BioTimer Assay, a new method for counting Staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm

The medical device-related infections are frequently a consequence of Staphylococcus biofilm, a lifestyle enhancing bacterial resistance to antibiotics. Antibiotic susceptibility tests are usually performed on planktonic forms of clinical isolates. Some methods have been developed to perform antibiotic susceptibility tests on biofilm. However, none of them counts bacterial inoculum. As antibiotic susceptibility is related to bacterial inoculum, the test results could be mistaken. Here, a new method, BioTimer Assay (BTA), able to count bacteria in biofilm without any manipulation of samples, is presented. Moreover, the BTA method is applied to analyze antibiotic susceptibility of six Staphylococcus strains in biofilm and to determine the number of viable bacteria in the presence of sub-inhibitory doses of four different antibiotics. To validate BTA, the new method was compared to reference methods both for counting and antibiotic susceptibility tests. A high agreement between BTA and reference methods is found on planktonic forms. Therefore, BTA was employed to count bacteria in biofilm and to analyze biofilm antibiotic susceptibility. Results confirm the high resistance to antibiotics of Staphylococcus biofilm. Moreover, BTA counts the number of viable bacteria in the presence of sub-inhibitory doses of antibiotics. The results show that the number of viable bacteria depends on sub-inhibitory doses, age of biofilm and type of antibiotic. In particular, differently to gentamicin and ampicillin, sub-inhibitory doses of ofloxacin and azithromycin reduce the number of viable bacteria at lower extent in young than in old biofilm. In conclusion, BTA is a reliable, rapid, easy-to-perform, and versatile method, and it can be considered a useful tool to analyze antibiotic susceptibility of Staphylococcus spp. in biofilm.     

Towards a new attenuating compound: a potentiometric, spectrophotometric and NMR equilibrium study on Fe(III), Al(III) and a new tetradentate mixed bisphosphonate-hydroxypyridinonate ligand

Coordination properties toward Fe(III) and Al(III) of a mixed bisphosphonate-hydroxypyridinonate ligand are presented. Potentiometric, spectrophotometric and NMR results allowed to conclude that Fe(III) and Al(III) coordination takes place on the pyridinone moiety. The high steric hindrance prevents the possibility of simultaneous coordination of both groups to the same metal ion. Quantum mechanical calculations confirm this finding allowing to determine the minimal length of the linker necessary for a stable conformation of complexes in which Fe(III) is coordinated both by pyridinone and bisphosphonate groups.     

Systems-wide Analysis of a Phosphatase Knock-down by Quantitative Proteomics and Phosphoproteomics

