Single Step Plasma Process for Covalent Binding of Antimicrobial Peptides on Catheters to Suppress Bacterial Adhesion

Catheter-associated biofilms are responsible for a large fraction of hospital acquired infections. Antimicrobial surface coating on catheters providing prevention at source is extensively studied to reduce bacterial adhesion. Antimicrobial peptides such as melimine and Mel4, covalently linked to surfaces have shown excellent potential in animal and human studies to suppress infection without toxicity. Covalent binding of the peptides on catheter surfaces improves efficacy but so far has been implemented using multi-step wet chemical coupling that will impede widespread adoption. Here we demonstrate plasma immersion ion implantation (PIII) as a single step treatment that covalently couples antimicrobial peptides to polyvinyl chloride (PVC). Strong antimicrobial activity was demonstrated by higher than 3 log kill of S. aureus. A variant of the process was demonstrated as an antimicrobial treatment for chemically inert glass surfaces. Covalent coupling was rigorously tested by stringent SDS washing. We further demonstrated that the plasma treatment can effectively functionalize both internal and external surfaces of catheter tubing, reducing 99% of bacterial adhesion. The process is feasible as a patient-safe treatment for treating various types of catheters and is suitable for commercial mass production. In a logical extension of the work, the process could be adapted to bone replacement scaffolds of all types including metallic, polymeric and ceramic

Radiation Damage to Polymer Optical Fibres

Recently, polymer optical fibre dosimeters have been proposed for use in radiation oncology. Prior to clinical acceptance the effects of radiation on the materials involved must be assessed. In this work, the effects of radiation on mechanical strength and power transmission of polymer optical fibres were investigated. The fibres were subject to high energy electrons and photons generated by a linear accelerator for a range of doses. The mechanical properties and melting point of the fibres after the radiation were evaluated using tensile and differential scanning calorimetry (DSC) tests. The power transmission and absorption spectrum to visible light and their dependence on radiation conditions were also estimated. The overall performance of these polymer optical fibres as a part of the dosimeter subjected to radiation is discussed

Small field in-air output factors: the role of miniphantom design and dosimeter type

The commissioning of treatment planning systems and beam modeling requires measured input parameters. The measurement of relative output in-air, Sc is particularly difficult for small fields. The purpose of this study was to investigate the influence of miniphantom design and detector selection on measured Sc values for small fields and to validate the measurements against Monte Carlo simulations. Measurements were performed using brass caps (with sidewalls) or tops (no sidewalls) of varying heights and widths. The performance of two unshielded diodes (60012 and SFD), EBT2 radiochromic film, and a fiber optic dosimeter (FOD) were compared for fields defined by MLCs (5-100 mm) and SRS cones (4-30 mm) on a Varian Novalis linear accelerator. Monte Carlo simulations were performed to theoretically predict Sc as measured by the FOD. For all detectors, Sc agreed to within 1% for fields larger than 10 mm and to within 2.3% for smaller fields. Monte Carlo simulation matched the FOD measurements for all size of cone defined fields to within 0.5%. Miniphantom design is the most important variable for reproducible and accurate measurements of the in-air output ratio, Sc, in small photon fields (less than 30 mm). Sidewalls are not required for fields less than or equal too 30 mm and tops are therefore preferred over the larger caps. Unlike output measurements in water, Scp, the selection of detector type for Sc is not critical, provided the active dosimeter volume is small relative to the field siz

Small field diode correction factors derived using an air core fibre optic scintillation dosimeter and EBT2 film

There is no commercially available real-time dosimeter that can accurately measure output factors for field sizes down to 4 mm without the use of correction factors. Silicon diode detectors are commonly used but are not dosimetrically water equivalent, resulting in energy dependence and fluence perturbation. In contrast, plastic scintillators are nearly dosimetrically water equivalent. A fibre optic dosimeter (FOD) with a 0.8 mm3 plastic scintillator coupled to an air core light guide was used to measure the output factors for Novalis/BrainLab stereotactic cones of diameter 4-30 mm and Novalis MLC fields of width 5-100 mm. The FOD data matched the output factors measured by a 0.125 cm3 Semiflex ion chamber for the MLC fields above 30 mm and those measured with the EBT2 radiochromic film for the cones and MLC fields below 30 mm. Relative detector readings were obtained with four diode types (IBA SFD, EFD, PFD, PTW 60012) for the same fields. Empirical diode correction factors were determined by taking the ratio of FOD output factors to diode relative detector readings. The diodes were found to over-respond by 3%-16% for the smallest field. There was good agreement between different diodes of the same model number

