ORIGINAL ARTICLE:RESEARCH
WAVE1regulates P-glycoprotein expression via Ezrin in leukemia cells
MING-HUA YANG 1,MING-YI ZHAO 1,ZHUO WANG 1,RUI KANG 1,4,YU-LEI HE 2,XIAO-CHENG YIN 3,LI-YING LIU 1,LIANG-CHUN YANG 1,CAI-XIA ZHAN 1,XIU-SHAN WU 5,DAO-LIN TANG 4,&LI-ZHI CAO 1
1
Department of Pediatrics,Xiangya Hospital,Central South University,Changsha,Hunan Province,People’s Republic of China,2Children’s Hospital of Chengdou,Chengdu,Sichuan Province,People’s Republic of China,3Department of Pediatrics,The First Af?liated Hospital of Nanhua University,Hengyang,Hunan Province,People’s Republic of China,4Hillman Cancer Center,University of Pittsburgh Cancer Institute,Pittsburgh,Pennsylvania,USA,and 5College of Life Sciences,Hunan Normal University,Changsha,Hunan Province,People’s Republic of China (Received 5September 2010;revised 1November 2010;accepted 4November 2010)
Abstract
For children with acute myeloblastic leukemia (AML),multidrug resistance (MDR)reduces treatment effectiveness,and often leads to poor patient survival.While a number of factors have been described that affect MDR,the mechanisms underlying this effect remain unclear.In this study,the role of WAVE1in MDR was investigated.Among 62children with AML,high levels of WAVE1were associated with poor patient outcomes.Proteomic techniques were used to identify novel WAVE1-interacting proteins from leukemia cells,one of which was the cytoskeleton regulator Ezrin.In leukemia cells,WAVE1co-localized with both Ezrin and P-glycoprotein (P-gp),a critical regulator of the MDR phenotype.Overexpression of WAVE1in K562leukemia cells up-regulated P-gp and Ezrin,and reduced K562cells’sensitivity to the chemotherapy drug adriamycin.The opposite effect was seen when WAVE1expression was reduced via RNA interference.Critically,overexpression of WAVE1in the absence of Ezrin did not affect P-gp levels or MDR.These data suggest that WAVE1affects P-gp and MDR of leukemia cells through Ezrin.
Keywords:WAVE1,drug resistance,Ezrin,P-glycoprotein
Introduction
The Wiskott–Aldrich syndrome family of proteins,including WAVE1,WAVE2,WAVE3,WASP,and N-WASP,transduces signals from transmembrane receptors to the actin cytoskeleton [1].Cytoskeleton regulation is essential for a myriad of cellular processes,including cell motility,morphogenesis,and apoptotic cell death [2].WAVE1is expressed most abundantly in murine brain tissue,but low levels of expression are also found in tissues such as the heart,liver,lung,kidney,pancreas,and periph-eral blood [3].Essential for central nervous system development and mammalian fertilization [4–6],WAVE1also interacts with mitochondrial proteins (i.e.glucokinase and protein kinase A).In this way,
WAVE1coordinates cell death and glycolysis [7].We have previously demonstrated that WAVE1is overexpressed in blood cancer cell lines,and in this context functions as a negative regulator of apoptosis [8].Based on ?ndings such as these,we analyzed WAVE1expression in children with acute myelo-blastic leukemia (AML).Evidence suggests that high levels of WAVE1in patient bone marrow correlate with unfavorable prognoses [9].Finally,we have shown that WAVE1is involved in multidrug resis-tance (MDR)of K562/A02leukemia cells [10].These ?ndings support a general role for WAVE1in the regulation of drug resistance and AML pathogenicity.
P-glycoprotein (P-gp)/Mdr1is a 170kDa glycosy-lated integral plasma membrane protein,belonging
Correspondence:Li-Zhi Cao,Department of Pediatrics,Xiangya Hospital,Central South University,Changsha,Hunan Province,410008,People’s Republic of China.Tel:t8613755167259.Fax:t86073184327402.E-mail:caolizhi318@https://www.doczj.com/doc/459111973.html,,caolizhi318@https://www.doczj.com/doc/459111973.html, Leukemia &Lymphoma ,February 2011;52(2):298–309
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to the adenosine triphosphate (ATP)-binding cas-sette transporter family.Expressed in a variety of human cell types,P-gp has been functionally associated with the MDR phenotype [11,12].How-ever,mechanisms underlying P-gp regulation and activity in this context remain unclear.Some evidence suggests that P-gp interacts with the actin cytoskeleton [11].Luciani et al .[12]found that P-gp associates with the actin cytoskeleton through Ezrin,Radixin,and Moesin,and that this interaction is essential for the MDR phenotype in human lym-phoid cells.Ezrin is a key signaling molecule that regulates cell survival,adhesion,migration,and invasion,and is believed to link plasma membrane proteins to the cytoskeleton [13].As such,high levels of Ezrin are correlated with metastatic behavior in tumors [14,15].A link has also been established between actin-and/or tubulin-binding proteins (in-cluding Ezrin)and resistance to vincristine,an anti-microtubule agent used in the treatment of childhood ALL [16].
Leukemia is a malignant disease of the bone marrow and blood,and the most common form of cancer in children.We have previously described a role for WAVE1in MDR [8–10],but WAVE1’s speci?c function in MDR of leukemia is not fully understood.In the studies presented here,WAVE1overexpression rendered leukemia cells resistant to anticancer drugs,whereas suppression of WAVE1through RNA interference increased their sensitivity.Furthermore,proteins found in a complex with WAVE1(including Ezrin)were identi?ed by im-munoprecipitation and mass spectrometry.Our data suggest that functional links exist between WAVE1,Ezrin,and P-gp that affect MDR in myeloblastic leukemia.
Materials and methods Patients
Between January 2005and December 2009,bone marrow samples were collected from 62children with newly diagnosed AML in three hospitals:Xiangya Hospital,Children’s Hospital of Chengdou,and The First Af?liated Hospital of Nanhua Uni-versity.This patient cohort had a median age at diagnosis of 6.21years (range 1–14years),and consisted of 39males and 23females.Patients were diagnosed based on FAB (French–American–British classi?cation)standards,and according to MIC (morphology,immunology,and cytogenetics classi-?cation).All patients with AML (M1,11;M2,30;M3,15;M4,four;and M5,two)were treated with protocols recommended by the Chinese Medical Association [17].Cases were monitored until July
2010,and subsequently divided into two groups:(1)refractory/relapsed group,and (2)complete contin-uous remission group (CCR).The refractory/re-lapsed group consisted of 25patients,including 18cases of recurrence (relapse after complete remis-sion)and seven cases of non-remission (no easing of bone marrow after three rounds of inductive treat-ment,or death).The median follow-up time was 8months (range 1–34months).Follow-up time was de?ned as the period from diagnosis to death or recurrence.The CCR group consisted of 37patients,with a median follow-up time of 25months (range 12–61months).In addition,samples were collected from 10healthy children to serve as controls for real-time quantitative polymerase chain reaction (qPCR)analysis.
Reagents and plasmids
Antibodies to WAVE1,P-gp,and Ezrin were obtained from Santa Cruz Biotechnology (Santa Cruz,CA,USA).Antibodies to glyceraldehyde 3-phosphate dehydrogenase (GAPDH)were obtained from Boster (Wuhan,Hubei,China).Adriamycin (ADM)was obtained from Sigma (Sigma-Aldrich,MO,USA).The pEFBOS-WAVE1expression vec-tor was a gift from Dr.Tadaomi Takenawa (Uni-versity of Tokyo,Tokyo,Japan).pcDNA3.1-WAVE1was constructed from pEFBOS-WAVE1.WAVE1-siRNA and Ezrin-siRNA were obtained from Santa Cruz Biotechnology.
Collection of peripheral blood and bone marrow Control peripheral blood samples (5mL)were collected from healthy children into acid citrate dextrose (ACD)or ethylenediaminetetra-acetic acid (EDTA)tubes.Control bone marrow material was obtained from patients who did not have leukemia or other malignancies,but were undergoing bone marrow aspirates as part of their clinical care.These control bone marrow samples were in EDTA tubes,and scheduled to be https://www.doczj.com/doc/459111973.html,e of discarded material was exempt from review by the Ethics Committee of Xiangya Hospital,Committee on Human Research Review.Patients with AML provided 2mL of aspirated bone marrow in EDTA.Parental consent was obtained using a consent form approved by the Ethics Committee of Xiangya Hospital.Bone marrow mononuclear cells (BMMCs)were isolated by Ficoll density gradient centrifugation.Isolated cells were counted and stored in preparation for experiments.BMMCs of newly diagnosed patients were analyzed by ?ow cytometry (CD45/side-scatter [SSC]gating)to de-termine the percentage of leukemic cells [18].
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Cell culture and protein preparation
Utilized cell lines included:HL-60(human acute promyelocytic leukemia),K562(chronic myelogen-ous leukemia),K562/A02(MDR K562)(ATCC,USA),and HL-60/ADM (Institute of Hematology &Blood Diseases Hospital,Chinese Academy of Medical Sciences &Peking Union Medical College,Beijing,China).Cells were cultured in RPMI 1640or Dulbecco’s modi?ed Eagles’medium (DMEM;Life Technologies,Carlsbad,CA,USA)with 10%heat-inactivated fetal bovine serum (FBS),in 5%CO2and 95%air.
