BADH1 and BADH2 are two homologous genes, encoding betaine aldehyde dehydrogenase in rice. In the present study, we scanned BADHs sequences of 295 rice cultivars, and 10 wild rice accessions to determine the polymorphisms, gene functions and domestication of these two genes. A total of 16 alleles for BADH1 and 10 alleles for BADH2 were detected in transcribed region of cultivars and wild species. Association study showed that BADH1 has significant correlation with salt tolerance in rice during germination stage, the SNP (T/A) in exon 4 is highly correlated with salt tolerance index (STI) (P<10−4). While, BADH2 was only responsible for rice fragrance, of which two BADH2 alleles (8 bp deletion in exon 7 and C/T SNP in exon 13) explain 97% of aroma variation in our germplasm. Theses indicate that there are no overlapping functions between the two homologous genes. In addition, a large LD block was detected in BADH2 region, however, there was no large LD blocks in a 4-Mb region of BADH1. We found that BADH2 region only showed significant bias in Tajima’s D value from the balance. Extended haplotype homozygosity study revealed fragrant accessions had a large LD block that extended around the mutation site (8 bp deletion in exon 7) of BADH2, while both of the BADH1 alleles (T/A in exon 4) did not show large extended LD block. All these results suggested that BADH2 was domesticated during rice evolution, while BADH1 was not selected by human beings. Spinacia oleracea L.), amaranth (Aramanthus spp.), barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.), BADH plays a role in abiotic stress tolerance through the accumulation of GB from betaine aldehyde (BA). Conversely, plant species such as tobacco (Nicotiana tabacum L.), tomato (Solanum lycopersicum L.) and rice (Oryza sativa L.) reportedly do not accumulate GB due to insufficient BA sh4 [rc [c [prog1 , GS3 [233 [badh2 .The badhvolution . The evoBADH1 and BADH2 gene from 295 cultivated rice and 10 wild rice by re-sequencing and (2) investigate the function of BADHs on the salt tolerance at germination stage and rice fragrance, and their overlapping functions. Lastly, we (3) investigated evolutionary path of BADHs in Asian rice.The goal of this study were to: (1) identify sequence polymorphism in the Two hundred and ninety-five rice varieties used for the present study were collected from RDA gene bank of Korea. Among the 295 accessions, 137 accessions were selected from worldwide 4,046 accessions, based on 15 SSRs by a heuristic approach using PowerCore software ,25, the Seeds of each accession were sterilized in sodium hypochlorite (1%) solution for 10 min and then washed twice with deionized distilled water. Then, the seeds were placed in petri dishes with two-layer filter paper and soaked in 200 mM NaCl solution, which was previously determined as an the optimal concentration (data not shown) to distinguish salt stress tolerance and sensitivity. The salt tolerance index (STI) was used to evaluate salt stress tolerance, where STI (%) =  × 100.The aroma of rice grains and leaves were measured by a sensory evaluation according to the method of Amarawathi et al. (2008) with minor modifications. Ten milled rice grains were placed in a 15 ml tube containing 8 ml of 1.7% KOH and incubated at 30°C water bath for 10 min with lids on. The lids were then opened and samples were smelled. The aroma of young leaves was checked by the same method to confirm the results for rice grain.https://github.com/najoshi/sickle). High-quality reads were aligned to the rice reference genome IRGSP-1.0 (http://rapdb.dna.affrc.go.jp/download/irgsp1.html) using the Burrows-Wheeler Aligner (BWA) (version 0.7.5a) with default parameters [http://broadinstitute.github.io/picard/). By using Genome Analysis Toolkit (GATK) (version 2.3.9 Lite) [Oryza rufipogon and 5 Oryza nivara) were downloaded from the National Centre for Biotechnology Information Sequence Read Archive 023116 [BADH1 and BADH2 were purified from whole-genome resequencing data.HiSeq 2000 and HiSeq 2500 were employed for whole-genome resequencing of the 295 rice accessions. Raw sequences were first processed to obtain an average of quality score (QS) per read ≤ 20 by trimming 3'-end of reads using SICKLE (rameters . The rea.9 Lite) , regionae 023116 . The polBADHs without any missing data. To get reliable results, P-value ≤ 0.001 was considered to have a significant effect on each trait.Genotypic and phenotypic data files were prepared and imported to TASSEL4.0  for assoBADH genes. Sliding windows of 1 kb and 3k were used to estimate nucleotide diversity for the 4-Mb region of each gene. In each window, the π and Tajima’s D value was calculated using VCFtools [To identify LD blocks in both wild and cultivated rice populations, we used Haploview software with “-dprime–minMAF 0.05 –hwcutoff 0.001 –blockoutput GAB –pairwiseTagging” parameters . AverageVCFtools . The patVCFtools .BADH1 and BADH2, were evaluated for salt stress index and rice fragrance. The average STI is 1.14. Among these, RWG-140 had the highest stress tolerance . However, RWG-222 had the lowest STI (0.26), which is salt stress sensitive. In the rice aroma test, total 12 accessions were evaluated to be fragrant rice , seven SNPs were found in the exon region, three SNPs and one insertion were in the untranslated region (UTR) in BADH1  one G/A SNP (P1141) in exon 1 leading to substitution from arginine to histidine; 2) one T/A SNP (P11483) in exon 4 leading to asparagine to lysine substitution; 3) one C/A SNP (P13605) in exon 11 leading to substitution from glutamine to lysine; 4) one T/C SNP (P13612) in exon 12 leading to substitution from isoleucine to threonine; 5) one G/T SNP (P13883) in exon 12 leading to substitution from glutamic acid to aspartic acid. Two non-synonymous mutations were represented in wild type as heterozygous SNPs. One is T/A SNP (P11232) in exon 3 leading to a substitution from aspartic acid to glutamic acid, the other was a G/T SNP (P13672) leading to a substitution from arginine to leucine in exon 11  8-bp deletion in exon 7 (P23036) leading to premature transcription termination; 2) one C/A SNP (P24488) in exon 10 leading to amino acid substitution from alanine to glutamic acid; 3) three base-pair deletion (P25240) in exon 12 leading to loss of one amino acid; 4) one C/T SNP (P25390) in exon 13 leading to substitution from alanine to valine; and 5) one base pair insertion in exon 14 resulting in frameshift and premature transcription termination have been detected. One synonymous SNP (P24528) in exon 10 has been found in two varieties. Two non-synonymous SNP were detected in wild rice, 1) C/G SNP (P2205) in exon 1 resulting to alanine to glycine substitution; and 2) C/T SNP (P22685) in exon 5 resulting to alanine to valine substitution. Those two alleles were heterozygous and initially detected in BADH2. In the UTR, three InDels, P214 (1 bp insertion), P236 (1 bp deletion) and P241 (insertion in the 5’UTR) and two SNPs (P224 C/T in the 3’UTR and P26038 in the 3’UTR) were detected.Eighteen alleles were detected in BADH1 and BADH2 region, but some accessions had low coverage or missing sequence in these gene regions. After removing accessions with missing sequence data, a total of 205 accessions remained. Among these 205 accessions, totally 19 SNPs and InDels were detected in BADH1 and BADH2 transcribed regions  of 0.12 between P11483/P13605/P14811 and STI (P<10−4) in BADH1  explain 97% of aroma variation in our germplasm. However, no association existed between aroma and BADH1  is the major allele which is associated with rice fragrance . Here we follow the method which was used by Kovach et al. (2009) for domestication study. Firstly, we divided the whole 205 accessions into two subgroups: the one that have 8-bp deletion in exon 7 (badh2.1), and without 8-bp deletion (Wild). Then we investigated the extended haplotype homozygosity of BADH1 and BADH2 regions to find the evidence for artificial selections. We calculated the EHH for BAHD2 gene by comparing the haplotypes of fragrant (badh2.1) and wild type  group. The badh2.1 had a large LD block that extended around the mutation site, while LD decreased rapidly around the wild type alleles  allele was significantly correlated with salt tolerance, we separated the 205 individuals into two subgroups, one included “T” allele (Allele_T group), another one included “A” allele (Allele_A group). STI values of the “T” allele subgroup were significantly higher than the “A” allele subgroup  and wide type (P11483 T). However, these two groups carrying A or T alleles did not exhibit large difference, both of them did not show large extended LD block, reducing rapidly around P11483  was significantly biased from the balance  suggested that BADH2 is also responsible for salt stress tolerance. The relationship between these two BADH genes and salt stress tolerance or fragrance in rice is unclear. Various studies have reported conflicting results [ in rice . As the  results . These iBADH1 and BADH2 genes. For BADH1, we found a total of 10 genetic variants, seven SNPs were in the exon region and two SNPs and one insertion were in the untranslated region , P13605 (C/A) in translated region and P14811 in the 3’UTR were highly correlated with STI with, P < 10−4 and R2 around 0.12  did show a difference between fragrant (badh2) and non-fragrant (BADH2) rice for the ability to produce mature seed in the presence of salt. Tang et al. (2014) proved that BADH1 was associated with rice salt (NaCl) tolerance at seedling stage by using RNAi-directed knock down of BADH1 [BADH1 and BADH2 are both suggested to be responsible for salt stress tolerance in rice, but, possibly, at different growth stages. Since our STI was tested at germination stage, we extended the conclusion that BADH1 is response for rice salt stress at germination and seedling stage, while BADH2 may be accountable at the reproductive stage.Fitzgerald et al. (2008) reported that the expression level of of BADH1 . Thus, B3036 (8 bp deletions) and P25390 (C/T) were the main alleles that explained the aroma variation with 74% and 23% respectively , demonstrating that BADH1 (and not BADH2) contributes to salt stress tolerance. However, we cannot exclude the possibility that these contradictions may be resulted from the different conditions undertaken.Singh et al. (2010) reported that there’s no association between salt tolerance and common BADH2 led by Kovach et al. (2009) suggested that BADH2 is one of the domesticated genes responsible for rice fragrance. In this study, we attempted to determine if BADH1 is also a domesticated gene.Domestication of crops has historically been critical for human reproduction and development. The domestication of rice were explained by several models . The rec1483, P13605 and P14811) in BADH1 highly correlated with STI. These three alleles were consistently present together in the rice genome. We selected P11483 (T/A) as the key allele for an evolution study of BADH1. To validate our method, we tested the domestication signal of BADH2 using the same method (EHH) as Kovach et al. (2009). Our EHH test result was similar to the results of Kovach et al. (2009). The fragrant accessions which carrying 8 bp deletions in exon 7 showed a large LD block extended around the mutation site, while the wild type alleles relatively reduced rapidly  over 7 kb, while fewer LD blocks were found in wild type group  found that the genetic diversity is strongly reduced in the aberrima . O. glab. sativa . As a ge. sativa . Our res. sativa . HoweverS1 Figbadh1.3, badh1.5 and badh1.6 were detected by previous study [badh1.1, badh1.2, badh1.4 and badh1.7 were detected in this study.(DOCX)Click here for additional data file.S2 Figbadh2.1~badh2.2 [badh2.11~badh2.12, badh2.14 [badh2.13 [badh2.15 [badh2.16~badh2.18 were detected in this study.~badh2.2 ; badh2.1badh2.14 ; badh2.1badh2.13 ; badh2.1(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(Excel)(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


In France during 2012, human enterovirus 71 (EV-A71) subgenogroup C4 strains were detected in 4 children hospitalized for neonatal fever or meningitis. Phylogenetic analysis showed novel and independent EV-A71 introductions, presumably from China, and suggested circulation of C4 strains throughout France. This observation emphasizes the need for monitoring EV-A71 infections in Europe. Picornaviridae, genus Enterovirus. On the basis of the 1D gene sequences encoding the VP1 capsid protein (1DVP1), EV-A71 has been classified into 3 genogroups (A–C) and 12 subgenogroups  (Human enterovirus 71 (EV-A71) is a member of the enterovirus species A in the family In the Asia–Pacific region, EV-A71 has emerged as a major public health concern over the past 15 years. Large outbreaks have been reported, associated with the emergence of new genogroups and subgenogroups, high rates of illness, and fatal cases of encephalitis (VP1), 12 of the 16 EV-A71 strains were assigned to subgenogroup C2, and 4 were assigned to subgenogroup C4.In France, EV infections diagnosed in hospital settings have been voluntarily reported to the National Institute for Public Health by a network of hospital laboratories since 2000 . Of the 4 patients, 3  had neonatal fever when medical care was sought, and 1 patient (4 years of age) had meningitis . The 21-VP1 sequences (VP1 sequences). The quick evolution of the 2 EV-A71 C4 strains from their common ancestor suggests local spread of the viruses. The other 2 strains displayed consistent clustering (posterior probability >0.8) with different strains isolated in China, suggesting independent introductions of virus.Phylogenetic analyses based on a Bayesian approach were performed with a set of 97 1Dequences . The chrIn 2012, EV-A71 C4 strains were detected in France in 4 children hospitalized for neonatal fever or meningitis. Although EV-A71 C4 strains have circulated extensively in China since 2008, this virus has rarely been detected in Europe. In France, 133 cases of EV-A71 infections were reported during January 2000–May 2013 (Many cases of fatal encephalitis have been associated with EV-A71 C4 infection outbreaks in China (Dated phylogeny inferred by using 97 enterovirus 71 1D gene sequences encoding the VP1 capsid proteins; taxon names of the strains selected for the phylogenetic analyses are shown.


