MgSOt test. P values <0.05 were considered significant. Survival analysis was assessed using Kaplan-Meier method and survival rate was compared by log-rank test.All statistics were performed using SPSS16.0 software. Measurement data was represented as mean ± SD. Comparison of the mean between groups was performed by Clinicopathologic profiles from the 139 cases selected for this study were reviewed in Hv1 expressed in immune cells is associated with “respiratory burst” As shown in P = 0.007), age (P = 0.021), tumor size (P = 0.000), lymph node status (P = 0.000), clinical stage (P = 0.000) and p53 status (P = 0.014) in patients who had high Hv1 expression compared with patients who had low Hv1 expression. There was no significant association between Hv1 expression and the other clinical features, such as gender, differentiation and Ki-67 expression. In addition, the correlations between the expression levels of p53, Ki-67, TopoII, GST-π and P-gp and clinicopathologic characteristics were showed in P = 0.011), Ki-67 to differentiation (P = 0.010), TopoII to differentiation (P = 0.010), and GST-π to tumor size (P = 0.007) and differentiation (P = 0.000). However, P-gp did not show statistical significance with clinicopathologic parameters.The correlations between Hv1 expression and clinicopathologic characteristics are summarized in P = 0.008, P = 0.008, P = 0.008) compared with patients who had low Hv1 expression  (P = 0.008). Univariate Cox regression analyses showed that Hv1 expression level was significantly associated with recurrence-free and overall survival, whereas other clinical characteristics lost their predictive significance. Furthermore, multivariate Cox regression analyses revealed that high expression of Hv1 was independent risk factor for overall survival  and recurrence-free survival  (P<0.008)  . Overall= 0.026) . PatientP<0.008) .To examine whether Hv1 is also expressed in human colorectal cancer cell lines, the expression of Hv1 in human colorectal cancer cell lines, SW620, HT29, LS174T, Colo205 and SW480, was detected by immunocytochemistry and real time RT-PCR. As shown in 22 . The time-dependent wound distances of SW620  + medium) that will drive an efflux of H+ ions. As shown in i occurred. In contrast to SW620 cells, the florescence intensity of BCECF at membrane depolarization almost did not change for SW480 cells ). These results revealed that the pHi recovery was due to active Hv1.The Hi), we measured the pHi in SW620 and SW480 cells using BCECF. Down-regulation of Hv1 expression by siRNA and inhibition of Hv1 activity by 100 µM ZnCl2 induced a decrease in intracellular pH in the highly metastatic colorectal cancer SW620 cells, but not the poorly metastatic colorectal cancer SW480 cells . Therefore, we conclude that Hv1 regulates intracellular pH (pHi) in the highly metastatic colorectal cancer cells.To elucidate the mechanism that the effect of Hv1 on colorectal cancer development and metastasis, the expression of Hv1 in colorectal cancer cell lines was also detected, and the role of Hv1 in migration and invasion of colorectal cancer cells has been assessed. We found that Hv1 is highly expressed in highly metastatic colorectal cell lines, but lowly in poorly metastatic colorectal cell lines. Down-regulation of Hv1 expression or inhibition of Hv1 activity notably decreases the migratory and invasive abilities of the highly metastatic colorectal cancer cells. Suppression of Hv1 activity in the highly metastatic colorectal cancer cells restrains the extrusion of intracellular protons and induces the reduction of intracellular pH (pHo) value than that of surrounding normal cells i regulatory mechanisms in tumor cells have been described, such as proton pumps, Na+/H+ exchangers, bicarbonate (HCO3−) transporters, and proton-lactate symporters, which have been shown to be involved in cancer progression and as promisingly therapeutic targets for future anticancer therapy +-ATPases (V-ATPases) +/H+ exchanger NHE1 +/lactate cotransporter Maintenance of cytosolic pH is vital for all biological processes in cells. Solid tumor cells often exist in a hypoxic microenvironment with an acidic extracellular pH (pHvia knockdown of the protein subunit ATP6L expression using RNA interfering technology can effectively retard the growth and metastasis of human hepatocellular carcinoma xenografts The importance of V-ATPases in cancer malignancy has been repeatedly demonstrated in several human cancer tumors and cell lines such as hepatocellular carcinoma In our previous work, we showed that Hv1 function relates to breast tumor growth and metastasis through proton extrusion The glycolysis and proton secretion in tumor cells are proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis In conclusion, we demonstrated here that Hv1 is over-expressed in patients with colorectal cancer and high Hv1 expression correlated with the disease progression and poor clinical outcome in colorectal cancer. Furthermore, Hv1 was proved to be a risk factor for tumor recurrence and an independent molecular marker of prognosis for colorectal cancer and may become a novel molecular target in the strategies for the prediction of tumor recurrence and prognosis or treatment of colorectal cancer.


Geobacillus kaustophilus strain GBlys was isolated along with the bacteriophage ϕOH2, which infects G. kaustophilus NBRC 102445T. Here we present a draft sequence of this strain’s genome, which consists of 216 contigs for a total of 3,541,481 bp, 3,679 predicted coding sequences, and a G+C content of 52.1%. Geobacillus is comprised of moderately thermophilic, neutrophilic, aerobic or facultatively anaerobic, motile, spore-forming rods , was isolated from pasteurized milk , only GVE2 is a lysogenic phage . Genomic DNA was then extracted and purified as described by Marmur (N50 contig size of 61,981 bp. Annotation of the obtained scaffolds was performed using Glimmer 3.02 software and BLAST searches against a nonredundant protein sequence database (G. kaustophilus strains GBlys and HTA426 using the BBH method with BLAST, we found that there are 2,943 orthologues with 98.52% identity.The genome of strain GBlys has a G+C content of 52.1%. Annotation using the GTPS, RDP, and SILVA databases with tRNAscan-SE v.1.21, and with further manual inspection, revealed 2,154 predicted coding regions, 88 tRNA genes, and 10 rRNA genes. The ϕOH2 prophage genome is situated entirely astride contigs GBL0024 and GBL0110, and the host genes encoding GroES, GroEL, and transposase are located adjacent to the ϕOH2 prophage genome. A putative integrase gene is located at the terminal of the ϕOH2 prophage. There are no excisionase gene homologues, and attachment sites adjacent to the integrase gene are unlike those of phage λ (G. kaustophilus GBlys genome sequence and annotation data have been deposited in the DDBJ/EMBL/GenBank under accession numbers BASG01000001 through BASG01000216.The


Canis latrans) and gray wolves (C. lupus) are historically sympatric in western North America. Following European settlement gray wolf range contracted, whereas coyote range expanded to include eastern North America. Furthermore, wolves with New World (NW) mitochondrial DNA (mtDNA) haplotypes now extend from Manitoba to Québec in Canada and hybridize with gray wolves and coyotes. Using mtDNA and 12 microsatellite markers, we evaluated levels of wolf-coyote hybridization in regions where coyotes were present  and absent historically . Wolves with NW mtDNA extended from central Saskatchewan  to northeastern Québec . On the Prairies, 6.3% of coyotes and 9.2% of wolves had genetic profiles suggesting wolf-coyote hybridization. In contrast, 12.6% of coyotes and 37.4% of wolves in Québec had profiles indicating hybrid origin. Wolves with NW and Old World (C. lupus) mtDNA appear to form integrated populations in both regions. Our results suggest that hybridization is more frequent in historically allopatric populations. Range shifts, now expected across taxa following climate change and other human influence on the environment, might therefore promote contemporary evolution by hybridization.Contemporary evolution through human-induced hybridization occurs throughout the taxonomic range. Formerly allopatric species appear especially susceptible to hybridization. Consequently, hybridization is expected to be more common in regions with recent sympatry owing to human activity than in areas of historical range overlap. Coyotes ( Landscape changes such as urbanization and agricultural development can cause rapid directional selection within species  are distributed across an extensive geographic area (Kurtén and Anderson C. latrans) were considered a western North American species with a range that extended eastward to Manitoba in Canada and Minnesota in the United States (Young and Jackson C. lupus) historically occurred throughout most of North America outside the southeastern US  mtDNA have been referred to as eastern wolves, and given species or subspecies status ( Goldman ). Wolves Goldman  and thus Goldman . Western Goldman . The evoWhereas coyotes benefited greatly from agricultural expansion and have extended their range Gier , gray woC. l. lycaon) a Species of Concern  and allopatry (Québec). We expected hybridization to be infrequent on the Prairies and more common in Québec. Subsequently, we compared areas within the allopatric region that have relatively well-known records of coyote colonization . We anticipated southern Québec canids to form a hybrid swarm, whereas west-central Québec was expected to be an active hybrid zone with greater genetic differentiation between canid types.We obtained tissue, blood, and hair samples of wolves and coyotes collected during 1990–2010. Samples were contributions from hunters, trappers, and collected from animals captured for radio collaring during other research projects. The samples originated from across Canada with most being collected in Québec and Manitoba see . Canids n = 34 blood samples), northeastern Ontario (n = 6 tissues), and northwestern Ontario (n = 6 tissues), coyotes from southern Ontario (n = 8 blood samples), and n = 6 tissue samples from western Canada for comparison , 1× reaction buffer, 2 mmol/L MgCl2, 0.25 mmol/L dNTP, 0.5 μmol/L of each primer, and 0.5 U Taq polymerase.We extracted DNA using a standard phenol-chloroform protocol, Quicklysis , 1× reaction buffer, 2 mmol/L MgCl2, 0.25 mmol/L dNTP, 0.5 μmol/L of primer R, 0.25 μmol/L of primer F labelled with a tail of M13, 0.5 μmol/L of M13-F tail labelled with fluorescent dye HEX or FAM, and 0.5 U Taq polymerase.Subsequently, we amplified a panel of 11 autosomal and one Y-chromosome microsatellite markers for individuals for whom ATP-8 amplification was successful. Autosomal markers amplified were PEZ15, PEZ19  initial denaturation at 94°C for 15 min, then 38 cycles of 94°C for 30 sec, 58°C for 90 sec, and 72°C for 60 sec, with a final extension at 72°C for 30 min  and (2) initial denaturation at 94°C for 60 sec, then 35 cycles of 94°C for 60 s, 52°C  or 58°C  for 45 sec, and 74°C for 60 sec, with a final extension at 72°C for 5 min. We subsequently ran PCR products stained with Bromophenol Blue and SYBR-green (Invitrogen Inc.) on a 3% agarose gel and determined amplification success using UV light.μL volume using the product concentrations and PCR conditions described above for the ATP-8 reaction. Genotyping and sequencing was performed using an ABI 3730xl automated sequencer supplied by Applied Biosystems . Microsatellite alleles were scored using GeneMarker1.71 . Each allele was scored three independent times and we reamplified and reanalysed 12% of the alleles to confirm the observed genotypes. Control-region mtDNA sequences (387–404 bp) were edited in CLC Sequence Viewer v.6 (http://www.clcbio.com/index.php?id=28) and compared to sequences previously published in GenBank  using GENETIX4.0  , Pritchard et al. 6 iterations and a burn-in period of 105, and obtained individual ancestry assignments  from the run with the highest probability and the lowest variance .We examined genetic population structure using STRUCTURE 2.3 , n = 77 Prairie wolves,n = 99 Québec wolves, n = 8 Algonquin wolves, n = 6 northeast Ontario wolves, n = 6 northwest Ontario wolves, n = 3 southern Ontario coyotes, and n = 6 Western Canadian wolves. Subsequently, we excluded some reference groups with ≤6 samples to clarify cluster representation (northwest and northeast Ontario wolves and southern Ontario coyotes) and performed analyses using n = 285 samples. STRUCTURE results showed that L(K) continued to increase with K, whereas the peak value for ΔK occurred at K = 2  in GenAlEx  v.6 , the Mixed Wood Plains , the Hudson Plains  and Taiga Shield . In Saskatchewan, we found OW and NW mtDNA on both sides of Prince Albert National Park . However, n = 25 fecal samples (likely from two wolf packs) excluded from our final data set owing to low microsatellite genotyping success showed only OW mtDNA in PANP. Eight of the 11 Manitoba wolves with NW mtDNA were found in and around Duck Mountain Provincial Park and Forest  and C13 (n = 1), and OW haplotype 16 (n = 2). One Saskatchewan coyote sequence was shorter than NW haplotype la31, but identical starting from bp 42. Wolves in northeastern Québec had OW haplotype C22 (n = 2), and NW haplotype C19 (n = 1). A coyote from Îles de la Madeleine  also showed haplotype C19. Elsewhere in Québec (n = 3) and NW haplotype C14 (n = 3) in wolves. In Québec coyotes we identified NW haplotypes C19 (n = 1) and C1 (n = 1). Two Québec coyotes also had a haplotype shorter than la18 but identical starting from bp 18.Wolves from central Saskatchewan had NW and OW mtDNA haplotypes ; we founn Québec  we foundST values between wolves and coyotes (ST = 0.056–0.121), and similar values were seen between Québec and Prairie wolves (FST = 0.048–0.072). We found low differentiation between wolves with NW and OW mtDNA both in Québec (FST = 0.020) and on the Prairies (FST = 0.025). We obtained successful profiles from all Ontario tissue samples, but only three from southeastern Ontario coyotes and eight from Algonquin wolves. We found OW and NW alleles  and higher variation in genetic profiles, which could indicate an active hybrid zone. Wolves appear to have been absent from the south shore of the St. Lawrence River in Québec for many years Banville  althoughn = 23 and n = 22) than for Prairie wolves and Québec coyotes (both n = 31). Nielsen et al. (n = 16) Québec coyotes further supports wolf-coyote hybridization. Most Québec wolves had OW Y-chromosome alleles, including wolves with NW mtDNA. Although our sample is limited, this suggests that admixture may be asymmetric between male and female canids. As Prairie (and western Great Lakes region) wolf-coyote hybridization seems relatively rare, the significance of our apparent limited ability to identify backcrosses with Prairie wolves is not clear and needs further evaluation. Few Prairie wolves had NW Y-chromosome alleles. However, most of our Prairie samples were collected in the RMNP region, and profiles from this area may not be fully representative for the Canadian Prairies.Our HYBRIDLAB results suggested variable probabilities of correctly identifying hybrids. The results appeared influenced by the sample sizes and identification probability for parental groups. The number of individuals available for simulation of Québec wolves and Prairie coyotes was lower  and more frequent where they were formerly allopatric . Future research could help elucidate why wolves with NW genetic material appear rarely to hybridize with western coyotes but seem well integrated with western wolves. Conversely, westward expansion of hybrids from eastern North America might promote dissolution of existing reproductive barriers, especially if hybridization with coyotes facilitates wolf adaptation to human-modified landscapes , one of the few protected areas for wolves in southern Québec (COSEWIC In southern Québec, the combination of frequent wolf-coyote hybridization and limited hunting and trapping regulations might threaten the ecological role of wolves. High human-caused mortality can disrupt wolf social structure and promote hybridization with coyotes (Rutledge et al.  COSEWIC . Despite COSEWIC .Wolves with NW and OW mtDNA appear to form integrated populations throughout much of southern Québec and the Canadian Prairies, which suggest that they interbreed within a shared ecological niche (Templeton


P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular , chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs , including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The “NOTCH signaling pathway” was the most significantly enriched term in the list (The online version of this article (doi:10.1007/s00401-013-1165-y) contains supplementary material, which is available to authorized users. Paragangliomas (PGLs), rare, weakly metastatic but invasive neoplasms of the paraganglia, provide an example of organoid tumorigenesis from neural crest-derived cells belonging to the autonomic nervous system. As paraganglia, PGLs can be catecholamine-secreting (chromaffin), mostly thoraco-abdominal (including pheochromocytomas), or non-chromaffin, mostly in the head and neck , 32. HeaSDHA, SDHB, SDHC, SDHD, and SDHAF2, encoding mitochondrial complex II components; and, with lower frequencies, VHL and PHD2 (EGLN1), that regulate HIFα; the MYC regulator MAX; RET, implicated in glial neurotrophic signaling; NF1, which controls glial tumorigenesis; TMEM127, associated with mTOR signaling, and KIF1Bβ, involved in mitochondrial transport and apoptosis [At least one-third of all PGLs have a hereditary basis, often blurred by incomplete penetrance or imprinting . The suspoptosis , 39, 47.This genetic heterogeneity contrasts with the substantially monotonous tumor phenotype, which mimics paragangliar histoarchitecture. In fact, PGLs are organized in interconnected cell clusters , composed of neurosecretory (chief) cells encircled by glial (sustentacular) cells, embedded in angiomatous stroma . At the Our goals were to identify candidate molecular pathway(s) commonly affected by genomic alterations in head and neck PGLs, characterize the expression patterns of the pertinent gene products and assess the possible involvement of microRNAs in their deregulation.G.d’Annunzio University. Blood and tumor samples were from consenting consecutive patients operated at the GruppoOtologico clinic, Piacenza, Italy. Only one patient reported PGL family history, only one was positive for metastases . Samples for nucleic acid analyses were stored at −80 °C in RNALater (Qiagen). Fresh samples were also obtained for immunofluorescence (IF), electron microscopy (EM) and cryoimmuno-electron microscopy (cryo-IEM). Overall, 28 cases with 29 tumors (one patient was affected with two synchronous PGLs) yielded nucleic acids adequate for the study . Based on quality/quantity of nucleic acids, 23 cases, with 24 independent tumors, were selected for CNV analysis, 14 samples from 13 independent tumors were used for miRNA expression profiling and 16 samples from 15 independent tumors for quantitative reverse-transcriptase real-time (qRT-PCR) . One tumor (case PTJ64) was used to establish primary cultures of PGL cells. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks qualitatively and quantitatively adequate for standard immunohistochemistry (IHC) could be retrieved for 22 prospectively collected tumors . In addition, 25 archival tumors  were studied by IHC only. Most cases were tympanic or tympano-jugular PGLs , that arise from Jacobson’s nerve (JN), the tympanic branch of the glossopharyngeal nerve (IX cn), from Arnold’s nerve, the auricular branch of the vagus (X cn), or from paraganglia of the jugular bulb [The study was approved by the Bioethical Committee of lar bulb . JN, remlar bulb , is the SDHB, SDHC, SDHD) was assessed according to published procedures [SDHAF2, which may contribute to PGL in patients negative for mutations in SDHB, SDHC and SDHD [SDHB was detected by CNV analysis and validated by orthogonal assays (as detailed below).Germline mutational status (point mutations and large deletions/rearrangements) of the three SDH complex genes strongly associated with PGL development , for 16 h. BeadChips were scanned with Illumina Iscan™ and image intensities were extracted and genotyped using Illumina’s Genome Studio 2011.1® software. The SNP genotyping call rate was >99.0 %, indicating high-quality data. Only autosomal SNPs were considered [Leaf [Leaf produced a list of the genes most significantly over-represented among those targeted by CNVs (P < 0.01 by Fisher’s exact test). This list was submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID), which uses fuzzy clustering to group genes into functionally related, statistically ranked classes, based on the similarity of the annotations (http://david.abcc.ncifcrf.gov) [Genomic DNA (gDNA) was extracted using DNeasy Blood and Tissue kit (Qiagen), checked by agarose electrophoresis, and quantitated by Qubit fluorometer (Life Technologies). Paired gDNAs (200 ng) from blood and tumor were processed according to the Infinium assay protocol and hybridized on HumanOmni1-Quad BeadChipsnsidered . The dated [Leaf , that ined [Leaf . Leaf prcrf.gov) .JAG2, HES5, CTBP1, AKIRIN1, IDUA and PHACTR4 genes. Each qPCR contained the FAM-labeled TaqMan probe for the gene of interest and the VIC-labeled TaqMan probe for the RNaseP reference . Each qPCR plate included three no template controls. The relative gene copy numbers were calculated according to manufacturer’s instructions. The Ct values were normalized versus the reference Ct (t) and the t method was computed using the mean of the normal samples as calibrator. The CNVs targeting NOTCH1, DVL1 and SDHB were validated by non-fluorescent multiplex-PCR coupled to high-performance liquid chromatography (NFMP-HPLC) [NOTCH1 , DVL1  and SDHB , together with a set of primers  targeting PCBD2, chosen as reference gene because it showed no CNVs in our dataset and in a previous study conducted by quantitative multiplex PCR of short fluorescent fragments [Commercial real-time qPCR assays (Life Technologies) were used to validate the CNVs in the MP-HPLC) , using pragments . The ampragments . At leasThe PGL cases were chosen for immunohistochemistry (IHC) after revision of all the standard FFPE blocks and hematoxylin–eosin-stained sections for sample quality and quantity. Overall, 47 FFPE tumors from 46 cases were rated as adequate for IHC. These included 22 of the 28 tumors that had been prospectively sampled and for which status at the relevant NOTCH-related genes was assessed by CNV and/or qPCR, and 25 retrospective tumors, for which status at the NOTCH-related genes was unknown . Immunostaining for NOTCH1  and JAG2 (Abnova), both diluted 1/50, was performed after heat-induced antigen retrieval . To characterize the cellular components of the tumors, step sections were incubated with antibodies against: the neuroendocrine marker synaptophysin, strongly expressed in both sustentacular and chief cells  ; the neuSDHB IHC, a surrogate marker for mutations in any of the PGL-associated SDH subunit genes , 63, was® . Positive and negative control slides were included for each antibody and in each staining batch. The controls for non-specific staining included blocking with normal secondary serum prior to staining with the primary antibody and substitution of normal serum or immunoglobulin G in place of the primary antibody. The results were evaluated both in terms of percentage of positive cells, counted in four high-magnification fields , and of intensity, scored on a semiquantitative scale . NOTCH1, JAG2, S100 and BCL2 were assessed in the three main PGL cell types ; synaptophysin in chief and sustentacular cells together, as these cell types were similarly and strongly labeled (endothelia were negative), vimentin in all cell types combined . Ki67 was evaluated in terms of percentage of positively stained nuclei (chief and/or sustentacular cells), counted in four high-magnification fields. The other routinely assessed clinicopathological variables included vascular invasion, bone infiltration and atypia.For all the study antibodies immunostaining was carried out on 5-μm-thick whole sections with 15 min incubation at room temperature, using a Bond Max Immunohistochemical Stainert test, or Fisher’s exact test, where appropriate.SDHB immunostaining was ranked positive when showing granular cytoplasmic labeling , and negative when weak/diffuse or absent, in the presence of positive internal controls (macrophages/monocytes and/or endothelia) , 63. TheTumor and/or tissue samples were fixed in 4 % buffered paraformaldehyde (PFA) at 4 °C for 5–10 h, followed by 2 % PFA at 4 °C until processing. Cells grown in CultureSlides plates (BD Biosciences) were fixed in 4 % PFA for 30 min at 4 °C, washed in PBS at 4 °C and processed within 2 days. IF was performed as described , using tSamples were fixed in 2 % glutaraldehyde in PBS for 24 h at 4 °C, post-fixed in 1 % OsO4 for 2 h, stained for 1 h in 1 % aqueous uranyl acetate, dehydrated with graded acetones and embedded in Epon-812 (Electron Microscopy Sciences). Semithin sections stained with 1 % methylene blue were used to select suitable areas of ultrastructural sectioning. Uranyl acetate/lead citrate-stained ultrathin sections were examined with a Philips CM10 transmission electron microscope (TEM) (FEI).Samples were fixed in 2 % PFA/0.2 % glutaraldehyde in 0.1 M PBS, pH 7.4, for 24 h at 4 °C, then in 2 % formaldehyde overnight at 4 °C. Tissue blocks were embedded into 10 % gelatin (Sigma-Aldrich) in 0.1 M PBS, pH 7.4, solidified on ice, infused in 2.3 M sucrose overnight at 4 °C, mounted on aluminum pins and frozen in liquid nitrogen. Ultrathin cryosections (60 nm) collected with 1 % methylcellulose in 1.15 M sucrose were immunolabeled with primary antibodies to NOTCH1, S100, chromogranin A, and vimentin, as described . Bound aP < 0.05 after FDR adjustment).Total RNA was purified using miRNeasy Mini Kit (Qiagen) and qualitatively checked using Experion (Bio-Rad Laboratories) and spectrophotometry. The RNA samples were selected based on relative quality index (RQI >4.5). MiRNA profiling was performed on 14 PGL samples from 13 independent tumors  and 13 Jacobson’s nerves . Because of the generally minute sample sizes and of the inherently low RNA yields, we were forced to pool the JN RNAs in 5 sets including 2–5 nerves, each contributing the same RNA quantity. The experiments were conducted using the Human v2 MicroRNA Expression Profiling Kit  and GoldenGate GT Universal BeadChips on an Illumina IScan™ reader. The data were processed through Illumina Genome Studio signal filtration and cleaning algorithms. The miRNAs differentially expressed between the PGL and the JN groups were identified using the differential analysis function of Genome Studio (http://www.mirbase.org/index.shtml). The stem loop RT primers were designed with a modification to include the Universal Probe Library (UPL) #21 sequence binding site [Total RNA from