HLA-DPA1 and HLA-DPB1 genes with metaP = 1.89×10−12 for rs3077 and metaP = 9.69×10−10 for rs9277542. We also found that the HLA-DPA1 and HLA-DPB1 genes were significantly associated with protective effects against chronic hepatitis B (CHB) in Japanese, Korean and other Asian populations, including Chinese and Thai individuals (metaP = 4.40×10−19 for rs3077 and metaP = 1.28×10−15 for rs9277542). These results suggest that the associations between the HLA-DP locus and the protective effects against persistent HBV infection and with clearance of HBV were replicated widely in East Asian populations; however, there are no reports of GWAS in Caucasian or African populations. Based on the GWAS in this study, there were no significant SNPs associated with HCC development. To clarify the pathogenesis of CHB and the mechanisms of HBV clearance, further studies are necessary, including functional analyses of the HLA-DP molecule.Hepatitis B virus (HBV) infection can lead to serious liver diseases, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC); however, about 85–90% of infected individuals become inactive carriers with sustained biochemical remission and very low risk of LC or HCC. To identify host genetic factors contributing to HBV clearance, we conducted genome-wide association studies (GWAS) and replication analysis using samples from HBV carriers and spontaneously HBV-resolved Japanese and Korean individuals. Association analysis in the Japanese and Korean data identified the  Overall, one-third of the world’s population (2.2 billion) is infected with hepatitis B virus (HBV), and about 15% of these are chronic carriers. About 75% of the chronic carriers live in the east-south Asia and east pacific area, and there are 1.3–1.5 million chronic carriers living in Japan HLA-DPA1 and HLA-DPB1 were identified using 786 Japanese individuals having CHB and 2,201 control individuals through a two-stage genome-wide association study (GWAS) HLA-DP locus and CHB, in addition to associations with another two SNPs located in the genetic region including the HLA-DQ gene HLA-DP variants with HBV infection were replicated in other Asian populations, including Thai and Han Chinese individuals In 2009, significant associations between chronic hepatitis B (CHB) and a region including Here, we performed a GWAS using samples from Japanese HBV carriers, healthy controls and spontaneously HBV-resolved individuals in order to confirm or identify the host genetic factors related to CHB and viral clearance. In the subsequent replication analysis, we validated the associated SNPs in the GWAS using two independent sets of Japanese and Korean individuals. In our study, healthy controls were randomly selected with clinically no evidence of HBV exposure, therefore, HBV-resolved individuals were prepared to clearly identify the host genetic factors related with CHB or HBV clearance.P≥0.001 in healthy controls. We identified significant associations of protective effects against CHB with two SNPs (rs3077 and rs9277542) using the allele frequency model, both of which are located in the 3′ UTR of HLA-DPA1 and in the sixth exon of HLA-DPB1, respectively . The association for rs9277542 reached a genome-wide level of significance in the GWAS panel ).In this study, we conducted a GWAS using samples from 181 Japanese HBV carriers (including asymptomatic carriers (ASC), CHB cases, LC cases and HCC cases, based on the criteria described in P = 2.70×10−8, OR  = 0.48 and rs9277542, P = 3.33×10−6, OR  = 0.54; replication-2 (Korean); rs3077, P = 2.08×10−6, OR  = 0.47 and rs9277542, P = 8.29×10−5, OR  = 0.54, hetP = 0.80 for rs3077 and 0.40 for rs9277542). metaP values were 4.40×10−19 for rs3077  = 0.39–0.54), and 1.28×10−15 for rs9277542 . Among the remaining 30 SNPs in the replication study, 27 SNPs were successfully genotyped by the DigiTag2 assay with SNP call rate ≥ 95% and HWE p-value ≥ 0.01. Two SNPs (rs9276431 and rs7768538), located in the genetic region including the HLA-DQ gene, were marginally replicated in the two sets of HBV carriers and healthy controls with Mantel-Haenszel P values of 2.80×10−7  and 1.09×10−7 , respectively, when using additive, two-tailed Cochran Mantel-Haenszel (CMH) fixed-effects model with no evidence of heterogeneity (hetP = 0.67 for rs9276431 and 0.70 for rs7768538) , were selected for replication in two independent sets of HBV carriers and healthy controls  .metaP = 3.94×10−45, OR  = 0.55 for rs3077, metaP = 1.74×10−21, OR  = 0.61 for rs9277535 and metaP = 1.69×10−15, OR  = 0.51 for rs9277542, with the SNP rs9277535 being located about 4-kb upstream from rs9277542 and showing strong linkage disequilibrium of r2 = 0.955 on the HapMap JPT  with clearance of HBV . The same two SNPs (rs3077 and rs9277542) showed strong associations in the allele frequency model : rs3077, P = 2.35×10−7, OR  = 0.41 and rs9277542, P = 4.97×10−6, OR  = 0.46; metaP = 1.56×10−4 for rs3077 , and 5.91×10−7 for rs9277542 . While there was evidence of heterogeneity between these studies for rs3077 (Phet = 0.03) and no evidence for rs9277542 (Phet = 0.19), significant associations with HBV clearance were observed with Mantel-Haenszel metaP = 3.28×10−12 for rs3077 and 1.42×10−10 for rs9277542, when using CMH fixed-effects model. Among the remaining 27 SNPs in the replication study, two SNPs (rs9276431 and rs7768538), located in a genetic region including HLA-DQ gene, were marginally replicated in the two sets of HBV carriers and HBV resolved individuals with Mantel-Haenszel P values of 2.10×10−5 (OR  = 0.59) and 1.10×10−5 (OR  = 0.56), respectively (hetP = 0.03 for rs9276431 and 0.04 for rs7768538), weak associations were observed with metaP = 0.03 for rs9276431 and 0.02 for rs7768538 by the random effects model meta-analysis.The above 32 SNPs, including the two associated SNPs (rs3077 and rs9277542), were selected for a replication study in two independent sets of HBV carriers and HBV resolved individuals . However, significant level of heterogeneity for rs3077 was observed with hetP = 9.57×10−6 across 5 independent studies, including our study.Meta-analysis across 6 independent studies, including 5 additional published data, showed s9277542 . As showhttps://gwas.lifesciencedb.jp/cgi-bin/gwasdb/gwas_top.cgi.The results of the present GWAS are registered at a public database: HLA-DPA1 and HLA-DPB1 genes were associated with chronic HBV infection in the Japanese and Thai population HLA-DPA1 and HLA-DPB1 and protective effects against CHB in Korean and Japanese individuals. Mata-analysis using the random effects model across 6 independent studies including our study suggested that, widely in East Asian populations, variants in antigen binding sites of HLA-DP contribute to protective effects against persistent HBV infection  on meta-analysis by adding replication-1 . Moreover, meta-analysis using GWAS and replication-2 showed significant association of metaP = 1.89×10−12, OR  = 0.43 for rs3077 with no evidence of heterogeneity (hetP = 0.75). Although the reason why heterogeneity was observed in replication-1 is unclear, one possible reason is the clinical heterogeneity due to different kits being used for antibody testing. The associations of HLA-DPA1/−DPB1 with CHB and HBV clearance showed the same level of significance in the comparison of HBV patients with HBV resolved individuals (OR  = 0.43 for rs3077 and 0.49 for rs9277542) as the one with healthy controls (OR  = 0.46 for rs3077 and 0.50 for rs9277542), when the replication-1 was excluded in the analysis  in the analysis . When th  = 0.56 . In our cation-1 . Despiteanalysis . The resanalysis . These rHLA-DPA1, HLA-DPB1 and HLA-DQB2), which were associated with CHB and HBV clearance, a weak linkage disequilibrium (r2<0.1) was observed between HLA-DQB2 locus and HLA-DPA1/−DPB1 loci in Japanese and Korean populations (2) were observed among three subgroups . Moreover, logistic regression analysis of HLA-DP (rs3077 and rs92775542) with use of HLA-DQ (rs9276431 and rs768538) as covariates showed that the same level of significant associations of HLA-DP with CHB and HBV clearance as shown in the single-point association analysis, while no associations of HLA-DQ with logP >0.05 were detected both in Japanese and in Korean , were collected at 20 multi-center hospitals (liver units with hepatologists) throughout Japan and Korea. The 19 hospitals in Japan were grouped into the following 8 areas: Hokkaido area , Tohoku area , Kanto area , Koshin area , Tokai area , Kinki area , Chugoku/Shikoku area  and Kyushu area . Korean samples were collected at Yonsei University College of Medicine.3, or a combination thereof. Histological confirmation by fine-needle biopsy of the liver was performed as required. Hepatocellular carcinoma (HCC) was diagnosed by ultrasonography, computerized tomography, magnetic resonance imaging, angiography, tumor biopsy or a combination thereof.HBV status was measured based on serological results for HBsAg and anti-HBc with a fully automated chemiluminescent enzyme immunoassay system . For clinical staging, inactive carrier (IC) state was defined by the presence of HBsAg with normal ALT levels over 1 year  and without evidence of portal hypertension. Chronic hepatitis (CH) was defined by elevated ALT levels  persisting over 6 months (at least by 3 bimonthly tests). Liver cirrhosis (LC) was diagnosed principally by ultrasonography , platelet counts <100,000/cmThe Japanese control samples from HBV-resolved subjects (HBsAg-negative and anti-HBc-positive) at Nagoya City University-affiliated healthcare center were used by comprehensive agreement (anonymization in an unlinkable manner) in this study. Some of the unrelated Japanese healthy controls were obtained from the Japan Health Science Research Resources Bank . One microgram of purified genomic DNA was dissolved in 100 µl of TE buffer (pH 8.0) , followed by storage at −20°C until use.P-value ≥0.001 for healthy controls P<0.0001 on association analyses in the allele frequency model were confirmed by visual inspection, and SNPs with ambiguous cluster plots were excluded.For GWAS, we genotyped a total of 550 individuals, including 181 Japanese HBV carriers, 184 Japanese healthy controls and 185 spontaneously HBV-resolved Japanese individuals (HBsAg-negative and anti-HBc-positive), using the Affymetrix Genome-Wide Human SNP Array 6.0 , in accordance with the manufacturer’s instructions. The average QC call rate for 550 samples reached 98.47% (95.00–99.92%), which had an average sample call rate of 98.91% (93.55–99.74%) by determining the genotype calls of over 900 K SNPs using the Genotyping Console v4.1 software  provided by the manufacturer P<0.0001 in the GWAS using HBV carriers and HBV-resolved individuals. SNP genotyping in two independent sets of 256 Japanese HBV carriers, 236 Japanese healthy controls and 150 Japanese HBV-resolved individuals  and P = 8.47×10−8 , respectively. For the replication study, 29 of 32 SNPs were successfully genotyped; therefore, we applied P = 0.0017 (0.05/29) as a significance level, and none of the 29 markers genotyped in the replication stage showed deviations from the Hardy-Weinberg equilibrium in healthy controls (P>0.01).The observed associations between SNPs and the protective effects on chronic hepatitis B or clearance of hepatitis virus B were assessed by chi-squared test with a two-by-two contingency table in allele frequency model. SNPs on chromosome X were removed because gender was not matched among HBV carriers, healthy controls and HBV-resolved individuals. A total of 597,789 SNPs and 590,278 SNPs passed the quality control filters in the GWAS stage; therefore, significance levels after Bonferroni correction for multiple testing were The genetic inflation factor λ was estimated by applying the Cochrane-Armitage test on all SNPs and was found to be 1.056 and 1.030 in the GWAS using HBV carriers and healthy controls, and using HBV carriers and HBV-resolved individuals, respectively . These rBased on the genotype data of a total of 1,793 samples including 1,192 Japanese samples and 601 Korean samples in both GWAS and replication stages, haplotype blocks were estimated using the Gabriel’s algorithm using the Haploview software (v4.2) . In the Figure S1GWAS using samples from HBV carriers with LC or HCC, and HBV carriers without LC and HCC.P values were calculated using chi-squared test for allele frequencies.