RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates   •WEE1 inhibition selectively kills H3K36me3-deficient cancer cells•These cells are killed through dNTP starvation because of RRM2 depletion•RRM2 is regulated by H3K36me3 through transcription and WEE1 via degradation•WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumors in vivo RRM2 transcription whereas WEE1 inhibition promotes RRM2 degradation via CDK activation.Pfister et al. show that WEE1 inhibition selectively kills H3K36me3-deficient cancer cells through dNTP starvation resulting from RRM2 depletion. Pfister et al. further show that H3K36me3 facilitates  Despite the high prevalence and poor prognosis associated with H3K36me3 loss, there is no therapy targeting H3K36me3-deficient cancers. Here we show these cancers can be targeted by inhibition of WEE1. We identify distinct roles for H3K36me3 and WEE1 in RRM2 regulation, nucleotide pool maintenance, and DNA replication. Our proposed therapy is based on synthetic lethality, which provides a less toxic and more effective treatment because it specifically targets cancer cells. H3K36me3 loss can be used as a predictive biomarker, which identifies multiple genetic mutations, and enables patient selection through immunohistochemistry. Finally, because the WEE1 inhibitor, for which we describe a target, is in clinical trials, we anticipate that these findings will be of immediate clinical relevance.Trimethylation of histone H3K36 (H3K36me3) is an epigenetic mark usually associated with actively transcribed genes , for whihttp://www.clinicaltrials.gov), but insights into the targeted use of this inhibitor are limited.The WEE1 kinase inhibits the activities of cyclin-dependent kinases CDK1 and CDK2 through tyrosine 15 phosphorylation . InhibitOne way to target genetic defects in cancer is through synthetic lethality. Synthetic lethality describes a genetic interaction between two genes or two pathways, where loss of either one alone has little effect on cell viability, but the simultaneous loss of both results in cell death. Therefore, synthetic lethal interactions can be exploited to selectively kill cancer cells that carry mutations in one of the genes in the interaction by chemically inhibiting the second gene product . Here weSchizosaccharomyces pombe. We therefore tested whether SETD2-deficient human cells can be selectively killed by inhibiting WEE1.From our unpublished results, we know that the loss of Set2 (a SETD2 ortholog) is synthetically lethal with the loss of Wee1 (a WEE1 ortholog) in 50] = 87 nM, LB996 IC50 = 68 nM versus RCC4 IC50 = 673 nM, U2OS IC50 = 712 nM) (p < 0.0001)   (p < 0.0001)   were hypersensitive to AZD1775  A. A498,  AZD1775 A and 1C. AZD1775 D and 1E. AZD1775 D. In bot 400 nM) F. Lastly 0.0001) H. This e 128 nM) D, and an 128 nM) I. Moreov 128 nM) E, and co 128 nM) F, confir 128 nM) G, and th 128 nM) H. TogethWe next examined the mechanism underlying this selective killing of SETD2-deficient cells, and observed a significant disturbance in S-phase. In particular, WEE1 inhibitor AZD1775 forced 32% of the CRISPR SETD2-knockout cells to accumulate as non-replicating S-phase cells , whereas it had no effect on U2OS parental cells A. The saTo understand the cause of S-phase arrest, we depicted the progression of individual replication forks using the DNA fiber assay. In U2OS cells, fork velocity was mildly reduced upon either SETD2 depletion or AZD1775 treatment (from an average of 0.6–0.8 kb/min to 0.4–0.6 kb/min in both cases) B, suggesTo study the molecular events at stalled replication forks, we used iPOND (isolation of proteins on nascent DNA) . In contWe next tested the possibility that p53 loss or homologous recombination (HR) deficiency were the cause of synthetic lethality with WEE1 inhibition. Previous publications showed that p53-deficient cells are killed by WEE1 inhibitors through mitotic catastrophe . HoweverS. pombe, the SETD2 ortholog Set2 is not required for HR (We and others have shown that SETD2-dependent H3K36me3 has a role in homologous recombination (HR) repair of DNA double-stranded breaks through facilitating LEDGF and CtIP binding to the chromatin . Howeverd for HR  yet it id for HR , but SETd for HR A. These We next investigated the cause of fork stalling and S-phase arrest. It is known that loss of ribonucleotide reductase (RNR) leads to fork stalling and S-phase arrest through deoxyribonucleoside triphosphate (dNTP) depletion . TherefoRRM2 mRNA levels were reduced by 60% by either SETD2 knockdown or CRISPR SETD2-knockout , where the RRM2 transcript and protein levels were 60% lower than that in SETD2 wild-type cells . To understand how SETD2 depletion reduces RRM2 protein levels (the first pathway), we performed qRT-PCR, and found that knockout B, suggespe cells C. Reducipe cells C.RRM2 promoter  mass spectrometry study found that 9 of 16 TBP-associated factors (TAFs) required for the initiation of transcription by RNA polymerase II (Pol2) are enriched at the histone mark H3K36me3 . Second,promoter D. Consispromoter D and S3Epromoter D and S3Eut cells F–S3H. Cout cells I and S3Jnt cells . Togethe50 = 157 nM versus siNT IC50 = 842 nM)  was the cause of the sensitivity toward the WEE1 inhibitor AZD1775, then RRM2 depletion or inhibition should resemble H3K36me3 loss, and also exhibit synthetic lethality with AZD1775. Indeed, depleting RRM2 by siRNA significantly sensitized SETD2 wild-type U2OS cells toward AZD1775 (siRRM2 IC 842 nM) E. Likewi 842 nM) F. MoreovTD2 cDNA K and S3LCyclin F is known to promote the ubiquitin-mediated proteolysis of RRM2 . WEE1 restrains the activity of CDK1/2 during S-phase , and CDK of RRM2 . FollowiFirst, we investigated the impact of WEE1 inhibition on RRM2 stability. Cycloheximide was used to block protein translation to allow measurement of RRM2 half-life in the presence or absence of the WEE1 inhibitor AZD1775. We found that AZD1775 treatment reduced RRM2 half-life from over 8 hr to less than 2 hr in both wild-type and CRISPR SETD2-knockout U2OS cells A. The reNext, we tested whether CDK activation (due to WEE1 inhibition) degrades RRM2 and kills SETD2-deficient cells. We found that in the presence of AZD1775, inhibiting CDK1 by RO3306 or inhibiting the NEDD8 activating enzyme with neddylation inhibitor MLN4924 (NAEi)  which bB and S4BTo further support our hypothesis that WEE1 regulates RRM2 degradation in S-phase by inhibiting CDK, we reasoned that inhibiting other negative regulators of CDK such as CHK1 and ATR, which also function in S-phase , may havCyclin F  that cannot be phosphorylated by CDK and degraded by SCFCyclin F . ExogenoCyclin F C, and suCyclin F C and S3KCyclin F-mediated degradation of RRM2.In addition to T33A, the toxicity of the WEE1 inhibitor was also suppressed by another RRM2 mutant: RxI/AxA, but not by wild-type RRM2 O. The RxAs expected, the reduction in RRM2 protein levels was reflected in reduced cellular dNTP levels. dATP levels were reduced by 50% following either SETD2 depletion or AZD1775, and by 70% when these treatments were combined D. T33A eWe have shown that RRM2 is transcriptionally regulated by H3K36me3 and is degraded by WEE1 inhibition. Because WEE1 inhibition also causes aberrant origin firing through CDK activation, which (like RRM2 depletion) exhausts the dNTP pool , we test50 for HU and GM versus over 5-fold difference in IC50 for AZD1775 in the same assay)  suggests that the traditional inhibitors of RRM2 would be less efficient in killing H3K36me3-deficient cells. Indeed, HU or GM (two inhibitors of ribonucleotide reductase) sensitized SETD2-knockout cells, but neither achieved a level of sensitivity comparable to the WEE1 inhibitor AZD1775 (2-fold or less difference in ICe assay) T and S4U3 versus 291.2 ± 40.0 mm3, p < 0.0001)    A and 5B. 0.0001) A. In con = 0.37) A and 5B.Consistent with the in vitro data, AZD1775-treated SETD2-deficient tumors showed significantly greater levels of replication stress (measured by γH2AX pan-nuclear staining) C and 5D,Finally, to identify patients who could benefit from this treatment, we sought a single biomarker to identify all the genetic mutations that lead to loss of H3K36me3. Using a monoclonal antibody against H3K36me3, we were able to distinguish SETD2-deficient from SETD2-proficient tumor xenografts by immunohistochemistry (IHC) G. ImportDespite the frequent loss of histone H3K36me3 in multiple cancer types and its association with poor patient outcome, there is no therapy targeting H3K36me3-deficient cancers. Although WEE1 inhibitors are currently in clinical trials, insights into determinants of sensitivity to WEE1 inhibitors are limited. Here, we identified a synthetic lethal interaction between loss of H3K36me3 and inhibition of WEE1. We established RRM2, a subunit of ribonucleotide reductase, to be the target of this synthetic lethal interaction, and showed it to be regulated by two pathways in this context. Disrupting the first pathway through H3K36me3 depletion reduces RRM2 expression and dNTP production. Disrupting the second pathway through WEE1 inhibition leads to aberrant CDK activity in S-phase, resulting in untimely RRM2 degradation, aberrant origin firing, and reduced dNTP pool levels. Simultaneous disruption of both pathways leads to critically low RRM2 levels, dNTP pool depletion, inhibition of DNA replication, and cell death .We have identified a role for SETD2-dependent H3K36me3 in facilitating RRM2-dependent nucleotide synthesis and efficient DNA replication. In all H3K36me3-deficient cell lines tested, RRM2 levels were consistently reduced. This is not simply a cell cycle effect because H3K36me3-depletion had no effect on the cell cycle . Nor is RRM2 and recruits transcription initiation factors . This finding is in accordance with the recent report of a role for murine SETD2 in transcriptional initiation of genes that are involved in endothelial differentiation  works together with RRM2 degradation to kill H3K36me3-deficient cells. We showed that depleting RRM2 without increasing origin firing , or increasing origin firing without depleting RRM2 , is less efficient in killing H3K36me3-deficient cells than combining aberrant origin firing with RRM2 depletion. These findings advance our current understanding of WEE1 inhibition, showing that it induces replication stress through RRM2 depletion as well as aberrant origin firing . The synIn summary, we define a synthetic lethality between H3K36me3 loss and WEE1 inhibition, and its underlying mechanisms, by a variety of experimental approaches in many cell lines. We further demonstrate that H3K36me3-deficient cancers can be targeted in vivo by exploiting this synthetic lethal relationship. The sensitivity to the WEE1 inhibitor results through DNA replication stress because of RRM2 depletion, suggesting that other cancers exhibiting such replication stress may be targeted similarly. Because the WEE1 inhibitor, AZD1775, is already in clinical trials, and because we have identified a testable predictive biomarker for loss of H3K36me3, we expect our findings to be of immediate clinical relevance.SETD2). The plasmid was transfected into U2OS cells using Fugene6 (Promega) and single-cell colonies of GFP-positive cells were selected and validated by western blotting and genomic PCR.The CRISPR plasmid was purchased from Horizon Discovery Group, and the plasmid (pD1301) contains the chimeric gRNA scaffold, Cas9 and DasherGFP. The target sequence of the gRNA is ACTCTGATCGTCGCTACCAT (first exon of 7 cells were treated with the AZD1775 or DMSO (24 hr) and pulsed with 10 μM EdU (10 min), after which they were either crosslinked , or washed and released into fresh medium containing 10 μM thymidine and AZD1775/DMSO for additional 90 min before crosslinking. The click reaction was performed according to the published protocol  to remove cellular debris. The supernatant was dried and dissolved in sterile water. Determination of the dNTP pool size was based on DNA polymerase-catalyzed incorporation of radioactive dNTP into synthetic oligonucleotide templates as described (Nucleotides were extracted by incubating cells in 60% methanol (1 hr) at −20°C. Samples were boiled (3 min) and centrifuged at 13,000 × escribed .6 cells per mouse in 50% v/v Matrigel, 14 mice for A498 and U2OS, 10 for LB996). When the mean tumor size reached 110 mm3, mice were divided into two equal groups. Group 1 received vehicle alone  for 12 days. Group 2 received AZD1775  for 12 days. Tumor size was determined by caliper measurements and calculated as (width × length × height) /2. All mice in the control group were killed on day 13, 24 hr after the last dose of vehicle. Three mice in the treatment group were killed on day 13, 24 hr after the last dose of AZD1775, and tumor re-growth in any remaining mice was followed until day 33, when all remaining mice were killed.Mice were given food and water ad libitum, and all animal procedures were carried out under a project license issued by the home office  following ethical review by the University of Oxford Committee on Animal Care, and performed according to United Kingdom Coordinating Committee on Cancer Research guidelines. BALB/C nude mice  were injected subcutaneously with A498, LB996, or U2OS tumor cells . Nephrectomies were collected between 1995 and 2004, with informed consent from all subjects to use of tissue in ethically approved research. Formalin-fixed paraffin-embedded tumor cores were used to construct renal cancer tissue microarrays, and 4 μm sections were stained on Bondmax Autostainer (Leica). Briefly, following heat-induced epitope retrieval  sections were stained with an antibody against H3K36me3  at 1:1,000 dilution for 1 hr. Antibody binding was identified using a Polymer Detection System (Leica), followed by 3,3′-diaminobenzidine staining.E.M., Y.J., and S.S. are co-second authors for this paper, having contributed equally to the paper. S.X.P., E.M., Y.J., S.S., M.W., L.P.Z., G.O., I.M., and N.D. designed and conducted the experiments with contributions from C.C.P.; C.V. and V.M. were involved in patient tissue microarray studies. S.X.P. and T.C.H. wrote the manuscript. G.D., S.Y., N.B.L.T., V.A., A.R., and T.C.H. supervised the work.


