free full text journal articles: biotechnology




Recent Articles in Biomedical Engineering Online

Anderson EJ, Knothe Tate ML
Open access to novel dual flow chamber technology for in vitro cell mechanotransduction, toxicity and pharamacokinetic studies.
Biomed Eng Online. 2007 Dec 4;6(1):46.
ABSTRACT: BACKGROUND: A major stumbling block for researchers developing experimental models of mechanotransduction is the control of experimental variables, in particular the transmission of the mechanical forces at the cellular level. A previous evaluation of state of the art commercial perfusion chambers showed that flow regimes, applied to impart a defined mechanical stimulus to cells, are poorly controlled and that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable. METHODS: This study provides a novel chamber design to provide both physiologically-based flow regimes, improvements in control of experimental variables, as well as ease of use compared to commercial chambers. This novel design achieves controlled stresses through five gasket designs and both single- and dual-flow regimes. RESULTS: The imparted shear stress within the gasket geometry is well controlled. In the 8 mm diameter circular area at the center of the chamber (where outcome measures are made), over 92% of the area is exposed to the target stress (+/-2.5%). In addition, other gasket geometries provide spatial gradients of stress that vary with distance from the chamber inlet. Bench-top testing of the novel chamber prototype shows improvements, in the ease of use as well as in performance, compared to the other commercial chambers. The design of the chamber eliminates flow deviations due to leakage and bubbles and allows actual flow profiles to better conform with those predicted in computational models. CONCLUSIONS: The novel flow chamber design provides predictable and well defined mechanical forces at the surface of a cell monolayer, showing improvement over previously tested commercial chambers. The predictability of the imparted stress improves both experiment repeatability as well as the accuracy of inter-study comparisons. Carefully controlling the stresses on cells is critical in effectively mimicking in vivo situations. Overall, the improved perfusion flow chamber provides the needed resolution, standardization and in vitro model analogous to in vivo conditions to make the step towards greater use in research and the opportunity to enter the diagnostic and therapeutic market. [Abstract/Link to Full Text]

Khezri M, Jahed M
Real-time intelligent pattern recognition algorithm for surface EMG signals.
Biomed Eng Online. 2007 Dec 3;6(1):45.
ABSTRACT: BACKGROUND: Electromyography (EMG) is the study of muscle function through the inquiry of electrical signals that the muscles emanate. EMG signals collected from the surface of the skin (Surface Electromyogram: sEMG) can be used in different applications such as recognizing musculoskeletal neural based patterns intercepted for hand prosthesis movements. Current systems designed for controlling the prosthetic hands either have limited functions or can only be used to perform simple movements or use excessive amount of electrodes in order to achieve acceptable results. In an attempt to overcome these problems we have proposed an intelligent system to recognize hand movements and have provided a user assessment routine to evaluate the correctness of executed movements. METHODS: We propose to use an intelligent approach based on adaptive neuro-fuzzy inference system (ANFIS) integrated with a real-time learning scheme to identify hand motion commands. For this purpose and to consider the effect of user evaluation on recognizing hand movements, vision feedback is applied to increase the capability of our system. By using this scheme the user may assess the correctness of the performed hand movement. In this work a hybrid method for training fuzzy system, consisting of back-propagation (BP) and least mean square (LMS) is utilized. Also in order to optimize the number of fuzzy rules, a subtractive clustering algorithm has been developed. To design an effective system, we consider a conventional scheme of EMG pattern recognition system. To design this system we propose to use two different sets of EMG features, namely time domain (TD) and time-frequency representation (TFR). Also in order to decrease the undesirable effects of the dimension of these feature sets, principle component analysis (PCA) is utilized. RESULTS: In this study, the myoelectric signals considered for classification consists of six unique hand movements. Features chosen for EMG signal are time and timefrequency domain. In this work we demonstrate the capability of an EMG pattern recognition system using ANFIS as classifier with a real-time learning method. Our results reveal that the utilized real-time ANFIS approach along with the user evaluation provides a 96.7% average accuracy. This rate is superior to the previously reported result utilizing artificial neural networks (ANN) real-time method [1]. CONCLUSIONS: This study shows that ANFIS real-time learning method coupled with mixed time and time-frequency features as EMG features can provide acceptable results for designing sEMG pattern recognition system suitable for hand prosthesis control. [Abstract/Link to Full Text]

Neofytou MS, Tanos V, Pattichis MS, Pattichis CS, Kyriacou EC, Koutsouris DD
A standardised protocol for texture feature analysis of endoscopic images in gynaecological cancer.
Biomed Eng Online. 2007 Nov 29;6(1):44.
ABSTRACT: BACKGROUND: In the development of tissue classification methods, classifiers rely on significant differences between texture features extracted from normal and abnormal regions. Yet, significant differences can arise due to variations in the image acquisition method. For endoscopic imaging of the endometrium, we propose a standardized image acquisition protocol to eliminate significant statistical differences due to variations in: (i) the distance from the tissue (panoramic vs close up), (ii) difference in viewing angles and (iii) color correction. METHODS: We investigate texture feature variability for a variety of targets encountered in clinical endoscopy. All images were captured at clinically optimum illumination and focus using 720x576 pixels and 24 bits color for: (i) a variety of testing targets from a color palette with a known color distribution, (ii) different viewing angles, (iv) two different distances from a calf endometrial and from a chicken cavity. Also, human images from the endometrium were captured and analysed. For texture feature analysis, three different sets were considered: (i) Statistical Features (SF), (ii) Spatial Gray Level Dependence Matrices (SGLDM), and (iii) Gray Level Difference Statistics (GLDS). All images were gamma corrected and the extracted texture feature values were compared against the texture feature values extracted from the uncorrected images. Statistical tests were applied to compare images from different viewing conditions so as to determine any significant differences. RESULTS: For the proposed acquisition procedure, results indicate that there is no significant difference in texture features between the panoramic and close up views and between angles. For a calibrated target image, gamma correction provided an acquired image that was a significantly better approximation to the original target image. In turn, this implies that the texture features extracted from the corrected images provided for better approximations to the original images. Within the proposed protocol, for human ROIs, we have found that there is a large number of texture features that showed significant differences between normal and abnormal endometrium. CONCLUSIONS: This study provides a standardized protocol for avoiding any significant texture feature differences that may arise due to variability in the acquisition procedure or the lack of color correction. After applying the protocol, we have found that significant differences in texture features will only be due to the fact that the features were extracted from different types of tissue (normal vs abnormal). [Abstract/Link to Full Text]

Baer GM, Small W, Wilson TS, Benett WJ, Matthews DL, Hartman J, Maitland DJ
Fabrication and in vitro deployment of a laser-activated shape memory polymer vascular stent.
Biomed Eng Online. 2007 Nov 27;6(1):43.
ABSTRACT: BACKGROUND: Vascular stents are small tubular scaffolds used in the treatment of arterial stenosis (narrowing of the vessel). Most vascular stents are metallic and are deployed either by balloon expansion or by self-expansion. A shape memory polymer (SMP) stent may enhance flexibility, compliance, and drug elution compared to its current metallic counterparts. The purpose of this study was to describe the fabrication of a laser-activated SMP stent and demonstrate photothermal expansion of the stent in an in vitro artery model. METHODS: A novel SMP stent was fabricated from thermoplastic polyurethane. A solid SMP tube formed by dip coating a stainless steel pin was laser-etched to create the mesh pattern of the finished stent. The stent was crimped over a fiber-optic cylindrical light diffuser coupled to an infrared diode laser. Photothermal actuation of the stent was performed in a water-filled mock artery. RESULTS: At a physiological flow rate, the stent did not fully expand at the maximum laser power (8.6 W) due to convective cooling. However, under zero flow, simulating the technique of endovascular flow occlusion, complete laser actuation was achieved in the mock artery at a laser power of ~8 W. CONCLUSIONS: We have shown the design and fabrication of an SMP stent and a means of light delivery for photothermal actuation. Though further studies are required to optimize the device and assess thermal tissue damage, photothermal actuation of the SMP stent was demonstrated. [Abstract/Link to Full Text]

Mueller HP, Unrath A, Sperfeld AD, Ludolph AC, Riecker A, Kassubek J
Diffusion tensor imaging and tractwise fractional anisotropy statistics: quantitative analysis in white matter pathology.
Biomed Eng Online. 2007 Nov 9;6(1):42.
ABSTRACT: BACKGROUND: Information on anatomical connectivity in the brain by measurements of the diffusion of water in white matter tracts lead to quantification of local tract directionality and integrity. METHODS: The combination of connectivity mapping (fibre tracking, FT) with quantitative diffusion fractional anisotropy (FA) mapping resulted in the approach of results based on group-averaged data, named tractwise FA statistics (TFAS). The task of this study was to apply these methods to group-averaged data from different subjects to quantify differences between normal subjects and subjects with defined alterations of the corpus callosum (CC). RESULTS: TFAS exhibited a significant FA reduction especially in the CC, in agreement with region of interest (ROI)-based analyses. CONCLUSIONS: In summary, the applicability of the TFAS approach to diffusion tensor imaging studies of normal and pathologically altered brains was demonstrated. [Abstract/Link to Full Text]

Sun H, Qu Z, Guo Y, Zeng G, Yang B
In vitro and in vivo effects of rat kidney vascular endothelial cells on osteogenesis of rat bone marrow mesenchymal stem cells growing on polylactide-glycoli acid (PLGA) scaffolds.
Biomed Eng Online. 2007 Nov 4;6(1):41.
ABSTRACT: It is well established that vascularization is critical for osteogenesis. However, dequate vascularization also remains one of the major challenges in tissue engineering of bone. This problem is further accentuated in regeneration of large volume of tissue. Although a complex process, vascularization involves reciprocal regulation and functional interaction between endothelial and osteoblast-like cells during osteogenesis. This prompted us to investigate the possibility of producing bone tissue both in vitro and ectopically in vivo using vascular endothelial cells because we hypothesized that the direct contact or interaction between vascular endothelial cells and bone marrow mesenchymal stem cells are of benefit to osteogenesis in vitro and in vivo. For that purpose we co-cultured rat bone marrow mesenchymal stem cells (MSC) and kidney vascular endothelial cells (VEC) with polylactide-glycolic acid scaffolds. In vitro experiments using alkaline phosphatase and osteocalcin assays demonstrated the proliferation and differentiation of MSC into osteoblast-like cells, especially the direct contact between VEC and MSC. In addition, histochemical analysis with CD31 and von-Willebrand factor staining showed that VEC retained their endothelial characteristics. In vivo implantation of MSC and VEC co-cultures into rat's muscle resulted in pre-vascular network-like structure established by the VEC in the PLGA. These structures developed into vascularized tissue, and increased the amount and size of the new bone compared to the control group (p<0.05). These results suggest that the vascular endothelial cells could efficiently stimulate the in vitro proliferation and differentiation of osteoblast-like cells and promote osteogenesis in vivo by the direct contact or interaction with the MSC. This technique for optimal regeneration of bone should be further investigated. [Abstract/Link to Full Text]

Naito S, Hiroma T, Nakamura T
Continuous negative extrathoracic pressure combined with high-frequency oscillation improves oxygenation with less impact on blood pressure than high-frequency oscillation alone in a rabbit model of surfactant depletion.
Biomed Eng Online. 2007 Oct 31;6(1):40.
ABSTRACT: Backgrounds: Negative air pressure ventilation has been used to maintain adequate functional residual capacity in patients with chronic muscular disease and to decrease transpulmonary pressure and improve cardiac output during right heart surgery. High-frequency oscillation (HFO) exerts beneficial effects on gas exchange in neonates with acute respiratory failure. We examined whether continuous negative extrathoracic pressure (CNEP) combined with HFO would be effective for treating acute respiratory failure in an animal model. METHODS: The effects of CNEP combined with HFO on pulmonary gas exchange and circulation were examined in a surfactant-depleted rabbit model. After induction of severe lung injury by repeated saline lung lavage, 18 adult white Japanese rabbits were randomly assigned to 3 groups: Group 1, CNEP (extra thoracic negative pressure, -10 cmH2O) with HFO (mean airway pressure (MAP), 10 cmH2O); Group 2, HFO (MAP, 10 cmH2O); and Group 3, HFO (MAP, 15 cmH2O). Physiological and blood gas data were compared among groups using analysis of variance. RESULTS: Group 1 showed significantly higher oxygenation than Group 2, and the same oxygenation with significantly higher mean blood pressure compared to Group 3. CONCLUSION: Adequate CNEP combined with HFO improves oxygenation with less impact on blood pressure than high-frequency oscillation alone in an animal model of respiratory failure. [Abstract/Link to Full Text]

Austin TM, Li L, Pullan AJ, Cheng LK
Effects of gastrointestinal tissue structure on computed dipole vectors.
Biomed Eng Online. 2007;639.
ABSTRACT: BACKGROUND: Digestive diseases are difficult to assess without using invasive measurements. Non-invasive measurements of body surface electrical and magnetic activity resulting from underlying gastro-intestinal activity are not widely used, in large due to their difficulty in interpretation. Mathematical modelling of the underlying processes may help provide additional information. When modelling myoelectrical activity, it is common for the electrical field to be represented by equivalent dipole sources. The gastrointestinal system is comprised of alternating layers of smooth muscle (SM) cells and Interstitial Cells of Cajal (ICC). In addition the small intestine has regions of high curvature as the intestine bends back upon itself. To eventually use modelling diagnostically, we must improve our understanding of the effect that intestinal structure has on dipole vector behaviour. METHODS: Normal intestine electrical behaviour was simulated on simple geometries using a monodomain formulation. The myoelectrical fields were then represented by their dipole vectors and an examination on the effect of structure was undertaken. The 3D intestine model was compared to a more computationally efficient 1D representation to determine the differences on the resultant dipole vectors. In addition, the conductivity values and the thickness of the different muscle layers were varied in the 3D model and the effects on the dipole vectors were investigated. RESULTS: The dipole vector orientations were largely affected by the curvature and by a transmural gradient in the electrical wavefront caused by the different properties of the SM and ICC layers. This gradient caused the dipoles to be oriented at an angle to the principal direction of electrical propagation. This angle increased when the ratio of the longitudinal and circular muscle was increased or when the the conductivity along and across the layers was increased. The 1D model was able to represent the geometry of the small intestine and successfully captured the propagation of the slow wave down the length of the mesh, however, it was unable to represent transmural diffusion within each layer, meaning the equivalent dipole sources were missing a lateral component and a reduced magnitude when compared to the full 3D models. CONCLUSION: The structure of the intestinal wall affected the potential gradient through the wall and the orientation and magnitude of the dipole vector. We have seen that the models with a symmetrical wall structure and extreme anisotropic conductivities had similar characteristics in their dipole magnitudes and orientations to the 1D model. If efficient 1D models are used instead of 3D models, then both the differences in magnitude and orientation need to be accounted for. [Abstract/Link to Full Text]

Doyle BJ, Callanan A, McGloughlin TM
A comparison of modelling techniques for computing wall stress in abdominal aortic aneurysms.
Biomed Eng Online. 2007 Oct 19;6(1):38.
ABSTRACT: BACKGROUND: Aneurysms, in particular abdominal aortic aneurysms (AAA), form a significant portion of cardiovascular related deaths. There is much debate as to the most suitable tool for rupture prediction and interventional surgery of AAAs, and currently, maximum diameter is used clinically as the determining factor for surgical intervention. Stress analysis techniques, such as, finite element analysis (FEA) to compute the wall stress in patient-specific AAAs have been regarded by some authors to be more clinically important than the use of a "one-size-fits-all" maximum diameter criterion, since some small AAAs have been shown to have higher wall stress than larger AAAs and have been known to rupture. METHODS: A patient-specific AAA was selected from our AAA database and 3D reconstruction was performed. The AAA was then modelled in this study using three different approaches, namely, AAA(SIMP), AAA(MOD) and AAA(COMP), with each model examined using linear and non-linear material properties. All models were analysed using the finite element method for wall stress distributions. RESULTS: Wall stress results show marked differences in peak wall stress results between the three methods. Peak wall stress was shown to reduce when more realistic parameters were utilised. It was also noted that wall stress reduced by 59% when modelled using the most accurate non-linear complex approach, compared to the same model without intraluminal thrombus. CONCLUSIONS: The results here show that using more realistic parameters affect resulting wall stress. The use of simplified computational modelling methods can lead to inaccurate stress distributions. Care should be taken when examining stress results found using simplified techniques, in particular, if the wall stress results are to have clinical importance. [Abstract/Link to Full Text]

Corovi? S, Pavlin M, Miklavcic D
Analytical and numerical quantification and comparison of the local electric field in the tissue for different electrode configurations.
Biomed Eng Online. 2007;637.
ABSTRACT: BACKGROUND: Electrochemotherapy and gene electrotransfer are novel promising treatments employing locally applied high electric pulses to introduce chemotherapeutic drugs into tumor cells or genes into target cells based on the cell membrane electroporation. The main focus of this paper was to calculate analytically and numerically local electric field distribution inside the treated tissue in two dimensional (2D) models for different plate and needle electrode configurations and to compare the local electric field distribution to parameter U/d, which is widely used in electrochemotherapy and gene electrotransfer studies. We demonstrate the importance of evaluating the local electric field distribution in electrochemotherapy and gene electrotransfer. METHODS: We analytically and numerically analyze electric field distribution based on 2D models for electrodes and electrode configurations which are most widely used in electrochemotherapy and gene electrotransfer. Analytical calculations were performed by solving the Laplace equation and numerical calculations by means of finite element method in two dimensions. RESULTS: We determine the minimal and maximal E inside the target tissue as well as the maximal E over the entire treated tissue for the given electrode configurations. By comparing the local electric field distribution calculated for different electrode configurations to the ratio U/d, we show that the parameter U/d can differ significantly from the actual calculated values of the local electric field inside the treated tissue. By calculating the needed voltage to obtain E > U/d inside the target tissue, we showed that better electric field distribution can be obtained by increasing the number and changing the arrangement of the electrodes. CONCLUSION: Based on our analytical and numerical models of the local electric field distribution we show that the applied voltage, configuration of the electrodes and electrode position need to be chosen specifically for each individual case, and that numerical modeling can be used to optimize the appropriate electrode configuration and adequate voltage. Using numerical models we further calculate the needed voltage for a specific electrode configuration to achieve adequate E inside the target tissue while minimizing damages of the surrounding tissue. We present also analytical solutions, which provide a convenient, rapid, but approximate method for a pre-analysis of electric field distribution in treated tissue. [Abstract/Link to Full Text]

Pavlopoulos S, Thireou T, Kontaxakis G, Santos A
Analysis and interpretation of dynamic FDG PET oncological studies using data reduction techniques.
Biomed Eng Online. 2007 Oct 3;6(1):36.
ABSTRACT: BACKGROUND: Dynamic positron emission tomography studies produce a large amount of image data, from which clinically useful parametric information can be extracted using tracer kinetic methods. Data reduction methods can facilitate the initial interpretation and visual analysis of these large image sequences and at the same time can preserve important information and allow for basic feature characterization. METHODS: We have applied principal component analysis to provide high-contrast parametric image sets of lower dimensions than the original data set separating structures based on their kinetic characteristics. Our method has the potential to constitute an alternative quantification method, independent of any kinetic model, and is particularly useful when the retrieval of the arterial input function is complicated. In independent component analysis images, structures that have different kinetic characteristics are assigned opposite values, and are readily discriminated. Furthermore, novel similarity mapping techniques are proposed, which can summarize in a single image the temporal properties of the entire image sequence according to a reference region. RESULTS: Using our new cubed sum coefficient similarity measure, we have shown that structures with similar time activity curves can be identified, thus facilitating the detection of lesions that are not easily discriminated using the conventional method employing standardized uptake values. [Abstract/Link to Full Text]

Frauenfelder T, Boutsianis E, Schertler T, Husmann L, Leschka S, Poulikakos D, Marincek B, Alkadhi H
Flow and wall shear stress in end-to-side and side-to-side anastomosis of venous coronary artery bypass grafts.
Biomed Eng Online. 2007;635.
PURPOSE: Coronary artery bypass graft (CABG) surgery represents the standard treatment of advanced coronary artery disease. Two major types of anastomosis exist to connect the graft to the coronary artery, i.e., by using an end-to-side or a side-to-side anastomosis. There is still controversy because of the differences in the patency rates of the two types of anastomosis. The purpose of this paper is to non-invasively quantify hemodynamic parameters, such as mass flow and wall shear stress (WSS), in end-to-side and side-to-side anastomoses of patients with CABG using computational fluid dynamics (CFD). METHODS: One patient with saphenous CABG and end-to-side anastomosis and one patient with saphenous CABG and side-to-side anastomosis underwent 16-detector row computed tomography (CT). Geometric models of coronary arteries and bypasses were reconstructed for CFD analysis. Blood flow was considered pulsatile, laminar, incompressible and Newtonian. Peri-anastomotic mass flow and WSS were quantified and flow patterns visualized. RESULTS: CFD analysis based on in-vivo CT coronary angiography data was feasible in both patients. For both types of CABG, flow patterns were characterized by a retrograde flow into the native coronary artery. WSS variations were found in both anastomoses types, with highest WSS values at the heel and lowest WSS values at the floor of the end-to-side anastomosis. In contrast, the highest WSS values of the side-to-side anastomosis configuration were found in stenotic vessel segments and not in the close vicinity of the anastomosis. Flow stagnation zones were found in end-to-side but not in side-to-side anastomosis, the latter also demonstrating a smoother stream division throughout the cardiac cycle. CONCLUSION: CFD analysis of venous CABG based on in-vivo CT datasets in patients was feasible producing qualitative and quantitative information on mass flow and WSS. Differences were found between the two types of anastomosis warranting further systematic application of the presented methodology on multiple patient datasets. [Abstract/Link to Full Text]

Greenwood MH, Sims RC, McLean JE, Doucette WJ
Temperature effect on tert-butyl alcohol (TBA) biodegradation kinetics in hyporheic zone soils.
Biomed Eng Online. 2007 Sep 19;6(1):34.
ABSTRACT: BACKGROUND: Remediation of tert-butyl alcohol (TBA) in subsurface waters should be taken into consideration at reformulated gasoline contaminated sites since it is a biodegradation intermediate of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-butyl formate (TBF).The effect of temperature on TBA biodegradation has not been not been published in the literature. METHODS: Biodegradation of [U 14C] TBA was determined using hyporheic zone soil microcosms. RESULTS: First order mineralization rate constants of TBA at 5oC, 15oC and 25oC were 7.84+/-0.14 x 10-3, 9.07+/-0.09 x 10-3, and 15.3+/-0.3 x 10-3 days-1, respectively (or 2.86+/-0.05, 3.31+/-0.03, 5.60+/-0.14 years-1, respectively). Temperature had a statistically significant effect on the mineralization rates and was modelled using the Arrhenius equation with frequency factor (A) and activation energy (Ea) of 154 day-1 and 23,006 mol/J, respectively. CONCLUSIONS: Results of this study are the first to determine mineralization rates of TBA for different temperatures. The kinetic rates determined in this study can be used in groundwater fate and transport modelling of TBA at the Ronan, MT site and provide an estimate for TBA removal at other similar shallow aquifer sites and hyporheic zones as a function of seasonal change in temperature. [Abstract/Link to Full Text]

Mwale F, Wang HT, Petit A, Girard-Lauriault PL, Hunter CJ, Ouellet JA, Wertheimer MR, Antoniou J
The effect of novel nitrogen-rich plasma polymer coatings on the phenotypic profile of notochordal cells.
Biomed Eng Online. 2007;633.
BACKGROUND: The loss of the notochordal cells from the nucleus pulposus is associated with ageing and disc degeneration. However, understanding the mechanisms responsible for the loss of these cells has been hampered in part due to the difficulty of culturing and maintaining their phenotype. Furthermore, little is known about the influence of the substratum on the molecular markers of notochordal cells. METHODS: Notochordal cells were isolated from lumbar spine of non-chondrodystrophoid dogs and cultured on N-rich plasma polymer layers, so-called "PPE:N" (N-doped plasma-polymerised ethylene, containing up to 36% [N]) surfaces, for 3, 7 or 14 days. Gene expression of vimentin (VIM), pleiotrophin (PTN), matrix Gla protein (MGP), cartilage oligomeric matrix protein (COMP), keratin 18 (KRT 18), aggrecan (AGG), collagen type 1 (COL1A2), collagen type 2 (COL2A1) was analyzed through semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Notochordal cells were maintained in culture on PPE:N for up to 14 days with no loss in cell viability. Except for VIM, gene expression varied depending on the culture periods and [N] concentration of the substratum. Generally, PPE:N surfaces altered gene expression significantly when cells were cultured for 3 or 7 days. CONCLUSION: The present study has shown that notochordal cells from dogs can attach to and grow on PPE:N surfaces. Analysis of the expression of different genes in these cells cultured on different N-functionalized surfaces indicates that cellular behaviour is gene-specific and time-dependent. Further studies are required to better understand the roles of specific surface functionalities on receptor sites, and their effects on cellular phenotypes. [Abstract/Link to Full Text]

Aberg MC, Wessberg J
Evolutionary optimization of classifiers and features for single-trial EEG discrimination.
Biomed Eng Online. 2007;632.
BACKGROUND: State-of-the-art signal processing methods are known to detect information in single-trial event-related EEG data, a crucial aspect in development of real-time applications such as brain computer interfaces. This paper investigates one such novel approach, evaluating how individual classifier and feature subset tailoring affects classification of single-trial EEG finger movements. The discrete wavelet transform was used to extract signal features that were classified using linear regression and non-linear neural network models, which were trained and architecturally optimized with evolutionary algorithms. The input feature subsets were also allowed to evolve, thus performing feature selection in a wrapper fashion. Filter approaches were implemented as well by limiting the degree of optimization. RESULTS: Using only 10 features and 100 patterns, the non-linear wrapper approach achieved the highest validation classification accuracy (subject mean 75%), closely followed by the linear wrapper method (73.5%). The optimal features differed much between subjects, yet some physiologically plausible patterns were observed. CONCLUSION: High degrees of classifier parameter, structure and feature subset tailoring on individual levels substantially increase single-trial EEG classification rates, an important consideration in areas where highly accurate detection rates are essential. Also, the presented method provides insight into the spatial characteristics of finger movement EEG patterns. [Abstract/Link to Full Text]

