Psychrobacter aquaticus strain CMS 56T, isolated from a cyanobacterial mat sample collected from a water body in the McMurdo Dry Valley region of Antarctica.We report the 3.2-Mb draft genome sequence of  Psychrobacter aquaticus strain CMS 56T is a Gram-negative, nonmotile, halotolerant, psychrophilic bacterium, isolated from a cyanobacterial mat sample collected from various water bodies in the McMurdo Dry Valley region of Antarctica  pyrosequencing platform, which yielded a raw sequence of 136,405,203 bp in 411,734 reads, which is 40× coverage of the genome. Assembly of the raw sequencing data was carried out using GS De Novo Assembler (v 2.8), which yielded a genome sequence of 3,216,409 bp in 38 contigs. All of the contigs were larger than 561 bp, with the longest contig being 535,134 bp long with a G+C mol% of 42.63%. Annotation of the assembled genome was performed using RAST (Rapid Annotation using Subsystem Technology)  were also predicted in the annotation.The http://www.theseed.org), revealed that Psychrobacter arcticus 273-4, Psychrobacter cryohalolentis K5, Moraxella catarrhalis RH4, and Enhydrobacter aerosaccus SK60, with similarity score values of 542, 521, 368, and 281, respectively, are the closest neighbors of Psychrobacter aquaticus CMS-56T. The whole-genome sequence of Psychrobacter aquaticus CMS-56T, isolated from Antarctica, will help in our understanding of the adaptation of psychrophiles to harsh conditions.The functional comparison of the genome sequence, performed using the SEED framework for comparative genomics (Psychrobacter aquaticus strain CMS 56T has been deposited in DDBJ/EMBL/GenBank under the accession number AUSW00000000. The version described in this paper is version AUSW01000000.The draft genome sequence of


E. coli (STEC), but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST) 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC). Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain , STEC , EAEC , and ESBL-production .Ruminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli.The faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28%) shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.While we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing  E. coli (STEC) O104:H4 strain, belonging to the HUSEC041 clone (HUS-associated enterohaemorrhagic E. coli) because of this specific serotype [E. coli (EAEC) of the same serotype, which was previously isolated from a patient in Africa. Therefore, this E. coli strain combines virulence traits of EAEC and STEC [The month of May 2011 marked the beginning of an outbreak of haemolytic uremic syndrome (HUS) caused by an unusual Shiga toxin-producing and STEC ,5-8.The principal reservoir of STEC strains are ruminants. However, they have not been reported to harbour STEC O104:H4 nor EAEC -11. To ath, 2011, during the time of the HUS outbreak, we visited a local abattoir and collected faecal samples of 100 animals, which were slaugh-tered on that day. As shown in Figure med = 2; Xarith = 2.94). After cultivation , 20 coliform colonies per animal  were picked according to the colony morphology and further investigated in a two-step-process: (i) testing the investigated colonies for the presence of the O104:H4 HUS outbreak strain, and (ii) testing the investigated colonies for the presence of STEC and EAEC as well as ESBL-positive strains using both PCR and phenotypic methods.On June 6E. coli isolates derived from the 100 faecal samples investigated, not a single one showed the O104:H4 HUS outbreak strain-specific combination of the genes stx2, terD, O104rfb, and H4 fliCdetected by this Multiplex-PCR. Thus, not a single animal shed the O104:H4 outbreak strain.In the first part of the present study, the strains were tested using the Multiplex-PCR developed by Bielaszewska et al. (2011) for rapid detection of the outbreak strain. Out of 2.000 stx1, stx2, bfpA, and escV; (ii) enteroaggregative E. coli (EAEC) properties, especially the occurrence of the genes pAA, aggR, but also the enteroaggregative adhesion pattern; and (iii) ESBL phenotype.The second part of the study focussed on the question, whether STEC, EAEC, or ESBL-positive strains are present in the investigated population. For this purpose, the strains were further tested for (i) STEC and EPEC properties, namely stx1 or stx2 or both . As for the bfpA gene, we were unable to find any positive isolate, thus no typical EPEC were identified.(i) Twenty-eight of the 100 faecal samples tested harboured STEC. One single sample could be positive for different STEC strains, that is, harbouring isolates either positive for pAA or aggR. In contrast, a large number of animals shed isolates that reacted positively for astA, the gene encoding an enterotoxin, which had originally been identified in EAEC, but is not specific for this pathotype. In summary, while STEC were shed by 28% of the investigated animals, not a single animal shed EAEC.(ii) None of the animals shed any isolates that showed typical genetic features of EAEC, as not a single sample could be identified, that harboured isolates reacting positive for E. coli as identified using the CHROMagar orientation supple-mented with cefotaxime. These animals originated from nine different farms. PCR based screening and subsequent sequence analysis revealed blaCTX-M-1 (n = 12) as the most common ESBL-resistance determinant. Additionally, we found a single blaTEM-positive isolate, which was identified as blaTEM52, and another ESBL-producing isolate positive for blaCTX-M-15. As outlined in Table blaCTX-M-15-positive strain IMT26356 harboured genes terD and astA. Also, four of the blaCTX-M1-positive strains were positive for H4fliC, the gene encoding H-antigen 4, but none possessed O104-encoding genes.(iii) Eleven of the one hundred animals tested shed a total of 14 ESBL-producing M-1 n = 1 as the mE. coli strains is more accurately reflected by its sequence type than by its serotype, we first sequenced the two housekeeping genes icd and mdh, the alleles of which are able to specifically determine strains of ST678. As the outbreak strain belongs to ST678, the unique combination of icd 136 and mdh 9 determines the existence of any phylogenetically related strain. The 86 strains were selected for MLST on the following bases: (i) ESBL-producing strains, (ii) strains positive for stx2, (iii) strains positive for H4fliC, and (iv) strains positive for terD. However, we did not find the unique ST678 allele combination of icd 136 and mdh 9 in any of these investigated strains. While icd allele 136 was never found, 18 strains contained mdh allele 9. Of these 18 strains that where potentially related to each other due to the common mdh allele, we identified 10 strains to be related to ST678 by Maximum Parsimony analysis  we tested these animals during the HUS outbreak ,14 and , while strain 17-2  [E. coli strain E2348/69  was used as an EPEC reference strain  and HUS; O3:H2)  served ath, 2011, faecal samples were taken from the gut of a total of 100 cattle, slaughtered on that day in one abattoir. The animals originated from 34 different farms in Northern Germany, the centre of the 2011 HUS outbreak. After diluting the samples in a ratio of 1:2 with PBS and vortexing, the samples were streaked on (i) CHROMagar Orientation , (ii) CHROMagar supplemented with cefotaxime (4 μg/ml) and (iii) Gassner agar plates . After 18 h of incubation at 37°C a total of 20 coliform colonies per animal were picked from these three plates based on colony morphology and streaked on blood agar, resulting in the total number of 2.000 coliform colonies that were further investigated. The next day, the colonies were grouped into 4 pools per animal, each pool containing material of five coliform colonies and after heat lysis treatment  the pools were analysed using three different Multiplex-PCRs (see below). If any gene detected in these Multiplex-PCRs yielded a positive result, the PCR was repeated with each single colony of the pool. Positive colonies were puri-fied and tested biochemically to confirm that these were E. coli after which each colony was now referred to as a bacterial strain.On June 6E. coli on CHROMagar agar plates supplemented with cefotaxime were also tested bio-chemically to confirm they were E. coli, and ESBL-production was confirmed according to the CLSI criteria [blaCTX-M, blaTEM and blaSHV [Colonies showing growth and the typical phenotype of criteria . Colonied blaSHV .stx2, terD, rfbO104, and fliCH4 [Two of the three Multiplex-PCRs utilized in this study have been published previously. In brief, for the detection and identification of the O104:H4 outbreak strain, we used the recently described Multiplex-PCR for d fliCH4 .E. coli (EPEC) were identified using a Multiplex-PCR including stx1 and stx2 (STEC) as well as escV and bfpA (EPEC) [pAA, aggR, and astA by a Multiplex-PCR based on published primer sequences and single PCR protocols, respectively. Briefly, primers for the detection of the virulence-associated factors pAA (5'-CTGGCGAAAGACTGTATCAT-3' and 5'-CAATGTATAGAAATCCGCTGTT-3') [aggR (5'-GTATACACAAAAGAAGGAAGC-3' and 5'-ACAGAATCGTCAGCATCAGC-3') [astA (5'-TGCCATCAACACAGTATATCC-3' and 5'-TAGGATCCTCAGGTCGCGAGTGACGGC-3') [2, 2U Taq DNA polymerase , 0.5 μl of