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Recent Articles in Biomedical Engineering Online

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Wellnhofer E, Goubergrits L, Kertzscher U, Affeld K
In-vivo coronary flow profiling based on biplane angiograms: influence of geometric simplifications on the three-dimensional reconstruction and wall shear stress calculation.
Biomed Eng Online. 2006;539.
BACKGROUND: Clinical studies suggest that local wall shear stress (WSS) patterns modulate the site and the progression of atherosclerotic lesions. Computational fluid dynamics (CFD) methods based on in-vivo three-dimensional vessel reconstructions have recently been shown to provide prognostically relevant WSS data. This approach is, however, complex and time-consuming. Methodological simplifications are desirable in porting this approach from bench to bedside. The impact of such simplifications on the accuracy of geometry and wall shear stress calculations has to be investigated. METHODS: We investigated the influence of two methods of lumen reconstruction, assuming circular versus elliptical cross-sections and using different resolutions for the cross-section reconstructions along the vessel axis. Three right coronary arteries were used, of which one represented a normal coronary artery, one with "obstructive", and one with "dilated" coronary atherosclerosis. The vessel volume reconstruction was performed with three-dimensional (3D) data from a previously validated 3D angiographic reconstruction of vessel cross-sections and vessel axis. RESULTS: The difference between the two vessel volumes calculated using the two evaluated methods is less than 1 %. The difference, of the calculated pressure loss, was between 2.5% and 8.5% for the evaluated methods. The distributions of the WSS histograms were nearly identical and strongly cross-correlated (0.91-0.95). The good agreement of the results was confirmed by a Chi-square test. CONCLUSION: A simplified approach to the reconstruction of coronary vessel lumina, using circular cross-sections and a reduced axial resolution of about 0.8 mm along the vessel axis, yields sufficiently accurate calculations of WSS. [Abstract/Link to Full Text]

Dou Y, Fan Y, Zhao J, Gregersen H
Longitudinal residual strain and stress-strain relationship in rat small intestine.
Biomed Eng Online. 2006;537.
BACKGROUND: To obtain a more detailed description of the stress-free state of the intestinal wall, longitudinal residual strain measurements are needed. Furthermore, data on longitudinal stress-strain relations in visceral organs are scarce. The present study aims to investigate the longitudinal residual strain and the longitudinal stress-strain relationship in the rat small intestine. METHODS: The longitudinal zero-stress state was obtained by cutting tissue strips parallel to the longitudinal axis of the intestine. The longitudinal residual stress was characterized by a bending angle (unit: degrees per unit length and positive when bending outwards). Residual strain was computed from the change in dimensions between the zero-stress state and the no-load state. Longitudinal stresses and strains were computed from stretch experiments in the distal ileum at luminal pressures ranging from 0-4 cmH2O. RESULTS: Large morphometric variations were found between the duodenum and ileum with the largest wall thickness and wall area in the duodenum and the largest inner circumference and luminal area in the distal ileum (p < 0.001). The bending angle did not differ between the duodenum and ileum (p > 0.5). The longitudinal residual strain was tensile at the serosal surface and compressive at the mucosal surface. Hence, the neutral axis was approximately in the mid-wall. The longitudinal residual strain and the bending angle was not uniform around the intestinal circumference and had the highest values on the mesenteric sides (p < 0.001). The stress-strain curves fitted well to the mono-exponential function with determination coefficients above 0.96. The alpha constant increased with the pressure, indicating the intestinal wall became stiffer in longitudinal direction when pressurized. CONCLUSION: Large longitudinal residual strains reside in the small intestine and showed circumferential variation. This indicates that the tissue is not uniform and cannot be treated as a homogenous material. The longitudinal stiffness of the intestinal wall increased with luminal pressure. Longitudinal residual strains must be taken into account in studies of gastrointestinal biomechanical properties. [Abstract/Link to Full Text]

Diaspro A, Bianchini P, Vicidomini G, Faretta M, Ramoino P, Usai C
Multi-photon excitation microscopy.
Biomed Eng Online. 2006;536.
Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments. [Abstract/Link to Full Text]

Busch MH, Vollmann W, Grönemeyer DH
Finite volume analysis of temperature effects induced by active MRI implants: 2. Defects on active MRI implants causing hot spots.
Biomed Eng Online. 2006;535.
BACKGROUND: Active magnetic resonance imaging implants, for example stents, stent grafts or vena cava filters, are constructed as wireless inductively coupled transmit and receive coils. They are built as a resonator tuned to the Larmor frequency of a magnetic resonance system. The resonator can be added to or incorporated within the implant. This technology can counteract the shielding caused by eddy currents inside the metallic implant structure. This may allow getting diagnostic information of the implant lumen (in stent stenosis or thrombosis for example). The electro magnetic rf-pulses during magnetic resonance imaging induce a current in the circuit path of the resonator. A by material fatigue provoked partial rupture of the circuit path or a broken wire with touching surfaces can set up a relatively high resistance on a very short distance, which may behave as a point-like power source, a hot spot, inside the body part the resonator is implanted to. This local power loss inside a small volume can reach (1/4) of the total power loss of the intact resonating circuit, which itself is proportional to the product of the resonator volume and the quality factor and depends as well from the orientation of the resonator with respect to the main magnetic field and the imaging sequence the resonator is exposed to. METHODS: First an analytical solution of a hot spot for thermal equilibrium is described. This analytical solution with a definite hot spot power loss represents the worst case scenario for thermal equilibrium inside a homogeneous medium without cooling effects. Starting with this worst case assumptions additional conditions are considered in a numerical simulation, which are more realistic and may make the results less critical. The analytical solution as well as the numerical simulations use the experimental experience of the maximum hot spot power loss of implanted resonators with a definite volume during magnetic resonance imaging investigations. The finite volume analysis calculates the time developing temperature maps for the model of a broken linear metallic wire embedded in tissue. Half of the total hot spot power loss is assumed to diffuse into both wire parts at the location of a defect. The energy is distributed from there by heat conduction. Additionally the effect of blood perfusion and blood flow is respected in some simulations because the simultaneous appearance of all worst case conditions, especially the absence of blood perfusion and blood flow near the hot spot, is very unlikely for vessel implants. RESULTS: The analytical solution as worst case scenario as well as the finite volume analysis for near worst case situations show not negligible volumes with critical temperature increases for part of the modeled hot spot situations. MR investigations with a high rf-pulse density lasting below a minute can establish volumes of several cubic millimeters with temperature increases high enough to start cell destruction. Longer exposure times can involve volumes larger than 100 mm3. Even temperature increases in the range of thermal ablation are reached for substantial volumes. MR sequence exposure time and hot spot power loss are the primary factors influencing the volume with critical temperature increases. Wire radius, wire material as well as the physiological parameters blood perfusion and blood flow inside larger vessels reduce the volume with critical temperature increases, but do not exclude a volume with critical tissue heating for resonators with a large product of resonator volume and quality factor. CONCLUSION: The worst case scenario assumes thermal equilibrium for a hot spot embedded in homogeneous tissue without any cooling due to blood perfusion or flow. The finite volume analysis can calculate the results for near and not close to worst case conditions. For both cases a substantial volume can reach a critical temperature increase in a short time. The analytical solution, as absolute worst case, points out that resonators with a small product of inductance volume and quality factor (Q V(ind) < 2 cm3) are definitely save. Stents for coronary vessels or resonators used as tracking devices for interventional procedures therefore have no risk of high temperature increases. The finite volume analysis shows for sure that also conditions not close to the worst case reach physiologically critical temperature increases for implants with a large product of inductance volume and quality factor (Q V(ind) > 10 cm3). Such resonators exclude patients from exactly the MRI investigation these devices are made for. [Abstract/Link to Full Text]

Leung JH, Wright AR, Cheshire N, Crane J, Thom SA, Hughes AD, Xu Y
Fluid structure interaction of patient specific abdominal aortic aneurysms: a comparison with solid stress models.
Biomed Eng Online. 2006;533.
BACKGROUND: Abdominal aortic aneurysm (AAA) is a dilatation of the aortic wall, which can rupture, if left untreated. Previous work has shown that, maximum diameter is not a reliable determinant of AAA rupture. However, it is currently the most widely accepted indicator. Wall stress may be a better indicator and promising patient specific results from structural models using static pressure, have been published. Since flow and pressure inside AAA are non-uniform, the dynamic interaction between the pulsatile flow and wall may influence the predicted wall stress. The purpose of the present study was to compare static and dynamic wall stress analysis of patient specific AAAs. METHOD: Patient-specific AAA models were created from CT scans of three patients. Two simulations were performed on each lumen model, fluid structure interaction (FSI) model and static structural (SS) model. The AAA wall was created by dilating the lumen with a uniform 1.5 mm thickness, and was modeled as a non-linear hyperelastic material. Commercial finite element code Adina 8.2 was used for all simulations. The results were compared between the FSI and SS simulations. RESULTS: Results are presented for the wall stress patterns, wall shear stress patterns, pressure, and velocity fields within the lumen. It is demonstrated that including fluid flow can change local wall stresses slightly. However, as far as the peak wall stress is concerned, this effect is negligible as the difference between SS and FSI models is less than 1%. CONCLUSION: The results suggest that fully coupled FSI simulation, which requires considerable computational power to run, adds little to rupture risk prediction. This justifies the use of SS models in previous studies. [Abstract/Link to Full Text]

Christov I, Dotsinsky I, Simova I, Prokopova R, Trendafilova E, Naydenov S
Dataset of manually measured QT intervals in the electrocardiogram.
Biomed Eng Online. 2006;531.
BACKGROUND: The QT interval and the QT dispersion are currently a subject of considerable interest. Cardiac repolarization delay is known to favor the development of arrhythmias. The QT dispersion, defined as the difference between the longest and the shortest QT intervals or as the standard deviation of the QT duration in the 12-lead ECG is assumed to be reliable predictor of cardiovascular mortality.The seventh annual PhysioNet/Computers in Cardiology Challenge, 2006 addresses a question of high clinical interest: Can the QT interval be measured by fully automated methods with accuracy acceptable for clinical evaluations? METHOD: The PTB Diagnostic ECG Database was given to 4 cardiologists and 1 biomedical engineer for manual marking of QRS onsets and T-wave ends in 458 recordings. Each recording consisted of one selected beat in lead II, chosen visually to have minimum baseline shift, noise, and artifact.In cases where no T wave could be observed or its amplitude was very small, the referees were instructed to mark a 'group-T-wave end' taking into consideration leads with better manifested T wave.A modified Delphi approach was used, which included up to three rounds of measurements to obtain results closer to the median. RESULTS: A total amount of 2*5*548 Q-onsets and T-wave ends were manually marked during round 1. To obtain closer to the median results, 8.58 % of Q-onsets and 3.21 % of the T-wave ends had to be reviewed during round 2, and 1.50 % Q-onsets and 1.17 % T-wave ends in round 3.The mean and standard deviation of the differences between the values of the referees and the median after round 3 were 2.43 +/- 0.96 ms for the Q-onset, and 7.43 +/- 3.44 ms for the T-wave end. CONCLUSION: A fully accessible, on the Internet, dataset of manually measured Q-onsets and T-wave ends was created and presented in additional file: 1 (Table 4) with this article. Thus, an available standard can be used for the development of automated methods for the detection of Q-onsets, T-wave ends and for QT interval measurements. [Abstract/Link to Full Text]

Niehof SP, Huygen FJ, van der Weerd RW, Westra M, Zijlstra FJ
Thermography imaging during static and controlled thermoregulation in complex regional pain syndrome type 1: diagnostic value and involvement of the central sympathetic system.
Biomed Eng Online. 2006;530.
BACKGROUND: Complex Regional Pain Syndrome type 1 (CRPS1) is a clinical diagnosis based on criteria describing symptoms of the disease.The main aim of the present study was to compare the sensitivity and specificity of calculation methods used to assess thermographic images (infrared imaging) obtained during temperature provocation. The secondary objective was to obtain information about the involvement of the sympathetic system in CRPS1. METHODS: We studied 12 patients in whom CRPS1 was diagnosed according to the criteria of Bruehl. High and low whole body cooling and warming induced and reduced sympathetic vasoconstrictor activity. The degree of vasoconstrictor activity in both hands was monitored using a videothermograph. The sensitivity and specificity of the calculation methods used to assess the thermographic images were calculated. RESULTS: The temperature difference between the hands in the CRPS patients increases significantly when the sympathetic system is provoked. At both the maximum and minimum vasoconstriction no significant differences were found in fingertip temperatures between both hands. CONCLUSION: The majority of CRPS1 patients do not show maximal obtainable temperature differences between the involved and contralateral extremity at room temperature (static measurement). During cold and warm temperature challenges this temperature difference increases significantly. As a result a higher sensitivity and specificity could be achieved in the diagnosis of CRPS1. These findings suggest that the sympathetic efferent system is involved in CRPS1. [Abstract/Link to Full Text]

