• Don't forget to check out Neurotransmitter.net's research index for a list of dozens of other resources created by Shawn Thomas.
• The articles are retrieved from PubMed Central and PubMed on a daily basis.
  
• Free full text articles are also available for the following subjects: biology, molecular and cellular biology, neuroscience, genetics and proteomics, psychiatry, pharmacology, endocrinology, microbiology, cancer, informatics, and miscellaneous.
  
• You can jump directly to articles from Biomedical Engineering Online, Journal of Biomedicine & Biotechnology, BMC Biotechnology, Journal of Nanobiotechnology, BMC Medical Imaging, and Biomagnetic Research and Technology.
  
• You can view other articles from these journals as well: skip 0, skip 30, skip 60, or skip 90 of the most recent articles.
  
• Confused by the terminology? Try searching The On-Line Medical Dictionary (OMD).

Web www.neurotransmitter.net

---

Recent Articles in Biomedical Engineering Online

---

Busch MH, Vollmann W, Schnorr J, Grönemeyer DH
Finite volume analysis of temperature effects induced by active MRI implants with cylindrical symmetry: 1. Properly working devices.
Biomed Eng Online. 2005;4(1):25.
BACKGROUND: Active Magnetic Resonance Imaging implants are constructed as resonators tuned to the Larmor frequency of a magnetic resonance system with a specific field strength. The resonating circuit may be embedded into or added to the normal metallic implant structure. The resonators build inductively coupled wireless transmit and receive coils and can amplify the signal, normally decreased by eddy currents, inside metallic structures without affecting the rest of the spin ensemble. During magnetic resonance imaging the resonators generate heat, which is additional to the usual one described by the specific absorption rate. This induces temperature increases of the tissue around the circuit paths and inside the lumen of an active implant and may negatively influence patient safety. METHODS: This investigation provides an overview of the supplementary power absorbed by active implants with a cylindrical geometry, corresponding to vessel implants such as stents, stent grafts or vena cava filters. The knowledge of the overall absorbed power is used in a finite volume analysis to estimate temperature maps around different implant structures inside homogeneous tissue under worst-case assumptions. The "worst-case scenario" assumes thermal heat conduction without blood perfusion inside the tissue around the implant and mostly without any cooling due to blood flow inside vessels. RESULTS: The additional power loss of a resonator is proportional to the volume and the quality factor, as well as the field strength of the MRI system and the specific absorption rate of the applied sequence. For properly working devices the finite volume analysis showed only tolerable heating during MRI investigations in most cases. Only resonators transforming a few hundred mW into heat may reach temperature increases over 5 K. This requires resonators with volumes of several ten cubic centimeters, short inductor circuit paths with only a few 10 cm and a quality factor above ten. Using MR sequences, for which the MRI system manufacturer declares the highest specific absorption rate of 4 W/kg, vascular implants with a realistic construction, size and quality factor do not show temperature increases over a critical value of 5 K. CONCLUSION: The results show dangerous heating for the assumed "worst-case scenario" only for constructions not acceptable for vascular implants. Realistic devices are safe with respect to temperature increases. However, this investigation discusses only properly working devices. Ruptures or partial ruptures of the wires carrying the electric current of the resonance circuits or other defects can set up a power source inside an extremely small volume. The temperature maps around such possible "hot spots" should be analyzed in an additional investigation. [Abstract/Link to Full Text]

Ismailov RM, Shevchuk NA, Khusanov H
Mathematical model describing erythrocyte sedimentation rate. Implications for blood viscosity changes in traumatic shock and crush syndrome.
Biomed Eng Online. 2005;4(1):24.
BACKGROUND: The erythrocyte sedimentation rate (ESR) is a simple and inexpensive laboratory test, which is widespread in clinical practice, for assessing the inflammatory or acute response. This work addresses the theoretical and experimental investigation of sedimentation a single and multiple particles in homogeneous and heterogeneous (multiphase) medium, as it relates to their internal structure (aggregation of solid or deformed particles). METHODS: The equation system has been solved numerically. To choose finite analogs of derivatives we used the schemes of directional differences. RESULTS: (1) Our model takes into account the influence of the vessel wall on group aggregation of particles in tubes as well as the effects of rotation of particles, the constraint coefficient, and viscosity of a mixture as a function of the volume fraction. (2) This model can describe ESR as a function of the velocity of adhesion of erythrocytes; (3) Determination of the ESR is best conducted at certain time intervals, i.e. in a series of periods not exceeding 5 minutes each; (4) Differential diagnosis of various diseases by means of ESR should be performed using the aforementioned timed measurement of ESR; (5) An increase in blood viscosity during trauma results from an increase in rouleaux formation and the time-course method of ESR will be useful in patients with trauma, in particular, with traumatic shock and crush syndrome. CONCLUSION: The mathematical model created in this study used the most fundamental differential equations that have ever been derived to estimate ESR. It may further our understanding of its complex mechanism. [Abstract/Link to Full Text]

Felice CJ, Madrid RE, Valentinuzzi ME
Amplifier spurious input current components in electrode-electrolyte interface impedance measurements.
Biomed Eng Online. 2005;4(1):22.
BACKGROUND: In Impedance Microbiology, the time during which the measuring equipment is connected to the bipolar cells is rather long, usually between 6 to 24 hrs for microorganisms with duplication times in the order of less than one hour and concentrations ranging from 10(1) to 10(7) [CFU/ml]. Under these conditions, the electrode-electrolyte interface impedance may show a slow drift of about 2%/hr. By and large, growth curves superimposed on such drift do not stabilize, are less reproducible, and keep on distorting all over the measurement of the temporal reactive or resistive records due to interface changes, in turn originated in bacterial activity. This problem has been found when growth curves were obtained by means of impedance analyzers or with impedance bridges using different types of operational amplifiers. METHODS: Suspecting that the input circuitry was the culprit of the deleterious effect, we used for that matter (a) ultra-low bias current amplifiers, (b) isolating relays for the selection of cells, and (c) a shorter connection time, so that the relays were maintained opened after the readings, to bring down such spurious drift to a negligible value. Bacterial growth curves were obtained in order to test their quality. RESULTS: It was demonstrated that the drift decreases ten fold when the circuit remained connected to the cell for a short time between measurements, so that the distortion became truly negligible. Improvement due to better-input amplifiers was not as good as by reducing the connection time. Moreover, temperature effects were insignificant with a regulation of +/- 0.2 [ degrees C]. Frequency did not influence either. CONCLUSION: The drift originated either at the dc input bias offset current (Ios) of the integrated circuits, or in discrete transistors connected directly to the electrodes immersed in the cells, depending on the particular circuit arrangement. Reduction of the connection time was the best countermeasure. [Abstract/Link to Full Text]

Foster KR
In memorium: Herman p. Schwan [1915-2005].
Biomed Eng Online. 2005;4(1):21.
Herman P. Schwan [1915-2005] was a distinguished scientist and engineer, and a founding father of the field of biomedical engineering. A man of integrity, Schwan influenced the lives of many, including his wife and children, and his many students and colleagues. Active in science until nearly the end of his life, he will be very much missed by his family and many colleagues. [Abstract/Link to Full Text]

Verhey JF, Wisser J, Warfield SK, Rexilius J, Kikinis R
Non-rigid registration of a 3D ultrasound and a MR image data set of the female pelvic floor using a biomechanical model.
Biomed Eng Online. 2005;4(1):19.
BACKGROUND: The visual combination of different modalities is essential for many medical imaging applications in the field of Computer-Assisted medical Diagnosis (CAD) to enhance the clinical information content. Clinically, incontinence is a diagnosis with high clinical prevalence and morbidity rate. The search for a method to identify risk patients and to control the success of operations is still a challenging task. The conjunction of magnetic resonance (MR) and 3D ultrasound (US) image data sets could lead to a new clinical visual representation of the morphology as we show with corresponding data sets of the female anal canal with this paper. METHODS: We present a feasibility study for a non-rigid registration technique based on a biomechanical model for MR and US image data sets of the female anal canal as a base for a new innovative clinical visual representation. RESULTS: It is shown in this case study that the internal and external sphincter region could be registered elastically and the registration partially corrects the compression induced by the ultrasound transducer, so the MR data set showing the native anatomy is used as a frame for the US data set showing the same region with higher resolution but distorted by the transducer CONCLUSION: The morphology is of special interest in the assessment of anal incontinence and the non-rigid registration of normal clinical MR and US image data sets is a new field of the adaptation of this method incorporating the advantages of both technologies. [Abstract/Link to Full Text]

Pressel T, Bouguecha A, Vogt U, Meyer-Lindenberg A, Behrens BA, Nolte I, Windhagen H
Mechanical properties of femoral trabecular bone in dogs.
Biomed Eng Online. 2005;4(1):17.
BACKGROUND: Studying mechanical properties of canine trabecular bone is important for a better understanding of fracture mechanics or bone disorders and is also needed for numerical simulation of canine femora. No detailed data about elastic moduli and degrees of anisotropy of canine femoral trabecular bone has been published so far, hence the purpose of this study was to measure the elastic modulus of trabecular bone in canine femoral heads by ultrasound testing and to assess whether assuming isotropy of the cancellous bone in femoral heads in dogs is a valid simplification. METHODS: From 8 euthanized dogs, both femora were obtained and cubic specimens were cut from the centre of the femoral head which were oriented along the main pressure and tension trajectories. The specimens were tested using a 100 MHz ultrasound transducer in all three orthogonal directions. The directional elastic moduli of trabecular bone tissue and degrees of anisotropy were calculated. RESULTS: The elastic modulus along principal bone trajectories was found to be 11.2 GPa +/- 0.4, 10.5 +/- 2.1 GPa and 10.5 +/- 1.8 GPa, respectively. The mean density of the specimens was 1.40 +/- 0.09 g/cm3. The degrees of anisotropy revealed a significant inverse relationship with specimen densities. No significant differences were found between the elastic moduli in x, y and z directions, suggesting an effective isotropy of trabecular bone tissue in canine femoral heads. DISCUSSION: This study presents detailed data about elastic moduli of trabecular bone tissue obtained from canine femoral heads. Limitations of the study are the relatively small number of animals investigated and the measurement of whole specimen densities instead of trabecular bone densities which might lead to an underestimation of Young's moduli. Publications on elastic moduli of trabecular bone tissue present results that are similar to our data. CONCLUSION: This study provides data about directional elastic moduli and degrees of anisotropy of canine femoral head trabecular bone and might be useful for biomechanical modeling of proximal canine femora. [Abstract/Link to Full Text]

Christov II, Iliev GL
Public access defibrillation: suppression of 16.7 Hz interference generated by the power supply of the railway systems.
Biomed Eng Online. 2005;4(1):16.
BACKGROUND: A specific problem using the public access defibrillators (PADs) arises at the railway stations. Some countries as Germany, Austria, Switzerland, Norway and Sweden are using AC railroad net power-supply system with rated 16.7 Hz frequency modulated from 15.69 Hz to 17.36 Hz. The power supply frequency contaminates the electrocardiogram (ECG). It is difficult to be suppressed or eliminated due to the fact that it considerably overlaps the frequency spectra of the ECG. The interference impedes the automated decision of the PADs whether a patient should be (or should not be) shocked.The aim of this study is the suppression of the 16.7 Hz interference generated by the power supply of the railway systems. METHODS: Software solution using adaptive filtering method was proposed for 16.7 Hz interference suppression. The optimal performance of the filter is achieved, embedding a reference channel in the PADs to record the interference. The method was tested with ECGs from AHA database. RESULTS: The method was tested with patients of normal sinus rhythms, symptoms of tachycardia and ventricular fibrillation. Simulated interference with frequency modulation from 15.69 Hz to 17.36 Hz changing at a rate of 2% per second was added to the ECGs, and then processed by the suggested adaptive filtering. The method totally suppresses the noise with no visible distortions of the original signals. CONCLUSION: The proposed adaptive filter for noise suppression generated by the power supply of the railway systems has a simple structure requiring a low level of computational resources, but a good reference signal as well. [Abstract/Link to Full Text]

Durka PJ
On the methodological unification in electroencephalography.
Biomed Eng Online. 2005;4(1):15.
BACKGROUND: This paper presents results of a pursuit of a repeatable and objective methodology of analysis of the electroencephalographic (EEG) time series. METHODS: Adaptive time-frequency approximations of EEG are discussed in the light of the available experimental and theoretical evidence, and applicability in various experimental and clinical setups. RESULTS: Four lemmas and three conjectures support the following conclusion. CONCLUSION: Adaptive time-frequency approximations of signals unify most of the univariate computational approaches to EEG analysis, and offer compatibility with its traditional (visual) analysis, used in clinical applications. [Abstract/Link to Full Text]

