Plasmodium proteasome system, relatively little investigation has been done to actually characterize the parasite degradation machinery. In this report, we provide an initial biological investigation of the ubiquitylating components of the endoplasmic reticulum-associated degradation (ERAD) system, which is a major pathway in targeting misfolded proteins from the ER to the cytosol for proteasome degradation. We are able to show that the ERAD system is essential for parasite survival and that the putative Plasmodium HRD1 (E3 ubiquitin ligase), UBC (E2 ubiquitin conjugating enzyme) and UBA1 (E1 ubiquitin activating enzyme) are able to mediate in vitro ubiquitylation. Furthermore, by using immunofluorescence, we report that Plasmodium HRD1 localizes to the ER membranes, while the Plasmodium UBC and UBA1 localize to the cytosol. In addition, our gene disruption experiments indicate that the Plasmodium HRD1 is likely essential. We have conducted an initial characterization of the ubiquitylating components of the Plasmodium ERAD system, a major pathway for protein degradation and parasite maintenance. In conjunction with promising proteasome inhibitor studies, we explore the possibility of targeting the Plasmodium ERAD system for future bottom-up drug development approaches.Ubiquitin-dependent protein degradation within malarial parasites is a burgeoning field of interest due to several encouraging reports of proteasome inhibitors that were able to confer antimalarial activity. Despite the growing interest in the  Plasmodium. Among them, Plasmodium falciparum is the deadliest human-infecting species. With the urgent need of developing new anti-malarial therapies, there has been much effort in understanding how the Plasmodium regulates its life cycle to subsequently identify potential new targets for drug development and therapeutic intervention.Malaria is one of the deadliest infectious diseases of the world, infecting up to half a billion and killing up to one million people each year P. falciparum’s 26S proteasome components via the 26S proteasome closely involves ubiquitylation of target substrates, very little work has been done regarding the characterization of the parasite’s ubiquitylating machinery acting upstream of the proteasome, possibly leading to the discovery of parasite-specific divergences that can be exploited for drug targeting. In eukaryotes, the endoplasmic reticulum-associated degradation (ERAD) pathway mediates protein degradation during quality control. Aberrant proteins are recognized by ER luminal chaperone proteins and protein disulfide isomerases to help discriminate properly folded proteins from misfolded proteins via the hierarchical intervention of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase that is usually involved in specific substrate recognition Plasmodium E3 ubiquitin ligases have been shown to have high levels of divergences that can be utilized for the development of new antimalarials In recent years, several studies showed that proteasome inhibitors have significant antimalarial properties P. falciparum ERAD system: one ubiquitin-activating E1 enzyme, an ubiquitin-conjugating E2 enzyme, and an ubiquitin E3 ligase. Our results show that the Plasmodium ERAD system is vital to the parasite and that recombinant E1, E2, and E3 enzymes promote ubiquitylation in vitro. Furthermore, immunofluorescence microscopy experiments also reveal that E1 and E2 localize to the cytosol whereas the E3 ubiquitin ligase is found within the ER membrane, consistent with their respective functions in the ERAD pathway. Finally, gene disruption experiments suggest that the ubiquitylating components of the Plasmodium ERAD system are essential to the parasite survival. This analysis contributes to our understanding of the mechanisms leading to protein degradation in the human malaria parasite and proposes the components of the ERAD pathway as potential targets for the ‘bottom-up’ development of new drugs.Here, we present the identification and biochemical characterization of the ubiquitylating components of the Plasmodium, we decided to test the inhibitory effects on P. falciparum cultures with Eeyarestatin I (ESI). ESI a known ERAD inhibitor which acts to block the release of ubiquitylated misfolded proteins from the Sec61 translocon at the ER membrane P. falciparum parasites were found to be highly sensitive to ESI, with an IC50 of 3.5415±1.0399 µM  long inal end . The shoinal end . Finallyinal end . The preinal end .in silico predictions, it is likely that these proteins have the necessary components to promote ubiquitylation and have enough homology to the human and yeast ERAD components to be part of the parasite’s ERAD system. In the following sections, we conduct some initial biological characterizations to validate our in silico data analyses.Based on our E. coli, fused to tandem GST and 6xHis tags, expressed, and purified , various human E2 (UBC) enzymes, and commercial purified ubiquitin . The previously observed pattern of ubiquitylation consisted of free poly-ubiquitin tails only  is capable of in vitro mono (27 kDa band) and di-auto-ubiquitylation (35 kDa band) activity when incubated with commercially available human UBA1  PFL1245w as a functional E1 ubiquitin-activating enzyme that can work in vitro with the E2 PFL0190w, and (ii) PFL0190w as a functional E2 ubiquitin-conjugating enzyme that can work in vitro with the E3 ligase PF14_0215.F14_0215 . PFL1245Plasmodium ERAD E1 and E2 enzymes, respectively, we investigated their localization using custom-made antibodies; antibody-specificity was verified by immunoblotting  are capable of binding to ubiquitin Plasmodium falciparum UBA1, UBC and HRD1  respectively and serve to ubiquitylate misfolded ER proteins for translocation from the ER lumen to the cytosol for proteasome degradation s are being screened for inhibitors that may confer anti-cancer properties. In fact, there are already several inhibitors of ubiquitin or ubiquitin-like enzymes that have shown effective results against cancer , with some already in clinical trials  for targeted protein degradation. Also, whether or not protein degradation in protozoan parasites has unique features compared to human and yeast models. However, this can only be uncovered with more targeted and vigilant examination. One aspect of parasite biology that we can be certain of is that protein degradation is necessary for parasite survival and has already been shown to be a good candidate for antimalarial targeting with reports of proteasome inhibitors conferring Plasmodium IC50 values within the µM to nM ranges Plasmodium ERAD system offers at least one parasite-specific drug target candidate, the ERAD E3 ubiquitin ligases, which have been characterized as being divergent from their human host counterparts. Though we have just begun to explore the machinery that is responsible for protein degradation in the human malaria parasite, we believe that the Plasmodium ERAD system may provide great drug target candidates that are both effective and parasite-specific.The biology of the I) (EMD Millipore Corporation) was reconstituted at 10 mM in DMSO. 3D7 parasites were introduced to a 3-fold serial dilution of ESI in complete culture medium in 96-well plates and incubated for 72 hours in a humid modular incubation chamber (Billups-rothenberg) after gassing at 1% O2/3% CO2/96% N2. Plates were frozen overnight then thawed and evaluated for relative parasite growth with SYBR Green dye and a Molecular Devices Gemini EM spectrofluorometer.Eeryarestatin I (ESI (10 µM), MG132 (0.1 µM), ESI (10 µM) + MG132 (0.1 µM) or DMSO only . After 8 hours incubation, parasites were isolated with 0.15% saponin and crude protein lysates were extracted. 0.5 µg of protein lysates from each condition were loaded in each well for SDS-PAGE. Immunoblots were probed with rabbit anti-ubiquitin antibodies  and goat anti-rabbit antibodies conjugated to HRP .3D7 parasites were synchronized with sorbitol treatment. During the early trophozoite stages, parasites were drugged with either of four different conditions: ESP. falciparum v8.0 was obtained from PlasmoDB (www.plasmodb.org) and was bioinformatically searched to identify ubiquitylating proteins using a previously published approach The translated genome of GATCGAGATCGATCTCGATC and ATCCATGGCCTTACCGCTGCTATGATGATGAT and inserted to replace the GST using ClaI and NcoI. The sequence coding for the RING domain in PF14_0215 was PCR-amplified from 3D7 strain cDNA using the primers AGGGGATCCCTTAAGCCGCGGCCTTTACATATGACAGCAGAT and AGGAAGCTTTTAACTAGTGCTAGCTTACTTTTGTGTTGTATCATTTTCTG. A sequence segment that contains the E1 ubiquitin-activating domain of PFL1245w was PCR-amplified using the primers AGGGGATCCCTTAAGCCGCGGGTGGTGAATATTTTTGGGTTGG and AGGAAGCTTTTAACTAGTGCTAGCCCAACCCAAAAATATTCACCAC. The entire gene of PFL0190w was amplified from 3D7 strain cDNA with the primers AGGGGATCCCTTAAGCCGCGGATGGCCCTTAAAAGAATAACAAAA and AGGAAGCTTTTAACTAGTGCTAGCTTATTGTGCATATTTTTGTGTCC. The amplified fragments were digested with SacII and SpeI and ligated into pGS-21a (6xHis-GST) or pGS-21aHis (6xHis only) where the gene was N-terminally tagged. Constructs were inserted into E. coli for expression.The plasmid pGS-21a (GenScript) and a modified version of it (designed as pGS-21aHis) were used as cloning vectors. The region coding for a GST was removed so that only the two 6xHIS tags subsist. The GST tag, along with the tandem 6xHIS tag, was removed by cutting pGS-21a with ClaI and NcoI. A new 6xHis-coding fragment was then amplified by PCR from purified pGS-21a using the primers E. coli cells were grown in LB at 25°C until OD600>0.5. IPTG 1 mM was added to the cultures, which were then incubated overnight at 12°C. Cells were collected by centrifugation at 14,000 g and 4°C and resuspended in lysis buffer , Mini EDTA-free Protease Inhibitor Cocktail (Roche), and AEBSF 1 mM). Cells were sonicated and lysates were cleared by centrifugation at 14,000 g and 4°C. Purifications of GST-tagged proteins were performed on glutathione agarose beads (Sigma-Aldrich). Glutathione-bound proteins were washed three times with GST wash buffer  and eluted with GST elution buffer . Purifications of His-tagged proteins were performed on Ni-NTA beads (Qiagen). Beads-bound proteins were washed several times with wash buffer  and subsequently eluted with elution buffer . Purified protein extracts were analyzed by immunoblots. GST-tagged proteins were detected using a goat anti-GST antibody  and a donkey anti-goat antibody conjugated to horseradish peroxidase . His-tagged proteins were detected using a mouse anti-His antibody  and a goat anti-mouse antibody conjugated to horseradish peroxidase .Transformed The following peptide sequences were used as antigens for the production of affinity-purified antibodies (Fisher Scientific or Genscript) in rabbits: RFKSFQKYRELTKNIETK for PF14_0215, KTDRTKYHQTAKAWTQKYAQ for PFL0190w, CSDQDLVDVLIPSIQFIYK for PFL1245w.P. falciparum 3D7  and D10 ACP(transit)-GFP  strains were grown in human O+ red blood cells according to a previously described protocol The nuclei visualization. Anti-plasmepsin V antibodies  were used to visualize the endoplasmic reticulum membrane Immunofluorescence assays were performed using the protocol described in Microscopy slides were mounted with slow-fade mounting medium  and viewed with an Olympus BX40 fluorescence microscope using a 100X objective lens (UPlanFI). Image capture was done with a CoolSNAP cf camera and processed by the Metavue software (Photometrics). Images were finally merged and background was reduced using ImageJ software.Plasmodium E3 ligase 1–12.5 µM was incubated in reaction buffer  for 2 h at 37°C. Alternatively, Human E1 and/or E2 were replaced with our purified Plasmodium proteins. Reactions were then analyzed by SDS-PAGE and anti-ubiquitin or anti-GST immunoblotting. Anti-ubiquitin immunoblots were probed with rabbit anti-ubiquitin antibodies  and goat anti-rabbit antibodies conjugated to HRP . Anti-GST immunoblots were probed with goat anti-GST antibodies  and donkey anti-goat antibodies conjugated to HRP .A mix containing free ubiquitin 50–200 µM (Boston Biochem), Human recombinant UBE1 E1 enzyme 0.05-0.2 µM (Boston Biochem), Human recombinant UBC E2 enzyme 1–5 µM (Boston Biochem), and purified Saccharomyces cerevisiae cytosine deaminase (ScCD) cassette for subsequent negative selection