each 10 mM dNTP, 0.1 μl (100 pmol) oligonucleotide primer pair, and 4 μl template DNA, supplemented with the appropriate volumes of double-distilled water. Reaction mixtures were subjected to the following temperature profiles: 5 min at 94°C; 25 cycles of 30 s at 94°C, 1 min at 56°C, 45 s at 72°C, with a final circle of 10 min at 72°C and a hold at 10°C.In a second part of the study, STEC and enteropathogenic A (EPEC) . The scrTGTT-3') , aggR (5CAGC-3') , as wellCGGC-3') , were chhttp://mlst.ucc.ie/mlst/dbs/Ecoli) can easily be identified by the analyses of two housekeeping genes only, namely icd and mdh, as the combination of icd allele 136 and mdh allele 9 specifically identifies this ST. Therefore, all identified STEC, phenotypic ESBL-producing E. coli and other isolates that harboured at least some of the genetic features common to the outbreak strain were further analysed by sequencing these two alleles.ST678, the ST of the current HUS outbreak strain, according to the scheme described by Wirth et al. (2006)  (http://http://www.megasoftware.net) and CLC Genomics Workbench 4.7 (http://www.clcbio.com). Based on these analyses, complete MLST was performed for all strains that seemed to be very closely related to ST678. MLST determination was carried out as described previously [All sequences generated for these two genes were concatenated and the phylogenetic relationship was inferred using the Maximum Parsimony method. Phylogenetic analyses were performed with Mega 5.05  for their adhesion pattern in HEp-2 cells, the gold standard for EAEC typing ,33. The 7 for theThe authors declare that they have no competing interests.Conceived and designed the study: LHW, AB, SG; Performed the experiments: AB, IE, BK, SG, EMA; Analyzed the data: LHW, TS, AB, LG; Wrote the paper: LHW, TS, AB, SG; Critically read the manuscript: HK, LG, EMA; Read and approved the final manuscript: LHW, TS, IE, EMA, BK, LG, HK, SG, ABMaximum Parsimony based clustering analysis of the concatenated sequences of the 7 genes used for MLST. Reference scale of tree is equal to 1 nucleotide substitution. Maximum Parsimony based clustering analysis of the concatenated sequences of the 7 genes used for MLST. Reference scale of tree is equal to 1 nucleotide substitution.Click here for fileCharacteristics of strains selected for cell adhesion assay (n = 27) and reference strains.Click here for file


The traditional Chinese medicine bufalin, extracted from toad’s skin, has been demonstrated to exert anticancer activities in various kinds of human cancers. The mechanisms of action lie in its capacity to induce apoptosis, or termed type I programmed cell death (PCD). However, type II PCD, or autophagy, participates in cancer proliferation, progression, and relapse, as well. Recent studies on autophagy seem to be controversial because of the dual roles of autophagy in cancer survival and death. In good agreement with previous studies, we found that 100 nM bufalin induced extensive HepG2 cell apoptosis. However, we also noticed bufalin triggered autophagy and enhanced Beclin-1 expression, LC3-I to LC3-II conversion, as well as decreased p62 expression and mTOR signaling activation in HepG2 cells. Blockage of autophagy by selective inhibitor 3-MA decreased apoptotic ratio in bufalin-treated HepG2 cells, suggesting a proapoptotic role of bufalin-induced autophagy. Furthermore, we investigated the underlying mechanisms of bufalin-induced autophagy. Bufalin treatment dose-dependently promoted AMPK phosphorylation while AMPK inhibition by compound C significantly attenuated bufalin-induced autophagy. Taken together, we report for the first time that bufalin induces HepG2 cells PCD, especially for autophagy, and the mechanism of action is, at least in part, AMPK-mTOR dependent. Hepatocellular carcinoma (HCC) is the most common type of liver cancer and causes more than 300,000 deaths in China each year . AlthougThe development and progression of HCC are believed to correlate with dysregulation of cancer cell proliferation and programmed cell death (PCD). Manipulating this balance would therefore provide significant clinical benefits . For insBufo bufo gargarizans Cantor, Bufo melanostictus Schneider, and Bufo raddei Sauch are species of toad found to be a source of bufalin, a soluble digoxin-like immunoreactive component also found in Chansu [In recent years, traditional Chinese medicines have attracted increasing interests because of a broad spectrum of biological activities. n Chansu . It has n Chansu . Severaln Chansu , gastricn Chansu , prostatn Chansu , liver cn Chansu , leukemin Chansu , melanomn Chansu , lung can Chansu , breast n Chansu , colon cn Chansu , and ostn Chansu . The mecn Chansu ,19. Althn Chansu ,20, the 50 of 143.2 nM  protein, and p62 (a selective target of autophagy) by western blotting. During autophagy, Beclin-1 was upregulated, LC3-I converted to LC3-II, and p62 was downregulated for the initiation and formation of autophagosomes . The expBecause autophagy is initially recognized as an adaptive response to nutrition deficient conditions, and breakdown of autophagosome contents allow non-essential proteins to be degraded and the amino acids recycled for the synthesis of essential proteins . In thesAMP-activated protein kinase (AMPK) is a principal energy-preserving intracellular enzyme activated in stress conditions that can induce autophagy through inhibition of the serine/threonine kinase mammalian target of rapamycin (mTOR), one major repressor of autophagy –35. The Bufalin, also termed 3-b, 14-Dihydroxy-5-beta-bufa-20,22-dienolide , was purMTT (methyl thiazolyl tetrazolium), DAPI (diamidino phenyl indole), 3-MA (an autophagy inhibitor), compound C  were purchased from Sigma-Aldrich . RPMI-1640 culture medium, and fetal bovine serum were purchased from Gibco . Rabbit anti-Beclin-1, LC3, p-mTOR, mTOR, p70S6K, p-p70S6K, p-AMPK, AMPK, β-actin polyclonal antibodies were purchased from Cell Signal Technology . HRP-conjugated goat anti-rabbit IgG were purchased from Abcam . Annexin V/PI apoptotic detection kit was purchased from Kehao Biotechnology .2 at 37 °C. In all experiments, exponentially growing cells were used.Human HCC HepG2 cells were purchased from the American Type Culture Collection (ATCC). Cells were routinely cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) in an atmosphere with 5% CO3 cells were incubated in 96-well plates in the presence of various concentrations of bufalin for 48 h. At the end of the treatment, 20 μL MTT (5 mg/mL dissolved in PBS) was added to each well and incubated for an additional 4 h at 37 °C. The purple-blue MTT formazan precipitate was dissolved in 200 μL of DMSO and the optical density was measured at 570 nm.Cell proliferation was assessed using the MTT assay. Briefly, 5 × 10DAPI, Annexin V, PI triple fluorescent staining for cancer cell apoptosis was performed as we described previously . BrieflyFor quantitative determination of apoptosis, cells were harvested with trypsinization at the 90% confluent after indicated treatment, stained by Annexin V-FITC and PI as have described, then assessed for cell apoptosis by flow cytometry . Each experiment was performed in triplicate.Autophagic HepG2 cells, in response to bufalin treatment, were detected through transmission electron microscopy as we have described . Cell peFor autophagic fluorescence observation, cells were grown on coverslips and treated with 100 nM bufalin with or without 1 mM 3-MA. After 48 h, cells were stained with 0.05 mM MDC at 37 °C for 1 h. The cellular fluorescence changes were observed using confocal laser scanning microscope .After indicated treatments, cells were collected and washed in ice-cold PBS, and lysed in RIPA buffer  for extraction of total protein. Protein concentrations were determined and equal amounts of sample were loaded on SDS-PAGE and transferred to nitrocellulose membranes at 400 mA for 1 h. Membranes were stained with 0.5% Ponceau in 1% acetic acid for confirmation. Blots were blocked for 2 h in TBST  containing 5% fat-free dried milk and then incubated with the primary antibodies overnight and then incubated with corresponding HRP-labeled secondary antibodies. β-actin was selected as an internal control. Protein bands were visualized by chemiluminescence with the enhanced chemiluminescent detection kit according to the manufacturer’s instructions.p < 0.05.Experiments presented in the figures are representative of three or more different repetitions. Values are expressed as mean ± SD. Clampfit software and ORIGIN6.1 software were used to analyze the data. One-way analysis of variance (ANOVA) was employed to determine the statistical significance between different groups. Significant difference was set at Collectively, the present study reports for the first time that bufalin induces apoptosis and autophagy in HCC HepG2 cells. The induction of autophagy by bufalin was associated with the activation of AMPK and the inhibition of mTOR signal pathway. These results suggest that bufalin may be applicable as a medical treatment for HCC. In addition, data from our study indicate that bufalin plays a proapoptotic role and facilitates apoptosis to promote PCD. The mechanisms of action are, at least in part, AMPK-mTOR signaling-dependent.


