Recent Biophysical Issues About the Preparation of Solute-Filled Lipid Vesicles

Here we report some recent biophysical issues on the preparation of solute-filled lipid vesicles and their relevance to the construction of “synthetic cells.” First, we introduce the “semi-synthetic minimal cells” as the liposome-based cell-like systems, which contain a minimal number of biomolecules required to display simple and complex biological functions. Next, we focus on recent aspects related to the construction of synthetic cells. Emphasis is given to the interplay between the methods of synthetic cell preparation and the physics of solute encapsulation. We briefly introduce the notion of structural and compositional “diversity” in synthetic cell populations.     

Prolonged tamoxifen exposure selects a breast cancer cell clone that is stable in vitro and in vivo

The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo.     

Cell-mediated immunological responses in cervical and vaginal cancer patients immunized with a lipidated epitope of human papillomavirus type 16 E7

Human papillomavirus (HPV) infection has been causally associated with cervical cancer. We tested the effectiveness of an HLA-A*0201-restricted, HPV-16 E7 lipopeptide vaccine in eliciting cellular immune responses in vivo in women with refractory cervical cancer. In a nonrandomized Phase I clinical trial, 12 women expressing the HLA-A2 allele with refractory cervical or vaginal cancer were vaccinated with four E786-93 lipopeptide inoculations at 3-week intervals. HLA-A2 subtyping was also performed, and HPV typing was assessed on tumor specimens. Induction of epitope-specific CD8+ T-lymphocyte (CTL) responses was analyzed using peripheral blood leukapheresis specimens obtained before and after vaccination. CTL specificity was measured by IFN-gamma release assay using HLA-A*0201 matched target cells. Clinical responses were assessed by physical examination and radiographic images. All HLA-A*0201 patients were able to mount a cellular immune response to a control peptide. E786-93-specific CTLs were elicited in 4 of 10 evaluable HLA-A*0201 subjects before vaccination, 5 of 7 evaluable HLA-A*0201 patients after two vaccinations, and 2 of 3 evaluable HLA-A*0201 cultures after all four inoculations. Two of three evaluable patients' CTLs converted from unreactive to reactive after administration of all four inoculations. There were no clinical responses or treatment toxicities. The ability to generate specific cellular immune responses is retained in patients with advanced cervical cancer. Vaccination with a lipidated HPV peptide epitope appears capable of safely augmenting CTL reactivity. Although enhancements of cellular immune responses are needed to achieve therapeutic utility in advanced cervical cancer, this approach might prove useful in treating preinvasive disease.     

Preparation and characterization of chitosan‐based dispersions

Complexation of chitosan in aqueous solutions by low molecular weight electrolytes is one of the simplest methods for the preparation of aqueous chitosan dispersions. In this work, the influence of storage time, sulfate concentration, method of preparation and surfactant content on some properties of the resultant chitosan dispersions (turbidity, viscosity and zeta potential) was analyzed. Turbidimetry was adequate to monitor the formation of particles, while viscometry was suitable to monitor changes in the dispersing phase. An analysis of the properties of these systems, mainly in terms of particle–particle and macromolecule–macromolecule interactions was carried out. Copyright © 2004 Society of Chemical Industry     

Study of the nozzle flow in a cross-flow turbine

An investigation of the flow inside the nozzle of a Cross-Flow turbine, which is a hydraulic turbine where the rectangular water jet issuing from the nozzle crosses the rotor blades twice, is presented here. Part of the investigation consisted in the experimental measurement of the static pressure distribution on the inside walls of two different nozzle configurations, both with the nozzle mounted alone and in the presence of a rotor. The tests performed in the presence of a rotor included the measurement of efficiency and covered a wide range of working conditions around the best efficiency point. The analysis of the results obtained in this way give us an indication of the influence of the turbine non-dimensional volume flow rate on the flow inside the nozzle and the way it affects the reaction degree of the machine and its efficiency level. Although most of the tests were carried out with a 25-blade rotor, one of the analysed nozzle configurations (that with an inside vane) was also tested with a 10-blade rotor, permitting the assessment of the effect the number of blades has on the flow in the nozzle.The flow inside the nozzle with no inside vane was numerically analysed using a method based on a Schwarz-Christoffel conformal transformation of variables. The numerical results show a fair agreement with the experimental data collected when the rotor was not present. A qualitative discussion of some of the losses occurring in the nozzle is advanced based on the computer results, and its conclusions are used for explaining the poor performance of the nozzle with no inside vane.     

