小麦条锈菌胞质游离钙离子动态检测方法的建立

胞质游离钙离子变化与植物病原真菌侵染寄主的动态过程具有重要的关联性。本研究以侵染小麦叶片的条锈菌31号生理小种(CYR31)为材料,以孵育法将Ca2+荧光探针Fluo-3-AM载入到小麦条锈菌细胞中,并结合激光共聚焦扫描显微技术,建立了测定侵染过程中条锈菌胞质游离Ca2+分布的试验方法。结果表明,采用10μmol/L Fluo-3-AM装载顺次进行低温4℃孵育1h,25℃孵育1h,可获得较为理想的条锈菌胞质游离Ca2+染色结果。该方法可用于检测不同侵染阶段的小麦条锈菌细胞质游离钙离子的分布变化,为进一步研究锈菌胞内钙离子动态与侵染寄主的关联性提供了技术支撑。     

条形柄锈菌吸器特异效应蛋白Hasp68抑制寄主免疫并与小麦组织蛋白酶B互作

作为活体营养专性寄生真菌,条形柄锈菌（小麦条锈病）在侵染过程中通过形成吸器向寄主细胞释放效应蛋白,干扰寄主的防卫反应,促进其侵染与致病。因此,条形柄锈菌效应蛋白的鉴定与功能研究对揭示其毒性机理具有重要意义。本实验室前期完成了条形柄锈菌CYR31生理小种吸器转录组分析,从中鉴定得到一个吸器特异诱导表达分泌蛋白Hasp68,利用农杆菌侵染在烟草细胞中瞬时表达该基因,能够抑制小鼠促细胞凋亡蛋白Bax诱导的细胞程序性死亡,鉴定为条形柄锈菌候选效应蛋白。Hasp68基因全长318bp,编码105_aa,N-端包含20_aa的信号肽,无保守结构域。BlastX分析表明Hasp68为条形柄锈菌特有效应蛋白,在其他真菌中无同源蛋白,且在条形柄锈菌16个菌系中呈较低的序列多态性,表明其在条形柄锈菌的进化过程中相对保守。借助荧光假单胞菌EtHAn的三型分泌系统,在小麦细胞中过表达Hasp68能够抑制由非致病细菌引起的PTI（PAMP-triggered immunity）相关胼胝质的积累;同时,也能抑制小麦与无毒条形柄锈菌互作中ETI（effector-triggered immunity）相关的活性氧爆发和过敏性坏死反应,表明效应蛋白Hasp68具有抑制寄主免疫反应的功能。利用酵母双杂交系统筛选Hasp68在小麦中的互作蛋白,发现其与组织蛋白酶B（cathepsin B）TaCTSB互作,双分子荧光技术进一步验证二者在烟草细胞中共表达存在互作,初步揭示了效应蛋白Hasp68的互作靶标。     

条形柄锈菌(小麦条锈病菌)侵染结构体外形成的观察

通过荧光显微镜和扫描电镜分别对条形柄锈菌夏孢子在寄主植物-小麦叶表和非寄主植物-水稻叶表以及小麦穗部和茎秆上的萌发过程进行了观察。结果发现,夏孢子在小麦叶片体表萌发产生芽管后,可依次分化形成气孔下囊、初生菌丝与吸器母细胞;在小麦颖片、稃片及茎秆部位表面,同样可观察到病菌在体外分化形成吸器母细胞;并且在水稻叶片上也观察到病菌侵染结构存在体外分化现象。经荧光染色发现,条形柄锈菌在体外与在小麦组织中形成的侵染结构没有明显的差别。观察结果可为条形柄锈菌侵染结构的离体诱导与调控机理研究提供依据。     