Cells were washed with cold phosphate buffered saline (PBS;Thermo Fisher Scienti?c,China),lysed in RIPA buffer (150mM NaCl,10mM Tris-HCl,pH 7.4,0.5%Triton X-100,and protease inhibi-tors),homogenized on ice,and centrifuged at 100006g at 48C for 20min.The supernatant was collected and stored at 7808C until use.Gene transfection and RNAi
Cell transfections were performed using the Fu-GENE HD Transfection Reagent (Roche Applied Science,Stockholm,Sweden)or Lipofectamine 2000(Invitrogen,Carlsbad,CA,USA),according to the manufacturer’s instructions.Human WAVE1-siR-NA or control-siRNA was transfected into cells using the X-tremeGENE siRNA Transfection Reagent (Roche Applied Science),according to the manu-facturer’s instructions.Real-time quantitative PCR
Total RNA was extracted from cells using Trizol (Invitrogen)and the total RNA concentration determined by measuring the 260nm absorbance.For each sample,1m g of the total RNA was used as input for ?rst-strand cDNA synthesis via the ReverTra Ace qPCR RT Kit (Toyobo,Osaka,Japan),according to the manufacturer’s recommen-dations.cDNAs were stored at 7208C until use.The following primers were used:forward P-gp :50-CC ATAGCTCGTGCCCTTGTTAGA-30,reverse P-gp :50-CGGTGAGCAATCACAATGCAG-30;for-ward WAVE1:50-TTTCTGCCTTGCCATTTAG-30,reverse WAVE1:50-ATGGATTACTACAGAA AATATGC-30;forward Ezrin :50-GTGTGGTAC TTTGGCCTCCACTATG-30,reverse Ezrin :50-GAACTTGGCCCGGAACTTGA-30;forward
GAPDH :50
-GAGAGACCCTCACTGCTG-30,re-verse GAPDH :50-GATGGTACATGACAAGGT GC-30.All primers were purchased from TaKaRa Biotechnology (Dalian,China).SYBR 1Premix Ex Taq TM (TaKaRa)was used for qPCR,with 20ng cDNA input.A blank sample with RNase-free water
was used for primer controls.Ampli?cation was done in a LightCycler (Roche Biotechnology,Chang sha,China)using a program of 45cycles at 958C for 10s and 52–558C for 30s for data collection.A melt curve was performed using a protocol of 1min at 958C,1min at 558C,and increasing the temperature in 0.58C increments for 80cycles of 10s each.Real-time PCR data were analyzed using the DD C t method,with GAPDH used as the endogenous control gene.The fold-change for each cDNA from the refractory/relapsed group samples relative to the control (CCR group)was calculated using the 27DD C t method [19],where DD C t ?D C t objective goup/period 7D C t control group/period,and D C t ?C t WAVE1/P-gp 7C t GAPDH .PCR was performed in triplicate.Immunoprecipitation
Cells were lysed at 48C in ice-cold lysis buffer (50mM Tris-HCl,pH 7.4,containing 150mM NaCl,1%NP-40,0.5%Na-deoxycholate,0.1%sodium dodecyl sulfate [SDS],and protease inhibitor cock-tail;Roche Biotechnology).Cell lysates were cleared by a brief centrifugation (120006g ,10min).Con-centrations of proteins in the supernatant were determined by bicinchoninic acid (BCA)assay.Before immunoprecipitation,samples containing equal amounts of proteins were pre-cleared with protein A/G agarose/sepharose (Santa Cruz)(48C,3h),and subsequently incubated with various irrelevant immunoglobulin G (IgG)or speci?c antibodies (5mg/mL)in the presence of protein A/G agarose/sepharose beads for 2h at room tempera-ture,or overnight at 48C with gentle shaking.After incubation,agarose/sepharose beads were washed extensively with PBS,and proteins eluted by boiling in a 26SDS sample buffer before SDS-polyacryla-mide gel electrophoresis (PAGE)electrophoresis.Proteomic analysis by mass spectrometry
WAVE1binding proteins were isolated by anti-WAVE1antibody immunoprecipitation from three leukemia cell types:BMMCs from a patient with refractory AML,HL-60/ADR cells,and K562/A02cells.The recovered protein complexes were sub-jected to SDS-PAGE.Three protein bands were cut from the gel,and 60mg of protein was used for mass spectrometry.Experiments were run in triplicate and samples were analyzed separately.Non-speci?c IgG or no antibody was used in negative controls.Samples were incubated for 30min in 5mM dithiothreitol (DTT),30min in 20mM iodoaceta-mide,and 10min in 40mM DTT.Samples were digested with 5mg trypsin,pH 8,at 378C overnight.
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Digestion was assessed by gel electrophoresis.Cation exchange chromatography was performed using a 1cc Oasis MCX cartridge (Waters Corp.,Milford,MA,USA).Samples were eluted in 90%methanol,10%NH4OH,at pH 12,and dried in vacuo .Microcapillary liquid chromatography-tandem mass spectrometry (MS/MS)was performed at the Beijing Proteome Research Center and Key Laboratory of Cancer Proteomics of the Chinese Ministry of Health,according to the manufacturer’s electrospray ionization tandem mass spectrometry operating instructions (Bruker,Bremen,Germany)[20].
Protein identi?cation
MS/MS spectra were searched using the SEQUEST search tool against a database composed of the International Protein Index human protein se-quences downloaded from the European Bioinfor-matics Institute.Bovine (TRYPBOVIN)and porcine trypsin (TRYPPIG)sequences from Swiss-Prot were appended to the human database.The database search results were validated using PeptideProphet software,which assigns to each peptide identi?cation a probability that it has been correctly identi?ed based upon its SEQUEST scores and features of the assigned peptide,including the number of tryptic https://www.doczj.com/doc/459111973.html,ing a second program,ProteinProphet,the assigned peptides were grouped according to the corresponding protein [20].Western blot analysis
Proteins were resolved on 6–10%SDS-PAGE gels,and transferred to polyvinylidene ?uoride membra-nes.After blocking the membranes at room tempera-ture for 2–4h,the membranes were incubated overnight at 48C with various primary antibodies.After incubation with peroxidase-conjugated second-ary antibodies for 1–2h at 258C,the blots were then developed by enhanced chemiluminescence (ECL;Pierce Biotechnology,Rockford,IL,USA).Dual immuno?uorescent histochemistry
Leukemia cells were ?xed in 4%formaldehyde for 30min at room temperature prior to cell permeabi-lization with 0.1%Triton X-100(48C,10min).Cells were spread on glass slides and blocked for 30min with normal goat serum (Boster),diluted 1:1in PBS.All incubations were performed at room temperature in a humidi?ed chamber.After removal of excess serum,the slides were incubated overnight with a mixture of goat anti-human WAVE1(Santa Cruz),and mouse anti-human Ezrin or a P-gp monoclonal antibody (Santa Cruz)at ?nal dilutions
of 1:10in PBS–0.5%Triton X-100.Primary anti-body incubation was followed by two 5min washes with PBS–0.5%Triton X-100,and two 5min washes with PBS.The WAVE1protein was detected by incubation for 30min with a ?uorescein isothio-cyanate (FITC)-conjugated rabbit anti-goat IgG (Sigma),diluted 1:100in PBS.Washes were as described above.Ezrin and P-gp were detected with a rhodamine-conjugated goat anti-mouse IgG anti-body (Sigma)[21].Fluorescent signals were ana-lyzed using an Olympus microscope (Tokyo,Japan).MTT cell viability assay
Leukemia cells were seeded in 96-well plates (4000cells/well)in DMEM with 10%FBS.The following day,cells were treated with ADM as indicated,and incubated for 72h.A 25m L aliquot of MTT [3-(4,5-dimethylthiazol–2-yl)-2,5-diphenyltetrazolium bro-mide;Sigma]was added to each well and incubated for 3.5h,followed by the addition of 100m L of N ,N -dimethylformamide (D4551;Sigma)solubilization solution.The plates were left at room temperature overnight to allow complete lysis of the cells,and read at 450nm the following day.Half-maximal inhibitory concentration (IC50),i.e.the drug con-centration at which 50%growth inhibition is achieved,was calculated using MS Excel.Statistical analysis
Data are expressed as the mean +https://www.doczj.com/doc/459111973.html,par-isons between groups were performed using the two-tailed Student’s t -test,or Fisher’s least signi?cant difference (LSD)test.A value of p 50.05was considered signi?cant.
Results
Refractory/relapsed group BMMCs contain high levels of WAVE1and P-gp
Bone marrow samples from 62children with AML were collected at the time of diagnosis.Patients were subsequently grouped based on response to treat-ment,and levels of WAVE1and P-gp determined in BMMCs.Both qPCR and Western blot analyses were used to provide gene expression and protein abun-dance data,respectively.qPCR revealed that non-responsive patients (refractory/relapsed group)had higher levels of WAVE1and P-gp mRNA in BMMCs than did patients in remission (CCR group).The 27DD C t value for WAVE1was 44.32(95%con?dence interval [CI],9.25–212.31)in patients in the refrac-tory/relapsed group,and 3.71(95%CI,1.32–11.16)in patients in the CCR group (p 50.001).The
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27DD C t value for P-gp was 33.36(95%CI,9.32–119.4)in patients in the refractory/relapsed group,and 0.83(95%CI,0.19–3.58)in patients in the CCR group (p 50.001)(Figure 1).Further statistical analysis revealed that WAVE1expression was corre-lated with P-gp mRNA levels (g ?0.444,p 50.01),as well as the percentage of leukemic cells in the BMMC samples (g ?0.456,p 50.01).
Similar results were obtained when WAVE1and P-gp protein levels were examined.Western blot analysis of the 62patient samples indicated that both proteins were elevated in refractory/relapsed group BMMCs relative to the CCR group.WAVE1protein levels
correlated with P-gp protein abundance (g ?0.601,p 50.01),and the percentage of leukemic cells in the BMMC sample (g ?0.338,p 50.01).Moreover,for both relapsed patients and the CCR group,WAVE1BMMC protein levels were lowest during the remis-sion stage (compared to the newly diagnosed or relapsed stage)(Figure 2).These data suggest that WAVE1levels correlate with AML development.WAVE1regulates P-gp expression in leukemia cell lines Both WAVE1and P-gp are associated with MDR and unfavorable outcomes in pediatric patients
with
Figure 1.Expression of WAVE1and P-gp mRNA in BMMCs of patients with AML.The range given for WAVE1/P-gp expression relative to the normal was determined by 27DD C t calculation.(A)WAVE1mRNA of BMMCs in the refractory/relapsed group of patients relative to that in the CCR group.Fold change is expressed as 27DD C t .(B)P-gp mRNA of BMMCs in the refractory/relapsed group of patients relative to that in the CCR group.Fold changes are expressed as 27DD C t
values.
Figure 2.WAVE1and P-gp protein levels in BMMCs of patients with AML.(A)Levels of both WAVE1and P-gp proteins were higher in the refractory/relapsed group than in the CCR group.Samples collected at the time of diagnosis (arbitrary units,AU).*p https://www.doczj.com/doc/459111973.html,R group.(B)WAVE1protein levels were lowest in complete continuous remission (arbitrary units,AU).*p 50.05vs.newly diagnosed;**p https://www.doczj.com/doc/459111973.html,plete continuous remission.
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AML.Consistent with this view,we found that WAVE1and P-gp levels were elevated in the MDR leukemia cell line K562/A02(relative to the non-drug resistant cell line K562).This was supported by both qPCR and Western blot analysis.The 27DD C t of WAVE1in K562/A02cells was 13.36(95%CI,12.12–14.60),while the 27DD C t of P-gp in K562/A02cells was 120.26(95%CI,119.22–121.5)(Table I).According to our Western blot analysis,WAVE1and P-gp protein levels were 50%and 521%higher,respectively,in K562/A02cells than in K562cells (Figure 3).