Sankaranarayanan M, Chua LP, Ghista DN, Tan YS
Computational model of blood flow in the aorto-coronary bypass graft.
Biomed Eng Online. 2005;4(1):14.
BACKGROUND: Coronary artery bypass grafting surgery is an effective treatment modality for patients with severe coronary artery disease. The conduits used during the surgery include both the arterial and venous conduits. Long- term graft patency rate for the internal mammary arterial graft is superior, but the same is not true for the saphenous vein grafts. At 10 years, more than 50% of the vein grafts would have occluded and many of them are diseased. Why do the saphenous vein grafts fail the test of time? Many causes have been proposed for saphenous graft failure. Some are non-modifiable and the rest are modifiable. Non-modifiable causes include different histological structure of the vein compared to artery, size disparity between coronary artery and saphenous vein. However, researches are more interested in the modifiable causes, such as graft flow dynamics and wall shear stress distribution at the anastomotic sites. Formation of intimal hyperplasia at the anastomotic junction has been implicated as the root cause of long- term graft failure.Many researchers have analyzed the complex flow patterns in the distal sapheno-coronary anastomotic region, using various simulated model in an attempt to explain the site of preferential intimal hyperplasia based on the flow disturbances and differential wall stress distribution. In this paper, the geometrical bypass models (aorto-left coronary bypass graft model and aorto-right coronary bypass graft model) are based on real-life situations. In our models, the dimensions of the aorta, saphenous vein and the coronary artery simulate the actual dimensions at surgery. Both the proximal and distal anastomoses are considered at the same time, and we also take into the consideration the cross-sectional shape change of the venous conduit from circular to elliptical. Contrary to previous works, we have carried out computational fluid dynamics (CFD) study in the entire aorta-graft-perfused artery domain. The results reported here focus on (i) the complex flow patterns both at the proximal and distal anastomotic sites, and (ii) the wall shear stress distribution, which is an important factor that contributes to graft patency. METHODS: The three-dimensional coronary bypass models of the aorto-right coronary bypass and the aorto-left coronary bypass systems are constructed using computational fluid-dynamics software (Fluent 6.0.1). To have a better understanding of the flow dynamics at specific time instants of the cardiac cycle, quasi-steady flow simulations are performed, using a finite-volume approach. The data input to the models are the physiological measurements of flow-rates at (i) the aortic entrance, (ii) the ascending aorta, (iii) the left coronary artery, and (iv) the right coronary artery. RESULTS: The flow field and the wall shear stress are calculated throughout the cycle, but reported in this paper at two different instants of the cardiac cycle, one at the onset of ejection and the other during mid-diastole for both the right and left aorto-coronary bypass graft models. Plots of velocity-vector and the wall shear stress distributions are displayed in the aorto-graft-coronary arterial flow-field domain. We have shown (i) how the blocked coronary artery is being perfused in systole and diastole, (ii) the flow patterns at the two anastomotic junctions, proximal and distal anastomotic sites, and (iii) the shear stress distributions and their associations with arterial disease. CONCLUSION: The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear-stress variation is observed around the distal anastomotic region. These results can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease. [Abstract/Link to Full Text]

Richerson SJ, Robinson CJ, Shum J
A comparative study of reaction times between type II diabetics and non-diabetics.
Biomed Eng Online. 2005;4(1):12.
BACKGROUND: Aging has been shown to slow reflexes and increase reaction time to varied stimuli. However, the effect of Type II diabetes on these same reaction times has not been reported. Diabetes affects peripheral nerves in the somatosensory and auditory system, slows psychomotor responses, and has cognitive effects on those individuals without proper metabolic control, all of which may affect reaction times. The additional slowing of reaction times may affect every-day tasks such as balance, increasing the probability of a slip or fall. METHODS: Reaction times to a plantar touch, a pure tone auditory stimulus, and rightward whole-body lateral movement of 4 mm at 100 mm/s2 on a platform upon which a subject stood, were measured in 37 adults over 50 yrs old. Thirteen (mean age = 60.6 +/- 6.5 years) had a clinical diagnosis of type II diabetes and 24 (mean age = 59.4 +/- 8.0 years) did not. Group averages were compared to averages obtained from nine healthy younger adult group (mean age = 22.7 +/- 1.2 years). RESULTS: Average reaction times for plantar touch were significantly longer in diabetic adults than the other two groups, while auditory reaction times were not significantly different among groups. Whole body reaction times were significantly different among all three groups with diabetic adults having the longest reaction times, followed by age-matched adults, and then younger adults. CONCLUSION: Whole body reaction time has been shown to be a sensitive indicator of differences between young adults, healthy mature adults, and mature diabetic adults. Additionally, the increased reaction time seen in this modality for subjects with diabetes may be one cause of increased slips and falls in this group. [Abstract/Link to Full Text]

Clayton RH, Holden AV
Dispersion of cardiac action potential duration and the initiation of re-entry: a computational study.
Biomed Eng Online. 2005;4(1):11.
BACKGROUND: The initiation of re-entrant cardiac arrhythmias is associated with increased dispersion of repolarisation, but the details are difficult to investigate either experimentally or clinically. We used a computational model of cardiac tissue to study systematically the association between action potential duration (APD) dispersion and susceptibility to re-entry. METHODS: We simulated a 60 x 60 mm2 D sheet of cardiac ventricular tissue using the Luo-Rudy phase 1 model, with maximal conductance of the K+ channel gKmax set to 0.004 mS mm(-2). Within the central 40 x 40 mm region we introduced square regions with prolonged APD by reducing gKmax to between 0.001 and 0.003 mS mm(-2). We varied (i) the spatial scale of these regions, (ii) the magnitude of gKmax in these regions, and (iii) cell-to-cell coupling. RESULTS: Changing spatial scale from 5 to 20 mm increased APD dispersion from 49 to 102 ms, and the susceptible window from 31 to 86 ms. Decreasing gKmax in regions with prolonged APD from 0.003 to 0.001 mS mm-2 increased APD dispersion from 22 to 70 ms, and the susceptible window from <1 to 56 ms. Decreasing cell-to-cell coupling by changing the diffusion coefficient from 0.2 to 0.05 mm2 ms(-1) increased APD dispersion from 57 to 88 ms, and increased the susceptible window from 41 to 74 ms. CONCLUSION: We found a close association between increased APD dispersion and susceptibility to re-entrant arrhythmias, when APD dispersion is increased by larger spatial scale of heterogeneity, greater electrophysiological heterogeneity, and weaker cell-to-cell coupling. [Abstract/Link to Full Text]

Zhong L, Ghista DN, Ng EY, Lim ST
Passive and active ventricular elastances of the left ventricle.
Biomed Eng Online. 2005;4(1):10.
BACKGROUND: Description of the heart as a pump has been dominated by models based on elastance and compliance. Here, we are presenting a somewhat new concept of time-varying passive and active elastance. The mathematical basis of time-varying elastance of the ventricle is presented. We have defined elastance in terms of the relationship between ventricular pressure and volume, as: dP = EdV + VdE, where E includes passive (Ep) and active (Ea) elastance. By incorporating this concept in left ventricular (LV) models to simulate filling and systolic phases, we have obtained the time-varying expression for Ea and the LV-volume dependent expression for Ep. METHODS AND RESULTS: Using the patient's catheterization-ventriculogram data, the values of passive and active elastance are computed. Ea is expressed as [formula: see text] Epis represented as: [formula: see text]. Ea is deemed to represent a measure of LV contractility. Hence, Peak dP/dt and ejection fraction (EF) are computed from the monitored data and used as the traditional measures of LV contractility. When our computed peak active elastance (Ea,max) is compared against these traditional indices by linear regression, a high degree of correlation is obtained. As regards Ep, it constitutes a volume-dependent stiffness property of the LV, and is deemed to represent resistance-to-filling. CONCLUSIONS: Passive and active ventricular elastance formulae can be evaluated from a single-beat P-V data by means of a simple-to-apply LV model. The active elastance (Ea) can be used to characterize the ventricle's contractile state, while passive elastance (Ep) can represent a measure of resistance-to-filling. [Abstract/Link to Full Text]

Valentinuzzi ME
Review of therapy, sensory integration and the autistic child.
Biomed Eng Online. 2005;4(1):9. [Abstract/Link to Full Text]

Sperelakis N, Ramasamy L
Gap-junction channels inhibit transverse propagation in cardiac muscle.
Biomed Eng Online. 2005;4(1):7.
The effect of adding many gap-junctions (g-j) channels between contiguous cells in a linear chain on transverse propagation between parallel chains was examined in a 5 x 5 model (5 parallel chains of 5 cells each) for cardiac muscle. The action potential upstrokes were simulated using the PSpice program for circuit analysis. Either a single cell was stimulated (cell A1) or the entire chain was stimulated simultaneously (A-chain). Transverse velocity was calculated from the total propagation time (TPT) from when the first AP crossed a Vm of -20 mV and the last AP crossed -20 mV. The number of g-j channels per junction was varied from zero to 100, 1,000 and 10,000 (Rgj of infinity, 100 MOmega, 10 MOmega, 1.0 MOmega, respectively). The longitudinal resistance of the interstitial fluid (ISF) space between the parallel chains (Rol2) was varied between 200 KOmega (standard value) and 1.0, 5.0, and 10 MOmega. The higher the Rol2 value, the tighter the packing of the chains. It was found that adding many g-j channels inhibited transverse propagation by blocking activation of all 5 chains, unless Rol2 was greatly increased above the standard value of 200 KOmega. This was true for either method of stimulation. This was explained by, when there is strong longitudinal coupling between all 5 cells of a chain awaiting excitation, there must be more transfer energy (i.e., more current) to simultaneously excite all 5 cells of a chain. [Abstract/Link to Full Text]

Sun Y, Chan KL, Krishnan SM
Life-threatening ventricular arrhythmia recognition by nonlinear descriptor.
Biomed Eng Online. 2005;4(1):6.
BACKGROUND: Ventricular tachycardia (VT) and ventricular fibrillation (VF) are ventricular cardiac arrhythmia that could be catastrophic and life threatening. Correct and timely detection of VT or VF can save lives. METHODS: In this paper, a multiscale-based non-linear descriptor, the Hurst index, is proposed to characterize the ECG episode, so that VT and VF can be recognized as different from normal sinus rhythm (NSR) in the descriptor domain. RESULTS: This newly proposed technique was tested using MIT-BIH malignant ventricular arrhythmia database. The relationship between the ECG episode length and the corresponding recognition performance was studied. The experiments demonstrated good performance of the proposed descriptor. An accuracy rate as high as 100% was obtained for VT/VF to be recognized from NSR; for VT and VF to be recognized from each other, the recognition accuracy varies from 84.24% to 100%. In addition, the results were compared favorably against those obtained using Complexity measure. CONCLUSIONS: There is strong potential for using the Hurst index for malignant ventricular arrhythmia recognition in clinical applications. [Abstract/Link to Full Text]

Valchinov ES, Pallikarakis NE
Design and testing of low intensity laser biostimulator.
Biomed Eng Online. 2005;4(1):5.
BACKGROUND: The non-invasive nature of laser biostimulation has made lasers an attractive alternative in Medical Acupuncture at the last 25 years. However, there is still an uncertainty as to whether they work or their effect is just placebo. Although a plethora of scientific papers published about the topic showing positive clinical results, there is still a lack of objective scientific proofs about the biostimulation effect of lasers in Medical Acupuncture. The objective of this work was to design and build a low cost portable laser device for stimulation of acupuncture points, considered here as small localized biosources (SLB), without stimulating any sensory nerves via shock or heat and to find out a suitable method for objectively evaluating its stimulating effect. The design is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the biostimulation effect of lasers used in Medical Acupuncture. METHODS: The proposed biostimulator features two operational modes: program mode and stimulation mode and two output polarization modes: linearly and circularly polarized laser emission. In program mode, different user-defined stimulation protocols can be created and memorized. The laser output can be either continuous or pulse modulated. Each stimulation session consists of a pre-defined number of successive continuous or square pulse modulated sequences of laser emission. The variable parameters of the laser output are: average output power, pulse width, pulse period, and continuous or pulsed sequence duration and repetition period. In stimulation mode the stimulus is automatically applied according to the pre-programmed protocol. The laser source is 30 mW AlGaInP laser diode with an emission wavelength of 685 nm, driven by a highly integrated driver. The optical system designed for beam collimation and polarization change uses single collimating lens with large numerical aperture, linear polarizer and a quarter-wave retardation plate. The proposed method for testing the device efficiency employs a biofeedback from the subject by recording the biopotentials evoked by the laser stimulus at related distant SLB sites. Therefore measuring of SLB biopotentials caused by the stimulus would indicate that a biopotential has been evoked at the irradiated site and has propagated to the measurement sites, rather than being caused by local changes of the electrical skin conductivity. RESULTS: A prototype device was built according to the proposed design using relatively inexpensive and commercially available components. The laser output can be pulse modulated from 0.1 to 1000 Hz with a duty factor from 10 to 90%. The average output power density can be adjusted in the range 24-480 mW/cm2, where the total irradiation is limited to 2 Joule per stimulation session. The device is controlled by an 8-bit RISC Flash microcontroller with internal RAM and EEPROM memory, which allows for a wide range of different stimulation protocols to be implemented and memorized. The integrated laser diode driver with its onboard light power control loop provides safe and consistent laser modulation. The prototype was tested on the right Tri-Heater (TH) acupuncture meridian according to the proposed method. Laser evoked potentials were recorded from most of the easily accessible SLB along the meridian under study. They appear like periodical spikes with a repetition rate from 0.05 to 10 Hz and amplitude range 0.1-1 mV. CONCLUSION: The prototype's specifications were found to be better or comparable to those of other existing devices. It features low component count, small size and low power consumption. Because of the low power levels used the possibility of sensory nerve stimulation via the phenomenon of shock or heat is excluded. Thus senseless optical stimulation is achieved. The optical system presented offers simple and cost effective way for beam collimation and polarization change. The novel method proposed for testing the device efficiency allows for objectively recording of SLB potentials evoked by laser stimulus. Based on the biopotential records obtained with this method, a scientifically based conclusion can be drawn about the effectiveness of the commercially available devices for low-level laser therapy used in Medical Acupuncture. The prototype tests showed that with the biostimulator presented, SLB could be effectively stimulated at low power levels. However more studies are needed to derive a general conclusion about the SLB biostimulation mechanism of lasers and their most effective power and optical settings. [Abstract/Link to Full Text]

Wu HI
A case study of type 2 diabetes self-management.
Biomed Eng Online. 2005;4(1):4.
BACKGROUND: It has been established that careful diabetes self-management is essential in avoiding chronic complications that compromise health. Disciplined diet control and regular exercise are the keys for the type 2 diabetes self-management. An ability to maintain one's blood glucose at a relatively flat level, not fluctuating wildly with meals and hypoglycemic medical intervention, would be the goal for self-management. Hemoglobin A1c (HbA1c or simply A1c) is a measure of a long-term blood plasma glucose average, a reliable index to reflect one's diabetic condition. A simple regimen that could reduce the elevated A1c levels without altering much of type 2 diabetic patients' daily routine denotes a successful self-management strategy. METHODS: A relatively simple model that relates the food impact on blood glucose excursions for type 2 diabetes was studied. Meal is treated as a bolus injection of glucose. Medical intervention of hypoglycaemic drug or injection, if any, is lumped with secreted insulin as a damping factor. Lunch was used for test meals. The recovery period of a blood glucose excursion returning to the pre-prandial level, the maximal reach, and the area under the excursion curve were used to characterize one's ability to regulate glucose metabolism. A case study is presented here to illustrate the possibility of devising an individual-based self-management regimen. RESULTS: Results of the lunch study for a type 2 diabetic subject indicate that the recovery time of the post-prandial blood glucose level can be adjusted to 4 hours, which is comparable to the typical time interval for non-diabetics: 3 to 4 hours. A moderate lifestyle adjustment of light supper coupled with morning swimming of 20 laps in a 25 m pool for 40 minutes enabled the subject to reduce his A1c level from 6.7 to 6.0 in six months and to maintain this level for the subsequent six months. CONCLUSIONS: The preliminary result of this case study is encouraging. An individual life-style adjustment can be structured from the extracted characteristics of the post-prandial blood glucose excursions. Additional studies are certainly required to draw general applicable guidelines for lifestyle adjustments of type 2 diabetic patients. [Abstract/Link to Full Text]

