Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg·L−1 starting concentration) over a range of 3%–10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability.  Equal volumes (130 μL) of chloroform and water were then added. The mixture was again shaken for 30 min and then centrifuged at 12,000 rpm for 30 min. The supernatant was recovered and dried. The pellet was resuspended in 300 μL of ammonium formate and 700 μL of acetonitrile (ACN) and was quantified by HPLC  using an Amide column . The mobile phase consisted of ACN and ammonium formate , and the flow rate was 0.8 mL·min −1. Detection was made at 210 nm with a UV detector, and the injection volume was 20 µL. The retention time of ectoine was determined by using a commercially available ectoine sample .The isolated strain was maintained on MSM containing 0.4% glucose, 0.2% yeast extract and different NaCl concentration. The cells were grown until late exponential phase, harvested by centrifugation and were lyophilized; then, the dry weight of the cells was determined . The celLmPH), catechol 1,2-dioxygenas gene (C12O) and catechol 2,3-dioxygenas (C23O). The genes encoding these enzymes were amplified by using the primers sets: LmPHf (5ʹ-CGCCAGAACCATTTATCGATC-3ʹ), LmPHr (5ʹ-AGGCATCAAGATCACCGACTG-3ʹ) [C12Of (5ʹ-ACCATCGARGGYCCSCTSTAY-3ʹ), C12Or (5ʹ-GTTRATCTGGGTGGTSAG-3ʹ) and C23Of (5ʹ-GARCTSTAYGCSGAYAAGGAR-3ʹ), C23Or (5ʹ-RCCGCTSGGRTCGAAGAARTA-3ʹ) [The isolated DNA of the isolated strain was screened for the presence of key phenol-degrading enzymes, including the largest subunit of multicomponent phenol hydroxylase (ACTG-3ʹ) ; C12Of (ARTA-3ʹ) .LmPH encoding genes consisted of one cycle of initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 35 s, extension at 72 °C for 1 min, followed by one additional cycle at 72 °C for 5 min and a final storage cycle at 4 °C. The PCR conditions for the amplification of C12O encoding genes consisted of an initial cycle of 5 min at 95 °C, followed by 35 cycles of: denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 1 min. Similar conditions were applied for the amplification of the C23O gene fragments, except that lower annealing temperatures were assayed (55 °C).The PCR conditions for the amplification of The GenBank accession number for the bacterial 16S rRNA gene sequence is KM894272, and those for the functional genes are KM975502–KM975503.Halomonas was isolated and characterized in terms of phenol-degrading ability at different salinities and intracellular concentrations of ectoine. Furthermore, functional genes involved in the phenol degradation process were detected. The high-throughput screening system was instructive for the rapid screening of degrading strains from the environment and contributed to the biological treatment of high-salinity phenolic wastewater.This study developed a high-throughput screening system for moderately halophilic phenol-degrading bacteria. The bacterial enrichments were cultivated from various habitats under different salt concentrations in 48 deep well microplates. Measurement of phenol concentrations was performed in 96-well microplates. Finally, a phenol-degrading strain related to


HMX1 is a homeobox-containing transcription factor implicated in eye development and responsible for the oculo-auricular syndrome of Schorderet-Munier-Franceschetti. HMX1 is composed of two exons with three conserved domains in exon 2, a homeobox and two domains called SD1 and SD2. The function of the latter two domains remains unknown. During retinal development, HMX1 is expressed in a polarized manner and thus seems to play a role in the establishment of retinal polarity although its exact role and mode of action in eye development are unknown. Here, we demonstrated that HMX1 dimerized and that the SD1 and homeodomains are required for this function. In addition, we showed that proper nuclear localization requires the presence of the homeodomain. We also identified that EPHA6, a gene implicated in retinal axon guidance, is one of its targets in eye development and showed that a dimerized HMX1 is needed to inhibit EPHA6 expression. HMX1 is a homeobox-containing transcription factor implicated in eye development. In 1992, Stadler et al. described a new homeobox gene called GH6. This gene was later renamed HMX1 and was assigned to the NKX5 family, the reason why HMX1 is also known as NKX5-3HMX2 (NKX5-2), HMX3 (NKX5-1) and, in chicken, zebrafish and medaka, SOHo-1HMX1, HMX2 and HMX3 contain two other conserved domains called SD1 and SD2, located immediately C-terminally to the homeobox Homeobox-containing transcription factors represent an important class of factors involved in the regulation of embryogenesis and other molecular programs. Hmx1 expression can be detected as early as E10.5, and transcripts are more specifically present in the lens and in the antero-medial part of the neural retina HMX1 expression starts 40 hours into development (stage 11) in the surface ectoderm surrounding the optic vesicle. At optic cup invagination (stage 14–15), it is expressed in the anterior/nasal side of the early retina hmx1 is first expressed in the entire eye at 10 somite-of-stage (ss), and is then repressed in the dorsal part at 18 ss. At 24 hours post fertilization (hpf), it is restricted to the nasal retina and, one day later, expression is restricted to the nasal part of the ganglion cell layer (GCL). At four and five days post fertilization, signal is also observed in the nasal part of the inner nuclear layer (INL). In the developing lens, expression is observed from 24 to 72 hpf Whereas Hmx2 and Hmx3 play a role in inner ear development, Hmx1 and SOHo-1 are mainly implicated in eye development. In the mouse eye, HMX1 leading to the oculo-auricular syndrome of Schorderet-Munier-Franceschetti (OMIM: 612109), characterized by microphthalmia, microcornea, nystagmus, cataract, coloboma, optic nerve dysplasia, RPE abnormalities, rod-cone dystrophy and deformation of the ear lobule Hmx1 has been described In vitro, HMX1 binds to a 5′-CAAGTG-3′ sequence, represses transcription from a luciferase reporter containing this binding site and can antagonizeNKX2.5, a cardiac homeo protein, which is activating this same reporter construct We recently reported a family with a 26-bp deletion in exon 1 of EPHA6 as a target of HMX1 and showed that HMX1 repressed the EPHA6 promoter in vitro. The inhibitory activity of HMX1 was associated with the presence of the SD1 and homeobox domains. Whereas the EPHA6 inhibition was lost with mutants of each of these 2 domains, the SD2 mutant showed a small activation of the EPHA6 promoter. Mutation of the three CAAG(TG) sequences of the promoter attenuated the repression by HMX1. This inhibition was confirmed in vivo in zebrafish embryos.In this study, we showed that HMX1 acted as a dimer and that the homeobox and the conserved domain SD1 were needed for dimerization to occur. SD2 was not involved in the dimerization process. We also identified Subcloning was performed according to standard protocols. Mutagenesis was performed using the QuickChange II Site-Directed Mutagenesis Kit . The sequence of the primers used in this study is available from the authors.2 atmosphere, in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose with stable glutamine , supplemented with 10% FBS , 100 U/ml penicillin and 100 µg/ml streptomycin . Transfection was performed using the Calcium Phosphate method .Human embryonic kidney (HEK) 293T cells were cultured at 37°C and in 5% CO2 excitation and 510 nm for emission. Cells expressing BRET2 donor (RLUC) alone were used to determine the fluorescence background. 200’000 cells with comparable fluorescence levels were distributed into white 96-well microplates for luminescence quantification. The luciferase substrate Coelenterazine 400A, DeepBlueC  was added to a final concentration of 5 µM. Filter sets were adapted to 410 nm for luciferase emission and 515 nm for GFP2 emission. The emitted fluorescence and luminescence were measured using an Envision 2103 Multilabel Reader , and analyzed with the Wallac Envision Manager V1.12 software .200’000 HEK 293T cells in DPBS were distributed into black 96-well microplates for fluorescence quantification. Filter sets were adapted to 485 nm for GFP200 µg of proteins were immunoprecipitated overnight at 4°C on a rotating wheel with 2.5 µl anti-Renilla Luciferase antibody . 20 µl of washed protein G plus agarose beads  were added and incubated 2 hrs at 4°C on the rotating wheel. After centrifugation at 4°C, the supernatants were kept as controls. The pellets were resuspended in 25 µl 2x SDS loading buffer and loaded on a 12% SDS-page gel, alongside with 20 µl of supernatant and 40 µg of proteins.Proteins were extracted from cell cultures using RIPA  and concentrations measured using the Micro BCA Protein Assay Kit  on a Multiplate Reader Synergy HT  with the KC4 software. The following antibodies were used: HA-Tag (6E2) Mouse mAb #2367 , GFP N-terminal G1544 , PARP (46D11) Rabbit mAb #9532 , p62/SQSTM1 P0067 , Ub (A-5) sc-166553  and α-Tubulin Clone B-5-1-2 T5168 .Cells were lysed in a non-denaturing lysis buffer . The protein concentrations were measured as described above. 5 µg were loaded on a Mini-Protean TGX precast gel 4–15%  with non-denaturing loading buffer  and migrated without denaturation in a running buffer without SDS.8 cells/ml Nuclei Isolation Buffer . Cells were then homogenized with a Potter and spread on a slide.Cells were analyzed 48 hrs post-transfection under a Zeiss Axiovert 200 microscope with filters adapted for excitation and emission at λex = 480 nm and λem = 510 nm, respectively, and the AxioVision 4.2 software. For nuclei isolation, cells were counted and resuspended at 10http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi.The mouse HMX1 sequence was entered in the NLS-Mapper software that can be found at M software .Immunofluorescence was performed 24 hrs post transfection. When necessary, 50 µM chloroquine were added for 16 hrs. The primary antibody  was diluted in 1x PBS +2% NGS +0.2% Triton X-100 and incubated overnight at 4°C in a humid chamber. The secondary antibody (Alexa Fluor 594 goat α-rabbit IgG (H+L) (A11012), Molecular Probes, LubioScience, Luzern, Switzerland) was diluted in the same buffer, and incubated 1 hr at RT in a humid chamber in the dark. Nucleic acids were stained with 100 µM DAPI  (1/1′500 in 1x PBS) for 10 min in a humid chamber in the dark. Cells were then mounted with Citifluor AF1 , and conserved at 4°C. The slides were analyzed under an Olympus BX61 microscope and the Cell20 mg/ml bisBenzimide H 33342 trihydrochloride  and 1 mg/ml Propidium Iodide  were diluted 1/2′000 into the culture medium. Cells were analyzed under a Zeiss Axiovert 200 microscope and the AxioVision 4.2 software.2HPO4 pH 7.8, 0.2% Triton X-100) were added. Cells were scraped at 4°C and centrifuged 3 min at 12′000 rpm at 4°C. 5 µl of supernatant were transferred to a transparent 96-well plate containing 50 µl 2x β-gal buffer . 