(PPTX)Click here for additional data file.Figure S2Estimation of linkage disequilibrium blocks in HBV patients, HBV resolved individuals and healthy controls in Japanese and Korean. The LD blocks (r2) were analyzed using the Gabriel’s algorithm.(PPTX)Click here for additional data file.Figure S3Quantile-quantile plot for test statistics  for GWAS results. Dots represent P values of each SNP that passed the quality control filters. Inflation factor λ was estimated to be: a) 1.056 in the analysis with HBV carriers and healthy controls; and b) 1.030 with HBV carriers and HBV-resolved individuals.(PPTX)Click here for additional data file.Figure S4Principal component analysis on a total of 550 individuals in GWAS, together with HapMap samples .(PPTX)Click here for additional data file.Table S1Results for 29 SNPs selected in replication study using samples of HBV carriers and healthy controls.aP values by chi-squared test for allelic model. bOdds ratio of minor allele from two-by-two allele frequency table. cMeta-analysis was tested using additive, two-tailed CMH fixed-effects model.(XLSX)Click here for additional data file.Table S2Results of meta-analysis for protective effects against persistent HB infection across 6 independent studies, including this study.aMinor allele frequency and minor allele in 198 healthy Japanese (ref#19). bOdds ratio of minor allele from two-by-two allele frequency table. cP value of Pearson’s chi-squared test for allele model. dHeterogeneity was tested using general variance-based method. eMeta-analysis was tested using the random effects model.(XLSX)Click here for additional data file.Table S3Results for 29 SNPs selected in replication study using samples from HBV carriers and HBV-resolved individuals.aP values by chi-squared test for allelic model. bOdds ratio of minor allele from two-by-two allele frequency table. cMeta-analysis was tested using additive, two-tailed CMH fixed-effects model.(XLSX)Click here for additional data file.Table S4Results of meta-analysis for clearance of HBV across 6 independent studies, including this study.aMinor allele frequency and minor allele in 198 healthy Japanese (ref#19). bOdds ratio of minor allele from two-by-two allele frequency table. cP value of Pearson’s chi-squared test for allele model. dHeterogeneity was tested using general variance-based method. eMeta-analysis was tested using the random effects model.(XLSX)Click here for additional data file.Table S5Logistic regression analysis of HLA-DP (rs3077 and rs9277542) and HLA-DQ (rs9276431 and rs7768538) with susceptibility to CHB and HBV clearance using the HLA-DQ genotypes individually as a covariate.(XLSX)Click here for additional data file.


Overexpression of RNF146 led to upregulated levels of matrix metalloproteinases 2 and 7 and enhanced lung cancer cell invasiveness, events that were mediated by the classical Wnt/β-catenin signaling pathway. In summary, the data in the present study indicate that RNF146 regulated the development and progression of NSCLC by enhancing cell growth, invasion, and survival, suggesting that RNF146 may be a potential treatment target in NSCLC.Studies have suggested a possible correlation between the newly identified E3 ubiquitin ligase ring finger protein 146 (RNF146) and tumor development. However, until now, studies on RNF146 have been restricted to poly(ADP-ribosyl)ation and ubiquitin ligation, whereas the role of RNF146 in tumor biology has rarely been reported. In the present study, the role of RNF146 in non-small cell lung cancer (NSCLC) was investigated. The results showed that the expression of RNF146 was increased in clinical lung cancer samples and cell lines. RNF146 expression correlated with tumor size, differentiation level, lymphatic metastasis, pTNM staging, and prognosis of patients in stage I. RNF146 expression was negatively correlated with Axin expression but positively correlated with the nuclear expression of β-catenin in NSCLC tissues. RNF146 downregulated the expression of Axin in lung cancer cell lines and induced the expression and nuclear distribution of β-catenin. Overexpression of RNF146 in NSCLC cell lines increased the levels of cyclinD1, cyclinE, and CDK4, promoted cell cycle G RNF146 gene is located on human chromosome 6q22, 33 E3 ubiquitin ligases play important roles in regulating cell functions including proliferation, cell cycle arrest, and apoptosis. They may also have additional functions that depend on the identity of their substrates. For example, if an E3 ubiquitin ligase targets an oncogene for degradation, it may be considered a tumor suppressor. Similarly, if an E3 uniquitin ligase degrades a tumor suppressor protein, it may be considered an oncogene. Many proteins containing RING-finger domains possess ubiquitin ligase activity, some of which participate in tumorigenesis and tumor metastasis. The newly identified E3 ubiquitin ligase RING finger protein 146 (RNF146) interacts with poly(ADP-ribose) (PAR) through a PAR-binding motif in the Trp-Trp-Glu (WWE) domain. The The Wnt signaling pathway is highly active in lung cancer cells, leading to metastasis and proliferation of these cells Despite many studies on RNF146, its exact role in tumorigenesis remains unclear. In the present study, the roles of RNF146 in lung cancer were investigated.Primary NSCLC samples and control tissues were collected from 133 patients. Normal lung samples were taken from lung tissue more than 5 cm from the cancer resection site. Procedures took place at the Fourth Affiliated Hospital of China Medical University. The patients did not receive any radiation or chemotherapy before the operation. NSCLC staging was based on the TNM Classification of Malignant Tumors, Seventh Edition The rabbit anti-human RNF146 polyclonal antibody was purchased from Abcam (Cambridge Science Park). Anti-Axin and anti-β-catenin antibodies were purchased from BD Transduction Laboratories . Anti-CyclinA, anti-CyclinB, anti-CyclinD1, and anti-pRB antibodies were from Cell Signaling Technology, Inc. . Anti-CDK4, Anti-CDK6, Anti-TIMP-1, Anti-CyclinE, siAxin, siβ-catenin, and siTCF4 were from Santa Cruz Biotechnology . Antibodies against RhoA, RhoB, RhoC, and Rock1 were from Proteintech . Antibodies against CDK2, P21, matrix metalloproteinase 2 (MMP2), MMP7, and MMP9 were from NeoMarkers . Anti-P27 and anti-P53 were from Thermo Fisher Scientific . The recombinant overexpression plasmids pEX4-hRNF146 and pGPHI-shhRNF146 and control empty plasmids were constructed by GenePharma . The immunohistochemical secondary antibody kit was purchased from Dako .2.The normal bronchial epithelial cell line HBE, the human lung adenocarcinoma cell lines A549 and H1299 were purchased from the American Type Culture Collection . The human NSCLC cell lines LK2, LH7, LTE, and SPC were purchased from the Shanghai Cell Bank of the Chinese Academy of Science. Cell lines were cultured in Kaighn’s Modification of Ham’s F-12 Medium, Roswell Park Memorial Institute (RPMI)-1640 or Dulbecco’s modified Eagle’s-F12 medium with 10% fetal bovine serum  containing 100 units/ml penicillin and 100 units/ml streptomycin  at 37°C with 5% COParaffin sections with a thickness of 4 µm were prepared and immunohistochemically stained with Envision as described previously Two pathology physicians assessed the staining results on a semiquantitative scale. Five high magnification fields were randomly selected from each section, and 100 tumor cells in each field were counted to evaluate the intensity and range of RNF146 staining. A negative result was given a score of 0 (no staining), faint yellow staining was recorded as a 1, yellow was scored as a 2, and deep brown staining was recorded as a 3. Percentage scores were assigned as follows: <5%, 0; 5–25%, 1; 26–50%, 2; 51–75%, 3; ≥75%, 4. Two independent scores were multiplied together to obtain the patient’s coloring coefficient, which was categorized as “low expression” for coloring coefficient <4 and “high expression” for coloring coefficient ≥4. Axin and β-catenin were scored as previously described 2, complete medium was added to the transfection mixture. Transfection efficiency was determined by Western blot.Cells were plated on 24-well plates at approximately 50% confluence and transfected with plasmid or siRNA 24 hours later. Transfection of lung cancer cell lines A549 and LTE was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s directions. For co-transfection experiments, 1 to 1.5 µg of co-transfected or empty vector and 45 pmol siRNA were added to the transfection mixture. After 5 h at 37°C and 5% CO5′-GGACGTCGCAGGAAGATTAAG-3′ and reverse: 5′-CAATGGAGGTGTCTGGTGCT-3′; Axin: forward: 5′-GACTTGCTGGACTTCTGGTT-3′ and reverse: 5′-TGTACTTTCGGTAGATGGCT-3′; β-catenin: forward: 5′-CTAAACAGGAAGGGATGGAAG-3′ and reverse: 5′-ACAGATGGCAGGCTCAGTGAT-3′; GAPDH: forward: 5′-GAAGGTCGGAGTCAACGGAT-3′ and reverse: 5′-CCTGGAAGATGGTGATGGG-3′. The PCR products for RNF146 (376 bp), Axin (109 bp), β-catenin (221 bp), and GAPDH (153 bp) were amplified with 30 PCR cycles.Total RNA was extracted using TRIzol (Invitrogen) according to the directions in the TaKaRa RNA PCR Kit. GAPDH expression served as the internal control. The primer sequences were: RNF146: forward: Cells were lysed in buffer containing 150 mM NaCl, 1% NP-40, 0.1% SDS, 2 µg/ml aprotinin, and 1 mM PMSF. Total protein was harvested, and 60 µg