Escherichia coli (STEC) strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m2, their infectivity dropped by 1–3 log10, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells.Shiga toxin-converting bacteriophages (Stx phages) are present as prophages in Shiga toxin-producing  Escherichia coli (STEC) strains, including their most dangerous subset, enterohemorrhagic E. coli (EHEC), depends on production of Shiga toxins [N-glycosidase activity which leads to modification and inactivation of rRNA [Virulence of Shiga toxin-producing a toxins ,2. These of rRNA ,4. Infec of rRNA . The hig of rRNA ,7,8.stx genes) are located on lambdoid prophages, called Shiga toxin-converting prophages or Stx prophages [stx genes, like vast majority of phage genes, are silent or expressed at low levels [stx genes are also activated. This leads to effective production of Shiga toxins which are released after phage-mediated lysis of the host cell [In all STEC strains investigated to date, genes coding for Shiga toxins  and 43 °C . When a lysate of bacteriophage λ was irradiated with UV light at the dose of 50 J/m2, no effects on infectivity of virions could be observed at both tested temperatures, 37 °C and 43 °C  number of pfu decreased by one order of magnitude or less, though the decrease was still statistically significant , a significant increase in the level of free phage DNA could be observed in experiments with Φ24B and P27 virions (B and P27), we recognized the results shown in The results presented in  virions . Note thB, in which no detectable and the most pronounced loss of infectivity and capsid damage were observed, respectively, for electron microscopic studies. In phage λ, UV irradiation at 50 J/m2 resulted in only a slight increase in the fraction of phage ghosts, with no other structural changes detected  virions were UV irradiated  and subjected to electron microscopic analyses. In the vast majority of lysates of tailed bacteriophages, apart from normal virions, those with empty (devoid of DNA) capsids occur at the levels from a few to several percent ,28; suchdetected , Table 2appeared , Table 2E. coli infected with equal number of untreated phages. To test this hypothesis, a sample of the lysate of either λ or Φ24B virions was divided to two halves of equal titer (determined as pfu/mL). One half was UV irradiated at 50 J/m2 while the second was not, and then E. coli cells were infected with either treated or untreated phages. Following infection, total RNA was isolated, and expression of N and cro genes , crucial for the lytic development [N and cro genes in the population of E. coli cells , as well as virion dysmorphology (identified in electron microscopic studies), indicated that damage of virions might be responsible for high UV sensitivity of Stx phages. This may have implications for the spreading of these phages and formation of new STEC strains. Although these processes might be effective in habitats devoid of UV, like in human or animal digestive tracts, one can expect their lower efficiency in other environments, outside of mammalian organism. It remains to be elucidated what structural properties of Stx virions make them labile upon UV irradiation at doses which do not cause any considerable damage of bacteriophage λ capsids.All the results presented in this report indicate that Stx virions are more sensitive to UV irradiation than those of bacteriophage λ. The process of inactivation of bacteriophages with UV light is a known phenomenon, however, UV doses used by others to decrease infectivity of various phages, including Stx phages, were generally significantly higher than those employed in this work ,33,34,35Escherichia coli MG1655 strain [B(∆stx2::cat) [stx2::catGFP), 22∆tox(∆stx2::catGFP), 27∆tox(∆stx2::catGFP), and 32∆tox(∆stx2::catGFP), referred to as Φ24B, 933W, P22, P27 and P32, respectively, in the text, were used in this work. Bacteriophages Φ24B, 933W, P22, P27 and P32 are Shiga toxin-converting phages. Φ24B and 933W have been used for many years as model phages from this group, while P22, P27 and P32 have been described as natural isolates [cat and/or GFP markers were described previously [5 strain  and bactx2::cat) , 933W∆toisolates . Derivateviously . Bacteri600 = 0.2. Sample of 1 mL was withdrawn and centrifuged . The pellet was suspended in 1 mL of 0.85% NaCl. Then, the sample was centrifuged again and the pellet was suspended in 1.2 mL of LB medium supplemented with MgSO4 and CaCl2 . Following incubation for 30 min at 37 °C, the volume of analyzed bacteriophage lysate, corresponding to m.o.i. of 5, untreated or treated with UV light , at 50 J/m2,  was E. coli MG1655 strain) as described previously [600 of 0.1. Prophage induction was provoked by mitomycin C which was added to final concentration of 1 μg/mL. After induction of phage lytic development, the culture was incubated for additional 6 to 10 h. Following 15 min agitation with chloroform (added in the volume of 600 μL per 10 mL), the culture was centrifuged (2000× g for 10 min at 4 °C) to remove bacterial debris, and supernatant was filtered using 0.22-μm-pore-size filters . Effect of UV irradiation on the phage lysates stability was determined simultaneously at 37 °C or 43 °C, using phage titration method performed according to the standard double agar overlay technique. At times 0 (before UV exposure) and 0.2, 1, 2, 3, 4, 5, 6 h after UV irradiation at 50 J/m2, samples of 0.5 mL were withdrawn, and prepared and titrated as described previously [Phage lysates were obtained after induction of the prophage from the host , one half of the phage lysate was incubated at 37 °C and the second half at 43 °C. Effect of UV irradiation on the amount of DNA released from capsids was determined simultaneously at both temperatures. Before (time zero) and after (the remaining times) exposure of phage lysate to UV light, DNA was quantified by staining with Qubit® dsDNA BR Assay Kit , according to the manufacturer’s protocol. At indicated time points after UV exposure, DNA contents were calculated relative to the DNA amount obtained at time zero which represented value of 1. Each experiment was repeated three times.Phage lysates were prepared as described above. Following UV exposure  were performed employing the Philips CM 100 electron microscope , by using negative staining with uranyl acetate, as described earlier [Virions were purified from phage lysates obtained after induction of the prophage from the host n method . Electro earlier .E. coli bacteria were cultured to A600 of 0.3 at 37 °C. Then, culture volume of 60 mL was centrifuged and the pallet was washed with 30 mL of 0.85% NaCl. Following additional centrifugation, the sample was suspended in 15 mL of LB medium enriched with MgSO4 and CaCl2 . After incubation at 37 °C for 30 min, the sample was chilled on ice. The appropriate volume of analyzed bacteriophage lysate, corresponding to m.o.i of 1 (treated or untreated with UV light at 50 J/m2) was added, and the mixture was incubated on ice. Next, infected bacterial cells were aerated in a water bath shaker at 37 °C. At indicated times  at final concentration of 10 mM, and harvested. The RNA and cDNA samples for real-time PCR assay were prepared as described previously [The experiments were performed according to the protocol described previously , with slnt rates ), 1 × 10eviously ,46 usingN and cro phage genes expression was determined by quantitative real-time reverse transcription-PCR (RT-qPCR) using the LightCycler® 480 Real-Time PCR System (Roche Applied Science) and cDNA samples obtained during bacteriophage infection experiment. According to a procedure described previously [icdA bacterial housekeeping gene. Expression of this gene was found to be stable also upon λ or Φ24B phage infection of E. coli MG1655 strain [® 480 SYBR Green I Master and following primers: pF_λ_N (5′-CTC GTG ATT TCG GTT TGC GA); pR_λ_N (5′-AAG CAG CAA ATC CCC TGT TG); pF_λ_cro (5′-ATG CGG AAG AGG TAA AGC CC); pR_λ_cro (5′-TGG AAT GTG TAA GAG CGG GG); pF_Φ24B_N (5′-AGG CGT TTC GTG AGT ACC TT); pR_Φ24B_N (5′-TTA CAC CGC CCT ACT CTA AGC); pF_Φ24B_cro (5′-CGA AGG CTT GTG GAG TTA GC); pR_ Φ24B_cro (5′-GTC TTA GGG AGG AAG CCG TT); pF_icdA (5′-CGA AGC GGC TGA CCT TAA TTG) and pR_icdA (5′-GTT ACG GTT TTC GCG TTG AT). The reaction components and amplification program were exactly the same as described previously [The level of eviously , transcr5 strain ,45. The eviously ,45,46.