50 µl of 2x ONPG  were added and the plate read at 412 nm of absorbance on a Multiplate Reader Synergy HT  with KC4 software. If the values were constant in all conditions, 5 µl of supernatants were transferred to a white 384-well plate, 20 µl of Luciferase Assay Reagent  were added and luminescence measured on the Multiplate Reader Synergy HT every 3 minutes until the peak of luciferase activity was reached. The obtained values were normalized using a β-gal reporter under the control of a CMV promoter. A mean between the 3 highest values was used for the luciferase/β-gal ratio. Each experiment was performed three times in duplicates. Only transfections with stable β-gal values between the different conditions, indicating similar transfection efficiency, were used. Two-tailed Student’s T-tests with unequal variance were used to determine statistical differences between the conditions.48 hrs post transfection cells were washed with 1x PBS, and 300 µl luciferase assay lysis buffer  twice and by shaking 15 min at RT. 12 µl 5M NaCl and 1 µl RNase A (10 mg/ml)  were added and the tubes were incubated 5 hrs at 67°C. The TIC samples were thawed and 100 µl transferred to new tubes. 500 µl IP elution buffer, 24 µl 5 M NaCl and 2 µl RNase A (10 mg/ml) were added, and the tubes were incubated 5 hrs at 67°C. After incubation, 2.5 volumes 100% EtOH were added for precipitation overnight at 4°C. The next day, the tubes were centrifuged and the pellets were dissolved in 100 µl TE 10∶1. 25 µl proteinase K buffer for ChIP  and 1 µl proteinase K  were added, and the tubes incubated 2 hrs at 45°C. 175 µl TE 10∶1 and 300 µl Phenol:Chlorophorm:Isoamyl Alcohol 25∶24∶1  were added, the tubes shaken and centrifuged. 30 µl of 5 M NaCl, 1 µl of 5 mg/ml glycogen and 750 µl of 100% EtOH were added to the supernatants, mixed and precipitated overnight at 4°C. The next day, the tubes were centrifuged, the supernatants were removed and the pellets resuspended in 30 µl TE 10∶1. PCR analysis was performed on 2 µl of samples.All experiments involving live animals were authorized by the Veterinary Service of the State of Valais under authorizations N° VS-13 and VS-19. The litter of four was housed with the mother and was anesthetized with isoflurane prior to being euthanized by cervical dislocation. Retinas from four 2-week-old wild-type C57Bl/6J mice were dissected, fixed, and homogenized. Glycine was added to a final concentration of 0.125 M before centrifugation. The pellet was resuspended in nuclei lysis buffer . The resulting chromatin was sonicated, snap-frozen in liquid nitrogen, and kept at −80°C. The next day, the tube was centrifuged, and the supernatant transferred to new eppendorf tubes containing 10 µl of Protein A-Agarose beads . 100 µl of supernatant were pre-cleared with the 10 µl of beads for 1 hr on a rotating wheel at 4°C. The tubes were centrifuged and the supernatants transferred to new tubes. A control tube containing 10 µl 5% BSA and a test tube with 2 µl Hmx1 antibody  were prepared. The tubes were incubated overnight at 4°C on a rotating wheel. After 10 µl of Protein A-Agarose beads were added to both tubes and another incubation, the tubes were centrifuged and the supernatants were kept at −80°C ( = TIC). The pellets were washed in IP wash buffer n°1 , 4 times in IP wash buffer n°2 , once in IP wash buffer n°3  and once in TE 10∶1 (10 mM Tris-HCl pH 7.5), 1 mM EDTA pH 8.0). The antibody was eluted from beads by adding 150 µl IP elution buffer (50 mM NaHCOhmx1 coding sequence was cloned downstream of the hsp70 promoter and the DNA construct together with the transposase mRNA were injected at the one-cell stage. Fish were raised to adulthood and the cardiac GFP expression was used as a marker for germline transmission. Experiments were done on F3 obtained from F2 that were intercrossed in order to increase the number of larvae carrying the transgene. Tg (hsp70: hmx1) and wt were heat shocked at 1 dpf during 30 min at 39°C, euthanized and fixed 4 hrs after.AB zebrafish were raised and kept under standard laboratory conditions at 28.5°C. Transgenesis was performed by generating Tol2 transposon constructs using the tol2kit in situ hybridization was performed at various stages. Hybridization reaction was done at 68°C for 14–18 hrs. Washing steps and antibody incubation were performed in an in situ machine . Templates used to generate DIG-labeled RNA probes included zebrafish hmx1 (ID: 797503), epha4b (ID: 64270) and pax6 (ID: 60639). In vitro transcription was done with the Roche RNA Labeling Kit .Standard one-color whole-mount 2 approach, whether HMX1 behaved similarly and formed dimers or oligomers. The BRET2 technique is based on the energy transfer occurring between the renilla luciferase (RLUC) and the green fluorescent protein (GFP) when they are in close proximity. The principle of the technique is to generate fusion proteins between proteins of interest and the RLUC and the GFP, and measure the energy transfer in culture conditions to determine if the proteins of interest are interacting.The ability to homo- or heterodimerize has been demonstrated for many transcription factors, including the NKX member NKX2-5, a cardiac homeobox gene that dimerizes through its homeodomain (HD) 2 and HMX1 were mixed and transfected in HEK 293T cells, a robust increase in the BRET2 ratio was observed with increasing concentrations of GFP2-HMX1, indicating that HMX1 dimerized , and two domains called SD1 and SD2, located 3′ to the HD and whose function is presently unknown. We deleted each of these domains separately by site-directed mutagenesis and repeated the BRET2 experiments. As shown in 2  and EPHA3 (chicken) are only expressed on the temporal side HMX1 and SOHO-1 are defining the EPHA3 expression domain in the developing chick retina EPHA6 promoter contains three such binding sites, the second being conserved between human and mouse (−39 relative to the ATG), whereas the EPHA5 promoter does not contain any. EPHA6 was also identified as a potential target of HMX1 using a predictive promoter model that we recently developed EPHA6 promoter. The technique we used allowed measuring the activity of the promoter by placing a luciferase reporter under its control. We subcloned a fragment spanning from −150 to +150 nucleotides relative to the EPHA6 translation initiation codon into a luciferase reporter vector. This fragment represented the minimal EPHA6 promoter with a 13-fold increased activity compared to pGL3-basic vector. Shorter fragments (−100 to +150 and −50 to +150) displayed reduced promoter activity  whereas the +1 to +150 fragment displayed no promoter activity (data not shown). The measured activity values were normalized using a β-gal reporter under the control of a CMV promoter. The subcloned fragment contained three potential binding sites for HMX1: one CAAGTG in the forward direction at position –39, one CAAG in the forward direction (−20) and one CAAG in the reverse direction (−65)  sequence on (−65) . We perf/6J mice , a technf epha4b , which w4b . Transfection experiments using these mutated constructs showed a reduction of the inhibitory activity of HMX1 from 42% to 22% . Moreover, PARP was not cleaved by Caspase 3 in GFP-HMX1 del HD transfections, indicating an absence of apoptosis (not shown). Thus, we do not know at this time what is the exact nature of these GFP-HMX1 del HD aggregates and if they represent pure HMX1 aggregates or a more complex structure.hmx1 had no effect on retinal patterning hmx1 was knocked-down on the nasal side of the retina, whereas the temporal part was unaffected by this procedure, and epha4b was able to play its role in the temporal retina.In a previous study, we showed that a morpholino-based knock-down of zebrafish EPHA6 as a target of HMX1. Identifying other targets will allow us to further understand the role of HMX1.In summary, we showed that HMX1 exerts its inhibitory activity through a dimer and identified


Cases with microcytosis not responding adequately to iron supplementation are diagnostic dilemma and have been reported to harbor alpha (α) thalassemia mutations. The aim of this study was to determine the common α globin gene deletions in cases with microcytic anemia.3.7, -α4.2, - -αSA and --αSEA) was done.Fifty four patients selected  had microcytic anemia  with raised TRBC (> 5M/mm3) but normal Hb HPLC. They had either low or normal Transferrin Saturation (TS). Gap-PCR for four common α-gene deletions  cases were found to have α gene mutations; Three homozygous and sixteen heterozygous cases including -αSA should be done even in the face of low iron stores in subjects who respond incompletely to iron supplementation.α gene mutations can confound iron deficiency anemia, but no RBC indices, or a discriminant function can identify it is presence Molecular studies have to be resorted to. Gap PCR for common α thalassemia mutation including –α  Iron deficiency anemia (IDA), β thalassemia trait (βTT) and anemia of chronic disease (ACD) are common causes of microcytosis that can be diagnosed accurately by Iron studies and Hb HPLC respectively. However, when HbA2 level is normal or low along with normal or low serum iron studies, microcytosis can be a diagnostic dilemma. Walford et al. and Pearson et al.3.7 is the commonest reported genotype which exists in α + (-α/αα) milder form.0- and α-thalassemia deletions in such cases when the Hemoglobin or microcytosis does not improve appropriately after iron supplementation.The α-thalassemia syndromes are among the most common single-gene disorders with more than 20% of the world population to be a carrier of some form of α–thalassemia, as estimated by The World Health Organization (WHO).The mutation spectrum for each population group is distinct characterized by a small number of founder mutations that reflect the predominant mutant alleles in carriers and affected individuals.3.7, -α 4.2,- - α SA and - - α SEAdeletions are done.Based on previous studies in India in our laboratory, gap PCR for - α Microcytic hypochromic anemia is the hallmark feature of α thalassemia, and the degree of microcytosis is directly proportional to the number of alpha genes deleted, thus, reflecting the rate of imbalance between α- and β-chain expression.This study was undertaken to identify and highlight the presence of common α thalassemia deletions in cases of microcytic anemia and its interaction with IDA.3. Only cases with standard Hb HPLC showing low or normal HbA2 (< 3.5 %) were taken to exclude βTT/Hemoglobinopathy.Fifty-four Patients, attending Hematology outpatient department, All India Institute of Medical Sciences, New Delhi were included in the study that was approved by institutional ethical committee. Duration of the study was 1.5 years between 2010 and 2011. The cases included had Hemoglobin <12 gm/dl, MCV<80fl and TRBC > 4.5 M/mm3 ml blood in EDTA for CBC and Hb HPLC and 1 ml blood in 3.2% sodium citrate for DNA studies was obtained after taking signed consent from the patients.Hematological parameters were analyzed by automated cell counter . Quantification of HbA2 was performed by High Performance Liquid Chromatography . Serum Iron studies were performed by standard laboratory methods and transferrin saturation (TS) <16% was taken to be iron-deficient.3.7 and -α 4.2 Baysal et al.