To characterize the functional relationship between WAVE1and P-gp,we expressed full-length human WAVE1in K562leukemia cells,and determined the effect on P-gp .The WAVE1overexpression protocol was effective,as the 27DD C t value for WAVE1in WAVE1-overexpressing K562cells was 53.82(95%CI,52.48–55.16),relative to non-transfected K562cells.At the same time,the 27DD C t value for P-gp in WAVE1-overexpressing K562cells was 35.26(95%CI,33.94–36.58)(Table I).Meanwhile,WAVE1and P-gp protein levels were increased by 110%and 279%,respectively,in the WAVE1-overexpressing
Table I.Expression of WAVE1and Mdr1mRNA in K562and K562/A02cells (n ?3).
Group
D C t
(WAVE1-GAPDH)D C t
(Mdr1-GAPDH)27DD C t
mean (WAVE1)
27DD C t
mean (Mdr1)K562
11.81+0.459.80+0.1211K562transfected with pcDNA3.1
11.38+0.099.51+0.41 1.35* 1.22*K562transfected with pcDNA3.1-WAVE1 6.06+0.42 4.66+0.4053.82*35.26*K562/A02
8.07+0.31 2.89+0.061{1{13.36*120.26*K562/A02transfected with control-siRNA 7.44+0.22 2.57+0.26 1.58{ 1.29{K562/A02transfected with WAVE1-siRNA
11.26+0.64
5.55+0.29
0.11{
0.16{
*p 50.05,relative to K562cells;{p 50.05,relative to K562/A02
cells.
Figure 3.WAVE1regulates P-gp levels in leukemia cells.(A)Western blot analysis of WAVE1,P-gp,and GAPDH in K562and K562/A02cell lines as indicated.(B)Quanti?cation of WAVE1and P-gp protein levels based on the relative optical intensity (in arbitrary units,AU)of Western blot bands.The legend indicates cell type.1:K562cells;2:K562cells transfected with pcDNA3.1;3:K562cells transfected with pcDNA3.1-WAVE1;4:K562/A02cells;5:K562/A02cells transfected with control-siRNA;6:K562/A02cells transfected with WAVE1-siRNA.*p 50.05vs.K562cells.#p 50.05vs.K562/A02cells.
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K562cells (Figure 3).Similar effects were seen with WAVE1loss-of-function.Transfection of WAVE1-siRNA into K562/A02leukemia cells to knock down WAVE1expression led to a signi?cant decrease in WAVE1mRNA levels (Figure 3).The 27DD C t value for WAVE1in WAVE1-siRNA K562/A02cells was 0.11(95%CI,70.71–0.93)relative to K562/A02cells.Meanwhile,the 27DD C t value for P-gp in WAVE1-siRNA K562/A02cells was 0.16(95%CI,0.66–0.98)relative to K562/A02cells (Table I).WAVE1and P-gp protein levels decreased to 18%and 56%of control values,respectively,in response to WAVE1-siRNA knockdown (Figure 3).These results clearly demonstrate that WAVE1acts as a positive regulator of P-gp in leukemia cells.
WAVE1is combined with Ezrin in myeloid leukemia cells To understand the mechanisms by which WAVE1affects P-gp levels and MDR in leukemia cells,we sought to isolate WAVE1binding proteins in this context.Three major protein bands were cut from the gel,in-gel digested,and analyzed by electrospray ionization-quadrupole-time-of-?ight (ESI-Q-TOF)(Figure 4).MS identi?ed 51WAVE1-interacting proteins.Twenty-one of these proteins are listed in Table II.Ezrin,a -tubulin,anaphase promoting complex subunit 1,T-cell receptor,IG heavy chain variable region,mitochondrial ribosomal protein L11,and ERCC-4were each found in both the patient sample and the leukemia cell lines.Mito-chondrial ribosomal protein L11and plasminogen activator inhibitor-1(PAI-1)mRNA-binding protein were identi?ed in two bands.T-cell receptor a chain was identi?ed in two bands,but with different
molecular weights.In total,the WAVE1-associated proteins could be divided into the following seven major groups based on function:tumor-associated proteins,cytoskeletal proteins,receptors,metabo-lism-related enzymes,immune-related proteins,membrane and ion channel proteins,and signaling pathway proteins (Table II).
Interactions between WAVE1and Ezrin or a -tubulin were con?rmed by co-immunoprecipita-tion and Western blot analysis.Both a -tubulin and Ezrin were con?rmed to co-precipitate with WAVE1in K562/A02,HL-60/ADR,and AML BMMCs.However,these interactions could not be detected in healthy children’s BMMCs.In each of the cell types tested,P-gp did not co-precipitate with WAVE1(Figure 5).
To verify a speci?c interaction between WAVE1and Ezrin,reverse immunoprecipitations were per-formed using an anti-Ezrin antibody.Western blot analysis of Ezrin immunoprecipitations con?rmed the presence of WAVE1.Importantly,the healthy child sample yielded no detectable WAVE1protein (Figure 5).These results con?rmed our MS results,demonstrating that both Ezrin and a -tubulin associ-ate with WAVE1in leukemia cells.WAVE1regulates P-gp and MDR via Ezrin
To determine whether WAVE1,Ezrin,and P-gp co-localize in vitro ,we used immuno?uorescence to label these proteins in leukemia cells.Indeed,the localization patterns of WAVE1and Ezrin were indistinguishable from one another in K562/A02leukemia cells.Similarly,WAVE1and P-gp co-localized in K562/A02leukemia cells (Figure
6).
Figure 4.Anti-WAVE1immunoprecipitations from leukemia cells.An SDS-PAGE gel visualized by Comas staining.A:K562/A02cells;B:HL-60/ADR cells;C:BMMCs of a child with AML;D:BMMCs of a healthy child;E:control mouse IgG.Arrows indicate bands that were chosen for analysis.
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Based on Ezrin’s association with WAVE1,we investigated whether Ezrin affects P-gp and MDR in this system.Overexpression of WAVE1in K562cells
increased levels of P-gp (as shown previously),increased Ezrin (Figure 7),and reduced the sensi-tivity of K562cells to ADM (Figure 8).Knockdown of either WAVE1or Ezrin by RNA interference decreased levels of P-gp in K562/A02cells (Figure 7),and restored the sensitivity of K562/A02cells to ADM (Figure 8).Critically,in the absence of Ezrin ,WAVE1overexpression did not affect P-gp levels in K562cells (Figure 7).As a result,loss of Ezrin restored the sensitivity of WAVE1-expressing K562cells to ADM (Figure 8).Discussion
Chemotherapy is the recommended treatment for nearly all types of childhood leukemia [22].MDR,however,often renders this therapy ineffective in patients with AML [23],and negatively impacts long-term event-free survival [24].It is important,therefore,to understand the relationship between WAVE1and MDR so that a new therapeutic target for AML can be identi?ed.
The mechanisms of play of drug transport resis-tance chie?y account for MDR [25,26].We demon-strated previously that high levels of WAVE1are
Table II.WAVE1binding proteins:peptide sequence tag identi?ed by Mascot software searching.
Protein band Protein name
Match peptide
Molecular weight 2a -Tubulin
R.AVFVDLEPTVIDEVR.T
50126K.TIGGGDDSFNTFFSETGAGK.H 1Actin related protein ARP4
K.QYTGINAINQK.K
453592DNA excision repair protein ERCC-4K.EIEAENKESEALGGPGQVLICASDDR.T 1032241Ezrin (fragment)(HL/ADR cells)K.EELMLRLQI.-195381Ezrin (fragment)(K562/A02cells)K.EELMLRLQI.-195381Ezrin (fragment)(BMMCs of patient)
K.EELMLRLQI.-and K.KFVIKPIDK.K 195383Electrogenic Na tbicarbonate cotransporter K.FEEKVEQGGER.W
751812erk2(EC 2.7.1.-)mutant
R.DLKPSNLLLNTTADLKICDFGLAR.V 386591Glucose-6-phosphate 1-dehydrogenase R.VQPNEAVYTKMMTK.K
548221Intercellular adhesion molecule-1?rst two domains,residues 1–185K.VILPRGGSVLVTCSTSCDQPK.L
204033Lysozyme precursor R.STDYGIFQINSR.Y 165443Lactoferrin R.YYGYTGAFR.C
305631Myosin IE
K.QLPLKFSNTLELK.L
1269822,3Mitochondrial ribosomal protein L11,K.AAAGIEKGARQTGKEVAGLVTLK.H 18194isoform b
K.EADLAAQEEAAK.K
1Myosin phosphatase Rho-interacting protein K.DQPDGSSLSPAQSPSQSQPPAASSLREPGLESK.E 1163742MLL protein (fragment)
R.EAGVVACLDSGPGAETFPSSLFS.-162571Sodium/potassium-transporting ATPase a -3chain
K.NLEAVETLGSTSTICSDK.T
1116631,2PAI-1mRNA-binding protein (PAI-RBP1)K.KEAGGGGVGGPGAK.S
449381Protein kinase ERK3
R.IEDEIDDIVLMAANQSQLSNWDTCSSR.Y
625842SRY (sex determining region Y)-box 1K.YSLAGGLLAAGAGGGGAAVAMGVGVGVGAAAVGQR.L 389981T-cell receptor a chain precursor V region K.ASQYVSLLIRDSQPSDSATYLCALR.D 112323T-cell receptor a chain -.CAASIGSGGGADGLTFGK.G
16252Tropomyosin
R.ARPAAPAAAPGPLAAATMDAIKK.K
34923
3
Voltage-gated L-type calcium channel a -1subunit
R.LPDSLSFGPSDDDRGTPTSSQPSVPQAGSNTHRR.G
Figure 5.Immunoprecipitation and Western blot analysis of the WAVE1interacting proteins a -tubulin and Ezrin.(A)Western blot analysis of a -tubulin in WAVE1-immunoprecipitate.(B)Western blot analysis of Ezrin in WAVE1-immunoprecipitate.(C)Western blot analysis of P-gp in WAVE1-immunoprecipitate.(D)Western blot analysis of WAVE1in Ezrin-immunoprecipitate.1:K562/A02cells;2:HL-60/ADR cells;3:BMMCs of a child with AML;4:BMMCs of a healthy child;5:control mouse IgG.