Regional citrate anticoagulation (RCA) is becoming the preferred method of extracorporeal anticoagulation in patients undergoing continuous renal replacement therapy (CRRT), however the ideal composition remains unclear. We have found protocolised hemofiltration with dilute solutions to be simple and safe, but commercially available solution options are limited. Recently the 14 mmol/L solution (Baxter Haemofiltration Fluid [C14]) was replaced in Australia with 18 mmol/L solution [C18], on the premise that the former provided inadequate anticoagulation. With the use of the C18 solution, a higher incidence of metabolic alkalosis was observed, prompting the design of a biochemically modified solution which would provide optimal anticoagulation, but less biochemical disturbance.This study primarily aimed to assess the changes in biochemistry occurring in patients undergoing CRRT using two trisodium citrate solutions, the C18  and a custom manufactured solution containing 15mmol/L (Baxter NamSol [C15]), delivered as a predilution fluid for hemofiltration.Secondary aims included duration of CRRT, filter life, length of ventilation, length of stay and mortality. The occurrence of significant adverse events was also noted.This is a single-blinded prospective randomised control trial, in a major regional adult intensive care unit. Patients were consented and allocated to one of 2 RCA fluids (C18 or C15) for CRRT. Progress was monitored using a standard daily panel of acid-base and biochemical tests. Analyses were performed days 1, 3 and 5.+] (144.7 mmol/L (SD 4.4) vs 140.1 mmol/L (SD 3.3), p = 0.0002) and Standard Base Excess (SBE) (7.6 (SD 3.5) vs 2.6 (SD 3.2), p = 0.005), both being higher in the C18 group . Four patients receiving C18 left the study by day 5 due to concerns regarding very high SBEs (> 12 meq/L).By Day 5, PAlbeit underpowered, there were no significant differences in secondary end-points, and no significant adverse events were noted in either group.For CRRT, the custom C15 solution provided equivalent acidosis recovery and filter-life to the C18 solution, but without the significant hypernatraemia and metabolic alkalosis. Neither solution was associated with any clinically evident adverse events. The C15 solution is a step closer to an ideal RCA replacement fluid, and should be considered for wider availability.


Here we demonstrate that CHI3L1 (aka Brp39) knockout (KO) mice treated with azoxymethane (AOM)/dextran sulphate sodium (DSS) developed severe colitis but lesser incidence of CAC as compared to that in wild-type (WT) mice. Highest CHI3L1 expression was found during the chronic phase of colitis, rather than the acute phase, and is essential to promote intestinal epithelial cell (IEC) proliferation in vivo. This CHI3L1-mediated cell proliferation/survival involves partial downregulation of the pro-apoptotic S100A9 protein that is highly expressed during the acute phase of colitis, by binding to the S100A9 receptor, RAGE (Receptor for Advanced Glycation End products). This interaction disrupts the S100A9-associated expression positive feedback loop during early immune activation, creating a CHI3L1hi S100A9low colonic environment, especially in the later phase of colitis, which promotes cell proliferation/survival of both normal IECs and tumor cells.Many host-factors are inducibly expressed during the development of inflammatory bowel disease (IBD), each having their unique properties, such as immune activation, bacterial clearance, and tissue repair/remodeling. Dysregulation/imbalance of these factors may have pathogenic effects that can contribute to colitis-associated cancer (CAC). Previous reports showed that IBD patients inducibly express colonic chitinase 3-like 1 (CHI3L1) that is further upregulated during CAC development. However, little is known about the direct pathogenic involvement of CHI3L1  The comparable risk of CAC has also been reported in longstanding Crohn's disease (CD) involving the colon, making CAC one of the major causes of death in IBD patients [The causal relationship between inflammation and cancer is now widely recognized. In the case of inflammatory bowel disease (IBD), patients are predisposed to a 2% risk of developing colitis-associated cancer (CAC) by the 10During the onset and/or flare of inflammation, innate immune activation provides the first response to counter pathogen exposure, releasing a milieu of molecules with anti-microbial and/or high reactive oxygen species (ROS) activities. Although these actions may be effective in promoting bacterial clearance, it also exerts high oxidative stress to intestinal epithelial cells (IECs) that may drive them into apoptosis and/or induce genomic mutation/instability. Hence, host IECs subsequently express pro-survival factors to counter effects of these harsh inflammatory events and promote epithelial restitution as part of recovery. However, promoting survival and proliferation of IECs that harbor genetic mutations induced as the consequence of the exaggerated inflammatory events will lead to tumor expansion. Since both inflammation and recovery can occur in the same region at the same time, a deeper understanding of how inducible specific factors promote intestinal homeostasis during the course of inflammation and how imbalance of these factors can exacerbate inflammation and/or tumorigenesis is needed.in vitro studies have suggested that CHI3L1 promotes proliferation in colon cancer cell lines and increases xenografted tumor load and angiogenesis [in vivo role of CHI3L1 during tumorigenesis, in particularly in an inflammation setting, has not been explored.Chitinase 3-like 1  is a pseudo-chitinase that is usually undetectable in the colon of healthy individuals, but is expressed by IECs and macrophages in inflamed intestines . CHI3L1 ogenesis . HoweverReceptor for Advance Glycation End product (RAGE) and activate NF-kB signaling that binds back onto the S100A9 promoter creating a transcription positive feedback loop [Like CHI3L1, the damage-associated molecular (DAMP) protein S100A9 is also usually undetectable in healthy colons, but can be induced in colonic mucosa during colitis . Intraceack loop . In thisack loop , 13. TheMycobacterium tuberculosis-infected CHI3L1 (Brp39) knockout (KO) mice, suggesting a potential cross-regulating mechanism that creates an inverse relationship between CHI3L1 and S100A9 expression [in vitro and in vivo. These anti-apoptotic and pro-proliferative effects of CHI3L1 are achieved though the downregulation of S100A9, by binding/blocking the RAGE that cuts the S100A9-RAGE positive feedback loop, resulting in a pro-proliferative CHI3L1hi S100A9low environment. This leads to the survival and proliferation of not only normal IECs under inflammatory conditions that facilitate the improvement of colitis, but also potential cancer progenitor cells that may promote CAC.Despite the fact that the expressions of both CHI3L1 and S100A9 are reportedly induced during inflammation, possible cross-regulation of these two proteins has not been explored. This hypothesis is based on previous reported genome-wide gene expression profiling that showed an upregulation of S100A9 in pression . In thisTo characterize the phenotypic role of CHI31L expression induced during chronic colitis and tumorigenesis, WT and Brp39 KO mice were given a single intraperitoneal injection of a carcinogen AOM and 3.5% DSS in the drinking water for five days and normal water for the subsequent 10 days for 3 cycles (AOM/DSS) . Brp39 KTo further investigate how CHI3L1 deficiency exacerbates chronic colitis, H&E sections of the mouse colon were examined microscopically. The Brp39 KO mice that had undergone AOM/DSS treatment showed partial mucosal erosions and atrophy, whereas much less epithelial damage was observed in the WT mice Figure . In addiTo delineate the roles of CHI3L1 expression in hematopoietic versus non-hematopoietic cells, we generated two models of bone marrow transplanted (BMT) chimeric mice: Brp39 KO BM-transplanted WT (Brp39 KO BM -> WT) mice and WT BM-transplanted Brp39 KO (WT BM-> Brp39 KO) mice. AOM/DSS treatment in the Brp39 KO BM -> WT BMT-chimeric mice resulted in a BW profile that is intermediate between WT and Brp39 KO mice, suggesting that the CHI3L1 expression in the non-hematopoietic cells, presumably IECs, can partially ameliorate colitis Figure . ConversTo explore the role of inducible CHI3L1 in CAC, WT- and Brp39 KO-AOM/DSS-treated mice was examined for tumor formation. Polypoid lesions were detected in the 88% of the AOM/DSS-treated WT mice, predominately in the distal part of the colon. In contrast, only 16% of AOM/DSS-treated Brp39 KO mice showed grossly detectable polypoid lesions Figure . NotablySince colonic CHI3L1 is known to be inducibly expressed in IECs during the course of IBD which is further overexpressed when CAC develops, quantifying CHI3L1 levels using easily accessible sample collection and methods may help to develop a new diagnostic strategy for predicting the neoplastic changes of IECs in IBD patients. Therefore, serum and stool samples were collected from untreated WT mice, WT mice that were given 1 cycle of DSS for 5 days (i.e. acute colitis), and WT mice that were given AOM/DSS treatment (i.e. chronic colitis and CAC). ELISA quantification showed a step-wise increase in CHI3L1 levels in both serum and stool of the untreated WT mice to WT mice that had undergone 1 cycle of DSS and to AOM/DSS treated WT mice, indicating that CHI3L1 levels are positively associated with the progression of colitis and CAC Figure . In addiSince CHI3L1 and S100A9 molecules are inducibly expressed in the intestine during IBD, but Brp39 KO mouse was previously reported to show an up-regulation of S100A9 expression during inflammation, we therefore examined the potential association between these two molecules . To do tOne of the major pathways that induce the endogenous expression of S100A9 is through binding of the RAGE receptor with its specific ligands, including AGEs and S100A9 itself. Thus, we hypothesize that CHI3L1 may interact with RAGE during inflammation and ultimately prevents the RAGE/RAGE-ligand interaction for the activation of S100A9 expression. To test this hypothesis, a ligand-binding assay was first performed by incubating total lysate from SW480 cells overexpressing His/Xpress-tagged CHI3L1 or LacZ (control) protein with overexpressed Flag-tagged RAGE protein. Subsequently, pulldown of His-tagged CHI3L1 protein, but not His-tagged LacZ control protein, with His-nickel beads co-precipitated Flag-tagged RAGE Figure  showed tSince both CHI3L1 and S100A9 expressions are induced during inflammation and both bind to their common receptor RAGE, we next hypothesized that CHI3L1 and S100A9 compete for the binding to RAGE. To test this hypothesis, a constant concentration of His-tagged rS100A9 protein was incubated with SW480 cells, which were transfected with pCDNA3.1 RAGE-expression plasmid, in the presence of various concentrations of purified CHI3L1 protein. Subsequently RAGE