In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten–eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme–product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme–substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases , indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme–product complex.Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G TDG excises thymine from G·T mispairs, an activity that is needed to protect against C→T transition mutations caused by deamination of mC to T ,2. It al-U (hmU) ,12. Huma-U (hmU) –18.cat; residues 111–308), with a tetrahydrofuran (THF) abasic site analog flipped into its active site  ,20; whicive site . This stive site . However mispair  and to a mispair , with nocat action on a G·hmU mispair in DNA (cat) (kmax = 2 min-1) (Escherichia coli MUG)   and the 2 min-1) , the sub2 min-1) . Notablyoli MUG) ,26, the oli MUG) . While uoli MUG) ,29, it boli MUG) ,31, whicoli MUG)  and TAG oli MUG) , were gecat G·hmU product complex also suggests that, in a discrete step after glycosidic bond cleavage, the hmU base rotates and moves 5 Å relative to its expected pre-cleavage position, forming new active-site contacts at O2, N3 and O4 (3A) are the same in the binary complex (TAG·m3A) and a ternary complex that also includes abasic DNA .,24.cat G3 and O4 ,34. For asic DNA ,35,36. Sasic DNA ,38 and 8asic DNA ,40. Thusasic DNA  would beN-glycosidic bond yields a discrete though short-lived oxacarbenium ion intermediate, followed by nucleophile addition  structures of product complexes generated from substrates including G·hmU, G·T, G·U, G·fC and G·caC. The new crystallization conditions yield structures of DNA-bound TDG that are of much higher resolution than those previously reported (up to 2.49 Å), revealing details that were not observed in previous structures. These improved crystallization conditions are expected to facilitate future structural studies of TDG. We also studied the product complex in aqueous solution, using nuclear magnetic resonance (NMR) spectroscopy and investigated the affinity of TDG for isolated nucleobases . The results define the constituents and nature of the product complex for TDGcat; residues 111–308), were expressed in E. coli and purified essentially as described  (C.I.L.)  overnight (∼16 h).Human TDG (full length), and its catalytic domain  ,50. BrieF and TF, respectively, were synthesized at Yale using phosphoramidites obtained from Glen Research (UF) or Link Technologies (TF)  were obtained (trityl-off) from the Keck Foundation Biotechnology Resource Laboratory, Yale University, or IDT. ODNs were purified by reverse phase HPLC using an XBridge OST C18 column (Waters Corp.), with mobile phases of 0.1 M TEAA pH 7.0, 5% acetonitrile (A) and 0.1 M TEAA pH 7 15% acetonitrile (B), a flow rate of 5 ml/min, and a gradient of 25 to 55% B over 16 min. Purified ODNs were exchanged into 0.02 M Tris–HCl pH 7.5, 0.04 M NaCl, and their concentration was determined by absorbance . ODNs coies (TF) . TDG bincleavage ,51–53, bCGTGAGCGATGGACAGCT, that pairs G with the abasic site. The target base (x) is in a CpG context (underlined), consistent with TDG specificity  PEG 4000, 0.2 M ammonium acetate, 0.1 M sodium acetate, pH 6.0. Crystals typically appeared within in a few days. Crystals were cryo-protected using mother liquor supplemented with 18% ethylene glycol (and other components as noted), and flash cooled in liquid nitrogen.Samples used for crystallization contained 0.35 mM TDG by HPLC . Crystalhttp://smb.slac.stanford.edu/facilities/software/xds). Whenever possible, we took advantage of the shape of these crystals (long thin blades) to collect multiple datasets that could later be merged to increase resolution. Resolution cutoff was determined based on CC1/2 values (cat as the search model (PDB ID: 4FNC). All other structures reported were determined by molecular replacement using our new structure (PDB ID: 4XEG) as the search model. Refinement was performed using BUSTER-TNT  was deemed amenable to anisotropic B factor refinement, given that it produced similar reduction in both R and Rfree and that the sphericity distribution was consistent with expectations as predicted by the PARVATI server .X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource . The images were processed and scaled using Mosflm  and Aiml2 values . The str2 values  and a prSTER-TNT  or REFMASTER-TNT , and modSTER-TNT . SpecifiSTER-TNT ,65, and I server . Final m15N-TROSY and 15N-HSQC experiments were collected on a 950 MHz Bruker Avance III NMR spectrometer, and the data were processed and analyzed using NMRPipe and NMRDraw (cat bound to DNA containing a pre-existing abasic (AP) site, the abasic site was generated by treating DNA containing a G·U mispair with a catalytic amount of UNG (1:500 molar ratio UNG:DNA). For all other E·P complexes, samples were generated by adding TDGcat to substrate DNA  and incubating sufficiently to ensure full conversion to product, as confirmed by HPLC , was analyzed using EMSAs, performed essentially as described . Samples contained 10 nM G·TF DNA, 5 nM to 1000 nM TDG and 10 mM of the nucleobase (where used). Samples were incubated at room temperature for 30 min, loaded onto a 6% native denaturing polyacrylamide gel and run for 60 min, 100 V at 4°C. Gels were imaged using a Typhoon 9400 variable mode imager .Equilibrium binding of TDG to a G·Tescribed . Note thcat) (cat:DNA), one subunit at the target site and a neighboring subunit bound to non-specific DNA  –24. Cryset al., by altering the DNA and screening for conditions at pH values above 4.6. We obtained high-quality crystals at pH 5.5 and 6.0, and optimized the conditions at pH 6.0, where TDG exhibits full glycosylase activity , is generally much better than that of any previously reported structure . As expected, these structures adopt a very similar overall fold relative to structures obtained from previous crystallization conditions   Figure  25). No. Noet alcat active site. Thus, hmU has dissociated from the product complex. The presence of acetate in the active site is likely explained by its interaction with three ordered water molecules and its high concentration (300 mM) in the mother liquor used for crystallization. Notably, the same acetate concentration was present in the mother liquor used by Hashimoto et al. for their structure of the E·P complex that was reported to contain the excised hmU base (PDB ID: 4FNC)  . The eleD: 4FNC)  validatiD: 4FNC)  Å, intercat on other substrates. Structures of E·P complexes generated from G·fC and G·caC substrates, solved at 2.02 Å and 2.45 Å resolution, respectively, give the same result .Given that hmU is clearly absent from the enzyme–product complex, we also determined the structure of product complexes resulting from the action of TDGcat with a G·T, G·U, or G·hmU substrate in buffer that also contained a 10 mM concentration of the relevant nucleobase . Importantly, the same concentration of the nucleobase was also present in the mother liquor for crystallization and the solution used to cryoprotect the crystals. The resulting structures reveal that the nucleobase is not bound in the product complex, despite its high concentration during sample preparation, crystallization and cryoprotection. This is clearly demonstrated by the E·P complex generated from a G·U substrate . The electron density demonstrates that the active site does not contain acetate or the excised uracil base ; the second sample was generated by incubating TDGcat with DNA substrate to give complete conversion to product (confirmed by HPLC). If the excised base dissociates from the product complex then there should be no substantial chemical shift differences because the resulting complexes would be identical (TDGcat bound to G·AP DNA). However, if the excised base is trapped in a ternary product complex and forms multiple interactions with active-site groups, as suggested . Remarkably, the NMR spectra reveal no significant differences, indicating that TDGcat has very weak affinity for these nucleobases, Kd >> 10 mM, even tough it readily excises them from DNA. We also investigated the potential interaction of these bases with full-length TDG, by examining their effect on TDG binding to DNA containing a G·TF mispair, where TF is an analog of deoxythymidine that flips into the active site but is not excised by TDG (F). Binding to G·T mispairs is weak relative to other substrates and is more sensitive to agents that perturb substrate binding  concentration . Together, the results indicate that free TDG has negligible affinity for U, T or hmU.Given that that the nucleobases excised by TDG do not bind with significant affinity to the enzyme–product complex, we investigated their potential binding to DNA-free TDG. We performed NMR experiments for TDGd by TDG ,48,51,52d by TDG  shows UF binding ,48,51,70The high-resolution structures reported here, which reveal hundreds of water molecules, indicate that the active site of DNA-bound enzyme is accessible to solvent. Specifically, the structures reveal a network of water molecules extending from the active site down along the target strand of the DNA toward the 5′ end Figure . The solδ of Asn140  in the active site impacts the equilibrium between the two potential anomeric forms of the abasic sugar produced by TDG. Abasic sites adopt several potential forms in free DNA Figure , includi0 Figure , the sid0 Figure . Interes complex . Our fincat yield structures of unprecedented resolution for this enzyme, maintain the 1:1 binding stoichiometry observed by Hashimoto et al. . These crystallographic findings are confirmed in solution by NMR, for product complexes generated from G·hmU, G·U and G·T substrates. Moreover, the same result is obtained even when the relevant base is supplemented to a concentration of 10 mM during sample preparation, crystal growth and cryoprotection. Our results are consistent with previous findings that the excised base (uracil) is not observed in a structure 2.35 Å) of the enzyme–product complex derived from a G·U substrate for MUG from E. coli , the mosE. coli . Our strubstrate . The cry.35 Å of Observation that the excised base is absent from the product complex raises the question of how it is released. One plausible mechanism is that the excised base escapes through the solvent-filled channel revealed by our new structures, which could involve movement of the enzyme or DNA (or both). Notably, MUG has a similar though seemingly smaller channel, and it releases the excised uracil base from an E·P complex . By contKd >> 10 mM). Likewise, enzyme–product complexes also lack significant affinity for free nucleobases. These results are consistent with previous findings that TDG is not inhibited by thymine, uracil, 5-fluorouracil and 3,N4-ethenocytosine (ϵC), at concentrations of 5 mM (Kd of about 0.1 mM (at pH 7.5) (3A with a Kd of 0.04 mM (Kd = 0.6 nM) relative to non-specific DNA (Kd = ∼0.3 uM)  ,82 and T 0.04 mM . Our fin∼0.3 uM) . Studiesectively . While sn of caC ,24, suggcat . Our new structures here indicate that release of the excised base (and the absence of acetate) is essential for adoption of the β anomer, and this idea is supported by structures of other DNA glycosylases. A structure of the MUG product complex (2.35 Å) shows that the excised uracil is absent and the abasic sugar adopts the β anomer  than the α anomer Figure , suggestβ anomer . Structuβ anomer ,34, whilβ anomer . Taken thttp://www.rcsb.org/) with accession numbers 4XEG, 4Z7B, 4Z7Z, 4Z47, 4Z3A and 5CYS.Coordinates and structure factors have been deposited in the Protein Data Bank (


