125IUdR靶点内照射与C6胶质瘤细胞的增殖抑制和细胞周期变化

目的研究125IUdR DNA靶点放疗对大鼠C6胶质瘤细胞增殖和细胞周期的影响.方法体外采用C6细胞单层指数生长模型,体内运用脑胶质瘤C6大鼠(肿瘤增殖高峰期局部缓慢三次注射药物,8.1×103kBq@次-1@天-1)30只,结合MTT法、平板克隆形成试验和流式细胞仪(FCM)检测肿瘤细胞的增殖情况和细胞周期分布,研究125IUdR治疗脑胶质瘤的作用机理.结果125IUdR可显著抑制C6细胞增殖,具有时间和浓度依赖性.细胞存活曲线呈无肩区曲线(直线),C37=9.06kBq/mL;MTT法中150kBq/mL125IUdR作用5 d后抑制率达93.06%;而Na125I和127IUdR对C6细胞生长无明显抑制作用;125IUdR治疗胶质瘤C6大鼠5 d后,肿瘤重量低于对照组(P＜0.01). 125lUdR可使C6细胞停滞于G0/G1期,G0/G1期比例上升,S期比例降低.体外经250 kBq/mL 125lUdR作用7 d,G0/G1期和S期分别占76.23%和12.84%,与空白组比较P＜0.01体内C6胶质瘤经125IUdR治疗后,G0/G1期和S期分别为85.19%和10.51%,与对照组及空白组比较.均P＜0.05.结论 125IUdR可显著抑制脑胶质瘤细胞的增殖,导致细胞周期调控紊乱,G0/G1期阻滞.125lUdR在治疗脑胶质瘤方面具有潜力.     

125IUdR靶点内照射与C6胶质瘤细胞的增殖抑制和细胞周期变化

目的　研究1 2 5IUdRDNA靶点放疗对大鼠C6 胶质瘤细胞增殖和细胞周期的影响。方法　体外采用C6 细胞单层指数生长模型 ,体内运用脑胶质瘤C6 大鼠 (肿瘤增殖高峰期局部缓慢三次注射药物 ,8 1× 10 3kBq·次- 1 ·天- 1 ) 3 0只 ,结合MTT法、平板克隆形成试验和流式细胞仪 (FCM)检测肿瘤细胞的增殖情况和细胞周期分布 ,研究1 2 5IUdR治疗脑胶质瘤的作用机理。结果　1 2 5IUdR可显著抑制C6 细胞增殖 ,具有时间和浓度依赖性。细胞存活曲线呈无肩区曲线 (直线 ) ,C37=9 0 6kBq/mL ;MTT法中 15 0kBq/mL1 2 5IUdR作用 5d后抑制率达 93 0 6% ;而Na1 2 5I和1 2 7IUdR对C6 细胞生长无明显抑制作用 ;1 2 5IUdR治疗胶质瘤C6 大鼠 5d后 ,肿瘤重量低于对照组 (P <0 0 1)。1 2 5IUdR可使C6 细胞停滞于G0 /G1 期 ,G0 /G1 期比例上升 ,S期比例降低。体外经 2 5 0kBq/mL1 2 5IUdR作用 7d,G0 /G1 期和S期分别占 76 2 3 %和 12 84% ,与空白组比较P <0 0 1:体内C6 胶质瘤经1 2 5IUdR治疗后 ,G0 /G1 期和S期分别为 85 19%和 10 5 1% ,与对照组及空白组比较 ,均P <0 0 5。结论　1 2 5IUdR可显著抑制脑胶质瘤细胞的增殖 ,导致细胞周期调控紊乱 ,G0 /G1 期阻滞。1 2 5IUdR在治疗脑胶质瘤方面具有潜力。     