Tebbe D, Thull R, Gbureck U
Influence of spacer length on heparin coupling efficiency and fibrinogen adsorption of modified titanium surfaces.
Biomed Eng Online. 2007;631.
BACKGROUND: Chemical bonding of the drug onto surfaces by means of spacer molecules is accompanied with a reduction of the biological activity of the drug due to a constricted mobility since normally only short spacer molecule like aminopropyltrimethoxysilane (APMS) are used for drug coupling. This work aimed to study covalent attachment of heparin to titanium(oxide) surfaces by varying the length of the silane coupling agent, which should affect the biological potency of the drug due to a higher mobility with longer spacer chains. METHODS: Covalent attachment of heparin to titanium metal and TiO2 powder was carried out using the coupling agents 3-(Trimethoxysilyl)-propylamine (APMS), N- [3-(Trimethoxysilyl)propyl]ethylenediamine (Diamino-APMS) and N1- [3-(Trimethoxy-silyl)-propyl]diethylenetriamine (Triamino-APMS). The amount of bound coupling agent and heparin was quantified photometrically by the ninhydrin reaction and the tolidine-blue test. The biological potency of heparin was determined photometrically by the chromogenic substrate Chromozym TH and fibrinogen adsorption to the modified surfaces was researched using the QCM-D (Quartz Crystal Microbalance with Dissipation Monitoring) technique. RESULTS: Zeta-potential measurements confirmed the successful coupling reaction; the potential of the unmodified anatase surface (approx. -26 mV) shifted into the positive range (> + 40 mV) after silanisation. Binding of heparin results in a strongly negatively charged surface with zeta-potentials of approx. -39 mV. The retaining biological activity of heparin was highest for the spacer molecule Triamino-APMS. QCM-D measurements showed a lower viscosity for adsorbed fibrinogen films on heparinised surfaces by means of Triamino-APMS. CONCLUSION: The remaining activity of heparin was found to be highest for the covalent attachment with Triamino-APMS as coupling agent due to the long chain of this spacer molecule and therefore the highest mobility of the drug. Furthermore, the adsorption of fibrinogen on the differently heparinised surfaces in real time demonstrated that with longer spacer chains the DeltaD/Deltaf ratios became higher, which is also associated with better biocompatible properties of the substrates in contact with a biosystem. [Abstract/Link to Full Text]

Burdío F, Berjano EJ, Navarro A, Burdío JM, Güemes A, Grande L, Sousa R, Subiró J, Gonzalez A, Cruz I, Castiella T, Tejero E, Lozano R, de Gregorio MA
RF tumor ablation with internally cooled electrodes and saline infusion: what is the optimal location of the saline infusion?
Biomed Eng Online. 2007;630.
BACKGROUND: Radiofrequency ablation (RFA) of tumors by means of internally cooled electrodes (ICE) combined with interstitial infusion of saline may improve clinical results. To date, infusion has been conducted through outlets placed on the surface of the cooled electrode. However, the effect of infusion at a distance from the electrode surface is unknown. Our aim was to assess the effect of perfusion distance (PD) on the coagulation geometry and deposited power during RFA using ICE. METHODS: Experiments were performed on excised bovine livers. Perfusion distance (PD) was defined as the shortest distance between the infusion outlet and the surface of the ICE. We considered three values of PD: 0, 2 and 4 mm. Two sets of experiments were considered: 1) 15 ablations of 10 minutes (n > or = 4 for each PD), in order to evaluate the effect of PD on volume and diameters of coagulation; and 2) 20 additional ablations of 20 minutes. The effect of PD on deposited power and relative frequency of uncontrolled impedance rises (roll-off) was evaluated using the results from the two sets of experiments (n > or = 7 for each PD). Comparisons between PD were performed by analysis of variance or Kruskal-Wallis test. Additionally, non-linear regression models were performed to elucidate the best PD in terms of coagulation volume and diameter, and the occurrence of uncontrolled impedance rises. RESULTS: The best-fit least square functions were always obtained with quadratic curves where volume and diameters of coagulation were maximum for a PD of 2 mm. A thirty per cent increase in volume coagulation was observed for this PD value compared to other values (P < 0.05). Likewise, the short coagulation diameter was nearly twenty five per cent larger for a 2 mm PD than for 0 mm. Regarding deposited power, the best-fit least square function was obtained by a quadratic curve with a 2 mm PD peak. This matched well with the higher relative frequency of uncontrolled impedance rises for PD of 0 and 4 mm. CONCLUSION: Saline perfusion at around 2 mm from the electrode surface while using an ICE in RFA improves deposition of energy and enlarges coagulation volume. [Abstract/Link to Full Text]

Rittgers SE, Oberdier MT, Pottala S
Physiologically-based testing system for the mechanical characterization of prosthetic vein valves.
Biomed Eng Online. 2007;629.
Due to the relatively limited amount of work done to date on developing prosthetic vein (as opposed to cardiac) valves, advances in this topic require progress in three distinct areas: 1) improved device design, 2) relevant device testing conditions, and, 3) appropriate parameters for evaluation of results. It is the purpose of this paper to address two of these issues (#2 and #3) by: 1) performing a study of normal volunteers to quantify the anatomy and hemodynamic features of healthy venous valves, 2) construction of a 2-step, in vitro testing procedure, which simulates both physiologic and postural conditions seen in the lower extremity venous system, and, 3) defining several modified and new parameters which quantify dynamic valve characteristics. [Abstract/Link to Full Text]

Rovati L, Salvatori G, Bulf L, Fonda S
Optical and electrical recording of neural activity evoked by graded contrast visual stimulus.
Biomed Eng Online. 2007;628.
BACKGROUND: Brain activity has been investigated by several methods with different principles, notably optical ones. Each method may offer information on distinct physiological or pathological aspects of brain function. The ideal instrument to measure brain activity should include complementary techniques and integrate the resultant information. As a "low cost" approach towards this objective, we combined the well-grounded electroencephalography technique with the newer near infrared spectroscopy methods to investigate human visual function. METHODS: The article describes an embedded instrumentation combining a continuous-wave near-infrared spectroscopy system and an electroencephalography system to simultaneously monitor functional hemodynamics and electrical activity. Near infrared spectroscopy (NIRS) signal depends on the light absorption spectra of haemoglobin and measures the blood volume and blood oxygenation regulation supporting the neural activity. The NIRS and visual evoked potential (VEP) are concurrently acquired during steady state visual stimulation, at 8 Hz, with a b/w "windmill" pattern, in nine human subjects. The pattern contrast is varied (1%, 10%, 100%) according to a stimulation protocol. RESULTS: In this study, we present the measuring system; the results consist in concurrent recordings of hemodynamic changes and evoked potential responses emerging from different contrast levels of a patterned stimulus.The concentration of [HbO2] increases and [HHb] decreases after the onset of the stimulus. Their variation shows a clear relationship with the contrast value: large contrast produce huge difference in concentration, while low contrast provokes small concentration difference. This behaviour is similar to the already known relationship between VEP response amplitude and contrast. CONCLUSION: The simultaneous recording and analysis of NIRS and VEP signals in humans during visual stimulation with a b/w pattern at variable contrast, demonstrates a strong linear correlation between hemodynamic changes and evoked potential amplitude. Furthermore both responses present a logarithmic profile with stimulus contrast. [Abstract/Link to Full Text]

Yoshioka S, Nagano A, Himeno R, Fukashiro S
Computation of the kinematics and the minimum peak joint moments of sit-to-stand movements.
Biomed Eng Online. 2007;626.
BACKGROUND: A sit-to-stand (STS) movement requires muscle strength higher than that of other daily activities. There are many elderly people, who experience difficulty when standing up from a chair. The muscle strength required (or the load on the joints) during a STS task is determined by the kinematics (movement pattern). The purpose of this study was to evaluate the kinematics and resultant joint moments of people standing up from a chair in order to determine the minimum peak joint moments required for a STS task. METHODS: This study consisted of three steps. In the first step, kinematic data of lower extremity joint angles (hip, knee and ankle) during STS movements were experimentally collected from human subjects. Eighty-five sets of STS kinematic data were obtained. In the second step, the experimentally collected kinematic data and a link segment model of the human body were used to generate more than 5,000,000 computed STS movements. In the third step, using inverse dynamics method, joint moments of the lower extremity were calculated for all movements obtained through the preceding steps. From the outputs of the third step, the optimal kinematics (movement pattern) in terms of minimized peak joint moment for the hip, knee and ankle was determined. RESULTS: The peak hip joint moment ranged from 0.24 to 1.92 N.m/kg. The peak knee joint moment ranged from 0.51 to 1.97 N.m/kg, and the peak ankle joint moment ranged from -0.11 to 1.32 N.m/kg. The optimal movement patterns differed depending on which minimized joint moment index was selected (hip, knee or ankle). However, the sum of the peak hip joint moment and peak knee joint moment was always approximately 1.53 N.m/kg regardless of which minimized joint moment index was selected. CONCLUSION: The most important finding of this study was that the relation between the peak joint moments at the hip and knee joints was complementary and the sum of those moments needed to be greater than 1.53 N.m/kg in order to perform a successful STS. A combined hip-knee value of 1.5 N.m/kg or lower may indicate the need for physical rehabilitation and/or exercise to increase muscular force. [Abstract/Link to Full Text]

Rebersek M, Faurie C, Kanduser M, Corovi? S, Teissié J, Rols MP, Miklavcic D
Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.
Biomed Eng Online. 2007;625.
BACKGROUND: Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses. METHODS: For this aim we used electroporator (EP-GMS 7.1) and developed new electrodes. We used finite-elements method to calculate and evaluate the electric field homogeneity between these new electrodes. Quick practical test was performed on confluent cell culture, to confirm and demonstrate electric field distribution. Then we experimentally evaluated the effectiveness of the new system and protocols on CHO cells. Gene transfection and cell survival were evaluated for different electric field protocols. RESULTS: The results of in-vitro gene electrotransfer experiments show that the fraction of transfected cells increases by changing the electric field direction between electrical pulses. The fluorescence intensity of transfected cells and cell survival does not depend on electric field protocol. Moreover, a new effect a shading effect was observed during our research. Namely, shading effect is observed during gene electrotransfer when cells are in clusters, where only cells facing negative electro-potential in clusters become transfected and other ones which are hidden behind these cells do not become transfected. CONCLUSION: On the basis of our results we can conclude that the new system can be used in in-vitro gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival. [Abstract/Link to Full Text]

Sadegh Zadeh K, Elman HC, Montas HJ, Shirmohammadi A
A finite element model for protein transport in vivo.
Biomed Eng Online. 2007;624.
BACKGROUND: Biological mass transport processes determine the behavior and function of cells, regulate interactions between synthetic agents and recipient targets, and are key elements in the design and use of biosensors. Accurately predicting the outcomes of such processes is crucial to both enhancing our understanding of how these systems function, enabling the design of effective strategies to control their function, and verifying that engineered solutions perform according to plan. METHODS: A Galerkin-based finite element model was developed and implemented to solve a system of two coupled partial differential equations governing biomolecule transport and reaction in live cells. The simulator was coupled, in the framework of an inverse modeling strategy, with an optimization algorithm and an experimental time series, obtained by the Fluorescence Recovery after Photobleaching (FRAP) technique, to estimate biomolecule mass transport and reaction rate parameters. In the inverse algorithm, an adaptive method was implemented to calculate sensitivity matrix. A multi-criteria termination rule was developed to stop the inverse code at the solution. The applicability of the model was illustrated by simulating the mobility and binding of GFP-tagged glucocorticoid receptor in the nucleoplasm of mouse adenocarcinoma. RESULTS: The numerical simulator shows excellent agreement with the analytic solutions and experimental FRAP data. Detailed residual analysis indicates that residuals have zero mean and constant variance and are normally distributed and uncorrelated. Therefore, the necessary and sufficient criteria for least square parameter optimization, which was used in this study, were met. CONCLUSION: The developed strategy is an efficient approach to extract as much physiochemical information from the FRAP protocol as possible. Well-posedness analysis of the inverse problem, however, indicates that the FRAP protocol provides insufficient information for unique simultaneous estimation of diffusion coefficient and binding rate parameters. Care should be exercised in drawing inferences, from FRAP data, regarding concentrations of free and bound proteins, average binding and diffusion times, and protein mobility unless they are confirmed by long-range Markov Chain-Monte Carlo (MCMC) methods and experimental observations. [Abstract/Link to Full Text]

Little MA, McSharry PE, Roberts SJ, Costello DA, Moroz IM
Exploiting nonlinear recurrence and fractal scaling properties for voice disorder detection.
Biomed Eng Online. 2007;623.
BACKGROUND: Voice disorders affect patients profoundly, and acoustic tools can potentially measure voice function objectively. Disordered sustained vowels exhibit wide-ranging phenomena, from nearly periodic to highly complex, aperiodic vibrations, and increased "breathiness". Modelling and surrogate data studies have shown significant nonlinear and non-Gaussian random properties in these sounds. Nonetheless, existing tools are limited to analysing voices displaying near periodicity, and do not account for this inherent biophysical nonlinearity and non-Gaussian randomness, often using linear signal processing methods insensitive to these properties. They do not directly measure the two main biophysical symptoms of disorder: complex nonlinear aperiodicity, and turbulent, aeroacoustic, non-Gaussian randomness. Often these tools cannot be applied to more severe disordered voices, limiting their clinical usefulness. METHODS: This paper introduces two new tools to speech analysis: recurrence and fractal scaling, which overcome the range limitations of existing tools by addressing directly these two symptoms of disorder, together reproducing a "hoarseness" diagram. A simple bootstrapped classifier then uses these two features to distinguish normal from disordered voices. RESULTS: On a large database of subjects with a wide variety of voice disorders, these new techniques can distinguish normal from disordered cases, using quadratic discriminant analysis, to overall correct classification performance of 91.8 +/- 2.0%. The true positive classification performance is 95.4 +/- 3.2%, and the true negative performance is 91.5 +/- 2.3% (95% confidence). This is shown to outperform all combinations of the most popular classical tools. CONCLUSION: Given the very large number of arbitrary parameters and computational complexity of existing techniques, these new techniques are far simpler and yet achieve clinically useful classification performance using only a basic classification technique. They do so by exploiting the inherent nonlinearity and turbulent randomness in disordered voice signals. They are widely applicable to the whole range of disordered voice phenomena by design. These new measures could therefore be used for a variety of practical clinical purposes. [Abstract/Link to Full Text]

Hassani K, Navidbakhsh M, Rostami M
Modeling of the aorta artery aneurysms and renal artery stenosis using cardiovascular electronic system.
Biomed Eng Online. 2007;622.
BACKGROUND: The aortic aneurysm is a dilatation of the aortic wall which occurs in the saccular and fusiform types. The aortic aneurysms can rupture, if left untreated. The renal stenosis occurs when the flow of blood from the arteries leading to the kidneys is constricted by atherosclerotic plaque. This narrowing may lead to the renal failure. Previous works have shown that, modelling is a useful tool for understanding of cardiovascular system functioning and pathophysiology of the system. The present study is concerned with the modelling of aortic aneurysms and renal artery stenosis using the cardiovascular electronic system. METHODS: The geometrical models of the aortic aneurysms and renal artery stenosis, with different rates, were constructed based on the original anatomical data. The pressure drop of each section due to the aneurysms or stenosis was computed by means of computational fluid dynamics method. The compliance of each section with the aneurysms or stenosis is also calculated using the mathematical method. An electrical system representing the cardiovascular circulation was used to study the effects of these pressure drops and the compliance variations on this system. RESULTS: The results showed the decreasing of pressure along the aorta and renal arteries lengths, due to the aneurysms and stenosis, at the peak systole. The mathematical method demonstrated that compliances of the aorta sections and renal increased with the expansion rate of the aneurysms and stenosis. The results of the modelling, such as electrical pressure graphs, exhibited the features of the pathologies such as hypertension and were compared with the relevant experimental data. CONCLUSION: We conclude from the study that the aortic aneurysms as well as renal artery stenosis may be the most important determinant of the arteries rupture and failure. Furthermore, these pathologies play important rules in increase of the cardiovascular pulse pressure which leads to the hypertension. [Abstract/Link to Full Text]

Hooks DA
Myocardial segment-specific model generation for simulating the electrical action of the heart.
Biomed Eng Online. 2007;621.
BACKGROUND: Computer models of the electrical and mechanical actions of the heart, solved on geometrically realistic domains, are becoming an increasingly useful scientific tool. Construction of these models requires detailed measurement of the microstructural features which impact on the function of the heart. Currently a few generic cardiac models are in use for a wide range of simulation problems, and contributions to publicly accessible databases of cardiac structures, on which models can be solved, remain rare. This paper presents to-date the largest database of porcine left ventricular segment microstructural architecture, for use in both electrical and mechanical simulation. METHODS: Cryosectioning techniques were used to reconstruct the myofibre and myosheet orientations in tissue blocks of size ~15 x 15 x 15 mm, taken from the mid-anterior left ventricular freewall, of seven hearts. Tissue sections were gathered on orthogonal planes, and the angles of intersection of myofibres and myosheets with these planes determined automatically with a gradient intensity based algorithm. These angles were then combined to provide a description of myofibre and myosheet variation throughout the tissue, in a form able to be input to biophysically based computational models of the heart. RESULTS: Several microstructural features were common across all hearts. Myofibres rotated through 141 +/- 18 degrees (mean +/- SD) from epicardium to endocardium, in near linear fashion. In the outer two-thirds of the wall sheet angles were predominantly negative, however, in the inner one-third an abrupt change in sheet angle, with reversal in sign, was seen in six of the seven hearts. Two distinct populations of sheets with orthogonal orientations often co-existed, usually with one population dominating. The utility of the tissue structures was demonstrated by simulating the passive and active electrical responses of two of the tissue blocks to current injection. Distinct patterns of electrical response were obtained in the two tissue blocks, illustrating the importance of testing model based predictions on a variety of tissue architectures. CONCLUSION: This study significantly expands the set of geometries on which models of cardiac function can be solved. [Abstract/Link to Full Text]

Nagano A, Komura T, Fukashiro S
Optimal coordination of maximal-effort horizontal and vertical jump motions--a computer simulation study.
Biomed Eng Online. 2007;620.
BACKGROUND: The purpose of this study was to investigate the coordination strategy of maximal-effort horizontal jumping in comparison with vertical jumping, using the methodology of computer simulation. METHODS: A skeletal model that has nine rigid body segments and twenty degrees of freedom was developed. Thirty-two Hill-type lower limb muscles were attached to the model. The excitation-contraction dynamics of the contractile element, the tissues around the joints to limit the joint range of motion, as well as the foot-ground interaction were implemented. Simulations were initiated from an identical standing posture for both motions. Optimal pattern of the activation input signal was searched through numerical optimization. For the horizontal jumping, the goal was to maximize the horizontal distance traveled by the body's center of mass. For the vertical jumping, the goal was to maximize the height reached by the body's center of mass. RESULTS: As a result, it was found that the hip joint was utilized more vigorously in the horizontal jumping than in the vertical jumping. The muscles that have a function of joint flexion such as the m. iliopsoas, m. rectus femoris and m. tibialis anterior were activated to a greater level during the countermovement in the horizontal jumping with an effect of moving the body's center of mass in the forward direction. Muscular work was transferred to the mechanical energy of the body's center of mass more effectively in the horizontal jump, which resulted in a greater energy gain of the body's center of mass throughout the motion. CONCLUSION: These differences in the optimal coordination strategy seem to be caused from the requirement that the body's center of mass needs to be located above the feet in a vertical jumping, whereas this requirement is not so strict in a horizontal jumping. [Abstract/Link to Full Text]

Dunster KR, Davies MW, Fraser JF
The use of chilled condensers for the recovery of perfluorocarbon liquid in an experimental model of perfluorocarbon vapour loss during neonatal partial liquid ventilation.
Biomed Eng Online. 2007;619.
BACKGROUND: Perfluorocarbon (PFC) vapour in the expired gases during partial liquid ventilation should be prevented from entering the atmosphere and recovered for potential reuse.This study aimed to determine how much PFC liquid could be recovered using a conventional humidified neonatal ventilator with chilled condensers in place of the usual expiratory ventilator circuit and whether PFC liquid could be recovered when using the chilled condensers at the ventilator exhaust outlet. METHODS: Using a model lung, perfluorocarbon vapour loss during humidified partial liquid ventilation of a 3.5 kg infant was approximated. For each test 30 mL of FC-77 was infused into the model lung. Condensers were placed in the expiratory limb of the ventilator circuit and the amounts of PFC (FC-77) and water recovered were measured five times. This was repeated with the condensers placed at the ventilator exhaust outlet. RESULTS: When the condensers were used as the expiratory limb, the mean (+/- SD) volume of FC77 recovered was 16.4 mL (+/- 0.18 mL). When the condensers were connected to the ventilator exhaust outlet the mean (+/- SD) volume of FC-77 recovered was 7.6 mL (+/- 1.14 mL). The volume of FC-77 recovered was significantly higher when the condenser was used as an expiratory limb. CONCLUSION: Using two series connected condensers in the ventilator expiratory line 55% of PFC liquid (FC-77) can be recovered during partial liquid ventilation without altering the function of the of the ventilator circuit. This volume of PFC recovered was just over twice that recovered with the condensers connected to the ventilator exhaust outlet. [Abstract/Link to Full Text]

Mabotuwana TD, Cheng LK, Pullan AJ
A model of blood flow in the mesenteric arterial system.
Biomed Eng Online. 2007;617.
BACKGROUND: There are some early clinical indicators of cardiac ischemia, most notably a change in a person's electrocardiogram. Less well understood, but potentially just as dangerous, is ischemia that develops in the gastrointestinal system. Such ischemia is difficult to diagnose without angiography (an invasive and time-consuming procedure) mainly due to the highly unspecific nature of the disease.Understanding how perfusion is affected during ischemic conditions can be a useful clinical tool which can help clinicians during the diagnosis process. As a first step towards this final goal, a computational model of the gastrointestinal system has been developed and used to simulate realistic blood flow during normal conditions. METHODS: An anatomically and biophysically based model of the major mesenteric arteries has been developed to be used to simulate normal blood flows. The computational mesh used for the simulations has been generated using data from the Visible Human project. The 3D Navier-Stokes equations that govern flow within this mesh have been simplified to an efficient 1D scheme. This scheme, together with a constitutive pressure-radius relationship, has been solved numerically for pressure, vessel radius and velocity for the entire mesenteric arterial network. RESULTS: The computational model developed shows close agreement with physiologically realistic geometries other researchers have recorded in vivo. Using this model as a framework, results were analyzed for the four distinct phases of the cardiac cycle--diastole, isovolumic contraction, ejection and isovolumic relaxation. Profiles showing the temporally varying pressure and velocity for a periodic input varying between 10.2 kPa (77 mmHg) and 14.6 kPa (110 mmHg) at the abdominal aorta are presented. An analytical solution has been developed to model blood flow in tapering vessels and when compared with the numerical solution, showed excellent agreement. CONCLUSION: An anatomically and physiologically realistic computational model of the major mesenteric arteries has been developed for the gastrointestinal system. Using this model, blood flow has been simulated which show physiologically realistic flow profiles. [Abstract/Link to Full Text]

Kudriavtsev V, Polyshchuk V, Roy DL
Heart energy signature spectrogram for cardiovascular diagnosis.
Biomed Eng Online. 2007;616.
A new method and application is proposed to characterize intensity and pitch of human heart sounds and murmurs. Using recorded heart sounds from the library of one of the authors, a visual map of heart sound energy was established. Both normal and abnormal heart sound recordings were studied. Representation is based on Wigner-Ville joint time-frequency transformations. The proposed methodology separates acoustic contributions of cardiac events simultaneously in pitch, time and energy. The resolution accuracy is superior to any other existing spectrogram method. The characteristic energy signature of the innocent heart murmur in a child with the S3 sound is presented. It allows clear detection of S1, S2 and S3 sounds, S2 split, systolic murmur, and intensity of these components. The original signal, heart sound power change with time, time-averaged frequency, energy density spectra and instantaneous variations of power and frequency/pitch with time, are presented. These data allow full quantitative characterization of heart sounds and murmurs. High accuracy in both time and pitch resolution is demonstrated. Resulting visual images have self-referencing quality, whereby individual features and their changes become immediately obvious. [Abstract/Link to Full Text]

Dragulescu D, Perdereau V, Drouin M, Ungureanu L, Menyhardt K
3D active workspace of human hand anatomical model.
Biomed Eng Online. 2007;615.
BACKGROUND: If the model of the human hand is created with accuracy by respecting the type of motion provided by each articulation and the dimensions of articulated bones, it can function as the real organ providing the same motions. Unfortunately, the human hand is hard to model due to its kinematical chains submitted to motion constraints. On the other hand, if an application does not impose a fine manipulation it is not necessary to create a model as complex as the human hand is. But always the hand model has to perform a certain space of motions in imposed workspace architecture no matter what the practical application does. METHODS: Based on Denavit-Hartenberg convention, we conceived the kinematical model of the human hand, having in mind the structure and the behavior of the natural model. We obtained the kinematical equations describing the motion of every fingertip with respect to the general coordinate system, placed on the wrist. For every joint variable, a range of motion was established. Dividing these joint variables to an appropriate number of intervals and connecting them, the complex surface bordering the active hand model workspace was obtained. RESULTS: Using MATLAB 7.0, the complex surface described by fingertips, when hand articulations are all simultaneously moving, was obtained. It can be seen that any point on surface has its own coordinates smaller than the maximum length of the middle finger in static position. Therefore, a sphere having the centre in the origin of the general coordinate system and the radius which equals this length covers the represented complex surface. CONCLUSION: We propose a human hand model that represents a new solution compared to the existing ones. This model is capable to make special movements like power grip and dexterous manipulations. During them, the fingertips do not exceed the active workspace encapsulated by determined surfaces. The proposed kinematical model can help to choose which model joints could be eliminated in order to preserve only the motions important for a certain application. The study shows that all models, simplified or not, exhibit a pronounced similitude with the real hand motion, validated by the fingertips' computed trajectories. The results were used to design an artificial hand capable to make some of the hand's functions with a reduced set of degrees of freedom. [Abstract/Link to Full Text]

Recent Articles in Journal of Biomedicine & Biotechnology

Dumonteil E
DNA Vaccines against Protozoan Parasites: Advances and Challenges.
J Biomed Biotechnol. 2007;2007(6):90520.
Over the past 15 years, DNA vaccines have gone from a scientific curiosity to one of the most dynamic research field and may offer new alternatives for the control of parasitic diseases such as leishmaniasis and Chagas disease. We review here some of the advances and challenges for the development of DNA vaccines against these diseases. Many studies have validated the concept of using DNA vaccines for both protection and therapy against these protozoan parasites in a variety of mouse models. The challenge now is to translate what has been achieved in these models into veterinary or human vaccines of comparable efficacy. Also, genome-mining and new antigen discovery strategies may provide new tools for a more rational search of novel vaccine candidates. [Abstract/Link to Full Text]

