Identification of additional proteins in differential proteomics using protein interaction networks

In this study, we developed a novel computational approach based on protein–protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell protein extracts that were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: (i) Compilation of a human protein–protein interaction network from public databases; (ii) calculation of interaction scores based on functional similarity; (iii) determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins; and (iv) ranking of the resulting 25 candidate proteins. Two of the three highest‐ranked proteins, beta‐arrestin 1, and beta‐arrestin 2, were experimentally tested, revealing that their abundance levels in human smooth muscle cell samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost‐effective means to identify additional proteins that remain elusive for current 2D gel‐based proteomic profiling techniques.     

SNaPaer: A Practical Single Nucleotide Polymorphism Multiplex Assay for Genotyping of Pseudomonas aeruginosa

Multilocus sequence typing (MLST) represents the gold standard genotyping method in studies concerning microbial population structure, being particularly helpful in the detection of clonal relatedness. However, its applicability on large-scale genotyping is limited due to the high cost and time spent on the task. The selection of the most informative nucleotide positions simplifies genomic characterization of bacteria. A simple and informative multiplex, SNaPaer assay, was developed and genotyping of Pseudomonas aeruginosa was obtained after a single reaction of multiplex PCR amplification and mini-sequencing. This cost-effective technique allowed the analysis of a Portuguese set of isolates (n = 111) collected from three distinct hospitals and the genotyping data could be obtained in less than six hours. Point mutations were shown to be the most frequent event responsible for diversification of the Portuguese population sample. The Portuguese isolates corroborated the epidemic hypothesis for P. aeruginosa population. SNaPaer genotyping assay provided a discriminatory power of 0.9993 for P. aeruginosa, by testing in silico several hundreds of MLST profiles available online. The newly proposed assay targets less than 0.01% of the total MLST length and guarantees reproducibility, unambiguous analysis and the possibility of comparing and transferring data between different laboratories. The plasticity of the method still supports the addition of extra molecular markers targeting specific purposes/populations. SNaPaer can be of great value to clinical laboratories by facilitating routine genotyping of P. aeruginosa.     

Phylogenetic analysis of ITS2 sequences suggests the taxonomic re‐structuring of Dunaliella viridis (Chlorophyceae,Dunaliellales)

We analyzed the ITS2 primary and secondary structure (including Compensatory Base Changes (CBCs)) of 17 new Dunaliella strains (11 D. viridis, two D. tertiolecta, and four Dunaliella sp.), and compared these with other Dunaliella sequences available from the ITS2 database to circumscribe their taxonomic position. The ITS2 primary and secondary structure analysis positioned the majority of D. viridis strains in four main clades, showing that D. viridis is polyphyletic. The detection of at least one CBC among these clades strongly suggests that they could correspond to different biological species. Unexpectedly, while D. viridis var. euchlora (CCAP19/21) was positioned within the subgenus Dunaliella, D. viridis var. palmelloides (CCAP11/34) was positioned clearly outside this subgenus, suggesting that this taxon may not be properly placed in Dunaliella. Furthermore, the detection of at least three compensatory base changes (CBCs) between D. viridis var. palmelloides (CCAP11/34) and the other strains analyzed, confirm that this strain is a different species. For these reasons we propose re‐naming D. viridis var. palmelloides (CCAP11/34) to incertae sedis, and D. viridis var. euchlora (CCAP19/21) to Dunaliella sp. Therefore, the ITS2 primary and secondary structure data suggest a taxonomic re‐structuring of D. viridis.     

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.     

