Analysis of the nuclear proteome of the resurrection plant Xerophyta viscosa in response to dehydration stress using iTRAQ with 2DLC and tandem mass spectrometry

Xerophyta viscosa Baker (family Velloziaceae) is a desiccation tolerant plant which survives extremes of dehydration down to 5% relative water content (RWC) and resumes full physiological activity within 80h of rehydration. The nuclear proteome of Xerophyta viscosa and its response to dehydration at 35% RWC as compared to fully hydrated plants was analysed using iTRAQ together with 2DLC and ESI-MS/MS. RWC at 35% is unique for desiccation tolerant species as it represents a distinct phase of the dehydration process where induction of late protection mechanisms are initiated. We reproducibly identified 122 proteins with confidence≥95% (ρ<0.05). In response to dehydration, 65% of the identified proteins had the same protein abundance as the hydrated, 22% were shown to be more abundant while 9.8% were less abundant. Classification of the nuclear proteins according to GO annotation showed that most proteins were part of cellular processes (77.43%) and had binding activity (85.47%) respectively. Ontological classification according to Interpro and Pfam databases categorized most nuclear proteins as part of gene regulation (21%) while the functions of the mapped proteins using MapMan showed involvement in protein synthesis (22%), degradation (9%), DNA structure (8%) and regulation (8%).     

Candida dubliniensis: an appraisal of its clinical significance as a bloodstream pathogen

A nine-year prospective study (2002-2010) on the prevalence of Candida dubliniensis among Candida bloodstream isolates is presented. The germ tube positive isolates were provisionally identified as C. dubliniensis by presence of fringed and rough colonies on sunflower seed agar. Subsequently, their identity was confirmed by Vitek2 Yeast identification system and/or by amplification and sequencing of the ITS region of rDNA. In all, 368 isolates were identified as C. dubliniensis; 67.1% came from respiratory specimens, 11.7% from oral swabs, 9.2% from urine, 3.8% from blood, 2.7% from vaginal swabs and 5.4% from other sources. All C. dubliniensis isolates tested by Etest were susceptible to voriconazole and amphotericin B. Resistance to fluconazole (≥8 μg/ml) was observed in 2.5% of C. dubliniensis isolates, 7 of which occurred between 2008-2010. Of note was the diagnosis of C. dubliniensis candidemia in 14 patients, 11 of them occurring between 2008-2010. None of the bloodstream isolate was resistant to fluconazole, while a solitary isolate showed increased MIC to 5-flucytosine (>32 μg/ml) and belonged to genotype 4. A review of literature since 1999 revealed 28 additional cases of C. dubliniensis candidemia, and 167 isolates identified from blood cultures since 1982. In conclusion, this study highlights a greater role of C. dubliniensis in bloodstream infections than hitherto recognized.     

Boosting the globalization of plant proteomics through INPPO: current developments and future prospects

The International Plant Proteomics Organization (INPPO) is a non-profit-organization consisting of people who are involved or interested in plant proteomics. INPPO is constantly growing in volume and activity, which is mostly due to the realization among plant proteomics researchers worldwide for the need of such a global platform. Their active participation resulted in the rapid growth within the first year of INPPO's official launch in 2011 via its website (www.inppo.com) and publication of the 'Viewpoint paper' in a special issue of PROTEOMICS (May 2011). Here, we will be highlighting the progress achieved in the year 2011 and the future targets for the year 2012 and onwards. INPPO has achieved a successful administrative structure, the Core Committee (CC; composed of President, Vice-President, and General Secretaries), Executive Council (EC), and General Body (GB) to achieve INPPO objectives. Various committees and subcommittees are in the process of being functionalized via discussion amongst scientists around the globe. INPPO's primary aim to popularize the plant proteomics research in biological sciences has also been recognized by PROTEOMICS where a section dedicated to plant proteomics has been introduced starting January 2012, following the very first issue of this journal devoted to plant proteomics in May 2011. To disseminate organizational activities to the scientific community, INPPO has launched a biannual (in January and July) newsletter entitled 'INPPO Express: News & Views' with the first issue published in January 2012. INPPO is also planning to have several activities in 2012, including programs within the Education Outreach committee in different countries, and the development of research ideas and proposals with priority on crop and horticultural plants, while keeping tight interactions with proteomics programs on model plants such as Arabidopsis thaliana, rice, and Medicago truncatula. Altogether, the INPPO progress and upcoming activities are because of immense support, dedication, and hard work of all members of the INPPO community, and also due to the wide encouragement and support from the communities (scientific and non-scientific).     