Santarém N, Silvestre R, Tavares J, Silva M, Cabral S, Maciel J, Cordeiro-da-Silva A
Immune response regulation by leishmania secreted and nonsecreted antigens.
J Biomed Biotechnol. 2007;2007(6):85154.
Leishmania infection consists in two sequential events, the host cell colonization followed by the proliferation/dissemination of the parasite. In this review, we discuss the importance of two distinct sets of molecules, the secreted and/or surface and the nonsecreted antigens. The importance of the immune response against secreted and surface antigens is noted in the establishment of the infection and we dissect the contribution of the nonsecreted antigens in the immunopathology associated with leishmaniasis, showing the importance of these panantigens during the course of the infection. As a further example of proteins belonging to these two different groups, we include several laboratorial observations on Leishmania Sir2 and LicTXNPx as excreted/secreted proteins and LmS3arp and LimTXNPx as nonsecreted/panantigens. The role of these two groups of antigens in the immune response observed during the infection is discussed. [Abstract/Link to Full Text]

Roussoulières A, McGregor B, Chalabreysse L, Cerutti C, Garnier JL, Boissonnat P, Bastien O, Scoazec JY, Thivolet-Bejui F, Sebbag L, L McGregor J
Expression of VE-Cadherin in Peritubular Endothelial Cells during Acute Rejection after Human Renal Transplantation.
J Biomed Biotechnol. 2007;2007(6):41705.
Genes involved in acute rejection (AR) after organ transplantation remain to be further elucidated. In a previous work we have demonstrated the under-expression of VE-Cadherin by endothelial cells (EC) in AR following murine and human heart transplantation. Serial sections from 15 human kidney Banff-graded transplant biopsies were examined for the presence of VE-Cadherin and CD34 staining by immunohistochemistry (no AR (n = 5), AR grade IA (n = 5), or AR grade IIA (n = 5)). Quantification of peritubular EC staining were evaluated and results were expressed by the percentage of stained cells per surface analysed. There was no difference in CD34 staining between the 3 groups. VE-Cadherin expression was significantly reduced in AR Grade IIA when compared to no AR (P = .01) and to AR grade IA (P = .02). This study demonstrates a reduced VE-Cadherin expression by EC in AR after renal transplantation. The down-regulation of VE-Cadherin may strongly participate in human AR. [Abstract/Link to Full Text]

Sioud S, Karray-Rebai I, Aouissaoui H, Aigle B, Bejar S, Mellouli L
Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro) Biosynthetic Pathway in Streptomyces sp. US24 Strain.
J Biomed Biotechnol. 2007;2007(6):91409.
We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro) diketopiperazine (DKP). To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp) was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS). The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro) biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide. [Abstract/Link to Full Text]

Zhao J, Pan R, He J, Liu Y, Li DF, He RQ
Eisenia fetida Protease-III-1 Functions in Both Fibrinolysis and Fibrogenesis.
J Biomed Biotechnol. 2007;2007(5):97654.
The fibrinolytic function of earthworm protease-III-1 (Ef P-III-1) has been studied in recent years. Here, we found that Ef P-III-1 acted not only in fibrinogenolysis, but also in fibrogenesis. We have used Ef P-III-1 to hydrolyze fibrinogen, and to activate plasminogen and prothrombin. Based on the N-terminal sequences of the hydrolytic fragments, Ef P-III-1 was showed to specifically recognize the carboxylic sites of arginine and lysine. Analyses by fibrinogenolysis mapping and amino acid sequencing revealed that the isozyme could cleave the alpha, beta, and gamma chains of fibrinogen, showing a high alpha-fibrinogenase, moderate beta-fibrinogenase, and low gamma-fibrinogenase activities. Interestingly, Ef P-III-1 activated plasminogen and released active plasmin, suggesting a tPA-like function. Furthermore, Ef P-III-1 showed a factor Xa-like function on prothrombin, producing alpha-thrombin. The function in both activating prothrombin and catalyzing fibrinogenolysis suggests that Ef P-III-1 may play a role in the balance between procoagulation and anticoagulation. [Abstract/Link to Full Text]

Cuna WR, Velasquez R, Riva J, Guachalla I, Rodríguez C
Enhancement of a t(h)1 immune response in amphotericin B-treated mucocutaneous leishmaniasis.
J Biomed Biotechnol. 2007;2007(5):96410.
In an attempt to investigate the effects of treatment of human leishmaniasis, the cytokines produced by peripheral blood mononuclear cells (PBMCs) of patients with cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) under treatment with amphotericin B were determined during the active disease from cocultures of cells and Leishmania (Viannia) braziliensis antigens. PBMC of these patients exhibited a nonsignificant marginal increased production of TNF-alpha upon antigen stimulation. However, under the same antigenic stimulus, patients with active MCL presented higher IFN-gamma production compared to patients with CL. This increased IFN-gamma production was accompanied by a drastically augmented IL-12 synthesis from cells of MCL patients. The highlighted T cell responses could be relevant for sound control measures of protozoan infections with emphasis on the combined usage of immunoenhancing agents and antiprotozoal drugs. [Abstract/Link to Full Text]

Ndam NT, Deloron P
Molecular Aspects of Plasmodium falciparum Infection during Pregnancy.
J Biomed Biotechnol. 2007;2007(5):43785.
Cytoadherence of Plasmodium-falciparum-parasitized red blood cells (PRBCs) to host receptors is the key phenomenon in the pathological process of the malaria disease. Some of these interactions can originate poor outcomes responsible for 1 to 3 million annual deaths mostly occurring among children in sub-Saharan Africa. Pregnancy-associated malaria (PAM) represents an important exception of the disease occurring at adulthood in malaria endemic settings. Consequences of this are shared between the mother (maternal anemia) and the baby (low birth weight and infant mortality). Demonstrating that parasites causing PAM express specific variant surface antigens (VSA(PAM)), including the P. falciparum erythrocyte membrane protein 1 (P f EMP1) variant VAR2CSA, that are targets for protective immunity has strengthened the possibility for the development of PAM-specific vaccine. In this paper, we review the molecular basis of malaria pathogenesis attributable to the erythrocyte stages of the parasites, and findings supporting potential anti-PAM vaccine components evidenced in PAM. [Abstract/Link to Full Text]

Ouaissi A
Regulatory cells and immunosuppressive cytokines: parasite-derived factors induce immune polarization.
J Biomed Biotechnol. 2007;2007(4):94971.
Parasitic infections are prevalent in both tropical and subtropical areas. Most of the affected and/or exposed populations are living in developing countries where control measures are lacking or inadequately applied. Although significant progress has been made in our understanding of the immune response to parasites, no definitive step has yet been successfully done in terms of operational vaccines against parasitic diseases. Evidence accumulated during the past few years suggests that the pathology observed during parasitic infections is in part due to deregulation of normal components of the immune system, mainly cytokines, antibodies, and immune effector cell populations. A large number of studies that illustrate how parasites can modify the host immune system for their own benefit have been reported in both metazoan and protozoan parasites. The first line of defense against foreign organisms is barrier tissue such as skin, humoral factors, for instance the complement system and pentraxin, which upon activation of the complement cascade facilitate pathogen recognition by cells of innate immunity such as macrophages and DC. However, all the major groups of parasites studied have been shown to contain and/or to release factors, which interfere with both arms of the host immune system. Even some astonishing observations relate to the production by some parasites of orthologues of mammalian cytokines. Furthermore, chronic parasitic infections have led to the immunosuppressive environment that correlates with increased levels of myeloid and T suppressor cells that may limit the success of immunotherapeutic strategies based on vaccination. This minireview briefly analyzes some of the current data related to the regulatory cells and molecules derived from parasites that affect cellular function and contribute to the polarization of the immune response of the host. Special attention is given to some of the data from our laboratory illustrating the role of immunomodulatory factors released by protozoan parasites, in the induction and perpetuation of chronic disease. [Abstract/Link to Full Text]

El-Abaseri TB, Hansen LA
EGFR Activation and Ultraviolet Light-Induced Skin Carcinogenesis.
J Biomed Biotechnol. 2007;2007(3):97939.
The epidermal growth factor receptor (EGFR) regulates the proliferation of keratinocytes through multiple mechanisms that differ depending on the localization of the cell within the skin. Ultraviolet (UV) irradiation, the main etiologic factor in the development of skin cancer, also activates the receptor. In this review, we discuss how the UV-induced activation of EGFR regulates the response of the skin to UV. UV-induced EGFR activation increases keratinocyte proliferation, suppresses apoptosis, and augments and accelerates epidermal hyperplasia in response to UV. Pharmacological inhibition of the UV-induced activation of EGFR in a genetically initiated mouse skin tumorigenesis model suppresses tumorigenesis and the activation of mitogen-activated protein (MAP) kinases and phosphatidyl inositol-3-kinase (PI3K)/AKT signaling pathways. EGFR has pleiotropic, complex, and cell-type-specific functions in cutaneous keratinocytes; suggesting that the receptor is an appropriate target for the development of molecularly targeted therapies for skin cancer and other pathologies. [Abstract/Link to Full Text]

Roeder I, Braesel K, Lorenz R, Loeffler M
Stem cell fate analysis revisited: interpretation of individual clone dynamics in the light of a new paradigm of stem cell organization.
J Biomed Biotechnol. 2007;2007(3):84656.
Many experimental findings on heterogeneity, flexibility, and plasticity of tissue stem cells are currently challenging stem cell concepts that assume a cell intrinsically predefined, unidirectional differentiation program. In contrast to these classical concepts, nonhierarchical self-organizing systems provide an elegant and comprehensive alternative to explain the experimental data. Here we present the application of such a self-organizing concept to quantitatively describe the hematopoietic stem cell system. Focusing on the analysis of individual-stem-cell fates and clonal dynamics, we particularly discuss implications of the theoretical results on the interpretation of experimental findings. We demonstrate that it is possible to understand hematopoietic stem cell organization without assumptions on unidirectional developmental hierarchies, preprogrammed asymmetric division events or other assumptions implying the existence of a predetermined stem cell entity. The proposed perspective, therefore, changes the general paradigm of thinking about stem cells. [Abstract/Link to Full Text]

Veness MJ
High-risk cutaneous squamous cell carcinoma of the head and neck.
J Biomed Biotechnol. 2007;2007(3):80572.
Nonmelanoma skin cancers (squamous cell and basal cell carcinomas) occur at an epidemic rate in many countries with the worldwide incidence increasing. The sun-exposed head and neck are the most frequent sites for these cancers to arise and in most patients diagnosed with a cutaneous squamous cell carcinoma, local treatment is usually curative. However, a subset is diagnosed with a high-risk cutaneous squamous cell carcinoma. High-risk factors include size (>2 cm), thickness/depth of invasion (>4 mm), recurrent lesions, the presence of perineural invasion, location near the parotid gland, and immunosuppression. These patients have a higher risk (>10-20%) of developing metastases to regional lymph nodes (often parotid nodes), and in some cases also of experiencing local morbidity (perineural invasion), based on unfavourable primary lesion and patient factors. Despite treatment, many patients developing metastatic cutaneous squamous cell carcinoma experience mortality and morbidity usually as a consequence of uncontrolled metastatic nodal disease. It is therefore important that clinicians treating nonmelanoma skin cancers have an understanding and awareness of these high-risk patients. The aim of this article is to discuss the factors that define a high-risk patient and to present some of the issues pertinent to their management. [Abstract/Link to Full Text]

Yoshida H, Miyazaki Y, Wang S, Hamano S
Regulation of Defense Responses against Protozoan Infection by Interleukin-27 and Related Cytokines.
J Biomed Biotechnol. 2007;2007(3):79401.
Cytokine-mediated immunity is crucial in the defense against pathogens. Recently, IL-23 and IL-27 were identified, which along with IL-12 belong to the IL-12 cytokine family. IL-27 is pivotal for the induction of helper T cell (Th) 1 responses while IL-23 is important for the proliferation of memory type Th1 cells. Recent studies revealed that IL-27 also has an anti-inflammatory property. In some protozoan infection, various proinflammatory cytokines were over produced causing lethal inflammatory responses in IL-27 receptor-deficient mice. The anti-inflammatory effect of IL-27 depends, at least partly, on inhibition of the development of Th17 cells, a newly identified Th population that is induced by IL-23 and is characterized by the production of the inflammatogenic cytokine, IL-17. IL-27 thus has a double identity as an initiator and as an attenuator of immune responses and inflammation. With the discoveries of the new IL-12-related cytokines and Th17 cells, Th development is facing a new paradigm. [Abstract/Link to Full Text]

Ouldim K, Natiq A, Jonveaux P, Sefiani A
Tetrasomy 15q11-q13 Diagnosed by FISH in a Patient with Autistic Disorder.
J Biomed Biotechnol. 2007;2007(3):61538.
We report the case of a Moroccan boy with mental retardation, hyperactivity, epilepsy, developmental problems and behavioural disorders. Cytogenetic analysis showed the presence of a supernumerary marker chromosome. Molecular cytogenetics allowed us to determine the marker as an inverted duplication of chromosome 15. It is the first case of a Moroccan patient with tetrasomy 15q in which fluorescence in situ hybridization (FISH) enabled us to specify the diagnosis. Interestingly, this patient has an infantile autism with cytogenetic abnormalities on chromosomal region 15q11-q13 as reported in patients with Autistic Disorder. [Abstract/Link to Full Text]

Chamekh M
CD40-CD40L Interaction in Immunity Against Protozoan Infections.
J Biomed Biotechnol. 2007;2007(3):59430.
Activation of the immune system against protozoan infections relies particularly on two specific signals provided by cognate interaction of T cells with antigen presenting cells (APCs). The first signal is attributed to binding of the T-cell receptor (TCR) to peptide/MHC complexes on the surface of APCs, whereas the second signal is triggered through binding of several costimulatory molecules on the surface of APCs with their corresponding receptors on T cells. Among these costimulatory signallings, CD40/CD40L interactions have been particularly investigated in protozoan infection models with regard to their potential to amplify cell-mediated immunity against intracellular parasites. This article reviews current studies of the potential role of CD40/CD40L interaction in the modulation of immune responses against some protozoan parasites and highlights recent developments regarding manipulation of this interaction for promoting control of parasite infections. [Abstract/Link to Full Text]

Pacifico A, Leone G
Role of p53 and CDKN2A Inactivation in Human Squamous Cell Carcinomas.
J Biomed Biotechnol. 2007;2007(3):43418.
p53 tumor suppressor gene is the most commonly mutated gene in human and mouse cancers. Disruption of the p53 and Rb pathways is a fundamental trend of most human cancer cells. Inactivation of CDKN2A can lead to deregulation of these two pathways. Genetic abnormalities in CDKN2A gene have been well documented in human melanoma but their involvement in human nonmelanoma skin cancer (NMSC) and in particular in squamous cell carcinoma (SCC) is less clear. Several studies have shown that human SCCs harbour unique mutations in the p53 gene as well as inactivation of the CDKN2A gene. While mutations in the p53 gene are induced by UV radiation and represent tumor initiating events, the majority of alterations detected in the CDKN2A gene do not appear to be UV-dependent. In conclusion, in addition to p53 mutations, silencing of the CDKN2A gene might play a significant role in SCC development. [Abstract/Link to Full Text]

Faber A, Roderburg C, Wein F, Saffrich R, Seckinger A, Horsch K, Diehlmann A, Wong D, Bridger G, Eckstein V, Ho AD, Wagner W
The Many Facets of SDF-1alpha, CXCR4 Agonists and Antagonists on Hematopoietic Progenitor Cells.
J Biomed Biotechnol. 2007;2007(3):26065.
Stromal cell-derived factor-1alpha (SDF-1alpha) has pleiotropic effects on hematopoietic progenitor cells (HPCs). We have monitored podia formation, migration, proliferation, and cell-cell adhesion of human HPC under the influence of SDF-1alpha, a peptide agonist of CXCR4 (CTCE-0214), a peptide antagonist (CTCE-9908), and a nonpeptide antagonist (AMD3100). Whereas SDF-1alpha induced migration of CD34(+) cells in a dose-dependent manner, CTCE-0214, CTCE-9908, and AMD3100 did not induce chemotaxis in this concentration range albeit the peptides CTCE-0214 and CTCE-9908 increased podia formation. Cell-cell adhesion of HPC to human mesenchymal stromal cells was impaired by the addition of SDF-1alpha, CTCE-0214, and AMD3100. Proliferation was not affected by SDF-1alpha or its analogs. Surface antigen detection of CXCR4 was reduced upon treatment with SDF-1alpha or AMD3100 and it was enhanced by CTCE-9908. Despite the fact that all these molecules target the same CXCR4 receptor, CXCR4 agonists and antagonists have selective effects on different functions of the natural molecule. [Abstract/Link to Full Text]

Wilkins-Port CE, Higgins CE, Freytag J, Higgins SP, Carlson JA, Higgins PJ
PAI-1 is a Critical Upstream Regulator of the TGF-beta1/EGF-Induced Invasive Phenotype in Mutant p53 Human Cutaneous Squamous Cell Carcinoma.
J Biomed Biotechnol. 2007;2007(2):85208.
The emergence of highly aggressive subtypes of human cutaneous squamous cell carcinoma (SCC) often reflects increased autocrine/paracrine TGF-beta synthesis and epidermal growth factor receptor (EGFR) amplification. Cooperative TGF-beta/EGFR signaling promotes cell migration and induces expression of both proteases and protease inhibitors that regulate stromal remodeling resulting in acquisition of an invasive phenotype. TGF-beta1+EGF stimulation increases the production of several matrix metalloproteinases (MMPs) in human SCC. Among the most prominent is MMP-10 which is known to be elevated in SCC in situ. Activation of stromal plasminogen appears to be critical in triggering downstream MMP activity. Paradoxically, PAI-1, the major physiological inhibitor of plasmin generation, is also up-regulated under these conditions and is an early event in progression of incipient epidermal SCC. A model is proposed in which TGF-beta1+EGF-dependent MMP-10 elevation directs focalized matrix remodeling events that promote epithelial cell plasticity and tissue invasion. Increased PAI-1 expression serves to temporally and spatially modulate plasmin-initiated pericellular proteolysis, further facilitating epithelial invasive potential. Defining the complex signaling mechanisms that maintain this elegant balance is critical to developing potential therapeutics for the treatment of human cutaneous malignancies. [Abstract/Link to Full Text]

Bennouar N, Allami A, Azeddoug H, Bendris A, Laraqui A, El Jaffali A, El Kadiri N, Benzidia R, Benomar A, Fellat S, Benomar M
Thermolabile Methylenetetrahydrofolate Reductase C677T Polymorphism and Homocysteine Are Risk Factors for Coronary Artery Disease in Moroccan Population.
J Biomed Biotechnol. 2007;2007(1):80687.
Increased plasma total homocysteine (tHcy) levels have been shown to be a risk factor for coronary artery disease (CAD). The common methylenetetrahydrofolate reductase C677T (MTHFR C677T) polymorphism has been reported to be a strong predictor of mild hyperhomocysteinaemia (HHcy). We assessed whether this mutation was associated with increased risk of CAD and plasma levels of tHcy. We also evaluated interactions between this polymorphism, mild elevated tHcy levels and conventional risk factors of CAD. Method. Using PCR-RFLP analysis, we studied the frequency of the C677T genotypes and its effect on CAD and on tHcy concentrations in 400 subjects without and with CAD angiographically confirmed. There were 210 subjects with CAD and 190 subjects without CAD. Results. The frequencies of the C677T genotypes were 53% (59.5% in controls versus 48.1% in cases), 34.8% (32.1 in controls versus 37.1 in cases), and 11.8% (8.4% in controls versus 14.8% in cases), respectively, for 677CC, 677CT, and 677TT. The genotype frequencies were significantly different between case and control groups (P < .05). The 677T allele enhances the risk of CAD associated to HHcy (P < .01). In multivariate analysis models, MTHFR C677T polymorphism effect on CAD was masked by other risk factors. HHcy was only and independently influenced by MTHFR polymorphism and smoking habits, and it is a strong predictor of CAD independently of conventional risk factors. Conclusion. Our data suggest that HHcy is strongly and independently associated to CAD risk increase; and MTHFR C677T polymorphism and smoking habits were the main predictors of tHcy levels. The CAD risk increase is mainly associated with mild HHcy in 677TT, whereas in 677CT and 677CC it is mainly associated with the conventional risk factors. [Abstract/Link to Full Text]

Ratajczak C, Duez C, Grangette C, Pochard P, Tonnel AB, Pestel J
Impact of lactic Acid bacteria on dendritic cells from allergic patients in an experimental model of intestinal epithelium.
J Biomed Biotechnol. 2007;2007(1):71921.
Lactic acid bacteria (LAB) are Gram positive nonpathogenic commensal organisms present in human gastrointestinal tract. In vivo, LAB are separated from antigen-presenting cells such as dendritic cells (DC) by the intestinal epithelial barrier. In this study, the impact of one LAB strain (Lactobacillus casei ATCC393) on human monocyte-derived DC from allergic and healthy donors was assessed by using a polarized epithelium model. Confocal and flow cytometer analyses showed that immature DC efficiently captured FITC-labelled L. casei through the epithelial layer. After interaction with L. casei, DC acquired a partial maturation status (i.e., CD86 and CD54 increase) and increased their interleukin (IL)-10 and IL-12 production. Interestingly, after activation by L. casei in the presence of experimental epithelium, DC from allergic patients instructed autologous naïve CD4(+) T cells to produce more interferon-gamma than without the epithelium. Thus by modulating human DC reactivity, LAB and intestinal epithelium might modify T cell immune response and regulate the development of allergic reaction. [Abstract/Link to Full Text]

Ayadi DZ, Chouayekh H, Mhiri S, Zerria K, Fathallah DM, Bejar S
Expression by Streptomyces lividans of the Rat alpha Integrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein.
J Biomed Biotechnol. 2007;2007(1):54327.
We already reported the use of a long synthetic signal peptide (LSSP) to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte beta2 integrin CD11/CD18 alpha M subunit (CD11b). This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the "LSSP" synthetic signal peptide. [Abstract/Link to Full Text]

Joubert O, Voegelin J, Guillet V, Tranier S, Werner S, Colin DA, Serra MD, Keller D, Monteil H, Mourey L, Prévost G
Distinction between Pore Assembly by Staphylococcal alpha-Toxin versus Leukotoxins.
J Biomed Biotechnol. 2007;200725935.
The staphylococcal bipartite leukotoxins and the homoheptameric alpha-toxin belong to the same family of beta-barrel pore-forming toxins despite slight differences. In the alpha-toxin pore, the N-terminal extremity of each protomer interacts as a deployed latch with two consecutive protomers in the vicinity of the pore lumen. N-terminal extremities of leukotoxins as seen in their three-dimensional structures are heterogeneous in length and take part in the beta-sandwich core of soluble monomers. Hence, the interaction of these N-terminal extremities within structures of adjacent monomers is questionable. We show here that modifications of their N-termini by two different processes, using fusion with glutathione S-transferase (GST) and bridging of the N-terminal extremity to the adjacent beta-sheet via disulphide bridges, are not deleterious for biological activity. Therefore, bipartite leukotoxins do not need a large extension of their N-terminal extremities to form functional pores, thus illustrating a microheterogeneity of the structural organizations between bipartite leukotoxins and alpha-toxin. [Abstract/Link to Full Text]

Garin G, Zibara K, Aguilar F, Lo M, Hurlstone A, Poston R, McGregor JL
6A3-5/Osa2 is an Early Activated Gene Implicated in the Control of Vascular Smooth Muscle Cell Functions.
J Biomed Biotechnol. 2006;2006(5):97287.
Vascular smooth muscle cells (VSMC) growth plays a key role in the pathophysiology of vascular diseases. However, the molecular mechanisms controlling gene transcription in VSMC remain poorly understood. We previously identified, by differential display, a new gene (6A3-5) overexpressed in proliferating rat VSMC. In this study, we have cloned the full-length cDNA by screening a rat foetal brain cDNA library and investigated its functions. The 6A3-5 protein shows 4 putative conserved functional motifs: a DNA binding domain called ARID (AT-rich interaction domain), two recently described motifs (Osa Homology Domain), and a nuclear localization signal. The deduced protein sequence was observed to be 85% identical to the recently described human Osa2 gene. Immunolabelling, using an anti-6A3-5/Osa2 monoclonal antibody, showed a nuclear localization of the 6A3-5/Osa2 protein. In addition, PDGF upregulated 6A3-5/Osa2 expression at both the transcript and protein levels in a dose and time-dependent fashion. The pattern of upregulation by PDGF was reminiscent of the early responsive gene c-fos. The PDGF-induced upregulation of 6A3-5/Osa2 and proliferation of VSMC were significantly inhibited in a dose and sequence-dependent fashion by an antisense, but not by sense, scrambled or mismatched oligonucleotides directed against 6A3-5/Osa2. In VSMC of aortas derived from hypertensive (LH) rats, 6A3-5/Osa2 is overexpressed as compared to that in normotensive (LL) rats. The 6A3-5/Osa2-gene expression is downregulated by an ACE inhibitor and upregulated by exogenous AngiotensinII in LH rats. In summary, these results indicate that 6A3-5/Osa2 is an early activated gene that belongs to a new family of proteins involved in the control of VSMC growth. [Abstract/Link to Full Text]

Toni M, Spisni E, Griffoni C, Santi S, Riccio M, Lenaz P, Tomasi V
Cellular prion protein and caveolin-1 interaction in a neuronal cell line precedes fyn/erk 1/2 signal transduction.
J Biomed Biotechnol. 2006;2006(5):69469.
It has been reported that cellular prion protein (PrPc) is enriched in caveolae or caveolae-like domains with caveolin-1 (Cav-1) participating to signal transduction events by Fyn kinase recruitment. By using the Glutathione-S-transferase (GST)-fusion proteins assay, we observed that PrPc strongly interacts in vitro with Cav-1. Thus, we ascertained the PrPc caveolar localization in a hypothalamic neuronal cell line (GN11), by confocal microscopy analysis, flotation on density gradient, and coimmunoprecipitation experiments. Following the anti-PrPc antibody-mediated stimulation of live GN11 cells, we observed that PrPc clustered on plasma membrane domains rich in Cav-1 in which Fyn kinase converged to be activated. After these events, a signaling cascade through p42/44 MAP kinase (Erk 1/2) was triggered, suggesting that following translocations from rafts to caveolae or caveolae-like domains PrPc could interact with Cav-1 and induce signal transduction events. [Abstract/Link to Full Text]

