双孢蘑菇子实体发育后期差异表达蛋白质分析

为探讨双孢蘑菇子实体发育后期的蛋白质表达变化,对双孢蘑菇As2796子实体采收期、成熟期和开伞期的蛋白质组进行了双向电泳(2-DE)分析,发现了16个表达差异明显的蛋白质。通过质谱分析(MALDI-TOF/TOF MS)和数据库检索,有14个差异蛋白质获得鉴定。其中磷酸烯醇式丙酮酸水合酶与能量代谢相关,T-蛋白复合体1、蛋白酶体、5-甲基四氢三谷氨酸-同型半胱氨酸甲基转移酶、1-吡咯琳-5-羧酸脱氢酶、精氨酸酶与氨基酸或蛋白质代谢直接相关,而GTP结合蛋白则参与细胞的多种生命活动,在细胞的生长发育过程中起着重要的作用。另外7个为功能未知的蛋白质。     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Patterns of DNA methylation in the normal colon vary by anatomical location,gender, and age

Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.     

An evaluation of analysis pipelines for DNA methylation profiling using the Illumina HumanMethylation450 BeadChip platform

The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.     

Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔV_Tot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔV_Tot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔV_Tot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔV_Tot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.     

Profiling and integrated analysis of differentially expressed circRNAs as novel biomarkers for breast cancer

Breast cancer (BC) is a globally common cancer with the highest and increasing morbidity and mortality among females. Novel biomarkers are warranted to be discovered for the early detection, treatment, and prognosis of BC. In this study, we investigated the profiles of differentially expressed (DE) circular RNAs (circRNAs) by competing endogenous RNAs (ceRNA) microarray to construct a genome-wide circRNA profile. Then, we performed Gene Ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis of the host genes (HGs) of circRNAs. A total of 4,370 DE circRNAs were detected and GO and KEGG analysis showed that they were significantly associated with cell cycle, DNA replication, BC, and familial BC. We validated the differential circRNAs and relevant HGs through quantitative real-time polymerase chain reaction and constructed a putative circRNA–microRNA–messenger RNA regulatory network. Eight circRNAs, including hsa_circ_0069094, hsa_circ_0062558, hsa_circ_0074026, hsa_circ_0079876, hsa_circ_0017536, hsa_circ_0023302, hsa_circ_0017650, and hsa_circ_0017545, were validated significantly DE in BC tissue and associated with TNM staging, lymph node infiltration, and Ki67. Hsa_circ_0069094, hsa_circ_0079876, hsa_circ_0017650, and hsa_circ_0017526 were upregulated in plasma. This study revealed the general expression characteristics of specific DE circRNAs in BC and hsa_circ_0069094, hsa_circ_0079876, hsa_circ_0017650, and hsa_circ_0017526 might be promising candidate targets.     

Characterization of the first adult de novo case of 46,X,der(Y)t(X;Y)(p22.3;q11.2)

Herein, we describe a case of an infertile man detected in postnatal diagnosis with FISH characterization and array-CGH used for genome-wide screening which allowed the identification of a complex rearrangement involving sex chromosomes, apparently without severe phenotypic consequences. The deletion detected in our patient has been compared with previously reported cases leading us to propose a hypothetical diagnostic algorithm that would be useful in similar clinical situations, with imperative multi disciplinary approach integrated with genetic counseling. Our patient, uniquely of reproductive age, is one of six reported cases of duplication of Xp22.3 (~ 8.4 Mb) segment and contemporary deletion of Yq (~ 42.9 Mb) with final karyotype as follows:

46,X,der(Y),t(X;Y)(Ypter → Yq11.221::Xp22.33 → Xpter).ish der(Y) (Yptel+,Ycen+,RP11-529I21+,RP11-506M9-Yqtel −,Xptel +). arrXp22.33p22.31(702–8,395,963, 8,408,289x1), Yq11.221q12 (14,569,317x1, 14,587,321–57,440,839x0)     

Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody

Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (bp 600–746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers, including those of prostate, breast, colon, lung, brain, and ovary, as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most nonmalignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation.     

Proteomics: A Paradigm Shift

ABSTRACT

Proteome—the protein complement of a genome—has become the protein renaissance and a key research tool in the post-genomic era. The basic technology involves the routine usage of gel electrophoresis and spectrometry procedures for deciphering the primary protein sequence/structure as well as knowing certain unique post-translational modifications that a particular protein has undergone to perform a specific function in the cell. However, the recent advancements in protein analysis have ushered this science to provide deeper, bigger and more valuable perspectives regarding performance of subtle protein-protein interactions. Applications of this branch of molecular biology are as vast as the subject is and include clinical diagnostics, pharmaceutical and biotechnological industries. The 21st century hails the use of products, procedures and advancements of this science as finer touches required for the grooming of fast-paced technology.