Engineering of protein secretion in yeast

The TLR2-MyD88-NOD2-RIPK2signalling axis regulates a balanced pro-inflammatory and IL-10-mediatedanti-inflammatory cytokine response to Gram-positive cell wallsLilian O.Moreira,1,2‡Karim C.El Kasmi,1†‡Amber M.Smith,1,2David Finkelstein,3Sophie Fillon,1†Yun-Gi Kim,4Gabriel Núñez,4Elaine Tuomanen 1and Peter J.Murray 1,2*1Department of Infectious Diseases,St.Jude Children’s Research Hospital,332North Lauderdale,Memphis,TN 38105,USA.2Department of Immunology,St.Jude Children’sResearch Hospital,332North Lauderdale,Memphis,TN 38105,USA.3Hartwell Center for Biotechnology and Bioinformatics,St.Jude Children’s Research Hospital,332North Lauderdale,Memphis,TN 38105,USA.4Department of Pathology,University of Michigan,1500East Medical Center Drive,Ann Arbor,MI 48109,USA.SummarySystemic infection with Streptococcus pneumoniae is associated with a vigorous pro-inflammatory response to structurally complex cell wall fragments (PnCW)that are shed during cell growth and antibiotic-induced autolysis.Consistent with previ-ous studies,inflammatory cytokine production induced by PnCW was dependent on TLR2but independent of NOD2,a cytoplasmic NLR protein.However,in parallel with the pro-inflammatory response,we found that PnCW also induced pro-digious secretion of anti-inflammatory IL-10from macrophages.This response was dependent on TLR2,but also involved NOD2as absence of NOD2-reduced IL-10secretion in response to cell wall and translated into diminished downstream effects on IL-10-regulated target gene expression.PnCW-mediated production of IL-10via TLR2required RIPK2a kinase required for NOD2function,and MyD88but differed from that known for zymosan in that ERK pathway activation was not detected.As mutations inNOD2are linked to aberrant immune responses,the temporal and quantitative effects of activation of the TLR2-NOD2-RIPK2pathway on IL-10secretion may affect the balance between pro-and anti-inflammatory responses to Gram-positive bacteria.IntroductionIL-10secretion from T cells,macrophages and dendritic cells is an essential regulatory mechanism to temper excessive inflammatory responses (Murray,2006).Mice lacking IL-10are extremely sensitive to a wide variety of pro-inflammatory stimuli including LPS,and systemic infections with bacteria and parasites that provoke a strong inflammatory response (Murray,2006).In all cases,IL-10is required to restrain the inflammatory response and protect the host against the damaging effects of pro-inflammatory cytokines.Systemic infection with S.pneumoniae provokes a generalized inflammatory response linked with devastating neurological sequelae and a fatality rate of ~15%(Tuomanen et al .,1995).Administration of purified pneumococcal cell wall (PnCW)recapitulates the majority of the inflammatory responses observed in systemic S.pneumoniae infection,suggest-ing that the pro-inflammatory properties of PnCW are a primary driver of inflammatory pathology (Moreillon and Majcherczyk,2003;Fillon et al.,2006;Orihuela et al .,2006).Because PnCW is mainly composed of peptidogly-can,teichoic acid,cell wall linked proteins and lipoteichoic acids,TLR2is considered especially important in the process of PnCW detection by macrophages and den-dritic cells that are then stimulated to produce inflam-matory cytokines (Yoshimura et al .,1999).IL-10can modulate PnCW induced inflammation (Orihuela et al .,2006),but the mechanism leading to IL-10production is entirely unclear.Modulation of inflammation has been suggested to involve NOD2,a cytoplasmic protein of the larger NLR family (Inohara and Nunez,2003;Inohara et al .,2005;Murray,2005;Strober et al .,2006;Kanneganti et al .,2007).For example,single nucleotide polymorphisms in NOD2have been observed at an increased frequency in Crohn’s disease (Lesage et al .,2002).However,severalReceived 2May,2008;revised 31May,2008;accepted 2June,2008.*For correspondence.E-mail peter.murray@;Tel.(+1)9014953219;Fax (+1)9014953099.†Present address:University of Colorado Health Sciences Center,Denver,CO,USA;‡Contributed equally to this work.Cellular Microbiology (2008)10(10),2067–2077doi:10.1111/j.1462-5822.2008.01189.xFirst published online 8July 2008©2008The AuthorsJournal compilation ©2008Blackwell Publishing Ltdloss-of-function mice in the Nod2gene do not have gut immunopathology when maintained under normal condi-tions(Pauleau and Murray,2003;Kobayashi et al.,2005; Maeda et al.,2005;Mariathasan et al.,2006;Barreau et al.,2007).These data are consistent with the fact that some people have mutations in one or both NOD2 alleles but have no clinical inflammatory bowel disease (Hugot et al.,2007).Thus NOD2likely plays a role in more complex pathways modulating pro-inflammatory responses(Strober et al.,2007;Xavier and Podolsky, 2007).NOD2-deficient mice,their immune cells and human cells bearing predicted loss-of-function NOD2alleles,are non-responsive to muramyl dipeptide(MDP),a‘minimal’component of bacterial peptidoglycan able to induce pro-inflammatory responses.A second pathway of MDP sensing appears to be involved in IL-1b processing and is mediated by NLR family members Cryopyrin(NLRP3)and Nalp1(NLRP1)(Faustin et al.,2007;Pan et al.,2007; Marina-Garcia et al.,2008).At this stage it is unclear whether human cells bearing two copies of the NOD2 frame-shift mutation are completely non-responsive to MDP because the truncated mutant protein should still be pared with other bacterial components such as LPS,peptidoglycan or CpG DNA,MDP is a very weak agonist of myeloid-derived cells,especially when considered by molar comparison.However,MDP syner-gizes with other TLR agonists to stimulate cytokine, chemokine and nitric oxide production.This effect is the basis of variants of the‘MDP synergy’assay,a common ‘readout’of NOD2function.In contrast to the lack of MDP responsiveness,macrophages from all NOD2-deficient mice have been generally reported to have‘normal’responses to highly defined TLR agonists.At present,the specific roles of MDP,the origin of MDP in mammalian infection systems and the link between NOD2and MDP remain unresolved.In the studies described here,we observed that TLR2 and NOD2were together responsible for IL-10production when macrophages were exposed to PnCW.By contrast, inflammatory cytokine production in response to PnCW was TLR2dependent and NOD2-independent.Our studies reveal an unexpected pathway of signal integra-tion in response to a complex microbial product that stimulates multiple signalling pathways in macrophages.ResultsWhen S.pneumoniae grow in vivo,cell wall components are released into the host environment by several pro-cesses:cell wall turnover that occurs during normal growth,antibiotics that induce autolysis,and subpopula-tions that undergo spontaneous autolysis as a mecha-nism of interbacterial gene transfer.The latter process lead in part to the discovery of DNA as the source of genetic information.Released cell wall fragments stimu-late a complex pathway of local and systemic inflamma-tory responses including neuronal damage and memory loss(Tuomanen et al.,1995).Because cell wall fragments derived from yeast(zymosan)induce robust IL-10secre-tion from myeloid-derived cells(Dillon et al.,2004;2006; Rogers et al.,2005;Slack et al.,2007),we tested if puri-fied PnCW(Fillon et al.,2006)also induced IL-10produc-tion from bone marrow-derived macrophages(BMDMs). PnCW induced previously unsuspected high amounts of IL-10(Fig.1).As purified PnCW is composed primarily of peptidoglycan,a TLR2agonist,this response was predict-ably TLR2dependent(Fig.1).In parallel experiments we also observed that IL-10and IL-10mRNA production in response to PnCW was also dependent on NOD2 (Figs1B,D and E).The reduction in IL-10production from PnCW-stimulated NOD-deficient macrophages was not to the same extent as Tlr2-/-BMDMs but nevertheless highly reproducible.We also observed that BMDMs derived from Tlr2-/-;Nod2-/-mice secreted undetectable IL-10,demonstrating that TLR2and NOD2might function in the same or related pathways for IL-10production (Figs1B,D and E).By contrast,IL-10production in response to LPS was indistinguishable in Tlr2-/-,Nod2-/-or Tlr2-/-;Nod2-/-cells(Fig.1C).We tested if NOD2was required for IL-10production in response to Pam3CSK4,a synthetic lipid that signals via exclusively TLR2.We observed that Nod2-/-BMDMs produced IL-10in response to Pam3CSK4similar to control cells(data not shown).In contrast,Pam3CSK4did not induce IL-10in Tlr2-/-or Tlr2-/-;Nod2-/-cells as expected(data not shown).NOD2was therefore not required for the‘canoni-cal’TLR2activation pathway stimulated by Pam3CSK4 but was important to the physiological PnCW stimulus. Because PnCW is a complex macromolecule,we tested its cellular fate after exposure to BMDMs.We visu-alized PnCW fragments in macrophages through the use of FITC-labelled PnCW(Fig.1F and G).Macroph-ages consumed all the PnCW over a period of~24h and thereafter,the PnCW remained internalized and appar-ently unchanged over7days when the experiments were paring these data with the kinetics of cytokine secretion,we suspect that macrophages are stimulated with early,external contact with PnCW.By contrast,phagocytosed PnCW appeared to persist within macrophages.Further experiments will be necessary to determine if macrophages can generate MDP from the PnCW and any signalling connections between NOD2 and in-dwelling PnCW.We next asked how broadly NOD2influenced overall macrophage transcriptional responses to PnCW.We per-formed transcriptome analysis of BMDMs derived from background-matched C57BL/6Nod2+/+or Nod2-/-mice2068L.O.Moreira et al.