PGLs and JNs was purified using TRIzol (Invitrogen) and quantified with Nanodrop 2000 (Thermo Scientific). The miRNA sequences were from miRBase  to identify the primers and the UPL probes . Total RNA (700 ng) was retro-transcribed with High Capacity cDNA Reverse Transcription Kit (Life Technologies). The real-time PCRs were performed using an Applied Biosystems 7900 instrument. MiRNA and mRNA levels were measured using Ct (threshold cycle). Target amount, normalized to an endogenous reference (RNU6 or ACTB) and relative to a calibrator, is given by 2−ΔΔCt and/or 2−ΔCt methods.The ) was acquired in 2007 and authenticated in January 2013 using AmpFlSTR-Identifiler-Plus Kit (Life Technologies). SH-SY5Y cells were cultured in RPMI 1640  with 10 % FBS, 2 mM l-glutamine, 100 IU penicillin, 100 μg/ml streptomycin. HEK293 cells were cultured in DMEM Low Glucose supplemented with 10 % FBS, 2 mM l-glutamine, 100 IU penicillin, 100 μg/ml streptomycin and 50 μg/ml Normocin (Invivogen). Primary PGL cell cultures from a prospectively sampled tympano-jugular PGL case  were established following a procedure previously described for primary rat carotid body cultures [2.The neuroblastoma cell line SH-SY5Y  of 50.MiRNA-expressing lentiviruses  were generated using Lentivector-based microRNA precursor constructs (System Biosciences), according to the manufacturer’s instructions. Control lentiviral particles (Cod. SBPMIRH000VA1) were purchased from System Biosciences. PTJ64p cells were seeded at 3.6 × 10® Assay (Promega) and the Toxilight bioassay kit , respectively, according to the manufacturer’s protocols, utilizing a VERITAS microplate luminometer (Turner BioSystems). All values were in triplicate and normalized to the controls, both untreated and infected with lentiviral control particles .Measurements of caspase activity and of adenylate kinase (AK) release were performed using the Caspase-Glo 3/7NOTCH1 was amplified using the primers in Supplementary Table 5 (Online Resource 1) and cloned downstream of Renilla luciferase in the psiCHECK2 vector (Promega). Substitutions into the miR-200 and miR-34 binding sites of the NOTCH1 3′UTR were introduced by Quick-Change site-directed mutagenesis (Stratagene) . The firefly luciferase activity of psiCHEK2 (Promega) was used as a reference. Transfection was conducted in 24-wells plates. Each well was co-transfected with psiCHECK2 (400 ng) and miRNA precursor or negative control (30 pmol) . Firefly and Renilla luciferase activities were measured 48 h after transfection using the Dual-Luciferase Report Assay (Promega). All experiments were replicated and performed at least in triplicate.MiRNA mimics precursor and negative control were from Life Technologies . MiRNAs and vectors were transfected with Lipofectamine 2000 (Life Technologies). After 48 h cells were collected for protein and RNA extraction. The 3′-untranslated region (UTR) of http://imagejdocu.tudor.lu/).Cells were collected from six-well plates using trypsin-EDTA and dissolved in lysis buffer  supplemented with complete protease  and phosphatase (Sigma-Aldrich) inhibitors. After electrophoresis and blotting, the primary antibodies  were incubated overnight at 4 °C. The peroxidase-conjugated anti-mouse or anti-rabbit antibodies were incubated for 1 h at room temperature and detected by chemiluminescence , β-actin or vinculin normalized loading. The digitalized signals were quantified in the linear range of the scanner using ImageJ 64 software  from 24 independent primary PGLs and paired blood from 23 patients  revealed a total of 19370 autosomal CNV calls (6777 in blood and 12593 in tumors). Figure analysis  of the dP < 0.01 by Fisher’s exact test). This highlighted 104 genes, of which 67 targeted by amplifications, 22 by deletions and 15 by both amplifications and deletions . None of these 104 genes has been previously associated with PGL. Some have been implicated in non-neoplastic diseases  [TMEM41B, top deleted gene), some are involved in organogenesis and oncogenesis  [To highlight the genes and the molecular pathways most frequently targeted by CNVs, we first identified the top affected genes, i.e., those showing the highest level of CNV concordances among tumors ( type I) , some hagenesis) .http://david.abcc.ncifcrf.gov) to identify the enriched biological themes and the functional-related gene groups among the top 104 CNV-affected genes. Only the term “NOTCH signaling pathway”, which included NOTCH1, HES5, JAG2, DVL1 and CTBP1, was statistically enriched after Bonferroni and Benjamini corrections . Leaf analysis [NOTCH1 or genes related to NOTCH signaling, although these terms were not statistically significant after Bonferroni or Benjamini corrections .We then used the DAVID bioinformatics resources , and for three deleted genes, AKIRIN1, PHACTR4 and SDHB. Overall, the orthogonal validations of these CNV hits yielded reproducible results . In particular, the CNV and the NFMP-HPLC assays were concordant in 77.5 % (NOTCH1), 84.0 % (DVL1) and 95.6 % (SDHB) of the tested samples. CNV and qPCR yielded concordances of 82.6 % (JAG2), 67.4 % (HES5), 76.6 % (CTBP1), 72.3 % (AKIRIN1), 85.1 % (IDUA) and 100 % (PHACTR4). The overall average concordance was 82.3 %. The orthogonal validation data are provided in Appendixes 1 and 2, Online Resource 3).The copy number assignments obtained with NOTCH1 mRNA was overexpressed in PGLs compared to control JNs. NOTCH1 expression was measured in 16 PGL samples  and compared to expression in 5 JNs. Overall, 10/16 PGL samples (56 %) showed significant NOTCH1 overexpression, with relative mRNA levels ranging from 2.27 to 15.27  showed NOTCH1 gains in the CNV analysis as well.To obtain evidence of possible in vivo deregulation, we tested whether Supplementary Fig. 2 in Online Resource 2 shows the histoarchitecture of PGL. We investigated three proteins belonging to the NOTCH pathway highlighted by CNV analysis, i.e., NOTCH1 (receptor), JAG2 (ligand) and CTBP1 . IHC on FFPE sections indicated that NOTCH1 was expressed in the three main PGL cell types . Diffuse NOTCH1 immunostaining in 100 % of each of the three main PGL cell types  was observed in all the PGLs examined (47/47), but the staining intensities differed significantly, in the following descending order: endothelial cells >sustentacular cells >chief cells  and also in the endothelial versus the chief cells (P < 0.001)  Table a, b. GivSDH gene defects, the PGL cases were analyzed for germline SDH mutations and/or tumor-associated loss of SDHB expression, a surrogate marker for mutations in any of the known PGL-related mitochondrial complex II genes. Germline SDH gene mutation analyses, performed in 34 cases, identified 13 mutation carriers, furthermore CNV analysis identified a large germline deletion/rearrangement of SDHB in one additional case . The overall frequency of germline mutations in SDHB, SDHC, SDHD and SDHAF2 was 14/35 (40 %); single gene mutation frequencies are detailed in Supplementary Table 9 (Online Resource 1). The germline SDH mutation frequencies in the clinically relevant subsets of the tested PGLs  were in substantial agreement with the literature data [SDH mutation status resulted associated to negative SDHB IHC, with 12 SDHB-negative tumors out of 14 tumors from identified SDH gene mutation carriers (85.7 %), versus 6 SDHB-negative tumors out of 20 tumors that tested negative for SDH genes mutations (30 %) .Considering that NOTCH signaling activation could be related to ure data , 39, 47.t test  resulted SDHB negative . Differences in NOTCH1 or JAG2 IHC intensities between the SDHB-positive and the SDHB-negative PGLs were not significant by two-tailed Student’s Cryo-IEM confirmed high NOTCH1 expression in the sustentacular cells and their elongated processes, identified by S100 labeling Fig. c–h. NotaCryo-IEM provided ultrastructural support for interpreting the punctate NOTCH1 cytoplasmic staining observed by IF Fig. b–d and tP < 0.05) downregulated and 3 miRNAs significantly upregulated in PGLs  versus JN control pools . We identified 16 miRNAs significantly  and was therefore excluded from further studies, although its concordance with the upregulation data analysis was greater than 80 %.Next, we validated the miRNA array data by qRT-PCR in 10 PGLs, 1 JN and SH-SY5Y cells. The Pearson correlation coefficient (R) was calculated for 11 miRNAs (5 downregulated and 6 upregulated), chosen irrespectively of the is Table . NotablyRemarkably, five of the most downregulated miRNAs, including miR-34b-5p, miR-34c-5p, miR-200a-3p, miR-200b-3p, and miR-200c-3p, were linked to the NOTCH pathway, although in different cellular contexts , 31, 70.NOTCH1, JAG2, CTBP1, HES5, DVL1 .Most of the tested PGLs showed marked downregulation of miR-34b-5p, miR-34c-5p, miR-200a-3p, miR-200b-3p, and/or miR-200c-3p , often co-occurring with genomic amplifications of NOTCH pathway genes, with the exception of 32PT, that had no or modest downregulation of the miRNAs targeting the NOTCH pathway and showed amplification of NOTCH1 3′UTR contains predicted binding