Abstract Endozoicomonas are prevalent, abundant bacterial associates of marine animals, including corals. Their role in holobiont health and functioning, however, remains poorly understood. To identify potential interactions within the coral holobiont, we characterized the novel isolate Endozoicomonas marisrubri sp. nov. 6c and assessed its transcriptomic and proteomic response to tissue extracts of its native host, the Red Sea coral Acropora humilis. We show that coral tissue extracts stimulated differential expression of genes putatively involved in symbiosis establishment via the modulation of the host immune response by E. marisrubri 6c, such as genes for flagellar assembly, ankyrins, ephrins, and serpins. Proteome analyses revealed that E. marisrubri 6c upregulated vitamin B1 and B6 biosynthesis and glycolytic processes in response to holobiont cues. Our results suggest that the priming of Endozoicomonas for a symbiotic lifestyle involves the modulation of host immunity and the exchange of essential metabolites with other holobiont members. Consequently, Endozoicomonas may play an important role in holobiont nutrient cycling and may therefore contribute to coral health, acclimatization, and adaptation.
The use of mass spectrometry to verify and quantify biomarkers requires the identification of the peptides that can be detectable. In this paper, we propose the use of genetic programming (GP) to measure the detection probability of the peptides. The new GP method is tested and verified on two different yeast data sets with increasing complexity and shows improved performance over other state-of-art classification and feature selection algorithms.
The physiology and central metabolism of a ppc mutant Escherichia coli were investigated based on the metabolic flux distribution obtained by 13C-labelling experiments using gas chromatography-mass spectrometry (GC-MS) and 2-dimensional nuclear magnetic resonance (2D NMR) strategies together with enzyme activity assays and intracellular metabolite concentration measurements. Compared to the wild type, its ppc mutant excreted little acetate and produced less carbon dioxide at the expense of a slower growth rate and a lower glucose uptake rate. Consequently, an improvement of the biomass yield on glucose was observed in the ppc mutant. Enzyme activity measurements revealed that isocitrate lyase activity increased by more than 3-fold in the ppc mutant. Some TCA cycle enzymes such as citrate synthase, aconitase and malate dehydrogenase were also upregulated, but enzymes of glycolysis and the pentose phosphate pathway were downregulated. The intracellular intermediates in the glycolysis and the pentose phosphate pathway, therefore, accumulated, while acetyl coenzyme A and oxaloacetate concentrations decreased in the ppc mutant. The intracellular metabolic flux analysis uncovered that deletion of ppc resulted in the appearance of the glyoxylate shunt, with 18.9% of the carbon flux being channeled via the glyoxylate shunt. However, the flux of the pentose phosphate pathway significantly decreased in the ppc mutant.
Aims The cancer stem cell concept proposes that tumor growth and recurrence is driven by a small population of cancer stem cells (CSCs). In this study we investigated the expression of induced-pluripotent stem cell (iPSC) markers and their localization in primary low-grade adenocarcinoma (LGCA) and high-grade adenocarcinoma (HGCA) and their patient-matched normal colon samples. Materials and methods Transcription and translation of iPSC markers OCT4, SOX2, NANOG, KLF4 and c-MYC were investigated using immunohistochemical (IHC) staining, RT-qPCR and in-situ hybridization (ISH). Results All five iPSC markers were detected at the transcriptional and translational levels. Protein abundance was found to be correlated with tumor grade. Based on their protein expression within the tumors, two sub-populations of cells were identified: a NANOG+/OCT4- epithelial subpopulation and an OCT4+/NANOG- stromal subpopulation. All cases were accurately graded based on four pieces of iPSC marker-related data. Conclusions This study suggests the presence of two putative sub-populations of CSCs: a NANOG+/OCT4- epithelial subpopulation and an OCT4+/NANOG- stromal subpopulation. Normal colon, LGCA and HGCA could be accurately distinguished from one another using iPSC marker expression. Once validated, novel combinations of iPSC markers may provide diagnostic and prognostic value to help guide patient management.