©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077stimulated for 0,1or 4h with PnCW at a concentration and conditions identical to that used for the experiments in Fig.1.We analysed the data first by principal component analysis (data not shown)and then by ordering gene expression according to degree of induction relative to the untreated (0h)time point data (Table 1).We observed that the absence of NOD2had limited effects on the overall inflammatory response to PnCW (Table 1,Fig.2A)consistent with the notion that other receptors,especially TLR2,mediate a strong cellular activation by PnCW.Of the mRNAs whose expression was altered,we noted that IL-10mRNA expression was reduced in NOD2-deficient cells relative to controls consistent with the ELISA and mRNA data shown in Fig.1.Because IL-10has essential autocrine-paracrine inhibitory effects on activated mac-rophages,we next asked if a panel of known downstream target genes of IL-10signalling (Lang et al .,2002;El Kasmi et al .,2007)were affected by the reduction of IL-10after PnCW stimulation of NOD2-deficient macrophages.We observed that expression of multiple IL-10target genes was lowered relative to controls in NOD2-deficient macrophages at 4h post stimulation (Fig.2B).This is consistent with the data that PnCW stimulates IL-10production in a NOD2-dependent way that leads,in part,to autocrine-paracrine downstream effects on IL-10-responsive gene expression.By contrast to the requirement for both TLR2and NOD2in IL-10production,we observed that alltestedbined signals from TLR2and NOD2are required for PnCW-induced IL-10production.A.Schematic representation of the composition of PnCW obtained by biochemical purification from whole lysed pneumococci.The upper part of the figure is cartoon of the bacteria cell membrane and wall indicating that the wall composed primarily of peptidoglycan,to which multiple proteins,lipopeptides and lipids are tethered,surrounds the cell membrane of pneumococci.Following purification,PnCW is predominantly peptidoglycan and teichoic acids.This fraction is particulate is nature,analogous to zymosan fractions from yeast cell walls.B.IL-10production from BMDMs stimulated with PnCW measured over time by ELISA.Results are combined from independent experiments (n =7).C.IL-10production from LPS stimulation of BMDMs from the same genotypes used in (B).D.Northern blotting analysis of IL-10mRNA over time (h)following PnCW stimulation of BMDMs from the indicated genotypes.Data from Nod2-/-;Tlr2-/-BMDMs were obtained from a different part of the gel as shown the separation from the other part of the gel.E.qRT-PCR analysis of IL-10mRNA production following PnCW stimulation.Data points are averages of duplicate samples.Data are representative of three independent experiments.F.Confocal microscopy to follow the fate of PnCW after contact with BMDMs.FITC-labelled PnCW was added to C57BL/6BMDMs andfollowed over time.Representative images at times 0,5,24and 48h are shown.At each time,cultures were fixed and labelled with phalloidin to visualize actin.Note that the FITC signal is not visible until sufficient PnCW particle have been phagocytosed and concentrated inside macrophages (24h).All images were collected using a 10¥objective,except the 48h time point (20¥).G.Higher power image of PnCW inside BMDMs at 48h (40¥objective).The TLR2-MyD88-NOD2-RIPK2signalling axis 2069©2008The AuthorsJournal compilation ©2008Blackwell Publishing Ltd,Cellular Microbiology ,10,2067–2077pro-inflammatory genes induced by PnCW were depen-dent on TLR2(Fig.3),but independent of NOD2as shown by the microarray data(Table1).These results are consistent with the notion that TLR2is the primary pathway by which macrophages detect PnCW and that NOD2plays a downstream role infine tuning the signal-ling response,in this case by regulating IL-10production. NOD2has been reported to form heterotypic com-plexes with another CARD domain protein,RIPK2(Kobayashi et al.,2002;Park et al.,2007;Hasegawa et al.,2008).Like NOD2-deficient cells,macrophages lacking RIPK2cannot respond to MDP,suggesting that NOD2and RIPK2function in the same pathway for MDP responsiveness(Park et al.,2007).Furthermore,RIPK2 is also required for NOD1function(which does not‘sense’MDP),suggesting that RIPK2is genetically and biochemi-cally linked to both NOD1and NOD2activity(Park et al., 2007).We therefore used macrophages derived from twoTable1.Gene expression increased by PnCW treatment.Gene Rank(WT)Fold(4h)Rank(Nod2-/-)Fold(4h)P(time)P(genotype) Gm19601222.141205.56 1.41E-130.5314Irg12124.622145.62 4.32E-110.8515Cxcl13102.854111.82 2.17E-110.3880Tnf482.043124.60 1.02E-110.8967Cxcl1578.065102.79 4.75E-110.3036Ccl3677.61676.38 4.39E-110.9913Saa3776.27854.01 2.37E-140.1997Ptgs2857.721629.27 4.13E-090.9574Ccl4954.471432.23 3.61E-120.6201Il1b1051.931532.008.30E-070.7485Gpr109a1151.731037.097.80E-140.2307Slc7a111251.721924.34 4.11E-140.6030Ptgs21351.634616.038.02E-080.1154Il1a1445.045015.37 6.39E-070.7571Il61544.031135.24 1.02E-060.3773Socs31643.811332.65 2.46E-110.2271Socs31740.921726.16 1.79E-110.4451Fpr11838.412621.24 2.66E-090.8110 Tnfaip31936.912024.25 6.30E-090.4238Cxcl22036.81760.84 6.79E-140.9650BB5485872136.547111.55 1.85E-060.7213Tnfsf92235.176412.63 5.17E-080.9962Socs32335.101825.02 1.24E-120.2296Ccl22434.012123.20 1.41E-090.5926Ccl72533.98942.028.91E-100.3433Fpr-rs22631.152721.21 3.19E-120.6983Il1rn2729.496911.71 2.74E-100.0624Ets22829.254915.50 3.77E-090.5326Ccrl22929.133019.91 2.75E-080.7250LOC6229763028.831233.45 6.43E-120.1185Il1rn3128.083218.99 4.52E-110.1198 Tnfaip33227.382421.82 1.42E-100.8902Zc3h12c3325.122322.497.12E-100.3216Ccl123425.125913.15 1.25E-080.2671Cdc42ep23524.164416.31 1.03E-080.7966Gpr843623.463119.54 3.15E-100.2886Il1rn3723.357311.359.55E-120.1113Nr4a33822.464815.73 2.80E-080.7223Ccl53922.153618.02 1.01E-070.2819Ch25h4021.836512.599.66E-110.2134Cd404121.426612.25 2.74E-110.8623Cd404221.315413.89 6.89E-120.3615Zc3h12c4321.072821.05 3.52E-100.7250Clec4e4420.752920.40 2.51E-090.9522Malt14520.223318.56 5.46E-120.8940 Hivep34619.932222.60 3.95E-150.4636 Ptges4719.705314.23 2.23E-100.2554Cd694819.603418.55 3.57E-070.5920Cd404919.486013.08 2.69E-120.7930Traf15019.184316.55 5.61E-080.2824 Probe sets from the top50induced targets in PnCW-stimulated control cells are ordered1through50based on fold induction relative to the untreated control cells at4h.The correlative rank of genes expressed in PnCW-stimulated NOD2-deficient macrophages relative to the control ranking is listed along with the P-values(ANOVA)by time relative to unstimulated cells and by genotype.2070L.O.Moreira et al.©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077strains of Ripk2-/-mice and mice lacking both NOD1and NOD2(Nod1-/-;Nod2-/-)to probe if IL-10production was inhibited.We observed by ELISA and qRT-PCR analysis that RIPK2-deficient cells had a similar phenotype to NOD2-deficient BMDMs(Fig.4)in terms of reduced IL-10 production in response to PnCW.Furthermore,macroph-ages from Nod1-/-;Nod2-/-mice,which have been reported to have similar phenotypes to Ripk2-/-macroph-ages(Park et al.,2007),also had a deficiency in IL-10production when stimulated with PnCW.Finally, when introduced into Nod2-/-macrophages by retroviral-mediated transduction,human NOD2rescued IL-10in response to PnCW and MDP responsiveness (Fig.4E–G).Analogous studies with NOD1were not pos-sible because human NOD1producer lines do not make virus at sufficiently high titre to infect stem cells.Collec-tively,these data provide evidence that NOD2and RIPK2 function in the same pathway that mediated IL-10produc-tion in response to PnCW.Zymosan,a complex cell wall fraction from yeast,has been shown to induce IL-10production by the TLR2-DECTIN1-SYK pathway and the TLR2-DECTIN1-ERK pathway(Dillon et al.,2004;2006;Rogers et al.,2005;Slack et al.,2007).We therefore asked if PnCW also stimulated ERK activation with the idea that ERK could be a common downstream mediator of multiple TLR2-driven pathways,including the TLR2-NOD2pathway.However, we observed that PnCW induced low or negligible ERK phosphorylation(Fig.5).Therefore,PnCW seems to stimulate IL-10production via pathways distinct from those induced by zymosan,even though both the DECTIN1-SYK and NOD2-RIPK2pathways depend on TLR2.Finally,IL-10production in response to PnCW was dependent on MyD88(Fig.5E),suggesting that the TLR2-MyD88pathway is absolutely necessary for PnCW-induced IL-10production.MyD88was not,however, required for MDP sensing(Fig.5F)as expected as shown by the use of a sensitive MDP stimulation assay where nitric oxide production is measured in response to MDP and IFN-g(Totemeyer et al.,2006).By contrast,MDP sensing was dependent on NOD2.DiscussionOur data suggest that NOD2is a component of a signalling module that regulates IL-10production in aFig.2.Absence of NOD2has a limited effecton PnCW-induced inflammatory transcription.A.Select inflammatory targets induced byPnCW in control or NOD2-deficient BMDMs.Data for each target mRNA is plotted as theaverage difference signal intensity.Data areaverages from independent(n=4)Affymetrixarrays performed per genotype and per time.No significant differences(paired t-test)weredetected between plete datasets for this experiment have been depositedin the GEO database.B.Expression of IL-10target genes isaffected in the absence of NOD2function.Signal intensity for IL-10(left most in thepanel)and selected genes whose expressionis dependent,in part,on autocrine-paracrineIL-10production.*P<0.05,paired t-testcomparing4h time points between controland Nod2-/-BMDMs(n=4).The TLR2-MyD88-NOD2-RIPK2signalling axis2071©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077stimulus-specific and cell type-specific way(Fig.6).Acti-vation of TLR2-MyD88by Pam3CSK4leads to ERK acti-vation and IL-10induction.In contrast,activation of TLR2-MyD88by PnCW invokes NOD2and RIPK2and bypasses ERK activation to produce IL-10.Activation of TLR2and DECTIN by zymosan leads to ERK activation and IL-10induction.It is well accepted that IL-10is essen-tial for regulating the amplitude of most,if not all inflam-matory responses in vivo.Therefore,mutant forms of NOD2may be a component of homeostatic regulation of IL-10in myeloid-derived cells.Notably,Netea and col-leagues have demonstrated that human monocyte-derived macrophages and dendritic cells isolated from people bearing NOD2mutations have reduced IL-10pro-duction in response to peptidoglycan and MDP(Netea et al.,2004;2005;Kullberg et al.,2008).We found parallels between PnCW stimulation of IL-10 production and the activation of dendritic cells by zymosan,a large fragmentary remnant of yeast cell walls. In both cases,immune cells are exposed to particulate fractions of cell walls of varying sizes and displaying a variety of different sugars,lipids and structural biopolymers.Zymosan stimulates IL-10production by a MyD88-independent pathway involving TLR2,DECTIN1, SYK and ERK(Dillon et al.,2004;2006;Rogers et al., 2005;Slack et al.,2007).PnCW stimulation of IL-10also requires TLR2,but does not seem to activate substantial ERK phosphorylation(Fig.5).Instead,MyD88,NOD2 and RIPK2are required.The PnCW and zymosan recog-nition pathways present a considerable contrast to the activation of immune cells with Pam3CSK4through TLR2, a pathway that has an absolute requirement for MyD88 but not NOD2.Therefore,there are different signalling routes to IL-10production following TLR2activation (Fig.6).Similarly,additional diversity of signalling for IL-10production has been observed for receptors such as the LPS-mediated pathway that requires TLR4and p38 (Park et al.,2005),and the immune complex pathway that enhances IL-10production via FcR ligation(Edwards et al.,2006).BTK also plays a key role in stimulus-specific IL-10production:the absence of BTK leads to a decrease in IL-10produced from macrophages and a correspond-ing increase in inflammatory mediators normally blocked by IL-10(e.g.IL-12,IL-6)(Kawakami et al.,2006;SchmidtFig.3.Pro-inflammatory cytokinetranscription in response to PnCW isdependent on TLR2.BMDMs from theindicated genotypes were stimulated withPnCW for the times shown.Cytokine mRNAswere measured by qRT-PCR.Data arerepresentative of three independentexperiments.2072L.O.Moreira et al.