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found in human blood cancer cell lines,while non-blood cancer cells are essentially WAVE1-negative [8].To determine whether WAVE1plays a role in membrane transport-associated MDR in children with AML,levels of WAVE1and P-gp were measured in BMMCs of 62patients with AML from three medical centers.Correlations between WAVE1/P-gp levels and clinical data were found.At the time of diagnosis,levels of WAVE1and P-gp were higher in the refractory/relapsed group than in the CCR group.Data from the same patient cohort also demonstrated that WAVE1mRNA and protein levels were lowest in the complete continuous remission phase (compared to the newly diagnosed or relapsed phase),suggesting that WAVE1con-tributes to the pathogenesis of AML.
As levels of WAVE1and P-gp were highly correlated in patient samples,we investigated whether WAVE1could affect P-gp expression and/or accumulation.To determine the relationship between WAVE1and P-gp,the leukemia cell line K562and its drug-resistant ancillary (K562/A02)were used as models.Expression of WAVEI was much higher in K562/A02cells than in K562cells.In our previous study,we reported that overexpression of WAVE1in K562cells signi?cantly increased ADM resistance,and that knockdown of WAVE1in K562/A02cells by RNA interference reduced the ADM resistance phenotype [10].Notably,in the present study,we showed that overexpression of WAVE1in K562cells increased P-gp levels,whereas knockdown of WAVE1expression in K562/A02cells by RNA interference decreased P-gp levels.These results suggest that WAVE1is involved in the P-gp-mediated MDR phenotype exhibited by K562/A02cells.
To understand how WAVE1affects the MDR phenotype,WAVE1binding proteins were identi?ed in BMMCs from patients with AML and two drug-resistant leukemia cell lines.Fifty-one proteins were identi?ed,which could be divided into seven main functional groups:tumor-associated proteins,cytos-keletal proteins,receptors,metabolism-related en-zymes,immune-related proteins,membrane and ion channel proteins,and signaling pathway proteins.Some of these proteins had been reported previously [6,27–32].These results provide novel avenues of inquiry that will help researchers decipher the role of WAVE1in leukemia.
The WAVE1-binding protein Ezrin was selected for further investigation.Ezrin is a member of the Ezrin–Radixin–Moesin (ERM)protein family [33],and links membrane proteins to the cytoskeleton.Ezrin participates in signaling through several growth factor receptors,promoting cell survival,differentia-tion,motility,invasion,and cell adhesion [33,34].Several studies have provided evidence that Ezrin promotes metastasis in tumor cells [35,36].Monni et al .[37]reported that Ezrin dysregulation promotes progression of preleukemic proerythroblasts
toward
Figure 6.Co-localization of WAVE1,Ezrin,and P-gp.(Top panel)Localization of WAVE1and Ezrin by dual immuno?uorescent histochemistry in K562/A02cells (6200).(a)WAVE1-speci?c antibody (FITC).(b)Ezrin-speci?c antibody (Rhodamine).(c)Merge.(Bottom panel)Localization of WAVE1and P-gp in K562/A02cells (6200).(a)WAVE1-speci?c antibody (FITC).(b)P-gp-speci?c antibody (Rhodamine).(c)Merge.
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malignancy.The authors demonstrate that Ezrin plays an important role in the oncogenic processes associated with activating mutations in tyrosine kinases.Luciani et al .[12]found that P-gp co-localized and co-immunoprecipitated with Ezrin.Furthermore,they demonstrated that antisense oligonucleotides to Ezrin/Radixin/Moesin restored drug susceptibility,consistent with inhibition of drug ef?ux,actin-P-gp association,and P-gp cellular redistribution.To date,more attention has been directed toward the actin–P-gp interaction in studies concerning MDR.WAVE1controls actin polymer-ization through the actin-related protein (Arp)-2/3complex [38],and some functions of WAVE1have been associated with ERM.For example,Sanchez et al .[39]considered the control of actin polymer-ization and branching via Moesin/WAVE1as a key function of estrogen receptor a in neurons.Until now,however,little attention has been given to the WAVE1/Ezrin interaction.
Although MDR is regulated by a number of factors,most studies have concluded that P-gp is one of the most important proteins in MDR of cancer cells,including leukemia [40].Our ?ndings suggest that Ezrin serves as the critical link between WAVE1and P-gp.In leukemia cells,WAVE1and Ezrin were found in the same protein complex (via immunopre-cipitation experiments)and co-localized at the cell membrane.As WAVE1binds Ezrin,and Ezrin has been shown to bind P-gp,this suggests that a WAVE1/Ezrin/P-gp complex can exist,and provides a plausible link between WAVE1and P-gp.Critical experiments presented here demonstrated that WAVE1’s involvement in P-gp-mediated MDR of K562/A02cells required Ezrin.Overexpression of WAVE1in K562cells increased both P-gp and Ezrin levels,whereas knockdown of either WAVE1or Ezrin via RNA interference decreased P-gp levels in K562/A02cells,and furthermore restored ADM sensitivity.Importantly,overexpression of WAVE1in the ab-sence of Ezrin (via Ezrin-siRNA)did not affect P-gp levels.These data are consistent with Ezrin acting as a downstream effector of WAVE1,and suggests that WAVE1regulates P-gp via Ezrin in leukemia cells.We should note that P-gp is not the only pathway through which WAVE1can regulate the
MDR
Figure 7.Interactions between WAVE1,Ezrin,and P-gp.(A)Western blot analysis of WAVE1,P-gp,Ezrin,and GAPDH in K562and K562/A02cell lines as indicated.(B)WAVE1levels were determined based on the relative optical intensity (in arbitrary units,AU)of Western blot bands.(C)P-gp levels were determined based on the relative optical intensity (in arbitrary units,AU)of Western blot bands.(D)Ezrin levels were determined based on the relative optical intensity (in arbitrary units,AU)of Western blot bands.1:K562cells;2:K562cells transfected with pcDNA3.1;3:K562cells transfected with pcDNA3.1-WAVE1;4:K562cells transfected with pcDNA3.1-WAVE1and Ezrin-siRNA;5:K562/A02cells;6:K562/A02cells transfected with control-siRNA;7:K562/A02cells transfected with WAVE1-siRNA;8:K562/A02cells transfected with Ezrin-siRNA.*p 50.05vs.K562cells,#p 50.05vs.K562cells transfected with pcDNA3.1-WAVE1;D p 50.05vs.K562/A02cells;'p 50.05vs.K562/A02cells transfected with WAVE1-siRNA.
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phenotype.For example,WAVE1regulates Bcl-2localization and phosphorylation in leukemia cells [10],and mass chromatographic analysis has shown that WAVE1interacts with tubulin and other proteins that mediate drug resistance.
Together,our data de?ne for the ?rst time a clear association between WAVE1and P-gp through the Ezrin protein,and demonstrate a dramatic inhibition of P-gp-mediated MDR after WAVE1loss-of-func-tion.Novel therapeutic strategies that target WAVE1and P-gp-mediated MDR to improve the ef?cacy of anti-leukemia treatments (especially in children)may stem from the results of this study.
Declaration of interest:This work was supported by grants from The National Natural Sciences Foundation of China (30571982,30772353,30973234to L.C.,30500485to D.T.),the Doctoral Program of Higher Education of China (20070533042to L.C.),and a grant from the University of Pittsburgh (to D.T.).References
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Figure 8.WAVE1and Ezrin affect ADM sensitivity of leukemia cells.Overexpression of WAVE1reduced ADM sensitivity of K562cells.In the absence of Ezrin ,WAVE1overexpression did not affect ADM sensitivity of K562cells.Suppression of either WAVE1or Ezrin restored ADM sensitivity of K562/A02cells.The ADM drug concentration that achieved 50%growth inhibi-tion was the half-maximal inhibitory concentration (IC50).1:K562cells;2:K562cells transfected with pcDNA3.1-WAVE1;3:K562cells transfected with pcDNA3.1-WAVE1and Ezrin-siRNA;4:K562/A02cells;5:K562/A02cells transfected with WAVE1-siRNA;6:K562/A02cells transfected with Ezrin-siRNA.*p 50.05vs .K562cells;#p 50.05vs.K562cells transfected with pcDNA3.1-WAVE1;D p 50.05vs.K562/A02cells.
