The alternative sigma factor sigma B of Staphylococcus aureus modulates virulence in experimental central venous catheter-related infections.

The impact of the alternative sigma factor sigma B (SigB) on pathogenesis of Staphylococcus aureus is not conclusively clarified. In this study, a central venous catheter (CVC) related model of multiorgan infection was used to investigate the role of SigB for the pathogenesis of S. aureus infections and biofilm formation in vivo. Analysis of two SigB-positive wild-type strains and their isogenic mutants revealed uniformly that the wild-type was significantly more virulent than the SigB-deficient mutant. The observed difference in virulence was apparently not linked to the capability of the strains to form biofilms in vivo since wild-type and mutant strains were able to produce biofilm layers inside of the catheter. The data strongly indicate that the alternative sigma factor SigB plays a role in CVC-associated infections caused by S. aureus.

The GAL genetic switch: visualisation of the interacting proteins by split-EGFP bimolecular fluorescence complementation

A split-EGFP bimolecular fluorescence complementation assay was used to visualise and locate three interacting pairs of proteins from the GAL genetic switch of the budding yeast, Saccharomyces cerevisiae. Both the Gal4p-Gal80p and Gal80p-Gal3p pairs were found to be located in the nucleus under inducing conditions. However, the Gal80p-Gal1p complex was located throughout the cell. These results support recent work establishing an initial interaction between Gal3p and Gal80p occurring in the nucleus. Labelling of all three protein pairs impaired the growth of the yeast strains and resulted in reduced galactokinase activity in cell extracts. The most likely cause of this impairment is decreased dissociation rates of the complexes, caused by the essentially irreversible reassembly of the EGFP fragments. This suggests that a fully functional GAL genetic switch requires dynamic interactions between the protein components. These results also highlight the need for caution in the interpretation of in vivo split-EGFP experiments.

A preliminary structural design procedure for laser beam welded airframe stiffened panels

Initial sizing procedures for aircraft stiffened panels that include the influence of welding fabrication residual process effects are missing. Herein, experimental and Finite Element analyses are coupled to generate knowledge to formulate an accurate and computationally efficient sizing procedure which will enable designers to routinely consider panel fabrication, via welding, accounting for the complex distortions and stresses induced by this manufacturing process. Validating experimental results demonstrate the need to consider welding induced material property degradation, residual stresses and distortions, as these can reduce static strength performance. However, results from fuselage and wing trade-studies, using the validated sizing procedure, establish that these potential reductions in strength performance may be overcome through local geometric tailoring during initial sizing, negating any weight penalty for the majority of design scenarios.

Halophilic microorganisms as a source of novel antibiofilm compounds

Objectives: There is great urgency for alternate sources of antibiotics to be identified. One relatively untapped source of novel bioproducts, including antimicrobials, is organisms derived from extreme environments. Halophiles (which require high salt concentrations) are one such group which is being increasingly explored for their biotechnological potential. The aim of this study was to identify halophilic environmental isolates which possessed in vitro and in vivo antimicrobial and antibiofilm activities. Methods: 73 halophilic bacteria and archaea were isolated from Kilroot salt mine in Northern Ireland. Culture extracts of each isolate were screened for antimicrobial and antibiofilm activity against numerous pathogenic bacteria, including Staphylococcus species and Pseudomonas aeruginosa, both model strains and clinical isolates. The methods used included disc diffusion assays of crude extracts, MIC screening, the MBEC assay, and an in vivo model based on the Greater Wax Moth (Galleria mellonella). Results: The assays indicated >50% of extracts displayed antimicrobial and antibiofilm activity against at least one pathogen, the majority being Staphylococcus species, but also E. coli and P. aeruginosa. Biofilms were either reduced or eradicated by halophile extracts when tested with the MBEC device. Further experiments demonstrated that these effects could be replicated in vivo, with extracts reducing the severity of infections and enhancing the survival of infected G. mellonella. Conclusions: The importance of extremophiles to pharmaceutical research should not be underestimated. While not yet fully characterised, based on the data obtained, the halophiles isolated during this study may provide a promising reservoir of novel antimicrobial and antibiofilm compounds.