ATXN1, DTNBP1, JARID2, and NHLRC1 that we propose may be responsible for ASDs and developmental delay.Interstitial deletions of the short arm of chromosome 6 are rare and have been associated with developmental delay, hypotonia, congenital anomalies, and dysmorphic features. We used array comparative genomic hybridization in a South Carolina Autism Project (SCAP) cohort of 97 subjects with autism spectrum disorders (ASDs) and identified an ~ 5.4 Mb deletion on chromosome 6p22.3-p23 in a 15-year-old patient with intellectual disability and ASDs. Subsequent database queries revealed five additional individuals with overlapping submicroscopic deletions and presenting with developmental and speech delay, seizures, behavioral abnormalities, heart defects, and dysmorphic features. The deletion found in the SCAP patient harbors  FOXC1 gene (MIM 01090) ) embody a group of behavioral abnormalities, including restricted and repetitive behaviors, and/or defects in social interaction and/or communication. Cytogenetic abnormalities were initially reported in 25-30% of individuals with ASDs and dysmorphic features , whereasUsing array CGH to search for CNVs that may be associated with ASDs in the patients from the SCAP collection, we identified a deletion at chromosome 6p22.3-p23 in a 15 year-old boy with ASDs. Further database [Medical Genetics Laboratories (MGL) and Signature Genomic Laboratories (SGL) diagnostic laboratories] and literature queries revealed additional five and 12 overlapping interstitial deletions, respectively. Patients 1, 2, and 4-6 had developmental and speech delays, features commonly seen in the published patients with the deletions in chromosome 6p22.3-p24.3   patient ATXN1 (deleted in 10 patients), JARID2 (deleted in eight patients), DTNBP1 (deleted in eight patients), and NHLRC1 (deleted in eight patients)  overlap with the deletion found in patient 1, who had a diagnosis of ASDs  had ASDs, speech delay, ADHD or hyperactivity, or other behavioral abnormalities , a neurodegenerative disorder with progressive cerebellar degeneration. ATXN1 is also proposed to function as a regulator of gene expression ), an autosomal recessive disease with features that include albinism, pulmonary fibrosis, and bleeding [Drosophila [DNTBP1 with schizophrenia (SCZD [MIM 181500]) [DTNBP1 has been connected with autistic features observed sometimes in patients with Duchenne muscular dystrophy (DMD [MIM 310200]) ) -54. DTNB310200]) .JARID2 is expressed in embryonic and adult human neurons ), an autosomal recessive disorder characterized by rapid and progressive adolescent-onset epilepsy, mental and motor deterioration, and short survival. NHLRC1 functions as an E3 ubiquitin ligase that mediates degradation of EPM2A (or LAFORIN). Mutations that disturb EPM2A degradation, leading to the accumulation of Lafora bodies, are also causative for MELF [Homozygous deletions, insertions, missense, or nonsense mutations in for MELF .Although most individuals with deletions in chromosome 6p22.3-p24.3 display developmental delay, speech delay, and ASDs or other behavioral abnormalities, there is no single locus that is deleted in all patients with 6p22-p24 deletion , the Institutional Review Board for Human Subject Research at Baylor College of Medicine (H-25466), and by the Institutional Review Board of Spokane (study number 1500) and were obtained for patients 1, 4 and 6, and patient 5, respectively.For patient 1, DNA was extracted from peripheral blood and lymphoblastoid cell line. DNA from the cell line was used for initial array CGH analysis at Baylor College of Medicine (BCM). DNA from peripheral blood was used to confirm the array CGH findings. For patients 2-4 and 6, DNA was extracted from whole blood using the Puregene DNA isolation kit . For patient 5, a blood specimen was sent to SGL, in Spokane, Washington for clinical array CGH analysis. Parental DNA was only tested by FISH.All arrays used in this study were designed and analyzed based on genome build hg18 . The coordinates found in tables and figures were converted to hg19 (GRCh 37), February 2009 using UCSC liftOver .® whole-genome custom microarray manufactured by Agilent for SGL [Patient 1 was tested for copy-number variants (CNVs) using array CGH with an Agilent custom designed exon targeted microarray with coverage for 294 ASDs candidate genes  . Patient for SGL . AdditioThe protocols for DNA digestion, labeling, purification, hybridization, array scan, and analysis of the clinical arrays followed the manufacturer's instructions with some modifications as previously described ,66,67.SNP genotyping for chromosome 6 was done in patient 1 and his mother using the commercially available Illumina Human 610-Quad BeadChip Kit . Arrays were scanned using the Illumina Iscan with Autoloader2. SNP genotyping and absence of heterozygosity analyses were performed using the Illumina GenomeStudio data analysis software.Confirmatory FISH analyses were performed in patients 2 and 4-6 using standard cytogenetic procedures with bacterial artificial chromosome (BAC) clones RP11-346 F18, RP11-637019, RP11-140A3, and RP11-127P7, respectively. The parental samples were tested for patients 2 and 5.® Flexi DNA Polymerase kit  with the forward primer 5' - TTTGGATTGGGAGGAATGAA - 3' and reverse primer 5' - GGGGAAAGAAACGGAACATC - 3' (Sigma-Aldrich Corp.). PCR products were analyzed using 1% agarose gel electrophoresis, purified from the agarose gel using the PCR Purification Kit , and then sent for Sanger dideoxy sequencing .PCR to confirm the 6p22.3-p23 deletion and to amplify the junction fragment in patient 1 was performed using Takara LA PCR kit  with the forward primer 5' - TGGTGTAGTTAAGGGGAAAGAGAGAGGAG - 3' and the reverse primer 5' - CTGCAGTATAAGCATACTACTACCCACTTAGGG - 3' . To test the patient's mother for a low level somatic mosaicism, which might have been missed by the array CGH assay, PCR was performed using GoTaqATXN1 gene, we followed the previously published procedures with minor modifications [To determine the size of the polyglutamine track in the ications -70. PCR ADHD: Attention deficit hyperactivity disorder; ASDs: Autism spectrum disorders; BAC: Bacterial artificial chromosome; BCM: Baylor College of Medicine; CGH: Comparative genomic hybridization; CNV: Copy-number variant; DECIPHER: Database of chromosomal imbalance and phenotype in humans using ensembl resources; FISH: Fluorescence in situ hybridization; MGL: Medical Genetics Laboratories; PCR: Polymerase chain reaction; SCAP: South Carolina autism project; SGL: Signature Genomic Laboratories.Many of the authors are faculty members in the Department of Molecular and Human Genetics at BCM, which offers extensive genetic laboratory testing including use of arrays for genomic copy-number analysis, and the Department derives revenue from this activity. JAR is an employee of Signature Genomics, a subsidiary of PerkinElmer, Inc.ATXN1 STR extension analysis. PBSC-S and PS performed the PCR and sequence analysis. JAR, S-HLK, SWC, and PS signed out the microarray-based hybridization analysis results and analyzed FISH results. CS, RS, JS, MMJN, DT, DC, GSP, LI, RM, and RES clinically examined the patients. PBSC-S and PS drafted the manuscript. All authors read and approved the final manuscript.ALB and PBSC-S designed the experiments. PBSC-S and PE performed the chromosomal microarray analysis. PBSC-S, AW, and JW performed the SNP and


Ginseng, an ancient and famous medicinal herb in the Orient, has been used as a valuable tonic and for the treatment of various diseases including hepatic disorders. Ginseng saponins, commonly known as ginsenosides, are principal constituents and have believed to be responsible for multiple ginseng health benefits. There are more 40 ginsenosides isolated from ginseng. To date, treatment options for common liver diseases such as cirrhosis, fatty liver, and chronic hepatitis remain problematic. In this regard, ginseng extracts and individual ginsenosides have shown a wide array of beneficial role in the regulation of regular liver functions and the treatment of liver disorders of acute/chronic hepatotoxicity, hepatitis, hepatic fibrosis/cirrhosis, hepatocellular carcinoma, and so on in various pathways and mechanisms. In this paper, we first outline the pharmacological effects of ginseng and ginsenosides on the liver functions. Panax ginseng C.A. Meyer , is one of the most valuable medicinal plants, particularly in Korea, China, and Japan [Botanical medicines have been applied for the treatment of various human diseases with thousands of years of history in Asia and are sharing a large market in the form of drugs, dietary supplements, and foods. In the west, botanical medicines are categorized as complementary/alternative medicines, dietary supplements, or foods. Ginseng, referred to as the root of nd Japan .1, Rb2, Rg1, Rd, and Re identified from ginseng up to date [in vitro, in vivo, and clinical models [Ginseng has been used as a valuable tonic and for the treatment of various diseases , 2. Trad to date , 5. Excel models . Having l models , 7, centl models , and imml models , 10. HowLiver diseases represent a major health burden worldwide, with liver cirrhosis being the ninth leading cause of death in western countries . Chronicin vitro and liver injury in various animal, and clinical models induced by a wide variety of hepatotoxins including hydrogen peroxide (H2O2) [4) [1 [2) [p-dioxin [ Acute/chronic hepatotoxicity suffers from drugs used, alcohol, contaminant, and poisoning and may be incidence of liver diseases including liver hepatitis, cirrhosis/fibrosis, and hepatocellular carcinoma (HCC). Ginseng extracts have been reported to show protective effects on hepatocytes e (H2O2) , alcohole (H2O2) , 18, car2O2) [4) , 19–22, ) [4) [1 , 24, fum) [4) [1 , cadmium) [1 [2) , 27, ben) [1 [2) , 2,3,7,8p-dioxin , lipopolp-dioxin , diethylp-dioxin , galactop-dioxin –34, bromp-dioxin , xenobiop-dioxin , and merp-dioxin . Ginsengp-dioxin  and virap-dioxin . Ginseng is beneficial in the treatment of acute graft-versus-host disease, a rare complication after liver transplantation with an extremely poor prognosis . On the in vivo [ Liver and adipose tissue play an important role in both lipid and sugar metabolism. It has been found that ginseng has effects on lipid and sugar when administrated to rats in vivo , 44. Hepin vivo . In anot1 