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Supplementary material available online Manufacturer’s information on protease inhibitor cocktail
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一级建造师和二级建造师哪个好 1、一级建造师和二级建造师都是全国统一命题、统一指定考试科目、统一考试， 在考生通过考试并取得建造师执业资格证并符合注册条件，经注册登记后才能获得一级或二级建 造师注册证书。 2、考试难度： 一级建造师考试难，二级建造师考试相对简单，大家可以从考试难易度入手，选择报考哪个科目。 3、考试科目： 一级建造师考试共有四个科目：建设工程经济、建设工程项目管理、建设工程法规 及相关知识、专业工程管理与实务（10个专业） 10个专业，分别为：建筑工程、公路工程、机电工程、市政公用工程、水利水电 工程、矿业工程、铁路工程、民航机场工程、通信与广电工程、港口与航道工程 二级建造师考试共有三个科目：建设工程施工管理、建设工程法规及相关知识、专业工程管理与实务（6个专业） 6个专业，分别为：建筑工程、公路工程、机电工程、市政公用工程、水利水电工 程、矿业工程 从考试专业来分析，一级建造师专业考试方向选择更多。 4、报考条件:
相较而言，二建报考条件要比一建低上许多，比如工作年限要求短，学历条件要求 低等等。于个人而言，肯定首先会满足二建条件，其次才会满足一建条件。若只满足二级建造师报考条件，又何来讨论一级建造师与二级建造师比较哪个更好？ 二级建造师报考条件：凡遵纪守法，具备工程类或工程经济类中等专科以上学历并从事建设工程项目施工管理工作满2年的人员，可报名参加二级建造师执业资格考试。 一级建造师报考条件： 1）、取得工程类或工程经济类大学专科学历，工作满6年，其中从事建设工程项目 施工管理工作满4年。 2）、取得工程类或工程经济类大学本科学历，工作满4年，其中从事建设工程项目 施工管理工作满3年。 3）、取得工程类或工程经济类双学士学位或研究生班毕业，工作满3年，其中从事 建设工程项目施工管理工作满2年。 4）、取得工程类或工程经济类硕士学位，工作满2年，其中从事建设工程项目施工 管理工作满1年。 5）、取得工程类或工程经济类博士学位，从事建设工程项目施工管理工作满1年 5、使用范围： 二级建造师：省内注册，全国通用
2017-11-17 在创业初期，资本匮乏的情况下，创业者们往往通过股权期权来对员工进行激励，一定程度上也起到集结公司上下的力量，众志成城，共同努力将创业项目落实成功的作用，但很多时候，前期没有分配好股权很容易在后期出现利益纠纷而导致公司人心涣散，影响企业运营效率，因此问题来了，如何分配好股权？本文便通过员工股权激励的进入退出机制叙说一二： 一、员工股权激励的进入机制 1、定时 有的创业者，在公司很初创阶段，就开始大量发放期权，甚至进行全员持股。我们的建议是，对于公司核心的合伙人团队，碰到合适的人，经过磨合期，就可以开始发放股权。 但是，对于非合伙人层面的员工，过早发放股权，一方面，股权激励成本很高，给单个员工三五个点股权，员工都可能没感觉；另一方面，激励效果很差，甚至会被认为是画大饼，起到负面激励效果。 因此，公司最好是走到一定阶段（比如，有天使轮融资，或公司收入或利润达到一定指标）后，发放期权的效果会比较好。 发放期权的节奏： 要控制发放的节奏与进度，为后续进入的团队预留期权发放空间（比如，按照上市前发4批计算）；全员持股可以成为企业的选择方向，但最
好是先解决第一梯队，再解决第二梯队，最后普惠制解决第三梯队，形成示范效应。这样既可以达到激励效果，又控制好激励成本；期权激励是中长期激励，激励对象的选择，最好先恋爱，再结婚，与公司经过一段时间的磨合期。 2、定人 股权激励的参与方，有合伙人，中高层管理人员（VP，总监等），骨干员工与外部顾问。 合伙人主要拿限制性股权，不参与期权分配。但是，如果合伙人的贡献与他持有的股权非常不匹配，也可以给合伙人增发一部分期权，来调整早期进行合伙人股权分配不合理的问题。 一般情况下，中高层管理人员是拿期权的主要人群。 3、定量 定量一方面是定公司期权池的总量，另一方面是定每个人或岗位的量。 公司的期权池，10-30%之间较多，15%是个中间值。期权池的大小需要根据公司情况来设定。 在确定具体到每个人的期权时，首先先考虑给到不同岗位和不同级别人员期权大小，然后再定具体个人的期权大小。在确定岗位期权量时可以先按部门分配，再具体到岗位。 公司总池子确定下来，再综合考虑他的职位、贡献、薪水与公司发展阶段，员工该取得的激励股权数量基本就确定下来了。同一个级别的技术大拿，在VC进来之前就参与创业、在VC进来后才加入公司、在C轮甚至IPO前夕加入公司，拿到的期权应该设计成区别对待。另外，公司也可以
Mathematica函数大全--运算符及特殊符号一、运算符及特殊符号 Line1;执行Line，不显示结果 Line1,line2顺次执行Line1，2，并显示结果 ?name关于系统变量name的信息 ??name关于系统变量name的全部信息 !command执行Dos命令 n! N的阶乘 !!filename显示文件内容
a-b减 a*b或a b 乘 a/b除 a^b 乘方 base^^num以base为进位的数 lhs&&rhs且 lhs||rhs或 !lha非 ++,-- 自加1，自减1 +=,-=,*=,/= 同C语言 >,<,>=,<=,==,!=逻辑判断（同c） lhs=rhs立即赋值 lhs:=rhs建立动态赋值 lhs:>rhs建立替换规则 expr//funname相当于filename[expr] expr/.rule将规则rule应用于expr expr//.rule 将规则rule不断应用于expr知道不变为止param_ 名为param的一个任意表达式（形式变量）param__名为param的任意多个任意表达式（形式变量） 二、系统常数 Pi 3.1415....的无限精度数值 E 2.17828...的无限精度数值 Catalan 0.915966..卡塔兰常数 EulerGamma 0.5772....高斯常数 GoldenRatio 1.61803...黄金分割数 Degree Pi/180角度弧度换算 I复数单位 Infinity无穷大
一建、二建知识点归拢便于记忆的顺口溜（另有免费资料分 享） 不得不说，一建和二建部分内容是重复的，而且是非常难记忆的，被同学们问到的问题，有时候也会抓瞎去翻书查看，下面对这些难记又拗口的知识点进行了总结和归纳，觉得游泳的朋友们可以收藏，祝大家顺利通过建造师考试！（文末有福利哦）一、塔吊操作规程“十不吊”顺口溜记忆：歪人看超信，器平加安力。（1）歪拉斜吊重物时不吊；（2）被吊物上有人或浮置物时不吊；（3）工作场地昏暗，无法看清场地、被吊物和指挥信号时不吊；（4）超载或被吊物重量不清不吊；（5）指挥信号不明确不吊；（6）容器内装的物品过满时不吊；（7）捆绑、吊挂不牢或不平衡，可能引起滑动时不吊；（8）被吊物棱角处与捆绑钢绳间未加衬垫时不吊；（9）结构或零部件有影响安全工作的缺陷或损伤时不吊；（10）遇有拉力不清的埋置物件时不吊。二、气割、电焊的“十不烧”规定顺口溜记忆：内飞盗名器，外围正动理。（1）焊工不了解焊件内部是否有易燃、易爆物时，不得进行焊、割；（2）用可燃材料保温层、冷却层、隔声、隔热设备的部位，或火星能飞溅到的地方，在未采取切实可靠的安全措施之前，不准焊、割；（3）有压力或密闭的管道、容器，不准焊、割；（4）附近有与明火作业相抵触的工种在作业时，不准焊、
割；（5）各种装过可燃气体、易燃液体和有毒物质的容器，未经彻底清洗，或未排除危险之前，不准进行焊、割；（6）与外单位相连的部位，在没有弄清有无险情，或明知存在危险而未采取有效的措施之前，不准焊、割；（7）焊工不了解焊、割现场的周围情况，不得进行焊、割；（8）焊工必须持证上岗，无证者不准进行焊、割作业；（9）属一、二、三级动火范围的焊、割作业，未经办理动火审批手续，不准进行焊割；（10）焊、割部位附近有易燃易爆物品，在未作清理或未采取有效的安全防护措施前，不准焊、割。三、安全检查的方式顺口溜记忆：专日季节，定开复设经。（1）专业性检查、专项检查；（2）日常巡查；（3）季节性检查；（4）节日前后的安全检查；（5）定期安全检查；（6）开工、复工安全检查；（7）设备、设施安全检查；（8）经常性检查。四、建筑工程费用顺口溜记忆：1、措施费：二环夜临大水，支架已安稳。（1）二：二次搬运；（2）环：环境保护；（3）夜：夜间施工；（4）临：临时措施；（5）大：大型机械；（6）水：降水、排水；（7）支：支模、模板费；（8）架：脚手架；（9）已：已完工程及设备保护费；（10）安：安全施工；（11）稳(文)：文明施工。2、规费：保定无公害。（1）保：社会保障；（2）定：工程定额测定；（3）无(污)：工程排污；（4）公：住房公积金；（5）害：危险意外伤害保险。3、企业管理费：公差使用保险金务工。（1）公：办公费；（2）差：差旅；（3）
建造师考试分为一级建造师和二级建造师，全国统一命题，统一考试，考试通过并取得建造师执业资格证书且符合注册条件的人员，经过注册登记后，即获得一级或二级建造师注册证书。到底有什么区别呢？下面一起看一下吧： 【区别】 1教材设置 一级、二级建造师考试及教材设置侧重点略有不同。 一级建造师专业科目有10个：建筑工程、公路工程、市政公用工程、 水利水电工程、矿业工程、机电工程、铁路工程、通信与广电工程、民航机场工程、港口与航道工程。 二级建造师专业科目只有6个：建筑工程、公路工程、市政公用工程、水利水电工程、矿业工程、机电工程。 教材设置而言：就法规和管理，两者差别并不很大，比如法规一级教材三百多页，二级只有二百多页，内容多了1/3。而最为关键的实务考试，一 级教材页数也明显多于二级，但在施工细节方面却略有不足。比如建筑中钢筋下料一级是不讲的，毕竟一级培养的大型工程项目经理，不是具体的施工员。而二级中对钢筋下料及公式换算有一定的篇幅，介绍也比较详细。2考试科目 一级建造师需要考四科：建设工程经济、建设工程项目管理、建设工程法规及相关知识、专业工程管理与实务 二级建造师需要考三科：建设工程法规及相关知识、建设工程施工管理、专业工程管理与实务 3考试时间 一级建造师考试时间大概在每年9月份 二级建造师考试时间大概在每年6月份 4考试通过率 一级建造师考试通过率6-8% 二级建造师一般在25-30%左右 不难看出，从考试难度上来说：一级建造师比二级建造师的考试难度要大的多，但是二级建造师却又是建筑行业入门不可或缺的敲门砖。