Desulfovibrio spp., Desulfonatronum spp., Desulforhabdus spp., and Desulfotomaculum spp. at 40°C, Thermodesulfovibrio spp. at 50°C, Thermodesulfovibrio spp. and Thermacetogenium spp. at 60°C, Thermacetogenium spp. and Archaeoglobus spp. at 70°C, and Archaeoglobus spp. at 80°C. None of these potential sulfate reducers exceeded 7% of the community in the untreated sample. Since no exogenous electron donor was provided during incubation, these sulfate reducers appeared to rely on the degradation of organic matter inherited from porewater and sediments. Aqueous chemistry indicated that fluids discharged in the region represented a mixture of saline formation water and low-salinity surface water; therefore, these lines of evidence suggest that deeply-sourced, thermophilic and surface-input, mesophilic sulfate-reducing populations entrapped along the subsurface fluid transport could respond rapidly once the ambient temperature is adjusted to a range close to their individual optima.Terrestrial hydrocarbon seeps are an important source of naturally emitted methane over geological time. The exact community compositions responsible for carbon cycling beneath these surface features remain obscure. As sulfate reduction represents an essential process for anoxic organic mineralization, this study collected muddy fluids from a high-temperature hydrocarbon seep in Taiwan and analyzed community structures of sulfate-supplemented sediment slurries incubated anoxically at elevated temperatures. The results obtained demonstrated that sulfate consumption occurred between 40°C and 80°C. Dominant potential sulfate reducers included  The fluids released into surface environments are generally considered to represent a mixture of deeplysourced formation water from a hydrocarbon reservoir and shallow-ranging meteoric water  are prominent surface geological features, in which gaseous fluids associated with unconsolidated sediments generated by tectonic pressurization, compaction dewatering, or clay dehydration are expelled , 40. Theic water , 78, thePrevious studies related to terrestrial hydrocarbon seeps or MVs mainly focused on identifying the origins of hydrocarbons, measuring the fluxes of methane emission, and determining the extent of secondary biodegradation , 20, 76.Hydrocarbon seeps in the Kuan-Tzu-Ling (KTL) region in southwestern Taiwan have been characterized by the continuous discharge of high-temperature, muddy fluids from the surface outcrops. Analyses of expelled fluids in our pilot surveys (unpublished data) and previous studies , 89 revein situ values obtained from previous studies  were measured on site using portable probes . Site KTLS was selected to conduct incubations and molecular analyses because this was the only site at which fluids were discharged directly from the fracture outcrop. Samples from sites KTL01 and KTL02 were collected from pools into which fluids drained from either the fracture outcrop or an exploration well.in situ community structures. All samples were shipped back to the laboratory within four h of sample collection.Samples for geochemical analyses were collected in centrifuge tubes and stored on ice. For incubation experiments, muddy fluids were diverted into sterilized serum bottles capped with thick butyl rubber stoppers. The serum bottles were filled until there was no headspace left and stored on ice. Fluids were also collected and stored on dry ice for analyses of g for 15 min immediately after being returned to the lab. The supernatant was filtered through a 0.22-μm pore-sized cellulose membrane. The filtrate was split into two portions, one of which was preserved in 1% HNO3 for cation and strontium isotopic analyses, while the other was stored in a −20°C freezer for anion and DOC analyses. The spun mud was dried overnight at 50°C and ground for later analyses of total organic carbon (TOC). Samples for molecular analyses were kept at −80°C until subsequent processing.Samples for aqueous geochemistry were centrifuged at 8,000×et al. (−1. The uncertainty for aqueous chemistry was ±2%.Cations were measured using an Ultima2 inductively coupled plasma (ICP)-optic emission spectrometer . Anions were determined using an ion chromatograph (IC) . Ammonia was determined via a colorimetric method . Dissolvet al. . Stronti2·6H2O, 0.3 g KCl, 0.15 g CaCl2·2H2O, 0.27 g NH4Cl, 0.2 g KH2PO4, 1 mL and 10 mL each of vitamin and trace metal solutions per L) at a volume ratio of 1:2 in an anaerobic glove bag on the same d of sample collection. The trace metal solution consisted of 1.5 g of nitrilotriacetic acid, 3 g of MgSO4·7H2O, 0.5 g of MnSO4·2H2O, 1 g of NaCl, 100 mg of FeSO4·7H2O, 180 mg of CoSO4·7H2O, 10 mg of CaCl2·2H2O, 180 mg of ZnSO4·7H2O, 10 mg of CuSO4·5H2O, 20 mg of KAl(SO4)2·12H2O, 10 mg of H3BO3, 10 mg of Na2MoO4·2H2O, 25 mg of NiCl2·6H2O, and 0.3 mg of Na2SeO3·5H2O per L. The vitamin solution was a mixture of 2 mg of biotin, 2 mg of folic acid, 10 mg of pyridoxine-HCl, 5 mg of thiamine-HCl·2H2O, 5 mg of ribo-flavin, 5 mg of nicotinic acid, 5 mg of D-Ca-pantothenate, 0.1 mg of vitamin B12, 5 mg of p-aminobenzoic acid, and 5 mg of lipoic acid in 1 L of deionized water. Trace metal and vitamin solutions were both adjusted to pH 7.5, 0.22-μm filtered, and sealed in sterilized serum bottles under anoxic conditions. The reducing agent (Na2S·9H2O) at a final concentration of 0.05% was added to decrease the redox potential and remove any trace amount of O2. The final mixture of the mud and sterilized basal salt solution was supplied with chloride at a final concentration of ~60 mM and sulfate at a final concentration of 1.3–2.0 mM in order to mimic in situ concentrations, dispensed into each serum vial with a total volume of 30 mL, sealed with a thick butyl rubber stopper, and purged with 0.22-μm filtered N2 in the headspace. The mixed slurries were incubated at temperatures ranging from 40°C to 90°C with a 10°C interval for 30 d. Aliquots (1 mL) of slurries were periodically withdrawn, preserved with a one-tenth volume of concentrated Zn-acetate (1 M) to remove H2S, centrifuged at 8,000×g for 5 min, and 0.22-μm filtered. The filtrate was stored at −20°C until further measurements of sulfate concentrations were conducted using the IC. At least 10 mL of muddy slurries were obtained at the end of the incubation for 16S rRNA gene analyses, and stored at −80°C.Muddy fluids collected from site KTLS were mixed with a basal salt solution  were used, with one containing 10 mM lactate and 15 mM bicarbonate, and the other one containing 10 mM lactate and 0.1% (w/w) yeast extract. The sulfate-free medium for fermentation was supplied with yeast extract, peptone, and tryptone, each at a final concentration of 0.1% (w/w). Parallel negative controls subjected to heat sterilization were used.The muddy fluids of KTLS were mixed with media, targeting fermentation and sulfate reduction at a volume ratio of 1:10, and incubated at 60°C and 80°C under a 100% NCell density and aqueous geochemistry in enrichments were periodically checked using a phase-contrast microscope and the methods described above. Once positive growth was confirmed, the enrichments were transferred to the same freshly prepared medium with a dilution factor of 10 several times to reduce the contribution of organic carbon inherited from the inoculated muddy fluids.et al.  according to the manufacturer’s instructions. The nearly full lengths of 16S rRNA genes were PCR-amplified on a Robocycler  using the primer pair B27F/U1492R  for bactet al. . The obtet al. , Mallardet al. , and aliet al. . Relativet al. . Phylogeet al. .Quantification of the gene abundance of total bacterial and archaeal 16S rRNA genes was performed on an iCycler thermo cycler . Each reaction mixture (20 μL) contained 1× iQ™ SYBR Green Supermix (Bio-Rad), 2 μL of template DNA, 100 nM of each primer, and DNase-free water. The primer pairs of B519F/B907R  and ArchSequences of the 16S rRNA genes from unincubated fluids, incubated samples, and enrichments have been deposited in the GenBank database under accession numbers from FJ638500 to FJ638610.h between −400 and −140 mV, pH between 7.5 and 8.1, and conductivities between 9 and 14 mS cm−1 (87Sr/86Sr) ranged between 0.71056 and 0.71142.Field surveys revealed that the collected fluids possessed temperatures ranging between 53°C and 78°C, E mS cm−1 . While d mS cm−1 . Dissolv−1 at 40°C to 5.4 μM h−1 at 50°C, 3.3 μM h−1 at 60°C, 9.7 μM h−1 at 70°C, and 13.9 μM h−1 at 80°C. Dissolved Mn, Fe, and ammonium were not detected at the end of the incubations.Sulfate was nearly exhausted within 30 d at temperatures ranging between 40°C and 80°C . No sulf5 and 6.0×104 copies g−1 of bacterial and archaeal 16S rRNA genes, respectively, for the unincubated sample  and were dominated by Syntrophobacterales, an order composed of microorganisms that use sulfate as an electron acceptor or that grow syntrophically with sulfate-reducing bacteria or methanogens , within which sequences related to sulfate-reducing Desulfovibrio alkalitolerans , a species known to be capable of reducing sulfate and sulfite  and two other Firmicutes ribotypes (41%)  possessed sequences resembling Thermacetogenium phaeum, which is known to either produce acetate from H2 and CO2 or oxidize acetate when coupled with the reduction of sulfate under thermophilic conditions  . One of hanogens . Other cFirmicutes  and Dictyoglomus thermophilum (22%)  , which aof human  and vari2% , whicof human , 74.Euryarchaeota were detected in all archaeal clone libraries, Thaumarchaeota were only present at 60°C. Four ribotypes were obtained in the unincubated sample  clone sequences retrieved from the Obsidian Pool of Yellowstone National Park in the USA  was related to Thermotoga hypogea at a 97% similarity. These two sequences were identical to those present in the incubation experiments. A sequence identical to KCL80a_coculture was detected in the sulfate-reducing enrichment supplied with lactate and bicarbonate at 80°C. Identical bacterial sequences (KCL_60b_Thermodesulfobacterium sp.) related to Thermodesulfobacterium commune (99% identity) were detected in both sulfate-reducing enrichments at 60°C.At 80°C, cocci and short rods were present at approximately equivalent abundances in the sulfate-reducing enrichment supplied with lactate and yeast, while cocci dominated over other morphotypes in the sulfate-reducing enrichment supplied with lactate and bicarbonate. After several transfers to ensure sulfate reduction, bacteria and archaea were both detected by PCR from enrichments supplied with lactate and yeast. Two clean sequences were obtained by directly sequencing these bacterial and archaeal amplicons without cloning. The archaeal sequence (KCL80a_coculture) was affiliated with Thermoanaerobacter brockii subsp. finnii (99% identity) and Thermotoga hypogea (97% identity) isolated from oil fields  related to l fields , respectWhen incubated, the overall bacterial and archaeal diversities, as reflected by the estimates of the Shannon-Wiener and Chao1 indices , decreasDesulfovibrio, Desulfonatronum, Desulforhabdus, and Desulfotomaculum at 40°C, Thermodesulfovibrio at 50°C and 60°C, and Thermacetogenium at 60°C and 70°C  and TOC (0.64% [w/w])) inherited from the sediments. Assuming that the DOC pool was more readily degraded than the TOC pool, a three-fold dilution of inoculated sediments should have resulted in 1.17 mM of DOC available for microbial utilization. This quantity of an electron donor could be used to reduce 0.59 mM of sulfate considering CHThermoanaerobacter sp. at 60°C and Thermotoga sp. at 80°C demonstrated the active fermentation of organic carbon from the media. Sequences identical to these two members also constituted high proportions of the incubated bacterial communities  for downstream sulfate reduction . Alterna 80°C; 2 , 57, alle.g. from aerobic to anaerobic catabolisms or from mesophilic to hyperthermophilic temperature ranges) suggest that the community assemblage in the unincubated sample represent a mixture of populations from various sources with contrasting geochemical and physical characteristics. To further constrain this interpretation, chloride and the 87Sr/86Sr ratios in the environmental samples were used as conservative tracers. Analyses revealed that the 87Sr/86Sr ratios of the fluids spanned from 0.71056 to 0.71142   , and theic water , 89. OveDesulfovibrio, Desulfonatronum, Desulforhabdus, Desulfotomaculum, Thermacetogenium, Thermodesulfovibrio, and Archaeoglobus, were dominant in slurries incubated at temperatures similar to their individual optima. The fast microbial turnovers of the substrate and community assemblage in response to temperature changes reflect the dynamic adaption of microbial communities to environmental fluctuations in high-temperature, terrestrial hydrocarbon seep and mud volcano ecosystems.This study demonstrated that the detection of key biogeochemical processes catalyzed by rare members could be achieved through combining incubation experiments with molecular analyses. Temperature appears to play an essential role in changing the structures of sulfate-reducing populations. Various potential sulfate reducers, including members affiliated with genera


