Using molecular simulation, FtsZ conformational changes were observed in both (S)- and (R)-rhodomyrtone binding states, compared with the three natural states of FtsZ . Calculations of free binding energy showed a higher affinity of FtsZ to (S)-rhodomyrtone  than the GDP substrate  while less affinity was observed in the case of (R)-rhodomyrtone . In vitro experiments further revealed that rhodomyrtone reduced FtsZ polymerization by 36% and inhibited GTPase activity by up to 45%. However, the compound had no effect on FtsZ localization in Bacillus subtilis at inhibitory concentrations and cells also did not elongate after treatment. Higher concentrations of rhodomyrtone did affect localization of FtsZ and also affected localization of its membrane anchor proteins FtsA and SepF, showing that the compound did not specifically inhibit FtsZ but rather impaired multiple divisome proteins. Furthermore, a number of cells adopted a bean-like shape suggesting that rhodomyrtone possibly possesses further targets involved in cell envelope synthesis and/or maintenance.Rhodomyrtone, a natural antimicrobial compound, displays potent activity against many Gram-positive pathogenic bacteria, comparable to last-defence antibiotics including vancomycin and daptomycin. Our previous studies pointed towards effects of rhodomyrtone on the bacterial membrane and cell wall. In addition, a recent molecular docking study suggested that the compound could competitively bind to the main bacterial cell division protein FtsZ. In this study, we applied a computational approach ( Rhodomyrtus tomentosa leaves, displays potent activity against many Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA)  . The act strains . Thus, rin silico screening for potential rhodomyrtone targets suggested that the compound could competitively bind to the main bacterial cell division protein FtsZ  and GTPase activity assays, we found that rhodomyrtone affected the function of purified FtsZ. Fluorescence light microscopy finally gave insights into the effect of rhodomyrtone on FtsZ in live bacteria.Staphylococcus aureus, (PDB entry 3VOA) as a molecular template. First, co-crystallized GDP in the structure was removed to obtain a ligand-free FtsZ starting structure. GDP, GTP, (S)-rhodomyrtone, and (R)-rhodomyrtone were docked to the ligand-free FtsZ protein in a molecular docking approach. GDP and GTP bound to FtsZ mimicked an in vivo state in S. aureus  and chloride (Cl−) ions were added, yielding 150 mM of KCl solution. The system was energy-minimized for 2,000 steps using the steepest descent algorithm to remove improper van der Waals contacts, and continued with a 500 ps canonical (NVT) ensemble at a temperature of 310 K (37 °C) using a time step of 1 fs. Harmonic potential was applied in the NVT simulation for positional restraint of the protein and ligand, using force constants of 200, 100, 50, 25, and 10 kcal mol−1 Å−2 in each 100 ps, respectively. After 500 ps, the restraint on the protein and ligand components was released and the system was switched into an isobaric-isothermal (NPT) simulation at a constant pressure of 1 atm, and 310 K with a time step of 2 fs for 150 ns. In the NVT simulation, the temperature was controlled by a Langevin thermostat  can be computed from energetic differences as follows  approach  was chos follows : \documeGLR, GL, and GR represent the free energy components of the ligand–receptor complex, ligand, and receptor respectively. The free energy of each state was derived from molecular mechanics energy, broken down into: Ebond, Eel, and EvdW are the MM energy values from the bonding terms , electrostatic, and van der Waals interactions, whereas Enpl and Epol are the nonpolar and polar contributions due to solvent solvation energy  implemented in an AMBER12 package. The total binding free energy of the ligands (rhodomyrtone and GDP) as well as their energy contribution determined and were used to assess the binding affinity of the ligand to FtsZ.Rhodomyrtus tomentosa by extraction with 95% ethanol as described by our group (Rhodomyrtone was isolated from leaves of ur group . Purity ur group . PurifieEscherichia coli BL21 (DE3) cells were freshly transformed with the plasmids pCXZ and pBS58 for co-expression of untagged B. subtilis FtsZ with E. coli FtsQAZ, the latter helping E. coli to survive stress induced by overexpression of the B. subtilis protein (600nm of 0.4 and induced with 1 mM IPTG (Sigma-Aldrich) for 4 h. Cultures were quickly cooled on slush ice, harvested by centrifugation, and washed once in 50 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA (Sigma-Aldrich). The dry cell pellet was flash frozen in liquid nitrogen and stored at −80 °C until further use. The pellet was dissolved in 60 mL 50 mM Tris pH 8.0, 50 mM KCl, 1 mM EDTA, 10 mM MgCl2, 1 mg/mL DNase (Sigma-Aldrich), and 1 complete mini protease inhibitor tablet (Roche). The cells were disrupted by French Press and cell debris was removed by centrifugation . The supernatant was subjected to ammonium sulfate precipitation as follows. 26.4 mL of a saturated ammonium sulfate solution were added drop by drop under continuous stirring at 4 °C, followed by further stirring for 20 min. Precipitated proteins were removed by centrifugation  and the FtsZ-containing supernatant was subjected to a second precipitation step by adding 9.5 mL of saturated ammonium sulfate solution as described above. After 10 min of stirring, FtsZ was spun down  and the pellet was dissolved in 45 mL 50 mM MES-KOH pH 6.5, 5 mM MgCl2 (buffer A), followed by ion exchange chromatography. The sample was loaded onto a 5 mL HiTrap Q HP column  equilibrated with 3 column volumes of buffer A. The column was washed with buffer A until reaching a stable baseline, followed by washing with 5% buffer B . After reaching a stable baseline again, FtsZ was eluted in a gradient up to 50% buffer B over 5 column volumes. FtsZ-containing fractions were pooled and concentrated with 10 kDa molecular weight cutoff filters (Amicon), if necessary. Glycerol was added to a final concentration of 10% prior to flash freezing. Samples were stored as single-use aliquots until further use.Calcium-competent  protein . Colonie2 and 1 mM GTP and monitored by observing light scattering over 2 min in a cuvette chamber at 37 °C. Measurements were performed with a PTI fluorometer operated under the control of FeliX32 software. Both the excitation and emission wavelengths were set at 350 nm and a slit width of 4 nm was used.FtsZ polymerization was monitored by 90° light scattering. FtsZ (10 µM) was added in a Tris buffer  in the absence (1% DMSO as a control) or presence of 10 µM rhodomyrtone, 20 µM rhodomyrtone, or 20 mM 3-methoxy-benzamide , respectively. FtsZ assembly was started by addition of 5 mM MgClTo visualise FtsZ filaments, 5 µl of sample were withdrawn immediately after starting the reaction and diluted 1:5 in the same buffer additionally containing 10% polyethylen glycol (Sigma-Aldrich) as a crowding agent. FtsZ filaments were visualised after 2 min of incubation at 30 °C using a Nikon Eclipse Ti microscope equipped with a CFI Plan Apochromat DM 100× oil objective, an Intensilight HG 130 W lamp, a C11440-22CU Hamamatsu ORCA camera, and NIS-Elements software, version 4.20.01.2 and 1 mM GTP. After 5 min of incubation, HClO4 (10% v/v) was added to quench the reaction. 100 µl of each sample was transferred to a 96-well plate, mixed and incubated with 40 µl of reaction agent for 2 min. Released inorganic phosphates were monitored by measuring the absorbance at 600 nm. The amount of released phosphate was calculated using a phosphate standard curve prepared with KH2PO4.The GTPase activity of FtsZ was analyzed using a malachite green/ammonium molybdate assay as described by B. subtilis 874 (gfp-sepF were grown overnight in LB broth at 30 °C in the presence of 50 µg/mL spectinomycin (Sigma-Aldrich). The overnight culture was diluted 1:100 into LB containing 0.5% xylose (FtsZ), 0.15% xylose (SepF), or 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (FtsA) to induce expression of GFP or YFP fusion proteins, and incubated at 30 °C until an OD600nm of 0.3. The cultures were then treated with 1xMIC , 2xMIC , and 4xMIC  of rhodomyrtone, 4xMIC  of 3-MBA, 100 µM CCCP (Sigma Aldrich), and 1% DMSO as a negative control. At various time pionts after treatment, samples were placed onto 1% agarose slides and images were taken using a Nikon Eclipse Ti microscope as specified above.To investigate the effect of the compound on Z-ring formation, FtsA, and MinD localization in living cells, ilis 874  expressiilis 874  expressiilis 874  expressiB. subtilis 168 , 2xMIC , and 4xMIC  of rhodomyrtone, 1xMIC , 2xMIC , and 4xMIC  of 3-MBA, and 1% DMSO as a negative control. After 1 h, 2 h, and 4 h of incubation, samples were placed onto 1% agarose slides and imaged with an Olympus BX60 microscope equipped with a Photometrics CoolSNAP fx digital camera. Images were analyzed with Image J.ilis 168  overnighS. aureus - and (S)-enantiomeric forms , with th. aureus  (PDB cod. aureus . (R)-rhoTo elucidate conformational changes in each region of the simulated proteins, time-averaged structures were created and compared with the starting crystal conformation . None ofπ–π stacking -rhodomyrtone than the GDP substrate. Interestingly, less affinity was observed in the case of (R)-rhodomyrtone.To estimate effective binding energy, energetic analysis was performed using the Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) method as summarized in stacking . The totTo observe motion in each FtsZ region, root-mean-square fluctuations (rmsf) were computed from trajectories of the last 50 ns simulations. Rmsf  indicatein vitro and in vivo. To this end, we used B. subtilis, a Gram-positive model organism for the study of cell division -rhodomyrtone and (R)-rhodomyrtone, reduced FtsZ assembly in a concentration-dependent manner by maximally 36%. Meanwhile, 3-MBA, a stabilizer of FtsZ polymers - and (R)-enantiomers, is not optimally efficient in inhibiting FtsZ. A similar phenomenon was observed with citronellal -citronellal caused disruption of animal and plant microtubules, while (−)-citronellal did not. Similarly, the (R)-enantiomer of N-benzyl-3-sulfonamidopyrrolidine was effective in inhibiting polymerization of E. coli FtsZ, whereas (S)-enantiomer did not have any effect -enantiomer with the nucleotide biding pocket. However, FtsZ is not the main in vivo target of the compound, which interferes with the localization of several divisome proteins and also seems to have an impact on the synthesis and/or maintenance of the cell envelope.We show that rhodomyrtone does inhibit FtsZ 10.7717/peerj.2962/supp-1Figure S1Black for ligand-free FtsZ, blue for GTP-FtsZ, green for GDP-FtsZ, red for (S)-rhodomyrtone-FtsZ, and yellow for (R)-rhodomyrtone. The regions with the most significant changes are labeled.Click here for additional data file.10.7717/peerj.2962/supp-2Data S1Click here for additional data file.10.7717/peerj.2962/supp-3Data S2Click here for additional data file.10.7717/peerj.2962/supp-4Data S3Click here for additional data file.