冷冻治疗对C6大鼠脑胶质瘤细胞增殖和凋亡的影响

目的探讨大鼠C6脑胶质瘤模型冷冻治疗后细胞凋亡水平和增殖活性的变化。方法借助于立体定位技术,建立大鼠C6皮层脑胶质瘤动物模型,待肿瘤生长至第15天、直径约6mm时,进行冷冻治疗。治疗后15d行细胞凋亡的末端标记法(TUNEL)和流式细胞仪(FCM)检测,以增殖细胞核抗原(PCNA)免疫组织化学及MRI检查了解肿瘤的增殖活性,测量肿瘤体积和抑瘤率。结果冷冻治疗后凋亡细胞密度及凋亡指数较对照组显著提高[前者分别为(36.73±9.54)/0.0625mm2和(4.87±2.80)/0.0625mm2;后者分别为(16.02±3.87)%和(1.70±1.74)%](P<0.01)。冷冻组肿瘤内PCNA阳性细胞数量较对照组显著下降[分别为(42.37±7.63)%和(73.93±9.60)%](P<0.01);肿瘤体积明显缩小[分别为(139.60±27.28)mm3和(414.40±69.01)mm3](P<0.01),增殖受抑制,抑瘤率为66.31%。结论大鼠C6脑胶质瘤冷冻治疗后除细胞坏死外,肿瘤细胞增殖水平下降和细胞凋亡增多也是冷冻治疗胶质瘤的机理之一。     

反义AKT2 cDNA治疗C6胶质瘤的体内外实验研究

目的研究反义AKT2(antisense AKT2，AS—AKT2)cDNA对鼠脑胶质瘤细胞系C6的生长抑制作用。方法将AS—AKT2 cDNA构建体转染鼠脑胶质瘤细胞系C6，原位杂交和蛋白印迹鉴定后，应用PCNA阳性率和MTT法检测细胞增殖能力，TUNEL法计算凋亡指数。应用立体定向技术将C6细胞和转染反义AKT2 cDNA的C6细胞种植到SD大鼠的右侧尾状核作为对照组和转染组；并对颅内已经形成C6胶质瘤的大鼠进行脂质体包裹的AS—AKT2 cDNA和空载体治疗；MRI动态监测大鼠颅内肿瘤生长情况，并检测标本AKT2和PCNA表达以及细胞的凋亡情况。结果转染AS—AKT2 cDNA后C6细胞AKT2表达显著抑制，增殖减慢，凋亡指数增加。反义治疗组和转染组大鼠生存时间明显延长；转染组和治疗组肿瘤标本AKT2表达下降或消失，PCNA阳性率降低，可见大量凋亡细胞，而对照组和空载组标本几乎没有凋亡细胞。结论体内外实验证明AS—AKT2 cDNA可以抑制肿瘤细胞增殖、诱导凋亡，AKT2可作为基因治疗胶质瘤的重要优选靶的。     

槲皮素对大鼠脑胶质瘤抑瘤作用的体内实验研究

目的观察槲皮素及顺铂对大鼠脑胶质瘤生长的抑制作用,初步探讨其作用机制。方法将30只颅内接种C6胶质瘤细胞的SD大鼠随机分为3组:分别给予顺铂、槲皮素及对照治疗7 d,观察各组肿瘤生长情况,于接种后17 d处死各组大鼠,收集肿瘤标本进行肿瘤大小、HE染色及电镜观察,并通过免疫组化检测肿瘤组织增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达。结果顺铂组[(66.2±5.9)mm3]、槲皮素组[(59.6±5.4)mm3]肿瘤体积低于对照组[(101.1±9.6)mm3]槲皮素组优于顺铂组(t=10.61,P<0.01),槲皮素组、顺铂组肿瘤抑制率分别为41.3%、34.8%。光镜观察槲皮素组、顺铂组肿瘤细胞密度降低,电镜显示有细胞凋亡。PCNA免疫组化结果显示槲皮素组(42.2%±5.1%)、顺铂组(50.0%±6.7%)肿瘤PCNA表达减少,与对照组(79.6%±8.6%)相比,具有统计学差异(F=416.12,P<0.01),两两比较,槲皮素组治疗效果优于顺铂组(q=55.25,P<0.01)。结论槲皮素和顺铂均可显著抑制大鼠颅内C6胶质瘤细胞生长,其机制可能与诱导肿瘤细胞凋亡和抑制肿瘤细胞增殖有关。     

