妊娠对TA2小鼠乳腺癌生长影响的初步研究

目的:通过检测妊娠环境对TA2小鼠乳腺癌生长的影响,初步探讨人妊娠相关乳腺癌发生发展的机制.方法:收集TA2自发乳腺癌小鼠84只,观察其生物学行为、形态及免疫表型特征;建立妊娠TA2小鼠乳腺癌移植瘤模型(n=20),检测妊娠环境下肿瘤生长;电化学法检测小鼠血清雌二醇与孕酮浓度,Real-time PCR检测妊娠组肿瘤和乳腺组织ERα和PR mRNA相对表达,免疫组织化学染色检测ERα、PR、p53、PCNA和CD31在各组瘤组织内的表达.结果:TA2自发乳腺癌均发生于妊娠期或哺乳期,易内脏转移,乳腺癌分化程度低,不表达ER和PR,表达p53.妊娠组肿瘤生长速度显著加快,肿瘤体积和重量均大于对照组(t=4.142,P<0.001),且癌组织PCNA表达水平增高.TA2孕鼠体内血清雌二醇和孕酮水平显著增高(t=-1.568,-8.927,P=0.168,0.001),而ERα和PR mRNA相对表达相对很低(t=12.245,10.933,P<0.001,0.001),肿瘤血管数量增多.结论:TA2自发乳腺癌与人妊娠相关乳腺癌相似,妊娠促进TA2乳腺癌生长,但雌二醇与孕酮对乳腺癌增殖的促进作用可能是间接实现的.     

纤维粘连蛋白在乳腺癌中的表达及与淋巴结转移的关系

目的 研究纤维粘连蛋白（ＦＮ）在乳腺浸润性癌中的表达及其与同侧腋窝淋巴结转移的关系。方法 应用免疫组化（ＳＰ）法检测５０例浸润性癌底膜及间质ＦＮ的表达。结果 癌巢周围基底膜ＦＮ的表达与癌细胞分化程度及淋巴结转移有关,而间质ＦＮ的表达与以上二者均无关,结论癌巢周围基底膜ＦＮ的表达对乳腺癌预后的评估有意义。     

小鼠三阴性乳腺癌干样细胞对EMT 发生及其生物学行为的影响*

目的:探讨TA2 小鼠三阴性乳腺癌中乙醛脱氢酶1+（ALDH1+）和CD133+表型的乳腺癌干样细胞在上皮间充质转化（EMT ）发生中的作用,及对其生物学行为的影响。方法:使用流式细胞术分析TA2 小鼠三阴性乳腺癌组织中ALDH1 及CD133 的表达量并分选出具有ALDH1+、ALDH1-、CD133+、CD133-表型的乳腺癌细胞,将其分别接种于TA2 小鼠,根据细胞表型不同设置为AL?DH1+、ALDH1-,CD133+、CD133-组,观察肿瘤生长情况,并制成组织切片,行三阴性乳腺癌中乳腺癌干细胞表面标记物ALDH1、CD133 与EMT 相关蛋白Twist1、E-cadherin 和VE-cadherin的免疫组织化学检测,分析其表达差异。结果:乳腺癌干细胞标记物ALDH1 及CD133 在TA2 小鼠三阴性乳腺癌中表达率分别为31.2% 和6.5% 。ALDH1+、CD133+组肿瘤生成能力明显强于ALDH1-、CD133-组。免疫组织化学结果显示ALDH1、Twist1、VE-cadherin在ALDH1+组的表达明显高于ALDH1-组（均P < 0.05）,E-cadherin在ALDH1+组的表达低于ALDH1-组（P < 0.05）。 CD133、Twist1、VE-cadherin在CD133+组的表达明显高于CD133-组（均P < 0.05）,E-cadherin 在CD133+组的表达低于CD133-组（P < 0.05）。 结论:TA2 小鼠三阴性乳腺癌中ALDH1+和CD133+表型的乳腺癌干样细胞可影响EMT 相关蛋白的表达,并促进三阴性乳腺癌的形成。      