C1Q deficiency was identified as causative risk for systemic lupus erythematosus (SLE), there are limited and inconsistent reports regarding the common polymorphisms of C1Q genes in SLE susceptibility. Furthermore, there are no reports concerning polymorphisms of C1S, C1R, and C1RL and whether they confer susceptibility to SLE. We therefore evaluated 22 SNPs across six C1-complex genes in two independent case-control cohorts, and identified four novel SNPs that confer protection from SLE. The four SNPs are all located in C1Q. Particularly, the variant rs653286 displayed an independent reduced risk on SLE susceptibility  and anti-dsDNA antibodies . By bioinformatics analysis, SNPs rs653286 and rs291985 displayed striking cis-eQTL effects on C1Q genes expression. Individuals homozygous for the ‘protective’ allele at four SNPs had significantly higher levels of serum C1q . To our knowledge, this is the first study to demonstrate that only C1Q polymorphisms are associated with SLE. The C1Q SNP rs653286 confers an independent protective effect on SLE susceptibility and affects transcript abundance.Although rare variant  Systemic lupus erythematosus (SLE) is a chronic and systemic autoimmune disease, clinically characterized by heterogeneous manifestations and production of multiple autoantibodies. The exact etiology of SLE is unknown, but it is generally acknowledged that the disease evolves a complex interplay between multiple environmental and genetic factors.C1QA, C1QB, and C1QC. The genes are arranged tandemly in order of A-C-B on a 24-kb stretch of DNA on human chromosome 1p361. Human C1S and C1R are closely located at a distance of approximately 9.5 kb on chromosome 12p13. Both C1S (10.5 kb) and C1R (11 kb) contain 12 exons, and are highly homologous in domain structure and function3. Furthermore, the complement C1r-like protein (C1RL) is a recently discovered homologue of C1R, located 2 kb centromeric to C1R4.Complement cascade molecules are the key component in innate immune activation and play an important role in SLE pathogenesis. The C1-complex, composed of three molecules C1q, C1r, and C1s, is the first component in activation of classical complement pathway. The C1q molecule is composed of three similar but different types of chains, namely A-, B-, and C-chains, which are encoded by three distinct genes, i.e. C1Q and C1S deficiencies were identified as causative genetic risks for SLE , such deficiencies are very rare. To date fewer than 90 individuals with C1 deficiencies have been reported worldwide: approximately 70 cases with C1Q deficiency and 19 cases with C1S deficiency6. There are limited and inconsistent reports regarding the common polymorphisms in C1Q cluster in relation to SLE susceptibility12. One publication evaluated the association of C1Q polymorphisms with SLE in Han population, using a candidate gene approach and focusing only on C1QA10. There are no reports on genetic association of C1S, C1R, and C1RL polymorphisms and susceptibility to SLE. We therefore undertook the current study to investigate the possible genetic association(s) between the six C1genes  and SLE susceptibility or clinical/serologic features. We report that C1Q polymorphisms, but not polymorphisms in C1S and C1R, are protective against SLE susceptibility and affect C1Q transcript abundance.Although C1QA, C1QC, C1QB, C1S, C1R, and C1RL loci and additional 4 SNPs known to be associated with SLE or related symptoms9 . In control groups, the allele frequencies of SNPs were similar to the data from HapMap CHB . A suggestive reduced risk effect was also found for C1QB rs291985 . Notably, the four SNPs are all resided in C1Q genes. No potential association was observed for other polymorphisms outside C1Q in SLE susceptibility  between the selected SNPs resided in C1-complex and SLE susceptibility in the discovery cohort. As shown in Tables To confirm the possible associations that we observed in the discovery cohort, the top 4 SNPs were replicated in an independent case-control cohort. Joint analysis was then performed by combining results from discovery and replication cohorts.p = 0.045, q = 0.090; rs291985: OR 0.75, P = 0.019, q = 0.076, respectively). A trend toward a protective effect was also observed for rs680123 and rs682658, although this negative association did not reach statistical significance.As shown in Tables combined = 0.86, P = 0.024, q = 0.070; rs291985: ORcombined = 0.87, P = 0.035, q = 0.070, respectively) and genotype levels  than the variants rs680123 and rs682658 did   and genotypic (dominant model: P = 2.16 × 10−3) levels. Subsequent additions of rs291985, rs682658 and rs680123 did not show significant association with SLE susceptibility.To test the independence of the four SNPs, a stepwise  logistic regression analysis was performed in the combined cohort. As shown in Table P = 0.024). However, no association was observed for other SLE manifestations (Table To investigate whether the independent protective SNP rs653286 predispose to any particular disease manifestation(s), we assessed the association(s) between SNP rs653286 and clinical/serologic features in a case-only cohort. Following stratification, we found SNP rs653286 was significantly associated with anti-dsDNA antibodies  effects on expression of C1QB , C1QA , and C1QC   binding sites or chromatin interactive regions, showing LD-proxies with rSNPs or overlapping with rCNVs, regulating TSS (transcription start site) or transcriptional enhancers, and/or association with mRNA abundance, etc. Fig. . By sear99) Fig. .Figure 12 = 0.96/0.97 in controls/cases, respectively), we defined individuals homozygous for minor alleles of the four SNPs as “m/m” genotype and individuals homozygous for the major alleles as “M/M” genotype, respectively. As shown in Fig. in silico functional annotations, individuals homozygous for the ‘protective’ allele “m/m” at all four SNPs had significantly higher levels of serum C1q expression, compared to individuals homozygous for the major allele “M/M”  or heterozygous “M/m”.Supported by the functional annotations, we next sought to determine whether the ‘protective’ alleles at the four SNPs had any impact on serum C1q levels. As SNPs rs680123–rs682658 and rs653286-rs291985 are almost in complete LD  of the six C1-complex genes, i.e. C1QA, C1QC, C1QB, C1S, C1R, and a novel human complement-related gene C1RL, with SLE susceptibility or its clinical/serologic manifestations. We demonstrate that only C1Q but not C1R, C1RL, and C1S polymorphisms, are negatively associated with SLE. The C1QC SNP, rs653286, has an independent protective effect on SLE susceptibility. Bioinformatics annotations and genotype-dependent expression analyses further support a potential regulatory role for each of the four C1Q variants.Although the rare deficiencies of C1Q polymorphisms to SLE susceptibility or sub-phenotypes12. Among those, Martens et al.8 reported that C1QB rs631090 was associated with SLE susceptibility, C1QA rs292001 and C1QC rs294183 was associated with more severe disease in 103 patients with SLE and their first degree relatives of a Caucasian cohort. Though the three SNPs were not tested in present study, they were captured by our candidate SNPs rs629409 , rs680123 , and rs653286 , respectively  underlying this genetic association remain unclear. However, by C1Q polymorphisms confer susceptibility to SLE. The four novel variants all confer reduced risk against SLE and affect its transcript abundance. The C1QC SNP, rs653286, has an independent protective effect on SLE susceptibility. Future association studies in other populations will be required to further confirm our findings. Future functional characterization of these polymorphisms is warranted to fully understand their contributions to SLE pathogenesis.In summary, to best of our knowledge, this is the first report to investigate the genetic association between the six C1-complex genes and susceptibility to SLE. Our data indicate that only 20. Autoantibodies, including anti-nuclear antibodies (ANA), anti-double stranded DNA (dsDNA) antibodies, anti-SSA/SSB antibodies, anti-Smith (Sm) antibodies, anti-cardiolipin antibodies (ACA), and anti-histone antibodies (AHA) were routinely measured and are detailed in the Supplementary Materials and Methods.A two-stage case-control study was conducted. Two independent cohorts, including 384 SLE patients and 384 healthy controls (discovery cohort), as well as 507 SLE patients and 645 controls (replication cohort), were enrolled in the study. Patients with SLE satisfied 1982 revised American College of Rheumatology classification criteria for a diagnosis of SLEThe SLE patients in the discovery and replication cohorts were recruited from the Department of Rheumatology at Peking University People’s Hospital and People’s Hospital of Xinjiang Province, respectively. The healthy controls were recruited from Health Care Centers of People’s Hospital. All patients and healthy controls were Han Chinese. The baseline demographic characteristics of patients and healthy controls were summarized in Table The study was approved by Medical Ethics Committee in Peking University People’s Hospital and written informed consent was obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations.C1QA, C1QC, C1QB, C1S, C1R, and C1RL loci and an additional 4 SNPs known to be associated with SLE or related symptoms  ≥5% using Haploview v4.2, according to the HapMap phase III Chinese Han Beijing (CHB) panel , and performed at Beijing SequeSci Co., Ltd. Briefly, DNA from study subjects was randomly assigned to the 96 well plates, and genotyping was performed blind to the status of all the samples. Genotyping was repeated in 5% of the samples for validation and quality control. The genotyping error rate was less than 0.1%. Individuals with genotyping success rates less than 90% were excluded from the analyses. Individual SNP markers with more than 10% missing genotypes were also removed from the analyses.A total of 342 genotyped SLE patients were quantified for serum C1q when sera were available. All the serum samples were derived from Peking University People’s Hospital. Human C1q Platinum ELISA kit (BMS2099) was used to measure serum C1q, according to the manufacturer’s instructions . In brief, an anti-human C1q monoclonal antibody is coated and adsorbed onto microwells. The absorbance was measured at 450 nm. The concentration of C1q in a serum sample was determined by matching its absorbance with the corresponding C1q concentration in the standard curve. Samples were run in duplicate and analyzed individually. All cases had genotyping data.http://regulome.stanford.edu/) and rVarBase (http://rv.psych.ac.cn/). RegulomeDB is a database that annotates SNPs with known and predicted regulatory features in the non-coding regions of the human genome. Known and predicted regulatory DNA elements include sites of DNAase hypersensitivity, binding sites for transcription factors, and promoter regions that have been biochemically characterized to regulate transcription. The sources of these data include public datasets from GEO, the ENCODE project, and published literature21. rVarBase annotates a variant’s regulatory features in following aspects: chromatin state of the region surrounding the variant, regulatory elements overlapped with the variant, and the variant’s potential target genes. rVarBase also provides additional extended annotations for variants, including: LD-proxies of known SNPs, SNP/CNV that are overlapped with or co-localize with the queried variant, and traits (disease and expression quantitative trait) associated with the variant22. The blood eQTL data were derived from Blood eQTL browser (http://genenetwork.nl/bloodeqtlbrowser/)24.The variant’s potential regulatory features were annotated according to RegulomeDB (www.cc-ims.net/RevMan) and carried out with the Mantel-Haenszel method. A significant I2 statistic  indicated heterogeneity for ORs across studies. The fixed-effects model was applied in current heterogeneity analyses.The HWE test was performed for each polymorphism, using Pearson’s goodness-of-fit chi-square test. The heterogeneity among study cohorts was evaluated using Review Manager 5 software . The Mann Whitney test was applied for the analysis of serum C1q levels between two genotypic groups. Statistical analyses were conducted using SPSS 13.0 software . The false discovery rate  was applied for the multiple testing corrections. P-value ≤ 0.05 was considered nominally statistical significant. A q-value ≤ 0.10 was considered statistically significant.The frequencies of alleles and genotypes were compared between cases and controls, and were assessed using Pearson chi-square test and logistic regression adjusting for age and sex, respectively. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate the relative risk for developing SLE or clinical/serologic manifestations. A stepwise  logistic regression analysis was performed to test the independence of the identified SNPs. LD and haplotype were calculated using online software SHEsis (Supplementary Information


Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1–3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1–3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1–3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1–3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1–3 loss-of-function is a mechanism of antifolate drug resistance.The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form  Trypanosoma brucei: T. brucei gambiense, an anthroponotic disease mainly affecting humans with a minor animal reservoir; and T. brucei rhodesiense, a zoonotic disease affecting mainly animals with humans inadvertently infected. Both forms of HAT clinically evolve in two stages, an early hemolymphatic stage and a second meningoencephalitic stage, with a fatality rate close to 100% if left untreated (T. brucei rhodesiense HAT is an acute disease that rapidly progresses to death within 6 months (T. brucei gambiense HAT has a more chronic course with an average duration of 3 years (T. brucei gambiense HAT (which accounts for 98% of HAT cases) by 2020 ,ntreated , 3. T. b6 months , whereas 3 years . Althoug 3 years , 8. In 2T. brucei gambiense HAT. Essential metabolic pathways of the parasite are being exploited to identify potential drug targets, and folate metabolism is one such pathway. T. brucei, like all trypanosomatids, is auxotrophic for folates  the plasma concentration in humans (e.g. DHFR-TS).In addition, the classical antifolates methotrexate (MTX) and raltitrexed (RTX) were found to have nanomolar potency against an serum , 14. Utin humans . The impT. brucei (p-aminobenzoylglutamate (pABA-Glu) in facilitating the uptake of non-classical antifolates and therefore their efficacy.We used a genome-wide RNA interference screening approach to identify potential antifolate resistance mechanisms, an approach that has been used successfully for other antitrypanosomal compounds , 20. Her. brucei , 14. We A and C). Under tetracycline induction, each T. brucei cell produces dsRNA from the integrated RNAi target fragment, and knockdown has the potential to confer a selective advantage under drug pressure. RIT-seq was subsequently used to generate a readout from the population that tolerated this regime. PCR products representing RNAi target fragments, derived from the RNAi screen, were separated on an agarose gel, and low-throughput RIT-seq of multiple fragments implicated the putative folate transporter genes  in both the MTX and RTX screens. These folate transporters share 96% nucleotide identity and are arranged in tandem, interrupted by an unrelated gene (Tb927.8.3640) on chromosome 8.A trypanosome RNAi library was exposed to a typically lethal dose of either the DHFR inhibitor, MTX, or the TS inhibitor, RTX , A and CFT1–3 and a truncated orphan folate transporter gene (Tb11.v5.0766) are the strongest “hits” in both the MTX and RTX screens, accounting for ∼90% of all mapped reads (B and D). We also note that 95% of the “MTX” and “RTX” reads mapped against a reference genome that included the T. brucei 427 telomeric variant surface glycoprotein expression site regions; in addition to encoding variant surface glycoproteins, these regions contain ESAG10 genes that also encode putative folate transporters related to FT1–3. A gene encoding a mitochondrial carrier protein  was also common to both screens , was specific to the RTX screen . A plot of the y axis intercepts (time 0) versus folate concentration is linear (B), suggesting nonspecific binding of folate to the trypanosome outer surface. The rate of uptake of folate was temperature-dependent (C) with a 12-fold higher rate at 23 °C (0.24 ± 0.03 pmol s−1 (108 cells)−1) compared with 4 °C (0.02 ± 0.01 pmol s−1 (108 cells)−1). Uptake rates for folate obeyed simple Michaelis-Menten kinetics (D) yielding a mK of 2.04 ± 0.53 μm and a Vmax of 0.20 ± 0.04 pmol s−1 (108 cells)−1. In the same way, MTX uptake was found to be linear over 150 s up to 20 μm (A) at 23 °C with evidence of nonspecific binding (B) and temperature dependence −1) at 23 and 4 °C, respectively). MTX uptake also obeyed simple Michaelis-Menten kinetics  yielding a mK of 18.4 ± 2.2 μm and Vmax of 0.76 ± 0.08 pmol s−1 (108 cells)−1.As the RIT-seq screens implicated folate transporters in antifolate drug resistance, transport studies of folate and MTX were undertaken to determine basic kinetic parameters. Folate uptake was found to be linear over 150 s and directly proportional to concentration up to 10 μat 23 °C A. A plots linear B, suggesependent C with a kinetics D yieldinto 20 μm A at 23 ° binding B and temA). RNA analysis using qRT-PCR confirmed FT1–3 mRNA knockdown after 3 days of induction (p = 0.02) and after 6 days of induction (p = 0.001) (B).To validate the RIT-seq analysis, we performed knockdown of FT1–3 in independent RNAi strains. Tetracycline-induced knockdown showed no significant change in growth over a 6-day period when compared with wild-type or un-induced controls A. RNA an= 0.001) B.Vmax of folate uptake at 3 and 6 days after induction (C). After 3 days, the Vmax of induced cells was 10- and 9-fold lower than wild-type and un-induced cells, respectively. Similarly by day 6, the Vmax of induced cells was 4-fold lower than wild-type and un-induced cells. No changes in mK values were detected, with mK values in the range 2.56–3.47 μm obtained for all three cell lines after 3 or 6 days of induction.RNAi knockdown of FT1–3 greatly reduced nduction C. After 50 determinations showed a >30-fold decrease in susceptibility for MTX and RTX after 3 and 6 days of FT1–3 knockdown, respectively . In contrast, uptake of folate was not inhibited by compounds lacking a pABA-Glu-like moiety, namely trimetrexate, nolatrexed, trimethoprim, pyrimethamine, or biopterin . Consistent with this observation, pABA-Glu itself substantially reduced folate uptake . MTX uptake was inhibited by the same classical antifolates or folic acid but not by the non-classical antifolates . As for folate, pABA-Glu also substantially reduced MTX uptake . Biopterin did not inhibit folate or MTX uptake, in agreement with the failure of the RIT-seq screens to identify the putative pteridine transporter genes on chromosomes 1 and 10.To further assess the interactions between trypanosomes and antifolates, we determined whether “folate-like molecules” had an effect on folate or MTX uptake. Folate uptake was inhibited by MTX, RTX, pemetrexed, and 5-methyl-THF A. In con50 values of folate analogues against uptake of either radiolabeled folate or MTX. MTX inhibited folate uptake with an IC50 of 3.4 ± 0.4 μm, and folic acid inhibited uptake of MTX with an IC50 of 1.9 ± 0.4 μm  (A). The resulting Lineweaver-Burk plot suggested either a competitive or mixed inhibition pattern. An F-test in GraFit established that these data fitted best to competitive inhibition, yielding a iK value for MTX of 0.48 ± 0.07 μm. A similar analysis revealed that folate is a competitive inhibitor of MTX transport with a iK value of 0.94 ± 0.21 μm (B). If two substrates compete for the same target or enzyme, in this case the folate transporter, 50 for any concentration of ligand. Applying experimental values to these equations yielded an IC50 for MTX of 2.2 μm against folate uptake  . To detectively) A. The re 0.21 μm B. If twoe uptake  and an Ie uptake . These vmentally , thus giT. brucei. The folate biopterin transporter (FBT) family is a novel class of membrane proteins and superfamily of transporters that were first characterized in Leishmania (T. brucei), malaria (Toxoplasma (T. brucei reference genome encodes eight FBT genes as follows: in tritrypdb.org (version 9.0), four of are annotated as putative folate transporters, with FT1–3 , clustered together on chromosome 8 and a truncated orphan gene on chromosome 11 (Tb11.v5.0766); and four annotated as putative pteridine transporters, with three  clustered together on chromosome 1 and an orphan gene on chromosome 10 (Tb927.10.9080). The putative folate transporters are predicted by the TMHMM server version 2.0 to contain 12 transmembrane helices consistent with other folate transporters. The orphan FT gene (Tb11.v5.0766) shares 97–100% sequence identity to FT1–3 (position 736–1899 of ORF), but this gene encodes a protein that lacks the first 245 amino acid residues that are predicted to encode the first four transmembrane segments and therefore is likely to be non-functional. There are also seven expression site-associated genes (ESAG10) in the T. brucei S427 strain that share 84–91% amino acid identity with FT1–3. ESAG10s are located in about half of the specialized telomeric transcription units (Bloodstream Expression Sites (BES)) devoted to expression of variant surface glycoproteins   . In contate/MTX) . From outhionine .RNAi knockdown of FT1–3 dramatically reduced folate transport, and competition assays with antifolates indicated that folate and classical antifolates share a common transport mechanism via folate transporters, which is not the case for non-classical antifolates. Indeed, classical antifolates bear a close structural resemblance to folate and use the same mechanism as folates for cell entry via specific transporters in human cells; in contrast, non-classical antifolates are lipophilic, do not contain a terminal glutamyl moiety, and enter cells by passive or facilitated diffusion –33. In o–C). Indeed the α- and γ-glutamate carboxyl groups of the classical antifolates are negatively charged at physiological pH and thus require carrier-mediated uptake for entry into cells  or a pABA-Glu-like moiety  compete for uptake into cells C. Indeedto cells . The addto cells , 36. Thisistance , 38 and sistance . A similishmania , 40 or mishmania  have beeT. brucei bloodstream form are quite similar to that found in Leishmania, where uptake is rapid and linear for 2–3 min in both parasites  and have a very high affinity for 5-methyl-THF, the principal form of folate in human serum  (mK of folate in T. brucei (2.1 μm). Likewise, T. brucei was shown to have a lower affinity for MTX uptake with a mK of 16 μm, as is the case for mammalian cells (range 2.3 to 26 μm) (The properties of folate transport in the arasites  The rate species . In contan serum . The Km  1–4 μm)  is compao 26 μm) .T. brucei  was a hit in both drug screens Mitochondrial carrier proteins are a group of structurally conserved proteins that regulate the transport of a variety of metabolic intermediates across the mitochondrial membrane; 24 mitochondrial carrier proteins have been identified in . brucei . Phyloge. brucei . One-car. brucei . Howeverrganelle . In addihondrial . Other fia major . NonetheN5,N10-methylene-THF in a two-step reaction  and [3H]MTX (38.2 Ci mmol−1) were purchased from Hartmann Analytical. DHFR-TS inhibitors were sourced as follows: MTX, trimethoprim and pyrimethamine from Sigma; nolatrexed, pemetrexed, and RTX from Sequoia Research Products; and trimetrexate from Tocris Bioscience. Melarsoprol was obtained from Rhone-Poulenc. Other chemicals and reagents used in this study were of the highest grade and purity available.Folic acid, biopterin, and T. brucei bloodstream-form “single marker” S427 (2 in HMI9-T medium. HMI9-T medium contains high concentrations of thymidine (∼160 μm) and folate (∼9 μm), the latter principally from Iscove's modified Dulbecco's medium and 10% Serum Plus components  .For routine culture, er” S427  and 2T1 er” S427  strains mponents . A mediuia (TBM) . Residua−1) and maintained under blasticidin (1 μg ml−1) and phleomycin (1 μg ml−1) selection at a minimum of 2.5 × 107 cells in 150 ml of media. Following induction for 24 h, 15 nm MTX or 10 nm RTX (∼3–4× EC50 for parasites cultured in TBM for 48 h) was added. Cultures were split and supplemented with fresh drug as required. At day 4, concentrations of MTX and RTX were increased to 30 nm. DNA was extracted from drug-resistant cells on day 10. RNAi target fragments were then amplified by PCR using the LIB2f and LIB2r primers. For low throughput identification of fragments, PCR products were separated on an agarose gel, and individual bands were excised and sequenced using the Lib2f and Lib2r primers  with Bowtie 2  in sterile water. 