Project description:With fast development of proteomic technology, the scale of missing proteins (MPs) has being continuously shrunk, approximately 1,470 MPs not explored yet. Discovery of MPs, on the other hand, is appearing more difficult. In some aneuploid cells, the abundance changes are proportional to the additional chromosome(s), while several MPs were found within them. Herein, we hypothesize that a stable aneuploid cell line with the increased chromosomes was a useful material that assists MP exploration. Ker-CT cell line with trisomy at chromosome 5 and 20 was selected. With a combination strategy of RNA-Seq and LC-M/MS, a total of 22,178 transcripts and 8,846 proteins were identified in Ker-CT. Although the transcripts corresponding to 31 and 32 MP genes located at chromosome 5 and 20 were detected, none of MPs was found in Ker-CT. Surprisingly, 3 MPs containing at least two unique non-nest peptides of length ≥9 amino acid were identified in Ker-CT, whose genes are located in chromosome 3 and 10. Furthermore, the 3 MPs were verified using the method of parallel reaction monitoring (PRM).
Project description:Gene expression profiles of Bacillus subtilis strain AG174 were compared as a function of steady-state external pH during growth with aeration. Aerated overnight cultures were diluted 1:500 and incubated in 250 mL baffled flasks containing potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM HOMOPIPES at pH 6.0, pH 7.0, and pH 9.0. Tubes were incubated at 37°C with aeration (260 rpm) to an optical density at 600 nm of 0.2. For each of the three pH conditions, RNA was isolated from five independent cultures. Overnight cultures were diluted 1:500 and incubated in 250 mL baffled flasks containing potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM HOMOPIPES at pH 6.0, pH 7.0, and pH 9.0. Cultures were incubated at 37°C with aeration (260 rpm) to an optical density at 600 nm of 0.2. Five replicate cultures were grown at each pH.
Project description:Understanding pathogen recognition and mechanisms in Atlantic cod are of significant importance for both basic research on wild populations and health management in aquaculture. A microarray approach was utilized to search for effects of viral (poly I:C/ polyinosinic acid:polycytidylic acid), bacterial (LPS/lipopolysaccharide) and polyclonal activator (PHA-L/phytohaemoagglutinin) stress in Atlantic cod head kidney cells. LPS cell activation increased mRNA expression of chemokine/Interleukin 8 (CXCL8/IL-8); interleukin -1? (IL-1?); cyclooxygenase 2 (COX2); leukocyte derived chemotaxin 2 (LECT2); LOC100698154, encoding a protein with unknown function; carboxyl-esterase 2 (CES2) and environmental biomarker cytochrome P450 1A (CYP1A). Mitogen activated protein kinase p38 (p38MAPK) and cathepsin F (CTSF) were downregulated by LPS. The antiviral responses induced by double stranded RNA (Poly I:C) clearly increased transcription of Toll like receptor 3 (TLR3) and interferon stimulating gene 15 (ISG15). The PHA response seemed to be more non-specific. Special for the PHA induction were the increase in Major histocompatibility complex class I (MHCI). CC chemokine type 2 (CK2) mRNA expression was increased by PHA, LPS and poly I:C, while p38MAPK and LECT2 was downregulated by PHA. Oxidative stress related genes like catalase and glutaredoxin (GLRX2) and the anti-apoptotic gene Bcl-2 showed no transcriptional changes compared to control in any of the treatments. Especially Poly I:C, but also LPS, induced leukotriene B4 (LTB4) and leukotriene B5 (LTB5) synthesis, while small amounts of prostaglandine E2 (PGE2) seemed to be constitutively produced in untreated cells, a production that was slightly elevated when exposing cells for LPS. This study reveals distinct signatures of bacteria and virus transcriptional responses in cod head kidney cells. In addition, the novel finding that Cyp1a was upregulated during the antibacterial response indicates a connection between immunity and aryl hydrocarbon receptor (AhR) activation in Atlantic cod.