Borrett DS, Jin F, Kwan HC
Evolutionary autonomous agents and the nature of apraxia.
Biomed Eng Online. 2005;4(1):1.
BACKGROUND: Evolutionary autonomous agents are robots or robot simulations whose controller is a dynamical neural network and whose evolution occurs autonomously under the guidance of a fitness function without the detailed or explicit direction of an external programmer. They are embodied agents with a simple neural network controller and as such they provide the optimal forum by which sensorimotor interactions in a specified environment can be studied without the computational assumptions inherent in standard neuroscience. METHODS: Evolutionary autonomous agents were evolved that were able to perform identical movements under two different contexts, one which represented an automatic movement and one which had a symbolic context. In an attempt to model the automatic-voluntary dissociation frequently seen in ideomotor apraxia, lesions were introduced into the neural network controllers resulting in a behavioral dissociation with loss of the ability to perform the movement which had a symbolic context and preservation of the simpler, automatic movement. RESULTS: Analysis of the changes in the hierarchical organization of the networks in the apractic EAAs demonstrated consistent changes in the network dynamics across all agents with loss of longer duration time scales in the network dynamics. CONCLUSION: The concepts of determinate motor programs and perceptual representations that are implicit in the present day understanding of ideomotor apraxia are assumptions inherent in the computational understanding of brain function. The strength of the present study using EAAs to model one aspect of ideomotor apraxia is the absence of these assumptions and a grounding of all sensorimotor interactions in an embodied, autonomous agent. The consistency of the hierarchical changes in the network dynamics across all apractic agents demonstrates that this technique is tenable and will be a valuable adjunct to a computational formalism in the understanding of the physical basis of neurological disorders. [Abstract/Link to Full Text]

Mohammed Y, Verhey JF
A finite element method model to simulate laser interstitial thermo therapy in anatomical inhomogeneous regions.
Biomed Eng Online. 2005;4(1):2.
BACKGROUND: Laser Interstitial ThermoTherapy (LITT) is a well established surgical method. The use of LITT is so far limited to homogeneous tissues, e.g. the liver. One of the reasons is the limited capability of existing treatment planning models to calculate accurately the damage zone. The treatment planning in inhomogeneous tissues, especially of regions near main vessels, poses still a challenge. In order to extend the application of LITT to a wider range of anatomical regions new simulation methods are needed. The model described with this article enables efficient simulation for predicting damaged tissue as a basis for a future laser-surgical planning system. Previously we described the dependency of the model on geometry. With the presented paper including two video files we focus on the methodological, physical and mathematical background of the model. METHODS: In contrast to previous simulation attempts, our model is based on finite element method (FEM). We propose the use of LITT, in sensitive areas such as the neck region to treat tumours in lymph node with dimensions of 0.5 cm - 2 cm in diameter near the carotid artery. Our model is based on calculations describing the light distribution using the diffusion approximation of the transport theory; the temperature rise using the bioheat equation, including the effect of microperfusion in tissue to determine the extent of thermal damage; and the dependency of thermal and optical properties on the temperature and the injury. Injury is estimated using a damage integral. To check our model we performed a first in vitro experiment on porcine muscle tissue. RESULTS: We performed the derivation of the geometry from 3D ultrasound data and show for this proposed geometry the energy distribution, the heat elevation, and the damage zone. Further on, we perform a comparison with the in-vitro experiment. The calculation shows an error of 5% in the x-axis parallel to the blood vessel. CONCLUSIONS: The FEM technique proposed can overcome limitations of other methods and enables an efficient simulation for predicting the damage zone induced using LITT. Our calculations show clearly that major vessels would not be damaged. The area/volume of the damaged zone calculated from both simulation and in-vitro experiment fits well and the deviation is small. One of the main reasons for the deviation is the lack of accurate values of the tissue optical properties. In further experiments this needs to be validated. [Abstract/Link to Full Text]

Cysarz D, Bettermann H, Lange S, Geue D, van Leeuwen P
A quantitative comparison of different methods to detect cardiorespiratory coordination during night-time sleep.
Biomed Eng Online. 2004 Nov 25;3(1):44.
BACKGROUND: The univariate approaches used to analyze heart rate variability have recently been extended by several bivariate approaches with respect to cardiorespiratory coordination. Some approaches are explicitly based on mathematical models which investigate the synchronization between weakly coupled complex systems. Others use an heuristic approach, i.e. characteristic features of both time series, to develop appropriate bivariate methods. OBJECTIVE: In this study six different methods used to analyze cardiorespiratory coordination have been quantitatively compared with respect to their performance (no. of sequences with cardiorespiratory coordination, no. of heart beats coordinated with respiration). Five of these approaches have been suggested in the recent literature whereas one method originates from older studies. RESULTS: The methods were applied to the simultaneous recordings of an electrocardiogram and a respiratory trace of 20 healthy subjects during night-time sleep from 0:00 to 6:00. The best temporal resolution and the highest number of coordinated heart beats were obtained with the analysis of 'Phase Recurrences'. Apart from the oldest method, all methods showed similar qualitative results although the quantities varied between the different approaches. In contrast, the oldest method detected considerably fewer coordinated heart beats since it only used part of the maximum amount of information available in each recording. CONCLUSIONS: The method of 'Phase Recurrences' should be the method of choice for the detection of cardiorespiratory coordination since it offers the best temporal resolution and the highest number of coordinated sequences and heart beats. Excluding the oldest method, the results of the heuristic approaches may also be interpreted in terms of the mathematical models. [Abstract/Link to Full Text]

Gowrishankar TR, Stewart DA, Martin GT, Weaver JC
Transport lattice models of heat transport in skin with spatially heterogeneous, temperature-dependent perfusion.
Biomed Eng Online. 2004 Nov 17;3(1):42.
BACKGROUND: Investigation of bioheat transfer problems requires the evaluation of temporal and spatial distributions of temperature. This class of problems has been traditionally addressed using the Pennes bioheat equation. Transport of heat by conduction, and by temperature-dependent, spatially heterogeneous blood perfusion is modeled here using a transport lattice approach. METHODS: We represent heat transport processes by using a lattice that represents the Pennes bioheat equation in perfused tissues, and diffusion in nonperfused regions. The three layer skin model has a nonperfused viable epidermis, and deeper regions of dermis and subcutaneous tissue with perfusion that is constant or temperature-dependent. Two cases are considered: (1) surface contact heating and (2) spatially distributed heating. The model is relevant to the prediction of the transient and steady state temperature rise for different methods of power deposition within the skin. Accumulated thermal damage is estimated by using an Arrhenius type rate equation at locations where viable tissue temperature exceeds 42 degrees C. Prediction of spatial temperature distributions is also illustrated with a two-dimensional model of skin created from a histological image. RESULTS: The transport lattice approach was validated by comparison with an analytical solution for a slab with homogeneous thermal properties and spatially distributed uniform sink held at constant temperatures at the ends. For typical transcutaneous blood gas sensing conditions the estimated damage is small, even with prolonged skin contact to a 45 degrees C surface. Spatial heterogeneity in skin thermal properties leads to a non-uniform temperature distribution during a 10 GHz electromagnetic field exposure. A realistic two-dimensional model of the skin shows that tissue heterogeneity does not lead to a significant local temperature increase when heated by a hot wire tip. CONCLUSIONS: The heat transport system model of the skin was solved by exploiting the mathematical analogy between local thermal models and local electrical (charge transport) models, thereby allowing robust, circuit simulation software to obtain solutions to Kirchhoff's laws for the system model. Transport lattices allow systematic introduction of realistic geometry and spatially heterogeneous heat transport mechanisms. Local representations for both simple, passive functions and more complex local models can be easily and intuitively included into the system model of a tissue. [Abstract/Link to Full Text]

Marx R, Jungwirth F, Walter PO
Threshold intensity factors as lower boundaries for crack propagation in ceramics.
Biomed Eng Online. 2004 Nov 17;3(1):41.
BACKGROUND: Slow crack growth can be described in a v (crack velocity) versus KI (stress intensity factor) diagram. Slow crack growth in ceramics is attributed to corrosion assisted stress at the crack tip or at any pre-existing defect in the ceramic. The combined effect of high stresses at the crack tip and the presence of water or body fluid molecules (reducing surface energy at the crack tip) induces crack propagation, which eventually may result in fatigue. The presence of a threshold in the stress intensity factor, below which no crack propagation occurs, has been the subject of important research in the last years. The higher this threshold, the higher the reliability of the ceramic, and consequently the longer its lifetime. METHODS: We utilize the Irwin K-field displacement relation to deduce crack tip stress intensity factors from the near crack tip profile. Cracks are initiated by indentation impressions. The threshold stress intensity factor is determined as the time limit of the tip stress intensity when the residual stresses have (nearly) disappeared. RESULTS: We determined the threshold stress intensity factors for most of the all ceramic materials presently important for dental restorations in Europe. Of special significance is the finding that alumina ceramic has a threshold limit nearly identical with that of zirconia. CONCLUSION: The intention of the present paper is to stress the point that the threshold stress intensity factor represents a more intrinsic property for a given ceramic material than the widely used toughness (bend strength or fracture toughness), which refers only to fast crack growth. Considering two ceramics with identical threshold limits, although with different critical stress intensity limits, means that both ceramics have identical starting points for slow crack growth. Fast catastrophic crack growth leading to spontaneous fatigue, however, is different. This growth starts later in those ceramic materials that have larger critical stress intensity factors. [Abstract/Link to Full Text]

Fan Y, Gregersen H, Kassab GS
A two-layered mechanical model of the rat esophagus. Experiment and theory.
Biomed Eng Online. 2004 Nov 1;3(1):40.
BACKGROUND: The function of esophagus is to move food by peristaltic motion which is the result of the interaction of the tissue forces in the esophageal wall and the hydrodynamic forces in the food bolus. The structure of the esophagus is layered. In this paper, the esophagus is treated as a two-layered structure consisting of an inner collagen-rich submucosa layer and an outer muscle layer. We developed a model and experimental setup for determination of elastic moduli in the two layers in circumferential direction and related the measured elastic modulus of the intact esophagus to the elastic modulus computed from the elastic moduli of the two layers. METHODS: Inflation experiments were done at in vivo length and pressure-diameters relations were recorded for the rat esophagus. Furthermore, the zero-stress state was taken into consideration. RESULTS: The radius and the strain increased as function of pressure in the intact as well as in the individual layers of the esophagus. At pressures higher than 1.5 cmH2O the muscle layer had a larger radius and strain than the mucosa-submucosa layer. The strain for the intact esophagus and for the muscle layer was negative at low pressures indicating the presence of residual strains in the tissue. The stress-strain curve for the submucosa-mucosa layer was shifted to the left of the curves for the muscle layer and for the intact esophagus at strains higher than 0.3. The tangent modulus was highest in the submucosa-mucosa layer, indicating that the submucosa-mucosa has the highest stiffness. A good agreement was found between the measured elastic modulus of the intact esophagus and the elastic modulus computed from the elastic moduli of the two separated layers. [Abstract/Link to Full Text]

Novak V, Yang AC, Lepicovsky L, Goldberger AL, Lipsitz LA, Peng CK
Multimodal pressure-flow method to assess dynamics of cerebral autoregulation in stroke and hypertension.
Biomed Eng Online. 2004 Oct 25;3(1):39.
BACKGROUND: This study evaluated the effects of stroke on regulation of cerebral blood flow in response to fluctuations in systemic blood pressure (BP). The autoregulatory dynamics are difficult to assess because of the nonstationarity and nonlinearity of the component signals. METHODS: We studied 15 normotensive, 20 hypertensive and 15 minor stroke subjects (48.0 +/- 1.3 years). BP and blood flow velocities (BFV) from middle cerebral arteries (MCA) were measured during the Valsalva maneuver (VM) using transcranial Doppler ultrasound. RESULTS: A new technique, multimodal pressure-flow analysis (MMPF), was implemented to analyze these short, nonstationary signals. MMPF analysis decomposes complex BP and BFV signals into multiple empirical modes, representing their instantaneous frequency-amplitude modulation. The empirical mode corresponding to the VM BP profile was used to construct the continuous phase diagram and to identify the minimum and maximum values from the residual BP (BPR) and BFV (BFVR) signals. The BP-BFV phase shift was calculated as the difference between the phase corresponding to the BPR and BFVR minimum (maximum) values. BP-BFV phase shifts were significantly different between groups. In the normotensive group, the BFVR minimum and maximum preceded the BPR minimum and maximum, respectively, leading to large positive values of BP-BFV shifts. CONCLUSION: In the stroke and hypertensive groups, the resulting BP-BFV phase shift was significantly smaller compared to the normotensive group. A standard autoregulation index did not differentiate the groups. The MMPF method enables evaluation of autoregulatory dynamics based on instantaneous BP-BFV phase analysis. Regulation of BP-BFV dynamics is altered with hypertension and after stroke, rendering blood flow dependent on blood pressure. [Abstract/Link to Full Text]

