Molecular BioSystems

Quanjun, Yang; Genjin, Yang; Lili, Wan; Bin, Li; Jin, Lu; Qi, Yu; Yan, Li; Yonglong, Han; Cheng, Guo; Junping, Zhang

doi: 10.1039/c3mb70134dpmid: 24056883

Cancer cachexia is a complex metabolic syndrome that cannot be fully reversed by conventional nutritional support, and leads to the progressive wasting of body tissues, particularly the loss of lean muscle mass. Formoterol, a highly selective β2-adrenoceptor therapeutic drug, gives potential anabolic responses in the context of skeletal muscles and was widely confirmed to possess anti-cachexia effects. However, the possible metabolic pathways and the metabolite changes that initiate and maintain these anabolic responses remain poorly understood. In the present study, a 1H NMR-metabonomics model was established to investigate the metabolic features of cancer cachexia and the contribution of formoterol to serum metabolites in a mouse model bearing CT26 carcinoma cells. Among the metabolic processes found in serum, the ones associated with cancer are glycolysis and lipid lipolysis. However, the citrate cycle and amino acid metabolism are the major metabolic characteristics of cachexia. Furthermore, formoterol stimulated skeletal muscle growth, increased the body weight and altered the metabolic profile. Amino acids, ketone bodies and citrate cycle metabolites are potential biomarkers associated with these functional pathways. Taking the pathways of cancer cachexia into account, formoterol could regulate the imbalance in glycolysis, the citrate cycle, and in lipid and amino acid metabolism. Collectively, these results indicate that formoterol partially reverses the metabolic disturbances associated with cachexia.

Li, Wan; Chen, Lina; Li, Xia; Jia, Xu; Feng, Chenchen; Zhang, Liangcai; He, Weiming; Lv, Junjie; He, Yuehan; Li, Weiguo; Qu, Xiaoli; Zhou, Yanyan; Shi, Yuchen

doi: 10.1039/c3mb70289h

Liu, Qi; Ullery, Jody; Zhu, Jing; Liebler, Daniel C.; Marnett, Lawrence J.; Zhang, Bing

doi: 10.1039/c3mb70114jpmid: 24056865

Reactive electrophiles produced during oxidative stress, such as 4-hydroxynonenal (HNE), are increasingly recognized as contributing factors in a variety of degenerative and inflammatory diseases. Here we used the RNA-seq technology to characterize transcriptome responses in RKO cells induced by HNE at subcytotoxic and cytotoxic doses. RNA-seq analysis rediscovered most of the differentially expressed genes reported by microarray studies and also identified novel gene responses. Interestingly, differential expression detection at the coding DNA sequence (CDS) level helped to further improve the consistency between the two technologies, suggesting the utility and importance of the CDS level analysis. RNA-seq data analysis combining gene and CDS levels yielded an informative and comprehensive picture of gradually evolving response networks with increasing HNE doses, from cell protection against oxidative injury at low dose, initiation of cell apoptosis and DNA damage at intermediate dose to significant deregulation of cellular functions at high dose. These evolving dose-dependent pathway changes, which cannot be observed by the gene level analysis alone, clearly reveal the HNE cytotoxic effect and are supported by IC50 experiments. Additionally, differential expression at the CDS level provides new insights into isoform regulation mechanisms. Taken together, our data demonstrate the power of RNA-seq to identify subtle transcriptome changes and to characterize effects induced by HNE through the generation of high-resolution data coupled with differential analysis at both gene and CDS levels.

Huang, Xiaoqin; Zheng, Fang; Zhan, Chang-Guo

doi: 10.1039/c3mb70231fpmid: 24057047

Homology modeling and molecular dynamics simulations have been carried out to model the detailed structures of the human neonatal Fc receptor (FcRn) binding with the wild-type Fc of human immunoglobulin G1 (IgG1) and its various mutants. Based on the modeled human FcRn–Fc binding structures, it has been proposed that the protein–protein binding interface is composed of three subsites. The first subsite is a hydrophobic core where residue I39 of human Fc can be accommodated very well, and the other two subsites are all composed of critical salt bridges between human FcRn and human Fc. All of the modeled structures and the calculated binding energies are qualitatively consistent with the available experimental data, suggesting that the modeled human FcRn–Fc binding structures are reasonable. The modeled human FcRn–Fc binding structure may be valuable for future rational design of novel mutants of human Fc and Fc-fused therapeutic proteins with a potentially higher binding affinity for human FcRn and, thus, a longer in vivo half-life in humans.

