Mass Spectrometric Characterization of Oligomers in Pseudomonas aeruginosa Azurin Solutions

We have employed laser-induced liquid bead ion desorption mass spectroscopy (LILBID MS) to study the solution behavior of Pseudomonas aeruginosa azurin as well as two mutants and corresponding Re-labeled derivatives containing a Re(CO)_(3)(4,7-dimethyl-1,10-phenanthroline)^+ chromophore appended to a surface histidine. LILBID spectra show broad oligomer distributions whose particular patterns depend on the solution composition (pure H_(2)O, 20−30 mM NaCl, 20 and 50 mM NaP_i or NH_(4)P_i at pH = 7). The distribution maximum shifts to smaller oligomers upon decreasing the azurin concentration and increasing the buffer concentration. Oligomerization is less extensive for native azurin than its mutants. The oligomerization propensities of unlabeled and Re-labeled proteins are generally comparable, and only Re126 shows some preference for the dimer that persists even in highly diluted solutions. Peak shifts to higher masses and broadening in 20−50 mM NaP_i confirm strong azurin association with buffer ions and solvation. We have found that LILBID MS reveals the solution behavior of weakly bound nonspecific protein oligomers, clearly distinguishing individual components of the oligomer distribution. Independently, average data on oligomerization and the dependence on solution composition were obtained by time-resolved anisotropy of the Re-label photoluminescence that confirmed relatively long rotation correlation times, 6−30 ns, depending on Re−azurin and solution composition. Labeling proteins with Re-chromophores that have long-lived phosphorescence extends the time scale of anisotropy measurements to hundreds of nanoseconds, thereby opening the way for investigations of large oligomers with long rotation times

Relaxation Dynamics of Pseudomonas aeruginosa Re^I(C)O_3(α-diimine)(HisX)^+ (X=83, 107, 109, 124, 126)Cu-^(II) Azurins

Photoinduced relaxation processes of five structurally characterized Pseudomonas aeruginosa Re^I(CO)_3(α-diimine)(HisX) (X = 83, 107, 109, 124, 126)Cu^(II) azurins have been investigated by time-resolved (ps−ns) IR spectroscopy and emission spectroscopy. Crystal structures reveal the presence of Re-azurin dimers and trimers that in two cases (X = 107, 124) involve van der Waals interactions between interdigitated diimine aromatic rings. Time-dependent emission anisotropy measurements confirm that the proteins aggregate in mM solutions (D2O, KPi buffer, pD = 7.1). Excited-state DFT calculations show that extensive charge redistribution in the ReI(CO)_3 → diimine ^3MLCT state occurs: excitation of this ^3MLCT state triggers several relaxation processes in Re-azurins whose kinetics strongly depend on the location of the metallolabel on the protein surface. Relaxation is manifested by dynamic blue shifts of excited-state ν(CO) IR bands that occur with triexponential kinetics: intramolecular vibrational redistribution together with vibrational and solvent relaxation give rise to subps, 2, and 8−20 ps components, while the ~10^2 ps kinetics are attributed to displacement (reorientation) of the Re^I(CO)_3(phen)(im) unit relative to the peptide chain, which optimizes Coulombic interactions of the Re^I excited-state electron density with solvated peptide groups. Evidence also suggests that additional segmental movements of Re-bearing β-strands occur without perturbing the reaction field or interactions with the peptide. Our work demonstrates that time-resolved IR spectroscopy and emission anisotropy of Re^I carbonyl−diimine complexes are powerful probes of molecular dynamics at or around the surfaces of proteins and protein−protein interfacial regions

Iatrogenic insertion of impression mould into middle ear and mastoid and its retrieval after 9 years: a case report

The magnitude of hearing loss in Pakistan is enormous. One in twelve children of Pakistan suffers from some form of hearing impairment. Many of them are unable to afford surgical procedures and resort to the use of cheap hearing aids fitted by untrained individuals or people lacking the required expertise. This predisposes the patients to significant complications during a process that is otherwise considered safe. We report the case of a child, where the process of making the mould for a hearing aid led to the perforation of the tympanic membrane and pouring of mould material into the middle ear, necessitating surgical intervention. During initial surgery it was thought that all mould had been removed from the middle ear but 9 years later this child underwent cochlear implantation at the same center and remaining part of ear mould was discovered from mastoid cavity

Reduced occupancy of hedgehogs (Erinaceus europaeus) in rural England and Wales: the influence of habitat and an asymmetric intra-guild predator

Agricultural landscapes have become increasingly intensively managed resulting in population declines across a broad range of taxa, including insectivores such as the hedgehog (Erinaceus europaeus). Hedgehog declines have also been attributed to an increase in the abundance of badgers (Meles meles), an intra-guild predator. The status of hedgehogs across the rural landscape at large spatial scales is, however, unknown. In this study, we used footprint tracking tunnels to conduct the first national survey of rural hedgehog populations in England and Wales. Single and two-species occupancy modelling was used to quantify hedgehog occupancy in relation to habitat and predator covariates. Hedgehog occupancy was low (22% nationally), and significantly negatively related to badger sett density and positively related to the built environment. Hedgehogs were also absent from 71% of sites that had no badger setts, indicating that large areas of the rural landscape are not occupied by hedgehogs. Our results provide the first field based national survey of hedgehogs, providing a robust baseline for future monitoring. Furthermore, the combined effects of increasing badger abundance and intensive agriculture may have provided a perfect storm for hedgehogs in rural Britain, leading to worryingly low levels of occupancy over large spatial scales

Excitation-wavelength Dependent Fluorescence of Ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate

The excitation wavelength dependence of the steady-state and time-resolved emission spectra of ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate (EAADCy) in tetrahydrofuran (THF) at room temperature has been examined. It is found that the ratio of the fluorescence intensity of the long-wavelength and short-wavelength fluorescence bands strongly depends on the excitation wavelength, whereas the wavelengths of the fluorescence excitation and fluorescence bands maxima are independent on the observation/excitation wavelengths. The dynamic Stokes shift of fluorophore in locally excited (LE) and intramolecular charge transfer (ICT) states has been studied with a time resolution about 30 ps. The difference between Stokes shift in the LE and ICT states was attributed to the solvent response to the large photoinduced dipole moment of EAADCy in the fluorescent charge transfer state. On this base we can state that, the relaxation of the polar solvent molecules around the fluorophore was observed

Close-packed dimers on the line: diffraction versus dynamical spectrum

The translation action of \RR^{d} on a translation bounded measure $\omega$ leads to an interesting class of dynamical systems, with a rather rich spectral theory. In general, the diffraction spectrum of $\omega$, which is the carrier of the diffraction measure, live on a subset of the dynamical spectrum. It is known that, under some mild assumptions, a pure point diffraction spectrum implies a pure point dynamical spectrum (the opposite implication always being true). For other systems, the diffraction spectrum can be a proper subset of the dynamical spectrum, as was pointed out for the Thue-Morse sequence (with singular continuous diffraction) in \cite{EM}. Here, we construct a random system of close-packed dimers on the line that have some underlying long-range periodic order as well, and display the same type of phenomenon for a system with absolutely continuous spectrum. An interpretation in terms of `atomic' versus `molecular' spectrum suggests a way to come to a more general correspondence between these two types of spectra.Comment: 14 pages, with some additions and improvement

Survival of ART restorations assessed using selected FDI and modified ART restoration criteria

A new set of criteria for assessing the quality of restorations using modern restorative materials, named FDI criteria, was recently introduced. This study tested the null hypothesis that there is no significant difference in survival estimate percentages of ART restorations assessed using selected FDI and modified ART criteria after 1 and 5 years. One operator placed a total of 60 class I and 30 Class II high-viscosity glass-ionomer ART restorations in ninety 14- to15-year-olds. Two calibrated and independent evaluators using both criteria evaluated restorations on diestone replicas at baseline and after 1 and 5 years. Statistical analyses were done using the Kaplan–Meier method and log-rank test. The survival results of ART restorations assessed using both sets of criteria after 1 and 5 years (p = 0.27) did not differ significantly. Three ART restorations were assessed as failures according to the ART criteria, while they were assessed as survived using the FDI criteria. We conclude that the modified ART criteria enable reliable assessment of ART restorations in permanent teeth from diestone replicas and that there was no significant difference in survival estimates of ART restorations assessed using both sets of criteria. The null hypothesis was accepted

Antithrombotic therapy in patients undergoing TAVI: An overview of Dutch hospitals

Purpose To assess current antithrombotic treatment strategies in the Netherlands in patients undergoing transcatheter aortic valve implantation (TAVI). Methods For every Dutch hospital performing TAVI (n =14) an interventional cardiologist experienced in performing TAVI was interviewed concerning heparin, aspirin, thienopyridine and oral anticoagulation treatment in patients undergoing TAVI. Results The response rate was 100 %. In every centre, a protocol for antithrombotic treatment after TAVI was available. Aspirin was prescribed in all centres, concomitant clopidogrel was prescribed 13 of the 14 centres. Duration of concomitant clopidogrel was 3 months in over twothirds of cases. In 2 centres, duration of concomitant clopidogrel was based upon type of prosthesis: 6 months versus 3 months for supra-annular and intra-annular prostheses, respectively. Conclusions Leaning on a small basis of evidence and recommendations, the antithrombotic policy for patients undergoing TAVI is highly variable in the Netherlands. As a standardised regimen might further reduce haemorrhagic complications, large randomised clinical trials may help to establish the most appropriate approach

Stereotactic body radiotherapy for stage I lung cancer and small lung metastasis: evaluation of an immobilization system for suppression of respiratory tumor movement and preliminary results

<p>Abstract</p> <p>Background</p> <p>In stereotactic body radiotherapy (SBRT) for lung tumors, reducing tumor movement is necessary. In this study, we evaluated changes in tumor movement and percutaneous oxygen saturation (SpO₂) levels, and preliminary clinical results of SBRT using the BodyFIX immobilization system.</p> <p>Methods</p> <p>Between 2004 and 2006, 53 consecutive patients were treated for 55 lesions; 42 were stage I non-small cell lung cancer (NSCLC), 10 were metastatic lung cancers, and 3 were local recurrences of NSCLC. Tumor movement was measured with fluoroscopy under breath holding, free breathing on a couch, and free breathing in the BodyFIX system. SpO₂levels were measured with a finger pulseoximeter under each condition. The delivered dose was 44, 48 or 52 Gy, depending on tumor diameter, in 4 fractions over 10 or 11 days.</p> <p>Results</p> <p>By using the BodyFIX system, respiratory tumor movements were significantly reduced compared with the free-breathing condition in both craniocaudal and lateral directions, although the amplitude of reduction in the craniocaudal direction was 3 mm or more in only 27% of the patients. The average SpO₂did not decrease by using the system. At 3 years, the local control rate was 80% for all lesions. Overall survival was 76%, cause-specific survival was 92%, and local progression-free survival was 76% at 3 years in primary NSCLC patients. Grade 2 radiation pneumonitis developed in 7 patients.</p> <p>Conclusion</p> <p>Respiratory tumor movement was modestly suppressed by the BodyFIX system, while the SpO₂level did not decrease. It was considered a simple and effective method for SBRT of lung tumors. Preliminary results were encouraging.</p