Double recombination-based gene disruption plasmids were constructed with the 5′ UTR and 3′UTR of PF14_0215 flanking a human dihydrofolate reductase (hDHFR) resistance cassette (for positive selection with WR99210) within the pCC1 vector that also contains a 2, 1 mM DTT, 1 mM AEBSF and cocktail protease inhibitor (Roche) (lysis buffer 2), and then incubated at 4°C with vigorous shaking for 20 minutes. After spinning, the supernatant was collected. The remaining pellet was then resuspended in lysis buffer 1 and then sonicated and spun down. Supernatant was collected and pooled with the previous two collected supernatants to create a mixture of extracted proteins. Extracted proteins were precleared with magnetic Protein A beads (Millipore) and then incubated with anti-PfHRD1 (PF14_0215) antibodies for 2 hours at 4°C with gentle shaking. Pre-washed magnetic Protein A beads were added to the samples and allowed to incubate overnight at 4°C with gentle shaking. Samples were washed several times with lysis buffer 1. Pulled-down PF14_0215 proteins were left bound to the beads.Isolated 3D7 parasites were resuspended in 20 mM HEPES pH7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM AEBSF (Fisher Scientific), 0.65% Igepal v/v and cocktail protease inhibitor (Roche) (lysis buffer 1). Parasites were allowed to lyse on ice for 10 minutes and then were spun down. Supernatant was collected. The pellet was then subsequently resuspended in 20 mM HEPES pH 7.9, 0.1 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgClPlasmodium recombinant PfUBC (PFL0190w) and pulled-down PfHRD1 were added to a solution containing 0.2 M HEPES pH7.9, 50 mM DTT, re-energizing buffer (Boston Biochem), 0.5 µg/µL final concentration of biotin conjugated ubiquitin (Boston Biochem), 0.5 µg/µL final concentration of ubiquitin (Boston Biochem), 5 mM AEBSF (Fisher Scientific) and cocktail protease inhibitor (Roche). The reactions were allowed to incubate at 37°C with gentle agitation for two hours. After incubation, reactions were terminated with Laemmli buffer and incubated at 95°C for 5 mins. Activity was visualized with affinity blots. Biotin affinity blots were probed with streptavidin conjugated with peroxidase . Immunoblot intensities were measured with ImageJ64 software.Human recombinant UBE1 (Boston Biochem), Figure S1Coomassie-stained SDS-PAGE gels of purified recombinant proteins. Coomassie-stained SDS-PAGE gels show the step-by step purification of recombinant (A) PF14_0215, (B) PFL1245w, and (C) PFL0190w that have been either tagged with HIS or with GST and HIS both. In each elution, a distinguishable band that represents the respective purified recombinant protein can be found at their expected molecular sizes: GST-HIS-PF14_0215(249–358 AA), 42.6 kDa; HIS-PFL1245w (506–799 AA), 41.4 kDa; HIS-PFL0190w (1–148 AA), 19.6 kDa.(TIF)Click here for additional data file.Figure S2Immunoblots showing custom antibody specificity. (A) The specificity of the custom-made antibody raised against PF14_0215 protein (59.8 kDa) was verified by western blot on crude parasite extract. (B) Using anti-PFL1245w custom antibodies on crude parasite protein extracts, a band at 131.8 kDa was detected on an immunoblot, which is the expected size for the PFL1245w protein. (C) Immunoblots were made using anti-PFL1245w custom antibodies on crude parasite protein extracts. A band at 131.8 kDa was detected, which is the expected size for the PFL1245w protein.(TIF)Click here for additional data file.Figure S3Knock-Out Attempt for PF14_0215. A PF14_0215 knockout vector was constructed with a human dihydrofolate reductase (hDHFR) selection cassette that is flanked by the 5′ UTR and 3′ UTR of the PF14_0215. A Saccharomyces cerevisiae cytosine deaminase (ScCD) cassette (not shown) was placed outside the PF14_0215 5′ UTR and 3′ UTR sections to be used for negative selection. Primers (indicated by red arrows) were designed to only amplify crossover events at either the 5′ or 3′ UTR of PF14_0215. PCR experiments show that only parasites that underwent a 3′ UTR crossover event, that still leaves the PF14_0215 intact, could be recovered. Double recombination that would excise the endogenous PF14_0215 gene was never recovered.(TIF)Click here for additional data file.Figure S4In vitro ubiquitylation activity of immunoprecipitated PfHRD1. Using anti-PfHRD1 (PF14_0215) antibodies, endogenous PfHRD1 was immunoprecipitated from 3D7 strains. In vitro ubiquitylation assays were performed using biotinylated-ubiquitin, thus only newly ubiquitylated products can be detected with streptavidin blots. Human recombinant UBE1 (lane 1) by itself was able to produce a single ubiquitylated product when incubated with biotin-ubiquitin. Aggregates of biotinylated ubiquitin (depicted by the arrow) were seen in all lanes  and are considered as background. Recombinant PfUBC (PFL0190w) (lane 2) and immunoprecitated PfHRD1 (lane 3) by themselves were unable to produce any ubiquitylated products. UBE1 and Plasmodium PfUBC together (lane 4) were able to generate significant levels of newly ubiquitylated products. With the addition of immunoprecipitated PfHRD1 (lane 5), we were able to detect around a 44% increased level of ubiquitylation when compared to lane 4.(TIF)Click here for additional data file.


Type 2 diabetes (T2D) is the most common form of diabetes in humans and is closely associated with dyslipidemia and obesity that magnifies the mortality and morbidity related to T2D. The genetic contribution to human T2D and related metabolic disorders is evident, and mostly follows polygenic inheritance. The TALLYHO/JngJ (TH) mice are a polygenic model for T2D characterized by obesity, hyperinsulinemia, impaired glucose uptake and tolerance, hyperlipidemia, and hyperglycemia.Apoa2 gene. Sequencing analysis revealed polymorphisms of Apoa2 in TH mice, suggesting Apoa2 as the candidate gene for the hyperlipidemia QTL. Gene expression analysis added novel information and aided in selection of candidates underlying the QTLs.In order to determine the genetic factors that contribute to these T2D related characteristics in TH mice, we interbred TH mice with C57BL/6J (B6) mice. The parental, F1, and F2 mice were phenotyped at 8, 12, 16, 20, and 24 weeks of age for 4-hour fasting plasma triglyceride, cholesterol, insulin, and glucose levels and body, fat pad and carcass weights. The F2 mice were genotyped genome-wide and used for quantitative trait locus (QTL) mapping. We also applied a genetical genomic approach using a subset of the F2 mice to seek candidate genes underlying the QTLs. Major QTLs were detected on chromosomes (Chrs) 1, 11, 4, and 8 for hypertriglyceridemia, 1 and 3 for hypercholesterolemia, 4 for hyperglycemia, 11 and 1 for body weight, 1 for fat pad weight, and 11 and 14 for carcass weight. Most alleles, except for Chr 3 and 14 QTLs, increased phenotypic values when contributed by the TH strain. Fourteen pairs of interacting loci were detected, none of which overlapped the major QTLs. The QTL interval linked to hypercholesterolemia and hypertriglyceridemia on distal Chr 1 contains Apoa2 gene are suggested to be responsible for the Chr 1 QTL linked to hypercholesterolemia and hypertriglyceridemia. Further, genetical genomic analysis led to potential candidate genes for the QTLs.We identified several genetic loci that affect the quantitative variations of plasma lipid and glucose levels and obesity traits in a TH × B6 intercross. Polymorphisms in  Type 2 diabetes (T2D) is the most common form of diabetes and is often associated with a collection of metabolic disorders including dyslipidemia and obesity which substantially magnifies the mortality and morbidity related to T2D  ad libitum with free access to water  under controlled temperature and humidity with a 12-hour light and dark cycle. All animal studies were carried out with the approvals of The University of Tennessee Animal Care and Use Committee and Marshall University Animal Care and Use Committee. Mice were euthanized by COg) at 4°C and plasma levels of glucose, true triglyceride, total cholesterol, and insulin and body weights were measured. At the end of the study, mice were killed and tissues including liver, skeletal muscle, pancreas, and adipose tissue  were collected, frozen in liquid nitrogen, and stored at -80°C for RNA isolation. Respective fat pad weight and carcass weight (body weight without the five fat pads) were also recorded during the dissection.Male TH mice were mated to female B6 mice. The resulting F1 hybrid mice were interbred to produce an F2 population. Male F2 mice were fasted for 4 hours at the beginning of the light cycle (6:00-7:00 AM) and blood was collected via the retro-orbital plexus using a heparinized microcapillary tube at 8, 12, 16, 20, and 24 weeks of age. Plasma was obtained by centrifugation , total cholesterol , and free and total glycerol  were determined using commercial colorimetric assays. Plasma true triglyceride concentrations were estimated by subtraction of free glycerol from total glycerol. Plasma insulin levels were determined using RIA .http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=markerQF. The thermal cycle consisted of 95°C for 2 min, followed by 49 cycles of 94°C (20 sec), 50°C (30 sec) and 72°C (40 sec) and a final extension at 72°C (7 min). Amplified products were electrophoretically separated on 3% metaphor /1% agarose  gels in 0.5 × tris-borate-EDTA buffer, pH 7.4. The DNA was visualized by ethidium bromide  staining.Genomic DNA was extracted from tail tips using proteinase K  and two Total RNA was isolated from liver, muscle (combined soleus and gastrocnemius), pancreas, and adipose tissue  using RNeasy Lipid Tissue Midi Kit  according to the manufacturer's instructions. For adipose tissue, muscle and pancreas, the entire tissue was homogenized and total RNA extracted, whereas approximately 50% of the liver was homogenized. Total RNA was further purified using RNeasy MinElute Cleanup Kit  for microarray analysis.® Mouse Genome 430 2.0 Array  following the standard protocol. The Mouse Genome 430 2.0 Array contains 45,000 probe sets on a single array to analyze the expression level of over 39,000 transcripts and variants from over 34,000 well-characterized mouse genes (Affymetrix). Total RNA isolated from adipose tissue, liver, muscle, and pancreas of a subset of F2 mice (n = 16) were used for microarray analysis, requiring 64 arrays. The 16 mice were chosen from the upper and lower tails (8 each) for plasma triglyceride distribution of all the male F2 mice, excluding overtly diabetic mice. The phenotypic values of these mice are presented in Table Hybridizations were performed at the University of Tennessee Affymetrix Facility  using Affymetrix GeneChipmice n = 6 were usCtrc (PPM39932A), 1700009P17Rik (PPM27997A), Sirpb1a (PPM39258A), Irs1 (PPM05117E), Cyp27a1 (PPM30124A), Mogat1 (PPM28787E), Usp37 (PPM60128A), Zfp69 (PPM40990E), Insig2 (PPM26980A), Pik3ca (PPM05112A), Agtr1b (PPM31347B), Ccdc79 (PPM35025A), Ccdc39 (PPM28741A), and Apoa2 (PPM05347B) . Oligonucleotide primers were also synthesized (Sigma) using sequences obtained from Primer Bank http://pga.mgh.harvard.edu/primerbank for Ccdc46, Igfbp2, Ccl3, Ccl9, Ccl6, Msi2, and Spag5 and from the published literature for 36B4 [Total RNA was isolated from adipose tissue, liver, muscle, and pancreas of B6 and TH male mice at 16 weeks of age as described above. Total RNA (2 μg) was reverse-transcribed with SUPERSCRIPT RT  using oligo d(T)12-18  as primer to synthesize first-strand cDNA in 20-μl volume according to manufacturer's instructions. The primers used for the real-time PCR were proprietary for for 36B4   containing 1 μl cDNA template diluate  and 6 pmol primers using ABI Prism 7700 or 7300 sequence detection system . For each sample, triplicate amplifications were performed and the average measurements used for data analysis.Apoa2 cDNA using the Expand Long Template PCR System . Full length coding sequence was amplified using Apoa2 specific primers F1 (5'-AGAATCGCAGCACTGTTCC-3') and R1 (5'-GGAGAAAACAGGCAGAAGG-3') derived from Mus Musculus Apoa2 gene mRNA sequences (NM_013474). PCR products were first electrophoresed on a 1.2 % agarose gel. Bands of interest were excised from the gel, and DNA fragments were isolated . The gel-purified PCR products were directly sequenced with primers originally used to amplify the PCR products. Sequencing was carried out automatically with fluorescent tags .Total RNA was extracted from pancreas of B6 and TH mice and the RNA (10 μg) was reverse-transcribed as described above. The single-strand cDNA was diluted with water , and 2 μl of the diluate was used to amplify QTL analysis was run using R/QTL  with comhttp://www.bioconductor.org, and the gcRMA (robust multi-array) process used to produce a signal measure for each gene. Statistical analysis was performed using SAS software . A mixed ANOVA model [http://www.ncbi.nlm.nih.gov/geo/ under the accession number GSE24637.Individual probe data were extracted using Bioconductor VA model  was run VA model  were conVA model . A total(ΔCt of TH mice - ΔCt of B6 mice) [Triplicate threshold cycle times were averaged for each mouse, and strain differences tested using a one-way ANOVA in SAS software , with the 36B4 control gene values as a covariate to avoid analysis of ratios. Data are presented as relative fold-change using B6 mice as the reference by 2B6 mice) , where ΔP < 0.05. All data are presented as mean ± SEM.Data analysis was conducted by ANOVA with StatView 5.0 . Differences were considered significant at TPS did phenotyping and genotyping of F2 mice and was in charge for all aspects of animal care. HYK did microarray analysis. AMS conducted statistical analysis of the genetic and genomic data. JHK conceived the study and was primarily responsible for its coordination and design and did qRT-PCR and sequencing analysis. AMS and JHK drafted the manuscript, tables and figures. All authors read and approved the final manuscript.Table S1 - Simple sequence length polymorphic markers for the genome-wide scans of the (B6 × TH) F2 mice. The file contains the list of genetic markers and their estimated genetic map positions used in this study.Click here for file


