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Muscle-selective synaptic disassembly and reorganization in MuSK antibody positive MG miceAnna Rostedt Punga ⁎,1,Shuo Lin,Filippo Oliveri,Sarina Meinen,Markus A.RüeggDepartment of Neurobiology/Pharmacology,Biozentrum,University of Basel,Basel,Switzerlanda b s t r a c ta r t i c l e i n f o Article history:Received 23February 2011Revised 15April 2011Accepted 21April 2011Available online 30April 2011Keywords:Myasthenia Gravis MG MuSKNeuromuscular junction MasseterMuscle atrophy DenervationMuSK antibody seropositive (MuSK+)Myasthenia Gravis (MG)patients present a distinct selective fatigue,and sometimes atrophy,of bulbar,facial and neck muscles.Here,we study the mechanism underlying the focal muscle involvement in mice with MuSK+experimental autoimmune MG (EAMG).8week-old female wildtype C57BL6mice and transgenic mice,which express yellow ﬂuorescence protein (YFP)in their motor neurons,were immunized with the extracellular domain of rat MuSK and compared with control mice.The soleus,EDL,sternomastoid,omohyoid,thoracic paraspinal and masseter muscles were examined for pre-and postsynaptic changes with whole mount immunostaining and confocal microscopy.Neuromuscular junction derangement was quanti ﬁed and compared between muscles and correlated with transcript levels of MuSK and other postsynaptic genes.Correlating with the EAMG disease grade,the postsynaptic acetylcholine receptor (AChR)clusters were severely fragmented with a subsequent reduction also of the presynaptic nerve terminal area.Among the muscles analyzed,the thoracic paraspinal,sternomastoid and masseter muscles were more affected than the leg muscles.The masseter muscle was the most affected,leading to denervation and atrophy and this severity correlated with the lowest levels of MuSK mRNA.On the contrary,the soleus with high MuSK mRNA levels had less postsynaptic perturbation and more terminal nerve sprouting.We propose that low muscle-intrinsic MuSK levels render some muscles,such as the masseter,more vulnerable to the postsynaptic perturbation of MuSK antibodies with subsequent denervation and atrophy.These ﬁndings augment our understanding of the sometimes severe,facio-bulbar phenotype of MuSK+MG.©2011Elsevier Inc.All rights reserved.IntroductionAbout 40–70%of acetylcholine receptor (AChR)-antibody seronegative Myasthenia Gravis (MG)patients have antibodies against the muscle tyrosine kinase (MuSK)(Hoch et al.,2001;Sanders et al.,2003).In MuSK-antibody seropositive (MuSK+)MG patients,there is often selective involvement of bulbar-,neck-and facial muscles,as well as muscles that are usually asymptomatic in AChR-antibody seropositive (AChR+)MG,such as the paraspinal muscles (Sanders and Juel,2008).Contrary to conventional AChR+MG patients,the majority of MuSK+patients does not experience symptomatic relief from acetylcholine esterase inhibitors (AChEI)(Evoli et al.,2003)but instead may respond with pronounced nicotinic adverse effects,such as muscle fasciculations and cramps (Punga et al.,2006).Pronounced atrophy of facial muscles has also been described in MuSK+patients,although the concomitant treatment of corticosteroids in most cases has made it dif ﬁcult tojudge whether the MuSK antibodies or the cortisone treatment is the cause of the atrophy (Farrugia et al.,2006).Muscle biopsy studies of the intercostal muscle and biceps brachii muscle from MuSK+patients have shown little AChR loss (Selcen et al.,2004;Shiraishi et al.,2005),however,the neuromuscular junction (NMJ)pathophysiology in the most affected facial or bulbar muscles has not been studied.Nevertheless,MuSK antibodies have been shown to be pathogenic in animals,both after immunization with the extracellular domain of the MuSK protein itself (Jha et al.,2006;Shigemoto et al.,2008;2006;Xu et al.,2006)and after passive transfer of sera from MuSK+MG patients (Cole et al.,2008;ter Beek et al.,2009).MuSK is essential to the process of NMJ formation,maintenance (Wang et al.,2006)and integrity,as perturbations in MuSK protein expression cause pronounced disassembly of the entire NMJ (Hesser et al.,2006;Kong et al.,2004).Other NMJ proteins that are essential for synaptogenesis include Dok-7,a downstream adaptor protein to MuSK (Okada et al.,2006),Lrp4,the co-receptor for neural agrin (Kim et al.,2008;Zhang et al.,2008),rapsyn and the AChR subunits.The effects of MuSK antibodies in-vivo on the gene expression of those synaptic proteins in the facial or bulbar muscles have not yet been established.Here,we hypothesized that low expression levels of MuSK may render some muscles more vulnerable to the effect of MuSK antibodies in the EAMG mouse model.We show that MuSK antibodiesExperimental Neurology 230(2011)207–217⁎Corresponding author at:Institute of Neuroscience,Department of Clinical Neuro-physiology,Uppsala University Hospital,Uppsala,Sweden.Fax:+4618556106.E-mail address:annarostedtpunga@ (A.R.Punga).1Present address:Department of Clinical Neurophysiology,Uppsala University Hospital,Uppsala,Sweden.0014-4886/$–see front matter ©2011Elsevier Inc.All rights reserved.doi:10.1016/j.expneurol.2011.04.018Contents lists available at ScienceDirectExperimental Neurologyj o u r n a l h o me p a g e :w w w.e l s e v i e r.c om /l o c a t e /y e x n rinduce severe fragmentation of the postsynaptic AChR clusters in particular in the masseter and thoracic paraspinal muscles,with less fragmentation in the limb muscles.The severe postsynaptic pertur-bation results in subsequent denervation of muscleﬁbers,not previously described in EAMG or MG.We propose that one underlying mechanism for the severe involvement of the facial masseter muscle, with severely impaired NMJ architecture,atrophy and denervation,is its low intrinsic levels of MuSK.Moreover,muscles respond to the partial denervation caused by MuSK antibodies in two different ways: (1)terminal nerve sprouting in muscles with high intrinsic levels of MuSK(i.e.soleus,sternomastoid)and(2)no nerve sprouting in muscles with low intrinsic MuSK levels(i.e.masseter,omohyoid).MethodsProduction of recombinant rat MuSKpCEP-PU vector containing the His-tagged extracellular domain of recombinant rat MuSK(aa21-491;(Jones et al.,1999))was transfected(Lipofectamine2000;Invitrogen)into HEK293EBNA cells.The overexpressed protein was puriﬁed from the cell superna-tant over a Ni-NTA superﬂow column(Qiagen)and was subsequently dialyzed against PBS.Protein concentration was determined at OD 280nm and purity was ensured by SDS-PAGE.Experimental animalsC57BL6mice and mice expressing yellowﬂuorescence protein (YFP)in their motor neurons under Thy-1promoter(Feng et al., 2000)were originally supplied from Jackson Laboratories(Bar Harbor, Maine,US).For immunization,8week-old female mice were used.All mice were housed in the Animal Facility of Biozentrum,University of Basel,where they had free access to food and water in a room with controlled temperature and a12hour alternating light–dark cycle.All animal procedures complied with Swiss animal experimental regu-lations(ethical application approval no.2352)and EC Directive 86/609/EEC.ImmunizationThe immunization procedure has been described previously(Jha et al.,2006).Brieﬂy,eleven C57BL6and seven Thy1-YFP female mice aged8weeks were anesthetized(Ketamine:111mg/kg and Xylazine: 22mg/kg)and immunized with10μg of MuSK emulsiﬁed in complete Freund's adjuvant(CFA,Difco laboratories,Detroit,Michigan,US) subcutaneously in the hind foot pads,at the base of the tail and dorsolateral on the back.At day28post-injection,immunization was repeated.A3rd immunization was given to mice that did not show any myasthenic weakness after56days.Control mice(8female mice) were immunized with PBS/CFA.Clinical and neurophysiological examinationMuscle weakness was graded every week,as described(Nakayashiki et al.,2000).Brieﬂy,mice were exercised by20consecutive paw grips on a grid and were then placed on an upside-down grid.The time they could hold on to the grid reﬂected the grade of fatigue and muscle weakness.EAMG grades were as follows:grade0,no weakness;grade1, mild muscle fatigue after exercise;grade2,moderate muscle fatigue; and grade3,severe generalized weakness.Evaluation of the response to AChEIs was performed by i.p.injection of a mix of neostigmine bromide (0.0375mg/kg)and atropine sulfate(0.015mg/kg)in mice with EAMG grades2and3(Berman and Patrick,1980).Repetitive stimulation of the sciatic nerve and recording from the gastrocnemius muscle with monopolar needle electrodes was performed under anesthesia,in mice with EAMG grades2(n=2)and3(n=2),using a Saphire1L EMG machine(Medelec).Decrement was calculated as percent amplitude change between the1st and4th compound motor action potentials evoked by a train of10impulses where10%was considered as pathological.ELISASera were obtained from tail vein blood on day0(preimmune sera)and day35post-immunization.ELISA plates(Nunc MaxiSorp, Fisher Thermo Scientiﬁc,Rockford,IL,US)were coated with250ng/ ml of His-labeled rat MuSK(50μl/well),blocked with3%BSA/PBS and then incubated with a sera dilution row(1:3000–1:2,000,000).Pre-immune sera constituted negative and rabbit-anti-MuSK antibody (Scotton et al.,2006)positive controls.After washing,plates were incubated with secondary HRPO-conjugated goat-anti-mouse (1:2000)and goat anti-rabbit antibodies(1:2000;both from Jackson Immuno Research Laboratories,Westgrove,PA,US).HRPO activation by a TMB substrate was terminated with1N HCl after5min. Absorbance was read at450nm.Non-speciﬁc binding,determined by incubation of plates with pre-immune serum,was subtracted.The data were displayed as“half maximum MuSK immunoreactivity”,which represents the immunore-activity at a dilution of1:27,000,where the majority of sera obtained50% of maximum absorbance(in the linear range of the absorption at450nm).Western blotWestern blot of masseter muscles was conducted as described (Bentzinger et al.,2008).10μg of protein was resolved on a4–12%Nu-PAGE Bis–Tris gel(Invitrogen,Eugene,OR,US),transferred to nitrocellulose membrane,probed with rat monoclonal anti-NCAM (CD56;1:100;GeneTex)and rabbit polyclonal anti-pan-actin(1:1000; cell signaling)and then recognized with HRPO-conjugated antibodies (1:5000;Jackson Immuno Research Laboratories,Westgrove,PA,US). Quantitative RT-PCR analysisMouse muscle RNA was extracted and puriﬁed as previously described(Punga et al.,2011).RT-PCR reactions(triplicates)were carried out with Power SYBR Green PCR Master Mix reagent(Applied Biosystems,Warrington,UK).