5报名条件 一级建造师报考条件： 可见，参加一建考试须取得工程类或工程经济类专科及以上学历才可报名； 二级建造师报考条件： 凡遵纪守法，具备工程类或工程经济类中等专科以上学历并从事建设工程项目施工管理工作满2年的人员，可报名参加二级建造师执业资格考试。6实务合格分数线 一级建造师96分(满分160) 二级建造师45-65分(满分120，具体分数线由各省控制，各专业也不同) 7就业方向 从使用范围上来说：一级建造师比二级建造师广，一级建造师是全国范围内通用的，二级建造师一般只能在省内通用。 一级建造师：可以建造师的名义担任建设工程项目施工的项目经理、从事其他施工活动的管理、从事法律、行政法规或国务院建设行政主管部门规定的其他业务。在行使项目经理职责时，一级注册建造师可以担任《建筑业企业资质等级标准》中规定的特级、一级建筑业企业资质的建设工程项目施工的项目经理。 大中型工程项目的项目经理必须逐步由取得建造师执业资格的人员担任;但取得建造师执业资格的人员能否担任大中型工程项目的项目经理，应由建筑业企业自主决定。 二级建造师：可以建造师的名义担任建设工程项目施工的项目经理、从事其他施工活动的管理、从事法律、行政法规或国务院建设行政主管部门规定的其他业务。在行使项目经理职责时，二级注册建造师可以担任二级建筑业企业资质的建设工程项目施工的项目经理。但取得建造师执业资格的人员能否担任大中型工程项目的项目经理，应由建筑业企业自主决定。 一建和二建考试七大不同之处，想好报哪个了吗？该考哪个看清楚了吗？
谈谈股权激励和期权激励 完善公司治理结构，建立所有者与经营者之间的制衡关系是现代公司制度的核心。为什么说它是现代公司的核心？这是因为，现代公司区别于传统公司的主要特征，是在所有与控制（经营）之间发生了分离。要想使现代公司经营得好，必须使经营者有职有权，与此同时，又必须将经营者的职权置于所有者利益的约束之下，避免出现“内部人控制”的弊病。对于这一矛盾，只有靠建立好的公司治理机制才能解决。 发达市场经济国家的理论和实距显示出以股东中心即增进股东价值（shareholder value）的公司理念越来越成为主流的趋向。薪酬激励是公司治理机制中的重要组成部分。在好的公司治理结构中，必然有精心设计的激励机制，用以协调所有者和经营者之间的矛盾，使二者利益趋向一致，达成追求股东价值最大化的目标。并且，它是由所有者的真正代表运用薪酬手段来保证激励的兼容性的，促使被激励者按照激励者的要求去活动。 薪酬组合中的每一项都有其针对性，比如，基本工资是保障员工的基本生活；奖金是对员工绩效的直接回报；长期激励的功能是解决所有者与经营者利益一致性，其主要形式股票期权对鼓励经理人员在任职期间努力工作可以起很好的作用；福利计划则是解决员工后顾之忧、弥补现金激励的不足。 在西方国家的大公司中，不同职位的人其薪酬组合的结构是不相同的。比如蓝领工人的长期激励部分相对较小，而高层经营管理人员的长期激励所占的比重很大。 由于高级经理人员的好坏决定了公司的成败，好的经理人员是具有特殊禀赋的人才，他们在市场上属于相对稀缺的资源。因此，为了保持高级经理人员的稳定，驱动他们除了保持一个一般良好的声誉和业绩之外，愿意承担一定的风险，不断地为股东博取更大的价值，利用股权激励，给他们带来可能的高收益，分享股东一部分剩余索取权是行之有效的办法。从经理人员的角度，如果他们薪酬只是基本工资和对已完成业绩的年度奖励，他势必只追求短期的利益，若要使他为企业的长远发展考虑，就必须有相应的激励手段。股权激励就是着眼于未来，把经理人员的可能收益和他对公司未来成绩的贡献联系起来。从已有的实践，股权激励是协调股东和经理人员根本利益的办法。 在上市公司中，对于经理人员的激励表现出两种偏向。一种是工资总额和等级标准，这种薪酬制度不能恰当地估计和承认经理人员的贡献，薪酬制度不足以吸引和激励人才；另一种则是在“年薪制”、“经理人员持股制”等试验中，在所有者虚位的情况下，经营者自定高额薪酬、浪费性职位消费、腐败等等，经理人员实行自我激励。 股票期权是公司所有者用以激励经理人员努力工作，提升股东价值的重要手段。因此，它必须掌握在所有者及其信任托管者董事会手中。这就是为什么美国十个大公司在介绍自己的激励制度时都强调了“不能够自己给自己定薪酬”的道理所
mathematica 运算符及特殊符号 一、运算符及特殊符号 Line1; 执行Line,不显示结果 Line1,line2 顺次执行Line1,2,并显示结果name 关于系统变量name 的信息 name 关于系统变量name 的全部信息 在c 语言中使用math 的函数 (*Note*) 程序的注释 #n 第n 个参数 ## 所有参数 rule& 把rule 作用于后面的式子 % 前一次的输出 %% 倒数第二次的输出 %n 第n 个输出 a+b 加 a-b 减 a*b 或a b 乘 a/b 除 a^b 乘方 base^^num 以base 为进位的数 lhs&&rhs 且
lhs||rhs 或 !lha 非 ++,-- 自加1,自减1 +=,-=,*=,/= 同C 语言 二、系统常数 Pi 3.1415....的无限精度数值 E 2.17828...的无限精度数值 Catalan 0.915966..卡塔兰常数EulerGamma 0.5772....高斯常数GoldenRatio 1.61803...黄金分割数 Degree Pi/180 角度弧度换算 I 复数单位 Infinity 无穷大 -Infinity 负无穷大 ComplexInfinity 复无穷大 Indeterminate 不定式 三、代数计算 Expand[expr] 展开表达式 Factor[expr] 展开表达式 Simplify[expr] 化简表达式 FullSimplify[expr] 将特殊函数等也进行化简PowerExpand[expr] 展开所有的幂次形式
ComplexExpand[expr,{x1,x2...}] 按复数实部虚部展开FunctionExpand[expr] 化简expr 中的特殊函数 Collect[expr, x] 合并同次项 Collect[expr, {x1,x2,...}] 合并x1,x2,...的同次项Together[expr] 通分 Apart[expr] 部分分式展开 Apart[expr, var] 对var 的部分分式展开 Cancel[expr] 约分 ExpandAll[expr] 展开表达式 ExpandAll[expr, patt] 展开表达式 FactorTerms[poly] 提出共有的数字因子 FactorTerms[poly, x] 提出与x 无关的数字因子FactorTerms[poly, {x1,x2...}] 提出与xi 无关的数字因子Coefficient[expr, form] 多项式expr 中form 的系数Coefficient[expr, form, n] 多项式expr 中form^n 的系数Exponent[expr, form] 表达式expr 中form 的最高指数Numerator[expr] 表达式expr 的分子 Denominator[expr] 表达式expr 的分母ExpandNumerator[expr] 展开expr 的分子部分ExpandDenominator[expr] 展开expr 的分母部分TrigExpand[expr] 展开表达式中的三角函数 TrigFactor[expr] 给出表达式中的三角函数因子
一建各科老师推荐 与你分享往届学员们的一建学习经验与心得，带你少走弯路，顺利 通关。 随着这几年出题越来越难，公共课只靠做会历年真题就能过的时代已经过去了。14 年法规异军突起，15 年经济独领风骚，16 年押题不是那么理想了，17年大家踏实把基础打好，加最后押题通过希 望才大。第一轮基础复习的时候，听讲的细，讲的透的老师，会是比较好的选择。 注：★评定主要从老师的形象与声音、讲课知识的系统性和条理性、与其它科目之间的联系性、每个章节间串讲真题的数量和解析深度、讲课内容的生动性以及最重要的帮助大家理解记忆的能力等几个方 面进行考量。 【管理】 朱俊文★★★★★管理没有特别牛的老师，但朱俊文毫无疑问是大家心里的NO.1 。娓娓道来容易让人接受，带你理解历年真题，并授以应试技巧。 赵进东★★★★很受欢迎的老师，重点突出，令人印象深，授课方式易于接受。复习时间不是很充裕的话，推荐第一轮就从赵进东入手。
肖国强★★★讲得比较细的老师，可是长相猥琐，还有点拽，加上口音，直接秒杀了一部分同学。但他会跟你强调一些比较重要的内容要看要背，就是篇幅过长，需要点耐心，刚开始复习小伙伴听起来会比较枯燥。 顾永才★★★讲课特点：语速快，很自信，听起来比较过瘾，老说书上这错那错的，但讲的内容范围过窄，听完之后发现没学到多少实质性内容，不适合打基础和应试。 龙炎飞★★★网上很多评论都说讲的很好，幽默，重点突出。个人听了没两节课就PASS过去了。感觉跳跃的内容过多，整个讲课的气氛让人容易犯困。习题讲解略肤浅。萝卜青菜吧。 【经济】 达江★★★★★听过他课的都觉得很棒，通俗易懂，深入浅出，举例很生动，教你建立整个章节的框架，把一个个知识点联系起来帮你记忆。零基础的听起来也比较轻松。 徐蓉★★★★★讲得很细，真题解析的比较透彻，会提醒你某些部分会与实务案例相联系。教你自己如何去归纳知识点。缺点：语速如切菜，凌厉的气势让一些小伙伴无法接受。 梅世强★★★★13年讲的重点都考到了，奠定了经济第一人的基础。讲课较为精简，基本内容以重点为主，零基础的听会觉得跳跃太快。比较适合冲刺的时候听。
建造师考试之一建与二建的区别 部分同学对一级建造师和二级建造师的区别很模糊，不知道究竟有什么不同，以至于不知道自己应该报考哪一个，今天小编就来给大家讲解一下他们的区别在哪里，方便大家了解，然后选择最适合自己的。 从考试时间上 一级建造师考试时间在每年的9月份左右（2018年9月15日、16日） 二级建造师考试时间在每年的5月份左右（2018年6月2日、3日） 从专业方向来说 1、一级建造师，是建设工程行业的一种执业资格，是担任大型工程项目经理的前提条件。建造师是指从事建设工程项目总承包和施工管理关键岗位的执业注册人员。建造师是懂管理、懂技术、懂经济、懂法规，综合素质较高的复合型人员，既要有理论水平，也要有丰富的实践经验和较强的组织能力。建造师注册受聘后，可以建造师的名义担任建设工程项目施工的项目经理、从事其他施工活动的管理、从事法律、行政法规或国务院建设行政主管部门规定的其他业务。建造师的职责是根据企业法定代表人的授权，对工程项目自开工准备至竣工验收，实施全面的组织管理。 2、二级建造师，是建筑类的一种执业资格，是担任项目经理的前提条件。注册建筑师以专业技术为依托，以工程项目管理为主业的注册执业人士。建造师注册受聘后，可以以建造师的名义担任建设工程项目施工的项目经理、从事其他施工活动的管理、从事法律、行政法规或国务院建设行政主管部门规定的其他业务。在行使项目经理职责时，二级注册建造师可以担任二级建筑业企业资质的建设工程项目施工的项目经理。但取得建造师执业资格的人员能否担任大中型工程项目的项目经理，应由建筑业企业自主决定。 从专业科目上说 一级的专业有10个：分别是建筑工程、公路工程、铁路工程、民航机场工程、港口与航道工程、水利水电工程、市政公用工程、通信与广电工程、矿业工程、机电工程。 二级的专业有6个：建筑工程、公路工程、水利水电工程、矿业工程、机电工程和市政公用工程。 从考试科目来说 一级建造师需要考四科：建设工程经济、建设工程项目管理、建设工程法规及相关知识、专业工程管理与实务 二级建造师需要考三科：建设工程法规及相关知识、建设工程施工管理、专业工程管理与实务
