EPRS

Abstract: In spite of succesful adoption of electronic patient records (EPR) by Norwegian GPs, what constitutes the actual benefits and effects of the use of EPRs in the perspective of the GPs and patients has not been fully characterized. We wanted to study primary care physicians' use of electronic patient record (EPR) systems in terms of use of different EPR functions and the time spent on using the records, as well as the potential effects of EPR systems on the clinician-patient relationship. A combined qualitative and quantitative study that uses data collected from focus groups, observations of primary care encounters and a questionnaire survey of a random sample of general practitioners to describe their use of EPR in primary care. The overall availability of individual patient records had improved, but the availability of the information within each EPR was not satisfactory. GPs' use of EPRs were efficient and comprehensive, but have resulted in transfer of administrative work from secretaries to physicians. We found no indications of disturbance of the clinician-patient relationship by use of computers in this study. Although GPs are generally satisfied with their EPRs systems, there are still unmet needs and functionality to be covered. It is urgent to find methods that can make a better representation of information in large patient records as well as prevent EPRs from contributing to increased administrative workload of physicians.

Abstract: Cytoplasmic aminoacyl-tRNA synthetases of higher eukaryotes acquired extra peptides in the course of their evolution. It has been thought that these appendices are related to the occurrence of the multiprotein complex con- sisting of at least eight different tRNA synthetase polypep- tides. This complex is believed to be a signature feature of metazoans. In this study, we used multiple sequence align- ments to infer the locations of the peptide appendices from human cytoplasmic tRNA synthetases found in the multisyn- thetase complex. The selected peptide appendices ranged from 22 aa of aspartyl-tRNA synthetase to 267 aa of methionyl- tRNA synthetase. We then made genetic constructions to investigate interactions between all 64 combinations of these peptides that were individually fused to nonsynthetase test proteins. The analyses identified 11 (10 heterologous and 1 homologous) interactions. The six peptide-dependent interac- tions paralleled what had been detected by crosslinking methods applied to the isolated multisynthetase complex. Thus, small peptide appendices seem to link together different synthetases into a complex. In addition, five interacting pairs that had not been detected previously were suggested from the observed peptide-dependent complexes. Proteins are molecular fossils that may help to unravel the history and mechanism of evolution. Aminoacyl-tRNA syn- thetases (ARSs) are one of the ancient proteins evolved to decode genetic information to amino acids. Although all of the tRNA synthetases catalyze the same chemical reactions, these enzymes have accumulated a wide range of sequence and structural diversity throughout evolution (1). In particular, the tRNA synthetases of higher eukaryotes have acquired a few features that are not present in those of other organisms. Most intriguing is the presence of noncatalytic peptide appendices, the roles of which are yet to be elucidated. It has been thought that these peptides are involved in protein-protein interactions among the tRNA synthetases that form the multiprotein complex. This complex is another characteristic of the cyto- plasmic tRNA synthetases of higher eukaryotes (2-4) and contains at least eight different tRNA synthetase polypeptides (Glu-Pro-tRNA synthetase (EPRS), IRS, LRS, MRS, QRS, RRS, KRS, and DRS; refs. 5-8), as well as three nonsynthetase proteins of 43 kDa, 38 kDa, and 18 kDa (5-11). Although these complexes have been known for more than two decades, the structural organization and interactions between the components have not been well understood. Structural analysis with electron microscopy has shown that the complex forms an elongated U shape (8). The complex structure has been studied extensively by stepwise dissociation of the components by using nonionic detergent (12, 13), changing salt concentration (14), and chemical crosslinking (15, 16). The multi-ARS complex has been proposed to consist of three subdomains. The base subdomain consists of EPRS, IRS, and LRS, which are the enzymes of higher molecular weights. There are two arm subdomains on the top of the base subdomain. Subdomain I contains a dimer of DRS and mono- mers of MRS and QRS; subdomain II is made of dimers of KRS and RRS (16). Most of the structural analyses on the multi-ARS complex have relied on biochemical approaches, as listed above. Be- cause these methods address only the physical relationships of the protein components, other approaches are necessary to determine the molecular mechanisms and the peptide regions involved in the assembly or maintenance of the complex. We thought that a genetic approach would be suitable to investi- gate these questions. A yeast two-hybrid system has proven useful to analyze protein-protein interactions (17). This method was employed previously to analyze the interactions of the repeated motifs of EPRS with the C-terminal repeated motifs of IRS (18), as well as with the N-terminal extension of RRS (19). The interaction between the repeats of EPRS and IRS was confirmed further by biochemical and biophysical methods (19). Interactions of p38 with the other ARS com- ponents recently have been reported by using two-hybrid analysis (11). The role of the peptide appendices in the formation of the multi-ARS complex has been studied in a few cases such as the N-terminal sequences of DRS (20, 21), RRS (22, 23), and KRS (6). However, systematic studies on the structural features and molecular interactions of these peptides in eukaryotic ARSs are needed to understand their functional significance. In the present study, we focused on the peptide appendices deduced from the complex-forming ARSs. We investigated whether these regions actually are involved in protein-protein interac- tions among the complex-forming ARSs and, if so, how they are connected to each other. Finally, the interactions between the unique peptides of complex-forming tRNA synthetases determined by the genetic method in this work were compared with those previously obtained by biochemical methods.

