Abstract:Autophagy which is a process that damaged organelles and cellular proteins are degraded by lysosomes plays a critical role in cellular homeostasis. Previous studies have demonstrated that autophagy influenced the vascular function, even involved in the pathophysiology of vascular diseases. In this review we focus on the effect of autophagy on the vascular function and vascular diseases such as atherosclerosis, abdominal aortic aneurysm, pulmonary hypertension and diabetes mellitus. Moreover, we try to provide a new way of vascular diseases treatment from the point of autophagy.

Abstract:CD4 CD25 regulatory T cells (Treg cells) play crucial roles in the maintenance of self-immune tolerance. Decreased numbers or functional deficiency of Treg cells result in development of a variety of autoimmune diseases like multiple sclerosis and typeⅠ diabetes. Moreover, Treg cells have been implicated in inflammation and transplantation settings. Two types of Treg cells including thymic-derived natural Treg (nTreg) and peripheral induced Treg (iTreg) cells have been identified based on their distinct developmental origin. nTreg cells are derived in the thymus and express high level of Forkhead transcription factor Foxp3, nTreg cells exhibit the suppressive function mainly in a cell-cell contact manner. nTreg cells develop in two steps largely shaped by TCR, co-stimulatory and cytokines in the thymus. In the present manuscript, we will mainly summarize recent advances regarding molecular and cellular regulation of nTreg development and differentiation.

Abstract:Analysis of the transcriptomes of single cells can provide a comprehensive understanding of the heterogeneous expression and gene regulatory networks within a population of cells. Through mRNA isolation, reverse transcription, amplification and deep-sequencing analysis, the transcriptional landscape can be obtained for subsequent detailed quantitative investigation of the levels and variation of gene expression underlying the relationship between the genotype and phenotype of a single cell. Not surprisingly, it has been applied within many fields such as developmental biology, basic medicine, clinical research and drug discovery. This review introduces the history, progress, and technological characteristics of whole transcriptome analyses of single cells, as well as detailed strategies and advantages of the methodology. The technical challenges, potential applications, and future developments are also discussed.

Abstract:Absolute quantification methodologies, which allow the determination of protein concentrations in biological samples such as cell, tissue and body fluid. Recently, protein quantification methodologies mainly are one which rely on the isotope dilution for absolute quantification of proteome and another one which rely on the statistic analysis of MS data called the lable-free technique. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy) or PrESTs (PrESTs-SILAC). Because isotope dilution can provide accurate and presice absolute quantification, so they are crucial for specific applications such as the evaluation of clinical biomarker candidates and understanding the biological function of proteins. In this review, we present a critical overview of these isotope dilution methodologies.

Abstract:Antibody surface display technology is critical for novel antibody screening and antibody affinity maturation. Currently most frequently used display methods are phage display and yeast display. Although Escherichia coli (E. coli) is easily cultured and genetically manipulated, thus is potentially an excellent display host, the display technology based on E. coli has not been widely used. One of the problems is lack of efficient display of antibodies on the surface of E. coli. Many proteins have been tested as display carriers in outer membrane display in E. coli. Display systems based on autotransporter protein (AT) and ice nucleation protein (INP) is the most extensively studied. Another problem is unstable survival rates of E. coli when antibody is displayed. In this study, we systematically examined display level, antigen-binding affinity of displayed antibody and survival rate of E. coli using Ag43β(β domain of Antigen43, an AT protein) and INPNC (fragment of N-terminal and C-terminal of INP) as carrier proteins and T7, lac, araBAD as promoters for antibody expression. We found that the antigen-binding ability of the Ag43β based display was superior to that of the INPNC based system. As expected, T7, lac and araBAD promoters drove high, medium and low expressions of antibody. The host survival rate using T7 promoter was extremely low (INPNC: 0.0033%, Ag43β: 0.02%, the host bearing araBAD promoter had the highest survival rate (INPNC: 37.80%, Ag43β: 90.23%), and the lac based system had a survival rate of 2.04% (INPNC) and 13.27% (Ag43β). Balancing the antigen-binding abilities, antibody expression levels and survival rates, a system using Ag43β as carrier protein and lac as promoter is the best choice for antibody display on E. coli.

Abstract:In addition to Smad pathway, our previous study has shown that BMP9 can induce osteogenic differentiation of mesenchymal stem cells (MSCs) through p38 MAPKs pathway. In this study, we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-indcued osteogenic differentiation of MSCs. BMP9 was introduced into MSCs by recombinant adenoviruses protocol, then, in vitro and in vivo assays were conducted to detect whether BMP9 can induce osteogenic differentiation of MSCs through JNKs kinase pathway. The results showed that BMP9 can activate JNKs kinase through increase the phosphorylated form of JNKs kinase. JNKs kinase inhibitor SP600125 can inhibit ALP activity, OPN and OCN expression, as well as calcium deposition induced by BMP9 in MSCs. Furthermore, SP600125 also led to a decrease in BMP9-induced Runx2 activity and canonical Smad signaling. Moreover, when JNKs kinase was silenced by RNA interference in MSCs, BMP9-induced osteogenic differentiation in vitro and ectopic bone formation in vivo were accordingly inhibited along with knockdown of JNKs. Taken together, those results intensively suggested that BMP9 can induce and regulate osteogenic differentiation of MSCs through activating JNKs kinase pathway.