This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as  As a nurocesses –12. Whilrocesses . Currentrocesses ,15. As Rrocesses , techniqin vitro transcription and radioactive 5′-end-labeling, the technique may be more suitable for studying nucleotide locations in RNA G4s, and not for in situ RNA G4 detection. Recent research has shown that nucleic acids that do not obey the classical description of sequences G2+NL1G2+NL2G2+NL3G2+ could also form G4 structures  are the most accurate techniques and have identified high-resolution structures of RNA G4s ,18. Thesructures –24. Howein living cells  and 610 nm (for CyT).PAGE was performed in 1× TBE buffer solution (0.09 M Tris-boric acid and 0.002 M EDTA) with 12% native gels. Oligonucleotides (5 μM) were loaded on the gel, and electrophoresis was run at 64 V for 2 h at room temperature. After electrophoresis, the gel was stained using either 20 μM CyT in Tris-KA549 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) contain 5% fetal bovine serum (FBS), streptomycin (0.1 mg/mL) and penicillin (100 U/mL), under 5% carbon dioxide at 37 °C. Cells grown on Petri dish were fixed for 5 min in cold methanol and 4% formaldehyde, rinsed twice with phosphate-buffered saline (PBS), and then permeabilized with 0.1% Triton X-100/PBS for 30 min at 37 °C. For enzymatic treatments, cells were incubated denaturing buffer and RNase T1 for 5 h at 37 °C after permeabilization. After treatment, cells were incubated with the CyT probe (5 μM) for 5 min. Cellular nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), before visualization under a confocal laser scanning microscope (CLSM) (OLYMPUS FV1000-IX81) equipped with an oil immersion 100X objective. CLSM images of DAPI and CyT were collected under excitation at 408 nm and 559 nm, respectively. For living cells, the CyT probe was incubated with the cells for 24 h and visualized, without washing, under the CLSM. Hoechst 33258 (8 μg/mL) was used to stained the nuclei.To demonstrate the feasibility of using a CyT probe for RNA G4 recognition, we compared the fluorescence intensity of CyT on its own or in the presence of various RNA sequences. The results of emission spectra are presented in Figure 5, (1.44 ± 0.29) × 105, (2.81 ± 0.63) × 105, (1.94 ± 0.29) × 105 and (3.49 ± 0.77) × 105 M−1, respectively. In comparison with RNA G4s, the binding strength to tRNA, ssAf17, ssAf22, HP18 and dsNC was much weaker, with binding constants (6.35 ± 2.26) × 104, (4.40 ± 0.38) × 104, (4.03 ± 0.36) × 104, (2.89 ± 0.28) × 104 and (2.42 ± 0.25) × 104 M−1, respectively. Of note, although the binding affinities of CyT to non-RNA G4s were obtained in the range of 104  was confirmed by the Job's plot analysis . The typical titration curves and fitting results according to the 1:1 binding model are presented in Supplementary Figure S2. The binding constants for CyT with Tel22, VEGF, TRF2, BCL-2 and NRAS are (1.17 ± 0.37) × 10ntensity .−1cm−1, while the fluorescence quantum yields of CyT bound to two equivalents of RNA G4s, such as Tel22, VEGF, TRF2, BCL-2 and NRAS, were 0.95, 0.92, 0.79, 0.74 and 0.58, respectively. The relatively high molar absorptivity and fluorescence intensity of the bound-state CyT may lead to CyT acting as a highly sensitive fluorescence probe for RNA G4 detection. Overall, the above results confirm that the CyT probe could be used for the recognition of RNA G4 structures with high specificity and sensitivity through fluorescence light-up.As molar absorptivity and fluorescence intensity are key factors influencing the sensitivity of a fluorescence probe, we evaluated the molar extinction coefficients and quantum yields of CyT bound to RNA G4s. The CyT monomer showed a relatively high molar extinction coefficient of 80 14;000 M+ and Li+ solutions. Figure + and Li+ conditions. As shown in Figure + and Li+, indicating the formation of parallel-type G4 structures. The CD intensity decreased under Li+ condition, compared to K+, which could be explained by the weaker G4 formation capability of Li+, compared to K+  and displayed a predominant absorption band at 650 nm, which was previously assigned to J-aggregates  was studied. A UV-vis titration was conducted by increasing the RNA G4 concentration (from 0.125 to 4 μM), with the CyT concentration fixed at 2 μM. As shown in Figure gregates . Of notegregates . These rgregates . The flu+) (J-aggregates) conditions, the fluorescence intensity of CyT was very weak. In contrast, significant fluorescence enhancement was induced by the bound-state CyT monomer, which can be considered as a unique signature in the recognition of RNA G4 structures.It is worth noting that the enhancement of monomeric CyT fluorescence intensity resulted from the bound-state CyT monomer, and not from the unbound-state monomer itself. As shown in Supplementary Figure S3, under methanol (unbound-state) and Tris-HCl  and subsequently stained using either 20 μM CyT Figure  or SYBR 2+NL1G2+NL2G2+NL3G2+ could also form G4 structures , double-stranded RNA (dsRNA), short hairpin RNA (shRNA) and RNA junction structures. Fluorescence assays showed that CyT strongly lit up RNA G4 structures and barely lit up the tested single-stranded, double-stranded and hairpin RNA structures, tRNAs, and three- and four-way junction structures. As a result we conclude that CyT, with its high specificity for RNA G4, is an excellent probe to stain and light up RNA G4 structures, and not other RNA structures, in the cells.Furthermore, given the structural similarities between DNA and RNA G4s, we determined whether DNA G4 detectors (such as TO) can also be applied as RNA G4 detectors. Although TO is widely used as a selective DNA G4 detector, our results showed that TO does not show any selectivity for RNA G4s, compared with other RNA forms. Therefore, this indicates that a DNA G4 probe may not be suitable for detecting or recognizing RNA G4s without validation.Additionally, our results showed that CyT emitted stronger fluorescence in the presence of RNA G4s, compared with DNA G4s with identical sequences . Although the CyT probe showed very high selectivity for RNA G4s, compared to other RNA forms, and thus can be used to distinguish RNA G4s from other RNA forms, CyT, at the present stage, may not be suitable for distinguishing RNA G4s and DNA G4s with different sequences.in vitro and in living human cells. The ability of CyT to distinguish between G4 and non-G4 RNA offers a promising tool for future RNA G4-based biomarker discovery, with potential diagnostic applications.The molecular probe CyT described in this study showed strong fluorescence enhancement in the presence of RNA G4 structures but did not show such a response toward other RNA forms. Therefore, our results demonstrate that CyT is an effective detector of RNA G4 structures both


SCN11A-encoded voltage-gated Na+ channel NaV1.9 cause severe pain disorders ranging from neuropathic pain to congenital pain insensitivity. However, the entire spectrum of the NaV1.9 diseases has yet to be defined. Applying whole-exome sequencing we here identify a missense change (p.V1184A) in NaV1.9, which leads to cold-aggravated peripheral pain in humans. Electrophysiological analysis reveals that p.V1184A shifts the voltage dependence of channel opening to hyperpolarized potentials thereby conferring gain-of-function characteristics to NaV1.9. Mutated channels diminish the resting membrane potential of mouse primary sensory neurons and cause cold-resistant hyperexcitability of nociceptors, suggesting a mechanistic basis for the temperature dependence of the pain phenotype. On the basis of direct comparison of the mutations linked to either cold-aggravated pain or pain insensitivity, we propose a model in which the physiological consequence of a mutation, that is, augmented versus absent pain, is critically dependent on the type of NaV1.9 hyperactivity.Gain-of-function mutations in the human  A mutation in the sodium channel Nav1.9 has been identified in a family and shown to associate with cold-aggravated pain. Here, the authors characterize the electrophysiological consequences of this mutation and propose a mechanism for the pain that the individuals experience. The family of mammalian NaV channels consists of nine members (NaV1.1–1.9) of which NaV1.7, NaV1.8 and NaV1.9 are the most abundant isoforms in nociceptive neuronsV1.7- and NaV1.8-mediated currents are the main contributors to the fast action potential upstroke in nociceptors, NaV1.9 channels modulate the threshold of nociceptor excitability by influencing the resting membrane potential (RMP)23Voltage-gated Nambers NaV.1–1.9 ofV channel functionSCN9A, encoding NaV1.7, result in inability to experience pain by impairing the electrical signalling of nociceptors6SCN9A result in hyperexcitability of nociceptors and cause debilitating pain disorders such as primary erythromelalgia and paroxysmal extreme pain disorder810V1.7 variants with increased activity have been associated with paroxysmal itchV1.8, encoded by SCN10A  that causes early onset cold-aggravated familial episodic pain. Electrophysiological evaluation reveals that the mutation enhances the activity of NaV1.9 by left shifting the voltage dependence of channel opening, thereby giving rise to hyperexcitability of nociceptors. In line with the temperature sensitivity of the patients' phenotype, p.V1184A-dependent nociceptor excitability is less attenuated by cold than the excitability of wild-type neurons, suggesting a temperature-dependent contribution of the novel NaV1.9 variant to nociceptor function.We here describe a NaWe studied a three-generation family of mixed European ancestry with severe episodic pain of unclear aetiology . Onset oSCN9A, SCN10A and TRPA1). Of the rare heterozygous genetic variants shared by both individuals, a missense change in SCN11A, c.3551T>C, p.V1184A , was the most likely candidate to cause the disorder. The variant was confirmed by Sanger sequencing  at both temperatures tested . However, at both temperatures the larger current densities of cells expressing mutant p.V1184A were accompanied by three major effects: mutated channels displayed (i) a strongly left-shifted voltage dependence of channel activation, effectively increasing Na+ influx at resting membrane voltages (Vm) from −52.0±2.6 to −69.5±2.9 mV at 30 °C  and from −47.5±2.5 to −67.0±2.7 mV at 20 °C . The associated slope factors (km), which reflect the voltage dependence of channel activation, were not affected by the mutation . The voltage dependence of opening and closing kinetics were compatible with a gating charge transfer of 2.8±0.5 e0 and 2.6±0.3 e0 and associated symmetry factors specifying the charge fraction assigned to channel opening of 0.53±0.08 and 0.47±0.02 at 30 and 20 °C, respectively. The Q10 temperature coefficients of the channel's opening kinetics, τa , and the closing kinetics, τd , were not markedly affected by the mutation , suggesting that temperature changes affect opening and closing of wild-type and mutant channels in a similar manner.Both, wild-type and mutant channels gave rise to currents with slow activation and inactivation kinetics typical for NaV1.9 . Compares tested . Mean cuvoltages , (ii) acvoltages  and . The half-maximal voltage of channel inactivation, Vh, measured after 500 ms conditioning episodes at various voltages, was