and urethane [ HCC is one of the most frequent tumor types worldwide. It is the fifth most common cancer and the third leading cause of cancer death . Ginsengurethane . Recentlurethane . The mechanisms which provide ginseng's hepatoprotective effects are closely attributed to antioxidation properties. Ginseng enhanced the antioxidant defense mechanism  and incrβ, IFN-γ) and chemokines  in CCl4-treated mice [α)-stimulated NF-kB activation and further suppressed the gene expression of iNOS and cyclooxygenase-2 (COX-2) in HepG-2 cells [ Inflammatory effects of ginseng have been responsible for the liver protection. Ginseng suppressed the production of inflammatory cytokines , matrix metalloproteinase (MMP)-2, and tissue inhibitor of metalloproteinase (TIMP)-1 in hepatic stellate cells, the major cause of liver fibrosis, at 10–80 μg/mL [1 is found to inhibit TNF-α-mediated NF-kB transcriptional activity in HepG2 cells with IC50 of 27.45 μM and gene expression of iNOS and COX-2 inducible inflammatory enzymes [Ginsenoisde Rb and AST , 60 and  and AST  and prot and AST . In a re80 μg/mL . More re enzymes .1 is beneficial against fatty/high cholesterol liver as well as hepatic triglycerides in rats fed on high-fat diet, supporting that ginsenoisde Rb1 may be involved in lipid metabolism by regulating the activity of microsomal CYP monooxygenase and 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase [Ginsenoisde Rbeductase –66.1, also one of the most abundant ginseng components of ginseng, significantly improves the extent of liver fibrosis in rat induced by thioacetamide as reducing the serum levels of fibrotic markers of ALT, AST, and alkaline phosphatase and hepatic hydroxyproline content [1 markedly inhibits cell proliferation, activation and formation of reactive oxygen species, and the NF-kB activity [1 is found to inhibit TNF-α-mediated NF-kB transcriptional activity in HepG2 cells with IC50 of 28.14 μM and gene expression of iNOS and COX-2 inducible inflammatory enzymes [Ginsenoisde Rg content . In addiactivity . Also, g enzymes .1 suppresses hepatic glucose production via AMP-activated protein kinase (AMPK) activation in HepG2 cells [Recently, ginsenoisde RgG2 cells .Yuan et al. reported that ginsenoisde Re (40 mg/kg) attenuates alcoholic fatty liver disease through regulation of AMPK and mitogen-activated protein kinase (MAPK) pathways in alcoholic-fed ICR mice . In anotα-mediated NF-kB transcriptional activity in HepG2 cells with IC50 of 12.05 μM and gene expression of iNOS and COX-2 inducible inflammatory enzymes [Ginsenoisde Rd is recently found to inhibit TNF- enzymes .4-induced acute hepatitis rats as monitored by the level of inducible hepatic enzymes of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase [Ginsenoisde Ro, an oleanane-type ginsenoisde,  shows the protective effects on GalN and CClsaminase .2 and Rg3 represent major components of red ginseng [1 or compound K is an intestine bacterial metabolite of the ginseng protopanaxadiol-type saponins. In a recent study, ginsenoisde Rg2 significantly inhibits hepatic glucose production in HepG2 cells via activation of AMPK pathway like ginsenoisde Re [3 and M1 have well evaluated for their cancer chemopreventative against several cancer cell lines and tumors in vitro and in vivo including leukemia, colon, and HCC [3 at 25–200 μg/mL shows significantly antiproliferative effects on liver cancer cells  and hepatocellular tumor growth in vivo by inhibiting cancer cell proliferation and promoting cancer cell apoptosis [1 exhibits antihepatocellular carcinoma activity by augmenting apoptosis via Bid-mediated mitochondrial pathways [1 has beneficial role against hyperlipidemia and obesity by attenuating hepatic lipid accumulation via AMPK activation in human hepatoma cells [3 and M1 are also involved in general hepatoprotection against hepatotoxins [Ginsenoisdes Rg ginseng . Ginsenooisde Re . Meanwhi and HCC , 72–74. poptosis , 76. In ma cells . In additotoxins , 79.Curcuma longa), green tea (Camellia sinensis), garlic (Allium sativa), licorice (Glycyrrhiza spp.), especially milk thistle (Silybum marianum) with have been well researched and recognized as phytotherapies for use in a wide variety of prevention and treatment of hepatic diseases [Liver disorders are among the most serious sicknesses in the population. Together with healthy lifestyle recommendation, therapies for hepatic diseases are necessary and need be more effective and economical . Botanicdiseases , 82.Based on compelling experimental evidence, ginseng has an interesting hepatoprotective profile. From a therapeutic standpoint, ginseng could provide an alternative phytomedicine and another option for the preventative therapy and treatments of common liver diseases such as cirrhosis, fatty liver, and chronic hepatitis in addition to a well-known tonic and in forms of healthy foods.Ginseng and its principal components, ginsenoisdes, have shown a wide array of pharmacological activities including beneficial role in the regulation of liver functions and the treatment of liver disorders of acute/chronic hepatotoxicity, hepatitis, hepatic fibrosis/cirrhosis, liver hepatectomy, liver transplantation, and even liver failure and HCC. The possible activity pathways of the actions have been also investigated. There is increasing attention to the effects of ginseng on the liver functions. However, more detailed molecular mechanisms of the activities of ginseng/ginsenoisdes as well as further efficacy and safety studies remain to be explored.It is another important area for further research and development to combine ginseng with other liver active drugs to investigate their possible synergic efficacy and preferably pharmacological properties. Taken together, accumulating evidence supports the potential of ginseng in the treatment of the hepatic diseases and further studies will facilitate their application so far.


Cyclobacterium qasimii strain M12-11BT, isolated from an Arctic marine sediment sample, is reported.A 6.29-Mb genome sequence of  Cyclobacterium qasimii strain M12-11BT is a Gram-negative, nonmotile, horseshoe-shaped bacterium isolated from a marine sediment sample collected from Kongsfjorden, Svalbard, the Arctic. The 16S rRNA gene sequence similarity revealed the type strains of Cyclobacterium amurskyense, Cyclobacterium marinum, and Cyclobacterium lianum to be most closely related to Cyclobacterium qasimii M12-11BT, with similarity score values of 98.2, 96.8, and 93.3%, respectively  was used for the assembly of the raw sequencing data, which yielded a genome of 6,293,689 bp in 205 contigs with a G+C mol% of 42.11%. The smallest contig was 500 bp long and the largest contig 248,907 bp long. RAST (Rapid Annotation using Subsystem Technology) (The Roche 454 (FLX Titanium) pyrosequencing platform was used to perform whole-genome sequencing of Cyclobacterium qasimii M12-11BT indicated that there are a total of 5,958 predicted protein-coding regions and 45 RNA features. There are 86 genes providing resistance against antibiotics and toxic compounds, including genes for resistance against copper, cobalt, zinc, cadmium, mercury, and arsenic. We also found 197 genes involved in membrane transport. There were 121 stress response genes in the annotation of the genome, including 12 genes for heat shock, 4 genes for cold shock, 62 genes for oxidative stress, and 10 genes involved in detoxification.The annotation of The whole-genome sequencing of this bacterium isolated from the extremely cold climatic conditions of the Arctic region will help in the understanding of their adaptation and evolution to survive under these conditions.Cyclobacterium qasimii strain M12-11BT has been deposited in DDBJ/EMBL/GenBank under the accession number ATNM00000000. The version described in this paper is version ATNM01000000.The draft genome sequence of


Cytochrome c release is generally considered as a pivotal step in apoptosis. Remarkably, overexpressed Zfra induces apoptosis via the mitochondrial pathway, which involves suppression of Bcl-2 and Bcl-xL expression (without causing cytochrome c release), counteracting the apoptotic function of tumor suppressor p53 and WWOX, and dissipation of mitochondrial membrane potential for ultimately leading to cell death. Control of cellular aging and apoptosis by Zfra, p53 and WWOX is discussed.Zfra ( WWOX gene is mapped to a common fragile site on chromosome ch16q23.3-24.1. Alteration of human WWOX gene has been found in breast, prostate and many types of cancers. WWOX/WOX1 possesses functional domains, including a nuclear localization sequence (NLS), two N-terminal WW domains (containing conserved tryptophan residues) and a C-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain. The WW domain participates in molecular interactions, signaling and apoptosis [In year 2000, we and two other groups have independently discovered a candidate tumor suppressor, named WW domain-containing oxidoreductase  ; reviewspoptosis . Whetherpoptosis . Also, spoptosis .in vivo and in vitro[in vivo[in vivo[Over the past 10 years of global research efforts, a big picture regarding the functional roles of WWOX/ WOX1 has been emerging. Areas of interests are the critical involvement of WWOX/WOX1 in 1) apoptotic and stress responses  in vitro,7, 2) reo[in vivo,7, 3) sio[in vivo-13, and o[in vivo-19.WWOX/WOX1 is known to increase the cytotoxic function of tumor necrosis factor (TNF) in killing cancer cells ,7,8. Ectin vivo and in vitro [in vivo [Tumor suppressor p53 is known to play a crucial role in aging -22. WWOXin vitro ,14-18. pin vitro ,24. p53 in vitro , and WWO[in vivo . That is[in vivo . Cumulat[in vivo -18,26, szinc finger-like protein that regulates apoptosis) [in vivo. Serine 8 (Ser8) is a conserved phosphorylation site. Overly expressed Zfra induces apoptosis in many types of cancer cells, whereas alteration of Ser8 abolishes its apoptotic function [To identify the possible presence of a common inhibitor of WWOX/WOX1 and p53, we carried out yeast two-hybrid cDNA library screen and identified a 31-amino-acid WOX1- binding protein, named Zfra (optosis) . The amifunction . The indfunction -29.The abundance of Zfra is very low; however, it is inducible under stress conditions (e.g. UV irradiation). Majority of the zinc finger proteins are capable of interacting with DNA and RNA to control gene tran-scription