Wnt家族成员在TA2小鼠乳腺癌生成过程中基因表达变化规律

  目的  研究乳腺癌生成过程中, Wnt家族成员表达的变化规律。  方法  收集自发乳腺癌小鼠42只, 记录其详细资料。采用基因芯片筛选正常TA2小鼠乳腺组织和自发乳腺癌TA2小鼠乳腺癌组织中的差异表达基因, 随后用Real-time PCR进行验证, 并检测TA2小鼠乳腺癌形成过程中相关基因表达的变化。  结果  TA2自发乳腺癌小鼠平均见瘤年龄为(336.706±85.05)d, 平均分娩次数为(3.767±1.79)次。分娩次数最多者为7次, 其平均见瘤年龄只有251.5 d。TA2小鼠基因芯片结果发现Wnt家族成员中, Wnt1、Wnt10b、Wnt5a、Wnt5b在正常乳腺组织和乳腺癌组织中表达具有差异。Real time PCR的结果证实了以上结果, 并发现Wnt1、Wnt10b、Wnt5a在癌前病变和乳腺癌组织中表达高于正常乳腺组织, 而Wnt5b在乳腺癌组织中表达低于正常乳腺组织。  结论  Wnt1、Wnt5a、Wmt10b在TA2小鼠乳腺癌生成过程中起促进作用, 而Wnt5b的作用可能是抑制小鼠乳腺癌的生成。      

TA2系小鼠乳腺癌肺转移(BCML-TA299)模型的建立及生物学特性的实验研究

目的:研究乳腺癌BCML-TA299生物学特性、DNA状况、相关基因蛋白的表达水平及其意义。方法:应用同系小鼠原位移植BCML-TA299细胞增殖动力学组织形态学、超微结构观察、流式细胞仪以及免疫组织化学染色技术检测TA2系小鼠乳腺癌移植瘤生物异质性、基因DNA状况和蛋白质的表达水平,研究其与细胞生物特性的关系。结果:用TA2系小鼠自发乳腺癌伴肺转移组织在同系小鼠乳腺原位建立了乳腺癌肺转移A型BCML-TA299模型。历时2年多,鼠间传至第47代,移植成功率为100%。未见自发消退,肿瘤生长特性稳定。鼠间荷瘤存活中位数为51天,平均为58.8±10.03天。肿瘤倍增时间为3.11天(指数生长期)。各代移植瘤病理形态学、超微结构观察证实保持了原代瘤的特征,转移瘤与原发瘤结构一致。染色体检查为鼠类肿瘤染色体,DNA含量经流式细胞仪检测显示为异倍体肿瘤。鼠间传代移植瘤与转移瘤保持p53、cerbB-2、nm23基因异常表达。经抗癌药物敏感实验,表明对CAP(CTX、ADM、DDP)联合化疗最为敏感,而单药紫杉醇作用差。结论:本模型可为研究乳腺癌生物异质性提供有价值的实验工具。     

妊娠及分娩后TA2小鼠T细胞亚群及IL-2水平变化在自发乳腺癌中的作用

目的:观察自发乳腺癌和妊娠及分娩后不同时间,TA2小鼠T细胞亚群及IL-2水平的变化,探讨妊娠及分娩在TA2小鼠自发乳腺癌中可能发挥的作用.方法:将TA2小鼠分为正常未妊娠组、自发瘤组、妊娠5、10、20天组和分娩后1、7、14、21天共9组,采用流式细胞术、ELISA等方法,测定小鼠脾脏CD3+、CD4+、CD8+T细胞亚群,计算CD4/CD8,并测定血清IL-2水平.结果:与正常未妊娠组比较,TA2小鼠妊娠及分娩后不同时间内CD3+T细胞亚群百分比均有下降(P<0.01),在妊娠10天下降至最低(P<0.01),CD4+细胞百分比、CD4+/CD8+在妊娠20天降至最低(P<0.01;P<0.05),而CD8+细胞百分比升至最高(P<0.01).与自发瘤组比较,除妊娠10天CD3+T细胞亚群百分比与荷瘤小鼠无明显差别外,其他各组均高于自发瘤组(P<0.05或P<0.01);妊娠期各组CD4+、CD8+、CD4/CD8均与自发瘤组无差别.IL-2在妊娠期间有所下降,以10天和20天组下降显著(P<0.05),分娩后1天恢复至正常未妊娠时水平,但在分娩后7、14、21天又逐渐下降,在分娩后21天明显低于正常未妊娠组(P<0.01),甚至与自发瘤组接近(P<0.01).结论:妊娠和分娩在一定程度上会影响TA2小鼠T细胞亚群分布和IL-2水平的变化,使其细胞免疫功能受抑制,成为TA2小鼠自发性乳腺癌发生的可能诱因之一.     