ligand binding assay and immunoblotting using anti-His antibody showed decreasing amounts of detectable His-tagged S100A9 that were bound with the RAGE in the presence of increasingly higher concentrations of CHI3L1 protein Figure . In contin vitro studies demonstrated that STAT3, β-catenin, NF-κB and AKT are the four main signaling pathways activated by CHI3L1 in IECs [In vivo, colonic sections from AOM/DSS treated WT mice clearly displayed strong nuclear localization of STAT3, β-catenin as well as NF-κB, whereas Brp39 KO mice predominately exhibited a cytoplasmic staining pattern of these transcriptional factors , or AOM/DSS (chronic colitis/CAC). Subsequently, Quantitative PCR and immunofluorescence microscopic analysis were performed to quantify and visualize the colonic expression levels of CHI3L1 and S100A9. During the onset of DSS-induced acute colitis, CHI3L1 expression is induced in WT mice that further up-regulated during the chronic phase  showed significant CHI3L1 expression in the lungs, but when these patients experienced acute exacerbations, CHI3L1 expression dropped to a level between those of IPF patients and the control group . In this effects . High coFurthermore, functions of CHI3L1 and S100A9 in cancer cells are also inversely correlated each other. For instance, S100A9 inhibits human cervical cancer cell metastasis . GastricListeria and Salmonella into epithelial cells exerted through its antimicrobial properties [When induced, the biological roles of CHI3L1 toward enteric microbes are distinct from those of S100A9. Overexpression of S100A9 downregulates the invasive abilities of operties . Howeveroperties , 24. In operties . All theAlthough S100A9 is pro-apoptotic at high concentration, it is still required for CAC development since S100A9 KO mice showed fewer incidences of AOM/DSS-induced CAC . High amIn summary, this study presents the essential role of CHI3L1 in promoting CAC by enhancing survival and proliferation of IECs and neoplastic cells through the downregulation of S100A9 and its effects during the preceding inflammation. We also show that the analysis of CHI3L1 levels in non-invasively collected feces serves as a convenient and reliable modality to predict the neoplastic changes during the course of chronic colitis.CHI3L1 KO mice on an inbred C57Bl/6 background were obtained from Drs. Anthony Coyle and Alexander Kozhick , originally generated by Drs. Chun Geun Lee and Jack A. Elias  and housed in the Massachusetts General Hospital specific pathogen-free facility, under an Institutional Animal Care and Use Committee approved protocol .CAC was induced in 8–10 week old Brp39 KO and C57Bl/6 wild-type (WT) mice by single injection of azoxymethane  at 12 mg/kg. Five days later, mice were treated with 3.5% dextran sulfate sodium (DSS) for 5 days and replaced with normal water for the next 10 days for 3 cycles. Body weight and clinical symptoms  were monitored daily. Mice were sacrificed on day 45 and gross macroscopic tumors were counted and measured under an Olympus SZ-PT dissection microscope . H&E sections of the colons were blindly scored by a pathologist with expertise in GI pathology based on previously reported criteria .5 SW480 cells were seeded on 6-well plate and transfected with 2.5 μg of His-Xpress tagged pCDNA4-CHI3L1- or -LacZ-, or 2.5 μg of Flag tagged pCDNA3.1-RAGE-expression plasmid, which was a gift from Dr. Lin Li  [4 × 10ore, MD) . Ligand ore, MD) .Colonic frozen sections (4 μm thick) were stained with the respective primary antibodies using the avidin-biotin-complex system . The following primary antibodies were used for staining: anti-Ki-67 and anti-β-catenin , anti-Annexin V , as well as anti-NF-κB p65 and anti-STAT3  antibodies.Mouse CHI3L1 levels in serum and stool (resuspended 10 mg/ml in PBS) were detected using ELISA kit purchased from R&D systems  and performed according to the manufacturer's instructions. Supernatant of SW480 cells that were transfected with pcDNA3.1-RAGE and stimulated with different concentrations of recombinant S100A9 (Life technologies) were collected and detected with human CHI3L1 using the same ELISA kit (R&D systems). Optical density was measured at 450 nm using Auto-Reader Model 680 . Human stool was collected from patients with IBD, colonic adenoma, sporadic colon cancer, or CAC at the Division of Gastroenterology and Division of Mucosal Immunology, Shiga University of Medical Science  from April 2007 to November 2009. Similarly, 10 samples from healthy individuals were obtained at the same University. One gram of fresh stool was collected and stored at −80°C until the stool CHI3L1 levels were measured by ELISA kit (R&D systems) as previously described .137Cs Gamma Cell source and intraperitoneally injected with 107 donor bone marrow cells. Four weeks later, mice were subjected to AOM/DSS treatment as described above.Bone marrow cells were aseptically isolated from the femurs and tibia of WT and Brp39 KO mice as previously described . Recipie2) for 15 minutes on ice and centrifuge at 3000 rpm for 10 min at 4°C. The pellet was then lysed with 1% NP-40 lysis buffer for 30 minutes on ice and supernatants were collected by centrifuging at 13,000 rpm for 30 minutes [Total proteins from RAGE and/or CHI3L1 expression plasmid transfected cells were extracted using 0.1% NP-40 lysis buffer as previously described . Nuclear minutes . SDS-PAG minutes .Colonic total RNA was extracted using Trizol reagent  according to the manufacturer's instructions. One microgram of RNA was reverse transcribed using iScript cDNA synthesis kit (Bio-rad). PCR reaction was set up using the iQTM SYBR Green Supermix (Bio-rad) and amplified the following primer pairs: human CHI3L1 , mouse CHI3L1 , human S100A9 , mouse S100A9 .SW480 cells were transfected with pcDNA3.1-RAGE or control expression plasmid for 48 hours, stimulated with different concentrations of purified-CHI3L1 protein  and/or rS100A9 (Life technologies) for the next 24 hours and then pulsed with 10 μmol/L 5-bromo-2-deoxyuridine  in cell culture medium for 1 hour. Cells were then trypsinised and cytocentrifuged using Shandon Southern Cytocentrifuge . Samples were then fixed in 100% acetone for 10 minutes and stained using anti-BrdU  or Annexin V (Thermo scientific) antibody.Mouse colonic cryosections were fixed in acetone for 10 minutes and air-dried at room temperature, blocked in 5% goat and horse serum for 30 minutes at room temperature and then co-stained with rabbit-anti-CHI3L1  and rat-anti-S100A9 (Abcam) antibodies at 4°C overnight. Sections were washed and stained with Alexa Fluor 647 goat-anti-rabbit IgG (Invitrogen) and FITC horse-anti-rat IgG (Vector Laboratories) secondary antibodies for 30 minutes at RT. The sections were washed with PBS, mounted in Vecta-shield (Vector) and analyzed under the Bio-Rad Radiance 2000 confocal microscope.T-test or One-Way and Two-Way ANOVA with post-hoc Tukey Test, when appropriate, was performed using the Graphpad Prism Software for multiple comparison experiments. Values were presented in mean ± SEM. P ≤ 0.05 was considered as statistically significant.Student's


Since March 2013, the emergence of an avian-origin influenza A (H7N9) virus has raised concern in China. Although most infections resulted in respiratory illness, some severe cases resulted in acute respiratory distress syndrome (ARDS), which is a severe form of acute lung injury (ALI) that further contributes to morbidity. To date, no effective drugs that improve the clinical outcome of influenza A (H7N9) virus-infected patients have been identified. Angiotensin-converting enzyme (ACE) and ACE2 are involved in several pathologies such as cardiovascular functions, renal disease, and acute lung injury. In the current study, we report that ACE2 could mediate the severe acute lung injury induced by influenza A (H7N9) virus infection in an experimental mouse model. Moreover, ACE2 deficiency worsened the disease pathogenesis markedly, mainly by targeting the angiotensin II type 1 receptor (AT1). The current findings demonstrate that ACE2 plays a critical role in influenza A (H7N9) virus-induced acute lung injury, and suggest that might be a useful potential therapeutic target for future influenza A (H7N9) outbreaks. Avian influenza A H7N9) virus is a viral subtype that was detected in birds previously. However, it had not been reported in either animals or humans prior to its identification in China in March 20131245N9 virus 89The rennin-angiotensin system (RAS) is a complex network that plays a major role in maintaining blood pressure, electrolyte and fluid homeostasis, and fluid and salt balance111218Four-week-old wild-type (WT) C57BL/6 (abbreviated B6) mice , and 4-week-old ACE2 knockout (abbreviated KO) mice  were housed in the animal facility at the Beijing Institute of Microbiology and Epidemiology in accordance with institutional guidelines.All experimental protocols were approved by the Institutional Animal Care and Use Committee of Academy Military Medical Sciences (ID: SYXK2010-005). Live-virus experiments were performed in Bio-safety Level 3 facilities in accordance with governmental and institutional guidelines.3.5 of the 50% tissue culture infectious dose (TCID50) of Hb01/H7N9 virus or mock-infected control allantoic fluid (AF). The survival, weight loss, acute pulmonary edema (wet-to-dry ratio), and histological measurements were performed as described previouslyThe influenza A H7N9 virus  used in this study was isolated from a confirmed H7N9-infected patient. The genomic sequences of Hb01/H7N9 are available in the Global Initiative on Sharing All Influenza Data (GISAID) database under the accession numbers EPI509120–EPI509127. Live virus experiments were performed in Biosafety Level 3 facilities in accordance with governmental and institutional guidelines. For influenza A H7N9 virus-induced acute lung injury, 4-week-old WT B6 mice were anesthetized with 50-μl 1% (w/v) pentobarbital sodium, and then inoculated intranasally (i.n.) with 2 × 10For inhibitor experiments, mice were injected intraperitoneally with the AT1 inhibitor losartan (15 mg/kg), the AT2 inhibitor PD123.319 (15 mg/kg), or PBS 30 min before Hb01/H7N9 virus infection.Ang II levels were detected as described previously3.5 TCID50 virus, and were then sacrificed at various days post-infection (DPI). The lungs of each group of mice (five mice per group) were fixed in formalin, and then embedded in paraffin. The number of inflammatory cells was counted, and the data are presented as the number of cell per 200× field.After being anesthetized with pentobarbital sodium, 4-week-old B6 mice were treated i.n. with 2 × 103.5 TCID50 virus. At 5 DPI, the wet weights of the lungs were then measured from eight mice per group. The lungs were then heated to 68°C overnight, and the dry weights were measured.To assess the extent of acute pulmonary edema, the lung wet-to-dry weight ratios were calculated. WT 4-week-old B6 mice were anesthetized using sodium pentobarbital, and were then inoculated intranasally with 2 × 103.5 TCID50 virus