Lausted CG, Johnson AT, Scott WH, Johnson MM, Coyne KM, Coursey DC
Maximum static inspiratory and expiratory pressures with different lung volumes.
Biomed Eng Online. 2006;529.
BACKGROUND: Maximum pressures developed by the respiratory muscles can indicate the health of the respiratory system, help to determine maximum respiratory flow rates, and contribute to respiratory power development. Past measurements of maximum pressures have been found to be inadequate for inclusion in some exercise models involving respiration. METHODS: Maximum inspiratory and expiratory airway pressures were measured over a range of lung volumes in 29 female and 19 male adults. A commercial bell spirometry system was programmed to occlude airflow at nine target lung volumes ranging from 10% to 90% of vital capacity. RESULTS: In women, maximum expiratory pressure increased with volume from 39 to 61 cmH2O and maximum inspiratory pressure decreased with volume from 66 to 28 cmH2O. In men, maximum expiratory pressure increased with volume from 63 to 97 cmH2O and maximum inspiratory pressure decreased with volume from 97 to 39 cmH2O. Equations describing pressures for both sexes are: Pe/Pmax = 0.1426 Ln( %VC) + 0.3402 R2 = 0.95 Pi/Pmax = 0.234 Ln(100 - %VC) - 0.0828 R2 = 0.96 CONCLUSION: These results were found to be consistent with values and trends obtained by other authors. Regression equations may be suitable for respiratory mechanics models. [Abstract/Link to Full Text]

Soleimani M
Electrical impedance tomography system: an open access circuit design.
Biomed Eng Online. 2006;528.
BACKGROUND: This paper reports a simple 2-D system for electrical impedance tomography EIT, which works efficiently and is low cost. The system has been developed in the Sharif University of Technology Tehran-Iran (for the author's MSc Project). METHODS: The EIT system consists of a PC in which an I/O card is installed with an external current generator, a multiplexer, a power supply and a phantom with an array of electrodes. The measurement system provides 12-bit accuracy and hence, suitable data acquisition software has been prepared accordingly. The synchronous phase detection method has been implemented for voltage measurement. Different methods of image reconstruction have been used with this instrument to generate electrical conductivity images. RESULTS: The results of simulation and real measurement of the system are presented. The reconstruction programs were written in MATLAB and the data acquisition software in C++. The system has been tested with both static and dynamic mode in a 2-D domain. Better results have been produced in the dynamic mode of operation, due to the cancellation of errors. CONCLUSION: In the spirit of open access publication the design details of this simple EIT system are made available here. [Abstract/Link to Full Text]

Anderson EJ, Falls TD, Sorkin AM, Knothe Tate ML
The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction.
Biomed Eng Online. 2006;527.
BACKGROUND: In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level. METHODS: To evaluate how well actual flow chambers meet their target stresses (set for 1 and 10 dyn/cm2 for this study) at a cellular level, computational models were developed to calculate flow velocity components and imparted shear stresses for a given pressure gradient. Computational predictions were validated with micro-particle image velocimetry (microPIV) experiments. RESULTS: Based on these computational and experimental studies, as few as 66% of cells seeded along the midplane of commonly implemented flow/perfusion chambers are subjected to stresses within +/-10% of the target stress. In addition, flow velocities and shear stresses imparted through fluid drag vary as a function of location within each chamber. Hence, not only a limited number of cells are exposed to target stress levels within each chamber, but also neighboring cells may experience different flow regimes. Finally, flow regimes are highly dependent on flow chamber geometry, resulting in significant variation in magnitudes and spatial distributions of stress between chambers. CONCLUSION: The results of this study challenge the basic premise of in vitro mechanotransduction studies, i.e. that a controlled flow regime is applied to impart a defined mechanical stimulus to cells. These results also underscore the fact that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable. [Abstract/Link to Full Text]

Mordon SR, Wassmer B, Zemmouri J
Mathematical modeling of endovenous laser treatment (ELT).
Biomed Eng Online. 2006;526.
BACKGROUND AND OBJECTIVES: Endovenous laser treatment (ELT) has been recently proposed as an alternative in the treatment of reflux of the Great Saphenous Vein (GSV) and Small Saphenous Vein (SSV). Successful ELT depends on the selection of optimal parameters required to achieve an optimal vein damage while avoiding side effects. Mathematical modeling of ELT could provide a better understanding of the ELT process and could determine the optimal dosage as a function of vein diameter. STUDY DESIGN/MATERIALS AND METHODS: The model is based on calculations describing the light distribution using the diffusion approximation of the transport theory, the temperature rise using the bioheat equation and the laser-induced injury using the Arrhenius damage model. The geometry to simulate ELT was based on a 2D model consisting of a cylindrically symmetric blood vessel including a vessel wall and surrounded by an infinite homogenous tissue. The mathematical model was implemented using the Macsyma-Pdease2D software (Macsyma Inc., Arlington, MA, USA). Damage to the vein wall for CW and single shot energy was calculated for 3 and 5 mm vein diameters. In pulsed mode, the pullback distance (3, 5 and 7 mm) was considered. For CW mode simulation, the pullback speed (1, 2, 3 mm/s) was the variable. The total dose was expressed as joules per centimeter in order to perform comparison to results already reported in clinical studies. RESULTS: In pulsed mode, for a 3 mm vein diameter, irrespective of the pullback distance (2, 5 or 7 mm), a minimum fluence of 15 J/cm is required to obtain a permanent damage of the intima. For a 5 mm vein diameter, 50 J/cm (15W-2s) is required. In continuous mode, for a 3 mm and 5 mm vein diameter, respectively 65 J/cm and 100 J/cm are required to obtain a permanent damage of the vessel wall. Finally, the use of different wavelengths (810 nm or 980 nm) played only a minor influence on these results. DISCUSSION AND CONCLUSION: The parameters determined by mathematical modeling are in agreement with those used in clinical practice. They confirm that thermal damage of the inner vein wall (tunica intima) is required to achieve the tissue alterations necessary in order to lead the vein to permanent occlusion. However, in order to obtain a high rate of success without adverse events, the knowledge of the vein diameter after tumescent anesthesia is recommended in order to use the optimal energy. As clearly demonstrated by our calculations, both pulsed and continuous mode operations of the laser can be efficient. An interesting observation in our model is that less amount of energy is required in pulsed mode than in continuous mode. Damaging the vein sequentially along its entire length may lead to permanent occlusion. However, the pulsed mode requires a very precise positioning of the fiber after each pullback and the duration of the treatment is much longer. For these reasons, continuous irradiation seems to be preferred by most clinicians. This model should serve as a useful tool to simulate and better understand the mechanism of action of the ELT. [Abstract/Link to Full Text]

Gao F, Watanabe M, Matsuzawa T
Stress analysis in a layered aortic arch model under pulsatile blood flow.
Biomed Eng Online. 2006;525.
BACKGROUND: Many cardiovascular diseases, such as aortic dissection, frequently occur on the aortic arch and fluid-structure interactions play an important role in the cardiovascular system. Mechanical stress is crucial in the functioning of the cardiovascular system; therefore, stress analysis is a useful tool for understanding vascular pathophysiology. The present study is concerned with the stress distribution in a layered aortic arch model with interaction between pulsatile flow and the wall of the blood vessel. METHODS: A three-dimensional (3D) layered aortic arch model was constructed based on the aortic wall structure and arch shape. The complex mechanical interaction between pulsatile blood flow and wall dynamics in the aortic arch model was simulated by means of computational loose coupling fluid-structure interaction analyses. RESULTS: The results showed the variations of mechanical stress along the outer wall of the arch during the cardiac cycle. Variations of circumferential stress are very similar to variations of pressure. Composite stress in the aortic wall plane is high at the ascending portion of the arch and along the top of the arch, and is higher in the media than in the intima and adventitia across the wall thickness. CONCLUSION: Our analysis indicates that circumferential stress in the aortic wall is directly associated with blood pressure, supporting the clinical importance of blood pressure control. High stress in the aortic wall could be a risk factor in aortic dissections. Our numerical layered aortic model may prove useful for biomechanical analyses and for studying the pathogeneses of aortic dissection. [Abstract/Link to Full Text]

Berjano EJ
Theoretical modeling for radiofrequency ablation: state-of-the-art and challenges for the future.
Biomed Eng Online. 2006;524.
Radiofrequency ablation is an interventional technique that in recent years has come to be employed in very different medical fields, such as the elimination of cardiac arrhythmias or the destruction of tumors in different locations. In order to investigate and develop new techniques, and also to improve those currently employed, theoretical models and computer simulations are a powerful tool since they provide vital information on the electrical and thermal behavior of ablation rapidly and at low cost. In the future they could even help to plan individual treatment for each patient. This review analyzes the state-of-the-art in theoretical modeling as applied to the study of radiofrequency ablation techniques. Firstly, it describes the most important issues involved in this methodology, including the experimental validation. Secondly, it points out the present limitations, especially those related to the lack of an accurate characterization of the biological tissues. After analyzing the current and future benefits of this technique it finally suggests future lines and trends in the research of this area. [Abstract/Link to Full Text]

Ewald A, Glückermann SK, Thull R, Gbureck U
Antimicrobial titanium/silver PVD coatings on titanium.
Biomed Eng Online. 2006;522.
BACKGROUND: Biofilm formation and deep infection of endoprostheses is a recurrent complication in implant surgery. Post-operative infections may be overcome by adjusting antimicrobial properties of the implant surface prior to implantation. In this work we described the development of an antimicrobial titanium/silver hard coating via the physical vapor deposition (PVD) process. METHODS: Coatings with a thickness of approximately 2 mum were deposited on titanium surfaces by simultaneous vaporisation of both metals in an inert argon atmosphere with a silver content of approximately 0.7-9% as indicated by energy dispersive X-ray analysis. On these surfaces microorganisms and eukaryotic culture cells were grown. RESULTS: The coatings released sufficient silver ions (0.5-2.3 ppb) when immersed in PBS and showed significant antimicrobial potency against Staphylococcus epidermis and Klebsiella pneumoniae strains. At the same time, no cytotoxic effects of the coatings on osteoblast and epithelial cells were found. CONCLUSION: Due to similar mechanical performance when compared to pure titanium, the TiAg coatings should be suitable to provide antimicrobial activity on load-bearing implant surfaces. [Abstract/Link to Full Text]

Mann CJ, Yu L, Kim MK
Movies of cellular and sub-cellular motion by digital holographic microscopy.
Biomed Eng Online. 2006;521.
BACKGROUND: Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. METHODS: A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. RESULTS: Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration. CONCLUSION: Digital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres. [Abstract/Link to Full Text]

Kleinstreuer C, Li Z
Analysis and computer program for rupture-risk prediction of abdominal aortic aneurysms.
Biomed Eng Online. 2006;519.
BACKGROUND: Ruptured abdominal aortic aneurysms (AAAs) are the 13th leading cause of death in the United States. While AAA rupture may occur without significant warning, its risk assessment is generally based on critical values of the maximum AAA diameter (> 5 cm) and AAA-growth rate (> 0.5 cm/year). These criteria may be insufficient for reliable AAA-rupture risk assessment especially when predicting possible rupture of smaller AAAs. METHODS: Based on clinical evidence, eight biomechanical factors with associated weighting coefficients were determined and summed up in terms of a dimensionless, time-dependent severity parameter, SP(t). The most important factor is the maximum wall stress for which a semi-empirical correlation has been developed. RESULTS: The patient-specific SP(t) indicates the risk level of AAA rupture and provides a threshold value when surgical intervention becomes necessary. The severity parameter was validated with four clinical cases and its application is demonstrated for two AAA cases. CONCLUSION: As part of computational AAA-risk assessment and medical management, a patient-specific severity parameter 0 < SP(t) < 1.0 has been developed. The time-dependent, normalized SP(t) depends on eight biomechanical factors, to be obtained via a patient's pressure and AAA-geometry measurements. The resulting program is an easy-to-use tool which allows medical practitioners to make scientific diagnoses, which may save lives and should lead to an improved quality of life. [Abstract/Link to Full Text]

Singare S, Li D, Liu Y, Wu Z, Wang J
The effect of latency on bone lengthening force and bone mineralization: an investigation using strain gauge mounted on internal distractor device.
Biomed Eng Online. 2006;518.
BACKGROUND: The purpose of this study was to investigate the effect of latency on the development of bone lengthening force and bone mineralization during mandible distraction osteogenesis. METHODS: Distraction tensions were investigated at different latency period in 36 rabbits using internal unilateral distractor. Strain gauges were prepared and attached to the distractor to directly assess the level of distraction tension during mandible lengthening. The tensile force environment of the mandible of rabbit during distraction was evaluated through in vivo experiments using two gauges. The animals were divided into 3 groups each containing 12 rabbits. Latency periods of 0, 4 and 7 days respectively were observed prior to beginning distraction. The distraction protocol consisted of a lengthening rate of 1 mm once daily for 8 days, followed by a consolidation phase of 2 weeks after which the animals were killed. Biopsies specimens were taken from the distracted area at the end of the distraction period. A non-distracted area of the mandible bone served as control. The specimens were analyzed by scanning electron microscopy to assess the ultrastructural pattern, and the bone mineralization. RESULTS: The resting tension acting on the distraction gap increases through distraction. The 7-day latency groups exhibit higher tension then those of 0-day and 4-days latency groups. Quantitative energy dispersive spectral analysis confirmed that immediate distractions were associated with lower calcium and phosphate atomic weight ratio. CONCLUSION: the latency periods could affect the bone lengthening tension and the bone mineralization process. [Abstract/Link to Full Text]