- - αSA deletion Shahji et al.,- - αSEA Chang et.al. was done.27Genomic DNA was prepared from peripheral blood by the standard phenol-chloroform extraction method. Deletion mutations were characterized by Gap-PCR. Detection for single deletion - α Though it was intended to be a cross-sectional study, the Follow-up data on Iron therapy available were evaluated in 27 (54%) cases.t-test was used for comparison of hematological parameters. Statistical significance was assessed as a test with p ≤ 0.05. The hematological parameters of three alpha thalassemia mutations, the four groups based on mutation status and TS </> 16 % were also compared, but the size in each group was too small for obtaining a valid statistical comparison.The following parameters were assessed: Hemoglobin, MCV, TRBC, RDW-CV, TS and HbA2. Mutation positive and negative groups were compared. Statistical analysis was carried out using the statistical package SPSS version,Of the 54 cases included in the study, 32 (57.4%) were males and 22 (42.6%) females and the median age was 19.5 years (13 months – 62 years). There were four pediatric cases (<18 years) in alpha positive genotype group and seven in wild alpha genotype group. The subjects were, for the most, anemic (median Hemoglobin was 9.6 g/dl (5.1–12g/dl), with RBC showing microcytosis (median MCV63.4fl (48–79 fl) and iron deficiency (median TS 10.2% (7.6–19.2%). They had TRBC median 5.2M/mm3 (4.5–6.2M/mm3), median RDWCV 19.3 %( 14–29%) and median HbA2 2.3 %( 1.6–2.8%).3.7 heterozygous, three were α 3.7 homozygous and one was - - α SA double deletion , compared to the median, 68.9fl (47.6–80fl) (29.6%), of the subjects -α3.7deletion heterozygous and to the value, 71.6fl, of the one subject (1.9%) –with α SA thalassemia.Nineteen out of 54 35.2 %) cases showed α thalassemia deletions and three genotypes were seen on gel electrophoresis. Fifteen were –αdeletion  and 2. C % cases There were high RDW-CV and low %TS indicating coexisting IDA in 10 of the 19 mutation-positive cases. On comparing the hematological parameters of α thalassemia mutation-positive and negative cases . The meaThe median Hemoglobin, MCV, and HbA2 were lower in both the iron deficient groups 1and three, but the values were relatively much lower in the mutation-negative group 1which was predominantly IDA. TRBC was high in both the groups. Between groups 2 & 4 which were both iron sufficient (i.e. TS > 16%) the median Hemoglobin, MCV, TRBC, and HbA2 were lower in group 4 (mutation positive) compared to group2. The RDW-CV was comparable indicating the coexisting IDA in both the groups. This shows that MCV is lower in α thalassemia compared to Normal. Follow up on Iron was available in 27(54.00%) cases. Response was seen predominantly (63.1%) in group 1 as expected but 36.8% in this group also failed to respond no details about the compliance or cause of refractoriness was available. Subjects of Group 2 did not require iron, but they needed to be investigated for other underlying Hemoglobinopathy, which was beyond the scope of this study. In mutation-positive group 3(50%) patients showed no or inadequate response to iron therapy while individuals in group 4 had % TS >16%and did not need iron supplementation. The family history too was significant only in individuals of the group 4.rd (35.2%-19/54cases) of the microcytic cases. These results agree with 50% α-thalassemia cases reported in non-anemic microcytosis cases by Borges et al.3.7 deletion was the commonest determinant in the study seen predominantly in the heterozygous state (31.7% of microcytic patients)..SA was detected incidentally.4.2 , -- α SEA deletion respectively have been reported in Indian subjects by Sarkar et al.7Fifty-four subjects with microcytosis but with normal Hb HPLC and normal or low Iron studies were evaluated for the presence of common α-thalassemia deletion mutations. Microcytosis was the defining criteria in the study, but it is also important to remember that normal RBC indices do not rule out α thalassemia carrier.3.7 deletion homozygous individuals compared to (29.6%)-α3.7 deletion heterozygous and one (1.9%) -- α SA thalassemia subjects. Though insufficient in number, the findings are largely in concordance with previous reports, where microcytosis has been explained on the basis of α-gene number.SA), nearly 1/3 (35.2%) of the 54 cases of microcytic anemia with or without IDA could be typed in the study. Finding of IDA should not be a deterrent for the screening of a coexisting α thalassemia mutation, which is particularly relevant in a country like India where both α thalassemia  and IDA occur with high frequency. Larger population studies, including other less common α thalassemia mutations, are required not only to identify the population group specific mutation spectrum but also to observe the interplaying of the two conditions on the RBC indices. In fact red cell indices could differentiate or indicate the coexistence of the two conditions. Until then molecular studies (gap-PCR) should be done for the detection of common α thalassemia mutations especially when the Hemoglobin or microcytosis does not improve appropriately on iron supplementation this can be useful in the countries where α thalassemia is not prevalent because as a consequence of population migrations alpha thalassemia has acquired a truly global distribution.Phenotypically all the 19 α thalassemia cases were very similar presenting with only mild anemia. None of the cases had jaundice or gall stone disease. Family history was positive in four individuals. Microcytosis was the defining criteria and was most pronounced in the three (5.6%) -αpared to .6%-α3.7 pared to 9.6%-α3.7


CVACO2 gap) has been proposed to better identify patients with persistent global hypoperfusion.The early postoperative period after liver transplantation is characterized by a complex hemodynamic situation, due to the interaction of multiple factors, as the persistence of the hyperdynamic circulation or the increase of lactate blood levels because of the impaired hepatic clearance.The central venous-to-arterial carbon dioxide difference , hemodynamic variables, and those related to graft function and postoperative complications were recorded.Data statistical analysis was done using SPSSâ software. Quantitative variables are expressed as mean with standard deviation (SD) and qualitative variables as absolute and relative frequencies. The statistical significance of the proportion comparison was addressed by the chi-square test or Student´s t-Test with a statistical significance p < 0.05.CVACO2 gap > 6 mmHg, simultaneously with a cardiac index >3 l/min/m2 and a mixed venous oxygen saturation >60%. Delayed extubation was associated with an elevated PCVACO2 gap in 30,8% of the cases vs. 0% in those with a normal PCVACO2 gap (p = 0.98). An elevated PCVACO2 gap was associated with longer ICU stay  and hospital stay .During the study period, 24 patients were admitted to the ICU after a liver transplantation  with a mean age of 55,9 ± 10 years. Mean MELD score was 15 ± 9; Mean Child-Pugh was 8 ± 3. Mean ICU stay was 8 ± 8 days, with an overall mortality of 8%. Mean hospital stay was 18 ± 8 days. Early graft dysfunction incidence was 12,5%. Delayed extubation (>24H) incidence was 16% meanwhile acute renal incidence was 58% . Two patients (8%) presented infectious complications and four 4(16%) developed seizures. During the 72 first hours of ICU stay, 13 patients (54% of the sample) presented a PCVACO2 gap is a feasible hemodynamic parameter that could predict a worse clinical outcome.In the early postoperative care after liver transplantation, the P