Guo Y, Eichler GS, Feng Y, Ingber DE, Huang S
Towards a holistic, yet gene-centered analysis of gene expression profiles: a case study of human lung cancers.
J Biomed Biotechnol. 2006;2006(5):69141.
Genome-wide gene expression profile studies encompass increasingly large number of samples, posing a challenge to their presentation and interpretation without losing the notion that each transcriptome constitutes a complex biological entity. Much like pathologists who visually analyze information-rich histological sections as a whole, we propose here an integrative approach. We use a self-organizing maps-based software, the gene expression dynamics inspector (GEDI) to analyze gene expression profiles of various lung tumors. GEDI allows the comparison of tumor profiles based on direct visual detection of transcriptome patterns. Such intuitive "gestalt" perception promotes the discovery of interesting relationships in the absence of an existing hypothesis. We uncovered qualitative relationships between squamous cell tumors, small-cell tumors, and carcinoid tumor that would have escaped existing algorithmic classifications. These results suggest that GEDI may be a valuable explorative tool that combines global and gene-centered analyses of molecular profiles from large-scale microarray experiments. [Abstract/Link to Full Text]

Stinghen ST, Moura JF, Zancanella P, Rodrigues GA, Pianovski MA, Lalli E, Arnold DL, Minozzo JC, Callefe LG, Ribeiro RC, Figueiredo BC
Specific immunoassays for placental alkaline phosphatase as a tumor marker.
J Biomed Biotechnol. 2006;2006(5):56087.
Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57-71) of hPLAP (ICA-PEP assay); the working range was 0.1-11 U/L and cutoff value was 0.2 U/L EA for nonsmokers. The intra- and interassay coefficients of variation were 3.7%-6.5% (ICA-PLAP assay) and 9.0%-9.9% (ICA-PEP assay). An insignificant cross-reactivity was noted for high levels of unheated intestinal alkaline phosphatase in ICA-PEP assay. A positive correlation between the regression of tumor size and EA was noted in a child with embryonal carcinoma. It can be concluded that ICA-PEP assay is more specific than ICA-PLAP, which is still useful to detect other PLAP/PLAP-like phenotypes. [Abstract/Link to Full Text]

Ebbesen M, Jensen TG
Nanomedicine: techniques, potentials, and ethical implications.
J Biomed Biotechnol. 2006;2006(5):51516.
Nanotechnology is concerned with materials and systems whose structures and components exhibit novel physical, chemical, and biological properties due to their nanoscale size. This paper focuses on what is known as nanomedicine, referring to the application of nanotechnology to medicine. We consider the use and potentials of emerging nanoscience techniques in medicine such as nanosurgery, tissue engineering, and targeted drug delivery, and we discuss the ethical questions that these techniques raise. The ethical considerations involved in nanomedicine are related to risk assessment in general, somatic-cell versus germline-cell therapy, the enhancement of human capabilities, research into human embryonic stem cells and the toxicity, uncontrolled function and self-assembly of nanoparticles. The ethical considerations associated with the application of nanotechnology to medicine have not been greatly discussed. This paper aims to balance clear ethical discussion and sound science and so provide nanotechnologists and biotechnologists with tools to assess ethical problems in nanomedicine. [Abstract/Link to Full Text]

Kim S, Soltani-Bejnood M, Quignard-Boulange A, Massiera F, Teboul M, Ailhaud G, Kim JH, Moustaid-Moussa N, Voy BH
The adipose Renin-Angiotensin system modulates systemic markers of insulin sensitivity and activates the intrarenal Renin-Angiotensin system.
J Biomed Biotechnol. 2006;2006(5):27012.
Background. The adipose tissue renin-angiotensin system (RAS) contributes to regulation of fat mass and may also impact systemic functions such as blood pressure and metabolism. Methods and results. A panel of mouse models including mice lacking angiotensinogen, Agt (Agt-KO), mice expressing Agt solely in adipose tissue (aP2-Agt/Agt-KO), and mice overexpressing Agt in adipose tissue (aP2-Agt) was studied. Total body weight, epididymal fat pad weight, and circulating levels of leptin, insulin, and resistin were significantly decreased in Agt-KO mice, while plasma adiponectin levels were increased. aP2-Agt mice exhibited increased adiposity and plasma leptin and insulin levels compared to wild type (WT) controls. Angiotensinogen and type I Ang II receptor protein levels were also elevated in kidney of aP2-Agt mice. Conclusion. These findings demonstrate that alterations in adipose RAS activity significantly impact both local and systemic physiology in a way that may contribute to the detrimental health effects of obesity. [Abstract/Link to Full Text]

Jacobsen F, Mertens-Rill J, Beller J, Hirsch T, Daigeler A, Langer S, Lehnhardt M, Steinau HU, Steinstraesser L
Nucleofection: a new method for cutaneous gene transfer?
J Biomed Biotechnol. 2006;2006(5):26060.
Background. Transfection efficacy after nonviral gene transfer in primary epithelial cells is limited. The aim of this study was to compare transfection efficacy of the recently available method of nucleofection with the established transfection reagent FuGENE6. Methods. Primary human keratinocytes (HKC), primary human fibroblasts (HFB), and a human keratinocyte cell line (HaCaT) were transfected with reporter gene construct by FuGENE6 or Amaxa Nucleofector device. At corresponding time points, beta-galactosidase expression, cell proliferation (MTT-Test), transduction efficiency (X-gal staining), cell morphology, and cytotoxicity (CASY) were determined. Results. Transgene expression after nucleofection was significantly higher in HKC and HFB and detected earlier (3 h vs. 24 h) than in FuGENE6. After lipofection 80%-90% of the cells remained proliferative without any influence on cell morphology. In contrast, nucleofection led to a decrease in keratinocyte cell size, with only 20%-42% proliferative cells. Conclusion. Related to the method-dependent increase of cytotoxicity, transgene expression after nucleofection was earlier and higher than after lipofection. [Abstract/Link to Full Text]

Thomassen GO, Røsok O, Rognes T
Computational Prediction of MicroRNAs Encoded in Viral and Other Genomes.
J Biomed Biotechnol. 2006;2006(4):95270.
We present an overview of selected computational methods for microRNA prediction. It is especially aimed at viral miRNA detection. As the number of microRNAs increases and the range of genomes encoding miRNAs expands, it seems that these small regulators have a more important role than has been previously thought. Most microRNAs have been detected by cloning and Northern blotting, but experimental methods are biased towards abundant microRNAs as well as being time-consuming. Computational detection methods must therefore be refined to serve as a faster, better, and more affordable method for microRNA detection. We also present data from a small study investigating the problems of computational miRNA prediction. Our findings suggest that the prediction of microRNA precursor candidates is fairly easy, while excluding false positives as well as exact prediction of the mature microRNA is hard. Finally, we discuss possible improvements to computational microRNA detection. [Abstract/Link to Full Text]

Sioud M, Haoudi A
RNA interference.
J Biomed Biotechnol. 2006;2006(4):89018. [Abstract/Link to Full Text]

Recent Articles in BMC Biotechnology

Mallory M, Chartrand K, Gauthier ER
Gadd153 expression does not necessarily correlate with changes in culture behavior of hybridoma cells.
BMC Biotechnol. 2007 Dec 10;7(1):89.
ABSTRACT: BACKGROUND: The acute sensitivity of some hybridoma cell lines to culture-related stresses severely limits their productivity. Recent developments in the characterization of the stress signals modulating the cellular phenotype revealed that the pro-apoptotic transcription factor Gadd153 could be used as a marker to facilitate the optimization of mammalian cell cultures. In this report, we analyzed the expression of Gadd153 in Sp2/0-Ag14 murine hybridoma cells grown in stationary batch culture and subjected to two different culture optimization paradigms: L-glutamine supplementation and ectopic expression of Bcl-xL, an anti-apoptotic gene. RESULTS: The expression of Gadd153 was found to increase in Sp2/0-Ag14 cells in a manner which coincided with the decline in cell viability. L-glutamine supplementation prolonged Sp2/0-Ag14 cell survival and greatly suppressed Gadd153 expression both at the mRNA and protein level. However, Gadd153 levels remained low after L-glutamine refeeding even as cell viability declined. Bcl-xL overexpression also extended Sp2/0-Ag14 cell viability, however, it did not prevent the increase in Gadd153 protein levels. Interestingly, L-glutamine supplementation prevented Gadd153 up-regulation in cells ectopically expressing Bcl-xL, but had no effect on cell viability. CONCLUSION: This study highlights important limitations to the use of Gadd153 as an indicator of cell stress in hybridoma cells. [Abstract/Link to Full Text]

Liebmann T, Rydholm S, Akpe V, Brismar H
Self-Assembling Fmoc Dipeptide Hydrogel for In Situ 3D Cell Culturing.
BMC Biotechnol. 2007 Dec 10;7(1):88.
ABSTRACT: BACKGROUND: Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale. RESULTS: Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms. CONCLUSIONS: Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology. [Abstract/Link to Full Text]

Hartshorn C, Eckert JJ, Hartung O, Wangh LJ
Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos.
BMC Biotechnol. 2007 Dec 7;7(1):87.
ABSTRACT: BACKGROUND: The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. RESULTS: We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. CONCLUSION: This paper describes the first example of multiplex RT-LATE-PCR and its utility, when combined with PurAmp sample preparation, for quantitative analysis of transcript levels in single cells. With this technique, copy numbers of different RNAs can be accurately measured independently from their relative abundance in a cell, a goal that cannot be achieved using symmetric PCR. The technique illustrated in this work is relevant to a wide array of applications, such as stem cell and cancer cell analysis and preimplantation genetic diagnostics. [Abstract/Link to Full Text]

Ryan BJ, O'Fagain C
Arginine-to-lysine substitutions influence recombinant horseradish peroxidase stability and immobilisation effectiveness.
BMC Biotechnol. 2007 Dec 5;7(1):86.
ABSTRACT: BACKGROUND: Horseradish Peroxidase (HRP) plays important roles in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Often, it is used in immobilised form. With conventional immobilisation techniques, the enzyme adheres in random orientation: the active site may face the solid phase rather than bulk medium, impeding substrate access and leading to sub-optimal catalytic performance. The ability to immobilise HRP in a directional manner, such that the active site would always face outwards from the insoluble matrix, would maximise the immobilised enzyme's catalytic potential and could increase HRP's range of actual and potential applications. RESULTS: We have replaced arginine residues on the face of glycan-free recombinant HRP opposite to the active site by lysines. Our strategy differs from previous reports of specific HRP immobilisation via an engineered affinity tag or single reactive residue. These conservative Arg-to-Lys substitutions provide a means of multipoint covalent immobilisation such that the active site will always face away from the immobilisation matrix. One triple and one pentuple mutant were generated by substitution of solvent-exposed arginines on the "back" of the polypeptide (R118, R159 and R283) and of residues known to influence stability (K232 and K241). Orientated HRP immobilisation was demonstrated using a modified polyethersulfone (PES) membrane; the protein was forced to orientate its active site away from the membrane and towards the bulk solution phase. Mutant properties and bioinformatic analysis suggested the reversion of K283R to improve stability, thus generating two additional mutants (K118/R159K and R118K/K232N/K241F/R283K). While most mutants were less stable in free solution than wild type rHRP, the quadruple revertant regained some stability over its mutant counterparts. A greater degree of immobilisation on CNBr-activated SepharoseTM was noted with increased lysine content; however, only marginal gains in solvent stability resulted from immobilisation on this latter matrix. CONCLUSIONS: Directional, orientated, immobilisation of rHRP mutants onto an activated, modified polyethersulfone membrane has been achieved with excellent retention of catalytic activity; however, re-engineering of acceptable stability characteristics into the "immobilisation mutants" will determine their applicability in diagnosis and biosensor development. [Abstract/Link to Full Text]

Albagli-Curiel O, Lecluse Y, Pognonec P, Boulukos KE, Martin P
A new generation of pPRIG-based retroviral vectors.
BMC Biotechnol. 2007 Nov 30;7(1):85.
ABSTRACT: BACKGROUND: Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring : i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences ; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c). RESULTS: The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS ("modular" PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R ("single color/resistance" PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively ("dual color/selection" PRIGs). Aditionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells. CONCLUSION: These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins ("modular" PRIGs), for multiple transductions and fluorescence analyses of transduced cells (" single color/resistance" PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells ("dual color/selection" PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings. [Abstract/Link to Full Text]

Imai-Nishiya H, Mori K, Inoue M, Wakitani M, Iida S, Shitara K, Satoh M
Double knockdown of alpha1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC.
BMC Biotechnol. 2007 Nov 30;7(1):84.
ABSTRACT: BACKGROUND: Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies. RESULTS: Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e., alpha1,6-fucosyltransferase (FUT8), GDP-mannose 4,6-dehydratase (GMD), and GDP-fucose transporter (GFT), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of FUT8 and GMD siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with GFT siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of FUT8 and GMD, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture. CONCLUSIONS: Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of FUT8 and GMD in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies. [Abstract/Link to Full Text]

Farlow SJ, Jerusalmi A, Sano T
Enhanced transduction of colonic cell lines in vitro and the inflamed colon in mice by viral vectors, derived from adeno-associated virus serotype 2, using virus-microbead conjugates bearing lectin.
BMC Biotechnol. 2007 Nov 28;7(1):83.
ABSTRACT: BACKGROUND: Virus-mediated delivery of therapeutic transgenes to the inflamed colon holds a great potential to serve as an effective therapeutic strategy for inflammatory bowel disease, since local, long-term expression of the encoded therapeutic proteins in the colorectal system is potentially achievable. Viral vectors, derived from adeno-associated virus (AAV), should be very useful for such therapeutic strategies, particularly because they can establish long-term expression of transgenes. However, few studies have been carried out to investigate the ability of AAV-based vectors to transduce the inflamed colon. RESULTS: AAV, derived from adeno-associated virus serotype 2 (AAV2), showed a limited ability to transduce colonic cell lines in vitro when used in free form. No appreciable enhancement of the transduction efficiency was seen when AAV2 particles were attached stably to the surfaces of microbeads and delivered to target cells in the form of AAV2-microbead conjugates. However, the transduction efficiency of these colonic cell lines was enhanced substantially when a lectin, concanavalin A (Con A), was co-attached to the microbead surfaces, to which AAV2 particles had been conjugated. This considerable infectivity enhancement of AAV2-microbead conjugates by the co-attachment of Con A may be derived from the fact that Con A binds to alpha-D-mannosyl moieties that are commonly and abundantly present in cell-surface carbohydrate chains, allowing the conjugates to associate stably with target cells. Intracolonical administration of free AAV2 or AAV2-microbead conjugates without Con A into a mouse colitis model by enema showed very poor transduction of the colonic tissue. In contrast, the delivery of AAV2 in the form of AAV2-microbead conjugates bearing Con A resulted in efficient transduction of the inflamed colon. CONCLUSIONS: AAV2-microbead conjugates bearing Con A can serve as efficient gene transfer agents both for poorly permissive colonic cell lines in vitro and for the inflamed colon in a mouse colitis model. This efficient transduction system for the inflamed colon should be useful for the development of gene therapy strategies for inflammatory bowel disease. [Abstract/Link to Full Text]

Mascellani N, Liu X, Rossi S, Marchesini J, Valentini D, Arcelli D, Taccioli C, Helmer Citterich M, Liu CG, Evangelisti R, Russo G, Santos JM, Croce CM, Volinia S
Compatible solutes from hyperthermophiles improve the quality of DNA microarrays.
BMC Biotechnol. 2007 Nov 23;7(1):82.
ABSTRACT: BACKGROUND: DNA microarrays are among the most widely used technical platforms for DNA and RNA studies, and issues related to microarrays sensitivity and specificity are therefore of general importance in life sciences. Compatible solutes are derived from hyperthermophilic microorganisms and allow such microorganisms to survive in environmental and stressful conditions. Compatible solutes show stabilization effects towards biological macromolecules, including DNA. RESULTS: We report here that compatible solutes from hyperthermophiles increased the performance of the hybridization buffer for Affymetrix GeneChip(R) arrays. The experimental setup included independent hybridizations with constant RNA over a wide range of compatible solute concentrations. The dependence of array quality and compatible solute was assessed using specialized statistical tools provided by both the proprietary Affymetrix quality control system and the open source Bioconductor suite. CONCLUSIONS: Low concentration (10 to 25 mM) of hydroxyectoine, potassium mannosylglycerate and potassium diglycerol phosphate in hybridization buffer positively affected hybridization parameters and enhanced microarrays outcome. This finding harbours a strong potential for the improvement of DNA microarray experiments. [Abstract/Link to Full Text]

Philibert P, Stoessel A, Wang W, Sibler AP, Bec N, Larroque C, Saven JG, Courtete J, Weiss E, Martineau P
A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm.
BMC Biotechnol. 2007 Nov 22;7(1):81.
ABSTRACT: BACKGROUND: Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus. RESULTS: We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the alpha-beta tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus. CONCLUSIONS: This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications. [Abstract/Link to Full Text]

Katashkina JI, Kuvaeva TM, Andreeva IG, Skorokhodova AY, Biryukova IV, Tokmakova IL, Golubeva LI, Mashko SV
Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization.
BMC Biotechnol. 2007 Nov 21;7(1):80.
ABSTRACT: BACKGROUND: RSF1010 is a well-studied broad-host-range plasmid able to mobilization to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the last case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet. RESULTS: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to exploiting of lambdaRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5-lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range. CONCLUSIONS: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements to the genetically modified organisms used in scale-up production. [Abstract/Link to Full Text]

Bannister SC, Wise TG, Cahill DM, Doran TJ
Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression.
BMC Biotechnol. 2007 Nov 19;7(1):79.
ABSTRACT: BACKGROUND: RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter. RESULTS: We identified and characterised the ch7SK promoter sequence upstream of the full-length 7SK small nuclear RNA (snRNA) sequence in the chicken genome and used this to construct vectors to express shRNAs targeting enhanced green fluorescent protein (EGFP). We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression. We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters. CONCLUSIONS: In this study we have demonstrated that the ch7SK promoter can express shRNAs capable of mediating efficient RNAi in a chicken cell line. However, our finding that RNAi driven by the ch7SK promoter is not more efficient than cU6 promoters contrasts previous comparisons of mammalian U6 and 7SK promoters. Since the ch7SK promoter is the first non-mammalian vertebrate 7SK promoter to be characterised, this finding may be helpful in understanding the divergence of pol III promoter activities between mammalian and non-mammalian vertebrates. This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells. [Abstract/Link to Full Text]

Liu JL, Anderson GP, Goldman ER
Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library.
BMC Biotechnol. 2007 Nov 19;7(1):78.
ABSTRACT: BACKGROUND: Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. RESULTS: A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. CONCLUSION: We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins. [Abstract/Link to Full Text]

Chaparro-Riggers JF, Loo BL, Polizzi KM, Gibbs PR, Tang XS, Nelson MJ, Bommarius AS
Revealing biases inherent in recombination protocols.
BMC Biotechnol. 2007 Nov 14;7(1):77.
ABSTRACT: BACKGROUND: The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing. RESULTS: We developed different test systems to compare and reveal biases from DNA shuffling and recombination-dependent PCR (RD-PCR), a StEP-like recombination protocol. An assay based on the reactivation of beta-lactamase was developed to simulate the recombination of point mutations. Both protocols performed similarly here, with slight advantages for RD-PCR. However, clear differences in the performance of the recombination protocols were observed when applied to homologous genes of varying DNA identities. Most importantly, the recombination-dependent PCR showed a less pronounced bias of the crossovers in regions with high sequence identity. We discovered that template variations, including engineered terminal truncations, have significant influence on the position of the crossovers in the recombination-dependent PCR. In comparison, DNA shuffling can produce higher crossover numbers, while the recombination-dependent PCR frequently results in one crossover. Lastly, DNA shuffling and recombination-dependent PCR both produce counter-productive variants such as parental sequences and have chimeras that are over-represented in a library, respectively. Lastly, only RD-PCR yielded chimeras in the low homology situation of GFP/mRFP (45% DNA identity level). CONCLUSION: By comparing different recombination scenarios, this study expands on existing recombination knowledge and sheds new light on known biases, which should improve library-creation efforts. It could be shown that the recombination-dependent PCR is an easy to perform alternative to DNA shuffling. [Abstract/Link to Full Text]

Mao F, Leung WY, Xin X
Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications.
BMC Biotechnol. 2007 Nov 9;7(1):76.
ABSTRACT: BACKGROUND: EvaGreen (EG) is a newly developed DNA-binding dye that has recently been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve analysis and several other applications. However, very little is known about the physicochemical properties of the dye and their relevance to the applications, particularly to qPCR and post PCR DNA melt curve analysis. In this paper, we characterized EG along with a widely used qPCR dye, SYBR Green I (SG), for their DNA-binding properties and stability, and compared their performance in qPCR under a variety of conditions. RESULTS: This study systematically compared the DNA binding profiles of the two dyes under different conditions and had these findings: a) EG had a lower binding affinity for both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) than SG; b) EG showed no apparent preference for either GC- or AT-rich sequence while SG had a slight preference for AT-rich sequence; c) both dyes showed substantially lower affinity toward ssDNA than toward dsDNA and even lower affinity toward shorter ssDNA fragments except that this trend was more pronounced for EG. Our result also demonstrated that EG was stable both under PCR condition and during routine storage and handling. In the comparative qPCR study, both EG and SG exhibited PCR interference when used at high dye concentration, as evident from delayed Ct and/or nonspecific product formation. The problem worsened when the chain extension time was shortened or when the amplicon size was relatively long (>500 bp). However, qPCR using EG tolerated a significantly higher dye concentration, thus permitting a more robust PCR signal as well as a sharper and stronger DNA melt peak. These differences in qPCR performance between the two dyes are believed to be attributable to their differences in DNA binding profiles. CONCLUSION: These findings suggest that an ideal qPCR dye should possess several DNA-binding characteristics, including a "just right" affinity for dsDNA and low or no affinity for ssDNA and short DNA fragments. The favorable DNA-binding profile of EG, coupled with its good stability and instrument-compatibility, should make EG a promising dye for qPCR and related applications. [Abstract/Link to Full Text]

Fluri DA, Daoud-El Baba M, Fussenegger M
Adeno-Associated Viral Vectors Engineered For Macrolide-Adjustable Transgene Expression In Mammalian Cells and Mice.
BMC Biotechnol. 2007 Nov 6;7(1):75.
ABSTRACT: BACKGROUND: Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. RESULTS: We designed recombinant adeno-associated virus (rAAV) vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF) into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7). Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression. CONCLUSION: Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice. [Abstract/Link to Full Text]

Allera-Moreau C, Delluc-Clavieres A, Castano C, Van den Berghe L, Golzio M, Moreau M, Teissie J, Arnal JF, Prats AC
Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle.
BMC Biotechnol. 2007 Oct 28;7(1):74.
ABSTRACT: BACKGROUND: : Electrotransfer of plasmid DNA into skeletal muscle is a promising strategy for the delivery of therapeutic molecules targeting various muscular diseases, cancer and lower-limb ischemia. Internal Ribosome Entry Sites (IRESs) allow co-expression of proteins of interest from a single transcriptional unit. IRESs are RNA elements that have been found in viral RNAs as well as a variety of cellular mRNAs with long 5' untranslated regions. While the encephalomyocarditis virus (EMCV) IRES is often used in expression vectors, we have shown that the FGF-1 IRES is equally active to drive short term transgene expression in mouse muscle. To compare the ability of the FGF-1 IRES to drive long term expression against the EMCV and FGF-2 IRESs, we performed analyses of expression kinetics using bicistronic vectors that express the bioluminescent renilla and firefly luciferase reporter genes. Long term expression of bicistronic vectors was also compared to that of monocistronic vectors. Bioluminescence was quantified ex vivo using a luminometer and in vivo using a CCD camera that monitors luminescence within live animals. RESULTS: : Our data demonstrate that the efficiency of the FGF-1 IRES is comparable to that of the EMCV IRES for long term expression of bicistronic transgenes in mouse muscle, whereas the FGF-2 IRES has a very poor activity. Interestingly, we show that despite the global decrease of vector expression over time, the ratio of firefly to renilla luciferase remains stable with bicistronic vectors containing the FGF-1 or FGF-2 IRES and is slightly affected with the EMCV IRES, whereas it is clearly unstable for mixed monocistronic vectors. In addition, long term expression more drastically decreases with monocistronic vectors, and is different for single or mixed vector injection. CONCLUSIONS: : These data validate the use of bicistronic vectors rather than mixed monocistronic vectors for long term expression, and support the use of the FGF-1 IRES. The use of a cellular IRES over one of viral origin is of particular interest in the goal of eliminating viral sequences from transgenic vectors. In addition, the FGF-1 IRES, compared to the EMCV IRES, has a more stable activity, is shorter in length and more flexible in terms of downstream cloning of second cistrons. Finally, the FGF-1 IRES is very attractive to develop multicistronic expression cassettes for gene transfer in mouse muscle. [Abstract/Link to Full Text]

Lopes Pinto F, Svensson H, Lindblad P
Webtag: a new web tool providing tags/anchors for RT-PCR experiments with prokaryotes.
BMC Biotechnol. 2007 Oct 25;7(1):73.
ABSTRACT: BACKGROUND: The Webtag is a tool providing oligonucleotide sequences (usually called tags or anchors) that are absent from a specified genome. These tags/anchors can be appended to gene specific primers for reverse transcriptase polymerase chain reaction experiments, circumventing genomic DNA contamination. RESULTS: The use of a relational database, in conjunction with a series of PHP scripts, allows the user to rapidly obtain tags that are: 1) suitable for a specific organism, and 2) compatible with other oligonucleotides to be used in the experimental procedures. CONCLUSIONS: This new web tool allows scientists to easily and rapidly obtain suitable tags for RT-PCR experiments, and is available at [Abstract/Link to Full Text]