only marginally shifted in the mutant from −69.1±3.9 to −64.8±3.3 mV at 30 °C and from −71.5±3.5 to −65.6±1.2 mV at 20 °C, without reaching the level of significance . The associated slope factors (kh) of wild-type (−10.0±1.1 mV) and mutant channels (−11.2±0.8 mV) characterizing the voltage dependence of channel inactivation were not significantly different at 20 °C; however, at 30 °C the mutation decreased kh from −9.3±1.2 to −12.8±1.0 mV , thus effectively increasing the availability of mutated channels  and 5.2 mV  at 30 and 20 °C, respectively, when compared with cells overexpressing NaV1.9 wild type (Vmin) at 30 °C by 4.4 mV  while they did not influence the after-hyperpolarization voltage at 20 °C. Other parameters characterizing the shapes of single evoked action potentials, that is, the voltage threshold for action potential firing (Vth), the action potential peak voltage (Vmax), the action potential duration (Width), as well as the time constant describing the relaxation of the action potential after-hyperpolarization back to the resting level (τAHP) were—at both temperatures—not significantly different between cells expressing NaV1.9 or NaV1.9-V1184A . However, lowering the temperature from 30 to 20 °C increased the duration of action potentials by factors of 1.92±0.32  and 1.52±0.36  when cells were transfected with NaV1.9 and NaV1.9-V1184A, respectively. Temperature-dependent changes were furthermore observed for the relaxation time constant τAHP, although statistically significant only for neurons expressing NaV1.9-V1184A .When overexpressed in DRG neurons, p.V1184A mutant channels depolarized the cell membrane at rest (RMP) by 5.8 mV  for mutant p.V1184A  at 30 °C and by 5.3±1.0 mV  at 20 °C compared with neurons expressing wild-type NaV1.9. Parameters characterizing the shape of single evoked action potentials were only marginally affected by p.L811P . Thus, p.L811P channels increase the probability of cumulative loss of action potential integrity in DRG neurons during extended firing periods.The phenotypes associated with mutation p.V1184A in Na p.L811P . Analysi p.L811P . As illuV1.9 as a cause for early onset cold-aggravated episodic peripheral pain. The mutation segregated with the disease phenotype in the affected family and showed slightly variable expressivity. Anti-inflammatory medication reduced the frequency of painful events, which was highest in childhood and gradually decreased with age. Similar age-dependent reduction of painful events as well as beneficial effects of anti-inflammatory drugs were reported for patients from two pedigrees suffering from NaV1.9-associated episodic peripheral painV1.9 under inflammatory conditions223V1.9 in mediating inflammatory pain.Our study identified a heterozygous missense mutation (p.V1184A) in NaV1.9 channels primarily by a hyperpolarizing shift in the voltage dependence of channel activation and a smaller voltage dependence of steady-state fast inactivation. The resulting increase in overlap of activation and inactivation of mutated channels predicts a larger steady-state window current23V and NaV channel structures262728V1.9 . Primary data were filtered according to signal purity with the illumina real-time analysis software v1.8. Subsequently, the reads were mapped to the human genome reference build hg19 using the Burrows-Wheeler alignment algorithm. GATK v.1.6 was used to mark duplicated reads, to perform a local realignment around short insertion and deletions, to recalibrate the base quality scores, and to call single nucleotide polymorphisms and short indels. Subsequent Sanger sequencing of SCN11A exons was performed using standard procedures. Primer sequences are available on request.For whole-exome sequencing, 1 μg of DNA from two family members with episodic pain was fragmented using sonication technology . The fragments were end-repaired and adaptor-ligated including incorporation of sample index barcodes. After size selection, the libraries were subjected to an enrichment process . Samples were sequenced on an Illumina HiSeq 2000 sequencing instrument. This resulted in 6.6  and 6.4  Gb of mapped sequences with a mean coverage of 77/78 and a 30 × coverage of 78/84% and a 10 × coverage of 95.8/96.4% of target sequences. For data analysis, the Varbank pipeline v.2.3 and interface was used  were taken from the volar forearm. The biopsies were fixed with Zamboni solution for immunohistochemistry and with 3.9% buffered glutaraldehyde for electron microscopy. For immunohistochemistry 40 μm cryostat sections were prepared and stained with anti-PGP9.5 primary antibody at 1:100 dilution , and Alexa Fluor 488-labelled goat anti-rabbit secondary antibody at 1:2,000 dilution V1.9 cDNA (NM_014139.2) in pcDNA3.1 was used as templateBlpI on both sites. Correct orientation of the BlpI-fragment was confirmed, and the coding sequence of SCN11A was verified by Sanger sequencing.Human NaV1.9 channels using the same batch of cells. After centrifugation (100 g for 3 min) both cell pellets were resuspended individually in 20 μl of P3 primary cell solution containing supplement 1 and 0.3 μg of a plasmid encoding the enhanced green fluorescent protein. Subsequently, one lot was supplemented with 1.2 μg of a NaV1.9-encoding plasmid, while the second lot was supplemented with 1.2 μg of a vector encoding mutant NaV1.9 variants. Transfection was performed using the electroporation protocol CA137 of the 4D-Neucleofector. After electroporation, 150 μl of low calcium Roswell Park Memorial Institute (Invitrogen) 1640 medium was added to each cell suspension and cells were allowed to recover for 10 min in a 10% CO2 incubator at 37 °C. The cell suspensions were then diluted in 300 μl DRG medium and immediately seeded on poly-D-lysin/laminin-coated glass coverslips, which were placed in the slots of a 24-well plate containing 1 ml of DRG medium per well. DRG medium contained 89.5% DMEM/F12  supplemented with 9.5% fetal calf serum and 1% penicillin/streptomycin (Invitrogen). Transfected cells were incubated in a 10% CO2 incubator at 37 °C and used for electrophysiological experiments 15–24 h after transfection. Current-clamp recordings were restricted to successfully transfected small-diameter (<25 μm) DRG neurons, which were identified visually by their green florescence using a 50 W HBO lamp as light source and a green fluorescent protein filter set.Animal care and experimental procedures followed the guidelines established by the animal welfare committee of the University of Jena. For action potential recordings, DRG from all levels of the spinal cord of 6–11-weeks-old wild-type C57BL6 mice were extracted and processed as described2 incubator at 37 °C. Cells were trypsinized, diluted with culture medium, and grown in 35 mm dishes. When grown to 30–50% confluence, cells were transfected with a 5:1 ratio of a DNA plasmid encoding either NaV1.9 or NaV1.9 mutants and a vector encoding the CD8 antigen using the Rotifect transfection kit  as described earlier2 incubator at 28 °C for up to 12 h. Voltage-clamp recordings were performed after an additional 1 h recovery period at 37 °C. Anti-CD8-coated Dynabeads  were used for visual identification of individual transfected cells.ND7/23 cells—a hybrid of mouse neuroblastoma and rat DRG neurons—, were maintained in 90% DMEM supplemented with 10% fetal calf serum in a 10% CO2, 1 MgCl2, 30 HEPES, 15 glucose (pH 7.4 with NaOH), and the pipette 125 KCl, 8 NaCl, 1 CaCl2, 1 MgCl2, 0.4 Na2-GTP, 4 Mg-ATP, 10 EGTA, 10 HEPES (pH 7.3 with KOH). Bath solution for voltage-clamp recordings contained (in mM) 150 NaCl, 2 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH) and was supplemented with 0.0003 tetrodotoxin to block endogenous Na+ currents in ND7/23 cells; the pipette contained (in mM) 35 NaCl, 105 CsF, 10 EGTA, 10 HEPES (pH 7.3 with CsOH). Temperature was controlled by continuous perfusion of cells with tempered bath solution using a SHM-8 in-line solution heater feedback-controlled by a TC-324B temperature controller .Current and voltage recordings were obtained in the whole-cell configuration of the patch-clamp method using an EPC-10 patch-clamp amplifier operated by PatchMaster software . Patch pipettes were fabricated from Kimax borosilicate glass of about 1.0–2.5 MΩ resistance and coated with room temperature vulcanization silicone adhesive  to reduce tip capacitance. Series resistance was corrected electronically up to 85% and all voltages were corrected for the liquid junction potential. Bath solution for current-clamp recordings contained (in mM) 120 NaCl, 3 KCl, 2.5 CaClCurrent-clamp recordings. The resting membrane voltage was measured by zero current injection directly after establishing the whole-cell configuration. Single action potentials were evoked at 30 or 20 °C by injecting a current of 100–200 pA for a period of 5 or 10 ms, respectively, followed by a 200-ms pulse without current injection. Sampling interval for voltage measurements was 50 μs. The voltage threshold of action potential firing, Vth, was defined as voltage at which dV/dt reached the level of 0.03 × (dV/dtmax−dV/dtmin)+dV/dtmin. Trains of action potentials were evoked repetitively by 2 s current injections increasing in steps of 20 pA.Voltage-clamp recordings. In voltage-clamp experiments with ND7/23 cells the holding potential was set to –137 mV. Data were low-pass filtered at 5 kHz and digitized with a sampling interval of 40 μs. Leak and capacitive currents were measured at –117 and –127 mV and subtracted manually.Channel activation. To measure channel activation, test depolarizations between –127 and 23 mV were applied in steps of 10 mV every 3 s. The voltage dependence of channel activation was estimated from mean current densities using the following formalism:Cm, conductance density (Γ), and the reversal potential (Erev). Vm is the half-maximal activation voltage and km the corresponding slope factor.with the cell capacitance Gating kinetics. The kinetics of activation and fast inactivation of NaV1.9 and NaV1.9-V1184A channels were analysed between –67 and –7 mV with a model assuming one activation gate and one inactivation gate:I0. m(t) and h(t) describe the kinetics of channel activation and inactivation, respectively; τm and τh are the associated time constants, and h∞ characterizes the fraction of non-inactivating current after infinite time.with the current amplitude Steady-state inactivation. NaV1.9 channels were activated with a first 50 ms test pulse to –47 mV followed by a conditioning interval of 500 ms at voltages ranging from –127 to –17 mV in steps of 10 mV. Peak currents of not inactivated channels were measured in a subsequent 50 ms test pulse. The repetition interval was 20 s. The current amplitude after conditioning (I500 ms) normalized to the control current amplitude before conditioning (I0) was described with the following Boltzmann formalism:hmax and hmin, the half-maximal inactivation voltage Vh and the corresponding slope factor kh.with the maximal and minimal channel availability Channel deactivation. Channels were first activated with a 25-ms depolarization to −47 mV. Subsequently, deactivation was triggered with a 150-ms repolarization period at voltages ranging from –117 to –77 mV in steps of 5 mV with a repetition interval of 10 s. The current decay during the repolarization period was fitted according to:I0 is the maximal current amplitude and I∞ the current remaining after infinite time. τd is the deactivation time constant. The voltage dependence of activation and deactivation time constants was described with a formalism assuming a one-step process for channel opening and closing:where τ0 is the voltage-independent, limiting speed of deactivation, and α0 is the rate at the equilibrium voltage Vm, and kT is the thermal energy. q is the total gating charge transfer and δ is the symmetry factor specifying the gating charge fraction associated to channel activation.Temperature dependence. Q10 temperature coefficients, specifying the change of reaction rates in response to a temperature change of 10 °C were calculated according to:k1 and k2 are the reaction rates obtained at temperatures T1 and T2, respectively.where n) with n being the number of independent experiments. Statistical comparisons of two groups of data were made using the two-tailed Student's t-test or the Z-Test; P values are given explicitly when appropriate.Data were analysed with FitMaster (HEKA Elektronik) and IgorPro  software. Data are presented as mean±s.e.m., .Supplementary Figure 1