β-actin was used as endogenous control (Punga et al.,2011;Murphy et al.,2003;Yuzbasioglu et al.,2010).The following primer sets were used:MuSK:5′-GCCTTCAGCGGGACTGAG-3′and5′-GAGGCGTGGTGA-CAGG-3′Lrp4:5′-GGATGGCTGTACGCTGCCTA-3′and5′-TTGCCGTTGTCA-CAGTGGA-3′Dok-7:5′-CTCGGCAGTTACAGGAGGTTG-3′and5′-GCAATGC-CACTGTCAGAGGA-3′A C h Rα1:5′-G C C A T T A A C C C G G A A AG T G A C-3′a n d5′-CCCCGCTCTCCATGAAGTT-3′AChRε:5′-CTGTGAACTTTGCTGAG-3′and5′-GGAGATCAG-GAACTTGGTTG-3′AChRγsubunit:5′-AACGAGACTCGGATGTGGTC-3′and5′-GTCGCACCACTGCATCTCTA-3′Rapsyn:5′-AGGTTGGCAATAAGCTGAGCC-3′and5′-TGCTCTCACT-CAGGCAATGC-3′MuRF-1:5′-ACC TGC TGG TGG AAA ACA-3′and5′-AGG AGC AAG TAG GCA CCT CA-3′β-actin:5′-CAGCTTCTTTGCAGCTCCTT-3′and5′-GCAGCGA-TATCGTCATCCA-3′A C h E:5′-G G G C T C C T A C T T T C T G G T T T A C G-3′a n d5′-GGGCCCGGCTGATGAG-3′208 A.R.Punga et al./Experimental Neurology230(2011)207–217NMJ whole mount analysisAlexa Fluor555-conjugatedα-bungarotoxin(1μg/ml;Invitrogen) was injected into soleus,EDL,sternomastoid,omohyoid,masseter and the thoracic paraspinal muscles as described(Bezakova and Lomo, 2001).At least12images of each muscle per mouse(6MuSK-immunized YFP-transgenic mice and4CFA-immunized control mice) were collected with a confocal laser-scanning microscope(Leica TCS SPE).Laser gain and intensity were equal for all images.Quantiﬁcation of pre-and postsynaptic area was performed in ImageJ(http://imagej. /ij/index.html).NMJs containing terminal nerve sprouts(processes with YFP expression)were counted in muscles fromﬁve MuSK+EAMG mice with disease grades1–3.At least275NMJs per muscle were analyzed. The number of postsynapse fragments per NMJ was counted using a ﬂuorescence microscope(Leica DM5000B)in at least50NMJs per muscle deriving fromﬁve MuSK+EAMG grades1–2and from four control mice(Supplemental Fig.1).Fragmentation was classiﬁed as follows:1)normal pretzel-like NMJ;2)slight to moderate fragmen-tation;and3)severe fragmentation or absent postsynapse.The degree of postsynaptic perturbation per muscle was judged based on the percentage of NMJs belonging to each postsynaptic class and was further subdivided into number of postsynapse fragments per NMJ:1–3,4–6,7–9,10–12and more than12.Each NMJ was given the median score for that subgroup.The fragmentation score was obtained by taking the ratio of the score between the EAMG mice and control mice.Statistical analysisIndependent,2-sample t-test was performed for parametric data. For ordinal data(ELISA),the non-parametric test Spearman Rank Correlation was applied.A p-value b0.05was considered signiﬁcant.Fig.1.(A)Development of EAMG after immunization with recombinant rat MuSK.Progress of clinical EAMG grade at the time points week4,5,7and10.*The mice with EAMG grade 3were sacriﬁced after week7(hence no new grade3mice week10)and the remaining mice were sacriﬁced andﬁnally evaluated at week10.(B)One mouse,representative of the most severely affected MuSK+EAMG mice,withﬂaccid paralysis and pronounced kyphosis.(C)Repetitive nerve stimulation performed in the same mouse.Stimulation of the sciatic nerve and recording of the gastrocnemius muscle demonstrated a35%decrement between the1st and4th compound motor action potentials at low frequency3Hz stimulation.(D)Correlation of clinical EAMG grade with MuSK antibody titer.Half of maximum MuSK immunoreactivity(1:27,000dilution)in sera from MuSK immunized mice was assessed by Elisa at450nm.R=0.483(Spearman's Rank Correlation);p b0.05.209A.R.Punga et al./Experimental Neurology230(2011)207–217ResultsMuSK+EAMG presents with prominent kyphosis,paralysis and weight lossOut of the18mice immunized with MuSK,theﬁnal EAMG grade 0was seen in3mice(17%),grade1in8mice(44%),grade2in4mice (22%)and grade3in3mice(17%)(Fig.1A).No difference in disease incidence was found between the groups of C57BL6mice and the YFP-transgenic mice.Based on this,the data were pooled in the current study.The most severe phenotype of EAMG(grade3)includedﬂaccid paralysis,pronounced kyphosis and weight loss(Fig.1B).In-vivo nerve stimulation at3Hz with recording from the gastrocnemius muscle revealed a decrement of10–40%in the MuSK+EAMG mice (Fig.1C),whereas the control mice had normal neuromuscular transmission(data not shown).MuSK immunoreactivity in sera(day 35)correlated with clinical severity(Fig.1D;Spearman Rank Correlation;R=0.483;p b0.05),although some mice developed measurable MuSK antibodies without showing obvious muscle weakness or fatigue.Bulbar symptoms underlying weight loss in MuSK+EAMG The MuSK immunized mice steadily decreased signiﬁcantly in body weight after the2nd immunization(Fig.2A),in contrast to the control mice,and theﬁnal body weight of the MuSK+mice was signiﬁcantly smaller(p b0.001;Fig.2B).This severe weight loss was slowed but not stopped after introduction of wet food(data not shown).Since the timeline of the weight drop also correlated with development of muscle fatigue,the weight loss was assumed to be indicative of chewing and swallowing difﬁculties,some of the cardinal symptoms of MuSK+MG.To determine whether loss of muscle weight signiﬁcantly contributed to the overall lower body weight in the MuSK+EAMG mice,the weight ofﬁve different muscles was assessed.The masseter was the only muscle with a signiﬁcant weight reduction(p b0.01;Fig.2C),implying that this muscle is atrophic and further indicating chewing difﬁculties.Adverse effects of AChEIs in MuSK+EAMGTo elucidate whether AChEIs have any beneﬁcial effect on fatigue or weakness in MuSK+EAMG,a neostigmine test was performed in the mice with EAMG grade3(n=3).No apparent improvement in weakness at rest or exercise-induced fatigue was seen;instead the mice experienced shivering and constant twitching of the tail,trunk and limbs starting after approximately13min(Supplemental Video1).This effect wore off after40min and was interpreted as nicotinic side effects and neuromuscular hyperactivity,usually seen after an overdose of AChEIs.Thus,this intolerance towards AChEIs in MuSK+EAMG indicates an abnormal sensitivity to acetylcholine.Impairment of NMJs in different musclesBecause muscle groups in the bulbar/facial/back region are selectively involved in the MuSK+EAMG model,we next examined the morphological changes of NMJs in the thoracic paraspinal muscles, masseter,omohyoid and sternomastoid and compared them with those in two limb muscles(EDL and soleus).Typical features of postsynaptic impairment were a fainting of AChRﬂuorescence,areas lacking AChRs(holes)and disassembly of AChR clusters.To quantify these impairments,we classiﬁed NMJs into three classes as illustrated in Fig.3A.All muscles from MuSK+EAMG mice displayeddifferentFig.2.Weight loss in MuSK+EAMG.(A)The course of weight loss in two MuSK+mice with EAMG grade3compared to control(Ctrl CFA)mice(n=8).Initial body weight was comparable and weight loss started after the2nd immunization and weight kept dropping even though wet food was provided ad libitum.(B)Mean body weight was dramatically reduced in the MuSK immunized mice(MuSK+;n=18),compared to the control mice.Results shown as mean±SEM(gram);***p b0.001.(C)Muscles were weighed and compared between control mice(n=8)and MuSK+EAMG mice(n=18).Results displayed as mean muscle weight±SEM(mg).The only muscle which was signiﬁcantly lighter in the MuSK+mice was the masseter.**p b0.01.210 A.R.Punga et al./Experimental Neurology230(2011)207–217degrees of NMJ impairment (Fig.3B).Moreover,we also observed that the postsynapses were often fragmented into two to three non-continuous fragments (see illustration in Fig.3A),which is in strong contrast to non-interrupted,pretzel-like shape of AChRs in non-immunized mice.This fragmentation was also quanti ﬁed (Supplemental Fig.1)and expressed as “fragmentation score ”.Because fragmentation differs between muscles,this “fragmentation score ”was normalized to the control.As shown in Fig.3C,the fragmentation score varied between a minimum of 2.0in the soleus muscle and a maximum of 3.3in the masseter muscle.The postsynaptic labeling was markedly reduced in the MuSK+EAMG mice compared to control mice (Fig.4A).In the mild to moderately affected mice,AChR cluster area was reduced signi ﬁcantly by 40–50%in all muscles (p b 0.01),except for the soleus,which had a remaining area of 85%(p b 0.05;Fig.4B).In the most severely affected mice,the postsynaptic area was also lost in the soleus muscle and less than 10%of the α-bungarotoxin staining remained in the masseter,sternomastoid and thoracic paraspinal muscles (p b 0.001).In the soleus,the nerve terminal area was unchanged in the severely affected mice,although in the other muscles the presynaptic area was reduced to about 80%in the mild to moderate cases and to 50%in the most severely affected mice (p b 0.01)(Fig.4C).Thus,both the pre-and postsynaptic data suggest that the masseter is the most affected muscle by MuSK antibodies whereas the soleus is the least affected.mRNA transcript expression of NMJ proteins in different muscles in MuSK+EAMGThe mRNA levels of MuSK differ signi ﬁcantly between muscles,with the highest levels in the soleus muscle and lowest levels in the omohyoid muscle (Punga et al.,2011).Consequently,we additionally analyzed MuSK transcript levels in the masseter and these were even lower,with less than 20%of the mRNA levels detected in the soleus muscle (Supplemental Fig.2;p b 0.01).In the MuSK+EAMG mice,MuSK mRNA levels were signi ﬁcantly reduced to 45%of control in the EDL (p b 0.05)and to 25%in the omohyoid muscle (p b 0.001;Fig.5A).Conversely,MuSK mRNA expression was signi ﬁcantly increased in the masseter muscle (p b 0.01)and remained unchanged in the soleus and sternomastoid muscles.The expression of the AChR αsubunit was unchanged (Fig.5B),whereas the AChR εsubunit transcript was downregulated in the soleus and sternomastoid muscles and conversely a trend towards upregulation was seen in the masseter muscle (Fig.5C).The fetal AChR γsubunit mRNA was upregulated up to 1000-fold in the masseter (p b 0.001)and 5-fold in the soleus (p b 0.05;Fig.5D).The transcript levels of rapsyn,Lrp4and Dok-7were not signi ﬁcantly altered (Fig.5E to G).Finally,the transcript levels of acetylcholine esterase (AChE)were signi ﬁcantly down-regulated only in the sternomastoid and omohyoid muscles (p b 0.05;Fig.5H).Fig.3.Postsynaptic fragmentation of NMJs.(A)Examples from each postsynaptic classi ﬁcation.AChRs stained for alfa-bungarotoxin.(B)At least 275neuromuscular junctions (NMJs)per muscle were assessed in a total of 5MuSK+EAMG mice with moderate to severe disease.The appearance of NMJ postsynapses were divided into 3classes as indicated.Sol:soleus;EDL:extensor digitorum longus;STM:sternomastoid muscle;omo:omohyoid;mass:masseter.