©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077et al .,2006).However,the reduced amounts of IL-10made in the absence of BTK,like the absence of NOD2,is insufficient to initiate the severe inflammatory bowel disease observed in the complete absence of IL-10(Schmidt et al .,2006)(L.M.and P .J.M.,unpubl.data).Therefore,NOD2and BTK both seem to function to tune stimulus-specific IL-10production.We found that neither TLR2nor NOD2were absolutely required for PnCW stimulation of IL-10production.IL-10was made,although in much lower amounts,in the absence of either TLR2or NOD2.However,cells lacking both TLR2and NOD2had a complete abrogation of IL-10production.These data suggest that TLR2and NOD2have a genetic interaction.A similar finding has been made in the ECOVA gut inflammation system where pathology observed in the absence of NOD2was reversed in the combined deficiency of both NOD2and TLR2(Watanabe et al .,2006).It is however,prematuretoFig.4.IL-10expression in response to PnCW is dependent on the RIPK2pathway.A.BMDMs from Ripk2-/-or Nod1-/-;Nod2-/-mice were stimulated with PnCW and IL-10measured over time by ELISA.B.As in (A),but following LPS stimulation.C and D.Analysis of BMDMs from an independent Ripk2mutant strain showing IL-10and IL-10mRNA produced in response to PnCW requires RIPK2.E and F.Reconstitution of Nod2-/-hematopoietic stem cells with human or mouse NOD2cDNAs rescues IL-10production in response to PnCW.Stem cells from Nod2-/-mice were infected with retroviruses containing hNOD2or mNOD2cDNAs and selected by cell sorting for YFP and plated into media containing CSF-1to drive differentiation into macrophages.After differentiation,macrophages were stimulated with LPS or LPS and MDP to measure the MDP synergy with TLR4signalling (E)or IFN-g signalling (F)or IL-10production after PnCW treatment (G).Data are representation of two independent infection studies.In the case of mNOD2,sufficient cells were obtained to perform the MDP-IFN-g synergy assay only.The TLR2-MyD88-NOD2-RIPK2signalling axis 2073©2008The AuthorsJournal compilation ©2008Blackwell Publishing Ltd,Cellular Microbiology ,10,2067–2077suggest that TLR2and NOD2function in a linear pathway where NOD2is downstream of TLR2.Instead,we suggest that cross-talk between the TLR2and NOD2pathways occurs in a stimulus-and cell-specific context.Cross-talk is not revealed when TLR2is stimulated with Pam 3CSK4but instead is exposed by agents such as PnCW and peptidoglycan,at least in the BMDMs used here.This idea may help resolve previous reports that suggested that NOD2was not involved in TLR2signalling (Kobayashi et al .,2005):dependence on both stimulus and cell type is needed to expose differences among pathways.Recent data from human monocytes bearing NOD2mutations has also revealed a complex interplay between NOD2and TLR2(Borm et al .,2008).Using transcriptome profiling,we also found that the absence of NOD2affected a surprisingly low number of mRNAs when BMDMs were stimulated with PnCW.These data are in keeping with published studies suggestingthatFig.6.Model of IL-10production myeloid lineage cells(macrophages and DCs)stimulated by agents that activate TLR2,alone or in combination with other cell surface molecules.Fig.5.PnCW activation of BMDMs does not activate the ERK or p38pathways but requires MyD88signalling.A.C57BL/6BMDMs were stimulated with Pam 3CSK4,LPS or PnCW over time and ERK phosphorylation measured byimmunoblotting for phosphorylated forms of ERK or reprobed for total ERK amounts.B.A similar experiment to (A)was performed using BMDMs from the genotypes indicated on the right.C.The same lysates from (B)were probed for phospho-p38.Note that PnCW does not activate ERK or p38in any of these experiments.D.BMDMs from control,Nod2-/-,Tlr2-/-or MyD88-/-mice were stimulated with PnCW for 8h and IL-10measured by ELISA.Data are average ϮSD from independent samples (n =3).E.BMDMs were stimulated with MDP orMDP +IFN-g overnight and nitrites measured by the Griess assay.Stimulation with IFN-g alone did not produce detectable nitrites (data notshown).2074L.O.Moreira et al.©2008The AuthorsJournal compilation ©2008Blackwell Publishing Ltd,Cellular Microbiology ,10,2067–2077loss of NOD2has minimal effects on pro-inflammatory TLR signalling(Pauleau and Murray,2003;Kobayashi et al.,2005;Park et al.,2007).However,PnCW is an example of a pathogen‘signal’that can activate multiple pathways including the TLR2pathway.It is not surprising that NOD2plays limited roles in the overall transcriptional response as the mammalian immune system most likely has developed multiple,overlapping ways to recognize and respond to such a complex material.Nevertheless, thefinding that IL-10was one of the most affected mRNAs,along with a cohort of IL-10-regulated genes, suggests that further work is warranted to determine the role of NOD2in IL-10production in humans bearing muta-tions in NOD2.It is tempting to speculate that IL-10regulation has key functional significance in chronic inflammatory states such as the inflammatory bowel diseases.Most research-ers agree that common pathways of inflammatory media-tor production,arrived at by a multitude of genetic and environmental mechanisms,drive disease and are the central focus of therapeutic intervention through anticy-tokine antagonists.IL-10plays an essential regulatory role in controlling the products of the common inflamma-tory pathway:IL-12,TNF-a,IL-6,etc.Therefore,small disturbances in temporal,anatomic or quantitative IL-10 amounts may translate into greater inflammation.Experimental proceduresMiceNod2-/-mice have been previously described(Pauleau and Murray,2003).Nod2-/-;Tlr2-/-mice on a C57BL/6background (n=5generations)have been described(Watanabe et al., 2006).Tlr2-/-mice were purchased from the Jackson Laboratories.Mice were bred and genotyped according to pub-lished protocols.For some experiments,Nod2-/-mice were backcrossed10generations to the C57BL/6background.All animal experiments were performed in accordance with protocols governed by the St Jude Animal Care and Use Committee(P.J. M.,Principal Investigator).Nod1-/-;Nod2-/-and Rip2k-/-mice have been described previously(Chin et al.,2002;Kobayashi et al.,2002;Park et al.,2007).Cell preparation and stimulus conditionsBone marrow-derived macrophages were isolated and cultured as described(Lang et al.,2002).Macrophages were stimulated with highly purified PnCW prepared as described previously (Tuomanen et al.,1985a,b).Briefly,S.pneumoniae unencapsu-lated strain R6were grown in C+Y medium,bacteria were boiled in SDS,mechanically broken by shaking with acid-washed glass beads,and treated sequentially with DNase,RNase,trypsin,LiCl, EDTA and acetone.PnCW was confirmed to be free of protein by analysis for non-cell wall amino acids using mass spectrometry. The absence of contaminating endotoxin was confirmedfirst by the Limulus test(Associates of Cape Cod)and then by ELISA measurement of hIL-8production by HEK293cells transfected with plasmids designed to express TLR4and MD2(a generous gift of Dr Doug Golenbock,UMass)in the presence of purified PnCW.Forfluorescence microscopy experiments,BMDMs were labelled with Phalloidin and4′,6-diamidino-3-phenylindole (DAPI),a nuclear stain.PnCW was directly labelled with 1mg ml-1FITC(Sigma-Aldrich)solution in carbonate buffer (pH9.2)for1h at room temperature in the dark and washed twice with PBS containing calcium and magnesium(Gosink et al., 2000).ELISAELISA was performed as previously described(El Kasmi et al., 2006).Capture and detection antibodies used were purchased from BD Pharmingen.Detection limits for the ELISAs were 50pg ml-1(IL-10)and10pg ml-1(IL-12p40).Northern blotting,RT-PCR and immunoblottingTotal RNA was isolated from primary macrophages using Trizol. Reverse transcription(qRT-PCR)was performed as described previously using primers and probes(Applied Biosciences)spe-cific for each cytokine mRNA(Lang et al.,2002).Immunoblotting was performed using the following rabbit polyclonal antibodies: antiphospho-p42,p44ERK and anti-phospho-p38from Cell Sig-naling Technology used at1:500final dilution.Anti-p42,p44ERK and anti-p38were from the same source and were used at afinal concentration of1:1000.Transcriptome analysisRNA samples from BMDM cultures representing four PnCW preparations,two genotypes(wild-type and the NOD2deficient) and three time points(0,1and4h)were arrayed on Affymetrix GeneChip analysis murine430v2GeneChip arrays.Signal was acquired with MAS5.0software and natural log transformed [ln(signal+20)]to stabilize variance and better conform the data to the normal distribution.Principal components analysis(Partek 6.1)revealed a batch effect which,being orthogonal to hour and genotype,was removed by capturing and mean adjusting residu-als of a one-way ANOVA-based on PnCW batch(STATA/SE9.2). The batch-corrected transformed signal was tested for exposure time and genotype effects in a two-way ANOVA using Partek.The P-values for the time effect were corrected for multiple compari-sons using the false discovery rate(Benjamini et al.,2001).Fold change calculations were based on the geometric means of the batch corrected data between the4h and0h time points for each genotype.Array data has been submitted to the GEO data-base(accession GSE8960).Retroviral-mediated transduction of stem cellsRetroviral transduction of murine bone marrow cells was per-formed as described previously(El Kasmi et al.,2006;Holst et al.,2006a,b).For the transduction experiments,we used fetal liver stem cells isolated from E14pregnant Nod2-/-female mice. Fetal liver cells were cultured for48h in complete DMEM with 20%FBS,20ng ml-1murine IL-3,50ng ml-1human IL-6and The TLR2-MyD88-NOD2-RIPK2signalling axis2075©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077。