using an RNeasy Mini Kit (Qiagen). Real-time RT-PCR was performed using an ABI 7500 Real-Time PCR system (Applied Biosystems) with primers and a TaqMan one-step RT-PCR master mix (Applied Biosystems). Primers that could detect the influenza NP gene specifically were used. The expression of target genes was normalized to that of the control gene GAPDH. Relative amounts of mRNA were calculated using the comparative CT method.RNA was extracted from the lungs of 4-week-old B6 mice infected with 2 × 1010 TCID50/g of lung tissue.Virus titers were measured in the supernatants of mouse lung homogenates from ACE2 KO or WT mice on day 5 post-infection. Briefly, samples were added to 96-well plates containing MDCK cells, and then diluted 10-fold. The infected cells were then cultured for 96 h. The viral titers were calculated using the Reed and Muench method, and data were expressed as logp < 0.05 was considered to indicate statistical significance.All data are presented as means ± SEM. Measurements at single time-points were analyzed using ANOVA, and survival data were analyzed using Kaplan-Meier survival analysis. All statistical analyses were performed using the GraphPad Prism 5 software. A value of Two research groups cloned the ACE homolog ACE2 independently in 2000p < 0.01) markedly. Similarly, there was also a significant increase in Ang II levels in the lungs of Hb01/H7N9 virus-infected mice  virus-induced ACI, we next examined the levels of Ang II in the plasma of H7N9-infected patients. Six patients who were PCR-positive for the 2013 influenza A (H7N9) virus were recruited from Beijing Chaoyang hospital and Quanzhou hospital, China. The characteristics underlying the conditions and the outcomes of the six patients are described in We next investigated whether ACE2 could mediate or protect mice from 2013 H7N9 influenza infection-induced ALI. ACE2 KO mice that were infected with influenza Hb01/H7N9 virus exhibited survival rates that were reduced significantly compared with WT mice . SpecifiWe next attempted to rescue the ALI in WT mice infected with influenza Hb01/H7N9 virus using specific AT1 and AT2 receptor blockers. Inhibiting AT1 alleviated the severity of influenza Hb01/H7N9 virus-induced lung injury significantly in WT mice, as determined by the lung wet-to-dry ratio and histopathology . SimilarSimilarly, the lung wet-to-dry ratio of H7N9 virus-infected ACE2 KO mice treated with At1 receptor blockers was decreased and the pathological lung alterations were improved . SimilarARDS is the most severe stage of ALI during pathogenic infectionsThe data presented here also demonstrated that ACE2 deficiency aggravated influenza A (H7N9) virus-induced ALI in mice. Nevertheless, further studies assessing the potential therapeutic effects of recombinant human ACE2 protein are required using different animal models to verify the protective effects of ACE2 against H7N9-induced lung pathologies. The combination of clinical findings and mice experiments has revealed a critical role for the RAS in the pathogenesis of influenza A (H7N9) virus-induced ALI, and demonstrated that ACE2 plays a key role in the development and progression of H7N9 influenza. In addition, the molecular basis by which influenza H7N9 virus infection decreases ACE2 expression should be investigated in future studies. Taken together, these data raise the intriguing possibility that treatments targeting ACE2 and RAS might be viable strategies for treating influenza A (H7N9) virus infections.P.Y., H.G., Z.Z., W.W., C.L., X.Y., L.Z., Y.D. and S.Z. contributed to experiment. P.Y., H.G. and B.C. analyzed the data. B.C., W.C., W.Z. and M.C. collected the samples. J.P., C.J. and X.W. designed the experiments and reviewed/edited the manuscript extensively. P.Y., C.J. and X.W. wrote the manuscript.SI


JMJD2C is a candidate oncogene that encodes a histone lysine demethylase with the ability to demethylate the lysine 9 residue of histone H3 (H3K9). The expression levels of JMJD2C are associated with tumor development and clinical outcome. Here we identify JMJD2C as a new substrate for caspase-3. JMJD2C is cleaved by caspase-3 at DEVD396G motif and then loses its demethylase activity. Additionally, we uncover D396N polymorphism (rs2296067) in the cleavage site of JMJD2C and establish its influence on the resistant to the cleavage by caspase-3. Importantly, we determined that D396N polymorphism is significantly associated with the prognosis of human breast cancer. We further found that the basal levels of DSB (double strand DNA break) repair proteins γ-H2AX (gamma-H2AX) increased when cells were treated with tumor necrosis factor-α (TNF-α) which activates caspase-3 activity. We also show that knockdown of JMJD2C expression results in up-regulation of basal γ-H2AX. We propose that D396N polymorphism of JMJD2C affects the prognosis of human breast cancer via altering the cleavage by caspase-3 and the ability of DSB repair which may contribute to therapy resistance. Also known as KDM4C and GASC1, JMJD2C is a member of the Jumonji domain-2 (JMJD2) family and encodes a protein with one JmjC domain, one JmjN domain, two PHD-type zinc fingers and two Tudor domains. JMJD2C has been proved to be a demethylase for H3K9 methylation , 6. The Caspase-3 is a primary effector of the cysteine protease family, which plays an essential role during apoptotic cell death by proteolytic cleaving a variety of key proteins required for cellular functioning and survival . For exaIn this report, we identify and describe a SNP rs2296067 (D396N) in caspase-3 cleavage site of JMJD2C that affects its cleavage by caspase-3. The cleavage of JMJD2C by caspase-3 increases the levels of H3K9me3 and basal levels of γ-H2AX . In addition, we observed that D396N polymorphism of JMJD2C is significantly associated with the prognosis of human breast cancer. This study demonstrates that D396N polymorphism makes JMJD2C resistant to caspase-3 cleavage, thereby revealing a functional connection between prognosis of breast cancer, caspase cleavage and DSB repair.Mr ~150K) were present in most samples. Meanwhile, the small fragments with relative molecular mass ~100,000 (Mr ~100K) were detected at low levels in most samples. Only the small fragment of JMJD2C was detected in the case 4 sample. By analyzing the amino acid sequence of JMJD2C, a caspase-3-like protease cleavage sequence DEVD396G  was found to be located downstream of the JmJC domain of JMJD2C . Interestingly, two specific fragments were found in several samples Fig. . High leD2C Fig. . The epiD2C Fig. . A300-88Mr 100K fragment of JMJD2C was detected using the A300-885A antibody treated with TNF-α or CHX alone for 6 h   for 6 h, whereas the abundance of the native Mr 150K fragment decreased markedly  /cycloheximide (CHX) to induce caspase-3-like protease activity. The ne) Fig. . Meanwhidly Fig. . In addirs) Fig. . These rWe isolated the full length of JMJD2C cDNA to do overexpression analysis with the cDNA from case 2 sample. After the sequencing, we got two kinds of clones with nucleotide bases A or G in the same site of JMJD2C cDNA (1188 site). The two different nucleotide bases (G/A) result in amino acid changes in the cleavage motif (DEVD/N396G) of JMJD2C. By searching the NCBI dbSNP database for polymorphisms in the sequences of JMJD2C gene, we found that a single nucleotide polymorphism rs2296067 (D396N) was responsible for this. By sequencing the exon-9 of JMJD2C gene that contains DEVD396 motif in the above samples, three types of genotype of SNP rs2296067 were shown in the above breast-tumor samples Fig. . FurtherMr 150K fragment and Mr 100K fragment were detected increased by using A300-885A antibody after the transfection  and the C-terminal cleavage fragments (Mr ~100K) of JMJD2C. In the presence of z-DEVD-fmk, the generation of C-terminal cleavage was inhibited. Meanwhile, PARP-1, a well-characterized caspase-3 substrate, was cleaved in a similar manner to JMJD2C  Fig. . These fH3K9me3 is involved in DSB repair by binding Tip60 acetyltransferase, which activates ATM and initiates a signaling cascade that regulates DSB repair , 11. TheNext we examined the distribution of SNP rs2296067 in breast cancer patients (n=223) and healthy controls n=162). SNP rs2296067 was found at relatively high frequency in the heterozygous state  and homozygous state  in the healthy controls  which results in the aspartate to asparagine acid substitution at position 396 of JMJD2C protein was found in JMJD2C gene. This SNP creates a substitution in the caspase-3 cleavage motif of JMJD2C (DEVD/N396G). Previous studies have shown that aspartate to asparagine acid mutation at position 214 (D/N214) in DEVD214G motif of PARP-1 renders it resistant to the cleavage by caspase-3. Similarly, the JMJD2C variant carrying DEVN396G motif is also resistant to the cleavage by caspase-3.JMJD2C was identified as a gene frequently amplified in different types of cancer such as oesophageal squamous cell carcinoma (ESCC) , primaryInterestingly, previous studies have shown that caspase-3 activity is critical to differentiation of ESCs (Embryonic stem cells) and the differentiation may depend on caspase-3-induced cleavage of Nanog in differentiating ESCs . H3K9 meIn many, but not all JMJD proteins, the JmjC domain has the catalytic activity and mediates the demethylation of histone lysine residues . A previIn this study, the JMJD2C SNP rs2296067 was found in the cleavage site of JMJD2C. The protein product of JMJD2C with the minor “A” allele in SNP rs2296067 is resistant to the cleavage by caspase-3. Accordingly, it seems like that the individuals carrying homozygous G/G genotype in SNP rs2296067 may suffer prolonged lysine 9 demethylation on histone H3. Thus, levels of H3K9me3 may be reduced in the individuals carrying homozygous G/G genotype in SNP rs2296067. Chemotherapeutic drugs and radiation cause DNA damage and induce signaling pathways for apoptotic cell death. Enhanced DNA damage repair processes play a role in the development of resistance to chemotherapy and radiation therapy . H3K9me3Our findings reveal a new mechanism for negative regulation of JMJD2C by caspase-3 cleavage. Moreover, the single nucleotide polymorphism rs2296067 of JMJD2C renders it resistant to the cleavage by caspase-3. Taken together, these results may lead to further insights into the regulation and function of JMJD2C in cancer and stem cells.2 atmosphere. Rabbit anti-JMJD2C (A300-885A) was from Bethyl Laboratories. Mouse anti-H3, anti-H3K9me3, anti-GAPDH, anti-FLAG and anti-HA were from Abmart Company. Rabbit anti-PARP-1 was obtained from Cell Signaling Technology. z-DEVD-fmk was from BD Bioscience. Cycloheximide was from Amresco. Recombinant Human TNF-α was from PeproTech. Polybrene and puromycin were from Sigma.The human breast cancer MCF-7 and MDA-MB-231 cell lines, 293T and HeLa cell lines were obtained from the American Type Culture Collection. MDA-MB-231 cell lines were maintained in continuous culture in L-15 medium supplemented with 10% fetal bovine serum (FBS). Other cells were