Oxidative stress-induced proteome alterations target different cellular pathways in human myoblasts

Although increased oxidative stress has been associated with the impairment of proliferation and function of adult human muscle stem cells, proteins either involved in the stress response or damaged by oxidation have not been identified. A parallel proteomics approach was performed for analyzing the protein expression profile as well as proteins preferentially oxidized upon hydrogen peroxide-induced oxidative stress. Fifteen proteins involved in the oxidative stress response were identified. Among them, protein spots identified as peroxiredoxins 1 and 6, glyceraldehyde-3-phosphate dehydrogenase, and α-enolase were shifted to a more acidic isoelectric point upon oxidative stress, indicating posttranslational modifications. Oxidized proteins were evidenced by immunodetection of derivatized carbonyl groups followed by identification by mass spectrometry. The carbonylated proteins identified are mainly cytosolic and involved in carbohydrate metabolism, cellular assembly, cellular homeostasis, and protein synthesis and degradation. Pathway analysis revealed skeletal and muscular disorders, cell death, and cancer-related as the main molecular networks altered. Interestingly, these pathways were focused on two distinct proteins: p53 for altered protein expression and huntingtin for increased protein carbonylation. This study emphasizes the importance of performing analysis addressing different aspects of the cellular proteome to have a more accurate view of their changes upon stress.     

Nucleoplasmin: a nuclear chaperone.

In this article, we briefly review the structural and functional information currently available on nucleoplasmin. Special emphasis is placed on the discussion of the molecular mechanism involved in the sperm chromatin remodelling activity of this protein. A model is proposed based on current crystallographic data, recent biophysical and functional studies, as well as in the previously available information.     

Paraquat, but not maneb, induces synucleinopathy and tauopathy in striata of mice through inhibition of proteasomal and autophagic pathways

SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau.     

Phylogenetic position of Dunaliella acidophila (Chlorophyceae) based on ITS and rbcL sequences

Dunaliella acidophila is one of the most extreme acidophiles on earth and is able to survive in highly acidic habitats. This characteristic has made this organism the universal model for the study of abiotic stress. Although D. acidophila is currently circumscribed to the subgenus Pascheria within Dunaliella Teodoresco (Chlorophyceae), its taxonomic position has stirred controversy. The comparison of D. acidophila CCAP19/35 internal transcribed spacers (including ITS2 secondary structure analysis) and RuBisCo large subunit (rbcL) sequences with other Dunaliella species confirms that D. acidophila should maintain its phylogenetic position within the genus Dunaliella, suggesting its inclusion within the subgenus Dunaliella. Furthermore, the ITS1 and ITS2 data revealed that D. acidophila was highly divergent from the other freshwater species assessed, D. lateralis, with which it barely shares a 56.8% similarity.     

The effects of repeated oral gavage on the health of male CD-1 mice

Oral gavage is a widely used method for administering substances to animals in pharmacological and toxicological studies. The authors evaluated whether oral gavage causes behavioral indicators of stress, increased mortality rate, alterations in food and water consumption and body weight or histological lesions in CD-1 mice. Gavage was carried out once per d for 5 d per week over 6 consecutive weeks. The mortality rate of mice in this study was 15%. Mice subjected to gavage did not undergo changes in food or water consumption during the study, and their mean body weights and relative organ weights were similar to those of mice in the control group. Serum cortisol levels at the time of euthanasia in mice in both groups were within the normal range. Histopathology showed acute esophagitis and pleurisy, indicative of perforation of the esophagus, in the two mice that died but no abnormalities in the other mice. The results suggest that animal stress and mortality related to oral gavage can be minimized when the procedure is carried out by an experienced technician.     

Tracheal telocytes

Recently, a novel type of stromal cell - the telocytes (TC) - was identified in mouse trachea. These cells are known to possess the ultrastructural characteristics, which support their role in intercellular signaling. We found TC in all stromal compartments of the tracheal wall. TC with long prolongations (telopodes, Tp) were lining longitudinally the collagen bundles, and were serially arranged (end-to-end connections of Tp were found). Noteworthy, Tp frequently establish stromal synapses with mast cells (MC). Primary cilia were also identified in TC. In conclusion, tracheal TC could be involved in the tracheal regulation (e.g. secretion, contractility). The tandem TC-MC deserves further investigations.