Signal transduction in metazoans regulates almost all aspects of biological function, and aberrant signaling is involved in many diseases. Perturbations in phosphorylation-based signaling networks are typically studied in a hypothesis-driven approach, using phospho-specific antibodies. Here we apply quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component. Drosophila cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) and the phosphatase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes, was knocked down by RNAi. In total we detected more than 10,000 phosphorylation sites in the phosphoproteome of Drosophila Schneider cells and trained a phosphorylation site predictor with this data. SILAC-based quantitation after phosphatase knock-down showed that apart from the phosphatase, the proteome was minimally affected whereas 288 of 6,478 high-confidence phosphorylation sites changed significantly. Responses at the phosphotyrosine level included the already described Ptp61F substrates Stat92E and Abi. Our analysis highlights a connection of Ptp61F to cytoskeletal regulation through GTPase regulating proteins and focal adhesion components.Information processing in biological systems relies heavily on activation and inactivation of proteins by phosphorylation. This key post-translational modification is involved in the regulation of most cellular processes and mediates many rapid responses as well as long-term gene expression changes in response to stimuli. Protein kinases and protein phosphatases coordinately regulate this highly dynamic and reversible modification. Phosphorylation is usually studied in a candidate-based approach by in vitro kinase assays or by immune techniques employing phospho-specific antibodies. Despite the success of this reductionist approach, it does not afford a systems-wide observation of the effects upon perturbations of signaling networks.Recent advances in MS-based¹ proteomics now allow the identification of thousands of phosphorylation sites from complex protein mixtures (1–3). Most large-scale phosphoproteomics studies have been qualitative rather than quantitative; however, isotope-based methods enable precise quantitation of phosphorylation sites between two or more cellular states (4–6). Our group has applied the metabolic labeling technology termed stable isotope labeling by amino acids in cell culture (SILAC) (7) for the quantitative comparison of phosphoproteomes. For example, we quantified phosphorylation dynamics in response to epidermal growth factor stimulation. Out of a measured phosphoproteome of several thousand sites only a minority (about 10%) was regulated by the signal, highlighting the importance of quantitation in pinpointing specific systems responses (8).Drosophila is a well established model system to study key players in cell signaling and development. Genetic studies have been performed for decades whereas more recently also RNA interference (RNAi) has been employed for gene function studies using a highly efficient silencing protocol (9). A further advantage of Drosophila as a model system is the lower degree of functional redundancy compared with higher vertebrates while maintaining a high level of conservation of human genes linked to disease (10).Two large-scale, non-quantitative Drosophila phosphoproteome studies were carried out in embryonic Kc167 cells (11) and embryos (12). Both studies identified more than 10,000 sites of the Drosophila phosphoproteome.We have recently adapted the SILAC methodology for quantitative proteomics to Drosophila. Schneider line 2 (SL2) cells were treated with either mock dsRNA or dsRNA against ISWI, a component of chromatin remodeling complexes. The combination of RNAi and SILAC allows the unbiased “phenotypization” of the gene knock-down directly at the proteome level (13).Here we determined a high-quality basal phosphoproteome in SL2 cells and characterized its structural and evolutionary properties. We compared kinase substrate motives between Drosophila and human and trained a Drosophila phosphorylation site predictor.To explore the potential of quantitative phosphoproteomics in a systems-wide manner, we focused on the Drosophila non-transmembrane tyrosine phosphatase Ptp61F. This phosphatase is the ortholog of mammalian PTB1B, which is thought to be involved in type 2 diabetes, obesity, and cancer (14), and which is the target of several ongoing drug development projects (15). Ptp61F is a negative regulator of JAK/STAT signaling (16, 17) and, together with the Ableson kinase (Abl), involved in the regulation of the Abl interacting protein (Abi) and lamella formation (18). Both PTP1B and Ptp61F are among the best studied protein tyrosine phosphatases in their respective organisms; however the characterization of their substrates is still far from complete. Two recent mass spectrometric studies employed substrate trapping to identify direct substrates of PTP1B and Ptp61F (19, 20). The PTP1B study was combined with phosphotyrosine peptide enrichment, which led to site-specific detection of potential PTP1B targets. PTP1B function was additionally investigated by quantitative phosphotyrosine proteomics comparing wild type and PTP1B-deficient fibroblasts. In contrast, the Ptp61F study identified potential substrates without site-specific information. One of these was PVR, the Drosophila homolog of VEGFR and PDGFR, suggesting that Ptp61F - like its mammalian counterpart - counteracts receptor tyrosine kinase signaling. Apart from Abi, further components of the SCAR/WAVE complex as well as its regulatory kinase Abl were identified as potential Ptp61F substrates. This supports an involvement of Ptp61F in the regulation of actin reorganization and remodeling.To study the role of Ptp61F in a global and unbiased approach we combined global quantitative phosphoproteome analysis with RNA interference. We profiled tyrosine, serine and threonine phosphorylation changes upon ablation of Ptp61F by RNAi. In parallel, we quantified changes in the proteome, which allowed us to normalize changes in phosphorylation sites to corresponding changes at the protein level. Interestingly, we observed increased tyrosine phosphorylation of the protein tyrosine kinase Abl which suggests an enhanced Abl activity upon Ptp61F RNAi. We additionally detected up-regulated phosphotyrosine sites on GTPase regulating proteins (like RhoGAP15B and Vav) and constituents of focal adhesions (like Paxillin and Lasp) which expand the proposed involvement of Ptp61F in the regulation of cytoskeleton organization. Our work represents proof-of-principle that the combination of large-scale phosphoproteomics and a loss-of-function approach can contribute significantly to elucidating the role of key players in phosphorylation-dependent signaling. Importantly, this systems-wide approach measures the net effect of the perturbation on the entire signaling network, without the need to define specific substrate-kinase or substrate -phosphatase relationships or other direct functional mechanisms.     