Abstract: Introduction: Abnormal status of gene expression plays an important role in tumorigenesis, progression and metastasis of breast cancer. Mechanisms of gene silence or activation were varied. Methylation of genes may contribute to alteration of gene expression. This study aimed to identify differentially expressed hub genes which may be regulated by DNA methylation and evaluate their prognostic value in breast cancer by bioinformatic analysis. Methods: GEO2R was used to obtain expression microarray data from GSE54002, GSE65194 and methylation microarray data from GSE20713, GSE32393. Differentially expressed-aberrantly methylated genes were identified by FunRich. Biological function and pathway enrichment analysis were conducted by DAVID. PPI network was constructed by STRING and hub genes was sorted by Cytoscape. Expression and DNA methylation of hub genes was validated by UALCAN and MethHC. Clinical outcome analysis of hub genes was performed by Kaplan Meier-plotter database for breast cancer. IHC was performed to analyze protein levels of EXO1 and Kaplan-Meier was used for survival analysis. Results: 677 upregulated-hypomethylated and 361 downregulated-hypermethylated genes were obtained from GSE54002, GSE65194, GSE20713 and GSE32393 by GEO2R and FunRich. The most significant biological process, cellular component, molecular function enriched and pathway for upregulated-hypomethylated genes were viral process, cytoplasm, protein binding and cell cycle respectively. For downregulated-hypermethylated genes, the result was peptidyl-tyrosine phosphorylation, plasma membrane, transmembrane receptor protein tyrosine kinase activity and Rap1 signaling pathway (All p< 0.05). 12 hub genes (TOP2A, MAD2L1, FEN1, EPRS, EXO1, MCM4, PTTG1, RRM2, PSMD14, CDKN3, H2AFZ, CCNE2) were sorted from 677 upregulated-hypomethylated genes. 4 hub genes (EGFR, FGF2, BCL2, PIK3R1) were sorted from 361 downregulated-hypermethylated genes. Differential expression of 16 hub genes was validated in UALCAN database (p<0.05). 7 in 12 upregulated-hypomethylated and 2 in 4 downregulated-hypermethylated hub genes were confirmed to be significantly hypomethylated or hypermethylated in breast cancer using MethHC database (p<0.05). Finally, 12 upregulated hub genes (TOP2A, MAD2L1, FEN1, EPRS, EXO1, MCM4, PTTG1, RRM2, PSMD14, CDKN3, H2AFZ, CCNE2) and 3 downregulated genes (FGF2, BCL2, PIK3R1) contributed to significant unfavorable clinical outcome in breast cancer (p<0.05). High expression level of EXO1 protein was significantly associated with poor OS in breast cancer patients (p=0.03). Conclusion: Overexpression of TOP2A, MAD2L1, FEN1, EPRS, EXO1, MCM4, PTTG1, RRM2, PSMD14, CDKN3, H2AFZ, CCNE2 and downregulation of FGF2, BCL2, PIK3R1 might serve as diagnosis and poor prognosis biomarkers in breast cancer by more research validation. EXO1 was identified as an individual unfavorable prognostic factor. Methylation might be one of the major causes leading to abnormal expression of those genes. Functional analysis and pathway enrichment analysis of those genes would provide novel ideas for breast cancer research.