线粒体凋亡途径在TA2小鼠自发性乳腺癌中的作用

比较TA2小鼠正常乳腺、乳腺癌前病变和乳腺癌组织中细胞增殖和凋亡的情况,通过检测线粒体凋亡途径中Bcl-2、Bax、Caspase-3和Caspase-9在乳腺组织中的表达,初步确定线粒体凋亡途径在TA2小鼠自发乳腺癌中可能发挥的作用。方法:收集正常TA2成年雌鼠的乳腺组织（NC组）和TA2自发性乳腺癌癌前病变组织（SBC-b组）和乳腺癌组织（SBC-t组）,采用免疫组化方法检测各组乳腺上皮细胞中PCNA、Bcl-2、Bax、Caspase-3和Caspase-9的表达并计算增殖指数（PI）;采用TUNEL方法检测细胞凋亡并计算凋亡指数（AI）;采用Real-time PCR和Western blot方法检测组织中Bcl-2、Bax、Caspase-3和Caspase-9蛋白表达及mRNA相对表达水平。结果:免疫组化染色,Real-time PCR以及Western blot结果一致显示,SBC-b组Bcl-2,Bax,Caspase-3和Caspase-9表达均明显高于NC（P<0.01或P<0.05）;SBC-t组中除Bcl-2高于NC外,其余蛋白表达均明显低于NC（P<0.01）,但bcl-2,bax和Caspase-3 mRNA表达均显著高于NC（P<0.01）,Caspase-9 mRNA略高于NC,但无统计学意义（P>0.05）。结论:线粒体途径可能参与了TA2鼠自发性乳腺癌的发生,其通过刺激部分乳腺上皮细胞凋亡,破坏了TA2小鼠乳腺上皮细胞增殖和凋亡的平衡,以致乳腺癌发生。      

L-选择素整合素αL及整合素β2 的表达与TA2 小鼠乳腺癌多器官转移的关系研究*

目的:探讨L-选择素、整合素α L 及整合素β 2 的表达与TA2 小鼠乳腺癌多器官转移的关系。方法:收集天津医科大学附属肿瘤医院病理科保存的高转移性（可发生多器官转移,A 组）、低转移性（无远处转移,B 组）TA2 小鼠乳腺癌及A 组肺、肝和脾转移瘤标本,观察肿瘤特征,利用免疫组化方法检测原发瘤及转移瘤组织中整合素α L、整合素β 2、L-选择素的表达情况,并利用Real-time PCR方法检测A 组和B 组原发瘤组织中上述分子mRNA 的相对表达水平。结果:A 组全组原发肿瘤均见明显出血和坏死,均为低分化或未分化癌,可观察到血管生成拟态,均出现了明显的肺、肝和脾转移。A 组和B 组原发肿瘤组织中整合素α L 和L-选择素的平均阳性细胞百分比分别为24.81% vs 10.47% 和39.81% vs 16.75% ,二者在A 组中的表达均显著高于B 组（Z=-2.31,Z=-2.14;P<0.05）,而整合素β2 的表达在A 组与B 组间无显著性差异;A 组原发瘤及其肺、肝和脾转移瘤中L-选择素平均阳性细胞百分比分别为39.81% 、39.07% 、62.09% 和55.51% ,其中肝转移瘤中平均阳性细胞百分比显著高于原发瘤（Z=-2.24,P=0.025）,而整合素α L、β 2 平均阳性细胞百分比在转移瘤与原发瘤间均无显著性差异。整合素α L 和L-选择素mRNA 的相对表达水平在A 组原发瘤显著高于B 组（Z=-3.47,Z=-3.32;P<0.01）。 结论:TA2 小鼠乳腺癌细胞可表达L-选择素、整合素α L 和β 2,其中L-选择素的表达可能与其肝转移有关。      