2T1 strains, containing a tetracycline repressor (−1) and hygromycin (2.5 μg ml−1) selection. Puromycin susceptibility (1 μg ml−1) was tested for full integration of the construct, and expression of stem-loop RNAi was induced with 1 μg ml−1 tetracycline.PCR primers were designed using RNAit  to generfor RNAi , 62. Conepressor , were elepressor  followinepressor . TransfoT. brucei RNA was isolated using an RNeasy purification kit (Qiagen) and cDNA-synthesized using a high capacity RNA-to-cDNA kit (Applied Biosystems). PCR primers were designed using the Premierbiosoft's Beacon Designer 6 to amplify a 117-bp region common to all three FT genes but distinct from the region targeted by RNAi . qRT-PCRs consisted of 1 μl (40 ng) of cDNA, 10 μl of Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies), 1 μl (500 nm) each of the forward and reverse primers, and 0.3 μl (30 nm) of reference dye and nuclease-free PCR grade-treated water. PCR was performed using an Agilent Mx3005P machine with the following cycling conditions: 95 °C for 3 min; 40 cycles of 95 °C for 20 s; then 60 °C for 20 s. The reference gene TERT  was used to provide a baseline of transcription levels for normalization of the data . Relative quantification in the tetracycline-induced FT1–3 knockdown cell line was normalized to the un-induced cell line using the ΔΔCt method, and a Student's unpaired t test was used to show significance on four experimental replicates. Statistical analyses were performed using Excel and GraFit 5.013 (Erithacus software).50 of antifolates in FT knockdown lines were determined after 3 and 6 days of induction with tetracycline in TBM. Serial doubling dilutions of antifolates (5–50 mm stocks prepared in DMSO) were prepared in 96-well microtiter plates in 100 μl of TBM, and trypanosomes (resuspended in the same medium) were added in 100 μl to give a final density of 2.5 × 103 cells ml−1 in 96-well plates. All wells, including controls, contained a final volume of 0.5% DMSO. Cultures were incubated for 72 h at 37 °C, 5% CO2 before cell density was determined using a resazurin-based assay  with a 3-parameter fit from triplicate readings. A weighted mean from three independent experiments was calculated.ECed assay . EC50 vaT. brucei cells were grown in TBM for 72 h at an initial seeding density of 5 × 103 cells ml−1. Cells were harvested by centrifugation , washed, and resuspended in transport buffer  . Microcentrifuge tubes were flash-frozen in liquid nitrogen, and the bottom of the tubes containing the cell pellets were cut off directly into scintillation vials. Pellets were solubilized in 1 m NaOH (150 μl) overnight and mixed with scintillation fluid (2 ml), and radioactivity was measured using a liquid scintillation counter (Beckman Coulter). pH 7.3)  at a deng et al. . Uptake 3H]folic acid (0.04 μm) was determined in the presence of varying concentrations of unlabeled folate. Similarly, uptake of [3H]MTX (0.07 μm) was determined in the presence of varying concentrations of unlabeled MTX. Uptake of both folate and MTX was determined at regular time intervals  at 23 °C and fitted using robust non-linear fitting to the linear equation y = mx + c. The nonspecific binding of radiolabeled ligand to trypanosomes at 4 °C was determined in a similar fashion. To calculate mK, results were fitted by non-linear regression to the Michaelis-Menten equation. A weighted mean from three independent experiments was calculated.Uptake of [m competing inhibitor with the standard assay mixture modified to contain folate (2.0 μm) or MTX (13 μm) at approximately [S] = mK. Inhibitor concentrations giving 50% inhibition (IC50) were then determined over a range of concentrations across 7–8-point serial doubling dilutions. Dose-response curves were fitted by non-linear regression to a two-parameter IC50 equation using GraFit 5.0 (Erithacus software). To determine the mode of inhibition of MTX on folate uptake and folate on MTX uptake, the linear rate of uptake was measured as before using four different substrate concentrations and three different inhibitor concentrations. The resulting data were plotted as a Lineweaver-Burk transformation, and the graphs were inspected to establish the most likely mode of inhibition (intersection on y axis). An F test confirmed the mode of inhibition as competitive rather than mixed. The entire data set was then globally fitted to the competitive Linear rates of uptake were measured as before in the presence of 100 μ50 for methotrexate ([M] = IC50M) is defined in The ICSubstituting 50 for folate, defined in vF/vM = 0.5,Likewise, the ICS. D. conducted the experiments, analyzed the results, and wrote the first draft of the paper. N. S. and H. B. O. designed the folate-deficient medium and assisted in the design of the transport experiments. R. J. W. and D. H. designed the RIT-seq experiments and helped analyze the data. A. H. F. conceived the project and analyzed all data. All authors contributed to writing the paper.


GluR2) expression in cortical neurons and enhances neuronal vulnerability to glutamate. However, the mechanism of this TBT-induced GluR2 decrease remains unknown. Therefore, we examined the effects of TBT on the activity of transcription factors that control GluR2 expression. Exposure of primary cortical neurons to 20 nM TBT for 3 h to 9 days resulted in a decrease in GluR2 mRNA expression. Moreover, TBT inhibited the DNA binding activity of nuclear respiratory factor-1 (NRF-1), a transcription factor that positively regulates the GluR2. This result indicates that TBT inhibits the activity of NRF-1 and subsequently decreases GluR2 expression. In addition, 20 nM TBT decreased the expression of genes such as cytochrome c, cytochrome c oxidase (COX) 4, and COX 6c, which are downstream of NRF-1. Our results suggest that NRF-1 inhibition is an important molecular action of the neurotoxicity induced by low-concentration TBT.Tributyltin (TBT), which has been widely used as an antifouling agent in paints, is a common environmental pollutant. Although the toxicity of high-dose TBT has been extensively reported, the effects of low concentrations of TBT are relatively less well studied. We have previously reported that low-concentration TBT decreases α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor subunit 2 ( Organotin compounds, especially tributyltin chloride (TBT), have been widely used as biocides, agricultural fungicides, and antifouling agents in paints. Because of its widespread use as an antifouling agent on the hulls of ships, toxic levels of TBT have been released into water and are polluting marine environments. Although the use of TBT in paints is currently internationally regulated, the issue of secondary exposure from contaminated fish, solids, and water remains, owing to its high bioaccumulation and long-term environmental persistence ,2. Indeecis retinoic acid (a well-known RXR ligand) induces gastropod imposex ). After being left for 15 min on ice, 0.5% NP-40 was added, and the cell suspension was vortexed and centrifuged at 10,000× g for 10 min. The supernatant was then transferred to a microtube (cytoplasmic extract). The cell pellet was subsequently resuspended in hypertonic buffer . After sonication, the mixture was centrifuged at 12,000× g for 10 min. The supernatant was then transferred to a microtube (nuclear extract).After TBT exposure, the cells were washed with PBS buffer and collected in a microtube. After centrifugation, the cell pellet was resuspended in hypotonic buffer  was added to the culture dish, and the cells were incubated for 20 min at 4 °C. Then, to stop the biotinylation reaction, PBS containing 100 mM glycine was added to the culture dish, and the cells were incubated for 30 min at 4 °C. The cells were then lysed in TNE buffer, and transferred to a microtube. The mixture was rotated at 4 °C and centrifuged at 15,000 rpm for 15 min. The supernatant was incubated with PBS containing avidin-agarose beads  at 4 °C overnight. After centrifugation at 15,000 rpm for 15 min, the pellet was resuspended in sample buffer, and denatured at 70 °C for 30 min to cleave the membrane protein–biotin bonds. The sample was then analyzed by western blotting.After the cells were washed with PBS buffer, 0.5 mg/mL (PBS (+)) EZ-LinkWestern blotting was performed as previously described  with somTotal RNA was extracted using an SV Total RNA Isolation System  according to the manufacture’s protocol. The total RNA was then reverse-transcribed at 42 °C for 1 h, and 75 °C for 15 min, in a reaction mixture containing 5× reaction buffer, RNase inhibitor, reverse transcriptase, and dNTP. Quantitative RT-PCR was performed using a QuantiTect SYBR Green PCR Kit . The thermocycling conditions were as follows: 95 °C for 3 min, then 50 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used for the PCR are shown in Detailed protocols for immunocytochemistry are included in Detailed protocols for ChIP assay are included in GluR2 promoter . The oligonucleotides were then separated from the unincorporated nucleotides by chromatography using a Micro Spin G-25 column . The purified probes were incubated with 5 μg of nuclear extract, 5× binding buffer (Promega), and gel loading buffer. For competition, oligonucleotides were incubated with the nuclear extract before adding the oligonucleotides. The probe/nuclear extract mixture was loaded onto a 4% polyacrylamide gel, and run at 200 V for 60–90 min. The results were visualized by autoradiography.Oligonucleotide probes containing the NRF-1 binding site on the promoter  were annFull-length HA-tagged NRF-1 was generated using the primers summarized in l-glutamine, 2 mg/mL NaHCO3, 100 units/mL penicillin , 100 μg/mL streptomycin , and 4.5 g/L at 37 °C in a humidified 5% CO2 incubator. For transfection of pcDNA3.1 and pAcGFP-C1 plasmids, FuGENE HD (Promega) was used as recommended by the manufacturer. After transfection for 48 h, cells were exposed to 20 nM TBT and lysed in TNE buffer.HEK 293T cells were cultured in DMEM (Nissui) supplemented with 10% FBS, 0.58 mg/mL Detailed protocols for immunoprecipitation are included in ® Luminescent Cell Viability Assay (Promega), according to the manufacture’s protocol.After exposure to TBT, the intracellular ATP content was measured using a Cell Titer-Glot-test. A value of p < 0.05 was considered to be indicative of significance.All the experiments were performed at least three times and representative data are shown. Data are expressed as mean + S.E.M. Statistical evaluation of the data was performed with ANOVA followed by Turkey’s test or with Student’s