maintained in continuous culture in MDEM medium supplemented with 10% FBS. These cell cultures were maintained in a 37°C incubator with a humidified 5% COGenomic DNA from 223 patient tumor samples with known clinical history was extracted manually using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. Genomic DNA from the blood leukocytes of 162 participants  was extracted Gentra's PureGene DNA Purification Kit (Gentra systems). Genotypes of SNP rs2296067 were determined by direct sequencing of exon-9 of JMJD2C gene from PCR-amplified genomic templates. Primer sequences used for PCR- amplification were 5'-TTCATATCCATTGCAGACATCCCGC-3' ; 5'-AGGCACTGCTGACTGCTTGTCT-3'. This study was approved in advance by the Institutional Review Board of the Cancer Hospital, Fudan University.Extraction and reverse transcription of the Total RNA were done with TRIzol reagent (Invitrogen) and PrimeScript RT reagent Kit (Takara Biotechnology). The pSG5 (2xFLAG epitope tag in N-terminus) plasmid is a gift from Jiemin Wong . The human full-length cDNA of JMJD2C carrying “G” allele in SNP rs2296067 was obtained from the cDNA in case 2 breast-tumor sample and then cloned into the vectors. In-Fusion HD cloning kit (Takara) was used for homologous recombination between amplified fragments and the vectors. The primers were: pcDNA3.1 , pSG5 . To overexpress the N-terminal cleavage JMJD2C in pSG5, the reverse primer was used: 5'- CGGGCGGCCGCTCGAGCTAATCG ACTTCATCTGACACTT-3'. The cDNA of JMJD2C were subcloned into lentiviral expression plasmid pCDH-CMV (from System Biosciences and adding n-1xHA or c-1xFLAG by our lab) to generate JMJD2C with C-terminal FLAG epitope tag and N-terminal HA epitope tag. The primers were: pCDH-CMV-c-FLAG , pCDH-CMV-n-HA . Lentiviral vectors (pLKO.1) containing shRNA sequences targeting JMJD2C  or non-silencing control scramble shRNA  were obtained from Open Biosystems. JMJD2C D396 was mutated to N396 using the primer 5'-GTCAGATGAAGTCAATGGGGCAGAGGTC-3' and the complementary primer 5'-GACCTCTGCCCCATTGACTTCATCTGAC-3' with the Site-directed Gene Mutagenesis Kit (Genesent) and the substitution was confirmed by sequencing.Transfections were done using Lipofectamine 2000 transfection reagents (Invitrogen) according to manufacturer instructions. The lentiviral expression vector was packed into HEK 293T cells to generate corresponding lentiviruses. The lentiviruses were added to cells with 8 μg/ml polybrene. 48 h later, the cells were changed to fresh puromycin-containing media (2.5μg/ml). After puromycin selection for 72 h, the lentivirus-free cells have completely died. The survived cells added with lentivirus were identified by western blotting analysis for the expression of JMJD2C.3VO4, and 5 mg/ml leupeptin. Then cell lysates were centrifuged at 14000×g for 10 min at 4 °C. Protein concentrations were estimated using protein-dye (Bio-Rad). Samples were separated by SDS-PAGE and then blotted onto a PVDF membrane (Millipore). After blotted with primary antibodies overnight, peroxidase conjugated secondary antibodies were incubated with the membrane. Bound antibodies were detected by Chemiluminescent HRP Substrate (Millipore) and photographed by LAS-3000 luminescent image system (Fujifilm).Cells were lysed for 20 min on ice in lysis buffer (Genesent) with 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, 10 mM NaF, 2m M NaANOVA and Student t tests were used to determine the statistical significance of differences between experimental groups. The Kaplan-Meier method was used to analyze breast cancer patient cumulative survival rate. A probability value of 0.05 or less was considered significant. Statistical analysis and graphs were created with GraphPad Prism (Graph-Pad Software).


Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.The  Ascaris, infect over a billion people worldwide and negatively affect growth, nutrition, cognition, and pregnancy (Bacillus thuringiensis (Bt) crystal (Cry) proteins to treat intestinal nematode infections in humans. B. thuringiensis is a Gram-positive, spore-forming bacterium that produces parasporal crystalline protein inclusions known as Cry proteins  parasites, including hookworms, whipworms, and regnancy . The Worregnancy . Resistaregnancy , and effproteins . The Cry hamsters infected with the zoonotic hookworm parasite Ancylostoma ceylanicum, and (iii) pigs infected with Ascaris  mice infected with the natural intestinal parasite  Ascaris , 7. Cry5 Ascaris , 8. The  elegans . The C-tproteins , 10.Lactococcus lactis has been used to express a variety of vaccines and biotherapeutics , potentially to target nematode parasites in the GIT. This study presents two systems in L. lactis MG1363 and KP1 were propagated aerobically without shaking at 30°C in Difco M17 medium  supplemented with 5% glucose (M17G). Escherichia coli DH5α  and XLI-Blue  were propagated with shaking in Luria-Bertani broth (LB) or on Bacto brain heart infusion (BHI) agar plates at 37°C (Becton Dickinson). Cultures were stored at −20°C in the appropriate medium supplemented with 10% glycerol. As necessary, growth media were supplemented with 1.5 or 150 μg/ml erythromycin (Em) for L. lactis or E. coli, respectively. Experiments for growth curves were performed in test tubes by using a Bausch and Lomb Spectronic 20 or in 96-well plates by using a FLUOstar Optima plate reader .Strains and plasmids used in this work are shown in Pfu Ultra II Fusion HS DNA polymerase . Restriction enzymes were obtained from Roche Applied Science . DNA fragments were gel purified using a QIAquick gel extraction kit, and PCR products were purified using a QIAquick PCR purification kit . DNA ligations were carried out with a Fast-Link DNA ligation kit . The ligation mixes were transformed into E. coli XL1-Blue or DH5α by using a Z-Competent E. coli transformation buffer set . Transformation isolates were selected on BHI agar plates with 150 μg/ml Em and propagated in LB with 125 μg/ml Em. Plasmid DNA was isolated using a QIAprep Spin miniprep kit. Cloned isolates were confirmed by sequencing at Eton Biosciences  or Davis Sequencing , directly from plasmid DNA or from PCR products obtained using Choice Taq Blue DNA polymerase . Plasmids were visualized in silico, and DNA sequences were aligned using the Clone Manager 9, professional edition , and Geneious Pro  software packages. Confirmed plasmids were electroporated into L. lactis as described previously  prior to the addition of buffer P2. The L. lactis clones were confirmed by sequencing of PCR products amplified from the plasmid DNA by using the M13 F and M13 R primers.Primers for PCR cloning were obtained from Integrated DNA Technologies . PCR was accomplished using the eviously . Plasmidcry5B gene were initially cloned into pTRK593 downstream of the constitutive P6 promoter (GTCGACAAGGAGAACGTATATGGCAACAATTAATGAGTTGTATC]), as well as downstream PstI sites  . The truncated version of Cry5B ends two amino acid residues after the box 5 feature of Cry5B, with the relevant coding sequence followed by a stop codon  and truncated  versions of the promoter . PCR pro2 bp and op codon . The cryLactococcus system was accomplished using vector pTRK1061. The vector was constructed from pTRK617 ; and reverse, Cry5B XhoI R (GATCCTCGAGTATCCAAGCTCAGCTAATTAAG). The reverse primer for truncated Cry5B was tCry5B XhoI R (GATCCTCGAGATCAGTCTATTGGATTTTTGGAAC). The Cry5B fragments and vector pTRK1061 were digested with PstI and XhoI and ligated.Cloning for Cry5B expression by the leaky  pTRK617  by usingcry5B genes were PCR amplified from pTRK1040 by using the following primers: cry5B SphI forward (GATCGCATGCGTGAGGAGAACGTATATGGCAACAATTAATGAGTTG) and cry5B BamHI reverse (GATCGGATCCGCAGTATCCAAGCTCAGCTAATTAAG). Truncated Cry5B was amplified with truncated cry5B BamHI reverse (GATCGGATCCGCAGTATCCAAGCTCAGCTAATTAAG). The PCR products were cloned into vector pSC-B by using a StrataClone Blunt PCR cloning kit, and from there into pMSP3535H3 by using the SphI and BamHI restriction sites encoded in the PCR primers.For nisin-induced expression of Cry5B by use of vector pMSP3535H3, full-length and truncated L. lactis cultures were propagated for 16 h and then transferred from overnight cultures and propagated in 10 ml M17G with Em (1.5 μg/ml) to an OD600 of 0.5 to 0.6 (log phase), followed by centrifugation to pellet cells. Supernatants and pellets were frozen separately at −80°C for storage. Cell supernatants were filtered through 30,000-molecular-weight-cutoff Amicon Ultra centrifugal filters following the manufacturer's instructions . The filters were then washed with 4 ml of phosphate-buffered saline (PBS), pH 7.4 , and the concentrated proteins were eluted with 50 μl of PBS. Cell pellets were resuspended in 1 ml PBS and then homogenized with a Mini BeadBeater 8, using 0.1-mm glass beads in 2-ml screw-cap tubes, for three 1-min intervals interspersed with 1 min on ice to produce cell lysates . Protein concentrations in all samples were determined using the Bradford reagent , and approximately 7 to 10 μg protein per lane was electrophoresed in Mini-Protein 7.5% or 4.5 to 10% TGX gels  and then transferred to polyvinylidene difluoride (PVDF) membranes by use of a Bio-Rad Trans-Blot Turbo transfer system following the manufacturer's instructions. The membranes were hybridized with Cry5B primary antibodies  and with goat anti-rabbit IgG(H+L)–horseradish peroxidase (HRP) conjugate (Bio-Rad) and developed with Pierce ECL Western blotting substrate  or Clarity Western ECL substrate (Bio-Rad) following the manufacturer's instructions.600) of 0.3 to 0.4 in M17G with Em after inoculation from 18-h cultures. Nisin was added at various dilutions to the cultures, and incubation was continued for 3 h before harvesting cells and supernatants for comparison of protein expression levels by Western blotting. For growth curve experiments, control strains were grown to an OD600 of 0.4 and then incubated for an additional 3 h without the addition of nisin .Stock solutions of 1 mg/ml nisin in 0.02 N HCl were prepared from a preparation of 2.5% nisin obtained from Sigma-Aldrich Co. . Ten-milliliter cultures were grown to an optical density at 600 nm (OD600 = 0.5) and transferred to a separate tube. A reagent mix of Tris, pH 6.8 (0.05 M), sodium pyruvate (0.1 M), and FDP (30 mM)  was aliquoted at 223 μl per well into the wells of a 96-well plate. Samples (20 μl) of cell supernatants were added to the wells in triplicate. NADH  was separately injected into each well, followed by 2 s of shaking and five consecutive OD340 readings spaced 30 s apart. The endpoints of the reactions were observed, and LDH activity was calculated from the slope of the kinetic curve for each well.Lactate dehydrogenase (LDH) activity was detected indirectly by measuring the oxidation of NADH in the presence of pyruvate and fructose 1,6-diphosphate (FDP) at 340 nm , 26. The690 of 0.3. One-hundred-microliter aliquots of adjusted cells were added in triplicate to the wells of a black 96-well plate  with 100 μl of an equal mixture of kit reagents A and