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

Chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL-BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo- and holo-cL-BABP but are lost immediately after the start of molecular dynamics. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Cellulose and hemicelluloses recovery from grape stalks

In this work, two mild chemical fractionation procedures were compared to separate and recover lignocellulosic components from grape stalks. The first method consisted of mild acid hydrolysis for hemicelluloses separation, followed by an alkaline/oxidative step for lignin solubilization, while in the second method the acid hydrolysis was preceded by an alkali steeping phase. Influence of the length of the first step of both methods (from 2 to 24 h) on monosaccharides and cellulose yields was investigated. The first method allowed a higher sugar recovery for longer times, and a slightly lower amount of cellulose. Cellulose residues from both the methods were comparable for cellulose content and thermal profile (studied by differential scanning calorimetry). Acid hydrolysis of the first step was carried out also in autoclave, showing that xylan degradation could be described by a first order kinetics where at higher temperature the presence of a fast reaction and a slow reacting fraction must be accounted for.     

Seroprevalence study of Tick-borne encephalitis, Borrelia burgdorferi, Dengue and Toscana virus in Turin Province

Tick borne encephalitis virus (TBEV) is present in some European countries and it is transmitted by a tick bite. Ixodes ricinus is the main vector of the infection in Italy, where fortunately clinical neurological manifestations, typical of the more serious phase of the disease, are very rarely observed. This behaviour is different from other endemic Euroasiatic areas where numerous cases of encephalitis are described. However TBE transmission has not been widely investigated in Italy and available epidemiological data have been obtained only by studies performed in Central and Northern Regions of the country. In addition seroepidemiological researches were made prevalently on subjects at high risk of tick bite, such as hunters or forest guards from Trentin and Central Italy. No precise information about TBE virus diffusion was available in the Piedmont before our investigations. We found that hunters and wild boar breeders seem to be particularly exposed to the risk of TBE virus infection in Turin Province and in particular in the Susa valley, although no neurological involvement was observed in our population. In particular a seroprevalence of about 5% was detected by the use of purified antigens ELISA test, amongst the subjects at high risk of tick bite. Moreover low risk individuals showed a seroprevalence of below 2%. In addition a parallel seroepidemiological study was performed in Turin Province for Borrelia burgdorferi, the aetiological agent of Lyme disease, also transmitted by tick bite (e.g. Ixodes ricinus), for Dengue and Toscana (TOS) arboviruses, respectively transmitted by Aedes mosquitoes and phlebotomes. Data reported here demonstrate only a sporadic presence in our population of antibodies against Borrelia and Dengue infection. Moreover using an ELISA test performed with viral purified nucleoprotein, we reported a total percentage of about 3% of subjects positive for TOSV.     

Role of the intracellular domain of IL-7 receptor in T cell development

Signals from the IL-7R are uniquely required for T cell development and maintenance, despite the resemblance of IL-7R to other cytokine receptors and the apparent sharing of common signaling pathways. This unique requirement could either reflect unique expression of IL-7R or IL-7, or it could indicate that the IL-7R delivers unique signals. To determine whether the IL-7R provided unique signals, we exchanged its intracellular domain with that of other cytokine receptors: IL-4R, IL-9R, and prolactin receptor (PRLR). Chimeric receptors were used to reconstitute development of IL-7R(-/-) hemopoietic progenitors by transducing the receptors in retroviral vectors. Whereas IL-7R(-/-) thymocytes are arrested at the double-negative stage, IL-4R, IL-9R, or PRLR all imparted some progression to the double-positive stage. IL-4R and PRLR gave only small numbers of thymocytes, whereas IL-9R gave robust alphabeta T cell development and reconstitution of peripheral CD4 and CD8 cells, indicating that it can duplicate many of the functions of IL-7R. However, IL-9R failed to reconstitute rearrangement of the TCRgamma locus or development of gammadelta T cells. Thus, the IL-7R signals required in the alphabeta T cell lineage (such as survival and proliferation) are not unique to this receptor, whereas rearrangement of the TCRgamma locus may require a signal that is not shared by other receptors.     

Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection

We found that the proteome of apoptotic T cells includes prominent fragments of cellular proteins generated by caspases and that a high proportion of distinct T cell epitopes in these fragments is recognized by CD8+ T cells during HIV infection. The frequencies of effector CD8+ T cells that are specific for apoptosis-dependent epitopes correlate with the frequency of circulating apoptotic CD4+ T cells in HIV-1-infected individuals. We propose that these self-reactive effector CD8+ T cells may contribute to the systemic immune activation during chronic HIV infection. The caspase-dependent cleavage of proteins associated with apoptotic cells has a key role in the induction of self-reactive CD8+ T cell responses, as the caspase-cleaved fragments are efficiently targeted to the processing machinery and are cross-presented by dendritic cells. These findings demonstrate a previously undescribed role for caspases in immunopathology.