Pseudomonas aeruginosa AES-1 exhibits increased virulence gene expression during chronic infection of cystic fibrosis lung

Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients. © 2011 Naughton et al

PPARγ agonists negatively regulate αIIbβ3 integrin outside-in signalling and platelet function through upregulation of protein kinase A activity

BACKGROUND: Agonists for the peroxisome proliferator activated receptor PPARγ, have been shown to have inhibitory effects on platelet activity following stimulation by GPVI and GPCR agonists. OBJECTIVES: Profound effects on thrombus formation led us to suspect a role for PPARγ agonists in the regulation of integrin αIIbβ3 mediated signalling. Both GPVI and GPCR signalling pathways lead to αIIbβ3 activation, and signalling through αIIbβ3 plays a critical role in platelet function and normal haemostasis. METHODS: The effects of PPARγ agonists on the regulation of αIIbβ3 outside-in signalling was determined by monitoring the ability of platelets to adhere and spread on fibrinogen and undergo clot retraction. Effects on signalling components downstream of αIIbβ3 activation were also determined following adhesion to fibrinogen by western blotting. RESULTS: Treatment of platelets with PPARγ agonists inhibited platelet adhesion and spreading on fibrinogen and diminished clot retraction. A reduction in phosphorylation of several components of αIIbβ3 signalling, including the integrin β3 subunit, Syk, PLCγ2, FAK and Akt was also observed as a result of reduced interaction of the integrin β3 subunit with Gα13. Studies of VASP phosphorylation revealed that this was a due to an increase in PKA activity following treatment with PPARγ receptor agonists. CONCLUSIONS: This study provides further evidence for anti-platelet actions of PPARγ agonists, identifies a negative regulatory role for PPARγ agonists in the control of integrin αIIbβ3 outside-in signalling, and provides a molecular basis by which the PPARγ agonists negatively regulate platelet activation and thrombus formation

Deep brain stimulation can suppress pathological synchronisation in parkinsonian patients

Background Although deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a highly effective therapeutic intervention in severe Parkinson's disease, its mechanism of action remains unclear. One possibility is that DBS suppresses local pathologically synchronised oscillatory activity.Methods To explore this, the authors recorded from DBS electrodes implanted in the STN of 16 patients with Parkinson's disease during simultaneous stimulation (pulse width 60 mu s; frequency 130 Hz) of the same target using a specially designed amplifier. The authors analysed data from 25 sides.Results The authors found that DBS progressively suppressed peaks in local field potential activity at frequencies between 11 and 30 Hz as voltage was increased beyond a stimulation threshold of 1.5 V. Median peak power had fallen to 54% of baseline values by a stimulation intensity of 3.0 V.Conclusion The findings suggest that DBS can suppress pathological 11-30 Hz activity in the vicinity of stimulation in patients with Parkinson's disease. This suppression occurs at stimulation voltages that are clinically effective

A Transiting Planet of a Sun-like Star

A planet transits an 11th magnitude, G1V star in the constellation Corona Borealis. We designate the planet XO-1b, and the star, XO-1, also known as GSC 02041-01657. XO-1 lacks a trigonometric distance; we estimate it to be 200+-20 pc. Of the ten stars currently known to host extrasolar transiting planets, the star XO-1 is the most similar to the Sun in its physical characteristics: its radius is 1.0+-0.08 R_Sun, its mass is 1.0+-0.03 M_Sun, V sini < 3 km/s, and its metallicity [Fe/H] is 0.015+-0.04. The orbital period of the planet XO-1b is 3.941534+-0.000027 days, one of the longer ones known. The planetary mass is 0.90+-0.07 M_Jupiter, which is marginally larger than that of other transiting planets with periods between 3 and 4 days. Both the planetary radius and the inclination are functions of the spectroscopically determined stellar radius. If the stellar radius is 1.0+-0.08 R_Sun, then the planetary radius is 1.30+-0.11 R_Jupiter and the inclination of the orbit is 87.7+-1.2 degrees. We have demonstrated a productive international collaboration between professional and amateur astronomers that was important to distinguishing this planet from many other similar candidates.Comment: 31 pages, 9 figures, accepted for part 1 of Ap

The chaperone protein HSP47: a platelet collagen binding protein that contributes to thrombosis and haemostasis

Objective: Heat shock protein 47 (HSP47) is an intracellular chaperone protein that is vital for collagen biosynthesis in collagen secreting cells. This protein has also been shown to be present on the surface of platelets. Given the importance of collagen and its interactions with platelets in triggering haemostasis and thrombosis, in this study we sought to characterise the role of HSP47 on these cells. Approach and Results: The deletion of HSP47 in mouse platelets or its inhibition in human platelets reduced their function in response to collagen and the GPVI agonist (CRP-XL), but responses to thrombin were unaltered. In the absence of functional HSP47, the interaction of collagen with platelets was reduced, and this was associated with reduced GPVI-collagen binding, signalling and platelet activation. Thrombus formation on collagen, under arterial flow conditions was also decreased following the inhibition or deletion of HSP47, in the presence or absence of the eptifibatide, consistent with a role for HSP47 in enhancing platelet adhesion to collagen. Platelet adhesion under flow to von Willebrand Factor was unaltered following HSP47 inhibition. Laser-induced thrombosis in cremaster muscle arterioles was reduced and bleeding time was prolonged in HSP47 deficient mice or following inhibition of HSP47. Conclusions: Our study demonstrates the presence of HSP47 on the platelet surface where it interacts with collagen, stabilises platelet adhesion and increases collagen mediated signalling and therefore thrombus formation and haemostasis