Wong LS, Johnson AT
Decrease of resistance to air flow with nasal strips as measured with the airflow perturbation device.
Biomed Eng Online. 2004 Oct 22;3(1):38.
BACKGROUND: Nasal strips are used by athletes, people who snore, and asthmatics to ease the burden of breathing. Although there are some published studies that demonstrate higher flow with nasal strips, none had directly measured the effect of the strips on nasal resistance using the airflow perturbation device (APD). The APD is an inexpensive instrument that can measure respiratory resistance based on changes in mouth pressure and rate of airflow. METHOD: This study tested forty-seven volunteers (14 men and 33 women), ranging in age from 17 to 51. Each volunteer was instructed to breathe normally into the APD using an oronasal mask with and without nasal strips. The APD measured respiratory resistance during inhalation, exhalation, and an average of the two. RESULTS: Results of a paired mean t-test comparing nasal strip against no nasal strip were statistically significant at the p = 0.05 level. The Breathe Right nasal dilator strips lowered nasal resistance by an average of 0.5 cm H20/Lps from an average nasal resistance of 5.5 cm H20/Lps. CONCLUSIONS: Nasal strips reduce nasal resistance when measured with the APD. The effect is equal during exhalation and during inhalation. [Abstract/Link to Full Text]

Mainardi LT, Corino VD, Lombardi L, Tondo C, Mantica M, Lombardi F, Cerutti S
Assessment of the dynamics of atrial signals and local atrial period series during atrial fibrillation: effects of isoproterenol administration.
Biomed Eng Online. 2004 Oct 22;3(1):37.
BACKGROUND: The autonomic nervous system (ANS) plays an important role in the genesis and maintenance of atrial fibrillation (AF), but quantification of its electrophysiologic effects is extremely complex and difficult. Aim of the study was to evaluate the capability of linear and non-linear indexes to capture the fine changing dynamics of atrial signals and local atrial period (LAP) series during adrenergic activation induced by isoproterenol (a sympathomimetic drug) infusion. METHODS: Nine patients with paroxysmal or persistent AF (aged 60 +/- 6) underwent electrophysiological study in which isoproterenol was administered to patients. Atrial electrograms were acquired during i) sinus rhythm (SR); ii) sinus rhythm during isoproterenol (SRISO) administration; iii) atrial fibrillation (AF) and iv) atrial fibrillation during isoproterenol (AFISO) administration. The level of organization between two electrograms was assessed by the synchronization index (S), whereas the degree of recurrence of a pattern in a signal was defined by the regularity index (R). In addition, the level of predictability (LP) and regularity of LAP series were computed. RESULTS: LAP series analysis shows a reduction of both LP and R index during isoproterenol infusion in SR and AF (RSR = 0.75 +/- 0.07 RSRISO = 0.69 +/- 0.10, p < 0.0001; RAF = 0.31 +/- 0.08 RAFISO = 0.26 +/- 0.09, p < 0.0001; LPSR = 99.99 +/- 0.001 LPSRISO = 99.97 +/- 0.03, p < 0.0001; LPAF = 69.46 +/- 21.55 LPAFISO = 55 +/- 24.75; p < 0.0001). Electrograms analysis shows R index reductions both in SR (RSR = 0.49 +/- 0.08 RSRISO = 0.46 +/- 0.09 p < 0.0001) and in AF (RAF = 0.29 +/- 0.09 RAFISO = 0.28 +/- 0.08 n.s.). CONCLUSIONS: The proposed parameters succeeded in discriminating the subtle changes due to isoproterenol infusion during both the rhythms especially when considering LAP series analysis. The reduced value of analyzed parameters after isoproterenol administration could reflect an important pro-arrhythmic influence of adrenergic activation on favoring maintenance of AF. [Abstract/Link to Full Text]

Twellmann T, Saalbach A, Gerstung O, Leach MO, Nattkemper TW
Image fusion for dynamic contrast enhanced magnetic resonance imaging.
Biomed Eng Online. 2004 Oct 19;3(1):35.
BACKGROUND: Multivariate imaging techniques such as dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) have been shown to provide valuable information for medical diagnosis. Even though these techniques provide new information, integrating and evaluating the much wider range of information is a challenging task for the human observer. This task may be assisted with the use of image fusion algorithms. METHODS: In this paper, image fusion based on Kernel Principal Component Analysis (KPCA) is proposed for the first time. It is demonstrated that a priori knowledge about the data domain can be easily incorporated into the parametrisation of the KPCA, leading to task-oriented visualisations of the multivariate data. The results of the fusion process are compared with those of the well-known and established standard linear Principal Component Analysis (PCA) by means of temporal sequences of 3D MRI volumes from six patients who took part in a breast cancer screening study. RESULTS: The PCA and KPCA algorithms are able to integrate information from a sequence of MRI volumes into informative gray value or colour images. By incorporating a priori knowledge, the fusion process can be automated and optimised in order to visualise suspicious lesions with high contrast to normal tissue. CONCLUSION: Our machine learning based image fusion approach maps the full signal space of a temporal DCE-MRI sequence to a single meaningful visualisation with good tissue/lesion contrast and thus supports the radiologist during manual image evaluation. [Abstract/Link to Full Text]

Beard BB, Kainz W
Review and standardization of cell phone exposure calculations using the SAM phantom and anatomically correct head models.
Biomed Eng Online. 2004 Oct 13;3(1):34.
We reviewed articles using computational RF dosimetry to compare the Specific Anthropomorphic Mannequin (SAM) to anatomically correct models of the human head. Published conclusions based on such comparisons have varied widely. We looked for reasons that might cause apparently similar comparisons to produce dissimilar results. We also looked at the information needed to adequately compare the results of computational RF dosimetry studies. We concluded studies were not comparable because of differences in definitions, models, and methodology. Therefore we propose a protocol, developed by an IEEE standards group, as an initial step in alleviating this problem. The protocol calls for a benchmark validation study comparing the SAM phantom to two anatomically correct models of the human head. It also establishes common definitions and reporting requirements that will increase the comparability of all computational RF dosimetry studies of the human head. [Abstract/Link to Full Text]

Verhey JF, Nathan NS
Feasibility of rapid and automated importation of 3D echocardiographic left ventricular (LV) geometry into a finite element (FEM) analysis model.
Biomed Eng Online. 2004 Oct 8;3(1):32.
BACKGROUND: Finite element method (FEM) analysis for intraoperative modeling of the left ventricle (LV) is presently not possible. Since 3D structural data of the LV is now obtainable using standard transesophageal echocardiography (TEE) devices intraoperatively, the present study describes a method to transfer this data into a commercially available FEM analysis system: ABAQUS. METHODS: In this prospective study TomTec LV Analysis TEE Software was used for semi-automatic endocardial border detection, reconstruction, and volume-rendering of the clinical 3D echocardiographic data. A newly developed software program MVCP FemCoGen, written in Delphi, reformats the TomTec file structures in five patients for use in ABAQUS and allows visualization of regional deformation of the LV. RESULTS: This study demonstrates that a fully automated importation of 3D TEE data into FEM modeling is feasible and can be efficiently accomplished in the operating room. CONCLUSION: For complete intraoperative 3D LV finite element analysis, three input elements are necessary: 1. time-gaited, reality-based structural information, 2. continuous LV pressure and 3. instantaneous tissue elastance. The first of these elements is now available using the methods presented herein. [Abstract/Link to Full Text]

Yin HM, Sun LZ, Wang G, Yamada T, Wang J, Vannier MW
ImageParser: a tool for finite element generation from three-dimensional medical images.
Biomed Eng Online. 2004 Oct 1;331.
BACKGROUND: The finite element method (FEM) is a powerful mathematical tool to simulate and visualize the mechanical deformation of tissues and organs during medical examinations or interventions. It is yet a challenge to build up an FEM mesh directly from a volumetric image partially because the regions (or structures) of interest (ROIs) may be irregular and fuzzy. METHODS: A software package, ImageParser, is developed to generate an FEM mesh from 3-D tomographic medical images. This software uses a semi-automatic method to detect ROIs from the context of image including neighboring tissues and organs, completes segmentation of different tissues, and meshes the organ into elements. RESULTS: The ImageParser is shown to build up an FEM model for simulating the mechanical responses of the breast based on 3-D CT images. The breast is compressed by two plate paddles under an overall displacement as large as 20% of the initial distance between the paddles. The strain and tangential Young's modulus distributions are specified for the biomechanical analysis of breast tissues. CONCLUSION: The ImageParser can successfully exact the geometry of ROIs from a complex medical image and generate the FEM mesh with customer-defined segmentation information. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Biomedicine & Biotechnology

---

Chen X, Murphy RF
Objective clustering of proteins based on subcellular location patterns.
J Biomed Biotechnol. 2005 Jun 30;2005(2):87-95.
The goal of proteomics is the complete characterization of all proteins. Efforts to characterize subcellular location have been limited to assigning proteins to general categories of organelles. We have previously designed numerical features to describe location patterns in microscope images and developed automated classifiers that distinguish major subcellular patterns with high accuracy (including patterns not distinguishable by visual examination). The results suggest the feasibility of automatically determining which proteins share a single location pattern in a given cell type. We describe an automated method that selects the best feature set to describe images for a given collection of proteins and constructs an effective partitioning of the proteins by location. An example for a limited protein set is presented. As additional data become available, this approach can produce for the first time an objective systematics for protein location and provide an important starting point for discovering sequence motifs that determine localization. [Abstract/Link to Full Text]

Bensmail H, Aruna B, Semmes OJ, Haoudi A
Functional clustering algorithm for high-dimensional proteomics data.
J Biomed Biotechnol. 2005 Jun 30;2005(2):80-6.
Clustering proteomics data is a challenging problem for any traditional clustering algorithm. Usually, the number of samples is largely smaller than the number of protein peaks. The use of a clustering algorithm which does not take into consideration the number of features of variables (here the number of peaks) is needed. An innovative hierarchical clustering algorithm may be a good approach. We propose here a new dissimilarity measure for the hierarchical clustering combined with a functional data analysis. We present a specific application of functional data analysis (FDA) to a high-throughput proteomics study. The high performance of the proposed algorithm is compared to two popular dissimilarity measures in the clustering of normal and human T-cell leukemia virus type 1 (HTLV-1)-infected patients samples. [Abstract/Link to Full Text]

Brylinski M, Konieczny L, Czerwonko P, Jurkowski W, Roterman I
Early-stage folding in proteins (in silico) sequence-to-structure relation.
J Biomed Biotechnol. 2005 Jun 30;2005(2):65-79.
A sequence-to-structure library has been created based on the complete PDB database. The tetrapeptide was selected as a unit representing a well-defined structural motif. Seven structural forms were introduced for structure classification. The early-stage folding conformations were used as the objects for structure analysis and classification. The degree of determinability was estimated for the sequence-to-structure and structure-to-sequence relations. Probability calculus and informational entropy were applied for quantitative estimation of the mutual relation between them. The structural motifs representing different forms of loops and bends were found to favor particular sequences in structure-to-sequence analysis. [Abstract/Link to Full Text]

Bensmail H, Haoudi A
Data mining in genomics and proteomics.
J Biomed Biotechnol. 2005 Jun 30;2005(2):63-4. [Abstract/Link to Full Text]

Williams M, Ouhtit A
Towards a Better Understanding of the Molecular Mechanisms Involved in Sunlight-Induced Melanoma.
J Biomed Biotechnol. 2005;2005(1):57-61.
Although much less prevalent than its nonmelanoma skin cancer counterparts, cutaneous malignant melanoma (CMM) is the most lethal human skin cancer. Epidemiological and biological studies have established a strong link between lifetime exposure to ultraviolet (UV) light, particularly sunburn in childhood, and the development of melanoma. However, the specific molecular targets of this environmental carcinogen are not known. Data obtained from genetic and molecular studies over the last few years have identified the INK4a/ARF locus as the "gatekeeper" melanoma suppressor, encoding two tumour suppressor proteins in human, p16 $^{\text{INK4a}}$ and p $14^{\text{ARF}}$ . Recent developments in molecular biotechnology and research using laboratory animals have made a significant gene breakthrough identifying the components of the p16 $^{\text{INK4a}}$ /Rb pathway as the principal and rate-limiting targets of UV radiation actions in melanoma formation. This review summarizes the current knowledge of the molecular mechanisms involved in melanoma development and its relationship to sunlight UV radiation. [Abstract/Link to Full Text]

Prakash S, Jones ML
Artificial Cell Therapy: New Strategies for the Therapeutic Delivery of Live Bacteria.
J Biomed Biotechnol. 2005;2005(1):44-56.
There has been rapid growth in research regarding the use of live bacterial cells for therapeutic purposes. The recognition that these cells can be genetically engineered to synthesize products that have therapeutic potential has generated considerable interest and excitement among clinicians and health professionals. It is expected that a wide range of disease modifying substrates such as enzymes, hormones, antibodies, vaccines, and other genetic products will be used successfully and will impact upon health care substantially. However, a major limitation in the use of these bacterial cells is the complexity of delivering them to the correct target tissues. Oral delivery of live cells, lyophilized cells, and immobilized cells has been attempted but with limited success. Primarily, this is because bacterial cells are incapable of surviving passage through the gastrointestinal tract. In many occasions, when given orally, these cells have been found to provoke immunogenic responses that are undesirable. Recent studies show that these problems can be overcome by delivering live bacterial cells, such as genetically engineered cells, using artificial cell microcapsules. This review summarizes recent advances in the therapeutic use of live bacterial cells for therapy, discusses the principles of using artificial cells for the oral delivery of bacterial cells, outlines methods for preparing suitable artificial cells for this purpose, addresses potentials and limitations for their application in therapy, and provides insight for the future direction of this emergent and highly prospective technology. [Abstract/Link to Full Text]

Brault MS, Kurt RA
Impact of Tumor-Derived CCL2 on Macrophage Effector Function.
J Biomed Biotechnol. 2005;2005(1):37-43.
Monocyte chemoattractant protein-1 (MCP-1, CCL2) is produced by many different types of cells. In the current investigation, the effect of tumor-derived CCL2 on macrophages was evaluated to determine the extent to which this chemokine influenced the innate immune response to cancer. To do this, we used the 4T1 murine mammary carcinoma cell line that constitutively expresses CCL2 and generated 4T1 expressing an antisense CCL2 transcript. The antisense-CCL2-expressing 4T1 produced no detectable CCL2. Macrophages from female BALB/c mice were exposed to supernatants from these tumor cells. The results showed that tumor-derived CCL2 was capable of modulating cytokine gene expression but not protein production in resting, activated, and tumor-associated macrophages. In addition, tumor-derived CCL2 did not affect phagocytic activity, nitric oxide production, or cytolytic activity of the macrophages. Overall, these data suggest that tumor-derived CCL2 does not directly influence macrophage-mediated antitumor activity. [Abstract/Link to Full Text]