sites for the miR-34 (miR-34ac/34c-5p/34b*/449abc/449c-5p) and miR-8 (miR-200bc/429/548a) gene families (http://www.targetscan.org/vert_61/), that include miRNAs that we found downregulated in PGLs . After comparing the list of the predicted miRNAs targeting NOTCH1 and the list of the miRNAs downregulated in PGL, we decided to investigate NOTCH1 as direct target of miR-34c-5p and miR-200b-3p in the SH-SY5Y background. Because of the sequence homology, we also considered miR-34b* (miR-34b-5p) and miR-200a (miR-200a-3p), both downregulated, although not predicted to target the NOTCH1 3′UTR. In addition, we studied miR-200c (miR-200c-3p), because it appears to target NOTCH pathway components, such as JAG1 and the mastermind-like coactivators MAML2 and MAML3 [P = 0.0739). None of these miRNAs map to the CNV regions detected in the tested PGL series (data not shown).The NOTCH1 3′UTRs, the predicted wild-type and mutant miR-200s and miR-34s target sites of the 3′UTR-NOTCH1 RNA were cloned downstream of the psiCHECK2 Renilla reporter. Then, either the wild type or the mutant forms of the psiCHECK2 3′UTR-NOTCH1 vectors were co-transfected with miRNA mimics into SH-SY5Y cells. Compared to controls, significant reductions in the luciferase activity of the psiCHECK vector carrying the wild-type NOTCH1–3′UTR were registered with miR-200b (~43 %), miR-200c (~29 %), miR-34b* (~64 %) and miR-34c (~29 %), but not with miR-200a, whereas in the mutated clones luciferase activity was not significantly downregulated by the tested miRNAs , miR-200c (~38 %), miR-34b* (~80 %) and miR-34c (~15 %) at 48 h from transfection, but, again, not by miR-200a  at 48 h from miRNA mimics transfection. In all the RNA samples tested HES1 and HES5 significantly decreased  and higher adenylate kinase release , as compared to the scramble control . More than 75 % of the cultured cells became GFP-positive after infection. Ectopic expression of the relevant miRNAs in PTJ64p  resulted in a clear reduction of NOTCH1 protein expression by IF, indicating that the miRNAs transduced by the lentivirus vector were functional Fig. a–l. ImpoThe genes involved in genetic susceptibility to PGL have been extensively studied, but little is known about the molecular pathways that drive PGL tumorigenesis at the somatic level , 18. PreP < 0.01) affected by CNVs. Gains were more frequent than losses in this top genes list. The genes were functionally diverse and had never been linked to PGL.Following a gene-centric approach, we next identified 104 genes that were more frequently (http://david.abcc.ncifcrf.gov). Canonical NOTCH signaling is a highly conserved contact-dependent intercellular signaling mechanism which, interacting with other molecular networks depending on cell/tissue contexts, controls a diversity of proliferation/differentiation processes, including embryofetal neurogenesis, gliogenesis and vasculogenesis, as well as physiological or pathological neoangiogenesis and glial homeostasis in the central and peripheral nervous systems [In the present work we focused on the most over-represented functional gene cluster, “NOTCH signaling” pathway, identified by submitting the list of the 104 genes with highly recurrent CNVs to the DAVID tool , JAG2 (14q32), HES5 (1p36.32), DVL1 (1p36), and CTBP1 (4p16). These CNVs were confirmed using orthogonal assays. NOTCH1, prototype of a family with four developmentally regulated and tissue-specific members (NOTCH1/4), encodes a transmembrane receptor that, after interaction with cognate ligand(s) expressed on adjacent cells, is converted into a transcription factor [In the PGL series genotyped for CNVs the high statistical significance of the “NOTCH signaling pathway” rested on five genes targeted by recurrent amplifications, i.e., n factor . Signal n factor . NICD1 tn factor  and its JAG2 [HES5, member of the HES (hairy enhancer of split) family of transcription factors, is a well-characterized transcriptional target of the NICD1 enhancer complex, implicated in neural stem cells induction [The other NOTCH1 pathway genes targeted by frequent copy number gains illuminate a molecular context that may constrain NOTCH signaling towards biological effects relevant for PGL tumorigenesis. JAG2 , one of JAG2 . HES5, mnduction . DVL1 monduction . Moreovenduction . CTBP1, nduction , links tnduction  and to snduction . In sum,Analysis of NOTCH1 and JAG2 expression by IHC in a series of 47 FFPE PGLs, including most of the cases genotyped by CNV analysis and a case for which we obtained primary cell cultures, provided a further level of evidence supporting the key role of NOTCH signaling in PGLs. These studies were complemented with IF and cryo-IEM, to define the cellular and subcellular localizations of the gene products and the relationships with known PGL-related markers. IHC demonstrated NOTCH1 immunostaining in all the PGLs analyzed, regardless of the individual or clinicopathological characteristics. Furthermore, NOTCH1 and JAG2 were highly expressed in tumors with or without evidence of CNV at the respective loci. This suggested that NOTCH1 signaling is a fundamental PGL pathway and that its activation may involve genomic amplification along with other mechanisms. Importantly, within tumors, NOTCH1 and JAG2 immunostaining was significantly correlated to cell type, being NOTCH1 higher in sustentacular relative to chief cells and highest in endothelial cells, while JAG2, undetectable in the endothelium, was particularly evident in sustentacular cells. In this respect the filamentous processes, characteristic of this glial cell type, are predicted to greatly increase the JAG2-expressing plasma membrane surface. Furthermore, these processes establish multiple contacts with the plasma membranes of other cells, even at remarkable distance. Thus, sustentacular cells might amplify JAG2-dependent, contact-mediated NOTCH1 activation in the PGL microenvironment. By cryo-IEM membrane contacts between sustentacular and chief cells demonstrated immunoultrastructural evidence of NOTCH1 internalization in both cell types. Remarkably, JAG2 immunostaining paralleled the distribution of S100, a Ca(2+)-binding protein highly expressed in sustentacular cells, that performs pro-inflammatory and trophic functions and suppresses P53-dependent apoptosis . This suAs outlined above, cooperative signaling involving NOTCH1, JAG2, DVL1 and CTBP1 may represent a key pro-angiogenic mechanism in PGL. In glioblastoma, NOTCH signaling was proposed to promote endothelial trans-differentiation of tumor cells , 23, 57.JAG1 and HES1 has been previously reported in association with VHL- and SDHx-related PGLs, that typically manifest a pseudo-hypoxic signature [SDH gene defects. No differences in NOTCH1 and JAG2 protein expression were observed in the subset of PGLs from ascertained germline SDH mutation carriers compared to cases putatively negative for constitutional SDH mutations. Furthermore, we found no differences in NOTCH1 and JAG2 protein expression in the subset of PGLs showing SDHB stain loss, indicative of mutations in SDH subunit genes [SDH gene defects.Overexpression of the NOTCH signaling-related genes ignature , 37. Theit genes , 63, comAdler et al.  reportedNOTCH mRNAs is negatively regulated by miRNAs [Copy number variation (CNV) and immunomorphological data concur in supporting core role(s) for NOTCH1 and related genes, yet the evidence of protein overexpression also in cases with no proof of relevant CNVs implies that independent mechanisms back up or replace genomic changes as drivers of NOTCH activation. Given that translation of y miRNAs , 34, we y miRNAs . We ideny miRNAs , 30 and y miRNAs . Importay miRNAs . Thus, iIn a previous study, Tombol et al.  reportedThe mechanisms underlying the reduced expression of the five presently reported miRNAs regulating NOTCH1 remain unclear. As none of these miRNAs were affected by CNVs, altered epigenetic regulation, such as aberrant DNA methylation or histone modifications, could represent a mechanism implicated in their downregulation, a possibility that will have to be investigated in future studies. Various other factors might play a role. JAG2, overexpressed in sustentacular cells, suppresses the miR-200 family in a murine model of metastatic adenocarcinoma . The S10In conclusion, whole-genome CNV analysis, miRNA profiling and immunomorphology converge in indicating that a NOTCH signaling axis involving angiogenic modulators is commonly activated in PGL. NOTCH1 signaling appears upregulated in all the three main PGL cell types, sustentacular , chief (neuroendocrine) and endothelial, possibly with a leading role of sustentacular cells, which overexpress ligand and receptor. NOTCH1 signaling is likely to be a key player in organoid PGL tumorigenesis and could be implicated in the resistance to radiotherapy and anti-angiogenic agents shown by this tumor type , 66. TheSupplementary material (PDF 282 kb)Supplementary material (PDF 7790 kb)Supplementary material (PDF 29 kb)Below is the link to the electronic supplementary material.


