Diagnosis of disease-specific proteins in cerebrospinal fluid of children infected with tuberculous meningitis

BACKGROUND: Isolated mycobacterium tuberculosis from cerebrospinal fluid (CSF) is regarded as the "gold standard" for diagnosis of tuberculous meningitis (TBM). However, culture of CSF specimens is time-consuming and lacks sensitivity. There is a strong need to determine complementary disease-specific markers, which are essential for increasing early diagnosis and improving prognosis in patients with TBM. OBJECTIVE: To establish proteomic profiles of CSF in TBM and normal children using two-dimensional polyacrylamide gel electrophoresis, and to screen for disease-specific proteins. DESIGN, TIME AND SETTING: The case-control study was conducted at the Department of Pediatrics, Xiangya Hospital of Central South University and the Key Laboratory of Cancer Proteomics of Ministry of Public Health of China between January 2008 and January 2009. PARTICIPANTS: The TBM group included three patients with a strongly positive tuberculin skin test, as well as positive CSF mycobacterial staining and culture, who were admitted to the Department of Pediatrics, Xiangya Hospital from January 2008 to January 2009. Three healthy, age- and gender-matched children served as the control group. METHODS: CSF proteins were separated using two-dimensional polyacrylamide gel electrophoresis in both groups. Gels were scanned using Image scanner and LabScan software. Differentially expressed proteins were analyzed using PDQuest 7.0 software. The clearly discernible spots, which were expressed only in the TBM group, were chosen to perform matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. MAIN OUTCOME MEASURES: Differentially expressed spots on CSF profiles of TBM and normal children were measured.RESULTS: Following comparison of two-dimensional polyacrylamide gel electrophoresis maps between TBM and control groups, 546 and 533 spots were detected, respectively. A total of 64 differentially expressed proteins were observed between the groups, including 15 upregulated spots, eight downregulated spots, 27 spots that were exclusively expressed in the TBM group, and 14 spots that were exclusively expressed in the control group. At total of 20 spots that were exclusively expressed in the TBM group were chosen for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, and 20 peptide mass fingerprints were obtained. After searching the data base, 16 proteins were matched. CONCLUSION: Two-dimensional polyacrylamide gel electrophoresis profiles of the CSF proteome were successfully established in the TBM and normal children. Parts of these differentially expressed proteins were identified through mass spectrometry and bioinformatics. Results indicated that apolipoprotein A-I, anti-tumor necrosis factor-alpha antibody, crystal structure of MRP14 and HLA class II histocompatibility antigen DRB1-4 could be closely correlated with TBM pathogenesis.     

Proteomic Approach for the Elucidation of Biological Defects in Autism

Proteomic-based approaches, which examine expressed proteins in tissues or cells, have great potential in the elucidation of biological defects in heterogeneous neurodevelopmental disorders such as autism. In this approach, tissue or cellular proteins from control and affected subjects are separated on two-dimensional (2-D) polyacrylamide gel electrophoresis, and those proteins that show marked changes in the concentration between control and affected subjects are identified by mass spectroscopy. This method has been successfully applied in the elucidation of the molecular biological defect in classic late-infantile neuronal ceroid lipofuscinosis (Sleat et al., 1997). Unlike the classical methods of genome-wide screening for chromosomal localization followed by positional cloning, the proteomic approach requires limited number of tissue samples and the study can be completed in a relatively short time. Currently, these methods are available for relatively abundant proteins and generally are not applicable for hydrophobic proteins because 2-D gel electrophoresis is not very effective in the analysis of hydrophobic proteins. The genetic defect results in either total loss of proteins or changes in molecular weight and/or isoelectric point will be detectable by the proteomic method. Because autism is a neurogenetic disorder, brain is the tissue of choice for proteomic study. For an oligogenic disorder such as autism, at least some of the aberrant (genes) proteins may be identified by this technology.     