Goh S, Camattari A, Ng D, Song R, Madden K, Westpheling J, Wong VV
An integrative expression vector for Actinosynnema pretiosum.
BMC Biotechnol. 2007 Oct 24;7(1):72.
ABSTRACT: BACKGROUND: The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One way to increase AP-3 production would be to constitutively express selected genes so as to relieve bottlenecks in the biosynthetic pathway; however, an integrative expression vector for A. pretiosum is lacking. The aim of this study was to construct a vector for heterologous gene expression in A. pretiosum. RESULTS: A series of integrative expression vectors have been made with the following features: the IS117 transposase from Streptomyces coelicolor, the constitutive ermE* promoter from Saccharopolyspora erythraea, different ribosome-binding site (RBS) sequences and xylE as a translational reporter. Positive E. coli clones and A. pretiosum transconjugants were assayed by catechol. pAP42, containing an E. coli consensus RBS, and pAP43, containing an asm19 RBS, gave strong and moderate gene expression, respectively. In addition, an operon construct capable of multi-gene expression was created. Plasmid integration sites in transconjugants were investigated and four different sites were observed. Although the most common integration site was within a putative ORF with sequence similarity to NADH-flavin reductase, AP-3 levels and cell growth of transconjugants were unaffected. CONCLUSIONS: A set of integrative vectors for constitutive gene expression in A. pretiosum has been constructed. Gene translation is easily determined by colorimetric assay on an agar plate. The vectors are suitable for studies relating to AP-3 biosynthesis as they do not affect AP-3 production. [Abstract/Link to Full Text]

Jaluria P, Betenbaugh M, Konstantopoulos K, Shiloach J
Enhancement of cell proliferation in mammalian cells by gene insertion of a cyclin-dependent kinase homolog.
BMC Biotechnol. 2007 Oct 18;7(1):71.
ABSTRACT: BACKGROUND: Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays. RESULTS: Two genes, cyclin-dependent kinase like 3 (cdkl3) and cytochrome c oxidase subunit (cox15), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in moderately elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15. Moreover, flow cytometric analysis indicated that cells with an insert of cdkl3 were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of cox15 was seen to moderately increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than cdkl3. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either cdkl3 or cox15. CONCLUSIONS: These results are consistent with previous studies on the functionalities of cdkl3 and cox15. For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to cox15 expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs. [Abstract/Link to Full Text]

Pavoni E, Monteriu G, Santapaola D, Petronzelli F, Anastasi AM, Pelliccia A, D'Alessio V, De Santis R, Minenkova O
Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells.
BMC Biotechnol. 2007 Oct 18;7(1):70.
ABSTRACT: BACKGROUND: There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients. METHODS: The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells. RESULTS: Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells. CONCLUSIONS: Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens in these patients. The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches. [Abstract/Link to Full Text]

Stougaard M, Lohmann JS, Zajac M, Hamilton-Dutoit S, Koch J
In situ detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS.
BMC Biotechnol. 2007 Oct 18;7(1):69.
ABSTRACT: BACKGROUND: In situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling. Whilst short probes have several advantages over long probes (e.g. higher resolution and specificity) they carry fewer labels per molecule and therefore require higher amplification for detection. Furthermore, short probes relying only on hybridization for specificity can result in non-specific signals appearing anywhere the probe attaches to the target specimen. One way to obtain high amplification whilst minimizing the risk of false positivity is to use small circular probes (e.g. Padlock Probes) in combination with target primed rolling circle DNA synthesis. This has previously been used for DNA detection in situ, but not until now for RNA targets. RESULTS: We present here a proof of principle investigation of a novel rolling circle technology for the detection of non-polyadenylated RNA molecules in situ, including a new probe format (the Turtle Probe) and optimized procedures for its use on formalin fixed paraffin embedded tissue sections and in solid support format applications. CONCLUSIONS: The method presented combines the high discriminatory power of short oligonucleotide probes with the impressive amplification power and selectivity of the rolling circle reaction, providing excellent signal to noise ratios in combination with exact target localization due to the target primed reaction. Furthermore, the procedure is easily multiplexed, allowing visualization of several different RNAs. [Abstract/Link to Full Text]

Donofrio G, Sartori C, Ravanetti L, Cavirani S, Gillet L, Vanderplasschen A, Taddei S, Flammini CF
Establishment of a bovine herpesvirus 4 based vector expressing a secreted form of the bovine viral diarrhoea virus structural glycoprotein E2 for immunization purposes.
BMC Biotechnol. 2007;768.
BACKGROUND: The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. RESULTS: A recombinant bovine herpesvirus 4 (BoHV-4CMV-IgKE2-14 Delta TK) expressing an enhanced secreted form of the bovine viral diarrhea virus (BVDV) structural glycoprotein E2 (gE2-14), obtained by the removal of the putative transmembrane domain and addition of a 14 amino acids peptide at its carboxyl terminal and an immunoglobulin K signal peptide to the amino terminal, was successfully constructed using a Recombineering (recombination -mediated genetic engineering) approach on BoHV-4 cloned as bacterial artificial chromosome. The galactokinase - based recombineering system was modified by the introduction of a kanamycin expression cassette and a kanamycin selection step that allowed a significant reduction of the untargeted background clones. BoHV-4CMV-IgKE2-14 Delta TK infected cell lines highly expressed gE2-14, which maintained native antigenic properties in a serum neutralization inhibition test. When rabbits and sheep were immunized with BoHV-4CMV-IgKE2-14 Delta TK, high levels of serum neutralized antibodies against BVDV were generated. CONCLUSION: This work highlights the engineerization of BoHV-4 genome as a vector for vaccine purposes and may provide the basis for BVDV vaccination exploiting the BoHV-4- based vector that delivers an improved secreted version of the BVDV structural glycoprotein E2. [Abstract/Link to Full Text]

Lin S, Xie X, Patel MR, Yang YH, Li Z, Cao F, Gheysens O, Zhang Y, Gambhir SS, Rao JH, Wu JC
Quantum dot imaging for embryonic stem cells.
BMC Biotechnol. 2007 Oct 9;7(1):67.
ABSTRACT: BACKGROUND: Semiconductor quantum dots (QDs) hold growing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate the in vivo multiplex and long-term imaging of mouse embryonic stem (ES) cells labeled with Qtracker delivered quantum dots (QDs). RESULTS: Murine ES cells were labeled with six different QDs delivered by Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P=NS). Afterward, labeled ES cells were injected separately and subcutaneously onto the back of athymic nude mice. These labeled ES cells can be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background)/exposure time in millisecond is 11+/-2 for cells labeled with QD 525, 12+/-9 for QD 565, 176+/-81 for QD 605, 176+/-136 for QD 655, 167+/-104 for QD 705, and 1,713+/-482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested. CONCLUSIONS: This is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will demonstrate greater utility for the long-term tracking of stem cells deep within deeper tissues. These results provide a promising, innovative tool for imaging stem cell therapy non-invasively in vivo. [Abstract/Link to Full Text]

Li F, Zhou X, Zhu J, Ma J, Huang X, Wong ST
High content image analysis for H4 human neuroglioma cells exposed to CuO nanoparticles.
BMC Biotechnol. 2007 Oct 9;7(1):66.
ABSTRACT: BACKGROUND: High content screening (HCS)-based image analysis is becoming an important and widely used research tool. Capitalizing this technology, ample cellular information can be extracted from the high content cellular images. In this study, an automated, reliable and quantitative cellular image analysis system developed in house has been employed to quantify the toxic responses of human H4 neuroglioma cells exposed to metal oxide nanoparticles. This system has been proved to be an essential tool in our study. RESULTS: The cellular images of H4 neuroglioma cells exposed to different concentrations of CuO nanoparticles were sampled using IN Cell Analyzer 1000. A fully automated cellular image analysis system has been developed to perform the image analysis for cell viability. A multiple adaptive thresholding method was used to classify the pixels of the nuclei image into three classes: bright nuclei, dark nuclei, and background. During the development of our image analysis methodology, we have achieved the followings: (1) The Gaussian filtering with proper scale has been applied to the cellular images for generation of a local intensity maximum inside each nucleus; (2) A novel local intensity maxima detection method based on the gradient vector field has been established; and (3) A statistical model based splitting method was proposed to overcome the under segmentation problem. Computational results indicate that 95.9% nuclei can be detected and segmented correctly by the image analysis system. CONCLUSIONS: Our automated image analysis system can effectively segment the cellular images of human H4 neuroglioma cells exposed to CuO nanoparticles. The computational results validated our biological finding that human H4 neuroglioma cells had a dose-dependent toxic response to the insult of CuO nanoparticles. [Abstract/Link to Full Text]

Krumpe LR, Schumacher KM, McMahon JB, Makowski L, Mori T
Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library.
BMC Biotechnol. 2007 Oct 5;7(1):65.
ABSTRACT: BACKGROUND: Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. METHODS: In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. RESULTS: We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. CONCLUSIONS: The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets. [Abstract/Link to Full Text]

Langlais C, Guilleaume B, Wermke N, Scheuermann T, Ebert L, LaBaer J, Korn B
A systematic approach for testing expression of human full-length proteins in cell-free expression systems.
BMC Biotechnol. 2007;764.
BACKGROUND: The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. RESULTS: In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. CONCLUSION: We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield. [Abstract/Link to Full Text]

Jaubert-Possamai S, Le Trionnaire G, Bonhomme J, Christophides GK, Rispe C, Tagu D
Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum.
BMC Biotechnol. 2007;763.
BACKGROUND: RNA interference (RNAi) is a powerful method to inhibit gene expression in a sequence specific manner. RESULTS: Here, we described the development of RNAi by micro-injection of double-stranded RNA (dsRNA) in the pea aphid Acyrthosiphon pisum. Injection of dsRNA into whole aphid body induced the silencing of two marker genes with different expression patterns: the ubiquitously expressed Ap-crt genes encoding a calreticulin and the gut specific Ap-cath-L gene encoding a cathepsin-L. Time-course analysis of the silencing showed similar temporal patterns for both genes: inhibition started at 1 day after injection, reached its maximum at 5 days and stopped at 7 days. A comparable 40% decrease of gene expression was observed for Ap-crt and Ap-cath-L. CONCLUSION: The pea aphid is the first Hemipteran insect for which genome sequence will be available soon. The gene knockdown technique developed in this study will be an essential post-genomic tool for further investigations in aphidology. [Abstract/Link to Full Text]

Yang CD, Liao JT, Lai CY, Jong MH, Liang CM, Lin YL, Lin NS, Hsu YH, Liang SM
Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes.
BMC Biotechnol. 2007;762.
BACKGROUND: Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. METHODS: We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s) of the capsid protein VP1 of foot-and-mouth disease virus (FMDV). The recombinant BaMV plasmid (pBVP1) was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164) of FMDV VP1. RESULTS: The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-gamma. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. CONCLUSION: Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles. [Abstract/Link to Full Text]

Gray DC, Hoeflich KP, Peng L, Gu Z, Gogineni A, Murray LJ, Eby M, Kljavin N, Seshagiri S, Cole MJ, Davis DP
pHUSH: a single vector system for conditional gene expression.
BMC Biotechnol. 2007 Sep 26;7(1):61.
ABSTRACT: BACKGROUND: Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector. RESULTS: Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer. CONCLUSION: We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells. [Abstract/Link to Full Text]

Chung BG, Park JW, Hu JS, Huang C, Monuki ES, Jeon NL
A hybrid microfluidic-vacuum device for direct interfacing with conventional cell culture methods.
BMC Biotechnol. 2007;760.
BACKGROUND: Microfluidics is an enabling technology with a number of advantages over traditional tissue culture methods when precise control of cellular microenvironment is required. However, there are a number of practical and technical limitations that impede wider implementation in routine biomedical research. Specialized equipment and protocols required for fabrication and setting up microfluidic experiments present hurdles for routine use by most biology laboratories. RESULTS: We have developed and validated a novel microfluidic device that can directly interface with conventional tissue culture methods to generate and maintain controlled soluble environments in a Petri dish. It incorporates separate sets of fluidic channels and vacuum networks on a single device that allows reversible application of microfluidic gradients onto wet cell culture surfaces. Stable, precise concentration gradients of soluble factors were generated using simple microfluidic channels that were attached to a perfusion system. We successfully demonstrated real-time optical live/dead cell imaging of neural stem cells exposed to a hydrogen peroxide gradient and chemotaxis of metastatic breast cancer cells in a growth factor gradient. CONCLUSION: This paper describes the design and application of a versatile microfluidic device that can directly interface with conventional cell culture methods. This platform provides a simple yet versatile tool for incorporating the advantages of a microfluidic approach to biological assays without changing established tissue culture protocols. [Abstract/Link to Full Text]

Recent Articles in Journal of Nanobiotechnology

Caldeira JC, Peabody DS
Stability and assembly in vitro of bacteriophage PP7 virus-like particles.
J Nanobiotechnology. 2007 Nov 26;5(1):10.
ABSTRACT: BACKGROUND: The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. The icosahedral capsid of the RNA bacteriophage PP7 is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes. This work determined the effects of these disulfides on the VLP's thermal stability. RESULTS: Measurements of the thermal denaturation behavior of PP7 VLPs in the presence and absence of a reducing agent show that disulfide cross-links substantially stabilize them against thermal denaturation. Although dimers in the capsid are linked to one another by disulfides, the two subunits of dimers themselves are held together only by non-covalent interactions. In an effort to confer even greater stability a new cross-link was introduced by genetically fusing two coat protein monomers, thus producing a "single-chain dimer" that assembles normally into a completely cross-linked VLP. However, subunit fusion failed to increase the thermal stability of the particles, even though it stabilized the isolated dimer. As a step toward gaining control of the internal composition of the capsid, conditions that promote the assembly of PP7 coat protein dimers into virus-like particles in vitro were established. CONCLUSIONS: The presence of inter-dimer disulfide bonds greatly stabilizes the PP7 virus-like particle against thermal denaturation. Covalently cross-linking the subunits of the dimers themselves by genetically fusing them through a dipeptide linker sequence, offers no further stabilization of the VLP, although it does stabilize the dimer. PP7 capsids readily assemble in vitro in a reaction that requires RNA. [Abstract/Link to Full Text]

Muller-Borer BJ, Collins MC, Gunst PR, Cascio WE, Kypson AP
Quantum dot labeling of mesenchymal stem cells.
J Nanobiotechnology. 2007 Nov 7;5(1):9.
ABSTRACT: BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, fat and muscle cells and are being investigated for their utility in cell-based transplantation therapy. Yet, adequate methods to track transplanted MSCs in vivo are limited, precluding functional studies. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. These nanoparticles are resistant to chemical and metabolic degradation, demonstrating long term photostability. Here, we investigate the cytotoxic effects of in vitro QD labeling on MSC proliferation and differentiation and use as a cell label in a cardiomyocyte co-culture. RESULTS: A dose-response to QDs in rat bone marrow MSCs was assessed in Control (no-QDs), Low concentration (LC, 5 nmol/L) and High concentration (HC, 20 nmol/L) groups. QD yield and retention, MSC survival, proinflammatory cytokines, proliferation and DNA damage were evaluated in MSCs, 24 -120 hrs post QD labeling. In addition, functional integration of QD labeled MSCs in an in vitro cardiomyocyte co-culture was assessed. A dose-dependent effect was measured with increased yield in HC vs. LC labeled MSCs (93 +/- 3% vs. 50% +/-15%, p<0.05), with a larger number of QD aggregates per cell in HC vs. LC MSCs at each time point (p<0.05). At 24 hrs >90% of QD labeled cells were viable in all groups, however, at 120 hrs increased apoptosis was measured in HC vs. Control MSCs (7.2% +/- 2.7% vs. 0.5% +/- 0.4%, p<0.05). MCP-1 and IL-6 levels doubled in HC MSCs when measured 24 hrs after QD labeling. No change in MSC proliferation or DNA damage was observed in QD labeled MSCs at 24, 72 and 120 hrs post labeling. Finally, in a cardiomyocyte co-culture QD labeled MSCs were easy to locate and formed functional cell-to-cell couplings, assessed by dye diffusion. CONCLUSION: Fluorescent QDs label MSC effectively in an in vitro co-culture model. QDs are easy to use, show a high yield and survival rate with minimal cytotoxic effects. Dose-dependent effects suggest limiting MSC QD exposure. [Abstract/Link to Full Text]

Yehia HN, Draper RK, Mikoryak C, Walker EK, Bajaj P, Musselman IH, Daigrepont MC, Dieckmann GR, Pantano P
Single-walled carbon nanotube interactions with HeLa cells.
J Nanobiotechnology. 2007;58.
ABSTRACT: This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports. [Abstract/Link to Full Text]

Uehara H, Kunitomi Y, Ikai A, Osada T
mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization.
J Nanobiotechnology. 2007 Oct 10;5(1):7.
ABSTRACT: BACKGROUND: The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. In situ hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells. RESULTS: In order to evaluate the SCN method, we compared the SCN method with in situ hybridization (ISH). First, we examined spatial beta-actin mRNA expression in single living cells with the SCN method, and then the same cells were subjected to ISH for beta-actin mRNA. In the SCN method, quantity of beta-actin mRNA were analysed by quantitative PCR, and in ISH we used intensity of ISH as a parameter of concentration of beta-actin mRNA. We showed that intensity of ISH is higher; quantity of beta-actin mRNA detected by the SCN method increased more. CONCLUSIONS: In this study, we compare the SCN method with the ISH. We examined b-actin mRNA expression in single cells using both methods. We picked up beta-actin mRNA from several loci of a single living cell using an AFM nanoprobe, and identical cells were subjected to ISH. The results showed a good correlation between the SCN method and ISH. The SCN method is suitable and reliable to examine mRNAs at medium or higher expression level. [Abstract/Link to Full Text]

Partha R, Lackey M, Hirsch A, Casscells SW, Conyers JL
Self assembly of amphiphilic C60 fullerene derivatives into nanoscale supramolecular structures.
J Nanobiotechnology. 2007;56.
ABSTRACT: BACKGROUND: The amphiphilic fullerene monomer (AF-1) consists of a "buckyball" cage to which a Newkome-like dendrimer unit and five lipophilic C12 chains positioned octahedrally to the dendrimer unit are attached. In this study, we report a novel fullerene-based liposome termed 'buckysome' that is water soluble and forms stable spherical nanometer sized vesicles. Cryogenic electron microscopy (Cryo-EM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) studies were used to characterize the different supra-molecular structures readily formed from the fullerene monomers under varying pH, aqueous solvents, and preparative conditions. RESULTS: Electron microscopy results indicate the formation of bilayer membranes with a width of ~6.5 nm, consistent with previously reported molecular dynamics simulations. Cryo-EM indicates the formation of large (400 nm diameter) multilamellar, liposome-like vesicles and unilamellar vesicles in the size range of 50-150 nm diameter. In addition, complex networks of cylindrical, tube-like aggregates with varying lengths and packing densities were observed. Under controlled experimental conditions, high concentrations of spherical vesicles could be formed. In vitro results suggest that these supra-molecular structures impose little to no toxicity. Cytotoxicity of 10-200 muM buckysomes were assessed in various cell lines. Ongoing studies are aimed at understanding cellular internalization of these nanoparticle aggregates. CONCLUSION: In this current study, we have designed a core platform based on a novel amphiphilic fullerene nanostructure, which readily assembles into supra-molecular structures. This delivery vector might provide promising features such as ease of preparation, long-term stability and controlled release. [Abstract/Link to Full Text]

Salata O
Nanotechnology in therapeutics: hydrogels and beyond.
J Nanobiotechnology. 2007;55.
Nanotechnology in Therapeutics: Current Technology and Applications, Edited by Nicholas A. Peppas, J. Zach Hilt and J. Brock Thomas (Horizon Bioscience, 2007) contains seventeen chapters written by leading specialists in the field of polymeric materials for drug delivery and holds wealth of background as well as state of the art material divided into four sections: "Intelligent Therapeutics and Responsive Delivery Systems for Improved Absorption and Delivery", "Therapeutic Micro- and Nanodevices", "Nanostructured Therapeutic Materials" and "Nanoparticulate Systems in Intelligent Therapy". This newly published volume provides a stimulating read and a good point of reference to researchers wishing to explore the interdisciplinary fusion of nnanotechnology and medical therapeutics. The following gives brief summary and critically reviews the book. [Abstract/Link to Full Text]

Mukherjee P, Bhattacharya R, Bone N, Lee YK, Patra CR, Wang S, Lu L, Secreto C, Banerjee PC, Yaszemski MJ, Kay NE, Mukhopadhyay D
Potential therapeutic application of gold nanoparticles in B-chronic lymphocytic leukemia (BCLL): enhancing apoptosis.
J Nanobiotechnology. 2007;54.
B-Chronic Lymphocytic Leukemia (CLL) is an incurable disease predominantly characterized by apoptosis resistance. We have previously described a VEGF signaling pathway that generates apoptosis resistance in CLL B cells. We found induction of significantly more apoptosis in CLL B cells by co-culture with an anti-VEGF antibody. To increase the efficacy of these agents in CLL therapy we have focused on the use of gold nanoparticles (GNP). Gold nanoparticles were chosen based on their biocompatibility, very high surface area, ease of characterization and surface functionalization. We attached VEGF antibody (AbVF) to the gold nanoparticles and determined their ability to kill CLL B cells. Gold nanoparticles and their nanoconjugates were characterized using UV-Visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). All the patient samples studied (N = 7) responded to the gold-AbVF treatment with a dose dependent apoptosis of CLL B cells. The induction of apoptosis with gold-AbVF was significantly higher than the CLL cells exposed to only AbVF or GNP. The gold-AbVF treated cells showed significant down regulation of anti-apoptotic proteins and exhibited PARP cleavage. Gold-AbVF treated and GNP treated cells showed internalization of the nanoparticles in early and late endosomes and in multivesicular bodies. Non-coated gold nanoparticles alone were able to induce some levels of apoptosis in CLL B cells. This paper opens up new opportunities in the treatment of CLL-B using gold nanoparticles and integrates nanoscience with therapy in CLL. In future, potential opportunities exist to harness the optoelectronic properties of gold nanoparticles in the treatment of CLL. [Abstract/Link to Full Text]

Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, Maitra A, Maitra A
Polymeric nanoparticle-encapsulated curcumin ("nanocurcumin"): a novel strategy for human cancer therapy.
J Nanobiotechnology. 2007;53.
BACKGROUND: Curcumin, a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa), has potent anti-cancer properties as demonstrated in a plethora of human cancer cell line and animal carcinogenesis models. Nevertheless, widespread clinical application of this relatively efficacious agent in cancer and other diseases has been limited due to poor aqueous solubility, and consequently, minimal systemic bioavailability. Nanoparticle-based drug delivery approaches have the potential for rendering hydrophobic agents like curcumin dispersible in aqueous media, thus circumventing the pitfalls of poor solubility. RESULTS: We have synthesized polymeric nanoparticle encapsulated formulation of curcumin - nanocurcumin - utilizing the micellar aggregates of cross-linked and random copolymers of N-isopropylacrylamide (NIPAAM), with N-vinyl-2-pyrrolidone (VP) and poly(ethyleneglycol)monoacrylate (PEG-A). Physico-chemical characterization of the polymeric nanoparticles by dynamic laser light scattering and transmission electron microscopy confirms a narrow size distribution in the 50 nm range. Nanocurcumin, unlike free curcumin, is readily dispersed in aqueous media. Nanocurcumin demonstrates comparable in vitro therapeutic efficacy to free curcumin against a panel of human pancreatic cancer cell lines, as assessed by cell viability and clonogenicity assays in soft agar. Further, nanocurcumin's mechanisms of action on pancreatic cancer cells mirror that of free curcumin, including induction of cellular apoptosis, blockade of nuclear factor kappa B (NFkappaB) activation, and downregulation of steady state levels of multiple pro-inflammatory cytokines (IL-6, IL-8, and TNFalpha). CONCLUSION: Nanocurcumin provides an opportunity to expand the clinical repertoire of this efficacious agent by enabling ready aqueous dispersion. Future studies utilizing nanocurcumin are warranted in pre-clinical in vivo models of cancer and other diseases that might benefit from the effects of curcumin. [Abstract/Link to Full Text]

Umemura K, Yamada T, Maeda Y, Kobayashi K, Kuroda R, Mayama S
Regulated growth of diatom cells on self-assembled monolayers.
J Nanobiotechnology. 2007;52.
We succeeded in regulating the growth of diatom cells on chemically modified glass surfaces. Glass surfaces were functionalized with -CF3, -CH3, -COOH, and -NH2 groups using the technique of self-assembled monolayers (SAM), and diatom cells were subsequently cultured on these surfaces. When the samples were rinsed after the adhesion of the diatom cells on the modified surfaces, the diatoms formed two dimensional arrays; this was not possible without the rinsing treatment. Furthermore, we examined the number of cells that grew and their motility by time-lapse imaging in order to clarify the interaction between the cells and SAMs. We hope that our results will be a basis for developing biodevices using living photosynthetic diatom cells. [Abstract/Link to Full Text]

Choi AO, Cho SJ, Desbarats J, Lovri? J, Maysinger D
Quantum dot-induced cell death involves Fas upregulation and lipid peroxidation in human neuroblastoma cells.
J Nanobiotechnology. 2007;51.
BACKGROUND: Neuroblastoma, a frequently occurring solid tumour in children, remains a therapeutic challenge as existing imaging tools are inadequate for proper and accurate diagnosis, resulting in treatment failures. Nanoparticles have recently been introduced to the field of cancer research and promise remarkable improvements in diagnostics, targeting and drug delivery. Among these nanoparticles, quantum dots (QDs) are highly appealing due to their manipulatable surfaces, yielding multifunctional QDs applicable in different biological models. The biocompatibility of these QDs, however, remains questionable. RESULTS: We show here that QD surface modifications with N-acetylcysteine (NAC) alter QD physical and biological properties. In human neuroblastoma (SH-SY5Y) cells, NAC modified QDs were internalized to a lesser extent and were less cytotoxic than unmodified QDs. Cytotoxicity was correlated with Fas upregulation on the surface of treated cells. Alongside the increased expression of Fas, QD treated cells had increased membrane lipid peroxidation, as measured by the fluorescent BODIPY-C11 dye. Moreover, peroxidized lipids were detected at the mitochondrial level, contributing to the impairment of mitochondrial functions as shown by the MTT reduction assay and imaged with confocal microscopy using the fluorescent JC-1 dye. CONCLUSION: QD core and surface compositions, as well as QD stability, all influence nanoparticle internalization and the consequent cytotoxicity. Cadmium telluride QD-induced toxicity involves the upregulation of the Fas receptor and lipid peroxidation, leading to impaired neuroblastoma cell functions. Further improvements of nanoparticles and our understanding of the underlying mechanisms of QD-toxicity are critical for the development of new nanotherapeutics or diagnostics in nano-oncology. [Abstract/Link to Full Text]