Verhey JF, Nathan NS, Rienhoff O, Kikinis R, Rakebrandt F, D'Ambra MN
Finite-element-method (FEM) model generation of time-resolved 3D echocardiographic geometry data for mitral-valve volumetry.
Biomed Eng Online. 2006;517.
INTRODUCTION: Mitral Valve (MV) 3D structural data can be easily obtained using standard transesophageal echocardiography (TEE) devices but quantitative pre- and intraoperative volume analysis of the MV is presently not feasible in the cardiac operation room (OR). Finite element method (FEM) modelling is necessary to carry out precise and individual volume analysis and in the future will form the basis for simulation of cardiac interventions. METHOD: With the present retrospective pilot study we describe a method to transfer MV geometric data to 3D Slicer 2 software, an open-source medical visualization and analysis software package. A newly developed software program (ROIExtract) allowed selection of a region-of-interest (ROI) from the TEE data and data transformation for use in 3D Slicer. FEM models for quantitative volumetric studies were generated. RESULTS: ROI selection permitted the visualization and calculations required to create a sequence of volume rendered models of the MV allowing time-based visualization of regional deformation. Quantitation of tissue volume, especially important in myxomatous degeneration can be carried out. Rendered volumes are shown in 3D as well as in time-resolved 4D animations. CONCLUSION: The visualization of the segmented MV may significantly enhance clinical interpretation. This method provides an infrastructure for the study of image guided assessment of clinical findings and surgical planning. For complete pre- and intraoperative 3D MV FEM analysis, three input elements are necessary: 1. time-gated, reality-based structural information, 2. continuous MV pressure and 3. instantaneous tissue elastance. The present process makes the first of these elements available. Volume defect analysis is essential to fully understand functional and geometrical dysfunction of but not limited to the valve. 3D Slicer was used for semi-automatic valve border detection and volume-rendering of clinical 3D echocardiographic data. FEM based models were also calculated. METHOD: A Philips/HP Sonos 5500 ultrasound device stores volume data as time-resolved 4D volume data sets. Data sets for three subjects were used. Since 3D Slicer does not process time-resolved data sets, we employed a standard movie maker to animate the individual time-based models and visualizations. Calculation time and model size were minimized. Pressures were also easily available. We speculate that calculation of instantaneous elastance may be possible using instantaneous pressure values and tissue deformation data derived from the animated FEM. [Abstract/Link to Full Text]

Rhodes SS, Camara AK, Ropella KM, Audi SH, Riess ML, Pagel PS, Stowe DF
Ischemia reperfusion dysfunction changes model-estimated kinetics of myofilament interaction due to inotropic drugs in isolated hearts.
Biomed Eng Online. 2006;516.
BACKGROUND: The phase-space relationship between simultaneously measured myoplasmic [Ca2+] and isovolumetric left ventricular pressure (LVP) in guinea pig intact hearts is altered by ischemic and inotropic interventions. Our objective was to mathematically model this phase-space relationship between [Ca2+] and LVP with a focus on the changes in cross-bridge kinetics and myofilament Ca2+ sensitivity responsible for alterations in Ca2+-contraction coupling due to inotropic drugs in the presence and absence of ischemia reperfusion (IR) injury. METHODS: We used a four state computational model to predict LVP using experimentally measured, averaged myoplasmic [Ca2+] transients from unpaced, isolated guinea pig hearts as the model input. Values of model parameters were estimated by minimizing the error between experimentally measured LVP and model-predicted LVP. RESULTS: We found that IR injury resulted in reduced myofilament Ca2+ sensitivity, and decreased cross-bridge association and dissociation rates. Dopamine (8 microM) reduced myofilament Ca2+ sensitivity before, but enhanced it after ischemia while improving cross-bridge kinetics before and after IR injury. Dobutamine (4 microM) reduced myofilament Ca2+ sensitivity while improving cross-bridge kinetics before and after ischemia. Digoxin (1 microM) increased myofilament Ca2+ sensitivity and cross-bridge kinetics after but not before ischemia. Levosimendan (1 microM) enhanced myofilament Ca2+ affinity and cross-bridge kinetics only after ischemia. CONCLUSION: Estimated model parameters reveal mechanistic changes in Ca2+-contraction coupling due to IR injury, specifically the inefficient utilization of Ca2+ for contractile function with diastolic contracture (increase in resting diastolic LVP). The model parameters also reveal drug-induced improvements in Ca2+-contraction coupling before and after IR injury. [Abstract/Link to Full Text]

Bertram JM, Yang D, Converse MC, Webster JG, Mahvi DM
Antenna design for microwave hepatic ablation using an axisymmetric electromagnetic model.
Biomed Eng Online. 2006;515.
BACKGROUND: An axisymmetric finite element method (FEM) model was employed to demonstrate important techniques used in the design of antennas for hepatic microwave ablation (MWA). To effectively treat deep-seated hepatic tumors, these antennas should produce a highly localized specific absorption rate (SAR) pattern and be efficient radiators at approved generator frequencies. METHODS AND RESULTS: As an example, a double slot choked antenna for hepatic MWA was designed and implemented using FEMLABtrade mark 3.0. DISCUSSION: This paper emphasizes the importance of factors that can affect simulation accuracy, which include boundary conditions, the dielectric properties of liver tissue, and mesh resolution. [Abstract/Link to Full Text]

Lin MC, Dung LR, Weng PK
An ultra-low-power image compressor for capsule endoscope.
Biomed Eng Online. 2006;514.
BACKGROUND: Gastrointestinal (GI) endoscopy has been popularly applied for the diagnosis of diseases of the alimentary canal including Crohn's Disease, Celiac disease and other malabsorption disorders, benign and malignant tumors of the small intestine, vascular disorders and medication related small bowel injury. The wireless capsule endoscope has been successfully utilized to diagnose diseases of the small intestine and alleviate the discomfort and pain of patients. However, the resolution of demosaicked image is still low, and some interesting spots may be unintentionally omitted. Especially, the images will be severely distorted when physicians zoom images in for detailed diagnosis. Increasing resolution may cause significant power consumption in RF transmitter; hence, image compression is necessary for saving the power dissipation of RF transmitter. To overcome this drawback, we have been developing a new capsule endoscope, called GICam. METHODS: We developed an ultra-low-power image compression processor for capsule endoscope or swallowable imaging capsules. In applications of capsule endoscopy, it is imperative to consider battery life/performance trade-offs. Applying state-of-the-art video compression techniques may significantly reduce the image bit rate by their high compression ratio, but they all require intensive computation and consume much battery power. There are many fast compression algorithms for reducing computation load; however, they may result in distortion of the original image, which is not good for use in the medical care. Thus, this paper will first simplify traditional video compression algorithms and propose a scalable compression architecture. CONCLUSION: As the result, the developed video compressor only costs 31 K gates at 2 frames per second, consumes 14.92 mW, and reduces the video size by 75% at least. [Abstract/Link to Full Text]

Spitzer P, Zierhofer C, Hochmair E
Algorithm for multi-curve-fitting with shared parameters and a possible application in evoked compound action potential measurements.
Biomed Eng Online. 2006;513.
BACKGROUND: Experimental results are commonly fitted by determining parameter values of suitable mathematical expressions. In case a relation exists between different data sets, the accuracy of the parameters obtained can be increased by incorporating this relationship in the fitting process instead of fitting the recordings separately. METHODS: An algorithm to fit multiple measured curves simultaneously was developed. The method accounts for parameters that are shared by some curves. It can be applied to either linear or nonlinear equations. Simulated noisy "measurement results" were created to compare the introduced method to the "straight forward" way of fitting the curves separately. RESULTS: The analysis of the simulated measurements confirm, that the introduced method yields more accurate parameters compared to the ones gained by fitting the measurements separately. Therefore it needs more computer time. As an example, the new fitting algorithm is applied to the measurements of the evoked compound action potentials (ECAP) of the auditory nerve: This leads to promising ideas to reduce artefacts generated by the measuring process. CONCLUSION: The introduced fitting algorithm uses the relationship between multiple measurement results to increase the accuracy of the parameters. Its application in the field of ECAP measurements is promising and should be further investigated. [Abstract/Link to Full Text]

Abi-Abdallah D, Chauvet E, Bouchet-Fakri L, Bataillard A, Briguet A, Fokapu O
Reference signal extraction from corrupted ECG using wavelet decomposition for MRI sequence triggering: application to small animals.
Biomed Eng Online. 2006;511.
BACKGROUND: Present developments in Nuclear Magnetic Resonance (NMR) imaging techniques strive for improved spatial and temporal resolution performances. However, trying to achieve the shortest gradient rising time with high intensity gradients has its drawbacks: It generates high amplitude noises that get superimposed on the simultaneously recorded electrophysiological signals, needed to synchronize moving organ images. Consequently, new strategies have to be developed for processing these collected signals during Magnetic Resonance Imaging (MRI) examinations. The aim of this work is to extract an efficient reference signal, from an electrocardiogram (ECG) that was contaminated by the NMR artefacts. This may be used for image triggering and/or cardiac rhythm monitoring. METHODS: Our method, based on sub-band decomposition using wavelet filters, is tested on various ECG signals recorded during three imaging sequences: Gradient Echo (GE), Fast Spin Echo (FSE) and Inversion Recovery with Spin Echo (IRSE). In order to define the most adapted wavelet functions to use according to the excitation protocols, noise generated by each imaging sequence is recorded and analysed. After exploring noise models along with information found in the literature, a group of 14 wavelets, members of three families (Daubechies, Coiflets, Symlets), is selected for the study. The extraction process is carried out by decomposing the contaminated ECG signals into 8 scales using a given wavelet function, then combining the sub-bands necessary for cardiac synchronization, i.e. those containing the essential part of the QRS energy, to construct a reference signal. RESULTS: The efficiency of the presented method has been tested on a group of quite representative signals containing: highly contaminated (mean SNR<--5 dB) simulated ECGs that replicate normal and pathological human heart beats, as well as some pathological and healthy rodents' actual ECG records. Despite the weak SNR of the contaminated ECG, the performances were quite satisfactory. When comparing the wavelet performances, one may notice that for a given sequence, some wavelets are more efficient for processing than others; for GE, FSE and IRSE sequence, good synchronisation condition is accomplished with coif5, sym8, and sym4 respectively. CONCLUSION: Sub-band decomposition proved to be very suitable for extracting a reference signal from a corrupted ECG for MRI triggering. An appropriate choice of the wavelet function, in accordance with the image sequence type, could considerably improve the quality of the reference signal for better image synchronization. [Abstract/Link to Full Text]

Rudman KE, Aspden RM, Meakin JR
Compression or tension? The stress distribution in the proximal femur.
Biomed Eng Online. 2006;512.
BACKGROUND: Questions regarding the distribution of stress in the proximal human femur have never been adequately resolved. Traditionally, by considering the femur in isolation, it has been believed that the effect of body weight on the projecting neck and head places the superior aspect of the neck in tension. A minority view has proposed that this region is in compression because of muscular forces pulling the femur into the pelvis. Little has been done to study stress distributions in the proximal femur. We hypothesise that under physiological loading the majority of the proximal femur is in compression and that the internal trabecular structure functions as an arch, transferring compressive stresses to the femoral shaft. METHODS: To demonstrate the principle, we have developed a 2D finite element model of the femur in which body weight, a representation of the pelvis, and ligamentous forces were included. The regions of higher trabecular bone density in the proximal femur (the principal trabecular systems) were assigned a higher modulus than the surrounding trabecular bone. Two-legged and one-legged stances, the latter including an abductor force, were investigated. RESULTS: The inclusion of ligamentous forces in two-legged stance generated compressive stresses in the proximal femur. The increased modulus in areas of greater structural density focuses the stresses through the arch-like internal structure. Including an abductor muscle force in simulated one-legged stance also produced compression, but with a different distribution. CONCLUSION: This 2D model shows, in principle, that including ligamentous and muscular forces has the effect of generating compressive stresses across most of the proximal femur. The arch-like trabecular structure transmits the compressive loads to the shaft. The greater strength of bone in compression than in tension is then used to advantage. These results support the hypothesis presented. If correct, a better understanding of the stress distribution in the proximal femur may lead to improvements in prosthetic devices and an appreciation of the effects of various surgical procedures affecting load transmission across the hip. [Abstract/Link to Full Text]

Valentinuzzi ME
Review of "Introduction to instrumentation and measurements" by Robert B. Northrop.
Biomed Eng Online. 2006 Feb 8;5(1):9.
ABSTRACT: NNN. [Abstract/Link to Full Text]

Ramon C, Schimpf P, Haueisen J
Influence of head models on EEG simulations and inverse source localizations.
Biomed Eng Online. 2006 Feb 8;5(1):10.
ABSTRACT: BACKGROUND: The structure of the anatomical surfaces, e.g., CSF and gray and white matter, could severely influence the flow of volume currents in a head model. This, in turn, will also influence the scalp potentials and the inverse source localizations. This was examined in detail with four different human head models. METHODS: Four finite element head models constructed from segmented MR images of an adult male subject were used for this study. These models were: (1) Model 1: full model with eleven tissues that included detailed structure of the scalp, hard and soft skull bone, CSF, gray and white matter and other prominent tissues, (2) the Model 2 was derived from the Model 1 in which the conductivity of gray matter was set equal to the white matter, i.e., a ten tissue-type model, (3) the Model 3 was derived from the Model 1 in which the conductivities of gray matter and CSF were set equal to the white matter, i.e., a nine tissue-type model, (4) the Model 4 consisted of scalp, hard skull bone, CSF, gray and white matter, i.e., a five tissue-type model. How model complexity influences the EEG source localizations was also studied with the above four finite element models of the head. The lead fields and scalp potentials due to dipolar sources in the motor cortex were computed for all four models. The inverse source localizations were performed with an exhaustive search pattern in the motor cortex area. The inverse analysis was performed by adding uncorrelated Gaussian noise to the scalp potentials to achieve a signal to noise ratio (SNR) of -10 to 30 dB. The Model 1 was used as a reference model. RESULTS: The reference model, as expected, performed the best. The Model 3, which did not have the CSF layer, performed the worst. The mean source localization errors (MLEs) of the Model 3 were larger than the Model 1 or 2. The scalp potentials were also most affected by the lack of CSF geometry in the Model 3. The MLEs for the Model 4 were also larger than the Model 1 and 2. The Model 4 and the Model 3 had similar MLEs in the SNR range of -10 dB to 0 dB. However, in the SNR range of 5 dB to 30 dB, the Model 4 has lower MLEs as compared with the Model 3. Discussions: These results indicate that the complexity of head models strongly influences the scalp potentials and the inverse source localizations. A more complex head model performs better in inverse source localizations as compared to a model with lesser tissue surfaces. The CSF layer plays an important role in modifying the scalp potentials and also influences the inverse source localizations. In summary, for best results one needs to have highly heterogeneous models of the head for accurate simulations of scalp potentials and for inverse source localizations. Key Words: EEG, Finite Element, Head Models, Inverse EEG, CSF and EEG, Source Localizations, EEG Simulations. [Abstract/Link to Full Text]