Here, we examined relationships between HIF-1α and VEGF expression and nocturnal IH in cutaneous melanoma (CM) tumor samples. Prospectively recruited patients with CM tumor samples were included and underwent overnight polygraphy. General clinical features, apnea–hypopnea index (AHI), desaturation index (DI4%), and CM characteristics were recorded. Histochemical assessments of VEGF and HIF-1α were performed, and the percentage of positive cells  was blindly tabulated for VEGF expression, and as 0, 0–5.9, 6.0–10.0, >10.0% for HIF-1α expression, respectively. Cases with HIF-1α expression >6% (high expression) were compared with those <6%, and VEGF expression >75% of cells was compared with those with <75%. 376 patients were included. High expression of VEGF and HIF-1α were seen in 88.8 and 4.2% of samples, respectively. High expression of VEGF was only associated with increasing age. However, high expression of HIF-1α was significantly associated with age, Breslow index, AHI, and DI4%. Logistic regression showed that DI4% [OR 1.03 (95% CI: 1.01–1.06)] and Breslow index [OR 1.28 (95% CI: 1.18–1.46)], but not AHI, remained independently associated with the presence of high HIF-1α expression. Thus, IH emerges as an independent risk factor for higher HIF-1α expression in CM tumors and is inferentially linked to worse clinical CM prognostic indicators.Epidemiological associations linking between obstructive sleep apnea and poorer solid malignant tumor outcomes have recently emerged. Putative pathways proposed to explain that these associations have included enhanced hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) cell expression in the tumor and altered immune functions  Cutaneous melanoma (CM) is a very aggressive type of skin cancer that is not only fraught with a high mortality rate, but is also a cancer type whose incidence has continued to increase over the last several decades . In an eSince intermittent hypoxia (IH)-induced alterations in tumor malignancy are driven, at least in part, by the transcriptional activity of hypoxia inducible factor-1α (HIF-1α) pathways –7, it isObstructive sleep apnea (OSA) is a highly prevalent disorder characterized by the presence of increased upper airway collapsibility during sleep that leads to either upper airway collapse or restricted airflow and consequent periodic hypoxia and hypercapnia events, usually terminated by arousal and restoration of airflow –24. The Based on aforementioned considerations, we hypothesized that semiquantitative assessments of HIF-1α and VEGF immunoreactivity in excised CM tumors may be associated with typical severity measures of OSA. Hence, the objective of the present study was to analyze potential associations between the expression of HIF-1α and VEGF and measures of respiratory disturbance during sleep in a large prospective series of patients suffering from CM.in situ,” respiratory failure, heart failure grades III–IV NYHA, pregnancy, or a prior diagnosis of SDB or CPAP treatment. The study was approved by the Ethical Committee of each participating center, and all patients signed the informed consent.We conducted a prospective, multicenter study in 29 Spanish University Hospitals. Consecutive patients diagnosed with CM by the dermatology or oncology departments were eligible for inclusion in this study. Patients were excluded if they had melanoma of an unknown primary cause, melanoma in mucosa or melanomas “2), and regression (presence vs. absence). Furthermore, the following clinical characteristics were also documented: tumor location , age at diagnosis, sex, body mass index , skin phototype, and stage at diagnosis  by the treating dermatologists and institutional pathologists. In addition, the staging of each tumor was confirmed by a multi-institutional tumor board that was unaware of the immunohistological findings described below. Other markers of melanoma aggressiveness, namely, the presence of ulceration and regression in biopsy tissue, histologic subtypes of melanoma, presence of metastasis, and the invasion of sentinel lymph nodes were also documented.All tumors were surgically resected and subjected to clinical staging. In addition to standard pathologic assessment in each center to establish the diagnosis of CM, all the specimens were re-evaluated in a melanoma reference center by an expert pathology panel whose members were blinded to any of the immunostaining findings. For each tumor, the following pathologic features were assessed by the panel: Breslow tumor thickness , ulceration (presence vs. absence), tumor mitotic rate  using the Leica Bond Polymer Refine Kit (Leica Biosystems). Melanoma samples were stained with a 1:2 dilution of anti-VEGF prediluted antibody  and with a 1:100 dilution of anti-HIF-1α antibody . The staining results were independently analyzed by two expert pathologists who were blinded to the staging and clinical features of the subjects. VEGF-specific staining was scored taking into account both the percentage of positive cells  and expression intensity  . Apnea was defined as complete cessation of oro-nasal flow for ≥10 s and was classified as either obstructive or central, based on the presence or absence of respiratory efforts. Hypopnea was defined as a 30–90% reduction in oro-nasal flow for ≥10 s followed by a ≥3% decrease in SpO2, while Tsat90 was defined as the percentage of recording time with SpO2 <90%. The recording time, desaturation indices at 4% and the mean baseline nocturnal, baseline daytime, and minimum nocturnal saturation were also recorded. Those tests in which the patients claimed to sleep less than 4 h, or in which there were less than 5 h of nocturnal recording, were repeated.All patients included in the study underwent a diagnostic sleep study by means of respiratory polygraphy, using a device validated against polysomnography (Embletta Gold in 37% of the patients and Alice PDX in 63% of patients) both validated against PSG, and following the Spanish Society of Pneumology and Thoracic Surgery Guidelines for OSA diagnosis and treatment. The time span between the initial appointment for suspicion of melanoma and the sleep study was always lower than 6 months. Every sleep study was manually scored by skilled staff. All the studies included continuous recording of the oro-nasal flow and pressure, respiratory movements, and oxyhemoglobin saturation  was used for all analyses. The data were expressed by using the mean and SD, or the median and the interquartile range for quantitative variables, according to whether or not they followed a normal distribution. The qualitative and dichotomic variables were expressed as the absolute value and the percentage with respect to the total. For analyses purposes, histochemical assessments of HIF-1α and VEGF expression variables were dichotomized: HIF-1α expression >6% (high expression) was compared with those samples with <6%, and VEGF expression >75% of cells was compared with those with <75%.t-test for independent means or the Mann–Whitney test, according to the distribution of the quantitative variables, while the chi-square test was used to compare the two percentages. The factors independently related to the presence of a high HIF-1α or VEGF expressions were determined by introducing into two multivariate logistical regression analyses those variables which, in the opinion of the researchers, could have clinical importance. Odds ratios with 95% confidence intervals were calculated for each molecule expression. Although a two-tailed p-value of <0.05 was initially considered as indicative of statistical significance, Bonferroni test was used to correct p-values for multiple comparisons as appropriate.The comparison between these two groups’ baseline variables was made with the Student’s 2, apnea–hypopnea index (AHI) of 14.9 (16.5) event/h, and DI4% of 10.2 (13.7) desaturations/h. Regarding the melanoma tumor, the mean Breslow index was 1.70 (2.5) mm, the most common anatomical site was the body (41.8%), and only four patients had distant metastasis at the time of the diagnosis.Of 476 eligible patients, 443 were prospectively recruited and provided informed consent to participate in this study and finally, 376 subjects had biopsy tissues available for analysis. The baseline clinical and tumor characteristics are depicted in Table The vast majority of melanoma samples presented high VEGF expression , and non-high HIF-1α expression . Only two samples (0.5%) presented no VEGF expression, and only 16 samples (4.2%) presented high HIF-1α expression, with only five patients presenting HIF-1α expression in more than 10% of cells. 6 (1.6%), 12 (3.4%), and 24 (6.4%) samples presented VEGF expression in less than 25%, between 25 and 50%, and between 51 and 75% of cells, respectively.Those patients with CM and high VEGF expression were most frequently females and showed significantly less mitotic index Table , higher p = 0.08) and nadir SpO2 values  and reflects the quartile-based categorical stratification approach undertaken here, as dictated by statistical considerations. Indeed, expansion of the number of staining intensity categories such as to offer more refined analyses would have required a much large cohort size to achieve adequate statistical power.Before we discuss the potential implications of our findings, several methodological points deserve mention. First, this is the first prospective study concurrently evaluating one type of cancer, namely CM, and the presence of sleep-disordered breathing. Indeed, most of the existing literature to date has relied on retrospective databases, or alternatively, due to cohort size limitations, has not investigated a specific type of malignancy , 35–41. via recruitment of HIF-1α signaling , Madrid, Spain; Manuel Sánchez de la Torre, Respiratory Department, Group of Translational Research in Respiratory Medicine, Hospital Universitari Arnau de Vilanova and Santa Maria, IRBLleida, Lleida, Catalonia, Spain, Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Esther de Eusebio, Dermatology Department, Hospital Universitario de Guadalajara, Spain; Pedro Landete, Pneumology Service, Hospital la Princesa, Madrid, Spain; Manuel Moragón Gordon, Dermatology Department, Hospital Universitario, San Juan de Alicante, Spain; Eva Arias, Respiratory Department, Hospital 12 de Octubre, Madrid, Spain; Fernando Masa, Respiratory Department, Hospital Universitario S. Pedro Alcántara, Cáceres, Spain, Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Carlos González Herrada, Dermatology Department, Hospital Universitario de Getafe, Madrid, Spain; Cristina Carrera, Dermatology Department, Hospital Clinic-IDIBAPS, Barcelona, Spain; Aida Muñoz Ferrer, Respiratory Department, Hospital Germans Trials y Pujol, Barcelona, Spain; Aram Boada, Dermatology Department, Hospital Germans Trials y Pujol, Barcelona, Spain; Ana Fortuna, Mercé Mayos, Respiratory Department, Hospital Santa Creu i Sant Pau, Barcelona, Spain; Jesús Gardeazabal García, Dermatology Department, Hospital Universitario Cruces, Bilbao, Spain.The study was approved by the human ethics committees at the various participating sites, and written informed consent was obtained from each participant in accordance with the Declaration of Helsinki. The following human subject committtes provided approval for the study: Central Ethics Committee: Comité ético de investigación biomédica of Hospital Politécnico y Universitario La Fe de Valencia. Protocol # Cod 2012/1048 . Comité ético de investigación clínica (CEIC) del Hospital Clinic de Barcelona. Protocol # 25042013. Comité ético de investigación clínica del Hospital General de Alicante. Agencia Valenciana de Salud. Protocol # 2013/030. Comité ético de investigación clínica del Hospital San Juan de Alicante. Agencia Valenciana de Salud. Código 12/304. Comité ético de investigación clínica y ensayos clínicos de Getafe. Protocol # A03-13. Comité ético de investigación clínica y ensayos clínicos de Guadalajara (SESCAM). Protocol # 19032013. Comitè d’Etica de la investigaciò del Hospital Germans Trials i Pujol. Protocol # PI13-074. Comitè d’Etica de la investigaciò del Hospital Santa Creu i Sant Pau. No protocol #. Comitè d’Etica de la investigaciò (CEIC) del Hospital Arnau de Vilanova de Lleida. Protocol # CEIC-1154. Comité ético de investigación clínica y ensayos clínicos de la Comunidad autónoma del País Vasco. Protocol # PI2014046. Informe del Comité local de ensatos clínicos. Valme. Protocol # 301012. Comité ético de investigación clínica y ensayos clínicos de Cáceres. No protocol #.IA, MM-G, FC-R, and DG participated in the conceptual framework of the project; MM-G., FC-R, ER-F, JR-P, EN, AM-C, LH-B, JR, EV, AS-T, JA-C, JM, AP-G, VC-N, IC-P, JC-P, TD-C, OM, and JD-A recruited patients and collected data and/or samples; MM-G, DG, IA, MM-G, FC-R, and RF interpreted data and drafted the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor is currently co-organizing a Research Topic with one of the authors IA and confirms the absence of any other collaboration.