Lohan J, Culligan K, Ohlendieck K
Deficiency in Cardiac Dystrophin Affects the Abundance of the $\alpha$ -/ $\beta$ -Dystroglycan Complex.
J Biomed Biotechnol. 2005;2005(1):28-36.
Although Duchenne muscular dystrophy is primarily categorised as a skeletal muscle disease, deficiency in the membrane cytoskeletal protein dystrophin also affects the heart. The central transsarcolemmal linker between the actin membrane cytoskeleton and the extracellular matrix is represented by the dystrophin-associated dystroglycans. Chemical cross-linking analysis revealed no significant differences in the dimeric status of the $\alpha$ -/ $\beta$ -dystroglycan subcomplex in the dystrophic mdx heart as compared to normal cardiac tissue. In analogy to skeletal muscle fibres, heart muscle also exhibited a greatly reduced abundance of both dystroglycans in dystrophin-deficient cells. Immunoblotting demonstrated that the degree of reduction in $\alpha$ -dystroglycan is more pronounced in matured mdx skeletal muscle as contrasted to the mdx heart. The fact that the deficiency in dystrophin triggers a similar pathobiochemical response in both types of muscle suggests that the cardiomyopathic complications observed in $x$ -linked muscular dystrophy might be initiated by the loss of the dystrophin-associated surface glycoprotein complex. [Abstract/Link to Full Text]

Sivakumar R, Ravindran G, Muthayya M, Lakshminarayanan S, Velmurughendran CU
Diabetic Retinopathy Analysis.
J Biomed Biotechnol. 2005;2005(1):20-27.
Diabetic retinopathy is one of the common complications of diabetes. Unfortunately, in many cases the patient is not aware of any symptoms until it is too late for effective treatment. Through analysis of evoked potential response of the retina, the optical nerve, and the optical brain center, a way will be paved for early diagnosis of diabetic retinopathy and prognosis during the treatment process. In this paper, we present an artificial-neural-network-based method to classify diabetic retinopathy subjects according to changes in visual evoked potential spectral components and an anatomically realistic computer model of the human eye under normal and retinopathy conditions in a virtual environment using 3D Max Studio and Windows Movie Maker. [Abstract/Link to Full Text]

Xanthopoulos JM, Romano AE, Majumdar SK
Response of Mouse Breast Cancer Cells to Anastrozole, Tamoxifen, and the Combination.
J Biomed Biotechnol. 2005;2005(1):10-19.
The murine breast cancer cells (4T1) grown both in female BALB/c mice and in culture were treated with anastrozole (50 $\mu$ g/mL), tamoxifen citrate (5 $\mu$ g/mL), and the combination of the two drugs in order to determine treatment efficacies, toxic potential, and the mechanism of cell death. The in vivo treatments were evaluated by monitoring tumor growth, development, and life span. The in vitro effects were measured through cell growth kinetics, cell proliferation, mitochondrial membrane potential disruption assay, and light and scanning electron microscopy. All drug treatments extended the mean life span of the 4T1-inoculated tumor-bearing mice; however, only tamoxifen and combination treatments statistically increased the life span when compared to untreated mice. Although the most drug inhibitory effect on cell multiplication was observed in the combination treatment, both anastrozole and tamoxifen individually inhibited cell proliferation significantly at most time periods in this mouse breast cancer cell line. The mitochondrial membrane potential disruption assay demonstrated significant increase in the percent of cells undergoing apoptosis in all treatment groups. However, the combination treatment was the most effective in inducing cell death via apoptosis. Light and scanning electron microscopy of the treated cells revealed characteristics such as rounding, clumping, and shrinkage of the cells as well as formation of cell surface blebbing and apoptotic bodies suggestive of cell death via apoptotic pathway. [Abstract/Link to Full Text]

Nour El-Dien FA, Zayed MA, Mohamed GG, El-Nahas RG
Two Spectrophotometric Assays for Dopamine Derivatives in Pharmaceutical Products and in Biological Samples of Schizophrenic Patients Using Copper Tetramine Complex and Tri-iodide Reagent.
J Biomed Biotechnol. 2005;2005(1):1-9.
Two simple, rapid, and sensitive spectrophotometric methods are proposed for the determination of levodopa (LD). The first method is based on coupling of 4-aminoantipyrine (4-AAP) with one of the dopamine derivatives (LD, CD) to give a new ligand that reacts with copper tetramine complex to give intensely colored chelates. The colored products are quantified spectrophotometrically at 525 and 520 nm for LD and CD, respectively. The optimization of the experimental conditions is described. The method has been used for the determination of $19.7$ - $69.0$ and $18.1$ - $54.3$ $\mu$ g mL ${}^{-1}$ of LD and CD, respectively. The accuracy of the method is achieved by the values of recovery ( $100\pm 0.2$ %) and the precision is supported by the low standard deviation (SD $=0.17$ - $0.59$ ) and relative standard deviation (CV $=0.4$ %- $1.54$ %) values. The second method is based on the formation of ion-pair iodinated inner sphere or outer sphere colored complexes between the LD and triiodide ions at pH 5 and room temperature ( $23\pm 3^\circ$ C). This method has been used for the determination of LD within the concentration range $39.44$ - $78.88$ $\mu$ g mL ${}^{-1}$ with SD $=0.22$ - $0.24$ and recovery percent $=100 \pm 0.3$ %. The sensitivity of the two methods is indicated by Sandell's sensitivity of $0.014$ - $0.019$ g cm ${}^{-2}$ . The results of the two methods are compared with those of the official method. The interference of common drug additives, degradation products, and excipients was also studied. The proposed methods were applied successfully to the determination of the LD-CD synthetic mixture and Levocare drug. The determination of LD in urine of some schizophrenic patients was applied with good precision and accuracy. The reliability of the methods was established by parallel determinations against the official British pharmacopoeia method. [Abstract/Link to Full Text]

Bitsch R, Netzel M, Sonntag S, Strass G, Frank T, Bitsch I
Urinary Excretion of Cyanidin Glucosides and Glucuronides in Healthy Humans After Elderberry Juice Ingestion.
J Biomed Biotechnol. 2004;2004(5):343-345.
In a pilot study with 6 females and 1 male, the metabolism of various cyanidin glucosides after oral administration of elderberry juice was investigated. The anthocyanin metabolites were detected in urinary excretion. After ingestion of a bolus quantity of $3.57$ g total anthocyanins in a $150$ mL elderberry juice concentrate, $0.053$ % of the administered dose was excreted in urine as glucosidically bound cyanidins within the first 5 hours. Only $0.003$ % of the ingested anthocyanin glucosides was excreted as cyanidin glucuronide, suggesting that this conversion step might be of minor importance in urinary excretion. [Abstract/Link to Full Text]

Miguel G, Fontes C, Antunes D, Neves A, Martins D
Anthocyanin Concentration of "Assaria" Pomegranate Fruits During Different Cold Storage Conditions.
J Biomed Biotechnol. 2004;2004(5):338-342.
The concentration of anthocyanins in fruits of "Assaria" pomegranate, a sweet Portuguese cultivar typically grown in Algarve (south Portugal), was monitored during storage under different conditions. The fruits were exposed to cold storage ( $5^{\circ}$ C) after the following treatments: spraying with wax; spraying with $1.5$ % CaCl(2); spraying with wax and $1.5$ % CaCl(2); covering boxes with 25 $\mu$ c thickness low-density polyethylene film. Untreated fruits were used as a control. The anthocyanin levels were quantified by either comparison with an external standard of cyanidin 3-rutinoside (based on the peak area) or individual calculation from the peak areas based on standard curves of each anthocyanin type. The storage time as well as the fruit treatment prior to storage influenced total anthocyanin content. The highest levels were observed at the end of the first month of storage, except for the fruits treated with CaCl(2), where the maximal values were achieved at the end of the second month. The anthocyanin quantification method influenced the final result. When total anthocyanin was calculated as a sum of individual pigments quantified based on standard curves of each anthocyanin type, lower values were obtained. [Abstract/Link to Full Text]

Miguel G, Dandlen S, Antunes D, Neves A, Martins D
The Effect of Two Methods of Pomegranate (Punica granatum L) Juice Extraction on Quality During Storage at $4^\circ$ C.
J Biomed Biotechnol. 2004;2004(5):332-337.
The effect of two extraction methods of pomegranate juice on its quality and stability was evaluated. The first method consisted of separation of the seeds from fruits and centrifugation. The second method consisted of squeezing fruit halves with an electric lemon squeezer. During a period of 72 hours of cold storage at $4^\circ$ C, the juices were evaluated for the presence of sugars, organic acids, and anthocyanins. Delphinidin 3-glucoside was identified to be the major anthocyanin present at the level of 45-69 mg/L. Among the organic acids, oxalic and tartaric acids dominated. The major sugars detected in pomegranate juice were glucose and sucrose. No significant differences in the content of sugars, organic acids, or anthocyanins in juices obtained through application of the two different extraction methods were detected, with the exception of the drastic decrease of cyanidin $3,5$ -diglucoside level in juice obtained by seed centrifugation. The pH did not show differences between treatments. Titrable acidity and the level of sugars expressed as ${}^{\circ}$ Brix decreased after 32 and 15 hours after extraction, respectively, when juice was obtained by centrifuging the seeds. [Abstract/Link to Full Text]

Liu X, Xiao G, Chen W, Xu Y, Wu J
Quantification and Purification of Mulberry Anthocyanins with Macroporous Resins.
J Biomed Biotechnol. 2004;2004(5):326-331.
Total anthocyanins in different cultivars of mulberry were measured and a process for the industrial preparation of mulberry anthocyanins as a natural food colorant was studied. In 31 cultivars of mulberry, the total anthocyanins, calculated as cyanidin 3-glucoside, ranged from $147.68$ to $2725.46$ mg/L juice. Extracting and purifying with macroporous resins was found to be an efficient potential method for the industrial production of mulberry anthocyanins as a food colorant. Of six resins tested, X-5 demonstrated the best adsorbent capability for mulberry anthocyanins (91 mg/mL resin). The adsorption capacity of resins increased with the surface area and the pore radius. Residual mulberry fruit juice after extraction of pigment retained most of its nutrients, except for anthocyanins, and may provide a substrate for further processing. [Abstract/Link to Full Text]

Hou DX, Fujii M, Terahara N, Yoshimoto M
Molecular Mechanisms Behind the Chemopreventive Effects of Anthocyanidins.
J Biomed Biotechnol. 2004;2004(5):321-325.
Anthocyanins are polyphenolic ring-based flavonoids, and are widespread in fruits and vegetables of red-blue color. Epidemiological investigations and animal experiments have indicated that anthocyanins may contribute to cancer chemoprevention. The studies on the mechanism have been done recently at molecular level. This review summarizes current molecular bases for anthocyanidins on several key steps involved in cancer chemoprevention: (i) inhibition of anthocyanidins in cell transformation through targeting mitogen-activated protein kinase (MAPK) pathway and activator protein 1 (AP-1) factor; (ii) suppression of anthocyanidins in inflammation and carcinogenesis through targeting nuclear factor kappa B (NF- $\kappa$ B) pathway and cyclooxygenase 2 (COX-2) gene; (iii) apoptotic induction of cancer cells by anthocyanidins through reactive oxygen species (ROS) / c-Jun NH(2)-terminal kinase (JNK)-mediated caspase activation. These data provide a first molecular view of anthocyanidins contributing to cancer chemoprevention. [Abstract/Link to Full Text]

Gould KS
Nature's Swiss Army Knife: The Diverse Protective Roles of Anthocyanins in Leaves.
J Biomed Biotechnol. 2004;2004(5):314-320.
Anthocyanins, the pigments responsible for spectacular displays of vermilion in the leaves of deciduous trees, have long been considered an extravagant waste of a plant's resources. Contemporary research, in contrast, has begun to show that the pigments can significantly influence the way a leaf responds to environmental stress. Anthocyanins have been implicated in tolerance to stressors as diverse as drought, UV-B, and heavy metals, as well as resistance to herbivores and pathogens. By absorbing high-energy quanta, anthocyanic cell vacuoles both protect chloroplasts from the photoinhibitory and photooxidative effects of strong light, and prevent the catabolism of photolabile defence compounds. Anthocyanins also mitigate photooxidative injury in leaves by efficiently scavenging free radicals and reactive oxygen species. Far from being a useless by-product of the flavonoid pathway, these red pigments may in some instances be critical for plant survival. [Abstract/Link to Full Text]

Lila MA
Anthocyanins and Human Health: An In Vitro Investigative Approach.
J Biomed Biotechnol. 2004;2004(5):306-313.
Anthocyanin pigments and associated flavonoids have demonstrated ability to protect against a myriad of human diseases, yet they have been notoriously difficult to study with regard to human health. Anthocyanins frequently interact with other phytochemicals to potentiate biological effects, thus contributions from individual components are difficult to decipher. The complex, multicomponent structure of compounds in a bioactive mixture and the degradation of flavonoids during harsh extraction procedures obscure the precise assignment of bioactivity to individual pigments. Extensive metabolic breakdown after ingestion complicates tracking of anthocyanins to assess absorption, bioavailability, and accumulation in various organs. Anthocyanin pigments and other flavonoids that are uniformly, predictably produced in rigorously controlled plant cell culture systems can be a great advantage for health and nutrition research because they are quickly, easily isolated, lack interferences found in whole fruits, can be elicited to provoke rapid and prolific accumulation, and are amenable to biolabeling so that metabolic fate can be investigated after ingestion. [Abstract/Link to Full Text]

Mateus N, Oliveira J, Haettich-Motta M, de Freitas V
New Family of Bluish Pyranoanthocyanins.
J Biomed Biotechnol. 2004;2004(5):299-305.
The use of anthocyanins has been investigated for the preparation of food and beverage natural colorants as they seem to have nontoxic effects. In this context, vinylpyranoanthocyanins were recently found to naturally occur in ageing red wine. This new family of anthocyanin-derived pigments may be obtained directly through the reaction between anthocyanin derivatives and other compounds. Some of these newly formed pigments have been found to exhibit a bluish color at acidic pH. The formation of bluish pigment was obtained through reaction between anthocyanin-pyruvic-acid adducts and flavanols in the presence of acetaldehyde. The formation of similar bluish pigments was attempted using other different precursors. The chromatic features of this kind of pigments bring promising expectations concerning the use of these naturally occurring blue pigments in the food industry. [Abstract/Link to Full Text]