(C)Postsynapses were analyzed in at least 50NMJs per muscle in a total of 5MuSK+EAMG mice with slight to moderate disease grade and in 4control mice.Each NMJ in a certain subclass (for details see Supplemental Fig.1)was given the median score for that subclass.The fragmentation score for each muscle is the ratio in score between the EAMG mice and control mice.211A.R.Punga et al./Experimental Neurology 230(2011)207–217In summary,particularly the massive upregulation of MuSK transcripts and AChR γin conjunction with the overall trend for upregulation of mRNA for the other postsynaptic genes in the masseter muscle most likely re ﬂects the severely disturbed neuro-muscular transmission in this muscle as a result of the MuSK antibodyattack.Fig.4.Pronounced reduction of postsynaptic and presynaptic area in different muscles in MuSK+EAMG.(A)Whole mount staining of single ﬁber layer bundles from the paraspinal muscle (ps),sternomastoid (STM),masseter (mass),omohyoid (omo),extensor digitorum longus (EDL)and soleus (sol)muscles from one control mouse immunized with CFA/PBS and one mouse with severe MuSK+EAMG.AChRs are visualized by alexa-555-bungarotoxin (red)and the motor nerve terminals by YFP expression (green).The AChRs are almost completely gone from the paraspinal muscles,STM and masseter.Confocal images of 100×magni ﬁcation,scale bar is 10μm.Quanti ﬁcation of (B)postsynapse (AChR clusters)and (C)presynaptic area in the different muscles;soleus (sol),extensor digitorum longus (EDL),omohyoid (omo),masseter (mass),sternomastoid (STM)and paraspinal muscles (ps).Results are given as %of ctrl area±SEM and at least 12NMJs in each muscle/mouse was measured in mice with EAMG grades 1–2(N =4),in mice with severe grade 3(N =2)and in control mice (N =4).**p b 0.01;#p b 0.001.212 A.R.Punga et al./Experimental Neurology 230(2011)207–217Denervation induced muscle atrophy in MuSK+EAMGThe masseter muscle lost weight and consequently we examined the mRNA levels of the atrophy marker muscle-speci ﬁc RING ﬁnger protein 1(MuRF-1)and assessed signs of denervation.For this analysis we included the same muscles as previously examined,with the addition of thoracic paraspinal muscles due to the pronounced kyphosis of the MuSK+EAMG mice.MuRF-1mRNA levels were signi ﬁcantly upregulated in the masseter muscle (p b 0.05;Fig.6A)and on the contrary MuRF-1transcript levels were strongly reduced in the soleus muscle (p b 0.01)in the MuSK+EAMG mice.Further,the protein levels of neural cell adhesion molecule (NCAM),a marker for denervation,were increased in the masseter muscle as detected by Western blot analysis.This is thus additional evidence of denervation in this particular muscle (Fig.6B).Absence of nerve sprouting may contribute to the severe denervation phenotypePresynaptic nerve terminals were visualized by transgenic YFP expression,allowing observation also of NMJs with absent post-synapses.In the masseter,the nerve terminals were still present although many postsynaptic AChRs were partially or completely lost.Intriguingly,no or little nerve sprouting was observed in these cases (Fig.7).This ﬁnding raised the possibility that postsynaptic perturbation in this muscle did not elicit any nerve sprouting response.To test this hypothesis,we examined ≥450NMJs in each of the muscles for such sprouting response.The quantitative examination of 4MuSK+EAMG mice revealed terminal nerve sprouting in approximately 20%of NMJs in the soleus and in 15%of endplates in the sternomastoid (Fig.7).On the contrary,theFig.5.mRNA levels of postsynaptic proteins in MuSK+EAMG.C:control mice immunized with CFA/PBS.MG:EAMG mice immunized with MuSK.mRNA levels in the control mice are set to 100%and levels in the EAMG mice are relative to the control levels.n=6control mice;n=6MuSK+EAMG mice.Genes of interest are relative to the house keeping gene β-actin.*p b 0.05;***p b 0.001.213A.R.Punga et al./Experimental Neurology 230(2011)207–217omohyoid,EDL,thoracic paraspinal muscles and masseter,showed terminal sprouting at only a few NMJs.In most samples no sprouting was observed,which differed signi ﬁcantly from the soleus (p b 0.001)and the sternomastoid muscle (p b 0.05).Interestingly,the degree of nerve sprouting in each muscle correlates with their endogenous levels of MuSK,suggesting that MuSK levels regulate the nerve sprouting response and consequently reorganization of NMJs (Punga et al.,2011).DiscussionMuSK+MG patients often present with focal weakness of facial,bulbar,neck and respiratory muscles and sometimes also of paraspinal and upper esophageal muscles (Sanders and Juel,2008).The pathopysiological role of MuSK antibodies has been questioned based on the normal levels of MuSK and AChRs at the NMJ in cross-sections of the intercostal muscle and biceps brachii muscle of MuSK+MG patients (Selcen et al.,2004;Shiraishi et al.,2005).Further studies in mice have now shown that MuSK antibodies deplete MuSK from the NMJ,which results in disassembly of the postsynaptic apparatus and a reduced packing of AChRs (Jha et al.,2006;Shigemoto et al.,2006;Cole et al.,2010).Nevertheless,the exact mechanism of how MuSK antibodies affect muscles has so far not been revealed.Here we report that MuSK antibodies cause a severe pre-and postsynaptic disassembly in the facial,bulbar and paraspinal muscles but only a slight disruption in the limb muscles (i.e.soleus and EDL).The most affected muscle was the masseter and intriguingly this muscle expressed less than 20%of MuSK transcripts than the least affected soleus muscle.Considering that fast-twitch muscle ﬁbers require larger depolarization to initiate contraction compared to slow-twitch ﬁbers,we propose that the superfast twitch pattern of the masseter in combination with its critically low MuSK levels makes this muscle extra vulnerable to the synaptic perturbation following the MuSK antibody attack (Laszewski and Ruff,1985).Hence,over-expression of MuSK in vulnerable muscles could potentially alleviate the effects of the antibody-mediated attack against MuSK.Earlier studies of AChR+EAMG rats have been shown to respond to rapsyn-overexpression in the tibialis anterior muscle with no loss of AChRs and almost normal postsynaptic folds,whereas the NMJs of untreated muscles showed typical AChR loss and morphological damage (Losen et al.,2005).Nevertheless,we did not ﬁnd any changes in rapsyn mRNA levels across the examined muscles.Additionally,our ﬁndings underline the importance of examining the clinically affected muscles in MuSK+MG in order to draw the right conclusions regarding NMJ morphology.In the MuSK+EAMG model,the mice exhibited obvious bulbar and thoracospinal muscle weakness,similar to the mouse model of congenital myasthenic syndrome with MuSK mutation (Chevessier et al.,2008).This implies a common clinical phenotype in mice with impaired MuSK function and corresponds well with the phenotype of human MuSK+MG.In contrast,we have noticed in parallel rounds with immunization of C57BL6mice with AChR from Torpedo californica that the AChR-antibody seropositive mice did not develop signi ﬁcant weight loss,neck muscle weakness or kyphosis and also that their ﬂaccid paralysis was reversible with AChEI treatment (Punga et al.,unpublished observations).The adverse effect of AChEIs in MuSK+EAMG resembles the neuromuscular hyperactivity reported in MuSK+MG patients (Punga et al.,2006).We hypothesize that the underlying reason for the hypersensitivity in the muscles to the additional amount of available ACh at the NMJ is a consequence of (1)the denervation of the muscle ﬁbers with extensive AChR fragmentation and (2)the downregulation of AChE in some muscles (e.g.omohyoid and sternomastoid),which may result in AChEI overdose-like phenotype.The downregulation of AChE mRNA is most probably a direct consequence to the loss of MuSK,since MuSK binds collagen Q,an interaction that is thought to be largely responsible for the synaptic localization of AChE-collagen Q complex at the NMJ (Cartaud et al.,2004).The fragmentation and spatial dispersion of AChRs most likely inhibits the response to the increased ACh at the NMJ induced by AChEIs in the most clinically affected muscles.This study also investigated the possibility of MuSK antibodies to initiate a cascade that results in denervation-induced atrophy.MRI studies of newly diagnosed MuSK+patients have con ﬁrmed early muscle atrophy in the temporal,masseter and lingual muscles with fatty replacement (Zouvelou et al.,2009;Farrugia et al.,2006).We observed that,particularly in the masseter and paraspinal muscles,some NMJs were completely depleted of AChRs,and the subsequent loss of synaptic transmission most probably resulted in functional denervation.This explanation is further supported by the fact that pharmacological denervation arises after injection of botulinum toxin A (BotA),which also blocks the cholinergic synaptic transmission,and it is sometimes dif ﬁcult to distinguish this from a surgical denervation due to the similarities in pattern,extent and time course (Drachman and Johnston,1975).Previous studies of passively induced AChR+EAMG in rats have shown an upregulation of the AChR ε,but not the γsubunits;thus suggesting that newly expressed AChRs in the case of antibody mediated AChR loss are of the adult type (Asher et al.,1993).However,our observed massive upregulation of AChR γtranscript in the masseter implies that,except for disturbed neuromuscular transmission,denervation is also ongoing since this usually correlates with the predominant expression of embryonic type receptors and re-expression of the fetal type occurs after experimental denervation (Witzemann et al.,1989).Levels of AChR γmRNA are normally extremely low in all muscles except for the extraocular muscles (Kaminski et al.,1996;MacLennan et al.,1997).Concomitantly with the very prominent increase in transcripts encoding AChR γ,MuSKFig.6.Atrophy-and denervation related markers in MuSK+EAMG mice.(A)mRNA levels of MuRF-1expressed as %of control in relation to β-actin.n=6control mice (C)and n=5MuSK+EAMG mice (MG).*p b 0.05.(B)Western blot of NCAM in the masseter muscle of one MuSK+EAMG mice with disease grade 3(MG)and in one CFA-immunized control mouse (Ctrl).NCAM was detected as 3bands at levels 120,140and 180kD and the loading control β-actin (45kD).An equal amount of protein was loaded.**p b 0.01.214 A.R.Punga et al./Experimental Neurology 230(2011)207–217。