Our results revealed that HUMSCs did not stimulate the proliferation of lymphocytes and did not induce allogeneic or xenogeneic immune cell responses. qRT-PCR demonstrated that the HUMSCs produced an immunosuppressive isoform of human leukocyte antigen (HLA-I) and did not express HLA-DR. Flow cytometry revealed that the HUMSCs did not express immune response-related surface antigens such as, CD40, CD40L, CD80 and CD86. IFN-γ secretion by human peripheral blood lymphocytes was reduced when the cells were co-cultured with HUMSCs. These results suggest that HUMSCs are tolerated by the host in an allogeneic transplant. We transplanted HUMSCs into diabetic rats, and the cells survived in the liver and pancreas. Hyperglycemia of the diabetic rats was improved and the destruction of pancreatic cells was partly repaired by HUMSC transplantation. Hyperglycemic improvement may be related to the immunomodulatory effects of HUMSCs. However, the exact mechanisms involved remain to be further clarified.Islet transplantation involves the transplantation of pancreatic islets from the pancreas of a donor to another individual. It has proven to be an effective method for the treatment of type 1 diabetes. However, islet transplantation is hampered by immune rejection, as well as the shortage of donor islets. Human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (HUMSCs) are an ideal cell source for use in transplantation due to their biological characteristics and their use does not provoke any ethical issues. In this study, we investigated the immunological characteristics of HUMSCs and their effects on lymphocyte proliferation and the secretion of interferon (IFN)-γ, and explored whether direct cell-to-cell interactions and soluble factors, such as IFN-γ were important for balancing HUMSC-mediated immune regulation. We transplanted HUMSCs into diabetic rats to investigate whether these cells can colonize  Type 1 diabetes is an insulin-dependent, autoimmune disorder characterized by the destruction of insulin-producing β-cells ,3. HowevStudies have indicated that bone marrow mesenchymal stem cells (BMSCs)  and embrHuman umbilical cord Wharton’s jelly is a new source of MSCs that exhibit a high degree of self-renewal capacity and multi-differentiation potential. Human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (HUMSCs) have a wider range of collection sources than BMSCs  or ESCs in vivo and differentiate into pancreatic β-cells, and examined whether the hyperglycemia of diabetic rats can be improved by HUMSC transplantation.Although the immunogenic behavior of BMSCs has been characterized ,14, the 2 flask in DMEM/F12  culture medium supplemented with 10% fetal bovine serum , 100 μg/ml penicillin/streptomycin , 1 g/ml amphotericin B , 5 ng/ml epidermal growth factor  and 5 ng/ml basic fibroblast growth factor . The cultures were left undisturbed for 5–7 days at 37°C, 5% CO2 to allow the migration of cells from the explants, after which the medium was replaced.Ethical approval was obtained from the Institutional Review Board of Shantou University Medical College, Shantou, China. Human umbilical cords from consenting patients (full-term caesarian sections) were collected immediately into a sterilized 50 ml tube, washed with phosphate-buffered saline (PBS) and cut into small 2–3-cm-thick sections. After dissecting the arteries and veins, the remaining tissue, the Wharton’s jelly, was diced into smaller fragments and transferred to a 75 cm6 HUMSCs at passage 3 were dispersed with trypsin and resuspended in PBS containing phycoerythrin (PE)-conjugated antibodies against CD40, CD40L, CD80 and CD86  for 60 min at 4°C. The cells were washed 3 times with PBS and incubated with PE-conjugated rabbit anti-mouse IgG  or FITC-conjugated goat anti-rat IgG (Santa Cruz Biotechnology) for 30 min at room temperature. After 3 washes, the cells were resuspended in 0.5 ml PBS and analyzed by flow cytometry with the use of Epics XL flow cytometer .Approximately 1×106/ml with RPMI-1640 medium  supplemented with 10% FBS. HUMSCs at passage 3 were harvested and adjusted to 1×103 cells/ml, 1×104 cells/ml, or 1×105 cells/ml in L-DMEM containing 10% FBS. A 100 μl suspension of HUMSCs was plated into 96-well plates. The plates were incubated for 72 h at 37°C, 5% CO2. After the cells reached 70–80% confluence, the medium was removed and 100 μl of fresh medium containing 2.5 μl of mitomycin C  were added for 30 min at 37°C to mitotically inactivate the HUMSCs. After the medium was removed, the inactivated HUMSCs were washed twice with PBS. HUMSCs were resuspended in 100 μl of lymphocyte medium (RPMI-1640 containing 10% FBS), co-cultured with 1×105 cells/l PBMC, and stimulated by PHA (10 mg/l) (Sigma-Aldrich) for 72 h at 37°C, 5% CO2. The cells were divided into the following groups: PBMCs + PHA (positive control); HUMSCs (1×105) + PBMCs + PHA; HUMSCs (1×104) + PBMCs + PHA; and HUMSCs (1×103) + PBMCs + PHA. Three ratios of HUMSCs to PBMCs were used: 1:1, 1:10 and 1:100. Each trial was repeated in triplicate. The CCK-8 kit  was used to assess the immunomodulatory impact of HUMSCs on PBMCs following stimulation with PHA. The procedure was carried out according to the manufacturer’s instructions. The inhibitory effects of HUMSCs on lymphocyte proliferation were evaluated by comparing the optical density (OD) in cells co-cultured with inactivated HUMSCs with the OD of lymphocytes cultured alone.Human peripheral blood lymphocytes (PBMCs) were isolated from healthy donors by Ficoll-Paque (1.077 g/ml) density gradient centrifugation. The cell concentration was adjusted to 1×105 cells/ml, and the cells were plated (1 ml) in 24-well plates. After the cells reached 70% confluence, 10 μl mitomycin-C (1 μg/μl) were added into each well. Following incubation for 1 h at 37°C, 5% CO2, the medium was removed and the cells were washed twice with PBS. PBMCs (1×105) in 1 ml of lymphocyte medium were added and co-cultured in the presence of 10 μl of PHA for 72 h at 37°C, 5% CO2. The groups were as follows: PBMCs + PHA (positive control); unstimulated PBMCs (negative control); and HUMSCs (1×105 cells) + PBMCs + PHA. The supernatants of each group were collected and IFN-γ expression was evaluated using the ELISA detection kit according to the manufacturer’s instructions (Invitrogen).Passage 3 HUMSCs were trypsinized, the concentrations adjusted to 1×10Total RNA was isolated from the HUMSCs. In addition, we extracted the pancreas of the diabetic rats after HUMSC transplantation, as well as the pancreas of diabetic rats without HUMSC transplantation using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was prepared using the Prime Script RT Reagent kit . cDNA samples were analyzed by quantitative PCR using SYBR premix (Takara) in an ABI 7300 system. The primers used for qRT-PCR analyses were as follows: human pancreatic and duodenal homeobox 1  forward, 5′-ttcacgagccagtatgaccttcac-3′ and reverse, 5′-gaagacagacctgggatgcaca-3′; human insulin (221 bp) forward, 5′-acccagccgcagcctttgtg-3′ and reverse, 5′-ttccacaatgcc acgcttctgc-3′; human glucagon (161 bp) forward, 5′-cagagctta ggacacagagcacatc-3′ and reverse, 5′-acgttgccagctgccttgta-3′; HLA-I (293 bp) forward, 5′-gcagacacggaatgtgaagg-3′ and reverse, 5′-gtaggctctcaactgctccg-3′; HLA-DR (350 bp) forward, 5′-tcttgtctgttctgcctcactc-3′ and reverse, 5′-ttccaggttggctttgtcc-3′; and β-actin (396 bp) forward, 5′-tggcaccacaccttctacaatgagc-3′ and reverse, 5′-gcacagcttctccttaatgtcacgc-3′.8 infection units/ml (IU/ml) of viruses was added into a 10-cm dish pre-seeded with 1×106 Ad293 cells  overnight. Following incubation for 30–48 h, the cells were harvested by scraping and centrifugation at 3,000 rpm for 10 min while the supernatant was saved for the following round of virus amplification. The harvested cells underwent 4 freeze/thaw cycles and were centrifuged at 12,000 × g for 10 min to obtain cell lysates. Serial dilutions of the supernatant and cell lysates were used to transduce Ad293 cells in a 96-well plate pre-seeded with 5,000 cells overnight. The viral titers (IU/ml) were determined by counting the EGFP-positive cells under a fluorescence microscope