第一章 绪论cellcell biology cell theory central dogma cytomics cytoplasmelectron microscope ，EM epigenetics gene细胞细胞生物学 细胞学说 中心法则 细胞组学 细胞质 电子显微镜 表观遗传 基因genomicshuman genome project,HGP medical cell biology model animal noncoding RNA proteomics stem cell biology translational medicine基因组学人类基因组计划医学细胞生物学 模式动物 非编码RNA 蛋白质组学 干细胞生物学 转化医学第二章细胞的概念和分子基础anticodon archaea archaebacteria bacteria cell membrane chlamydia codon cytoplasm cytosol deoxyribonucleic acid,DNAenzyme eukarya eukaryotic cell glycolipids glycoproteins message RNA ， mRNAmicroRNA, miRNA mycoplasma反密码子 古菌域 古细菌 细菌 细胞膜 衣原体 密码子 细胞质 细胞质溶胶 脱氧核糖核酸 酶 真核域 真核细胞 糖脂 糖蛋白 信使核糖核酸 微小RNA 支原体 nucleoid nucleus peptide plasmid prion prokaryotic cell protoplasm ribonucleic acid ，RNA ribosomeribosome RNA ， rRNA ribozyme RNA silencingsmall nuclear RNA, snRNA structural domains transfer RNA, tRNA viroid virus 拟核细胞核 肽 质粒 朊病毒 原核细胞 原生质 核糖核酸 核糖体 核糖体RNA 核酶RNA 沉默 小核RNA 结构域 转运RNA 类病毒 病毒第三章细胞生物学研究方法Abbe limitagaroseatomic force microscope,AFM autoradiographybiochipcell culturecell free systemcell linecell strainChIPAbbe 限度 琼脂糖 原子力显微镜放射性自显影技术 生物芯片 细胞培养 非细胞体系 细胞系 细胞株 染色质免疫沉淀技术 CLIPcolumn chromatography cytochemistry technique differential centrifugation diffraction pattern DNA denaturation dNTPelectrospray ionization mass spectrometry,ESI-MS embedding紫外交联免疫沉淀技术 柱层析细胞化学技术 差速离心 X-射线衍射图 DNA 变性4种脱氧核苷酸 电喷雾电离质谱 包埋emission lightemzyme cytochemistryequilibrium sedimentation excitation lightfixationflow cytometerfluorescence microscope fluorescence resonanceemergy transfer,FRETfreeze-etchfreeze-fracturegene chipgreen fluorescentprotein,GFPhigh performance liquidchromatography,HPLCimmunocytochemistry,ICC immunomagnetic microsphere immuno-precipitation,IPin situ hybridization,ISHisoelectric focusinglaser capturemicrodissection,LCMmatrix-assisted laserdesorptionionization/time-of-flight ， MALDI-TOF-MSmetal shadowingNorthern blotnuclear magnetic resonance spectroscopy,NMR over-expression phage display发射光 酶细胞化学技术 平衡沉降激发光 固定 流式细胞仪 荧光显微镜荧光共振能量转移 冰冻蚀刻 冰冻断裂 基因芯片 绿色荧光蛋白 高效液相层析 免疫细胞化学技术 免疫磁珠免疫沉淀 原位杂交技术 等点聚焦 激光俘获显微切割电泳 基质辅助激光解吸/电离飞行时间质谱金属投影法 Northern 印迹杂交 核磁共振光谱过表达 噬菌体展示polymerase chainreaction,PCR primary cultureprobeprotein chipproteomeproteomicsquantitative PCR ，qPCR radioisotoperenaturationresolutionrestriction nucleasescanning electronmicroscope,SEM scanning tunnelingmicroscope,STM SDS polyacrylamid gel electrophoresissecondary culturesectionsingle molecular fluorescence imaging southern blot stainingtandem massspectrometry,MS/MStransfectiontransmission electron microscope,TEM velocity sedimentation yeast two-hybridzation phase contrast microscope聚合酶链式反应原代培养 探针 蛋白质芯片 蛋白质组 蛋白质组学 荧光实时定量PCR 反应放射性同位素 复性 分辨率 限制性内切酶 扫描电子显微镜扫描隧道显微镜SDS-聚丙烯酰胺凝胶电泳 传代培养 切片 单分子荧光成像 Southern 印迹杂交染色 串联质谱 转染透射电子显微镜速度沉降 酵母双杂交 相差显微镜第四章细胞膜与物质的穿膜运输active transport adaptinantiportaquaporin ，AQPbilayerbiomembranecarrier proteincell coatcell membrane主动运输衔接蛋白 反向运输 双分子层 水孔蛋白 生物膜 载体蛋白 细胞外被 细胞膜 channel proteincholesterol clathrin coated pits coated vesicle constitutive secretion endocytosis exocytosis extrinsic protein通道蛋白 网格蛋白 胆固醇 有被小窝 有被小泡 连续性分泌 胞吞作用 胞吐作用 膜外在蛋白facilitated diffusion fluid mosaic model glycolipid intrinsic proteinligand-gated channel lipid raftslipid anchored protein lipid-linked protein lamella structure model membrane lipid membrane proteinmembrane transport protein micellepassive diffusion passive transport 易化扩散 流动镶嵌模型 糖脂膜内在蛋白 配体门控通道 脂筏脂锚定蛋白 脂连接蛋白 片层结构模型 膜脂 膜蛋白 膜运输蛋白 球状分子团 被动扩散 被动运输 peripheral protein phagocytosis phospholipid pinocytosis plasma membrane regulated secretion simple diffusionstress-activated channel symporttransmembrane protein unit membraneunit membrane model vesicular transport voltage-gated channel外周蛋白 吞噬作用 磷脂 胞饮作用 质膜 受调分泌 简单扩散 应力激活通道 同向运输 穿膜蛋白 单位膜 单位膜模型 小泡运输 电压门控通道第五章内膜系统annulate lamellaeautophagic lysosomecalreticulincis Golgi networkcisternaeclathrin-coated vesiclecontinuous secretioncotranslation insertiondiscontinuous secretionendoplasmic reticulumgated transportglucose regulated protein94,GRP94 glycosylationGolgi complexheavy-chain bindingprotein,BiP heterophagic lysosome internal signal peptideinternal start-transfer peptidelysosomemedial Golgi stackmolecular chaperonemyeloid bodyN-linked glycosylationphagolysosome孔环状片层 自噬溶酶体 钙网素 顺面高尔基网 扁平囊泡 网格蛋白有被囊泡 连续分泌 共翻译插入 非连续分泌 内质网 门孔运输 葡萄糖调节蛋白94糖基化 高尔基复合体 重链结合蛋白 异噬溶酶体内信号肽 内开始转移肽 溶酶体 高尔基中间膜囊 分子伴侣 髓样体 N-连接糖基化 异噬溶酶体 phospholipid exchangeproteins,PEP primary lysosome protein disulfide isomerase,PDI retention protein reticulo-plasmin rough endoplasmic reticulum ，RER sarcoplasmic reticulum secondary lysosome signal hypothesis signal patch signal peptide signal recognition particle,SRPSRP-receptor,SRP-R smooth endoplasmic reticulum ，SERtarget-SNAREs,t-SNAREs tertiary lysosome trans Golgi network translocontransmembrane transport unfolded protein response, UPR磷脂交换蛋白初级溶酶体蛋白二硫键异构酶驻留蛋白 网质蛋白 糙面内质网肌质网 次级溶酶体 信号肽假说 信号斑 信号肽信号识别颗粒信号识别颗粒受体 光面内质网靶SNAREs 三级溶酶体 反面高尔基网 转运体 穿膜运输未折叠蛋白反应vacuoles vesicle vesicles 大囊泡囊泡小囊泡vesicle-SNAREs,v- SNAREsvesicular transport囊泡SNAREs囊泡转运第六章线粒体与细胞的能量转换ATP synthase complex biological oxidation cellular oxidation cellular respiration chemiosmotic coupling hypothesiscristaeelementary particle glycolysisinner membrane intercristae space intermembrane space intracristae spacematrixmatrix spacematrix-targeting sequence，MTS ATP合酶复合体生物氧化细胞氧化细胞呼吸化学渗透假说嵴基粒糖酵解内膜嵴间腔膜间腔嵴内空间基质基质腔基质导入序列mitochondrial disordersmitochondrial phaseouter membraneoxidative phosphorylationpostmitochondrial phasepremitochondrial phasereactive oxygen species, ROSsubstrate-levelphosphorylationtranslocation contact sitetranslocon of the innermembrane，Timtranslocon of the outermembrane，Tomtricarboxylic acid cycle，TCAcycle线粒体疾病线粒体期外膜氧化磷酸化线粒体后期线粒体前期活性氧底物水平磷酸化转位接触点内膜转位子外膜转位子三羧酸循环第七章细胞骨架与细胞的运动actincell cortexcochicine contractile ring cytochalasin B cytoskeletondynamic instability model dyneinfilopodiaintegrin intermediate filaments, IF kinesin lamellipodia microfilaments , MF microfilament associated protein,MAP 肌动蛋白细胞皮层秋水仙素收缩环细胞松弛素B细胞骨架非稳态动力学模型动力蛋白丝状伪足整合蛋白中间纤维驱动蛋白片状伪足微丝微丝结合蛋白microtubule organizing center,MTOCmicrotubules, MTmicrotubule-associadedprotein ，MAPmyosinnucleation phasephalloidinpolymerization phasesliding filament modelsteady state phasetaxoltreadmilling modeltubulinvinblastine微管组织中心微管微管结合蛋白肌球蛋白成核期鬼笔环肽聚合期滑动丝模型稳定期紫杉醇踏车模型管蛋白长春新碱第八章细胞核acrocentric chromosome annular subunitbandcentral domaincentral plug centromerechromatidchromatin chromosomecolumn subunit constitutive heterochromatin cytoplasmic ringdense fibrillar component，DFCeuchromatinexportin facultative heterochromatinfibrillar center，FCfish-trapgenomegranular component，GC heterochromatin nucleosomenucleusouter nuclear membrane pairing domain perinuclear space primary constriction replication origin satellitescaffold radial loop structure model 近端着丝粒染色体环状亚单位带型中央结构域中央栓着丝粒染色单体染色质染色体柱状亚单位组成性异染色质胞质环致密纤维组分常染色质输出蛋白兼性异染色质纤维中心捕鱼笼基因组颗粒中心异染色质核小体细胞核外核膜配对结构域核周间隙主溢痕复制源随体染色体骨架-放射环模型histonekaryophilic proteinsecondary constrictionsolenoidkaryotypekinetochorekinetochore domainluminal subunitmetacentric chromosomeminibandmultiple coiling modelnuclear localization signal,NLSnuclear matrixnuclear matrix associatedproteinnuclear matrix proteinnuclear porenuclear pore complex，NPCnuclear ringnucleolar cyclenucleolar matrixnucleolar organizernucleolar organizing regionnucleolusnucleosomal histonespokesubmetacentric chromosomesupersolenoidtelocentric chromosometelomere组蛋白亲核蛋白次溢痕螺线管核型动粒动粒结构域腔内亚单位中着丝粒染色体微带多级螺旋化模型核内定位信号核基质核基质结合蛋白核基质蛋白核孔核孔复合体核质环核仁周期核仁基质核仁组织者核仁组织区核仁核小体组蛋白辐亚中着丝粒染色体超螺线管端着丝粒染色体端粒第九章基因信息的传递与蛋白质合成activating domain adaptive expression alternative splicing aminoacyl site aminoacyl-tRNA antibiotics anticodon antisense strand 转录激活域适应性表达可变剪接A位点氨酰-tRNA抗生素反密码子反义链CAAT boxcentral dogmachaperoninschromatin remoldingcis-acting elementcoding regioncoding strandcodonCAAT盒中心法则伴侣素染色质重塑顺式作用元件编码区编码链密码子commalessconstitutive expressionconstitutive splicingdegeneracydirectionDNA binding domainenhancerexonGC boxgenegene clustergene familygeneral transcription factor genetic codegenomehelix-loop-helix ，HLHhelix-turn-helix ，HTHheterogeneous nuclear RNA ，hnRNAhighly repetitive sequence inducible geneintronleucine zippermiddle repetitive sequence negative regulationnon-coding regiontranslocationtranspeptidaseubiquitin-proteasomeunique sequenceuniversalwobblezinc finger连续性 组成性表达 常规剪接 简并性 方向性 DNA 结合域 增强子 外显子 GC 盒 基因 基因簇 基因家族 通用转录因子遗传密码 基因组 螺旋-环-螺旋 螺旋-转角-螺旋 不均一核RNA高度重复序列可诱导基因 内含子 亮氨酸拉链 中等重复序列负性调控 非编码区 转位 转肽酶 泛素- 蛋白酶体 单一序列 通用性 摆动性 锌指 operonpeptide formation peptidyl-tRNA site polyribosome positive regulation pribnow box Promoter Registerregulatory gene repetitive sequence repressible gene repressor remodelerribosome circulation sense strandShine-Dalgarno sequence silencer small nuclearribonucleoprotein particle ，snRNPspatial specificity spliceosome split gene structural gene sumoylation TATA box template strand temporal specificity terminatortrans-acting factor transcriptiontranscription factor translation操纵子 成肽 P 位点多聚核糖体 正性调控 Pribnow 盒 启动子 进位 调控基因 重复序列 可阻遏基因 阻遏蛋白 染色质重建子 核糖体循环 有意义链 SD 序列 沉默子核糖核蛋白颗粒空间特异性 剪接体 断裂基因 结构基因 sumo 化 TATA 盒 模板链 时间特异性 终止子反式作用因子 转录 转录因子 翻译第十章细胞连接与细胞黏附adhering junctionadhesion beltanchoring junctioncadherincell adhesioncell adhesion molecule ，CAMcell junctionchemical synapse黏着连接 黏着带 锚定连接 钙黏着蛋白 细胞黏附 细胞黏附分子 细胞连接 化学突触 claudincommunicating junction connexon desmosomedesmosome junction electric coupling electronic synapse filamin密封蛋白 连接连接子 桥粒 桥粒连接 电耦联 电突触 细丝蛋白focal adhesiongap junction hemidesmosome heterophilic binding homophilic binding immunoglobin-superfamily, Ig-SF inside outintegrinintracellular anchor protein linker-dependent binding metabolic coupling epithelial-mesenchymal 黏着斑间隙连接半桥粒异亲型结合同亲型结合免疫球蛋白超家族由内向外整联蛋白细胞内锚定蛋白连接分子依赖性结合代谢耦联上皮-间质转型通讯neural cell adhesionmolecule，N-CAMoccludinoccluding junctionoutside inplectinselectinsynapsetalintransmembrane adhesionprotein神经细胞黏附分子闭合蛋白封闭连接由外向内网蛋白选择素突触踝蛋白穿膜黏连蛋白transition,EMT第十一章细胞外基质及其与细胞的相互作用anchorage dependent growth anoikisbasal lamina，basement membrane，BM chondroitin sulfate，CS collagencollagen disease collagenasecore proteindermatan sulfate，DS elastaseelastinextracellular matrix, ECM fibronectin, FN glycosaminoglycan, GAG 锚定依赖性生长失巢凋亡基膜硫酸软骨素胶原胶原病胶原酶核心蛋白硫酸皮肤素弹性蛋白酶弹性蛋白细胞外基质纤连蛋白糖胺聚糖glycosyltransferasesheparan sulfate，HSheparinhyaluronic acid，HAkeratan sulfate，KSlaminin，LNmatrix metalloproteinases，MMPnidogen，entactinperlecanproteoglycan，PGsyndecantriple helixtropoelastin糖基转移酶硫酸乙酰肝素肝素透明质酸硫酸角质素层粘连蛋白基质金属蛋白酶巢蛋白渗滤素蛋白聚糖连接素三股螺旋可溶性弹性蛋白原第十二章细胞的信号转导adenylate cyclase, AC calmodulin，CaM cAMP-dependent protein kinase A，PKA cascadecGMP depedent protein kinase G，PKG cyclic AMP，cAMP