Bitsch R, Netzel M, Frank T, Strass G, Bitsch I
Bioavailability and Biokinetics of Anthocyanins From Red Grape Juice and Red Wine.
J Biomed Biotechnol. 2004;2004(5):293-298.
In a comparative study, 9 healthy volunteers ingested a single oral dose of 400 mL red grape juice or red wine with dose-adjusted anthocyanin content ( $283.5$ mg or $279.6$ mg, resp.) in crossover. The content of anthocyanin glucosides was detected in plasma and urinary excretion. Additionally, the plasmatic antioxidant activity was assessed after intake. Based on the plasma content, biokinetic criteria of the single anthocyanins were calculated, such as AUC, $\mathrm{c}_{\mathrm{max}}$ , $\mathrm{t}_{\mathrm{max}}$ , and the elimination rate $\mathrm{t}_{1/2}$ . The urinary excretion of total anthocyanins differed significantly and amounted to $0.18$ % (red wine) and $0.23$ % (red grape juice) of the administered dose. Additionally, the plasmatic antioxidant activity increased to higher levels after juice ingestion compared to wine. The intestinal absorption of the anthocyanins of red grape juice seemed to be improved compared to red wine, suggesting a possible synergistic effect of the glucose content of the juice. The improved absorption resulted in an enhanced plasmatic bioactivity. [Abstract/Link to Full Text]

Konczak I, Okuno S, Yoshimoto M, Yamakawa O
Caffeoylquinic Acids Generated In Vitro in a High-Anthocyanin-Accumulating Sweet potato Cell Line.
J Biomed Biotechnol. 2004;2004(5):287-292.
Accumulation of phenolic compounds has been monitored in a suspension culture of anthocyanin-accumulating sweet potato cell line grown under the conditions of modified Murashige and Skoog high-anthocyanin production medium (APM) over a period of 24 days. Tissue samples extracted with 15% acetic acid were analysed using HPLC at a detection wavelength of 326 nm. Among others, the following derivatives of caffeoylquinic acids were detected: 4,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and 3,4,5-tricaffeoylquinic acid. Their total amount reached a maximum of 110 mg/gFW between the 4th and the 15th day of culture growth on APM. The major compound of the phenolic mixture was 3,5-dicaffeoylquinic acid with maximum accumulation level of 80 mg/100 gFW. The potential effects of targeted phenolic compounds on the nutraceutical qualities of in vitro produced anthocyanin-rich extracts are discussed. [Abstract/Link to Full Text]

Terahara N, Konczak I, Ono H, Yoshimoto M, Yamakawa O
Characterization of Acylated Anthocyanins in Callus Induced From Storage Root of Purple-Fleshed Sweet Potato, Ipomoea batatas L.
J Biomed Biotechnol. 2004;2004(5):279-286.
Four anthocyanins were isolated from a highly pigmented callus induced from the storage root of purple-fleshed sweet potato (Ipomoea batatas L) cultivar Ayamurasaki. The anthocyanins were respectively identified as cyanidin 3- $O$ -(2- $O$ -(6- $O$ -( $E$ )-caffeoyl- $\beta$ -D-glucopyranosyl)- $\beta$ -D-glucopyranoside)-5- $O$ - $\beta$ -D-glucopyranoside, cyanidin 3- $O$ -(2- $O$ -(6- $O$ -( $E$ )- $p$ -coumaroyl- $\beta$ -D-glucopyranosyl)-6- $O$ -( $E$ )-caffeoyl- $\beta$ -D-glucopyranoside)-5- $O$ - $\beta$ -D-glucopyranoside, cyanidin 3- $O$ -(2- $O$ -(6- $O$ -( $E$ )- $p$ -coumaroyl- $\beta$ -D-glucopyranosyl)-6- $O$ -( $E$ )- $p$ -coumaroyl- $\beta$ -D-glucopyranoside)-5- $O$ - $\beta$ -D-glucopyranoside, and peonidin 3- $O$ -(2- $O$ -(6- $O$ -( $E$ )- $p$ -coumaroyl- $\beta$ -D-glucopyranosyl)-6- $O$ -( $E$ )- $p$ -coumaroyl- $\beta$ -D-glucopyranoside)-5- $O$ - $\beta$ -D-glucopyranoside by chemical and spectroscopic analyses. These anthocyanins were examined with respect to the stability in neutral aqueous solution as well as the radical scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. These acylated anthocyanins exhibited both higher stability and higher DPPH radical scavenging activity than corresponding nonacylated cyanidin and peonidin 3- $O$ -sophoroside-5- $O$ -glucosides. [Abstract/Link to Full Text]

Clifton PM
Effect of Grape Seed Extract and Quercetin on Cardiovascular and Endothelial Parameters in High-Risk Subjects.
J Biomed Biotechnol. 2004;2004(5):272-278.
Grape seed extract (GSE) has in vitro antioxidant activity but whether or not it works in vivo is not clear. In a fully randomised, crossover trial with 4-week treatment periods on 36 men and women with above-average vascular risk, we aimed to demonstrate that 2 g/day of GSE (1 g of polyphenols) alone, or with 1 g/day of added quercetin in yoghurt, favourably alters vascular function, endothelial function, and degree of oxidative damage in comparison to a control yoghurt. GSE alone improved flow-mediated dilatation determined ultrasonically by an absolute $1.1$ % compared with control. There was no effect of the combination of GSE with quercetin. No other blood or urine measure was altered. Thus sufficient polyphenols from GSE appear to be absorbed to influence endothelial nitric oxide production, and GSE has the potential to favourably influence vascular function. [Abstract/Link to Full Text]

Zhang W, Franco C, Curtin C, Conn S
To Stretch the Boundary of Secondary Metabolite Production in Plant Cell-Based Bioprocessing: Anthocyanin as a Case Study.
J Biomed Biotechnol. 2004;2004(5):264-271.
Plant cells and tissue cultures hold great promise for controlled production of a myriad of useful secondary metabolites on demand. The current yield and productivity cannot fulfill the commercial goal of a plant cell-based bioprocess for the production of most secondary metabolites. In order to stretch the boundary, recent advances, new directions and opportunities in plant cell-based bioprocessing, have been critically examined for the 10 years from 1992 to 2002. A review of the literature indicated that most of the R&D work was devoted predominantly to studies at an empirical level. A rational approach to molecular plant cell bioprocessing based on the fundamental understanding of metabolic pathways and their regulations is urgently required to stimulate further advances; however, the strategies and technical framework are still being developed. It is the aim of this review to take a step forward in framing workable strategies and technologies for molecular plant cell-based bioprocessing. Using anthocyanin biosynthesis as a case study, an integrated postgenomic approach has been proposed. This combines the functional analysis of metabolic pathways for biosynthesis of a particular metabolite from profiling of gene expression and protein expression to metabolic profiling. A global correlation not only can thus be established at the three molecular levels, but also places emphasis on the interactions between primary metabolism and secondary metabolism; between competing and/or complimentary pathways; and between biosynthetic and post-biosynthetic events. [Abstract/Link to Full Text]

Zhou Y, Singh BR
Effect of Light on Anthocyanin Levels in Submerged, Harvested Cranberry Fruit.
J Biomed Biotechnol. 2004;2004(5):259-263.
Anthocyanins are a group of plant antioxidants known for their therapeutic use. The effects of natural light, red light, and far-red light on individual as well as total anthocyanin content in cranberry fruit (Vaccinium macrocarpon Ait) were examined in an experimental setting designed to mimic water-harvesting conditions. The reversed-phase high performance liquid chromatography (HPLC) method was used to separate and analyze the anthocyanins. In contrast to the case of the control sample that was kept in the dark, natural light increased the total anthocyanin level by $75.3$ % and $87.2$ % after 24 and 48 hours water immersion, respectively. Red light and far-red light increased the total anthocyanin level by $41.5$ % and $34.7$ %, respectively. The amount of each individual anthocyanin increased differently under natural light, red light, and far-red light, suggesting that expressions of enzymes that catalyze the anthocyanin biosynthesis are regulated differently by environments. [Abstract/Link to Full Text]

Blando F, Gerardi C, Nicoletti I
Sour Cherry (Prunus cerasus L) Anthocyanins as Ingredients for Functional Foods.
J Biomed Biotechnol. 2004;2004(5):253-258.
In the recent years many studies on anthocyanins have revealed their strong antioxidant activity and their possible use as chemotherapeutics. The finding that sour cherries (Prunus cerasus L) (also called tart cherries) contain high levels of anthocyanins that possess strong antioxidant and anti-inflammatory properties has attracted much attention to this species. Here we report the preliminary results of the induction of anthocyanin biosynthesis in sour cherry callus cell cultures. The evaluation and characterization of the in vitro produced pigments are compared to those of the anthocyanins found in vivo in fruits of several sour cherry cultivars. Interestingly, the anthocyanin profiles found in whole fruit extracts were similar in all tested genotypes but were different with respect to the callus extract. The evaluation of antioxidant activity, performed by ORAC and TEAC assays, revealed a relatively high antioxidant capacity for the fruit extracts (from 1145 to 2592 $\mu $ mol TE/100 g FW) and a lower one for the callus extract (688 $\mu $ mol TE/100 g FW). [Abstract/Link to Full Text]

Lohachoompol V, Srzednicki G, Craske J
The Change of Total Anthocyanins in Blueberries and Their Antioxidant Effect After Drying and Freezing.
J Biomed Biotechnol. 2004;2004(5):248-252.
This study examined the effects of freezing, storage, and cabinet drying on the anthocyanin content and antioxidant activity of blueberries (Vaccinium corymbosum L). Fresh samples were stored for two weeks at $\5^\circ$ C while frozen samples were kept for up to three months at $-20^\circ$ C. There were two drying treatments, one including osmotic pretreatment followed by cabinet drying and the other involving only cabinet drying. Total anthocyanins found in fresh blueberries were $7.2 \pm 0.5$ mg/g dry matter, expressed as cyanidin 3-rutinoside equivalents. In comparison with fresh samples, total anthocyanins in untreated and pretreated dried blueberries were significantly reduced to $4.3 \pm 0.1$ mg/g solid content, 41% loss, and $3.7 \pm 0.2$ mg/g solid content, 49% loss, respectively. Osmotic treatment followed by a thermal treatment had a greater effect on anthocyanin loss than the thermal treatment alone. In contrast, the frozen samples did not show any significant decrease in anthocyanin level during three months of storage. Measurement of the antioxidant activity of anthocyanin extracts from blueberries showed there was no significant difference between fresh, dried, and frozen blueberries. [Abstract/Link to Full Text]

Nakajima JI, Tanaka I, Seo S, Yamazaki M, Saito K
LC/PDA/ESI-MS Profiling and Radical Scavenging Activity of Anthocyanins in Various Berries.
J Biomed Biotechnol. 2004;2004(5):241-247.
Anthocyanin extracts of two blueberries, Vaccinium myrtillus (bilberry) and Vaccinium ashei (rabbiteye blueberry), and of three other berries, Ribes nigrum (black currant), Aronia melanocarpa (chokeberry), and Sambucus nigra (elderberry), were analyzed by high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization - mass spectrometry (LC/PDA/ESI-MS). Both bilberry and rabbiteye blueberry contained 15 identical anthocyanins with different distribution patterns. Black currant, chokeberry, and elderberry contained 6, 4, and 4 kinds of anthocyanins, respectively. The radical scavenging activities of these berry extracts were analyzed by using 2,2-diphenyl-1-picrylhydrazyl (DPPH). All these extracts showed potent antiradical activities. [Abstract/Link to Full Text]

Konczak I, Zhang W
Anthocyanins-More Than Nature's Colours.
J Biomed Biotechnol. 2004;2004(5):239-240. [Abstract/Link to Full Text]

Tuteja N, Chandra M, Tuteja R, Misra MK
Nitric Oxide as a Unique Bioactive Signaling Messenger in Physiology and Pathophysiology.
J Biomed Biotechnol. 2004;2004(4):227-237.
Nitric oxide (NO) is an intra- and extracellular messenger that mediates diverse signaling pathways in target cells and is known to play an important role in many physiological processes including neuronal signaling, immune response, inflammatory response, modulation of ion channels, phagocytic defense mechanism, penile erection, and cardiovascular homeostasis and its decompensation in atherogenesis. Recent studies have also revealed a role for NO as signaling molecule in plant, as it activates various defense genes and acts as developmental regulator. In plants, NO can also be produced by nitrate reductase. NO can operate through posttranslational modification of proteins (nitrosylation). NO is also a causative agent in various pathophysiological abnormalities. One of the very important systems, the cardiovascular system, is affected by NO production, as this bioactive molecule is involved in the regulation of cardiovascular motor tone, modulation of myocardial contractivity, control of cell proliferation, and inhibition of platelet activation, aggregation, and adhesion. The prime source of NO in the cardiovascular system is endothelial NO synthase, which is tightly regulated with respect to activity and localization. The inhibition of chronic NO synthesis leads to neurogenic and arterial hypertensions, which later contribute to development of myocardial fibrosis. Overall, the modulation of NO synthesis is associated with hypertension. This review briefly describes the physiology of NO, its synthesis, catabolism, and targeting, the mechanism of NO action, and the pharmacological role of NO with special reference to its essential role in hypertension. [Abstract/Link to Full Text]

---

Recent Articles in BMC Biotechnology

---

Herrmann L, Bockau U, Tiedtke A, Hartmann MW, Weide T
The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications.
BMC Biotechnol. 2006;621.
BACKGROUND: Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. RESULTS: Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein. CONCLUSION: This system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications. [Abstract/Link to Full Text]