Soleimani M
Electrical impedance tomography imaging using a priori ultrasound data.
Biomed Eng Online. 2006;58.
BACKGROUND: Different imaging systems (e.g. electrical, magnetic, and ultrasound) rely on a wide variety of physical properties, and the datasets obtained from such systems provide only partial information about the unknown true state. One approach is to choose complementary imaging systems, and to combine the information to achieve a better representation. METHODS: This paper discusses the combination of ultrasound and electrical impedance tomography (EIT) information. Ultrasound reflection signals are good at locating sharp acoustic density changes associated with the boundaries of objects. Some boundaries, however, may be indeterminable due to masking from intermediate boundaries or because they are outside the ultrasound beam. Conversely, the EIT data contains relatively low-quality information, but it includes the whole region enclosed by the electrodes. RESULTS: Results are shown from a narrowband level-set method applied to 2D and 3D EIT incorporating limited angle ultrasound time of flight data. CONCLUSION: The EIT reconstruction is shown to be faster and more accurate using the additional edge information from both one and four transducer ultrasound systems. [Abstract/Link to Full Text]

Dunster KR, Davies MW, Fraser JF
An advanced expiratory circuit for the recovery of perfluorocarbon liquid from non-saturated perfluorocarbon vapour during partial liquid ventilation: an experimental model.
Biomed Eng Online. 2006;57.
BACKGROUND: The loss of perfluorocarbon (PFC) vapour in the expired gases during partial liquid ventilation should be minimized both to prevent perfluorocarbon vapour entering the atmosphere and to re-use the recovered PFC liquid. Using a substantially modified design of our previously described condenser, we aimed to determine how much perfluorocarbon liquid could be recovered from gases containing PFC and water vapour, at concentrations found during partial liquid ventilation, and to determine if the amount recovered differed with background flow rate (at flow rates suitable for use in neonates). METHODS: The expiratory line of a standard ventilator circuit set-up was mimicked, with the addition of two condensers. Perfluorocarbon (30 mL of FC-77) and water vapour, at concentrations found during partial liquid ventilation, were passed through the circuit at a number of flow rates and the percentage recovery of the liquids measured. RESULTS: From 14.2 mL (47%) to 27.3 mL (91%) of the infused 30 mL of FC-77 was recovered at the flow rates studied. Significantly higher FC-77 recovery was obtained at lower flow rates (ANOVA with Bonferroni's multiple comparison test, p < 0.0001). As a percentage of the theoretical maximum recovery, 64 to 95% of the FC-77 was recovered. Statistically significantly less FC-77 was recovered at 5 Lmin(-1) (ANOVA with Bonferroni's multiple comparison test, p < 0.0001). Amounts of perfluorocarbon vapour recovered were 47%, 50%, 81% and 91% at flow rates of 10, 5, 2 and 1 Lmin(-1), respectively. CONCLUSION: Using two condensers in series 47% to 91% of perfluorocarbon liquid can be recovered, from gases containing perfluorocarbon and water vapour, at concentrations found during partial liquid ventilation. [Abstract/Link to Full Text]

Toro J
Review of "Medical Image Analysis Methods" by Lena Costaridou.
Biomed Eng Online. 2006 Feb 2;5(1):6.
ABSTRACT: [Abstract/Link to Full Text]

Bilgen M
Inductively-overcoupled coil design for high resolution magnetic resonance imaging.
Biomed Eng Online. 2006;53.
BACKGROUND: Maintaining the quality of magnetic resonance images acquired with the current implantable coil technology is challenging in longitudinal studies. To overcome this challenge, the principle of 'inductive overcoupling' is introduced as a method to tune and match a dual coil system. This system consists of an imaging coil built with fixed electrical elements and a matching coil equipped with tuning and matching capabilities. Overcoupling here refers to the condition beyond which the peak of the current in the imaging coil splits. METHODS: The combined coils are coupled inductively to operate like a transformer. Each coil circuit is electrically represented by equivalent lumped-elements. A theoretical analysis is given to identify the frequency response characteristics of the currents in each coil. The predictions from this analysis are translated into experiments and applied to locally image rat spinal cord at 9.4 T using an implantable coil as the imaging coil and an external volume coil as the matching coil. RESULTS: The theoretical analysis indicated that strong coupling between the coils divides the resonance peaks on the response curves of the currents. Once these newly generated peaks were tuned and matched to the desired frequency and impedance of operation, in vivo images were acquired from the rat spinal cord at high quality and high resolution. CONCLUSION: After proper implementation, inductive overcoupling provides a unique opportunity for tuning and matching the coil system, and allows reliable and repeatable acquisitions of magnetic resonance data. This feature is likely to be useful in experimental studies, such as those aimed at longitudinally imaging the rat following spinal cord injury. [Abstract/Link to Full Text]

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Recent Articles in Journal of Biomedicine & Biotechnology

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Higgins PJ
The TGF-beta1/Upstream Stimulatory Factor-Regulated PAI-1 Gene: Potential Involvement and a Therapeutic Target in Alzheimer's Disease.
J Biomed Biotechnol. 2006;2006(3):15792.
Amyloid peptide (A beta) aggregates, derived from initial beta-site proteolytic processing of the amyloid precursor protein (APP), accumulate in the brains of Alzheimer's disease patients. The plasmin-generating cascade appears to serve a protective role in the central nervous system since plasmin-mediated proteolysis of APP utilizes the alpha site, eventually generating nontoxic peptides, and plasmin also degrades A beta. The conversion of plasminogen to plasmin by tissue-type plasminogen activator in the brain is negatively regulated by plasminogen activator inhibitor type-1 (PAI-1) resulting in attenuation of plasmin-dependent substrate degradation with resultant accumulation of A beta. PAI-1 and its major physiological inducer TGF-beta1, moreover, are increased in models of Alzheimer's disease and have been implicated in the etiology and progression of human neurodegenerative disorders. This review highlights the potential role of PAI-1 and TGF-beta1 in this process. Current molecular events associated with TGF-beta1-induced PAI-1 transcription are presented with particular relevance to potential targeting of PAI-1 gene expression as a molecular approach to the therapy of neurodegenerative diseases associated with increased PAI-1 expression such as Alzheimer's disease. [Abstract/Link to Full Text]

Keller JN
Interplay between oxidative damage, protein synthesis, and protein degradation in Alzheimer's disease.
J Biomed Biotechnol. 2006;2006(3):12129.
Protein synthesis and protein degradation are highly regulated cellular processes that are essential to maintaining cell viability. Numerous studies now indicate that protein synthesis and protein degradation are significantly altered in Alzheimer's disease (AD), with impairments in these two processes potentially contributing to AD pathogenesis. Alterations in steady state protein regulation may be a particularly important factor in regulating whether cells maintain homeostasis in response to oxidative damage, or conversely whether oxidative stress is induced by oxidative damage. The focus of this review is to discuss recent findings on each of these topics, and to discuss their importance to the onset and progression of AD. [Abstract/Link to Full Text]

Castillo-Cadena J, Tenorio-Vieyra LE, Quintana-Carabia AI, García-Fabila MM, Juan ER, Madrigal-Bujaidar E
Determination of DNA damage in floriculturists exposed to mixtures of pesticides.
J Biomed Biotechnol. 2006;2006(2):97896.
The aim of the study was to determine possible DNA damage in floriculturists chronically exposed to pesticides. Leukocytes from 52 workers, 46 environmentally exposed, and 38 control individuals were evaluated with the comet assay. Serum from all individuals was also analyzed for pesticides using gas chromatography coupled to mass spectrometry. A statistically significant difference in DNA fragmentation in the pesticide exposed group compared to the other two groups (P < .001) was found. No differences between environmentally exposed and control individuals were detected. The statistical analysis showed no significant correlation between DNA damage and sex, age, drinking or smoking habits, as well as years of exposure. One or more pesticides were detected in 50% of the floriculturists, while in the rest of the individuals, a chemical related with the preparation of pesticides, such as additives, plasticizers, or solvents, was found. Our study shows that chronic exposure to pesticides produces DNA damage in floriculturists. It also suggests that this type of monitoring could be valuable in recommending preventive measures. [Abstract/Link to Full Text]

Granfeldt D, Harbecke O, Björstad A, Karlsson A, Dahlgren C
Neutrophil Secretion Induced by an Intracellular Ca(2+) Rise and Followed by Whole-Cell Patch-Clamp Recordings Occurs Without any Selective Mobilization of Different Granule Populations.
J Biomed Biotechnol. 2006;2006(2):97803.
We have investigated calcium-induced secretion in human neutrophils, using a whole-cell patch-clamp technique. Mobilization of subcellular granules to the cell membrane was followed as the change in membrane capacitance ( big up tri, openC(m)). Both the magnitude and the kinetics of the response differed between low and high concentrations of Ca(2+). A sustained secretion following a short lag phase was induced by high concentrations of Ca(2+) (100muM and higher). A stable plateau was reached after 5-7 minutes at big up tri, openC(m) values corresponding to values expected after all specific as well as azurophil granules have been mobilized. Capacitance values of the same magnitude could be obtained also at lower Ca(2+) concentrations, but typically no stable plateau was reached within the measuring time. In contrast to previous studies, we were unable to detect any pattern of secretion corresponding to a distinct submaximal response or selective mobilization of granule subsets specified by their Ca(2+)-sensitivity. [Abstract/Link to Full Text]

Torres A, Nieto JJ
Fuzzy logic in medicine and bioinformatics.
J Biomed Biotechnol. 2006;2006(2):91908.
The purpose of this paper is to present a general view of the current applications of fuzzy logic in medicine and bioinformatics. We particularly review the medical literature using fuzzy logic. We then recall the geometrical interpretation of fuzzy sets as points in a fuzzy hypercube and present two concrete illustrations in medicine (drug addictions) and in bioinformatics (comparison of genomes). [Abstract/Link to Full Text]

Klees RF, De Marco PC, Salasznyk RM, Ahuja D, Hogg M, Antoniotti S, Kamath L, Dordick JS, Plopper GE
Apocynin derivatives interrupt intracellular signaling resulting in decreased migration in breast cancer cells.
J Biomed Biotechnol. 2006;2006(2):87246.
Cancer cells are defined by their ability to divide uncontrollably and metastasize to secondary sites in the body. Consequently, tumor cell migration represents a promising target for anticancer drug development. Using our high-throughput cell migration assay, we have screened several classes of compounds for noncytotoxic tumor cell migration inhibiting activity. One such compound, apocynin (4-acetovanillone), is oxidized by peroxidases to yield a variety of oligophenolic and quinone-type compounds that are recognized inhibitors of NADPH oxidase and may be inhibitors of the small G protein Rac1 that controls cell migration. We report here that while apocynin itself is not effective, apocynin derivatives inhibit migration of the breast cancer cell line MDA-MB-435 at subtoxic concentrations; the migration of nonmalignant MCF10A breast cells is unaffected. These compounds also cause a significant rearrangement of the actin cytoskeleton, cell rounding, and decreased levels of active Rac1 and its related G protein Cdc42. These results may suggest a promising new route to the development of novel anticancer therapeutics. [Abstract/Link to Full Text]

Moussa LB, Werner S, Coraiola M, Colin DA, Keller D, Sanni A, Serra MD, Monteil H, Prévost G
Site-Directed Mutagenesis to Assess the Binding Capacity of Class S Protein of Staphylococcus aureus Leucotoxins to the Surface of Polymorphonuclear Cells.
J Biomed Biotechnol. 2006;2006(2):80101.
Staphylococcal leucotoxins result from the association of class S components and class F component inducing the activation and the permeabilization of the target cells. Like alpha-toxin, the leucotoxins are pore-forming toxins with more than 70% beta-sheet. This was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. In addition, threonine 28 of a predicted and conserved beta-sheet at the N-terminal extremity of class S proteins composing leucotoxins aligns with histidine 35 of alpha-toxin, which has a key role in oligomerization of the final pore. Flow cytometry was used to study different aminoacid substitutions of the threonine 28 in order to evaluate its role in the biological activity of these class S proteins. Finally, results show that threonine 28 of the leucotoxin probably plays a role similar to that of histidine 35 of alpha-toxin. Mutations on this threonin largely influenced the secondary interaction of the class F component and led to inactive toxin. [Abstract/Link to Full Text]