Sun, Meng; Gao, Xueqin; Zhang, Dongwei; Ke, Chaofu; Hou, Yan; Fan, Lijun; Zhang, Ruoxi; Liu, Haixia; Li, Kang; Yu, Bo

doi: 10.1039/c3mb70216bpmid: 24061630

Unstable angina (UA) is one of the most dangerous types of coronary heart disease and has high mortality and morbidity rates worldwide. However, the diagnostic accuracy for UA is unsatisfactory in clinical practice. In this study, we investigated the application of plasma metabolomics in discovering potential biomarkers for the diagnosis of UA. Plasma samples from 45 UA and 43 atherosclerosis (AS) in-patients were collected and analyzed using rapid resolution liquid chromatography quadrupole time-of-flight mass spectrometry (RRLC-QTOF/MS) in both positive and negative ion modes. Good separations were observed between the UA patients and AS controls. Tandem mass spectrometry experiments were carried out to identify biomarker candidates that contributed most to the discrimination (VIP > 1.2 and p < 0.05). Sixteen potential endogenous biomarkers for UA were identified, and those could perform a satisfactory diagnostic accuracy for discrimination between UA and AS patients (AUC = 0.9143). In the UA patients compared to the AS controls, the plasma concentrations of 12 metabolites were higher while the concentrations of four metabolites were lower. In conclusion, our study demonstrated that plasma metabolomics analyzed by RRLC-QTOF/MS had great potential in biomarker discovery for UA. These biomarkers could not only be helpful for the diagnosis of patients with UA, but also provide more information for further understanding of the metabolic processes of UA.

Breydo, Leonid; Mikheeva, Larissa M.; Madeira, Pedro P.; Zaslavsky, Boris Y.; Uversky, Vladimir N.

doi: 10.1039/c3mb70329kpmid: 24072065

In an aqueous two-phase system (ATPS), the partitioning of a protein is defined by the differential interactions of the protein with aqueous media in the two phases. Our study shows that partitioning of proteins in a set of ATPSs of different ionic compositions can be used to quantify structural differences between α-synuclein, its variants and several globular proteins. Since application of ATPSs implies the use of high concentrations of two polymers in water when a certain threshold concentration of the polymers is exceeded, and since these levels of polymer concentrations are similar to those commonly used to mimic the effects of macromolecular crowding on proteins, we used circular dichroism spectroscopy to evaluate the structural consequences of placing proteins in solutions with high polymer concentrations and various ionic compositions.

Chen, Qing-yong; Jiao, De-min; Wu, Yu-quan; Wang, Lishan; Hu, Hui-zhen; Song, Jia; Yan, Jie; Wu, Li-jun

doi: 10.1039/c3mb70288jpmid: 24077187

Metastasis is a common feature of lung cancer, involving relationships between genes, proteins and miRNAs. However, lack of early detection and limited options for targeted therapies are weaknesses that cantribute to the dismal statistics observed in lung cancer metastasis. In this paper, gene expression profiling analysis for genes differentially expressed between high- (95D) and low-metastatic lung cancer cell lines (95C) was performed using gene annotation, pathway analysis, literature mining, and the integrated regulatory network as well as motif analysis of miRNA–DEG and TF–DEG. In addition, the expression of EGR-1 (early growth reponse-1) in surgically resected lung squamous carcinomas, adenocarcinomas and normal lung tissue was detected by immunohistochemistry to reveal the relationships between EGR-1 and lung cancer metastasis. A total of 570 different expressed genes (DEGs) were screened, the vast majority of up-regulated DEGs were connected to cell adhesion and focal adhesion. EGR-1 was observed in the center node of the regulatory network, which seems to play a role in the process of cancer metastasis, and further immunohistochemistry detection confirmed this reasoning. Besides EGR-1, several significant module-related DEGs were enriched in the pathway within cancer and focal adhesion according to KEGG pathway enrichment analysis of network modules. The construction of an integrated regulatory network and the functional prediction of EGR-1 provided us with the cytological basis of lung cancer metastasis research and an understanding of the mechanism of metastasis in lung cancer. EGR-1 should be considered as a potential target gene in therapeutic agent for lung cancer metastasis.