A device to investigate the axial strain dependence of the critical current density in superconductors

We have developed an instrument to study the behavior of the critical current density (J(c)) in superconducting wires and tapes as a function of field (mu(0)H), temperature (T), and axial applied strain (epsilon(a)). The apparatus is an improvement of similar devices that have been successfully used in our institute for over a decade. It encompasses specific advantages such as a simple sample layout, a well defined and homogeneous strain application, the possibility of investigating large compressive strains and the option of simple temperature variation, while improving the main drawback in our previous systems by increasing the investigated sample length by approximately a factor of 10. The increase in length is achieved via a design change from a straight beam section to an initially curved beam, placed perpendicular to the applied field axis in the limited diameter of a high field magnet bore. This article describes in detail the mechanical design of the device and its calibrations. Additionally initial J(c)(epsilon(a)) data, measured at liquid helium temperature, are presented for a bronze processed and for a powder-in-tube Nb3Sn superconducting wire. Comparisons are made with earlier characterizations, indicating consistent behavior of the instrument. The improved voltage resolution, resulting from the increased sample length, enables J(c) determinations at an electric field criterion E-c=10 muV/m, which is substantially lower than a criterion of E-c=100 muV/m which was possible in our previous systems. (C) 2004 American Institute of Physics.

Conventional in vivo irradiation procedures are insufficient to accurately determine tumor responses to non-uniform radiation fields

Purpose: To determine differences in overall tumor responses measured by volumetric assessment and bioluminescence imaging (BLI) following exposure to uniform and non-uniform radiation fields in an ectopic prostate tumor model.

Materials and methods: Bioluminescent human prostate tumor xenografts were established by subcutaneous implantation into male mice. Tumors were irradiated with uniform or non-uniform field configurations using conventional in vivo irradiation procedures performed using a 225 kVp generator with custom lead shielding. Tumor responses were measured using Vernier calipers and by BLI using an in vivo imaging system. Survival was defined as the time to quadroupling of pre-treatment tumor volume. 

Results: The correlation between BLI and tumor volume measurements was found to be different for un-irradiated (R = 0.61), uniformly irradiated (R = 0.34) and partially irradiated (R = 0.30) tumors. Uniformly irradiated tumors resulted in an average tumor growth delay of 60 days with median survival of 75 days, compared to partially irradiated tumors which showed an average growth delay of 24 days and median survival of 38 days. 

Conclusions: Correlation between BLI and tumor volume measurements is lower for partially irradiated tumors than those exposed to uniform dose distributions. The response of partially irradiated tumors suggests non-uniformity in response beyond physical dose distribution within the target volume. Dosimetric uncertainty associated with conventional in vivo irradiation procedures prohibits their ability to accurately determine tumor response to non-uniform radiation fields and stresses the need for image guided small animal radiation research platforms.

Comparison of three molecular techniques for typing Pseudomonas aeruginosa isolates in sputum samples from patients with cystic fibrosis

Monitoring the emergence and transmission of Pseudomonas aeruginosa strains among cystic fibrosis (CF) patients is important for infection control in CF centers internationally. A recently developed multilocus sequence typing (MLST) scheme is used for epidemiologic analyses of P. aeruginosa outbreaks; however, little is known about its suitability for isolates from CF patients compared with that of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). As part of a prevalence study of P. aeruginosa strains in Australian CF clinics, we compared the discriminatory power and concordance of ERIC-PCR, PFGE, and MLST among 93 CF sputum and 11 control P. aeruginosa isolates. PFGE and MLST analyses were also performed on 30 paired isolates collected 85 to 354 days apart from 30 patients attending two CF centers separated by 3,600 kilometers in order to detect within-host evolution. Each of the three methods displayed high levels of concordance and discrimination; however, overall lower discrimination was seen with ERIC-PCR than with MLST and PFGE. Analysis of the 50 ERIC-PCR types yielded 54 PFGE types, which were related by ≤ 6 band differences, and 59 sequence types, which were classified into 7 BURST groups and 42 singletons. MLST also proved useful for detecting novel and known strains and for inferring relatedness among unique PFGE types. However, 47% of the paired isolates produced PFGE patterns that within 1 year differed by one to five bands, whereas with MLST all paired isolates remained identical. MLST thus represents a categorical analysis tool with resolving power similar to that of PFGE for typing P. aeruginosa. Its focus on highly conserved housekeeping genes is particularly suited for long-term clinical monitoring and detecting novel strains.

Identification of systemic immune response markers through metabolomic profiling of plasma from calves given an intra-nasally delivered respiratory vaccine