Alajez NM, Eghtesad S, Finn OJ
Cloning and expression of human membrane-bound and soluble engineered T cell receptors for immunotherapy.
J Biomed Biotechnol. 2006;2006(2):68091.
We report here the design and construction of several gene vectors for expression in mammalian cells of membrane-bound and soluble human T cell receptors (TR). We designed a vector (TR-ALPHA-IRES-TR-BETA pEF4) that encodes high-level expression of the full-length TR on the surface of T cells. Furthermore, we engineered TR that does not require the presence of endogenous CD3 molecules for surface expression and thus expression is not limited to T cells. We also constructed a vector encoding a single-chain TR (scTR) as a fusion protein of V-ALPHA-V-BETA-C-BETA with CD3Z. Since it is encoded and expressed as a single molecule, this scTR is well suited for gene therapy. Lastly, we successfully used a mammalian expression vector for generation of soluble human TR. The approaches we used here for manipulation of a human tumor-specific TR can be useful for other investigators interested in TR-based immunotherapy. [Abstract/Link to Full Text]

García VP, Valdés F, Martín R, Luis JC, Afonso AM, Ayala JH
Biosynthesis of antitumoral and bactericidal sanguinarine.
J Biomed Biotechnol. 2006;2006(2):63518.
A simple, rapid, and reliable TLC method for the separation and determination of sanguinarine has been established. This intensively studied biologically active alkaloid has a wide range of potentially useful medicinal properties, such as antimicrobial, antiinflammatory, and antitumoral activities. Sanguinarine has also been incorporated into expectorant mixtures and has a strong bactericidal effect upon gram-positive bacteria, particularly Bacillus anthracis and staphylococci. These medicinal properties are due to the interaction of sanguinarine with DNA. A fibre-optic-based fluorescence instrument for in situ scanning was used for quantitative measurements. The sanguinarine was determined over the range 5-40 ng and a detection limit of 1.60 ng. The method was applied to the quantification of sanguinarine in tissue culture extracts of Chelidonium majus L. [Abstract/Link to Full Text]

Bourdillon MC, Randon J, Barek L, Zibara K, Covacho C, Poston RN, Chignier E, McGregor JL
Reduced atherosclerotic lesion size in p-selectin deficient apolipoprotein e-knockout mice fed a chow but not a fat diet.
J Biomed Biotechnol. 2006;2006(2):49193.
P-selectin. We investigated the role of P-selectin on the development of vascular lesions in an ApoE(-/-) male mice. Double-knockout (ApoE(-/-), P-selectin(-/-); DKO) were compared to single-knockout (ApoE(-/-); SKO) mice. They were fed a chow or fat diet for 3, 6, 15, and 20 weeks, without any differences in cholesterol levels. DKO mice fed a chow diet exhibited a ratio of lesion area over media lower than SKO mice, for 3 (P < .03) , 6 (P < .001), and 15 (P < .02) weeks. DKO mice fed a fat diet showed a lower ratio only at 3 weeks. P-selectin deficiency in ApoE(-/-) mice has a protective effect in atherosclerotic lesions development. Reduction of lesion size depends on diet type and duration. A fat diet could neutralize the beneficial effects of P-selectin deficiency, inducing atherosclerotic lesions via probably other adhesion molecules. [Abstract/Link to Full Text]

Crawford BH, Hussain AA, Jideama NM
Evidence of a Genomic Biomarker in Normal Human Epithelial Mammary Cell Line, MCF-10A, That Is Absent in the Human Breast Cancer Cell Line, MCF-7.
J Biomed Biotechnol. 2006;2006(2):43181.
This study investigated the use of DNA amplification fingerprinting (DAF) to identify biomarkers useful in the elucidating genetic factors that lead to carcinogenesis. The DNA amplification fingerprinting (DAF) technique was used to generate fingerprint profiles of a normal human mammary epithelial cell line (MCF-10A) and a human breast cancer cell line (MCF-7). When compared with one another, a polymorphic biomarker gene (262 base pairs (bps)) was identified in MCF-10A but was not present in MCF-7. This gene was cloned from the genomic DNA of the MCF-10A cell line, and subjected to Genbank database analysis. The analysis of the nucleotide sequence polymorphic marker (Genbank account: AC079630) shows that this biomarker has 100% homology with the nucleotide sequence of human chromosome 12 BAC RP11-476D10 (bps 19612-19353). The nucleotide sequence was used for possible protein translation product and the result obtained indicated that the gene codes for hypothetical protein XF2620. In order to evaluate the effects that the 262 bps biomarker would have on the morphology of MCF-7 cells, it was transfected into MCF-7 cells. There were observable changes in the morphology of the transfected cells. These changes included an increase in cell elongation and a decrease in cell aggregation. [Abstract/Link to Full Text]

Latham JR, Wilson AK, Steinbrecher RA
The mutational consequences of plant transformation.
J Biomed Biotechnol. 2006;2006(2):25376.
Plant transformation is a genetic engineering tool for introducing transgenes into plant genomes. It is now being used for the breeding of commercial crops. A central feature of transformation is insertion of the transgene into plant chromosomal DNA. Transgene insertion is infrequently, if ever, a precise event. Mutations found at transgene insertion sites include deletions and rearrangements of host chromosomal DNA and introduction of superfluous DNA. Insertion sites introduced using Agrobacterium tumefaciens tend to have simpler structures but can be associated with extensive chromosomal rearrangements, while those of particle bombardment appear invariably to be associated with deletion and extensive scrambling of inserted and chromosomal DNA. Ancillary procedures associated with plant transformation, including tissue culture and infection with A tumefaciens, can also introduce mutations. These genome-wide mutations can number from hundreds to many thousands per diploid genome. Despite the fact that confidence in the safety and dependability of crop species rests significantly on their genetic integrity, the frequency of transformation-induced mutations and their importance as potential biosafety hazards are poorly understood. [Abstract/Link to Full Text]

Ouaissi M, Ouaissi A
Histone deacetylase enzymes as potential drug targets in cancer and parasitic diseases.
J Biomed Biotechnol. 2006;2006(2):13474.
The elucidation of the mechanisms of transcriptional activation and repression in eukaryotic cells has shed light on the important role of acetylation-deacetylation of histones mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. Another group belonging to the large family of sirtuins (silent information regulators (SIRs)) has an (nicotinamide adenine dinucleotide) NAD(+)-dependent HDAC activity. Several inhibitors of HDACs (HDIs) have been shown to exert antitumor effects. Interestingly, some of the HDIs exerted a broad spectrum of antiprotozoal activity. The purpose of this review is to analyze some of the current data related to the deacetylase enzymes as a possible target for drug development in cancer and parasitic diseases with special reference to protozoan infections. Given the structural differences among members of this family of enzymes, development of specific inhibitors will not only allow selective therapeutic intervention, but may also provide a powerful tool for functional study of these enzymes. [Abstract/Link to Full Text]

Schulz WA
L1 retrotransposons in human cancers.
J Biomed Biotechnol. 2006;2006(1):83672.
Retrotransposons like L1 are silenced in somatic cells by a variety of mechanisms acting at different levels. Protective mechanisms include DNA methylation and packaging into inactive chromatin to suppress transcription and prevent recombination, potentially supported by cytidine deaminase editing of RNA. Furthermore, DNA strand breaks arising during attempted retrotranspositions ought to activate cellular checkpoints, and L1 activation outside immunoprivileged sites may elicit immune responses. A number of observations indicate that L1 sequences nevertheless become reactivated in human cancer. Prominently, methylation of L1 sequences is diminished in many cancer types and full-length L1 RNAs become detectable, although strong expression is restricted to germ cell cancers. L1 elements have been found to be enriched at sites of illegitimate recombination in many cancers. In theory, lack of L1 repression in cancer might cause transcriptional deregulation, insertional mutations, DNA breaks, and an increased frequency of recombinations, contributing to genome disorganization, expression changes, and chromosomal instability. There is however little evidence that such effects occur at a gross scale in human cancers. Rather, as a rule, L1 repression is only partly alleviated. Unfortunately, many techniques commonly used to investigate genetic and epigenetic alterations in cancer cells are not well suited to detect subtle effects elicited by partial reactivation of retroelements like L1 which are present as abundant, but heterogeneous copies. Therefore, effects of L1 sequences exerted on the local chromatin structure, on the transcriptional regulation of individual genes, and on chromosome fragility need to be more closely investigated in normal and cancer cells. [Abstract/Link to Full Text]

Graham T, Boissinot S
The genomic distribution of l1 elements: the role of insertion bias and natural selection.
J Biomed Biotechnol. 2006;2006(1):75327.
LINE-1 (L1) retrotransposons constitute the most successful family of retroelements in mammals and account for as much as 20% of mammalian DNA. L1 elements can be found in all genomic regions but they are far more abundant in AT-rich, gene-poor, and low-recombining regions of the genome. In addition, the sex chromosomes and some genes seem disproportionately enriched in L1 elements. Insertion bias and selective processes can both account for this biased distribution of L1 elements. L1 elements do not appear to insert randomly in the genome and this insertion bias can at least partially explain the genomic distribution of L1. The contrasted distribution of L1 and Alu elements suggests that postinsertional processes play a major role in shaping L1 distribution. The most likely mechanism is the loss of recently integrated L1 elements that are deleterious (negative selection) either because of disruption of gene function or their ability to mediate ectopic recombination. By comparison, the retention of L1 elements because of some positive effect is limited to a small fraction of the genome. Understanding the respective importance of insertion bias and selection will require a better knowledge of insertion mechanisms and the dynamics of L1 inserts in populations. [Abstract/Link to Full Text]

Mätlik K, Redik K, Speek M
L1 antisense promoter drives tissue-specific transcription of human genes.
J Biomed Biotechnol. 2006;2006(1):71753.
Transcription of transposable elements interspersed in the genome is controlled by complex interactions between their regulatory elements and host factors. However, the same regulatory elements may be occasionally used for the transcription of host genes. One such example is the human L1 retrotransposon, which contains an antisense promoter (ASP) driving transcription into adjacent genes yielding chimeric transcripts. We have characterized 49 chimeric mRNAs corresponding to sense and antisense strands of human genes. Here we show that L1 ASP is capable of functioning as an alternative promoter, giving rise to a chimeric transcript whose coding region is identical to the ORF of mRNA of the following genes: KIAA1797, CLCN5, and SLCO1A2. Furthermore, in these cases the activity of L1 ASP is tissue-specific and may expand the expression pattern of the respective gene. The activity of L1 ASP is tissue-specific also in cases where L1 ASP produces antisense RNAs complementary to COL11A1 and BOLL mRNAs. Simultaneous assessment of the activity of L1 ASPs in multiple loci revealed the presence of L1 ASP-derived transcripts in all human tissues examined. We also demonstrate that L1 ASP can act as a promoter in vivo and predict that it has a heterogeneous transcription initiation site. Our data suggest that L1 ASP-driven transcription may increase the transcriptional flexibility of several human genes. [Abstract/Link to Full Text]

Lyon MF
Do LINEs Have a Role in X-Chromosome Inactivation?
J Biomed Biotechnol. 2006;2006(1):59746.
There is longstanding evidence that X-chromosome inactivation (XCI) travels less successfully in autosomal than in X-chromosomal chromatin. The interspersed repeat elements LINE1s (L1s) have been suggested as candidates for "boosters" which promote the spread of XCI in the X-chromosome. The present paper reviews the current evidence concerning the possible role of L1s in XCI. Recent evidence, accruing from the human genome sequencing project and other sources, confirms that mammalian X-chromosomes are indeed rich in L1s, except in regions where there are many genes escaping XCI. The density of L1s is the highest in the evolutionarily oldest regions. Recent work on X; autosome translocations in human and mouse suggested failure of stabilization of XCI in autosomal material, so that genes are reactivated, but resistance of autosomal genes to the original silencing is not excluded. The accumulation of L1s on the X-chromosome may have resulted from reduced recombination or late replication. Whether L1s are part of the mechanism of XCI or a result of it remains enigmatic. [Abstract/Link to Full Text]

Chen JM, Férec C, Cooper DN
LINE-1 Endonuclease-Dependent Retrotranspositional Events Causing Human Genetic Disease: Mutation Detection Bias and Multiple Mechanisms of Target Gene Disruption.
J Biomed Biotechnol. 2006;2006(1):56182.
LINE-1 (L1) elements are the most abundant autonomous non-LTR retrotransposons in the human genome. Having recently performed a meta-analysis of L1 endonuclease-mediated retrotranspositional events causing human genetic disease, we have extended this study by focusing on two key issues, namely, mutation detection bias and the multiplicity of mechanisms of target gene disruption. Our analysis suggests that whereas an ascertainment bias may have generally militated against the detection of autosomal L1-mediated insertions, autosomal L1 direct insertions could have been disproportionately overlooked owing to their unusually large size. Our analysis has also indicated that the mechanisms underlying the functional disruption of target genes by L1-mediated retrotranspositional events are likely to be dependent on several different factors such as the type of insertion (L1 direct, L1 trans-driven Alu, or SVA), the precise locations of the inserted sequences within the target gene regions, the length of the inserted sequences, and possibly also their orientation. [Abstract/Link to Full Text]