WhiB2/Rv3260c, a cell division-associated protein of Mycobacterium tuberculosis H37Rv, has properties of a chaperone

whiB-like genes have been found in all actinomycetes sequenced so far. The amino-acid sequences of WhiB proteins of Mycobacterium?tuberculosis H37Rv are highly conserved and participate in several cellular functions. Unlike other WhiB proteins of M.?tuberculosis that have properties of protein disulfide reductases, WhiB2 showed properties like a chaperone as it suppressed the aggregation of several model substrates (e.g. citrate synthase, rhodanese and luciferase). Suppression of aggregation of the model substrates did not require ATP. Four cysteine residues of WhiB2 form two intramolecular disulfide bonds; however, chaperone function was unaffected by the redox state of the cysteines. WhiB2 also restored the activity of chemically denatured citrate synthase and did not require either ATP or a co-chaperone for refolding. The results indicate that WhiB2, which has been shown to be associated with cell division in mycobacteria and streptomyces, has evolved independently of other WhiBs, although it retains basic properties of this group of proteins. This is the first report to show that a WhiB protein has chaperone-like function; therefore, this report will have major implications in attempts to understand the role of WhiB proteins in mycobacteria, particularly in cell division.     

Chiral HPLC Separation and Modeling of Four Stereomers of DL‐Leucine‐DL‐Tryptophan Dipeptide on Amylose Chiral Column

Chiral high‐performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL‐leucine‐tryptophan DL‐dipeptide on AmyCoat‐RP column are described. The mobile phase applied was ammonium acetate (10 mM)‐methanol‐acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL‐, DD‐, DL‐, and LD‐ stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL‐leucine‐DL‐tryptophan in unknown matrices. Chirality 28:642–648, 2016. © 2016 Wiley Periodicals, Inc.     

Minor contribution of mutations at iniA codon 501 and embC-embA intergenic region in ethambutol-resistant clinical Mycobacterium tuberculosis isolates in Kuwait

Background

Mycoplasma pneumoniae and Chlamydophila pneumoniae are major causes of lower and upper respiratory infections that are difficult to diagnose using conventional methods such as culture. The ProPneumo-1 (Prodesse, Waukesha, WI) assay is a commercial multiplex real-time PCR assay for the simultaneous detection of M. pneumoniae and/or C. pneumoniae DNA in clinical respiratory samples.

Objective

The aim of this study was to evaluate the sensitivity and specificity of the ProPneumo-1, a newly developed commercial multiplex real-time PCR assay.

Methods

A total of 146 clinical respiratory specimens, collected from 1997 to 2007, suspected of C. pneumoniae or M. pneumoniae infections were tested retrospectively. Nucleic acid was extracted using an automated NucliSense easyMag (bioMerieux, Netherlands). We used a "Home-brew" monoplex real-time assay as the reference method for the analysis of C. pneumoniae and culture as the reference method for the analysis of M. pneumoniae. For discordant analysis specimens were re-tested using another commercial multiplex PCR, the PneumoBacter-1 assay (Seegene, Korea).

Results

Following discordant analysis, the sensitivity of the ProPneumo-1 assay for pathogens, C. pneumoniae or M. pneumoniae, was 100%. The specificity of the ProPneumo-1 assay, however, was 100% for C. pneumoniae and 98% for M. pneumoniae. The limits of detection were 1 genome equivalent (Geq) per reaction for pathogens, M. pneumoniae and C. pneumoniae. Due to the multipex format of the ProPneumo-1 assay, we identified 5 additional positive specimens, 2 C. pneumoniae in the M. pneumoniae-negative pool and 3 M. pneumoniae in the C. pneumoniae-negative pool.

Conclusion

The ProPneumo-1 assay is a rapid, sensitive and effective method for the simultaneous detection of M. pneumoniae and C. pneumoniae directly in respiratory specimens.     