especially during embryogenesis, suggesting that Zfra may possess gene regulatory functions by binding with DNA and RNA.N-terminal first WW domain and the C-terminal SDR domain of WOX1. Upregulation of TNF and inflammatory cytokines is shown during normal or pathogenic aging processes [Due to its small in size, Zfra physically interacts with many proteins in the TNF signaling ,28. Alsorocesses . Dependirocesses ,28. TNF rocesses ,32. Casprocesses -34.We determined that TNF increases the expression of Zfra and enhances its binding with TRADD at the plasma membrane ,28. We sN-terminal first WW domain and the C-terminal SDR domain of WOX1. Without phosphorylation of Tyr33 in the first WW domain, Zfra could not interact with WOX1 [Zfra binds downstream proteins in the TNF signaling. TNF induces the binding of Zfra with activated WOX1, JNK1, and NF-κB ,28. Zfraith WOX1 ,35,36. Zith WOX1 . SequestMany C2H2 zinc fingers proteins are involved in the regulation of gene transcription, growth suppression, and/or apoptosis -39. NuclThe specific threonine/serine kinase(s), which phosphorylates Zfra, is unknown and remains to be identified. A likely candidate for phosphorylating Zfra is JNK1. JNK1 plays a central role in the MAPK signaling, and it integrates many routes of signaling pathways . TNF andde novo formation of the Zfra-p53-WOX1 complex for relocating to the nuclei. Whether the endogenous Zfra blocks the apoptotic function of p53 and WOX1 remains to be determined.Interestingly, phospho-Zfra undergoes rapid de-phosphorylation and degradation, suggesting that Zfra may affect the functional activation and turnover of its binding proteins. During UV irradiation, Zfra is shown to physically interact with activated p53 and WOX1. That is, UV induces the Zfra exhibits a unique function in modulating mitochondrial apoptosis. When cells are exposed to inducers of mitochondrial pathway of apoptosis (e.g. staurosporine or betulinic acid), Zfra becomes phosphorylated at Ser8 and relocates to the mitochondria . Alteratc release, which is very unusual and intriguing. Cytochrome c release from the mitochondria is a hallmark event in apoptosis. A likely scenario is that Zfra directly binds cytochrome c and blocks its release from the mitochondria  in the intermembrane space of mitochondria requires leakage of outer mitochondrial membrane. Bcl-2 and Bcl-xL provide a homeostatic control against the pore forming activity of Bax and Bak [Normally, release of proapoptotic proteins  dissipation [Although Zfra may block cytochrome sipation . Alteratsipation . Also, aNumerous proteins are shown to relocate to the mitochondria during apoptosis. WOX1, p53 and Zfra are known molecules, which participate in apoptosis via the mitochondrial pathway. Whether relocation of WOX1, p53 and Zfra occurs as a tri-molecular complex is not known. Nonetheless, there is a close functional relationship among these proteins. For example, Zfra blocks WOX1-induced cytochrome c release   by physically interacting with receptorassociated adaptor TRADD and downstream NF-κB, JNK1, and WOX1 . Remarka


We found that hippocampus was the area where gene expression changes due to Pb exposure were more pronounced. To recapitulate gestational Pb exposure in vitro, we differentiated mouse embryonic stem cells (ESC) into neurons and treated ESC-derived neurons with Pb for the length of the differentiation process. These neurons expressed the characteristic neuronal markers Tubb3, Syp, Gap43, Hud, Ngn1, Vglut1 (a marker of glutamatergic neurons), and all the glutamate receptor subunits, but not the glial marker Gafp. Importantly, several of the changes observed in Pb-exposed mouse brains in vivo were also observed in Pb-treated ESC-derived neurons, including those affecting expression of Ngn1, Bdnf exon IV, Grin1, Grin2D, Grik5, Gria4, and Grm6. We conclude that our ESC-derived model of toxicant exposure during neural differentiation promises to be a useful model to analyze mechanisms of neurotoxicity induced by Pb and other environmental agents. Exposure to environmental toxicants during embryonic life causes changes in the expression of developmental genes that may last for a lifetime and adversely affect the exposed individual. Developmental exposure to lead (Pb), an ubiquitous environmental contaminant, causes deficits in cognitive functions and IQ, behavioral effects, and attention deficit hyperactivity disorder (ADHD). Long-term effects observed after early life exposure to Pb include reduction of gray matter, alteration of myelin structure, and increment of criminal behavior in adults. Despite growing research interest, the molecular mechanisms responsible for the effects of lead in the central nervous system are still largely unknown. To study the molecular changes due to Pb exposure during neurodevelopment, we exposed mice to Pb  Lead, a non-biodegradable metal, is one of the most ubiquitous persistent toxicants present in the environment. Unlike other heavy metals, like iron, copper or manganese, lead has no known biological functions , but, beOccupational and accidental lead poisoning have been described since the time of the ancient Romans ,3. Pb inMacaca fascicularis monkeys showed elevated expression of Alzheimer disease-related genes, including β-amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 (BACE1), and an increase of total amyloid plaques in the cortex [-3A, and methyl CpG binding protein-2 levels, and modifications of histone marks critical for the regulation of gene expression [Danio rerio), where Pb altered neurological development pathways and caused neurotoxicity [There is a significant body of evidence in support of this contention. Twenty-three years after exposure to Pb during infancy, e cortex . Signifipression ,18. Simipression ,20, and toxicity ,22. 2+/calmodulin dependent protein kinase II (CaMKII), phosphorylation of transcription factor CREB, and expression and translocation of brain-derived neurotrophic factor (BDNF) [Recent studies using primary neuronal cultures indicate that lead exposure during childhood may negatively modify important neuronal pathways implicated in the late effects observed during adult life, especially affecting pathways implicated in synaptic plasticity, learning, memory, and cell survival, including modification of the N-Methyl-D-Aspartate (NMDA) receptor architecture ,24, chanr (BDNF) ,26. Pb er (BDNF) . in utero and found that these mice presented alterations in the expression of several genes, including neural markers, neurotrophins, transcription factors, and glutamate-related genes. We also induced mouse ESCs to differentiate into neurons and treated them with Pb during the differentiation process and observed that some of the changes induced by Pb in the brains of mice were also found in vitro. This in vitro model may serve as a powerful tool to study the mechanisms of lead and other environmental neurotoxicants.In this study we have initiated a test of the hypothesis that the Pb-induced alterations that occurs during neural development may be responsible for the behavior and cognitive impairment observed in adult life. We exposed mice to Pb C57BL/6J mice were housed in the vivarium at Cincinnati Children's Hospital Medical Center under controlled conditions of temperature, humidity, and lighting, and provided standard mouse chow and water ad libitum. All experimental procedures conducted with these animals have been approved by the University of Cincinnati and Cincinnati Children's Animal Care and Use Committees. Female mice were exposed to 0 or 3 ppm of lead acetate in drinking water from 8 weeks prior to mating, through gestation and until postnatal day PD10. This treatment regimen insured steady state lead levels in the dam at the time of gestation. From each treatment group, brain tissues of 3 male offspring, including cortex, hippocampus, and thalamus were collected at PD60.2, and passaged every two days. Neural differentiation followed protocols previously described by others [6 undifferentiated mESCs were plated in bacteriological dishes for 8 days using CA medium, consisting of DMEM supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 550 nM βME, 0.1 mM NEAA, 100 U/ml penicillin, and 100 µg/ml streptomycin. On day 4, the CA medium was made to contain 5 μM retinoic acid (Sigma-Aldrich). To obtain a cellular suspension, cells were trypsinized and plated at 2 x 105 cells/cm2 in 24-well or 6-well plates coated with poly-DL-ornithine (Sigma-Aldrich) and laminin (Roche), using N2 medium, consisting of a 1:1 mixture of DMEM and DMEM nutrient mixture F-12 ham (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 50 μg/ml BSA, 100 nM putrescine, 20 nM progesterone, 30 nM sodium selenite, 25 μg/ml insulin, 25 μg/ml transferrin, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 48 hours, the N2 medium was replaced with complete medium, consisting of DMEM supplemented with 2 mM L-glutamine, 50 μg/ml BSA, 1X serum-free medium supplement B27 (GIBCO), 30 nM sodium selenite, 25 μg/ml insulin, 25 μg/ml transferrin, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2. When needed, cells were treated with lead acetate (Sigma-Aldrich) at different concentrations for the duration of neural differentiation.Undifferentiated C57BL/6-C2 mouse mESC  were culy others . BrieflyWestern immunoblots were performed as previously described . Briefly5 cells/cm2. After 3 days in culture, cells, termed 3-DIV cells for 3-days-in-vitro, were fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed twice with PBS and cells were permeabilized and blocked in a blocking solution containing 0.05% (v/v) Triton X-100 and 2% (w/v) BSA in PBS. Primary antibodies were incubated in blocking solution for 2 hours at room temperature at 1:500 for TUBB3, 1:500 for vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems), and1:1000 for GFAP. Samples were washed twice in a washing solution containing 0.05% (v/v) Triton X-100 in PBS and incubated with the appropriate secondary antibodies in blocking solution at room temperature for 1 hour at 1:100 for Alexa 488 anti-mouse, and 1:100 for Alexa 488 anti-rabbit (Molecular Probes). After the second antibody was removed, samples were washed twice with washing solution and incubated with 2 μg/ml Hoechst in PBS for 10 minutes. Coverslips were mounted onto slides using 44% glycerol in PBS. Micrographs were taken at 40X