Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.     

虹膜定位技术在斜视手术中的应用研究

目的 以虹膜定位技术为客观定量检查方法,结合临床观察斜视患者手术前、后眼球的客观旋转状态,探讨其在手术中的作用和意义,为手术改善和完善、测量标准提供新的依据.方法 收集2009年10月至2011年1月入院的共同性外斜视、上斜肌麻痹的患者各40例,并随机分为显微手术组、非显微手术组,使用WaveScan波前像差仪取坐位行虹膜识别,获得虹膜图像和数据,使用美国威视公司VISX Star S4-IR准分子激光系统行仰卧位定位,记录术前、术后虹膜(眼球)旋转的角度.结果 主斜眼、主视眼手术前、后眼球的旋转度进行配对t检验,主斜眼、主视眼的非显微手术组与显微手术组进行两样本t检验.①共同性外斜视非显微手术组:主视眼手术前、后眼球旋转度分别为(4.88±2.55)°、(4.76±2.62)°,差异无统计学意义.主斜眼手术前、后眼球旋转度分别为(2.70±2.36)°、(6.00±2.76)°,差异有统计学意义.显微手术组:主视眼手术前、后眼球旋转度分别为(2.86±2.28)°、(3.12±2.17)°,差异无统计学意义.主斜眼手术前、手术后眼球旋转度分别为(2.08±1.86)°、(3.28±2.04)°,差异有统计学意义.主斜眼非显微手术组与显微手术组进行两样本t检验,术前差异无统计学意义,术后差异有统计学意义.②上斜肌麻痹非显微手术组:主视眼手术前、手术后眼球旋转度分别为(2.88±3.58)°、(2.08±2.36)°,差别无统计学意义.主斜眼手术前、手术后眼球旋转度分别为(2.48±2.51)°、(5.73±1.98)°,差别有统计学意义.显微手术组:主视眼手术前、手术后眼球旋转度分别为(4.90±3.60)°、(4.56±1.12)°,差异无统计学意义.主斜眼手术前、手术后眼球旋转度分别为(3.12±3.07)°、(4.26±1.98)°,差异有统计学意义.主斜眼非显微手术组与显微手术组进行两样本f检验,术前两组差异无统计学意义,术后两组差异有统计学意义.结论 ①显微手术可以提高斜视手术的精细度.②虹膜定位技术可以客观地描述斜视患者的眼球旋转状态,减少手术源性旋转,并且为临床手术效果做出评价.     

Whole Exome Sequencing Reveals Uncommon Mutations in the Recently Identified Fanconi Anemia Gene SLX4/FANCP

Fanconi anemia (FA) is a rare genetic disorder characterized by congenital malformations, progressive bone marrow failure (BMF), and susceptibility to malignancies. FA is caused by biallelic or hemizygous mutations in one of 15 known FA genes, whose products are involved in the FA/BRCA DNA damage response pathway. Here, we report on a patient with previously unknown mutations of the most recently identified FA gene, SLX4/FANCP. Whole exome sequencing (WES) revealed a nonsense mutation and an unusual splice site mutation resulting in the partial replacement of exonic with intronic bases, thereby removing a nuclear localization signal. Immunoblotting detected no residual SLX4 protein, which was consistent with abrogated interactions with XPF/ERCC1 and MUS81/EME1. This cellular finding did not result in a more severe clinical phenotype than that of previously reported FA‐P patients. Our study additionally exemplifies the versatility of WES for the detection of mutations in heterogenic disorders such as FA.     

Mutations in the COPII Vesicle Component Gene SEC24B are Associated with Human Neural Tube Defects

Neural tube defects (NTDs) are severe birth malformations that affect one in 1,000 live births. Recently, mutations in the planar cell polarity (PCP) pathway genes had been implicated in the pathogenesis of NTDs in both the mouse model and in human cohorts. Mouse models indicate that the homozygous disruption of Sec24b, which mediates the ER‐to‐Golgi transportation of the core PCP gene Vangl2 as a component of the COPII vesicle, will result in craniorachischisis. In this study, we found four rare missense heterozygous SEC24B mutations (p.Phe227Ser, p.Phe682Leu, p.Arg1248Gln, and p.Ala1251Gly) in NTDs cases that were absent in all controls. Among them, p.Phe227Ser and p.Phe682Leu affected its protein stability and physical interaction with VANGL2. Three variants (p.Phe227Ser, p.Arg1248Gln, and p.Ala1251Gly) were demonstrated to affect VANGL2 subcellular localization in cultured cells. Further functional analysis in the zebrafish including overexpression and dosage‐dependent rescue study suggested that these four mutations all displayed loss‐of‐function effects compared with wild‐type SEC24B. Our study demonstrated that functional mutations in SEC24B might contribute to the etiology of a subset of human NTDs and further expanded our knowledge of the role of PCP pathway‐related genes in the pathogenesis of human NTDs.     

Assessment of naturally occurring covalent and total dimer levels in human IgG1 and IgG2

Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally.     

Differential expression and subcellular localization of Prohibitin 1 are related to tumorigenesis and progression of non-small cell lung cancer

Lung cancer remains the leading cause of cancer-related deaths worldwide and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer. With a variety of biological functions, Prohibitin1 (PHB1) has been proved tumor-associated. But there are conflicting data regarding the involvement of PHB1 in tumorigenesis and few studies regarding the role of PHB1 in lung cancer. The studies reported herein used a combination of clinical observations and molecular methods to investigate the possible role of PHB1 in NSCLC tissues and cell lines. PHB1 expression was evaluated by RT-PCR, RT-qPCR, Western blotting and immunohistochemistry analysis. Flow cytometric analysis was used to determine the surface expression profiles of PHB1 in lung cell lines. The results showed that PHB1 expression were generally increased in lung cancer tissues when compared with matched noncancerous tissues and closely related with tumor differentiation and lymph node invasion. PHB1 expression levels was also increased in three lung cancer cell lines (SK-MES-1, NCI-H157 and NCI-H292) as compared with BEAS-2B cells. Moreover, there were various subcellular localization of PHB1 in different lung cancer cells and the presence of PHB1 on the surface of lung cancer cells was significantly reduced. In conclusion, PHB1 expression is increased in NSCLC and the up-regulation of PHB1 is associated with clinically aggressive phenotype. The different subcellular localization of PHB1 in NSCLC cells and the loss of the membrane-associated PHB1 probably related to the tumorigenesis and progression of NSCLC and suggests that PHB1 may play different roles in various types of NSCLC.