基于靶向低密度脂蛋白受体蛋白1的载药嵌段共聚物抗胶质瘤作用的体外实验研究

目的研究靶向低密度脂蛋白受体蛋白1(LRP1)的载药嵌段共聚物(ANPs/CPT)对胶质瘤细胞的增殖抑制和促进凋亡的作用。方法采用Western blotting技术检测LRP1在大鼠胶质瘤细胞(C6细胞)和脑毛细血管内皮细胞(BCECs)的表达水平。用激光共聚焦显微镜测定C6细胞和BCECs对Angiopep-2修饰的靶向未载药嵌段共聚物(ANPs)和非靶向对照NPs的摄取量;采用噻唑蓝(MTT)比色法检测ANPs/CPT抑制C6细胞和BCECs增殖的作用;采用流式细胞仪分析不同CPT终浓度(10μg/mL,20μg/mL)的ANPs/CPT促C6细胞凋亡作用。结果 LRP1在C6和BCECs细胞膜表面高表达。C6细胞对ANPs的摄取量显著高于NPs(P<0.01);ANPs/CPT对C6细胞的增殖抑制作用显著高于NPs/CPT(P<0.05),但对BCECs的增殖抑制作用与NPs/CPT无显著差异(P> 0.05)。CPT终浓度10μg/mL、20μg/mL的ANPs/CPT与NPs/CPT作用于C6细胞48 h后的凋亡率分别为(16.1±0.9)%、(30.4±...     

125IUdR与胶质瘤治疗的研究概况

125I是一种俄歇电子(Auger dectron，AE)释放体，掺人细胞DNA后具有显著的细胞毒性。IUdR特异性掺入S期细胞DNA，是125I的良好转运载体。大量动物实验已证明肿瘤局部直接持续和间隔反复注射125IUdR的抗肿瘤作用显著，疗效与肿瘤增殖动力学有关。由于神经组织的自身特点，125IUdR在脑胶质瘤治疗方面具有独特优势，能选择性杀伤肿瘤细胞，尤其对肿瘤残灶而言，故可作为外科手术的一项有益辅助治疗手段，提高胶质瘤患者的生存率。     

苦参碱对胶质瘤大鼠模型中Fas表达的调节作用的实验研究

目的 探讨苦参碱应用前后C6脑胶质瘤大鼠模型中Fas因子的表达变化及意义.方法采用脑立体定向技术,将体外培养的C6胶质瘤细胞注入大鼠尾状核区制备胶质瘤大鼠模型,并根据是否用药及用药量的多少分为空白对照组、冰片组、苦参碱低剂量组、苦参碱高剂量组、苦参碱低剂量+冰片组、苦参碱高剂量+冰片组.通过大鼠生存状态、标本的大体所见、MRI、HE染色观察苦参碱对胶质瘤大鼠模型生存质量及胶质瘤体积的影响,用免疫组织化学方法检测苦参碱对胶质瘤大鼠模型肿瘤细胞中Fas表达的影响.结果 大鼠生存状态、标本的大体所见、MRI及HE染色显示苦参碱可显著提高胶质瘤大鼠模型的生存质量,抑制胶质瘤细胞增殖.免疫组化结果显示,苦参碱低剂量+冰片组(98.16±11.82)、苦参碱高剂量+冰片组(1 12.80±12.12)Fas表达高于空白对照组(39.09±7.79)、冰片组(46.87±7.43)、苦参碱低剂量组(42.41±7.83)、苦参碱高剂量组(44.20±7.47),苦参碱高剂量+冰片组Fas表达高于苦参碱低剂量+冰片组,差异均有统计学意义(P＜0.05).结论 苦参碱能增加胶质瘤细胞中Fas的表达,抑制胶质瘤细胞增殖.     

铜绿假单胞菌制剂对大鼠C6胶质瘤细胞作用的体外机制研究

[摘要] 目的 初步探讨铜绿假单胞菌制剂（PA-MSHA）对体外培养的大鼠C6胶质瘤细胞的增殖抑制机制。方法 体外培养大鼠C6胶质瘤细胞, MTT比色法测定不同时间、不同浓度PA-MSHA对C6胶质瘤细胞的增殖抑制率。Hochest 33258染色,荧光显微镜下检测细胞凋亡,流式细胞仪检测PA-MSHA对C6细胞周期的影响。结果MTT比色法结果提示,细胞生长抑制率与药物作用时间和剂量正相关。24小时和48小时IC50值分别为(5.80±1.79)× 108cfu/ml和(3.90±2.14) × 108cfu/ml。Hochest33258染色可见明显的细胞凋亡形态学改变,流式细胞仪检测,药物作用组可见典型的亚二倍体峰即凋亡峰（AP）,且随浓度加大S期比例明显增多。 结论 铜绿假单胞菌(PA-MSHA)制剂对体外培养的C6胶质瘤细胞具有增殖抑制作用,且呈时间剂量依赖性。其机制可能与诱导细胞凋亡及S期阻滞有关。     