A global minimum search using a genetic algorithm and density functional theory calculations confirms that such onion-like three-shell structures are the ground states for these A21B12 binary clusters. All of these icosahedral matryoshka clusters, including two previously found ones, i.e., [As@Ni12@As20]3− and [Sn@Cu12@Sn20]12−, follow the 108-electron rule, which originates from the high hI symmetry and consequently the splitting of superatom orbitals of high angular momentum. More interestingly, two magnetic matryoshka clusters, i.e., Sn@Mn12@Sn20 and Pb@Mn12@Pb20, are designed, which combine a large magnetic moment of 28 µB, a moderate HOMO-LUMO gap, and weak inter-cluster interaction energy, making them ideal building blocks in novel magnetic materials and devices.We propose a series of icosahedral matryoshka clusters of A@B Interst12@As20]3−, isolated as its n-Bu4P+ salt and structurally characterized by X-ray diffractionhI) symmetrical three-shell matryoshka doll structure consisting of an outer As20 regular dodecahedron encapsulating an Ni12 icosahedron, which in turn encapsulates an As3− trianion . This [Sn@Cu12@Sn20]12− anion is exactly isoelectronic with the [As@Ni12@As20]3− anion with 108 valence electrons beyond the filled d10 shells of the tin and copper components. Similar bonding models appear to be applicable to both the [As@Ni12@As20]3− and [Sn@Cu12@Sn20]12− systems. Furthermore, the observation of the icosahedral matryoshka doll structure in both the soluble [As@Ni12@As20]3− anion and the [Sn@Cu12@Sn20]12− anion in the A12Cu12Sn21 intermetallics suggests that such structures are particularly favorable in clusters where 108 total valence electrons are available from the elements composing the three layers of the matryoshka structure.Initially the icosahedral matryoshka 3− and [Sn@Cu12@Sn20]12−, all of these clusters show high stability and alignment of superatom orbitals. In particular, the Sn@Mn12@Sn20 and Pb@Mn12@Pb20 clusters possess a giant magnetic moment of 28 μB, which can be retained in cluster assemblies. Our results thus extend the scope of icosahedral matryoshka clusters and provide novel building blocks for cluster-based materials and devices exhibiting fascinating novel magnetic and optical properties.Stimulated by the above-mentioned progresses, here we propose a series of icosahedral matryoshka clusters, i.e., A@B12@A20 clusters  are summarized in xBy, its formation energy is defined by: Ef (AxBy) = E(AxBy) – xE(A) – yE(B), where E(AxBy) is the energy of AxBy cluster, E(A) or E(B) is the energy per atom of the constituent element A or B in the standard states, x or y is the number of A or B atoms in the cluster. Detailed information on geometry parameters and on-site charges for these clusters are given in The binding energies, formation energies, HOMO-LUMO gaps, and optical absorption gaps for icosahedral matryoshka A@B38, 0.281 eV/atom for Sn38, 0.576 eV/atom for Mg38, 0.497 eV/atom for Zn38, and 0.385 eV/atom for Cd38 clusters, respectively. Using global optimization combined with DFT calculations, Ferrando's group13@Mg20 that is isostructural to the clusters considered here. However, the formation energy of this Ni13@Mg20 cluster is as high as 0.468 eV/atom from our DFT calculation. The more favorable formation energies of the present icosahedral matryoshka superatoms clearly demonstrate the stabilization effect of electronic structure, which will be discussed in detail later.All the clusters explored here show reasonably low formation energy , indicating that they are easy to form thermodynamically. For comparison, we consider several 38-atom elementary metal clusters with a fcc-like truncated octahedron structure20 (3.073 Å) and Sn20 (2.891 Å) cages from our DFT optimization. Therefore, the enlarged Pb20 and Sn20 cages are stabilized by the encapsulated C@B12 icosahedron , as previously found12@As20]3−. It is also interesting to compare the structural parameters of Sn@Zn12@Sn20 with its isoelectronic counterpart, i.e., [Sn@Cu12@Sn20]12−. The theoretical Sn-Sn bond lengths in Sn@Zn12@Sn20 of 3.278 Å are comparable to that of 3.290 Å for [Sn@Cu12@Sn20]12− computed with the same level of theory. Note that the experimental Sn-Sn bond lengths of [Sn@Cu12@Sn20]12− in the condensed phase obtained by X-ray crystallography range between 3.076 to 3.133 ÅNote that the lengths of Pb-Pb (3.317 to 3.578 Å) and Sn-Sn (3.176 to 3.463 Å) bonds listed in According to the on-site population analysis see , the B a21B12 clusters with B = Mg, Zn, Cd range from 1.27 to 1.54 eV, comparable to those of [As@Ni12@As20]3− (1.44 eV) and [Sn@Cu12@Sn20]12− (1.39 eV) using the same computational scheme. Previous DFT calculations yielded similar band gaps for [As@Ni12@As20]3− (1.44 eV12@Sn20]12− (1.34 eVog) and the associated energy levels are summarized in og by TD-DFT ranges from 1.72 to 1.94 eV , making them potentially useful as nanoscale building blocks for optoelectronic devices25As shown in − 1.44 eV and [Sn@− 1.34 eV. The hig12@As20]3− and [Sn@Cu12@Sn20]12−, are exactly isoelectronic with 108 valence electrons beyond the filled d12@A20 clusters considered here, noting that Mg has no d electrons. However, 108 does not explicitly belong to any existing magic numbers for various models, e.g., the shell model within the jellium approximationAs aforementioned, the experimentally found icosahedral matryoshka clusters, i.e., 3−, [Sn@Cu12@Sn20]12−, Sn@Mg12@Sn20, and Sn@Zn12@Sn20, are compared in The energy levels for the four icosahedral matryoshka clusters, including − cluster, iso-structural to the current icosahedral matryoshka, was also predicted to possess a giant magnetic moment of 44 μB12@Au20 is 0.795 eV/atom, about twice of Sn21Mn12. Again, this indicates that design of highly stable superatoms requires the consideration of electronic structures, i.e., closure of electronic shell or subshell. Also note that Sun et al.12@Au42 in which the magnetic moment on Fe atom (3 μB) is enhanced over bulk value, while Wu and Jena12C6 (14 μB), Mn12C6 (38 μB), and Mn24C18 (70 μB).Previously, an [Mn@Mn12] single molecular magnet, i.e., Mn12O12(CH3COO)16(H2O)4, which possesses a magnetic moment of 20 μB21Mn12 and Pb21Mn12 can be regarded as a kind of ligand-free single molecular magnet of [Mn12] with slightly enhanced magnetic moment (28 μB vs. 20 μB). The high symmetries, appreciable HOMO-LUMO gaps, and giant magnetic moments makes Sn21Mn12 and Pb21Mn12 clusters promising building units for novel cluster-assembled magnetic materials and devices, which have potential applications in high-density information storageIn addition, a [Mn812@Sn20 and Pb@Mn12@Pb20. The atomic structure and binding curve for [Pb@Mn12@Pb20]2 dimer are shown in d = 3.787 Å for [Sn@Mn12@Sn20]2 and d = 4.153 Å for [Pb@Mn12@Pb20]2, see 12@Sn20]2 and 0.291 eV for [Pb@Mn12@Pb20]2. Such weak cluster-cluster interaction might be attributed to the moderate HOMO-LUMO gap as well as the sphere-like icosahedral geometry of the clusters. Most excitingly, the total magnetic moments of both cluster dimers are exactly twice of the individual ones, i.e., 56 μB. Indeed, the on-site local moments in the cluster dimers are barely affected by the neighboring cluster  binary clusters. In addition, the high stabilities of these spherical matryoshka clusters are demonstrated by their large HOMO-LUMO gaps and low formation energies. The molecular orbitals for different icosahedral matryoshka clusters share the same pattern of superatom orbitals. The “magic number” of 108 electrons can be attributed to the high hI symmetry and consequently the splitting of superatom orbitals of high angular momentum. Such “108-electron rule” should be extendable to other icosahedral matryoshka superatoms with different charge states, which might lead to future experimental discoveries of new metal clusters in solution as well as novel solid-state intermetallic compounds. Two related magnetic icosahedral matryoshka clusters, namely Sn@Mn12@Sn20 and Pb@Mn12@Pb20, are predicted to exhibit large magnetic moments (28 μB) and moderate HOMO-LUMO gaps. More impressively, after formation of cluster assemblies, these two clusters are able to keep their identities and retain their magnetic moments due to weak inter-cluster interaction. These novel binary clusters with unique icosahedral nesting doll geometry, high thermodynamic stabilities, and interesting physical properties , are expected to be useful building blocks in future nanoscale materials and devices.A series of three-shell icosahedral matryoshka clusters related to the experimentally known [As@NiAb initio calculations were performed using the spin-polarized density functional theory as implemented in the DMol3 program12@A20  clusters to obtain the optical adsorption spectra and optical gaps.21B12 clusters , we performed an unbiased global minimum search of two representative clusters (Sn21Mg12 and Sn21Zn12) using a genetic algorithm (GA) incorporated with DFT calculations implemented in the DMol3 package, which was described in our previous publication12@Sn20 and Sn@Zn12@Sn20 clusters as the ground state. In addition, some metastable isomers (To confirm that the three-shell icosahedral configuration in  isomers  are founJ.Z. designed the models and calculations. X.H., J.Z. and Y.S. did the calculations. X.H. and J.Z. prepared all the figures. J.Z., Z.C. and R.B.K. wrote the manuscript. All the authors discussed the results and commented on the manuscript.Supplementary information


Hypersensitivity to H2O2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1.Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H For colony formation assay, 200 cells from exponentially growing culture were seeded into 60-mm dishes. After incubation for 3 h, H2O2 was added to each dish and treated for 30 min at 37°C. After incubation for 7 d, colonies were fixed with methanol, stained with 2% Giemsa solution and counted. Colony numbers were normalized to that of mock controls.HFour hundred log-phase cells in Eagle's MEM were seeded into a 100-mm dish. After incubation for 3 h, cells were exposed to X-rays at 0.5, 1, 2, 4 and 6 Gy  at a dose rate of 0.465 Gy/min. After incubation for 7 d, colonies were fixed with methanol, stained with 2% Giemsa solution, and counted under a microscope. Colony numbers were normalized to that of mock-irradiated controls.6 cells/ml) in PBS(-) was mixed with a 1/5 volume of 1.2% TritonX-100-containing PBS(-). After centrifugation at 3000×g for 5 min at 4°C, the nuclei precipitate was washed with PBS(-) containing 0.2% TritonX-100 and incubated in the presence of RNaseA (50 μg/ml) at 37°C for 30 min. After centrifugation at 3000×g for 5 min at 4°C, the nuclei pellet was washed with PBS(-) containing 0.2% TritonX-100 and resuspended and stained in PBS(-) containing 0.2% TritonX-100 and 10 μg/ml PI at 37°C for 20 min. After passing through nylon mesh, nuclei were analyzed using a FACS Calibur .Cells were incubated in Eagle's MEM without serum for 24 h and then in Eagle's MEM for 15 h. Mitotic cells were collected by mitotic shake off, and the cell suspension was centrifuged at 1000×g for 5 min. After washing with PBS(-), the cells were resuspended in Eagle's MEM and incubated for 3 or 10 h to obtain a G1 or S phase-dominant population, respectively. The synchronized cell suspension  and 59% in mouse L cells (L cell-KD), compared with control cells with empty vectors  heterotrimer and each monomer protein of 9-1-1 stimulate hNEIL1 activity and interact with the hNEIL1 C-terminal domain . With rein vitro and in vivo, and interaction of the C-terminal region of hNEIL1 and the BRCT domain of PARP1 inhibits hNEIL1 incision activity in a concentration-dependent manner. These results suggest that hNEIL1 is not a simple glycosylase/AP-lyase, but plays multiple roles in a highly organized DNA damage control system.Several BER proteins, including DNA polymerase β, XRCC1, DNA ligase IIIα and FEN-1, have also been shown to interact with hNEIL1 , 24, 26,2O2  Grant-in Aid for Scientific Research (KAKENHI) Grants No. 20510054 and No. 25340036.