Martin SL
The ORF1 Protein Encoded by LINE-1: Structure and Function During L1 Retrotransposition.
J Biomed Biotechnol. 2006;2006(1):45621.
LINE-1 or L1 is an autonomous non-LTR retrotransposon in mammals. Retrotransposition requires the function of the two L1-encoded polypeptides, ORF1p and ORF2p. Early recognition of regions of homology between the predicted amino acid sequence of ORF2 and known endonuclease and reverse transcriptase enzymes led to testable hypotheses regarding the function of ORF2p in retrotransposition. As predicted, ORF2p has been demonstrated to have both endonuclease and reverse transcriptase activities. In contrast, no homologs of known function have contributed to our understanding of the function of ORF1p during retrotransposition. Nevertheless, significant advances have been made such that we now know that ORF1p is a high-affinity RNA-binding protein that forms a ribonucleoprotein particle together with L1 RNA. Furthermore, ORF1p is a nucleic acid chaperone and this nucleic acid chaperone activity is required for L1 retrotransposition. [Abstract/Link to Full Text]

Farkash EA, Prak ET
DNA damage and l1 retrotransposition.
J Biomed Biotechnol. 2006;2006(1):37285.
Barbara McClintock was the first to suggest that transposons are a source of genome instability and that genotoxic stress assisted in their mobilization. The generation of double-stranded DNA breaks (DSBs) is a severe form of genotoxic stress that threatens the integrity of the genome, activates cell cycle checkpoints, and, in some cases, causes cell death. Applying McClintock's stress hypothesis to humans, are L1 retrotransposons, the most active autonomous mobile elements in the modern day human genome, mobilized by DSBs? Here, evidence that transposable elements, particularly retrotransposons, are mobilized by genotoxic stress is reviewed. In the setting of DSB formation, L1 mobility may be affected by changes in the substrate for L1 integration, the DNA repair machinery, or the L1 element itself. The review concludes with a discussion of the potential consequences of L1 mobilization in the setting of genotoxic stress. [Abstract/Link to Full Text]

Prak NL, Haoudi A
LINE-1 Retrotransposition: Impact on Genome Stability and Diversity and Human Disease.
J Biomed Biotechnol. 2006;2006(1):37049. [Abstract/Link to Full Text]

Horman SR, Svoboda P, Prak ET
The potential regulation of l1 mobility by RNA interference.
J Biomed Biotechnol. 2006;2006(1):32713.
The hypothesis that RNA interference constrains L1 mobility seems inherently reasonable: L1 mobility can be dangerous and L1 RNA, the presumed target of RNAi, serves as a critical retrotransposition intermediate. Despite its plausibility, proof for this hypothesis has been difficult to obtain. Studies attempting to link the L1 retrotransposition frequency to alterations in RNAi activity have been hampered by the long times required to measure retrotransposition frequency, the pleiotropic and toxic effects of altering RNAi over similar time periods, and the possibility that other cellular machinery may contribute to the regulation of L1s. Another problem is that the commonly used L1 reporter cassette may serve as a substrate for RNAi. Here we review the L1-RNAi hypothesis and describe a genetic assay with a modified reporter cassette that detects approximately 4 times more L1 insertions than the conventional retrotransposition assay. [Abstract/Link to Full Text]

Soifer HS
Do Small RNAs Interfere With LINE-1?
J Biomed Biotechnol. 2006;2006(1):29049.
Long interspersed elements (LINE-1 or L1) are the most active transposable elements in the human genome. Due to their high copy number and ability to sponsor retrotransposition of nonautonomous RNA sequences, unchecked L1 activity can negatively impact the genome by a number of means. Substantial evidence in lower eukaryotes demonstrates that the RNA interference (RNAi) machinery plays a major role in containing transposon activity. Despite extensive analysis in other eukaryotes, no experimental evidence has been presented that L1-derived siRNAs exist, or that the RNAi plays a significant role in restricting L1 activity in the human genome. This review will present evidence showing a direct role for RNAi in suppressing the movement of transposable elements in other eukaryotes, as well as speculate on the role RNAi might play in protecting the human genome from LINE-1 activity. [Abstract/Link to Full Text]

Asada K, Kotake Y, Asada R, Saunders D, Broyles RH, Towner RA, Fukui H, Floyd RA
LINE-1 Hypomethylation in a Choline-Deficiency-Induced Liver Cancer in Rats: Dependence on Feeding Period.
J Biomed Biotechnol. 2006;2006(1):17142.
Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation. [Abstract/Link to Full Text]

Matsutani S
Links between repeated sequences.
J Biomed Biotechnol. 2006;2006(1):13569.
L1 and Alu elements are long and short interspersed retrotransposable elements (LINEs and SINEs) in humans, respectively. Proteins encoded in the autonomous L1 mediate retrotransposition of the nonautonomous Alu and cellular mRNAs. Alu is the only active SINE in the human genome and is derived from 7SL RNA of signal recognition particle. In the other eukaryotic genomes, various tRNA- and 5S rRNA-derived SINEs are found. Some of the tRNA- and 5S rRNA-derived SINEs have partner LINEs of which 3' sequences are similar to those of the SINEs. One of the tRNA-derived SINEs is shown to be mobilized by its partner LINE. Many copies of tRNA and 5S rRNA pseudogenes are present in the human genome. These pseudogenes may have been generated via the retrotransposition process using L1 proteins. Although there are no sequence similarities between L1 and Alu, L1 functionally links with Alu and even cellular genes, impacting on our genome shaping. [Abstract/Link to Full Text]

Haoudi A
Review of "protein microarrays" edited by mark schena.
J Biomed Biotechnol. 2005;2005(4):385. [Abstract/Link to Full Text]

Burrowes OJ, Diamond G, Lee TC
Recombinant Expression of Pleurocidin cDNA Using the Pichia pastoris Expression System.
J Biomed Biotechnol. 2005;2005(4):374-84.
This research utilized the Pichia pastoris expression system for recombinant expression of cDNA of pleurocidin, a small (2.7 kd) antimicrobial peptide isolated from winter flounder (Pleuronectes americanus). The Pichia vector contains the alcohol oxidase gene promoter (AOX 1), which under the induction of methanol allows for expression of heterologous protein gene inserted downstream in the vector. Two strains of P pastoris were used as host cells, the wild type (P pastoris X-33((mut(+)))) and the mutant (P pasatoris KM71H((mut(s))) ). Polymerase chain reaction (PCR) and DNA sequencing showed that pleurocidin cDNA was successfully integrated into the P pastoris genome. Reverse transcription (RT)-PCR showed that pleurocidin was transcribed by both Pichia host strains. Affinity chromatography, SDS-PAGE, and immunological techniques were used for purification and detection of recombinant peptide. Although there was strong evidence of transcription of pleurocidin cDNA, the Pichia system requires further optimization to obtain detectable levels of this small peptide. [Abstract/Link to Full Text]

Villar-Suárez V, Colaço B, Calles-Venal I, Bravo IG, Fernández-Alvarez JG, Fernández-Caso M, Villar-Lacilla JM
Effects of extracellular matrix on the morphology and behaviour of rabbit auricular chondrocytes in culture.
J Biomed Biotechnol. 2005;2005(4):364-73.
Isolated chondrocytes dedifferentiate to a fibroblast-like shape on plastic substrata and proliferate extensively, but rarely form nodules. However, when dissociation is not complete and some cartilage remnants are included in the culture, proliferation decreases and cells grow in a reticular pattern with numerous nodules, which occasionally form small cartilage-like fragments. In an attempt to reproduce this stable chondrogenic state, we added a cartilage protein extract, a sugar extract, and hyaluronan to the medium of previously dedifferentiated chondrocytes. When protein extract was added, many cartilaginous nodules appeared. Hyaluronan produced changes in cell phenotype and behaviour, but not nodule formation. Protein extract has positive effects on the differentiation of previously proliferated chondrocytes and permits nodule formation and the extensive production of type-II collagen. A comparison with incompletely dissociated chondrocyte cultures suggests that the presence of some living cells anchored to their natural extracellular matrix provides some important additional factors for the phenotypical stability of chondrocytes on plastic surfaces. In order to elucidate if it is possible that the incidence of apoptosis is related to the results, we also characterized the molecular traits of apoptosis. [Abstract/Link to Full Text]

Tsigelny I, Burton DW, Sharikov Y, Hastings RH, Deftos LJ
Coherent expression chromosome cluster analysis reveals differential regulatory functions of amino-terminal and distal parathyroid hormone-related protein domains in prostate carcinoma.
J Biomed Biotechnol. 2005;2005(4):353-63.
Parathyroid hormone-related protein (PTHrP) has a number of cancer-related actions. While best known for causing hypercalcemia of malignancy, it also has effects on cancer cell growth, apoptosis, and angiogenesis. Studying the actions of PTHrP in human cancer is complicated because there are three isoforms and many derived peptides. Several peptides are biologically active at known or presumed cell surface receptors; in addition, the PTHrP-derived molecules can exert effects at the cell nucleus. To address this complexity, we studied gene expression in a DU 145 prostate cancer cell line that was stably transfected with control vector, PTHrP 1-173 and PTHrP 33-173. With this model, regulatory effects of the amino-terminal portion of PTHrP would result only from transduction with the full-length molecule, while effects pertaining to distal sequences would be evident with either construct. Analysis of the expression profiles by microarrays demonstrated nonoverlapping groups of differentially expressed genes. Amino-terminal PTHrP affected groups of genes involved in apoptosis, prostaglandin and sex steroid metabolism, cell-matrix interactions, and cell differentiation, while PTHrP 33-173 caused substantial increases in MHC class I antigen expression. This work demonstrates the distinct biological actions of the amino-terminus compared to distal mid-molecule or carboxy-terminal sequences of PTHrP in prostate carcinoma cells and provides targets for further study of the malignant process. [Abstract/Link to Full Text]

Kleter GA, Peijnenburg AA, Aarts HJ
Health considerations regarding horizontal transfer of microbial transgenes present in genetically modified crops.
J Biomed Biotechnol. 2005;2005(4):326-52.
The potential effects of horizontal gene transfer on human health are an important item in the safety assessment of genetically modified organisms. Horizontal gene transfer from genetically modified crops to gut microflora most likely occurs with transgenes of microbial origin. The characteristics of microbial transgenes other than antibiotic-resistance genes in market-approved genetically modified crops are reviewed. These characteristics include the microbial source, natural function, function in genetically modified crops, natural prevalence, geographical distribution, similarity to other microbial genes, known horizontal transfer activity, selective conditions and environments for horizontally transferred genes, and potential contribution to pathogenicity and virulence in humans and animals. The assessment of this set of data for each of the microbial genes reviewed does not give rise to health concerns. We recommend including the above-mentioned items into the premarket safety assessment of genetically modified crops carrying transgenes other than those reviewed in the present study. [Abstract/Link to Full Text]

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Recent Articles in BMC Biotechnology

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Pimkin M, Caretti E, Canutescu A, Yeung JB, Cohn H, Chen Y, Oleykowski C, Bellacosa A, Yeung AT
Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection.
BMC Biotechnol. 2007;729.
BACKGROUND: The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria. RESULTS: We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites. CONCLUSION: The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies. [Abstract/Link to Full Text]

Rautio JJ, Bailey M, Kivioja T, Söderlund H, Penttilä M, Saloheimo M
Physiological evaluation of the filamentous fungus Trichoderma reesei in production processes by marker gene expression analysis.
BMC Biotechnol. 2007;728.
BACKGROUND: Biologically relevant molecular markers can be used in evaluation of the physiological state of an organism in biotechnical processes. We monitored at high frequency the expression of 34 marker genes in batch, fed-batch and continuous cultures of the filamentous fungus Trichoderma reesei by the transcriptional analysis method TRAC (TRanscript analysis with the aid of Affinity Capture). Expression of specific genes was normalised either with respect to biomass or to overall polyA RNA concentration. Expressional variation of the genes involved in various process relevant cellular functions, such as protein production, growth and stress responses, was related to process parameters such as specific growth and production rates and substrate and dissolved oxygen concentrations. RESULTS: Gene expression of secreted cellulases and recombinant Melanocarpus albomyces laccase predicted the trends in the corresponding extracellular enzyme production rates and was highest in a narrow "physiological window" in the specific growth rate (micro) range of 0.03-0.05 h-1. Expression of ribosomal protein mRNAs was consistent with the changes in mu. Nine starvation-related genes were found as potential markers for detection of insufficient substrate feed for maintaining optimal protein production. For two genes induced in anaerobic conditions, increasing transcript levels were measured as dissolved oxygen decreased. CONCLUSION: The data obtained by TRAC supported the usefulness of focused and intensive transcriptional analysis in monitoring of biotechnical processes providing thus tools for process optimisation purposes. [Abstract/Link to Full Text]

Geyer BC, Fletcher SP, Griffin TA, Lopker MJ, Soreq H, Mor TS
Translational control of recombinant human acetylcholinesterase accumulation in plants.
BMC Biotechnol. 2007;727.
BACKGROUND: Codon usage differences are known to regulate the levels of gene expression in a species-specific manner, with the primary factors often cited to be mRNA processing and accumulation. We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content. RESULTS: We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in Nicotiana benthamiana plants expressing the optimized gene as compared to the native human sequence. Both transient expression assays and stable transformants demonstrated conspicuously increased accumulation levels. Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA. CONCLUSION: Our findings demonstrate that codon usage differences may regulate gene expression at different levels and anticipate translational control of acetylcholinesterase gene expression in its native mammalian host as well. [Abstract/Link to Full Text]