Alishewanella sp. WH16-1 (= CCTCC M201507) is a facultative anaerobic, motile, Gram-negative, rod-shaped bacterium isolated from soil of a copper and iron mine. This strain efficiently reduces chromate (Cr6+) to the much less toxic Cr3+. In addition, it reduces sulfate (SO42−) to S2−. The S2− could react with Cd2+ to generate precipitated CdS. Thus, strain WH16-1 shows a great potential to bioremediate Cr and Cd contaimination. Here we describe the features of this organism, together with the draft genome and comparative genomic results among strain WH16-1 and other Alishewanella strains. The genome comprises 3,488,867 bp, 50.4 % G + C content, 3,132 protein-coding genes and 80 RNA genes. Both putative chromate- and sulfate-reducing genes are identified.The online version of this article (doi:10.1186/s40793-016-0169-3) contains supplementary material, which is available to authorized users. Alishewanella was established by Vogel et al., in 2000 with Alishewanella fetalis as the type species. It belongs to the family Alteromonadaceae of the class Gammaproteobacteria [. So far, Alishewanella contains six species: A. fetalis, Alishewanella aestuarii, Alishewanella jeotgali, Alishewanella agri and Alishewanella tabrizica and Alishewanella solinquinati [Alishewanella are Gram-negative, rod-shaped and positive for oxidase and catalase [Alishewanella strains were able to degrade pectin which is applicable in bioremediation of food industrial wastes [Alishewanella strains  have been sequenced and the pectin degradation pathway was found in their genomes [Alishewanella were reported to tolerate arsenic [Alishewanella strains to resist or transform other heavy metal(loids) have not been reported.The genus bacteria . So far,nquinati –6. The ccatalase –6. Some l wastes –11. Thre genomes –11. Some arsenic , 13, butAlishewanella sp. WH16-1 was isolated from mining soil in 2009. This strain could resist to multiple heavy metals. During cultivation, it could efficiently reduce the toxic chromate (Cr6+) to the much less toxic and less bioavaliable Cr3+. It could also reduce sulfate (SO42−) to S2−. When Cd2+ was present, the S2− reacted with Cd2+ and precipitated as CdS. These characteristics made strain WH16-1 a great potential for bioremediate Cr and Cd contamination. In pot experiments of rice, tobacco and Chinese cabbage, with the addition of the bacterial culture, the amount of Cr and Cd in the plants decreased significantly [Alishewanella genomes would provide a means of establishing the molecular determinants required for chromate/sulfate reduction, heavy metal resistance and pectin degradation, and for better application of these strains. Here we report the high quality draft genomic information of strain WH16-1 and compare it to the three sequenced Alishewanella genomes.ficantly . SequencA. agri BL06T (99.7 %) and A. fetalisTCCUG 30811 (99.1 %)  sequences   in 36 h and remove 60 μmol/L Cd2+ (added as CdCl2) in 60 h . A NanoDrop Spectrophotometer 2000 was used to determine the quality and quantity of the DNA. Six micrograms of DNA was sent to Majorbio Bio-pharm Technology Co., Ltd  for sequencing.The genome sequencing of strain WH16-1 was performed on an Illumina Hiseq2000  and asse+ [The draft genome of WH16-1 was annotated through the NCBI PGAP, which combines the gene caller GeneMarkS+  with the+  with E-v+ . Signal + . The tra+  and the + . Interna+ , 31.The whole genome of strain WH16-1 is 3,488,867 bp in length, with an average G + C content of 50.4 %, and is distributed in 149 contigs (>200 bp). The genome properties and statistics are summarized in Table 42− reduction pathway according to the KEGG analysis, including CysPUWA, CysN, CysD, CysC, CysH and CysIJ  by sulfate adenylyltransferases CysN and CysD [32−) by PAPS reductase CysH [32− is finally reduced to sulfide (S2−) by sulfite reductase CysIJ [2+ most probably due to the reaction between S2− and Cd2+ to form the precipitated CdS [6+ reduction, a putative chromate reductase YieF was found  and the ABC transport permease genes, respectively. The chromate resistance levels were both decreased significantly in the chrA and ABC transport permease gene mutant strains (data not shown).Strain WH16-1 has the genes for a complete SOthe cell ; 2) the and CysD ; 3) the ase CysH  and, 5) se CysIJ . Strain ated CdS . For Cr6ytoplasm . An indi2+, Pb2+ and Zn2+ are found [2+, As3+ and Hg2+ resistance determinants are also present, such as Cu transporter ATPase [2+ resistance system CopABCD [In addition, various heavy metal transformation and resistance determinants are identified in the genome of strain WH16-1 Several transporters  that might be involved in the efflux of Cdre found –39. Cu2+r ATPase , Cu2+ re CopABCD , Ars [42 CopABCD  systems  CopABCD  , A. aestuarii B11T (3.59 Mbp) and A. agri BL06T (3.49 Mbp) [Alishewanella strains . Strain WH16-1 shares 2,474 proteins with the other three Alishewanella genomes and has 217 strain-specific proteins , similar to 22429T 3.4 Mbp, A..49 Mbp)  9 Mbp, sii B11T 3.9 Mbp andA. agri BL06T, A. jeotgaliTKCTC 22429 and A. aestuarii B11T were all reported to have the ability of degrading pectin and possess pectin degradation genes [T, TKCTC 22429 and B11T, strain WH16-1 was unable to degrade pectin and the pectin degradation genes are not found in its genome. Since strain WH16-1 was isolated from a heavy metal rich environment, it may be more relevant for bioremediation of heavy metal contamination. The pectin degradation genes may be lost during the evolution.In addition, on genes –11. HoweAlishewanella sp. WH16-1 reveal correlation between the gene types and some phenotypes. The strain harbors various genes responsible for sulfate transport and reduction, chromate reduction and resistance of multi-heavy metals. These observations provide insights into understand the molecular mechanisms of heavy metals. In addition, all of the analyzed Alishewanella genomes have putative sulfate and chromate reduction genes, which indicates that sulfate and chromate reduction may be the important characters of the Alishewanella strains. Thus, these strains have a great potential for application in bioremediation of heavy metal or other industrial wastes.The genomic results of


C-reactive protein (C-RP) and the complex SC5b-9 were detected in the plasma using commercial kits. A total of 83 patients were included. The laboratory results and clinical histories showed that 26 patients had sepsis; 14 were VPT, and 12 were PT. The levels of C-RP, SC5b-9 (innate immune response mediators), and IL-10 or IL-4 (anti-inflammatory cytokines) were elevated during sepsis in both groups. IFN-γ, TNF-α, and IL-6 (proinflammatory cytokines) were differentially elevated only in PT neonates. The VPT neonates with sepsis presented increases in C-RP, SC5b-9, and anti-inflammatory cytokines but not in proinflammatory cytokines, whereas PT neonates showed increases in all studied mediators of inflammation.The response of the adaptive immune system is usually less intense in premature neonates than term neonates. The primary objective of this study was to determine whether immunological parameters vary between preterm (PT) neonates  and very preterm (VPT) neonates . A cross-sectional study was designed to prospectively follow PT and VPT neonates at risk of developing sepsis. Plasma concentrations of IFN- Neonatal sepsis affects up to 20% of very low birth weight premature newborns, causing death in up to 18% of these newborns. The health of neonates can deteriorate quickly, and the newborns can develop septic shock and die before agent identification and antimicrobial sensitivity tests are ready. Because inflammation is an important event in the sepsis process, many studies have focused on measuring inflammatory mediators to permit an early diagnosis of sepsis ; howeverα, and TNF-α production in monocytes and conventional dendritic cells (cDCs) of preterm neonates are attenuated after stimulation via Toll-like receptors (TLRs) [Discrepancies might originate from the fact that gestational age is usually not considered when the mediators of inflammation are analysed, even when the immune response in neonates is likely to be immature. For example, IL-6, IFN-s (TLRs) .γ/δ and mature β-1a receptors predominate in neonates at <32 weeks of gestational age compared with term neonates who express Tα/β and β-1b receptors on their lymphocyte cells [Furthermore, it is important to consider that the immune system develops throughout the foetal period. Research regarding the immune system throughout foetal development indicates that the adaptive immune response in the preterm neonate exhibits more vulnerability because the T cells are limited by a lack of clonal expansion because of the absence of contact with the antigens. Therefore, at birth, immunological memory does not exist, and there is poor costimulatory receptor expression, which results in poor lymphocyte stimulation with low cytokine and antibody production. The limitations of the adaptive immune system are more marked in premature newborns than in neonates at term , 10. Ontte cells . The dif Streptococcus group B colonisation, and fever during labour or prolonged premature rupture of membranes; consent from the newborns' parents was required to participate in the study. Infants at the neonatal intensive care unit (NICU) were also included when they showed three or more clinical signs of sepsis such as apnoea, tachypnoea (>60/min), nasal flaring, retraction, cyanosis or respiratory distress-bradycardia (<100/min), tachycardia (>180/min), hypotonia, seizures, poor skin colour, irritability, or lethargy. We excluded neonates with haemotransfusion, herpetiform dermatitis, atopic eczema, congenital malformations, and/or laboratory confirmed TORCH  infections or those receiving gamma globulin IV. All participants were born at the Instituto Nacional de Perinatología (INPer), Mexico City. The Human Investigation Committee of the INPer approved this study. Because neonates in the NICU are continuously monitored, the results were identified as presepsis if the sample was taken a week before clinical or proven sepsis, sepsis if the neonate had clinical or proven sepsis while being studied, including samples taken within a range of 2 days, and postsepsis if the sample was provided a week after clinical or proven sepsis  and very preterm  neonates at risk of developing sepsis. Thus, a cross-sectional study was designed to follow neonates of mothers with chorioamnionitis, urinary infection,n sepsis . The diaBlood cultures were performed at the microbiology laboratory using standard techniques via the automated vac+/alert system . Bacterial identification was performed using the Vitek automated system .™ Plus kit .C-RP levels were analysed using a MininephThe complex C5b-9, which is formed when complement is activated by any of the four routes, is then coupled to the S protein to form the soluble complex SC5b-9, which was detected in the serum using a commercial kit  according to a standard enzyme-linked immunosorbent assay (ELISA) procedure. Colour development in the plates was read using a spectrophotometer  at 405 nm.γ, and TNF-α were detected simultaneously using a human cytometric bead array (CBA) kit from BD . Fifty microliters of plasma or the provided standard cytokines was added to the premixed microbeads in 12 mm × 75 mm Falcon tubes (BD). After the addition of 50 μL of a mixture of PE-conjugated antibodies against the cytokines, the mixture was incubated for 2 h in the dark at room temperature. This mixture was washed and centrifuged at 500 g for 5 min, and the pellet was resuspended in 300 μL of wash buffer. A FACS Aria II (BD) was used to read the samples. A standard curve including 9 concentrations was created for each determination. A typical sample mixture included 10 μL of capture beads, 50 μL of plasma, and 50 μL of the detector. The samples were mixed for 2 h in the dark and were then washed and read in the cytometer. The results were analysed using FACS Diva™ BD software.The concentrations of IL-4, IL-6, IL-10, IFN-t-test for independent samples at a 95% confidence level, the χ2 test, or Fisher's exact test for categorical variables. Intragroup comparisons based on a one-way analysis of variance with repeated measures (ANOVA) were initially performed. When Levene's homogeneity of variance test indicated significant differences, the Friedman test was used. Statistical analyses were performed using XL-STAT 2013, version 5.06, for Windows .The clinical characteristics of the participants are presented using descriptive statistics . Individual data were compared to normal reference ranges using Student's P < 0.05, Student's t-test, for both cases); the sex relationship was similar between the groups . Two deaths occurred, one in each sepsis group. Early-onset sepsis was observed in 6/12 PT neonates and in 6/14 VPT neonates , Escherichia coli (n = 4), followed by Candida spp. (n = 2), Enterobacter spp. (n = 2), Staphylococcus aureus (n = 1), Staphylococcus spp. coagulase-negative (n = 2), and group B Streptococcus (n = 1). No differences in the number of isolated microorganisms were found between the PT and VPT groups ; however, the VPT group showed more infections with gram-positive microorganisms (n = 5) and only two cases with gram-negative microorganisms, whereas the PT group presented more frequent infections with gram-negative microorganisms (n = 8).The blood culture was positive in 16/26 sepsis cases. The most frequent microorganisms wereP = 0.403, ANOVA); these values did not differ from the presepsis, sepsis, and postsepsis values of 14.5 ± 4.95, 76.2 ± 119.19, and 12.4 ± 10.83 mg/dL, respectively, for the VPT . During sepsis, the values were similar for both PT and VPT of the sepsis points  values for C-RP were 19.5 ± 21.2, 85.57 ± 99.18, and 55.2 ± 60.36 mg/dL for presepsis, sepsis, and postsepsis, respectively, for PT newborns . For the VPT neonates, the values were 821.5 ± 163.34, 1,502 ± 643.59, and 1,181 ± 807.27 ng/mL , respectively. No differences were observed between the PT and VPT groups during sepsis  presepsis value was 1.147 ± 1.08 pg/mL, and the postsepsis mean was 0.289 ± 0.36 pg/mL. At