Computational Analysis of Phosphopeptide Binding to the Polo-Box Domain of the Mitotic Kinase PLK1 Using Molecular Dynamics Simulation

The Polo-Like Kinase 1 (PLK1) acts as a central regulator of mitosis and is over-expressed in a wide range of human tumours where high levels of expression correlate with a poor prognosis. PLK1 comprises two structural elements, a kinase domain and a polo-box domain (PBD). The PBD binds phosphorylated substrates to control substrate phosphorylation by the kinase domain. Although the PBD preferentially binds to phosphopeptides, it has a relatively broad sequence specificity in comparison with other phosphopeptide binding domains. We analysed the molecular determinants of recognition by performing molecular dynamics simulations of the PBD with one of its natural substrates, CDC25c. Predicted binding free energies were calculated using a molecular mechanics, Poisson-Boltzmann surface area approach. We calculated the per-residue contributions to the binding free energy change, showing that the phosphothreonine residue and the mainchain account for the vast majority of the interaction energy. This explains the very broad sequence specificity with respect to other sidechain residues. Finally, we considered the key role of bridging water molecules at the binding interface. We employed inhomogeneous fluid solvation theory to consider the free energy of water molecules on the protein surface with respect to bulk water molecules. Such an analysis highlights binding hotspots created by elimination of water molecules from hydrophobic surfaces. It also predicts that a number of water molecules are stabilized by the presence of the charged phosphate group, and that this will have a significant effect on the binding affinity. Our findings suggest a molecular rationale for the promiscuous binding of the PBD and highlight a role for bridging water molecules at the interface. We expect that this method of analysis will be very useful for probing other protein surfaces to identify binding hotspots for natural binding partners and small molecule inhibitors

Ribavirin Enhances IFN-α Signalling and MxA Expression: A Novel Immune Modulation Mechanism during Treatment of HCV

The nucleoside analogue Ribavirin significantly increases patient response to IFN-α treatment of HCV, by directly inhibiting viral replication. Recent studies indicate that Ribavirin also regulates immunity and we propose that Ribavirin enhances specific interferon sensitive gene (ISG) expression by amplifying the IFN-α-JAK/STAT pathway. We found that IFN-α-induced STAT1 and STAT3 phosphorylation was increased in hepatocytes co-treated with Ribavirin and IFN-α, compared to IFN-α alone. Ribavirin specifically enhanced IFN-α induced mRNA and protein of the anti-viral mediator MxA, which co-localised with HCV core protein. These novel findings indicate for the first time that Ribavirin, in addition to its viral incorporation, also enhances IFN-α-JAK/STAT signalling, leading to a novel MxA-mediated immuno-modulatory mechanism that may enhance IFN-α anti-viral activity against HCV

Single-cell analysis reveals individual spore responses to simulated space vacuum

Outer space is a challenging environment for all forms of life, and dormant spores of bacteria have been frequently used to study the survival of terrestrial life in a space journey. Previous work showed that outer space vacuum alone can kill bacterial spores. However, the responses and mechanisms of resistance of individual spores to space vacuum are unclear. Here, we examined spores’ molecular changes under simulated space vacuum (~10−5 Pa) using micro-Raman spectroscopy and found that this vacuum did not cause significant denaturation of spore protein. Then, live-cell microscopy was developed to investigate the temporal events during germination, outgrowth, and growth of individual Bacillus spores. The results showed that after exposure to simulated space vacuum for 10 days, viability of spores of two Bacillus species was reduced up to 35%, but all spores retained their large Ca2 +-dipicolinic acid depot. Some of the killed spores did not germinate, and the remaining germinated but did not proceed to vegetative growth. The vacuum treatment slowed spore germination, and changed average times of all major germination events. In addition, viable vacuum-treated spores exhibited much greater sensitivity than untreated spores to dry heat and hyperosmotic stress. Among spores’ resistance mechanisms to high vacuum, DNA-protective α/β−type small acid-soluble proteins, and non- homologous end joining and base excision repair of DNA played the most important roles, especially against multiple cycles of vacuum treatment. Overall, these results give new insight into individual spore’s responses to space vacuum and provide new techniques for microorganism analysis at the single-cell level

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay

Desmoglein 2 is a substrate of kallikrein 7 in pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>In a previous report we have demonstrated that the chymotryptic-like serine protease kallikrein 7 (<it>KLK7</it>/hK7) is overexpressed in pancreatic cancer. In normal skin, hK7 is thought to participate in skin desquamation by contributing in the degradation of desmosomal components, such as desmogleins. Thus, the ability of hK7 to degrade desmogleins was assessed and the effect of hK7 expression on desmoglein 2 was examined in cultured pancreatic cancer cells.</p> <p>Methods</p> <p>The expression of Dsg1, Dsg2, and Dsg3 in pancreatic tissues was examined by immunohistochemistry and their expression in two pancreatic cancer cell lines, BxPC-3 and Panc-1, was determined by western blot analysis. The ability of hK7 to degrade Dsg1 and Dsg2 was investigated using <it>in vitro </it>degradation assays. BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2.</p> <p>Results</p> <p>The levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues. Among the desmosomal proteins examined, Dsg2 exhibited robust expression on the surface of BxPC-3 cells. When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells.</p> <p>Conclusion</p> <p>A reduction in the amount of the cell adhesion components Dsg1 and Dsg2 in pancreatic tumors suggests that loss of these desmosomal proteins may play a role in pancreatic cancer invasion. Using <it>in vitro </it>degradation assays, both Dsg1 and Dsg2 could be readily proteolyzed by hK7, which is overexpressed in pancreatic adenocarcinomas. The enforced expression of hK7 in BxPC-3 cells that express significant amounts of Dsg2 resulted in a marked increase in the shedding of soluble Dsg2, which is consistent with the notion that aberrant expression of hK7 in pancreatic tumors may result in diminished cell-cell adhesion and facilitate tumor cell invasion.</p

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration

A selective retinoid X receptor agonist bexarotene (LGD1069, targretin) inhibits angiogenesis and metastasis in solid tumours

The present study determined the influence of a retinoid X receptor agonist bexarotene on angiogenesis and metastasis in solid tumours. In the experimental lung metastasis xenograft models, treatment with bexarotene inhibited the development of the lung tumour nodule formation compared to control. In vivo angiogenesis assay utilising gelfoam sponges, bexarotene reduced angiogenesis in sponges containing vascular endothelial growth factor, epidermal growth factor and basic fibroblast growth factor to various extent. To determine the basis of these observations, human breast and non-small-cell lung cancer cells were subjected to migration and invasion assays in the presence of bexarotene. Our data showed that bexarotene decrease migration and invasiveness of tumour cells in a dose-dependent manner. Furthermore, bexarotene inhibited angiogenesis by directly inhibiting human umbilical vein endothelial cell growth and indirectly inhibiting tumour cell-mediated migration of human umbilical vein endothelial cells through Matrigel matrix. Analysis of tumour-conditioned medium indicated that bexarotene decreased the secretion of angiogenic factors and matrix metalloproteinases and increased the tissue inhibitor of matrix metalloproteinases. The ability of bexarotene to inhibit angiogenesis and metastasis was dependent on activation of its heterodimerisation partner peroxisome proliferator-activated receptor γ. Collectively, our results suggest a role of bexarotene in treatment of angiogenesis and metastasis in solid tumours