Articles Online (Volume 12, Issue 6)

Protocol

Systematic Characterization of Cell Cycle Phase-dependent Protein Dynamics and Pathway Activities by High-content Microscopy-assisted Cell Cycle Phenotyping

Christopher Bruhn,Torsten Kroll,Zhao-Qi Wang

Cell cycle progression is coordinated with metabolism, signaling and other complex cellular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping (hiMAC), which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hiMAC is compatible with cell types from any species and allows for statistically powerful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular localization at all cell cycle stages within a single sample. For illustration, we provide a hiMAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3−4- day protocol, which can be adjusted to any other cell cycle stage-dependent analysis.
细胞周期进程协调着细胞生理代谢、信号传导等复杂生物学功能。解析细胞周期特异的生物学机制是现代分子细胞生物学研究的一个热点。通常,为鉴别不同细胞周期需通过药物来诱导细胞周期同步化并对细胞采用细胞周期特异分子进行免疫荧光染色。但是,细胞同步化药物对细胞造成的非生理性损伤,不同类型细胞对药物敏感性的差异,加之细胞周期特异性抗体在实验选择和应用上的局限,均妨碍精准研究细胞活性及机制。在本研究中,我们创建了一种高通量细胞周期分析方法(hiMAC,全称为高通量显微细胞周期分析术)。该技术可在非同步化细胞中,通过免疫荧光显微镜技术实现高分辨率细胞周期分析。hiMAC技术可以运用在各种细胞类型中, 对同一样品和多样本同步在不同细胞周期进行蛋白-蛋白相互作用、蛋白质修饰及其亚细胞定位等检测,并且可对结果进行高效的统计学分析。基于该技术平台,我们开发了分析程序包,可以在3-4天内完成对不同细胞周期中细胞DNA损伤应答反应和基因组稳定性分析。同时,该程序包也适用于其他细胞周期特异性的功能研究。
Der Zellzyklus wird mit Signaltransduktionswegen, Stoffwechsel, und anderen komplexen Zellfunktionen koordiniert. Die Aufklärung mechanistischer Zusammenhänge zwischen Zellzyklusphase und verschiedenen zellulären Vorgängen ist das Ziel vieler molekularer und zellbiologischer Studien. Zellzyklus-Synchronisation und Immunfärbung von Zellzyklusmarkern ermöglichen derartige Analysen, sind jedoch in ihrem Nutzen begrenzt durch die Induktion von unphysiologischem experimentellen Stress, durch die Abhängigkeit vom untersuchten Zelltyp, und oft durch geringe Flexibilität. Hier beschreiben wir „high-content microscopy-assisted cell cycle phenotyping“ (hiMAC), eine Methode, welche die Generierung hochauflösender Zellzyklus-Profile von asynchronen Zellpopulationen mit Immunfluoreszenz-Mikroskopie zusammenführt. hiMAC ist mit Zelltypen beliebiger Spezies kompatibel und ermöglicht statistisch aussagekräftige, objektive und simultane Analysen der Interaktion, Modifikation und subzellulären Lokalisierung von Proteinen in allen Zellzyklusphasen in einer einzigen Probe. Zur Veranschaulichung stellen wir eine hiMAC Pipeline zur Verfügung, welche auf die Analyse der DNA-Schadensantwort und der genomischen Instabilität in einem 3-4-Tage-Protokoll zugeschnitten ist und auf beliebige andere Zellzyklus-abhängige Analysen angepasst werden kann.

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Research Article

In vitro Transcriptome Analysis of Two Chinese Isolates of Streptococcus suis Serotype 2

Dake Zhang,Nan Du,Sufang Ma,Qingtao Hu,Guangwen Lu,Wei Chen,Changqing Zeng

The Streptococcus suis serotype 2 (S. suis 2) isolates 05ZYH33 and 98HAH33 have caused severe human infections in China. Using a strand-specific RNA-seq analysis, we compared the in vitro transcriptomes of these two Chinese isolates with that of a reference strain (P1/7). In the 89K genomic island that is specific to these Chinese isolates, a toxin–antitoxin system showed relatively high levels of transcription among the S. suis. The known virulence factors with high transcriptional activity in these two highly-pathogenic strains are mainly involved in adhesion, biofilm formation, hemolysis and the synthesis and transport of the outer membrane protein. Furthermore, our analysis of novel transcripts identified over 50 protein-coding genes with one of them encoding a toxin protein. We also predicted over 30 small RNAs (sRNAs) in each strain, and most of them are involved in riboswitches. We found that six sRNA candidates that are related to bacterial virulence, including cspA and rli38, are specific to Chinese isolates. These results provide insight into the factors responsible for the difference in virulence among the different S. suis 2 isolates.