Koch TG, Heerkens T, Thomsen PD, Betts DH
Isolation of mesenchymal stem cells from equine umbilical cord blood.
BMC Biotechnol. 2007;726.
BACKGROUND: There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood. RESULTS: Cord blood was collected from 7 foals immediately after foaling. The mononuclear cell fraction was isolated by Ficoll density centrifugation and cultured in a DMEM low glucose based media at 38.5 degrees C in humidified atmosphere containing 5% CO2. In 4 out of 7 samples colonies with MSC morphology were observed. Cellular morphology varied between monolayers of elongated spindle-shaped cells to layered cell clusters of cuboidal cells with shorter cytoplasmic extensions. Positive Alizarin Red and von Kossa staining as well as significant calcium deposition and alkaline phosphatase activity confirmed osteogenesis. Histology and positive Safranin O staining of matrix glycosaminoglycans illustrated chondrogenesis. Oil Red O staining of lipid droplets confirmed adipogenesis. CONCLUSION: We here report, for the first time, the isolation of mesenchymal-like stem cells from fresh equine cord blood and their differentiation into osteocytes, chondrocytes and adipocytes. This novel isolation of equine cord blood MSCs and their preliminary in vitro differentiation positions the horse as the ideal pre-clinical animal model for proof-of-principle studies of cord blood derived MSCs. [Abstract/Link to Full Text]

Maeda T, Vardar G, Self WT, Wood TK
Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803.
BMC Biotechnol. 2007;725.
BACKGROUND: Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H+ + 2e- <--> H2 (g). RESULTS: Hydrogen yields were enhanced up to 41-fold by cloning the bidirectional hydrogenase (encoded by hoxEFUYH) from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone (small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild-type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. This appears to be the first enhanced hydrogen production by cloning a cyanobacterial enzyme into a heterologous host. CONCLUSION: Enhanced hydrogen production in E. coli cells expressing the cyanobacterial HoxEFUYH is by inhibiting hydrogen uptake of both hydrogenase 1 and hydrogenase 2. [Abstract/Link to Full Text]

Lonergan W, Whistler T, Vernon SD
Comparison of target labeling methods for use with Affymetrix GeneChips.
BMC Biotechnol. 2007;724.
BACKGROUND: Several different commercial one-cycle labeling kits are available for preparation of the target for use with the Affymetrix GeneChip platform. However, there have been no evaluations of these different kits to determine if comparable results were generated. We report on the cRNA target synthesis, labeling efficiency and hybridization results using the One-Cycle Target Labeling Assay (Affymetrix), the BioArray RNA Amplification and Labeling System (Enzo Life Sciences), and the Superscript RNA Amplification System (Invitrogen Life Technologies). RESULTS: The only notable difference between kits was in the yield of cRNA target synthesized during in vitro transcription, where the BioArray assay had to be repeated several times in order to have sufficient target. However, each kit resulted in comparable signal and detection calls when hybridized to the Affymetrix GeneChip. CONCLUSION: These 3 one-cycle labeling kits produce comparable hybridization results. This provides users with several kit options and flexibility when using the Affymetrix system. [Abstract/Link to Full Text]

Liu JZ, Wang M
Improvement of activity and stability of chloroperoxidase by chemical modification.
BMC Biotechnol. 2007;723.
BACKGROUND: Enzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water. These limit the industrial applications of enzymes. RESULTS: In order to improve the activity and stability of chloroperoxidase, chloroperoxidase was modified by citraconic anhydride, maleic anhydride or phthalic anhydride. The catalytic activities, thermostabilities and organic solvent tolerances of native and modified enzymes were compared. In aqueous buffer, modified chloroperoxidases showed similar Km values and greater catalytic efficiencies kcat/Km for both sulfoxidation and oxidation of phenol compared to native chloroperoxidase. Of these modified chloroperoxidases, citraconic anhydride-modified chloroperoxidase showed the greatest catalytic efficiency in aqueous buffer. These modifications of chloroperoxidase increased their catalytic efficiencies for sulfoxidation by 12%~26% and catalytic efficiencies for phenol oxidation by 7%~53% in aqueous buffer. However, in organic solvent (DMF), modified chloroperoxidases had lower Km values and higher catalytic efficiencies kcat/Km than native chloroperoxidase. These modifications also improved their thermostabilities by 1~2-fold and solvent tolerances of DMF. CD studies show that these modifications did not change the secondary structure of chloroperoxidase. Fluorescence spectra proved that these modifications changed the environment of tryptophan. CONCLUSION: Chemical modification of epsilon-amino groups of lysine residues of chloroperoxidase using citraconic anhydride, maleic anhydride or phthalic anhydride is a simple and powerful method to enhance catalytic properties of enzyme. The improvements of the activity and stability of chloroperoxidase are related to side chain reorientations of aromatics upon both modifications. [Abstract/Link to Full Text]

Schmeisser F, Weir JP
Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes.
BMC Biotechnol. 2007;722.
BACKGROUND: Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. RESULTS: Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. CONCLUSION: Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. [Abstract/Link to Full Text]

Haddad F, Qin AX, Giger JM, Guo H, Baldwin KM
Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR.
BMC Biotechnol. 2007;721.
BACKGROUND: The ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase H- RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs. RESULTS: Our findings on cellular RNA and in vitro synthesized RNA suggest that these products are likely the results of RNA self-priming to generate random cDNA products, which contributes to the loss of strand specificity. The use of RNase H+ RT enzyme and carrying the RT reaction at high temperature (50 degrees C) greatly improved the strand specificity of the RT-PCR detection. CONCLUSION: While RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when both sense and antisense RNA are expressed. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls. [Abstract/Link to Full Text]

Hermant B, Desroches-Castan A, Dubessay ML, Prandini MH, Huber P, Vittet D
Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis.
BMC Biotechnol. 2007;720.
BACKGROUND: Angiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis. RESULTS: Here, we have optimized an angiogenesis model based on ES cell differentiation for screening experiments. We have established conditions leading to angiogenic sprouting of embryoid bodies during ES cell differentiation in type I three-dimensional collagen gels. Immunostaining experiments carried out during these cultures showed the formation of numerous buds comprising CD31 positive cells, after 11 days of culture of ES cells. Moreover, this one-step model has been validated in response to activators and inhibitors of angiogenesis. Sprouting was specifically stimulated in the presence of VEGF and FGF2. Alternatively, endothelial sprouting induced by angiogenic activators was inhibited by angiogenesis inhibitors such as angiostatin, TGFbeta and PF4. Sprouting angiogenesis can be easily quantified by image analysis after immunostaining of endothelial cells with CD31 pan-endothelial marker. CONCLUSION: Taken together, these data clearly validate that this one-step ES differentiation model constitutes a simple and versatile angiogenesis system that should facilitate, in future investigations, the screening of both activators and inhibitors of angiogenesis. [Abstract/Link to Full Text]

Lasken RS, Stockwell TB
Mechanism of chimera formation during the Multiple Displacement Amplification reaction.
BMC Biotechnol. 2007;719.
BACKGROUND: Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. RESULTS: Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2-21 nucleotides (nts) in the new templates. CONCLUSION: Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications. [Abstract/Link to Full Text]

Kopsidas G, Carman RK, Stutt EL, Raicevic A, Roberts AS, Siomos MA, Dobric N, Pontes-Braz L, Coia G
RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution.
BMC Biotechnol. 2007;718.
BACKGROUND: In protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties. One basic approach to protein maturation is the introduction of random DNA mutations into the target gene sequence to produce a library of variants that can be screened for the preferred protein properties. Unfortunately, the capability of this approach has been restricted by deficiencies in the methods currently available for random DNA mutagenesis and library generation. Current DNA based methodologies generally suffer from nucleotide substitution bias that preferentially mutate particular base pairs or show significant bias with respect to transitions or transversions. In this report, we describe a novel RNA-based random mutagenesis strategy that utilizes Qbeta replicase to manufacture complex mRNA libraries with a mutational spectrum that is close to the ideal. RESULTS: We show that Qbeta replicase generates all possible base substitutions with an equivalent preference for mutating A/T or G/C bases and with no significant bias for transitions over transversions. To demonstrate the high diversity that can be sampled from a Qbeta replicase-generated mRNA library, the approach was used to evolve the binding affinity of a single domain VNAR shark antibody fragment (12Y-2) against malarial apical membrane antigen-1 (AMA-1) via ribosome display. The binding constant (KD) of 12Y-2 was increased by 22-fold following two consecutive but discrete rounds of mutagenesis and selection. The mutagenesis method was also used to alter the substrate specificity of beta-lactamase which does not significantly hydrolyse the antibiotic cefotaxime. Two cycles of RNA mutagenesis and selection on increasing concentrations of cefotaxime resulted in mutants with a minimum 10,000-fold increase in resistance, an outcome achieved faster and with fewer overall mutations than in comparable studies using other mutagenesis strategies. CONCLUSION: The RNA based approach outlined here is rapid and simple to perform and generates large, highly diverse populations of proteins, each differing by only one or two amino acids from the parent protein. The practical implications of our results are that suitable improved protein candidates can be recovered from in vitro protein evolution approaches using significantly fewer rounds of mutagenesis and selection, and with little or no collateral damage to the protein or its mRNA. [Abstract/Link to Full Text]

Corral T, Ver LS, Mottet G, Cano O, García-Barreno B, Calder LJ, Skehel JJ, Roux L, Melero JA
High level expression of soluble glycoproteins in the allantoic fluid of embryonated chicken eggs using a Sendai virus minigenome system.
BMC Biotechnol. 2007;717.
BACKGROUND: Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs. RESULTS: Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5-10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach. CONCLUSION: The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies. [Abstract/Link to Full Text]

Liao J, Warmuth MK, Govindarajan S, Ness JE, Wang RP, Gustafsson C, Minshull J
Engineering proteinase K using machine learning and synthetic genes.
BMC Biotechnol. 2007;716.
BACKGROUND: Altering a protein's function by changing its sequence allows natural proteins to be converted into useful molecular tools. Current protein engineering methods are limited by a lack of high throughput physical or computational tests that can accurately predict protein activity under conditions relevant to its final application. Here we describe a new synthetic biology approach to protein engineering that avoids these limitations by combining high throughput gene synthesis with machine learning-based design algorithms. RESULTS: We selected 24 amino acid substitutions to make in proteinase K from alignments of homologous sequences. We then designed and synthesized 59 specific proteinase K variants containing different combinations of the selected substitutions. The 59 variants were tested for their ability to hydrolyze a tetrapeptide substrate after the enzyme was first heated to 68 degrees C for 5 minutes. Sequence and activity data was analyzed using machine learning algorithms. This analysis was used to design a new set of variants predicted to have increased activity over the training set, that were then synthesized and tested. By performing two cycles of machine learning analysis and variant design we obtained 20-fold improved proteinase K variants while only testing a total of 95 variant enzymes. CONCLUSION: The number of protein variants that must be tested to obtain significant functional improvements determines the type of tests that can be performed. Protein engineers wishing to modify the property of a protein to shrink tumours or catalyze chemical reactions under industrial conditions have until now been forced to accept high throughput surrogate screens to measure protein properties that they hope will correlate with the functionalities that they intend to modify. By reducing the number of variants that must be tested to fewer than 100, machine learning algorithms make it possible to use more complex and expensive tests so that only protein properties that are directly relevant to the desired application need to be measured. Protein design algorithms that only require the testing of a small number of variants represent a significant step towards a generic, resource-optimized protein engineering process. [Abstract/Link to Full Text]

Papagianni M, Avramidis N, Filioussis G
High efficiency electrotransformation of Lactococcus lactis spp. lactis cells pretreated with lithium acetate and dithiothreitol.
BMC Biotechnol. 2007;715.
BACKGROUND: A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria. RESULTS: Electrotransformation efficiencies of up to 105 transformants per mug DNA have been reported in the literature for L. lactis spp.lactis LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 +/- 52.5 x 107 transformants per mug DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 +/- 0.5 x 105 transformants per mug DNA. Results of the same trend were obtained with L. lactis ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 1010 cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined. Both host-vector systems proved to be reproducible and highly efficient. CONCLUSION: This investigation sought to improve still further transformation efficiencies and to provide a reliable high efficiency transformation system for L. lactis spp. lactis. The applied methodology, tested in two well-known strains, allows the production of large numbers of transformants and the construction of large recombinant libraries. [Abstract/Link to Full Text]

Hust M, Jostock T, Menzel C, Voedisch B, Mohr A, Brenneis M, Kirsch MI, Meier D, Dübel S
Single chain Fab (scFab) fragment.
BMC Biotechnol. 2007;714.
BACKGROUND: The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. RESULTS: Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabDeltaC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. CONCLUSION: A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection. [Abstract/Link to Full Text]

?o? M, Golec P, ?o? JM, Weglewska-Jurkiewicz A, Czyz A, Wegrzyn A, Wegrzyn G, Neubauer P
Effective inhibition of lytic development of bacteriophages lambda, P1 and T4 by starvation of their host, Escherichia coli.
BMC Biotechnol. 2007;713.
BACKGROUND: Bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful. RESULTS: Here, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, lambda, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats. CONCLUSION: Bacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively. [Abstract/Link to Full Text]

Marusic C, Nuttall J, Buriani G, Lico C, Lombardi R, Baschieri S, Benvenuto E, Frigerio L
Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells.
BMC Biotechnol. 2007;712.
BACKGROUND: Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco. RESULTS: We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. CONCLUSION: We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue. [Abstract/Link to Full Text]