In vivo, ophiobolin O suppressed tumor growth and showed little toxicity in mouse xenograft models. Overall, these findings provide theoretical basis for the therapeutic use of ophiobolin O.Ophiobolin O is a member of ophiobolin family, which has been proved to be a potent anti-tumor drug candidate for human breast cancer. However, the anti-tumor effect and the mechanism of ophiobolin O remain unclear. In this study, we further verified ophiobolin O-induced G1 phase arrest in human breast cancer MCF-7 cells, and found that ophiobolin O reduced the phosphorylation level of AKT and GSK3β, and induced down-regulation of cyclin D1. The inverse docking (INVDOCK) analysis indicated that ophiobolin O could bind to GSK3β, and GSK3β knockdown abolished cyclin D1 degradation and G1 phase arrest. Pre-treatment with phosphatase inhibitor sodium or thovanadate halted dephosphorylation of AKT and GSK3β, and blocked ophiobolin O-induced G1 phase arrest. These data suggest that ophiobolin O may induce G1 arrest in MCF-7 cells through interaction with AKT/GSK3β/cyclin D1 signaling.  Aspergillus ustus 094102 [Ophiobolins belong to the family of natural sesquiterpenes that are characterized by a tricyclic (5-7-5) ring system. Ophiobolins as secondary metabolites that are produced by pathogenic fungi, show a broad spectrum of inhibitory activity against nematodes, fungi, bacteria and cancer cells ,2,3. OphIn previous study, we found that the low concentration of ophiobolin O (0.1 μM) down-regulated the expression of resistance-related protein P-Glycoprotein , which makes Adriamycin-resistant human breast carcinoma (MCF-7/ADR) cells more sensitive to Adriamycin treatment. Therefore, we demonstrated that ophiobolin O reversed Adriamycin resistance, and suggested ophiobolin O acted as a potential agent to reverse chemotherapy drug resistance . On the In the current study, we further verified ophiobolin O-induced G1 phase arrest in MCF-7 cells. Then we investigated the putative protein targets of ophiobolin O using INVDOCK program. Three apoptotic or G1 phase arrest-related proteins were extracted. Two of them were closely related to cell apoptosis, and could validate our previous study . The resAspergillus ustus 094102 inhibited the growth of human breast cancer cells [50 value of 13.45 ± 1.26 μM. The IC50 values were 84.96 ± 11.73 μM, 16.48 ± 2.68 μM and 13.55 ± 1.01 μM when ophiobolin O treated HUVEC, DU145 and NCI-H460 cells, respectively. It is worth noting that normal human umbilical vein endothelial cells (HUVEC) are less sensitive to ophiobolin O compared with other cancer cell lines  analysis was applied to identify the possible targets of ophiobolin O. Using the INVDOCK program, 90 putative proteins were extracted from the Protein Data Bank. Of these, three proteins were closely related to apoptosis and G1 phase arrest . TNF  was detected in control cells, and showed marked time-dependent decrease after ophiobolin O treatment A, consisAll above strongly suggest that ophiobolin O may target AKT/GSK3β/cyclin D1 signaling. The increase of active form of GSK3β accelerates cyclin D1 degradation, and inventually induces G1 phase arrest in MCF-7 cells. Cyclin D1 regulation and the probable mechanisms of ophiobolin O-induced G1 phase arrest are characterized in DMEM, fetal bovine serum, penicillin and streptomycin were purchased from Biowest . Cell cycle detection kits were provided by Nanjing KeyGen Biotech Co. Ltd. . GAPDH was obtained from Tianjin Sungene Biotech . Antibodies against cyclin D1 (#2926), p-cyclin D1 , GSK3β (#9315) p-GSK3β , CDK4 (#2906) and CDK6 (#3136) were purchased from Cell Signaling Technology . siRNA used for silence GSK3β gene was purchased from Shanghai Gene Pharma Co., Ltd .Aspergillus ustus 094102. The compound was dissolved in 100% DMSO and stored at −20 °C, then diluted with cell culture media before using. The final DMSO concentration did not exceed 0.1%. Human breast cancer cell line MCF-7 was purchased from the cell bank of the Shanghai Institute of Cell Biology ,and was cultured in DMEM with 10% fetal bovine serum and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin) in a humidified 5% CO2 incubator at 37 °C.Ophiobolin O is a natural compound that has been isolated from Cells were seeded into 96-well plates at 4 × 104 cells/mL, incubated for 24 h, and then treated with the indicated concentrations of ophiobolin O  for 48 h. Cell viability was determined using MTT assay.Cells were harvested, washed twice with PBS, fixed in 70% ethanol and stored at 4 °C overnight, then washed with PBS, incubated with RNase at 37 °C for 30 min, and stained with PI (1 mg/mL) in the presence of RNase A for 30 min. Cell cycle phase analysis was performed by using a FACScalibur flow cytometer.g centrifugation for 15 min, the protein content of supernatant was quantified using BCA protein assay kit . Equal amounts of the protein samples were separated by SDS-PAGE, and transferred to nitrocellulose membranes using an electro-blotting apparatus . Then the membranes were blocked in blocking buffer (TBST plus 5% skimmed milk), and incubated with primary antibodies overnight at 4 °C. After that the membranes were washed with TBST and incubated with HRP (horseradish peroxidase)-conjugated secondary antibodies for 1.5 h at 4 °C. Protein expression was visualized using the chemoluminescence reagent  and detected on photographic film.Cells were lysed in Western blotting lysis buffer  at 4 °C for 30 min. After 12,000× Activity of GSK3β was determined by the GSK3β enzyme activity detection kit  according to manufacturer’s instructions. Briefly, the samples of drug-treated cells were rinsed by reagent A and then re-suspended with the extraction buffer reagent B. After 30 min incubation on ice, the mixture was centrifuged at 4 °C. The supernatant was collected and then assayed for enzyme activity, by mixing 65 μL reagent C, 10 μL reagent D, 10 μL reagent E and 10 μL reagent F and incubation at 30 °C for 3 min. Immediately after the addition of 5 μL supernatant to the reagent mixture, the optical density was measured at 340 nm using a Microplate Reader at 0 min and 5 min. The activity was measured as the difference between the absorbance value at 0 and 5 min [To verify the proteins related to possible Ophiobolin O targets, a flexible ligand-protein inverse docking program, INVDOCK was used to identify putative protein targets for Ophiobolin O. The 3D structure of Ophiobolin O was input into the INVDOCK program. The software automatically searched for protein cavities derived from 3D structures of all candidate proteins. An energy value statistically derived from the analysis of a large number of PDB ligand-protein complexes was used as a threshold for screening likely binders. A human protein was considered as a putative target of Ophiobolin O if the molecule could be docked into the protein and the binding satisfied a molecular-mechanics based criterion for chemical complementarity .Cells were plated in six-well plates, grown for 24 h, then transfected with 80 pmol of siRNA for 12 h using Lipofectamine 2000 reagent and OPTIMEM reduced serum medium . The cells were assayed within 72 h after transfection.6 cells s.c. into the right armpit of five-week old BALB/c female athymic mice . The mice were randomized into vehicle control and treatment groups when xenografts were palpable after 10 days. Vehicle or drugs were administered i.v. every four days until sacrifice; body weight and tumor size were measured and recorded at the same time. Tumor size was measured using electronic caliper, and the tumor volumes were calculated using the formula: length × width2/2. On 50 days, mice were sacrificed; tumors were collected, weighed, and photographed. Tumor inhibition effect was calculated using the following equation: tumor suppression (%) = (1 − T/C) × 100, where T is the average tumor weight of the treated group and C is that of the control group.The mouse xenograft model was established by injection of 5 × 10In vivo, ophiobolin O suppressed tumor growth and showed little toxcity in mouse xenograft models.Ophiobolin O induces G1 phase arrest in human breast cancer MCF-7 cells. It targets GSK3β and interacts with GSK3β/cyclin D1 signaling pathway to induced growth inhibition.