using an Axioplan Zeiss fluorescent microscope equipped with an AxioCam ERc5s and Zeiss’ Zen Microscopy suite application. Cellular aggregates were dissociated with trypsin and the cells were grown on coverslips at 2 x 10Gapdh mRNA as the normalization standard. Primers used are shown in t, where ΔΔCt = (CtGene - CtGapdh)Assay.Total RNA was isolated from undifferentiated and differentiated cells by using TRIzol reagent (Ambion) and from tissues by using a RNeasy Minikit (Qiagen). Reverse transcription was performed using random hexamer primers and SuperScript III transcriptase (Invitrogen) as previously described . Quantitg for 5 minutes. After washing twice with PBS, active caspase-3 was assayed with a FITC Active Caspase-3 Apoptosis Kit (BD Pharmigen) following the manufacturer’s protocols and processed by flow cytometry. Undifferentiated ESC and cellular aggregates were trypsinized to obtain isolated cells and collected by centrifugation at 200xp-value < 0.05 was considered statistically significant.All differentiation experiments were done using at least three independent cultures. Data are shown as the mean ± SEM. Group comparisons were made using one-way ANOVA followed by post hoc Tukey test. A Prenatal and early childhood lead exposure are associated with adverse cognitive, neurobehavioral and motor outcomes, suggesting altered brain structure and function and, by extension, altered gene expression patterns in the CNS . These aSyn1, Vamp1, and Syp), neurite and axon growth , axon maturation and synaptogenesis (Reln), mRNA stabilization (Hud), BDNF vesicle transport (Htt), and transcription factors involved in neuronal differentiation  (blue bars). We also examined the expression of the neurotrophins Bdnf, Ngf, Nt3 and Nt4 (orange bars), which are produced and secreted by neural and glial cells and are implicated in cell proliferation, maturation survival, axonal outgrowth, and neural plasticity [pink bars), and glutamate-related genes (green bars), including the different subunits of the glutamate receptors Grin1, Grin2A-D, and Grin3A-B, for the glutamate N-methyl-D-aspartate (NMDA) receptors, Grik1-5 for the kainate receptors, Gria1-4 for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, Grm1-8 for the metabotropic receptors, and Vglut1-2 for the vesicular glutamate transporters. Of the three tissues tested, hippocampus showed more Pb-induced gene expression changes than the others. The neural markers Hud, Syn1, Htt, Vamp1, and Ngn1, the member of the NF-κβ family crel, and the glutamate-related genes Grin2A, Grin3B, Grik2, Grm4, and Grm8 were up-regulated, while the neural markers NeuroD1 and Sox3, the neurotrophins Bdnf exon IX and Ngf, the transcription factor Creb1 and Creb2, the members of the NF-κβ family Nfκb2, Rela, and Relb, and the subunits for glutamate receptors Grin1, Grin2D, Gria1, Gria4, and Grm6 were down-regulated  to measure the expression of several neural markers implicated in vesicle release  following cellular aggregate disaggregation, immunoblot analyses showed that 3-DIV cells expressed high levels of TUBB3 and no detectable GFAP , axon growth and maturation and synaptogenesis , transcription factors , BDNF vesicle transport (Htt), and mRNA stabilization (Hud). All the neural genes tested were significantly up-regulated, although to different extents . The neural marker Ngn1 was up-regulated after Pb treatment, whereas Syp, Htt, Gap43, and Reln were marginally down-regulated in the presence of Pb, although the difference was not statistically significant (blue bars). We also examined the differentiated neurons for expression of the neurotrophins and the transcription factors analyzed in the mouse brain tissues. Bdnf exon IV was down-regulated in cells treated with 0.1 µM Pb, whereas Bdnf exon IX was up-regulated (orange bars). Creb1 was slightly down-regulated, with no significant difference to their expression in control neurons, and Rela was up-regulated by Pb. The rest of the genes analyzed did not change their expression (pink bars). To complete the analysis, we measured the expression of subunits for NMDA, kainate, AMPA, and metabotropic receptors for glutamate, and for the glutamate transporters Vglut1 and Vglut2. Pb down-regulated Grin1 and Grin2D of the NMDA receptor subunits, Grik1 and Grik5 of the kainate receptor subunits, Gria4 of the AMPA receptor subunits, Grm1 and Grm6 of the metabotropic receptor, and Vglut1 of the glutamate transporters. Grin2A, Grin2B, Grin2C, Grin3B, Gria2, and Grm4 were marginally different from their expression in control neurons (yellow bars). These results show that Pb exhibits a repressive effect on the expression of glutamate receptors, leading to the conclusion that Pb treatment during neural differentiation process of ESC in vitro causes changes in gene expression patterns that affect multiple neuronal transport processes, including calcium signaling pathways.The neurons resulting from Pb treatment of ESC during the differentiation process are TUBB3 and VGLUT1 positive, morphologically indistinguishable from untreated control neurons, as determined by immunofluorescence analysis . ExpressNgn1 in vitro and in hippocampus, while it down-regulates the glutamate receptor subunits Grin1, Grin2D, Gria4, and Grm6. Ngn1 is also up-regulated by Pb in the thalamus. Bdnf is down-regulated in vitro and in the cortex and the glutamate receptor subunits Grm3 and Grm4 subunits are down-regulated in vitro and in thalamus , followed by thalamus  and cortex . After a side-by-side comparison of the effects of Pb on the gene expression in ESC-derived neurons and brain tissues, we observed that Ngn1 was up-regulated and Grin1, Grin2D and Grm6 were down-regulated in vitro and in hippocampus, being Ngn1 also up-regulated in thalamus. Bdnf exon IV and Grik5 were down-regulated in vitro and in cortex and thalamus, respectively. Finally, Gria4 was repressed in vitro, in hippocampus, and thalamus. In this study we have found that mice exposed to Pb in vitro model, Reln was up-regulated in the cortex after gestational exposure to Pb, without any alteration in the other brain tissues analyzed. Decreases in synaptophysin (SYP) phosphorylation as a result of Pb exposure have been described recently in hippocampal primary cultures, suggesting that this disturbance may produce impairment in vesicular release in synapses [Ngn1), implicated in neural differentiation programs and cell fate [in vitro and in vivo models. Although we do not detect any morphological alterations when the cells are treated with Pb, our gene expression data suggest that the differentiation process may be compromised.Recently, it has been described that reelin (RELN), implicated in axon growth and maturation, is decreased in zebrafish telencephalon  when expsynapses ,27. The synapses . None ofell fate , are sigG-protein-coupled metabotropic receptor for glutamate are involved in excitatory transmission with important effects on synaptic plasticity that are implicated in neuronal processes such as learning and memory [in vivo studies, Grin2A was up-regulated in hippocampus and thalamus of mice treated gestationally with Pb, but we did not observe any expression changes in cortex or in cultures of glutamatergic neurons. Additionally, Grin1 was down-regulated in hippocampus and ESC-derived neurons exposed to Pb. Calcium conductance increases when the kainate receptors contain the Grik5 subunit, while when they contain Grik1 and Grik2 they are less permeable to calcium [The ionotropic receptors NMDA, kainate, and AMPA, and the d memory . Recentld memory ,24. Our  calcium ,49. AlthBdnf transcripts containing exon IV [Bdnf exon IX transcripts are repressed after Pb treatment in both hippocampus and cortex, being not affected in thalamus and increased in the neurons obtained from neural differentiation of mESCs. These discrepancies suggest that other non-coding exon(s) may play a role in the maintenance of Bdnf expression. The transcripts of the coding exon IV are down-regulated in cortex and our in vitro model, and not affected in hippocampus and thalamus. BDNF expression is highly controlled by different regulatory mechanisms, including epigenetic mechanisms, and mitogen-activated protein kinases, phosphoinositide-3 kinase and phospholipase-γ pathways [Bdnf exon IV and Grin1 are down-regulated in cortex and hippocampus of mice treated gestationally with Pb and in 3-DIV neurons exposed to Pb during the neural differentiation process, suggesting that these changes may extend into adulthood, beyond the time when the organism was exposed to the agent.Calcium signaling through the glutamate receptor has been shown to increase the level of CREB-dependent  exon IV , an effe exon IV . In our pathways . As the pathways . InteresPb toxicity has been associated with apoptosis  and showin vitro model that uses mouse ESC differentiation to recapitulate neural gestational exposure to lead, which may serve to characterize the molecular mechanisms of lead neurotoxicity.It is paramount to determine the molecular mechanisms by which Pb produces neurotoxicity. Although the use of lead has been reduced in the last few decades, its non-biodegradable nature and ubiquitous presence make it an environmental agent of general concern. Developmental or early-life exposure to Pb may produce CNS disorders detectable later in adulthood. The theory of the fetal origin of adult disease states that during development, exposure to environmental agents such as heavy metals, or to stressful situations like poor nutrition, may adversely contribute to adult pathogenesis ,59, possFigure S1Quantification of TUBB3 and VGLUT1 positive cells. After fluorescence detection of TUBB3 and VGLUT1 neurons obtained from mESC, the percentage of TUBB3 and VGLUT1 positive cells was calculated as the Number of immunopositive cells/Total number of cells x100. At least five micrographs from different assays were analyzed. (*) p<0.05.(TIF)Click here for additional data file.Figure S2Effects of Pb in mouse ESC cell number. Mouse ES cells were treated with the indicated concentration of Pb during ten days. Cell numbers were recorded every other day at the time that the cells were passaged. Cell numbers were normalized to the number of cells plated on day 0.(TIF)Click here for additional data file.Table S1List of primers used for real-time RT-PCR.(PDF)Click here for additional data file.