H9N2 influenza A viruses have undergone extensive reassortments in different host species, and could lead to the epidemics or pandemics with the potential emergence of novel viruses.To understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals.Our results demonstrated that most of the 15 isolates were reassortants and generated four novel genotypes (B62-B65), which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 epidemic (genotypes B55 and B65) were lowly infectious, while two representative viruses of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08  was able to replicate well in mouse lungs with high virus titers but caused mild clinical signs.Several lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses. H9N2 influenza viruses are panzootic in birds worldwide. Statistical analysis of the host range and location of H9N2 subtype influenza A viruses in the NCBI database according to HA gene revealed that approximately 60% of all the H9N2 influenza viruses were isolated from chickens, with the remainder from wild birds 16.8%), ducks (8.9%), turkeys (6.7%), and other domestic avian populations 3.7%) (data not shown). In addition, the overwhelming majority 94.2%) of H9N2 influenza viruses were isolated in Asia, with > 65% coming from mainland and Hong Kong of China (data not shown). Since in China the H9N2 virus was first time isolated in 1994 while approximately 74 different genotypes have been observed till now and new lineages and genotypes continuously identified throughout China  and NP [Genbank: DQ064447] genes from an early North American lineage (represented by Ty/WI/1/66) [H9N2 influenza viruses can also generate novel reassortment or genotype viruses that carry gene segments with wide evolutionary distances. It has been reported that H9N2 strain A/swine/Korea/S190/2004 was a reassortant between a Eurasian lineage and a North American lineage viruses [DQ023146], A/swine/Shandong/2/03 (H5N1) , and A/environment/Qinghai/1/2008 (H5N1) . Interestingly, one H5N1 reassortment virus, A/plateau pika/Qinghai/04/2007 (H5N1), was closely related to Ck/SD/LY-1/08 in its PB2 (98.4% homology) [GenBank: FJ390058], PA (98.5% homology) [GenBank: FJ390060], and NP (99.6% homology) [GenBank: FJ390062] gene segments  H5N1 virus in 1997, and the Qa/HK/G1/97(H9N2)-like viruses were hypothesized to have been involved in the generation of the H5N1 virus . PreviouH9N2 viruses of different genotypes and reassortment patterns could have huge differences in their pathogenicity and transmission in mammals (BALB/c mice and swine) under experimental conditions ,9. In thIn light of the persistent evolution of H9N2 viruses with high evolution rate, it is highly necessary to monitor the evolution and evaluate the virulence of novel avian H9N2 viruses to mammal animals, which may discover and prohibit the potential threat to human by novel viruses, and provide the key clues for preventing new pandemic influenza.Here, we described the genetic and pathogenic characteristics of the representative H9N2 viruses isolated from chicken farms in northern China over a 12-year span. Phylogenetic analyses revealed that H9N2 influenza viruses evolved quickly in China, and new genotypes were frequently generated in chicken flocks. The present study identified two stable H9N2 lineages (genotype B55 and B65) that have been circulating in China in recent years. In addition, different H9N2 viruses possessed diverse replication capacity and pathogenicity in mice.In summary, our study provides evidences to further understand the genetic evolution and inter-species transmission of H9N2 influenza A viruses. Their different pathogenicitiy behavior and host adaptations to mice may likely be associated with both the gene reassortments and key amino acid mutations. Thus, surveillance followed by large-scale sequencing efforts and animal experiments are critically important to detect the virulent variants and provide insight into the key features for emergence of pandemic viruses.Surveillance was conducted in northern China from 1998 to 2010. Tracheal, cloacal and fecal swabs, and lung tissues were collected from sick or dead chickens from chicken farms. The tissue homogenates of clinical specimens were centrifuged for 10 minutes at 8, 000 × g, and after removing bacterial contaminants by micropore membrane filters (0.22 μm) the supernatant was incubated in the allantoic cavities of 10-day-old specific pathogen free (SPF) chicken eggs  at 37°C for 72 h for viral isolation, finally the allantoic fluids was harvested and stored at -80°C. The purification of all the strains was done by the end-point dilution assay [Viral RNA was extracted from infected allantoic fluids using Trizol reagent (Invitrogen). Reverse transcription (RT) was performed by using the Uni12 primer (AGCAAAAGCAGG), and specific primers were designed amplifying the eight full-length gene segments of the virus. The PCR products were purified and sequenced from the Beijing Genomics Institute using synthetic oligonucleotides.http://www.megasoftware.net via Neighbor-Joining algorithm and the reliability of the trees was assessed by bootstrap analysis with 1, 000 replications.To determine the molecular evolutionary characteristics of H9N2 viruses isolated in China, eight gene segments from each of 15 isolates were phylogenetically analyzed together with all the full length sequences of the representative viruses of China available in GenBank. The nucleotide and amino acid sequences of each gene segment were analyzed by DNAMAN  and DNASTAR  softwares. The nucleotide sequence-based phylogenetic trees were generated with MEGA 4.1 Genotype analysis was performed systematically for each of the eight gene segments based on the distribution of lineages in phylogenetic trees, and the genes sharing over 95% homology in the same lineage were considered as one genotypic group . H9N2 vi7 EID50 of H9N2 virus in a volume of 50 μl. Mock-infected control mice were inoculated i.n. with 50 μl phosphate-buffered saline (PBS). Three mice were humanely euthanized at 3 d.p.i., and their lungs were collected, ground and homogenized in cold PBS under sterile conditions. Then the solid debris was pelleted by centrifugation at 5, 000 × g for 10 min, and the homogenates were used for virus titrations in 10-day-old embryonated chicken eggs. Virus titers were reported in units of log10EID50 per 0.1 ml. Nose, trachea, and part of left lung lobes were fixed in 10% buffered formalin, embedded in paraffin, and the tissues were stained with hematoxylin-eosin for histopathological evaluation. The rest of the mice were monitored daily for general behavior and clinical signs, including food intake, body weight, inactivity, and mortality, for 14 days.H9N2 influenza viruses from each genotype were selected to assess their potential pathogenicity to mammal. BALB/c mice  were purchased from Vital River Laboratories, Beijing. Mice in each group (n = 7) were lightly anesthetized with Zoletil  and inoculated i.n. with 10The use of all laboratory animals and animal subjects in this study was approved by the Beijing Association for Science and Technology, with approval ID SYXK (Beijing) 2007-0023, and followed by the Beijing Laboratory Animal Welfare and Ethical Guidelines of the Beijing Administration Committee of Laboratory Animals.JF795035-JF795148).The nucleotide sequences of 15 H9N2 influenza viruses isolated in this study have been submitted to the GenBank database and have assigned the accession numbers  (A), NA (20-1387 nt) (B), M (26-1007 nt) (C), NS (27-849 nt) (D), PB2 (28-2289 nt) (E), PB1 (25-2242 nt) (F), PA (25-2175 nt) (G), and NP (46-1526 nt) (H) genes of H9N2 influenza A viruses. Trees were generated by the Maximum Composite Likelihood model of Neighbor-Joining algorithm with MEGA 4.1 http://www.megasoftware.net. The reliability of the trees was assessed by bootstrap analysis with 1, 000 replicates and only bootstrap values ≥ 90% were shown. Different lineages were indicated by different colors. The viruses obtained in the present study were marked with red circles, and the representative strains in each lineage were marked with yellow squares.Click here for fileTable S1. Comparison of amino acid sequences of HA, NA, and PB2 genes of representative H9N2 viruses from northern China.Click here for fileTable S2. The clinical information of representative avian H9N2 influenza viruses isolated from northern China.Click here for file


Morocco is a medium-level epidemic country for Hepatitis B Virus (HBV). However, little is known about clinical, virologic and phylogenetic features of HBV infection. The aim of the present study is to determine the HBV genetic variability, and its association with clinical outcome and severity of disease in Moroccan HBV carriers.The study included 250 chronic HBV infection patients at different stages of liver disease . Serum samples were tested for serological markers and HBV DNA levels. The HBV Surface and core promoter/precore regions were amplified and directly sequenced. The clinical, virologic and phylogenetic characteristics were investigated.adw2. In genotypes D strains, subtypes ayw2 (91.7%), ayw3 (3.3%) and ayw4 (3.3%) were identified. A significance prevalence of mutations in the Major Hydrophilic Region (MHR) of HBsAg was found with P120T/S the most frequent. In the core promoter region, the most frequent mutations are G1757A (48.9%), T1773C (42.8%), C1766GÂ /T (40.8%), T1753V (30.7%) and A1762T/G1764A (24.4%). In the precore region, the most common mutations are G1896A (55.1%) and G1899A (34.6%). Double mutation in the core promoter A1762T/G1764A was found more frequently in HCC patients than that in non HCC patients . In addition, the prevalence of C1653T, T1753V, and G1862T mutations was significantly higher in HCC patients compared with non HCC. However, the prevalence of the G1896A precore mutation was not different between patients with HCC and HBV carriers without HCC .The mean age of patients was 44 ± 12.2. Most of them were HBeAg negative (90%). The mean HBV DNA was 475104 ± 160591 UI/mL. Phylogenetic analysis identified 90% isolates in genotype D and 10% in genotype A. Most genotypes D isolates belonged to subgenotype D7 (80%) followed by subgenotype D1 (25%) and one isolate belonged to subgenotype D2. All genotype A strains belonged to subgenotype A2 and specified subtype We described for the first time that Subgenotype D7/ayw2 is the most predominant in Morocco. The high frequency of mutations in the core promoter in patients with HCC indicates their association with severity of infection.