人脑胶质瘤中cyclin E的表达及其对细胞增殖活性的影响

目的研究人脑胶质瘤中细胞周期素E(cyclin E)表达与胶质瘤细胞恶性程度及细胞增殖活性的关系.方法采用免疫组化方法检测52例人脑胶质瘤和8例正常脑组织标本中cyclin E和增殖细胞核抗原(PCNA)的表达水平,观察并分析各病理等级中两者表达的阳性率和标记指数.结果正常脑组织中无cyclin E或PCNA表达.随着人脑胶质瘤病理分级的增高,cyclin E的阳性率和平均标记指数均明显升高,在高、低度恶性度肿瘤之间也存在明显差异(P<0.05).双元相关性分析发现cyclin E与PCNA的平均标记指数呈明显正相关(Pearson相关系数r=0.576,P<0.01).结论人脑胶质瘤中cyclin E的表达与细胞增殖活性和病理学分级关系密切,很可能对肿瘤细胞的增殖和恶性转化产生重要的推动作用.     

视频脑电图在小儿癫痫诊断中的应用

目的评价视频脑电图(video-EEG)在小儿癫诊断中的应用价值。方法对126例具有发作性症状的患儿进行连续8h的包括清醒、睡眠、诱发试验及必要的认知测验的视频脑电图监测。结果经发作期视频脑电图证实,39例初诊为癫性发作的患儿中14例(35%)为非癫性发作;15例其他症状发作中13例(86%)为非癫性发作。64例样放电患儿中51例(80%)确定发作类型,22例(34%)确定癫类型。视频脑电图可发现短暂轻微的癫发作及样放电引起的一过性认知损伤。结论视频脑电图在排除非癫性发作、确定癫性发作的类型、评价脑电-临床关系方面可提供准确可靠的证据,进一步提高癫的临床诊断水平。     

Neuroprotective properties of memantine in different in vitro and in vivo models of excitotoxicity

The pathogenesis of stroke, trauma and chronic degenerative diseases, such as Alzheimer's disease (AD), has been linked to excitotoxic processes due to inappropriate stimulation of the N-methyl-D-aspartate receptor (NMDA-R). Attempts to use potent competitive NMDA-R antagonists as neuroprotectants have shown serious side-effects in patients. As an alternative approach, we were interested in the anti-excitotoxic properties of memantine, a well-tolerated low affinity uncompetitive NMDA-R antagonist presently used as an anti-dementia agent. We explored in a series of models of increasing complexity, whether this voltage-dependent channel blocker had neuroprotective properties at clinically relevant concentrations. As expected, memantine protected neurons in organotypic hippocampal slices or dissociated cultures from direct NMDA-induced excitotoxicity. However, low concentrations of memantine were also effective in neuronal (cortical neurons and cerebellar granule cells) stress models dependent on endogenous glutamate stimulation and mitochondrial stress, i.e. exposure to hypoxia, the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) or a nitric oxide (NO) donor. Furthermore, memantine reduced lethality and brain damage in vivo in a model of neonatal hypoxia-ischemia (HI). Finally, we investigated functional rescue (neuronal capacity to migrate along radial glia) by memantine in cerebellar microexplant cultures exposed to the indirect excitotoxin 3-nitropropionic acid (3-NP). Potent NMDA-R antagonists, such as (+)MK-801, are known to block neuronal migration in microexplant cultures. Interestingly, memantine significantly restored the number of neurons able to migrate out of the stressed microexplants. These findings suggest that inhibition of the NMDA-R by memantine is sufficient to block excitotoxicity, while still allowing some degree of signalling.     