were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes . Proteins on PVDF membranes were blocked with 5% bovine serum albumin (BSA) in phosphate buffered saline with tween (PBST), incubated with primary antibody at 4°C overnight, and then incubated with secondary antibodies at room temperature for 2 hours. Membranes were developed with enhanced chemical liquid (ECL) (Thermo Fisher Scientific), and the results were recorded using a bioimaging system . Protein expression levels were evaluated using β-actin as a loading control.Cells grown on glass cover slips were fixed with 4% paraformaldehyde and treated with 0.2% Triton X-100. After washing with PBS, cells were blocked with nonimmunized animal serum at 37°C for 30 minutes, and then incubated with primary antibodies anti-RNF146 (1∶100), anti-Axin (1∶100), and anti-β-catenin (1∶100) at 4°C overnight. Negative and positive controls were included in all experiments. Cells were incubated with FITC or TRITC-labeled secondary antibodies , at 37°C for 30 minutes. After washing with PBS, cells were incubated with 0.1% 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen) at 37°C for 10 minutes. Cells were washed with PBS and observed under a fluorescence microscope.3 cells per well and cultured for 24 hours. Cell proliferation was detected with the cell counting Kit-8 solution  according to the manufacturer’s instructions. The 3--2,5-diphenyltetrazolium bromide (MTT) assay was repeated on 4 consecutive days. Optical density values were measured using a microplate reader  at an absorbance of 550 nm.Transfected cells and control cells were plated on 96-well plates at 105 cells, cultured for 24 hours, and scratched with a spearhead. Cells were washed with PBS and cultured in serum-free medium at 37°C and 5% CO2. Samples were harvested and photographs were taken at 0, 6, 12 and 24 hours. Each experiment was repeated at least three times and average values were calculated.Each well of a 6-well plate was seeded with 5×106 cells/ml were added to the upper chamber of a transwell insert, and 600 µl medium containing 10% fetal bovine serum was placed in the lower chamber. A polycarbonate microporous membrane with a diameter of 8 µm separated the upper and the lower chambers. Cells were cultured at 37°C and 5% CO2 for 6 hours. After washing with PBS, cells were fixed with methanol at room temperature for 15 minutes. Finally, the treated cells were stained with hematoxylin, photographs were taken, and stained cells were counted. To measure the invasive capacity of cells, precooled medium without serum and Matrigel  were mixed at a volume ratio of 1∶7 and were added into the upper chamber. The volume of the mixture liquid was 100 µl. The cells were treated for 3 hours at room temperature, and the results were obtained after the cells were cultured for 24 hours.Cell suspensions of 100 µl containing 2.5×106 cells per 6 cm culture dish, cultured for 12 hours, and transfected. Cells were synchronized by serum starvation for 20 hours. Cells were maintained in medium containing 10% serum for 24 hours. Cells were harvested and fixed in cold 70% ethanol. After washing with PBS, cells were incubated with 0.1 mg/ml RNaseA  at 37°C for 30 minutes and treated with 5 mg/ml propidium iodide for 30 minutes. Finally, the treated cells were analyzed by flow cytometry.Cells were seeded at 5×102χ test and Fisher test. Kaplan-Meier survival curves and Log-rank were used to detect and evaluate differences. Other data were compared by t-test. Data from three independent experiments were shown as ±s. A P<0.05 was considered statistically significant.Databases were set up using Excel. Data analysis was conducted using SPSS17.0. Relationships between the expression of RNF146, Axin, β-catenin, and clinical and pathological factors were evaluated by the P = 0.030)   . The res= 0.000) , consist= 0.000) . The expP = 0.044), histological type (P = 0.005), poor differentiation (P = 0.002), lymphatic metastasis (P = 0.009), and pTNM stage (P = 0.015). However, RNF146 expression was not correlated with the age or gender of NSCLC patients (P = 0.016). There was no correlation between RNF146 expression and survival time in all patients (P = 0.616) or those patients with stage II (P = 0.437), or stage III NSCLC (P = 0.520)  .P = 0.003), but it was not correlated with the decreased expression of β-catenin in membranes or the increased expression of β-catenin in the cytoplasm (P = 0.524). High RNF146 expression was positively correlated with nuclear β-catenin staining (P = 0.047)  .LTE cell lines were transiently transfected with the pEX4-RNF146 overexpression plasmid or the control pEX4 empty plasmid. Experiments confirmed that RNF146 was upregulated. A549 and H1299 cell lines were transiently transfected with RNF146-shRNA1, RNF146-shRNA2 or control shNC, and experiments confirmed that RNF146 expression was reduced . Further0/G1 phase LTE cells and increased the percentage of S phase cells. In contrast, knockdown of RNF146 in A549 and H1299 cells increased the percentage of cells in the G0/G1 phase and decreased the percentage of cells in S stage /adenomatous polyposis coli (APC)/Casein kinase I (CKI) degradation complex, which enhances the degradation of β-catenin and inhibits tumor proliferation, invasion, and metastasis We found that RNF146 was negatively correlated with the expression of Axin and positively correlated with nuclear expression of β-catenin. Immunofluorescent staining demonstrated that RNF146 regulated the distribution and nuclear accumulation of β-catenin. Previous studies have demonstrated that nuclear β-catenin expression leads to the loss of cell adhesion. Moreover, this protein increased the transcription of target genes, including c-myc, cyclinD1, VEGF, and MMP7, by interacting with the T cell factor/lymphoid enhancer factor, thereby leading to the proliferation and metastasis of tumor cells 1 phase progression 1/S transition Our MTT and cell cycle experiments showed that RNF146 not only regulated cell proliferation and cell cycle progression but also affected the expression levels of proteins related to the cell cycle. Cyclin D1 mainly regulates GOur data also showed that RNF146 affected the migration and invasion of lung cancer cells, suggesting that RNF146 has multiple functions. The scratch test and transwell assay results showed that RNF146 enhanced the migration and invasion of lung cancer cells. MMPs degrade various proteins in the extracellular matrix, disrupt tissue barriers to tumor cell invasion, and play key roles in the invasion and metastasis of tumors Prior to our study, the roles of RNF146 in the Wnt signaling pathway were confined to the degradation of Axin and the promotion of the nuclear aggregation of β-catenin. Because β-catenin interacts with TCF-4 after it enters the nucleus However some caveats need to be considered in the potential mechanism of RNF146 in Wnt signaling pathway. First, Wnt is a very complex process and lots of proteins are involved in Wnt regulation. Wnt signaling is regulated by altered expression of various regulatory proteins in lung cancer and RNF146 may be the only one of those regulators. So, further studies are required to verify the potential mechanism of RNF146 in Wnt signaling pathway. Second, RNF146 was confirmed to be the novel E3 ubiquitin ligase 1, however whether the biologic function of RNF146 in tumors is dependent or independent on ubiquitin ligase activity remains unknown. This potential link needs to be fully investigated.In summary, we observed high expression levels of RNF146 in clinical NSCLC samples, consistent with the finding that RNF146 overexpression enhanced the proliferation and invasion of lung cancer cells. In addition to regulating the growth and survival of tumor cells, RNF146 played tumor-enhancing roles and affected the migration and invasion of tumor cells in NSCLC. These effects probably depended on key factors, including Axin and β-catenin in the Wnt signaling pathway. The results reveal new roles for RNF146 in the development and progression of NSCLC and identify RNF146 as a potential target for lung cancer treatment.Figure S1Silencing of RNF146 suppressed cell growth and regulated cell cycle progression. (A) Silencing of RNF146 in A549 and H1299 cells. Both cell lines were transfected with shNC, shRNF146-1, or shRNF146-2. After 48 hours, the effective knockdown of RNF146 was confirmed by Western bolt. (B) MTT assay of A549 and H1299 cells showed decreased number of viable cells by shRNA-mediated knockdown of RNF146. **P<0.05. (C) Effects of RNF146 depletion on cell cycle analyzed by flow cytometry.(TIF)Click here for additional data file.Figure S2Knockdown of RNF146 inhibited cell migration. Wound-healing assay was carried out. Confluent monolayer of A549 cells (A) or H1299 cells (B) was scratched using a sterile pipette. At 24 hours after wounding, migration of cells into the scraped area was photographed. The percentages of migrated cells were quantified. **P<0.05.(TIF)Click here for additional data file.Figure S3Effects of RNF146 on migration and invasion in A549 and H1299 cells. Cell migration and invasion assays of A549 and H1299 cells transfected with shNC, RNF146-specific shRNA were carried out using a 12-well Transwell. The images show representative density of cells that migrated  or invasion . The bar graph depicts quantification of migration or invasion cells. **P<0.05.(TIF)Click here for additional data file.


In addition to the eight genotypes of classic human astroviruses, seven new genotypes have been reported from two novel clades, MLB and VA. However, the epidemiology of these highly diverse astroviruses remains largely unknown. We report here the complete genome sequences of two MLB1 strains from Bhutanese children with diarrhea. Each year, 700,000 children aged <5 years die throughout the world due to diarrhea . RotavirIn 2009, astrovirus clade MLB1 was detected during an outbreak of diarrhea in Australian children . SubsequThe complete genome sequence of MLB1 was determined using primers which were designed using sequences reported previously , 8 from Astroviridae. The genome has three open reading frames (ORFs), ORF1a, ORF1b, and ORF2, which encode protease, RNA-dependent RNA polymerase, and capsid protein, respectively.Astroviruses are nonenveloped viruses with a positive-sense RNA genome that belong to the family The complete genome sequences of Bhutanese MLB1s contain 6,171 nucleotides (nt), excluding the poly(A) tail. ORF1a contains 2,364 nt from nt 15 to 2378, ORF1b contains 1,536 nt from nt 2315 to 3859 nt, and ORF2 contains 2,268 nt from nt 3843 to 6110. Both strains have a 30-nt 5′-untranslated region and heptameric slippery sequence, which are conserved in astroviruses (AB823731 and AB823732, respectively.The genome sequences of BtnMLB1-40 and BtnMLB1-86 appear in the DDBJ/EMBL/GenBank databases with accession no.