B , each diluted 3:2,000 (vol/vol) in H2O. After mixing, the plates were held for 15 min in the dark and then read at 490/530- and 490/600-nm excitation/emission settings on a FLUOstar Optima plate reader. Ratios of SYTO 9 signals to PI signals were determined, and the results were used to calculate the percentage of undamaged cells based on the standard curve.To assay cell permeability, a LIVE/DEAD BacLight bacterial viability kit  was used according to the manufacturer's instructions. The kit uses two nucleic acid-binding stains to differentiate cells with intact membranes (live cells) from those with compromised membranes (dead cells). SYTO 9 is a green, cell-permeant dye that labels all bacteria, regardless of membrane integrity. Propidium iodide (PI) is a red dye which labels cells with compromised or damaged cell membranes. A standard curve was prepared according to the manufacturer's instructions, using standard ratios of untreated log-phase cells and log-phase cells treated with 70% isopropyl alcohol for 1 h. For the assay, 6 to 10 ml of untreated or treated log-phase cells was centrifuged, resuspended in 4 ml of 0.85% NaCl, and adjusted to an ODC. eleganssek-1 (km4) strain was used in a bioactivity assay  was maintained using standard techniques, with E. coli strain OP50 as a food source  first-life-stage larvae and 0.2 μl 5-mg/ml cholesterol (in ethanol) as the food source. Assays were performed in 48-well plates, with two wells per condition, in each of three independent trials. Images of C. elegans were captured using an Olympus BX-60 microscope at 48 h of incubation. The images shown in A ty assay . SEK-1 ie, NSY-1 , 29. C. d source . Assays d source , except E. coli and Lactococcus with a high copy number. The genes encoding the Bt Cry proteins Cry5B and tCry5B were cloned behind a strong, constitutive promoter, P6, positioned upstream of a multiple-cloning site in pTRK593. Plasmids pTRK1040 (pTRK593::cry5B) and pTRK1041 (pTRK593::tcry5B) were first obtained in E. coli and then transformed into L. lactis strains MG1363 and KP1.Vector pTRK593 , which iLactococcus backgrounds , and L. lactis subsp. lactis ATCC 11454 , with nisin added at 0, 50, and 200 ng/ml  was shown to externalize tetanus toxin fragment C (TTFC) expressed from pTRK617 (15A10) and encodes Tac31A, a phage transcriptional activator. The leaky phenotype depends on the presence in the lactococcal host strain of the plasmid-encoded activator as well as a resident prophage which carries a phage promoter homologous to P15A10. Low-level activation of the resident prophage holin and lysin gene cassette in trans from the plasmid produces the leaky phenotype  assays were used to investigate whether or not Cry5B was released into the cell lysate through the normal process of cell lysis in a bacterial population. Normally, LDH is an intracellular enzyme and is not found in cell supernatants unless cell lysis occurs . LDH expL. lactis, bioactivity assays of cell lysates of L. lactis MG1363 strains producing Cry5B and tCry5B were performed using a laboratory roundworm model, C. eleganssek-1 (km4). sek-1 (km4) animals carry a mutation in the MAPKK pathway which leads to hypersusceptibility of this C. elegans strain to Cry5B (–sek-1 (km4) worms treated with the host strain MG1363 show no effect of the added bacterial cell lysate. Similarly, lysates from MG1363 with the constitutive vector (pTRK593) or with constitutively expressed tCry5B (pTRK1041) did not show an effect (sek-1 (km4) C. elegans worms, likely due to the worms' sensitivity to nisin, which was added to the culture of the L. lactis control strain prior to cell lysate preparation. However, there was a clear additional effect of both nisin-induced Cry5B (from pTRK1068) (sek-1 (km4) C. elegans strain. In addition, these results suggest the presence of biological activity from the processed version of full-length Cry5B shown in the immunoblot in To assess the biological activity of Cry5B expressed in to Cry5B –29. Reprn effect  and D. Hn effect  were cleTRK1068)  and nisiTRK1068) . These aB. thuringiensis Cry protein Cry5B (140 kDa) and a truncated form, tCry5B (79 kDa), can be expressed intracellularly in and released extracellularly from L. lactis. This is the first step toward the use of L. lactis as a delivery vehicle for Cry5B in the treatment of mammalian helminth infections.In this study, we demonstrated that the L. lactis is a food-safe, generally recognized as safe (GRAS) organism which can be engineered to deliver Cry proteins safely and economically for treatment of helminth infections.New anthelminthics must be inexpensive and produced in large quantities for delivery under adverse environmental conditions . IdeallyL. lactis and can be used as a natural preservative. Nisin induction has been used widely in L. lactis and other bacteria for expression of heterologous proteins, and it is described as being simple to use, with high yields  worms demonstrated that the nisin-induced Cry5B and tCry5B proteins effectively halted worm development.In this study, Cry5B was overexpressed by using nisin induction. Nisin is an antimicrobial peptide secreted by various strains of h yields . The L. of nisin . NisI apof nisin . The nisml nisin . Cry5B wStaphylococcus aureus cell supernatants was done by comparing wild-type cells with mutants in the major autolysin Atl  activity of L. lactis KP1 was detected in the cell supernatant, with no discernible effect on cell growth, lysis, or membrane integrity (S. aureus autolysin-controlled system (The leaky ntegrity . The lead system , is seled system , 40. NonL. lactis KP1 (L. lactis MG1363 carries prophage sequences, β-Gal was not externalized from this strain, suggesting that Tac31A is not an activator of a holin/lysin promoter for this strain (L. lactis strains (Lactococcus system.The Tac31A activator regulates a late phage promoter of a prophage in ctis KP1 . While Ls strain , 41. Invs strain . KP1 hol strains . Activat strains . FurtherIn vivo challenges to C. elegans worms with cell lysates of L. lactis expressing Cry5B and tCry5B demonstrated biological activity and abated worm development. Nisin-induced expression of both Cry toxins showed distinct biological activity, in synergism with or in addition to the effect of nisin, whereas only Cry5B showed activity expressed from the base constitutive expression plasmid, pTRK593.L. lactis cells, both constitutively and through nisin induction. Both forms of Cry5B are found intracellularly and in culture supernatants from log-phase L. lactis MG1363 and KP1 cells when expressed constitutively. The externalization of Cry5B occurs without any detectable disruption in cell growth or membrane integrity and in the absence of increased cell death. After nisin induction of log-phase L. lactis MG1363, increasing amounts of Cry5B are detected intracellularly with increasing concentrations of nisin. However, Cry5B is not detected in the cell supernatants, perhaps because the 3-h assay is not long enough to allow for externalization. When Cry5B is expressed constitutively in L. lactis KP1 by use of the leaky lactococcus system, virtually all of the protein is found in the cell supernatants, and none is detected intracellularly. Together, these systems provide multiple options for expression of active Cry5B and tCry5B proteins from L. lactis for possible use as oral anthelminthics.We have shown here that active full-length and truncated Bt Cry5B and tCry5B proteins can be expressed in


Mutational data identified a key glutamate residue of Emp46p that serves as the pH-sensing switch of their oligomer formation. Our findings elucidate the mechanisms of the dynamic cargo receptor interactions in the secretory pathway and the design framework of the environment-responsive molecular assembly and disassembly systems.Emp46p and Emp47p are yeast putative cargo receptors that recycle between the endoplasmic reticulum and the Golgi apparatus. These receptors can form complexes in a pH-dependent manner, but their molecular mechanisms remain unclear. Here, we successfully reproduced their interactions  Eukaryotic cells are equipped with a vesicular transport system for sorting and translocating cargo proteins in the secretory pathway. Protein transport in the early secretory pathway is mediated by COPII-coated vesicles, which bud from the endoplasmic reticulum (ER) and travel to the Golgi compartment . Growing2+- and pH-dependent manners [2+-binding protein MCFD2, which cooperates with ERGIC-53 in the vesicular transport of blood coagulation factors V and VIII [In mammalian cells, ERGIC-53 has been identified as a cargo receptor that recycles between the ER and the Golgi apparatus and contributes to the anterograde transport of several glycoproteins, including the lysosomal glycoproteins cathepsin C and cathepsin Z and blood coagulation factors V and VIII [ manners . In ERGIand VIII .2+ and their coiled-coil segments contain no cysteine residue. Immunoprecipitation of differently tagged recombinant proteins expressed in the emp46Δemp47Δ cells indicated that, in the ER, Emp46p and Emp47p are monomeric and homo-oligomeric, respectively, and capable of interacting with each other [Emp46p and Emp47p, the yeast homologs of ERGIC-53, are considered putative cargo receptors recycling the ER and the Golgi apparatus because disruption of both proteins significantly impairs the secretion of a subset of glycoproteins, although their client cargos remain unidentified as yet –11. Likech other . These iCC (252–355) and Emp47pCC (271–274) were subjected to circular dichroism (CD) spectroscopic analysis, which confirmed the α-helical structures of both recombinant proteins  and mass spectroscopy (MS) analyses. SV-AUC data showed that Emp46pCC exhibited two species, with peaks at 1.2 S and 1.9 S, from which their molecular weight (Mw) were estimated to be 12.7 and 21.2 kDa, respectively  and polar (H) residues, and are assembled to bury their hydrophobic H/H surface. A glutamate residue (Glu303) is located in the hydrophobic surface of Emp46pCC  [CC (252–355) or Emp47pCC (254–374) produced by amplification of polymerase chain reaction were constructed and cloned for their expression as a fusion protein with hexahistidine-tagged ubiquitin (His6-Ub) using a pET28a(+) vector (Novagene) or a His6-tagged protein using a pET28b(+) vector (Novagene). These vectors were transformed into the Escherichia coli strain BL21-CodonPlus. The cells were grown at 37°C in Luria—Bertani medium containing 15 μg/mL of kanamycin. Protein expression was induced by adding 0.5 mM isopropyl-βD-thiogalactopyranoside when absorbance reached 0.8 at 600 nm. After 3 h, cells were harvested, suspended in 50 mM Tris—HCl (pH 8.0), and subsequently, disrupted by sonication. After centrifugation, the His6-Ub-tagged or His6-tagged proteins were purified in a Chelating Sepharose Fast Flow column . The Emp46pCC and Emp47pCC were enzymatically cleaved from the His6–Ub moiety using recombinant glutathione S-transferase-tagged yeast ubiquitin hydrolase-1, as described