小麦条锈菌钙调素依赖蛋白激酶基因Pscamk的功能

【目的】克隆小麦条锈菌钙调素依赖蛋白激酶基因Pscamk,并分析其在条锈菌侵染小麦过程中的表达特征及初步功能。【方法】基于本实验室已测序的小麦条锈菌基因组序列,利用RT-PCR方法,从小麦条锈菌生理小种CYR32中克隆Pscamk基因的cDNA序列,并利用网络数据库和生物信息学工具预测该基因编码蛋白的基本特征和保守结构;运用qRT-PCR技术分析Pscamk在不同发育及侵染阶段的表达水平,进一步通过钙调素依赖蛋白激酶(CaMK)的免疫抑制剂KN-93处理小麦条锈菌夏孢子,观察其萌发状况。【结果】获得1个1620 bp的小麦条锈菌CaMK基因Pscamk;序列分析发现,Pscamk编码蛋白包含CaMK蛋白的保守结构域,并与小麦杆锈菌该类蛋白序列相似性最高。qRT-PCR分析表明,Pscamk在条锈菌侵染初期过程中的芽管发育、初生菌丝侵染及吸器形成时期呈显著上调表达,且在条锈菌接种6 h时表达量最高,为对照夏孢子的20.74倍。在专一性免疫抑制剂KN-93处理后,随着KN-93施加浓度的增加,条锈菌夏孢子萌发率逐渐降低,当浓度为1.4μmol/L时夏孢子萌发率为8.02%,仅为对照的12%。【讨论】推测Pscamk基因参与了小麦条锈菌夏孢子萌发、芽管发育以及初期侵染结构的形成。本研究为进一步探索条锈菌细胞钙信号传导机理和致病机制奠定了基础。     

三裂叶豚草锈菌侵染寄主叶片的显微和超微观察

【背景】三裂叶豚草是我国重要的外来入侵植物之一,其传播速度快,已给我国造成巨大的经济损失。近年来发现的三裂叶豚草锈菌是一种对其具有生物防治潜力的病原菌。【方法】本文利用显微技术研究了三裂叶豚草锈菌的侵染过程及其对寄主结构的影响。【结果】三裂叶豚草锈菌菌丝可从多处侵入同一个叶肉细胞,胞间菌丝与叶肉细胞相接触可使部分细胞壁增厚。锈菌侵染使三裂叶豚草叶脉末梢导管分枝增多,造成三裂叶豚草水分代谢失调;叶片细胞内膜系统破碎化,细胞器结构受到不同程度的破坏,导致细胞内膜系统紊乱,细胞器结构稳定性降低。【结论与意义】豚草锈菌侵染破坏了三裂叶豚草叶片的细胞结构。本研究为深入研究豚草锈菌的致病机理奠定了基础。     

食线虫真菌侵染性胞外酶和生防应用

食线虫真菌作为重要的植物寄生线虫的生物防治资源,深入了解它们的侵染方式、毒力因子是了解食线虫真菌侵染的分子机理和开发高效、稳定的生物杀线虫制剂的关键。目前的研究表明,食线虫真菌能分泌具有降解线虫体壁或线虫卵壳的胞外酶,它们在食线虫真菌侵染线虫的过程中起着非常重要的作用。对这些侵染性胞外水解酶的深入研究将促进人们对食线虫真菌的侵染过程和侵染机制的了解以及高效生防制剂的开发。综述了近年来食线虫真菌侵染性胞外酶的研究概况,对食线虫真菌胞外丝氨酸蛋白酶进行同源性分析,对以后食线虫真菌侵染性胞外酶的研究和高效生防制剂开发进行了评述。     