Pinus massoniana) forests in Chongqing, southwest China, have increasingly declined. Soil acidification was believed to be an important cause. Liming is widely used as a measure to alleviate soil acidification and its damage to trees, but little is known about long-term effects of liming on the health and growth of declining Masson pine forests. Soil chemical properties, health condition (defoliation and discoloration), and growth were evaluated following application of limestone powder (0 (unlimed control), 1, 2, 3, and 4 t ha−1) in an acidified and declining Masson pine stand at Tieshanping (TSP) of Chongqing. Eight years after liming, in the 0–20 cm and 20–40 cm mineral soil layers, soil pH values, exchangeable calcium (Ca) contents, and Ca/Al molar ratios increased, but exchangeable aluminum (Al) levels decreased, and as a result, length densities of living fine roots of Masson pine increased, with increasing dose. Mean crown defoliation of Masson pines  decreased with increasing dose, and it linearly decreased with length densities of living fine roots. However, Masson pines (Kraft classes 1–3) in all treatments showed no symptoms of discoloration. Mean current-year twig length, twig dry weight, needle number per twig, needle length per twig, and needle dry weight per twig increased with increasing dose. Over 8 years, mean height increment of Masson pines (Kraft classes 1–3) increased from 5.5 m in the control to 5.8, 6.9, 8.3, and 9.5 m in the 1, 2, 3, and 4 t ha−1 lime treatments, and their mean DBH (diameter at breast height) increment increased from 3.1 to 3.2, 3.8, 4.9, and 6.2 cm, respectively. The values of all aboveground growth parameters linearly increased with length densities of living fine roots. Our results show that liming improved tree health and growth, and these effects increased with increasing dose.In the last decades, the Masson pine ( Atmospheric emissions of acidifying compounds resulting from anthropogenic activities since the beginning of the industrial revolution have led to extensive soil acidification in terrestrial ecosystems Pinus massoniana) is one of the most widespread forest species across subtropical China and it is highly sensitive to acid deposition Masson pine  . TSP, covering about 1200 ha, is an almost pure Masson pine forest located on a sandstone ridge approximately 25 km northeast from the urban centre of Chongqing  in southeast Sichuan Basin, southwest China. Masson pines have been planted since the early 1960s. The study site has a subtropical humid climate, with a mean annual air temperature of 18°C, a mean annual precipitation of 1100 mm that mainly occurs from April to October, and a mean annual relative humidity of 80%. TSP has been experiencing a long-term severe acid deposition, with low mean annual pH (4.0–4.2) and high frequency of acid rain  Schima superba, Cunninghamia lanceolata, Schoepfia jasminodora, and Lithocarpus glaber grew. The density of Masson pines for this stand was 1192 trees ha−1. In early June 2004, the mean defoliation, discoloration, height, and DBH of Masson pines  for this stand were 34%, 1%, 13.3 m, and 15.5 cm, respectively.In early May 2004, we selected a 26-year-old Masson pine stand, damaged (defoliation >25%) by soil acidification −1) and three replicates. There were total fifteen plots. Each plot measured 10 m×10 m, with about 1 m buffer strips around each plot. The overall size of the stand within which all plots were installed was about 0.3 ha. Limestone powder was broadcast one time and evenly on the forest floor by hand in early June 2004.The trial was established in this stand as a randomized complete block experiment with five limestone powder  treatments (0 (unlimed control), 1, 2, 3, and 4 t haIn mid June 2012, two dominant Masson pines (Kraft class 1) were selected near the centre of each plot for sampling. Humus and soil were collected from the east, south, west, and north directions 1.0 m away from the main stem of each selected tree. Samples of the humus layer and the 0–20 cm, 20–40 cm and 40–60 cm mineral soil layers were taken using a cylindrical auger with an inner diameter of 10 cm and a height of 25 cm. The four samples collected around each selected tree in each plot were pooled to produce one composite sample per layer. In all, there were 120 composite samples representing five treatments and three field replicates. All composite samples were placed in plastic bags and transported to the laboratory, where they were bulked and crumbled manually. Living roots of different diameters of Masson pine were picked out according to the characteristics of appearance, color, elasticity, and odor −3) of living fine roots were calculated from all samples according to the volumes of the humus and soil samples. During sampling and measurements, polypropylene gloves were used to avoid contamination. In this study, we chose to analyze the soil chemical properties and fine root growth of Masson pine in the 0–20 cm and 20–40 cm mineral soil layers where the roots were mainly distributed Soil pH was determined in a 1∶2.5 slurry of soil∶1 M KCl solution using a combination glass electrode, after shaking for 0.5 h and then equilibrating for 0.5 h. The accuracy was 0.01 pH. Exchangeable Ca and Al were extracted with 1 M KCl solution and determined using an atomic absorption spectrometer . The Ca/Al molar ratios were calculated from exchangeable Ca and Al contents of all humus and soil samples. Lengths of living fine roots (diameter ≤2 mm) were measured using an automated root scanning and analysis system . Length densities , height, and DBH were performed twice on all dominant, codominant and subdominant Masson pines (Kraft classes 1–3) per plot according to the ICP–Forests manual At TSP, current-year needles make up the majority of tree foliage. In early June 2012, five current-year twigs with needles were sampled from the upper third of the crown on the south side of each of the two selected trees near the centre of each plot. The five current-year twigs with needles collected from each selected tree per plot were pooled to form a sample. Thirty samples were transported in clean plastic bags to the laboratory, where for each sample we separated needles from twigs, and measured twig length, twig dry weight, needle number per twig, needle length per twig, and needle dry weight per twig. Twig and needle lengths were measured to the nearest millimeter. The samples were ovendried at 60°C for about one week to a constant weight and weighed. Mean twig lengths, mean twig dry weights, mean needle numbers per twig, mean needle lengths per twig, and mean needle dry weights per twig were calculated from all samples.P<0.05 was considered significant.The data were expressed as mean ± standard deviation (SD). They were assessed using one-way analysis of variance (ANOVA) with the SPSS 12.0 software package. The fixed model was used in ANOVA. Multiple comparisons of means among treatments were performed using Duncan test. −1>3 t ha−1>2 t ha−1>1 t ha−1>0 t ha−1, while the exchangeable Al exhibited the reverse pattern. Overall, there were no statistical differences in the soil chemical parameters in the same layers between the 0 and 1 t ha−1 lime treatments, whereas significant differences were observed among the 1, 2, 3, and 4 t ha−1 lime treatments (P<0.05).Liming increased the pH, exchangeable Ca, and Ca/Al molar ratio, but decreased the exchangeable Al in the 0–20 cm and 20–40 cm mineral soil layers . The pH,−1>3 t ha−1>2 t ha−1>1 t ha−1>0 t ha−1. There were no statistical differences in the length density in the same layers between the 0 and 1 t ha−1 lime treatments, whereas significant differences were found among the 1, 2, 3, and 4 t ha−1 lime treatments (P<0.05). The length densities of living fine roots were significantly positively correlated with the pH values, exchangeable Ca contents, and Ca/Al molar ratios, but significantly negatively with the exchangeable Al levels in both soil layers  in the 4 t ha−1 lime treatment was 25%, which is the lower limit of the moderate defoliation range (>25%–60%). However, the mean discoloration of Masson pines (Kraft classes 1–3) in all treatments lay within no discoloration range (0–10%). The mean defoliation was significantly negatively correlated with the length densities of living fine roots in both soil layers, but the values of current-year twig and needle growth parameters were significantly positively correlated with them (Liming decreased the defoliation of Masson pines (Kraft classes 1–3), but increased their current-year twig and needle growth . The meaith them .−1 lime treatment was the greatest in terms of both the mean height increment and the mean DBH increment of Masson pines (Kraft classes 1–3), it was followed by, in decreasing order, the 3, 2, 1, and 0 t ha−1 lime treatments. The mean height increments of Masson pines (Kraft classes 1–3) were 5.5±0.4, 5.8±0.5, 6.9±0.5, 8.3±0.6, and 9.5±0.6 m in the 0, 1, 2, 3, and 4 t ha−1 lime treatments, and their mean DBH increments 3.1±0.2, 3.2±0.2, 3.8±0.3, 4.9±0.4, and 6.2±0.4 cm, respectively. There were no statistical differences in both parameters between the 0 and 1 t ha−1 lime treatments, but significant differences were detected among the 1, 2, 3, and 4 t ha−1 lime treatments (P<0.05). The mean height increment and mean DBH increment were significantly positively correlated with the length densities of living fine roots in the 0–20 cm and 20–40 cm mineral soil layers (Liming increased the height and DBH of Masson pines (Kraft classes 1–3) from early June 2004 to early June 2012. Among all treatments, the 4 t hal layers .−1 lime treatment the soil pH values increased to 4.48 and 4.92, respectively  while Ca contents in 20–40 cm depth were unaffected. Guckland et al. P. abies, F. sylvatica, Quercus petraea/Q. robur, and Pinus sylvestris 2–9 years after one-time liming with 3–5 t ha−1 lime , and they suggested that the element input, CEC, C stock of the forest floor, and groundwater recharge were the most important parameters connected with the change of Ca and Mg stocks after liming in 0–10 cm, but in 10–20 and 20–40 cm, the C concentration of this depth and the year after first liming gained more influence. In our study conducted at TSP in the humid subtropical zone, in the 0–40 cm mineral soil depth of the limed Masson pine stand 8 years after one-time liming with 2–4 t ha−1 limestone powder, significant effects of liming were observed on the chemical properties and the growth of fine roots  of plants is coordinated with the growth of belowground part (roots) In summary, in the acidified and declining Masson pine stand of Chongqing, southwest China, application of limestone powder proved to be effective for ameliorating the soil acidity, improving the crown health condition, and stimulating the tree growth; these effects increased as the amount of applied limestone powder increased.