after 30 h of culture. HUMSCs at passages 3–5 were seeded at a density of 1×105 cells/well in 6-well plates. Following 24 h of culture, the medium was replaced with 1 ml of serum-free medium containing indicated adenoviruses at a multipicity of infection (MOI) of 50 for 4 h.The E1-deleted adenovirus (serotype 5) carrying the CMV promoter/EGFP hybrid gene was purchased from Vector Gene Technology Company . For amplification of the adenoviruses, 1×10Ethical approval for the animal experiments was obtained from the Institutional Review Board of Shantou University Medical College. A total of 20 rats received an intraperitoneal injection of streptozotocin  at 70 mg/kg in order to induce diabetes. Blood glucose levels were monitored every 3 days. Rats with blood glucose levels >16.7 mmol/l for 3 measurements were diagnosed with type 1 diabetes.6 HUMSCs suspended in 0.1 ml of normal saline were injected through the tail vein. The control group underwent the same procedure, but was only injected with PBS. Body weight, blood glucose and serum insulin levels were recorded before and after cell transplantation. Blood was collected from the tail vein and blood glucose levels were measured using a blood glucose meter .The rats were divided into 3 groups, with 6–8 rats/group. After blood glucose spontaneously increased to 16.7 mmol/l, the rats were restrained and 5×10The results are expressed as the means ± standard deviation (SD). The statistical significance of the differences was assessed by the analysis of variance. In all comparisons, a value of P<0.05 was considered to indicate a statistically significant difference.in vitro for 7–10 days. After 2 weeks, some adherent cells had dissociated around the adherent tissue sections and were visible under an inverted microscope  + PBMCs + PHA, 1.37±0.024 ; HUMSCs (1×104) + PBMCs + PHA, 1.81±0.31 ; HUMSCs (1×103) + PBMCs + PHA, 1.71±0.28 ; ; . ELISA a5, n=3); .in vivo. Green fluorescence was detected by laser scanning microscopy of frozen sections of the rat pancreas and liver. The pancreas and liver of the rats transplanted with HUMSCs stained positive for PDX1 and glucagon, but the control groups showed negative staining  to severe hyperglycemic levels (24.04±2.84 mM). EGFP-positive HUMSCs were infused into diabetic rats on the 10th day. To avoid the aggregation of these cells and to ensure reproducible delivery, the cells were injected into the rats through the caudal vein. The rat pancreas and liver sections were analyzed by fluorescence microscopy 4 weeks after transplantation to observe whether the HUMSCs colonized staining . The PDXstaining . Blood gstaining  decreasestaining , body westaining  and the staining , and islstaining  comparedin vivo, and whether these are useful in the treatment of diabetes, requires further research.MSCs can not only be derived from bone marrow, but also from blood, spleen, amniotic fluid, placenta, tendon, adipose tissue, synovial fluid, thymus cancellous bone, umbilical cord blood, skin, pulp, lungs and umbilical cord –18. The + T cells  cells . The HLA T cells . In this T cells . This in T cells . The ful T cells . Our resin vivo transplantation.In recent years, a number of studies have focused on the influence of MSC-regulated immune cells, particularly T cells. MSCs can suppress the proliferation of lymphocytes ,28. It hAdult stem cells, which include HUMSCs, exhibit two important biological characteristics: first, injected adult stem cells can migrate to the target affected areas in the body after intravenous injection, and second, stem cells can be induced to differentiate into appropriate cells required for the repair of damaged tissue. This phenomenon is termed ‘site-specific differentiation’ . We founIn conclusion, we found that HUMSCs did not stimulate the proliferation lymphocytes and did not induce allogeneic or xenogeneic immune cell responses. qRT-PCR revealed that the HUMSCs produced an immunosuppressive isoform of HLA-I, and did not express HLA-DR. Flow cytometry revealed that the expression of immune response-related surface antigens, such as CD40, CD40L, CD80 and CD86 was absent on the HUMSCs. These results suggest that HUMSCs may be tolerated in an allogeneic transplant. HUMSCs were transplanted into diabetic rats, and these cells survived in the liver and pancreas. The hyperglycemia of diabetic rats was improved and the damage to pancreatic cells was partly reversed by HUMSC transplantation. Hyperglycemic improvement may be related to the immunomodulatory effects of HUMSCs, although the exact mechanisms involved remain to be clarified.


AbstractMegapulvinaria maxima (Green) was known from China. However, a new species Megapulvinaria beihaiensis Wang & Feng, sp. n. is described below and Megapulvinaria maxima is redescribed. A key is provided for the five species now placed in this genus.Prior to this study, only  Coccidae is the third largest family after Diaspididae and Pseudococcidae within the superfamily Coccoidea , Megapulvinaria orientalis  and Megapulvinaria maskelli  have been subsequently added .Megapulvinaria maxima ; China , Thailand, India, Indonesia, Philippines, Sri Lanka, Vietnam, Papua New Guinea, Chuuk Islands.Megapulvinaria burkilli ; India.Megapulvinaria orientalis ; Thailand.Megapulvinaria maskelli ; Australia.Megapulvinaria beihaiensis sp. n.; China (Guangxi).GenusYoung, 1982http://species-id.net/wiki/MegapulvinariaMegapulvinariaPulvinaria maxima Green, 1904. By original designation and monotype.PageBreak Young, 1982: 162. Type species: Adult female. Body elongate oval to broad oval; stigmatic clefts distinct. Dorsum. Dorsal setae spinose or conical. Dorsal submarginal tubercles absent. Preopercular pores present or absent. Dorsal tubular ducts present or absent. Eyespots generally displaced onto dorsum . Anal plates together quadrate, each plate with 2 spinose and/or truncate setae along inner margin, a similar seta on apex and a spinose seta present in discal position (possibly on outer margin of Megapulvinaria maskelli). Anal ring with 6 setae. Margin. Marginal setae stout, apex truncate or bidentate, and with 2 types present, one shorter and broader than other (about same length and one slightly broader than other both in Megapulvinaria maskelli and Megapulvinaria beihaiensis); broader setae on head and posterior margins of abdomen (0–3 broader setae present between two stigmatic clefts in Megapulvinaria beihaiensis). Stigmatic clefts deep or shallow, each with 3–12 stigmatic spines. Venter. Antennae 7–9 (mostly 8) segmented. Legs well-developed, each with a tibio-tarsal articulation and an articulatory sclerosis, each claw with a denticle on the widest part. Pregenital setae 2 pairs. Spiracular disc-pores each mainly with 5 loculi. Pregenital disc-pores each mainly with 10 loculi, restricted to abdominal segments. Ventral tubular ducts of three types, with a submarginal band of small tubular ducts; median area of head, thorax, and anterior 1–3 abdominal segments with large ducts each with both outer and inner ductules broad or stout ; posterior abdominal segments of moderately tubular ducts.Oriental and Australian regions.http://species-id.net/wiki/Megapulvinaria_maximaPulvinaria maximaPulvinaria thespesiaeEriochiton formosaeMegapulvinaria maxima (Green), PageBreakCajanus cajan (L.) Millsp., Leguminosae), Xiao-Ze Chen (NWAFU).5 adult females, CHINA, Yunnan, Jingdong, 18. x. 1976 on Pigeonpea  large and stout setae, each 17–38 µm long, with nearly parallel sides, and with either a truncate or a bifid apex, all with well-developed basal sockets, each socket with 1 or 2 small pores; with 96–110 setae between anterior clefts, 36–46 setae on each side between stigmatic clefts, and 84–98 setae between each posterior stigmatic cleft and anal cleft; and 2) quite broad and short setae, each 14–24 µm long, with parallel sides and a truncate, flattened apex, and with a larger basal socket about twice as broad as that of type 1), each socket with 3–8 small pores; latter type of marginal setae only distributed on anterior and posterior ends, with 16–22 setae anteriorly on head and prothorax, 5–12 setae on either side of abdomen