The family of RecQ helicases is evolutionary conserved from bacteria to humans and play key roles in genome stability. The budding yeast RecQ helicase Sgs1 has been implicated in several key processes during the repair of DNA damage by homologous recombination as part of the STR complex (Sgs1-Top3-Rmi1). Limited information on how is Sgs1 recruited and regulated at sites of damage is available. Recently, we and others have uncover a direct link between the Smc5/6 complex and Sgs1. Most roles of Sgs1 during recombination, including DNA end resection, Holiday junction dissolution, and crossover suppression, are regulated through Mms21-dependent SUMOylation. Smc5/6 first acts as a recruiting platform for STR and then SUMOylates STR components to regulate their function. Importantly, the assembly of STR is totally independent of Smc5/6. Here, we provide a brief overview of STR regulation by Smc5/6.The online version of this article (doi:10.1007/s00294-016-0648-5) contains supplementary material, which is available to authorized users. DNA damage, originates from endogenous and exogenous sources, and is constantly challenging genome integrity. To overcome this threat, cells have evolved a complex network of intertwined DNA damage repair pathways. Failures in these lifeguard systems compromise genome integrity, which can lead to genome instability, cancer predisposition, and ageing. The RecQ helicases are highly conserved family of proteins from bacteria to humans with critical roles in the maintenance of genome stability over generations. Humans have five distinct RecQ members: BLM, WRN, RECQL1, RECQL4, and RECQL5, whereas only two RecQ-like helicases have been described in budding yeast, Sgs1, and Hrq1 [reviewed in , a genetic disorder characterized by a strong growth deficiency, reduced fertility, diabetes, and cancer predisposition  complex in humans and the Sgs1-Top3-Rmi1 (STR) complex in budding yeast are key players in various processes of DNA metabolism, such as replication, DNA repair, and telomere maintenance. These functions have been extensively reviewed in several journals  process, where it carries both pro- and anti-recombinogenic functions. First, BLM/Sgs1, together with DNA2 and EXO1, promotes DSB resection  and two subunits of the STR complex (Sgs1 and Top3) . Pull-downs of Rmi1 resulted in a strong co-immunoprecipitation of Top3-6HA Fig. a. This ills Fig. a. This rlls Fig. . Intereslls Fig. b. Next, lls Fig. . Intereslls Fig. c, d. TheBased on the fact that under DNA damage conditions, we observed a significant increase in the interaction between Sgs1 and the Smc5/6 complex Fig. e, which BLM/Sgs1 has many roles in the repair of DSBs and replication forks (Larsen and Hickson sgs1-3KR) show an accumulation of SCJs when exposed to MMS and show a higher proportion of crossovers and an impaired DNA end resection upon DSBs  supplemented with glucose at 2 % final concentration. To induce DNA damage, cells were treated with 0.033 % MMS for 2 h before collecting them.−8 M until >95 % had been arrested in G1. The release from G1 was conducted by washing cells twice with pre-warmed medium and resuspending them in medium containing 0.1 mg/ml pronase from Streptomyces griceus .To synchronize cells in G1, exponentially growing cultures were treated with α factor (Insight Biotechnologies) at a final concentration of 10Co-immunoprecipitation analyses were performed as previously described in Below is the link to the electronic supplementary material.


Tph2 knockout female mice. Sexually naive Tph2−/− female mice failed to recognize olfactory cues regarding sex, genetic relatedness, and social hierarchy despite exhibiting normal olfactory discrimination. However, reproduction-experienced Tph2−/− female mice recovered sexual olfactory preferences, as did sexually naive Tph2+/+ females. Meanwhile, both the estrogen receptor α and oxytocin receptor in the amygdala of reproduction-experienced Tph2−/− females presented upregulated expression at the mRNA level and an upward tendency at the protein level vs. sexually naive Tph2−/− females. Intracerebroventricular administration of a combination of estrogen receptor α and oxytocin receptor agonists, but not either agent alone, could restore the sexual olfactory preferences of sexually naive Tph2−/− female mice to some degree. We speculate that estrogen receptor α and oxytocin receptor activation in the amygdala after reproductive experiences restores sexual olfactory recognition in Tph2−/− female mice.The central 5-hydroxytryptamine system impairs sociosexual behaviors and olfaction preferences in sexually naive mice. However, it remains unknown whether reproductive experiences impart an effect on the sexual olfactory preferences of female mice lacking central serotonin. Here, we aimed at examining such effects and the underlying mechanisms using  In rodents, choice of mate relies heavily on olfactory signals, of which urine odor is one of the most important signals with which to communicate sex, social status, kinship, and individual identity , 2, 3. FSexual experiences are able to guide animals toward stimuli that are predictive of sexual reward and reproductive success , 10, 11.Tph2) is the initial and rate-limiting enzyme on the pathway from tryptophan to central 5-HT [Tph2 knockout mice generated by deleting exon 5 that encodes the tryptophan hydroxylase domain only have minute amounts of central 5-HT and show strong deficiencies, such as growth retardation, 50% lethality in the first 4 weeks after birth, loss of sexual preference evidenced by the paradigms of mating choice assay, genital odor preference assay, bedding preference assay, and lordosis assay, exaggerated aggression, and decreased anxiety [As one of the most important neurotransmitters in the brain, serotonin  has a widespread and profound effect on information processing, cognition, memory, stress adaption, anxiety, depression, aggression, sexual preference, and maternal care in rodents , 18, 19.ral 5-HT , 16. Tph anxiety , 18, 19.Tph2 knockout on the sexual olfactory preference. Furthermore, females are often the choosy sex that could discriminate amongst competitive males inmate choice for high fitness [Tph2 knockout female mice prefer female urine odor more than the male one? The right strain? The dominant male odor?Evidence for both the disinhibitory effect of reproductive experiences and the inhibitory effect of central 5-HT deficiency on sexual preferences of both male and female mice has been published , 14, 15. fitness . Then, rTph2 knockout mice, a sophisticated interplay among 5-HT, estrogens and oxytocin (OT) has been shown to affect sexual performance [Even though there are still no direct evidences of how ERα, ERβ, OT and OTR changed in formance , 23, 24.formance , 23. Furformance , 26. ERβformance .Tph2 knockout female mice prefer female urine odor more than the male one? The right strain? The dominant male odor? And, the role of ERα, ERβ, OT and OTR played in such processes?Here, we examined whether reproductive experiences could make the Tph2 line generated from C57BL/6 mice was a generous gift from the laboratory of Dr. Yi Rao . It was then maintained and genotyped in our laboratory, as previously described [The Twenty male and 24 female C57BL/6 mice at an age of 40 weeks were used as urine donors for sex. The estrous status of the female C57BL/6 mice was determined by microscopic examination of vaginal epithelium. An additional 20 male C57BL/6 mice and 20 male Balb/c mice at an age of 12 weeks were used as urine donors for genetic relatedness. Nine pairs of male C57BL/6 mice with a stable dominance–submission relationship were selected as urine donors for social hierarchy at the age of 17 weeks. The formation of a stable dominance–submission relationship was ensured by use of a dyadic encountering test in a neutral area between paired males over the course of 21 consecutive days at the age of 13 weeks. In brief, weight-matched male mice were paired and simultaneously placed into a clean mouse cage for 10 min of continuous recording beginning with initial aggressive behavior  .ad libitum.The plastic cage was 27 × 12 × 17 cm in size. The housing room was kept under a reversed 14L: 10D light/dark photoperiod (lights on at 7:00 pm), and the temperature was maintained at 23 ± 2 °C. Commercial standard rat/mouse pellet chow  and water were provided All of the mice experiments were reviewed and approved by the Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences . Adequate measures were taken to minimize discomfort for mice and to ensure that the entire experimental protocol complied with the Institutional Guidelines for Animal Use and Care at the Institute of Zoology, Chinese Academy of Sciences.Tph2+/+ female mice (aged 19–20 weeks) and 27 Tph2−/− female mice (aged 12 weeks) were separately brought into the home cage of Tph2+/+ male mice of the same age for sexual intercourse. When there was a visible sign of pregnancy, the male mice were removed from the cage. The gestation period showed no difference between Tph2+/+ and Tph2−/− females. Then, the female mice were allowed to experience parturition and lactation. In spite of being fertile and producing milk, Tph2−/− female mice present poor maternal care leading to a low survival rate of their pups, most of which die on Day 2 or 3 after birth. If the pups died during lactation, the corpses were immediately removed from the cage. If the pups were still alive, they were removed from the cage on the day following 21 days of lactation. Whether or not there were living pups, the female mice had to go all the way through to 21 days of lactation. Female mice experienced the processes including mating, pregnancy, parturition, and lactation were classified as reproduction-experienced (R-E) females. R-E female mice were housed singly for 1–2 weeks before the start of behavioral tests.Eight sexually naive As previously described , during Binary choice tests using the capillary method were carried out to explore urinary preference , 6. The Tph2 line, female mice used in these binary tests were taken from different generations. In Figs Tph2−/− female mice and sexually naive Tph2+/+ female mice was Generation 1 (at the age of 