Loxodonta africana and nyala Tragelaphus angasii in the critically endangered Sand Forest, which is restricted to east southern Africa, and is conserved mainly in small reserves with high herbivore densities. Replicated experimental treatments (400 m2) in a single forest patch were used to exclude elephant, or both elephant and nyala. In each treatment, all woody individuals were identified to species and number of stems, diameter and height were recorded. Results of changes after two years are presented. Individual tree and stem densities had increased in absence of nyala and elephant. Seedling recruitment (based on height and diameter) was inhibited by nyala, and by elephant and nyala in combination, thereby preventing recruitment into the sapling stage. Neither nyala or elephant significantly reduced sapling densities. Excluding both elephant and nyala in combination enhanced recruitment of woody species, as seedling densities increased, indicating that forest regeneration is impacted by both mega- and mesoherbivores. The Sand Forest tree community approached an inverse J-shaped curve, with the highest abundance in the smaller size classes. However, the larger characteristic tree species in particular, such as Newtonia hildebrandtii, were missing cohorts in the middle size classes. When setting management goals to conserve habitats of key importance, conservation management plans need to consider the total herbivore assemblage present and the resulting browsing effects on vegetation. Especially in Africa, where the broadest suite of megaherbivores still persists, and which is currently dealing with the ‘elephant problem’, the individual effects of different herbivore species on recruitment and dynamics of forests and woodlands are important issues which need conclusive answers.Herbivory by megaherbivores on woody vegetation in general is well documented; however studies focusing on the individual browsing effects of both mega- and mesoherbivore species on recruitment are scarce. We determined these effects for elephant  Different-size herbivores have different feeding preferences While numerous studies have focused on megaherbivore impact on woody communities e.g. , and on Here we focus on the impacts on seedling and sapling recruitment by a mega- and mesoherbivore within the critically endangered Sand Forest community Loxodonta africana and nyala Tragelaphus angasii became locally abundant in protected Sand Forest areas after (re)introductions of these species in the early 1990s.The dynamics of Sand Forest are poorly understood Aepyceros melampus have been linked to recruitment limitation Conservation of the Sand Forest community is of critical importance, and it is therefore imperative to assess potential drivers affecting the tree community and its low recruitment rates. Management questions have been raised regarding the impact of herbivores, in particular elephants 2  conservation area in Maputaland, northern KwaZulu-Natal, South Africa. The reserve includes a wide range of habitat types, such as western Maputaland sandy bushveld as well as several patches of the endemic Sand Forest Phinda Private Game Reserve (hereafter Phinda) is a 180 kmGiraffa camelopardalis (154), kudu Tragelaphus strepsiceros (188), impala (1690), red duiker Cephalophus natalensis (23), common duiker Sylvicapra grimmia (no count available) and suni Neotragus moschatus (no count available). Counts in parentheses are approximate and reflect the 2007 annual helicopter game count.Before Phinda was created in 1991, the area consisted of private and small game farms. Game was introduced following the establishment of the park Balanites maughamii, Cleistanthus schlechteri, Cola greenwayi, Croton pseudopulchellus, Dialium schlechteri, Drypetes arguta, Hymenocardia ulmoides, Newtonia hildebrandtii and Pteleopsis myrtifoliaonly mega- and mesoherbivore utilising the Sand Forest patches (Lagendijk pers. obs.).This study was conducted in the endemic Sand Forest, which occurs in the northern section of Phinda. Sand Forests occur on acidic, sandy soils with very little clay 2 of the 5.2 km2 Sand Forest patch  unfenced area available to all herbivores (open access “+E+N”); (2) area fenced to exclude only elephant ; (3) area fenced to exclude both nyala and elephants (full exclosure “−E−N”), but providing access to smaller herbivores. There were a total of 12 replicates of this set of three treatments. Distance between replicates ranged between 0.12 km and 2.75 km.The effects of elephant and nyala on Sand Forest recruitment were tested using exclosures. In November 2005, elephants were excluded from part of the Sand Forest using electrified (7000 volts per second) high tension galvanized wires (2.4 mm thick) erected at 1.8 m and 2 m above the ground, enclosing 3.09 kmst patch . To deteA base-line study was conducted in 2005. In each quadrat (n = 36) all woody individuals  were identified to species and counted. Diameters above the buttress swelling of all stems (including seedlings and saplings) and all tree heights were recorded. Diameters and the heights of trees to 2 m were measured, the heights of trees between 2 m and 4 m estimated to the nearest 20 cm, and the heights of taller trees estimated to the nearest 50 cm using the height of an observer as a scale following Shannon et al. Following recommendations by Clarke and Warwick 2 to 1 ha. Individual trees and stems were allocated to seven height classes , which roughly correspond to the limits at which browsing by different-size herbivores occurs.Because there are two possible demographic responses to browsing viz. mortality or a coppicing response  and sapling (>0.5 m and ≤1.5 m in height) abundance, as well as stem densities in these height classes, among treatments were also analysed using ANOVA. Pair-wise comparisons of individual overall tree densities and seedlings per treatment between 2005 and 2007 were also analysed using ANOVA.2 = 0.73)). A G-test was used to determine whether size distributions differed among treatments for the pooled data. To prevent compounding of Type 1 errors from running three pair-wise G-tests, alpha of 0.05 was Bonferroni-adjusted to 0.017.Tree populations are regenerating when the population structure displays an inverse J-shaped frequency distribution Salacia leptoclada, Uvaria caffra and Tricalysia junodii) and on three characteristic Sand Forest trees  to determine the effect of elephant and/or nyala on recruitment. Seedling and sapling abundance were analysed separately among treatments per focus species using a two-way ANOVA. When ANOVA assumptions were not met, densities were analysed with a Kruskal-Wallis test. Size class distributions (SCD), which reflect population structures, were analysed for each of these six species using linear regressions (cf. i) for the class midpoint (Mi). These variables were ‘ln+1’ – transformed prior to regression analyses. All size classes up to the largest size class containing individuals were included in the analyses. We used SCD slopes to interpret population structures. An inverse J-shaped curve is represented by a steep negative slope, while species with little regeneration show a negative slope close to zero.At the tree species level, we focused our analyses on the three most common Sand Forest species in our study area  were followed-up with Tukey's post-hoc tests. All statistical analyses were performed using SPSS 15.0 .S. leptoclada, T. junodii, and U. caffra made up 49.9% of all trees, compared to 53.4% in 2005.In 2005, 12638 individual plants from 95 woody species, and in 2007, 17825 individual trees and 143 woody species were recorded in all treatments. In 2007, the dominant Sand Forest species 0.05(2)2,33 = 0.363, P = 0.698), sapling (F0.05(2)2,33 = 0.944, P = 0.399), and overall  tree and stem densities (F0.05(2)2,33 = 0.842, P = 0.440 and F0.05(2)2,33 = 1.905, P = 0.165 respectively). Note that when all species present were included in this analysis, there were still no significant differences for any of these contrasts (seedling: F0.05(2)2,33 = 1.044, P = 0.363; sapling: F0.05(2)2,33 = 0.758, P = 0.464; tree density for all size classes combined: F0.05(2)2,33 = 0.883, P = 0.444; stem density for all size classes combined: F0.05(2)2,33 = 2.734, P = 0.080).The twelve replicates of the experiment were considered to be homogeneous in 2005 as, when only taking the more abundant species (n = 26) into account, there were no significant differences among treatments for the seedling (F0.05(2)2,33 = 5.180, P = 0.011) and the overall stem densities (F0.05(2)2,33 = 4.426, P = 0.020), with densities in the full exclosure (−E−N) being significantly greater than in the open access treatment (+E+N) . The overall abundance of individual trees in the partial exclosure (−E+N), were not significantly different from those in the open access (+E+N) or in the full exclosure (−E−N) treatment. However stem densities were greater in the full exclosure than in the partial exclosure, although this was marginally not significant (Tukey: P = 0.056). Pair-wise comparisons between 2005 and 2007 showed a significant increase in the full exclosure for overall tree densities (−E−N: F0.05(2)1,22 = 7.387, P = 0.013). Differences in overall stem densities per treatment between 2005 and 2007 were not significant (open access (+E+N): F0.05(2)1,12 = 0.599, P = 0.454; partial exclosure (−E+N): F0.05(2)1,22 = 0.537, P = 0.471; full exclosure (−E−N): F0.05(2)1,22 = 2.401, P = 0.136). This indicated that recruitment was taking place within the full exclosure (−E−N).In contrast to this, for the 26 species in 2007 there were significant differences among the treatments in both the overall tree densities (F0.05(2)2,33 = 3.582, P = 0.039; 0.05(2)2,33 = 5.104, P = 0.012), with greater seedling densities in the full exclosure (−E−N) than in the open access treatment 1,22 = 3.186, P = 0.088). However, there was a significant increase in seedling densities in the open access treatment (+E+N: F0.05(2)1, 12 = 5.386, P = 0.039) and the full exclosure between 2005 and 2007 (−E−N: F0.05(2)1,22 = 9.755, P = 0.005; For the 26 species, pair-wise comparisons of seedling (≤0.5 m in height) density between 2005 and 2007 was not significantly different within the partial exclosure (−E+N: F0.05(2)2,33 = 1.421, P = 0.256; 0.05(2)2,33 = 0.123, P = 0.884; For the 26 species, sapling densities in 2007 were not significantly different among treatments (F0.05(2)2,33 = 5.030, P = 0.012; 0.05(2)2,33 = 0.146, P = 0.865; Seedling stem densities in 2007 were significantly different among the three treatments . In all treatments, the highest abundance was found in the smallest size (≤1.0 cm) class  class . PopulatS. leptoclada, U. caffra and T. junodii approached the inverse J-shaped curve characteristic of increasing populations, which is supported by the strong negative SCD slopes for these species  and D. schlechteri (in the full exclosure (−E−N)).Both seedling and sapling densities of each of the six selected focus species did not significantly differ among treatments  (U. caffra does not occur in TEP) Neither seedlings nor saplings of the three common and three characteristic focus species showed a significant effect from browsing. Elephants have been found to select for D. schlechteri, N. hildebrandtii and P. myrtifolia) had size classes missing in the middle size cohorts, which may be explained by previous human utilisation of stems. In the last 25 years, the human population in the region All three large tree species , this experiment shows that by manipulating disturbance factors (e.g. herbivory), changes in recruitment can be demonstrated within a short time interval cf. ; 3 yearsWe emphasise here the need to consider all possible factors influencing tree communities, and not only the “obvious” or “political” ones. In the case of Sand Forest, while fencing elephants from the Sand Forest will provide a reduction in damage to larger trees Since tourism revenues are an important source of income for most parks, the creation of botanical reserves within the protected area can be a lucrative management strategy. This type of management approach could also be applicable to other natural systems.Table S1Regression analyses for tree abundance vs. tree diameter.(DOC)Click here for additional data file.


As a precursor to overt heart failure, diabetics experience a unique alteration in myocardial architecture marked by diffuse interstitial fibrosis. We hypothesized that the myocardial extracellular volume fraction (MECVF) could be used to detect myocardial matrix remodeling and its relationship to ventricular structure and function in diabetics without cardiovascular disease.Diabetics without any history of MI  or heart failure underwent 3T CMR imaging and assessment of MECVF and assessment of biochemical parameters of insulin sensitivity. 10 volunteers without diabetes, hypertension, or clinical cardiac disease were used for comparison. Cine SSFP, LGE imaging, and myocardial T1 quantitation using a modified Look-Locker sequence were performed. For measurement of MECVF, segmental myocardial T1 was measured once pre-Gd and at least 3 times over 3-20 minutes post-Gd . Regression of myocardial R1 (=1/T1) on R1 in the blood pool was used to determine the Gd partition coefficient, which when multiplied by (1-hematocrit/100), gave MECVF. Left atrial volume index (LAVI) and emptying function, as well as LV and RV volumes and function, were determined by standard techniques.55 recruited patients were predominantly male (62%), with an average age of 54±8 years, systolic blood pressure 126±14 mmHg, body mass index (BMI) 32.4±4 kg/m2, fasting glucose 108±25 mg/dl, and triglycerides 128±69 mg/dl. CMR LVEF averaged 60±5%, LV mass index 45±11 g/m2, LVEDV index 68±14 ml/m2, LAVI 35±9 ml/ m2 . MECVF in diabetics was 0.36±0.05 . MECVF correlated with LAVI , but not with LV mass index, fasting glucose or insulin levels, insulin sensitivity, triglycerides, LVEF, age, BMI, LA function or systolic blood pressure.Well-controlled diabetics with normal ventricular structure, function, and no prior cardiac disease have diffuse interstitial myocardial fibrosis by CMR. Diffuse fibrosis is associated with LAVI, a marker of diastolic function, but not with other markers of clinical or cardio-metabolic risk. Diffuse myocardial damage occurs early in the course of diabetic cardiomyopathy independent of hypertension and obesity, and may be a potential target for early intervention in at-risk diabetic patients with subclinical myocardial disease.RVS was supported by a Post-Doctoral Fellowship Grant from the American Heart Association (11POST000002). RYK, MJ, GKA are supported by grants from the National Institutes of Health.


To examine the association between brace compliance and outcome.495  patients with late onset juvenile and adolescent idiopathic scoliosis were examined prospectively, before bracing, and at least 2 years after brace weaning. One spine surgeon examined all patients. 381  answered a standardised questionnaire, and 355 had radiological examination after median 24 years. Compliance was defined as brace wear > 20 hours daily until weaning. Main outcomes were curve progression and surgery.At weaning, 76/389 compliers and 59/106 non-compliers had curve progression ï‚³ 6° . At long-term, the numbers were 68/284 and 46/71 , and 10/284 versus 17/71 had been operated .We conclude that the risk for curve progression, and surgery, are reduced in patients with good brace compliance.