Trefoil factor 1 (TFF1) mediates mucosal repair and belongs to a highly conserved trefoil factor family proteins which are secreted by epithelial cells in the stomach or colon mucous membrane. TFF1 forms a homodimer via a disulphide linkage that affects wound healing activity. Previous recombinant expressions of TFF1 were too low yield for industrial application. This study aims to improve the expression level of bioactive recombinant TFF1 (rTFF1) and facilitate application potency.rtff1 was synthesized, expressed by Escherichia coli and secreted by Brevibacillus choshinensis. The rTFF1s were purified. The polymeric patterns and wound healing capacities of purified rTFF1s were checked.The rTFF1 gene Escherichia coli, 21.08 mg/L rTFF1 was stably expressed as monomer, dimer and oligomer in soluble fraction. In Brevebacillus choshinensis, the rTFF1 was secreted extracellularly at high level (35.73 mg/L) and formed monomer, dimer and oligomer forms. Both proteins from different sources were purified by Ni-NTA chromatography and exhibited the wound healing activities. The rTFF1 produced by B. choshinensis had better wound healing capability than the rTFF1 produced by E. coli. After pH 2.4 buffer treatments, the purified rTFF1 formed more oligomeric forms as well as better wound healing capability. Glycosylation assay and LC-MS/MS spectrometry experiments showed that the rTFF1 produced by B. choshinensis was unexpectedly glycosylated at N-terminal Ser residue. The glycosylation may contribute to the better wound healing capacity.In B. choshinensis were more efficient than rTFF1 produced by E. coli.This study provides a potent tool of rTFF1 production to be applied in gastric damage protection and wound healing. The protein sources from The online version of this article (doi:10.1186/s12896-015-0149-5) contains supplementary material, which is available to authorized users. Gastric mucosal defense systems assist the gastric mucosa to withstand frequent exposure to damaging factors. Mucosal defense in the gastrointestinal tract includes local gastric mucosal defense mechanisms and neurohormonal regulation. The surface epithelial cells secrete mucus, bicarbonate and generate prostaglandins, heat shock proteins, trefoil factor peptides (TFFs), cathelicidins, and defenses to defend against or regulate mucosal damage . The TFFin vitro or by in vivo animal models [Among TFF family, trefoil factor 1 (TFF1) had been identified in human, mouse, rat and canine  and was l models ,11. The l models .Escherichia coli [Bacillus subtilis system [Pichia pastoris [B. subtilis system. In Pichia pastoris secretion system, the yield was about 20 mg/L but the TFF1 dimer form wasn’t observed. In E. coli HB10, dimer forms of TFF1 was expressed in cytosol, but the yield was low (16 mg/L) and the purification procedure was more complicated. The B. choshinensis was suggested to be a good candidate host to express disulfide bond containing proteins [B. choshinensis expression system to be considered as a good choice for extracellular expression of disulfide bond containing proteins, such as TFF1. The secretion capacity of B. choshinensis was expected to simplify the purification steps to obtain rTFF1 more easily. In this study, attempts were tried to elevate expression of recombinant human TFF1 by E. coli cytoplasmic and B. choshinensi secretion expression systems. The expressions and biological activities were compared.Oral trefoil factors in gastric injury rats and treatment with trefoil factors in gastric cancer cell lines showed the protection against gastric damage and promoted wound healing, respectively . Therefohia coli , Bacillus system , and Picpastoris . There wproteins -19. CharE. coli BL21(DE3) (pET-TFF1) expressed rTFF1 in cytosol by IPTG, inducing the T7 promoter. The optimal expression was 21.08 mg/L at 12 h cultivation  expressed rTFF1 extracellularly by using the constitutive P2 promoter and SPSec signal peptide. TFF1 continued to secrete and stably expressed until 5 days  and analyzed. Significant conformation differences were observed between rTFF1/EC and rTFF1/BC. The rTFF1 from all sources had the same monomer protein forms prior to and after pH treatment on a native gel with reducing agent (DTT). Alternatively, the rTFF1/EC exhibited more tetrameric forms on native gel without DTT. The rTFF1/BC exhibited monomeric, dimeric, and trimeric forms on native gel without DTT. Pretreatment with pH 2.4 buffer promoted more trimeric forms Figure b.To further evaluate the polymerization pattern of rTFF1s, the molecular weights of rTFF1s were determined by MALDI-TOF mass spectrometry to compare with the observed polymerization patterns on native gel. The purified rTFF1s from both sources were treated with different buffers  and examined. The different peak valleys were presented in buffers  with or without DTT treatment  enhanced the capability of wound healing  observation. The minimum concentration of 400 ng/mL rTFF1 was recommended for the g Figure .Figure 5To mimic gastric conditions, purified rTFF1s were treated with buffer (pH 2.4), and then preceded the wound healing assay on AGS cells. The results showed that rTFF1 treated with pH 2.4 buffer enhanced cell mobility, especially for rTFF1/BC Figure a. Measur2+ to replace the Ca2+ elevated the rTFF1 productivity in trypton-based media , but not in soytone-based medium (Broth C/A)  Figure a. The re) Figure a. Variou) Figure b.Figure B. choshinensis expressing vector pNCMO2-TFF1, the rTFF1 was constructed in which Sec signal peptide was fused in front of the rTFF1 and His•Tag sequence was in the C-terminal of rTFF1. These constructions were predicted to express functional rTFF1 of molecular mass 7.6 kDa and 7.7 kDa in cytoplasm and extracellularly, respectively. These two vectors successfully expressed rTFF1 in the cytoplasm of E. coli and secreted rTFF1 extracellularly by B. choshinensis, respectively  of rTFF1 was achieved 10 hours after induction, but the rTFF1 was unstable and degraded into lower molecular weight fragments after 34 hours of induction. In the B. choshinensis system, continuous expression of the pNCMO2-TFF1 vector was driven by the P2 promoter, and secreted by SPSec signal peptide. After cleavage of the signal peptide during the translocation process, the secreted rTFF1 was observed at 12 hours in the culture medium fraction. The highest yield (36 mg/L) of rTFF1 was achieved after 108 hours in the culture extracellular fraction, higher than the E. coli system of this study and previously reported levels ranging from 3.5 mg/L to 20 mg/L by E. coli [B. choshinensis system was examined. The extracellular expression was expected to be more efficient for industrial scale-up production [In this study, human TFF1 sequence was synthesized and cloned into expressing vector pET-TFF1 in which the C-terminal of rTFF1 contained His•Tag. In the  E. coli  and othe E. coli ,16,20. Ioduction -19.E. coli was estimated by NMR spectra previously. A short α-helix packed against a two-stranded antiparallel β-sheet and three closely-packed loop structure was determined [E. coli and B. choshinensis systems were about 45%, 32%, 15%, and 9% on SDS-PAGE gel, respectively. Using glutaraldehyde as a crosslinker can detect the existence of the correct folding and inter-disulfide bonds cross-linking between free thiol of rTFF1 or the interactions of mere proximity of rTFF1.The 3D structure of the recombinant human TFF1 from termined . In anottermined . In our 2+ ions enhance interaction [. The mobility shift between TFF1/EC and TFF1/BC on native gel was slightly different. Reducing treatment with DTT exhibited monomer forms for all the rTFF1s. However, different mobility shift, differences between polymeric forms of rTFF1/EC and rTFF1/BC on native PAGE gel and the unexpected mass increase of rTFF1/BC all indicated that other factors may contribute to the observed polymerization difference. It is possible that post-translational modification can occur on rTFF1/BC. Results of LC-MS/MS spectrometry and glycoprotein stained ; supplements of the extra yeast extract (Broth C) covered the effect of metal ions. In the trypton-based medium, the effect of ions weren’t significant. The Broth G supplement with extra yeast extract showed better productivity at 48 hrs of culturing. Results suggested that a nitrogen source was more effective than metal ions in rTFF1 productivity. Among nitrogen sources, soytone based media  were better than tryptone-based media  in improving rTFF1 productivity; the extra yeast extract (15 g/L) improved rTFF1 productivity (Broth C at 84 hrs and Broth G at 24 hrs of culturing)  achieved the highest secretion productivity of rTFF1 (170 mg/L) at 48 hr of culturing; enhanced productivity and less culture time to achieve optimal production were both achieved.To improve rTFF1 secretion productivity, various media were tested to optimize yields. All the growth curves of ) Figure a. It is B. choshinensis, superior to that of E. coli. The rTFF1 purified from B. choshinensis was unexpectedly glycosylated at the N-terminal serine residue and enhanced wound healing capacity. The rTFF1 purified from B. choshinensis also promoted better wound healing capacity after the pH 2.4 buffer treatment; this character suggests effective application potency in stomach acid conditions. The extracellular production by the B. choshinensis system was suggested to facilitate industrial production as well as ease of downstream processing. Production of active eukaryotic proteins through bacterial expression systems still remains “an art” [High levels extracellular production of recombinant human trefoil factor 1 (rTFF1) were achieved by “an art” . This stE. coli BL21 (DE3)  and B. choshinensis SP3  were used for protein expression. E. coli JM109  was used for DNA manipulation . The novel rtff1 gene was designed according to the preferred codons of Bacillus subtilis and can be found in a patent . The protein expression vectors pET-TFF1 and pNCMO2-TFF1 were derived from pET-29a(+) and pNCMO2, respectively. To generate pET-TFF1 and pNCMO2-TFF1, primer pairs were used  to obtain the fragment of rtff1. The PCR amplified product of TFF1 and pET-29a(+) were digested with Nde I and Hind III  respectively, and ligated  to obtain pET-TFF1, and then electro-transformed into E. coli JM109 to manipulate the plasmid. The desired plasmid was checked and electro-transformed into E. coli BL21(DE3) and checked again to obtain the desired transformant. A similar protocol was used to construct pNCMO2-TFF1. PCR reaction was carried out over 35 cycles  followed by a 10 min extension at 72°C in 50 μl reaction buffer. The PCR amplified rtff1 and pNCMO2 were digested with BamHI and Hind III , and then ligated and electro-transformed into E. coli JM109 to manipulate pNCMO2-TFF1. The desired plasmid was transformed into B. choshinensis SP3 with the Tris/PEG method as described . All constructs were confirmed by restrict enzyme digestion and DNA sequencing .The gene E. coli BL21(DE3) (pET-TFF1) were cultivated at 37°C in 2 × TY medium  supplement with 30 μg/mL kanamycin. At OD600 about 1.0, a final concentration of 1 mM IPTG was added into the medium and then cultured for 4 hours. Transformant B. choshinensis (pNCMO2-TFF1) was cultivated at 30°C in 2SY medium  supplemented with 50 μg/mL neomycin with 120 rpm shaking for 5 days. E. coli BL21(DE3) (pET-TFF1) cells were harvested by centrifugation and suspended in lysis buffer . The cell suspension was disrupted by ultrasonic treatment, and the extract was clarified by centrifugation at 13,000 × g for 20 min. The supernatant was mixed with Ni-NTA affinity resin  for further purification. Transformant B. choshinensis (pNCMO2-TFF1) cells were centrifuged and removed; the supernatant was treated with 60% ammonium sulfate to precipitate the secreted proteins. The precipitated proteins were separated by centrifugation at 10,000 × g for 20 min, resuspended in 10 mL Tris  and dialyzed against the same buffer . The protein suspensions were then