Levi N, Hantgan RR, Lively MO, Carroll DL, Prasad GL
C60-fullerenes: detection of intracellular photoluminescence and lack of cytotoxic effects.
J Nanobiotechnology. 2006;414.
We have developed a new method of application of C60 to cultured cells that does not require water-solubilization techniques. Normal and malignant cells take-up C60 and the inherent photoluminescence of C60 is detected within multiple cell lines. Treatment of cells with up to 200 microg/ml (200 ppm) of C60 does not alter morphology, cytoskeletal organization, cell cycle dynamics nor does it inhibit cell proliferation. Our work shows that pristine C60 is non-toxic to the cells, and suggests that fullerene-based nanocarriers may be used for biomedical applications. [Abstract/Link to Full Text]

Gilbert L, Toivola J, Välilehto O, Saloniemi T, Cunningham C, White D, Mäkelä AR, Korhonen E, Vuento M, Oker-Blom C
Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles.
J Nanobiotechnology. 2006;413.
Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles. [Abstract/Link to Full Text]

Baldessari F, Santiago JG
Electrophoresis in nanochannels: brief review and speculation.
J Nanobiotechnology. 2006;412.
The relevant physical phenomena that dominate electrophoretic transport of ions and macromolecules within long, thin nanochannels are reviewed, and a few papers relevant to the discussion are cited. Sample ion transport through nanochannels is largely a function of their interaction with electric double layer. For small ions, this coupling includes the net effect of the external applied field, the internal field of the double layer, and the non-uniform velocity of the liquid. Adsorption/desorption kinetics and the effects of surface roughness may also be important in nanochannel electrophoresis. For macromolecules, the resulting motion is more complex as there is further coupling via steric interactions and perhaps polarization effects. These complex interactions and coupled physics represent a valuable opportunity for novel electrophoretic and chromatographic separations. [Abstract/Link to Full Text]

Patra CR, Bhattacharya R, Patra S, Basu S, Mukherjee P, Mukhopadhyay D
Inorganic phosphate nanorods are a novel fluorescent label in cell biology.
J Nanobiotechnology. 2006;411.
We report the first use of inorganic fluorescent lanthanide (europium and terbium) ortho phosphate [LnPO4.H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC), 786-O cells, or renal carcinoma cells (RCC). The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS), differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM). At concentrations up to 50 microg/ml, the use of [3H]-thymidine incorporation assays, apoptosis assays (TUNEL), and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes. [Abstract/Link to Full Text]

Funnell WR, Maysinger D
Three-dimensional reconstruction of cell nuclei, internalized quantum dots and sites of lipid peroxidation.
J Nanobiotechnology. 2006;410.
BACKGROUND: The purpose of the study was to develop and illustrate three-dimensional (3-D) reconstruction of nuclei and intracellular lipid peroxidation in cells exposed to oxidative stress induced by quantum dots. Programmed cell death is characterized by multiple biochemical and morphological changes in different organelles, including nuclei, mitochondria and lysosomes. It is the dynamics of the spatio-temporal changes in the signalling and morphological adaptations which will ultimately determine the 'shape' and fate of the cell. RESULTS: We present new approaches to the 3-D reconstruction of organelle morphology and biochemical changes in confocal live-cell images. We demonstrate the 3-D shapes of nuclei, the 3-D intracellular distributions of QDs and the accompanying lipid-membrane peroxidation, and provide methods for quantification. CONCLUSION: This study provides an approach to 3-D organelle and nanoparticle visualization in the context of cell death; however, this approach is also applicable more generally to investigating changes in organelle morphology in response to therapeutic interventions, stressful stimuli and internalized nanoparticles. Moreover, the approach provides quantitative data for such changes, which will help us to better integrate compartmentalization of subcellular events and to link morphological and biochemical changes with physiological outcomes. [Abstract/Link to Full Text]

Prina-Mello A, Diao Z, Coey JM
Internalization of ferromagnetic nanowires by different living cells.
J Nanobiotechnology. 2006;49.
The ability of living cells, either adherent or suspended, to internalize nickel nanowires is demonstrated for MC3T3-E1, UMR106-tumour and Marrow-Stromal cells. Nanowires were produced by electrodeposition, 20 mum long and 200 nm in diameter. Cell separation and manipulation was achieved for the three cell types. Applied magnetic field successfully oriented the internalized nanowires but no clear anisotropy is induced on the adherent cells. Nanowires tend to bind to cytoplasm metalloproteins and trigger lysosome reorganization around the nucleus. This work demonstrates the applications of nanowires in adherent and suspended cells for cell separation and manipulation, and further explore into their role in nanobiotechnology. [Abstract/Link to Full Text]

Kouassi GK, Irudayaraj J
A nanoparticle-based immobilization assay for prion-kinetics study.
J Nanobiotechnology. 2006;48.
Magnetic and gold coated magnetic nanoparticles were synthesized by co-precipitation of ferrous and ferric chlorides, and by the micromicelles method, respectively. Synthesized nanoparticles were functionalized to bear carboxyl and amino acid moieties and used as prion protein carriers after carbodiimide activation in the presence of N-hydroxysuccinimide. The binding of human recombinant prion protein (huPrPrec) to the surface of these nanoparticles was confirmed by FTIR and the size and structures of the particles were characterized by transmission electron microscopy. Findings indicate that the rate of prion binding increased only slightly when the concentration of prion in the reaction medium was increased. Rate constants of binding were very similar on Fe3O4@Au and Fe3O4-LAA when the concentrations of protein were 1, 2, 1.5, 2.25 and 3.57 microg/ml. For a 5 microg/ml concentration of huPrPrec the binding rate constant was higher for the Fe3O4-LAA particles. This study paves the way towards the formation of prion protein complexes onto a 3-dimensional structure that could reveal obscure physiological and pathological structure and prion protein kinetics. [Abstract/Link to Full Text]

Matsumura K, Orita K, Wakamoto Y, Yasuda K
Phagocytic response to fully controlled plural stimulation of antigens on macrophage using on-chip microcultivation system.
J Nanobiotechnology. 2006;47.
To understand the control mechanism of innate immune response in macrophages, a series of phagocytic responses to plural stimulation of antigens on identical cells was observed. Two zymosan particles, which were used as antigens, were put on different surfaces of a macrophage using optical tweezers in an on-chip single-cell cultivation system, which maintains isolated conditions of each macrophage during their cultivation. When the two zymosan particles were attached to the macrophage simultaneously, the macrophage responded and phagocytosed both of the antigens simultaneously. In contrast, when the second antigen was attached to the surface after the first phagocytosis had started, the macrophage did not respond to the second stimulation during the first phagocytosis; the second phagocytosis started only after the first process had finished. These results indicate that (i) phagocytosis in a macrophage is not an independent process when there are plural stimulations; (ii) the response of the macrophage to the second stimulation is related to the time" delay from the first stimulation. Stimulations that occur at short time intervals resulted in simultaneous phagocytosis, while a second stimulation that is delayed long enough might be neglected until the completion of the first phagocytic process. [Abstract/Link to Full Text]

Yin Y, Yin J
Geometric conservation laws for cells or vesicles with membrane nanotubes or singular points.
J Nanobiotechnology. 2006;46.
On the basis of the integral theorems about the mean curvature and Gauss curvature, geometric conservation laws for cells or vesicles are proved. These conservation laws may depict various special bionano structures discovered in experiments, such as the membrane nanotubes and singular points grown from the surfaces of cells or vesicles. Potential applications of the conservation laws to lipid nanotube junctions that interconnect cells or vesicles are discussed. [Abstract/Link to Full Text]

Müller F, Houben A, Barker PE, Xiao Y, Käs JA, Melzer M
Quantum dots--a versatile tool in plant science?
J Nanobiotechnology. 2006;45.
An optically stable, novel class of fluorophores (quantum dots) for in situ hybridisation analysis was tested to investigate their signal stability and intensity in plant chromosome analyses. Detection of hybridisation sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide. Comparison of quantum dots (QDs) with conventional detection systems (Alexa 488) in immunolabeling experiments demonstrated greater sensitivity than the conventional system. In contrast, detection of QDs in in situ hybridisation of several plant chromosomes, using several high-copy sequences, was less sensitive than Alexa 488. Thus, semiconductor nanocrystal fluorophores are more suitable for immunostaining but not for in situ hybridisation of plant chromosomes. [Abstract/Link to Full Text]

Mondalek FG, Zhang YY, Kropp B, Kopke RD, Ge X, Jackson RL, Dormer KJ
The permeability of SPION over an artificial three-layer membrane is enhanced by external magnetic field.
J Nanobiotechnology. 2006;44.
BACKGROUND: Sensorineural hearing loss, a subset of all clinical hearing loss, may be correctable through the use of gene therapy. We are testing a delivery system of therapeutics through a 3 cell-layer round window membrane model (RWM model) that may provide an entry of drugs or genes to the inner ear. We designed an in vitro RWM model similar to the RWM (will be referred to throughout the paper as RWM model) to determine the feasibility of using superparamagnetic iron oxide (Fe3O4) nanoparticles (SPION) for targeted delivery of therapeutics to the inner ear.The RWM model is a 3 cell-layer model with epithelial cells cultured on both sides of a small intestinal submucosal (SIS) matrix and fibroblasts seeded in between. Dextran encapsulated nanoparticle clusters 130 nm in diameter were pulled through the RWM model using permanent magnets with flux density 0.410 Tesla at the pole face. The SIS membranes were harvested at day 7 and then fixed in 4% paraformaldehyde. Transmission electron microscopy and fluorescence spectrophotometry were used to verify transepithelial transport of the SPION across the cell-culture model. Histological sections were examined for evidence of SPION toxicity, as well to generate a timeline of the position of the SPION at different times. SPION also were added to cells in culture to assess in vitro toxicity. RESULTS: Transepithelial electrical resistance measurements confirmed epithelial confluence, as SPION crossed a membrane consisting of three co-cultured layers of cells, under the influence of a magnetic field. Micrographs showed SPION distributed throughout the membrane model, in between cell layers, and sometimes on the surface of cells. TEM verified that the SPION were pulled through the membrane into the culture well below. Fluorescence spectrophotometry quantified the number of SPION that went through the SIS membrane. SPION showed no toxicity to cells in culture. CONCLUSION: A three-cell layer model of the human round window membrane has been constructed. SPION have been magnetically transported through this model, allowing quantitative evaluation of prospective targeted drug or gene delivery through the RWM. Putative in vivo carrier superparamagnetic nanoparticles may be evaluated using this model. [Abstract/Link to Full Text]

Williams DN, Ehrman SH, Pulliam Holoman TR
Evaluation of the microbial growth response to inorganic nanoparticles.
J Nanobiotechnology. 2006;43.
In order to enhance the utilization of inorganic nanoparticles in biological systems, it is important to develop a fundamental understanding of the influence they have on cellular health and function. Experiments were conducted to test silica, silica/iron oxide, and gold nanoparticles for their effects on the growth and activity of Escherichia coli (E. coli). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the morphology and quantify size distribution of the nanoparticles, respectively. TEM was also used to verify the interactions between composite iron oxide nanoparticles and E. coli. The results from DLS indicated that the inorganic nanoparticles formed small aggregates in the growth media. Growth studies measured the influence of the nanoparticles on cell proliferation at various concentrations, showing that the growth of E. coli in media containing the nanoparticles indicated no overt signs of toxicity. [Abstract/Link to Full Text]

Singh P, Destito G, Schneemann A, Manchester M
Canine parvovirus-like particles, a novel nanomaterial for tumor targeting.
J Nanobiotechnology. 2006;42.
Specific targeting of tumor cells is an important goal for the design of nanotherapeutics for the treatment of cancer. Recently, viruses have been explored as nano-containers for specific targeting applications, however these systems typically require modification of the virus surface using chemical or genetic means to achieve tumor-specific delivery. Interestingly, there exists a subset of viruses with natural affinity for receptors on tumor cells that could be exploited for nanotechnology applications. For example, the canine parvovirus (CPV) utilizes transferrin receptors (TfRs) for binding and cell entry into canine as well as human cells. TfRs are over-expressed by a variety of tumor cells and are widely being investigated for tumor-targeted drug delivery. We explored whether the natural tropism of CPV to TfRs could be harnessed for targeting tumor cells. Towards this goal, CPV virus-like particles (VLPs) produced by expression of the CPV-VP2 capsid protein in a baculovirus expression system were examined for attachment of small molecules and delivery to tumor cells. Structural modeling suggested that six lysines per VP2 subunit are presumably addressable for bioconjugation on the CPV capsid exterior. Between 45 and 100 of the possible 360 lysines/particle could be routinely derivatized with dye molecules depending on the conjugation conditions. Dye conjugation also demonstrated that the CPV-VLPs could withstand conditions for chemical modification on lysines. Attachment of fluorescent dyes neither impaired binding to the TfRs nor affected internalization of the 26 nm-sized VLPs into several human tumor cell lines. CPV-VLPs therefore exhibit highly favorable characteristics for development as a novel nanomaterial for tumor targeting. [Abstract/Link to Full Text]

Muys JJ, Alkaisi MM, Melville DO, Nagase J, Sykes P, Parguez GM, Evans JJ
Cellular transfer and AFM imaging of cancer cells using Bioimprint.
J Nanobiotechnology. 2006;41.
A technique for permanently capturing a replica impression of biological cells has been developed to facilitate analysis using nanometer resolution imaging tools, namely the atomic force microscope (AFM). The method, termed Bioimprint, creates a permanent cell 'footprint' in a non-biohazardous Poly (dimethylsiloxane) (PDMS) polymer composite. The transfer of nanometer scale biological information is presented as an alternative imaging technique at a resolution beyond that of optical microscopy. By transferring cell topology into a rigid medium more suited for AFM imaging, many of the limitations associated with scanning of biological specimens can be overcome. Potential for this technique is demonstrated by analyzing Bioimprint replicas created from human endometrial cancer cells. The high resolution transfer of this process is further detailed by imaging membrane morphological structures consistent with exocytosis. The integration of soft lithography to replicate biological materials presents an enhanced method for the study of biological systems at the nanoscale. [Abstract/Link to Full Text]

Kumar S, Chaudhury K, Sen P, Guha SK
Atomic force microscopy: a powerful tool for high-resolution imaging of spermatozoa.
J Nanobiotechnology. 2005 Sep 27;39.
Atomic force microscopy (AFM) has emerged as the only technique capable of real-time imaging of the surface of a living cell at nano-resolution. Since AFM provides the advantage of directly observing living biological cells in their native environment, this technique has found many applications in pharmacology, biotechnology, microbiology, structural and molecular biology, genetics and other biology-related fields. AFM has also proved to be a valuable tool for reproductive biologists. An exhaustive review on the various applications of AFM to sperm cells is presented. AFM has been extensively applied for determining the structural and topological features of spermatozoa. Unstained, unfixed spermatozoa in their natural physiological surroundings can be imaged by this technique which provides valuable information about the morphological and pathological defects in sperm cells as three-dimensional images with precise topographical details. Sperm head defects and the acrosome at the tip of the head responsible for fertilization, can be examined and correlated with the lack of functional integrity of the cell. Considerable amount of work is reported on the structural details of the highly condensed chromatin in sperm head using AFM. Detailed information on 3D topographical images of spermatozoa acquired by AFM is expected to provide a better understanding of various reproductive pathways which, in turn, can facilitate improved infertility management and/or contraceptive development. [Abstract/Link to Full Text]

Durán N, Marcato PD, Alves OL, Souza GI, Esposito E
Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains.
J Nanobiotechnology. 2005 Jul 13;38.
Extracellular production of metal nanoparticles by several strains of the fungus Fusarium oxysporum was carried out. It was found that aqueous silver ions when exposed to several Fusarium oxysporum strains are reduced in solution, thereby leading to the formation of silver hydrosol. The silver nanoparticles were in the range of 20-50 nm in dimensions. The reduction of the metal ions occurs by a nitrate-dependent reductase and a shuttle quinone extracellular process. The potentialities of this nanotechnological design based in fugal biosynthesis of nanoparticles for several technical applications are important, including their high potential as antibacterial material. [Abstract/Link to Full Text]

Thibault C, Le Berre V, Casimirius S, Trévisiol E, François J, Vieu C
Direct microcontact printing of oligonucleotides for biochip applications.
J Nanobiotechnology. 2005 Jul 1;3(1):7.
BACKGROUND: A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips. RESULTS: Contrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane) material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene. CONCLUSION: The microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting technology. [Abstract/Link to Full Text]

Elechiguerra JL, Burt JL, Morones JR, Camacho-Bragado A, Gao X, Lara HH, Yacaman MJ
Interaction of silver nanoparticles with HIV-1.
J Nanobiotechnology. 2005 Jun 29;36.
The interaction of nanoparticles with biomolecules and microorganisms is an expanding field of research. Within this field, an area that has been largely unexplored is the interaction of metal nanoparticles with viruses. In this work, we demonstrate that silver nanoparticles undergo a size-dependent interaction with HIV-1, with nanoparticles exclusively in the range of 1-10 nm attached to the virus. The regular spatial arrangement of the attached nanoparticles, the center-to-center distance between nanoparticles, and the fact that the exposed sulfur-bearing residues of the glycoprotein knobs would be attractive sites for nanoparticle interaction suggest that silver nanoparticles interact with the HIV-1 virus via preferential binding to the gp120 glycoprotein knobs. Due to this interaction, silver nanoparticles inhibit the virus from binding to host cells, as demonstrated in vitro. [Abstract/Link to Full Text]

von Nickisch-Rosenegk M, Ehrentreich-Forster E, Strehlow R, Christmann A, Bier FF
Chemically synthesized zinc finger molecules as nano-addressable probes for double-stranded DNAs.
J Nanobiotechnology. 2005 Jun 29;3(1):5.
Our experiments describe an alternative method of dsDNA recognition using zinc finger (ZF) molecules which bind DNA specifically and with high affinity. Our aim was to develop zinc finger probes which are able to bind to dsDNA molecules at predetermined sites. In our basic approach we used pairs of complementary oligonucleotides to form dsDNAs, containing one of the three SP1-transcription factor motifs as a zinc finger recognition site. Two zinc finger probes of the SP1 motif were chemically synthesized and modified with a Dy-633 fluorophore. The SP1 peptides were folded into functional zinc fingers using zinc chloride. The addressable dsDNAs were immobilized on optical fibres, and the kinetics and binding rates of the artificial zinc finger probes were measured by a fluorescence detecting device (photomultiplying tube). The two zinc fingers and their corresponding DNA recognition sites served as specific probes and controls for the matching site and vice versa. Our experiments showed that a variety of dsDNA-binding probes may be created by modification of the amino acid sequence of natural zinc finger proteins. Our findings offer an alternative approach of addressing dsDNA molecules, for example for use in a nanoarray device. [Abstract/Link to Full Text]

Kojima K, Kaneko T, Yasuda K
Stability of beating frequency in cardiac myocytes by their community effect measured by agarose microchamber chip.
J Nanobiotechnology. 2005 May 31;3(1):4.
To understand the contribution of community effect on the stability of beating frequency in cardiac myocyte cell groups, the stepwise network formation of cells as the reconstructive approach using the on-chip agarose microchamber cell microcultivation system with photo-thermal etching method was applied. In the system, the shapes of agarose microstructures were changed step by step with photo-thermal etching of agarose-layer of the chip using a 1064-nm infrared focused laser beam to increase the interaction of cardiac myocyte cells during cultivation. First, individual rat cardiac myocyte in each microstructure were cultivated under isolated condition, and then connected them one by one through newly-created microchannels by photo-thermal etching to compare the contribution of community size for the magnitude of beating stability of the cell groups. Though the isolated individual cells have 50% fluctuation of beating frequency, their stability increased as the number of connected cells increased. And finally when the number reached to eight cells, they stabilized around the 10% fluctuation, which was the same magnitude of the tissue model cultivated on the dish. The result indicates the importance of the community size of cells to stabilize their performance for making cell-network model for using cells for monitoring their functions like the tissue model. [Abstract/Link to Full Text]

Recent Articles in BMC Medical Imaging

Karaçali B, Vamvakidou AP, Tözeren A
Automated recognition of cell phenotypes in histology images based on membrane- and nuclei-targeting biomarkers.
BMC Med Imaging. 2007;77.
BACKGROUND: Three-dimensional in vitro culture of cancer cells are used to predict the effects of prospective anti-cancer drugs in vivo. In this study, we present an automated image analysis protocol for detailed morphological protein marker profiling of tumoroid cross section images. METHODS: Histologic cross sections of breast tumoroids developed in co-culture suspensions of breast cancer cell lines, stained for E-cadherin and progesterone receptor, were digitized and pixels in these images were classified into five categories using k-means clustering. Automated segmentation was used to identify image regions composed of cells expressing a given biomarker. Synthesized images were created to check the accuracy of the image processing system. RESULTS: Accuracy of automated segmentation was over 95% in identifying regions of interest in synthesized images. Image analysis of adjacent histology slides stained, respectively, for Ecad and PR, accurately predicted regions of different cell phenotypes. Image analysis of tumoroid cross sections from different tumoroids obtained under the same co-culture conditions indicated the variation of cellular composition from one tumoroid to another. Variations in the compositions of cross sections obtained from the same tumoroid were established by parallel analysis of Ecad and PR-stained cross section images. CONCLUSION: Proposed image analysis methods offer standardized high throughput profiling of molecular anatomy of tumoroids based on both membrane and nuclei markers that is suitable to rapid large scale investigations of anti-cancer compounds for drug development. [Abstract/Link to Full Text]

Syvänen S, Eriksson J, Genchel T, Lindhe O, Antoni G, Långström B
Synthesis of two potential NK1-receptor ligands using [1-11C]ethyl iodide and [1-11C]propyl iodide and initial PET-imaging.
BMC Med Imaging. 2007;76.
BACKGROUND: The previously validated NK1-receptor ligand [O-methyl-11C]GR205171 binds with a high affinity to the NK1-receptor and displays a slow dissociation from the receptor. Hence, it cannot be used in vivo for detecting concentration changes in substance P, the endogenous ligand for the NK1-receptor. A radioligand used for monitoring these changes has to enable displacement by the endogenous ligand and thus bind reversibly to the receptor. Small changes in the structure of a receptor ligand can lead to changes in binding characteristics and also in the ability to penetrate the blood-brain barrier. The aim of this study was to use carbon-11 labelled ethyl and propyl iodide with high specific radioactivity in the synthesis of two new and potentially reversible NK1-receptor ligands with chemical structures based on [O-methyl-11C]GR205171. METHODS: [1-11C]Ethyl and [1-11C]propyl iodide with specific radioactivities of 90 GBq/mumol and 270 GBq/mumol, respectively, were used in the synthesis of [O-methyl-11C]GR205171 analogues by alkylation of O-desmethyl GR205171. The brain uptake of the obtained (2S,3S)-N-(1-(2- [1-11C]ethoxy-5-(3-(trifluoromethyl)-4H-1,2,4-triazol-4-yl)phenyl)ethyl)-2-phenylpiperidin-3-amine (I) and (2S,3S)-2-phenyl-N-(1-(2- [1-11C]propoxy-5-(3-(trifluoromethyl)-4H-1,2,4-triazol-4-yl)phenyl)ethyl)piperidin-3-amine (II) was studied with PET in guinea pigs and rhesus monkeys and compared to the uptake of [O-methyl-11C]GR205171. RESULTS: All ligands had similar uptake distribution in the guinea pig brain. The PET-studies in rhesus monkeys showed that (II) had no specific binding in striatum. Ligand (I) had moderate specific binding compared to the [O-methyl-11C]GR205171. The ethyl analogue (I) displayed reversible binding characteristics contrary to the slow dissociation rate shown by [O-methyl-11C]GR205171. CONCLUSION: The propyl-analogue (II) cannot be used for detecting changes in NK1-ligand levels, while further studies should be performed with the ethyl-analogue (I). [Abstract/Link to Full Text]

Dauer LT, Casciotta KA, Erdi YE, Rothenberg LN
Radiation dose reduction at a price: the effectiveness of a male gonadal shield during helical CT scans.
BMC Med Imaging. 2007;75.
BACKGROUND: It is estimated that 60 million computed tomography (CT) scans were performed during 2006, with approximately 11% of those performed on children age 0-15 years. Various types of gonadal shielding have been evaluated for reducing exposure to the gonads. The purpose of this study was to quantify the radiation dose reduction to the gonads and its effect on image quality when a wrap-around male pediatric gonad shield was used during CT scanning. This information is obtained to assist the attending radiologist in the decision to utilize such male gonadal shields in pediatric imaging practice. METHODS: The dose reduction to the gonads was measured for both direct radiation and for indirect scattered radiation from the abdomen. A 6 cm3 ion chamber (Model 10X5-6, Radcal Corporation, Monrovia, CA) was placed on a Humanoid real bone pelvic phantom at a position of the male gonads. When exposure measurements with shielding were made, a 1 mm lead wrap-around gonadal shield was placed around the ion chamber sensitive volume. RESULTS: The use of the shields reduced scatter dose to the gonads by a factor of about 2 with no appreciable loss of image quality. The shields reduced the direct beam dose by a factor of about 35 at the expense of extremely poor CT image quality due to severe streak artifacts. CONCLUSION: Images in the direct exposure case are not useful due to these severe artifacts and the difficulties in positioning these shields on patients in the scatter exposure case may not be warranted by the small absolute reduction in scatter dose unless it is expected that the patient will be subjected to numerous future CT scans. [Abstract/Link to Full Text]

