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Journal Cover Journal of Integrated OMICS
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  This is an Open Access Journal Open Access journal
   ISSN (Print) 2182-0287 - ISSN (Online) 2182-0287
   Published by Proteomass Homepage  [1 journal]
  • Vol 7, No 1 (2017)

    • Authors: José Luís Capelo Martínez
      PubDate: 2017-06-07
      Issue No: Vol. 7 (2017)
       
  • Identification of NEK3 interacting proteins and functional
           characterization of its signaling mechanisms

    • Authors: Vanessa B Cardoso, Talita Hanchuk, Edmarcia de Souza, Priscila Papa, Gabriela Meirelles, Jorg Kobarg
      Abstract: NEKs (NIMA-related kinases) are a group of kinases that share high amino acid sequence identity to NIMA (Never in mitosis gene A), which exists as a single member in the fungi Aspergillus nidulans and is functionally involved in the initiation of mitosis. NEK3 is a 506 amino acid serine/threonine kinase, localizes both to the nucleus and cytoplasm, and its gene localizes to 13q14.2 chromosome. It has an N-terminal catalytic domain and a C-terminal regulatory domain, which contains Thr475 in its PEST domain, which gets phosphorylated upon activation. Expression of mutants without Thr475 or PEST domain causes changes in cellular morphology and polarity of neuronal cells. NEK3 is also involved in cell motility and invasiveness of breast cancer tumor cells through interaction with regulators of the Rho GTPases Rac1 and RhoA, mediated by prolactin induced association of NEK3 to the human Prolactin Receptor (PRLR). Using the Matchmaker Gold Yeast Two-Hybrid system, a screening for interaction partners wasperformed and 65 clones were obtained, which cDNAs encode 27 different proteins. The identified candidate interacting proteins are functionally involved in sumoylation, ubiquitinylation, transcriptional regulation, DNA repair, RNA processing, and the regulation of cell proliferation, invasiveness and metastasis.Some interaction partners for NEK3 are located in the nucleus and plasma membrane but most of them localize to the cytoplasm. One of the cytoplasmatic interactors for NEK3, RhoGDI2, is a regulator of RhoGTPases and inhibits Rac1 and RhoA activation levels. In our pull down assay, NEK3 overexpression increases Rac1-GTP while concomittant overexpression of RhoGDI2 reducesit to non-detectable levels. Our data suggests that NEK3 interaction with RhoGDI2 is a important new regulatory elements in Rac1 signaling pathways.
       
      PubDate: 2017-05-06
      Issue No: Vol. 7 (2017)
       
  • A novel extraction method for the preparation of heparinized chicken
           (Gallus gallus domesticus) and horse (Equus caballus) whole blood for
           1H-NMR metabolomics using Drabkin’s reagent

    • Authors: Jennifer N Niemuth, Michael K Stoskopf
      Abstract: Despite the ease of collection, heparinized whole blood (HWB) is underutilized in 1H-NMR-based metabolomics particularly because of the lack of sample homogeneity.  Drabkin’s reagent (DR), an aqueous solution of potassium ferricyanide, potassium cyanide, and sodium bicarbonate, causes hemolysis and has been used for quantification of hemoglobin.  The objectives of this study were to determine if the use of DR with HWB for metabolomics samples would result in consistent hemolysis, while being invisible to 1H-NMR and quenching metabolic activity.  HWB from a chicken (Gallus gallus domesticus) and a horse (Equus caballus) was used.  All HWB samples were mixed 1:10 volume:volume HWB:DR.  Spectrophotometric evaluation of incubated DR treated samples revealed little change in absorbance after approximately 10-15 min incubation at room temperature (20-21 °C); all following samples were incubated for 10 min, except where noted.  Samples subjected to five repeated extractions with DR showed a decrease in absorbance of  >95% after the first extraction; all subsequent samples were extracted with a single aliquot of DR.  Lyophilized DR rehydrated with a 100% deuterium oxide solution was invisible to 1H-NMR.  Standard (10 min incubation, 20 min centrifugal filtration) and delayed (120 min incubation, 20 min centrifugal filtration) samples were prepared for both species and did not appear considerably different upon visual inspection with the exception of minor differences in the major peaks of the metabolites 3-methylhistidine and betaine in the chicken samples.  Comparison of standard and delayed samples via two-sample Kolmogorov-Smirnov tests found no significant differences with either species (chicken p = 1, horse p = 0.9887).  Use of DR resulted in consistent, complete hemolysis, while being invisible to 1H-NMR and quenching metabolic activity for at least 140 min at room temperature.  This protocol should be considered when HWB is the only sample type available and/or if the investigator is interested in questions specific to erythrocyte metabolism.
      PubDate: 2017-04-12
      Issue No: Vol. 7 (2017)
       
  • Comparative integrated omics approach sterically understanding hepatic
           metabolic dynamics in mouse model

    • Authors: Shoko Takahashi, Huijuan Jia, Kenji Saito, Hisanori Kato
      Abstract: Currently, the general approach of analysis is using a single omics technique, however the combined analysis of data by employing multiple omics analyses can enable a more fundamental understanding of the biological phenomena. Multi-layered combination of multiple omics technologies involves generation of a large amount of data, which leads to increased complexity and makes comprehension of bio-information more difficult. The objective of this study was to investigate the utility of incorporating multiple omics technologies in a multi-layered fashion. Transcriptomic, proteomic, and metabolomic analyses were carried out using a mouse model of diet-induced obesity. The present study reported the comprehensiveness of three omics analyses and the utility of using multiple omics analyses. Uniform changes were observed among changes at all stages but the majority of these specific to the omics approach. This data supports the fact that various molecules progress through the central dogma at differing speeds. Since the time axis differs for each molecule, combining multiple omics analyses makes it possible to investigate the reactions in organisms three-dimensionally. At first glance, it simply appears that combining a number of very large data sets produces even more complexity but, if multi-layered omics data are treated with an awareness of their meaning, benefits, and limitations, then the combination of multiple omics analyses can be extremely useful for research in molecular biology.
      PubDate: 2017-01-18
      Issue No: Vol. 7 (2017)
       
 
 
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