小麦叶锈菌休眠与萌发夏孢子的差异表达

【目的】小麦叶锈菌引起的小麦叶锈病是小麦的重要病害之一,孢子萌发和侵入气孔是保证叶锈菌正常侵染寄主的重要前提。本研究旨在研究小麦叶锈菌夏孢子萌发的差异表达特性,为揭示萌发的机制及其与致病的关系提供依据。【方法】利用小麦叶锈菌致病类型THTT的休眠夏孢子和萌发夏孢子进行RNA-seq分析。【结果】在萌发夏孢子中筛选出相比休眠夏孢子上调表达的基因为4400个,下调基因5325个。GO富集分析发现,上调差异基因主要涉及细胞进程、有机物代谢、信号传导、单个有机体过程、催化酶活性等;下调基因主要涉及单组织过程、单个有机体细胞过程、有机物代谢过程、催化活性调节等过程。利用KEGG分析发现,差异基因共参与109条通路,从中筛选出两条与孢子萌发相关的通路——丝裂原活化蛋白激酶(MAPK)信号通路和囊泡运输中的SNARE蛋白交流。10个基因的定量分析结果与基因数字表达谱分析结果一致。【结论】研究获得了叶锈菌孢子萌发及侵入气孔过程中的重要差异基因,研究结果为研究叶锈菌致病机制奠定基础。     

细胞骨架在植物抗锈病中的作用

锈菌是真菌中一个很大的类群,由锈菌侵染引起的病害可造成世界范围内大多数重要作物锈病的大流行和严重的产量损失。细胞骨架包括微管和微丝,在植物生命活动中担负着复杂的生理功能。越来越多的实验证明,细胞骨架在植物抗锈病中起着重要的作用。该文着重对国内外有关植物与锈菌相互作用过程中细胞骨架重组、活性氧积累、过敏性坏死反应发生、细胞骨架结合蛋白功能、细胞信号转导方面的研究进展进行综述,为深入了解植物抗锈性遗传机制并最终应用于植物锈病的防治奠定基础。     

鞘锈菌属一新种

本文报道了寄生在山牛蒡上鞘锈菌一新种，山牛蒡鞘锈菌。其形态特征有拉丁文和汉文描述，主模式标本存放在东北林业大学，等模式标本放在中国科学院微生物研究所真菌标本室。     

石楠锈孢锈菌重寄生拟盘多毛孢的分离鉴定及抑菌活性

重寄生拟盘多毛孢因其特殊生境,可产生区别于内生或病原拟盘多毛孢的次生代谢产物,是有待开发的真菌资源。为了获得活性较好的石楠锈孢锈菌重寄生菌,采用组织分离法,从患有锈病的球花石楠叶片上分离纯化出3株疑似拟盘多毛孢真菌,它们形态特征差异明显,通过回接实验证实其均为石楠锈孢锈菌的重寄生菌。对3菌株在不同培养基上的培养性状和显微特征作了描述,并对ITS序列进行了扩增和序列分析。形态学和分子鉴定结果证实这3株真菌均为拟盘多毛孢属真菌。抑菌实验结果表明,3株重寄生菌除了对石楠锈孢锈菌的锈孢子有破坏作用外,对其他10种常见植物病原真菌也有一定的抑制作用。     

Impact of histology on the treatment outcome of metastatic or recurrent renal cell carcinoma

Among 107 renal cell carcinoma (RCC) patients with histopathologic subtype diagnosis who were treated at Albert Einstein Cancer Center with cytokines over a 10-year period, seven patients had sarcomatoid histology, 63 had clear cell carcinoma, and 10 patients had mixed histology (sarcomatoid and clear cell). Regardless of their histology, patients with a disease free interval of 2 years or more had significantly better survival. None of the patients with sarcomatoid histology responded to therapy. However, several patients with mixed and clear cell histology achieved partial or complete responses following high dose interleukin-2 (IL-2) therapy. The median survival of patients with sarcomatoid histology was the shortest (13.8 months), whilst that of patients with mixed and clear cell histology was much longer (34.8 months and 39.1 months, respectively). This result was statistically significant in both univariate and multivariate survival analysis (P<0.001 andP<0.01, respectively). Patients with mixed and clear cell histology had no significant difference in survival, and their median survival combined was significantly longer than that of patients with sarcomatoid histology (P<0.0001 in univariate analysis,P<0.01 in multivariate analysis). This study suggests that survival with a diagnosis of RCC is predicted by tumor histology and disease free interval, and this impacts on the ability to respond to standard therapy. Patients with mixed and clear cell histology respond to cytokine therapy. Other therapies should be sought for patients with sarcomatoid RCC.     

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs^1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.     