Adiponectin-induced ERK and Akt Phosphorylation Protects against Pancreatic Beta Cell Apoptosis and Increases Insulin Gene Expression and Secretion

The functional impact of adiponectin on pancreatic beta cells is so far poorly understood. Although adiponectin receptors (AdipoR1/2) were identified, their involvement in adiponectin-induced signaling and other molecules involved is not clearly defined. Therefore, we investigated the role of adiponectin in beta cells and the signaling mediators involved. MIN6 beta cells and mouse islets were stimulated with globular (2.5 μg/ml) or full-length (5 μg/ml) adiponectin under serum starvation, and cell viability, proliferation, apoptosis, insulin gene expression, and secretion were measured. Lysates were subjected to Western blot analysis to determine phosphorylation of AMP-activated protein kinase (AMPK), Akt, or ERK. Functional significance of signaling was confirmed using dominant negative mutants or pharmacological inhibitors. Participation of AdipoRs was assessed by overexpression or siRNA. Adiponectin failed to activate AMPK after 10 min or 1- and 24-h stimulation. ERK was significantly phosphorylated after 24-h treatment with adiponectin, whereas Akt was activated at all time points examined. 24-h stimulation with adiponectin significantly increased cell viability by decreasing cellular apoptosis, and this was prevented by dominant negative Akt, wortmannin (PI3K inhibitor), and U0126 (MEK inhibitor). Moreover, adiponectin regulated insulin gene expression and glucose-stimulated insulin secretion, which was also prevented by wortmannin and U0126 treatment. Interestingly, the data also suggest adiponectin-induced changes in Akt and ERK phosphorylation and caspase-3 may occur independent of the level of AdipoR expression. This study demonstrates a lack of AMPK involvement and implicates Akt and ERK in adiponectin signaling, leading to protection against apoptosis and stimulation of insulin gene expression and secretion in pancreatic beta cells.     

The recent evolutionary origin of the phenylalanine-sensitive isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in the enteric lineage of bacteria

Summary Evolutionary events that generated the three regulatory isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase present in contemporary strains ofEscherichia coli have been proposed recently [Ahmad et al. (1986) J Bacteriol 165:146–154]. The phylogenetic subdivision of gram-negative prokaryotes studied (Superfamily B) includes enteric bacteria, anOceanospirillum cluster, pseudomonad Group I (e.g.,Pseudomonas aeruginosa), pseudomonad Group V (e.g.,Xanthomonas), and theAcinetobacter grouping. DAHP synthase-phe, a regulatory isozyme subject to allosteric control byl-phenylalanine, was the last member of the isozyme family to evolve. Thus, DAHP synthase-phe is absent throughout Superfamily B except within the enteric lineage. Bacteria that make up the enteric lineage (Escherichia, Klebsiella, Erwinia, Serratia, Proteus, Aeromonas, andAlteromonas) were examined in detail; DAHP synthasephe was present in each of these organisms. Therefore, the isozyme originated between the separation of the enteric andOceanospirillum lineages, prior to the divergence ofAlteromonas putrefaciens (44% homology withE. coli by DNA:rRNA hybridization) from the rest of the enteric lineage. DAHP synthase-tyr and DAHP synthase-trp were uniformly present within the enteric lineage, although it was often necessary to derepress DAHP synthase-trp by physiological manipulation in order to demonstrate its presence.     

The stable phylogenetic distribution of the recently evolvedl-phenylalanine-inhibited isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in enteric bacteria

Escherichia coli and some other enteric bacteria possess three regulatory isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, each of which is inhibited by one aromatic amino acid. Thel-phenylalanine-sensitive isozyme of DAHP synthase has evolved most recently since it is absent in all other members of the Gram-negative cluster that contains enteric bacteria as a subcluster. A comprehensive survey of enteric genera was carried out to determine whether the newly evolved isozyme is a stable, conserved trait. The results obtained show that all the genera of the contemporaryEnterobacteriaceae family possess the recently evolved phenylalanine-sensitive isozyme in addition to the tyrosine-and tryptophansensitive isozymes of DAHP synthase. However, physiological manipulation was usually necessary to derepress the tryptophan-sensitive DAHP synthase in order to demonstrate its presence.Florida Experiment Station Journal Series No. 9603.