Basal-like型乳腺癌临床特征与生存分析

目的分析Basal-like型乳腺癌所占比例、临床特征以及生存情况,为Basal-like型乳腺癌临床诊治提供参考依据。方法回顾性分析我院经手术治疗、资料完整、经病理确诊的女性乳腺癌患者171例。Basal-like型乳腺癌的判定采用Nielsen标准。比较Basal-like型乳腺癌与非Basal-like型乳腺癌的临床病理特征、复发转移及生存情况。结果Basal-like型乳腺癌26例,占15.3%。Basal-like型乳腺癌具有组织学分级高（Basal-like型与非Basal-like型乳腺癌组织学分级Ⅲ级的比例分别为：69.23%和3034%,P＜0.001）,淋巴结转移阳性率低（34.62%和58.62%,P=0.023）,淋巴结转移率低（41.38%和65.38%,P=0.038）的特点。与非Basal-like型乳腺癌相比,Basal-like型乳腺癌肺转移发生率较高（15.38%和3.45%,P=0.012）。Basal-like型乳腺癌和非Basal-like型乳腺癌的5年生存率分别为703%和87.9%（P=0.042）,5年无病生存率分别为59.5%和80.3%（P=0.013）。结论本组患者并没有发现回、汉族患者Basal-like型乳腺癌的发病率差别。本组Basal-like型乳腺癌患者具有组织学分级高、淋巴结转移阳性率低、淋巴结转移率低、易于发生肺转移和预后较差的特点。     

乳腺癌PCNA表达与肥大细胞密度的关系

 目的　探讨乳腺癌组织中PCNA表达与MC密度的关系。方法　用免疫组织化学方法和俾士麦棕法分别检测100例乳腺癌组织中增殖细胞核抗原(PCNA)和肥大细胞(MC)。结果　1.PCNA阳性表达与乳腺癌分化程度和淋巴结转移相关,高分化癌阳性表达低于中分化癌和未分化癌,P<0.05;淋巴结有转移者PCNA阳性表达高于无转移者,P<0.01。2. PCNA阳性者,MC计数较低;PCNA阴性者,MC计数较高,乳腺癌PCNA表达与癌周MC密度呈负相关。结论　PCNA是判断乳腺癌预后的有效指标,MC可能通过影响PCNA的表达发挥其抗肿瘤作用。     

Combined treatment with exogenous estradiol and progesterone increases the incidence of breast cancer in TA2 mice without ovaries

TA2 mice have a high incidence of spontaneous breast cancer without chemical stimulus. There are two proposed explanations for this phenomenon: one is gravidity and the frequency of pregnancies, and the other is related to the presence of the mouse mammary tumor virus (MMTV). MMTV is hormonally regulated and indirectly promotes tumor formation by leading to the activation of Wnt oncogenes through insertional mutagenesis. In order to clarify the relationship between estrogen, progesterone, MMTV, Wnt oncogenes and breast cancer, ovaries from virgin female TA2 mice were removed and the mice were treated with exogenous estradiol and progesterone in different patterns. This study found that the combination of exogenous estradiol and progesterone induced breast cancer formation in TA2 mice without ovaries. MMTV-LTR mRNA exhibited the highest expression in tumor tissue from the combination treatment group (CT). Mammary tissue from mice in the CT group had the highest Wnt1, Wnt5a, Wnt5b and Wnt10b mRNA expression levels. These results indicate that estradiol and progesterone act in a synergistic manner to upregulate MMTV, which subsequently induces breast cancer in TA2 mice. Various members of the Wnt gene family may play specific roles in different stages of carcinogenesis in TA2 mice.     