Storage of lipofuscin in neurons in mucopolysaccharidosis

Summary A histochemical and ultrastructural study was made on the brain of a 23-year-old man with Sanfilippo's syndrome. In accordance with previous reports the cortical nerve cells contained a PAS-positive lipid storage substance. This showed intense autofluorescence in UV-light and was positive with various stains for lipofuscin. The storage material appeared ultrastructurally as inclusion bodies composed of short lamellated membranes, granular material, and vacuoles. In addition, concentrically and transversely lamellated membranous cytoplasmic bodies were observed in the nerve cells. It is concluded that the PAS-positive lipid storage material in the neurons was composed partly of lipofuscin in addition to other lipids presumably glycosphingolipids.Supported by a grant from the Expressen Prenatal Research Foundation     

脑电图预测痫性发作研究进展

癫痫(epilepsy)是由脑部神经元高度同步化异常放电所致的临床综合征,系神经系统的常见病,困扰着全世界约1%的人群.每次神经元的阵发性放电或短暂的脑功能异常称为痫性发作(seizures).     

Role of vincristine in the inhibition of angiogenesis in glioblastoma

Objective: Vincristine, a microtubule-destabilizing drug, was found to exhibit anti-angiogenic effects and anti-tumoral activity. However, the precise mechanism by which vincristine inhibits angiogenesis in glioblastomas is not well understood. Our aim was to investigate whether vincristine affects vascular endothelial growth factor (VEGF) expression in glioblastoma cells and determine whether it is mediated by the downregulation of hypoxia-inducible factor-1α (HIF-1α).

Methods: We investigated the expression of HIF-1α in glioblastoma tissues resected from patients and in human glioblastoma cell lines using immunohistochemistry, Western blot analysis, and immunocytochemistry. In addition to an MTT assay assessing the effect of vincristine on cell proliferation and viability, the effects of vincristine on VEGF mRNA expression and HIF-1α protein were examined using real-time RT-PCR and Western blot analysis under 1% O₂ (hypoxia).

Results: HIF-1α was expressed in the majority of glioblastoma tissues and was detected mainly in the nucleus. Strong immunoreactivity for HIF- 1 α was found often in the hypercellular zones. Under hypoxic conditions, HIF-1α protein levels in the glioblastoma cell lines increased, primarily localizing into the nucleus similar to glioblastoma tissues. Exposure of glioblastoma cells to vincristine resulted in enrichment of the G₂-M fraction of the cell cycle, which suggests that vincristine-mediated growth inhibition of glioblastoma is correlated with mitotic inhibition. Using doses lower than those found to reduce the viability and proliferation of cells by 50% (IC₅₀), vincristine decreased both the expression of VEGF mRNA and the level of HIF-1α protein in hypoxic glioblastoma cells. In addition, following exposure to vincristine, the expression of VEGF mRNA was correlated with HIF-1α protein levels.

Conclusions: Our results suggest that the mechanism by which vincristine elicits an anti-angiogenic effect in glioblastomas under hypoxic conditions might be mediated, in part, by HIF-1α inhibition.     

Midazolam in treatment of various types of seizures in children

Midazolam is a recently developed water-soluble benzodiazepine that shares anxiolytic, muscle relaxant, hypnotic and anticonvulsant actions with other members of this class. There are limited studies that midazolam can be used successfully to treat seizures in adults and children. In this study, 0.2 mg/kg intramuscular (IM) midazolam was administered to 11 children (eight boys and three girls), aged 3 days to 4 years (mean age 1.8±1.4 years), with seizures of various types. In all but one child, seizures stopped in 15 s–5 min after injection. No side effects were observed. These results suggest that IM administration of midazolam may be useful in a variety of seizures during childhood, especially in case of intravenous (IV) line problem.     

The Burden of Agoraphobia in Worsening Quality of Life in a Community Survey in Italy

ObjectiveCurrent nosology redefined agoraphobia as an autonomous diagnosis distinct from panic disorder. We investigated the lifetime prevalence of agoraphobia, its association with other mental disorders, and its impact on the health-related quality of life (HR-QoL). MethodsCommunity survey in 2,338 randomly selected adult subjects. Participants were interviewed with the Advanced Neuropsychiatric Tools and Assessment Schedule (ANTAS), administered by clinicians. The diagnoses were based on the ICD-10 criteria. The Short-Form Health Survey (SF-12) was used to quantify HR-QoL. ResultsIn the sample, 35 subjects met the criteria for agoraphobia (1.5%), with greater prevalence among women (2.0%) than men (0.9%): odds ratio (OR) 2.23; 95% CI: 1.0-5–2. Agoraphobia was more often seen among those with (n=26; 1.1%) than without (n=9; 0.4%) panic disorder: OR=8.3; 2.9–24.4. Co-morbidity with other mental disorders was substantial. The mean score of SF-12 in people with agoraphobia was 35.2±7.8, with similar levels of HR-QoL in people with (35.3±7.9) or without (34.8±7.3) panic disorder: ANOVA: F(1;33)=0.0; p=1.00. ConclusionOne out of seventy people may suffer from agoraphobia in their lifetime. The attributable burden in terms of HR-QoL is substantial and comparable to the one observed for chronic mental disorders such as major depression, post-traumatic stress disorder, or obsessive-compulsive disorder.     