Hasterok R, Dulawa J, Jenkins G, Leggett M, Langdon T
Multi-substrate chromosome preparations for high throughput comparative FISH.
BMC Biotechnol. 2006;620.
BACKGROUND: A modification of a standard method of fluorescence in situ hybridisation (FISH) is described, by which a combination of several substrates and probes on single microscope slides enables more accurate comparisons of the distribution and abundance of chromosomal sequences and improves the relatively low throughput of standard FISH methods. RESULTS: The utility and application of multi-colour, multi-substrate FISH is illustrated by the simultaneous physical mapping of retrotransposon sequences to three species of Avena, and single locus BAC (bacterial artificial chromosome) clones and rDNA probes to three species of Brachypodium, demonstrating how this would enable better understanding of complex phylogenetic relationships among some of the species belonging to these two genera. CONCLUSION: The results show that use of multi-substrate chromosome preparations significantly increases the utility of FISH in comparative analyses of the distribution and abundance of chromosomal sequences in closely related plant species. [Abstract/Link to Full Text]

Pham Trung N, Fitches E, Gatehouse JA
A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects.
BMC Biotechnol. 2006;618.
BACKGROUND: Despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. Snowdrop lectin (Galanthus nivalis agglutinin; GNA) is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. Recombinant proteins containing GNA expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of GNA-mediated uptake. RESULTS: A gene encoding a toxin, ButaIT, from the red scorpion (Mesobuthus tamulus) was synthesised and assembled into expression constructs. One construct contained ButaIT alone, whereas the other contained ButaIT fused N-terminally to a GNA polypeptide (ButaIT/GNA). Both recombinant proteins were produced using the yeast Pichia pastoris as an expression host, and purified. Recombinant ButaIT and ButaIT/GNA were acutely toxic when injected into larvae of tomato moth (Lacanobia oleracea), causing slow paralysis, leading to mortality or decreased growth. ButaIT/GNA was chronically toxic when fed to L. oleracea larvae, causing decreased survival and weight gain under conditions where GNA alone was effectively non-toxic. Intact ButaIT/GNA was detected in larval haemolymph from insects fed the fusion protein orally, demonstrating transport of the linked polypeptide across the gut. Proteolysis of the fusion protein was also observed. ButaIT/GNA was significantly more toxic that GNA alone when fed to the homopteran Nilaparvata lugens (rice brown planthopper) in liquid artificial diet. CONCLUSION: The ButaIT/GNA recombinant fusion protein is toxic to lepidopteran larvae both when injected and when fed orally, showing the utility of GNA as a carrier to transport potentially toxic peptides and proteins across the insect gut. Although ButaIT has been claimed to be lepidopteran-specific, the fusion protein has more wide-ranging insecticidal activity. Fusion proteins based on plant lectins have potential applications in crop protection, both as exogenously applied treatments and as endogenous products in transgenic plants. [Abstract/Link to Full Text]

Weide T, Herrmann L, Bockau U, Niebur N, Aldag I, Laroy W, Contreras R, Tiedtke A, Hartmann MW
Secretion of functional human enzymes by Tetrahymena thermophila.
BMC Biotechnol. 2006;619.
BACKGROUND: The non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen. RESULTS: We have been able to express a human enzyme in T. thermophila using expression modules that encode a fusion protein consisting of the endogenous phospholipase A1 precursor and mature human DNaseI. The recombinant human enzyme is active, indicating that also disulfide bridges are correctly formed. Furthermore, a detailed N-glycan structure of the recombinant enzyme is presented, illustrating a very consistent glycosylation pattern. CONCLUSION: The ciliate expression system has the potential to become an excellent expression system. However, additional optimisation steps including host strain improvement as wells as measures to increase the yield of expression are necessary to be able to provide an alternative to the common E. coli and yeast-based systems as well as to transformed mammalian cell lines. [Abstract/Link to Full Text]

Tolmachov O, Palaszewski I, Bigger B, Coutelle C
RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA.
BMC Biotechnol. 2006;617.
BACKGROUND: Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. RESULTS: We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3', which was previously shown to be an inefficient partner in Cre-mediated recombination. CONCLUSION: Using transient RecET-driven recombination we inserted a single copy of the araC controlled Cre gene into the lacZ gene on the chromosome of E. coli recA- strain HB101. The resultant recA- minicircle DNA producer strain HB101Cre was used to obtain pure minicircle DNA, consisting of monomeric and multimeric minicircle forms. The obtained recA- minicircle DNA producer strain is expected to decrease the risk of undesired deletions within minicircle producer plasmids and, therefore, to improve production of the therapeutic minicircle vectors. [Abstract/Link to Full Text]

McKeown L, Robinson P, Greenwood SM, Hu W, Jones OT
PIN-G--a novel reporter for imaging and defining the effects of trafficking signals in membrane proteins.
BMC Biotechnol. 2006;615.
BACKGROUND: The identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. Unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. Powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. However, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells. RESULTS: Consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter--pIN-G--consisting of a simple, type I integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein (GFP), cMyc and HA epitope tags. Fluorescence imaging, flow cytometry and biochemical assays of transfected HEK293 cells, confirm the size, topology and surface expression of PIN-G. Moreover, a pIN-G fusion construct, containing a Trans-Golgi Network (TGN) targeting determinant, internalises rapidly from the cell surface and localises to the TGN. Additionally, another PIN-G fusion protein and its mutants reveal trafficking determinants in the cytoplasmic carboxy terminus of Kv1.4 voltage-gated potassium channels. CONCLUSION: Together, these data indicate that pIN-G is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics. [Abstract/Link to Full Text]

Bloquel C, Trollet C, Pradines E, Seguin J, Scherman D, Bureau MF
Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee.
BMC Biotechnol. 2006;616.
BACKGROUND: Optical imaging is an attractive non-invasive way to evaluate the expression of a transferred DNA, mainly thanks to its lower cost and ease of realization. In this study optical imaging was evaluated for monitoring and quantification of the mouse knee joint and tibial cranial muscle electrotransfer of a luciferase encoding plasmid. Optical imaging was applied to study the kinetics of luciferase expression in both tissues. RESULTS: The substrate of luciferase (luciferin) was injected either intraperitonealy (i.p.) or in situ into the muscle or the knee joint. Luminescence resulting from the luciferase-luciferin reaction was measured in vivo with a cooled CCD camera and/or in vitro on tissue lysate. Maximal luminescence of the knee joint and muscle after i.p. (2.5 mg) or local injection of luciferin (50 microg in the knee joint, 100 microg in the muscle) were highly correlated. With the local injection procedure adopted, in vivo and in vitro luminescences measured on the same muscles significantly correlated. Luminescence measurements were reproducible and the signal level was proportional to the amount of plasmid injected. In vivo luciferase activity in the electrotransfered knee joint was detected for two weeks. Intramuscular electrotransfer of 0.3 or 3 microg of plasmid led to stable luciferase expression for 62 days, whereas injecting 30 microg of plasmid resulted in a drop of luminescence three weeks after electrotransfer. These decreases were partially associated with the development of an immune response. CONCLUSION: A particular advantage of the i.p. injection of substrate is a widespread distribution at luciferase production sites. We have also highlighted advantages of local injection as a more sensitive detection method with reduced substrate consumption. Besides, this route of injection is relatively free of uncontrolled parameters, such as diffusion to the target organ, crossing of biological barriers and evidencing variations in local enzymatic kinetics, probably related to the reaction medium in the targeted organ. Optical imaging was shown to be a sensitive and relevant technique to quantify variations of luciferase activity in vivo. Further evaluation of the effective amount of luciferase in a given tissue by in vivo optical imaging relies on conditions of the enzymatic reaction and light absorption and presently requires in vitro calibration for each targeted organ. [Abstract/Link to Full Text]

Langenbach KJ, Elliott JT, Tona A, McDaniel D, Plant AL
Thin films of Type 1 collagen for cell by cell analysis of morphology and tenascin-C promoter activity.
BMC Biotechnol. 2006;614.
BACKGROUND: The use of highly reproducible and spatiallyhomogeneous thin film matrices permits automated microscopy and quantitative determination of the response of hundreds of cells in a population. Using thin films of extracellular matrix proteins, we have quantified, on a cell-by-cell basis, phenotypic parameters of cells on different extracellular matrices. We have quantitatively examined the relationship between fibroblast morphology and activation of the promoter for the extracellular matrix protein tenascin-C using a tenascin-C promoter-based GFP reporter construct. RESULTS: We find that when considering the average response from the population of cells, cell area correlates with tenascin-C promoter activity as has been previously suggested; however cell-by-cell analysis suggests that cell area and promoter activity are not tightly correlated within individual cells. CONCLUSION: This study demonstrates how quantitative cell-by-cell analysis, facilitated by the use of thin films of extracellular matrix proteins, can provide insight into the relationship between phenotypic parameters. [Abstract/Link to Full Text]

Colwill K, Wells CD, Elder K, Goudreault M, Hersi K, Kulkarni S, Hardy WR, Pawson T, Morin GB
Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.
BMC Biotechnol. 2006;613.
BACKGROUND: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. RESULTS: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. CONCLUSION: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics. [Abstract/Link to Full Text]

Cabrita LD, Dai W, Bottomley SP
A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production.
BMC Biotechnol. 2006;612.
BACKGROUND: In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. RESULTS: The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA) and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease) of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. CONCLUSION: The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high-throughput screening. [Abstract/Link to Full Text]

Krom YD, Fallaux FJ, Que I, Lowik C, van Dijk KW
Efficient in vivo knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA.
BMC Biotechnol. 2006;611.
BACKGROUND: Adenovirus (Ad) mediated gene transfer is a well-established tool to transiently express constructs in livers of mice in vivo. In the present study, we determined the specificity and efficiency of Ad vectors expressing short hairpin (sh) RNA constructs to knock-down the estrogen receptor alpha (ERalpha). RESULTS: Two different shRNA constructs derived from the murine ERalpha coding sequence were designed (shERalpha). In vitro, transfection of three mouse cell lines with pSUPER-shERalpha constructs resulted in up to 80% reduction of endogenous ERalpha activity. A single mismatch in the target sequence eliminated the reduction of ERalpha activity, demonstrating the specificity of shERalpha. The subsequently generated Ad.shERalpha vectors were equally effective in vitro. In vivo, intravenous administration of Ad.shERalpha resulted in 70% reduced hepatic mouse ERalpha mRNA levels. Co-injection of Ad.shERalpha with an Ad vector containing a luciferase (luc) gene driven by an estrogen responsive element (ERE) containing promoter resulted in a significant (90% on day five) down-regulation of hepatic luciferase activity, as determined by non-invasive optical imaging. Down-regulation was sustained up to day seven post-injection. CONCLUSION: Ad mediated transfer of shERalpha expression constructs results in efficient and specific knockdown of endogenous ERalpha transcription both in vitro and in vivo. [Abstract/Link to Full Text]

Ungrin MD, Harrington L
Strict control of telomerase activation using Cre-mediated inversion.
BMC Biotechnol. 2006;610.
BACKGROUND: Human cells appear exquisitely sensitive to the levels of hTERT expression, the telomerase reverse transcriptase. In primary cells that do not express hTERT, telomeres erode with each successive cell division, leading to the eventual loss of telomere DNA, an induction of a telomere DNA damage response, and the onset of cellular senescence or crisis. In some instances, an average of less than one appropriately spliced hTERT transcript per cell appears sufficient to restore telomerase activity and telomere maintenance, and overcome finite replicative capacity. RESULTS: To underscore this sensitivity, we showed that a widely used system of transcriptional induction involving ecdysone (muristerone) led to sufficient expression of hTERT to immortalize human fibroblasts, even in the absence of induction. To permit tightly regulated expression of hTERT, or any other gene of interest, we developed a method of transcriptional control using an invertible expression cassette flanked by antiparallel loxP recombination sites. When introduced into human fibroblasts with the hTERT cDNA positioned in the opposite orientation relative to a constitutively active promoter, no telomerase activity was detected, and the cell population retained a mortal phenotype. Upon inversion of the hTERT cDNA to a transcriptionally competent orientation via the action of Cre recombinase, cells acquired telomerase activity, telomere DNA was replenished, and the population was immortalized. Further, using expression of a fluorescent protein marker, we demonstrated the ability to repeatedly invert specific transcripts between an active and inactive state in an otherwise isogenic cell background. CONCLUSION: This binary expression system thus provides a useful genetic means to strictly regulate the expression of a given gene, or to control the expression of at least two different genes in a mutually exclusive manner. [Abstract/Link to Full Text]

Kachel V, Sindelar G, Grimm S
High-throughput isolation of ultra-pure plasmid DNA by a robotic system.
BMC Biotechnol. 2006;69.
BACKGROUND: With the availability of complete genomes, a systematic inventory of cellular processes becomes achievable. This requires assessing the function of all individual genes. Transfection of plasmid DNA into cell culture cells is an essential technique for this aim as it allows functional overexpression or downregulation of genes. While many robotic systems isolate plasmids for sequencing purposes, for more demanding applications such as transfections there is a shortage of robots for the high-throughput isolation of plasmid DNA. RESULTS: Here we describe a custom-made, automated device, which uses a special protocol to isolate plasmid DNAs with a purity sufficient for efficient transfections into mammalian cells. Approximately 1,600 ultra pure plasmids can be isolated in a 96-well plate format within 12 hours. As a unique feature the robot comprises the integration of a centrifuge instead of expensive columns, the use of a custom-made pipetting head with a movable gripper, especially designed shaking platforms and an acetone wash facility. CONCLUSION: Using this robot we demonstrate how centrifugation steps with multiple precipitations, most notably through a precipitation step of SDS in isopropanol, lead to high purity plasmid DNA and make possible high-throughput transfections into mammalian cells for functional gene annotations. [Abstract/Link to Full Text]

Ioannou Y, Giles I, Lambrianides A, Richardson C, Pearl LH, Latchman DS, Isenberg DA, Rahman A
A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli.
BMC Biotechnol. 2006;68.
BACKGROUND: The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (beta2GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. METHODS: Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR) to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b) and expressed in BL21(DE3) Escherichia coli (E. coli). By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. RESULTS: Purified, soluble his-tagged DI in yields of 750 microg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of beta2GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. CONCLUSION: By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the eukaryotic protein DI of beta2GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production. [Abstract/Link to Full Text]