Moncayo R, Rudisch A, Diemling M, Kremser C
In-vivo visualisation of the anatomical structures related to the acupuncture points Dai mai and Shen mai by MRI: a single-case pilot study.
BMC Med Imaging. 2007;74.
BACKGROUND: The concept of acupuncture point localisation in Traditional Chinese Medicine (TCM) is based on millenary practical experience. Modern imaging methods such as PET, MRI and SPECT have been used primary for the investigation of the mechanisms of action of acupuncture. In this pilot single-case study we have evaluated the technical possibilities for in-vivo imaging of the anatomical relations of acupuncture points using state of the art MRI. METHODS: Preliminary experiments relating to the quality of acupuncture needles under the setting of MRI were done both with stainless steel and gold needles. In a second step, in-vivo imaging was carried out. A licensed acupuncture practitioner (RM) chose two points belonging to the so-called extraordinary vessels. In 2 sequential, separate procedures, he inserted himself gold acupuncture needles using a neutral technique (known as Ping Bu Ping Xie) into the Dai mai and Shen mai points, i.e. gall bladder 26 and bladder 62. Imaging was done on a Siemens Magnetom Avanto MR scanner using a head array and body coil. Mainly T1-weighted imaging sequences, as routinely used for patient exams, were used to obtain multi-slice images. RESULTS: In the preliminary experiments only acupuncture needles made of gold showed enough stability in order to be used for further imaging procedures. Using an onion and a banana as an object, further studies showed that the gold needles produced a void defect that corresponds to the tip of the inserted needle, while at the same time an artefactually increased diameter was observed. The in-vivo experiments showed that the Dai mai point was in relation to the abdominal internal oblique muscle. The Shen mai point artefact showed up close to the longus and brevis peroneal tendons at the fibular malleolus. Side effects related to heating or burning were not observed. Improved anatomical recognition was obtained using 3D-volume rendering techniques. CONCLUSION: Through an adequate choice of acupuncture material (gold needles) as well as of ideal MRI imaging sequences it has been possible to visualize the anatomical characteristics at the acupuncture points Dai mai and Shen mai in-vivo. At the selected sites the needles showed a relation to tendino-fascial and muscular structures. These anatomical structures fit well into the recently described WOMED concept of lateral tension in which these acupuncture points play a regulatory role. [Abstract/Link to Full Text]

Al-Attar SA, Pollex RL, Robinson JF, Miskie BA, Walcarius R, Little CH, Rutt BK, Hegele RA
Quantitative and qualitative differences in subcutaneous adipose tissue stores across lipodystrophy types shown by magnetic resonance imaging.
BMC Med Imaging. 2007;73.
BACKGROUND: Lipodystrophies are characterized by redistributed subcutaneous fat stores. We previously quantified subcutaneous fat by magnetic resonance imaging (MRI) in the legs of two patients with familial partial lipodystrophy subtypes 2 and 3 (FPLD2 and FPLD3, respectively). We now extend the MRI analysis across the whole body of patients with different forms of lipodystrophy. METHODS: We studied five subcutaneous fat stores (supraclavicular, abdominal, gluteal, thigh and calf) and the abdominal visceral fat stores in 10, 2, 1, 1 and 2 female subjects with, respectively, FPLD2, FPLD3, HIV-related partial lipodystrophy (HIVPL), acquired partial lipodystrophy (APL), congenital generalized lipodystrophy (CGL) and in six normal control subjects. RESULTS: Compared with normal controls, FPLD2 subjects had significantly increased supraclavicular fat, with decreased abdominal, gluteal, thigh and calf subcutaneous fat. FPLD3 subjects had increased supraclavicular and abdominal subcutaneous fat, with less severe reductions in gluteal, thigh and calf fat compared to FPLD2 subjects. The repartitioning of fat in the HIVPL subject closely resembled that of FPLD3 subjects. APL and CGL subjects had reduced upper body, gluteal and thigh subcutaneous fat; the APL subject had increased, while CGL subjects had decreased subcutaneous calf fat. Visceral fat was markedly increased in FPLD2 and APL subjects. CONCLUSION: Semi-automated MRI-based adipose tissue quantification indicates differences between various lipodystrophy types in these studied clinical cases and is a potentially useful tool for extended quantitative phenomic analysis of genetic metabolic disorders. Further studies with a larger sample size are essential for confirming these preliminary findings. [Abstract/Link to Full Text]

Karaçali B, Tözeren A
Automated detection of regions of interest for tissue microarray experiments: an image texture analysis.
BMC Med Imaging. 2007;72.
BACKGROUND: Recent research with tissue microarrays led to a rapid progress toward quantifying the expressions of large sets of biomarkers in normal and diseased tissue. However, standard procedures for sampling tissue for molecular profiling have not yet been established. METHODS: This study presents a high throughput analysis of texture heterogeneity on breast tissue images for the purpose of identifying regions of interest in the tissue for molecular profiling via tissue microarray technology. Image texture of breast histology slides was described in terms of three parameters: the percentage of area occupied in an image block by chromatin (B), percentage occupied by stroma-like regions (P), and a statistical heterogeneity index H commonly used in image analysis. Texture parameters were defined and computed for each of the thousands of image blocks in our dataset using both the gray scale and color segmentation. The image blocks were then classified into three categories using the texture feature parameters in a novel statistical learning algorithm. These categories are as follows: image blocks specific to normal breast tissue, blocks specific to cancerous tissue, and those image blocks that are non-specific to normal and disease states. RESULTS: Gray scale and color segmentation techniques led to identification of same regions in histology slides as cancer-specific. Moreover the image blocks identified as cancer-specific belonged to those cell crowded regions in whole section image slides that were marked by two pathologists as regions of interest for further histological studies. CONCLUSION: These results indicate the high efficiency of our automated method for identifying pathologic regions of interest on histology slides. Automation of critical region identification will help minimize the inter-rater variability among different raters (pathologists) as hundreds of tumors that are used to develop an array have typically been evaluated (graded) by different pathologists. The region of interest information gathered from the whole section images will guide the excision of tissue for constructing tissue microarrays and for high throughput profiling of global gene expression. [Abstract/Link to Full Text]

Schweitzer W, Häusler M, Bär W, Schaepman M
Evaluation of 3D surface scanners for skin documentation in forensic medicine: comparison of benchmark surfaces.
BMC Med Imaging. 2007;71.
BACKGROUND: Two 3D surface scanners using collimated light patterns were evaluated in a new application domain: to document details of surfaces similar to the ones encountered in forensic skin pathology. Since these scanners have not been specifically designed for forensic skin pathology, we tested their performance under practical constraints in an application domain that is to be considered new. METHODS: Two solid benchmark objects containing relevant features were used to compare two 3D surface scanners: the ATOS-II (GOM, Germany) and the QTSculptor (Polygon Technology, Germany). Both scanners were used to capture and process data within a limited amount of time, whereas point-and-click editing was not allowed. We conducted (a) a qualitative appreciation of setup, handling and resulting 3D data, (b) an experimental subjective evaluation of matching 3D data versus photos of benchmark object regions by a number of 12 judges who were forced to state their preference for either of the two scanners, and (c) a quantitative characterization of both 3D data sets comparing 220 single surface areas with the real benchmark objects in order to determine the recognition rate's possible dependency on feature size and geometry. RESULTS: The QTSculptor generated significantly better 3D data in both qualitative tests (a, b) that we had conducted, possibly because of a higher lateral point resolution; statistical evaluation (c) showed that the QTSculptor-generated data allowed the discrimination of features as little as 0.3 mm, whereas ATOS-II-generated data allowed for discrimination of features sized not smaller than 1.2 mm. CONCLUSION: It is particularly important to conduct specific benchmark tests if devices are brought into new application domains they were not specifically designed for; using a realistic test featuring forensic skin pathology features, QT Sculptor-generated data quantitatively exceeded manufacturer's specifications, whereas ATOS-II-generated data was within the limits of the manufacturer's specifications. When designing practically constrained specific tests, benchmark objects should be designed to contain features relevant for the application domain. As costs for 3D scanner hardware, software and data analysis can be hundred times as high compared to high-resolution digital photography equipment, independent user driven evaluation of such systems is paramount. INDEX TERMS: Forensic pathology, Rough surfaces, Surface Scanning, Technology Assessment. [Abstract/Link to Full Text]

Bilgen M
Imaging corticospinal tract connectivity in injured rat spinal cord using manganese-enhanced MRI.
BMC Med Imaging. 2006;615.
BACKGROUND: Manganese-enhanced MRI (MEI) offers a novel neuroimaging modality to trace corticospinal tract (CST) in live animals. This paper expands this capability further and tests the utility of MEI to image axonal fiber connectivity in CST of injured spinal cord (SC). METHODS: A rat was injured at the thoracic T4 level of the SC. The CST was labeled with manganese (Mn) injected intracortically at two weeks post injury. Next day, the injured SC was imaged using MEI and diffusion tensor imaging (DTI) modalities. RESULTS: In vivo MEI data obtained from cervical SC confirmed that CST was successfully labeled with Mn. Ex vivo MEI data obtained from excised SC depicted Mn labeling of the CST in SC sections caudal to the lesion, which meant that Mn was transported through the injury, possibly mediated by viable CST fibers present at the injury site. Examining the ex vivo data from the injury epicenter closely revealed a thin strip of signal enhancement located ventrally between the dorsal horns. This enhancement was presumably associated with the Mn accumulation in these intact fibers projecting caudally as part of the CST. Additional measurements with DTI supported this view. CONCLUSION: Combining these preliminary results collectively demonstrated the feasibility of imaging fiber connectivity in experimentally injured SC using MEI. This approach may play important role in future investigations aimed at understanding the neuroplasticity in experimental SCI research. [Abstract/Link to Full Text]

Petushi S, Garcia FU, Haber MM, Katsinis C, Tozeren A
Large-scale computations on histology images reveal grade-differentiating parameters for breast cancer.
BMC Med Imaging. 2006;614.
BACKGROUND: Tumor classification is inexact and largely dependent on the qualitative pathological examination of the images of the tumor tissue slides. In this study, our aim was to develop an automated computational method to classify Hematoxylin and Eosin (H&E) stained tissue sections based on cancer tissue texture features. METHODS: Image processing of histology slide images was used to detect and identify adipose tissue, extracellular matrix, morphologically distinct cell nuclei types, and the tubular architecture. The texture parameters derived from image analysis were then applied to classify images in a supervised classification scheme using histologic grade of a testing set as guidance. RESULTS: The histologic grade assigned by pathologists to invasive breast carcinoma images strongly correlated with both the presence and extent of cell nuclei with dispersed chromatin and the architecture, specifically the extent of presence of tubular cross sections. The two parameters that differentiated tumor grade found in this study were (1) the number density of cell nuclei with dispersed chromatin and (2) the number density of tubular cross sections identified through image processing as white blobs that were surrounded by a continuous string of cell nuclei. Classification based on subdivisions of a whole slide image containing a high concentration of cancer cell nuclei consistently agreed with the grade classification of the entire slide. CONCLUSION: The automated image analysis and classification presented in this study demonstrate the feasibility of developing clinically relevant classification of histology images based on micro- texture. This method provides pathologists an invaluable quantitative tool for evaluation of the components of the Nottingham system for breast tumor grading and avoid intra-observer variability thus increasing the consistency of the decision-making process. [Abstract/Link to Full Text]

Mukerji N, Wallace D, Mitra D
Audit of the change in the on-call practices in neuroradiology and factors affecting it.
BMC Med Imaging. 2006;613.
BACKGROUND: On call practices had recently changed at the Newcastle General Hospital to accommodate increasing CT scan requests and reduce the workloads of the radiologists. In the new system, the person responsible for dealing with the out of hours requests for imaging changed from the neuroradiologist to the neuroradiographer. This audit was conducted to assess any change in the departmental workload as a result of this change. METHODS: The audit was carried out over a period of six months and data was collected from the on-call booklets which the neuroradiographers maintained and the log books maintained in the department of neuroradiology. Details of the imaging requested; the source of the request, the reason for the request and the results of the scans were recorded and analysed using Microsoft Excel. RESULTS: The number of CT scans requested from the A&E went up by 73.4% after the change in practice and majority of these increases were due to increased requests for scans on head injuries which increased by 122%. Although this was not statistically significant due to lack of study power, it is clinically relevant. CONCLUSION: The increase in the number of CT scans for head injuries reflects a general change in practice in management of head injuries in the UK. Changing the gatekeeper from radiologist to radiographer was associated with an increase in CT rate, particularly for head injuries. Other factors such as clinician seniority and a greater awareness of the NICE guidelines may have also contributed. [Abstract/Link to Full Text]

Wood BR, Bambery KR, Evans CJ, Quinn MA, McNaughton D
A three-dimensional multivariate image processing technique for the analysis of FTIR spectroscopic images of multiple tissue sections.
BMC Med Imaging. 2006;612.
BACKGROUND: Three-dimensional (3D) multivariate Fourier Transform Infrared (FTIR) image maps of tissue sections are presented. A villoglandular adenocarcinoma from a cervical biopsy with a number of interesting anatomical features was used as a model system to demonstrate the efficacy of the technique. METHODS: Four FTIR images recorded using a focal plane array detector of adjacent tissue sections were stitched together using a MATLAB routine and placed in a single data matrix for multivariate analysis using Cytospec. Unsupervised Hierarchical Cluster Analysis (UHCA) was performed simultaneously on all 4 sections and 4 clusters plotted. The four UHCA maps were then stacked together and interpolated with a box function using SCIRun software. RESULTS: The resultant 3D-images can be rotated in three-dimensions, sliced and made semi-transparent to view the internal structure of the tissue block. A number of anatomical and histopathological features including connective tissue, red blood cells, inflammatory exudate and glandular cells could be identified in the cluster maps and correlated with Hematoxylin & Eosin stained sections. The mean extracted spectra from individual clusters provide macromolecular information on tissue components. CONCLUSION: 3D-multivariate imaging provides a new avenue to study the shape and penetration of important anatomical and histopathological features based on the underlying macromolecular chemistry and therefore has clear potential in biology and medicine. [Abstract/Link to Full Text]

Al-Attar SA, Pollex RL, Robinson JF, Miskie BA, Walcarius R, Rutt BK, Hegele RA
Semi-automated segmentation and quantification of adipose tissue in calf and thigh by MRI: a preliminary study in patients with monogenic metabolic syndrome.
BMC Med Imaging. 2006;611.
BACKGROUND: With the growing prevalence of obesity and metabolic syndrome, reliable quantitative imaging methods for adipose tissue are required. Monogenic forms of the metabolic syndrome include Dunnigan-variety familial partial lipodystrophy subtypes 2 and 3 (FPLD2 and FPLD3), which are characterized by the loss of subcutaneous fat in the extremities. Through magnetic resonance imaging (MRI) of FPLD patients, we have developed a method of quantifying the core FPLD anthropometric phenotype, namely adipose tissue in the mid-calf and mid-thigh regions. METHODS: Four female subjects, including an FPLD2 subject (LMNA R482Q), an FPLD3 subject (PPARG F388L), and two control subjects were selected for MRI and analysis. MRI scans of subjects were performed on a 1.5T GE MR Medical system, with 17 transaxial slices comprising a 51 mm section obtained in both the mid-calf and mid-thigh regions. Using ImageJ 1.34 n software, analysis of raw MR images involved the creation of a connectedness map of the subcutaneous adipose tissue contours within the lower limb segment from a user-defined seed point. Quantification of the adipose tissue was then obtained after thresholding the connected map and counting the voxels (volumetric pixels) present within the specified region. RESULTS: MR images revealed significant differences in the amounts of subcutaneous adipose tissue in lower limb segments of FPLD3 and FPLD2 subjects: respectively, mid-calf, 15.5% and 0%, and mid-thigh, 25.0% and 13.3%. In comparison, old and young healthy controls had values, respectively, of mid-calf, 32.5% and 26.2%, and mid-thigh, 52.2% and 36.1%. The FPLD2 patient had significantly reduced subcutaneous adipose tissue compared to FPLD3 patient. CONCLUSION: Thus, semi-automated quantification of adipose tissue of the lower extremity can detect differences between individuals of various lipodystrophy genotypes and represents a potentially useful tool for extended quantitative phenotypic analysis of other genetic metabolic disorders. [Abstract/Link to Full Text]

Adebiyi AA, Ogah OS, Aje A, Ojji DB, Adebayo AK, Oladapo OO, Falase AO
Echocardiographic partition values and prevalence of left ventricular hypertrophy in hypertensive Nigerians.
BMC Med Imaging. 2006;610.
BACKGROUND: Left ventricular hypertrophy (LVH) is a well known independent risk factor for cardiovascular events. It has been shown that combination of left ventricular mass (LVM) and relative wall thickness (RWT) can be used to identify different forms of left ventricular (LV) geometry. Prospective studies have shown that LV geometric patterns have prognostic implications, with the worst prognosis associated with concentric hypertrophy. The methods for the normalization or indexation of LVM have also recently been shown to confer some prognostic value especially in obese population. We sought to determine the prevalence of echocardiographic lLVH using eight different and published cut-off or threshold values in hypertensive subjects seen in a developing country's tertiary centre. METHODS: Echocardiography was performed in four hundred and eighty consecutive hypertensive subjects attending the cardiology clinic of the University college Hospital Ibadan, Nigeria over a two-year period. RESULTS: Complete data was obtained in 457 (95.2%) of the 480 subjects (48.6% women). The prevalence of LVH ranged between 30.9-56.0%. The highest prevalence was when LVM was indexed to the power of 2.7 with a partition value of 49.2 g/ht2.7 in men and 46.7 g/ht2.7 in women. The lowest prevalence was observed when LVM was indexed to body surface area (BSA) and a partition value of 125 g/m2 was used for both sexes. Abnormal LV geometry was present in 61.1%-74.0% of our subjects and commoner in women. CONCLUSION: The prevalence of LVH hypertensive patients is strongly dependent on the cut-off value used to define it. Large-scale prospective study will be needed to determine the prognostic implications of the different LV geometry in native Africans. [Abstract/Link to Full Text]

Kaltenthaler EC, Walters SJ, Chilcott J, Blakeborough A, Vergel YB, Thomas S
MRCP compared to diagnostic ERCP for diagnosis when biliary obstruction is suspected: a systematic review.
BMC Med Imaging. 2006;69.
BACKGROUND: Magnetic resonance cholangiopancreatography (MRCP) is an alternative to diagnostic endoscopic retrograde cholangiopancreatography (ERCP) for investigating biliary obstruction. The use of MRCP, a non-invasive procedure, may prevent the use of unnecessary invasive procedures. The aim of the study was to compare the findings of MRCP with those of ERCP by the computation of accuracy statistics. METHODS: Thirteen electronic bibliographic databases, covering biomedical, science, health economics and grey literature were searched. A systematic review of studies comparing MRCP to diagnostic ERCP in patients with suspected biliary obstruction was conducted. Sensitivity, specificity, likelihood ratios, acceptability and adverse events were reported. RESULTS: 25 studies were identified reporting several conditions including choledocholithiasis (18 studies), malignancy (four studies), obstruction (three studies), stricture (two studies) and dilatation (five studies). Three of the 18 studies reporting choledocholithiasis were excluded from the analysis due to lack of data, or differences in study design. The sensitivity for the 15 studies of choledocholithiasis ranged from 0.50 to 1.00 while specificity ranged from 0.83 to 1.00. The positive likelihood ratio ranged: from 5.44-47.72 and the negative likelihood ratio for the 15 studies ranged from 0.00-0.51. Significant heterogeneity was found across the 15 studies so the sensitivities and specificities were summarised by a Receiver Operating Characteristic (ROC) curve. For malignancy, sensitivity ranged from 0.81 to 0.94 and specificity from 0.92 to 1.00. Positive likelihood ratios ranged from 10.12 to 43 and negative likelihood ratios ranged from 0.15 to 0.21, although these estimates were less reliable. CONCLUSION: MRCP is a comparable diagnostic investigation in comparison to ERCP for diagnosing biliary obstruction. [Abstract/Link to Full Text]

Graat ME, Hendrikse KA, Spronk PE, Korevaar JC, Stoker J, Schultz MJ
Chest radiography practice in critically ill patients: a postal survey in the Netherlands.
BMC Med Imaging. 2006;68.
BACKGROUND: To ascertain current chest radiography practice in intensive care units (ICUs) in the Netherlands. METHODS: Postal survey: a questionnaire was sent to all ICUs with > 5 beds suitable for mechanical ventilation; pediatric ICUs were excluded. When an ICU performed daily-routine chest radiographs in any group of patients it was considered to be a "daily-routine chest radiography" ICU. RESULTS: From the number of ICUs responding, 63% practice a daily-routine strategy, in which chest radiographs are obtained on a daily basis without any specific reason. A daily-routine chest radiography strategy is practiced less frequently in university-affiliated ICUs (50%) as compared to other ICUs (68%), as well as in larger ICUs (> 20 beds, 50%) as compared to smaller ICUs (< 20 beds, 65%) (P > 0.05). Remarkably, physicians that practice a daily-routine strategy consider daily-routine radiographs helpful in guiding daily practice in less than 30% of all performed radiographs. Chest radiographs are considered essential for verification of the position of invasive devices (81%) and for diagnosing pneumothorax, pneumonia or acute respiratory distress syndrome (82%, 74% and 69%, respectively). On demand chest radiographs are obtained after introduction of thoracic drains, central venous lines and endotracheal tubes in 98%, 84% and 75% of responding ICUs, respectively. Chest films are also obtained in case of ventilatory deterioration (49% of responding ICUs), and after cardiopulmonary resuscitation (59%), tracheotomy (58%) and mini-tracheotomy (23%). CONCLUSION: There is notable lack of consensus on chest radiography practice in the Netherlands. This survey suggests that a large number of intensivists may doubt the value of daily-routine chest radiography, but still practice a daily-routine strategy. [Abstract/Link to Full Text]

Tossios P, Müller-Ehmsen J, Schmidt M, Scheid C, Unal N, Moka D, Schwinger RH, Mehlhorn U
No evidence of myocardial restoration following transplantation of mononuclear bone marrow cells in coronary bypass grafting surgery patients based upon cardiac SPECT and 18F-PET.
BMC Med Imaging. 2006;67.
BACKGROUND: We tested the hypothesis, that intramyocardial injection of mononuclear bone marrow cells combined with coronary artery bypass grafting (CABG) surgery improves tissue viability or function in infarct regions with non-viable myocardium as assessed by nuclear imaging techniques. METHODS: Thus far, 7 patients (60 +/- 10 [SD] years) undergoing elective CABG surgery after a myocardial infarction were included in this study. Prior to sternotomy, bone marrow was harvested by sternal puncture. Mononuclear bone marrow cells were isolated by gradient centrifugation and resuspended in 2 ml volume of Hank's buffered salt solution. At the end of CABG surgery 10 injections of 0.2 ml each were applied to the core area and borderzones of the infarct. Global and regional perfusion and viability were evaluated by ECG-gated 99mTc-tetrofosmin myocardial single-photon emission computed tomograph (SPECT) imaging and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) in all study patients < 6 days before and 3 months after the intervention. RESULTS: Non-viable segments indicating transmural defects were identified in 5 patients. Two patients were found to have non-transmural defects before surgery. Concomitant surgical revascularisation and bone marrow cell injection was performed in all patients without major complications. The median total injected mononuclear cell number was 7.0 x 10(7) (range: 0.8-20.4). At 3 months 99mTc-tetrofosmin SPECT and 18F-FDG-PET scanning showed in 5 patients (transmural defect n = 4; non-transmural defect n = 1) no change in myocardial viability and in two patients (transmural defect n = 1, non-transmural defect n = 1) enhanced myocardial viability by 75%. Overall, global and regional LV ejection fraction was not significantly increased after surgery compared with the preoperative value. CONCLUSION: In CABG surgery patients with non-viable segments the concurrent use of intramyocardial cell transfer did not show any clear improvement in tissue viability or function by means of non-invasive bioimaging techniques. [Abstract/Link to Full Text]

Hujoel P, Hollender L, Bollen AM, Young JD, Cunha-Cruz J, McGee M, Grosso A
Thyroid shields and neck exposures in cephalometric radiography.
BMC Med Imaging. 2006;66.
BACKGROUND: The thyroid is among the more radiosensitive organs in the body. The goal of this study was twofold: (1) to evaluate age-related changes in what is exposed to ionizing radiation in the neck area, and (2) to assess thyroid shield presence in cephalometric radiographs METHODS: Cephalometric radiographs at one academic setting were sampled and neck exposure was related to calendar year and patient's gender and age. RESULTS: In the absence of shields, children have more vertebrae exposed than adults (p < 0.0001) and females have more neck tissue exposed inferior to the hyoid bone than males (p < 0.0001). The hyoid bone-porion distance increased with age (p <0.01). Thyroid shields were visible in 19% of the radiographs and depended strongly on the calendar year during which patient was seen (p-value <0.0001). Compared to adults, children were less likely to wear thyroid shields, particularly between 1973 and 1990 (1.8% versus 7.3% -p-value < 0.05) and between 2001 and 2003 (7.1% versus 42.9% -p-value < 0.05). CONCLUSION: In the absence of a thyroid shield, children have more neck structure exposed to radiation than adults. In agreement with other reports, thyroid shield utilization in this study was low, particularly in children. [Abstract/Link to Full Text]

Mainardi LT, Passera KM, Lucesoli A, Potepan P, Setti E, Musumeci R
A method for dynamic subtraction MR imaging of the liver.
BMC Med Imaging. 2006;65.
BACKGROUND: Subtraction of Dynamic Contrast-Enhanced 3D Magnetic Resonance (DCE-MR) volumes can result in images that depict and accurately characterize a variety of liver lesions. However, the diagnostic utility of subtraction images depends on the extent of co-registration between non-enhanced and enhanced volumes. Movement of liver structures during acquisition must be corrected prior to subtraction. Currently available methods are computer intensive. We report a new method for the dynamic subtraction of MR liver images that does not require excessive computer time. METHODS: Nineteen consecutive patients (median age 45 years; range 37-67) were evaluated by VIBE T1-weighted sequences (TR 5.2 ms, TE 2.6 ms, flip angle 20 degrees , slice thickness 1.5 mm) acquired before and 45s after contrast injection. Acquisition parameters were optimized for best portal system enhancement. Pre and post-contrast liver volumes were realigned using our 3D registration method which combines: (a) rigid 3D translation using maximization of normalized mutual information (NMI), and (b) fast 2D non-rigid registration which employs a complex discrete wavelet transform algorithm to maximize pixel phase correlation and perform multiresolution analysis. Registration performance was assessed quantitatively by NMI. RESULTS: The new registration procedure was able to realign liver structures in all 19 patients. NMI increased by about 8% after rigid registration (native vs. rigid registration 0.073 +/- 0.031 vs. 0.078 +/- 0.031, n.s., paired t-test) and by a further 23% (0.096 +/- 0.035 vs. 0.078 +/- 0.031, p < 0.001, paired t-test) after non-rigid realignment. The overall average NMI increase was 31%. CONCLUSION: This new method for realigning dynamic contrast-enhanced 3D MR volumes of liver leads to subtraction images that enhance diagnostic possibilities for liver lesions. [Abstract/Link to Full Text]