Characteristics of nociceptors in the periodontium--an in vitro study in rats

Recent studies have indicated that nociceptors can be classified into various types according to their physiological properties. These studies have clarified that the frequency distribution of various nociceptor types is different among body sites and animal species. In the present study, we investigated the physiological properties of rat's periodontal nociceptors in an in vitro jaw-nerve preparation. Responses were recorded from functional single filaments in the inferior alveolar nerve. To determine the nociceptor type, calibrated von Frey filaments, heat, and bradykinin (BK) stimuli were used. We found five subtypes of nociceptors in the periodontal ligaments of the lower incisor: Adelta-high threshold mechanonociceptors (Adelta-HTM, n=28), Adelta-mechanoheat nociceptors (Adelta-MH, n=6), Adelta-polymodal nociceptors (Adelta-POLY, n=26), C-high threshold mechanonociceptors (C-HTM, n=3) and C-polymodal nociceptors (C-POLY, n=4). Most nociceptors were Adelta-innervated, while only a small number of C-innervated nociceptors were found. The present results suggest that periodontal nociceptors transmit mainly fast pain, and may thus play a role in rapid detection of injure-related stimuli during mastication.     

自噬参与帕金森病发病的研究进展

近年来,蛋白质的降解障碍被认为是帕金森病（Parkinson’Sdisease,PD）发病过程中的重要因素,人们已经公认泛素一蛋白酶体系统（ubiquitin--pro—teasomesystem,UPS）功能异常或衰竭能够导致细胞内异常蛋白蓄积、细胞功能障碍,甚至细胞凋亡。与此同时,蛋白降解的另一条途径——自噬-溶酶体途径（autophagy—lysosomepathway,ALP）也已成为了生命科学领域的研究热点,自噬与神经变性疾病,尤其是PD的关系日益受到人们的重视。     

Developmental effects of in vivo and in vitro inhibition of nitric oxide synthase in neurons

The diffusible chemical messenger nitric oxide (NO) is involved in neuronal plasticity and it is, therefore, supposed to play a role in brain development. A shortage of NO during the critical period of brain maturation may theoretically have long-lasting consequences on the organization of the adult brain. We have performed in neonatal rats a chronic inhibition of the enzyme responsible for NO production, nitric oxide synthase (NOS), from postnatal day 3 to postnatal day 23, through administration of the competitive antagonist N-nitro-L-arginine methylester (L-NAME). The calcium-dependent catalytic activity resulted almost completely inhibited throughout the period of treatment and it took more than 4 days after its suspension to get a full recovery. The expression of the neuronal isoform of the enzyme (nNOS), revealed by immunoblotting, was unchanged during the treatment and after it. The histochemical reaction for NADPH diaphorase was reduced at the end of the treatment and recovered in concomitance with the recovery of the catalytic NOS activity. No gross structural alterations were detected in brain morphology. The levels of three neurotransmitter-related and one astrocytic marker were unchanged in the cerebellum, hippocampus and cortex of 60-day-old rats which had been neonatally treated. A similar lack of significant effects on neurochemical brain maturation was also noticed in a parallel series of experiments, in which a short pulse of NOS inhibition was performed at a critical prenatal time of brain development, from gestational day 14 to gestational day 19. In vitro, chronic exposure of cerebellar granule cells to L-NAME (500 microM) resulted in slight decrease of surviving neurons after 8 days in culture and in better resistance to the challenge of stressful culture conditions. The present results suggest that the basic plan of brain organization can be achieved despite an almost complete NOS inhibition during the maturation period. In vitro, NOS inhibition may bring to more pronounced consequences on neuronal viability and function.