Hoxb7 inhibits transgenic HER-2/neu-induced mouse mammary tumor onset but promotes progression and lung metastasis

Our previous studies have shown that HOXB7 mRNA is overexpressed in approximately 50% of invasive breast carcinomas and promotes tumor progression in breast cancer cells grown as xenografts in mice. In silico analysis of published microarray data showed that high levels of HOXB7 predict a poor outcome in HER-2-positive (P = 0.046), but not in HER-2-negative breast cancers (P = 0.94). To study the function of HOXB7 in vivo in the context of HER-2 overexpression, we generated mouse mammary tumor virus (MMTV)-Hoxb7 transgenic mice, and then crossed them with MMTV-HER-2/neu transgenic mice. In the mice carrying both Hoxb7 and HER-2/neu transgenes, Hoxb7 plays a dual role in mammary tumorigenesis. In double transgenic mice, overexpression of Hoxb7 delayed tumor onset and lowered tumor multiplicity. However, consistent with the clinical data, once the tumors appeared, their growth was faster and metastasis to the lungs occurred at a higher frequency. Our data show, for the first time, that deregulated expression of Hoxb7 in mammary tumor cells can significantly modulate HER-2/neu-oncogene induced tumorigenesis in vivo.     

Stromal immunoglobulin κC expression is associated with initiation of breast cancer in TA2 mice and human breast cancer

The initiation of spontaneous breast cancer (SBC) in Tientsin Albino 2 (TA2) mice is related to mouse mammary tumor virus (MMTV) infection, and MMTV amplification is hormonally regulated. To explore the insertion site of MMTVLTR in TA2 mouse genome, reverse PCR and nested PCR were used to amplify the unknown sequence on both sides of the MMTV‐LTRSAG gene in SBC and normal breast tissue of TA2 mice. Furthermore, the clinicopathological significance of the insertion site was evaluated in 43 samples of normal breast tissue, 46 samples of breast cystic hyperplasia, 54 samples of ductal carcinoma in situ, 142 samples of primary breast cancer and 47 samples of lymph node metastatic breast cancer by RNA in situ hybridization. We confirmed that the insertion site of the MMTV‐LTRSAG gene was located between Ig κ v2‐112 and Ig κ v14‐111 in chromosome 6 of TA2 mouse. IG κ C was localized in the stromal cells of TA2 mouse with SBC and in human breast cancer tissues. Tumor cells were negative for IG κ C in RNA in situ hybridization. The positive staining index of IG κ C in stromal cells was the highest in lymph node metastatic breast cancer, followed by primary breast cancer, ductal carcinoma in situ, and breast cystic hyperplasia. Furthermore, the positive staining index of IG κ C was related to the expression of ER, PR, HER2 and Ki‐67. Our findings showed that stromal IG κ C expression was associated with the initiation of SBC in TA2 mice. IG κ C may be a high‐risk factor for the initiation and progression of human breast cancer.     

Celecoxib,a selective cyclooxygenase 2 inhibitor,protects against human epidermal growth factor receptor 2 (HER-2)/neu-induced breast cancer

Cyclooxygenase 2 (HER-2) (Cox-2), an inducible form of Cox, is overexpressed in HER-2/neu-positive human breast cancers. The aim of this study was to determine whether celecoxib, a selective Cox-2 inhibitor, protected against HER-2/neu-induced experimental breast cancer. Cox-2 protein was detected in breast carcinomas from mouse mammary tumor virus (MMTV)/neu mice. Treatment with celecoxib (500 ppm) significantly reduced the incidence of mammary tumors in MMTV/neu mice (P = 0.003) and caused about a 50% reduction in mammary prostaglandin E2 (PGE2) levels. Because mammary glands from MMTV/neu mice expressed all four PGE2 receptor subtypes, we speculate that signaling through PGE2 receptors is important for mammary tumorigenesis. These results strengthen the rationale for developing clinical trials to determine whether selective Cox-2 inhibitors possess anticancer properties in humans at risk for breast cancer.     

Mouse mammary tumor-like virus is associated with p53 nuclear accumulation and progesterone receptor positivity but not estrogen positivity in human female breast cancer.