12–14 weeks). The batch of reproduction-experienced Tph2−/− female mice was Generation 2 (at the age of 20–21 weeks). The order of these binary tests was as follows: 1) male C57BL/6 mice urine vs. female C57BL/6 mice (estrous) urine; 2) dominant male C57BL/6 mice urine vs. subordinate male C57BL/6 mice urine; 3) male C57BL/6 mice urine vs. male Balb/c mice urine. Each test recipient female mouse was given 1 d of rest between each test for repeated use. In Tph2−/− female mice used for intracerebroventricular injection was Generation 3 (at the age of 24–36 weeks). Each of the mice was given approximately 1 week of rest before the next drug administration and the following binary test for repeated use.As it was difficult to maintain the Tph2−/− female mice  in order to investigate olfactory discrimination ability [Habituation–dishabituation tests were conducted via one capillary using sexually naive  ability , 29. In After all of the behavioral tests described above, the amygdala (approximately from −0.22 mm to −2.54 mm relative to the bregma) of all the female mice were dissected in a mouse brain matrix on ice . The tis® RT reagent Kit with gDNA Eraser  (Takara), following the manufacturer’s instructions. PCR reactions were performed using RealMasterMix (SYBR Green)  using the a Mx3005P quantitative PCR system . Thermal cycling conditions were: 95 °C for 2 min followed by 40 cycles of 95 °C for 20 s, 60 °C for 20 s, and 68 °C for 40 s. To exclude the interference of unspecific products, a melting curve analysis was conducted using high-resolution data collection during an incremental temperature change from 60 to 95 °C with a ramp rate of 0.2 °C /s. β-actin was chosen as a reference. Primer sequences were: β-actin, forward-TCCATCATGAAGTGTGACGT, reverse-GAGCAATGATCTTGATCTTCAT; ERα, forward-GCTGGCCTGACTCTGCA, reverse-TCTGGCTGGGCTCCTCT; ERβ, forward-TTGCTCCAGACCTCGTT, reverse-CATCTGTCACTGCGTTCA; OTR, forward-CGTCAATGCGCCCAAAG, reverse-CGAGCAGAGCAGCAGAGGAA. Data were calculated using the 2C-ΔΔT formula.Quantitative real-time PCR was performed, as previously described , 30. BriWestern blot analysis was carried out as previously described , 32. TheDimethyl sulfoxide  and artificial cerebrospinal fluid (ACSF) were selected as the solvent. The vehicle and the final concentration of each drug were as given below: ERα agonist-PPT , 38% DMSO/ACSF, 2.5 μg/μL; OTR agonist-WAY 267464 dihydrochloride , 35% DMSO/ACSF, 1.25 μg/μL.Tph2−/− female mice were anesthetized using sodium pentobarbital (80 mg/kg) by intraperitoneal injection and then placed in a stereotaxic frame  with blunt ear bars. One stainless-steel guide cannula  was implanted into the brain, targeting the lateral ventricle at the following coordinates (mm) anteroposterior (AP)/mediolateral (ML)/dorsoventral (DV): −0.58/−1.0/−2.0, respectively, relative to Bregma [Tph2−/− female mice was in the following order (the control group received an injection of the same amount of corresponding solvent): 1) ERα agonist plus OTR agonist in combination; 2) OTR agonist; 3) ERα agonist. During the course of this procedure, one sexually naive and two reproduction-experienced Tph2−/− female mice belonging to the drug group died.As described previously , anothero Bregma . We havet-test, while non-normally distributed data were examined using a Wilcoxon matched-pairs signed-rank test. For others analyses, independent-samples t-test was used for normally distributed data and a Mann-Whitney U test was used for the nonparametric test. All statistical analyses were conducted using SPSS 16.0 software  with the critical value of a = 0.05.The Kolmogorov–Smirnov test was used to determine parametric or nonparametric tests. For binary choice tests, normally distributed data were tested using a paired-samples Tph2−/− female mice were habituated to repeated exposure to the urine odor of male C57BL/6 mice .Nevertheless, sexually naive Tph2+/+ female mice, the Tph2−/− female mice with reproductive experiences preferred urine odor from male C57BL/6 mice to female C57BL/6 mice  expression (independent-samples  females .Tph2−/− females, the mRNA expression of ERα (independent-samples t-test, t = 3.348, df = 10, P = 0.007, t-test, t = 2.486, df = 10, P = 0.032, Tph2−/− females. Similarly, compared with sexually naive Tph2−/− females, the protein expression of OTR (independent-samples t-test, t = 2.252, df = 8, P = 0.054, marginal significance, Tph2−/− females. Moreover, there was a clear trend that the protein expression of ERα in the amygdala of reproduction-experienced Tph2−/− females was higher than in sexually naive Tph2−/− females (0.6374 ± 0.2385 vs. 1.4734 ± 0.4743, independent-samples t-test, t = 1.575, df = 8, P = 0.154, Tph2−/− females, ERβ gene expression in the amygdala of reproduction-experienced Tph2−/− females presented an upward tendency at the mRNA level (independent-samples t-test, t = 1.886, df = 9, P = 0.092, marginal significance, However, relative to sexually naive Tph2+/+ female mice, the mRNA expression of ERα (independent-samples t-test, t = 2.878, df = 10, P = 0.016, Tph2−/− females was lower, while the mRNA expression of ERβ also had a downward tendency (independent-samples t-test, t = 2.159, df = 9, P = 0.059, marginal significance, Tph2+/+ female mice, the expression of ERα (Mann-Whitney U test, Z = 2.402, N = 10, P = 0.016, U test, Z = 2.193, N = 10, P = 0.028, Tph2−/− females.Furthermore, as compared with sexually naive Tph2−/− females to choose the urine odor of C57BL/6 males over that of C57BL/6 females to some extent (Wilcoxon rank sum test, Z = 1.947, N = 7, P = 0.051, marginal significance, t = 0.778, df = 6, P = 0.466, Binary choice tests revealed that infusion of OTR and ERα agonists in combination could elicit sexually naive Tph2−/− female mice could not recognize urine odor related to sex, corroborated previous studies that deficiency in central 5-HT could bring the loss of sexual preference [Tph2−/− female mice could not recognize urine odor not only related to sex but also related to genetic relatedness and social hierarchy despite possessing normal olfactory discrimination extended previous observations [It is well known that 5-HT is closely related to central information processing, stress adaption, anxiety, depression, aggression, sexual preference, and maternal care in rodents , 35, 36.eference , 15. Addrvations , 15, 37.Tph2 knockout and may, thereby, have guided Tph2−/− female mice toward male urine odor.As evident from extensive literature in this area, sexual preference can be affected by previous sexual experiences in both early life and adulthood in many species of rodents, such as hamsters, lemmings, prairie voles, and mice , 41, 42.Tph2−/− female mice gained sexual olfactory preference, implying that some facilitative mechanisms may exist. The pathways through which estrogens and serotonin exert their effects appear to be linked, although each has an independent effect [Tph2−/− female mice were higher than in sexually naive Tph2−/− females at the mRNA level. Meanwhile, at the protein level, relative to the sexually naive Tph2−/− females, the expressions of ERα and OTR in the amygdala of reproduction-experienced Tph2−/− female mice also exhibited an upward tendency. Moreover, OTR and ERα agonists in combination, but not singly, had a tendency to rescue the sexual olfactory preference of sexually naive Tph2−/− female mice to some degree.Reproduction-experienced t effect , 23. A ft effect , 50. In t effect , 52. HerTph2−/− female mice together with the pharmacological effect of ERα and/or OTR agonist on sexual olfactory preference of Tph2−/− female mice, we speculate that ERα and OTR might act in a combined way to exert their effects. Such findings confirm and further expand on the view of Young that ERα and OTR in the amygdala are necessary for the regulation of social identification and recognition [Consequently, from the upregulated expression of ERα and OTR in the amygdala of reproduction-experienced ognition . Furtherognition . The preTph2 line, we only have a limited sample size for each batch and each test. This small sample size may have caused some results to present marginal significance or no statistical difference, even though the tendency was suggestive. Furthermore, the fact that the estrous cycles were not determined in any test for recipient female mice was really a limitation and might account for the high variability and lack of statistical significance for some outcomes. Also, different batches used in binary choice tests was another limitation and might be the most important reasons that the investigation time seem to vary considerably between reproduction-experienced Tph2−/− female mice in Figs However, as stated above, it should be pointed out that there were some weaknesses in the current study. As it is difficult to maintain the Tph2 knockout affected the ability of olfactory recognition rather than olfactory discrimination, which confirms and expand on previous findings. For the first time, reproductive experiences have been shown to mitigate the disruptive effect of Tph2 knockout on the sexual olfactory preference of female mice. Furthermore, ERα and OTR activation might constitute the neural substrates that are involved in the facilitative mechanisms.In conclusion, our results indicate that