cyclic GMP，cGMP diacylglycerol，DAG effector protein 腺苷酸环化酶钙调蛋白cAMP依赖蛋白激酶A级联反应cGMP依赖蛋白激酶G环磷酸腺苷环磷酸鸟苷二酰基甘油效应蛋白first messengerG proteinG protein linked receptorguanylate cyclase，GCinositol trisphosphate，IP3insulinion channel receptorligandprotein tyrosine kinase，PTKreceptorsecond messenger第一信使G蛋白G蛋白偶联受体鸟苷酸环化酶三磷酸肌醇胰岛素离子通道受体配体酪氨酸蛋白激酶受体第二信使serine/threonine kinases ，STKsignal transduction丝氨酸/苏氨酸蛋白激酶 信号转导 signaling networktyrosine-specific protein kinase receptor ，TPKR信号网络酪氨酸蛋白激酶型受体第十三章细胞分裂与细胞周期amitosis anaphase anti oncogene aster bivalentCdk inhibitor ，CKI cell cycle cell divisioncellular oncogene ，C-onc centrosome chalone checkpoint chiasmachiasma terminalization contractile ring cyclincyclin-dependent kinase ，CDKcytostatic factor,CSF direct division growth factor无丝分裂 后期 抑癌基因 星体 二价体Cdk 激酶抑制物 细胞周期 细胞分裂 细胞癌基因 中心体 抑素 检测点 交叉 交叉端化 收缩环 细胞周期蛋白细胞周期蛋白依赖性激酶细胞静止因子 直接分裂 生长因子indirect divisioninterphasekinetochore microtubule maturation promotingfactor ，MPF meiosismetaphasemitosismitosis,Mmitotic apparatuspolar microtubuleprophaseproto-oncogenerecombination nodulespindlesynapsissynaptonemal complex ，SC telophasetetradV-oncogene ，V-onc间接分裂 分裂间期 动粒微管成熟促进因子减数分裂 中期 有丝分裂 分裂期 有丝分裂器 极微管 前期 原癌基因 重组小结 纺锤体 联会 联会复合体 末期 四分体 癌基因 第十五章细胞分化apical ectodermal cap cell determination cell differentiation cellular reprogramming cleavagecombinatory control compensatory regeneration dedifferentiation differential expression ectodermembryonic induction endoderm顶端外胚层帽 细胞决定 细胞分化 细胞重编程 卵裂 组合调控补偿性再生 去分化 差异表达 外胚层 胚胎诱导 内胚层epimorphosis regeneration gastrulation genomic imprinting grafting experiment histone code homeobox homeobox gene homeodomain homeodomain protein homeosis homeotic gene housekeeping gene微变态再生 原肠形成 基因组印记 胚胎移植实验 组蛋白密码 同源异形框 同源异形框基因 同源异形结构域 同源异形域蛋白 同源异形转变 同源异形基因 管家基因induced pluripotent stem cells juxtacrine interaction lateral inhibitionlocus control region,LCR long non-coding RNA，lncRNAluxury geneluxury proteinmaster control gene maternal effect gene,MEG mesoderm metamorphosis microRNA, miRNA morphallaxis regeneration non-coding RNA诱导多能干细胞,iPS细胞近分泌相互作用侧向抑制基因座控制区长链非编码RNA奢侈基因奢侈蛋白细胞分化主导基因母体效应基因中胚层变态微小RNA变形再生非编码RNAorganogenesisparacrine factorpluripotent cellregenerationregeneration blastemasmall interfering RNA, siRNAsomatic recombinationspatial specificitystage specificitytemporal specificitytotipotent celltotipotent nucleustransdeterminationtransdifferentiationunipotency器官发生旁分泌因子多能（干）细胞再生再生胚芽小干扰RNA体细胞重组阶段特异性空间特异性时间特异性全能（干）细胞全能性细胞核转决定转分化单能第十六章细胞衰老与细胞死亡anoikisanti-apoptosis gene apoptosis apoptotic bodies autophagosome autophagycell deathcell senescencecell shrinkage chromatin condensation ClassIIIPI3Kcytochrome C, cyt C death receptor, DR DNA ladders 失巢凋亡抗凋亡基因细胞凋亡凋亡小体自噬泡细胞自噬细胞死亡细胞衰老细胞皱缩染色质凝聚III型磷脂酰肌醇三磷酸激酶细胞色素C死亡受体DNA梯状条带DNA stainabilityendonucleaseHayflick life spaninterleukin-1β convertingenzyme, ICEnecrosisnerve growth factor receptor,NGFRpermeability transition pores,PT poresphosphatidylserine, PSprogrammed cell death, PCDreactive oxygen species, ROStumor necrosis factor receptor,TNFRDNA可染性核酸内切酶Hayflick 界限白细胞介素-1β转换酶细胞坏死神经生长因子受体渗透转变孔磷脂酰丝氨酸程序性细胞死亡活性氧类物质肿瘤坏死因子受体第十七章干细胞与组织的维持与再生adult stem cell asymmetry division cancer stem cell, CSC chimeracorneal stem cell directly induced differentiation 成体干细胞不对称分裂癌干细胞嵌合体角膜干细胞直接诱导分化dedifferentiationembryoid body, EBembryonic carcinoma cell，ECembryonic germ cell，EGembryonic stem cell，ESepidermal stem cell去分化类胚体胚胎癌细胞胚胎生殖细胞胚胎干细胞皮肤干细胞hematopoiesis stem cell ，HSCinner cell mass ，ICMintegral membrane protein induced pluripotent stem cellintegrinintestinal stem cellliver stem celllung stem cellmarrow cavitymesenchymal stem cell, MSCmultipotent stem cellmyogenic stem cellneural stem cells, NSCpancreatic stem cellplasticitypluripotent stem cell造血干细胞 内细胞团 整合膜蛋白诱导多能干细胞，iPS细胞 整联蛋白 小肠干细胞 肝干细胞 肺脏干细胞 骨髓腔 间充质干细胞 专能干细胞 肌肉干细胞 神经干细胞 胰腺干细胞 可塑性 多能干细胞 pluripotenyprogenitor cell renal stem cell self-maintenance somatic stem cellspermatogonial stem cell stage-specific embryonic antigen,SSEA stem cell stem cell niche symmetry division tissue specific stem cell totipotencytotipotent stem cell transdetermination transdifferentiation unipotency多能 前体细胞 肾脏干细胞 自稳定性 成体干细胞 精原干细胞胚胎阶段特异性抗原干细胞 干细胞巢 对称分裂组织特异性干细胞 全 能 全能干细胞 转决定 转分化 专 能。

4 Sample Sample must must must be be be treated treated treated with with with some some some organic organic organic solvent, solvent, solvent, previous previous previous to to to or or or simultaneously simultaneously simultaneously with with saponification or extraction process, in order to disrupt the structures where vitamin E can be associated to (membranes, lipoproteins, fat droplets . . . ), to eliminate interferences from big molecules molecules such such such as as as proteins or proteins or carbohydrates, carbohydrates, that that that are are are non-soluble non-soluble non-soluble in in in organic organic organic phases, phases, phases, and and to provide a medium in which analytes can be freely soluble. 样品在皂化或提取的时候，样品在皂化或提取的时候，或在此之前，或在此之前，必须用某些有机溶剂进行处理，必须用某些有机溶剂进行处理，其目的是为了破坏其目的是为了破坏VE 和细胞膜、脂蛋白、脂肪滴等之间的结合；消除蛋白质或碳水化合物等大分子的干扰，这些大分子在有机相中都是不可溶的；以及为被分析物提供能在其中充分溶解的介质。

2023最新大学英语四级模拟试题（一）Part ⅠWriting (30 minutes)Directions: For this part, you are allowed 30 minutes to write a short essay entitled The Popularity of Getting Certificates on Campus. You should write at least 150 words following the outline given below.1.大学校园内多种证书旳报考十分火热2.大学生考证旳利弊3.考证面前，我旳选择The Popularity of Getting Certificates on CampusPart ⅡReading Comprehension (Skimming and Scanning) (15 minutes) Directions: In this part, you will have 15 minutes to go over the passage quickly and answer the questions on Answer Sheet 1.For questions 1-7, choose the best answer from the four choices marked A), B), C) and D). For questions 8-10, complete the sentences with the information given in the passage.Main Energies for the BodyA balanced diet is one that provides an adequate intake of energy and nutrients for maintenance of the body and therefore good health. A diet can easily be adequate for normal bodily functioning, yet may not be a balanced diet.CarbohydratesCarbohydrates are a rapid source of energy, they are the body's fuel. The bulk of a balanced diet should be made from carbohydrates. If eaten in an excess of the dietary requirements carbohydrates are easily stored as fats in the cells, although carbohydrate is the first source of energy in the body. An average adult requires about 12,000kJ of energy a day, most of this is supplied by the respiration of carbohydrates in the cells.Carbohydrates are used principally as a respiratory substrates, i.e. to be oxidized to release energy for active transport, macromolecule synthesis, cell division and muscle contraction. Carbohydrates are digested in the duodenum and ileum and absorbed as glucose into cells. Sources of carbohydrates such as starch are rice, potatoes, wheat and other cereals. Sugars are also carbohydrates, sources of sugars are refined sugar - sucrose, which is a food sweetener and preservative and fruit sugars - fructose. If the diet lacks carbohydrate stores of fat are mobilized and used as an energy source.ProteinsProtein is not a direct source of energy in the body, it is used primarily for growth and repair of body tissues while remaining an energy source as a last resort. Proteins fulfill a wide variety of roles in the body. They are broken down in the stomach and intestines to amino acids which are then absorbed. The body can only form 8 amino acids to build proteins from, the diet must provide Essential Amino Acids (EAAs) which are synthesized into proteins which can be structural, i.e. collagen in bone, keratin in hair, myosin and actinin muscle; metabolic enzymes, hemoglobin, protective antibodies and communicative hormones.Sources of protein include meat, fish, eggs and pulses. The diet needs to provide 8 EAAs as the body is unable to synthesis proteins without these molecules. 2 other amino acids are synthesized from EAAs so if the diet lacks the original EAAs these other two will not be present either. Phenylalanine is converted to tyrosine and methionine is converted to cysteine. Cells draw upon a pool of amino acids for protein synthesis which either come from dietary protein digested and absorbed in the gut and the breakdown of body protein such as muscle. However, unlike fats and carbohydrates there is no store of amino acids for cells to draw on, any amino acid in excess of immediate bodily requirements is broken down into urea and excreted. It is therefore important to maintain the dietary intake of protein everyday. If the body lacks protein, muscle wasting occurs as muscle is broken down.If protein is lacked in a diet a person develops kwashiorkor which is caused when high levels of carbohydrates are eaten to overcome the lack of protein in the diet. One symptom of kwashiorkor is the abnormal collection of fluid around the abdomen due to the lack of protein in the blood. The body cannot retain water by osmosis and fluid accumulates in tissues causing them to become waterlogged.Vitamin CategoriesVitamins cannot be synthesized by the body so must be supplied by diet. Vitamins have no common structure or function but are essential in small amounts for the body to be able to utilize other dietary components efficiently.Vitamins fall into two categories, fat soluble vitamins such as vitamin A, D, E and K which are ingested with fatty foods and water soluble vitamins such as the B group vitamins and vitamin C. Vitamins are known as micronutrients because only small quantities are required for a healthy diet, in fact fat soluble vitamins can be toxic in high concentrations, for example the body stores vitamin A, or retinol, in the liver as it is toxic if kept in high concentrations in the blood stream, a dose of more than 3300mg of vitamin A can be considered toxic. Water soluble vitamins such as vitamin C and B groups vitamins can be excreted in the urine if in excess in the diet.Vitamins AVitamin A is essential to the proper functioning of the retina in the eye and the epithelial tissues. A lack of vitamin A results in dry, rough skin, inflammation of the eyes, a drying or scarring of the cornea - xerophthalmia, which occurs when the secretion of lubricating tears is stopped, the eyelids become swollen and sticky with pus. Mucous surfaces of the eye may become eroded allowing infection to set in, leading to ulceration and destruction of the cornea. Night blindness - an inability to see in dim light can also occur. Rod cells in the retina of the eye detect light of low intensity, they convert vitamin A into a pigment, rhodopsin, which is