认清期权和股票的区别，投资者很多只认识股票不认识股权，只是单纯的认为期权其实就是高杠杆的配资，期权其实就是股票。这里其实是很多投资者在一些非专业的人员的宣传下产生了一下误解和概念性的模糊。那么我们来了解一下期权和股票之间的区别，两者有着什么样的联系。 期权和股票概念 期权是指一种合约，该合约赋予持有人在某一特定日期或该日之前的任何时间以固定价格购进或售出一种资产的权利。 股票（stock）是股份公司发行的所有权凭证，是股份公司为筹集资金而发行给各个股东作为持股凭证并借以取得股息和红利的一种有价证券。每股股票都代表股东对企业拥有一个基本单位的所有权。每家上市公司都会发行股票。 期权和股票的区别： 1.期权和股票投资对象不同： 期权是一种能在未来特定时间以特定价格买进或卖出一定数量的特定资产的权利。 股票是对上市的企业进行投资; 2.期权和股票投资期限： 期权的期限一般建议在1月以上是属于中、长期投资; 股票的期限是灵活的，实施T+1交易制度，买入的股票在第二个工作日就可以开始交易。当然根据投资者的投资偏好也可以进行长期投资; 3.期权和股票投资回报不同： 期权;购买的是标的未来上涨空间的无限收益， 股票更多的是赚取买入卖出的股价中间差; 4.期权和股票投资性质： 期权是属于策略性衍生品的投资; 股票是公开交易的;
5.期权和股票投资起点： 期权门槛一般都是百万级别的（名义本金）; 股票的最低门槛可以很低，就是看投资者选购哪只股票了，只要100股起买，低价股100块就可以投资了。 综上所述，期权是一种选择的权利，买方有权利无义务，有利就行权，不利就放弃行权，亏损权利金。股票是单纯性的线性交易，收益与风险相对。期权包括股票期权，利率期权，商品期货期权，股指期权、外汇期权等。所有期权是一种套利保险，降低线性交易中的下跌风险与股票有本质上的区别。 了解更多50ETF期权基础知识或操作技巧，请微信搜索“期权魔方”公众号（注册邀请码：5006）
【4句话告诉你】一建与二建真正的差别 1、从使用范围上来说 一级建造师比二级建造师广一些，一级建造师是全国范围内通用的，二级建造师只能在本省使用。 2、从考试难度上来说 二级建造师是根据通过率来计算合格分数线的，每年都会降分；一级建造师的合格线是按总分的60%也就是60分和72分。一级建造师比二级的难很多，尤其是实务。 3、从专业科目上来说 一级的专业有10个，分别是建筑工程、公路工程、铁路工程、民航机场工程、港口与航道工程、水利水电工程、市政公用工程、通信与广电工程、矿业工程、机电工程。二级的专业只有6个：建筑工程、公路工程、水利水电工程、矿业工程、机电工程和市政公用工程。 4、从从业方向上来说 一级建造师：可以建造师的名义担任建设工程项目施工的项目经理、从事其他施工活动的管理、从事法律、行政法规或国务院建设行政主管部门规定的其他业务。在行使项目经理职责时，一级注册建造师可以担任《建筑业企业资质等级标准》中规定的特级、一级建筑业企业资质的建设工程项目施工的项目经理。大中型工程项目的项目经理必须逐步由取得建造师执业资格的人员担任；但取得建造师执业资格的人员能否担任大中型工程项目的项目经理，应由建筑业企业自主决定。一建二建课件+面授+精准押题Q/微信673881898
二级建造师：可以建造师的名义担任建设工程项目施工的项目经理、从事其他施工活动的管理、从事法律、行政法规或国务院建设行政主管部门规定的其他业务。在行使项目经理职责时，二级注册建造师可以担任二级建筑业企业资质的建设工程项目施工的项目经理。但取得建造师执业资格的人员能否担任大中型工程项目的项目经理，应由建筑业企业自主决定。
期权和权证的区别 期权和权证的区别 交易双方的地位不同。期权的双方一般而言都是普通的投资者，他们约定的标的物是某种权益凭证，例如在某时间以某价格买卖某只股票。而权证，是上市公司跟一般投资者之间的约定。相同点主要是：投资买入期权或权证后，（无论是买权或卖权），他们都有选择是否执行该权利。而卖方只有等待买方的执行与否，没有任何权利，但可以收取卖出权利的费用。首先，期权是一种在交易所交易的标准化合约，其行权价格、到期日等条款已标准化，不利于在经常变动的市场中灵活调整，也缺乏创新的空间；相反，与权证挂钩的相关资产的数量远较期权的选择多。通常的情况是活跃股票的权证品种多，而活跃股票的期权则较少，投资者往往较难找到合适的期权。而每当市场出现一些新的持续性热点，备兑权证发行人看准时机，就可发行新的备兑证，在符合香港交易所《上市规则》的前提下，适时为投资者提供更多的选择，满足市场的需求。另一方面，备兑证市场可创新的空间很大，针对各种不同市况以及针对一些有较明确观点的投资群体，发行人可以适时推出带有特殊结构的备兑证，如锁定回报权证、跨价权证、一揽子股票权证等，方便投资者选择符合自己观点的备兑证进行投资。 其次，期权市场可运用的策略远较权证市场丰富。投资者参与期权投资，除了可以像买卖权证一样买入认购（或认沽）期权（又称为“长仓”），还可以卖出认购（或认沽）期权（又称为“短仓”），赚取权利金；而在权证市场，只有发行人才可以卖出备兑证收取权利金，投资者只能付出权利金买入认购证或认沽证。例如，买入认沽期权和卖出认购期权两种操作，表面上看都是看跌的策略，但实际的投资效果大不一样，所适合的市况也有差异，如果再把买进和卖出期权进行组合，其策略就更加丰富，可以达到根据自己的观点量身定做一个投资工具的效果，给投资者的满足感更大。 第三，期权是一种标准化合约，只要有成交就会产生一份期权合约，因此理论上，期权成交的数量可以是无限的。而单只权证的发行量通常会在发行文件中给出一个上限，并在一定的条件下可以增加发行量。但就香港市场而言，备兑证市场的发行量及交易量都远大于期权市场，其原因主要在于市场机制上有所差别。由于期权类产品的定价不像股票定价单纯，其中涉及高等数学及深奥的金融工程知识，所以香港和全球大部分市场一样都实行“庄家制”（国内称为权证一级交易商制度），通过专业金融机构同时提供买盘和卖盘报价，满足市场对流动性的要求。期权市场是属于交易所的产品，由多家证券公司一起提供报价，而权证市场则由单一庄家（通常是发行人）报价。期权市场由于实行多家庄家共管的制度，经营好坏难以与庄家的声誉挂钩，反而形成三个和尚没水喝的局面，有时一个期权的叫买价和叫卖价之间相差达到10％~15％，而且报盘可能只有几张。相反，权证发行人为了吸引投资者参与自己所发行的权证（赚取权证的时间价值），除了会积极进行市场推广，让投资大众有更多的机会接触和了解权证之外，也会提供更多实质的售后服务，例如，在正常情况下倾向开出较有竞争性的价格、维持较窄的买卖差价和足够数量的买卖接盘（流动性），提供方便而友善的咨询查询服务等。这是因为每一只权证都有一个证券代码，都代表着惟一的发行人，而香港市场的报盘都会揭示席位号，每一位发行人都有公开的专用席位号，所以谁挂出了买卖接盘一目了然。发行人为建立自己的品牌，扩大竞争优势，有动力投入更多的资源。归根到底，权证是属于产品差异化很低、进入壁垒很低的产品，因而竞争激烈。依笔者之见，单一庄家制度是香港权证市场赖以发展壮大的关键因素之一。 问题补充：是不是期权与权证的区别是物与股票? 还有股票期权是不是权证? 权证就是期权的一种，大概就像你理解的那样 股票期权可以是权证，也就是单独交易的一个证券产品也可以是一种对公司高管人员的奖励手段，比如我们经常听到的公司的股权激励，这类期权是不能交易的，期权是指在未来一定时期可以买卖的权力，是买方向卖方支付一定数量的金额（指权利金）后拥有的在未来一段时间内（指美式期权）或未来某一特定日期（指欧式期权）以事先规定好的价格（指履约价格）向卖方购买或出售一定数量的特定标的物的权力，但不负有必须买进或卖出的义务。 1）看涨期权：1月1日，标的物是铜期货，它的期权执行价格为1850美元/吨。A买入这个权利，付出5美元；B卖出这个权利，收入5美元。2月1日，铜期货价上涨至1905美元／吨，看涨期权的价格涨至55美元。A可采取两个策略： 行使权利一一A有权按1 850美元/吨的价格从B手中买入铜期货；B在A提出这个行使期权的要求后，必须予以满足，即便日手中没有铜，也只能以1905美元吨的市价在期货市场上买入而以1850美元／吨的执行价卖给A，而A可以1905美元/吨的市价在期货币场上抛出，获利50美元（1 905一1850一5）。B则损失50美元（1850一1905+5）。 售出权利一一A可以55美元的价格售出看涨期权、A获利50 美元（55一5）。 如果铜价下跌，即铜期货市价低于敲定价格1850美元／吨，A就会放弃这个权利，只损失5美元权利金，B则净赚5美元。 权证的举例:1） 4）：A公司发行以该公司股票为标的证券的权证，假定发行时股票市场价格为15元，发行公告书列举的发行条件如下：发行日期：2005年8月8日；存续期间：6个月；权证种类：欧式认购权证；发行数量：5000万份；发行价格：0.66元；行权价格：18.00元；行权期限：到期日；行权结算方式：证券给付结算；行权比例：1：1。 上述条款告诉投资者由A公司发行的权证是一种股本认购权证，该权证每份权利金是0.66元，发行总额为5000万份，权证可以在六个月内买卖，但行权则必须在六个月后的到期日进行。如果到期时A公司股票市场价格为20元，高于权证的行权价18
第8章Mathematica中的常用函数8.1 运算符及特殊符号 Linel 执行Line，不显示结果 Linel,line2 顺次执行Line1，Line2，并显示结果 ?name 关于系统变量name的信息 ??name 关于系统变量name的全部信息 !command 执行Dos命令 N! N的阶乘 !!filename 显示文件内容 <