SCN9A, the gene that encodes Nav1.7, are associated with a syndrome of congenital insensitivity to pain, gain-of-function mutations are linked to the debiliting chronic pain conditions erythromelalgia and paroxysmal extreme pain disorder, and upregulated expression of Nav1.7 accompanies pain in diabetes and inflammation. Since Nav1.7 has been implicated as playing a critical role in pain pathways, we examined by immunocytochemical methods the expression and distribution of Nav1.7 in rat dorsal root ganglia neurons, from peripheral terminals in the skin to central terminals in the spinal cord dorsal horn.Sodium channel Nav1.7 has emerged as a target of considerable interest in pain research, since loss-of-function mutations in Nav1.7 is robustly expressed within the somata of peptidergic and non-peptidergic DRG neurons, and along the peripherally- and centrally-directed C-fibers of these cells. Nav1.7 is also expressed at nodes of Ranvier in a subpopulation of Aδ-fibers within sciatic nerve and dorsal root. The peripheral terminals of DRG neurons within skin, intraepidermal nerve fibers (IENF), exhibit robust Nav1.7 immunolabeling. The central projections of DRG neurons in the superficial lamina of spinal cord dorsal horn also display Nav1.7 immunoreactivity which extends to presynaptic terminals.The expression of Nav1.7 in DRG neurons extends from peripheral terminals in the skin to preterminal central branches and terminals in the dorsal horn. These data support a major contribution for Nav1.7 in pain pathways, including action potential electrogenesis, conduction along axonal trunks and depolarization/invasion of presynaptic axons. The findings presented here may be important for pharmaceutical development, where target engagement in the right compartment is essential. SCN9A, the gene that encodes Nav1.7, are associated with congenital insensitivity to pain [Voltage-gated sodium channels are critical participants in neuronal excitability and transmition of electrical impulses along pain pathways, and have emerged as major targets for therapeutic intervention in pain disorders -4. Of th to pain -13 and p to pain -16. In a to pain -19.Nav1.7 is a tetrodotoxin-sensitive (TTX-S), fast-activating and fast-inactivating sodium channel that recovers (reprimes) slowly from fast-inactivation . Nav1.7 Nav1.7 is preferentially expressed in dorsal root ganglia (DRG) and sympathetic neurons ,24-27, aIn the present report, the expression and distribution of Nav1.7 in unmyelinated and myelinated DRG neurons along the entire trajectory from peripheral to central terminals is described. The results demonstrate that Nav1.7 is highly-expressed in small diameter DRG and their peripherally- and centrally-directed processes from the skin to the CNS. Notably, Nav1.7 is clearly present in both peripheral axon terminals of DRG neurons and also in their centrally-directed axons within the dorsal horn, extending to central perterminal and terminal regions of these pain-signalling neurons. Moreover, Nav1.7 is robustly expressed at nodes of Ranvier in a subpopulation of small diameter myelinated fibers. These observations are consistent with critical roles for Nav1.7 channels at multiple sites within nociceptive DRG neurons and their processes.Sodium channels play important roles in nociception and chronic pain syndromes ,6, with DRG were triple-labeled with antibodies to peripherin, a C-fiber marker , neurofi+  and CGRP+  DRG neurons exhibited Nav1.7 immunolabeling.C-fiber DRG neurons, which are predominantly nociceptive, can be divided into subpopulations of peptidergic, which express neuropeptides as substance P and calcitonin gene-related protein (CGRP), and non-peptidergic, which bind isolectin B4 (IB4) and lack neuropeptide expression, neurons . To deteDRG neurons are psuedo-unipolar cells whose single process bifurcates, sending one branch peripherally to terminate in somatic targets and one branch centrally to terminate centrally in CNS. To determine the expression of Nav1.7 in peripherally-directed DRG fibers, sections of sciatic nerves were immunoreacted with Nav1.7, peripherin, to label unmyelinated fibers , and cas+ axons exhibited Nav1.7 immunoreactivity along the myelinated fibers, and, when present, the immunosignal was of low intensity. In addition, Nav1.7 immunoreactivity was generally not exhibited at nodes of Ranvier. However, a subset of small diameter (<1 μm at nodes) myelinated axons displayed robust Nav1.7 labeling at their nodes  of NF/75 of NFWithin the skin, intraepidermal nerve fibers (IENF) branch perpendicularly from bundles of C- and Aδ-fibers that run parallel and subjacent to the dermis/epidermis boundary and ascend within layers of the epidermis. Peptidergic fibers generally terminate in the stratum spinosum layer, while non-peptidergic fibers terminate in stratum granulosum . SubepidThe central processes of DRG neurons form dorsal roots along their projection to synaptic terminations in the spinal cord. Dorsal roots exhibited extensive peripherin labeling of unmyelinated sensory fibers, as well as neurofilament-positive myelinated fibers Figure . PeripheTo determine whether small diameter myelinated fibers in dorsal roots display Nav1.7 immunolabeling at nodes, similar to that observed in sciatic nerves, dorsal root sections were immunoreacted with Nav1.7 and caspr antibodies. As in sciatic nerve, a subset of small diameter (<1 μm) myelinated fibers exhibited nodal Nav1.7 immunolabeling Figure . The nodAs demonstrated in Figure The labeling pattern of Nav1.7 with IB4 and CGRP in the dorsal horn strongly suggests that Nav1.7 is localized within pre-synaptic central terminals of nociceptive DRG neurons. To address this suggestion, we co-labeled spinal cord sections with synaptophysin, a marker of synapses , and NavThe present results demonstrate that Nav1.7 is robustly expressed in the somata of virtually all small diameter (<30 μm) DRG neurons, which predominantly give rise to nociceptive C- and Aδ-fibers ,41. In aThe Nav1.7 channel has recently garnered substantial interest in pain research, due to its association with human pain disorders, including inherited channelopathies, diabetic neuropathy, small fiber neuropathy, neuromas and inflammation. Loss-of-function mutations in Nav1.7 are associated with congenital insensitivity to pain (CIP) , while gThe activity of wild-type Nav1.7 channels is also implicated in chronic human pain syndromes. Injury to peripheral nerves can result in the formation of painful neuromas, tangles of proliferating connective tissue and blind-ending axons, which often exhibit spontaneous ectopic activity ,44. Nav1Animal studies provide additional support for a major contribution of Nav1.7 in nociception and chronic pain. Experimentally-induced diabetes results in mechanical allodynia and thermal hyperalgesia that is accompanied by an upregulation of Nav1.7 in DRG neurons -52. SignA major role for Nav1.7 in inflammatory pain is supported by knock-out studies. Deletion of Nav1.7 in Nav1.8-expressing DRG neurons, which are principally nociceptive, greatly attenuated or eliminated behavioral responses to a range of inflammatory agents . InteresThe Nav1.7 sodium channel exhibits fast-activation and fast-inactivation, similar to the other tetrodotoxin-sensitive (TTX-S) channels, Nav1.1, Nav1.2, Nav1.3, and Nav1.6, expressed in nervous tissue ,20. HoweNav1.6 is the predominant sodium channel isoform aggregated at nodes of Ranvier in adult tissue . HoweverWe demonstrated the co-expression of Nav1.7 with CGRP and IB4 in lamina I/IIo and IIi, respectively, of spinal cord dorsal horn. We further demonstrated co-localization of Nav1.7 with synaptophysin, a marker of pre-synaptic terminals, and a lack of Nav1.7 labeling in NeuN labeled post-synaptic neurons in the superficial layers of dorsal horn. These results support the observations of Minett et al.  in whichIn summary, our results demonstrate that Nav1.7 is expressed by nociceptive DRG neurons along their entire trajectory, with expression extending from the peripheral terminals of IENF in the skin to preterminal projections of these axons with the dorsal horn, and to central synaptic terminals in the spinal cord. The expression of Nav1.7 at central and peripheral terminals, as well as along the peripherally- and centrally-directed trunk of nociceptive fibers, suggests functional contributions of Nav1.7 at multiple foci within pain pathways, extending from peripheral axons within the skin to central preterminal axons and axon terminals in the dorsal horn. These observations may have important implications for development of pain pharmacotherapy, where target engagement in the right compartment is essential.ad libitum access to water and food. The experimental procedures were approved by the VA Connecticut Healthcare System Institutional Animal Care and Use Committee, in accordance with NIH guidelines.Sprague–Dawley male rats  were housed under a 12 hr light/dark cycle in a pathogen-free area with v1.7 protein and cell-specific markers as described previously [Rats were deeply anesthetized with ketamine/xylazine  and transcardially perfused with 0.01 M PBS (pH 7.4) followed by ice-cold 4% paraformaldehyde in 0.14 M Sorensen’s phosphate buffer (pH 7.4). Tissues  were removed, immersion-fixed for an additional 20 min  and cryoprotected with 30% (w/v) sucrose in PBS overnight at 4°C. Hindpaw glabrous skin was immersion-fixed in Zamboni’s fixative for 8 hours at 4°C, which yielded more robust labeling of intraepidermal nerve fibers than paraformaldehyde fixation, prior to cryoprotection. Ten-μm thick cryosections were mounted on slides  and processed for detection of Naeviously . In brieeviously ; mouse aeviously ) and mouImages of dorsal roots (3 sections each for n=4 rats), sciatic nerves (3 sections each for n=4 rats) and DRG (3 sections each for n=5 rats) were acquired, yielding 12, 12, and 15 separate images of dorsal root, sciatic nerve and DRG tissue, respectively, for quantification.For determination of co-localization of Nav1.7, peripherin, and neurofilament in sciatic nerve and dorsal root, a line was placed on the images orthogonal to the axis of the fibers, which extended from edge to edge of the tissue (~350-500 μm). Nav1.7 (red)-, peripherin (green)- and NF (blue)-positive fibers (at least 10 μm in length) that intersected the line were counted separately and merged (i.e. Nav1.7 and peripherin = yellow). Percentage of peripherin- or neurofilament-positive fibers expressing Nav1.7 was calculated as total number of peripherin- or neurofilament-positive fibers co-localized with Nav1.7  divided by the total number of peripherin or neurofilament-positive fibers (i.e. green or blue).For determination of Nav1.7 immunolabeling at nodes of Ranvier, sections of sciatic nerve and dorsal root were reacted with antibodies to Nav1.7 and caspr, which is a marker of paranodes . Images To determine the number of DRG neurons expressing Nav1.7 (red), IB4 (green),CGRP (blue), peripherin (green) and neurofilament (blue) in triple-labeled sections , the number of positive neurons for each channel was assessed. The numbers of neurons exhibiting co-localization of Nav1.7/IB4, Nav1.7/CGRP, Nav1.7/peripherin and Nav1.7/neurofilament were then counted and the percentage of neurons displaying colocalization was calculated.Caspr: Contactin associated protein; CGRP: Calcitonin gene-related protein; CIP: Chronic insensitivity to pain; DRG: Dorsal root ganglia; ERK1/2: Extracellular signal-regulated kinase 1/2; HSV: Herpes simplex virus; IB4: Isolectin B4; IENF: Intraepidermal nerve fiber; MAP kinase: Mitogen-activated protein kinase; NeuN: Neuronal nuclei; PEPD: Paroxysmal extreme pain syndrome; TTX-S: Tetrodotoxin-sensitive; WT: Wild-type.The authors declare that they have no competing interests.JAB designed immunocytochemical experiments, acquired, analyzed and interpreted data, and participated in writing the manuscript. NF performed immunocytochemical experiments and acquired, analyzed and interpreted data. SDH and SGW participated in design of experiments and edited the manuscript. All authors read and approved the final manuscript.


Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries. Familial aggregation in CRC is also important outside syndromic forms and, in this case, a polygenic model with several common low-penetrance alleles contributing to CRC genetic predisposition could be hypothesized. Mucins and GALNTs  are interesting candidates for CRC genetic susceptibility and have not been previously evaluated. We present results for ten genetic variants linked to CRC risk in previous studies (previously identified category) and 18 selected variants from the mucin gene family in a case-control association study from the Spanish EPICOLON consortium.ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and 18 selected variants in the mucin gene family.CRC cases and matched controls were from EPICOLON, a prospective, multicenter, nationwide Spanish initiative, comprised of two independent stages. Stage 1 corresponded to 515 CRC cases and 515 controls, whereas stage 2 consisted of 901 CRC cases and 909 controls. Also, an independent cohort of 549 CRC cases and 599 controls outside EPICOLON was available for additional replication. Genotyping was performed for ten previously identified SNPs in P-value < 0.05 in EPICOLON stage 1 [rs698 in ADH1C , rs1800795 in IL6 , rs3803185 in ARL11 , and rs2102302 in GALNTL2 . In the joint analysis for both stages, results were only significant for rs3803185  and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts .None of the 28 SNPs analyzed in our study was found to be associated with CRC risk. Although four SNPs were significant with a ARL11, ADH1C, GALNTL2 and IL6 genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC studies. Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries . FamiliaFrom the EPICOLON consortium , severalADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11) have been analyzed in previous independent genetic association studies and they are a priori attractive candidates for genetic susceptibility to CRC [APC), alcohol metabolism (ADH1C), inflammation , cell cycle regulation (CCDN1), energy balance , methylation (MTHFR), vitamin D (VDR), and the RAS superfamily (ARL11). On the other hand, mucins are protein constituents of the mucous barrier that protects human epithelia from adverse conditions, and they are highly glycosylated by GALNT proteins. Mucins and GALNTs, members of the mucin gene family, can be found deregulated in CRC and other neoplasms and, although also interesting candidates, they have not been previously evaluated for CRC genetic susceptibility [SNPs in genes selected in the previously identified category  was available for further replication of putative positive hits in EPICOLON. Cases and controls were matched for sex and age (± 5 years) and controls were negative for personal and family cancer history. All samples were obtained with informed consent reviewed by the ethical board of the corresponding hospital.Studied subjects were mainly from EPICOLON, a prospective, multicenter, nationwide Spanish initiative 6&grupo=4, compris6&grupo=4. EPICOLOADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11. For the mucin gene family, 1-2 SNPs were selected from each of the following genes: MUC7, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC21, GALNT1, GALNTL2, GALNT10, GALNT14, GALNT4 and OVGP1. Mucin gene selection did not intentionally include GALTN12. This gene is located on the 9q22 region and it was studied in our candidate-gene approach for regions with positive linkage in CRC families . SNP selection in the mucin gene family was performed using Pupasuite, a web tool for the selection of genetic variants with potential phenotypic effect (pupasuite.bioinfo.cipf.es) [Selected SNPs were included in the previously identified category if they corresponded to genetic variants linked to CRC risk by previously published independent studies, or in the mucin gene family if located on genes encoding for mucins and GALNT proteins. Mucins are protein constituents of the mucous barrier highly glycosylated by GALNT proteins. One relevant previously identified SNP was studied in each of the following genes: cipf.es) . SNPs wehttp://www.cegen.org, and at the Genome Analysis Platform of the CIC-BioGUNE http://www.cicbiogune.es.High-throughput genotyping in EPICOLON cohorts was performed according to manufacturer's instructions with the TaqMan allelic discrimination and SNPlex™ systems , and single-base primer extension chemistry matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) genotyping platform . Genotyping in the MCC-Spain cohort was performed using the VeraCode technology . Genotyping was performed at the Santiago de Compostela and Barcelona nodes of the Spanish National Genotyping Centre P > 0.01), supporting absence of genotyping artifacts. There was no sign of underlying population stratification in EPICOLON as tested by an independent study [P = 0.0125 for four SNPs). Study power was estimated with CaTS software [As quality control, genotyping success was set above 90% for SNPs. Allelic frequency description and Hardy-Weinberg equilibrium test were performed using SNPator, a web-based tool offered by the Bioinformatics division of the Spanish National Genotyping Centre (bioinformatica.cegen.upf.es) . All SNPnt study . Genotypnt study . Inter-gsoftware .P-value < 0.05 in any of the tested inheritance models: rs698 in ADH1C , rs1800795 in IL6 , rs3803185 in ARL11 , and rs2102302 in GALNTL2 .Twenty-eight SNPs, ten from the previously identified category and 18 from the mucin gene family, were successfully genotyped in EPICOLON stage 1. Results in EPICOLON stage 1 are shown in Additional File P-value threshold  was used to avoid false-negative results. We then validated statistically-significant stage 1 results by replicating them in another independent CRC cohort (EPICOLON stage 2). Further replication in stage 2 was performed only for significant SNPs in stage 1. Results for rs698, rs1800795, rs3803185, and rs2102302 in EPICOLON stage 2 are shown in Additional File P-value = 0.01, recessive model, AA-AG vs GG). In order to improve statistical power, results for EPICOLON stages 1 and 2 were also analyzed jointly for these four SNPs. Results were only significant for rs3803185  and borderline significant for rs698 and rs2102302 .In order to further investigate rs3803185, we were able to genotype it additionally outside EPICOLON in a cohort from a Spanish multicase-control population study for common neoplasms (MCC-Spain). This variant was not significantly associated with CRC risk in this independent cohort. Pooled analysis for rs3803185 in the EPICOLON and MCC-Spain cohorts still showed borderline significance  (Table P = 0.03). However, it should be commented that weak associations observed in our study would have not been present if Bonferroni correction for multiple testing was applied and, therefore, they should be considered as not statistically significant.Associations of rs698, rs1800795, rs3803185 and rs2102302 were also evaluated in 2 cohorts described in a previous GWAS [ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and 18 selected variants in the mucin gene family were evaluated with a genetic association strategy. CRC cases and matched controls collected in two independent stages within the EPICOLON consortium were genotyped in order to evaluate its potential association with CRC risk. Mucins and GALNTs, members of the mucin gene family, have not been previously evaluated for CRC genetic susceptibility.Ten previously identified SNPs in APC [CCDN1 [IL6 and IL8 [IRS1 [MTHFR [PPARG [VDR [MTHFR and CCDN1 were additionally supported by meta-analyses of several individual studies [Significant results for previously identified SNPs in independent cohorts regarding CRC risk were previously reported for APC ,12, CCDNC [CCDN1 ,14, IL6  and IL8 ,16, IRS1L8 [IRS1 , MTHFR [1 [MTHFR -20, PPARR [PPARG , and VDRARG [VDR ,23. Rema studies . HoweverADH1C, rs1800795 in IL6, rs3803185 in ARL11, and rs2102302 in GALNTL2), but only rs3803185 achieved statistical significance in EPICOLON stage 2. In the joint analysis for both stages, results were only significant for rs3803185 and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts.In our study, four SNPs were significant in EPICOLON stage 1 , is a tumor suppressor gene that belongs to the ARF family of the Ras superfamily of small GTPases that are known to be involved in multiple regulatory pathways altered in human carcinogenesis. It has been suggested that ARL11 SNPs, especially rs3803185, may act as low penetrance variants in several neoplasms including CRC [ADH1C gene encodes for class I alcohol dehydrogenase, gamma subunit, which is a member of the alcohol dehydrogenase family. Members of this enzyme family metabolize a wide variety of substrates, including ethanol. There is a noticeable association between alcohol consumption and CRC risk [GALNTL2 , also known as GALNT15, is ubiquitously expressed in human tissues [IL6 (interleukin 6) gene, among others, has been analyzed in previous independent genetic association studies and it is an a priori attractive candidate for genetic susceptibility to CRC [ding CRC ,24,32. TCRC risk , and it  tissues . This ge tissues . Chronicy to CRC .Finally, as limitations of our study, it should be commented that our cohort sample size may probably be not large enough to reach stronger conclusions for the analyzed variants. However, our study  had an estimated 80% power to detect an OR as low as 1.3 with a MAF of 0.30, and 1.34 for a MAF down to 0.20, assuming a log-additive model and α = 0.05. Also, since we did not follow a GWAS strategy, our results apply only for the selected SNPs. Nevertheless, gene/SNP selection was biased to include those with previously published positive association results for CRC risk or those mucin SNPs with a putative functional effect.ARL11, ADH1C, GALNTL2 and IL6 may have an effect on CRC risk. Mucins and GALNTs, included in the mucin gene family, have been found deregulated in CRC and other neoplasms, and they are interesting candidates for CRC genetic susceptibility [ARL11, ADH1C, GALNTL2 and IL6 variants in CRC susceptibility.In summary, none of the 28 SNPs analyzed in our study could be associated with CRC risk. However, variants in tibility . Since tARL11, ADH1C, GALNTL2 and IL6 genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC cohorts.None of the 28 SNPs analyzed in our study could be associated with CRC risk. However, CRC: colorectal cancer; SNP: single nucleotide polymorphism; GWAS: genome-wide association study; GALNT: N-acetylgalactosaminyltransferase; MCC: multicase-control; MAF: minor allele frequency; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; 95% CI: 95% confidence interval; OR: odds ratio; ARLTS1: ADP-ribosylation factor-like tumor suppressor gene 1; GALNTL2: UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 2; Chr: Chromosome; UTR: untranslated region; I5: Illumina HumanHap550; I3 Illumina HumanHap300; AIC: Akaike information content.The authors declare that they have no competing interests.Samples were collected within the EPICOLON by XB, DG, JCu, JCl, JDM, JR, ML, FF-B, EP, SR, RJ, XLl, VM, AC and MA and other members of this consortium. Study was designed by SCB and CRP. Data acquisition was performed by all authors. Quality control was performed by AA, CFR, VG, XB, RJ, XLL, MA and SCB. Data was analyzed and interpreted by AA, CFR, CRP and SCB. Statistical analysis was performed by AA, CV, VM and SCB. Manuscript was prepared by AA and SCB. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/339/prepubResults for previously identified and mucin SNPs in EPICOLON stage 1. SNPassoc results for previously identified and mucin SNPs in EPICOLON stage 1. P-values for some SNPs are highlighted in bold if significant (P < 0.05).Click here for fileResults for previously identified and mucin SNPs in EPICOLON stage 2. SNPassoc results for previously identified and mucin SNPs in EPICOLON stage 2. P-values for some SNPs are highlighted in bold if significant (P < 0.05).Click here for file