Sle.Sle2.Sle3 (B6.TC) model of lupus, the majority of marginal zone B cells are located outside the marginal zone and inside the follicles. Genetic alterations of this strain have shown a correlation between autoimmune pathogenesis and the presence of intrafollicular marginal zone B cells. This study was designed first to strengthen our original observations and to determine how the marginal zone B cells from the lupus-prone mice respond to stimulations and interact with T cells.Marginal zone B cells have been implicated in the production of autoantibodies in murine models of lupus. It has been suggested that they contribute to lupus immunopathogenesis through their enhanced effector functions and their repertoire that is biased toward autoreactive specificities. In the B6.NZM2410.+ T cells. Two different autoreactive B cell receptor (BCR) transgenic models showed that the expression of the Sle susceptibility loci enhances the presence of MZB cells inside the follicles. In vitro, B6.TC MZB cells were better effectors than B6 MZB cells with enhanced proliferation and antibody (Ab) production, including anti-DNA Ab, in response to stimulation with TLR ligands, immune complexes or anti-CD40. Furthermore, B6.TC MZB and CD4+ T cells showed a reciprocally enhanced activation, which indicated that their contacts inside B6.TC follicles have functional consequences that suggest an amplification loop between these two cell types.The intrafollicular location of B6.TC MZB cells starts before disease manifestations and puts MZB cells in direct contact with CD4These results showed that the NZM2410 susceptibility loci induce MZB cells to locate into the follicles, and that this breach of follicular exclusion occurs early in the development of the autoimmune pathogenesis. The enhanced responses to stimulation and increased effector functions of MZB cells from lupus-prone mice as compare to non-autoimmune MZB cells provide a mechanism by which the failure of MZB cell follicular exclusion contributes to the autoimmune process. Margina in vivo , showingZB cells . These oZB cells ,13), hasZB cells . The expZB cells -17, but ZB cells ,19. HoweSle1, Sle2 and Sle3 susceptibility loci [We have shown that in lupus-prone B6.TC mice that express the NZM2410-derived ity loci , a largeity loci . On the ity loci . Furtherity loci . Overall+ T cells. We have used the anti-DNA 56R [Sle susceptibility loci favors the recruitment of the Tg MZB cells to the follicles. In vitro, B6.TC MZB cells proliferated more and produced more IgM than B6 MZB cells in response to TLR, immune complex (IC) and CD4+ T cell stimulation. Finally, B6.TC MZB cells activated CD4+ T cells more than either B6 MZB cells or FOB cells. Overall, our results demonstrate that autoreactive MZB cells have a greater propensity for intrafollicular location, due at least in part to their enhanced responsiveness to a variety of stimuli. Our results also suggest that B6.TC MZB cells contribute to autoimmune pathogenesis through an enhanced mutual relationship with CD4+ T cells that they encounter in the follicles.In this report, we show that a large proportion of B6.TC MZB cells enter the follicles early in the disease process, before autoAb are secreted, and these intrafollicular MZB cells establish contact with CD4-DNA 56R  and rheu-DNA 56R  heavy ch-DNA 56R  and Morehi B220+ cells are CD21+ CD23-, which indicates that CD1d can be used to track MZB cells by immunoflurorescence [hi B220+ cells accounted for about 50% of the B cells in the follicles of 3 mo old B6.TC mice, and that this number increased to about 75% in 10 mo old mice  of IgMa cells express the AM14 HC Tg, but some IgMa+ cells express the endogenous IgHa HC. However, there was no difference between IgHb and IgHa MZB cell distribution on either non-Tg B6 or B6.TC background (data not shown). This indicated that the endogenous non-transgenic B cells do not contribute to the results reported below, and that the Tg B cells accounted for the differences in MZB cell distribution between non-Tg and AM14 Tg mice. A large number of IgMa cells expressed CD1d in both B6.TC.AM14.IgHa/b and B6.AM14.IgHa/b spleens , and the proportion of CD1d+ IgMa cells with an intrafollicular location was significantly higher in B6.TC.AM14.IgHa/b than in B6.AM14.IgHa/b spleens . Overall these results show in two models that Tg B cells expressing an autoreactive HC are not only preferentially selected to the MZB compartment, but they are also more likely to breach follicular exclusion. Moreover, expression of the Sle susceptibility loci enhances the migration of the HC Tg MZB cells into the follicles, more strongly when the three loci are expressed than with just Sle2.When paired with endogenous Vκ8, the AM14 HC Tg encodes for anti-IgG2a IgMa RF . In B6.Ts Figure , indicate Figure  and 2C. a B cells in the CD23hi CD21hi subset, which contains the precursors of the Tg MZB cells, was reduced in both B6.Sle2.56R and B6.TC.AM14.IgHa/b mice as compared to their B6 controls . The proportion and number of Tg MZB cells was significantly higher in B6.Sle2.56R than in B6.56R mice  provided by B6.TC T cells stimulating B6.TC MZB cells  or in response to CXCL13 or S1P [The two models of autoreactive HC Tg B cells that we used, 56R and AM14, are directed against prototypic lupus antigens, DNA  and IgG2atin ICs . As prevatin ICs , the AM13 or S1P  may contin vitro experiments have confirmed established results: First, B6.TC B cells have greater effector functions than B6 B cells [+ T cells [NZW on MZB cells has not been reported, in general and in the B6.TC strain . The strong response of B6.TC MZB cells to chromatin ICs strongly suggest that these cells are enriched for anti-chromatin autoreactive specificities. Loss of tolerance to chromatin represents a critical step in the initiation of systemic autoimmunity in lupus [+ T cells, indicating that these MZB cells have the potential to activate non-autoreactive T cells. The strongest responses were however observed between B6.TC MZB and B6.TC CD4+ T cells, which are likely to share autoreactive specificities, such as chromatin [+ T cells in which MZB cells respond aberrantly to TLR or IC stimulation, secrete autoAb and move inside the follicles where they encounter CD4+ T cells. Upon these encounters, the CD4+ T cells become activated themselves and can in turn provide further activation signals to MZB cells or other cell types to participate in the autoimmune process.Our  B cells ,34, and  B cells ,36 or to B cells ,10, and  T cells  may contin lupus ,39, and hromatin . This suThis study showed that the NZM2410 susceptibility loci induce MZB cells to locate into the follicles, and that this breach of follicular exclusion occurs early in the development of the autoimmune pathogenesis. The enhanced responses to stimulation and increased effector functions of MZB cells from lupus-prone mice as compare to non-autoimmune MZB cells provide a mechanism by which the failure of MZB cell follicular exclusion contributes to the autoimmune process. Overall, our results suggest a novel tolerance checkpoint in regulating systemic tolerance consisting of containing hyperactive MZB cells in the marginal zone away from T cells.Sle.Sle2.Sle3  [Sle2.56R and B6.56R breeders were a generous gift from Chandra Mohan (UTSW). B6.TC.AM14.IgHa/b and B6.AM14.IgHa/b were obtained by breeding B6.TC and B6 to B6.AM14.lpr, which carry the AM14 heavy chain (HC) transgene (Tg)  and C57BL/6J-Cg-IghaThy1aGpila/J (B6a) mice. The presence of the IgHa allele was screened by flow cytometry with allotype-specific anti-IgMa (Igh6a). The presence of the AM14 HC Tg was detected by PCR as previously described [in vitro experiments used 2 to 3 mo old females before any manifestation of clinical disease. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida.The triple congenic B6.NZM2410.s B6.TC)  and C57Ba-Biotin-SA-PB or B220-PB (RA3-6B2). Quantitation was performed with a MetaMorph 7.5 image analysis station, which quantifies the number of pixels for a given color corresponding to a specific stain combination. For each B6.TC and B6 mice, the numbers of CD1d+ B220+ cells and CD1d- B220+ were computed in four areas with homogeneous staining selected on each side of the Moma-1+ ring on 100X or 200X amplification images. Five mice were evaluated for each strain and age group.Immunofluorescence staining was performed on frozen sections as previously described , with Mo+ CD23- CD21hi MZB and B220+ CD23+ CD21int FOB cells were purified on a FACSAria cell sorter, yielding 95-98% purity in both strains. We used the anti-CD21 7E9 Ab which binds equally the B6 and NZW alleles of CD21 [+ T cells were enriched by negative selection with magnetic beads (Myltenei). CFSE-labeled MZB or FOB cells (105 per well) were cultured with or without CFSE-labeled CD4+ T cells in RPMI 1640 containing 10% FCS for either 3 or 5 d with LPS , ODN1826 CpG , anti-CD40 , anti-chromatin or protein ICs . ICs were prepared as previously described [+ T cells (1×106/ml) were pre-activated overnight with 1 μg/ml of PMA and 1 μM ionomycin. In each assay, 2-3 mice per strain were used and each result reported in this study is representative of at least 2 independent assays. Culture supernatants were assayed at 1:10 dilution for total IgM, anti-ssDNA and anti-dsDNA IgM by ELISA carried out as previously described [6 negatively selected total splenic B cells.B220 of CD21 . CD4+ T escribed . Brieflyescribed . SpontanThe following conjugated mAbs were used: CD23 (B3B4), CD1d (1B1), CD21 (7E9), CD69 (H1.2 F3), CD62L (MEL14), CD138 (281-2), B220 (RA3-6B2), IgM (II/41), CXCR5 (2G8), all, except 7E9, purchased from BD Pharmingen. Stained cells were analyzed on FACSCalibur cytometer (BD Biosciences). Dead cells were excluded based on forward and side scatters characteristics.Data were analyzed with the GraphPad Prism 4.0 software package with the appropriate tests as indicated in the text.Sle.Sle2.Sle3 or BcN/LmoJ; Ab: antibody; HC: immunoglobulin heavy chain; Tg: transgenic; IC: immune complex; B6: C57BL/6J; SEM: standard error of the mean.SLE: systemic lupus erythematosus; MZ: marginal zone; BCR: B cell receptor; FO: follicular; B6.TC: B6.NZM2410.The authors declare that they have no competing interests.in vitro experiments and the morphometric analysis; HN performed the immunofluorescence histology and assisted with the in vitro experiments; YYZ performed the migration assays, LM conceived the study, performed the statistical analysis and wrote the manuscript. All authors read and approved the final manuscript.ZZ participated in the study design, performed the


Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs. Epigenetic silencing of various genes by aberrant promoter hypermethylation is a common feature of human cancer cells The mechanisms for the establishment and the maintenance of gene-specific hypermethylation during cancer development have not been fully elucidated yet. However, several candidate factors have been identified which might play an important role in recruiting DNA methyltransferases to specific genomic loci in cancer cells. Therefore, dysregulation of recruiting factors or alterations of gene-specific chromatin modifications involved in recruitment could potentially lead to a hypermethylation phenotype in cancer. In agreement with this notion, it has been shown that genes that are associated with components of the Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells are frequently hypermethylated in cancer In human cells, DNA methylation is catalyzed by DNMT1, DNMT3A and DNMT3B In line with these findings, DNMT3B has recently been proposed to act as a bona fide oncogene in human cancer cell lines by correlating DNMT3B gene amplification with resistance to DNA demethylating drugs Cell lines have been obtained from different providers: HCT-116, Caco-2, HT-29, SW-480 from the German Collection of Microorganisms and Cell Cultures ; HCT-15 and SW-620 from the NCI 60-Panel; DLD-1 and WI-38 cells from the ATCC. Human non-tumorigenic hTERT-immortalized mammary epithelial cells with reduced levels of p53 (HMEC-T53) were generated at Bayer Healthcare Pharmaceuticals, Berlin, Germany . HCT-116, Caco-2, and HT29 cells were grown in DMEM/Ham's F12 medium supplemented with L-glutamine (Biochrom) and 10% fetal calf serum (PAA). SW-480, HCT-15, SW-620, and DLD-1 cells were cultured in RPMI 1640 medium supplemented with L-glutamine (Biochrom) and 10% fetal calf serum (PAA). WI-38 cells were maintained in MEM Earle's medium supplemented with L-glutamine (Biochrom) and 10% fetal calf serum (PAA), and HMEC-T53 cells were grown in Mammary Epithelial Cell Growth Medium (Promocell: C-21010) and SupplementMix (Promocell: C-39115). HCT-116 DNMT3B knockout (3BKO), HCT-116 DNMT1 knockout (1KO) and HCT-116 DNMT1; DNMT3B double knockout (DKO) cells were kindly provided by Bert Vogelstein 5′-CTGGAAATGTTTGCATCGAAGA-3′) and LAMIN_B1_rev (5′-GCCTCCCATTGGTTGATCC-3′). The DNMT3B expression levels normalized to GAPDH or Lamin B1 were calculated with the deltaCt method.Information on tissue-specific DNMT3B mRNA expression levels was obtained from the Array Northern database which is based on data of mRNA hybridized to Affymetrix arrays For immunoblot analysis, cell pellets were resuspended in PBS (pH 7.4) containing Complete protease inhibitor cocktail (Roche) and 25 U Benzonase Nuclease (Novagen). Cells were disrupted using the Bioruptor Sonication system (Diagenode). Equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Whatman). Proteins were detected using antibodies directed against DNMT3B  and β-actin . Horseradish peroxidase-conjugated secondary antibodies were visualized by enhanced chemiluminescence  and protein expression was quantified using the ImageJ software 5′-TTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAA-3′, shDNMT3B#1: 5′-GACGGATGCCTAGAGTTTATTCAAGAGATAAACTCTAGGCATCCGTC-3′, shDNMT3B#2: 5′-TGGAGATGGAGACAGTTCATTCAAGAGATGAACTGTCTCCATCTCCA-3′. For lentivirus production and gene transfer, HCT-116 cells were grown in DMEM/Ham's F12 medium supplemented with L-glutamine and 10% fetal calf serum (PAA). Selection was performed in the presence of 1 µg/ml puromycin (Sigma). Lentiviruses were produced by co-transfecting the respective pGT396-Puro construct with lentiviral Packaging Mix (Invitrogen) into HEK-293FT cells (Invitrogen). Viral supernatants were collected and concentrated by ultracentrifugation . Viral titers were determined using a HIV p24 ELISA (Perkin Elmer). Transductions were carried out using p24 concentrations of 1 µg/ml for 6 h at 37°C.siRNA duplexes and non-targeting control siRNAs were obtained from Qiagen . We confirmed in silico that DNMT3B siRNAs target all mRNA isoforms of the active DNMT3B enzyme. 24 hours after cell plating, cells were transfected with 5 nM siRNAs for 72 hours using Lipofectamine RNAiMAX reagent (Invitrogen). Transfection was repeated every 24 hours to increase knockdown efficiency. For the generation of stable shDNMT3B clones, preselected shRNAs were cloned into the lentiviral pGT396-Puro vectors. The shRNA sequences of the forward oligonucleotides were as follows: sh_nontargeting: Cell viability was measured in 96-well plates using the CellTiter-Glo assay (Promega) according to the manufacturer's instructions. The TECAN Infinite 200 microplate reader was used for detection of luminescence signals. For the quantitative detection of caspase 3/7 activities, the Caspase-Glo 3/7 Assay (Promega) was used according to the manufacturer's protocol. Assays were performed in 96-multiwell plates and analyzed by a TECAN Infinite 200 microplate reader. For the adjustment of cell numbers, cell viability was determined in parallel and used for normalization. Each sample was analyzed in quadruplicates; data are reported as mean ± SD.4 cells was acquired and analyzed with CellQuest Pro software (Becton Dickinson). Annexin V-positive cells (as early marker of apoptosis) as well as Annexin V-positive and propidium iodide (PI)-positive cells (as early and late markers of apoptosis) were quantified.Apoptotic and necrotic cells were analyzed with the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). Cells were transfected with 20 nM of indicated siRNAs, 24 hours after cell plating. Transfection was repeated once after 24 hours to increase knockdown efficiency. Cells were washed with cold PBS and trypsinised 96 hours after the first transfection. After two washes with PBS, cells were stained according to the manufacturer's protocols. Samples were analyzed with a BD FACSCalibur™ flow cytometer. The fluorescence of 1×10P≥0.05 were excluded from the analysis. Loci were scored as hypermethylated if the AVB was greater than or equal to 0.8. Since changes in AVB greater than 0.2 can be detected with 95% statistical confidence, we used this value as a threshold to identify significant methylation changes in our analyses www.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-719.Array-based gene-specific DNA methylation analysis was performed using the Infinium HumanMethylation27 and HumanMethylation450 bead chip technology (Illumina) according to the manufacturer's instructions. Shortly, genomic DNA of different cell lines and siRNA-experiments (n = 1) was isolated with the DNeasy Blood and Tissue Kit (Qiagen) and deaminated using the EZ DNA Methylation Kit (Zymo Research). Results were analyzed using Illumina's BeadStudio software, version 3.1.3.0. The methylation status of specific cytosines is indicated by an average beta value (AVB) where 1 corresponds to complete methylation and 0 to no methylation. Signals of probes with Results were analyzed using using Illumina's BeadStudio software, version 3.1.3.0 and the R stats package, version 2.14.1 To determine the mRNA expression of DNMT3B in various human tissues and cancers, we analyzed the expression profiles in the Array Northern database Using quantitative real-time PCR and immunoblotting, we screened a panel of human colon cancer cell lines for DNMT3B expression on the mRNA and protein level . The resTo analyze whether DNMT3B-overexpressing cells require DNMT3B for proliferation and survival, we performed short-term siRNA knockdown experiments and subsequently analyzed cell proliferation with cell viability assays. To account for the fact that DNMT3B is expressed in many isoforms and to exclude off-target effects, we used six different siRNAs that specifically target different regions of the DNMT3B mRNA. Knockdown efficiency was analyzed by quantitative RT-PCR and confirmed to be in the range of 65% to 90% . The knoDnmt3b in the murine intestine has been shown to cause DNA hypermethylation at various loci high Caco-2 cells were completely unmethylated at the five CIMP markers, whereas CpG islands of the same genes were hypermethylated in the DNMT3Blow DLD-1 cells  being hypermethylated . Knockdown efficiency for cell viability and caspase assays was analyzed by quantitative RT-PCR in (A) Caco-2, (B) HCT-116, (C) HT-29, and (D) DLD-1 cells. Expression values are means of triplicates and were calculated relative to Lamin B1 expression. Error bars represent standard errors. Normalized expression values in control siRNA transfected cells were set as 1.0.(TIF)Click here for additional data file.Figure S2Infinium 27 k methylation analysis of HCT-116 cells. (A–C) Comparison of Infinium 27 k methylation profiles between HCT-116 cells transfected with either DNMT3B siRNA #3, #5, or #6, and HCT-116 cells transfected with control siRNAs, respectively. (D) Comparison of Infinium 27 k methylation profiles between HCT-116 cells stably transduced with lentiviruses containing DNMT3B shRNA #2 or transduced with control shRNA.(TIF)Click here for additional data file.Figure S3Analysis of DNMT3B knockdown for Infinium methylation arrays. (A) 72 h after transfection with the indicated siRNAs, DNMT3B mRNA levels were determined by quantitative RT-PCR analysis. Expression values are means of triplicates and were calculated relative to Lamin B1 expression. Error bars represent standard errors. Normalized expression values in HCT-116 control siRNA transfected cells were set as 100%. (B) Efficient depletion of DNMT3B protein in stably shRNA-transduced HCT-116 cells. DNMT3B protein levels were determined by immunoblot analysis using ß-actin as a loading control. The double band presumably reflects the expression of two (or more) DNMT3B isoforms.(TIF)Click here for additional data file.