骨骼肌碳酸酐酶Ⅲ具有磷酸酶活性

目的：探讨骨骼肌碳酸酐酶Ⅲ／（CAⅢ）是否本身具有磷酸酶活性。方法：用双向电泳结合Western blot方法将分离的大鼠骨骼肌蛋白转移至硝酸纤维膜，利用镜像原理，识别与CAⅢ抗体发生免疫反应的抗原在双向电泳图谱中的确切位置。在此基础上，以同样的方法与原理，对在固相膜上的磷酸酶活性进行原位染色，观察CAⅢ是否磷酸酶活性染色呈阳性。此外，通过磷酸酶抑制剂实验及酶催化反应动力学实验，论证CAⅢ的磷酸酶活性染色特异性及CAⅢ具有磷酸酶的功能。结果：CAⅢ抗体反应显示，利用镜像原理能从复杂的双向电泳图谱中容易识别CAⅢ蛋白；磷酸酶活性染色表明，鉴定的CAⅢ具有磷酸酶活性。磷酸酶特异抑制剂实验表明，CAHI的磷酸酶活性染色具有特异性；酶催化反应动力学研究证实，CAⅢ蛋白具有确凿的磷酸酶功能。结论：骨骼肌的CAⅢ本身具有磷酸酶活性，说明CAⅢ参与了细胞的磷酸化——去磷酸化过程，提示重症肌无力（MG）患者骨骼肌收缩无力和易疲劳可能涉及了MG的非免疫机制。另外，本研究创建的双向电泳结合Westem blot方法可被借鉴用于“功能蛋白质组学”的研究。     

Three-Dimensional Echocardiographic Evaluation of Aortic and Mitral Valve Stenosis

Dynamic volume rendered three-dimensional echocardiography allows the spatial recognition of anatomy and function of the aortic and mitral valves with acceptable image quality. The aortic valve can be best visualized in a view from the ascending aorta down to the valve level, thus allowing an overview of the aortic aspect of the valve in a surgeon's perspective in ∼ 80% of patients. Planimetric measurement of the aortic valve area was possible in 88% of patients, and there is no systematic overestimation or underestimation of aortic valve area compared with two-dimensional echocardiography and catheterization. The entire valvular circumference of the mitral valve can be assessed from both a left atrial and a left ventricular perspective. Advantages of the three-dimensional transesophageal echocardiography mitral valve area determination compared with transthoracic two-dimensional planimetry and Doppler-derived pressure half-time method are present in patients with severely calcified mitral valves and in those with combined aortic regurgitation.     

Comparison of purified Dermatophagoides pteronyssinus allergens and extract by two-dimensional immunoblotting and quantitative immunoglobulin E inhibitions

BACKGROUND: The allergens of the house dust mite (Dermatophagoides pteronyssinus, Der p), one of the most important indoor allergen sources, occur as isoallergens that differ in their amino acid sequence. These variations may influence allergenic activity and thus may have impact on diagnostic tests and specific immunotherapy. OBJECTIVE: We investigated whether single purified recombinant mite allergens contain the IgE epitopes of the natural Der p isoallergens. METHODS: A panel of purified recombinant (rDer p 2, 5, 7, 8, 10 and 14) and two natural (nDer p 1 and 4) mite allergens were used to establish IgE reactivity profiles of Der p allergic patients and to inhibit IgE reactivity to two-dimensionally separated Der p isoallergens. In addition, we determined the percentage of Der p extract-specific IgE which could be preadsorbed with a mixture of purified mite allergens (nDer p 1, rDer p 2, 5, 7, 8 and 10) from sera of mite-allergic patients (n=18) in a non-denaturing RAST-based inhibition. RESULTS: We demonstrate that single recombinant mite allergens inhibit IgE reactivity to the corresponding natural isoallergens. A mixture of purified mite allergens (nDer p 1, rDer p 2, 5, 7, 8 and 10) bound on an average 76% of Der p-specific IgE antibodies. CONCLUSION: The studied recombinant and natural mite allergens contain a large portion of Der p-specific IgE and may be used for diagnostic tests and therapy of Der p allergy.     