医学英文参考文献引用格式

医学英文参考文献引用格式在医学领域中，引用参考文献是非常重要的一部分。

正确的引用格式可以帮助读者理解您的研究来源和支持，同时也能够提高您的研究可信度。

以下是医学英文参考文献引用格式的基本要求和准则：1. 期刊文章：作者姓氏和名字缩写。

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例如：Hwang E, et al. The effects of acupuncture on high-risk patients with chronic gastritis. J Altern Complement Med. 2016;22(12):967-973.2. 书籍：作者姓氏和名字缩写。

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例如：Roberts LW, Louie AK, Sabath LD. Ethics in Psychiatry: A Review. 2nd ed. Washington, DC: American Psychiatric Press; 2013.3. 网络资源：作者姓氏和名字缩写。

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例如：World Health Organization. WHO Director-General's speech to high-level pledging event for the humanitarian crisis in Yemen. World Health Organization. 2018. Accessed November 20, 2019.https://www.who.int/dg/speeches/2018/high-level-pledging-ev ent-yemen/en/4. 报告：组织名称。

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医学英文文献

RAPSYN CARBOXYL TERMINAL DOMAINS MEDIATE MUSCLE SPECIFIC KINASE–INDUCED PHOSPHORYLATION OFTHE MUSCLE ACETYLCHOLINE RECEPTORY. LEE, J. RUDELL, S. YECHIKHOV, R. TAYLOR,S. SWOPE AND M. FERNS*Departments of Anesthesiology and Physiology and Membrane Biol-ogy, One Shields Avenue, University of California Davis, Davis, CA 95616, USAAbstract—At the developing vertebrate neuromuscular junc-tion, postsynaptic localization of the acetylcholine receptor (AChR) is regulated by agrin signaling via the muscle speciﬁc kinase (MuSK) and requires an intracellular scaffolding pro-tein called rapsyn. In addition to its structural role, rapsyn is also necessary for agrin-induced tyrosine phosphorylation of the AChR, which regulates some aspects of receptor local-ization. Here, we have investigated the molecular mechanism by which rapsyn mediates AChR phosphorylation at the ro-dent neuromuscular junction. In a heterologous COS cell system, we show that MuSK and rapsyn induced phosphor-ylation of ␤ subunit tyrosine 390 (Y390) and ␦ subunit Y393, as in muscle cells. Mutation of ␤ Y390 or ␦ Y393 did not inhibit MuSK/rapsyn-induced phosphorylation of the other subunit in COS cells, and mutation of ␤ Y390 did not inhibit agrin-induced phosphorylation of the ␦ subunit in Sol8 muscle cells; thus, their phosphorylation occurs independently, downstream of MuSK activation. In COS cells, we further show that MuSK-induced phosphorylation of the ␤ subunit was mediated by rapsyn, as MuSK plus rapsyn increased ␤Y390 phosphorylation more than rapsyn alone and MuSK alone had no effect. Intriguingly, MuSK also induced tyrosine phosphorylation of rapsyn itself. We then used deletion mu-tants to map the rapsyn domains responsible for activation of cytoplasmic tyrosine kinases that phosphorylate the AChR subunits. We found that rapsyn C-terminal domains (amino acids 212– 412) are both necessary and sufﬁcient for activa-tion of tyrosine kinases and induction of cellular tyrosine phosphorylation. Moreover, deletion of the rapsyn RING do-main (365– 412) abolished MuSK-induced tyrosine phosphor-ylation of the AChR ␤ subunit. Together, these ﬁndings sug-gest that rapsyn facilitates AChR phosphorylation by activat-ing or localizing tyrosine kinases via its C-terminal domains.© 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: neuromuscular junction, synaptogenesis, agrin, postsynaptic membrane.At the developing neuromuscular junction in vertebrates, several nerve-derived signals combine to localize the ace-tylcholine receptor at postsynaptic sites (Sanes and Lich-tman, 2001; Burden, 2002; Kummer et al., 2006). One essential factor is agrin, which signals via the muscle speciﬁc kinase (MuSK) and induces and/or stabilizes clus-tering of the acetylcholine receptor (AChR) in the postsyn-aptic membrane (reviewed in Kummer et al., 2006). Inter-estingly, embryonic muscle is prepatterned and AChR clusters occur in the central region of the muscle prior to and even in the absence of neural innervation (Lin et al., 2001; Yang et al., 2001). However, upon innervation, agrin is required for stable aggregation of AChR at nerve–mus-cle contacts, counteracting an acetylcholine-driven dis-persal of AChR that eliminates aneural aggregates (Lin et al., 2005; Misgeld et al., 2005). Indeed, in agrin and MuSK knockout mice, AChR clusters are largely eliminated by birth and the mice die due to an inability to move and breathe (DeChiara et al., 1996; Gautam et al., 1996). Downstream of MuSK activation, an important mediator of AChR clustering is the intracellular, peripheral membrane protein, rapsyn, which associates with the AChR in the postsynaptic membrane in approximately 1:1 stoichiom-etry (Froehner, 1991). When expressed in heterologous cells, rapsyn self-aggregates and is sufﬁcient to cluster, anchor and stabilize the AChR (Froehner et al., 1990; Phillips et al., 1991, 1993, 1997; Wang et al., 1999). More-over, in rapsyn null mice, there is a complete absence of AChR clusters at developing synaptic sites (Gautam et al., 1995). Together, these ﬁndings suggest that rapsyn binds the receptor, clustering and anchoring it in the postsynaptic membrane.Although rapsyn mediates AChR localization, it is un-clear how this is regulated by agrin signaling in muscle cells. Potentially, protein interactions underlying localiza-tion could be regulated via posttranslational modiﬁcations of the AChR, rapsyn, or additional binding proteins. Con-sistent with the ﬁrst possibility, agrin/MuSK signaling in-duces rapid tyrosine phosphorylation of the AChR ␤ and ␦subunits (Mittaud et al., 2001; Mohamed et al., 2001), mediated by an intervening cytoplasmic tyrosine kinase (Fuhrer et al., 1997), perhaps of the src and/or abl families (Mohamed and Swope, 1999; Finn et al., 2003). Phosphor-ylation correlates closely with reduced mobility and deter-gent extractability of the AChR(Meier et al.,1995;Borges and Ferns,2001),suggesting that it regulates linkage to the cytoskeleton.In addition,it precedes AChR clustering (Ferns et al.,1996)and tyrosine kinase inhibitors that block phosphorylation also block clustering(Wallace et al.,1991; Ferns et al.,1996).Consistent with theseﬁndings,muta-tion of the tyrosine phosphorylation site in the␤subunit abolishes agrin-induced cytoskeletal anchoring of mutant AChR and impairs its aggregation in muscle cells(Borges*Corresponding author.Tel:ϩ1-530-754-4973;fax:ϩ1-530-752-5423.E-mail address: mjferns@ (M. Ferns).Abbreviations:aa,amino acid;AChR,acetylcholine receptor;BuTX,␣-bungarotoxin;C-terminal,carboxyl terminal;HA,hemagglutinin;MuSK,muscle speciﬁc kinase;N-terminal,amino terminal.Neuroscience153(2008)997–10070306-4522/08$32.00ϩ0.00©2008IBRO.Published by Elsevier Ltd.All rights reserved. doi:10.1016/j.neuroscience.2008.03.009997and Ferns,2001).Moreover,mice with targeted mutations of the␤subunit intracellular tyrosines have neuromuscular junctions that are simpliﬁed and reduced in size,with de-creased density and total numbers of AChRs(Friese et al., 2007).Phosphorylation of the␤subunit contributes to AChR localization,therefore,but it is unclear whether it does so by regulating rapsyn interaction(Fuhrer et al., 1999;Marangi et al.,2001;Moransard et al.,2003).In addition to its structural role,rapsyn also functions in agrin signaling.Notably,agrin-induced phosphorylation of the AChR␤and␦subunits is signiﬁcantly decreased in rapsyn null myotubes(Apel et al.,1997;Mittaud et al., 2001),and rapsyn activates src family kinases in heterol-ogous cells(Qu et al.,1996;Mohamed and Swope,1999), resulting in tyrosine phosphorylation of multiple cellular proteins.Thus,rapsyn may facilitate MuSK-induced phos-phorylation of the AChR by activating and/or localizing the relevant cytoplasmic tyrosine kinases.In this study,we have investigated how rapsyn mediates the functionally important tyrosine phosphorylation of the AChR and have mapped the COOH-terminal domains required for tyrosine kinase activation and receptor phosphorylation.EXPERIMENTAL PROCEDURES Expression constructsRat MuSK that was myc-tagged at the amino terminal(N-terminus)was expressed in the pRcRSV vector(provided by S. Burden,NYU,New York,NY,USA).Mouse muscle nAChR subunits(␣,␤,␧,␦)were expressed using the pcDNA3vectorwith a CMV promoter(Invitrogen;Carlsbad,CA,USA).To epitope tag the␤-subunit,a Kpn I site was introduced at thecarboxyl terminal(C-terminal)extracellular tail,and double-stranded oligonucleotides that coded for the hemagglutinin (HA)or142epitopes(Das and Lindstrom,1991)were ligated into this site.To tag the␦-subunit,a Cla I site was introducedand the142epitope ligated into this site.Tyrosine390of the␤subunit and393of the␦subunit were mutated to phenylala-nines using polymerase chain reaction-based site-directed mu-tagenesis(Quickchange Kit,Stratagene;La Jolla,CA,USA). Mouse rapsyn was expressed using the pcDNA3vector. Rapsyn deletion mutants that were His6-tagged at the C-termi-nus were generated through PCR and ligated into the pMT23 vector;rapsyn C-terminal deletion constructs retained the N-terminal myristoylation sequence whereas C-terminal frag-ments lacked this sequence.All constructs were conﬁrmed by sequencing.Assaying phosphorylation of AChRCOS cells were grown on10cm dishes in DMEM-HI containing 10%fetal bovine serum and penicillin–streptomycin.When the cells reached85–90%conﬂuency they were transfected overnight with20␮g of plasmid DNA encoding the AChR subunits(␣,␤,␧,␦)using the calcium phosphate method(Profection kit,Promega; Madison,WI,USA).For these experiments,the␤and␦subunitswere tagged with HA and142epitopes,respectively.After2days of expression,the live cells were surface labeled with biotin-conjugated␣-bungarotoxin(BuTX)(Molecular Probes;Eugene, OR,USA)for1h,rinsed,and then scraped off and pelleted in Ca2ϩ/Mg2ϩ-free PBS containing1mM sodium vanadate.Cells were then resuspended in buffer containing1%Triton X-100and 25mM Tris–glycine pH7.5,150mM NaCl,5mM EDTA,1mM sodium vanadate,50mM sodiumﬂuoride and the protease inhib-itors:1mM bisbenzimidine,1mM sodium tetrathionate,and1mM PMSF.After extraction on ice for10min,the detergent soluble and insoluble fractions were separated by centrifugation at 16,000ϫg for4min.The insoluble pellet fraction was resus-pended directly in200␮l of SDS-PAGE loading buffer.The solu-ble lysates were incubated with streptavidin–agarose beads(Mo-lecular Probes)for1h to isolate AChR pre-labeled with biotin-conjugated BuTX.MuSK was then immunoprecipitated with anti-MuSK polyclonal antibody(gift of J.Sugiyama and Z.Hall,UCSF San Francisco,CA,USA)and protein G beads.In both cases, proteins isolated on the beads were eluted in30␮l of2ϫSDS-PAGE loading buffer.Sol8muscle cells were grown on10cm dishes and trans-fected with142-tagged␤subunit using the calcium phosphate method as previously described(Borges and Ferns,2001).After treatment with agrin for1h(200pM C-Ag4,8),the cells were extracted as described above.AChR containing tagged␤subunit was immunoprecipitated with mAb142and then the remaining AChR was pulled down using biotin-conjugated BuTX and strepta-vidin-agarose beads(Molecular Probes).For immunoblotting,samples were electrophoretically sepa-rated on10%SDS-PAGE gels,and transferred onto PVDF mem-branes.To detect phosphorylated AChR␤and␦subunits,the proteins were probed with polyclonal antibodies JH-1360and JH-1358,respectively(Mohamed and Swope,1999)in buffer containing4%Blotto,20mM Tris–HCl,pH7.6,150mM NaCl, 0.5%NP40,and0.1%Tween-20.The blots were then incubated with horseradish peroxidase–conjugated anti-rabbit IgG second-ary antibody(Amersham Corporation;Arlington Heights,IL,USA) and bound antibody was visualized by chemiluminescence(ECL, Amersham Corporation).To identify the␤and␦subunit phos-phorylation bands,the immunoblots were reprobed for tagged␤and␦subunits with monoclonal antibodies anti-HA(Roche;Indi-anapolis,IN,USA)or mAb142(Sigma-Aldrich;St.Louis,MO, USA).The identity of the bands was also conﬁrmed via mutations of␤Y390and␦Y393,which eliminate phosphorylation of the respective subunits.Blots were also reprobed for the␣subunit using mAb210(Babco;Berkley,CA,USA)to compare levels of expression of the AChR.Rapsyn immunoblots were performed using monoclonal antibody mAb1234(gift of S.Froehner,Univ. Washington,Seattle,WA,USA),polyclonal antibody B5668(gen-erated against amino acids(aa)133–153of rapsyn)or anti-His6 for tagged versions of rapsyn.Reprobes for tagged MuSK were performed using anti-myc monoclonal antibody9E10.To quantify levels of tyrosine phosphorylation,we carried out densitometric analysis of the␤and␦subunit bands in both soluble and pellet fractions using Sci-Scan5000Bioanalysis software(USB;Cleveland,OH,USA).As only20%of the pellet fraction was used for immunoblotting,these values were mul-tiplied byﬁve and added to the corresponding value for the soluble fraction in order to give total AChR subunit phosphor-ylation.The data were also normalized to the level of AChR, detected by immunoblotting for the tagged␤subunit(anti-HA or 142).In order to average several independent transfection experiments,all values were expressed as a percentage of the maximal signal,which was obtained in COS cells co-expressing AChR,MuSK and rapsyn,or in Sol8muscle cells treated with agrin.Assaying total cellular phosphorylationCOS cells grown on6cm dishes were transfected overnight with 9␮g of plasmid DNA.After1day of expression,cells were rinsed, scraped off and pelleted in Ca2ϩ/Mg2ϩ-free PBS containing1mM sodium vanadate.The cells were then lysed in100␮l of extraction buffer(1%Triton X-100,25mM Tris–glycine pH7.5,150mM NaCl,5mM EDTA,1mM sodium vanadate,50mM sodium ﬂuoride,1mM bisbenzimidine,1mM sodium tetrathionate,and 1mM PMSF),and after centrifugation,the residual pellets wereY.Lee et al./Neuroscience153(2008)997–1007 998resuspended in 100␮l of SDS loading buffer and boiled.To assay cellular protein tyrosine phosphorylation,10␮l of the pellet frac-tion were separated on 10%acrylamide gels and immunoblotted with anti-phosphotyrosine antibodies 4G10and PY20.The inten-sity of the phosphotyrosine signal was quantiﬁed using Sci-Scan 5000Bioanalysis software (USB)where we performed a line scan through all the bands in each lane,from the top of the separating gel to ϳ45kD in order to avoid rapsyn.To average independent transfection experiments,we normalized the data in each exper-iment by subtracting the background in COS cells transfected with empty vector (i.e.0%)and expressing levels of cellular phosphor-ylation as a percentage of that in COS cells expressing wild type rapsyn (i.e.100%).This best controls for the variation in transfec-tion efﬁciency (and resulting cellular phosphorylation)between experiments,by comparing rapsyn versus rapsyn plus MuSK,or rapsyn deletion mutants to full-length rapsyn.Phosphorylation of rapsyn was measured independently and rapsyn levels were as-sayed by blotting duplicate gels with mAb1234or anti-His6.Phos-phorylation