bleached when light enters the eye. Rod cellsresynthesis rhodopsin, but if there is a deficiency of the vitamin, rod cells can no longer function and the result is night blindness. Epithelial cells use retinol to make retinoic acid, an intracellular messenger used in cell differentiation and growth. Without retinoic acid epithelial cells are not maintained properly and the body becomes susceptible to infections, particularly measles and infections of the respiratory system and gut.Xenophthalmia is common among children who's diets consist of mainly cereals with little meat or fresh vegetables, this is common in Indonesia, Bangladesh, India and the Philippines.Vitamins DVitamin D, or calciferol, is another fat soluble steroid vitamin which functions to stimulate calcium uptake from the gut and its deposition in bone. vitamin D acts as a hormone when converted by enzymes in the gut and liver into an active form of "active vitamin D", which stimulates epithelial cells in the intestine to absorb calcium. vitamin D is therefore essential in growing children's diets to enable the growth of strong bones. Without adequate amounts of vitamin D children can develop rickets, which is the deformation of the legs caused when they lack calcium to strengthen the bones. In adults a lack of vitamin D in the diet can lead to osteomalacia, a progressive softening of the bones which can make them highly susceptible to fracture.Vitamin D is made by the body when exposed to sunlight and is stored in the muscles, however, if the skin is rarely exposed to the sunlight or is dark little vitamin D is produced. Foods such as eggs and oily fish are all rich in vitamin D.Vitamins KVitamin K, phylloquinone, is found in dark green leafy vegetables such as spinach and kale. It is a fat soluble vitamin which is involved in the clotting process of blood. In the intestines bacteria synthesize a number of important clotting factors which need vitamin K. Without vitamin K cuts can fail to heal and internal bleeding can occur.Vitamins CVitamin C is a water soluble vitamin, known chemically as ascorbic acid. It is found in citrus fruits such as oranges and lemons, and also in potatoes and tomatoes. The main function of vitamin C is the formation of connective tissues such as collagen. It is also known to be an antioxidant which helps to remove toxins and aids the immune system. A lack of vitamin C leads to Scurvy, a condition experienced by sailors on long journeys when they did not have fruit in their diets. Scurvy causes painful, bleeding gums. As vitamin C is water soluble, it is not toxic in high doses as it can be excreted in the urine, very high doses can however cause diarrhea.Vitamins BB group vitamins have a wide range of roles acting as co-enzymes in metabolic pathways. They are found in most plant and animal tissues involved in metabolism,therefore foods such as liver, yeast and dairy products are all rich in B group vitamins. Deficiency of B group vitamins include dermatitis, fatigue and malformation of red blood cells.1. An adult needs about 12,000kJ of energy a day from ________.A. the cellB. the respiring process of carbohydratesC. fats in the cellD. a balanced diet2. Carbohydrates are ultimately absorbed into cells in the process of _______.A. digestionB. respirationC. oxidizationD. mobilization3. The Essential Amino Acids which build part of proteins can be obtained from______.A. stomachB. body tissuesC. the bodyD. the diet4. The ultimate cause of kwashiorkor is lack of ________.A. proteinB. carbohydratesC. vitaminsD. diet5. Vitamins are called “micronutrients” in that _________.A. excessive fat soluble vitamins can be excreted in the urineB. the body only requires small amount of vitaminsC. a dose of 3300mg of vitamins can be considered toxicD. the high concentrations of water soluble vitamins are toxic6. Night blindness is a disease normally caused by lack of __________.A. fat soluble vitaminsB. water soluble vitaminsC. vitamin AD. innate disability7. The main function of vitamin D is to prevent adults from ________.A. the growth of strong bonesB. fractureC. a progressive softening of the bonesD. calcium uptake from the gut8. Although the human body produces vitamin D normally, it fails to do so if there is not enough ______________.9. The reason why vitamin C is seen as an antioxidant is that it drives__________ out of the body.10. If you are in lack of B group vitamins, you should turn to _______________. Part III Listening Comprehension (35 minutes)Section ADirections: In this section, you will hear 8 short conversations and 2 long conversations. At the end of each conversation, one or more questions will be asked about what was said. Both the conversation and the questions will be spoken only once. After each question there will be a pause. During the pause, you must read the four choicesmarked A), B), C) and D), and decide which is the best answer. Then mark the corresponding letter on Answer Sheet 2 with a single line through the centre.11. A) He thinks he’s very o rganized.B) He doesn’t want to join the display.C) He doesn’t think he should lead the study group.D) He knows someone who can lead the study group.12. A) He doesn’t know where his brother keeps his computer.B) The woman should buy a used computer.C) He doesn’t know how much computers cost.D) His brother paid too much for the computer.13. A) It’s been to warm to wear the jacket.B) The jacket is too big for him.C) He doesn’t like cold weather.D) He didn’t buy the jacket until cooler weather arrived.14. A) He started the semester in a bad mood.B) He’s not usually bad-tempered.C) He has few responsibilities.D) He doesn’t like the man.15. A) He forgot to cancel the reservation.B) They can go to the restaurant after the woman has finished working.C) He has to work late tonight.D) They don’t have a reservation at the restaurant.16. A) Use bleach on his socks.B) Buy new white socks.C) Wash his red T-shirt again.D) Throw away his pink socks.17. A) He isn’t satisfied with his progress.B) He wants to move up more quickly than he’s presently doing.C) He has advance quickly enough in his career.D) He feels frustrated as he tries to move up the ladder.18. A) Try on a smaller sweater.B) Look for another style at a different store.C) Give the sweater away as a gift.D) Exchange the sweater for a bigger one.Questions 19 to 22 are based on the conversation you have just heard.19. A) She's unable to attend the study session.B) She has seen a doctor recently.C) She's concerned about medical care.D) She mentions the need for some medical tests.20. A) To improve the study skills of university students.B) To suggest changes in the student government.C) To give people the opportunity to speak with a politician.D) To discuss graduation requirements for political science majors.21. A) Graduate school application procedures.B) Funding for university education.C) Winning the confidence of voters.D) Preparing for an important test.22. A) Tell her what to study for the history test.B) Write a favorable letter of recommendation.C) Advise her about how to run an election campaign.D) Suggest a topic for a research paper.Questions 23 to 25 are based on the conversation you have just heard.23. A) Boston schools.B) Frontier life.C) Teaching requirements.D) Immigration patterns.24. A) She was a famous author.B) Her family later became famous landowners.C) She exemplifies the immigrant spirit.D) She invented some labor-saving farm equipment.25. A) To the library.B) To the movies.C) To a bookstore.D) To a travel bureau.Section BDirections: In this section, you will hear 3 short passages. At the end of each passage, you will hear some question. Both the passage and the questions will be spoken only once. After you hear a question, you must choose the best answer from the four choices marked A),B),C),and D). Then mark the corresponding letter on the Answer Sheet with a single line through the center.Passage OneQuestions 26 to 29 are based on the passage you have just heard.26. A) They were drawing pictures. B) They were watching TV.C) They were making a telephone call. D) They were tidying up the drawing room.27. A) They locked the couple up in the drawing room.B) They seriously injured the owners of the house.C) They smashed the TV set and the telephone.D) They took away sixteen valuable paintings.28. A) He accused them of the theft.B) He raised the rents.C) He refused to prolong their land lease.D) He forced them to abandon their traditions.29. A) They wanted to protect the farmers’ interests.B) They wanted to extend the reservation area for birds.C) They wanted to steal his valuable paintings.D) They wanted to drive him away from the island.Passage TwoQuestions 30 to 32 are based on the passage you have just heard.30. A) Through food. B) Through air.C) Through insects. D) Through body fluids.31. A) They ran a high fever. B) They died from excessive bleeding.C) Their nervous system was damaged. D) They suffered from heart-attack.32. A) To see what happened to the survivors of the outbreak.B) To study animals that can also get infected with the disease.C) To find out where the virus originates.D) To look for the plants that could cure the disease.Passage ThreeQuestions 33 to 35 are based on the passage you have just heard.33. A) To determine whether the Earth’s temperature is going up.B) To study the behavior of some sea animals.C) To measure the depths of the ocean.D) To measure the movement of waves in the ocean.34. A) They were frightened and distressed.B) They swam away when the speaker was turned on.C) They swam closer to “examine” the speaker when it was turned off.D) They didn’t seem to be frightened and kept swimming near the speaker.35. A) To attract more sea animals to the testing site.B) To drive dangerous sea animals away from the testing site.C) To help trace the sea animals being tested.D) To determine how sea animals communicate with each other.Section CDirections: In this section, you will hear a passage three times. When the passage is read for the first time, you should listen carefully for its general idea. When the passage is read for the second time, you are required to fill in the blanks numbered from 36 to 43 with the exact words you have heard. For blanks numbered from 44 to 46 you are required to fill in the missing information. For these blanks, you can either use the exact words you have just heard or write down the main points in your own words. Finally, when the passage is read for the third time, you should check what you have written.Part IV Reading