-Infinity 负无穷大 Complexlnfinity 复无穷大 Indeterminate 不定式 8.3 代数计算 Expand[expr] 展开表达式 Factor[expr] 展开表达式 Simplify[expr] 化简表达式 FullSimplify[expr] 将特殊函数也进行化简PowerExpand[expr] 展开所有的幂次形式ComplexExpand[expr,{x1，x2…}] 按复数实部虚部展开FunctionExpand[expr] 化简表达式中的特殊函数 Collect[expr,x] 合并同次项 Collect[expr,{x1，x2，…}] 合并x1，x2,...的同次项 Together[expr] 通分 Apart[expr] 部分分式展开 Apart[expr,var] 对var的部分分式展开 Cancel[expr] 约分 ExpandAll[expr] 展开表达式 ExpandAll[expr,patt] 展开表达式 FactorTermsrpoly] 提出共有的数字因子 FactorTerms[poly，x] 提出与x无关的数字因子 FactorTerms[poly,(x1,x2…)] 提出与xi无关的数字因子 Coefficient[expr,form] 多项式expr中form的系数 Coefficient[expr,form，n] 多项式expr中form^n的系数 Exponent[expr,form] 表达式expr中form的最高指数 Numerator[expr] 表达式expr的分子 Denominator[expr] 表达式expr的分母 ExpandNumerator[expr] 展开expr的分子部分 8.4 解方程 Solve[eqns,vats] 从方程组eqns中解出Vats Solve[eqns,vats,elims] 从方程组eqns中削去变量elims，解出vats DSolve[eqn,y,x] 解微分方程，其中、y是x的函数 DSolve[{eqnl,eqn2,…}，{y1，y2…},] 解微分方程组，其中yi是x的函数DSolve[eqn,y,{x1,x2…}]解偏微分方程 Eliminate[eqns，Vats] 把方程组eqns中变量vars约去SolveAlways[eqns，vars] 给出等式成立的所有参数满足的条件Reduce[eqns，Vats] 化简并给出所有可能解的条件LogicalExpand[expr] 用&&和，，将逻辑表达式展开InverseFunction[f] 求函数f的反函数 Root[f,k] 求多项式函数的第k个根
d(f,x) f对x方向的微分 1. 使用d算符来计算一个变量对另一个变量的导数，如： d(T,x)指变量T对x求导,而d(u^2,u)=2*u等； 2. 如果模型中含有任何独立变量，建模中使用d算符会使 模型变为非线性； 3. 在解的后处理上使用d算符，可以使用一些预置的变 量，如：uxx,d(ux,x),d(d(u,x),x)都是等效的； 4. pd算符与d算符类似，但对独立变量不使用链式法则； 5. d(E,TIME)求解表达式E的时间导数； 6. dtang算符可以计算表达式在边界上的切向微分（d算 符无法计算），在求解域上使用dtang等价于d，dtang 只求解对坐标变量的微分，但需要注意的是并不是所有 的量都有切向微分。 pd(f,x) f对x方向的微分 pd和d的区别： d(u+x,x)=ux+1，d(u,t)=ut，u和x,t等有关 pd(u+x,x)=1，pd(u,t)=0，u是独立的和x,t无关 dtang(f,x) 边界上f对x的切向微分 在边界上d(u,x)不能定义，但是可以使用dtang(u,x)，dtang 付出基本的微分法则，如乘积法则和链式法则，但是需要指 出的是，dtang(x,x)不一定等于1。 test(expr) 试函数 用于方程弱形式的算符，test(F(u,?u))等价于： var(expr,fieldnam 变异算子 e1, 用于弱形式，它和test算符功能相同，但是仅用于某些特
fieldname2, ...) 定的场中； 如var(F(u,?u, v,?v),a)，变量u是a场的变量，而v不 是。 试函数之只作用于变量u。 nojac(expr) 对Jacobian矩阵没有贡献 将表达式排除在Jacobian计算外，这对那些对Jacobian贡 献不大，但是计算消耗很大的变量是否有效； k-e 湍流模型就是利用 nojac算符来提高计算性能的例子。up(expr) 上邻近估算表达式 up，down，mean算符只能用在边界上，对于一个表达式或变 量在边界处两边不连续，COMSOL通常显示边界的平均值，使 用up，down可计算某个方向上的值。 down(expr) 下邻近估算表达式 mean(expr) 邻近边界上的平均值 depends(expr) 查看某个表达式是否依赖于求解结果 isdefined(variabl 变量是否定义 e) dest(expr) 在目标端计算积分耦合表达式 dest算符强制将source points上的表达式用在 destination points上。 例如：u/((dest(x)-x)^2+(dest(y)-y)^2) if(cond,expr1,exp 条件表达式 r2) 例如：if(x==0,1,sin(x)/x) isinf(expr) 表达式的值是否是无穷大 islinear(expr) 解是否是线性函数 isnan(expr) 表达式是否是非数 with 调用某个解
建造师执业资格考试分为一级建造师和二建建造师两个等级，两个等级考试科目、考试专业、报考条件包括以后的执业范围等各项因素都不一样。本次我们为您解答一建和二建有什么区别： 考试科目： 一级建造师需要考四科：建设工程经济、建设工程项目管理、建设工程法规及相关知识、专业工程管理与实务。 二级建造师需要考三科：建设工程法规及相关知识、建设工程施工管理、专业工程管理与实务。 考试专业： 一级建造师专业有10个：分别是建筑工程、公路工程、铁路工程、民航机场工程、港口与航道工程、水利水电工程、市政公用工程、通信与广电工程、矿业工程、机电工程。 二级建造师专业只有6个：分别是建筑工程、公路工程、水利水电工程、矿业工程、机电工程和市政公用工程。 就业方向： 一级建造师注册受聘后，可以建造师的名义担任建设工程项目施工的项目经理、从事其他施工活动的管理、从事法律、行政法规或国务院建设行政主管部门规定的其他业务。在行使项目经理职责时，一级注册建造师可以担任《建筑业企业资质等级标准》中规定的特级、一级建筑业企业资质的建设工程项目施工的项目经理。大中型工程项目的项目经理必须逐步由取得建造师执业资格的人员担任；但取得建造师执业资格的人员能否担任大中型工程项目的项目经理，应由建筑业企业自主决定。 二级建造师注册受聘后，可以建造师的名义担任建设工程项目施工的项目经理、从事其他施工活动的管理、从事法律、行政法规或国务院建设行政主管部门规定的其他业务。在行使项目经理职责时，二级注册建造师可以担任二级建筑业企业资质的建设工程项目施工的项目经理。但取得建造师执业资格的人员能否担任大中型工程项目的项目经理，应由建筑业企业自主决定。 使用方面： 一级建造师可以在全国范围内进行注册，全国均可执业，而二级建造师则是在所在考区省内在注册，省内执业。 证书： 一级、二级建造师执业资格考试合格人员，分别获得《中华人民共和国一级建造师执业资格证书》、《中华人民共和国二级建造师执业资格证书》。
权证与期权的区别 (1)有效期。认股权证的有效期(即发行日至到期日之间的期间长度)通常比股票期权的有效期长：认股权证的有效期一般在一年以上，而股票期权的有效期一般在一年以内。股票期权的有效期偏短，并不是因为不能设计成长有效期，而是交易者自然选择的结果。就像期货合约一样，期货合约的交易量一般聚集在近月合约上，远月合约的交易量一般很少，不如不挂。 (2)标准化。认股权证通常是非标准化的，在发行量、执行价、发行日和有效期等方面，发行人通常可以自行设定，而交易所交易的股票期权绝大多数是高度标准化的合约。当然，当前随着IT技术的发展，柜台市场与交易所场内市场出现融合的趋势，柜台市场上的非标准化的股票期权也开始进入交易所场内市场。(3)卖空。认股权证的交易通常不允许卖空，即使允许卖空，卖空也必须建立在先借入权证实物的基础上。如果没有新发行和到期，则流通中的权证的数量是固定的。而股票期权在交易中，投资者可以自由地卖空，并且可以自由选择开平仓，股票期权的净持仓数量随着投资者的开平仓行为不断变化。(4)第三方结算。在认股权证的结算是在发行人和持有人之间进行，而股票期权的结算，是由独立于买卖双方的专业结算机构进行结算。因此，交易股票期权的信用风险要低于交易认股权证的信用风险。 (5)做市商。认股权证的做市义务通常有发行人自动承担，即使是没有得到交易所的正式指定，发行人也通常需要主动为其所发行的认股权证的交易提供流动性。而股票期权的做市商必须是经由交易所正式授权。上面已经看到，全球股票期权的交易量远远大于衍生权证的交易量。现在通过这种比较，容易理解为什么会是这样。显然，在可交易性、信用风险等方面，股票期权要优于认股权证。大致上可以这么来说，交易所交易的股票期权就好比是现在的工业化大生产下的高度标准化、正规化的产品，而认股权证则像是手工作坊阶段量身定制的产品。虽然，对个别人来讲，量身定制的产品要好于标准化的产品，但是从适用面上就显得窄。但是另外一方面，高度标准化的产品也不能完全取代量身定制的产品。 首先,交易双方的地位不同。期权的双方一般而言都是普通的投资者，他们约定的标的物是某种权益凭证。例如，在某时间以某价格买卖某只股票。而权证，是上市公司跟一般投资者之间的约定。相同点主要是：投资买入期权或权证后（无论是买权或卖权），他们都有选择是否执行该权利。而卖方只有等待买方的执行与否，没有任何权利，但可以收取卖出权利的费用。首先，期权是一种在交易所交易的标准化合约，其行权价格、到期日等条款已标准化，不利于在经常变动的市场中灵活调整，也缺乏创新的空间；相反，与权证挂钩的相关资产的数量远较期权的选择多。通常的情况是活跃股票的权证品种多，而活跃股票的期权则较少，投资者往往较难找到合适的期权。而每当市场出现一些新的持续性热点，备兑权证发行人看准时机，就可发行新的备兑证，在符合香港交易所《上市规则》的前提下，适时为投资者提供更多的选择，满足市场的需求。另一方面，备兑证市场可创新的空间很大，针对各种不同市况以及针对一些有较明确观点的投资群体，发行人可以适时推出带有特殊结构的备兑证，如锁定回报权证、跨价权证、一揽子股票权证等，方便投资者选择符合自己观点的备兑证进行投资。 其次，期权市场可运用的策略远较权证市场丰富。投资者参与期权投资，除了可以像买卖权证一样买入认购（或认沽）期权（又称为“长仓”），还可以卖出认购（或认沽）期权（又称为“短仓”），赚取权利金。而在权证市场，只有发行人才可以卖出备兑证收取权利金，投资者只能付出权利金买入认购证或认沽证。例如，买入认沽期权和卖出认购期权两种操作，表面上看都是看跌的策略，但实际的投资效果大不一样，所适合的市况也有差异，如果再把买进和卖出期权进行组合，其策略就更加丰富，可以达到根据自己的观点量身定做一个投资工具的效果，给投资者的满足感更大。 第三，发行数量的不同。期权是一种标准化合约，只要有成交就会产生一份期权合约。因此理论上，期权成交的数量可以是无限的。而单只权证的发行量通常会在发行文件中给出