Hanhineva KJ, Kärenlampi SO
Production of transgenic strawberries by temporary immersion bioreactor system and verification by TAIL-PCR.
BMC Biotechnol. 2007;711.
BACKGROUND: Strawberry (Fragaria x ananassa) is an economically important soft fruit crop with polyploid genome which complicates the breeding of new cultivars. For certain traits, genetic engineering offers a potential alternative to traditional breeding. However, many strawberry varieties are quite recalcitrant for Agrobacterium-mediated transformation, and a method allowing easy handling of large amounts of starting material is needed. Also the genotyping of putative transformants is challenging since the isolation of DNA for Southern analysis is difficult due to the high amount of phenolic compounds and polysaccharides that complicate efficient extraction of digestable DNA. There is thus a need to apply a screening method that is sensitive and unambiguous in identifying the different transformation events. RESULTS: Hygromycin-resistant strawberries were developed in temporary immersion bioreactors by Agrobacterium-mediated gene transfer. Putative transformants were screened by TAIL-PCR to verify T-DNA integration and to distinguish between the individual transformation events. Several different types of border sequence arrangements were detected. CONCLUSION: This study demonstrates that temporary immersion bioreactor system suits well for the regeneration of transgenic strawberry plants as a labour-efficient technique. Small amount of DNA required by TAIL-PCR is easily recovered even from a small transformant, which allows rapid verification of T-DNA integration and detection of separate gene transfer events. These techniques combined clearly facilitate the generation of transgenic strawberries but should be applicable to other plants as well. [Abstract/Link to Full Text]

Khanam S, Rajendra P, Khanna N, Swaminathan S
An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes.
BMC Biotechnol. 2007;710.
BACKGROUND: Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. RESULTS: This work stems from the emergence of (i) the DEN virus envelope (E) domain III (EDIII) as the most important region of the molecule from a vaccine perspective and (ii) the adenovirus (Ad) as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd) vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. CONCLUSION: Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has implications for the development of safe and effective tetravalent dengue vaccines. [Abstract/Link to Full Text]

Woraratanadharm J, Rubinchik S, Yu H, Dong JY
Novel system uses probasin-based promoter, transcriptional silencers and amplification loop to induce high-level prostate expression.
BMC Biotechnol. 2007;79.
BACKGROUND: Despite several effective treatment options available for prostate cancer, it remains the second leading cause of cancer death in American men. Thus, there is a great need for new treatments to improve outcomes. One such strategy is to eliminate cancer through the expression of cytotoxic genes specifically in prostate cells by gene therapy vectored delivery. To prevent systemic toxicity, tissue- and/or cancer-specific gene expression is required. However, the use of tissue- or cancer-specific promoters to target transgene expression has been hampered by their weak activity. RESULTS: To address this issue, we have developed a regulation strategy that includes feedback amplification of gene expression along with a differentially suppressible tetracycline regulated expression system (DiSTRES). By differentially suppressing expression of the tetracycline-regulated transcriptional activator (tTA) and silencer (tTS) genes based on the cell origin, this leads to the activation and silencing of the TRE promoter, respectively. In vitro transduction of LNCaP cells with Ad/GFPDiSTRES lead to GFP expression levels that were over 30-fold higher than Ad/CMV-GFP. Furthermore, Ad/FasL-GFPDiSTRES demonstrated cytotoxic effects in prostate cancer cells known to be resistant to Fas-mediated apoptosis. CONCLUSION: Prostate-specific regulation from the DiSTRES system, therefore, serves as a promising new regulation strategy for future applications in the field of cancer gene therapy and gene therapy as a whole. [Abstract/Link to Full Text]

Branham WS, Melvin CD, Han T, Desai VG, Moland CL, Scully AT, Fuscoe JC
Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements.
BMC Biotechnol. 2007;78.
BACKGROUND: Environmental ozone can rapidly degrade cyanine 5 (Cy5), a fluorescent dye commonly used in microarray gene expression studies. Cyanine 3 (Cy3) is much less affected by atmospheric ozone. Degradation of the Cy5 signal relative to the Cy3 signal in 2-color microarrays will adversely reduce the Cy5/Cy3 ratio resulting in unreliable microarray data. RESULTS: Ozone in central Arkansas typically ranges between approximately 22 ppb to approximately 46 ppb and can be as high as 60-100 ppb depending upon season, meteorological conditions, and time of day. These levels of ozone are common in many areas of the country during the summer. A carbon filter was installed in the laboratory air handling system to reduce ozone levels in the microarray laboratory. In addition, the airflow was balanced to prevent non-filtered air from entering the laboratory. These modifications reduced the ozone within the microarray laboratory to approximately 2-4 ppb. Data presented here document reductions in Cy5 signal on both in-house produced microarrays and commercial microarrays as a result of exposure to unfiltered air. Comparisons of identically hybridized microarrays exposed to either carbon-filtered or unfiltered air demonstrated the protective effect of carbon-filtration on microarray data as indicated by Cy5 and Cy3 intensities. LOWESS normalization of the data was not able to completely overcome the effect of ozone-induced reduction of Cy5 signal. Experiments were also conducted to examine the effects of high humidity on microarray quality. Modest, but significant, increases in Cy5 and Cy3 signal intensities were observed after 2 or 4 hours at 98-99% humidity compared to 42% humidity. CONCLUSION: Simple installation of carbon filters in the laboratory air handling system resulted in low and consistent ozone levels. This allowed the accurate determination of gene expression by microarray using Cy5 and Cy3 fluorescent dyes. [Abstract/Link to Full Text]

Olichon A, Schweizer D, Muyldermans S, de Marco A
Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains.
BMC Biotechnol. 2007;77.
BACKGROUND: Recombinant antibodies from Camelidae (VHHs) are potentially useful tools for both basic research and biotechnological applications because of their small size, robustness, easy handling and possibility to refold after chemio-physical denaturation. Their heat tolerance is a particularly interesting feature because it has been recently related to both high yields during recombinant expression and selective purification of folded protein. RESULTS: Purification of recombinant RE3 VHH by heat treatment yielded the same amount of antibody as purification by affinity chromatography and negligible differences were found in stability, secondary structure and functionality. Similar results were obtained using another class of thermotolerant proteins, the single domain VH scaffold, described by Jespers et al. However, thermosensitive VHs could not withstand the heat treatment and co-precipitated with the bacterial proteins. In both cases, the thermotolerant proteins unfolded during the treatment but promptly refolded when moved back to a compatible temperature. CONCLUSION: Heat treatment can simplify the purification protocol of thermotolerant proteins as well as remove any soluble aggregate. Since the re-folding capability after heat-induced denaturation was previously correlated to higher performance during recombinant expression, a unique heating step can be envisaged to screen constructs that can provide high yields of correctly-folded proteins. [Abstract/Link to Full Text]

Zhou X, Symons J, Hoppes R, Krueger C, Berens C, Hillen W, Berkhout B, Das AT
Improved single-chain transactivators of the Tet-On gene expression system.
BMC Biotechnol. 2007;76.
BACKGROUND: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. RESULTS: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used. CONCLUSION: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used. [Abstract/Link to Full Text]

Shi X, Harrison RL, Hollister JR, Mohammed A, Fraser MJ, Jarvis DL
Construction and characterization of new piggyBac vectors for constitutive or inducible expression of heterologous gene pairs and the identification of a previously unrecognized activator sequence in piggyBac.
BMC Biotechnol. 2007;75.
BACKGROUND: We constructed and characterized several new piggyBac vectors to provide transposition of constitutively- or inducibly-expressible heterologous gene pairs. The dual constitutive control element consists of back-to-back copies of a baculovirus immediate early (ie1) promoter separated by a baculovirus enhancer (hr5). The dual inducible control element consists of back-to-back copies of a minimal cytomegalovirus (CMVmin) promoter separated by a synthetic operator (TetO7), which drives transcription in the presence of a mutant transcriptional repressor plus tetracycline. RESULTS: Characterization of these vectors revealed an unexpected position effect, in which heterologous genes adjacent to the 3'- terminal region ("rightward" genes) were consistently expressed at higher levels than those adjacent to the 5'-terminal region ("leftward" genes) of the piggyBac element. This position effect was observed with all six heterologous genes examined and with both transcriptional control elements. Further analysis demonstrated that this position effect resulted from stimulation of rightward gene expression by the internal domain sequence of the 3'-terminal region of piggyBac. Inserting a copy of this sequence into the 5'- terminal repeat region of our new piggyBac vectors in either orientation stimulated leftward gene expression. Representative piggyBac vectors designed for constitutive or inducible expression of heterologous gene pairs were shown to be functional as insect transformation vectors. CONCLUSION: This study is significant because (a) it demonstrates the utility of a strategy for the construction of piggyBac vectors that can provide constitutive or inducible heterologous gene pair expression and (b) it reveals the presence of a previously unrecognized transcriptional activator in piggyBac, which is an important and increasingly utilized transposable element. [Abstract/Link to Full Text]

Vidi PA, Kessler F, Bréhélin C
Plastoglobules: a new address for targeting recombinant proteins in the chloroplast.
BMC Biotechnol. 2007;74.
BACKGROUND: The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures) to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary. RESULTS: Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34) as the carrier. Similar to adipocyte differentiation related protein (ADRP) in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism. CONCLUSION: Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming. [Abstract/Link to Full Text]

Bäckström BT, Brockelbank JA, Rehm BH
Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein.
BMC Biotechnol. 2007;73.
BACKGROUND: Fluorescence activated cell sorting (FACS) is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA) granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis. RESULTS: Hybrid genes were constructed, which encode either the mouse interleukin-2 (IL2) or the myelin oligodendrocyte glycoprotein (MOG) fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin) and the respective sera were analysed using MOG-displaying PHA granules and FACS analysis showing a specific and sensitive detection of antigen-specific antibodies within a wide dynamic range. CONCLUSION: E. coli can be genetically engineered to produce PHA granules displaying correctly folded eukaryotic proteins and which can be applied as beads in FACS based diagnostics. Since PHA granule formation and protein attachment occurs in one step already inside the bacterial cell, microbial production could be a cheap and efficient alternative to commercial beads. [Abstract/Link to Full Text]

Bins AD, van Rheenen J, Jalink K, Halstead JR, Divecha N, Spencer DM, Haanen JB, Schumacher TN
Intravital imaging of fluorescent markers and FRET probes by DNA tattooing.
BMC Biotechnol. 2007;72.
BACKGROUND: Advances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals. However, the generation of transgenic mice is a lengthy process and intravital imaging requires specialized knowledge and equipment. Here, we report a rapid and undemanding intravital imaging method using generally available equipment. RESULTS: By DNA tattooing we transfect keratinocytes of living mice with DNA encoding fluorescent biosensors. Subsequently, the behavior of individual cells expressing these biosensors can be visualized within hours and using conventional microscopy equipment. Using this "instant transgenic" model in combination with a corrected coordinate system, we followed the in vivo behavior of individual cells expressing either FRET- or location-based biosensors for several days. The utility of this approach was demonstrated by assessment of in vivo caspase-3 activation upon induction of apoptosis. CONCLUSION: This "instant skin transgenic" model can be used to follow the in vivo behavior of individual cells expressing either FRET- or location-based probes for several days after tattooing and provides a rapid and inexpensive method for intravital imaging in murine skin. [Abstract/Link to Full Text]

Rinne J, Albarran B, Jylhävä J, Ihalainen TO, Kankaanpää P, Hytönen VP, Stayton PS, Kulomaa MS, Vihinen-Ranta M
Internalization of novel non-viral vector TAT-streptavidin into human cells.
BMC Biotechnol. 2007;71.
BACKGROUND: The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT47-57-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes. RESULTS: By confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid) (PPAA), nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs. CONCLUSION: This study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells. [Abstract/Link to Full Text]

Muratori C, D'Aloja P, Superti F, Tinari A, Sol-Foulon N, Sparacio S, Bosch V, Schwartz O, Federico M
Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way.
BMC Biotechnol. 2006;652.
BACKGROUND: The availability of cell lines releasing fluorescent viral particles can significantly support a variety of investigations, including the study of virus-cell interaction and the screening of antiviral compounds. Regarding HIV-1, the recovery of such biologic reagents represents a very hard challenge due to the intrinsic cytotoxicity of many HIV-1 products. We sought to overcome such a limitation by using a cell line releasing HIV-1 particles in an inducible way, and by exploiting the ability of a HIV-1 Nef mutant to be incorporated in virions at quite high levels. RESULTS: Here, we report the isolation and characterization of a HIV-1 packaging cell line, termed 18-4s, able to release valuable amounts of fluorescent HIV-1 based Virus-Like Particles (VLPs) in an inducible way. 18-4s cells were recovered by constitutively expressing the HIV-1 NefG3C mutant fused with the enhanced-green fluorescent protein (NefG3C-GFP) in a previously isolated inducible HIV-1 packaging cell line. The G3C mutation creates a palmitoylation site which results in NefG3C-GFP incorporation into virions greatly exceeding that of the wild type counterpart. Upon induction of 18-4s cells with ponasterone A and sodium butyrate, up to 4 mug/ml of VLPs, which had incorporated about 150 molecules of NefG3C-GFP per viral particle, were released into the culture supernatant. Due to their intrinsic strong fluorescence, the 18-4s VLPs were easily detectable by a novel cytofluorometric-based assay developed here. The treatment of target cells with fluorescent 18-4 VLPs pseudotyped with different glycoprotein receptors resulted in these becoming fluorescent as early as two hours post-challenge. CONCLUSION: We created a stable cell line releasing fluorescent HIV-1 based VLPs upon induction useful for several applications including the study of virus-cell interactions and the screening of antiviral compounds. [Abstract/Link to Full Text]

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