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Research Article

Identification of Differentially-expressed Genes in Intestinal Gastric Cancer by Microarray Analysis

Shizhu Zang, Ruifang Guo,Rui Xing,Liang Zhang,Wenmei Li,Min Zhao,Jingyuan Fang,Fulian Hu,Bin Kang,Yonghong Ren,Yonglong Zhuang,Siqi Liu, Rong Wang,Xianghong Li,Yingyan Yu , Jing Cheng,Youyong Lu

Gastric cancer (GC) is one of the most frequent malignant tumors. In order to systematically characterize the cellular and molecular mechanisms of intestinal GC development, in this study, we used 22 K oligonucleotide microarrays and bioinformatics analysis to evaluate the gene expression profiles of GC in 45 tissue samples, including 20 intestinal GC tissue samples, 20 normal appearing tissues (NATs) adjacent to tumors and 5 noncancerous gastric mucosa tissue samples. These profiles allowed us to explore the transcriptional characteristics of GC and determine the change patterns in gene expression that may be of clinical significance. 1519 and 1255 differentially-expressed genes (DEGs) were identified in intestinal GC tissues and NATs, respectively, as determined by Bayesian analysis (P < 0.001). These genes were associated with diverse functions such as mucosa secretion, metabolism, proliferation, signaling and development, which occur at different stages of GC development.

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Research Article

Expression Profiling and Structural Characterization of MicroRNAs in Adipose Tissues of Hibernating Ground Squirrels

Cheng-Wei Wu, Kyle K. Biggar, Kenneth B. Storey

MicroRNAs (miRNAs) are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 miRNAs in brown (BAT) and white adipose tissue (WAT) during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six miRNAs including let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 was significantly downregulated in WAT (P < 0.05), which was 16%–54% of euthermic non-torpid control squirrels, whereas expression of three miRNAs including miR-143, miR-200a and miR-519d was found to be upregulated by 1.32–2.34-fold. Similarly, expression of more miRNAs was downregulated in BAT during torpor. We detected reduced expression of 6 miRNAs including miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 (48%–70% of control), while only expression of miR-138 was significantly upregulated (2.91 ± 0.8-fold of the control, P < 0.05). Interestingly, miRNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas miRNAs with altered expression in BAT during torpor were linked to mitochondrial β-oxidation. miRPath target prediction analysis showed that miRNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase (MAPK) signaling, while the miRNAs upregulated in WAT were linked to transforming growth factor β (TGFβ) signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-miRNAs for the miRNAs used in this study, suggesting no structure-influenced changes in pre-miRNA processing efficiency in the squirrel. As well, the expression of miRNA processing enzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of miRNA expression in adipose tissues may be linked to distinct biological roles in WAT and BAT during hibernation and may involve the regulation of signaling cascades.

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Letter

Association Between rs1344706 of ZNF804A and Schizophrenia: A Meta-analysis

Meiyan Zhu,Tongyang Liu, Jihong Zhang,Shuting Jia,Wenru Tang, Ying Luo

Schizophrenia is one of the most serious mental diseases found in humans. Previous studies indicated that the single nucleotide polymorphism (SNP) rs1344706 in the gene ZNF804A encoding zinc finger protein 804A was associated with schizophrenia in Caucasian population but not in Chinese Han population. However, current results are conflicting in Asian population. In the present study, a meta-analysis was performed to revisit the association between rs1344706 and the risk of schizophrenia in Asian, Caucasian and other populations. Electronic search of PubMed database identified 25 case–control studies with available genotype frequencies of rs1344706 for the meta-analysis, involving a total of 15,788 cases and 22,654 controls. A pooled odds ratio (OR) with 95% confidence interval (CI) was used to assess the association. The current meta-analysis showed an association between rs1344706 and schizophrenia in Caucasian populations (P = 0.028, OR = 1.138, 95% CI: 1.014–1.278; P = 0.004 for heterogeneity) and Asian populations (P = 0.008, OR = 1.092, 95% CI: 1.023–1.165; P = 0.001 for heterogeneity), but not in other populations (P = 0.286, OR = 1.209, 95% CI: 0.853–1.714, P = 0.120 for heterogeneity). Egger’s test (P > 0.05) and Begg’s test (P > 0.05) are both suggestive of the lack of publication bias for the included studies. Thus, the absence of association in other populations suggests a genetic heterogeneity in the susceptibility of schizophrenia and demonstrates the difficulties in replicating genome-wide association study findings regarding schizophrenia across different ethnic populations. To validate the association between rs1344706 and schizophrenia, further studies with larger participant populations worldwide are needed.

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