clinical sepsis, the value was 3.095 ± 2.71 pg/mL . The IFN-γ values remained high for approximately three to five days during clinical sepsis; however, the values for the VPT infants throughout the sepsis process were 0.381 ± 0.74 pg/mL, 0.117 ± 0.31 pg/mL, and 0.234 ± 0.30 pg/mL at presepsis, sepsis, and postsepsis, respectively . Individual comparisons between both the PT and VPT neonates with regard to the IFN-γ values during sepsis showed significant differences  or a week after clinical sepsis (postsepsis), the values for IFN-α during the sepsis process of the neonates was similar to that of INF-γ and depended on the time of sepsis and the gestational age of the infant. The values for the PT neonates were 3.362 ± 3.18 pg/mL, 4.995 ± 4.12 pg/mL, and 2.574 ± 1.64 pg/mL at presepsis, sepsis, and postsepsis, respectively . The presepsis, sepsis, and postsepsis values for the VPT neonates were 1.138 ± 1.10 pg/mL, 1.646 ± 1.12 pg/mL, and 2.079 ± 2.16 pg/mL, respectively . The clinical sepsis times between the PT and VPT groups differed . For the VPT group, the presepsis, sepsis, and postsepsis values were 48.58 ± 95.38 pg/mL, 21.5 ± 22.80 pg/mL, and 16.3 ± 25.56 pg/mL, respectively . A difference was observed between PT and VPT groups with regard to the sepsis time points . The values for the VPT group were 3.09 ± 2.65 pg/mL, 13.08 ± 16.85 pg/mL, and 1.17 ± 0.78 pg/mL at presepsis, sepsis, and postsepsis, respectively . The values at the sepsis time point for the VPT group were slightly higher than those for the PT group, although no difference was detected . The values for the VPT group were 5.13 ± 7.14 pg/mL, 31.6 ± 68.90 pg/mL, and 11.37 ± 20.22 pg/mL at presepsis, sepsis, and postsepsis, respectively . No differences were observed between the PT and VPT groups with regard to IL-10 at sepsis . The type of isolated microorganism differed between the groups, with more frequent gram-positive microorganisms in the VPT group and more gram-negative microorganisms in the PT group . The immunological parameters were analysed as positive or negative with respect to the cut-off value. They were considered as positive when at least one innate immunity, proinflammatory, or anti-inflammatory cytokine result tested positive at sepsis. The innate immunity factors C-RP and active complement SC5b-9 were identified in 8/10 patients with sepsis in the PT group and in 5/5 patients with sepsis in the VPT group, resulting in 13/15 patients with sepsis identified. No differences were observed between groups . The proinflammatory cytokines IFN-γ, TNF-α, and IL-6 were elevated in 9/11 PT infants with sepsis but only in 1/12 VPT infants with sepsis , resulting in 10/23 infants with sepsis identified. The anti-inflammatory cytokines IL-4 and IL-10 were elevated in 8/11 PT infants with sepsis and in 7/12 VPT infants with sepsis , resulting in 15/23 identified infants with sepsis.In summary, the identification of microorganisms using blood culture  was possHuman studies , 18, 19 This study is the first to report that proinflammatory cytokine production in sepsis can differentially distinguish between the dynamics of the innate, proinflammatory, or anti-inflammatory response that are most likely related to the foetal development of the immune system. Here, neonates with sepsis had elevated levels of mediators of innate defences including C-RP and SC5b-9, independent of gestational age. This result is in agreement with the presepsin (another innate component recently studied) detection in neonates <32 weeks of GA .α/β receptor but display predominant expression of the Tγ/δ receptor, which is a receptor observed in mucosal immunity and has a limited repertoire for antigen recognition. Cytokine production differs between both types of cells [γ, TNF-α, and IL-6 were differentially elevated only in PT neonates but not in VPT neonates in whom the values were low. This finding is important and should be considered when understanding sepsis diagnoses based on proinflammatory cytokine production.During foetal development, the innate defences develop first. For example, granulocytes mature during the 12th week of gestation, monocytes/macrophages during the 16th week of gestation, and NK cells during the 21st week gestation; however, the adaptive defences, including T and B lymphocytes, appear first as immature forms beginning in the 7th week of gestation but are not yet ready to fully respond. The lymphocytes follow a maturation process that includes the differential expression of several surface molecules that provide unique characteristics and the abilities to tolerate “self-antigens” and to respond to external antigens. Lymphocytes are not fully mature until the 32nd gestational week. Before that, T lymphocytes lack the expression of the Tof cells . Cytokinγ/δ cell type 2 innate lymphoid cells (ILC2) that differentiate earlier than Tα/β cells and could produce IL-4 before 32 gestational weeks of age [IL-4 was elevated in both groups; however, recent advances have shown that during T cell differentiation, IL-4 is predominantly produced by a subset of Ts of age .IL-10 levels did not differ between the two groups. IL-10 is another cytokine that could be produced early during foetal development because it can be produced by B-1a cells , which i in vitro stimulation. When monocytes and cDCs from preterm neonatal mice were stimulated to produce cytokines via TLR induction, a reduction in the levels of TNF-α, IFN-α, or IL-6 was observed. The authors also reported equivalent levels of IL-10 in both the preterm and at term groups [The low response of the neonate immune cells has been additionally identified in animal models followingm groups .The aetiological agent identification in this study coincides with other studies that identified agents for early- and late-onset sepsis . Few casThe measurement of the inflammatory mediators for sepsis diagnosis is extremely relevant for VPT neonates, who rarely provide plenty of blood for cultures. Thus, it is very important to distinguish the types of mediators that are candidates for sepsis diagnosis. SC5b-9 is a likely candidate because it increases in both PT and VPT neonates during sepsis. In addition, it is advantageous in that it can be detected in an under 1 : 10 dilution; this condition was recently used to measure cytokines in a mouse model of sepsis  and shouα, IFN-γ, and IL-6) increase differentially in neonates with sepsis based on gestational age. As expected, these cytokine values were high for neonates ≥32 weeks of GA during sepsis; however, they were negligible for neonates born <32 weeks of GA. We therefore suggest that the interpretation of proinflammatory cytokines should consider the gestational age of the neonate. The levels of the anti-inflammatory cytokines IL-4 and IL-10 as well as the components of innate immunity C-RP and SC5b-9 were high during sepsis in both gestational age groups, unlike the proinflammatory cytokines.The proinflammatory cytokines (TNF-


ZFPM2, encoding a zinc finger protein and abundantly expressed in the brain, uterus and smooth muscles, plays important roles in cardiac and gonadal development. Abnormal expression of ZFPM2 in ovarian tumors and neuroblastoma has been reported but hitherto its genetic association with cancer and effects on gliomas have not been studied. In the present study, the hexamer insertion-deletion polymorphism rs71305152, located within a large haplotype block spanning intron 1 to intron 3 of ZFPM2, was genotyped in Chinese cohorts of glioma (n = 350), non-glioma cancer (n = 354) and healthy control (n = 463) by direct sequencing and length polymorphism in gel electrophoresis, and ZFPM2 expression in glioma tissues (n = 69) of different grades was quantified by real-time RT-PCR. Moreover, potential natural selection pressure acting on the gene was investigated. Disease-association analysis showed that the overall genotype of rs71305152 was significantly associated with gliomas (P = 0.016), and the heterozygous genotype compared to the combined homozygous genotypes was less frequent in gliomas than in controls (P = 0.005) or non-glioma cancers (P = 0.020). ZFPM2 mRNA expression was negatively correlated with the grades of gliomas (P = 0.002), with higher expression levels in the low-grade gliomas. In the astrocytoma subtype, higher ZFPM2 expression was also correlated with the rs71305152 heterozygous genotype (P = 0.028). In addition, summary statistics tests gave highly positive values, demonstrating that the gene is under the influence of balancing selection. These findings suggest that ZFPM2 is a glioma susceptibility gene, its genotype and expression showing associations with incidence and severity, respectively. Moreover, the balancing selection acting on ZFPM2 may be related to the important roles it has to play in multiple organ development or associated disease etiology. ZFPM2) gene, also known as friend of GATA-2 (FOG2), encodes a transcriptional cofactor of members of the GATA-binding family that regulates expression of key genes essential for the development of multiple organs [The zinc finger protein multitype 2 . The control cohort consisted of healthy volunteers recruited by Hong Kong Red Cross, and Beijing volunteers. Leukemia, lymphoma and lung cancer cohorts were unrelated Chinese Han individuals recruited from Shanghai Changhai Hospital and Guangzhou Nanfang Hospital. The demographic characterizations of all the samples are described in The various cohorts in this study were enrolled from Beijing, Shanghai, Guangzhou and Hong Kong. The glioma cohort were unrelated Chinese fication ,30, namePeripheral white blood cells, formalin-fixed paraffin-embedded (FFPE) glioma tissues, and fresh glioma tissues were collected for DNA and/or RNA extraction. Glioma U87 cells  were harvested for RNA extraction. DNA was extracted from 5 ml peripheral blood by the phenol-chloroform method. DNA was extracted from FFPE samples with xylene, PCR buffer and Proteinase K, and mRNA was isolated from ~100 mg samples of frozen glioma tissue or glioma U87 cells with TRIzol solution (Invitrogen).An insertion-deletion polymorphism locus , previously amplified by the AluScan method  and veri5-TAAAGCAGCTGTCAGATCACATCC-3’ and reverse primer ‘5-AAGTTATTGTGCAGGAACATGGC-3’. Each 15 μl PCR reaction mixture contained 1.5 μl 10x long PCR buffer , 4 mM MgCl2, 20 μM dNTP mix, 0.1 μM forward primer and reverse primer, 0.5 unit Taq polymerase and 40–100 ng genome DNA. The PCR amplification was performed at 95°C 5 min, 35 cycles of 95°C 30 s, 69.3°C 30 s, and 72°C 1.5 min, and finally another 5 min at 72°C.Nested PCR was used to amplify the gene fragment containing rs71305152. For this purpose two pairs of PCR primers were designed, and their specificities were checked with PerlPrimer software version 1.1.19. The fragment size of the first PCR was 1,114 bp, and was amplified using the forward primer ‘5-GTCGACTTTGACGGTAATGTCCT-3’ and reverse primer ‘5-GAGGTAAGAGTATAATCCAGAAGAC-3’ contained in 15 μl PCR mixture, which also included 1.5μl 10 x long PCR buffer, 4 mM MgCl2, 20 μM dNTP mix, 0.5 unit Taq polymerase and 3 μl purified first PCR products. PCR amplification was conducted at 95°C 5 min, 35 cycles of 95°C 30 s, 67°C 30 s, and 72°C 1.5 min, and finally another 5 min at 72°C. For each sample, 3 μl PCR products were mixed with 2 μl 6x DNA loading dye and loaded on 0.7% ethidium bromide stained agarose gel in 1x Tris-acetate-EDTA (TAE) buffer. Following electrophoresis, the presence of products of 1,009 bp was confirmed under UV.The second PCR reaction was performed with 0.1 μM forward primer ‘5-AAAATTTTCATCTTGA-3’ and reverse primer ‘5- ATTCTCATCCCGTAT -3’ to amplify a 44-bp fragment in ZFPM2. In each instance, 15 μl PCR reaction was conducted containing 0.1 μM of each primer, 1.5 μl 10x long PCR buffer, 4 mM MgCl2, 20 μM dNTP mix, 0.5 unit Taq polymerase and 100 ng DNA. PCR amplification was carried out at 95°C 5 min, 35 cycles of 95°C 30 s, 51°C 20 s, and 72°C 10 s, and finally another 3 min at 72°C. PCR products (10 μl) of each sample were mixed with 2 μl 6x DNA loading dye and loaded on 5% ethidium bromide stained agarose gel in 1x TAE buffer. Following electrophoresis, the presence of the 44-bp product was confirmed under UV.In order to distinguish between the three rs71305152 genotypes by gel electrophoresis, PCR was performed with the forward primer ‘5-GCTGTTAAATGTGTATACTT-3’, and 3 μl purified PCR products. Reaction was performed at 96°C 1 min, followed by 25 cycles of 96°C 10 s, 50°C 5 s, 60°C 4 min. The sequencing was carried out using the 3130xl Genetic Analyzer, and the three different genotypes were determined using the BioEdit biological sequence alignment editor software version 7.0.5.The genotypes of rs71305152 for each sample were also identified by direct DNA sequencing. Each 15 μl sequencing reaction contained 0.75 μl BigDye Terminator v3.1 (Applied Biosystems), 3 μl BigDye