previously [6-segment using thrombin protease , and then purified by chromatography using a gel filtration column .The coiled-coil segments of Emp46p and Emp47p were identified using COILS/PCOILS (/pcoils) . PlasmidCC and Emp47pCC (0.1 mg/mL) were dissolved in 50 mM Tris—HCl buffer (pH 7.6) containing 0.15 M NaCl. Measurements of CD spectra were performed in a 1-mm quartz cuvette at room temperature using a spectropolarimeter . After subtraction of the spectrum of the buffer alone, data were represented as mean residue ellipticities.The purified Emp46pCC protein (70 μM), either alone or in the presence of Emp46pCC or its E303A mutants, was subjected to gel filtration using a Superose 12 column , equilibrated with 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl or 50 mM acetate buffer (pH 5.0) containing 150 mM NaCl.The purified Emp47pCC (or its E303A mutant) and Emp47pCC proteins were dialyzed against 100 mM carbonate/bicarbonate buffer (pH 8.9). Next, dimethyl suberimidate dihydrochloride (DMS) was added to derive a final concentration of 0.1 mg/mL and incubated for 60 or 120 min at room temperature. The cross-linking reaction was quenched by adding 500 mM sodium phosphate buffer (pH 6) to adjust pH.The purified Emp46pCC (75 μM), Emp47pCC (75 μM), and their mixtures at varying protein concentrations were measured. Runs were conducted at 60,000 rpm, at a temperature of 20°C using 12-mm aluminum double-sector centerpieces and a four-hole An60 Ti analytical rotor that was equilibrated to 20°C. The sedimentating boundaries were monitored using Rayleigh interferometric detection at 655 nm. In total, 150 scans were obtained between 5.9 cm and 7.25 cm from the center of the rotation axis. All SV-AUC raw data were analyzed using the continuous C (s) distribution model provided by the SEDFIT11.71 software program. The parameters used for the analysis, that is, the partial specific volume of the protein calculated from amino acid composition , buffer density (ρ = 1.00530 g/ cm3), and buffer viscosity (η = 1.0193 cP) were estimated using SEDNTERP ver.1.09 [SV-AUC experiments were performed in 10 mM mmol/L phosphate buffer (pH 7.4) containing 150 mM NaCl using a Proteomelab XL-I Analytical Ultracentrifuge (Beckman—Coulter). The samples of Emp46pver.1.09 .CC (or its E303A mutant) and Emp47pCC proteins (100 μM) were buffer-exchanged into 200 mM ammonium acetate at pH 7.4 by passing through a Bio-Spin 6 column (Bio-Rad). The samples (43 μM) were immediately analyzed by nanoflow electrospray using in-house made gold-coated glass capillaries . The mixtures of Emp47pCC (33 μM) and 17, 33, and 67 μM Emp46pCC were incubated at 25°C for 30 min, and analyzed by electron spray ionization MS after the buffer exchange. Spectra were recorded on a SYNAPT G2-Si HDMS mass spectrometer  in positive ionization mode at 1.63 kV with 150 V sampling cone voltage and source offset voltage, 0 V trap and transfer collision energy, and 5 mL/min trap gas flow. The spectra were calibrated using 1 mg/mL cesium iodide and analyzed by Mass Lynx software (Waters).The purified Emp46pS1 FigCC, Emp47pCC, and their mixture of Emp46pCC and Emp47pCC were individually incubated with DMS for 60 min. The reaction mixtures were analyzed using SDS-PAGE.Emp46p(PDF)Click here for additional data file.S2 FigCC. (B) Emp47pCC. Solid line: 150 μM, dashed line: 75 μM, dotted line: 15 μM.(A) Emp46p(PDF)Click here for additional data file.S3 FigCC (top). Emp47pCC (bottom). Peaks corresponding to the monomer and dimer of Emp46pCC are indicated by purple and red dots with charge states, respectively. Peaks corresponding to the monomer, trimer, and tetramer of Emp47pCC are indicated by blue, yellow, and green dots with charge states, respectively.Emp46p(PDF)Click here for additional data file.S4 FigCC was fixed and that of Emp46pCC was varied at the molar ratios indicated. Peaks corresponding to 1:3 and 2:2 heterotetramers of Emp46pCC and Emp47pCC are indicated by magenta and cyan dots with charge states, respectively. Peaks corresponding to the homotetramer of Emp47pCC are indicated by a green dot with charge states.The concentration of Emp47p(PDF)Click here for additional data file.S5 FigCC (right). (B) DMS cross-linking of the wild-type and E303A-mutated Emp46pCC in the presence or absence of Emp47pCC. The proteins and their mixtures were individually incubated with DMS for 60 min. The reaction mixtures were analyzed using SDS-PAGE.(A) CD spectra of the wild-type (left) and E303A-mutated Emp46p(PDF)Click here for additional data file.S6 FigCC was fixed and that of the mutated Emp46pCC was varied at the molar ratios indicated. Peaks corresponding to 1:3 heterotetramers of Emp47pCC and the E303A-mutated Emp46pCC are indicated by magenta dots with charge states. Peaks corresponding to the homotetramer of Emp47pCC are indicated by a green dot with charge states.The concentration of Emp47p(PDF)Click here for additional data file.


Limited data exist regarding impact of IDH mutations in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs). Prognostic significance of IDH mutations was asessed in 184 Ph-negative MPN patients - 107 essential thrombocythemia (ET) and 77 primary myelofibrosis (PMF).High-resolution melting (HRM) analysis was used to detect IDH1 and IDH2 mutations.PMF and ET patients showed no significant difference for prevalence of IDH mutations. Mutant IDH (IDH1 or IDH2) was documented in five of PMF (6.5%) and two of ET patients (1.9%). IDH mutations in ET patients included one IDH1 R132C and one IDH2 R140Q. Of the five IDH-mutated PMF patients, four (80%) displayed IDH1 (three IDH1 R132C and one IDH1 R132S) and one (20%) carried IDH2 (IDH2 R140Q) mutation. Sixty percent (three in five) of IDH-mutated PMF patients carried JAK2V617F with following allele burdens: 31-50%, 5-12.5% and 31-50%, respectively. Three of 77 PMF patients (3.9%) simultaneously harbored IDH and JAK2V617F mutations. IDH mutations in PMF showed a trend towards higher rate in females . Bleeding complications were significantly higher in IDH-mutated PMF patients compared to IDH wild-type counterparts. Trend towards a lower prevalance of acetylsalicylic acid (ASA) use was present in IDH mutant PMF patients compared to wild-type counterparts . Death rate was higher in IDH-mutated PMF patients compared to IDH wild-type PMF patients (60% and 15.3%). In univariate analysis, a significantly shorter leukemia-free survival (LFS) was observed in IDH-mutated PMF patients.We conclude that IDH mutations indicate a risk for leukemic transformation in PMF. IDH1 (located on chromosome 2q33.3) and IDH2 (located on chromosome 15q26.1) encode two seperate different isocitrate dehydrogenase enzymes. These enzymes catalyze oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). IDH mutations are located on exon 4 and involve a change of a single aminoacid in the arginine residue. IDH mutations affect three specific arginine residues: R132 (IDH1), R172  and R140 (IDH2) . The accIntegrated genomic analysis identified recurrent mutations in the active site of IDH1 in 12% of glioblastoma multiforme patients . SubsequIDH1 and IDH2 mutations were also identified in chronic myeloid neoplasms, including MDS and myeloproliferative neoplasms (MPNs) . IDH mutIn the current study, we set out to explore the prevalence and prognostic significance of IDH1 and IDH2 mutations in a relatively large cohort of Turkish MPN patients (n = 184). Clinical and hematologic parameters were also analyzed.-, or 11q23 rearrangement [The study was approved by local ethics committee of Istanbul University Istanbul Medical Faculty and performed according to the principles of the Declaration of Helsinki. All participants provided informed written consent for study sample collection and permission for use in research. The study includes 184 patients with Ph-negative MPNs - 107 essential thrombocythemia (ET) and 77 primary myelofibrosis (PMF) patients - diagnosed according to the 2008 WHO criteria recruited between May 1995 and July 2013 . Data obangement . CytogenIDH1/2 mutations were assessed at the Molecular Hematology Laboratory of Istanbul University. Genomic DNA was extracted from peripheral blood granulocytes using a high pure polymerase chain reaction (PCR) template preparation kit . The concentration of DNA was measured using a Nano-Drop-2000 spectrophotometer .2 concentration, 50 ng/μL of genomic DNA and sufficient amount of dH2O to complete the volume to 20 μL. PCR mix was dispensed into individual wells of the 96-well plate. The reaction mix was subjected to an initial denaturation of 95 °C for 10 min, followed by 45 cycles of amplification consisting of denaturation at 95 °C for 10 s, annealing at 54 °C for 15 s, and extension at 72 °C for 25 s. Melting was performed with a denaturation step at 95 °C for 1 min, followed by an annealing step at 40 °C for 1 min and a melt from 65 to 95 °C at a ramp rate of 0.02 °C/s. Melting curves from the samples were automatically normalized and analyzed by direct comparison with LightCycler 480 Software, version 1.5.0 SP3 (Roche Diagnostics). All of the patient samples with curves that differed in shape and/or melting temperature were considered as potential variant carriers. The results were confirmed by direct sequence in both directions on ABI 3730 × l DNA analyzers, including PCR primers . HRM curve analyses for IDH1 (R132), IDH2 (R140) and IDH2 (R172) are shown in IDH mutation analysis was performed using high-resolution melting (HRM) in the LightCycler 480 real-time PCR system (Roche Diagnostics), using primers as described below. The primer sequences used to amplify IDH1 exon 4 (R132) were: 5’-CGGTCTTCAGAGAAGCCATT-3’ (forward), and 5’-CACATTATTGCCAACATGAC-3’ (reverse) and IDH2 exon 4 (R140) were: 5’-GCTGAAGAAGATGTGGAA-3’ (forward), and 5’- TGATGGGCTCCCGGAAGA-3’ (reverse), respectively as previously described . IDH2 exJAK2 MutaScreen kit  was used for the detection of JAK2V617F status and quantitative JAK2V617F allele burden in genomic DNA using TaqMan allelic discrimination , 30. TheSPSS version 16  was used for all stastistical calculations. Numerical variables were summarized as mean (SD). The Chi-square statistics were used to compare categorical variables between the groups. Comparison between categorical and continuous variables was performed using the Mann-Whitney U test (two groups) or Kruskal-Wallis test (more than two groups). The odds ratios (ORs) are accompanied by Cornfield 95% confidence limits (CIs). A two-tailed P value of less than 0.05 was considered significant. Parametric and nonparametric correlation analyses were used to detect associations with IDH mutations. Kaplan-Meier estimates were used to plot OS and LFS in patients with PMF. The curves of OS and LFS were compared by log-rank test.IDH (IDH1 or IDH2) mutations were identified in five (6.5%) PMF and two (1.9%) ET patients. Of the five IDH-mutated PMF patients, three displayed the IDH1 R132C mutation, one harbored the IDH1 R132S substitution and one carried the IDH2 R140Q mutation . In summPathogenic