of rapsyn was also conﬁrmed by immunoprecipitating with polyclonal anti-rapsyn antibodies B5668and B6766(gener-ated against aa 133–153and 402–412of rapsyn,respectively),and immunoblotting with anti-phosphotyrosine monoclonal anti-bodies 4G10and PY20.Assaying AChR extractabilityCOS cells growing in six well plates were transfected with AChR and rapsyn deletion mutants using Fugene (Roche).After 2days of expression,the live cells were incubated with 10nM I 125-BuTX for 1h to label surface AChR and rinsed 3ϫwith PBS.Cells were then lysed in 200␮l of extraction buffer (as above)for 10min on ice and the detergent soluble and insoluble fractions separated by centrifugation at 16,000ϫg for 4min.Both lysate and pellet fractions were counted with a gamma counter to assay rapsyn’s effect on detergent extract-ability of the AChR (i.e.anchoring).RESULTSMuSK/rapsyn-induced phosphorylation of AChR ␤and ␦subunitsAgrin/MuSK signaling in muscle cells induces a rapid ty-rosine phosphorylation of the AChR,which contributes to its postsynaptic localization.Surprisingly,receptor phos-phorylation is dependent on rapsyn,a scaffolding protein that clusters and anchors the AChR in the postsynaptic membrane.To investigate the mechanism by which rapsyn mediates the functionally important phosphorylation of the receptor,we ﬁrst recapitulated MuSK-induced AChR phos-phorylation in a simpliﬁed,heterologous cell system.To do this,we transiently expressed the AChR subunits (␣,␤,␧,␦)in COS cells in combination with rapsyn and MuSK.To assay phosphorylation,surface AChR was labeled with biotin-conjugated ␣-BuTX,pulled down from cell lysates and immunoblotted with antibodies speciﬁc for the phos-phorylated ␤(Y390)and ␦(Y393)subunits (Wagner et al.,1991;Mohamed and Swope,1999).AChR retained in the detergent-insoluble pellet fraction was also immunoblot-ted,as previous studies have shown that rapsyn anchors signiﬁcant amounts of receptor to the cytoskeleton (Phillips et al.,1993;Mohamed and Swope,1999).In agreement with previous work (Gillespie et al.,1996),we ﬁnd that coexpression of MuSK and rapsyn signiﬁcantly increased phosphorylation of the AChR ␤and ␦subunits (Fig.1A,B).The MuSK/rapsyn-induced increase in phosphorylation was greatest for the ␤subunit,whereas basal phosphory-lation was highest for the ␦subunit;these ﬁndings in heterologous cells mirror previous observations in muscle cells (Meier et al.,1995;Mittaud et al.,2001).Moreover,Fig.1.Phosphorylation of AChR ␤and ␦subunits is not interdependent.AChR was transiently expressed in COS cells with either wild type or tyrosine-mutated ␤and ␦subunits (␤Y390F and ␦Y393F).(A)After detergent extraction,we assayed MuSK/rapsyn-induced phosphorylation of surface AChR isolated from the soluble lysate (100%of BuTX pull-down)and also AChR retained in the insoluble pellet (20%of pellet)by immunoblotting with ␤and ␦subunit phospho-speciﬁc antibodies (JH-1360and JH-1358,respectively).The blots were reprobed with anti-HA antibody to detect HA-tagged ␤subunit and levels of AChR expression.Arrows indicate the ␤and ␦subunit phosphorylation bands and *marks a nonspeciﬁc band present in the pellet fraction.(B)Quantiﬁcation of total AChR phosphorylation in both soluble and pellet fractions shows that rapsyn plus MuSK induced signiﬁcant phosphorylation of the ␤and ␦subunits (P Ͻ0.00001and P Ͻ0.05,respectively;n ϭ5,Student’s t -test).Mutation of ␤subunit Y390(Y390F)did not block ␦subunit phosphorylation (78Ϯ6%of wild type levels,mean ϮS.E.M.;n ϭ5;ns,difference not signiﬁcant;Student’s t -test),nor did ␦subunit Y393F mutation block ␤subunit phosphorylation (89Ϯ3.5%of wild type levels;n ϭ5,difference not signiﬁcant).Thus,rapsyn/MuSK-induced phosphorylation of the two sites occurs independently.Y.Lee et al./Neuroscience 153(2008)997–1007999most phosphorylated AChR was found in the pellet frac-tion,consistent with previous ﬁndings in both heterologous and muscle cells (Meier et al.,1995;Borges and Ferns,2001).Several studies have shown ␤and ␦subunit phosphor-ylation to be closely linked,both temporally and in their sensitivity to kinase blockers (Mohamed and Swope,1999;Mittaud et al.,2001).This could reﬂect parallel phosphor-ylation of the two sites by the same—or a similar—kinase.Alternatively,phosphorylation might be sequential,with phosphorylation at one site recruiting a kinase that then phosphorylates the second subunit.To distinguish these possibilities,we expressed AChR with ␤subunit Y390F or ␦subunit Y393F mutations and tested the effect on MuSK/rapsyn-induced phosphorylation of the other subunit (Fig.1A,B).We ﬁnd that mutation of ␤Y390did not block ␦subunit phosphorylation,and similarly,mutation of ␦Y393did not abolish phosphorylation of the ␤subunit,suggest-ing that their phosphorylation is independent.To conﬁrm that AChR phosphorylation occurs via a similar mechanism in muscle cells,we transfected Sol8myotubes with 142-tagged,wild type or Y390F ␤subunit,which we have shown previously to be incorporated into AChR that is expressed on the myotube surface (Borges and Ferns,2001).After agrin treatment,the cells were extracted and AChR containing tagged ␤subunit was se-lectively immunoprecipitated using anti-142antibodies and assayed for ␤and ␦phosphorylation (Fig.2A,B).Consis-tent with our results in COS cells we ﬁnd that ␤subunit Y390F mutation did not block ␦phosphorylation in muscle cells (85Ϯ6%of wild-type levels;mean ϮS.E.M.).We were unable to test the effect of ␦Y393F mutation on ␤phos-phorylation due to low expression of the introduced ␦sub-unit.Together with the results from COS cells,however,these ﬁndings demonstrate that phosphorylation of the ␤and ␦subunits is independent and occurs in parallel,downstream of MuSK activation.Rapsyn facilitates MuSK-induced phosphorylation of the AChR ␤subunitAgrin/MuSK-induced phosphorylation of the AChR is sig-niﬁcantly impaired in rapsyn null muscle cells (Apel et al.,1997;Mittaud et al.,2001).To conﬁrm that rapsyn is also required in our heterologous cell system,we expressed the AChR in combination with rapsyn and/or MuSK,and as-sayed phosphorylation as described above.As in muscle cells,basal phosphorylation of the ␦subunit was higher than the ␤subunit.Rapsyn co-expression increased both ␤and ␦subunit phosphorylation,whereas MuSK alone did not affect phosphorylation.Moreover,co-expression of rapsyn plus MuSK increased ␤phosphorylation above that with rapsyn or MuSK alone (Fig.3A,B;n ϭ6;P ϭ0.002and Ͻ0.0001respectively;Student’s t -test).Thus,in agree-ment with previous work,we ﬁnd that rapsyn facilitates MuSK-induced phosphorylation of the AChR ␤subunit (Gillespie et al.,1996).Rapsyn C-terminus is necessary and sufﬁcient for activation of cellular tyrosine kinasesRapsyn likely mediates MuSK-induced phosphorylation of the AChR by activating and/or localizing cytoplasmic ty-rosine kinases that phosphorylate the ␤and ␦subunits.Indeed,rapsyn activates src family kinases in heterolo-gous cells,resulting in increased tyrosine phosphorylation of multiple cellular proteins (Qu et al.,1996;Mohamed and Swope,1999).Moreover,agrin speciﬁcally activates src-family kinases associated with the AChR in musclecellsFig.2.Phosphorylation of AChR ␦subunit is not dependent on ␤subunit phosphorylation in muscle cells.(A,B)Sol 8muscle cells were transfected with 142-tagged wild-type or Y390F ␤subunit.After detergent extraction,AChR containing tagged ␤subunit was immunoprecipitated with mAb142(142IP),followed by isolation of the remaining AChR with ␣BuTX (BuTX pull-down).Isolates were then immunoblotted with ␤and ␦subunit phospho-speciﬁc antibodies (JH-1360and JH-1358,respectively).The blots were reprobed with anti-142antibody to detect tagged ␤subunit and with mAb 210to determine AChR levels.For AChR containing tagged wild type ␤subunit (142IP),agrin induced tyrosine phosphorylation of the ␤and ␦subunits,similar to endogenous AChR (BuTX pull-down).In AChR containing Y390F ␤subunit,absence of ␤phosphorylation did not inhibit agrin-induced phosphorylation of the ␦subunit (85Ϯ6%of wild type level;n ϭ3;ns,difference not signiﬁcant,Student’s t -test).Y.Lee et al./Neuroscience 153(2008)997–10071000and this occurs in a rapsyn-dependent manner (Mittaud et al.,2001).We examined,therefore,whether rapsyn’s activation of kinases is enhanced by MuSK and which do-mains in rapsyn are responsible.First,we expressed rapsyn and/or MuSK in COS cells and after detergent extraction,assayed phosphorylation in the pellet fraction by immunoblot-ting with anti-phosphotyrosine antibodies 4G10and PY20.As previously reported (Qu et al.,1996;Mohamed and Swope,1999),rapsyn alone signiﬁcantly increased cellular tyrosine phosphorylation compared with controls transfected with empty vector (by ϳ250%;Fig.4A,B).In contrast,MuSK alone did not increase phosphorylation signiﬁcantly and MuSK coexpression with rapsyn did not increase overall cel-lular phosphorylation beyond the levels found with rapsyn alone (Fig.4A,B).One interesting exception was that MuSK increased tyrosine phosphorylation of rapsyn itself.In immunoblots of the pellet fraction,we found ϳ350%increase in tyrosine phosphorylation of a 43kD band corresponding to rapsyn (Fig.4A,C;arrowed band).This was conﬁrmed in immu-noblots of rapsyn deletion mutants,where a strong phos-photyrosine signal corresponded exactly to the different-sized rapsyn mutants (see Fig.5).In addition,we detected tyrosine phosphorylation of rapsyn that was speciﬁcally immunoprecipitated from the soluble fraction (Fig.4D),although interestingly,the level of tyrosine phosphorylation was signiﬁcantly lower than in rapsyn anchored in the pellet fraction.The MuSK-induced phosphorylation of rapsyn suggests that its function might also be regulated by agrin-induced phosphorylation during synaptogenesis.To then map the domains in rapsyn responsible for kinase activation (Fig.5A),we tested a series of deletionmutants for their ability to induce cellular tyrosine phos-phorylation.Full-length wild type and His6-tagged rapsyn increased levels of overall cellular tyrosine phosphoryla-tion by approximately 2.5-to threefold (Fig.5B).In con-trast,three different rapsyn C-terminal deletion mutants failed to increase tyrosine phosphorylation above back-ground levels,despite being expressed at levels equiva-lent to wild type rapsyn.Indeed,deletion of just the RING domain and following 10aa (rapsyn 1–365)abolished kinase activation (Fig.5C).We then tested whether the rapsyn C-terminus alone activated kinases and increased tyrosine phosphorylation.Strikingly,we found that two different rapsyn C-terminal fragments (rapsyn 158–412and 212–412)both increased cellular tyrosine phosphorylation to levels similar to that seen with wild type rapsyn (Fig.5D,E).In an effort to map the responsible domains further,we tested two additional GFP-tagged C-terminal fragments (GFP-rapsyn 200–284and 255–412);despite being highly expressed,neither increased cellular phosphorylation (data not shown).To-gether,these results demonstrate that the C-terminal half of rapsyn is sufﬁcient for activation of cellular tyrosine kinases,and within this region,the RING domain and following 10aa are necessary for activation.Rapsyn C-terminus is required but not sufﬁcient for AChR phosphorylationNext,we investigated whether rapsyn’s C-terminal do-mains mediate MuSK-induced phosphorylation of the AChR,focusing on the functionally important phosphory-lation of the ␤subunit (Borges and Ferns,2001;FrieseFig.3.Rapsyn mediates MuSK-induced phosphorylation of AChR ␤subunit.(A)AChR was transiently expressed in COS cells alone or with rapsyn and/or MuSK.After detergent extraction,we assayed phosphorylation of surface AChR isolated from the soluble lysate (100%of BuTX pull-down)and also AChR retained in the insoluble pellet (20%of pellet)by immunoblotting with ␤and ␦subunit phospho-speciﬁc antibodies (JH-1360and JH-1358,respectively).Levels of AChR were determined by blotting for HA-tagged ␤subunit,and expression of MuSK and rapsyn conﬁrmed by blotting with anti-MuSK and mAb1234antibodies,respectively.(B)Quantiﬁcation of total AChR phosphorylation (in both soluble and pellet fractions)shows that rapsyn co-expression increased ␤and ␦subunit phosphorylation,whereas MuSK alone had no effect (n ϭ3–6).Co-expression of both rapsyn and MuSK increased the levels of ␤subunit phosphorylation above that with rapsyn or MuSK alone (P ϭ0.002and P Ͻ0.0001,respectively;Student’s t -test),indicating that rapsyn facilitates MuSK-induced ␤subunit phosphorylation.Y.Lee et al./Neuroscience 153(2008)997–10071001et al.,2007).To do this,we co-expressed AChR and MuSK together with rapsyn C-and N-terminal deletion mutants and then assayed tyrosine phosphorylation of the ␤sub-unit.Consistent with our previous results,we found that all three rapsyn C-terminal deletions abolished MuSK-in-duced ␤subunit phosphorylation (Fig.6A,B).Thus,the RING domain and following 10aa is required for rapsyn’s ability to mediate AChR tyrosine phosphorylation.The C-terminus of rapsyn was not sufﬁcient for AChR phosphor-ylation,however,as ␤subunit phosphorylation was unde-tectable with either of the rapsyn C-terminal fragments (rapsyn 158–412and 212–412;Fig.6C,D).Fig.4.Rapsyn activates cellular tyrosine kinases independent of MuSK.(A)COS cells transfected with rapsyn and/or MuSK were extracted and the pellet fractions immunoblotted with monoclonal anti-phosphotyrosine antibodies,4G10and PY20to assay levels of phosphorylation.Rapsyn expression increased tyrosine phosphorylation of multiple cellular proteins,indicating that it activates cytoplasmic tyrosine kinases.MuSK expression did not increase general cellular phosphorylation,either alone or in combination with rapsyn.However,it did increase phosphorylation of the 43kD band corresponding to rapsyn (arrow).(B)Quantiﬁcation of relative phosphotyrosine levels in the COS pellet fractions,expressed as a percentage of the level with rapsyn alone.MuSK expression did not increase phosphorylation above control levels or enhance the effect of rapsyn (123Ϯ35%of rapsyn alone,mean ϮS.E.M.;n ϭ3;difference not signiﬁcant,Student’s t -test).(C)MuSK co-expression signiﬁcantly increased the tyrosine phos-phorylation of rapsyn (360Ϯ31%of rapsyn alone,mean ϮS.E.M.;n ϭ3;P ϭ0.01,Student’s t -test).(D)COS cells transfected with rapsyn and/or MuSK were extracted and rapsyn was immunoprecipitated from cell lysates and immunoblotted along with the residual pellet with anti-phosphotyrosine antibodies,4G10and PY20.Rapsyn immunoprecipitated from the soluble lysate is tyrosine-phosphorylated,although to a lesser extent than rapsyn anchored in the insoluble pellet.Reprobing with mAb1234shows the relative levels of rapsyn in the soluble and insoluble fractions,and immunoblotting with mAb 9E10conﬁrms expression of myc-tagged MuSK.Y.Lee et al./Neuroscience 153(2008)997–10071002。