Vaccination procedures within the cattle industry are important disease control tools to minimize economic and welfare burdens associated with respiratory pathogens. However, new vaccine, antigen and carrier technologies are required to combat emerging viral strains and enhance the efficacy of respiratory vaccines, particularly at the point of pathogen entry. New technologies, specifically metabolomic profiling, could be applied to identify metabolite immune-correlates representative of immune protection following vaccination aiding in the design and screening of vaccine candidates. This study for the first time demonstrates the ability of untargeted UPLC-MS metabolomic profiling to identify metabolite immune correlates characteristic of immune responses following mucosal vaccination in calves. Male Holstein Friesian calves were vaccinated with Pfizer Rispoval® PI3 + RSV intranasal vaccine and metabolomic profiling of post-vaccination plasma revealed 12 metabolites whose peak intensities differed significantly from controls. Plasma levels of glycocholic acid, N-[(3α,5β,12α)-3,12-Dihydroxy-7,24-dioxocholan-24-yl]glycine, uric acid and biliverdin were found to be significantly elevated in vaccinated animals following secondary vaccine administration, whereas hippuric acid significantly decreased. In contrast, significant upregulation of taurodeoxycholic acid and propionylcarnitine levels were confined to primary vaccine administration. Assessment of such metabolite markers may provide greater information on the immune pathways stimulated from vaccine formulations and benchmarking early metabolomic responses to highly immunogenic vaccine formulations could provide a means for rapidly assessing new vaccine formulations. Furthermore, the identification of metabolic systemic immune response markers which relate to specific cell signaling pathways of the immune system could allow for targeted vaccine design to stimulate key pathways which can be assessed at the metabolic level.

A multi-proxy chronological reconstruction of palaeosalinity in the eastern Limfjord, Denmark

The Jutland peninsula in northern Denmark is home to the Limfjord, one of the largest estuarine bodies of water in the region. Human inhabitance of the Limfjord’s surrounding coastlines stretches back further than 7,800 cal BP, with anthropogenic influence on the landscape beginning approximately 6,000 cal BP. Understanding how the Limfjord as a system has changed throughout time is useful in comprehending subsistence patterns and anthropogenic influence. This research is part of a larger project aimed at discerning subsistence patterns and environmental change in the region. Following the Younger Dryas, as the Fennoscandian ice sheet began to melt, Denmark experienced isostatic rebound, which contributed to the complex sea level history in the region. Between ice melt and isostatic rebound, the Jutland peninsula experienced many transgression and regression events. Connections to surrounding seas have shifted throughout time, with most attention focused on the western connection of the Limfjord with the North Sea, which has experienced numerous closures and subsequent re-openings throughout the Holocene. Furthermore, the Limfjord-North Sea connection has been the focal point of research because of the west to east water flow in the system, and the present day higher salinity in the west compared to the east. Little to no consideration has been paid to the influence of the Kattegat and Baltic on the Limfjord until now. A 10m sediment core was taken from Sebbersund (near Nibe, Limfjord), along the connection between the Limfjord and the Kattegat in the east to understand how the eastern part of the system has changed and differed from changes observed in the west. The Sebbersund sequence spans a majority of the Holocene, from 9600 cal BP to 1030 cal BP, determined via radiocarbon dating of terrestrial macrofossils and bulk sediment. Over this time period palaeoenvironmental conditions were reconstructed through the use of geochemical analyses (13C, 15N, C:N), physical sediment analyses, dinoflagellate cyst abundances and molluscan analyses. apart from two instances of low salinity, one at the top and one at the bottom of the core, the sequence has a strong marine signal for a majority of the Holocene. Radiocarbon dating of bulk sediment samples showed the presence of old carbon in the system, creating an age offset between 1,300 ± 200 and 2,800 ± 200 calibrated 14C years compared to the age-depth curve based on the terrestrial macrofossils. This finding, along with the strong marine influence in the system, discerned through geochemical data, dinoflagellate cyst and mollusc counts, is important for obtaining accurate radiocarbon ages in the region and stresses the importance of understanding both the marine and freshwater reservoir effects. The marine dominance in the eastern Limfjord differs from the west, which is characterized by a number of freshwater events when the North Sea connection was closed off during the Holocene. The eastern connection was open to the Kattegat throughout a large portion of the Holocene, with influx of open ocean water entering the system during periods of higher sea level.

FEM Analysis to Optimally Design End Mill cutters for Milling of Ti-6Al-4V

This paper presents an FEM analysis conducted for optimally designing end mill cutters through verifying the cutting tool forces and stresses for milling Titanium alloy Ti-6Al-4 V. Initially, the theoretical tool forces are calculated by considering the cutting edge on a cutting tool as the curve of an intersection over a spherical/flat surface based on the model developed by Lee & Altinas [1]. Considering the lowest tool forces the cutting tool parameters are taken and optimal design of end mill is decided for different sizes. Then the 3D CAD models of the end mills are developed and used for Finite Element Method to verify the cutting forces for milling Ti-6Al-4 V. The cutting tool forces, stress, strain concentration (s), tool wear, and temperature of the cutting tool with the different geometric shapes are simulated considering Ti-6Al-4 V as work piece material. Finally, the simulated and theoretical values are compared and the optimal design of cutting tool for different sizes are validated. The present approach considers to improve the quality of machining surface and tool life with effects of the various parameters concerning the oblique cutting process namely axial, radial and tangential forces. Various simulated test cases are presented to highlight the approach on optimally designing end mill cutters.