Comprehension (Reading in Depth) (25 minutes)Section ADirections: In this section, there is a passage with ten blanks. You are required to select one word for each blank from a list of choices given in a word bank following the passage. Read the passage through carefully before making your choices. Each choice in the bank is identified by a letter. Please mark the corresponding letter for each item on Answer Sheet 2 with a single line through the centre. You may not use any of the words in the bank more than once.Questions 47 to 56 are based on the following passage.Dreams are a way for the subconscious to communicate with the __47__ mind. Dreaming of something you’re worried about, researchers say, is the brain’s way of helping you rehearse for a disaster in case it occurs. Dreaming of a challenge, like giving a presentation at work or playing sports, can enhance your __48__. And cognitive neuroscientists have discovered that dreams and the rapid eye movement (REM) that happens while you’re dreaming are __49__ to our ability to learn and remember. Dreaming is a “mood regulatory system,” says Rosalind Cartwright, PhD, chairman of the psychology __50__ at Rush University Medical Center in Chicago. She’s found that dreams help people work through the day’s emotional quandaries. “It’s like having a built-in therapist,” says Cartwright. While we sleep, dreams __51__ new emotional experience to old memories, creating plaid-like patterns of old images laid on top of new ones. As she puts it, “You may wake up and think, What was Uncle Harry doing in my dream? I haven’t seen him for 50years. But the old and new images are __52__ related.” It’s the job of the conscious mind to figure out the relationship. In fact, dream emotions can help real therapists treat patients __53__ traumatic (创伤旳) life events. In a new study of 30 recently __54__ adults, Cartwright tracked their dreams over a five-month period, measuring their feelings toward their ex-spouses. She discovered that those who were angriest at the spouse while dreaming had the best chance of successfully coping with divorce. “If their dreams were bland,” Cartwright says, “they hadn’t started to work through their emotions and __55__ with the divorce.” For therapists, this finding will help __56__ whether divorced men or women need counseling or have already dreamed their troubles away.A. dealB. physicallyC. wakeD. performanceE. makingF. undergoingG. experienceH. divorcedI. determineJ. compareK. departmentL. consciousM. presentationN. linkedO. emotionallySection BDirections: There are 2 passages in this section. Each passage is followed by some questions or unfinished statements. For each of them there are four choices marked A), B), C) and D). You should decide on the best choice and mark the corresponding letter on Answer Sheet 2 with a single line through the centre.Passage OneQuestions 57 to 61 are based on the following passage.A few years ago a young mother watched her husband diaper (给…换尿布) their firstborn son. “You do not have to be unhappy about it,” she protested. “You can talk to him and smile a little.” The father, who happened to be a psychologist, answered firmly, “He has nothing to say to me, and I have nothing to say to him.”Psychologist now know how wrong that father was. From the moment of birth, a baby has a great deal to say to his parents, and they to him. But a decade or so ago, these experts were describing the newborn as a primitive creature who reacted only by reflex, a helpless victim of its environment without capacity to influence it. And mothers acceptedthe truth. Most thought (and some still do) that a new infant could see only blurry (模糊旳) shadows, that his other senses were undeveloped, and that all he required was nourishment, clean diapers, and a warm bassinet.Today university laboratories across the country are studying newborns in their first month of life. As a result, psychologists now describe the new baby as perceptive, with remarkable learning abilities and an even more remarkable capacity to shape his or her environment including the attitudes and actions of his parents. Some researchers believe that the neonatal period may even be the most significant four weeks in an entire lifetime.Far from being helpless, the newborn knows what he likes and rejects what he doesn’t. He shut out unpleasant sensations by closing his eyes or averting his face. He is a glutton for novelty. He prefers animate things over inanimate and likes people more than anything.When a more nine minutes out, an infant prefers a human face to a head-shaped outline. He makes the choice despite the fact that, with delivery room attendants masked and gowned, he has never seen a human face before. By the time he’s twelve hours old, his entire body moves in precise synchrony (同步发生) to the sound of a human voice, as if he were dancing. A non-human sound, such as a tapping noise, brings no such response.57. The author points out that the father diapering his first-born son was wrong because________.A) he believed the baby was not able to hear himB) he thought the baby didn’t have the power of speechC) he was a psychologist unworthy of his professionD) he thought the baby was not capable of any response58. According to the passage, which of the following is TRUE?A) A new infant can see only blurry shadows.B) A new infant’s senses are undevelopedC) All a new infant requires is nourishment, clean diapers, and a warm bassinet.D) A new infant is actually able to influence his or her environment59. What does the sentence “He is a glutton for novelty” probably mean?A) The newborn is greedy for new food.B) The newborn tends to overeat.C) The newborn always loves things that are new to him.D) The newborn’s appetite is a constant topic in no vels.60. According to the passage, it’s groundless to think that newbornsprefer________.A) a human face to a head-shaped outlineB) animate things to inanimate onesC) human voice to non-human soundsD) nourishment to a warm bassinet61. What is the passage mainly discussing about?A) What people know about newborns.B) How wrong parents are when they handle their babies.C) How much newborns have progressed in about a decade’s time.D) Why the first month of life is the most significant four weeks in a lifetime.Passage TwoQuestions 62 to 66 are based on the following passage.Mobile office is the mutual product of economic，scientific，and social progress.Mobile office has become a solution that provides users with convenient, prompt, safe, reliable, and reasonably priced communications and office faculty anywhere anytime via the support of mobile interconnection platform(MIP)and its applications systems. Using mobile office and WAP technology, people can do their work anywhere anytime, can send and receive data via terminals such as mobile phone, and palm computer, and can surf the Internet.When you leave your office to attend meetings or travel on business，what would happen to your business routine?Of course, faxes and e-mails would be still sent to your fax machine or e-mail box, but you cannot read them and make prompt reaction timely. When your clients need you to make some urgent modifications on your work and you are neither in the office nor carrying relevant documents, what can you do?Maybe you have to say “sorry” to the clients. But, your business will be affected，the clients will be unhappy and disappointed because of your delay，and you will lose a lot of business opportunities.In fact, very frequently, you need to check, reply, distribute, modify, or read some materials when you are not in your office. You must get out of this dilemma. The best solution to normally handle your business anywhere anytime and not to disappoint your clients is to let your office “move” with you. With the development of communications technology, mobile office has become simpler and smaller, and even can be realized via one mobile phone with data communications function. Thus, mobile office has already been put into your pocket, and office mobility has been realized.Mobile office has provided people with convenient, casual working environment, but at the same time it still has some unsatisfactory aspects such as mismatching equipment interface and inadequate battery. Nevertheless, we believe that with technical progress, people can certainly overcome all kinds of difficulties. Mobile office will realize the dream of completely free communication. Users will enjoy more colorful life and better working environment, and users’ living standard, working efficiency, and even enterprises’ production efficiency will certainly be immensely raised.注意：此部分试题在答题卡2上作答62. According to the passage, mobile office help you with the following except ________ .A) keeping update with the latest newsB) checking e-mails any time one wantsC) conducting internet surfingD) finding one’s true love in life63. Which of the following best expresses the main idea of the second paragraph?A) You would lose a lot of business opportunities if you always delay your work.B) You should read and reply faxes and e-mail timely.C) When you leave your office your business routine might be damaged.D) When you cannot meet the need of your clients you should immediately say sorry.64. When you let your office “move” with you, you __________ .A) will never let your clients downB) you don’t have to stay at office anymoreC) you then find the best way to handle your business anywhere anytimeD) you no longer face the dilemma between work and life65. It can be inferred from the passage that __________ .A) mobile office communication is very cost-consumingB) with the development of science, mobile office has eventually come to our lifeC) people had no convenient and reliable communications and office faculty beforeD) economic factors are essential in the operation of mobile office66. According to the author, mobile office _________ .A) would help achieve complete communication mobilityB) is too expensive to afford by small companiesC) has some fatal defects impossible to modifyD) is too complicated to operate in everyday businessPart V Cloze (15 minutes)Directions: There are 20 blanks in the following passage. For each blank there are four choices marked A), B), C) and D) on the right side of the paper. You should choose the ONE that best fits into the passage. Then mark the corresponding letter on Answer Sheet 2 with a single line through the centre.For many people today, reading is no longer relaxation. To keep up their work, they have to read all kinds of materials. In _67_a job or advancing in one, the ability to read and comprehend _68_can mean the difference between success and failure. Yet the unfortunate fact is that most of us are _69_readers.Most of us develop poor reading _70_at an early age, and never get over them.The main deficiency _71_in the actual component of language itself-words. Take individually, words have _72_meaning until they are put together into phrases, sentences and paragraphs._73_, however, the untrained reader does not read groups of words. He laboriously reads one word at a time, often turning back to _74_words or passages.。