mixed with Ni-NTA affinity resin  for further purification. Both mixtures of Ni-NTA affinity resin and proteins were gently agitated in the presence of 10 mM imidazole for 2 h at 4°C. The mixed resin was then washed repeatedly with wash buffer , and then eluted with 250 mM imidazole-containing wash buffer. The concentration of eluted protein was determined by the Bradford method using bovine serum albumin (BSA) as standard [To express protein, transformant standard .Various culture media containing various carbon sources  and various nitrogen sources (soytone or tryptone) were used to optimize the protein secretion production. Culture media were incubated in baffled shake flasks at 30°C for 120 rpm. The tested media were listed in Table E. coli BL21(DE3) (pET-TFF1) and B. choshinensis (pNCMO2-TFF1) at various time expression intervals were analyzed by Tricine-SDS-PAGE [TM version 3.0 . The electrophoresis gel was then transferred into PVDF membranes . The mouse anti- His•tag  and goat anti-mouse HRP  were used to immunize the rTFF1. The presentations of rTFF1s were then stained with Western LightningTM Plus-ECL , analyzed by Fusion-Capt advance analysis software .The rTFF1 produced by SDS-PAGE . The yie2HPO4-citric acid buffer (pH 2.4) [To examine the existence of dimer forms, the proteins (0.103 mg/mL) were treated with 0.05% glutaraldehyde at room temperature for 1 h and then were analyzed with 16.5% Tricine-SDS-PAGE. To examine the rTFF1 polymeric forms, the proteins were treated with phosphate buffer (pH 7.0) and Na(pH 2.4) , and the(pH 2.4) . For com2. The confluent cultures were washed with Ham’s F-12 K Medium (serum free), and the cells (5.0 × 104) were loaded into culture inserts  within culture dishes (35 mm × 10 mm) which incubated in Ham’s F-12 K Medium containing 10% FBS. After 18 h of culturing, culture inserts were removed and various concentrations of rTFF1s were added and cultures were maintained in Ham’s F-12 K Medium (with or without 10% FBS) and incubated at 37°C in an atmosphere of 5% CO2. Photographs were made approximately every 24 h after starting at 0 h . The complete experiment was done in 48 h; wound closure was monitored and photographed at 0, 24 and 48 h with a Nikon TS100 inverted-microscope and a Nikon D5100 camera .AGS cells were maintained in Ham’s F-12 K Medium  containing 10% FBS, 100 U/mL Penicillin, 0.25 μg/mL Amphotericin B, and 100 μg/mL Streptomycin . The cells were incubated at 37°C in an atmosphere of 5% COB. choshinensis (pNCMO2-TFF1) were confirmed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF)  . Additionally, the modification of rTFF1 was also confirmed by LC-MS/MS .To examine the existence of polymeric forms, the purified rTFF1/ECBL21(DE3) (pET-TFF1) and B. choshinensis (pNCMO2-TFF1) was confirmed with a glycoprotein stain kit .To advance the determination of post-translational modification of rTFF1, the protein from p < 0.05 were considered significant.The results were expressed as the mean ± SD of data obtained from quadruplicate experiments. The statistical significance was estimated using a SPSS 12.0 Tukey HSD. Values with


Soil N2O emission was measured using closed chamber method and a separate incubation experiment using acetylene inhibition method was carried out to determine denitrification fluxes and the contribution of nitrification and denitrification to N2O emissions between Jul. and Oct. 2012. An NH4NO3 addition of 50 kg N/ha/yr significantly increased N2O and N2 emissions, but their “pulse emission” induced by N addition only lasted for two weeks. Mean nitrification-derived N2O to denitrification-derived N2O ratio was 0.56 in control plots, indicating higher contribution of denitrification to N2O emissions in the study area, and this ratio was not influenced by N addition. The N2O to (N2+N2O) ratio was 0.41–0.55 in control plots and was reduced by N addition at one sampling time point. Based on this short term experiment, we propose that N2O and denitrification rate might increase with increasing N deposition at least by the same fold in the future, which would deteriorate global warming problems.Nitrogen (N) deposition has increased significantly globally since the industrial revolution. Previous studies on the response of gaseous emissions to N deposition have shown controversial results, pointing to the system-specific effect of N addition. Here we conducted an N addition experiment in a temperate natural forest in northeastern China to test how potential changes in N deposition alter soil N However, very limited data exist on this ratio or the denitrification rate in temperate forest ecosystems NPinus koraiensis Siebold & Zucc) mixed forest, which is the dominant natural forest type in northeastern China. The system was previously thought to be N-limited but may be experiencing N saturation currently 2O fluxes, ratio of nitrification derived to denitrification derived N2O, and denitrification rate, for the first time in the study area; 2) to examine the treatment effect of N addition on these fluxes; 3) to investigate the influences of environmental factors on these variables. The hypotheses of this study are: 1) N addition would increase soil N2O emissions and denitrification rate; 2) soil temperature, moisture, WFPS, pH, and soil texture play an important role in soil N2O emissions and denitrification.This study was carried out in a broad-leaved Korean pine (The study was carried out within the research forest of Changbai Forest Ecosystem Research Station (CBFERS) established in 1979 . No specific permits were required for the described field studies. Research activities within that research forest do not need any specific permissions from any government levels, but need to inform Professor Han . The field studies did not involve endangered or protected species.The study region is characterized by a typical temperate climate, with long and cold winters, and warm summers. Mean annual temperature is 3.6 °C, with the highest temperature in July, and the lowest in January. Mean annual precipitation is 745 mm, mainly falling between May and September. The growing season is from Jun. to Sep., with mean temperature at 16.7 °C. Natural Korean pine and broad-leaved mixed forest is distributed from approximately 750 to 1100 m asl. The soil is a dark brown soil developed from volcanic ash (Albic Luvisol).−1 yr−1, with higher values in eastern China −1 yr−1−1 yr−1−1 yr−1 in the treatment as a reasonable estimate of future change. Six sampling plots (25 m×50 m) were established: three replicates for both control and N addition treatment. Plots were separated by 10 m buffer strips. Vegetation was homogeneous based on visual observation.Present N deposition rate in China is 12.89–63.53 kg N ha4NO3 solution has been evenly sprayed for N addition treatment monthly from May to September at a rate of 10 kg N ha−1 (starting on May 17th), giving an annual N addition rate of 50 kg N ha−1 yr−1. The same volume (40 L) of water was sprayed on the control plots.Starting in 2008, NH2O emission was measured using closed chamber method Soil N4NO3 was sprayed on the day 17th each month, gas samples were collected in three periods: Period one (Jul. 17th–Aug. 16th): 7, 9, 13, 18, and 27 days after N addition; Period two (Aug. 17th–Sep. 16th): 4, 8, 15, 19, 24, and 28 days after N addition; Period three (Sep. 17th to Oct. 18th): 3, 9, 17, 22, 26, and 31 days after N addition. Samples were taken between 9:00 and 11:00 hours and between 15:00 and 17:00 hours because during these two time slots the flux can represent daily average 2O concentration was measured by gas chromatography (HP 5890-II). The temperature of the chromatographic column was 55°C and the carrying gas was high purity N2.We sampled for three N addition periods in year 2012, 5–6 times for each period. Samples were taken once every 5–6 days. Given that NH2O based on the following principle: high concentration of acetylene (10 kPa) could inhibit both nitrification and the reduction of N2O to N2 by denitrifiers, so that the otherwise emitted N2 remained as N2O. When the acetylene concentration was low (10–100Pa), only nitrification was inhibited 2O could be from three sources: N2Oden (N2O produced by denitrification); N2ON2; N2Onit (N2O produced by nitrification). In each sampling plot, soils were incubated in situ in three tightly sealed bottles injected with different concentrations of C2H2 (purified by H2SO4 and distilled water): 0 (nothing inhibited), 60 Pa (nitrification inhibited), and 10 kPa (nitrification and the reduction of N2O to N2 inhibited). Correspondingly, measured N2O of the three incubation bottles were N2Onit+N2Oden, N2Oden, and N2Oden+N2ON2, respectively. Therefore, nitrification-derived N2O, denitrification-derived N2O, and denitrification-derived N2 can be calculated from these three measured N2O fluxes. Denitrification rate is equal to N2Oden+N2ON2.Acetylene inhibition method 2H2 was used for low concentration treatment as recommended in previous experiment in the study area 60 Pa C2O in situ incubation method to reduce some potential bias Although the acetylene inhibition technique has not always been satisfactory due to incomplete or non-specific inhibition 2H2. The bottles were then buried in situ and 60 ml headspace samples were taken after 24 hours incubation and analyzed for N2O concentration by gas chromatography (HP 5890-II). Gas samples were collected on Aug. 18th, Aug. 30th, Sep. 10th, Sep. 16th, Sep. 23rd, Sep. 30th, Oct. 7th, and Oct. 15th, 2012.Three sampling points were selected within each sampling plot. Nine soil cores (5 cm diameter×10 cm depth) were sampled by PVC tubes at each point. There were 25 holes (0.6 cm diameter) on each PVC tube to ensure acetylene circulation. At each sampling point, every three soil cores were assigned to one of the three incubation bottles. Therefore, we had three soil cores in each incubation bottle at each plot. Before the incubation, the headspace air was replaced by purified Cin situ at each sampling time of both closed chamber experiment and acetylene inhibition experiment using TP3001 thermometer   . Precipi, China) . Bulk de4+ and NO3-. Standard oven-drying method was used to measure soil gravimetric water content, which was expressed as kg H2O/kg dry soil. WFPS was calculated based on Eqn.1:One soil sample (0–10 cm) in each plot was collected at each sampling time to determine pH, soil moisture content, and soil NHSoil pH was measured on a 1∶2.5 soil solution in de-ionized water using a pH meter. Soil texture was determined by the pipette-sedimentation method Repeated measures ANOVA was used to examine the effects of N addition using N treatment method as the between subject factor and sampling time as the within subject factor. All analyses were carried out in SPSS −3 and 0.587±0.113 g cm−3 respectively). Soil moisture and WFPS were not significantly different between N-treated plots (101.90±26.94 kg H2O kg−1 dry soil and 76.07±20.15% respectively) and control plots (94.81±21.99 kg H2O kg−1 dry soil and 70.48±15.88% respectively). Mean soil NO3- concentration was significantly increased in N-treated plots (22.56±26.26 mg N kg−1 soil) compared to control plots (19.11±23.87 mg N kg−1 soil) based on repeated measures ANOVA, but mean soil NH4+ concentration was not changed . This difference between N-treated and control plots declined with time and became minimal on the 15th day after N addition. Similarly, for the next sampling period, mean N2O emission in N-treated plots was 414.07 µg N m−2 h−1 three days after N addition, compared with 36.88 µg N m−2 h−1 in control plots. Only 17 days later, N2O flux was similar in N-treated (44.13 µg N m−2 h−1) and control (85.38 µg N m−2 h−1) plots.N-treated plots had significantly higher N 2 and 3 . For exa2O emission was positively correlated with soil NO3- (r = 0.225), soil total inorganic N (r = 0.210), and soil temperature (r = 0.253)   . N2O emi= 0.727) .