Decompressive laminectomy is the most common operation performed to treat spinal stenosis. This study was performed in order to evaluate surgical outcomes between laminectomy at the L3/L4 level compared with L4/ L5.The patients diagnosed with one level stenosis at L3/L4 or L4/ L5 who were candidate for surgery entered into this cross-sectional study. The outcome measures were the Neurogenic Claudication Outcome Score (NCOS), the Japanese Orthopaedic Association (JOA) Score, subjective walking distance, and Visual Analog Score (VAS) of leg pain/numbness. T-test tests were used to analyze the comparisons of outcomes between the L3/L4 and L4/ L5 single-level laminectomy.Ninety-four patients were eligible to enter the study during the four- year courses of study. Patients were aged 39 to 79 years (mean age 63.7 ± 9.83 years) and were followed up for at least one year. Thirty-one L3/4 and 63 L4/L5 laminectomies were performed. No significant difference was observed in the clinical indications, JOA, NCOS, duration of symptoms or VAS of leg pain/numbness between the two groups. The difference between pre and postoperative was statistically significant (P less than 0.0001).The findings of this study suggest that no statistically signiﬁcant difference exists between L3/L4 and L4/ L5 laminectomies in terms of preoperative and postoperative outcomes. Spinal stenosis, Laminectomy, Single-level, Outcome measures


Pulse pressure variation (PPV) has been shown to predict preload fluid responsiveness in mechanically ventilated patients . InhalanP < 0.05).Twenty-four anesthetized and mechanically ventilated pigs were randomly assigned into three groups of eight animals: desfluorane (DESF), sevofluorane (SEVO) and isofluorane (ISO). Static hemodynamic parameters and PPV, measured by pulmonary artery and femoral arterial catheters, were assessed at baseline (T1) using 1 MAC of the volatile agent; T2 (1.25 MAC); T3 (1 MAC) and T4 (1.0 MAC associated with a 30% hemorrhage of estimated average volemia). Two-way ANOVA and Tukey test were used for statistical analysis (P < 0.001); SEVO  and ISO . No statistical difference between groups was found for PPV. Mean arterial pressure (MAP) decreased after 25% increment of MAC (T2) and after hemorrhage. At T4, MAP decreased significantly lower than basal values (T1) in groups DESF (P < 0.001), SEVO (P < 0.001) and ISO (P < 0.001). Cardiac index (CI) decreased in T2 compared with T1: DESF , SEVO  and ISO . The CI drop after hemorrhage showed no statistical difference when compared with T1.At T2 there was an increase in PPV in all groups but not statistically significant compared with T1 or among groups. At T4 the increase in PPV was significant compared with basal values in the three groups: DESF (11 ± 4 vs. 7 ± 2%, PPV behaved similarly with different inhaled anesthetics. Although PPV did not reflect the hemodynamic depression of incrementing MAC values, it increased after bleeding 30% of estimated volemia.


Sox2 is a key component of the transcription factor network that maintains the pluripotent state of embryonic stem cells (ESCs). Sox2 is regulated by multiple post-translational modifications, including ubiquitination, sumoylation, acetylation and phosphorylation. Here we report that Sox2 is in association with and methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein arginine methyltransferase that plays a pivotal role in ESCs. We found that CARM1 facilitates Sox2-mediated transactivation and directly methylates Sox2 at arginine 113. This methylation event enhances Sox2 self-association. Furthermore, the physiological retention of Sox2 on chromatin restricts the Sox2 methylation level. Our study reveals the direct regulation of Sox2 by CARM1 that sheds lights on how arginine methylation signals are integrated into the pluripotent transcription factor network. We also show that methylated Sox2 is more prone to self-associate than its unmethylated counterpart. In addition, the methylation level of Sox2 is restricted by the tight association of Sox2 with chromatin. Our data suggest a direct relationship between arginine methylation and Sox2 function.fgf4 and utf1 are typical Sox2 target genes fgf4 and utf1 respectively, to enrich endogenous Sox2 from P19 embryonal carcinoma cells Because CARM1 is required for the identity maintenance of ESCs, we supposed that CARM1 might directly regulate the core pluripotent transcription factor network. To verify this hypothesis, we first examined the interaction of CARM1 with Sox2, one of the most critical transcription factors during embryogenesis To investigated the impact of CARM1 on Sox2-meditated transactivation, we constructed a Sox2 reporter plasmid 8×S/O-luc  to inhibit These results suggest that Arg113 methylation promotes Sox2 self-association but does not affect Sox2 subcellular localization or degradation.Because CARM1 localizes primarily to the cytoplasm and Sox2-mNLS1 displays an increased co-localization with CARM1 in MCF7 cells , which iIn this work we demonstrate that CARM1 is in association with Sox2 and facilitates Sox2-mediated transactivation. We identify Sox2 as a novel substrate for CARM1. Methylated Sox2 has increased self-association, but displays no obvious change in protein stability or subcellular localization. However, the Sox2 methylation level is restricted by the tight association of Sox2 with chromatin.CARM1 has been shown to act as a coactivator of a number of transcription factors through direct modification of histone arginines, and subsequently, it increases local chromatin accessibility for transcriptional machinery The structure of the HMG box and the residues flanking methylation site of Sox2 are conserved among Sox family members . The Sox2 coding sequence was subcloned into EGFP-C1 to construct GFP-Sox2. Myc-CARM1 was constructed by the insertion of the CARM1 CDS into pCMV-tag-3B.To obtain the 8×(S/O)-luciferase reporter construct, four copies of annealed oligonucleotides containing two Cells were transfected using the Vigofect reagent  in accordance with the manufacturer's instructions.t-test.For the firefly luciferase activity analysis, the CMV-Renilla plasmid was used as an internal control to normalize for transfection efficiency. The statistical significance  was calculated by unpaired Student's For CARM1 knock down in P19 cells, nineteen base-pair of oligo-RNA segments  were transfected with Lipofectamine 2000 (Invitrogen) into P19 cells. The cells were harvested after 36 hours for RNA extraction.Total RNA was purified with Trizol reagent (Invitrogen) according to the manufacturer's instructions. One microgram of total RNA was used for cDNA synthesis with a Reverse transcription kit (Invitrogen). Quantitative real-time PCR was performed using the ExTaq Mix kit (Takara), and the primer sequences used are listed as follow: TCCATGACCAGCTCGCAGA; R: GAGGAAGAGGTAACCACGGGSox2, F: TCACTCACATCGCCAATCA; R: GTAGCCTCATACTCTTCTCGTTOct3/4, F: GGCAGCCCTGATTCTTCTAC; R: CGCTTGCACTTCATCCTTTNanog, F: CGGCCAAAAGGGAAGAACAT; R: CATTCCTTGGCATGATGGTCTLin28, F: GGCTTCGGCGGCTCTACT; R: AGGATTCGTAGGCGTTGTAGTTGFgf4, F: TCCTCTTACGAGCACCGACAC; R: GCAACGCGGTATTCAACGAUtf1, F: TGTGGCTGGAATGCCTACTG; R: TTGGACAATGCCCGTGCTCARM1, F: GAAGGTGAAGGTCGGAGTC; R: GAAGATGGTGATGGGATTTGAPDH, F: E. coli BL21 and purified as per Pharmacia's instructions. Methylation assays were performed as described 3H]S-adenosylmethionine (SAM) or unlabeled 100 µM SAM and brought up to a total reaction volume of 50 µl. Reactions were incubated at 30°C for one hour. Samples were resolved by SDS-PAGE and stained with Coomassie brilliant blue. After destaining, gels with radioactively labeled samples were visualized by fuorography with EN3HANCE (PerkinElmer) according to the manufacturer's instructions. Gel bands corresponding to methylated substrates with unlabeled SAM were excised, cut into small cubes, digested in-gel with trypsin and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis . MS/MS spectra were analyzed using Bioworks 3.1 software.Protein coding sequences of PRMTs and Sox2 were inserted into the pGEX-6p-1 plasmid individually, and GST-fusion proteins were expressed in 2, 10 mM KCl, 0.5 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and left on ice for 5 minutes. NP-40 was added to 0.3%, and samples were immediately pipetted up and down to disrupt cell membranes. Samples were centrifuged at 500 g for 5 min at 4°C. Supernatants were then centrifuged at maximum speed  for 15 min at 4°C, and supernatants were stored as Cytoplasmic Extract ‘CE’. Nuclei pellets obtained as above were then lysed in 500 µl RIPA buffer  for 30 minutes with gentle rotation. Samples were centrifuged at maximum speed  for 15 min at 4°C, and the supernatants were stored as ‘nucleosol’, mainly containing the physiologically soluble nuclear material. To obtain the ‘chromatin’ fraction, the pellets from above were washed twice with RIPA buffer, then resuspended in 500 µl RIPA buffer (containing protease inhibitors) with NaCl up to 500 mM, and extracted for an hour. The supernatants were then collected.Fractions of MCF7 cells were prepared as previously described GST Pull-down, co-immunoprecipitation, immunoblotting and immunostaining assays were carried out as previously described Cell permeabilization assays were conducted as previously described fgf4 or utf1 were incubated with 1 mg whole cell extracts of P19 cells and streptavidin-conjugated agarose beads (Millipore) overnight at 4°C. Then the beads were collected and washed five times with lysis buffer and prepared for SDS-PAGE and detected by immunoblotting. The sequences of oligo-DNA are indicated as below:DNA pull-down assays were performed as previously described fgf4, sense strand:biotin-CTGAAAGAAAACTCTTTGTTTGGATGCTAATGGGATACTAAGCTGA;antisense strand:biotin-TCAGCTTAGTATCCCATTAGCATCCAAACAAAGAGTTTTCTTTCAG.utf1, sense strand:biotin-CTGAAAGATGAGAGCCCTCATTGTTATGCTAGTGAAGTGCCAAGCTGA;antisense strand:biotin-TCAGCTTGGCACTTCACTAGCATAACAATGAGGGCTCTCATCTTTCAG.Rdi-methylPRRKTKC (asymmetric di-methylarginines are underlined) and purified using a peptide-affinity column.Antibodies are listed as follows: anti-GFP , anti-CARM1 , anti-FLAG , anti-Gapdh , anti-Lamin-B , and anti-Sox2 . A polyclonal rabbit antibody against di-methylated Arg113 of Sox2 (anti-methyl-Sox2) was raised using the methylated peptide KEHPDYXYFigure S1Schematic representation of 8×S/O-luc reporter plasmid. 8×S/O-luc carries eight copies of tandem fgf4 enhancer elements consisting of neighboring binding sites for Sox2 and Oct3/4.(JPG)Click here for additional data file.Figure S2The effect of CARM1 overexpression on the expression of pluripotency-related genes in P19 cells. The relative mRNA levels of pluripotency markers were normalized to Gapdh.(JPG)Click here for additional data file.Figure S3The effect of CARM1 knockdown with synthesized siRNA on the expression of pluripotency-related genes in P19 cells. The relative mRNA levels of pluripotency markers were normalized to Gapdh. Control: scrambled siRNA.(JPG)Click here for additional data file.Figure S4MS/MS spectrum of the Sox2 peptide EHPDYKYRdi-methylPR. GST-Sox2 methylated by GST-CARM1 was digested with trypsin and subjected to LC-MS/MS analysis. The MS/MS spectrum of a peptide corresponding to residues 106-115 of Sox2 showed that Arg113 was di-methylated. An asterisk indicates the methylated arginine.(JPG)Click here for additional data file.Figure S5Specifically recognition of Sox2 peptide methylated at Arg113, by anti-methyl-Sox2 antibody. Un-methylated peptide KEHPDYXYRPRRKTKC (Arg113 is underlined) designated as ‘R113me0’, and the peptide asymmetrically di-methylated at Arg113 named as ‘R113me2’ were used in dot blot analysis.(JPG)Click here for additional data file.Figure S6Schematic representation of Sox2 nuclear localization signals. The key residues of NLSs are underlined, and an asterisk indicates the identified methylation site Arg113. The residues that are mutated in Sox2-mNLS1 are also shown.(JPG)Click here for additional data file.Figure S7CARM1-mediated methylation may be a regulation mechanism shared by certain other Sox family members. Alignment of Sox members highly conserved in sequences flanking Arg113 was shown. NLS2 was underlined, Arg113 and its counterparts in other Sox proteins were pointed out with asterisks, conserved residues were indicated in shadow.(JPG)Click here for additional data file.