Spermatogonia survival in young ram lambs following irradiation, Busulfan or thermal treatment

Many alternatives to surgical castration have been explored to induce short or long term infertility in male animals. Comparatively few have been carefully evaluated in very young production animals. A comparison of four treatments known to deplete testicular cells in rodents (heating, cooling, chemotherapy, radiation) was undertaken using ram lambs. Neither testicular cooling (0 °C) nor heating (45 °C) affected testis weights, tubule diameters or germ cell numbers. Low dose chemotherapy (Busulfan 4 mg/kg) treatment caused dramatic falls in white blood cells and platelets numbers which recovered within 3 weeks. Spermatogonia numbers were not significantly reduced (27% change). The impact of irradiation doses (0–15 Gy) delivered with high precision to the testis by a 6 MV photon beam was assessed by serial biopsies. Sertoli cell numbers were depleted by 90% at 3 weeks in 15 Gy treated testes. Spermatogonia were depleted 8 weeks after irradiation with 9 Gy, 12 Gy and 15 Gy. By 13 weeks, only in the 15 Gy treated testes were spermatogonia and Sertoli cell numbers lower. At 13 weeks testis atrophy resulted in 3/6 lambs irradiated with 12 Gy or 15 Gy. Irradiation of very young lambs clearly compromised testis function, thermal treatment was ineffective and busulfan treatment resulted in minimal effects

Profiling of the secretome of human cancer cells : preparation of supernatant for proteomic analysis

Secretomic analysis requires removal of serum proteins from cell-culture media. We evaluate the proteins washed from cells prepared in bovine serum-supplemented medium. PBS and serum-free-medium (SFM) were the washing solutions. A Bradford assay was used for total protein concentration and a 1D gel and LC-MS/MS, to assign the protein to human or bovine origin. For both wash solutions, all bovine protein had been removed by the third wash, without compromising the number of living cells. Further washes reduced the number of living cells, especially when using PBS. Proteomic analysis of wash supernatant showed that SFM induced greater lysis of dead cells. Three washes were sufficient to minimize the effects on cell viability, while still removing serum proteins. Washing in SFM resulted in contamination of the wash supernatant with lysed dead cell proteins. Washed cells were incubated in SFM and exposed to ionizing radiation. Analysis of the supernatant showed an increase in human cytoplasmic, plasma membrane, and nuclear protein following irradiation. Secreted proteins were also detected, but in smaller quantities. The significance of these findings extend to in vitro studies of bystander phenomena, since the proteins of lysed dead cells may participate in driving bystander responses.8 page(s

Irradiation Enhances the Efficiency of Testicular Germ Cell Transplantation in Sheep

Testis germ cell transplantation in livestock has the potential for production of transgenic genotypes and for use as an alternative to artificial insemination in animal breeding systems. In a pilot experiment, we investigated a workable protocol for testis germ cell transplantation in sheep, including donor cell isolation, rete testis injection, and microsatellite detection of donor spermatozoa in recipient semen. In a second experiment, the effect of depletion of endogenous stem cells with a single irradiation dose of 9 Gy (n = 5) or 15 Gy (n = 5) on the outcome of germ cell transplantation was investigated. Irradiation of recipient testes with a single dose of 15 Gy, followed by transplantation 6 wk after depletion, may be most advantageous because it resulted in all recipients (five of five) producing donor-derived spermatozoa, while the 9-Gy and control groups had limited success rates (two of five and one of three, respectively). Using microsatellite markers to detect the presence of donor DNA, 10 rams were identified that produced spermatozoa of donor origin. The proportion of donor DNA was between 1% and 30% of total ejaculate DNA. When three of these positive rams were used in breeding experiments, four donor-derived offspring (four of 50 [8% of progeny])resulted from a recipient in Merino to Merino transplantation. Six lambs (six of 41 [15% of progeny]) were sired by donor-derived Border Leicester sperm produced in a Merino recipient ram; however, no donor-derived offspring were detected among 34 progeny from a second Border Leicester to Merino combination. These results confirm that preparation of recipient animals with a correct dose of irradiation not only enhances the success rate of the transplantation procedure but also increases the proportion of donor spermatozoa in recipient semen. This study represents the first report of the production of live progeny following testis germ cell transplantation using irradiated recipients in a livestock species