A two-dimensional electrophoresis reference map of human ovary

The ovary plays a central role in oogenesis and gonadal hormone secretion. Proteomic analysis is a valuable approach for gaining an increased understanding of the molecular nature of the ovary. In this work, two-dimensional electrophoresis for protein separation followed by matrix-assisted laser desorption/ionization mass spectrometry and database searches, identified 231 protein spots corresponding to 138 individual proteins that were found in gels representing both the follicular and luteal phases. The data were used to construct a database online (). The identified proteins were functionally classified into seven groups: (1) cell signaling/communication, (2) cell division, (3) gene/protein expression, (4) metabolism, (5) cell structure and motility, (6) cell/organism defense, and (7) unclassified. Among the proteins identified, 47% had not been previously reported in the human ovary. In addition, a number of disease-related proteins were identified in this protein map, including some cancer- and polycystic ovarian syndrome-related proteins. Two proteins with phosphorylation were verified by Western blot analysis. Comparison of protein abundance between follicular and luteal stages produced seven protein spots that had been identified in our database. This study provides a preliminary reference map of normal human ovary that will form a basis for comparative studies on normal and pathological conditions of the human ovary and may serve as a potential tool for clinical diagnosis, therapeutics, and prognosis.Electronic Supplementary Material Supplementary material is available in the online version of this article at L. Wang and Y.-F. Zhu contributed equally to this work     

Intracellular antibodies for proteomics

The intracellular antibody technology has many applications for proteomics studies.

The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen–antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology.

We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins.

The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.     

Genetic regulation of thymic involution

In this review, we have summarized our work using combined complex statistical genetics, bioinformatics, and functional genomics to determine the genetic basis of the age-related thymic involution in C57BL/6J X DBA/2J recombinant inbred mice and the parental B6 and D2 mice. We have shown that these mice provided a valuable genetic model that can permit resampling of thymuses from different aged but genetically identical animals and determination of the relative significance of age-associated changes in the thymus. Our results suggest that the quantitative trait loci (QTL) regulating the Con A-induced thymocyte proliferative response were mapped to mouse chromosome Chr 11 (D11Mit51 at 18 cM), a region that harbors the IL-12b gene. The importance of IL-12b in maintaining thymic integrity and function during the aging process was confirmed by a more rapid involution of the thymus in IL-12b knockout (IL-12b-/-) mice compared to wild-type (WT) mice. Functionally, IL-12 provided a strong synergistic effect to augment the IL-7 or IL-2 induced thymocyte proliferative response, especially in both aged WT and IL-12b-/- mice, but not in normal young mice. In contract to the proliferative response, the age-related decline in the total number of thymocytes was determined at different age, and mapped to loci on Chr 9, 62 cM and Chr 10, 32 cM. Using matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS), increased expression of peroxiredoxin was found to be correlated with thymic involution. Our results suggest the possibility to identify the complex molecular network that can be associated with the regulation of thymic involution in aged mice using a high-dimensional functional genomics approach.     

Systems biology: integrating technology,biology, and computation

The Human Genome Project has changed the worlds of biology and medicine-helping to catalyze two major paradigm changes: systems biology and predictive, preventive and personalized medicine. These two themes will dominate 21st century biology and medicine. I will discuss these changes and indicate how they may interface with with the process of aging.     

Obesity,metabolic health and omics: Current status and future directions

The growing obesity epidemic is becoming a major public health concern, and the associated costs represent a considerable burden on societies. Among the most common complications of severe obesity are the development of hypertension, dyslipidemia, type 2 diabetes, cardiovascular disease, and various types of cancer. Interestingly, some obese individuals have a favorable metabolic profile and appear to be somehow protected from the detrimental effects of excessive adipose tissue accumulation. These individuals remain normoglycemic, insulin sensitive, and hypotensive with proper blood lipid levels, despite their high body mass index and/or waist circumference. Multiple independent observations have led to the concept of the metabolically healthy obese (MHO) phenotype, yet no consensus has been reached to date regarding a universal definition or the main mechanism behind this phenomenon. Recent technological advances and the use of high-throughput analysis techniques have revolutionized different areas of biomedical research. A multi-omics approach, which is used to investigate changes at different molecular levels in an organism or tissue, may provide valuable insights into the interplay between the molecules or pathways and the roles of different factors involved in the mechanisms underlying metabolic health deterioration. The aim of this review is to present the current status regarding the use of omics technologies to investigate the MHO phenotype, as well as the results of targeted analyses conducted in MHO individuals.     

Proteomics reveals a global phenotypic shift of NK cells in HCV patients treated with direct-acting antivirals

Chronic hepatitis C virus (HCV) infections compromise natural killer (NK)-cell immunity. Direct-acting antivirals (DAA) effectively eliminate HCV, but the long-term effects on NK cells in cured patients are debated. We conducted a proteomic study on CD56⁺ NK cells of chronic HCV-infected patients before and 1 year after DAA therapy. Donor-variation was observed in NK-cell proteomes of HCV-infected patients, with 46 dysregulated proteins restored after DAA therapy. However, 30% of the CD56⁺ NK-cell proteome remained altered 1 year post-therapy, indicating a phenotypic shift with low donor-variation. NK cells from virus-negative cured patients exhibited global regulation of RNA-processing and pathways related to “stimuli response”, “chemokine signaling”, and “cytotoxicity regulation”. Proteomics identified downregulation of vesicle transport components (CD107a, COPI/II complexes) and altered receptor expression profiles, indicating an inhibited NK-cell phenotype. Yet, activated NK cells from HCV patients before and after therapy effectively upregulated IFN-γ and recruited CD107a. Conversely, reduced surface expression levels of Tim-3 and 2B4 were observed before and after therapy. In conclusion, this study reveals long-term effects on the CD56⁺ NK-cell compartment in convalescent HCV patients 1 year after therapy, with limited abundance of vesicle transport complexes and surface receptors, associated with a responsive NK-cell phenotype.     

苍耳子对小鼠脾淋巴细胞蛋白质组影响的双向电泳分析

目的观察苍耳子对致敏小鼠脾淋巴细胞蛋白质表达的影响.方法NIH小鼠正常组腹腔注射生理盐水,造模组和治疗组均注射羊红细胞(SRBC)致敏,治疗组经致敏后以苍耳子水煎剂灌胃,处理4 d后,分离小鼠脾淋巴细胞;提取总蛋白,双向凝胶电泳(2-DE)后,以计算机图像分析软件分析电泳图谱.结果致敏会引起多种蛋白质表达发生变化,而苍耳子可逆转其中部分蛋白质的变化.结论苍耳子可改善由致敏引起的小鼠脾淋巴细胞中部分蛋白表达的变化.     

蛋白质组学技术及其在中药复杂体系研究中的应用

系统生物学在中药复杂体系研究中的可行性和合理性已经获得了中药研究者的认可。在后基因组时代,蛋白质组学是系统生物学研究的重要组成部分,蛋白质组学在中药研究中的应用也已经有了一些成功的实例。作者在近年来部分研究工作的基础上,对蛋白质组学的主要研究技术及蛋白质组学在中药复杂体系研究中的应用思路进行了综述和探讨。