Terminator v3.1 5xSequencing Buffer (Applied Biosystems), 0.24 μM sequencing primer ‘ZFPM2 expression levels in glioma tissue were measured by real-time quantitative reverse transcription PCR . Total RNA was firstly reverse transcribed to cDNA through reverse transcription-polymerase chain reaction (RT-PCR) with TaqMan Reverse Transcription Kit (Applied Biosystems). Each 20 μl RT-PCR contained 2 μl 10x RT buffer, 2 μl 10x RT Random Primers, 80 mM 25x dNTP Mix, 1 unit MultiScribe Reverse Transcriptase (Applied Biosystems), 40 unit RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) and 1 μg mRNA. RT-PCR was carried out at 25°C 10 min, 37°C 120 min, and finally 85°C 5 min. Real-time qRT-PCR was subsequently performed with two housekeeping genes hypoxanthine phosphoribosyltransferase 1 (HTRP1) and TATA box binding protein (TBP), which are appropriate for glioma tissue gene expression analysis [HTRP1 and TBP primers were as described [ZFPM2 forward primer ‘5-ATTCTTTGAAGACAAAGGCTCAG-3’ within exon 5 and reverse primer ‘5-GGTACATCCCTTCTGTGAGAG-3’ within exon 7 were designed using the PerlPrimer software version 1.1.19. Each 10 μl Real-Time qRT-PCR mixture contained 0.1 μM per primer, 4 μl SYBR Premix Ex Taq (2x) (TAKARA), 0.2 μl ROX Reference Dye II (50x), and 0.04 μg sample RNA cDNA product. The reaction was performed on the 7500 Fast Real-Time PCR System (Applied Biosystem) at 95°C 30 s, 40 cycles of 95°C 3 s, 60°C 30 s, and one cycle of 95°C 15 s, 60°C 1 min, 95°C 15 s. The ZFPM2 expression levels were determined from CT values, and batch-to-batch experimental variations correction was carried out utilizing mRNA of U87 cells as calibrator. The expression levels were further normalized against the geometric mean of the expression levels of the two housekeeping genes described above.analysis . The HTRescribed . The ZFPP value was less than 0.05. The genotype frequency deviation from Hardy-Weinberg equilibrium was tested based on Pearson’s Chi-square test through the online program (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Pearson’s Chi-square test in the program UNPHASED version 3.1.2 was employed for analyzing overall allele and genotype frequency difference between the case and control cohorts. The same test in SPSS version 17.0  was employed for comparison of the frequency difference between the case and control cohorts for each genotype sub-group. Student’s t-test and ANOVA in SPSS version 17.0 were used to test the significance of correlation of the three glioma grades, survival status, or genotypes with ZFPM2 mRNA expression levels in the glioma tissues. Kaplan-Meier non-parametric test [The statistical tests were regarded as significant when ric test  and Cox ZFPM2 gene (genomic region 106400 kb—106886 kb) on chromosome 8 was retrieved from Phase III of the HapMap Project and used as input for the program DnaSP [Phased haplotype data for the am DnaSP  to yieldam DnaSP , Fu and am DnaSP  for eachZFPM2 , according to the Hardy-Weinberg equilibrium test. In addition, there was no statistical difference in genotype frequencies between the control cohorts from two different geographical regions, Hong Kong and Beijing. The allele frequency difference was analyzed between different cancer-control cohorts, and neither insertion allele nor deletion allele was found to be associated with any type of cancer (Tables P = 0.016)  . The inc= 0.016) , no geno= 0.016) .P = 0.021). In comparing the frequencies of the homozygous deletion (DD) and heterozygous ID genotypes, the DD frequency was significantly increased in the glioma cohort, relative to either the control cohort , or the non-glioma cancer cohort . Furthermore, in comparing the frequencies of the combined homozygous genotypes and the ID genotype, the ID frequency was substantially decreased in the glioma cohort relative to either the control cohort (P = 0.005) or the non-glioma cancer cohort (P = 0.020) (P = 0.020), but not in the female glioma patients (P = 0.148). As shown in viz. A II (n = 44), A III—IV (n = 162), O + OA II (n = 89), and AO + AOA III (n = 55) (P = 0.956).Pearson’s Chi-square test was conducted as a post-hoc test on the frequency difference of any two genotypes between the glioma cohort on the one hand, and control cohort or non-glioma cancer cohort on the other. As shown in = 0.020) . When thZFPM2 mRNA expression levels of 69 glioma patients were examined using real-time qRT-PCR  (ZFPM2 expression in grade II tumors were significantly higher than that in grade IV tumors (P = 0.003). Moreover, there was no significant correlation found between ZFPM2 expression with either age or gender . When ANOVA was employed to compare the different grades in the individual glioma subtype, significant difference in ZFPM2 expression between grades was found for astrocytoma (P = 0.006), but not for oligodendroglial tumors where grade IV tumors are absent  . Post-hoe absent .ZFPM2 expression levels, the 69 patients were separated into two subgroups for analysis, those with homozygous (both II and DD) genotypes and those with the ID genotype. Significant difference in ZFPM2 expression between the two genotype subgroups was observed for astrocytomas  as determined using Student’s t-test, with lower expression in the homozygous group, but no difference was found for oligodendroglial tumors  (To determine the possible association between genotypes and = 0.347) .P = 0.339) (Upon follow up of 62 patients comprising 54 astrocytomas (including 50 GBMs), and 8 oligodendroglial tumors, 11 survived for a median of 91 (range 65–338) weeks, and 51 were deceased with a median survival of 40 (range 2–282) weeks. Based on the log rank test of Kaplan-Meier, there was no significant association between the genotypes and survival time of the 47 deceased GBM patients (= 0.339) .P = 0.077)  weeks up to the study end point for 15 patients with expression data . Cox regression analysis using expression level as covariate indicated that expression exerted a moderate ‘protective’ effect on survival (exp(B) = 0.949; = 0.077) .ZFPM2 is under evolutionary pressure, data for all single nucleotide polymorphisms (SNPs) within the gene were retrieved from the HapMap Project database and used for summary statistics tests, including Tajima’s D, Fu and Li’s D* and Fu and Li’s F*, and these statistics were analyzed to determine whether they were significantly different from expected values under a standard neutral model by performing coalescent simulation assuming no recombination. The 11 populations sampled in Phase III of the HapMap Project all gave significantly positive values for the three summary statistics tested . This association was not previously identified in GWAS studies, possibly because the effective sample size of the polymorphism is too small for detection in such genome-wide approaches, and/or the six-nucleotide deletion itself was not included in the probe set in the array platforms employed in the GWAS. Nevertheless, the P value of Hardy-Weinberg equilibrium test, which has been validated as furnishing evidence for association [P = 0.0015), thereby supporting ZFPM2 as a glioma susceptibility gene. Compared to the control cohort, both the homozygous insertion genotype (P = 0.021) and the homozygous deletion genotype (P = 0.015) showed a moderate increase in gliomas (P = 0.005), or non-glioma cancers (P = 0.020), suggests a protective role being played by the heterozygous genotype in gliomas and this is a characteristic of loci that are under the influence of balancing selection [Association analysis showed that rs71305152 was strongly associated with gliomas , but not in female patients (P = 0.148). Epidemiological studies have shown a higher incidence of gliomas in males than females  [ZFPM2 could also be a contributing factor to the higher incidence of gliomas observed for males. Further studies with increased sample size would be most valuable in this regard.Interestingly, in 1.4–1.6) ,43, and 1.4–1.6) . The preZFPM2 plays an essential role in lung development [ZFPM2-related FOG gene is strongly expressed in immature hematopoietic cell lineages [P = 0.629), leukemia (P = 0.528) or lymphoma (P = 0.228). However, the explicit disassociation between rs71305152 and these cancer types need to be further tested with large sample sizes in the future. In addition, the possible association between this marker and more types of cancer need to be examined, especially for ovary tumor and neuroblastoma, where abnormal ZFPM2 expression has been reported, in order to determine the extent of specificity of the rs71305152-glioma association.As many susceptibility SNPs have been associated with more than one type of cancer , the prelineages , Table 2ZFPM2 were significantly different between the glioma grades  = 0.949; P = 0.077, ZFPM2 expression and spontaneous regression also has been reported for neuroblastoma [Based on ANOVA analysis, the expression levels of ZFPM2 expression in astrocytomas (P = 0.028) as indicated in P = 0.339, ZFPM2 expression, but not the progression of glioma to severity. Nevertheless, all expression data was obtained from only a small sample set, and further studies with increased sample size are warranted.Significant association was observed between patients’ genotypes and ZFPM2 was revealed in P = 0.005), suggesting a potentially protective role played by the heterozygous genotype in gliomas. This is corroborated by the fact that the heterozygous genotype was the major genotype in the control cohort (46.65%), in contrast to its being a minor genotype in the glioma cohort (36.86%) as shown in ZFPM2 for the three summary statistics Tajima’s D, Fu and Li’s D* and Fu and Li’s F* indeed indicated an excess of heterozygotes. Although this excess of intermediate-frequency alleles in a population can result from either balancing selection or population bottlenecks, given that these statistics were significantly positive across all the tested populations and not only to specific populations, it is unlikely for demographic effects to be a major factor. Both lines of evidence therefore provide strong evidence for balancing selection acting on the ZFPM2 gene. Balancing selection is an important evolutionary force and its targets include genes in key functional systems such as the immune system and the reproductive system [ZFPM2 may likewise be associated with its important roles in multiple organ development, including the reproductive organs.Most human genetic mutations are neutral or deleterious, but some are advantageous and persist in the population either to fixation or maintained at high frequencies. The latter variations contribute to advantageous population diversity and are evolutionarily maintained by balancing selection . A classe system ,49,50. TZFPM2 on gliomas, the most prevalent brain tumor, and balancing selection in the gene have been examined. The genetic association results clearly indicate that rs71305152 is a susceptibility marker for gliomas in the Han Chinese population, especially for males. That the genotypes of ZFPM2 could be a contributing factor to the greater prevalence of gliomas in males highlights the importance of the polymorphism. In addition, the relative stability of the polymorphism against somatic mutation and ease of its genotyping using gel electrophoresis underlies the potential of rs71305152 as a clinically useful marker. Moreover, ZFPM2 expression levels were negatively correlated with glioma grades, suggesting that ZFPM2 expression can be a potentially useful indicator of disease severity for gliomas, as well as a significant factor in glioma pathogenesis, in which case ZFPM2 could provide a valuable target for glioma therapeutics. Finally, that balancing selection was found to act on ZFPM2 highlights its evolutionary importance and further investigation is merited to understand how its fundamental functions contribute to its myriad involvement in organ development and disease phenotypes.In summary, the present study represents the first instance where effects of S1 FigA II and A III—IV represent low grade (II), and high grades (III—IV) astrocytomas. O + OA II represents grade II oligodendroglial tumors (oligodendrogliomas and anaplastic oligoastrocytomas). AO + AOA III represents grade III oligodendroglial tumors (anaplastic oligodendrogliomas and anaplastic oligoastrocytomas). Dark grey bars, light grey bars and white bars represent frequency of homozygous insertion (II), heterozygous (ID) and homozygous deletion (DD) genotypes, respectively. The sample size of each genotype is shown on each bar.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableP values determined from 104 coalescent simulations with no recombination are shown in the column next to each statistic.The values for the summary statistics Tajima’s D, Fu and Li’s D* and Fu and LI’s F* are given for the 11 HapMap populations. Nominal (PDF)Click here for additional data file.