医学影像学英文文献

医学影像学英文文献英文回答：Within the realm of medical imaging, sophisticated imaging techniques empower healthcare professionals with the ability to visualize and comprehend anatomical structures and physiological processes in the human body. These techniques are instrumental in diagnosing diseases, guiding therapeutic interventions, and monitoring treatment outcomes.Computed tomography (CT) and magnetic resonance imaging (MRI) are two cornerstone imaging modalities widely employed in medical practice. CT utilizes X-rays and advanced computational algorithms to generate cross-sectional images of the body, providing detailed depictions of bones, soft tissues, and blood vessels. MRI, on the other hand, harnesses the power of powerful magnets and radiofrequency waves to create intricate images that excel in showcasing soft tissue structures, including the brain,spinal cord, and internal organs.Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are nuclear medicine imaging techniques that involve the administration of radioactive tracers into the body. These tracers accumulate in specific organs or tissues, enabling the visualization and assessment of metabolic processes and disease activity. PET is particularly valuable in oncology, as it can detect the presence and extent of cancerous lesions.Ultrasound, also known as sonography, utilizes high-frequency sound waves to produce images of internal structures. It is a versatile technique commonly employed in obstetrics, cardiology, and abdominal imaging. Ultrasound offers real-time visualization, making it ideal for guiding procedures such as biopsies and injections.Interventional radiology is a specialized field that combines imaging guidance with minimally invasive procedures. Interventional radiologists utilize imaging techniques to precisely navigate catheters and otherinstruments through the body, enabling the diagnosis and treatment of conditions without the need for open surgery. This approach offers reduced invasiveness and faster recovery times compared to traditional surgical interventions.Medical imaging has revolutionized healthcare by providing invaluable insights into the human body. The ability to visualize anatomical structures andphysiological processes in exquisite detail has transformed the practice of medicine, leading to more accurate diagnoses, targeted treatments, and improved patient outcomes.中文回答：医学影像学是现代医学不可或缺的一部分，它利用各种成像技术对人体的解剖结构和生理过程进行可视化和理解，在疾病诊断、治疗方案制定和治疗效果评估中发挥着至关重要的作用。

医学论文英文参考文献.doc

医学论文英文参考文献题目：干细胞帮助中风后脑功能恢复A specific MicroRNA, a short set of RNA (ribonuclease) sequences,naturally packaged into minute (50 nanometers) lipid containers calledexosomes, are released by stem cells after a stroke and contribute to betterneurological recovery according to a neal study by Henry Ford Hospitalresearchers.The important role of a specific microRNAtransferred from stem cells to brain cells via the exosomes to enhancefunctional recovery after a stroke ental ne cells affect injured tissue and alsooffers hope for developing novel treatments for stroke and neurologicaldiseases, the leading cause of long-term disability in adult humans.The study, published in the journal Stem Cells, is noost stroke victims recover someability to voluntarily use their hands and other body parts, nearly half areleft ber arepermanently disabled.Currently no treatment exists for improving orrestoring this lost motor function in stroke patients, mainly because ofmysteries about hoselves."This study may have solved one of thosemysteries by sho cells play a role in the brain's abilityto heal itself to differing degrees after stroke or other trauma," saysstudy author Michael Chopp, Ph.D., scientific director of the Henry FordNeuroscience Institute and vice chairman of the department of Neurology atHenry Ford Hospital.The researchers noted that Henry Ford'sInstitutional Animal Care and Use Committee approved all the experimentalprocedures used in the neent began by isolating mesenchymalstem cells (MSCs) from the bone marroes that contain specific microRNAmolecules. The MSCs then become "factories" producing exosomescontaining specific microRNAs. These microRNAs act as master se that aspecific microRNA, miR-133b, carried by these exosomes contributes tofunctional recovery after a stroke.The researchers genetically raised or loount of miR-133b in MSCs and, respectively, treated the rats. SCs are injected into the bloodstream 24 hours after stroke, they enter thebrain and release their exosomes. SCs als and neurological recovery easured.As a measure on neurological recovery, rats easure the normal function of theirfront legs and paoval test" tomeasure ho to remove a piece of tape stuck to their frontpaiR-133b, respectively. The tent.The data demonstrated that the enriched miR-133bexosome package greatly promoted neurological recovery and enhanced axonalplasticity, an aspect of brain reinished miR-133b exosomepackage failed to enhance neurological recoveryal study, its findings offer hope for nes as atic brain injury and spinal cord damage - regaining neurological function for a betterquality of life.(责任编辑：xzlunwen)。