Hordeum vulgare L.), including stable stand establishment, water and nutrients uptake, biotic resistance and abiotic stresses, and can influence yield and quality. However, the genetic mechanisms underlying seedling characteristics in barley are largely unknown and little research has been done. In the present work, 21 seedling-related characteristics are assessed in a barley double haploid (DH) population, grown under hydroponic conditions. Of them, leaf age (LAG), shoot height (SH), maximum root length (MRL), main root number (MRN) and seedling fresh weight (SFW) were investigated at the 13th, 20th, 27th, and 34th day after germination. The objectives were to identify quantitative trait loci (QTLs) underlying these seedling characteristics using a high-density linkage map and to reveal the QTL expression pattern by comparing the QTLs among four different seedling growth stages.Seedling characteristics play significant roles in the growth and development of barley , 3H (two regions) and 7H (three regions) involved 39 QTLs (55.71%), each simultaneously influenced more than one trait. Meanwhile, 9 co-localized genomic regions involving 22 QTLs for five seedling characteristics  at the 13th, 20th, 27th and 34th day-old seedling were common for two or more growth stages of seedling. QTL in the vicinity of Six QTL cluster regions associated with 16 seedling-related characteristics were observed on chromosome 2H, 3H and 7H. The majority of the 29 regions identified for five seedling characteristics were selectively expressed at different developmental stages. The genetic effects of 9 consecutive expression regions displayed different developmental influences at different developmental stages. These findings enhanced our understanding of a genetic basis underlying seedling characteristics in barley. Some QTLs detected here could be used for marker-assisted selection (MAS) in barley breeding.The online version of this article (10.1186/s12863-017-0562-y) contains supplementary material, which is available to authorized users. Hordeum vulgare L.) is not only widely used as human food and animal feed, but is also an ideal model of genetic research because it is early maturing, diploid, self- fertilizing and has a short growth period [As the earliest domesticated crop and the fourth most important cereal crop in the world, barley  but FILW and FOLW  were detected in 17 of the 21 seedling characteristics using ICIM mapping Table . Ten co-qLAG2–228) and SFW (qSFW2–202) explaining more than 20% of the phenotypic variation at more than one stage of seedling growth was identified on chromosome 2H. We analyzed the temporal-dynamics contribution of QTLs detected at any of the four stages to the proportion of explained phenotypic variation for five seedling characteristics  at four developmental stages identified 8 QTLs for LAG, 14 QTLs for SH, 8 QTLs for SFW, 8 QTLs for MRL and 4 QTLs for MRN Table ; Fig. 2.ics Fig. . The resqSLDW7–45. The major QTL qSLFW2–202 was detected for SLFW on chromosome 2H around 127/129 cM and co-located with QTLs for SFW at S1, S3 and S4 (qSFW2–202), MRN at S2 (qMRN2–199) and SLDW at S4 (qSLDW2–202). Another major QTL was identified on chromosome 2H around 137/139 cM and had effect on MRN at S3 and S4 (qMRL2–214), RFW at S4 (qRFW2–209) and RDW at S4 (qRDW2–214).We assessed four seedling characteristics  at S4 stage, and detected one QTL for SLFW, 2 QTLs for RFW, 3 QTLs for SLDW and 2 QTLs for RDW , a total of 21 QTLs on chromosome 2H (nine QTLs), chromosome 3H (three QTLs), chromosome 6H (one QTL) and on 7H (eight QTLs) were identified. The LOD value ranged from 3.17 to 27.07, and individual QTL accounted for 5.01%–77.78% of the phenotypic variation  to compare the genetic markers with those results present in this study, we found that some QTLs identified in our study are likely same to the QTLs reported in the past research. Such as, qSH3–44 for SH is likely the same to the QTL QHei.S42IL-3H reported by Hoffmann et al. [QSdw.S42.2H.c for SLDW on chromosome 2H was linked with the marker bPb-8143, and since the bPb-8143 is near the morphological marker Vrs1, as inferred from GrainGenes3.0 (http://wheat.pw.usda.gov/GG3/), we suggested that a co-localized QTLs for SLDW, SFW, SLFW, MRN, FILA, SELW, SELA and FOLA identified in our study is likely the same to the QTL QSdw.S42.2H.c. This QTL may be an important locus for controlling stem-and-leaf related traits in barley. The SNP marker 2HL_34260490 may be useful for marker-assisted selection (MAS) in barley breeding.In previous studies, QTLs conferring seedling height (SH) , 30, roon et al.  reportedqSH7–184) on chromosome 7H and QTL for RFW (qRFW2–90 and qRFW2–209) on chromosome 2H, are likely different from the QTL reported by Xu et al. [qMRL2–113 and qMRL3–117), QTL for RDW (qRDW2–130 and qRDW2–214) and QTL for MRN (qMRN3–120) are likely different from those QTLs reported previously [qMRL7–27 (S1 and S2), located on chromosome 7H, is different from the QRl.S42IL-7H.a and QRl.S42IL-7H.b, as reported by Hoffmann et al. [qMRL2–214 (S3 and S4) for MRL, mapped together with qRDW2–214 (S4) for RDW and qRFW2–209 (S4) for RFW on chromosome 2H, are different from those QTLs on chromosome 2H, as reported previously [In addition, some new QTLs were also detected in this study. QTL for SH  has a pleiotropic effect on multiple traits [Vrs1 locus had a pleiotropic effect on thousand grain weight, spike length, plant height, spikelet number on main spike and grain number per plant. Furthermore, some malting quality traits, such as grain-protein-percentage, diastatic power and soluble/total protein ratio, were also coincident with the Vrs1 locus reported by Marquez-Cedillo et al. [Vrs1 locus reported in two-row × six-row crosses might be pleiotropic effect of Vrs1 locus. Our study also showed a possible pleiotropic effect of the Vrs1 locus and provided evidence to further support previous suggestions.Five regions on chromosome 2H were observed to simultaneously affect multiple seedling characteristics. Of these, a significant region with pleiotropic or tightly linkage effects on SFW, SLFW, SLDW, MRN, SELW, FILA, SELA and FOLA was close to both the SNP marker, 2HL_34260490 and the morphological marker, rs1 Fig. ; Table 3e traits –63. For e traits  and Wango et al. . Sato ethttp://wheat.pw.usda.gov/GG3/) revealed that the marker bPb-6504 is near to the marker Bmag13, indicating that this QTL is likely the same to the qSPL3c associated with spikes per line. In the previous studies using the same DH population, QTL for heading date (Qhd3–13), culm length (Qcl3–13), two internode length(Qitw3–13) and three internode length (Qith3–13), as reported by Ren et al. [qGs3–13) and transpiration rate (qTr3–13) reported by Liu et al. [qRWC7–9) for relative water content and QTL (qSPD7–9) for relative chlorophyll content, as reported by Liu et al. [On chromosome 3H and 7H, we also found that some regions showed pleiotropic effects on different seedling characteristics  for LAG, SH, MRL, MRN, SFW, SLFW, SLDW, RFW, RDW, FOLL, SELW, FILA, SELA and FOLA with contribution of 12.05%–32.39%  in barley breeding.In this research, a number of genomic regions containing many seedling-related QTLs involved in different traits and diverse seedling growth stages were detected. The two parents used for mapping population construction are different in row types (six-rowed dwarfing barley cultivar Huaai 11 and two-rowed barley cultivar Huadamai 6). We found that chromosome 2H close to the morphological marker −2 s−1 during the 16 h light period, and relative humidity of 65% for 24 h. Barley seeds of the two parents and 122 lines were surface sterilized for 15 min in a 5% solution of sodium hypochlorite, rinsed thoroughly with distilled water, soaked in deionised water for 2 h, and then germinated for 5 days on plastic board floating over the deionized water at room temperature. After 5 days, 6 uniform seedlings from each line were selected for transplanting into foam board (polystyrene) suspended in the plastic tanks  with nutrient solution. Each seedling was anchored to holes 1.5 cm diameter in foam board with sterilized sponge. Each foam board had 5 rows of holes, and each row had 6 holes for a total of 30 seedlings per board. The distance between two seedlings was 3.6 cm (between rows) and 4.0 cm (within a row), respectively. Each tank contained eight liters of revised Hoagland’s nutrient solution [The barley double haploid (DH) population used in the current study was derived by anther culture from a cross between Huaai 11 and Huadamai 6 in our research group. This population consisted of 122 DH lines, have been described in our previous studies , 49–52. solution , which w2) were measured in four leaves according to the leaf order of seedling. LL was measured for first leaf length , second leaf length , third leaf length  and fourth leaf length . LW was measured for first leaf width , second leaf width , third leaf width  and fourth leaf width . LA as measured for first leaf area , second leaf area , third leaf area  and fourth leaf area . The methods of measurement are listed in Table The phenotypes measured included leaf age (LAG), seedling height , maximum root length , main roots number (MRN) and seedling fresh weight  at four growth stages  on the same six seedlings for each line. Root fresh weight , stem-and-leaf fresh weight , root dry weight  and stem-and-leaf dry weight  were only measured at the 34th day after germination. For convenience, we used S1, S2, S3 and S4 to represent the 13th, 20th, 27th and 34th day after germination, respectively. In addition, leaf length , leaf width  and leaf area . The heritability was estimated [http://www.originlab.com). P-value higher than 0.05 was deemed as significance level.The mean phenotypic values of the 21 seedling characteristics obtained from hydroponic culture were subjected to statistical analysis. Normality of distribution was tested using the method of Shapiro-Wilk. The descriptive statistics analyses were performed using SPSS programs  was used to compare the maker information.The high-density genetic linkage map constructed by Ren et al.  for the Additional file 1: Figure S1.P value of Shapiro-Wilk test for each stage was shown, the hypothesis of normal distribution was accepted when P > 0.05 (significant at P = 0.05), and the trend lines of the accepted normal distribution were shown. Trait abbreviations refer to the Table Frequency distribution of 21 seedling characteristics in DH population. Additional file 2: Figure S2.QTL likelihood map for 17 seedling characteristics in the DH population grown under hydroponic culture conditions using inclusive composite interval mapping. Genetic maps  of barley linkage groups were shown in the abscissa and LOD scores of each trait in the ordinate. The significant LOD threshold was determined to be 3.0 by 1000 permutations test. Trait abbreviations refer to the Table Additional file 3: Figure S3.QTL locations for previous studies and current studies detected in the Huaai 11 × Huadamai 6 DH population. QTLs were mapped to chromosomes using peak position and nearest marker. (JPEG 4137 kb)