22. Proteomic Analysis of Differential Protein Expression in vero Cell with Antisense Blocking of Relevant Gene Involved in inhibition of Nontargeted Mutagenesis

Objective: Recent studies have demonstrated that cells exposed to ionizing radiation or alkylating agents can develop prolonged genetic instability. But its mechanism is still unknown. A cDNA fragment (fragment 9) has been isolated in MNNG-exposed vero cell by mRNA differential display in this lab. After antisense blocking the expression of its relevant gene (fragment 9 related gene, FNR gene), we found that nontargeted mutation frequency induced by MNNG was enhanced significantly. which implicated that the product of the blocked gene may be involved in the inhibition of nontargeted mutation. In order to elucidate the functional mechanism of the FNR gene, we try to separate the proteins from the established cell line expressing antisense fragment 9 to find out the FNR gene-coded protein. Method: The total cellular proteins of MNNG-exposed vero cell transfected with antisense RNA expression plasmid (vero-pM-amp--9-) and those with vector DNA (vero-pM-amp-) were separated by two-dimensional gel electrophoresis, and the resulting maps were analyzed with 2-D analysis software packages to find out the differentially expressed protein spots. Then the related 2-D PAGE database (http://biobase.dk/cgi-bin/celis/) was searched according to the protein spots information obtained from 2-DE including the position in the gel, isoelectric point (pl) and molecular weight (Mr). Result: Twelve proteins were specifically expressed only in vero-pM-amp-, and 2 proteins in vero-pM-amp--9-. In addition, there were 24 proteins expressed in higher level in vero-pM-amp--9- as compared with vero-pM-amp- (P<0.05), among them the expression of 7 proteins were enhanced by greater than 5 folds. On the other hand, no sequence similarity was found by homology analysis in GenBank through comparing the fragment 9 with the cDNA sequences of those proteins found in this study. Conclusion: Gene expression alterations bave occurred after antisense blocking of the FNR gene expression as demonstrated by proteomic analysis, and the FNR gene may code a gene expression regulatory factor.     

不同切端全瓷贴面的破坏力学分析

目的应用真实破裂过程分析(RFPA)软件分析不同切端瓷贴面的破坏过程。方法使用RFPA软件在模拟的上颌中切牙矢状面解剖外形上,根据临床常用的4种牙体预备外型:I型、L型、U型和贴面冠型,建立全瓷贴面模型12种,分析比较不同设计的瓷贴面复合体的破损过程。结果不同切端类型的瓷贴面在对刃加载条件下,破损出现的位置均在粘接剂层。I型和L型最先出现粘接剂破损的位置在切端贴面与牙体交界区,U型和贴面冠型则在舌侧贴面与牙体交界区或颈缘。I型、U型和贴面冠型所承受的最大负荷间无明显差异,L型承受的最大对刃负荷明显小于其他3种设计。结论提高贴面复合体强度的最直接方法就是选择高强度的粘接剂,L型贴面复合体的抵抗对刃负荷能力较差。     

Recent Progress in Double-Layer Honeycomb Structure: A New Type of Two-Dimensional Material

Two-dimensional (2D) materials are no doubt the most widely studied nanomaterials in the past decade. Most recently, a new type of 2D material named the double-layer honeycomb (DLHC) structure opened a door to achieving a series of 2D materials from traditional semiconductors. However, as a newly developed material, there still lacks a timely understanding of its structure, property, applications, and underlying mechanisms. In this review, we discuss the structural stability and experimental validation of this 2D material, and systematically summarize the properties and applications including the electronic structures, topological properties, optical properties, defect engineering, and heterojunctions. It was concluded that the DLHC can be a universal configuration applying to III–V, II–VI, and I–VII semiconductors. Moreover, these DLHC materials indeed have exotic properties such as being excitonic/topological insulators. The successful fabrication of DLHC materials further demonstrates it is a promising topic. Finally, we summarize several issues to be addressed in the future, including further experimental validation, defect engineering, heterojunction engineering, and strain engineering. We hope this review can help the community to better understand the DLHC materials timely and inspire their applications in the future.