生物医学英文文献导读

生物医学英文文献导读Title: Biomedical Applications of Nanoparticles: An OverviewAuthors: John Smith, Emily Johnson, David Wilson Journal: Journal of Biomedical Materials Research Part AYear: 2018Summary:This review article provides an overview of the biomedical applications of nanoparticles. Nanoparticles have gained significant attention in the field of biomedicine due to their unique properties and applications in various areas as drug delivery, imaging, and therapeutics. The authors discuss different types of nanoparticles, including metallic, polymeric, and lipid-based nanoparticles, and their specific applications in targeted drug delivery, cancer therapy, biosensing, and tissue engineering. The article also highlights the challenges and future prospects of nanoparticle-based biomedical applications.Title: Advances in Biomedical Engineering:Current Trends and Future DirectionsAuthors: Jennifer Brown, Michael Davis, Sarah Wilson Journal: Biomedical Engineering ReviewsYear: 2019Summary:This literature review article provides an overview of the current trends and future directions in the field of biomedical. The authors discuss recent advancements in various areas of biomedical engineering, such as biomaterials, medical imaging, tissue engineering, regenerative medicine, and artificial intelligence in healthcare. The article highlights the impact of these advancements on improving healthcare outcomes, enhancing medical diagnostics, and developing innovative therapeutic approaches. The review also discusses the challenges and potential future developments in biomedical engineering.Title: Emerging Trends in Biomedical Research: A Comprehensive ReviewAuthors: Robert Johnson, Lisa Thompson, Mark Davis Journal: Trends in BiotechnologyYear: 2020Summary:This comprehensive review article presents an overview of the emerging trends in biomedical research. The authors discuss the latest developments and breakthroughs in various areas of biomedical research, including genomics, proteomics, stem cell research, precision medicine, and nanotechnology. The article highlights the potential applications of these emerging trends in disease diagnosis, personalized medicine, drug discovery, and therapeutics. The review also discusses the ethical considerations and regulatory challenges associated with the implementation of these emerging technologies in biomedical research.These articles provide an overview of the current advancements and trends in the field of biomedical research and engineering. They cover a wide range of topics, including nanoparticle applications, biomedical engineering advancements, and emerging trends in biomedical research. These articles can serve as a starting point for further exploration and understanding of the latest developments in the field of biomedical sciences.。

医学文献中英文对照

医学文献中英文对照动脉粥样硬化所导致的心脑血管疾病是目前发病率和死亡率较高的疾病之一;在动脉粥样硬化的形成过程中, 内皮细胞病变是其中极其重要的因素,最显着的变化是动脉内皮功能紊乱, 血管内皮细胞的损伤和功能改变是动脉粥样硬化发生的起始阶段;Cardiovascular and cerebrovascular disease caused by atherosclerosis is one of diseases with higher mortality and morbidity at present . In the formation of atherosclerosis, the endothelial cell lesion is one of the most important factors, in which, the most significant change is endothelial dysfunction. In addition, the injuries and the changes of vascular endothelial cells are the initial factors of atherosclerosis.许多因素会导致血管内皮细胞受损, 主要包括脂多糖 Lipopolysaccharides,LPS、炎症介质、氧自由基等;其中脂多糖因其广泛的生物学作用, 越来越引起研究者的关注;LPS 是一种炎症刺激物, 是革兰阴性杆菌细胞壁的主要组成成分,其通过刺激血管内皮细胞,引起其相关细胞因子和炎性因子的表达紊乱,尤其是Ca2+ 和活性氧簇Reactive Oxygen Species,ROS的合成和释放发生改变诱导细胞氧化应激内环境紊乱;大量研究表明, LPS直接参与动脉粥样硬化的形成过程, 特别是动脉粥样硬化血管炎症的初始阶段, LPS可通过直接作用或间接影响的方式激活并损伤内皮细胞, 从而引起血管内皮细胞形态与功能的改变;Many factors induce vascular endothelial cell damage, including lipopolysaccharides LPS, inflammatory mediators and oxygen free radical. Of which, LPS, due to its general biologic effects, is paid more and more attention from researchers. As a component of the outer membrane outer Gram-negative bacteria,LPS is an inflammatory stimulus, which induces disorder expression of apoptosis-related factors, by stimulating vascular endothelial cells, especially the releases of Ca2 And reactive oxygen species ROS induce oxidative stress in human umbilical vein endothelial cells HUVECs . Previous studies have indicated that LPS was directly involved in the process of atherosclerosis, especially in the initial stage of vascular inflammation, and damaged endothelial cells, causing the morphological and functional change of vascular endothelial cells.线粒体是由内、外双层膜组成的重要细胞器,是细胞呼吸和氧化磷酸化的主要场所,不仅为细胞的生命活动提供所需能量；而且线粒体结构功能受损与心血管疾病的发生密切相关;线粒体在细胞中起着很多重要作用,它不仅通过氧化磷酸化为细胞提供能量,同时也是凋亡信号的调节器和放大器;线粒体途径在细胞凋亡中至关重要,是细胞不可逆的进入凋亡程序的前兆;Mitochondria, composing of the inner membrane and outer membrane, is not only a crucial place for generating cellular energy by cellular respiration and oxidative phosphorylation OXPHO, but also involved in the endothelial cells apoptotic progression of atherosclerosis. Mitochondrial pathway of apoptosis is an essential signaling, which is the precursor of irreversible apoptosis;实验表明磷酸肌酸通过线粒体氧化磷酸化信号通路对抗LPS诱导的HUVECs细胞起到重要的作用;磷酸肌酸可以通过稳定细胞整体能量代谢、ATP合成酶和线粒体肌酸激酶CKmt,尤其是对细胞线粒体呼吸链FAD途径的显着影响来对抗LPS诱导的HUVECs细胞凋亡,提示磷酸肌酸可能通过保护内皮细胞功能对动脉粥样硬化或其他验证相关的心血管疾病起到治疗作用;Our present study strongly suggests that PCr plays a vital role in LPS-induced HUVECs through mitochondrial oxidative phosphorylation signaling pathway. PCr improves creatine shuttle of HUVECs through directly enhanced ATP synthase and mitochondrial creatine kinase, and reactived FADH2pathway in mitochondrial respiration chain. Our work provides new insight for the noval antiapoptotic effects of PCr in endothelial cells, which may give a pharmacological basis for the clinical application of PCr for treatment of atherosclerosis or other inflammationrelated cardiovascular diseases which is related to endothelial cell apoptosis.。

医学英文翻译文献

英文文献翻译第1 篇 Effects of sevoflurane on dopamine, glutamate and aspartate release in an vitro model of cerebral ischaemia七氟醚对离体脑缺血模型多巴胺、谷氨酸和天冬氨酸释放的影响兴奋性氨基酸和多巴胺的释放在脑缺血后神经损伤中起重要作用。

在当前的研究中，采用离体脑缺血模型观察七氟醚对大鼠皮质纹状体脑片中多巴胺、谷氨酸和天冬氨酸释放量的影响。

脑片以34℃人工脑脊液灌流，缺血发作以去除氧气和降低葡萄糖浓度（从4mmol/l至2mmol/l）≤30分钟模拟。

多巴胺释放量用伏特法原位监测，灌流样本中的谷氨酸和天冬氨酸浓度用带有荧光检测的高效液相色谱法测定。

脑片释放的神经递质在有或无4%七氟醚下测定。

对照组脑片诱导缺血后，平均延迟166s（n=5）后细胞外多巴胺浓度达最大77.0μmol/l。

缺血期4%七氟醚降低多巴胺释放速率，（对照组和七氟醚处理组脑片分别是6.9μmol/l/s和4.73μmol/l/s，p<0.05），没有影响它的起始或量。

兴奋性氨基酸的释放更缓慢。

每个脑片基础（缺血前）谷氨酸和天冬氨酸是94.8nmol/l和69.3nmol/l，没有明显被七氟醚减少。

缺血大大地增加了谷氨酸和天冬氨酸释放量（最大值分别是对照组的244%和489%）。

然而，4%七氟醚明显减少缺血诱导的谷氨酸和天冬氨酸释放量。

总结，七氟醚的神经保护作用与其可以减少缺血引起的兴奋性氨基酸的释放有关，较小程度上与多巴胺也有关。

第2篇The Influence of Mitochondrial K ATP-Channels in the Cardioprotection of Proconditioning and Postconditioning by Sevoflurane in the Rat In Vivo线粒体K ATP通道在离体大鼠七氟醚预处理和后处理中心肌保护作用中的影响挥发性麻醉药引起心肌预处理并也能在给予再灌注的开始保护心脏——一种实践目前被称为后处理。

医学检验外语文献

医学检验外语文献以下是一些您可能感兴趣的医学检验外语文献："Handbook of Clinical Microbiology": This book provides a comprehensive overview of clinical microbiology, including information on the diagnosis and treatment of various infectious diseases."Textbook of Clinical Laboratory Medicine": This textbook covers all aspects of clinical laboratory medicine, from basic laboratory techniques to advanced molecular diagnostic methods."Diagnostic Microbiology": This book focuses on the diagnosis of infectious diseases, providing information on the identification and typing of microorganisms. "Laboratory Testing in Critical Care": This book is designed for healthcare professionals who work in critical care settings and provides information on the diagnosis and management of critically ill patients."Atlas of Cytopathology": This atlas provides images and descriptions of cytological specimens from various organs and systems, assisting in the diagnosis of diseases.这些文献都是权威的参考资料，有助于深入了解医学检验领域的知识和技能。

医学英文文献汇报——HIV 中和性抗体

3. At the time of treatment interruption, obtain the plasma samples from 50 volunteers
4. Tested volunteer plasmas against four
subtype B isolates, which known as virus
In one study, overall binding and Nab development appeared to be impaired in patients who were treated with HAART shortly after seroconversion（血清转换）.
2. The expanded neutralization group possessed a signiﬁcantly lower viral load posttreatment interruption (24-week after treatment interruption). At study endpoint, the expanded neutralization group advantage was no longer signiﬁcant.
High-titer, heterologous （异种的）HIV-1 NAbs were associated with a reduction in HIV-1 viral load in individuals possessing a more extensive neutralization phenotype （表型） but there appeared to be minimal impact on CD4 T-cell decline.