−1 h−1) than in control plots (0.37 µg N kg soil−1 h−1) (2O to (N2+N2O) ratios in N-treated plots (0.29–0.45) were all lower than those in control plots (0.41–0.55), although the effect was not statistically significant (th and Sep. 17th did not affect this pattern (2Onit: N2Oden ratio or a significant effect of N addition on the ratio (2Onit: N2Oden ratio in N-treated plots had a wider range (0.17–4.47) than that in control plots (0.19–0.94).One day after N addition in August, denitrification rate was significantly higher in N-treated plots (3.95 µg N kg soill−1 h−1) . The effl−1 h−1) . The N2Onificant . N-addit pattern . We did he ratio . The N2O2 flux had a significantly positive correlation with WFPS (r = 0.344 and 0.353 respectively). Neither the N2O to (N2+N2O) ratio nor the N2Onit: N2Oden ratio correlated with WFPS or any other measured soil variables  −1 yr−1) could increase N2O fluxes to up to 501 µg N m−2 h−1 and the monthly sum of N2O emission in N-treated plots were five-times higher than that in control plots  than that in control plots (0.19–0.94). Mean N2Onit: N2Oden ratio was 0.56 and 1.46 in control and N-treated plots respectively, which is lower than what Papen & Butterback-Bahl 2O was emitted from denitrification. The N2Onit: N2Oden ratio was not significantly correlated with any soil factors we examined  and there is no denitrification in the other months −2 yr−1. This is close to the upper end of the modelled average value (0.156–0.368 g N m−2 yr−1) in global temperate and boreal forest ecosystems by Bai et al. In situ denitrification data from temperate forest are very limited. Dannenmann et al. −2 yr−1 in a mountainous beech forest using seasonally weighted means, although their direct up-scaling of plot means got much higher values (1.4–9.37 g N m−2 yr−1). Wolf & Brumme −2 yr−1in situ denitrification rates from beech forest floor and mineral soils. Our estimation is comparable to these above reported values.Denitrification in the control plots were between 0.126 and 5.460 µg N kgsoil h−1 . Bulk de h−1 (−3  with a m−1 soil h−1 (−1 yr−1−1 yr−1 (2.2 fold) has caused approximately a 2.5 fold increase in denitrification. Therefore, we predict that with the increase of N deposition, denitrification will increase correspondingly, at least by the same fold.N addition significantly increased denitrification rates to a mean of 5.539 µg N kgsoil h−1 , Fig. 3a2 is emitted after N addition compared with N2O because the N2O to (N2+N2O) ratio was lower in N-treated plots (2O to (N2+N2O) ratios were between 0.41–0.55 and 0.29–0.45 in control and N-treated plots respectively. The N2O to (N2+N2O) ratio has been used to estimate denitrification rate because N2O was easier to measure 3- has been found to increase the N2O to (N2+N2O) ratio because of the greater affinity of NO3- as terminal electron acceptor 2O to (N2+N2O) ratio in our study, and further exploration is needed.For the two gaseous products of denitrification, more Ned plots . The N2O2 emission, but not with N2O fluxes (2 fluxes is unidirectional, while bell-shaped relationship between WFPS and N2O is more common WFPS was positively correlated with NO fluxes , Table 32O and denitrification fluxes. However, the effect only lasted for two weeks. Based on this short term experiment, we propose that denitrification rate might increase at least by the same fold as the increase of N deposition in the future. N2O may also increase with increasing N deposition in our studied forest, deteriorating global warming problems. Future studies on the underlying mechanism 2O emissions following N addition are needed to better understand the processes contributing to increasing N2O emissions.In conclusion, N addition did not change soil pH or soil bulk density in the study area. Mainly due to the increase of substrate availability, N addition significantly increased N


Acute traumatic coagulopathy (ATC) identified in the first hour of trauma patients is associated with worse outcomes and increased risk of death . IdentifBoth wild type (WT) and modified Knock-in TM pro/pro mice (TMKI) were subjected to trauma  and haemorrhage (40± 5% of estimated blood volume) to a target mean arterial blood pressure of 30 ± 5 mmHg. This model was developed to elicit coagulopathy as a response to trauma and bleeding thus mimicking the coagulopathy present in the injured patient. The animals were kept euthermic and were not resuscitated. After 60 minutes blood samples were taken and analysed via thrombelastometry tests.All animals were shocked when compared with controls . After trauma and haemorrhagic shock (THS), WT mice developed a significant coagulopathy, as measured by a reduction in clotting amplitude at 5 minutes (CA5’)  and in maximal clot firmness (MCF) . In the TMKI group, which entails a 1000 fold reduction in aPC production there was a correction of the coagulopathy (CA 5’: TMKI time 0 vs time 60 = 42.6± 2.3 vs 41.6±1.5 mm, p<0.0002 and MCF (TMKI t0 vs t60 =56.8±2 mm vs 55.1± 1 mm, p<0.001.Severe trauma shock induced a significant coagulopathy characterised by the generation of fragile clots. The reduction in activated protein C generation encompassed an improvement in the experimental coagulopathy therefore the protein C pathway seems to play an important role in acute traumatic coagulopathy.


CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter.The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct  NPFs), the nitrate transporters (NRT2s) and the nitrate assimilation related genes (NAR2s)NRT2 family is part of the major facilitator superfamily (MFS). The Arabidopsis thaliana  genome harbors the seven NRT2 genes, referred to as AtNRT2.1 through 2.7NRT2 homologs are also known in other plant species: five have been identified in barleyChlamydomonas reinhardtiiNRT2 was initially identified as a putative high affinity transport gene in barleyA. thaliana910AtNRT2s have been classified into three types: those which are inducible , those which are constitutively transcribed , and the repressible gene 2.5AtNRT2.1 encodes a high affinity nitrate transporter which functions at low external nitrate concentrations, acting as either a nitrate sensor or a signal transducer in the rootAtNRT2.1 product is an important contributor to the inducible component of high affinity nitrate transportPlants have evolved both a high (HATS) and a low (LATS) affinity transport system, which act together to determine nitrate uptake from the soil. The genes involved in HATS and LATS belong to three distinct gene families, namely the nitrate transporter/peptide transporters  is a leading ornamental species, particularly in China, where the heavy application of nitrogenous fertilizer is a common practice pursued by growers, despite its known deleterious effect on the environment. So far, only one CmNRT2 (AB921547 renamed CmNRT2.4 in this study) gene has been isolated; its product interacts with CmNAR2CmNRT2s and a more detailed analysis of the function of CmNRT2.1 has been given. In particular, its inducibility by both nitrate and ammonium has been characterized, and its interaction with CmNAR2 further detailed. Finally, a demonstration is given that the heterologous expression of CmNRT2.1 in A. thaliana resulted in an increased rate of nitrate uptake into the root.Chrysanthemum (CmNRT2s using 3′ RACE. The outcome of the procedure was the successful identification of seven distinct 3′ untranslated regions (3′ UTRs), including that of CmNRT2.4  share two conserved introns, whereas 2.5 harbors two introns sited in a different part of the gene, and 2.7 lacks any introns. A phylogenetic tree based on peptide sequences is shown in A peptide level sequence comparison between the set of AtNRT2s and the single previously known CmNRT2  revealed the presence of two conserved regions, namely A1 [FGMRGRLW(N/T/A/I/G)(L/W)W] and A2 [(H/Y)FPQWG(S/G)M(F/C)] . Based oCmNRT2.4 , which iCmNRT2.1 transcription showed that the gene was constitutively transcribed throughout the plant except in the flower, with the root being the site where its transcript was most abundant  were used to study the transgene’s impact on plant growth and nitrate uptake. Both root and shoot fresh weight (FW) of the two lines were significantly higher than those of the wild type (WT) control and vector-transformed lines when the growing medium contained 0.25 mM nitrate. With respect to root FW, there was no significant difference in performance between the transgenics and the controls when the medium contained 10 mM nitrate, but shoot FW was enhanced in RT-2 . Phenotypically uniform seedlings at the eight leaf stage were grown in a pH 6.5 medium containing 5 mM NH3 for 4 h, RNA was extracted from the roots using the RNAiso reagent , following the manufacturer’s protocol, then treated with RNase-free DNaseI . The concentration and the integrity of the extract were assessed following Gu et al.−) , following the manufacturer’s protocol. Two degenerate primers A1f and A2-2f . The genes’ deduced polypeptide sequences were used in a BLASTp search to identify homologs. An alignment of the seven derived CmNRT2s was performed using DNAman v 5.2.2 software . A phylogenetic analysis of the CmNRT2s and heterologous NRT2s was finally performed using MEGA 5 softwareFor the gene isolation experiment, seedlings were grown in the solution  for four weeks, then starved of nitrogen by removing them to a nitrogen-free version of the same medium for one week. After exposing the seedlings to 5 mM KNO3) or 5 mM NH4Cl, the roots were harvested over a time period , and the RNA extracted as above. RNA was also extracted from various parts of 90-day old plants grown as described in the part of plant materials and growing conditions. Aliquots (1 μg) of the total RNA were reverse transcribed using SuperScript III reverse transcriptase  based on oligo(dT) primers. The subsequent qRT-PCRs were formulated with SYBR® Premix Ex Taq™ II (TaKaRa). The reactions were initially denatured (95 °C/60 s), then subjected to 40 cycles of 95 °C/15 s, 55 °C/30 s, 72 °C/30 s. A melting curve was obtained by heating the amplicon from 55 °C to 95 °C at 0.5 °C s−1. The chosen reference gene was CmPsaA (AB548817)CmNRT2.1 were qCmNRT2.1-F (5′-GTAACACCTCCGAGCAACAC-3′) and qCmNRT2.1-R (5′-TTTCACAATGCAATAGCATG-3′)  for four weeks. After providing a variable amount of nitrate  . CmPsaA-  and transformed as described by Gu et al.CmNRT2.1 cDNA was first amplified using Phusion® HS DNA polymerase  based on the primer pair pPR3-N-2.1-JS/pPR3-N-2.1-JX (CmNRT2.1 and CmNAR2 fusions were then co-transformed into yeast strain NMY51 (MATa his3 trp1 leu2 ade2 LYS2::HIS3 ura3::lacZ ade2::ADE2 GAL4) using a DS yeast transformation kit  following Gu et al.546 of the cultures reached 1.0, the cultures were serially diluted . A 5 μL aliquot of each dilution was spotted on to both SD-LW and SD-AHLW solid medium and incubated at 30 °C for three days. Positive clones were assayed for β-galactosidase activity.The existence of a CmNRT2.1/CmNAR2 interaction was tested using the mating-based split-ubiquitin system  and a BiFC assay. The necessary fusion gene p2.1-Bi-X  and the N-2.1-JX , and theCmNRT2.1 ORF was amplified using the primer pair CmNRT2.1-1301-F/CmNRT2.1-1301-R  (MS) medium containing 20 mg/L hygromycin and 25 mg/L ampicillin. Positive transformants were validated by observing GUS expression in the leafCmNRT2.1 transcription using the primer pair CmNRT2.1s/CmNRT2.1x (AtUBQ (NM_116771.5) assayed using the primer pair AtUBQs/AtUBQx 2) or 10 mM nitrate (5 mM KNO3 and 2.5 mM Ca(NO3)2). The nitrates were replaced by the same molarity of chloride salts to produce a nitrogen deficient medium. Each of three replicated treatments comprised a set of 50 plants. Shoot and root FW was measured after 14 days. The uptake of labeled nitrate (15NO3) was assayed as described elsewhere4 for 1 min, then to a complete nutrient solution containing 0.2 mM 15NO3-for 5 min and finally to 0.1 mM CaSO4 for 1 min. The root homogenate was dried overnight at 80 °C. The content of labeled nitrate was analyzed using a PDZ Europa ANCA-MS device . The recorded measurements represent the mean of three biological repeats.Seedlings were raised on vertical MS agar plates two weeks, then transferred to a growth culture conditions mediumStatistical analysis was performed by the one-way analysis of variance (ANOVA) using SPSS 11.5 software , and Duncan’s multiple range test was employed to detect differences between means.How to cite this article: Gu, C. et al. Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1. Sci. Rep.6, 23462; doi: 10.1038/srep23462 (2016).