STEP: Satellite Test of the Equivalence Principle. Report on the phase A study

During Phase A, the STEP Study Team identified three types of experiments that can be accommodated on the STEP satellite within the mission constraints and whose performance is orders of magnitude better than any present or planned future experiment of the same kind on the ground. The scientific objectives of the STEP mission are to: test the Equivalence Principle to one part in 10(exp 17), six orders of magnitude better than has been achieved on the ground; search for a new interaction between quantum-mechanical spin and ordinary matter with a sensitivity of the mass-spin coupling constant g(sub p)g(sub s) = 6 x 10(exp -34) at a range of 1 mm, which represents a seven order-of-magnitude improvement over comparable ground-based measurements; and determine the constant of gravity G with a precision of one part in 10(exp 6) and to test the validity of the inverse square law with the same precision, both two orders of magnitude better than has been achieved on the ground

Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib

The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side-effects of ibrutinib. The second generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with Non-Hodgkin Lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide (CRP-XL) and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, while size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited SFKs and that SFKs have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of vWF and FVIII ex vivo by addition of intermediate purity FVIII (haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma vWF and FVIII

Three Stages of Lysozyme Thermal Stabilization by High and Medium Charge Density Anions

Addition of high and medium charge density anions (phosphate, sulfate, and chloride) to lysozyme in pure water demonstrates three stages for stabilization of the protein structure. The first two stages have a minor impact on lysozyme stability and are probably associated with direct interaction of the ions with charged and partial charges on the protein’s surface. There is a clear transition between the second and third stages; in the case of sodium chloride, disodium sulfate and disodium hydrogen phosphate this is at 550, 210, and 120 mM, respectively. Stabilization of lysozyme can be explained by the free energy required to hydrate the protein as it unfolds. At low ion concentrations, the protein’s hydration layer is at equilibrium with the bulk water. After the transition, bulk water is depleted and the protein is competing for water with the ions. With competition for water between the protein and the ions at higher salt concentrations, the free energy required to hydrate the interior of the protein rises and it is this that stabilizes the protein structure

A De Novo Mutation in the Sodium-Activated Potassium Channel KCNT2 Alters Ion Selectivity and Causes Epileptic Encephalopathy.

Early infantile epileptic encephalopathies (EOEE) are a debilitating spectrum of disorders associated with cognitive impairments. We present a clinical report of a KCNT2 mutation in an EOEE patient. The de novo heterozygous variant Phe240Leu SLICK was identified by exome sequencing and confirmed by Sanger sequencing. Phe240Leu rSlick and hSLICK channels were electrophysiologically, heterologously characterized to reveal three significant alterations to channel function. First, [Cl-]i sensitivity was reversed in Phe240Leu channels. Second, predominantly K+-selective WT channels were made to favor Na+ over K+ by Phe240Leu. Third, and consequent to altered ion selectivity, Phe240Leu channels had larger inward conductance. Further, rSlick channels induced membrane hyperexcitability when expressed in primary neurons, resembling the cellular seizure phenotype. Taken together, our results confirm that Phe240Leu is a "change-of-function" KCNT2 mutation, demonstrating unusual altered selectivity in KNa channels. These findings establish pathogenicity of the Phe240Leu KCNT2 mutation in the reported EOEE patient

Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation

Background Accurate protein quantification is a vital prerequisite for generating meaningful predictions when using systems biology approaches, a method that is increasingly being used to unravel the complexities of sub cellular interactions and as part of the drug discovery process. Quantitative proteomics, flow cytometry and western blotting have been extensively used to define human platelet protein copy numbers, yet for mouse platelets, a model widely used for platelet research, evidence is largely limited to a single proteomic dataset in which the total amount of proteins were generally comparatively higher than those found in human platelets. Objectives To investigate the functional implications of discrepancies between levels of mouse and human proteins in the GPVI signalling pathway using a systems pharmacology model of GPVI Methods The protein copy number of mouse platelet receptors was determined using flow cytometry. The Virtual Platelet, a mathematical model of Glycoprotein VI (GPVI) signalling, was used to determine the consequences of protein copy number differences observed between human and mouse platelets. Results and conclusion Despite the small size of mouse platelets compared to human platelets they possessed a greater density of surface receptors alongside a higher concentration of intracellular signalling proteins. Surprisingly the predicted temporal profile of Syk activity was similar in both species with predictions supported experimentally. Super resolution microscopy demonstrates that the spatial distribution of Syk is similar between species, suggesting that the spatial distribution of receptors and signalling molecules in activated platelets, rather than their copy number, is important for signalling pathway regulation