Taxman DJ, Livingstone LR, Zhang J, Conti BJ, Iocca HA, Williams KL, Lich JD, Ting JP, Reed W
Criteria for effective design, construction, and gene knockdown by shRNA vectors.
BMC Biotechnol. 2006;67.
BACKGROUND: RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized. RESULTS: To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings. CONCLUSION: The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors. [Abstract/Link to Full Text]

McLaggan D, Adjimatera N, Sepci? K, Jaspars M, MacEwan DJ, Blagbrough IS, Scott RH
Pore forming polyalkylpyridinium salts from marine sponges versus synthetic lipofection systems: distinct tools for intracellular delivery of cDNA and siRNA.
BMC Biotechnol. 2006;66.
BACKGROUND: Haplosclerid marine sponges produce pore forming polyalkylpyridinium salts (poly-APS), which can be used to deliver macromolecules into cells. The aim of this study was to investigate the delivery of DNA, siRNA and lucifer yellow into cells mediated by poly-APS and its potential mechanisms as compared with other lipofection systems (lipofectamine and N4,N9-dioleoylspermine (LipoGen)). DNA condensation was evaluated and HEK 293 and HtTA HeLa cells were used to investigate pore formation and intracellular delivery of cDNA, siRNA and lucifer yellow. RESULTS: Poly-APS and LipoGen were both found to be highly efficient DNA condensing agents. Fura-2 calcium imaging was used to measure calcium transients indicative of cell membrane pore forming activity. Calcium transients were evoked by poly-APS but not LipoGen and lipofectamine. The increases in intracellular calcium produced by poly-APS showed temperature sensitivity with greater responses being observed at 12 degrees C compared to 21 degrees C. Similarly, delivery of lucifer yellow into cells with poly-APS was enhanced at lower temperatures. Transfection with cDNA encoding for the expression enhanced green fluorescent protein was also evaluated at 12 degrees C with poly-APS, lipofectamine and LipoGen. Intracellular delivery of siRNA was achieved with knockdown in beta-actin expression when lipofectamine and LipoGen were used as transfection reagents. However, intracellular delivery of siRNA was not achieved with poly-APS. CONCLUSION: Poly-APS mediated pore formation is critical to its activity as a transfection reagent, but lipofection systems utilise distinct mechanisms to enable delivery of DNA and siRNA into cells. [Abstract/Link to Full Text]

Gendron D, Carriero S, Garneau D, Villemaire J, Klinck R, Elela SA, Damha MJ, Chabot B
Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals.
BMC Biotechnol. 2006;65.
BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. RESULTS: We show that an oligonucleotide with a 5' tail containing the human beta-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. CONCLUSION: Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology. [Abstract/Link to Full Text]

Martin P, Albagli O, Poggi MC, Boulukos KE, Pognonec P
Development of a new bicistronic retroviral vector with strong IRES activity.
BMC Biotechnol. 2006;64.
BACKGROUND: Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies. RESULTS: We observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function. CONCLUSION: From these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP). [Abstract/Link to Full Text]

Mori Y, Hirano T, Notomi T
Sequence specific visual detection of LAMP reactions by addition of cationic polymers.
BMC Biotechnol. 2006;63.
BACKGROUND: Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP). RESULTS: Oligo DNA probes labeled with different fluorescent dyes were prepared for multiple nucleic acid templates, and the templates were amplified by the LAMP reactions under the existence of the probes. At completion of the LAMP reaction, an optimal amount of low molecular weight polyethylenimine (PEI) was added, resulting in the precipitation of the insoluble LAMP amplicon-PEI complex. The fluorescently labeled Oligo DNA probes hybridized to the LAMP product were incorporated into the precipitation, and the precipitate emitted fluorescence corresponding to the amplified nucleic acid templates. The color of emitted fluorescence can be detected easily by naked eye on a conventional UV illuminator. CONCLUSION: The presence or absence of minute amount of nucleic acid templates could be detected in a simple manner through visual assessment for the color of the LAMP amplicon-PEI complex precipitate. We conclude that this detection method may facilitate development of small and simple g-POCT device. [Abstract/Link to Full Text]

Mota RM, Moreira JL, Souza MR, Horta MF, Teixeira SM, Neumann E, Nicoli JR, Nunes AC
Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines.
BMC Biotechnol. 2006;62.
BACKGROUND: The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. RESULTS: Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. CONCLUSION: Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases. [Abstract/Link to Full Text]

McIntyre GJ, Fanning GC
Design and cloning strategies for constructing shRNA expression vectors.
BMC Biotechnol. 2006;61.
BACKGROUND: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). RESULTS: We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20-40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. CONCLUSION: In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process. [Abstract/Link to Full Text]

Saini D, Kala M, Jain V, Sinha S
Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor.
BMC Biotechnol. 2005;533.
BACKGROUND: The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP) is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP). The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. RESULTS: Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP) and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP. CONCLUSION: The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations. [Abstract/Link to Full Text]

Rotino GL, Acciarri N, Sabatini E, Mennella G, Lo Scalzo R, Maestrelli A, Molesini B, Pandolfini T, Scalzo J, Mezzetti B, Spena A
Open field trial of genetically modified parthenocarpic tomato: seedlessness and fruit quality.
BMC Biotechnol. 2005;532.
BACKGROUND: Parthenocarpic tomato lines transgenic for the DefH9-RI-iaaM gene have been cultivated under open field conditions to address some aspects of the equivalence of genetically modified (GM) fruit in comparison to controls (non-GM). RESULTS: Under open field cultivation conditions, two tomato lines (UC 82) transgenic for the DefH9-RI-iaaM gene produced parthenocarpic fruits. DefH9-RI-iaaM fruits were either seedless or contained very few seeds. GM fruit quality, with the exception of a higher beta-carotene level, did not show any difference, neither technological (colour, firmness, dry matter, degrees Brix, pH) nor chemical (titratable acidity, organic acids, lycopene, tomatine, total polyphenols and antioxidant capacity - TEAC), when compared to that of fruits from control line. Highly significant differences in quality traits exist between the tomato F1 commercial hybrid Allflesh and the three UC 82 genotypes tested, regardless of whether or not they are GM. Total yield per plant did not differ between GM and parental line UC 82. Fruit number was increased in GM lines, and GM fruit weight was decreased. CONCLUSION: The use in the diet of fruits from a new line or variety introduces much greater changes than the consumption of GM fruits in comparison to its genetic background. Parthenocarpic fruits, produced under open field conditions, contained 10-fold less seeds than control fruits. Thus parthenocarpy caused by DefH9-RI-iaaM gene represents also a tool for mitigating GM seeds dispersal in the environment. [Abstract/Link to Full Text]

Burns MJ, Nixon GJ, Foy CA, Harris N
Standardisation of data from real-time quantitative PCR methods - evaluation of outliers and comparison of calibration curves.
BMC Biotechnol. 2005;531.
BACKGROUND: As real-time quantitative PCR (RT-QPCR) is increasingly being relied upon for the enforcement of legislation and regulations dependent upon the trace detection of DNA, focus has increased on the quality issues related to the technique. Recent work has focused on the identification of factors that contribute towards significant measurement uncertainty in the real-time quantitative PCR technique, through investigation of the experimental design and operating procedure. However, measurement uncertainty contributions made during the data analysis procedure have not been studied in detail. This paper presents two additional approaches for standardising data analysis through the novel application of statistical methods to RT-QPCR, in order to minimise potential uncertainty in results. RESULTS: Experimental data was generated in order to develop the two aspects of data handling and analysis that can contribute towards measurement uncertainty in results. This paper describes preliminary aspects in standardising data through the application of statistical techniques to the area of RT-QPCR. The first aspect concerns the statistical identification and subsequent handling of outlying values arising from RT-QPCR, and discusses the implementation of ISO guidelines in relation to acceptance or rejection of outlying values. The second aspect relates to the development of an objective statistical test for the comparison of calibration curves. CONCLUSION: The preliminary statistical tests for outlying values and comparisons between calibration curves can be applied using basic functions found in standard spreadsheet software. These two aspects emphasise that the comparability of results arising from RT-QPCR needs further refinement and development at the data-handling phase. The implementation of standardised approaches to data analysis should further help minimise variation due to subjective judgements. The aspects described in this paper will help contribute towards the development of a set of best practice guidelines regarding standardising handling and interpretation of data arising from RT-QPCR experiments. [Abstract/Link to Full Text]

Schaaf A, Tintelnot S, Baur A, Reski R, Gorr G, Decker EL
Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells.
BMC Biotechnol. 2005;530.
BACKGROUND: Efficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. A common practice is to use the native secretory signal peptide of the heterologous protein to be produced. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition. RESULTS: Aiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss (Physcomitrella patens) proteins. We therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhVEGF and GST, respectively. Our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides. CONCLUSION: From the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle (SRP-cycle) as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. In this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. Though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems. [Abstract/Link to Full Text]

Rambabu KM, Rao SH, Rao NM
Efficient expression of transgenes in adult zebrafish by electroporation.
BMC Biotechnol. 2005;529.
BACKGROUND: Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3-6 month old adult zebrafish. RESULTS: Electroporation parameters such as number of pulses, voltage and amount of plasmid DNA were optimized and it was found that 6 pulses of 40 V.cm(-1) at 15 microg of plasmid DNA per fish increased the luciferase expression 10-fold compared to controls. Similar enhancement in transgene expression was also observed in Indian carp (Labeo rohita). To establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in A2 cells and adult zebrafish after electroporation. The relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. GFP fluorescence in tissues after electroporation was also studied by fluorescence microscopy. CONCLUSION: Electroporation after DNA injection enhances gene expression 10-fold in adult zebrafish. Electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. Enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish. [Abstract/Link to Full Text]

Hytönen VP, Määttä JA, Kidron H, Halling KK, Hörhä J, Kulomaa T, Nyholm TK, Johnson MS, Salminen TA, Kulomaa MS, Airenne TT
Avidin related protein 2 shows unique structural and functional features among the avidin protein family.
BMC Biotechnol. 2005;528.
BACKGROUND: The chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin. RESULTS: In the present study, we have employed multiple biochemical methods to better understand the structure-function relationship of AVR proteins focusing on AVR2. Firstly, we have solved the high-resolution crystal structure of AVR2 in complex with a bound ligand, D-biotin. The AVR2 structure reveals an overall fold similar to the previously determined structures of avidin and AVR4. Major differences are seen, especially at the 1-3 subunit interface, which is stabilized mainly by polar interactions in the case of AVR2 but by hydrophobic interactions in the case of AVR4 and avidin, and in the vicinity of the biotin binding pocket. Secondly, mutagenesis, competitive dissociation analysis and differential scanning calorimetry were used to compare and study the biotin-binding properties as well as the thermal stability of AVRs and avidin. These analyses pinpointed the importance of residue 109 for biotin binding and stability of AVRs. The I109K mutation increased the biotin-binding affinity of AVR2, whereas the K109I mutation decreased the biotin-binding affinity of AVR4. Furthermore, the thermal stability of AVR2(I109K) increased in comparison to the wild-type protein and the K109I mutation led to a decrease in the thermal stability of AVR4. CONCLUSION: Altogether, this study broadens our understanding of the structural features determining the ligand-binding affinities and stability as well as the molecular evolution within the protein family. This novel information can be applied to further develop and improve the tools already widely used in avidin-biotin technology. [Abstract/Link to Full Text]

Cheng EY, Collins C, Berru M, Shulman MJ
A system for precise analysis of transcription-regulating elements of immunoglobulin genes.
BMC Biotechnol. 2005;527.
BACKGROUND: Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state. RESULTS: As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin mu heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer. CONCLUSION: This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci. [Abstract/Link to Full Text]

Trieschmann L, Navarrete Santos A, Kaschig K, Torkler S, Maas E, Schätzl H, Böhm G
Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy.
BMC Biotechnol. 2005;526.
BACKGROUND: The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrPSc) in brain tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods. RESULTS: We developed a highly sensitive method for detecting prion protein aggregates that takes advantage of kinetic differences between seeded and unseeded polymerization of prion protein monomers. Detection of the aggregates was carried out by flow cytometry. In the presence of prion seeds, the association of labelled recombinant PrP monomers in plasma and serum proceeds much more efficiently than in the absence of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down to a concentration of approximately 10(-8) nM [0.24 fg/ml]. A specific signal was detected in six out of six available serum samples from BSE-positive cattle. CONCLUSION: We have developed a method based on seed-dependent PrP fibril formation that shows promising results in differentiating a small number of BSE-positive serum samples from healthy controls. This method may provide the basis for an ante mortem diagnostic test for prion diseases. [Abstract/Link to Full Text]

Dong Y, Friedrich M
Nymphal RNAi: systemic RNAi mediated gene knockdown in juvenile grasshopper.
BMC Biotechnol. 2005;525.
BACKGROUND: Grasshopper serves as important model system in neuroscience, development and evolution. Representatives of this primitive insect group are also highly relevant targets of pest control efforts. Unfortunately, the lack of genetics or gene specific molecular manipulation imposes major limitations to the study of grasshopper biology. RESULTS: We investigated whether juvenile instars of the grasshopper species Schistocerca americana are conducive to gene silencing via the systemic RNAi pathway. Injection of dsRNA corresponding to the eye colour gene vermilion into first instar nymphs triggered suppression of ommochrome formation in the eye lasting through two instars equivalent to 10-14 days in absolute time. QRT-PCR analysis revealed a two fold decrease of target transcript levels in affected animals. Control injections of EGFP dsRNA did not result in detectable phenotypic changes. RT-PCR and in situ hybridization detected ubiquitous expression of the grasshopper homolog of the dsRNA channel protein gene sid-1 in embryos, nymphs and adults. CONCLUSION: Our results demonstrate that systemic dsRNA application elicits specific and long-term gene silencing in juvenile grasshopper instars. The conservation of systemic RNAi in the grasshopper suggests that this pathway can be exploited for gene specific manipulation of juvenile and adult instars in a wide range of primitive insects. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Nanobiotechnology

---

No recent articles are currently available.

---

Recent Articles in BMC Medical Imaging

---

No recent articles are currently available.

---

Recent Articles in Biomagnetic Research and Technology

---

No recent articles are currently available.