Unlike the well-established picture for the entry of enveloped viruses, the mechanism of cellular entry of non-enveloped eukaryotic viruses remains largely mysterious. Picornaviruses are representative models for such viruses, and initiate this entry process by their functional receptors. Here we present the structural and functional studies of SCARB2, a functional receptor of the important human enterovirus 71 (EV71). SCARB2 is responsible for attachment as well as uncoating of EV71. Differences in the structures of SCARB2 under neutral and acidic conditions reveal that SCARB2 undergoes a pivotal pH-dependent conformational change which opens a lipid-transfer tunnel to mediate the expulsion of a hydrophobic pocket factor from the virion, a pre-requisite for uncoating. We have also identified the key residues essential for attachment to SCARB2, identifying the canyon region of EV71 as mediating the receptor interaction. Together these results provide a clear understanding of cellular attachment and initiation of uncoating for enteroviruses.The online version of this article (doi:10.1007/s13238-014-0087-3) contains supplementary material, which is available to authorized users. Cell entry of non-enveloped viruses can be accomplished through two distinct pathways: (a) the endocytic pathway that employs clathrin-coated vesicles or caveolae; or (b) the formation of a pore on the cell surface Hogle, . PicornaEnterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are representative members of the enterovirus (EV) genus of Picornaviruses and are two major causative agents of hand-foot-and-mouth disease (HFMD) in the Asia-Pacific region  (hereafter denoted as nSCARB2) and acidic conditions (pH 4.8) (hereafter denoted as aSCARB2) Fig. .Figure 1The structure of aSCARB2 (pH 4.8) is similar to the recently published pH 5.5 structure -labeled sphingosine with excess recombinant SCARB2-His-tag protein and Ni-NTA beads under acidic conditions (pH 5.0). The mixture was incubated at 37°C and aliquots were withdrawn at different time intervals for analysis. SCARB2 or SCARB2-EV71 complexes were removed from the samples by low-speed centrifugation. The Ni-NTA bead-bound His-tagged protein was sedimented upon centrifugation. The supernatant containing EV71 particles was ultracentrifuged to pellet EV71 particles. Radioactivity associated with EV71 particles was measured and the relative protein content of EV71 capsid was evaluated by Western blot.To prove that SCARB2 initiates uncoating of EV71 by removing the “pocket factor” from EV71 virions, we incubated mature virions with excessive [3H]-labeled sphingosine to attain saturation in the capsid. However, under acidic conditions (pH 5.0), addition of recombinant SCARB2 to EV71 containing [3H]-labeled sphingosine followed by incubation of the mixture at 37°C for 1–4 h, resulted in a rapid decrease in [3H]-labeled sphingosine in virions when compared to the amount of radioactively labeled sphinghosine associated with similar amount of virions incubated under identical conditions but without SCARB2  studies on EV71 alone and in the presence of SCARB2. Mature virions sediment at ~160 S regardless of the pH Fig.  interferE. coli and tested for their ability to bind SCARB2 using a GST pull-down assay. The results showed that only peptides VP1-2, VP1-6, VP1-8, VP2-2 and VP3-4 could bind to recombinant SCARB2  that a peptide corresponding to residues 146–166 of SCARB2 interacted with EV71 mature virions tro Fig. B. Residutro Fig. , these rN-linked glycoproteins to remove glycans . 293A-hSCARB2 cells pretreated with PNGase F showed similar expression of SCARB2 on the cellular membrane when compared to the untreated control cells . N325 is located at the bottom of a cleft formed between helices α11 and α12. Man8GlcNA2 protrudes to the surface out of this chasm and stations itself in vicinity of the cap. Residues from all three domains interact with this glycan. Notably, the sugar moieties adjacent to α4 and α5 are stabilized by P314, E145 and H150  and aSCARB2 were collected at beam line BL5A and BL17A of the Photon Factory (PF) synchrotron facility in Japan with the highest resolution being 3.0 Å, 2.8 Å and 3.65 Å, belonging to space groups of 3H]-labeled sphingosine. For each assay, 400 μL of purified EV71 mature virions (20 μg/mL) were incubated with 8 μL [3H]-labeled sphingosine  at room temperature for the indicated times  in PBS buffer (pH 7.4). Then EV71 virions were pelleted, washed for 3 times using PBS buffer (pH 7.4) and the radioactivity associated with them was measured. Radioactivity data were further normalized to the one of incubation for 1 h, which was considered as 100%. All reactions reported here were repeated triple times.EV71 production and purification can be found in the Extend Experimental Procedures. Competitive displacement of natural lipid from EV71 capsid proteins using [3H]-labeled sphingosine (5 μmol/L) for 60 h were incubated with Ni-NTA beads in the absence or presence of mammalian expressed SCARB2 with an His tag at a molar ratio of 60:1 (SCARB2:Virus) in acidic pH buffer (pH 5.0) at 37°C for the indicated times . Centrifuged at 3000 rpm for 3 min to collect Ni-NTA beads, the supernatant was then used to pellet EV71 particles at 45,000 rpm for 1.5 h. The radioactivity of pelleted EV71 particles was determined, meanwhile, the relative protein contents were estimated by Western blot. Radioactivity data were further normalized to the one of the control group (in the absence of recombinant SCARB2 for 0 h), which was considered as 100%. All reactions reported here were repeated triple times.400 μL of purified EV71 mature virions (20 μg/mL) pretreated with 8 μL [280nm absorption of about 0.4. Samples were loaded into a conventional double-sector quartz cell and mounted in a Beckman four-hole An-60 Ti rotor. Data were collected at 12,000 rpm at a wavelength of 280 nm. Interference sedimentation coefficient distributions were calculated from the sedimentation velocity data using the SEDFIT software program .EV71 full particles were mixed with mammalian expressed SCARB2 at a molar ratio of 1:60 at pH 7.4 and pH 5.0, respectively and then incubated at 37°C for 1 h. The full particles alone were analyzed under identical conditions as a control. NLD, an inhibitor of EV71 which can prevent viral uncoating was also used in this experiment with pre-incubation with EV71 full particles at a molar ratio of 240:1 (NLD:Virus) at room temperature for 4 h and then addition of SCARB2 as mentioned above. Sedimentation velocity experiments were performed on a Beckman XL-I analytical ultracentrifuge at 20°C. Different preprocessed virus samples were diluted with corresponding buffer (PBS pH 7.4 or Na-Citrate pH 5.0) to 400 μL at an Ain vitro by Pull-down assay can be found in the Extended Experimental Procedures.293A-hSCARB2 cell line, which stably expressed human SCARB2 on the cell surface of 293A cells was established in our lab previously. EV71-GFP virus was prepared with enhanced green fluorescent protein (EGFP) gene inserted into EV71 genome between virus 5′ UTR and coding region such that GFP gene can be transcribed and translated during viral replication only. 293A-hSCARB2 cells were seeded in polylysine pretreated 96-well plate and cultured in complete DMEM. One day later, the cells were treated with 0, 4 or 40 μg/mL PNGase F at 37°C for 1 h. After washing with PBS, EV71-GFP virus was added to pretreated cells at 0.1 MOI. Sixteen hours post incubation, the cells were photographed under fluorescence microscopy, and then digested with trypsin and fixed with 4% paraformaldehyde (PFA). The GFP positive cells were analysed by flow cytometry, which can be found in the Extended Experimental Procedures. Deglycosylation of SCARB2 by PNGase F and the interaction between SCARB2 and EV71 Protein-protein docking was used to obtain an ensemble of complexes between deglycosylated aSCARB2 and capsid proteins (VP1–3) of EV71. The results of binding interface mapping provide directions for EV71-SCARB2 docking. More details can be found in the Extended Experimental Procedures.Highly purified EV71 mature virions and the synthesized peptide (aa 146–166 of SCARB2) were in ITC buffer (PBS buffer pH 7.4). The SCARB2 peptide  was loaded into the syringe of the microcalorimeter (GE Company), with EV71 mature virions in the cell at a concentration of 7 μmol/L. Injections of 10 μL of the peptide were made into the cell at 25°C. Data were analysed by the Origin 7 software.Coordinates and structure factors have been deposited with RCSB accession codes: 4TVZ, 4TW0, 4TW2.Supplementary material 1 (PDF 1473 kb)Below is the link to the electronic supplementary material.