Huang, Chao; Ba, Qian; Yue, Qingxi; Li, Junyang; Li, Jingquan; Chu, Ruiai; Wang, Hui

doi: 10.1039/c3mb70342hpmid: 24085322

Artemisinin and related compounds (artemisinins), as a frontline treatment for malaria, have been used to save millions of lives. Their potential application in cancer treatment is promising. Nevertheless, the precise mechanisms of action of artemisinins are still controversial. In particular, the system-level influence of artemisinins on protein interactions and regulatory networks remains unknown, limiting progress in development of this class of compounds as anticancer drugs. In the present study, we investigated the mechanism of action of artemisinins in cancer therapy through an analysis based on biological networks. According to experimental evidence from more than 400 literature studies, 558 key proteins were derived and the artemisinins-rewired protein interaction network was constructed. Topological properties were analyzed to show that the protein network was a scale-free biological system. And the modularity analysis and pathway identification were performed. Five key pathways including PI3K-Akt, T cell receptor, Toll-like receptor, TGF-beta and insulin signaling pathways were involved in artemisinins-mediated anticancer effects; their identification was confirmed by microarray data. Based on these results, predictions were made about the targets of artemisinins in various pathways. These results provide a deeper understanding of the molecular mechanisms of action of artemisinins and will contribute to the development and application of this class of compounds in cancer treatment.

Patkari, Minal; Mehra, Sarika

doi: 10.1039/c3mb70341jpmid: 24100886

Soil organisms exhibit resistance to a wide range of antibiotics as they either need to protect themselves from endogenous antibiotics or from those present in their soil environment. The soil could serve as a reservoir for resistance mechanisms that have already emerged or have the potential to emerge in clinically important bacteria. Streptomyces coelicolor, a non-pathogenic soil-dwelling organism, is thus used as a model for the study of intrinsic resistance. Preliminary screening of several compounds showed that S. coelicolor had high intrinsic resistance for the fluoroquinolone group of antibiotics. We subjected the bacteria to sub-inhibitory concentrations of ciprofloxacin and studied the transcriptomic response using microarrays. The data were supported with various biochemical and phenotypic assays. Ciprofloxacin treatment leads to differential expression of many genes with enhanced mRNA expression of its target, DNA gyrase gene. High induction of DNA repair pathways was also observed and many transporters were upregulated. Ciprofloxacin was found to induce ROS formation in a dose dependent manner. Reduction of ROS via anti-oxidants increased the effective MIC of the drug in the bacteria. The regulation of antibiotic resistance in S. coelicolor was studied systematically and contribution of different mechanisms in the development of resistance was assessed. Our data suggest that multiple mechanisms work in coordination to facilitate the cell to combat the stress due to ciprofloxacin.

Chandraseelan, Jerome G.; Oliveira, Samuel M. D.; Häkkinen, Antti; Tran, Huy; Potapov, Ilya; Sala, Adrien; Kandhavelu, Meenakshisundaram; Ribeiro, Andre S.

doi: 10.1039/c3mb70203kpmid: 24104727

We studied the behaviour of the repressilator at 28 °C, 30 °C, 33 °C, and 37 °C. From the fluorescence in each cell over time, we determined the period of oscillations, the functionality (fraction of cells exhibiting oscillations) and the robustness (fraction of expected oscillations that occur) of this circuit. We show that the oscillatory dynamics differs with temperature. Functionality is maximized at 30 °C. Robustness decreases beyond 30 °C, as most cells exhibit ‘failed’ oscillations. These failures cause the distribution of periods to become bimodal, with an ‘apparent period’ that is minimal at 30 °C, while the true period decreases with increasing temperature. Based on previous studies, we hypothesized that the failures are due to a loss of functionality of one protein of the repressilator, CI. To test this, we studied the kinetics of a genetic switch, formed by the proteins CI and Cro, whose expression is controlled by PRM and PR, respectively. By probing the activity of PRM by in vivo detection of MS2-GFP tagged RNA, we find that, beyond 30 °C, the production of the CI-coding RNA changes from sub-Poissonian to super-Poissonian. Given this, we suggest that the decrease in efficiency of CI as a repressor with temperature hinders the robustness of the repressilator beyond 30 °C. We conclude that the repressilator is sensitive but not robust to temperature. Replacing CI for a less temperature-dependent protein should enhance robustness.