Helicoverpa armigera esterases, known as Clade 001, was implicated in organophosphate and pyrethroid insecticide resistance due to their overabundance in resistant strains. In this work, a novel carboxylesterasegene 001D of H. armigera from China was cloned, which has an open reading frame of 1665 nucleotides encoding 554 amino acid residues. We used a series of fusion proteins to successfully express carboxylesterase 001D in Escherichia coli. Three different fusion proteins were generated and tested. The enzyme kinetic assay towards 1-naphthyl acetate showed all three purified fusion proteins are active with a Kcat between 0.35 and 2.29 s−1, and a Km between 7.61 and 19.72 μM. The HPLC assay showed all three purified fusion proteins had low but measurable hydrolase activity towards β-cypermethrin and fenvalerate insecticides (specific activities ranging from 0.13 to 0.67 μM·min−1·(μM−1·protein)). The enzyme was stable up to 40 °C and at pH 6.0–11.0. The results imply that carboxylesterase 001D is involved in detoxification, and this moderate insecticide hydrolysis may suggest that overexpression of the gene to enhance insecticide sequestration is necessary to allow carboxylesterases to confer resistance to these insecticides in H. armigera.Carboxylesterasesare an important class of detoxification enzymes involved in insecticide resistance in insects. A subgroup of  Helicoverpa armigera, is a major crop pest in many parts of the world [The cotton bollworm, he world ,2,3,4. Ihe world ,6,7,8. Che world ,12,13,14he world ,16,17,18he world ,21,22,23H. armigera, a correlation between enhanced esterase activity and pyrethroid resistance was found in Australia, Asia, and Africa [H. armigera GR strain, and they were figured into a lot of subgroups according to a phylogenetic analysis [H. armigera esterases derived from the susceptible GR strain, eight carboxylesterases belonging to the CCE Clade 001 were expressed in Sf9 insect cells, which showed tight binding to OPs and some hydrolytic activity towards OPs [H. armigera YGF strain from China, two unique isozyme bands in native polyacrylamide gel electrophoresis (PAGE) were matched with four carboxylesterase genes, 001A, 001D, 001I, 001J by mass spectrometry analysis, shown to be overexpressed up to 90-fold, as compared to the susceptible SCD strain [H. armigera strain (MonoR) from China also showed that the overexpression of six Clade 001 enzymes, including 001D, was associated well with monocrotophos resistance [H. armigera were involved in resistance to OP and pyrethroid insecticides via enhanced sequestration, favoured by overexpression. Nearly all published studies focusing on the hydrolytic activity of H. armigera carboxylesterases towards OP and pyrethroid insecticides used the enzymes heterologously expressed in insect Sf9 cells with the baculovirus expression system [Lucilia cuprina and the juvenile hormone esterase gene (Nljhe) from Nilaparvata lugens have also been expressed in the baculovirus expression system [E. coli system is a preferred expression system because of its extensive characterization and ease of handling [E. coli [H. armigera carboxylesterases from the susceptible GR strain showed no esterase activity when expressed in E. coli, using the Gateway-compatible expression vector with their native start and stop codons [In d Africa ,25,26,27d Africa . Thirty-analysis . In mostards OPs ,30. Howeards OPs ,30. In tD strain . Additiosistance .They shon system ,30,31. In system ,32. Howehandling . Some in[E. coli ,35,36. Ip codons .001D) encoding a carboxylesterase from an insecticide-susceptible H. armigera strain from the Wuhan region of China (WH). In order to obtain active enzyme, we heterologously expressed three fusion proteins containing three different solubility/affinity tags in the E. coli cells. We purified each fusion protein and evaluated their hydrolytic activities towards a model substrate  and two pyrethroid insecticides . We demonstrated that all three purified proteins of 001D were capable of metabolizing two real insecticide substrates. Moreover, the effect of temperature and pH on the enzyme activity was also explored, and the mechanism of 001D involved in pyrethroids resistance is further discussed.In this study, we sequenced a cDNA (001D was cloned from a cDNA library of the midgut of H. armigera from the susceptible Wuhan (WH) strain. The novel cDNA sequence of 001D (GenBank® accession number KT345935) has an open reading frame of 1665 nucleotides, encoding 554 amino acid residues with a molecular weight of 62.6 kDa, and an isoelectric point of 5.27. It includes a signal peptide containing a cleavage site between the 16th and 17th amino acids. The alignment showed that carboxylesterase 001D has highly conserved residues of a catalytic triad (S202-H443-E330) and a pentapeptide termed the nucleophilic elbow (G200-S201-S202-A203-G204) (® accession number ADF43460) from Australia [® accession numbers ADE05550 and ADE05555) from China, respectively [Thecarboxylesterase gene 03-G204) . 001D al03-G204)  showed tustralia . Additioectively .001D gene without signal peptide was inserted in three types of expression vectors, pE1, pET30a, and pET32a, and expressed in the E. coli BL21 (DE3) strain. Three different fusion proteins, 6×His/001D (pE1), 6×His/S-tag/001D (pET30a), and Trx/6×His/S-tag/001D (pET32a) were purified using Ni2+-Nitrilotriacetic acid (NTA) resin .The coding sequence of the A) resin . The purA) resin A. The anA) resin B. By conKm of 7.61 μM and a kcat of 2.29 s−1, showing it has a high affinity and turn-over to the substrate compared to those of the recombinant 001D from pE1 and pET30a. The fusion protein of 001D from pE1 had a very poor affinity and low turn-over to the substrate. In contrast, the purified protein of 001D from pET30a showed moderate esterase activity towards 1-NA with a specific activity of 0.57 μM·s−1·(μM−1·protein). The results suggest that the three types of fusion tags have different effects on enzymatic activity of the carboxylesterase 001D, among them, the larger fusion tag, Trx-tag, has strongest enhancing effect on the esterase activity of 001D towards the model substrate.Activities of the purified recombinant enzymes towards the 1-naphthyl acetate (1-NA) were determined and shown in β-cypermethrin and fenvalerate substrates. The purified fusion proteins of 001D from pET32a showed relatively lower hydrolase activity towards the two real insecticides, as compared to the protein from pET30a. However, the specific activities of 001D from pET30a are higher, 0.41 and 0.67 μM·min−1·μM−1 towards the two pyrethroid substrates, respectively. For the β-cypermethrin substrate, the hydrolysis rate is 2-fold higher than that from pET32a and pE1. For the fenvalerate substrate, the hydrolysis rate is 2- and 5-fold higher than that from pET32a and pE1, respectively  of the enzyme [H. armigera may not occur through the first mechanism. Our results show the 001D can catalyze the pyrethroid substrates with a slow velocity, which probably suggests the overexpression of the carboxylesterase genes is indispensable to allow carboxylesterases to confer resistance to pyrethroid insecticides in H. armigera.In this work, the results of pyrethroid hydrolysis analysis show that the purified proteins of 001D can metabolize the two real insecticide substrates, e enzyme ,30, sugg2+-NTA affinity purification, pET30a-001D showed a high purity as only a single band was observed in the SDS-PAGE and Western blot assay  and the hydrolytic activity towards the real insecticides (pyrethroids) are mainly due to the differences in their chemical structure between the model substrate and the real insecticides [However, among the three fusion proteins, the fusion protein 6×His/S-tag/001D (pET30a) was expressed at a higher level compared to other two fusion proteins, Trx/6×His/S-tag/001D (pET32a) and 6×His/001D (pE1), and after a single step of Niot assay . Its hydot assay . The rescticides ,16,43.Cydia pomonella [In most cases, enzyme activity can be easily influenced by the variation of temperature and pH value due to each enzyme only functioning efficiently within a suitable temperature and pH range. In the present study, the purified enzyme 001D showed high activity and stability over a broad range of temperatures. Its residual activity was 46.8% when it was incubated at 45 °C for 1h. It appears more stable than the carboxylesterase CpCE-1 from omonella . Howeveromonella .H. armigera WH strain expressed in E. coli are different from those of the carboxylesterase 001D of the susceptible GR strain from Australia and the susceptible YG strain from China, expressed in the baculovirus system [β-cypermethrin is lower than that of the 001D from the GR strain, but it is to some extent closer to that of 001D from the YG strain (the specific activity is about 0.76 and 2.60 μM·min−1·(μM−1·protein)) towards the insecticidal pyrethroid isomers, 1R trans-αS and 1R cis-αS, respectively [−1·(μM−1·protein),which is between the GR strain (0.81 μM·min−1·(μM−1·protein)) and the YG strain (0.0 μM·min−1·(μM−1·protein)) [E. coli cells, and the activity assays were performed using highly purified fusion proteins. We further observed that different fusion strategies have effects on protein expression and enzymatic activity. Furthermore, in previous work, the insecticides used in the hydrolytic activity assay were single isomers of cypermethrin and fenvalerate, but in this work both β-cypermethrin and fenvalerate substrates are in a complex of four isomers.We also noted that the pyrethroid hydrolase activities of the carboxylesterase 001Dof the susceptible s system ,30,31. Brotein)) ,31. In tβ-cypermethrin  and fenvalerate (pEASY®-Blunt E1 vector (pE1) was obtained from TransGen Biotech , pET30a and PET32a were purchased from Novagen .The insecticides valerate  were of H. armigera strain from the Wuhan region of China (WH strain) was originally collected from the Wuhan region of the Hubei Province in China over 30 years ago and was maintained on an artificial diet without outcrossing at 28 °C ± 1 °C, 60% ± 10% relative humidity, and a photoperiod of 16:8 (Light:Dark). Adult moths were held under the same temperature and supplied with a 5% sugar solution.The susceptible ® accession number FJ997295). The 50 μL PCR reaction mixture contained 2 μL of template cDNA, 3.0 μL of 10 μM of each primers, 4.0 μL of 2.5 mM dNTP, 0.5 μL of PrimeSTAR HS DNA Polymerase, and 10 μL of 5× PrimeSTAR Buffer (Takara). The thermocycler program was 98 °C for 30 s, followed by 30 cycles of 98 °C for 12 s, 60 °C for 25 s, 72 °C for 2 min, and with a final extension of 72 °C for 10 min. The PCR products were gel-purified, ligated into