We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution. The classical paradigm that protein functionality requires a pre-formed three-dimensional structure has significantly eroded over the last decade under mounting evidence regarding the role of intrinsically disordered proteins (IDPs), inherently devoid of a defined three-dimensional structure Here we demonstrate for the first time the systematic use of cell-free protein synthesis (CFPS) E. coli-based batch CFPS In vivo, regions of low-complexity and disorder of overexpressed heterologous proteins are often prone to proteolytic degradation, which may decrease the yields or even prohibit protein production. In CFPS, the proteolysis is circumvented by short expression time and by adding protease inhibitors directly into the reaction CFPS is an established tool for producing functional protein samples E. coli-based batch CFPS system 15N/13C labeled protein samples. The most expensive ingredient of the reaction (72% of the costs) is the mixture of double-labeled amino acids (1 mg/mL). With the high level of amino acid incorporation in our CFPS We use a low-cost, Chemical shifts report on IDP structure and interactions Validation of the results of complex analysis such as protein sequential assignment or structure calculation is an important problem In this work, the quality of the obtained assignments and existence of alternative solutions are evaluated with a newly presented data resampling method. The statistics on multiple assignments is obtained by repeating steps (ii–iv) of the TA procedure described above, on a number of data subsets, each containing 70% of the randomly selected points acquired in the triple resonance experiments. This jackknife-type statistical procedure addresses the uncertainties originating from the experimental data and reveals errors and ambiguities in the peak lists and potential instabilities of the downstream analysis.de novo backbone assignments obtained using the presented CFPS-TA approach. These include five out of six TCR and BCR cytosolic domains, as well as mutated and modified forms of the domains. The experiments were performed in “native” (phosphate buffer), denaturing (6 M urea), and helix-promoting  conditions. Depending on the sample, the TA measurement time required for the backbone assignment varied from 0.8 to 47 hours.The lowest protein concentration, for which assignment was obtained, is 30 µM. It should be noted that apart from the relatively low protein concentrations the assignments were obtained despite of low signal dispersion and strong peak overlap, which is typical for IDPs. The signal separation in the spectra of our samples was significantly lower than the average of the BMRB database  for the de novo assignments for six CD79a samples were necessary due to rearrangement of the signal positions exacerbated by the strong peak overlap. The previously published 13C secondary chemical shifts of CD79a in native and TFE states referenced versus the so-called intrinsic random coil shifts Herein, we have presented an efficient methodology for extensive experimental characterization of highly disordered proteins in solution. Rapid and economical sample preparation and backbone assignment has been demonstrated for several TCR and BCR cytosolic domains. The technique allows chemical shifts perturbation analysis in multitude of IDP samples including site-specific mutants, chemical modification, different solution compositions, and in the presence of molecular interactions. The approach is demonstrated for five 6–13 kDa cytosolic domains of T- and B-cell receptors. In favorable cases, the three-dimensional BEST-TROSY spectra provide sufficient resolution for assigning IDPs up to 30 kDa 13C/15N labeled amino acids were from CIL.All chemicals were from Sigma-Aldrich except where stated otherwise. TOP10 and BL21(DE3)-Rosetta 2 cells were from Life Technologies and Merck, respectively. Constructs for the human ITAM-containing cytosolic subunits were cloned. The cDNAs for CD79a , CD79b , CD3δ , CD3ε , CD3γ  and ξ  were purchased from Geneservice and subcloned into pEXP5-NT (Invitrogen), resulting in constructs with a 6xHis-tag fused to the N-terminus, followed by a TEV protease recognition site. The double mutant constructs of CD79a (K4C/C35S and Y25E/Y36E) were made by two rounds of QuickChange II XL Site-Directed Mutagenesis (Agilent Technologies). All clones were verified by sequencing. Cleavage with TEV protease resulted in 2 non-native residues (S and L) at the N terminus of the constructs. The vectors were transformed into One Shot® TOP10 chemically competent cells (Invitrogen). 100 ml cultures were grown overnight at 37°C, 200 rpm, and plasmids were purified with the Plus Maxi Kit (Qiagen).E. coli S12 extract was used for production of the peptides, as described 15N/13C labelled amino acids. 7 ml of the reaction was sufficient for producing 0.4 mg of pure CD79a/CD3ε. The identity and integrity of all expressed proteins have been confirmed using either mass spectrometry or NMR assignment. The cell-free synthesized human cytosolic subunits were purified for further analysis. The lysate from the centrifugation step was separated from the pellet and mixed 1∶2 with 20 mM Tris buffer pH 8.1, 8 M urea for 10 minutes. Ni-NTA resin (Qiagen) was equilibrated using buffer A  and mixed with the protein-Tris buffer solution. Incubation at 4°C for 1.5 hours was followed by gravity flow purification with extensive washing using buffer A and buffer B . For protein elution, one-hour incubation with four times the gel column volume of buffer A (with 250 mM imidazole) was used. Imidazole was removed with PD10 desalting columns . The protein samples were mixed (10∶1) with his-tagged TEV protease prepared as described A batch format cell-free expression system based on an 2O and 2 mM DTT for cysteine-containing IDPs). For attachment of MTSL -methanethiosulfonate  the NMR samples were treated with 10 mM DTT. A PD10 desalting column  was used to remove DTT and to change buffer to 10 mM NaPi, pH 7.0. 3–5 fold molar excess of MTSL dissolved in acetone was added and incubated overnight at 4°C. Unreacted MTSL was removed with a PD10 column followed by lyophilization of the sample. Reduced MTSL was obtained by adding 2 mM ascorbic acid to the solution.Lyophilized fractions were dissolved in NMR buffer , 7–15% D15N chemical shift evolution was set to the corresponding constant time period for the 13C-15N magnetization transfer; acquisition times for 13Cα/13Cβ and 13CO evolution were set to 12 ms and 20 ms, respectively.nussampler from MDDNMR software package 13Cα and 13Cβ evolution according to the single-bond 13C -13C homonuclear coupling constant of 35 Hz. The bias for the 13CO spectral dimension was set according to assumed transverse relaxation time of 20 ms.The NUS schedules were prepared by program 13Cα, 13Cβ and 13CO resonances were calculated by subtracting intrinsic random coil chemical shifts Secondary chemical shifts (SCS) for TANSY (Targeted Acquisition NMR Spectroscopy) procedure, which we introduce in this work, combines several techniques to achieve high efficiency of data collection and analysis. For the backbone assignment, it uses a set of fast-pulsing triple resonance BEST-TROSY experiments http://python.org/) with QT (http://qt.nokia.com/) based graphical interface. PAM uses MDDNMR http://cara.nmr.ch/) for manual inspection of the assignment and further analysis. TANSY software is available from the authors upon request.TANSY software consists of two separate modules: the acquisition module (AM) and the processing and analysis module (PAM). The former is running on an NMR spectrometer and organizes the stepwise incremental data collection. AM is implemented using macro and graphical interface features of VnmrJ  and TopSpin (Bruker Biospin) spectrometer software. AM runs a set of NMR experiments using a NUS schedule. The acquired data are continuously saved to computer disk and become immediately available for the downstream processing by PAM. The PAM can operate in real-time with the AM or can be used as an off-line processing and analysis tool. PAM is implemented in the Python programming language +I sin(ωN t).cos(ω The 13CO time domain shapes are presented as products of known frequency CO t)+I sin(ωCO t)cos CD79a, (B) CD79b, (C) CD3ε, (D) CD3γ. Crowded regions are shown as inserts.(EPS)Click here for additional data file.Figure S2Peak separation index (PSI) versus protein size. PSI is calculated as a sum of ten minimal pairwise distances in 2D 1H-15N plane between assigned amide groups for each protein: Hδ and ΔNδ are ppm distances in amide proton and nitrogen dimensions, respectively. (yellow) PSIs of 10% of randomly chosen proteins from full BMRB database; (blue) 65 protein entries found in the IDP subset of BMRB; (red) twelve TCR and BCR cytosolic domains constructs: (1) CD3γ; (2) CD79b; (3) CD3ε, 20% TFE; (4) CD3ε in 6 M urea; (5) CD3ε; (6) CD79a TFE 20%, (7) CD79a and CD79a in 6 M Urea; (8) CD79a MTSL; (9) CD79a Y25E/Y36E and K4C/C33S; (10) TCRζ.(EPS)Click here for additional data file.Figure S3CD spectra of CD79a samples. Far-UV circular dichroism spectra (190–250 nm) were recorded at 25°C on a ChiraScan CD spectrometer (Applied Photophysics) with 0.38 mg/ml CD79a using a 0.1 cm path-length quartz cuvette. 5 mM NaPi pH 6.7 buffer with 0.5 mM DTT was used for sample dilution. The sample blank was subtracted and 5 scans averaged for the protein. (thick line) CD spectra for CD79a in buffer, (thin line) 10% trifluoroethanol (TFE), (grey line) 20% TFE and (dashed line) 30% TFE. Under native condition, CD79a shows a characteristic random coil curve with the minimum around 200 nm. As TFE concentration increases, the CD spectra gradually adopt alpha helical features . The presented CD spectrum for the native condition is similar to the spectrum reported in (EPS)Click here for additional data file.Table S1The NMR experimental parameters for backbone assignments.(PDF)Click here for additional data file.Table S2Measurement times (min) needed for reaching 90% completeness of the sequential backbone assignment for CD79a samples at four different protein concentrations.(PDF)Click here for additional data file.