优化教学活动,提高教学质量——组织胚胎学教学方法的探索与实践

对组织胚胎学课程的教学改革模式进行分析,并根据多年教学实践经验,结合中医院校的教学现状,提出要从优化教学内容、创新教学手段、采用多种教学方法等方面入手,以进一步提高组织胚胎学课程的教学质量.     

Correlation between AgNOR count and subjective AgNOR pattern assessment score in cytology and histology of breast lumps.

OBJECTIVE: To find the correlation of argyrophilic nucleolar organizer region (AgNOR) count and subjective AgNOR pattern assessment (SAPA) score in cytology and histology of breast lumps. STUDY DESIGN: The study group consisted of 73 patients (46 malignant, 27 benign) with breast lumps. In all cases, fine needle aspiration cytology (FNAC) samples and histologic specimens were studied by conventional and silver staining for AgNORs. RESULTS: AgNOR count and SAPA score were helpful in differentiating benign from malignant tumors in both the cytologic smear and histologic specimen. AgNOR count was 6.94+/-2.74 in FNAC and 6.57+/-2.73 in histology of malignant tumors, while in benign tumors it was 2.75+/-0.74 in FNAC and 2.68+/-0.77 in histology. SAPA score was 9.02+/-4.60 in FNAC and 8.76+/-2.34 in histology in malignant tumors and 5.87+/-0.93 in FNAC and 5.82+/- 0.83 in histology in benign tumors. CONCLUSION: Both AgNOR count and SAPA score gave similar results, but SAPA score is a more convenient, reproducible and rapid method of AgNOR evaluation.     

Oviduct development and its relation to other aspects of reproduction in domesticated canaries

The histology and development of the oviduct of unpaired female canaries during the natural breeding season were examined. The histology resembles that of the fowl. The tubular glands develop by invagination of the epithelial cells, and albumen granules subsequently form in their cytoplasm. The epithelium differentiates into ciliated columnar cells and goblet cells. The histology of the magnum is uniform along its length at all stages of development.
Oviduct development is closely correlated with that of the ovary. In its early stages there appears to be little correlation between oviduct development and that of the brood patch or of nest-building. The formation of albumen coincides with the enlargement of one ovarian follicle more than the rest, the completion of defeathering, and intensive nest-building behaviour.     

A study of diagnostic methods for Marteilioides chungmuensis infections in the Pacific oyster Crassostrea gigas

The eggs of the Pacific oyster, Crassostraea gigas, become infertile when infected by the parasite Marteilioides chungmuensis. Histologically, M. chungmuensis infects the oyster oocyte cytoplasm, and the ovaries take on a "lumpy" appearance once infected, which lowers commercial value of the oyster. This has a negative economic impact on oyster farms in South Korea and Japan. In this study, we compared traditional diagnostic methods (histology) with two molecular-based methods (polymerase chain reaction [PCR] amplification and in situ hybridization [ISH]) to identify M. chungmuensis-infected oysters. The efficacy of PCR and ISH to identify M. chungmuensis-infected oysters was compared to that of routine histology in 100 oysters. Thirty infections were identified using PCR and 16 using histology, whereas 31 infections were identified using ISH. The ISH and PCR assays were more sensitive compared to using histology with standard epidemiological methods. We strongly recommend that early parasitic invasion should be monitored with PCR/ISH methodologies as a basis for developing effective diagnostic techniques to identify M. chungmuensis-infected oysters.     

Cytohormonal and histomorphologic patterns in endometrium in symptomatic premenopausal women.

Endometrial histology in the premenopausal symptomatic group revealed hyperplastic endometrium in 29.1 percent of the cases. Cytohormonal evaluation has shown hyperesterin activity reflected by a high Karyopyknotic Index in cystic glandular and adenomatous hyperplasia. A fairly good positive correlation is seen between colpocytology and endometrial histology. In few patients vaginal cytology has revealed regressive activity which was not evident on histology alone. It is felt that cases showing regressive smears are the ones where treatment including estrogens should be most effective.