near anal cleft. Stigmatic clefts deep; stigmatic spines bluntly spinose and mostly straight, with 4–8 spines in each anterior cleft and 5–10 in each posterior cleft; length of each 42–96 µm, with median 1–3 spines much longer than the lateral spines.Venter. Derm membranous. Antennae 8 segmented, each 505–586 µm long; third segment longest; with 2 pairs of long setae and 1–3 pairs of short interantennal setae. Clypeolabral shield 198–232 µm long, 205–240 µm wide; labium 90–106 µm long, 113–144 µm wide. Legs well-developed, each with a tibio-tarsal articulation and articulatory sclerosis; claws with a denticle on widest part, claw digitules broad and expanded apically, tarsal digitules slender, knobbed and longer than claw digitules; trochanter+femur 239–405 µm, tibia 180–245 µm and tarsus 96–122 µm. With 2 pairs of long pregenital setae present in both segments VI & VII; submarginal setae present in a single row; other setae slender, each 4–10 µm long, quite sparsely distributed. Spiracles normal, spiracular disc-pores each with 5 loculi, present in a broad band between stigmatic cleft and each spiracle. Pregenital disc-pores each mainly withPageBreak 10 loculi, present around the vulva and on posterior 4 abdominal segments. Ventral microducts scattered. Ventral tubular ducts of 3 types present: 1) a duct with a short outer ductule and a fine inner filament, with a minute terminal gland, present in a PageBreakcomplete submarginal band; 2) a duct with outer and inner ductules both broad and with a well-developed terminal gland, present medially on head, thorax and anterior 1 or 2 abdominal segments; and 3) a duct with a moderately long outer ductule and a thin inner ductule slightly longer than outer ductule, with a flower-shaped terminal gland, present medially on posterior abdominal segments and extending and mingling with marginal band of type 1) ducts.China , Thailand, India, Indonesia, Philippines, Sri Lanka, Vietnam, Papua New Guinea, Chuuk Islands.Megapulvinaria maxima, it had been described and illustrated by many authors, such as Since Megapulvinaria burkilli (Green) , but it can be distinguished from the latter by the following features (character states of Megapulvinaria burkilli in brackets): (1) the much larger body size in comparison to the latter ; and (2) with well-developed dermal areolations present (absent).This species is close to Wang & Fengsp. n.urn:lsid:zoobank.org:act:56E4CA5F-6C56-431C-AD2B-6A54776BC16Bhttp://species-id.net/wiki/Megapulvinaria_beihaiensisHolotype: adult female. CHINA, Guangxi, Beihai, Haibin Park. 26. vii. 2010, on Cinnamomum sp., (Lauraceae), Bin Zhang (NWAFU)3 adult females, the data same as holotype.The measurements are based on all 4 specimens.Description.Adult female.Unmounted material. Adult female yellowish brown or dark brown, elongate oval and with a longitudinal dorsal ridge in dorsal straight median area . The specimens collected on the lamina of the host plant.Mounted material. Body elongate oval, about 2.1–3.2 mm long, 1.3–1.7 mm wide. Anal cleft approximately 1/8 of the body length. Stigmatic clefts deep.Dorsum. Derm membranous. Dermal areolations well-developed, each with a dorsal microduct. Dorsal setae conical, with a well-developed basal socket, each 6–11 µm long, scattered throughout. Dorsal simple pores each with a slightly sclerotized margin, randomly distributed. Dorsal microducts each with a very short outer ducPageBreaktule and a long, fairly broad inner filamentous ductule, sparsely located in each dorsal areaolation. Dorsal tubular ducts each with a short outer ductule and a fine inner ductule with a minute terminal gland, sparsely distributed. Preopercular pores absent. PageBreakAnal plates together quadrate, dorsal surface with reticulations on anterior two-thirds; posterior margin subequal to or slightly longer than anterior margin, outer angle a right-angle; each plate with a blunt spinose seta in discal position, each 34–42 µm long, a large spinose or spatulate seta apically, each 48–54 µm long, and with 2 spinose setae along posterior 1/3rd of the inner margin, each 32–44 µm long, length of plates 146–167 µm, width of single plate 74–88 µm. Ano-genital fold with 1 pair of long setae and 1 pair of short setae along anterior margin and 2 or 3 pairs lateral margin. Anal ring subcircular, with 2 or 3 rows of translucent pores and 6 anal ring setae. Eyespots present some way onto dorsum, each 42–60 µm wide.Margin. Marginal setae of 2 types: 1) stout setae, each 18–30 µm long; each seta with nearly parallel sides and with either a truncate or a bifid apex, all with well-developed basal sockets, each socket with 1 or 2 small pores; with 101–111 setae between anterior clefts, 34–42 setae on each side between stigmatic clefts, and 74–85 setae between each posterior stigmatic cleft and anal cleft; 2) quite strong setae, subequal in length with type 1) but slightly broader; each seta with parallel sides, with a truncate and flattened apex, and with a large basal socket about twice as broad as that of type 1), each socket with 2–8 small pores; with 10–16 setae anteriorly on head and prothorax, 0–3 setae between stigmatic clefts, and 4–10 setae on either side of abdomen near anal cleft. Stigmatic clefts deep; stigmatic spines bluntly spinose and mainly curved apically, with 4 or 5 spines in each anterior cleft and 5–8 spines in each posterior cleft; length of each 34–62 µm, and the median 1–3 spines longer than the lateral spines.Venter. Derm membranous. Antennae 8 segmented, each 346–378 µm long, the third segment longest; with 2 pairs of long setae and 2 or 3 pairs of short interantennal setae. Clypeolabral shield 138–160 µm long, 160–172 µm wide; labium 96–112 µm long, 84–112 µm wide. Legs well-developed, each with a tibio-tarsal articulation and articulatory sclerosis; claws with a denticle on widest part, claw digitules both broad and expanded apically; tarsal digitules slender, knobbed and longer than claw digitules; trochanter+femur 212–245 µm, tibia 136–188 µm and tarsus 54–75 µm. With 2 pairs of long pregenital setae present in both segments VI & VII; submarginal setae present in a single row; other setae slender, 6–20 µm long, quite sparsely distributed. Spiracles normal; spiracular disc-pores each mainly with 5 loculi, present in a broad band between stigmatic cleft and each spiracle. Pregenital disc-pores each mainly with 10 loculi, present around the vulva and on posterior 5 abdominal segments but becoming progressively less frequent anteriorly. Ventral microducts scattered. Ventral tubular ducts of 3 types present: 1) a duct with a short outer ductule and a fine inner filament with a minute terminal gland, present in a complete submarginal band; 2) a duct with a broad outer ductule, a stout inner ductule (as broad as outer ductule in some specimens) and with a well-developed terminal gland, present medially on thorax and anterior abdominal segments; and 3) a duct with a moderately long outer ductule, a thin inner ductule slightly longer than outer ductule, with a flower-shaped terminal gland, present medially on posterior abdominal segments and extending and mingling with marginal band of type 1) ducts.China (Guangxi).PageBreakThe specific epithet is taken from the type locality Beihai.Megapulvinaria maskelli (Olliff) (data from Megapulvinaria beihaiensis can be distinguished by following features (character states of Megapulvinaria maskelli in brackets): (1) more than 3 stigmatic spines in each stigmatic cleft (only 3); (2) having dorsal tubular ducts (absent); (3) lacking preopercular pores (present); (4) eyespots displaced onto the dorsum (on the margin); and (5) the marginal setae of much broader basal socket often present between stigmatic clefts (absent).This new species resembles Megapulvinaria maskelli, currently only known from the Australian region, is the only non-Oriental species in this genus and has some distinctive characteristics within Megapulvinaria. It differs from other species in having: (1) only 3 stigmatic spines in each stigmatic cleft; (2) eyespots located on margin; and (3) the discal setae possibly on outer margin of anal plates.