Cyclic AMP pathway activation and extracellular zinc induce rapid intracellular zinc mobilization in Candida albicans

LK was supported by Innovation Fund Denmark, DK (4019-00019B). Pcovery ApS received funding from Wellcome Trust, Research Councils, UK (100480/Z/12), Novo Seeds, DK and Boehringer Ingelheim Venture Fund, D. DW is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (102549/Z/13/Z), the Medical Research Council and University of Aberdeen (MR/N006364/1) and received support from a Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology (097377/Z/11/Z). The funders had no part in study design, data collection and interpretation, or the decision to submit the work for publication.Peer reviewedPublisher PD

Design, Construction and Cloning of Truncated ORF2 and tPAsp-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector

Background: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. Objectives: The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. Materials and Methods: A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. Results: Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunofluorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations

Erratum to : Analysis of the mitochondrial maxicircle of Trypanosoma lewisi, a neglected human pathogen

BACKGROUND The haemoflagellate Trypanosoma lewisi is a kinetoplastid parasite which, as it has been recently reported to cause human disease, deserves increased attention. Characteristic features of all kinetoplastid flagellates are a uniquely structured mitochondrial DNA or kinetoplast, comprised of a network of catenated DNA circles, and RNA editing of mitochondrial transcripts. The aim of this study was to describe the kinetoplast DNA of T. lewisi. METHODS/RESULTS In this study, purified kinetoplast DNA from T. lewisi was sequenced using high-throughput sequencing in combination with sequencing of PCR amplicons. This allowed the assembly of the T. lewisi kinetoplast maxicircle DNA, which is a homologue of the mitochondrial genome in other eukaryotes. The assembly of 23,745 bp comprises the non-coding and coding regions. Comparative analysis of the maxicircle sequence of T. lewisi with Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma brucei and Leishmania tarentolae revealed that it shares 78 %, 77 %, 74 % and 66 % sequence identity with these parasites, respectively. The high GC content in at least 9 maxicircle genes of T. lewisi (ATPase6; NADH dehydrogenase subunits ND3, ND7, ND8 and ND9; G-rich regions GR3 and GR4; cytochrome oxidase subunit COIII and ribosomal protein RPS12) implies that their products may be extensively edited. A detailed analysis of the non-coding region revealed that it contains numerous repeat motifs and palindromes. CONCLUSIONS We have sequenced and comprehensively annotated the kinetoplast maxicircle of T. lewisi. Our analysis reveals that T. lewisi is closely related to T. cruzi and T. brucei, and may share similar RNA editing patterns with them rather than with L. tarentolae. These findings provide novel insight into the biological features of this emerging human pathogen

Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

  IntroductionMass spectrometry (MS) is a powerful technology for study of PTMs, including protein phosphorylation. Due to the low abundance of many phosphoproteins and the relatively poor ionization efficiency of phosphopeptides, specific enrichment of phosphopeptides prior to MS analysis is necessary. At present, numerous phosphopeptide enrichment approaches have been established and applied to complex biological samples. We and others have reported that multi-step phosphopeptide purification methods enable better recovery of phosphopeptide and achieve higher selectivity and sensitivity than stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation.MethodPlant plasma membranes were isolated from Arabidopsis thaliana (Col-0) leaves using a two-phase partitioning system. The concentration of plasma membrane protein was determined by Bradford assay. Protein was digested with Lys-C for 4 hours and then by trypsin overnight. The peptide mixture was purified with IMAC, TiO2, CPP, SIMAC (IMAC+TiO2), the combination of CPP and IMAC, and the combination of CPP and TiO2, respectively.Nano-LC-MS was performed using LTQ-Orbitrap XL and LTQ-Orbitrap-XL/ETD mass spectrometer (Thermo Electron, Bremen, Germany) connected to an EASY nano-LC system (Proxeon Biosystems, Odense, Denmark). In CID mode, multi-stage activation (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany).ResultsWe first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A-score algorithm. Among 890 unique peptides, 389 of them were identified as phosphopeptides from IMAC enrichment. From TiO2 enrichment, 131 of 240 identified peptides were phosphopeptides. Since the results are not so satisfactory, we further investigated these samples using the combination of CPP and TiO2 enrichment methods. 1024 phosphopeptides were identified from the combined method, with a efficiency of 90% in this combined method. The results produced from the 3 enrichment experiments were carefully analyzed, and we conclude that the combined method gives better phosphopeptide recovery and higher selectivity. The overlap between the 3 enrichment experiments was quite small. We are currently investigating further combination of enrichment methods: SIMAC enrichment and the combination of CPP and IMAC enrichment. Samples will be analyzed by LTQ-Orbitrap-ETD MS, and the behavior of phosphopeptides on CID mode and ETD mode will be compared. Innovative aspects Combination of different phosphopeptide enrichment methods</p

Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

  IntroductionMass spectrometry (MS) is a powerful technology for study of PTMs, including protein phosphorylation. Due to the low abundance of many phosphoproteins and the relatively poor ionization efficiency of phosphopeptides, specific enrichment of phosphopeptides prior to MS analysis is necessary. At present, numerous phosphopeptide enrichment approaches have been established and applied to complex biological samples. We and others have reported that multi-step phosphopeptide purification methods enable better recovery of phosphopeptide and achieve higher selectivity and sensitivity than stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation.MethodPlant plasma membranes were isolated from Arabidopsis thaliana (Col-0) leaves using a two-phase partitioning system. The concentration of plasma membrane protein was determined by Bradford assay. Protein was digested with Lys-C for 4 hours and then by trypsin overnight. The peptide mixture was purified with IMAC, TiO2, CPP, SIMAC (IMAC+TiO2), the combination of CPP and IMAC, and the combination of CPP and TiO2, respectively.Nano-LC-MS was performed using LTQ-Orbitrap XL and LTQ-Orbitrap-XL/ETD mass spectrometer (Thermo Electron, Bremen, Germany) connected to an EASY nano-LC system (Proxeon Biosystems, Odense, Denmark). In CID mode, multi-stage activation (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany).ResultsWe first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A-score algorithm. Among 890 unique peptides, 389 of them were identified as phosphopeptides from IMAC enrichment. From TiO2 enrichment, 131 of 240 identified peptides were phosphopeptides. Since the results are not so satisfactory, we further investigated these samples using the combination of CPP and TiO2 enrichment methods. 1024 phosphopeptides were identified from the combined method, with a efficiency of 90% in this combined method. The results produced from the 3 enrichment experiments were carefully analyzed, and we conclude that the combined method gives better phosphopeptide recovery and higher selectivity. The overlap between the 3 enrichment experiments was quite small. We are currently investigating further combination of enrichment methods: SIMAC enrichment and the combination of CPP and IMAC enrichment. Samples will be analyzed by LTQ-Orbitrap-ETD MS, and the behavior of phosphopeptides on CID mode and ETD mode will be compared. Innovative aspects Combination of different phosphopeptide enrichment methods</p

RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack

In plants, the protein Rin4 acts with the plasma membrane H+-ATPase to regulate pathogen entry and the innate immune response, in part, through the regulation of stomatal closure

Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

  IntroductionMass spectrometry (MS) is a powerful technology for study of PTMs, including protein phosphorylation. Due to the low abundance of many phosphoproteins and the relatively poor ionization efficiency of phosphopeptides, specific enrichment of phosphopeptides prior to MS analysis is necessary. At present, numerous phosphopeptide enrichment approaches have been established and applied to complex biological samples. We and others have reported that multi-step phosphopeptide purification methods enable better recovery of phosphopeptide and achieve higher selectivity and sensitivity than stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation.MethodPlant plasma membranes were isolated from Arabidopsis thaliana (Col-0) leaves using a two-phase partitioning system. The concentration of plasma membrane protein was determined by Bradford assay. Protein was digested with Lys-C for 4 hours and then by trypsin overnight. The peptide mixture was purified with IMAC, TiO2, CPP, SIMAC (IMAC+TiO2), the combination of CPP and IMAC, and the combination of CPP and TiO2, respectively.Nano-LC-MS was performed using LTQ-Orbitrap XL and LTQ-Orbitrap-XL/ETD mass spectrometer (Thermo Electron, Bremen, Germany) connected to an EASY nano-LC system (Proxeon Biosystems, Odense, Denmark). In CID mode, multi-stage activation (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany).ResultsWe first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A-score algorithm. Among 890 unique peptides, 389 of them were identified as phosphopeptides from IMAC enrichment. From TiO2 enrichment, 131 of 240 identified peptides were phosphopeptides. Since the results are not so satisfactory, we further investigated these samples using the combination of CPP and TiO2 enrichment methods. 1024 phosphopeptides were identified from the combined method, with a efficiency of 90% in this combined method. The results produced from the 3 enrichment experiments were carefully analyzed, and we conclude that the combined method gives better phosphopeptide recovery and higher selectivity. The overlap between the 3 enrichment experiments was quite small. We are currently investigating further combination of enrichment methods: SIMAC enrichment and the combination of CPP and IMAC enrichment. Samples will be analyzed by LTQ-Orbitrap-ETD MS, and the behavior of phosphopeptides on CID mode and ETD mode will be compared. Innovative aspects Combination of different phosphopeptide enrichment methods</p