PURPOSE: The purpose is to compare the presence of proteins with known associations with breast cancer-progesterone receptor (PgR), estrogen receptor, and p53, with the prevalence of mouse mammary tumor virus (MMTV)-like DNA sequences in human female breast cancers. EXPERIMENTAL DESIGN: A cohort of 128 Australian female breast cancers were screened for MMTV-like DNA sequences using PCR. The presence of PgR, estrogen receptor, and nuclear accumulation of p53 protein was assessed in the same samples using immunohistochemical staining. RESULTS: Nuclear accumulation of p53 was significantly more prevalent (P = 0.05) in archival human breast cancers containing MMTV-like DNA sequences. The presence of progesterone receptor was significantly higher in MMTV-positive than MMTV-negative breast cancers (P = 0.01). No correlation between estrogen receptor and MMTV-like DNA sequences was found. CONCLUSIONS: MMTV causes breast cancer in mice, and hormones up-regulate expression of virus in mice mammary tissue. It is unknown if this is the case in human breast cancers shown to contain DNA of MMTV-like viruses. The positive association between MMTV-like DNA sequences and PgR indicates hormones and MMTV may play a role in human breast cancer. Mutations of the tumor suppressor gene p53 are common in human breast cancer and are associated with higher grades of cancer. The association of MMTV-like DNA sequences with higher grades of cancer, and the positive association between p53 and MMTV-like DNA sequences clearly warrant additional investigation.     

Understanding and treating triple-negative breast cancer

Triple-negative breast cancer is a subtype of breast cancer that is clinically negative for expression of estrogen and progesterone receptors (ER/PR) and HER2 protein. It is characterized by its unique molecular profile, aggressive behavior, distinct patterns of metastasis, and lack of targeted therapies. Although not synonymous, the majority of triple-negative breast cancers carry the "basal-like" molecular profile on gene expression arrays. The majority of BRCA1-associated breast cancers are triple-negative and basal-like; the extent to which the BRCA1 pathway contributes to the behavior of sporadic basal-like breast cancers is an area of active research. Epidemiologic studies illustrate a high prevalence of triple-negative breast cancers among younger women and those of African descent. Increasing evidence suggests that the risk factor profile differs between this subtype and the more common luminal subtypes. Although sensitive to chemotherapy, early relapse is common and a predilection for visceral metastasis, including brain metastasis, is seen. Targeted agents, including epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and poly (ADP-ribose) polymerase (PARP) inhibitors, are currently in clinical trials and hold promise in the treatment of this aggressive disease.     

Agonists and antagonists of GnRH-I and -II reduce metastasis formation by triple-negative human breast cancer cells in vivo

Metastasis to bone is a frequent problem of advanced breast cancer. Particularly breast cancers, which do not express estrogen and progesterone receptors and which have no overexpression/amplification of the HER2-neu gene, so called triple-negative breast cancers, are considered as very aggressive and possess a bad prognosis. About 60% of all human breast cancers and about 74% of triple-negative breast cancers express receptors for gonadotropin-releasing hormone (GnRH), which might be used as a therapeutic target. Recently, we could show that bone-directed invasion of human breast cancer cells in vitro is time- and dose-dependently reduced by GnRH analogs. In the present study, we have analyzed whether GnRH analogs are able to reduce metastases of triple-negative breast cancers in vivo. In addition, we have evaluated the effects of GnRH analogs on tumor growth. To quantify formation of metastasis by triple-negative MDA-MB-435 and MDA-MB-231 human breast cancers, we used a real-time PCR method based on detection of human-specific alu sequences measuring accurately the amount of human tumor DNA in athymic mouse organs. To analyze tumor growth, the volumes of breast cancer xenotransplants into nude mice were measured. We could demonstrate that GnRH analogs significantly reduced metastasis formation by triple-negative breast cancer in vivo. In addition, we could show that GnRH analogs significantly inhibited the growth of breast cancer into nude mice. Side effects were not detectable. In conclusion, GnRH analogs seem to be suitable drugs for an efficacious therapy for triple-negative, GnRH receptor-positive human breast cancers to prevent metastasis formation.     

Constitutive activation of nuclear factor-κB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines

Constitutive nuclear factor (NF)-κB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-κB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-κB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-κB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF-κB activation by expression of IκBα super-repressor reduced proliferation of the basal-like subtype cell lines. Expression levels of mRNA encoding NF-κB-inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference-mediated knockdown of NIK reduced NF-κB activation in a subset of the basal-like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF-κB activation, partially dependent on NIK, is preferentially involved in proliferation of basal-like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer. ( Cancer Sci 2009; 100: 1668–1674)