Thermococcus kodakarensis KOD1 (KOD), as well as SSB proteins from Thermus thermophilus (TTH), Escherichia coli, and Sulfolobus Solfataricus P2 (SSOB), abbreviated kod, tth, bl21, and ssob, respectively. These SSB proteins could bind ssDNA and viral RNA. bl21 resisted heat treatment for more than 9 h, Ssob and kod could withstand 95°C for 10 h and retain its ssDNA- and RNA-binding ability. Four SSB proteins promoted the specificity of the DNA polymerase in PCR-based 5- and 9-kb genome fragment amplification. kod also increased the amplification of a 13-kb PCR product, and SSB protein–bound RNA resisted Benzonase digestion. The SSB proteins could also enter the host cell bound to RNA, which resulted in modulation of viral RNA metabolism, particularly ssob and bl21.The accumulation of single-stranded DNA-binding (SSB) proteins is essential for organisms and has various applications. However, no study has simultaneously and systematically compared the characteristics of SSB proteins. In addition, SSB proteins may bind RNA and play an unknown biological role in RNA metabolism. Here, we expressed a novel species of SSB protein derived from  Nucleic acid replication, repair, recombination, transcription, and translation play a critical role in genome metabolism in organisms Escherichia coli has the following functions; targeting restriction endonuclease digestion to any restriction enzyme site in cloned ssDNA via DNA oligo splints SSB proteins are present in all three branches of organisms and in viruses E. coli remain unknown. In this study, we compared the biological functions of SSB proteins derived from the thermophilic bacteria TTH, SSOB, and KOD, as well as E. coli BL21 (DE3). Overall, our results increase our understanding of the SSB proteins.Although numerous applications of SSB proteins have been reported, a systematic functional comparative analysis of SSB proteins derived from different species has not yet been performed. Thus, it remains possible that SSB proteins bind single-stranded RNA. The SSB proteins may also have novel applications based on other biological characteristics. Also, differences between the SSB proteins derived from thermophilic bacteria and Thermus thermophilus HB8 (AP008226.1), Sulfolobus Solfataricus P2 (AE006641.1), and Thermococcus kodakarensis KOD1 (AP006878.1), and one SSB protein from Escherichia coli BL21 (AM946981) termed tth, ssob, kod and bl21, respectively. To express these four SSB proteins, the coding sequences of tth, ssob and kod were synthesised by Biovisualab Ltd  with a His-tag at their N-termini, and were inserted into the pET11a expression vector . The open reading frame encoding bl21 was cloned by PCR using E. coli BL21 (DE3) chromosomal DNA as template with primers . A high-fidelity DNA polymerase (HiFiFast)  was used. The PCR product with a His-tag was inserted into a pET21a expression vector  after digestion with NdeI and XhoI . After sequence confirmation, the correct plasmids were used for SSB protein expression. Based on the supporting rare codon, tth was expressed in BL21 (DE3) competent cells and the remaining kod, ssob, and bl21 were expressed in Transetta (DE3) chemically competent cells .We selected three SSB proteins derived from the thermophilic bacteria 600 of 0.8. IPTG was then added to a final concentration of 1 mM. The cells were harvested after 4 h induction by centrifugation at 3,000 rpm for 30 min in a Hitachi centrifuge , and the pellet was resuspended in 50 mL of buffer , and then disrupted by sonication. Insoluble debris was removed by centrifugation at 12,000 rpm for 20 min in a Hitachi centrifuge . The supernatant was heat-treated at 70°C for 30 min and denatured mesophilic proteins were discarded by centrifugation at 12,000 rpm for 20 min in a Hitachi centrifuge . The clarified supernatant was loaded onto a Ni2+ Sepharose column , and bound proteins were washed and eluted with 5–120 mM imidazole. The eluted fractions containing protein were detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Target proteins were collected and dialysed against 40 mM Tris–HCl (pH 8.0) and 200 mM NaCl. The concentration of purified protein was determined using a spectrophotometer  with the absorption coefficient at 280 nm. A series of quantitative albumin was used as a reference. After filtration through a 0.45-mm Durapore polyvinylidene fluoride (PVDF) membrane filter , 50% (v/v) glycerol was added to each of the SSB proteins and then stored at −20°C for further use.Cells were grown at 37°C in LB medium containing ampicillin (80 mg/L) or chloramphenicol (34 mg/L) to an ODTo confirm the protein molecular weight and purity, 5 µg of each SSB protein was applied to 12.5% running gel containing 0.1% SDS under reducing conditions for SDS-PAGE analysis. To verify the polymerisation form of these SSB proteins, 5 µg of each SSB protein was loaded onto 3.5–5–12% non-denaturing gradient polyacrylamide gels for electrophoresis with a NativeMarker™ protein marker . ssob is a strongly alkaline protein, it’s isoelectric point = 8.464, with positive charge at PH 8.3 buffer and will move to anode. To visualize the ssob in a native-PAGE, we lowered the pH of the running buffer and gel to 7.4 and reversed the negative and positive pole. All gels were stained with Coomassie brilliant blue R-250 .3 staining since this method stains both DNA and protein. The gels were fixed with 10% acetic acid for 20 min, rinsed three times with water for 2 min, impregnated with AgNO3 (1 g/L) and HCOH (1.5 mL/L) for 30 min, rinsed again with water briefly, and visualised using developing solution  for 2–10 min. The ssDNA was clearly stained red To verify the ssDNA binding activity of the four SSB proteins, a 146- or 537-bp PCR product was denatured at 100°C for 10 min and cooled on ice rapidly to obtain ssDNA. ssDNA (250 ng) was incubated with 1–5 µg of each of the SSB proteins in 100 mM Tris-HCl (pH 8.0) and 0.2 mM EDTA binding buffer at room temperature for 5 min. Then, the protein ssDNA mix was analysed by native-PAGE analysis. To visualise the ssDNA–SSB protein complex, we first performed AgNOin vitro transcribed hepatitis C virus (HCV) RNA was incubated with 1.5 µg each of the four SSB proteins in 100 mM Tris-HCl (pH 8.0) and 0.2 mM EDTA binding buffer at room temperature for 5 min. The denatured HCV RNA was obtained by heating the RNA at 65°C for 5 min. RNA–SSB protein complexes were applied to a 1.2% TAE agarose gel. After electrophoresis, RNA or RNA protein complexes were stained with ethidium bromide.For RNA binding analysis, 120 ng of To determine their heat resistance, the SSB proteins were incubated in water at 95°C for 5, 120, 240, or 600 min. Their binding activity was re-confirmed, as described above.in vitro, we examined the threshold dose of Benzonase  with 120 ng of HCV RNA, according to the manufacturer’s protocol. A total of 3.2×10−3 U of Benzonase could digest 120 ng of HCV RNA at room temperature within 30 min, while 3.2×10−2 U could completely digest the HCV RNA. To challenge the protection effect, 1.5 µg of each of the SSB proteins were added to bind RNA or denatured RNA. Next, 3.2×10−3 U and 3.2×10−2 U of Benzonase were added and incubated at room temperature for 30 min. The protection effect was visualised by 1.2%TAE agarose gel electrophoresis.To determine if the SSB proteins could protect the RNA against nuclease attack 5′-GGCCAGC AGCTGCTGTGCTCAATTCCTCGC-3′, reverse primer: 5′- GTGTGCAGTTCAAGGCCCTGTAGTTGCT G-3′), 9 kb  and a 13-kb  PCR fragment. The HiFiFast DNA polymerase  without any modifications was selected to verify the effect of SSB proteins on PCR specificity. The PCR reaction conditions were optimised as follows; 100 ng of template DNA, 200 nM each of the dNTPs, 200 nM each of the primers, 2.5 µM MgCl2, and 2.5 U of HiFiFast DNA polymerase . The PCR cycling conditions were 2 min initial denaturation at 98°C, followed by 35 cycles of 20 s at 98°C, 3 min at 68°C, and 5 min final elongation at 72°C. To evaluate the SSB protein effect on PCR specificity, 0.5–2 ng of ssob, bl21, kod or tth was added to the PCR mix. The effect of SSB proteins was confirmed by 1% TBE agarose gel electrophoresis.To evaluate the effect of SSB proteins on PCR specificity, the human beta globin gene was used as template to amplify a 5-kb  was denatured with XbaI, and the 5′overhangs were removed by treatment with mung bean nuclease. Templates were then purified by digestion with proteinase K, two rounds of phenol-chloroform extraction, and ethanol precipitation, and then resuspended at 1 µg/µL. RNA transcription reactions contained 20 mM Tris-HCl (2-amino-2-(hydroxymethyl)-1,3-propanediol), pH 7.5; 5 mM sodium chloride, 9 mM magnesium chloride, 3 mM each nucleoside triphosphate, 20 units T7 RNA polymerase , 12 units SUPERasin , and 0.05 µg/µL linear template. Following a 30 min synthesis at 37°C, RNAs were purified and DNase-treated with RNeasy minicolumns  and eluted in 2 mM sodium citrate, pH 6.4. RNA concentrations were measured using spectrophotometry and adjusted to 100 ng/µL. Aliquots (10 µL) of RNA were stored frozen at −80°C until use.7 cells/well 24 h prior to transfection and were ∼80% confluent at the time of transfection. DMRIE-C (4 µL) transfection reagent (Invitrogen) was added to 0.5-mL DMEM in a sterile polystyrene tube and mixed gently. Viral RNA (500 ng–1 µg) was then added, mixed and immediately added to the cells washed twice by DMEM prior to the transfection. The cells were incubated for 6 h at 37°C and in 5% CO2, and then the medium containing the transfection complexes was removed and replaced with complete growth medium (DMEM with 10% serum) for a further 48–72 h. To verify the effects of SSB proteins on viral RNA expression, 10 or 20 ng of each SSB protein was added to the transfection mix. To reduce systemic and technical errors, all transfections were performed in parallel.Huh7 cells  were maintained in complete growth medium  containing 10% heat-inactivated foetal calf serum (Invitrogen) and 0.1 mM non-essential amino acids. For RNA transfection, Huh7 cells were plated in 1.0×10To confirm the effects of SSB proteins on viral RNA expression, the cells were harvested with 0.25% Trypsin-EDTA (Invitrogen) 48 h after the transfection. The cells were washed twice with PBS and subsequently fixed with 3.7% formaldehyde in PBS for 30 min at room temperature. After washing the cells again with PBS, the cells were blocked with PBS containing 2% normal serum for 1 h. The cells were then incubated with HCV-infected human serum (1∶50)  for 2 h at room temperature. Cells were washed three times with PBS and then incubated with FITC-labelled goat anti-human IgG  for 1 h at room temperature. After washing the cells twice with ice-cold PBS, the cells were submitted for FACS  to determine the number of HCV protein–positive cells.To determine if the SSB proteins could co-enter the Huh7 cells together with the transfection complex, we performed immunocellularchemistry analysis in parallel with the FACS analysis. After transfection (48 h), the cells were washed twice with PBS and subsequently fixed with 3.7% formaldehyde in PBS for 30 min at room temperature. After washing the cells again with PBS, the cells were blocked with PBS containing 2% normal serum for 1 h. The cells were then incubated with a mouse anti-His antibody (Invitrogen) for 2 h at room temperature. Cells were washed three times with PBS and then incubated with FITC-labelled goat anti-mouse IgG  for 1 h at room temperature. After glass coverslips were coated with fluorescent mounting medium (Dako), SSB proteins in the host cell were examined using a confocal fluorescent microscope .t test or analysis of variance with post hoc Scheffe testing when appropriate. p<0.05 was considered statistically significant.The results are expressed as mean±standard deviation (SD) from at least 3 independent experiments. The statistical analysis was performed using Student http://mafft.cbrc.jp/alignment/server/). bl21 contained a ssDNA binding domain, a glycine and proline-rich region, and a C-terminal tail, as reported previously   were aligned using a multiple alignment program for amino acid or nucleotide sequences  . Meanwhi:1EYG_D) . tth and:1EYG_D) .Four SSB proteins encoding genes with a 6× his-tag at the 5′ terminus were inserted into the pET expression system. Suitable competent cells were transformed with the expression plasmids, and robust and stable expression clones were selected to yield SSB proteins after IPTG induction. SSB proteins were purified by affinity chromatography after cell lysis at 70°C for 20 min, excluding bl21, which did not undergo the heating stage.To visualise their purity and molecular weight, all SSB proteins were applied to SDS-PAGE analysis in a 12.5% polyacrylamide gel containing 0.1% SDS under reducing conditions. The molecular weights of ssob, tth, and bl21 were 20, 29.8, and 19.8 kDa, respectively , which iTo confirm that SSB proteins bind DNA, PCR products (537 or 146 bp) were heated at 100°C for 10 min and cooled on ice rapidly to create ssDNA, Non-denaturing gel electrophoresis (3.5–5–12%) was used to confirm SSB protein-binding activity. The relative dsDNA was used as a binding specificity control. All SSB proteins displayed ssDNA binding characteristics. Due to ssob is a strongly alkaline protein, it’s isoelectric point = 8.464, with positive charge at pH 8.3 running buffer moving to anode, so we could not view the protein bands together with other SSBs. While, when ssob bound to the 537-bp ssDNA, the complex migrated around the 1048 kDa which suggested ssob could specifically bind to ssDNA and the isoelectric point was changed when ssob bound to ssDNA . For tthE. coli. To confirm their heat-resistance, the four proteins were heated at 95°C for1, 2, 3, 4, 5, 120, 240, and 600 min, respectively, and their binding activity was assessed by native PAGE analysis. As reported by Perales et al., tth could not withstand heat treatment for more than 5 min E. coli could withstand heat treatment for more than 9 h.SSB proteins derived from thermophilic bacteria are thought to be heat resistant, as opposed to SSB proteins from SSB proteins are known to enhance target DNA synthesis and reduce PCR by-products in vitro transcribed HCV RNA was used to interact with the four SSB proteins. The binding effect was visualised by 1.2% TAE agarose gel electrophoresis. Unexpectedly, when four SSB proteins associated with non-denatured RNA, the mobility was clearly slower than that of RNA alone (nude RNA), which suggested that SSB proteins bound ssRNA  was transfected into 1×106 Huh7 cells with or without 10 or 20 ng SSB proteins, Bull serum albumin (BSA) was used as a random protein control. After a 72 h incubation, viral RNA transduction and expression was analysed using an immunocellularchemistry assay. A well-verified chronic HCV–infected patient serum was used as the primary antibody to probe HCV proteins expressed by transfected HCV RNA. After conjugation with FITC-labelled goat anti-human secondary antibody, the numbers of positive cells and mean fluorescence intensity were determined using FACS. As shown in Since SSB proteins could bind and protect viral RNA, we evaluated their influence on viral RNA metabolism. To evaluate whether the SSB proteins could enhance the viral protein expression, the mean fluorescence intensity of HCV protein positive cells were calculated. As shown in To confirm that SSB proteins were transfected into host cells along with the viral RNA, cells transfected with 500 ng HCV RNA and SSB proteins were analysed using the immunocellularchemistry assay 24 h after transfection. SSB proteins could be detected by the primary anti-His antibody followed by a Alexa Fluor 568-labelled secondary antibody in cells transfected with SSB proteins, which suggested that SSB proteins bound viral RNA and co-entered host cells with the transfection complex .in vitro–transcribed viral RNA. In vitro–transcribed HCV RNA acted as mRNA for viral protein translation in transfected host cells The electrostatic, hydrogen-bonding and stacking between SSB proteins and nucleoside bases, sugars and phosphates of ssDNA was believed to form the basis for ssDNA binding and specificity Thermococcus kodakarensis KOD1 is a novel thermophilic bacterium The functional unit of bl21 was reported to be tetrameric E. coli (NCBI LOCUS: 1EYG_D), which suggested that although TTH is a thermophilic bacterium, its SSB shares structural features with that of E. coli. kod is similar to Sulfolobus solfataricus, which suggested that these proteins from both thermophilic bacteria are relatively similar. Protein derived from non-extremophiles showed moderate heat-resistance. Interestingly, protein derived from thermophilic bacteria was not always heat-resistant.The four SSB proteins were divided into two groups based on primary sequence analysis. tth and bl21 were similar to that of 3. Thus, we performed a series of experiments to explore the characteristics of ssDNA and ssDNA-binding patterns by AgNO3 staining. In this report, we used Coomassie brilliant blue-stained gels to represent a series of experiments since the binding activity could be reflected by mobility changes of the protein ssDNA complex. Regarding the effects on PCR specificity and enhancement of reverse transcriptions, SSB proteins were mixed with DNA polymerases and reverse transcriptases in vitro. We found that the SSB proteins did not interact with the DNA polymerase or reverse transcriptase (data not shown). Thus, the effects of SSB proteins on PCR specificity and enhancement of reverse transcriptions is likely accomplished through other mechanisms, and not by a direct interaction with the DNA polymerase and reverse transcriptase.Although ssRNA could be stained with ethidium bromide in a TAE gel, visualisation of the ssDNA protein complex was difficult. Ethidium bromide could not efficiently stain the ssDNA, but ssDNA was stained red by AgNOE. coli SSB was found to increase the processivity of DNA unwinding by the HCV helicase, SSB stabilizes the helicase at the unwinding junction and prevents its dissociation In summary, we present here a systematic functional analysis of a novel SSB protein derived from KOD1 and three reported SSB proteins. We found that all SSB proteins bound to ssDNA and viral RNA. Also, ssob and bl21 resisted heat-treatment for more than 4 h, and kod could withstand 95°C for at least 10 h. When SSB proteins bound to RNA, they both protected the RNA against nuclease attack and accelerated viral RNA processes in the host cell. Our results may provide a basis for future studies of the novel functions of SSB proteins.


G. parvulum species complex were established from seven different sites in the Faroe Islands, Sweden, Germany, Mexico and Korea, and were studied in detail. Eight morphodemes were identified which corresponded to the descriptions of the cosmopolitan taxon G. parvulum (Kützing) Kützing sensu lato: its nominate variety (var. parvulum), G. parvulum var. exilissimum Grunow and G. parvulum f. saprophilum Lange-Bertalot & Reichardt, G. [parvulum var.] lagenula Kützing plus four unidentifiable morphodemes. The concatenated analysis of the sequences of the markers 18SV4, rbcL, and ITS as well as morphological data resulted in a separation of four taxa based on their biogeography in Mexico, Korea, central Continental Europe and Northern Atlantic Europe. Mantel tests showed a significant correlation between molecular and geographical distances. The diagnoses of two taxa, G. parvulum sensu stricto, and G. lagenula, were emended, G. saprophilum elevated to species rank and epitypes designated. One species was newly described.Diatom cultures of the  Gomphonema parvulum belongs, have been discussed by Lange-Bertalot Diatoms are eukaryotic unicellular microalgae present in all types of water bodies and therefore often considered to be cosmopolitan and even ubiquitous. With their cell sizes ranging mainly between 10 µm and 200 µm, they seem to fit the hypothesis that microorganisms smaller than 1 mm have a cosmopolitan distribution in which the environment – here the ecology of the water – determines the occurrence of a taxon Molecular methods are playing an increasingly important role in the discovery and delimitation of diatom species and are revealing a hidden diversity, i.e. many taxa contain cryptic species, resulting in the description of many more species Gomphonema parvulum (Kützing) Kützing is a slender diatom of about 25 µm length and 6 µm width. It has long been recognized as a cosmopolitan, ubiquitous, and morphologically highly variable taxon for which many varieties have been described www.algaebase.org) includes 28 taxon names with 23 varieties and 4 forms of which 12 are currently accepted taxonomically. Gomphonema parvulum s.l. has been reported worldwide as an abundant benthic species occurring over a wide range of freshwater environmental conditions, including a few saline lakes and brackish waters. In addition, cells from the Gomphonema parvulum group seem to respond sensitively to water-quality changes. Some (infra-specific) taxa in this species complex, therefore, were identified as indicators of environmental conditions in both ecological and paleoecological studies Gomphonema parvulum group from different ends of the Eurasian holarctic region can be differentiated by their morphological and molecular data, and if these in turn are different to taxa from the neotropics. Besides possible biogeographical distribution patterns, given the tangled taxonomy of this group mentioned above, it is necessary to clarify if morphological identification is supported by molecular data, and to assess potential cryptic species, as well as infraspecific morphological variation. These questions are tested with a number of morphometric features and three molecular markers on 21 cultures from 7 sites from the Faroe Islands, Sweden, Germany, Mexico and Korea. The recently-published paper and its available molecular and morphological data for several Gomphonema parvulum strains The aim of this study is to determine whether taxa of the Gomphonema parvulum taxa were established by micropipette isolation, for details see Romero & Jahn Freshwater samples were collected from Northern Germany, Southern Sweden, Faroe Islands, Korea, and Mexico between 2004 and 2005 , Fig. 1.The harvested cultures were transferred to 1.5 ml tubes. DNA was isolated using either Dynal® DynaBeads  or Qiagen® Dneasy Plant Mini Kit  following the respective product instructions. DNA concentrations were checked using gel electrophoresis (1.5% agarose gel) and Nanodrop® . DNA samples were stored at −20°C until further use. Long-term storage of samples will take place at −80°C within the DNA bank network rbcL locus was amplified in two overlapping parts using two different primer pairs; Diat-rbcL-F (5′-AAGTGACCGTKACGAATCTGG-3′) and Diat-rbcL-iR (5′-AAGAAWCGYTCTCTCCAACG-3′) as well as Diat-rbcL-iF (5′-AAGGHTTAAAAGGTGGTTTAGA-3′) and Diat-rbcL-R (5′-RTARAAACCTTTAATCA-3′). These primers were developed by Birgit Gemeinholzer at the BGBM. The polymerase