The genetics of primary CoQ10 deficiency has been described in several studies, whereas the influence of common genetic variants on CoQ10 status is largely unknown. Here we tested for non-synonymous single-nucleotidepolymorphisms (SNP) in genes involved in the biosynthesis , reduction  and metabolism (apoE3/4) of CoQ10 and their association with CoQ10 status. For this purpose, CoQ10 serum levels of 54 healthy male volunteers were determined before (T0) and after a 14 days supplementation (T14) with 150 mg/d of the reduced form of CoQ10.Coenzyme Q0, the CoQ10 level of heterozygous NQO1P187S carriers were significantly lower than homozygous S/S carriers . For this polymorphism a structure homology-based method (PolyPhen) revealed a possibly damaging effect on NQO1 protein activity. Furthermore, CoQ10 plasma levels were significantly increased in apoE4/E4 genotype after supplementation in comparison to apoE2/E3 genotype . Likewise heterozygous CoQ3G272S carriers had higher CoQ10 plasma levels at T14 compared to G/G carriers but this difference did not reach significance .At TP187S and apoE polymorphisms influence CoQ10 status in humans.In conclusion, our pilot study provides evidence that NQO1 Synthesized in the mitochondrial inner membrane, CoQ10 is comprised of a ubiquinone head group attached to a trial of 10 five-carbon isoprenoid units, that anchors the molecule to the membranes [10, however it can also be acquired through the diet and dietary supplements [10 acts in the respiratory chain and is necessary for pyrimidine biosynthesis as well as a cofactor of uncoupling proteins [10 has been also identified as a modulator of gene expression [Coenzyme Qplements . CoQ10 aproteins . CoQ10 hpression -6, inflapression -9 and appression .10 biosynthetic pathway comprises 10 steps, including methylations, decarboxylations, hydroxylations and isoprenoid synthesis and transfer [E. coli and S. cerevisiae [10 synthesis causes mitochondrial dysfunction, as CoQ10 carries electrons from complex I and complex II to complex III in the mitochondrial respiratory chain. Several forms of human CoQ10 deficiencies were characterized by infantile encephalomyopathy, renal failure, cerebellar ataxia or myopathy [The CoQtransfer . The elurevisiae ,14. In hmyopathy -17.10 biosynthesis suggests that genetic defects in different biosynthetic enzymes or regulatory proteins may cause different clinical syndromes. Although several studies have been undertaken to look into primary CoQ10 deficiency, the influence of common genetic variants on CoQ10 status is largely unknown. Therefore a proof of principle study in humans was performed to associate single nucleotide polymorphisms (SNPs) in genes encoding proteins of CoQ10 biosynthesis, reduction and metabolism with CoQ10 status before and after supplementation.The complexity of CoQ10  daily in form of three capsules with each principal meal for 14 days. Fasting blood samples were taken before (T0) and after (T14) supplementation with ubiquinol from all study participants. The participants, aged 30.1 ± 6.7 years, had an average Body Mass Index (BMI) of 24.1 ± 2.5, no history of gastrointestinal, hepatic, cardiovascular or renal diseases, a habit of non- or occasional smoking (≤ 3 cigarettes/day) and maintenance of usual nutrition habits. The study was approved by the ethics committee of the Medical Faculty of Kiel University, Germany, and was conformed to Helsinki Declaration. All volunteers gave written informed consent.Sample characteristics of subjects and study design have been recently described . In shorGenomic DNA was isolated from whole blood samples. Genotyping of all SNPs investigated Table  was perf10 analysis was based on the method of high-pressure liquid chromatography (HPLC) with electrochemical detection and internal standardisation using ubihydroquinone-9 and ubiquinone-9 as standards and has been described elsewhere [CoQlsewhere .t-test and additionally for CoQ6M406V the χ2 -test in a dominant genetic model. To determine statistical significance between all genotypes, test for linear trend in one way analysis of variance (ANOVA) was performed. P-values ≤ 0.05 were considered statistically significant and all statistical analyses were computed using SPSS (Version 13.0). In order to analyze the impact of non-synonymous SNPs on the structure and function of proteins, PolyPhen server [Data are expressed as means ± SD. Differences in the characteristics of the study population between two genotype groups were examined using the Student n server  was used10 status, we searched in the HapMap data base for non-synonymous variants in genes which are involved in CoQ10 biosynthesis and metabolism. As shown in table 10 biosynthesis. Functional variants [10. Furthermore they protect cells from oxidative damage by catalyzing reduction of carcinogenic quinone compounds to their hydroquinone forms [In order to identify common SNPs which may be associated with the CoQvariants ,21 in thne forms . Two SNPne forms ,24, athene forms , inflammne forms , lipid pne forms  and longne forms .G272S polymorphism revealed 38 homozygous for G/G (73%), 13 heterozygous for G/S (25%) and 1 homozygous for S/S (2%), while 1 sample failed genotyping. Analysis of the CoQ6M406V genotype showed 19 homozygous for M/M (36%), 24 heterozygous for M/V (44%) and 11 homozygous for V/V (20%). Genotyping of CoQ7M103T polymorphism revealed 25 M/M (48%), 17 M/T (33%) and 10 T/T (19%) carriers. Two samples failed genotyping. Concerning the distribution of the NQO1P187S SNP, 30 persons are carriers of two P/P alleles (56%), 22 persons were heterozygous with one P and one S allele (41%) and two participants were carriers of two S/S alleles (3%). NQO2L47F genotyping displayed 35 participants were homozygous L/L carriers (65%), 15 participants were heterozygous for L/F (28%) and 4 participants were homozygous F/F carriers (7%). The genotype distribution of apoE was as follows: 1 person with E2/E2 genotype (2%), 7 persons with E2/E3 (14%), 29 persons with E3/E3 (58%), 11 persons with E3/E4 (22%) and 2 persons with E4/E4 (4%). For 4 persons, genotyping of one or both SNPs respectively failed. Thus, the Apo E genotype distribution in our cohort of 54 healthy men was comparable with previously published data [The selected SNPs were genotyped in 54 healthy male volunteers. The obtained genotype distribution Figure  and 2 we10 daily in form of three capsules with each principal meal for 14 days. This supplementation led to a significant 4-fold increase in total CoQ10 plasma levels at T14 (4.60 ± 1.55 μmol/L) compared to T0 (0.96 ± 0.31 μmol/L) [10 levels. Trend analysis (ANOVA) over all genotype variants of CoQ7M103T and NQO2L47F revealed p values >0.05 and were therefore considered as not significant.As previously described , 54 heal μmol/L) . As show10 [10 levels at T0, no significant differences between genotypes could be revealed. Yet at T14, G/S carriers in CoQ3G272S genotype had a higher total CoQ10 content (5.30 ± 0.96 μmol/L) after supplementation compared to G/G carriers (4.42 ± 1.67 μmol/L) with borderline significance .The COQ3 gene encodes an O-methyltransferase required for two steps in the biosynthetic pathway of CoQ10 . Analysi10 biosynthesis for incorporation of oxygen to the benzoquinone ring [10 plasma levels were not significantly changed within genotype distribution of CoQ6 rs8500 SNP before (T0) and after (T14) supplementation. However, considering total CoQ10 distribution at T0 in a chi-square cross tabulation as a function of CoQ6 rs8500 genotype . This disclosed a total of 898 individuals are required to receive 95% power.CoQ6 is mapped to human chromosome 14q24.3 and encodes a monooygenase, which is required in CoQone ring . CoQ10 pP187S SNP was associated with CoQ10 levels at T0 . Thus, this pilot study indicates that Pro187Ser SNP in NQO1 gene could participate in abnormal CoQ10 metabolism. SNP prediction of functional effects of human nsSNPs with structure homology-based method (PolyPhen) revealed a possibly damaging effect of NQO1P187S SNP with a score of 0.215. However, genotype distribution of the S/S genotype was low (n = 2), which reflects the ethnic variation of this polymorphism with the highest prevalence of the S allele in East Asian populations  and the lowest prevalence in Caucasians (4%) [10 metabolism in an East Asian population may be preferable.It has been shown, that NQO1 can generate and maintain the reduced state of ubiquinones in membrane systems and liposomes, thereby promoting their antioxidant function ,34. NQO1ans (4%) . Furtherans (4%)  found a 10 status. At T0, total CoQ10 levels were higher in E4/E4 carriers as compared to all other genotype groups, however p values did not reached significance  in E4/E4 carriers after supplementation (T14), which has to the best of our knowledge not been shown so far. Thus, E4/E4 carriers may be more responsive towards a dietary CoQ10 supplementation than non E2/E3 carriers. The underlying physiological and/or molecular mechanisms for this finding still need to be elucidated.Apolipoprotein E (apoE) is a polymorphic multifunctional protein with three common isoforms in humans . Presence of the apoE4 allele is associated with a 40-50% higher risk of cardiovascular disease . There iP187S and apoE polymorphisms may influence CoQ10 status in humans. According to our results and power calculation, larger cohorts are needed in further studies to determine the association between single nucleotide polymorphisms in genes encoding proteins of CoQ10 biosynthesis, reduction and metabolism and CoQ10 status.Taken together, our pilot study with 54 volunteers provides evidence that NQO1The authors declare that they have no competing interests.10 measurements. FD was responsible for the concept and design of the study and the writing of the paper. All authors read and approved the final manuscript.AF analysed the data and wrote the manuscript. CS participated in the design of the study, acquired and analysed the data. GR participated in the design of the study and critically revised the manuscript. PN and TM carried out the CoQ