A genome-wide association study (GWAS) for these traits and their calculated changes was conducted to identify quantitative trait loci (QTL) using a 600 K SNP panel.Heat stress in poultry results in considerable economic losses and is a concern for both animal health and welfare. Physiological changes occur during periods of heat stress, including changes in blood chemistry components. A highly advanced intercross line, created from a broiler (heat susceptible) by Fayoumi (heat resistant) cross, was exposed to daily heat cycles for seven days starting at 22 days of age. Blood components measured pre-heat treatment and on the seventh day of heat treatment included pH, pCO3, TCO2, ionized Ca, hematocrit, hemoglobin, and sO2, and significant decreases in pCO2 and glucose after 7 days of heat treatment. Heritabilities ranged from 0.01-0.21 for pre-heat measurements, 0.01-0.23 for measurements taken during heat, and 0.00-0.10 for the calculated change due to heat treatment. All blood components were highly correlated within measurement days, but not correlated between measurement days. The GWAS revealed 61 QTL for all traits, located on GGA  1, 3, 6, 9, 10, 12–14, 17, 18, 21–28, and Z. A functional analysis of the genes in these QTL regions identified the Angiopoietin pathway as significant. The QTL that co-localized for three or more traits were on GGA10, 22, 26, 28, and Z and revealed candidate genes for birds’ response to heat stress.There were significant increases in pH, base excess, HCOThe results of this study contribute to our knowledge of levels and heritabilities of several blood components of chickens under thermoneutral and heat stress conditions. Most components responded to heat treatment. Mapped QTL may serve as markers for genomic selection to enhance heat tolerance in poultry. The Angiopoietin pathway is likely involved in the response to heat stress in chickens. Several candidate genes were identified, giving additional insight into potential mechanisms of physiologic response to high ambient temperatures.The online version of this article (doi:10.1186/s12864-016-2601-x) contains supplementary material, which is available to authorized users. Climate change has increased the frequency of severe heat waves and the global temperature is projected to become increasingly warmer . Heat stTo reduce core body temperature during periods of heat stress, blood flow to internal organs decreases and blood flow to the combs and other surface tissues increases in chickens . During During high ambient temperatures, chickens reduce feed intake by as much as 17 %, which reduces growth . However2. Respiratory alkalosis occurs in broilers during heat stress and is associated with reduced growth rate [A major change in blood components is caused by heat-induced increased respiration, which results in respiratory alkalosis, a disturbance in the acid base balance characterized by an increase in blood pH accompanied by a decrease in pCOwth rate . Metabolwth rate .Electrolyte balance is essential for acid base balance, maintenance of cellular homeostasis, synthesis of tissue protein, electrical potential of cell membranes, enzymatic reactions, and maintaining osmotic pressure . Alterin2, pO2, base excess (BE), HCO3, TCO2, K, Na, ionized Ca (iCa), hematocrit (Hct), hemoglobin (Hb), sO2, and glucose (Glu). In a commercial egg laying population, developmental measures have been established with hopes of using measures of blood chemistry components for selection [The goal of the current study was to identify the physiological changes and genomic regions associated with response to heat stress in chickens as characterized by the blood chemistry components, including pH, pCOelection . To dateelection –18. We u3, TCO2, iCa, Hct, Hb, and sO2 significantly increased while pCO2 and glucose significantly decreased. There were no significant changes in pO2, K, and Na due to heat treatment.Phenotypic means and heritabilities are given in Table Heritabilities ranged from 0.01-0.21 for pre-heat measurements, 0.01-0.23 for measurements taken during heat, and 0.00-0.10 for the calculated change due to heat treatment.Correlations between blood components at each measurement phase are given in Fig. Of the 480 genotyped birds, 458 Advanced Intercross Line (AIL) and all 12 parental line birds passed the whole animal DishQC criterion. Of the 580,961 SNPs on the array, filtering based on SNP call rate ≥ 95 % removed a small proportion , whereas filtering based on MAF removed a much larger proportion , yielding 210,117 SNPs for subsequent analyses.The results from the GWAS for each trait are depicted in Fig. Six QTL for pH phenotypes were identified: three for pH20 with two on GGA18 and one on GGA28, one for pH28 on GGA12, and two for pH28-20 with one each on GGA6 and GGA10.2 measurements were identified: one for pCO220 on GGA28, four for pCO228 located on GGA1, 9, 10, and 27, and four for pCO228-20 on GGA3, 10, 23, and 28. No QTL were identified for pO220 or for pO228-20. One QTL was identified for pO228 on GGA13.Nine QTL for pCO2 traits: none for TCO220; eight for TCO228 one each on GGA6 and GGA26, and six on GGAZ, and one for TCO228-20 on GGA10. No QTL were identified for HCO320 or HCO328-20, while seven were revealed for HCO328 with one on GGA6 and six on GGAZ.A total of five QTL were identified for BE traits: two for BE20 on GGA18, three for BE28 with one each on GGA1, 21, and 27, and none for BE28-20. Nine QTL were identified for TCOFive QTL for K traits were identified: four for K20 with two on GGA10, one on GGA12, and one on GGA26, none for K28 and one for K28-20 located on GGA12. No QTL were identified for the Na phenotypes. A single QTL was identified for ionized Ca phenotypes: for iCa28 on GGA26.2 phenotypes: none for sO220, two for sO228 located on GGA24 and GGA25, and one for sO228-20 on GGA17.We identified five QTL for Hct measurements: none for Hct20 or Hct28-20, and five for Hct28 located one each on GGA1, 10, 14, 22, and two on GGA28. Seven QTL were identified for Hb: none for Hb20, six for Hb28 located one each on GGA1, 10, 14, 22, and two on 28, and one for Hb28-20 on GGA22. There were three QTL for sOFour QTL were identified for Glu: one for Glu20 on GGA10, and three for Glu28 with one on GGA22 and two on GGAZ.P ≤ 0.05) canonical pathways for each group are listed in Table The pathway analysis of all annotated genes within significant QTL regions across all measured traits, and separately for genes in the regions of QTL co-localization, and the top 20 significant  using a novel AIL of chickens under heat stress and a 600 K SNP panel for genotyping. The blood components measured were within the accepted range reported for chicken [The aim of this study was to identify and estimate the effect of QTL, and to perform a functional analysis using positional candidate genes, for blood components .To our knowledge, QTL for blood pH, pCO3, and base excess, and can be induced in growing layers by high levels of calcium in feed [Metabolic alkalosis occurs when there is a disturbance in the fixed acids and bases in the extracellular fluid . Imbalan in feed .Base excess is considered a comprehensive measure of the metabolic components of bases, which reflects the nonrespiratory contribution to changes in acid–base disturbances . Base ex3 is the most abundant buffer in the blood, is primarily regulated by the kidneys, and is a metabolic component of acid–base balance [3 due to heat treatment. These results contrasted with a previous study using broilers at 28 days of age in which blood HCO3 significantly decreased in panting birds under acute heat stress [3 after a heat stress at 32 °C for 10 h [2 also increased in response to heat treatment. It was unexpected to observe a decrease in base excess, consistent with metabolic alkalosis, while HCO3 and TCO2 increased, because the traits are highly positively correlated within all treatment phases (r ≥ 0.95).HCO balance . We obset stress , and anofor 10 h . TCO2 al3 between 0.03-0.23, and of TCO2 between 0.01-0.13. In broiler chickens at 22 days of age reared under cold stress conditions, blood HCO3 and TCO2 heritability were both estimated at 0.19 [We estimated heritability of base excess between 0.00-0.10, of HCO at 0.19 .3, and TCO2, which are related to metabolic alkalosis. QTL for base excess are located on separate chromosomes for all measurement phases, indicating a strong genetics by environmental (G x E) temperature interaction. The phenotypic correlations for base excess between measurement phases were both very low (r = 0.03). The QTL for base excess on GGA18 overlap with pH measured at thermoneutrality and were highly correlated (r = 0.78). Surprisingly, QTL for HCO3 were only identified during heat treatment and were on GGA6 and GGAZ. Ten of the eleven QTL for TCO2 measured during heat co-localized with QTL for HCO3 and these co-localized regions were located on GGA6, 26, and Z.We are the first to report QTL in chickens for blood base excess, HCOBlood K and Na levels numerically increased and iCa statistically increased in response to heat treatment. This is in disagreement with previous reports of decreasing levels of both K and Na in response to heat stress, likely due to increased water intake which results in decreased concentrations of electrolytes within the blood , 13, 38.Heritability of K and Na blood levels in humans has been estimated to be very low, 0.03 and 0.04, respectively , in agrer = 0.10). No significant QTL for Na were identified in the current study and a single QTL for ionized Ca was located on GGA26 for the measurement taken during heat.This research is the first to describe QTL for the electrolyte-balance traits of blood K, Na, and ionized Ca in the chicken. In swine, QTL have been identified for these traits . QTL for2 is a measure of oxyhemoglobin in relation to total hemoglobin that is able to bind oxygen [Changes in blood volume and oxygen carrying capacity occur in chickens during periods of heat stress . Both hed oxygen , and thi2 were very low (0.01-0.03), which is in general agreement with a previously reported value of 0.07 in cold-stressed broiler chickens at 22 days of age [The heritability of Hct was estimated as very low at 0.01 and 0.02 for pre-heat and the differential, respectively, while during heat was moderately heritable at 0.21. Heritability has been estimated for hematocrit at 0.39 in domestic fowl . The incs of age .www.animalgenome.org). In a broiler by layer F2 intercross, QTL for hematocrit were located on GGA1, 2, 6, and 14 [r ≥ 0.99). We identified novel QTL for sO2 on GGA17, 24, and 25, none of which overlapped between measurement phases, indicating separate genetic control of this trait dependent upon environmental temperature. A previous study using a commercial broiler line identified one on GGA16 [2 appear to be population specific.Seven QTL for haematocrit have been identified in chickens . QTL for hemoglobin and hematocrit represent 3  regions of co-localization and there is a positive linear relationship between hematocrit and heart weight in chickens under heat stress [The co-localized regions resulted in many significant canonical pathways and the top 20 pathways are found Table The QTL regions that co-localized for three or more traits were further investigated for positional, functional candidate genes to give further insight into the biological mechanisms involved in the response of blood components to heat stress. The identified genes are located in Additional file 228, and TCO228-20. With 2 of these 3 traits associated with CO2 concentration, CA12 (carbonic anhydrase) is a likely candidate gene involved in the CO2 response to heat stress. Carbonic