chain reaction (PCR) for the V4 region was conducted following Zimmermann et al. 2O, 2.5 µl 10× buffer S, 1.5 µl MgCl2, 5 µl pecGOLD dNTPs (each 1.25 mM), 2.5 µl Betaine , 2 µl of each primer (20 pm/µl), 0.15 µl HotStarTaq® polymerase (Qiagen), and 1 µl DNA sample. Our ITS PCR regime followed Amato et al. rcbL the PCR mix (25 µl) consisted of 12.9 µl HPLC H2O, 2.5 µl 10× buffer S, 1.5 µl MgCl2, 2.5 µl pecGOLD dNTPs (each 1.25 mM), 2.5 µl Betaine , 1 µl of each primer (20 pm/µl), 0.15 µl HotStarTaq® polymerase (Qiagen), and 1 µl DNA sample. The PCR regime for rbcL initial denaturation 2 min at 94°C, 40 cycles of denaturation 1 min at 94°C, annealing 45 s at 52.8°C (rbcL-F/rbcL-iR) resp. 38.6°C (rcbL-iF/rbcL-R) and elongation 1 min 30 s at 72°C, followed by a final elongation of 10 min at 72°C.The samples were all amplified with the primer pair M13F-D512for 18S/M13F-D978rev 18S PCR products were visualised in a 1.5% agarose gel and cleaned with MSB Spin PCRapace®  following standard procedure. DNA content was measured using Nanodrop (PeqLab Biotechnology). The samples were normalised to a total DNA content >200 ng using the spectrophotometer Nanodrop® ND1000 (PeqLab Biotechnology).The Sanger sequencing was conducted by Starseq® . As sequencing primers M13 tails were used for the V4 region, following Ivanova et al. The sequences were edited in ChromasPro  aligned using ClustalW 2O2. The hydrogen peroxide was removed by repeated centrifugation and washing events using distilled water. Cleaned subsamples were dispersed on cover glasses, dried, and embedded in the diatom resin Naphrax® for further microscopic observation. Photomicrographs of the cleaned valves were taken, using a Zeiss Axioscope-microscope with Differential Interference Contrast (DIC) and an AXIOAM MRc camera. The morphology of cleaned frustules was examined and photographed using both LM and scanning electron microscopy (SEM Philips 515).Diatom frustules were cleaned in the laboratory at 80°C in HIdentification of the cleaned valves was based on Krammer & Lange-Bertalot Valve length, width, density of striae and density and number of areolae of at least 10 valves per strain were measured using ImageJ 1.37v software . The valrbcL individual datasets  in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS. Uncorrected p-distances were calculated for each marker individually as well as a concatenated data set using PAUP 4.0b10 A matrix of direct line geographic distances (km) and genetic distances Gomphonema parvulum: lanceolate or linear-lanceolate heteropolar valves with more or less subrostrate or rostrate heads. The central area is asymmetrical with a single long stria on one side terminating in a distinct stigma, and a single short stria on the other side. Striae are almost perpendicular to the raphe. Such characteristics, associated with the length (17–29 µm), breadth (5–8 µm), length/breadth ratio (3–5) and 12–21striae in 10 µm, allowed us to group all studied specimens into Gomphonema parvulum s.l.The morphometric data  and morpparvulum sensu stricto , exilissimum (D12_022), lagenula , saprophilum . The remaining four morphodemes, as yet unnamed, did not correspond to named taxa. These morphodemes were .In LM observations , four ofÜbergangsformen) between the outlines. Some of the proposed differentiating micro-morphological features are present within the same strain such as raphe fissures  is the phylogenetically most informative dataset. The splits supported by the combined dataset are the same as in the individual subsets: however not all subsets support all splits. The splits that are unambiguously supported by the combined dataset following SAMS are indicated by arrows in rbcL gene  are strong (>75) for the splits (a), (b), (c) and (e) . BL postbcL gene .rbcL analysis show it is sister to another clade from Mayotte (Clade 1 is composed of strains from the Faroe Islands (Gomphonema exilissimum (Grunow) Lange-Bertalot & Reichardt 1996 in Lange-Bertalot & Metzeltin rbcL, other markers too much data missing) and only very little  and southern Sweden  (rbcL (Gomphonema parvulum var. parvulum f. saprophilumD20_027) ; there aD20_027) /C1 and o7) (rbcL /C3 and a7) , Another strain from Mayotte (TCC500), being identical with TCC429 in ITS and rbcL  from the other strains  is restricted in our study to the Faroe Islands as can be seen in exilissimum is found in only one culture on the Island Vagar (D12_022) on the Faroe Islands  is found only in the northern central Continental European clade  is observed only within the Mexican clade  Kützing var. parvulum  Lange-Bertalot & Reichardt 1996 Heterotypic synonyms: Lectotype designated here from the syntypes designated by Krammer & Lange-Bertalot Type locality: Falaise, France.Epitype (designated here): B 40 0040914 (strain D16_045); see Type locality of the epitype (designated here): Faroe Islands, Viöoy, Waterfall below church, Lat 62.360335°, Lon −6.542833° ±20 m, 04.08.2004, leg. J. Bansemer.Emended species description:This species is highly variable in its valve shape: lanceolate, linear-lanceolate or oval heteropolar valves with more or less subrostrate or rostrate head. The central area is asymmetrical with a single long stria on one side terminating in a distinct stigma, and a single short stria (sometimes two) on the other side. Striae slightly radiate or parallel, not distinctly punctuate. Length 22–29 µm, breadth 5–7.5 µm, striae 12–20 in 10 µm.Gomphonema saprophilum  Abarca, R. Jahn, J. Zimmermann & Enke comb. nov. : fig.76: 11 in Krammer & Lange-Bertalot 1991 5-Werten um 20 mg O2/L (15.1.1975).Type locality: Kommunale Kläranlage der Stadt Frankfurt a.M.; “voll” gereinigtes Abwasser mit einer Restbelastung von BSBEpitype (designated here): B 40 0040919 (strain D36_003), see Epitype locality: Germany, Berlin, Tiergarten, Landwehrkanal, Lat 52.511°, Lon 13.339°, ±20 m, 11.06.2005, leg. W.-H. Kusber.G. parvulum based mostly on the valve-shape; the taxon G. saprophilum has a rather rhomboidal shape with slightly rostrate ends, the head pole and the valve are on average also wider than those of G. parvulum  Frenguelly 1923.Homotypic synonym: Lectotype designated by Krammer & Lange-Bertalot Type locality: Trinidad, Tobago.Epitype (designated here): B 40 0040918 (strain D33_024), see Epitype locality (designated here): Mexico, Ixtlán de los Hervores, 06.12.2004, leg. N. Abarca.Emended species description:G. parvulum based on the shape of the head pole, which is consistently more rostrate to capitate. This taxon is differentiated from G. parvulum by its rostrate to capitate valve shape with strongly protruded valve ends and areolae divided by struts in the alveolus. This taxon has a broadly lanceolate shape with markedly rostrate to slightly subcapitate polar ends : B 40 0040917 (strain D23_012); see G. parvulum based on the shape. Frustule in girdle view narrow rectangular, hardly perceptible cuneate. Lanceolate to linear-lanceolate, almost naviculoid with only moderate rostrate head pole in comparison to the foot-pole . Also for the central Continental European Gomphonema strains there was a positive correlation . No association between geographic and genetic distance was found for the strains within the Faroe Islands .The Mantel test revealed a positive correlation between genetic and geographic distances separating the G. parvulum s.l. has long been recognized as a highly variable species complex with broad ecological tolerances and many varieties. Many references suggest there is great richness of morphologies that seem to inter-grade with each other and form a morphologically diverse but single taxon G. parvulum is due to the varying conditions of the environment in which these organisms are found, and synonymized many varieties under the single taxon G. parvulum. In recent years, studies on the taxonomy of this group have resulted in the splitting off of many new varieties and forms, and old names synonymized in the past have been restored in order to accommodate some of the morphological variations G. parvulum s.l. is based on morphological criteria alone. But this current taxonomic concept is only partly supported by our study on the morphological variability of strains  between 11 strains (see details in results). We have chosen D16_045 as the epitype of Gomphonema parvulum var. parvulum. In this main group though, we found one strain with apparently typical specimens of G. exilissimum (D12_022) with characters hardly separable using the light microscope  in oligosaprobic and mesosaprobic waters independent of their trophic situation. The wide variability of G. parvulum Kützing has also been recorded as producing a morphological oddity, the Janus cell In our study, we found Clade 1  to be mocroscope . But in parvulum ; interesG. parvulum-group  which do not show up in the main um-group  show mucum-group . Neverthum-group , none ofGomphonema parvulum are hard to unravel since it has been used as a collective name for the entire species complex for two centuries; being characteristic of organically polluted waters, but also found in samples from a wide range of water qualities sensu stricto is really ubiquitous and cosmopolitan or if it is restricted to specific environments or geographies.Reported occurrences for G. saprophilum form a well-supported clade. Although the three cultures were sampled from three different sites, they are morphologically and molecularly almost homogeneous. This clade has a combined distance of 1.0–1.5%  and Portugal , named clade B and B′ respectively in the corresponding paper is clade /C7. But Interestingly, this clade, the combined data of our findings (our Clade 4) plus the data of Kermarrec et al G. parvulum complex contradict the hypothesis of cosmopolitanism of microorganisms G. parvulum sensu stricto, G. saprophilum, G. lagenula, and G. narodoense, which are on average only 25 µm long, are not distributed randomly but hint at a biogeographical or phylogeographical pattern ; S1/B1 18SV4: MP strict consensus tree, LB, PB and PP given, black arrows indicate SAMS support, clades named according to S1/B2 ITS: MP strict consensus tree, LB, PB and PP given, black arrows indicate SAMS support, clades named according to S1/B3 rbcL: MP strict consensus tree, LB, PB and PP given, black arrows indicate SAMS support, clades named according to S1/B4 18SV4 with Genbank sequences: Bootstrapped (10 000 replicates) MP strict consensus tree, PB given, black arrows indicate SAMS support, clades named according to S1/B5 18SV4 with Genbank sequences: Bootstrapped (10 000 replicates) RAxML best scoring ML tree, LB given, black arrow indicate SAMS support,clades named according to S1/B6 18SV4 with Genbank sequences: Majority rule consensus tree from MrBayes, PP given, black arrows indicate SAMS support, clades named according to S1/B7 ITS with Genbank: MP strict consensus tree, LB, PB and PP given, black arrows indicate SAMS support, clades named according to S1/B8 rbcL with Genbank: MP strict consensus tree, LB, PB and PP given, black arrows indicate SAMS support, clades named according to S1/C Uncorrected P-Distances; S1/C1 18SV4; S1/C2 ITS; S1/C3 rbcL; S1/C4 18SV, ITS and rbcL combined; S1/C5 18SV, ITS and rbcL combined, compared to Genbank sequences; S1/C6 Clade 2: Individual p-Distances for 18SV, ITS and rbcL compared to Genbank sequences; S1/C7 Clade 3: Individual p-Distances for 18SV, ITS and rbcL compared to Genbank sequences; S1/C8 Clade 4: Individual p-Distances for 18SV, ITS and rbcL compared to Genbank sequences.(PDF)Click here for additional data file.