the pGEM-T easy vector after an adenine was added at the 3′ terminus, and used to transform JM109 cells. White clones were sequenced  and were found to contain a 001DcDNA that differed by 11 amino acid residues from the closest homologue in the database. The sequence of this new allele was deposited in the NCBI database (GenBank® accession number KT345935).Total RNA was extracted from the midguts of fifth-instarlarvae by using the SV Total RNA Isolation System Kit (Promega). First-strand cDNA was synthesized from 1 μg of DNA-free total RNA, using RevertAid M-MuL V Reverse Transcriptase . Primers  for amplifying the open reading frame (ORF) of the gene were designed based on the sequence deposited in NCBI database  containing EcoRI and XhoI restriction endonuclease recognition sites (underlined). The amplicons were purified using the Universal DNA Purification Kit , digested with the corresponding endonuclease and ligated into either pET30a or PET32a using T4 DNA ligase. Plasmids were verified by restriction digestion and DNA sequencing (Invitrogen™). The amplicons were also directly ligated into pE1 Expression Vector according to the manufacturer’s protocol, and then verified by sequencing. The plasmids (E. coli BL21 (DE3) to produce recombinant proteins, each containing a 6×His-tag at N-terminus for purification.The carboxylesterase ORF encoding the mature enzyme lacking a signal peptide sequence was amplified with specific primer sequences (HdF2: 5′CCGplasmids  were traE. coli was used to inoculate 5 mL of Luria-Bertani (LB) broth medium containing 50 μg·mL−1 kanamycin. Cells were cultured overnight at 37 °C with shaking at 220 rpm. 4 mL cell suspension was used to inoculate 400 mL of LB medium containing 1% casein hydrolysate , 17 mM KH2PO4 and 72 mM K2HPO4, 50 μg·mL−1 kanamycin [d-thiogalactopyranoside (IPTG) for 48 h at 18 °C with shaking at 200 rpm. Cells were harvested when cell growth reached the exponential phase (OD600 = 0.5–0.6).For protein expression, a colony from freshly transformed anamycin , and indg  at 4 °C for 30 min and further clarified by passing through a 0.22 μm filter. For purification of 6×His-tagged recombinant proteins, 1 mL of Ni2+-NTA agarose gel column (TransGen Biotech) was equilibrated with 10 mL of balance buffer . Supernatant was added to the column and then washed with 6 mL of balance buffer. Bound proteins were eluted from the affinity resin with 5 mL elution buffer . Fractions were collected and analyzed on a 12% SDS-PAGE gel and stained with Coomassie Blue R250 solution . The recombinant fusion protein was then dialyzed against 25 mM Tris-HCl (pH 8.0), and aliquots (50–100 μL) were stored at −70 °C for later use. The purified target proteins were verified via Western blot analysis with anti-6×His monoclonal antibody , followed by staining with goat anti-mouse IgG horseradish peroxides (HRP) conjugate . Antibody binding was detected with a diaminobenzidine kit according to the manufacturer’s protocol . Protein concentrations were determined using the Bradford method with bovine serum albumin as a standard [Bacterial cells were harvested by centrifugation and stored at −70 °C until further processing. Cell pellets were re-suspended in 25 mM Tris-HCl (pH 8.0) at a concentration of 1 mL per 200 mg cell wet weight and then lysed by sonication. Supernatant was harvested after centrifugation at 17,400× standard .et al. [et al. [v/v) ethanol and 3 mM Fast Blue RR salt at a range of substrate concentrations (16–400 μM·L−1) spanning the Km (based on preliminary assays). Formation of 1-naphthol was monitored by recording the change in absorbance at 450 nm for 10 min in a microplate reader  and quantified using 1-naphthol standard curves. Values for kcat and Km were estimated using “Hyper32” hyperbolic regression software [The kinetics of carboxylesterase activities against 1-naphthyl acetate were determined by the method of Han et al.  and Tees [et al.  with a mβ-cypermethrin and fenvalerate were carried out by monitoring substrate loss by a reversed-phase high performance liquid chromatography unit  attached to a dual λ absorbance detector (Waters 2487) as described by Teese et al. [−1 β-cypermethrinorfenvalerate and 50 μL of diluted purified enzyme (1.75–2.4 μg). Reactions were stopped by the addition of 100 μL of acetonitrile and vortexing. Samples were then prepared by passing through a 0.22 μm filter before transferring into a 2 mL brown sample vial . An aliquot (10 μL) of each reaction was injected on a Symmetry C18 column  and monitored at 210 nm, the mobile phase contained a mixture of acetonitrile and water  with a 1.0 mL·min−1 flow rate. For all substrates, control reactions were performed with the purified enzyme of 001D from pET30a inactivated by boiling for 10 min.Assays for the hydrolysis of e et al.  with somThe optimal temperature of the enzyme was investigated by incubating the enzyme and substrate (1-naphthyl acetate) at different temperatures (15 °C–60 °C) in 100 mM phosphate buffer (pH 7.0) for 5 min. The thermostability of the enzymes was determined by incubating the purified recombinant protein at temperatures from 15 to 60 °C for 1 h. The residual activity was measured and expressed as a percentage of the highest activity obtained in this case. The optimal pH of the enzyme activity was determined using the following 25 mM buffers: citric acid buffer (pH 4.0–6.0), phosphate buffer (pH 7.0) and Tris-HCl buffer (pH 8.0–11.0). The determination of pH stability was performed by pre-incubating the enzyme in a corresponding buffer for 1 h at 30 °C. Then, enzyme activity was measured as above in 100 mM phosphate buffer (pH 7.0). The fusion protein 6×His/S-tag/001D (pET30a) was used in all above assays. The highest activity obtained in this casewas used as the control (100%).001D gene from the susceptible H. armigera WH strain, and it contains the highly-conserved residues of catalytic triad and other active subsites in its amino acid sequence. We successfully expressed the carboxylesterase 001D fused with three different solubility/affinity tags in the E. coli cells. We purified the fusion proteins and evaluated their hydrolytic activities towards a model substrate and two pyrethroid insecticides. The results showed that carboxylesterase 001D was active and involved in detoxification, which probably implicated that overexpression of the genes to allow for effective sequestration of the insecticides is necessary to allow carboxylesterases to confer resistance to these insecticides in H. armigera.Here we cloned the carboxylesterase


The interest in organic–inorganic hybrid molecules as molecular probes for biological systems has been growing rapidly. Such hybrid molecules exhibit unique biological activities. Herein, copper(II) bis(diethyldithiocarbamate) (Cu10) was found to activate the transcription factor NF-E2-related factor 2 (Nrf2), which is responsible for regulating antioxidant and phase II xenobiotic enzymes, in vascular endothelial cells. The copper complex rapidly accumulated within cells and induced nuclear translocation of Nrf2, leading to upregulation of the expression of downstream proteins without cytotoxic effects. However, while copper bis(2-hydroxyethyl)dithiocarbamate activated Nrf2, copper ion, diethyldithiocarbamate ligand with or without zinc or iron failed to exhibit this activity. Intracellular accumulation of Cu10 was higher than that of Cu(II) and Cu(I). While the accumulation of copper(II) bis(dimethyldithiocarbamate) was reduced by small interfering RNA (siRNA)-mediated knockdown of the copper transporter CTR1, the knockdown did not affect Cu10 accumulation, indicating that Cu10 rapidly enters vascular endothelial cells via CTR1-independent mechanisms. In addition, copper and iron complexes with other ligands tested could not activate Nrf2, suggesting that the intramolecular interaction between copper and dithiocarbamate ligand is important for the activation of the transcription factor. Cu10 induced the expression of heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and γ-glutamylcysteine synthetase, downstream proteins of Nrf2. It was suggested that Cu10-induced activation of Nrf2 was due to proteasome inhibition as well as binding to Kelch-like ECH-associated protein 1. Since the effects of Cu10 on vascular endothelial cells are unique and diverse, the copper complex may be a good molecular probe to analyze the functions of the cells.The online version of this article (doi:10.1007/s00775-016-1337-z) contains supplementary material, which is available to authorized users. NF-E2-related factor 2 (Nrf2) is a transcription factor that belongs to Cap‘n’Collar transcription factor family and has a basic leucine zipper domain . Under bNrf2 mainly regulates the gene expression of antioxidant and phase II xenobiotic metabolizing enzymes such as heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and γ-glutamylcysteine synthetase, by binding to the ARE of the promoter region of the genes. Induction of heme oxygenase-1 protects cells from oxidative injury by catalyzing heme to biliverdin, carbon monoxide, and iron . NAD(P)Hl-cysteinato)zincate(II) serves as a specific zinc donor to the metal response element-binding transcription factor-1, a transcription factor containing six C2H2 zinc finger domains bismuthane—exhibits vascular endothelial cell-specific toxicity : C, 45.78; H, 7.68; N, 5.93 and found: C, 46.04; H, 7.80; N, 5.65. Copper(II) bis(dibenzyldithiocarbamate) (Cu19) was prepared analogously using zinc(II) bis(dibenzyldithiocarbamate) (92 % yield). Elemental analysis calculated for [C30H28CuN2S4]: C, 59.23; H, 4.64; N, 4.60 and found: C, 59.64; H, 4.68; N, 4.24. The elemental analyses were recorded on a Yanaco CHN recorder MT-6 at the Chemical Instrumental Center, Research Center for Materials Science, Nagoya University.ocedures –16. Copp2 for 1 h. The membrane was blocked with 5 % skim milk in 20 mM Tris–HCl buffer containing 150 mM NaCl and 0.1 % Tween-20, pH 7.5 and incubated with primary antibodies (1:200) at 4 °C overnight. The membrane was washed with 20 mM Tris–HCl buffer solution containing 150 mM NaCl and 0.1 % Tween 20 (pH 7.5), and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence and scanned by LAS3000 . Separately, vascular endothelial cells were treated with Cu10 at 0.1, 0.5, 1, 5, or 10 µM for 3 or 6 h and the nuclear fraction was prepared from the cell layer using the NE-PER Nuclear Cytoplasmic Extraction Reagents (Thermofisher Scientific). Nuclear protein concentration was determined by a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific). The samples (8 µg protein) were mixed with 50 mM Tris–HCl solution containing 8 % glycerol, 2 % sodium dodecyl sulfate, 2-mercaptoethanol, and 0.005 % bromophenol blue, pH 6.8 and incubated at 95 °C for 3 min. These samples were analyzed by western blotting as described above.Confluent cultures of vascular endothelial cells in 35-mm culture dishes were incubated at 37 °C for 1, 2, 3, 4, 6, 8, 12, or 24 h with Cu10 or other compounds at 0.1, 0.5, 1, 2, 5, or 10 µM. The cells were washed twice with ice-cold calcium- and magnesium-free phosphate buffered saline; total cellular proteins obtained by lysis in sodium dodecyl sulfate sample buffer  were incubated at 95 °C for 5 min. The protein concentration was determined using a bicinchoninic acid protein assay reagent kit . 