alterations in the IDH gene were detected in two of 107 ET cases (1.9%). Of two IDH-mutated ET patients, one displayed the IDH1 R132C substitution and the other showed the IDH2 R140Q mutation . The ET There was no significant difference in the prevalence of IDH mutation (IDH1 or IDH2) in ET and PMF patients . ET and PMF patients showed no significant difference in the prevelance of combined JAK2V617F and IDH mutations .A total of seven IDH mutations (3.8%) were detected: five in PMF (6.5%) and two in ET patients (1.9%). All alterations in IDH gene were missense mutations. The most common variation was IDH1 R132C, which comprised four of seven IDH mutations (57.1%), followed by IDH2 R140Q  and IDH1 R132S . Of the seven IDH-mutated patients, five displayed IDH1 (71.4%) and two IDH2 (28.6%) mutations. Among five PMF patients with IDH mutations, four (80%) carried IDH1 mutations  and one patient harbored IDH2 mutation . One ET patient with IDH mutation harbored IDH1 R132C while the other patient carried IDH2 R140Q substitution.Two of 107 ET patients (1.9%) displayed IDH mutations. IDH mutations included one IDH1 R132C and one IDH2 R140Q. The ET patient with IDH2 R140Q mutation harbored JAK2V617F mutation with an allele burden of 5% while the other patient with IDH1 R132C mutation did not harbor JAK2V617F mutation. All ET patients carrying IDH mutations were exposed to hydroxyurea treatment and were alive at the end of follow-up. The distribution of IDH mutations in patients with ET is outlined in In 77 consecutive PMF patients, mutant IDH was detected in five patients (6.5%): four IDH1 (three IDH1 R132C and one IDH1 R132S) and one IDH2 (IDH2 R140Q). The distribution of IDH mutations in PMF patients is outlined in Clinical and laboratory features of PMF patients according to the IDH mutational status are listed in In PMF, the presence of IDH mutations was not influenced by age at recording, sampling and diagnosis . IDH-mutated PMF patients showed a trend towards a higher rate in female patients . There were no significant differences in levels of hemoglobin (Hb), hematocrit (HCT), total leukocyte count and platelet count between IDH mutant and wild-type PMF patients. In addition, IDH-mutated PMF patients showed similar levels of LDH with wild-type counterparts.The mean spleen size and degree of splenomegaly did not differ between IDH-mutated and wild-type PMF patients. Similar results were documented for the two groups with respect to the need for red blood cell transfusion and the presence of risk factors for cardiovascular diseases. The prevalence of total thrombotic events, arterial thrombosis and venous thrombosis did not significantly differ among IDH-mutated and wild-type PMF patients .IDH mutant PMF patients demonstrated higher prevalence of bleeding complications than IDH wild-type PMF patients . As regards the localization of bleeding events among PMF patients, 20% of IDH mutant PMF patients experienced gastrointestinal bleeding and 20% intracranial hemorrhage, while gastrointestinal hemorrhage occurred in 5.6% and intracranial hemorrhage in 1.4% of IDH wild-type PMF patients (P = 0.061). There was a trend towards severe bleeding in IDH-mutated PMF patients compared to wild-type PMF patients (P = 0.061). A trend towards a lower prevalance of acetylsalicylic acid (ASA) use was present in the IDH mutant PMF patients compared to wild-type counterparts . This can be explained by the increased frequency of bleeding complications in IDH-mutated PMF patients.No significant difference was observed in the frequency of JAK2V617F mutation between IDH-mutated and wild-type PMF patients . Similarly, the quantitative JAK2V617F allele burdens showed no difference among PMF patients with or without IDH mutation (P = 0.704). Likewise, the prevalence of JAK2V617F-positive patients with mutant allele burden in upper quartile ranges was not different in IDH mutant PMF patients compared to IDH wild-type PMF patients . Three of 58 (5.2%) JAK2V617F-positive and two of 19 (10.5%) JAK2V617F-negative PMF patients displayed IDH mutations (exon 4) (P = 0.36). Similarly, JAK2V617F-positive and JAK2V617F-negative PMF patients showed no significant difference for the frequency of IDH1 and IDH2 mutations . As regards the JAK2V617F allele burden, the prevalence of IDH mutation did not differ across all three groups: 10.5% (two of 19) in JAK2V617F wild-type, 7.5% (three of 40) in patients with low JAK2V617F allele burden and 0 in patients with high JAK2V617F allele burden (P = 0.401). The prevalence of IDH1 mutations was 10.5% in JAK2V617F wild-type PMF patients, 5% in PMF patients with low JAK2 allele burden and 0 in PMF patients with high JAK2 allele burden while 0 of JAK2V617F wild-type PMF patients, 2.5% of PMF patients with low JAK2 allele burden and 0 of PMF patients with high JAK2 allele burden displayed IDH2 mutations . With respect to IDH mutation status, PMF patients showed no significant difference in the prevalence of hydroxyurea use, use of other medical treatments, rate of AHSCT and history of splenectomy (P > 0.05).DIPSS-plus risk stratification was similar for PMF patients with and without IDH mutations (P = 0.889). Among IDH-mutated and wild-type PMF patients, the distribution of karyotype abnormalities was not different (P = 0.671). The prevalence of normal karyotype, favorable and unfavorable karyotype abnormalities in IDH mutant and wild-type PMF patients were as follows: 100%, 0 and 0; 86.1%, 9.7% and 4.2%, respectively (P = 0.671).Higher rates of death were documented in IDH-mutated PMF patients compared to wild-type PMF patients . The rate of leukemic transformation was higher in IDH-mutated PMF patients than wild-type counterparts but with no statistical significance .In PMF patients, the presence of IDH mutation did not correlate with HCT level, total leukocyte count, platelet count, LDH level, mean spleen size, total thrombotic events, arterial thrombosis and venous thrombosis (r < 0.2). There was a weak positive correlation between IDH mutation and bleeding events (r = 0.27). In PMF, the copresence of JAK2V617F and IDH mutations did not correlate with HCT level, total leukocyte count, platelet count, LDH level, bleeding complications, total thrombotic events, arterial thrombosis, venous thrombosis and death (r < 0.2).Although the number of IDH-mutated PMF patients was small, Kaplan-Meier plots were used to calculate OS and LFS. OS did not differ between IDH mutant and wild-type PMF patients  . UnivariIDH1 and IDH2 catalyze an essential step in the Krebs cycle that converts isocitrate to α-KG. Mutant IDH not only loses their normal activity in catalyzing NADP+-dependent oxidative decarboxylation of isocitrate into α-KG, but also gains a new function: catalyzing the α-KG to 2-HG (2-4). 2-HG is found elevated in the serum of patients with gliomas and AML harboring IDH mutations, suggesting that 2-HG could be a biomarker for IDH-mutated glioma and AML , 5.Although many investigators performed mutational analysis of large AML cohorts, limited number of studies previously assessed the prevalence of IDH1 and IDH2 mutations in Ph-negative MPNs , 15, 24.Several studies reported that HRM curve analysis is as reliable as direct nucleotide sequencing for assessing IDH mutations in AML and MPN patients , 35. AlsCandidate gene resequencing of IDH1 and IDH2 in a very large cohort of MPN patients demonstrated that mutations in IDH1/2 occur most commonly at the IDH2 R140 residue in MPNs, followed by IDH1 R132 and IDH2 R172 . The samA limited number of studies have investigated the association of gender and age with IDH mutational status in Ph-negative MPNs , 11. In To the best of our knowledge, only one previous study had compared the clinical and laboratory characteristics of IDH mutant PMF patients with those of wild-type counterparts . ConsistTo our knowledge, no previous study of MPNs has assessed the relationship of IDH mutation with thrombosis, bleeding events and LDH levels. In our study, IDH mutant and IDH wild-type PMF patients showed no significant difference with respect to LDH levels, rate of total thrombotic events, arterial thrombosis and venous thrombosis. However, the prevalence of bleeding complications was significantly higher in our IDH mutant PMF patients compared to wild-type PMF patients . Also, in our PMF patients, a weak positive correlation was found between IDH mutation and bleeding complications. As regards the localization of bleeding complications among PMF patients, 20% of IDH mutant PMF patients experienced gastrointestinal bleeding and 20% intracranial hemorrhage, whereas gastrointestinal hemorrhage occurred in 5.6% and intracranial hemorrhage in 1.4% of IDH wild-type PMF patients. Consequently, there was a trend towards severe bleeding in IDH mutant PMF patients compared to wild-type PMF patients.In a series of 200 MPN patients, IDH mutations were reported at a frequency of 21% in blast-phase MPN and 4% in PMF . In thatA limited number of studies have assessed the cytogenetic risk categories of IDH mutant MPN patients , 11, 24.In our study group, all IDH mutations detected in PMF patients were associated with normal karyotype yet predicted inferior LFS. This finding suggests that IDH mutations may represent an independent prognostic biomarker in PMF.Abdel-Wahab et al reported that DH1 mutations were most commonly observed in MPN/AML patients with wild-type JAK2V617F . The freThe incidence of IDH mutation was higher in our PMF patients  than ET patients , yet the difference did not reach statistical significance. During follow-up, one of the five IDH mutant PMF patients (20%) underwent leukemic transformation. Our findings support the possibility that IDH mutations are early genetic events in Ph-negative MPN patients and increase leukemic transformation in PMF. Thus, it can be inferred that IDH mutations may play a role in prediction of leukemic transformation in PMF patients.In our study, approximately 2.3% (one in 43) of JAK2V617F-negative ET patients harbored the IDH mutations. The frequency of IDH mutations in our JAK2V617F-negative PMF patients was 10.5% (two in 19). Based on our findings, it may be deduced that analysis of the IDH genes provided an additional 2.3% increase in ET and a 10.5% increase in PMF patients with a detected molecular marker of clonality.In conclusion, IDH mutations had significant impact on LFS in PMF patients. Also, IDH mutations were associated with high rate of death but did not influence OS in PMF. Despite the limited number of IDH-mutated PMF patients included, our observations support that IDH mutations provide genetic events in the pathogenesis and prognosis of PMF patients. Clearly, screening for IDH mutation might be added to future clinical trials and prospective observational studies in PMF. Given the small number of IDH-mutated ET patients in our study group, we could not determine the impact of these genetic alterations on patient characteristics. Larger number of ET patients are needed to detect the prognostic importance of IDH mutations in ET patients.