Abstract:Our research selected the RNF149, the novel ubiquitin ligase which owned the high identity to GRAIL and belonged to the typeⅠ transmembrane protein, as our object. By the confocal laser scanning microscope, it was demonstrated that RNF149 is located at lysosome and the RNF149 is co-located with CD9. The interactions between RNF149 and CD9 were demonstrated by immune co-precipitation.RNF149 polyubiquitinates CD9 via ubiquitin Lys-48. The HeLa cells were co-transfected with the same quantity of the CD9 plasmids and the gradient increase quantity of the RNF149 plasmids. We found that the exogenous quantity of CD9 was decreasing with the increased expression of the exogenous RNF149. In HEK293T cells, the knocking down RNF149 by shRNA led to the increase of the endogenous CD9. All these evidence suggested that CD9 maybe regulated by RNF149. In addition, the knocking down RNF149 by shRNA led to the inhibition of the cell proliferation in HEK293T cells. This phenomenon suggested that the RNF149 possibly could be considered as the regulatory factor of the cell proliferation.

Abstract:Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an extracellular enzyme capable of producing cyclodextrins through an intramolecular transglycosylation reaction. With the application of cyclodextrins expanding in the industries related to food, pharmaceuticals, cosmetic, etc, CGTase has become the focus of scientific research nowadays. Calcium binding sites widely exit in α-amylase family. Previous studies indicated that these sites had very important roles for α-amylase. It was known that CGTases also possess two or three calcium binding sites. However, their structure and function are not very clear. In the present study, structure and function of calcium binding sites in CGTases were analyzed. Sequence comparisons were performed using the ClustalX 1.8 sequence alignment program. Based on the results and crystal structure analysis, it was found that calcium binding sites CaⅠ and CaⅡ exist commonly in CGTase. Most amino acids at these two calcium binding sites are highly conserved, but the residue 29 at CaⅠ and residue 199 at CaⅡ have significant differences between different types of CGTases. The residue 29 in α-CGTase primarily producing α-cyclodextrin or γ-CGTase primarily producing γ-cyclodextrin is Asp, while others are Asn. The residue 199 in γ-CGTase is Ser, while others are Asp. Calcium binding site CaⅢ only exists in few CGTases. The site consists of residues 315 and 577. In addition, site-directed mutagenesis was used to investigate the functions of calcium binding sites in CGTases. The replacement of Asp29 by Asn and Arg resulted in 23% and 35% increase in β-cyclization activity, respectively. Mutant D29R and D315A showed higher stability than wild-type CGTase at 60℃. Moreover, the mutant D315A had higher β- and γ-cyclodextrin specificity. These results suggested that calcium binding sites might be related to cycling activity, thermal stability, and product specificity of CGTases, which provided the directions for further revealing biological functions of calcium binding sites of CGTases.

Abstract:Amnestic mild cognitive impairment (aMCI) patients show impairment in both item and associative memory. The current study administered the widely used associative and item memory tests from the Clinical Memory Scale to investigate whether aMCI patients are more impaired in associative memory than item memory. In addition, we aimed to analyze an associative memory test (the paired-associate learning test) in order to investigate in detail the nature of associative memory impairment in aMCI patients. Twenty-five individuals with aMCI and 28 healthy controls took part in the study. Two associative memory tests (paired-associate learning test and associative recall), two item memory tests (free recall of pictures and recognition of meaningless figures), and a set of neurocognitive tests were administered to the participants. aMCI patients performed significantly worse than healthy older adults in both associative memory tests, even when controlling for impairment in the item memory tests. In addition, ROC curve analysis indicated that discriminative power was higher in associative than in item memory tests. Further, the results of the paired-associate learning test indicated that, compared to normal older adults, aMCI patients showed more deficits in remembering the easy word pairs, relative to the difficult word pairs. Our results confirm that aMCI patients demonstrate greater deficits in associative memory than in item memory. In addition to the impairment in creating memory links between items, aMCI patients may have deficits in using the semantic information presented by the items. The associative memory tests showed higher discriminative power than item memory tests in the identification of aMCI patients. Including associative memory testing in future neurocognitive assessment protocols could be helpful to improve the "hit rate" of aMCI detection.

Abstract:Current pathway enrichment method is mainly based on the gene that are differentially expressed, and no enrichment method considers pathway variability (variance). We observed that in the phenotype of disease, some pathways have a significant increase or decrease in variability describing appropriate statistics. Therefore, in this article, we hypothesize that the variation of single pathway is significantly different between two phenotypes. We designed fourteen types of statistics coupled with their test methods to analyze pathways variation and the pathways enrichment significance between two phenotypes, and we compared the results with those obtained by document retrieval. At the same time, the results of five different data preprocessing methods on data were investigated. The results show that RMA is stable in the five gene expression data preprocessing methods. The pathway variation is different between the two phenotypes. According to the literature research results, the permutation test coupled with the variance of Euclidean distance of each gene (the eleventh method) can identify significant pathways more efficiently than GSEA. In conclusion, pathway enrichment analysis strategy based on the pathway variation is feasible, which could be a theoretical guideline for enrichment analysis and a new biological insights of study in human diseases.