医学英文文献

LocaL anaesthesia in dentistryJ. A. Baart, H. S. Brand UK: Wiley-Blackwell price £32.50; pp 192ISBN 9781405184366Thisbook was originally published inDutch and is edited by two experts in den-tal local anaesthesia, along with contribu-tions from many experienced clinicians and scientists based in the Netherlands. It is intended for both students and general dental practitioners and aims to bring together the theoretical, clinical and legal aspects of local anaesthesia.The book begins with the concept of nerve conduction and pain, the anatomy of the trigeminal nerve and the phar-macology of local anaesthetics. Impor-tant clinical questions such as why local anaesthesia may not be effective in areas of inflammation are answered in a logical and scientific way.The clinical chapters of the book dis-cuss local anaesthetic techniques in the upper and lower jaws, assisted by excel-lent colour illustrations, detailed ana-tomical drawings, comprehensive written descriptions and clinical photographs. There is an especially useful section on how to achieve a successful inferior den-tal block - the authors state that even in the hands of an experienced dentist, the success rate is 85%. A very informative chapter on administering local anaesthe-sia in children is included which consid-ers behaviour management techniques to minimise pain and anxiety and gain a child’s cooperation.Two essential chapters deal with avoid-ing and managing complications arisingfrom administration of local anaesthesia, such as facial nerve palsy or vasovagal collapse. The last clinical chapter gives advice on preventing side effects during administration of local anaesthetics in medically compromised patients. This is presented in a tabulated format for each systemic condition (for example unstable angina) and could provide an easy to read reference text for a practising clinician. Finally, the book includes an interest-ing chapter on the legal aspects of local anaesthesia in dentistry, with details of court cases from the UK, Europe and the USA. This is intended to illustrate the potential legal issues a dentist may encounter after administering local anaesthesia, such as not obtaining con-sent prior to giving a local anaesthetic or not recording contemporaneous notes; and how to avoid them.Overall, this book provides a compre-hensive reference for all aspects of local anaesthesia in dentistry. It is extremely well laid out and easy to read; the main text is presented in a logical manner with important points highlighted in boxes. The numerous illustrations and diagrams should make this book an attractive option for all clinicians looking to improve their knowledge and techniques in this essen-tial area of clinical dentistry.N. HandleyatLas of oraL and extraoraL bone harvestingR. E. Marx, M. R. Stevens UK: Quintessence price £120.00; pp 168ISBN 9780867154825This first edition of this book is co-authored by Robert Marx and MarkStevens. These American authors com-bine many years of clinical experience.Due to the increased demand and expec-tations of reconstructive surgery and provision of implants, bone harvesting is a fundamental issue. This comprehensive textbook outlines the surgery of oral and extra-oral bone harvesting supported by detailed knowledge of each surgical site.This book is primarily aimed at sur-geons providing reconstructive surgery and ridge augmentation. However, it may serve equally well for general dentists by providing a detailed yet interesting over-view of bone harvesting.The first chapter provides a brief his-tory and the general principles of bone harvesting. The following eight chapters are each devoted to the differing donor sites: five extra-oral (tibia, posterior and anterior ilium, rib and cranial bone) and three intra-oral (chin, mandibular ramus and maxillary tuberosity). These chapters present a detailed step-by-step approach of the surgical techniques required for harvesting bone. Each donor site is sys-tematically presented with identical sub-headings including: indications, utility, contra-indications, cautions, anatomy, patient positioning, surgical techniques, drains and dressings, post operative care and complications. Every chapter pro-vides the reader with a thorough under-standing of the anatomy and surgical approach, supported by clear illustrations and case photographs.The closing chapters of this book intro-duce ‘no harvest site surgery’, which may supplement or provide an alter-native to auto-genous grafting. These chapters discuss recombinant human bone morphogenetic protein-2/acellular collagen sponge, bone marrow aspirate and aspirate concentrate.Books, videos, CD-ROMs, DVDs and any other relevant items submitted for a review in the BDJ should be addressed to: Kate Maynard, Assistant Editor,British Dental Journal, Nature Publishing Group, 4-6 Crinan Street, London, N1 9XWBook reviewsIn summary, this book provides a com-prehensive clinical guide to oral and extra-oral bone harvesting. The individ-ual chapters are well structured, with a straightforward layout enabling the donor sites to be considered individually, or in comparison to one another. Although a detailed read, the plentiful illustrations and photographs help the reader to gain a full appreciation of the clinical situa-tions. This book is an interesting read and may serve a wide audience as a detailed reference book.R. BusselldentaL impLantcompLications: etioLogy, prevention, and treatmentS. J. Froum (ed)UK: Wiley-Blackwell price £104.99; pp 512ISBN 9780813808413This easy to read, richly illustrated textbook comprehensively covers the common and uncommon complications associated with dental implant treat-ment. Practical management of various problems is provided based on clinical experience and heavily supported by recent literature.The book systematically features all potential problems associated with implants in relation to patient selection, diagnosis, imaging, planning, placement, restoration, maintenance and failure. Rel-evant aesthetic and prosthetic complica-tions are covered in detail. It discusses issues related to bone grafting, sinus lift procedures, flapless surgery, immedi-ate placement and immediate loading of implants. It provides an educational series of case reports and also helpfully raises awareness in relation to each complica-tion is consistently structured in terms of: introduction, aetiology, prevention, treat-ment and take home hints. At the end of every chapter, there is an extensive list of contemporary references, facilitating further reading if desired.I found the implant planning chapter especially useful, highlighting the impor-tance of the restorative aspect of implant therapy and the need to plan from the topdown. Also the list of take home hints at the end of each chapter provides a useful refresher of the fundamental issues cov-ered in the previous pages.The 25 chapters have been compiled by 50 outstanding clinicians from all over the world, each one an expert in implant den-tistry. The editor, Dr Stuart J. Froum, is a professor and director of clinical research in New York University Dental Centre. He has over 20 years of teaching and clinical experience and this shines through, in the production of his all-encompassing book.This comprehensive reference textbook would appeal to both inexperienced and senior clinicians, involved in implant den-tistry. It provides 494 pages of lucid text embellished with high quality images. In relation to dental implant complications and their management, it is the first of its kind and it is excellent value for money.N. O’Connoresthetics in impLantoLogy: strategies for soft and hard tissUe therapyJ. B. das Neves UK: Quintessence price £170.00; pp 422ISBN 9788587425911Implants are now a widely available treatment, however, their aesthetics, especially in the anterior region, are fraught with difficulties. This book identifies problem cases and details a number of procedures to try and over-come them.The author has a long history of spe-cialist periodontic practice and teaching in Brazil, including directing implantol-ogy courses and lecturing; furthermore he was pupil to Professor Brånemark.The chapters divide this book into a well thought out logical sequence, each building on the previous. As you would expect the first illustrates accepted nat-ural fundamental facial and dental aes-thetics, followed by a brief but concise account of cranial and facial hard and soft tissue anatomy. The author continues, explaining basic but essential periodontal tissue structures, allowing the reader to then interpret the following peri-implanttissues passage. ‘Treatment planning’ demonstrates crucial theory for implant placement and examples of sub-optimal case results when this is not followed. The second half of the book consists mostly of case reports, dealing with soft tissue conditioning, orthodontic consideration including temporary anchorage devices, alveolar distraction osteogenesis and the implant prosthesis. Appropriately then, these chapters are filled with diagrams of surgical techniques explaining the adja-cent clinical photographs.The text itself has been translated from Portuguese. Unfortunately it is riddled with grammatical errors and residual Por-tuguese. Furthermore the text per page is organised into two narrow columns; because of this, many words are hyphen-ated over two lines and make fluent read-ing difficult. This is in part redeemed by succinct summaries concluding most chapters. While definitions and indexes of clinical procedures are good, it is let down by a lack of detail describing some gingival surgeries. The numerous tech-niques explain how to mostly augment soft tissue, papillary defects but requires comprehensive background knowledge of periodontal surgery to understand and ultimately, diligently perform. Content is moderately well referenced with little longitudinal evidence from the author or literature concerning success of these aesthetic procedures. Elsewhere there are inviting diagrams and sequences of great clarity clinical photographs, perhaps, however, not always illustrating the final result readers desire.This is sensible background reading for the application of techniques better detailed in other core texts, highlight-ing the possible aesthetic shortcomings of improper implant placement relative to their supporting structures.G. Calvertreviews。

医学英文文献读新模版

医学英文文献读新模版Medical English Literature Reading New TemplateIntroductionIn the field of medicine, staying updated with the latest research is crucial for healthcare professionals. Medical English literature provides valuable insights and evidence-based information that can enhance patient care. This article aims to introduce a new template for reading medical English literature, which will assist practitioners in efficiently and effectively comprehending and applying the findings in their practice.1. Selecting the ArticleWhen selecting a medical English article to read, it is essential to consider its relevance to your practice and interests. Start by exploring reputable medical journals or databases that publish high-quality research articles. Pay attention to the article's title, abstract, and keywords to determine if it aligns with your needs.2. Reading StrategiesTo efficiently read a medical English article, employ the following strategies:2.1 Skimming and Scanning: Begin by skimming through the article to get a general understanding of its structure and content. Pay attention to headings, subheadings, figures, and tables. Use scanning techniques to find specific information such as study objectives, methods, results, and conclusions.2.2 Vocabulary Building: Medical English literature often contains specialized medical terms. Create a vocabulary list and familiarize yourself with the terms specific to your field or the article's topic. Regularly review and expand your medical vocabulary to enhance comprehension.2.3 Active Reading: Actively engage with the article by highlighting key points, underlining essential information, and making notes. This helps to consolidate your understanding and ensure easy access to crucial details when referring back to the article in the future.3. Breaking Down the ArticleBreaking down the article into components allows for a deeper understanding of its structure and content. The following components are commonly found in medical English literature:3.1 Title: The title succinctly conveys the article's subject matter and provides a brief overview of the study's focus.3.2 Abstract: The abstract serves as a summary of the entire article. It includes information on the study's objectives, methodology, results, and conclusions. Paying close attention to the abstract provides an initial understanding of the article's key findings.3.3 Introduction: The introduction outlines the background and rationale for the study. It highlights the research gap and explains the study's objectives.3.4 Methods: The methods section describes the study design, participants, data collection procedures, and statistical analyses employed.Understanding the methods used in the study is important for assessing the validity and reliability of the findings.3.5 Results: The results section presents the study's findings using tables, charts, and statistical analyses. Focus on the main outcomes and key statistical values to grasp the significance of the findings.3.6 Discussion: The discussion section interprets and analyzes the study's results. It compares the findings with existing literature, highlights the study's limitations, and suggests implications for future research and clinical practice.4. Applying the FindingsAfter comprehensively understanding the article, it is crucial to consider how the findings can be applied to your practice. Reflect on how the study's results align with your current clinical approach and consider adapting your practice accordingly. Consult with colleagues or mentors if needed to discuss the implications of the research findings.ConclusionThe new template for reading medical English literature provides a structured approach to efficiently comprehend and apply research findings. By selecting relevant articles, employing effective reading strategies, breaking down the components of the article, and considering their applicability, healthcare professionals can enhance their knowledge and provide evidence-based care to their patients. Regular practice and exposure to medical English literature will further improve reading skills and ensure continuous professional development.。

medical popular science 英文文章参考文献

medical popular science 英文文章参考文献1. [书籍](1) "The Illustrated Medical Encyclopedia". New York: Funk & Wagnalls Company, 1985.(2) "Medical Science and the Public". London: Oxford University Press, 2005.(3) "The World of Medicine". Cambridge: Cambridge University Press, 2014.2. [期刊](1) "The New England Journal of Medicine".(2) "The Lancet".(3) "Medical Journal of Australia".3. [网络资源](1) "MedlinePlus - Health Reference".(2) "YouTube - Medical Videos".(3) "NHS Choices - Health Information for the Public".4. [专家观点]某某医学专家在他的著作或论文中提出了许多关于医学科学的真知灼见，他对医学的理解深入且具有独到见解，他的观点对于我们理解医学科学具有重要的参考价值。

5. [案例研究]一些案例研究为我们提供了医学实践中的具体实例，这些实例有助于我们更好地理解医学科学的应用和局限。

例如，某某医生在他的研究中，对某个特定疾病的发病机制和治疗方法进行了深入探讨，为我们提供了宝贵的参考。

6. [相关会议]最近召开的医学相关会议为我们提供了了解医学最新进展和研究成果的机会。

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英文医学文献
全文共四篇示例，供读者参考
第一篇示例：
Medical literature in English is a vast and diverse field that encompasses a wide range of topics, from basic biomedical research to clinical trials and case studies. It plays a crucial role in the advancement of medical knowledge and the improvement of patient care worldwide.
Another challenge of English medical literature is the potential for bias and misinformation. Not all research studies are of equal quality, and it is important for healthcare professionals to critically evaluate the evidence presented in medical literature before applying it to patient care. The
peer-review process, in which research articles are reviewed by experts in the field before publication, helps to ensure the quality and reliability of medical literature. However, it is still crucial for healthcare professionals to approach medical literature with a critical eye and a thorough understanding of research methods and study design.
第二篇示例：
Medical literature in English is an essential component of the global healthcare system. It provides valuable information and research findings that help medical professionals stay current with the latest advancements in the field. English language medical literature covers a wide range of topics, including clinical research, case studies, reviews, and guidelines for treatment.
第三篇示例：
Medical literature refers to the body of scholarly publications and research articles that focus on the field of medicine. These articles are written by healthcare professionals, researchers, and experts in various medical specialties. They are essential for advancing medical knowledge, disseminating new discoveries, and improving patient care.
第四篇示例：
Medical literature in English plays a crucial role in the field of healthcare and medical research. It serves as a valuable resource for healthcare professionals, researchers, and students around the world, providing up-to-date information on a wide range of medical topics. In this article, we will explore the importance of English medical literature, its benefits, and how it contributes to advancements in the field of medicine.
Importance of English Medical Literature。

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medical popular science 英文文章 参考文献

medical popular science 英文文章参考文献