2-Mercaptoethanol and bromophenol blue (1.67 % each) were added to the proteins (10 μg). The proteins were separated by SDS–polyacrylamide gel electrophoresis on 10 % polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane at 2 mA/cm4, sodium diethyldithiocarbamate trihydrate (Na01), zinc(II) bis(diethyldithiocarbamate) (Zn01), iron(III) tris(diethyldithiocarbamate) (Fe05), copper(II) bis(2-hydroxyethyl)dithiocarbamate (Cu09), CuSO4 [Cu(II)], CuSO4 with 1 mM ascorbate [Cu(I)]  , or Cu10c method  to normaVascular endothelial cells were cultured and siRNAs  for the copper transporter CTR1 were transfected using RNAiMAX reagent , as described previously . BrieflyE. coli cells and purified using a ProBond nickel-resin. The BPM-labeling assay was performed according to the method described by Toyama et al. [5-maleimide was added to the samples and the samples were incubated at 37 °C for 30 min. The samples were electrophoresed on 10 % sodium dodecyl sulfate–polyacrylamide gel in 50 mM Tris–HCl containing 2 % SDS, 8 % glycerol, and 0.005 % bromophenol blue (pH 6.8) without 2-mercaptoethanol and were incubated at 37 °C for 30 min. They were then subjected to immunoblotting as described above.The mouse recombinant Keap1 construct was prepared as described previously , 20. Thet test when possible. P values less than 0.01 were considered statistically significant.The data were analyzed for statistical significance by Student’s Figure In order to examine whether copper in the Cu10 molecule is critical to the activation of endothelial Nrf2, the effects of zinc and iron complexes with the same ligand of Cu10 on Nrf2 activation were investigated. In this experiment, copper sulfate, Na01 as the ligand of Cu10, and Cu09 were investigated. The structures of the tested metal complexes and Na01 are shown in Fig. Since Cu10 and Cu09 highly accumulated within vascular endothelial cells and enhanced Nrf2 expression, Cu(II) may have been reduced to Cu(I) by thiol groups in the thiocarbamate ligands and Cu(I) ion released from the Cu10 molecule efficiently entered the cells through the copper transporter CTR1 that mediated Cu(I) uptake . To exam4, and Na01 is shown in S1.It is possible that copper complexes in general activate endothelial Nrf2. To examine this possibility, we evaluated copper complexes with various ligands. In this experiment, compounds containing iron were also examined. The compounds used in this experiment are shown in Fig. The activation of Nrf2 by Cu10 was compared with that by sulforaphane  may be reduced to Cu(I) by thiol groups in thiocarbamate ligands, and the Cu(I) ion released from the Cu10 molecule efficiently entered the cells via the copper transporter CTR1 . In otheUbiquitin–proteasome system is responsible for the degradation of numerous proteins, including Nrf2. The 26S proteasome consists of two complexes—the 20S proteolytic core and the 19S regulatory complex . In thisA compound that has a specific target biomolecule is an excellent tool to analyze the role of the biomolecule in the regulation of some biological systems. However, a compound that has multiple targets is also useful to analyze the relationship among the targets. Cu10 appears to belong to the latter case. In fact, we have analyzed vascular endothelial cell functions using Cu10. Metallothionein is a low-molecular-weight, cysteine-rich, metal-containing, inducible protein, which protects cells from heavy metals and oxidative stress . Since cSupplementary material 1 (PDF 190 kb)


A gene not only control tiller and plant height, but also regulate panicle structure by QTL dissection in rice.qPPB3 for primary panicle branch number. With a BC3F2 population derived from a backcross between a resequenced RIL carrying PA64s allele and 93-11, qPPB3 was fine mapped to a 34.6-kb genomic region. Gene prediction analysis identified four putative genes, among which Os03g0203200, a previously reported gene for plant height and tiller number, Dwarf 88 (D88)/Dwarf 14 (D14), had three nucleotide substitutions in 93-11 compared with PA64s. The T to G substitution resulted in one amino acid change from valine in 93-11 to glycine in PA64s. Real-time PCR analysis showed expression level of D88 was higher in 93-11 than PA64s. The expression of APO1 and IPA1 increased, while GN1a and DST decreased in 93-11 compared with PA64s. Therefore, D88/D14 is not only a key regulator for branching, but also affects panicle structure.An ideal panicle structure is important for improvement of plant architecture and rice yield. In this study, using recombinant inbred lines (RILs) of PA64s and 93-11, we identified a quantitative trait locus (QTL), designated The online version of this article (doi:10.1007/s00299-014-1661-0) contains supplementary material, which is available to authorized users. MOC1  for primary panicle branch number was detected on chromosome 3. The region of qPPB3 was narrowed by map-based cloning strategy with BC3F2 population derived from a chromosome segment substitution line (CSSL). Based on sequencing and expression analysis, the predicted ORF for qPPB3 encoding a protein of the α/β-fold hydrolase superfamily (D88/D14), was proved to be not only a key regulator for plant shoot branching  using maximum likelihood interval mapping (MIM) based on a permutation test  for each dataset to confirm the LOD threshold. It was considered as a major effect QTL when its LOD was larger than 2.5. QTLs were named according to McCouch  V8.01. QTL analysis was performed with the MultiQTL1.6 package  identified between 93-11 and PA64s . Real-time PCR amplification mixtures (10 μl) contained 50 ng template cDNA, 2× SYBR Green PCR Master Mix (10 μl) (Applied Biosystems), and 200 nM forward and reverse primers. Reactions were run on an ABI PRISM® 7900HT Sequence Detector (Applied Biosystems). The relative expression level of each transcript was obtained by comparing to the expression of the OsACTIN1 gene. Primers for D88, APO1, IPA1, GN1a, DST, and Actin are listed in Table Total RNA was isolated from panicles before booting stage with the micro RNA extraction kit (Axygen). DNase treatment, cDNA synthesis, primer design and SYBR Green I Real-time PCR were carried out as described  (Table Rice panicle branch traits include primary panicle branch number (PPB) and secondary panicle branch number (SPB). And they directly affect spikelet number per panicle (SN), a crucial factor for rice grain yield. Our study showed PPB was significantly related to SPB and SN, with correlation coefficient 0.5204 and 0.4062, respectively. SPB was significantly related to SN (8) Table .Table 2SNP markers, covered all 12 chromosomes, were used to construct a high-resolution genetic linkage map with total genetic distance of 1,382 cM and an average distance of 0.53 cM between two adjacent SNP markers, as described in Gao et al. .qPPB3 and qPPB8. The qPPB3 explained 9.4 % phenotypic variation with additive effect came from PA64s. And qPPB8 explained 15.3 % phenotypic variation came from 93-11. Two QTLs for SPB were identified. The qSPB1 was mapped on chromosome 1 and explained 10 % phenotypic variation with additive effect came from 93-11. The positive effect of qSPB9 was from 93-11, explained 9.6 % phenotypic variation. PPB and SPB are two key factors for SN determined by panicle architecture. Two QTLs, qSN8 and qSN9 for SN were mapped on chromosomes 8 and 9 with additive effects of 35.5 and 33.2, respectively . With 1,522 RIL populations, qPPB3 was fine mapped in the region between markers P1 and P2. Total 1,105 BC3F2 individuals were utilized to further mapping, and 17 lines were selected from BC3F2 progenies derived from the cross between GH18 and 93-11. Parent 93-11 and GH18 were designated as the controls L1 and L2, respectively. Out of ten lines with high PPB similar to L2 (genotype of PA64s), there were five recombinant events between P4 and qPPB3, four between P5 and qPPB3, one between P7 and qPPB3, one between P6 and qPPB3, four between P3 and qPPB3. On the other hand, among seven lines with low PPB similar to L1 (genotype of 93-11), there were two recombinant events between P4 and qPPB3, one between P5 and qPPB3, one between P3 and qPPB3, and P6 and P7 co-segregated with qPPB3. Finally, qPPB3 was delimited to an interval of 34.6-kb region between markers P6 and P7 on the BAC clone AC146702 , and four corresponding genes  were found. After comparing sequence of the region between parents, only two genes, Os03g0203200 and Os03g0203700, had three SNPs and one InDel between two parents, respectively. However, product of Os03g0203700 was similar to Calcium-transporting ATPase 2, which has been found irrelevant to plant branching so far, and its expression showed no significant difference between two parents. Instead, Os03g0203200 (D88) encoded esterase D14/D88 homologous to the α/β-fold hydrolase superfamily protein, which has been reported affecting plant branching and significant difference in its expression level was detected here between 93-11 and PA64s. Only one substitution (T959 to G959) resulted in amino acid change from valine in 93-11 to glycine in PA64s  gene was suggested to be a candidate gene for qPPB3.To screen candidate genes in the critical 34.6-kb genomic region of Nipponbare genome  for PPB and another QTL (QSbn3a) for SPB were detected in the same genomic region of qPPB3  existed. The translational product of Os03g0203200 was D88/D14, an esterase that confirmed to control rice tillering, with d88/d14 mutants exhibits high tillers but dwarfism . Here, two QTLs were mapped for SPB, qPPB3 was fine mapped to a region covering the D88 gene, whose product believed to be an important receptor in strigolactone signaling , cytokinin (CK), auxin and strigolactone (SL)  for rice in the future.Dense panicle structure has been a major target for improvement of rice grain yield because of its relationship with grain number. The utilization of QTLs panicle structure, such as Supplementary material 1 (PPT 150 kb)Below is the link to the electronic supplementary material.