anhydrases catalyse the reaction of CO2 and H2O to form HCO3 and H+, and thus may stabilize blood acid base balance during heat stress. Another strong functional candidate in this region is HSP40, a member of the heat shock protein family that functions as a molecular chaperone to prevent cellular damage during heat stress [GCNT3, a glucosamine acetyl transferase which is associated with glucose metabolism in humans [There are 51 genes in the region on GGA10 between 3–6 Mb that contained QTL for Glu20, pCOn humans .ALDH6  which functions to convert aldehydes to carboxylic acids. This gene may function to maintain blood acid base balance during heat stress. Another gene in this region is IGF1 (insulin like growth factor 1), which has many roles and is a biomarker for growth [Fourteen genes were identified on GGA10 between 16–17 Mb, where QTLs co-localized for pH28-20, Hct28, Hb28, and K20. Many QTL in chicken have been identified in this region including those related to growth , 53–55, r growth .TGFA (pretransforming growth factor) and ADRA1A (adrenergic receptor) both regulate cell growth. It is known that metabolic changes occur during periods of heat in chickens that contribute to reduction in growth, independent upon feed intake [Four genes were identified on GGA22 between 3–4 Mbs, where QTL were co-localized for Hct28, Hb28, Hb28-20, and Glu28. To our knowledge, no QTL have been reported in this region. Because all traits were measured during heat treatment or as the differential, we propose these to be heat specific QTL. Candidate genes d intake .228, K20, and iCa28. Notably, a QTL for tibia bone mineral density identified in a commercial broiler and layer cross is located within this region [There are 48 genes in the 1 Mb region on GGA26 between 3–4 Mbs, where QTL co-localized for TCOs region . This co220, and pCO228-20. A QTL for heart weight, relating to susceptibility of pulmonary hypertension [SLC39A3, SLC25A42 and SLC35E1 were identified, as well as CHERP and CIB3, involved in calcium homeostasis. Transcription-related genes include SUGP1, which is involved in RNA splicing; RFXANK, a DNA-binding protein; NR2C2AP, a nuclear receptor protein; DDX49, an RNA helicase; ELL, an RNA polymerase II elongation factor; and SIN3B a transcriptional regulator.There are 86 genes in the 2 Mb region on GGA28 between 3–5 Mb where QTLs co-localize for pH20, Hb28, Hct28, pCOrtension  co-local328, and TCO228. The only reported QTL near this region is for antibody response to KLH antigen [KIAA1328, involved in chromosomal integrity during mitosis; and TPGS2, involved in tubulin formation.On GGAZ, 2 genes were identified between 5–7 Mbs, where QTL co-localize for Glu28, HCO antigen . Heat st antigen , and thi antigen  and we o328, and TCO228. The one QTL that is near this region was identified in a previous generation of the same AIL as the current study, and is for bone mineral density [XPA, which is a DNA repair protein, FOXE3 which is part of the forkhead box, and SNORA66 which is small nuclear RNA. Additionally, microRNAs gga-mir-2954, gga-mir-2131, and gga-mir-1583 were identified in this region. An additional gene of interest identified was DNAJA1, which is part of the heat shock family of proteins.On GGAZ, 21 genes were identified between 69–71 Mbs, where QTL co-localize for Glu28, HCO density . A recen density . In huma density . Althoug2 in the current study were located on GGA1, 3, 9, 10, 23, 27, and 28. In swine, QTL for blood pCO2 are on chromosomes 6, 7, 8, 9, and X [2 QTL and several orthologous genes including FUNDC1, EFHC2, NDP, and MAOA. Another region of synteny exists between chicken chromosome 10, 1–4 Mb, and pig chromosome 7, 53–65 Mb  in chicken, which is involved in calcium metabolism, but this gene maps on pig chromosome 1, a chromosome on which no QTL have been reported for blood chemistry measurements. The region on GGA10, 1–4 Mb, contains QTL for Glu20, pCO228, pCO228-20, and TCO228-20. The syntenic region in swine contains co-localized QTL for pCO2, HCO3, TCO2, and base excess [QTL for blood pCO9, and X . We iden Mb Fig. , which ce excess .Fig. 3SyIGF-1.A QTL for blood K level mapped to syntenic regions in chicken GGA10, 16–17 Mb, in our line and swine chromosome 1, 63–226 Mb Fig.  in a preThe results of this study contribute to the currently sparse knowledge of levels and heritabilities of several blood components under thermoneutral and heat stress conditions in chickens. Most blood components changed in response to heat treatment. Mapped QTL may serve as markers for genomic selection to enhance heat tolerance in poultry and several candidate genes were identified which may give additional insight into mechanisms of physiologic response to high ambient temperatures.Animal experiments were approved by the Institutional Animal Care and Use Committee of Iowa State University: Log #4-11-7128-G.We used the F18 and F19 generations of an AIL between chicken lines divergent for thermotolerence created by crossing a single broiler sire to six highly inbred Fayoumi dams . Birds wA total of 631 birds from four hatches (two hatches in each of the two generations) were used for independent heat stress experiments (four replicates). At 17 days of age, birds were transferred to environmentally controlled chambers and acclimated for five days. Multiple chambers, each containing 6 pens, were used per replicate. Ten to 12 birds were placed in each pen. From day 22 to 28 of age, the chambers heated to 35 °C for 7 h per day and remained at 25 °C at all other times.2, pO2, base excess, HCO3, TCO2, K, Na, ionized Ca, hematocrit, hemoglobin, sO2, and glucose.Blood was collected from the wing vein on day 20 (pre-heat) and day 28 (during heat) using a heparinized syringe and needle, and analysed immediately using an iSTAT Portable Clinical Analyser . The iSTBlood was collected from the wing vein by using an EDTA-coated syringe and needle, and stored at −20 °C. DNA was extracted using a salting out method. Briefly, whole blood was incubated with lysis buffer containing proteinase K. Proteins were precipitated out using 5 M NaCl while the supernatant remained. The supernatant was combined with 70 % ethanol to precipitate out DNA. The DNA isolated from 468 AIL, 6 broiler, and 6 Fayoumi chickens was genotyped on the Affymetrix 600 K chicken SNP axiom array  by GeneSP value ≤ 0.05 using JMP statistical software [Calculations of means and standard errors, fixed effects and covariates for the GWAS were calculated based on ANOVA , and significant terms were fit as fixed effects with a software . Heritabsoftware .Parameters for inclusion of SNP genotypes included SNP call rate ≥ 95 % and minor allele frequency ≥ 5 %. Genotyping console (Affymetrix) software was used to create genotyping calls and quality control based on whole animal DishQC score ≥ 0.7. The SNPolisher (Affymetrix) R package was used for quality control of individual SNP in all animals with passing DishQC scores.The GWAS of phenotypic traits with SNP genotypes was done using GenSel software . Bayes By = vector form of phenotypes, X = incidence matrix to account for fixed effects on phenotypes, b = vector of fixed effects, zj = vector of genotypes for SNP j based on the number of B alleles , αj = allele substitution effect for SNP j, δj = whether SNP j was included in the Markov chain Monte Carlo (MCMC) chain, and ε is the error associated with the analysis.Where j was set so that π = 0.9978 to avoid fitting more SNPs than number of animals in a given iteration. In a true infinitesimal model, each window is expected to explain 0.1 % (100 %/1001) of the genetic variation; therefore, a 1 Mb window was considered significant if it explained ≥ 0.5 % of the total genetic variation, corresponding to 5 times more observed than expected.The genomic markers were split into 1001 non-overlapping 1 Mb windows across the genome. A total of 41,000 MCMC iterations were run for each analysis and the first 1000 iterations were discarded (burn in). The δP ≤ 0.05) canonical pathways and the top 20 significant pathways were reported. Additionally, a gene list was created using the regions of QTL co-localization (3 or more traits) and analysed as described for all QTL regions.To further investigate QTL regions, we conducted a pathway analysis using Ingenuity Pathway Analysis (IPA) software. All annotated genes within significant (explaining ≥ 0.05 % of the genetic variation) 1 Mb windows for any measured trait were identified using Ensemble biomart. This gene list was used as input into IPA and a core analysis was completed using default parameters to identify significant . All genes within the region were identified using ENSEMBL biomart .To identify syntenic regions for reported QTL for the same blood chemistry component measurements between chicken and pig, the Comparative Genomics option was used in Ensembl .http://www.animalgenome.org/cgi-bin/QTLdb/GG/pubtails?PUBMED_ID=ISU0082. The phenotypic dataset supporting the conclusions of this article is included within the article as an additional file  repository and can be found at


Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 , produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain. Bacillus subtilis, a well-characterized gram-positive bacterium, has been widely used for the production of heterologous proteins. This species and some of its close relatives have excellent protein secretory capability and are generally recognized as safe (GRAS), making them important hosts for the production of antibiotics, medicinal proteins, and industrial enzymes. B. subtilis 168 is a model laboratory strain that carries many mutations that have occurred during its modification via irradiation and selectionB. subtilis strains commonly used for recombinant protein production, such as WB600 and WB800, were constructed on the basis of B. subtilis 168B. subtilis ATCC 6051a is superior to that of B. subtilis 168, it has been widely applied to the production of industrial enzymes3B. subtilis ATCC 6051a has some undomesticated properties that hamper the extracellular production of recombinant proteins. In particular, it can produce large amounts of foam, highly resistant spores, multiple types of extracellular protease, and high level of amylase during fermentation, which related to srfCspoIIACnprEaprEamyEB. subtilis ATCC 6051a is poorly transformable, compared with laboratory strains, because it harbours an 84-kb endogenous plasmid pBS32, which encodes a single-pass trans-membrane protein ComI that inhibits the competence of DNA uptakeB. subtilis ATCC 6051a is difficult and require a highly efficient genome editing method. The genome sequence of B. subtilis ATCC 6051a was recently determined by Jeong et al.Because the recombinant protein productivity of Streptococcus pyogenes type II CRISPR/Cas system14Escherichia coliStreptococcus pneumoniaSaccharomyces cerevisiaeLactobacillus reuteriBombyx moriDrosophilaClustered regularly interspaced short palindromic repeat (CRISPR) systems, which are composed of CRISPR RNAs (crRNA), trans-activating CRISPR RNAs (tracrRNAs) and CRISPR-associated (Cas) proteins constitute an immune system in bacteria and archaea that efficiently cleaves foreign DNA entering the cell, including phages and plasmidsB. subtilis using a CRISPR/Cas9 system. This study describes the establishment and optimization of a CRISPR/Cas9 system in B. subtilis ATCC 6051a. We used this system to disrupt five genes in the B. subtilis ATCC 6051a genome  that hamper its use during industrial fermentation. Compared with B. subtilis ATCC 6051a, this mutant strain, named BS5, produces much less foam at the level of control, exhibits great resistance to spore formation, and secretes 