Thrombotic thrombocytopenic purpura and hemolytic uremic syndrome (TTP/HUS). Description of a series of 35 patients

Subsequent to the implementation of a severity marker stamp in case notes, an audit was performed in 86 admissions with acute asthma to a specialist centre over a 12 month period. Compared to previous audit the documentation of severity markers was significantly better (PEFR: 52% vs 83% p = 0.001, Respiratory rate: 44% vs 81% p = 0.001, ABG: 72% vs 80% p = 0.04, air entry: 58% vs 86% p = 0.001, speech: 27% vs 86% p = 0.001, exhaustion: 4% vs 86% p = 0.001). In contrast to the previous audit where no patient received FiO2 > 0.35, 66% of the cases in the repeat audit received FiO2 0.60 (p = 0.001). The mean duration of admission was five days and showed highest partial correlation (r = 0.6) to the time in hours for the pulse to fall to 80/min. Multiple linear regression showed that this was the only variable best predicting the duration of admission (R2 = 0.3). Admission pulse rate (p = 0.04) and serum K+ (p = 0.04) best discriminated between patients admitted for over and under five days. Logistic regression identified only the admission pulse as significant in calculating the odds of the patient staying in the hospital for > 5 days.     

Electric field effects on roto-vibrational transitions of13CD3OH1

We used a Stark-Optoacoustic cell and hybrid waveguide resonators to perform an Infrared and Far Infrared Stark Spectroscopy study on some transitions of¹³CD₃OH. Different behaviours of the transitions in the presence of a d.c. electric field were observed. The Stark splittings of six FIR laser lines ranging from 34 to 136 MHz/kVcm^?1 were determined. The analysis of the behaviour of the IR and FIR transitions in the presence of the external electric fields gives important and exclusive information on the levels involved in the transitions.     

Data collection: evaluation of a pilot model

The overall purpose of this study was to explore nurses' feelings about the applicability and adequacy of a pilot model of nursing assessment (PMNA) developed for coronary care units (CCU) in order to obtain data that could help in establishing a definitive model. The evaluation, performed by 11 CCU nurses, showed that they considered the development and implementation of PMNA as valuable, and that its design was adequate for interviewing cardiac patients. These results will be employed in the elaboration of a definitive model of nursing assessment.     

The role of hydrodynamic stress on the phenotypic characteristics of single and binary biofilms of Pseudomonas fluorescens.

This study investigates the phenotype of turbulent (Re = 5,200) and laminar (Re = 2,000) flow-generated Pseudomonas fluorescens biofilms. Three P. fluorescens strains, the type strain ATCC 13525 and two strains isolated from an industrial processing plant, D3-348 and D3-350, were used throughout this study. The isolated strains were used to form single and binary biofilms. The biofilm physiology (metabolic activity, cellular density, mass, extracellular polymeric substances, structural characteristics and outer membrane proteins [OMP] expression) was compared. The results indicate that, for every situation, turbulent flow-generated biofilms were more active (p < 0.05), had more mass per cm(2) (p < 0.05), a higher cellular density (p < 0.05), distinct morphology, similar matrix proteins (p > 0.1) and identical (isolated strains -single and binary biofilms) and higher (type strain) matrix polysaccharides contents (p < 0.05) than laminar flow-generated biofilms. Flow-generated biofilms formed by the type strain revealed a considerably higher cellular density and amount of matrix polysaccharides than single and binary biofilms formed by the isolated strains (p < 0.05). Similar OMP expression was detected for the several single strains and for the binary situation, not dependent on the hydrodynamic conditions. Binary biofilms revealed an equal coexistence of the isolated strains with apparent neutral interactions. In summary, the biofilms formed by the type strain represent, apparently, the worst situation in a context of control. The results obtained clearly illustrate the importance of considering strain variation and hydrodynamics in biofilm development, and complement previous studies which have focused on physical aspects of structural and density differences.