2007年,英国牛津大学的刘骥陇等在研究果蝇U 小体和P 小体(U 小体和P 小体是真核生物细胞质中的无膜细胞器)的功能关系时,用4种针对Cup (P 小体中的一种蛋白质)的抗体,对雌性果蝇的卵巢组织进行免疫组织化学染色,染色结果除了预期标记上的P 小体外,还标记出了长条形的丝状结构[1]。

这种结构的形状和数量与纤毛很相似,导致当时以为在果蝇中找到了有纤毛的新细胞类型。

但后来的一系列实验表明,该结构与纤毛没有关系,于是将其命名为“细胞蛇”。

最初是抗Cup 抗体不纯产生假象,意外发现的细胞蛇,而采用亲和层析纯化后的抗Cup 抗体无法再DOI:10.16605/ki.1007-7847.2020.10.0258细胞蛇的研究进展收稿日期:2020-10-22;修回日期:2020-11-19;网络首发日期:2021-07-27基金项目:宁夏自然科学基金项目(2020AAC03179);国家自然科学基金资助项目(31560329)作者简介:李欣玲(1999—),女,广西贵港人,学生;*通信作者:俞晓丽(1984—),女,宁夏银川人,博士,副教授,主要从事干细胞与生殖生物学研究,E-mail:********************。

李欣玲,张樱馨,李进兰,潘文鑫,王彦凤,杨丽蓉,王通,俞晓丽*(宁夏医科大学生育力保持教育部重点实验室临床医学院基础医学院,中国宁夏银川750000)摘要:细胞蛇是近年来细胞生物学研究的热门方向之一,由于其在细胞的增殖、代谢和发育上具有一定的生物学功能,因此,对一些疾病如癌症等的临床诊断或治疗具有一定的指导意义。

细胞蛇是由三磷酸胞苷合成酶(cytidine triphosphate synthetase,CTPS)聚合而成的无膜细胞器,其形成过程及功能在不同类型的细胞中不尽相同。

例如:细胞蛇能促进癌细胞增殖,并使患者病情恶化;过表达的细胞蛇可抑制神经干细胞增殖,影响大脑皮层发育;在卵泡细胞中,细胞蛇相当于CTPS 的存储库,在卵子发生过程起到促进细胞增殖和代谢的作用。

硕士学位论文基于单核细胞靶向递送宾达利对动脉粥样硬化的治疗作用研究殷露萁指导教师 李晓辉 教授导师组成员 张建祥教授、贾乙教授、周兴教授、邓有才副教授培养单位 陆军军医大学药学与检验医学系申请学位类别硕士专业学位 专业名称 药学（药剂学） 答辩委员会主席吕万良教授 答辩委员于超教授 胡厚源教授 论文答辩时间2020年11月 论文提交时间 2020年9月二〇二〇年十一月分类号 R94 密级 公开 学号 2002017165 学校代码 91012目录缩略语表 (1)英文摘要 (2)中文摘要 (6)论文正文基于单核细胞靶向递送宾达利对动脉粥样硬化的治疗作用研究 (9)第一章前言 (9)第二章宾达利酵母微囊的制备与表征 (11)2.1 酵母微囊的制备及其表征 (11)2.2 宾达利正电荷纳米粒的制备及表征 (13)2.3 酵母微囊负载宾达利纳米粒的制备及表征 (16)2.4 本章小结 (19)第三章宾达利酵母微囊靶向特性评价 (20)3.1 宾达利酵母微囊的体外靶向特性评价 (20)3.2 宾达利酵母微囊的体内靶向特性评价 (29)3.3 本章小结 (37)第四章宾达利酵母微囊对动脉粥样硬化的治疗作用研究 (39)4.1 口服宾达利酵母微囊治疗动脉粥样硬化斑块部位炎症改善的评价 (39)4.2 口服宾达利酵母微囊治疗动脉粥样硬化降脂评价 (42)4.3 口服宾达利酵母微囊治疗对动脉粥样硬化斑块面积的影响 (45)4.4 口服宾达利酵母微囊对主要脏器组织功能的影响 (47)4.5 本章小结 (51)全文结论 (52)参考文献 (53)文献综述以转运蛋白为靶标的新型纳米给药系统的研究进展 (58)参考文献 (63)攻读学位期间发表的文章 (66)致谢 (68)缩略语表Monocyte-based targeted delivery of bindarit forprevention of atherosclerosisAbstractAtherosclerosis (AS) is a high risk factor leading to fatal coronary heart disease, stroke, peripheral vascular disease and other diseases. As a chronic inflammatory disease, studies have shown that the development process of AS is closely related to Monocyte chemotactic protein-1 (MCP-1). In the early stage of atherosclerosis, monocytes migrate into the arterial intima and become macrophages under the control of MCP-1. Macrophages adhere to vascular endothelial cells and migrate under the endothelium to take up lipids and transform them into foam cells, thereby forming plaques. The accumulation of a large number of macrophages in the plaque is the main sign of active inflammation. Therefore, inhibiting the secretion of MCP-1 to inhibit the migration and infiltration of monocytes is expected to be an effective strategy for early treatment of atherosclerosis. Bindarit (BIN) has been proven to be an effective selective oral inhibitor of MCP-1/CC chemokine CC motif ligand 2 (CCL2), but it still has oral formulation disadvantages. The drug retention rate in the lesions caused by specific distribution is low, and long-term, high-dose administration is required. Therefore, how to achieve targeted delivery of BIN to AS plaques through oral administration to reduce the dose and increase the concentration of drug retention in the lesion is the core issue of our research.After a preliminary research, our group found that through the bionic approach, the yeast capsules (YCs) with the contents removed can be targeted to the tumor site and acute inflammation site through the oral route. Therefore, in this project, based on similar principles, imitating the way bacteria infect the body through the intestine, using yeast capsules as a carrier, an oral BIN targeted delivery system for the treatment of AS was constructed.Method1. Preparation of yeast capsulesA certain amount of yeast was taken, washed twice with isopropanol after acid-base treatment, and then dried to obtain blank yeast capsules.2. Preparation and characterization of bindarit nanoparticlesA certain amount of bindarit (BIN) and polythene imide (PEI) was weighed and dissolved in dimethyl sulfoxide (DMSO), then put into dialysis bag. The Bindarit/ polythene imide (BIN/PEI) nanoparticles were obtained by dialysis in pure water for 8 hours. The particle size and surface potential were measured by a Nano Zetasizer (Nano ZS) and the morphology of the nanoparticles was observed by a transmission electron microscope. (TEM).3. Preparation and characterization of yeast capsules loaded bindarit nanoparticlesA certain amount of YC was weighed and BIN/PEI NPs was added, and the BIN/PEI mass ratio was 1:1. The two were mixed evenly, the mass ratio of YC and (bin-PEI) was 1:1, and the mixture was incubated at 37℃for 3h. After freeze-drying, the yeast capsules loaded with bindarit nanoparticles were obtained, and the drug loading was measured by high performance liquid chromatography. The transmission electron microscope and laser confocal microscope were used to observe the embedding of the nanoparticles in the yeast capsules.4. In vitro release evaluation of yeast capsules loaded bindarit nanoparticlesPrepare a pH 7.4 phosphate buffer solution, mix the drug-loaded yeast capsules into physiological saline (PBS), and take the supernatant at 9 time points (1 h, 2 h, 4 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h). The release degree of BIN was determined by high performance liquid chromatography, the cumulative release percentage was calculated, and the cumulative release curve of drug was plotted.5. Study on the targeting effect of oral yeast capsules on atherosclerotic plaqueThe fluorescent cyanine dye 5.5 (Cyanine 5.5,Cy5.5) is chemically bonded to the surface of the PEI through a chemical reaction, and the Cy5.5-PEI nanoparticles (CY5.5-PEI NPs) are prepared by dialysis in pure water against light. Repeat the above BIN and PEI synthesis steps to obtain BIN/PEI-Cy5.5 NPs. Then YC was incubated with BIN/PEI-Cy5.5 NPs to obtain fluorescently labeled yeast capsules (BIN-Cy5.5/YCs). Apolipoprotein E-deficient mice (ApoE-/-) were given a high-fat diet to establish an atherosclerosis model, and were given Cy5.5-PEI NPs and BIN-Cy5.5/YCs by gavage for 4 consecutive days. After euthanasia, the aorta and major organ tissues were stripped, and the fluorescence intensity was determined bya Living Imaging system.6. Pharmacodynamic evaluation of oral drug-loaded yeast capsules in the treatment of atherosclerosisApoE-/- mice were used to establish an AS model. They were given saline, BIN, BIN NPs, and BIN/YCs, and gavage every three days. After 1 month of treatment, other major organs were dissected and the aorta was removed. Stain with Oil Red O to evaluate the development of plaque. Immunohistochemical staining was used to observe the distribution of plaques in mouse aortic root sections by CLSM. The proportion of mononuclear macrophages in the whole blood of mice was determined by flow cytometry.7. Preliminary safety study on the treatment of atherosclerosis with drug-loaded yeast capsulesAfter hematoxylin-eosin staining, observe the structure of organs and gastrointestinal tissues of each group of mice pathological changes. After hematoxylin-eosin staining, the structure of organs and gastrointestinal tissues in each group of mice pathological changes were observed.Result:The yeast capsules whose outer wall is mainly composed of β-1,3-D-glucan are successfully obtained after acid-base treatment based on yeast. And using the electrostatic interaction between the positively charged nanoparticles and the yeast capsules, they gather on the surface of the yeast capsules and then enter the yeast capsules to form BIN/YCs. After assembling polyethyleneimine and bindarit to form nanoparticles, the results of particle size analyzer and potential analyzer show that the size of the nanoparticles is uniform, with an average particle size of 25.24 ± 2.62 nm and a Zeta potential of 34.70 ± 0.30 mV. Using static electricity-mediated spontaneous deposition, through a simple suspension process, the BIN/PEI positively charged nanoparticles were successfully loaded into YC. TEM observation results showed that the nanoparticles were dispersed inside the yeast capsules. The average particle size is 3451.45±443.45 nm, and the Zeta potential is 0.01±0.02 mV. The positively charged bindarit nanoparticles and yeast-loaded bindarit nanoparticles are completely released within 8 hours at pH 7.4. In vivo imaging results showed that after oral administration of BIN-Cy5.5/YCs, the accumulation of Cy5.5 in atherosclerotic plaques can be significantly increased. The analysis of the fluorescence intensity of various organs and tissues showed that the fluorescence intensity of lymph-related mesenteric lymph nodes, small intestine and aortic plaques all have a significant increase. The results of oil red O staining showed that the proportion of AS plaques in ApoE-/- mice was significantly reduced after oral administration ofBIN/YCs, and the level of related pro-inflammatory factors in the serum was significantly reduced. The preliminary safety evaluation showed that there were no significant differences in blood physiological indicators, liver and kidney functions, and no obvious pathological changes in organs and tissues.Conclusion:Using electrostatic adsorption, positively charged bindarit nanoparticles can be successfully loaded inside YC. After oral administration, BIN/YCs can be targeted to atherosclerotic plaques in mice, and this process is closely related to the lymphatic system and intestinal tissues. At the same time, mononuclear macrophages may play an important role in the targeting process. Oral BIN/YCs can effectively reduce the growth of AS plaques without obvious side effects.To sum up, this project constructed an oral targeted BIN delivery system for treating AS by constructing oral targeted agents to the inflammatory site and using yeast microcapsules AS drug carrier, which is expected to provide theoretical and experimental basis for the development of a new type of AS drug.Keywords: Atherosclerosis, Oral targeting, yeast capsules, Drug delivery, Fluorescence imaging基于单核细胞靶向递送宾达利对动脉粥样硬化的治疗作用研究摘要近年来，心脑血管疾病已成为人类健康的头号杀手。