2.5 times more β-cyclodextrin glycosyltransferase (β-CGTase) into the fermentation medium. β-CGTase is widely used in the production of β-cyclodextrin and mainly produced from wild strains and E. coliBacillus subtilis can largely reduce the cost of β-cyclodextrin production, which shows the high industrial value of BS5.Although there have been several successful implementations of the CRISPR/Cas9 system in microbial systems, there have not been any reports of genome editing in B. subtilis , which originates from B. amyloliquefaciens. The P43 promoter is a constitutively expressed promoterB. subtilis. The homologous repair template was obtained through overlap extension PCR of regions upstream and downstream of the target locus. The length of the upstream and downstream regions ranged from 450 to 550 bp, and they introduced an Xho I site into the target locus. The knockout plasmids and plasmid pHY300PLK-β-CGTase encode ampicillin resistance in E. coli and tetracycline resistance in both E. coli and B. subtilis.We assembled a complete CRISPR/Cas9 system in a single knockout plasmid that could be used for highly efficiently genome editing in subtilis . The sixB. subtilis ATCC 6051a as the initial strain in which to perform genetic manipulations. Fermentation of B. subtilis ATCC 6051a in a 3 L fermenter in our laboratory produced a massive amount of foam that requires large quantities of antifoam agent  continuously; BS1 produced much less foam at a controllable level that needed 60 ul antifoam. The foam height of B. subtilis ATCC 6051a and BS1 was similar after 42 h, while the foam of BS1 shows great sensitive to antifoam , while the sporulation efficiency of BS2 was 0% (0 colonies/182 colonies), which shows that mutant BS2 has great resistant to spore formation.In order to cope with limiting nutrient sources and high cell density, th Xho I . The homth Xho I  to confinprE and aprE genes encode alkaline and neutral extracellular proteases, respectively, in B. subtilis. We constructed the knockout plasmids pHYcas9dnpr and pHYcas9dapr and used them to sequentially disrupt nprE and aprE gene of mutant BS2 using the CRISPR/Cas9 method described above. Amplification of the upstream and downstream regions, and then digesting the PCR products with Xho I allowed us to select the appropriate mutant, named BS3 with disruption efficiency of 53% ± 6%  have been disrupted, as a host for extracellular recombinant protein expression, the ability of mutant BS5 to produce β-CGTase was compared with that of B. subtilis ATCC 6051a. Both expression plasmid pHY300PLK-β-CGTase and empty expression plasmid pHY300PLK were transferred into B. subtilis ATCC 6051a and mutant BS5, and the resulting strains were used in an expression study. After 48 h of cultivation in TB medium, the culture supernatants of B. subtilis ATCC 6051a and BS5 that harbouring empty expression vector PHY300PLK show no β-CGTase activity. After 80 h of cultivation in 3 L fermenter, the highest β-CGTase activity of B. subtilis ATCC 6051a that harbours expression plasmid pHY300PLK-β-CGTase was 110.8 U/ml in 56 h, and the highest dry cell weight (DCW) was 68.8 g/L in 66 h; the highest β-CGTase activity of BS5 that harbours expression plasmid pHY300PLK-β-CGTase was 277.8 U/ml in 70 h, which was 2.5 times of B. subtilis ATCC 6051a, and the highest DCW was 70.3 g/L in 75 h  did not contain a homologous repair template; therefore, a homologous repair template was transferred into B. subtilis ATCC 6051a in the form of PCR fragment, along with the knockout plasmid. This attempt did not meet the efficiency required for genetic editing, perhaps because B. subtilis ATCC 6051a was unable to simultaneously take up the knockout plasmid and the homologous repair PCR fragment.The transformation efficiency in The target recognition of the sgRNA mainly depends on the last 12 bp of the guide sequence; thus, the existence of highly homologous regions in chromosomal DNA may result in off-target effectsBacillus strains, B. subtilis produces surfactin, which contains a peptide moiety and a β-hydroxy fatty acid side chain40B. subtilis ATCC 6051a produces a large amount of foam during fermentation, which has an extreme affect on fermentation process control and may lead to contamination. Compared with its parent strain, the mutant BS1 produces much less foam and at the level of control. This shows that surfactin may be a major mediator of foam formation, and that there is more work to be done to thoroughly inhibit foam production.Like many B. subtilis is governed by multiple RNA polymerase sigma factorsSpoIIAC gene, controls the forespore, sigma E controls the early stage of sporulation, and then sigmas G and K control later stages32SpoIIAC nonsense mutations can block the processing of sigma E precursor protein P31 to sigma E and prevent transcription of spoIID6B. subtilis.Spore development in Bacillus species produce many different types of extracellular protease to degrade heterologous extracellular proteins7B. subtilis WB600, which is deficient in six extracellular proteases due to knockouts of the nprB, nprE, aprE, mpr, bpr and epr genes. This strain displays a level of recombinant protein secretion higher than that of its parent strainB. amyloliquefaciens strain lacking six extracellular protease genes was constructed. This strain displays improved production of levan and α-amylaseB. subtilis releases multiple extracellular enzymes during the post-exponential period; α-amylase is among the major proteins releasedB. subtilis makes the isolation and purification of recombinant proteins difficult on an industrial scale. In addition, the massive expression of endogenous α-amylase increases secretion stress and influences the production of recombinant proteinsB. subtilis ATCC 6051a, which is an undomesticated strain with favorable growth characteristics. To improve the usefulness of B. subtilis ATCC 6051a as an industrial expression host, we disrupted the srfC, spoIIAC, nprE, aprE and amyE genes with an efficiency of 33% to 53%. Compared with B. subtilis ATCC 6051a, the final mutant (BS5) forms less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5 times more β-CGTase. Thus, the CRISPR system developed here can be used to modify industrially relevant strains with high efficiency, and mutant BS5 can be applied as a superior expression host.In summary, we established a CRISPR/Cas9 system in the poorly transformable strain Escherichia coli JM109 was used for plasmid construction. Plasmid pHY300PLK-β-CGTase was previously constructed in our laboratory by inserting the β-CGTase gene of Bacillus circulans 251 into the B. subtilis-E. coli shuttle expression vector pHY300PLK B. subtilis ATCC 6051a was purchased from the American Type Culture Collection (ATCC).All bacterial strains and plasmids used in this study are described in Dpn I, T4 DNA ligase, In-Fusion HD Cloning Plus kit and vector (pMD18-T) were purchased from Takara . The plasmid mini-prep kit, PCR purification kit, and the agarose gel DNA purification kit were purchased from Tiangen Co. Ltd . DNA sequencing and DNA primer synthesis were performed by Shanghai RuiDi Biological Technology Co. Ltd. . Tryptone and yeast extract were obtained from Oxoid . β-cyclodextrin was purchased from Sigma-Aldrich . Non-fat powdered milk was purchased from BBI Life Sciences . Other chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd. .PrimeSTAR polymerase, restriction enzymes, calf intestinal alkaline phosphatase, B. subtilis mutants, E. coli, B. subtilis and B. subtilis derivatives were cultured in LB medium  at 37 °C with shaking at 200 rpm. To evaluate spore formation, B. subtilis mutants BS1 and BS2 were cultured at 40 °C with shaking at 200 rpm for 48 h in a medium containing 30 g/L dextrin, 30 g/L bean peptone, 1 g/L CaCl2 and 0.5 g/L NaCl. Ampicillin (100 mg/L) and tetracycline (20 mg/L) were added as needed.For routine construction of plasmids and 3PO4 and NH4OH, its temperature was adjusted to 37 °C, and it was added with 30 ml 166.7 g/L glucose. After 2 h of inoculation, the inducer of 30 ml 333.3 g/L lactose was added into fermentation culture as needed. The feed solution (Seed culture was obtained by inoculating 100 ul frozen glycerol stock (stored at −80 °C) into 100 ml LB medium and then incubating 12 h at 37 °C with shaking at 200 rpm. As for Shake-flask cultivation, a portion of 5% (v/v) seed culture (2.5 ml) was used to inoculate 50 ml of TB medium , which wsolution  was fed srfC fused with the sgRNA sequence, and a temperature-sensitive replicon PE194 for plasmid curing, was synthesized and ligated into the cloning vector pMD18-T. This fragment was amplified using the primer pair P07/P08. Fragment Three includes an E. coli replication origin (p15A ori), an ampicillin-resistance marker (ampR) and the α-amylase promoter (PamyQ) from B. amyloliquefaciens. This fragment was amplified from plasmid pHY300PLK-β-CGTase using primers P02 and P05. Fragment Four includes a tetracycline resistance marker (TcR) and a terminator from B. amyloliquefaciens. This fragment was amplified from plasmid pHY300PLK-β-CGTase using primers P01and P06. We amplified the upstream and downstream regions of the target locus using B. subtilis ATCC 6051A genomic DNA as a template and two primer pairs: P09/P10 and P11/P12. The amplified fragments were ligated together using overlap extension PCR, forming homologous repair template. To form the final knockout plasmid, pHYcas9dsrf1, the appropriate homologous repair template was inserted into the Xba I site of pHYcas9d , P19/P20 and P21/P22 (for pHYcas9dspo), P25/P26 and P27/P28 (for pHYcas9dnpr), P31/P32 and P33/P34 (for pHYcas9dapr), and P37/P38 and P39/P40 (for pHYcas9damy). Then the appropriate homologous repair template was inserted into the modified pHYcas9dsrf1 by digesting the PCR product and the modified plasmid with Xba I and ligating the appropriate fragments.The other five knockout plasmids  were constructed from pHYcas9dsrf1 by changing the 20-nt guide sequence (except pHYcas9dsrf2) and replacing the homologous repair template. The 20-nt guide sequence was changed using inverse PCR with the four primer pairs P17/P18 (for pHYcas9dspo), P23/P24 (for pHYcas9dnpr), P29/P30 (for pHYcas9dapr), and P35/P36 (for pHYcas9damy), which hanging the new modified 20-nt guide sequence at the 5′ end. The homologous repair templates were created by overlap extension PCR of sequences upstream and downstream of the 20-nt guide sequence. These sequences were amplified from the B. subtilis competent cells were made by the method of Anagnostopoulos and Spizizencas9 gene. Using transformant genomic DNA as the template, verification PCR reactions were carried out using specific primers . The culture was incubated at 37  °C, and then streaked onto LB plates that were subsequently incubated overnight at 51  °CAfter cultivation in a medium conducive to spore formation at 40  °C for 48 h, Cells of mutants BS1 and BS2 were diluted and divided into two same amount of portions, respectively, one of which was heat at 75  °C for 20 minTo detect the protease activity of mutants BS2, BS3 and BS4, strains were dropped onto a 5% non-fat powdered milk plate at 37  °C and incubated for 36 hThe α-amylase expression of mutants BS4 and BS5 was detected by dropping the strains onto LB plates containing 1% soluble starch at 37  °C for 24 h, then staining the plates with iodineThe β-cyclodextrin-forming activity was determined using the observation that β-cyclodextrin forms a stable, colourless inclusion complex with phenolphthaleinHow to cite this article: Zhang, K. et al. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system. Sci. Rep.6, 27943; doi: 10.1038/srep27943 (2016).