Abstract Adoptive transfer of in vitro activated, tumor-specific CTL is a useful strategy for cancer immunotherapy. We compared in vitro culture conditions for ability to generate tumor-specific CTL that reject tumours in vivo, and for expression of molecular markers associated with effective anti-tumor activity. Specific CTL were obtained in short-term in vitro cultures of naïve CD8+ T cells and bone marrow DC loaded with peptide antigen, and were expanded in IL-2 for 2 days before transfer in vivo. The conditions of DC activation did not appear to affect the recovery, phenotype or effector function of cultured CTL, but had a substantial effect on the CTL’s ability to prevent tumour growth in vivo. Proteomic analysis of the total and surface proteome of different CTL populations was carried out using liquid chromatography and tandem mass spectrometry. Among more than 1,000 identified proteins, several were expressed with different abundance in CTL populations with varying anti-tumour activity. The functional significance of proteins showing the greatest differential expression is currently being examined using a combination of gene silencing and in vivo transfer studies. The identification of markers associated with effective anti-tumor function will provide useful means to understand and predict the functional activity of CTL populations for in vivo transfer.
The cancer stem cell (CSC) concept proposes that cancer recurrence and metastasis are driven by CSCs. In this study, we investigated whether cells from colon adenocarcinoma (CA) with a CSC-like phenotype express renin-angiotensin system (RAS) components, and the effect of RAS inhibitors on CA-derived primary cell lines. Expression of RAS components was interrogated using immunohistochemical and immunofluorescence staining in 6 low-grade CA (LGCA) and 6 high-grade CA (HGCA) tissue samples and patient-matched normal colon samples. Primary cell lines derived from 4 HGCA tissues were treated with RAS inhibitors to investigate their effect on cellular metabolism, tumorsphere formation and transcription of pluripotency genes. Immunohistochemical and immunofluorescence staining showed expression of AT2R, ACE2, PRR, and cathepsins B and D by cells expressing pluripotency markers. β-blockers and AT2R antagonists reduced cellular metabolism, pluripotency marker expression, and tumorsphere-forming capacity of CA-derived primary cell lines. This study suggests that the RAS is active in CSC-like cells in CA, and further investigation is warranted to determine whether RAS inhibition is a viable method of targeting CSCs.
Tandem mass spectrometry (MS/MS) is currently the most commonly used technology in proteomics for identifying proteins in complex biological samples. Mass spectrometers can produce a large number of MS/MS spectra each of which has hundreds of peaks. These peaks normally contain background noise, therefore a preprocessing step to filter the noise peaks can improve the accuracy and reliability of peptide identification. This paper proposes to preprocess the data by classifying peaks as noise peaks or signal peaks, i.e., a highly-imbalanced binary classification task, and uses genetic programming (GP) to address this task. The expectation is to increase the peptide identification reliability. Meanwhile, six different types of classification algorithms in addition to GP are used on various imbalance ratios and evaluated in terms of the average accuracy and recall. The GP method appears to be the best in the retention of more signal peaks as examined on a benchmark dataset containing 1, 674 MS/MS spectra. To further evaluate the effectiveness of the GP method, the preprocessed spectral data is submitted to a benchmark de novo sequencing software, PEAKS, to identify the peptides. The results show that the proposed method improves the reliability of peptide identification compared to the original un-preprocessed data and the intensity-based thresholding methods.
For coniferous gymnosperms, few data exist as to the contribution of the membrane-associated proteome to cell wall and wood formation. In this study, we begin to address this knowledge deficiency by examining the proteomic profile of Golgi-enriched membrane preparations derived from developing Pinus radiata compression wood. These membrane preparations were generated by a combination of discontinuous sucrose gradient centrifugation and Triton X-114-based phase separation. Fractionation by phase separation removed contaminating proteins associated with the cytoskeleton and enabled the discrimination between soluble and membrane-bound/integral proteins. The proteomic analysis of the resulting aqueous and detergent phases using high-performance liquid chromatography–tandem mass spectrometry resulted in the identification of 175 proteins. The majority of the identified proteins were membrane bound/integral and originated from cellular components such as the nucleus, plastids, endoplasmic reticulum, plasma membrane and Golgi vesicles. On the basis of bioinformatic analysis, many of the identified proteins were predicted to be involved either in the regulation of wood formation or in cell wall biosynthesis, which indicated that the proteomic analysis of non-cytosolic proteins in developing xylem is a useful strategy to investigate the molecular aspects of wood formation in pine.
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