Haidekker MA, Boettcher LW, Suter JD, Rone R, Grant SA
Influence of gold nanoparticles on collagen fibril morphology quantified using transmission electron microscopy and image analysis.
BMC Med Imaging. 2006;64.
BACKGROUND: Development of implantable biosensors for disease detection is challenging because of poor biocompatibility of synthetic materials. A possible solution involves engineering interface materials that promote selfassembly and adhesion of autologous cells on sensor surfaces. Crosslinked type-I collagen is an acceptable material for developing engineered basement membranes. In this study, we used functionalized gold nanoparticles as the crosslinking agent. Functionalized nanoparticles provide sites for crosslinking collagen as well as sites to deliver signaling compounds that direct selfassembly and reduce inflammation. The goal of this study was to obtain a quantitative parameter to objectively determine the presence of crosslinks. METHODS: We analyzed TEM images of collagen fibrils by two methods: Run length analysis and topology analysis after medial axis transform. RESULTS: Run length analysis showed a significant reduction of the interfibril spaces in the presence of nanoparticles (change of 40%, P < 0.05), whereas the fibril thickness remained unchanged. In the topological network, the number of elements, number of branches and number of sides increased significantly in the presence of nanoparticles (P < 0.05). Other parameters, especially the number of loops showed only a minimal and nonsignificant change. We chose a ratiometric parameter of the number of branches normalized by the number of loops to achieve independence from gross fibril density. This parameter is lower by a factor of 2.8 in the presence of nanoparticles (P < 0.05). CONCLUSION: The numerical parameters presented herein allow not only to quantify fibril mesh complexity and crosslinking, but also to help quantitatively compare cell growth and adhesion on collagen matrices of different degree of crosslinking in further studies. [Abstract/Link to Full Text]

Burbridge B, Stoneham G, Szkup P
Percutaneous subclavian artery stent-graft placement following failed ultrasound guided subclavian venous access.
BMC Med Imaging. 2006;63.
BACKGROUND: Ultrasound guidance for central and peripheral venous access has been proven to improve success rates and reduce complications of venous cannulation. Appropriately trained and experienced operators add significantly to diminished patient morbidity related to venous access procedures. We discuss a patient who required an arterial stent-graft to prevent arterial hemorrhage following inadvertent cannulation of the proximal, ventral, right subclavian artery related to unsuccessful ultrasound guided access of the subclavian vein. CASE PRESENTATION: During pre-operative preparation for aortic valve replacement and aorto-coronary bypass surgery an anesthetist attempted ultrasound guided venous access. The ultrasound guided attempt to access the right jugular vein failed and the ultrasound guided attempt at accessing the subclavian vein resulted in inappropriate placement of an 8.5 F sheath in the arterial system. Following angiographic imaging and specialist consultations, an arterial stent-graft was deployed in the right subclavian artery rather than perform an extensive anterior chest wall resection and dissection to extract the arterial sheath. The patient tolerated the procedure, without complication, despite occlusion of the right internal mammary artery and the right vertebral artery. There were no neurologic sequelae. There was no evidence of hemorrhage after subclavian artery sheath extraction and stent-graft implantation. CONCLUSION: The attempted ultrasound guided puncture of the subclavian vein resulted in placement of an 8.5 F subclavian artery catheter. Entry of the catheter into the proximal subclavian artery beneath the medial clavicle, the medial first rib and the manubrium suggests that the operator, most likely, did not directly visualize the puncture needle enter the vessel with the ultrasound. The bones of the anterior chest impede the ultrasound beam and the vessels in this area would not be visible to ultrasound imaging. Appropriate training and supervised experience in ultrasound guided venous access coupled with quality ultrasound equipment would most likely have significantly diminished the likelihood of this complication. The potential for significant patient morbidity, and possible mortality, was prevented by implantation of an arterial stent-graft. [Abstract/Link to Full Text]

Delaney A, Carter A, Fisher M
The prevention of anaphylactoid reactions to iodinated radiological contrast media: a systematic review.
BMC Med Imaging. 2006;62.
BACKGROUND: Anaphylactoid reactions to iodinated contrast media are relatively common and potentially life threatening. Opinion is divided as to the utility of medications for preventing these reactions. We performed a systematic review to assess regimes for the prevention of anaphylactoid reactions to iodinated contrast media. METHODS: Searches for studies were conducted in the Medline, EMBASE, CINAHL and CENTRAL databases. Bibliographies of included studies and review articles were examined and experts were contacted. Randomised clinical trials that examined agents given prior to iodinated contrast material for the prevention of anaphylactoid reactions were included in the review. The validity of the included studies was examined using a component approach. RESULTS: Six studies met the inclusion criteria, but only one of these fulfilled all of the validity criteria. There were four studies that examined the use of H1 antihistamines, each was used to prevent anaphylactoid reactions to ionic contrast. The random effects pooled relative risk demonstrated a significant reduction in the overall rate of anaphylactoid reactions (RR = 0.4, 95% CI 0.18-0.9, p = 0.027). There were insufficient studies to produce a pooled statistic for the use of corticosteroids, however regimes of steroids (methylprednisolone 32 mg) given at least six hours and again two hours prior to the administration of contrast suggested a reduction in the incidence of anaphylactoid reactions. CONCLUSION: In conclusion, there are few high quality randomised clinical trials that have addressed the question of the optimal methods to prevent allergic type reactions to iodinated radiological contrast media. Allowing for these limitations, the results suggest that H1 antihistamines given immediately prior to the administration of ionic contrast may be useful in preventing reactions to ionic contrast and are suggestive of a protective effect of corticosteroids when given in two doses at least six hours prior and again two hours prior to the administration of contrast, both ionic and non-ionic. These agents should be considered for use in patients who are at high risk of an anaphylactoid reaction to contrast media and for who prophylactic therapy is considered necessary. Further research is needed before definitive recommendations can be made. [Abstract/Link to Full Text]

Persson A, Dahlström N, Smedby O, Brismar TB
Three-dimensional drip infusion CT cholangiography in patients with suspected obstructive biliary disease: a retrospective analysis of feasibility and adverse reaction to contrast material.
BMC Med Imaging. 2006;61.
BACKGROUND: Computed Tomography Cholangiography (CTC) is a fast and widely available alternative technique to visualise hepatobiliary disease in patients with an inconclusive ultrasound when MRI cannot be performed. The method has previously been relatively unknown and sparsely used, due to concerns about adverse reactions and about image quality in patients with impaired hepatic function and thus reduced contrast excretion. In this retrospective study, the feasibility and the frequency of adverse reactions of CTC when using a drip infusion scheme based on bilirubin levels were evaluated. METHODS: The medical records of patients who had undergone upper abdominal spiral CT with subsequent three-dimensional rendering of the biliary tract by means of CTC during seven years were retrospectively reviewed regarding serum bilirubin concentration, adverse reaction and presence of visible contrast media in the bile ducts at CT examination. In total, 153 consecutive examinations in 142 patients were reviewed. RESULTS: Contrast media was observed in the bile ducts at 144 examinations. In 110 examinations, the infusion time had been recorded in the medical records. Among these, 42 examinations had an elevated bilirubin value (>19 micromol/L). There were nine patients without contrast excretion; 3 of which had a normal bilirubin value and 6 had an elevated value (25-133 micromol/L). Two of the 153 examinations were inconclusive. One subject (0.7%) experienced a minor adverse reaction - a pricking sensation in the face. No other adverse effects were noted. CONCLUSION: We conclude that drip infusion CTC with an infusion rate of the biliary contrast agent iotroxate governed by the serum bilirubin value is a feasible and safe alternative to MRC in patients with and without impaired biliary excretion.In this retrospective study the feasibility and the frequency of adverse reactions when using a drip infusion scheme based on bilirubin levels has been evaluated. [Abstract/Link to Full Text]

Klein A, Mensh B, Ghosh S, Tourville J, Hirsch J
Mindboggle: automated brain labeling with multiple atlases.
BMC Med Imaging. 2005 Oct 5;57.
BACKGROUND: To make inferences about brain structures or activity across multiple individuals, one first needs to determine the structural correspondences across their image data. We have recently developed Mindboggle as a fully automated, feature-matching approach to assign anatomical labels to cortical structures and activity in human brain MRI data. Label assignment is based on structural correspondences between labeled atlases and unlabeled image data, where an atlas consists of a set of labels manually assigned to a single brain image. In the present work, we study the influence of using variable numbers of individual atlases to nonlinearly label human brain image data. METHODS: Each brain image voxel of each of 20 human subjects is assigned a label by each of the remaining 19 atlases using Mindboggle. The most common label is selected and is given a confidence rating based on the number of atlases that assigned that label. The automatically assigned labels for each subject brain are compared with the manual labels for that subject (its atlas). Unlike recent approaches that transform subject data to a labeled, probabilistic atlas space (constructed from a database of atlases), Mindboggle labels a subject by each atlas in a database independently. RESULTS: When Mindboggle labels a human subject's brain image with at least four atlases, the resulting label agreement with coregistered manual labels is significantly higher than when only a single atlas is used. Different numbers of atlases provide significantly higher label agreements for individual brain regions. CONCLUSION: Increasing the number of reference brains used to automatically label a human subject brain improves labeling accuracy with respect to manually assigned labels. Mindboggle software can provide confidence measures for labels based on probabilistic assignment of labels and could be applied to large databases of brain images. [Abstract/Link to Full Text]

Goodacre S, Sampson F, Thomas S, van Beek E, Sutton A
Systematic review and meta-analysis of the diagnostic accuracy of ultrasonography for deep vein thrombosis.
BMC Med Imaging. 2005 Oct 3;56.
BACKGROUND: Ultrasound (US) has largely replaced contrast venography as the definitive diagnostic test for deep vein thrombosis (DVT). We aimed to derive a definitive estimate of the diagnostic accuracy of US for clinically suspected DVT and identify study-level factors that might predict accuracy. METHODS: We undertook a systematic review, meta-analysis and meta-regression of diagnostic cohort studies that compared US to contrast venography in patients with suspected DVT. We searched Medline, EMBASE, CINAHL, Web of Science, Cochrane Database of Systematic Reviews, Cochrane Controlled Trials Register, Database of Reviews of Effectiveness, the ACP Journal Club, and citation lists (1966 to April 2004). Random effects meta-analysis was used to derive pooled estimates of sensitivity and specificity. Random effects meta-regression was used to identify study-level covariates that predicted diagnostic performance. RESULTS: We identified 100 cohorts comparing US to venography in patients with suspected DVT. Overall sensitivity for proximal DVT (95% confidence interval) was 94.2% (93.2 to 95.0), for distal DVT was 63.5% (59.8 to 67.0), and specificity was 93.8% (93.1 to 94.4). Duplex US had pooled sensitivity of 96.5% (95.1 to 97.6) for proximal DVT, 71.2% (64.6 to 77.2) for distal DVT and specificity of 94.0% (92.8 to 95.1). Triplex US had pooled sensitivity of 96.4% (94.4 to 97.1%) for proximal DVT, 75.2% (67.7 to 81.6) for distal DVT and specificity of 94.3% (92.5 to 95.8). Compression US alone had pooled sensitivity of 93.8 % (92.0 to 95.3%) for proximal DVT, 56.8% (49.0 to 66.4) for distal DVT and specificity of 97.8% (97.0 to 98.4). Sensitivity was higher in more recently published studies and in cohorts with higher prevalence of DVT and more proximal DVT, and was lower in cohorts that reported interpretation by a radiologist. Specificity was higher in cohorts that excluded patients with previous DVT. No studies were identified that compared repeat US to venography in all patients. Repeat US appears to have a positive yield of 1.3%, with 89% of these being confirmed by venography. CONCLUSION: Combined colour-doppler US techniques have optimal sensitivity, while compression US has optimal specificity for DVT. However, all estimates are subject to substantial unexplained heterogeneity. The role of repeat scanning is very uncertain and based upon limited data. [Abstract/Link to Full Text]

Razifar P, Sandström M, Schnieder H, Långström B, Maripuu E, Bengtsson E, Bergström M
Noise correlation in PET, CT, SPECT and PET/CT data evaluated using autocorrelation function: a phantom study on data, reconstructed using FBP and OSEM.
BMC Med Imaging. 2005 Aug 25;55.
BACKGROUND: Positron Emission Tomography (PET), Computed Tomography (CT), PET/CT and Single Photon Emission Tomography (SPECT) are non-invasive imaging tools used for creating two dimensional (2D) cross section images of three dimensional (3D) objects. PET and SPECT have the potential of providing functional or biochemical information by measuring distribution and kinetics of radiolabelled molecules, whereas CT visualizes X-ray density in tissues in the body. PET/CT provides fused images representing both functional and anatomical information with better precision in localization than PET alone. Images generated by these types of techniques are generally noisy, thereby impairing the imaging potential and affecting the precision in quantitative values derived from the images. It is crucial to explore and understand the properties of noise in these imaging techniques. Here we used autocorrelation function (ACF) specifically to describe noise correlation and its non-isotropic behaviour in experimentally generated images of PET, CT, PET/CT and SPECT. METHODS: Experiments were performed using phantoms with different shapes. In PET and PET/CT studies, data were acquired in 2D acquisition mode and reconstructed by both analytical filter back projection (FBP) and iterative, ordered subsets expectation maximisation (OSEM) methods. In the PET/CT studies, different magnitudes of X-ray dose in the transmission were employed by using different mA settings for the X-ray tube. In the CT studies, data were acquired using different slice thickness with and without applied dose reduction function and the images were reconstructed by FBP. SPECT studies were performed in 2D, reconstructed using FBP and OSEM, using post 3D filtering. ACF images were generated from the primary images, and profiles across the ACF images were used to describe the noise correlation in different directions. The variance of noise across the images was visualised as images and with profiles across these images. RESULTS: The most important finding was that the pattern of noise correlation is rotation symmetric or isotropic, independent of object shape in PET and PET/CT images reconstructed using the iterative method. This is, however, not the case in FBP images when the shape of phantom is not circular. Also CT images reconstructed using FBP show the same non-isotropic pattern independent of slice thickness and utilization of care dose function. SPECT images show an isotropic correlation of the noise independent of object shape or applied reconstruction algorithm. Noise in PET/CT images was identical independent of the applied X-ray dose in the transmission part (CT), indicating that the noise from transmission with the applied doses does not propagate into the PET images showing that the noise from the emission part is dominant. The results indicate that in human studies it is possible to utilize a low dose in transmission part while maintaining the noise behaviour and the quality of the images. CONCLUSION: The combined effect of noise correlation for asymmetric objects and a varying noise variance across the image field significantly complicates the interpretation of the images when statistical methods are used, such as with statistical estimates of precision in average values, use of statistical parametric mapping methods and principal component analysis. Hence it is recommended that iterative reconstruction methods are used for such applications. However, it is possible to calculate the noise analytically in images reconstructed by FBP, while it is not possible to do the same calculation in images reconstructed by iterative methods. Therefore for performing statistical methods of analysis which depend on knowing the noise, FBP would be preferred. [Abstract/Link to Full Text]

Brunet-Imbault B, Lemineur G, Chappard C, Harba R, Benhamou CL
A new anisotropy index on trabecular bone radiographic images using the fast Fourier transform.
BMC Med Imaging. 2005 May 31;54.
BACKGROUND: The degree of anisotropy (DA) on radiographs is related to bone structure, we present a new index to assess DA. METHODS: In a region of interest from calcaneus radiographs, we applied a Fast Fourier Transform (FFT). All the FFT spectra involve the horizontal and vertical components corresponding respectively to longitudinal and transversal trabeculae. By visual inspection, we measured the spreading angles: Dispersion Longitudinal Index (DLI) and Dispersion Transverse Index (DTI) and calculated DA = 180/(DLI+DTI). To test the reliability of DA assessment, we synthesized images simulating radiological projections of periodic structures with elements more or less disoriented. RESULTS: Firstly, we tested synthetic images which comprised a large variety of structures from highly anisotropic structure to the almost isotropic, DA was ranging from 1.3 to 3.8 respectively. The analysis of the FFT spectra was performed by two observers, the Coefficients of Variation were 1.5% and 3.1 % for intra-and inter-observer reproducibility, respectively. In 22 post-menopausal women with osteoporotic fracture cases and 44 age-matched controls, DA values were respectively 1.87 +/- 0.15 versus 1.72 +/- 0.18 (p = 0.001). From the ROC analysis, the Area Under Curve (AUC) were respectively 0.65, 0.62, 0.64, 0.77 for lumbar spine, femoral neck, total femoral BMD and DA. CONCLUSION: The highest DA values in fracture cases suggest that the structure is more anisotropic in osteoporosis due to preferential deletion of trabeculae in some directions. [Abstract/Link to Full Text]

Razifar P, Lubberink M, Schneider H, Långström B, Bengtsson E, Bergström M
Non-isotropic noise correlation in PET data reconstructed by FBP but not by OSEM demonstrated using auto-correlation function.
BMC Med Imaging. 2005 May 13;5(1):3.
BACKGROUND: Positron emission tomography (PET) is a powerful imaging technique with the potential of obtaining functional or biochemical information by measuring distribution and kinetics of radiolabelled molecules in a biological system, both in vitro and in vivo. PET images can be used directly or after kinetic modelling to extract quantitative values of a desired physiological, biochemical or pharmacological entity. Because such images are generally noisy, it is essential to understand how noise affects the derived quantitative values. A pre-requisite for this understanding is that the properties of noise such as variance (magnitude) and texture (correlation) are known. METHODS: In this paper we explored the pattern of noise correlation in experimentally generated PET images, with emphasis on the angular dependence of correlation, using the autocorrelation function (ACF). Experimental PET data were acquired in 2D and 3D acquisition mode and reconstructed by analytical filtered back projection (FBP) and iterative ordered subsets expectation maximisation (OSEM) methods. The 3D data was rebinned to a 2D dataset using FOurier REbinning (FORE) followed by 2D reconstruction using either FBP or OSEM. In synthetic images we compared the ACF results with those from covariance matrix. The results were illustrated as 1D profiles and also visualized as 2D ACF images. RESULTS: We found that the autocorrelation images from PET data obtained after FBP were not fully rotationally symmetric or isotropic if the object deviated from a uniform cylindrical radioactivity distribution. In contrast, similar autocorrelation images obtained after OSEM reconstruction were isotropic even when the phantom was not circular. Simulations indicated that the noise autocorrelation is non-isotropic in images created by FBP when the level of noise in projections is angularly variable. Comparison between 1D cross profiles on autocorrelation images obtained by FBP reconstruction and covariance matrices produced almost identical results in a simulation study. CONCLUSION: With asymmetric radioactivity distribution in PET, reconstruction using FBP, in contrast to OSEM, generates images in which the noise correlation is non-isotropic when the noise magnitude is angular dependent, such as in objects with asymmetric radioactivity distribution. In this respect, iterative reconstruction is superior since it creates isotropic noise correlations in the images. [Abstract/Link to Full Text]

Hynes A, Scott DA, Man A, Singer DL, Sowa MG, Liu KZ
Molecular mapping of periodontal tissues using infrared microspectroscopy.
BMC Med Imaging. 2005 May 12;5(1):2.
BACKGROUND: Chronic periodontitis is an inflammatory disease of the supporting structures of the teeth. Infrared microspectroscopy has the potential to simultaneously monitor multiple disease markers, including cellular infiltration and collagen catabolism, and hence differentiate diseased and healthy tissues. Therefore, our aim was to establish an infrared microspectroscopy methodology with which to analyze and interpret molecular maps defining pathogenic processes in periodontal tissues. METHODS: Specific key cellular and connective tissue components were identified by infrared microspectroscopy and using a chemical imaging method. RESULTS: Higher densities of DNA, total protein and lipid were revealed in epithelial tissue, compared to the lower percentage of these components in connective tissue. Collagen-specific tissue mapping by infrared microspectroscopy revealed much higher levels of collagen deposition in the connective tissues compared to that in the epithelium, as would be expected. Thus inflammatory events such as cellular infiltration and collagen deposition and catabolism can be identified by infrared microspectroscopy. CONCLUSION: These results suggest that infrared microspectroscopy may represent a simple, reagent-free, multi-dimensional tool with which to examine periodontal disease etiology using entirely unprocessed tissue sections. [Abstract/Link to Full Text]

Brownbill RA, Ilich JZ
Measuring body composition in overweight individuals by dual energy x-ray absorptiometry.
BMC Med Imaging. 2005 Mar 4;5(1):1.
BACKGROUND: Dual energy x-ray absorptiometry (DXA) is widely used for body composition measurements in normal-weight and overweight/obese individuals. The limitations of bone densitometers have been frequently addressed. However, the possible errors in assessing body composition in overweight individuals due to incorrect positioning or limitations of DXA to accurately assess both bone mineral density and body composition in obese individuals have not received much attention and are the focus of this report. DISCUSSION: We discuss proper ways of measuring overweight individuals and point to some studies where that might not have been the case. It appears that currently, the most prudent approach to assess body composition of large individuals who cannot fit under the scanning area would be to estimate regional fat, namely the regions of thigh and/or abdomen. Additionally, using two-half body scans, although time consuming, may provide a relatively accurate measurement of total body fat, however, more studies using this technique are needed to validate it. SUMMARY: Researchers using bone densitometers for body composition measurements need to have an understanding of its limitations in overweight individuals and address them appropriately when interpreting their results. Studies on accuracy and precision in measurements of both bone and soft tissue composition in overweight individuals using available densitometers are needed. [Abstract/Link to Full Text]

Gregory JS, Stewart A, Undrill PE, Reid DM, Aspden RM
Identification of hip fracture patients from radiographs using Fourier analysis of the trabecular structure: a cross-sectional study.
BMC Med Imaging. 2004 10 6;4(1):4.
BACKGROUND: This study presents an analysis of trabecular bone structure in standard radiographs using Fourier transforms and principal components analysis (PCA) to identify contributions to hip fracture risk. METHODS: Radiographs were obtained from 26 hip fracture patients and 24 controls. They were digitised and five regions of interest (ROI) were identified from the femoral head and neck for analysis. The power spectrum was obtained from the Fourier transform of each region and three profiles were produced; a circular profile and profiles parallel and perpendicular to the preferred orientation of the trabeculae. PCA was used to generate a score from each profile, which we hypothesised could be used to discriminate between the fracture and control groups. The fractal dimension was also calculated for comparison. The area under the receiver operating characteristic curve (Az) discriminating the hip fracture cases from controls was calculated for each analysis. RESULTS: Texture analysis of standard radiographs using the fast Fourier transform yielded variables that were significantly associated with fracture and not significantly correlated with age, body mass index or femoral neck bone mineral density. The anisotropy of the trabecular structure was important; both the perpendicular and circular profiles were significantly better than the parallel-profile (P < 0.05). No significant differences resulted from using the various ROI within the proximal femur. For the best three groupings of profile (circular, parallel or perpendicular), method (PCA or fractal) and ROI (Az = 0.84 - 0.93), there were no significant correlations with femoral neck bone mineral density, age, or body mass index. PCA analysis was found to perform better than fractal analysis (P = 0.019). CONCLUSIONS: Both PCA and fractal analysis of the FFT data could discriminate successfully between the fracture and control groups, although PCA was significantly stronger than fractal dimension. This method appears to provide a powerful tool for the assessment of bone structure in vivo with advantages over standard fractal methods. [Abstract/Link to Full Text]

Recent Articles in Biomagnetic Research and Technology

Golestani-Rad L, Elahi B, Rashed-Mohassel J
Investigating the effects of external fields polarization on the coupling of pure magnetic waves in the human body in very low frequencies.
Biomagn Res Technol. 2007;53.
In this paper we studied the effects of external fields' polarization on the coupling of pure magnetic fields into human body. Finite Difference Time Domain (FDTD) method is used to calculate the current densities induced in a 1 cm resolution anatomically based model with proper tissue conductivities. Twenty different tissues have been considered in this investigation and scaled FDTD technique is used to convert the results of computer code run in 15 MHz to low frequencies which are encountered in the vicinity of industrial induction heating and melting devices. It has been found that external magnetic field's orientation due to human body has a pronounced impact on the level of induced currents in different body tissues. This may potentially help developing protecting strategies to mitigate the situations in which workers are exposed to high levels of external magnetic radiation. [Abstract/Link to Full Text]

Zhao H, Gagnon J, Häfeli UO
Process and formulation variables in the preparation of injectable and biodegradable magnetic microspheres.
Biomagn Res Technol. 2007;52.
The aim of this study was to prepare biodegradable sustained release magnetite microspheres sized between 1 to 2 microm. The microspheres with or without magnetic materials were prepared by a W/O/W double emulsion solvent evaporation technique using poly(lactide-co-glycolide) (PLGA) as the biodegradable matrix forming polymer. Effects of manufacturing and formulation variables on particle size were investigated with non-magnetic microspheres. Microsphere size could be controlled by modification of homogenization speed, PLGA concentration in the oil phase, oil phase volume, solvent composition, and polyvinyl alcohol (PVA) concentration in the outer water phase. Most influential were the agitation velocity and all parameters that influence the kinematic viscosity of oil and outer water phase, specifically the type and concentration of the oil phase. The magnetic component yielding homogeneous magnetic microspheres consisted of magnetite nanoparticles of 8 nm diameter stabilized with a polyethylene glycole/polyacrylic acid (PEG/PAA) coating and a saturation magnetization of 47.8 emu/g. Non-magnetic and magnetic microspheres had very similar size, morphology, and size distribution, as shown by scanning electron microscopy. The optimized conditions yielded microspheres with 13.7 weight% of magnetite and an average diameter of 1.37 microm. Such biodegradable magnetic microspheres seem appropriate for vascular administration followed by magnetic drug targeting. [Abstract/Link to Full Text]

Barnes AL, Wassel RA, Mondalek F, Chen K, Dormer KJ, Kopke RD
Magnetic characterization of superparamagnetic nanoparticles pulled through model membranes.
Biomagn Res Technol. 2007;51.
BACKGROUND: To quantitatively compare in-vitro and in vivo membrane transport studies of targeted delivery, one needs characterization of the magnetically-induced mobility of superparamagnetic iron oxide nanoparticles (SPION). Flux densities, gradients, and nanoparticle properties were measured in order to quantify the magnetic force on the SPION in both an artificial cochlear round window membrane (RWM) model and the guinea pig RWM. METHODS: Three-dimensional maps were created for flux density and magnetic gradient produced by a 24-well casing of 4.1 kilo-Gauss neodymium-iron-boron (NdFeB) disc magnets. The casing was used to pull SPION through a three-layer cell culture RWM model. Similar maps were created for a 4 inch (10.16 cm) cube 48 MGOe NdFeB magnet used to pull polymeric-nanoparticles through the RWM of anesthetized guinea pigs. Other parameters needed to compute magnetic force were nanoparticle and polymer properties, including average radius, density, magnetic susceptibility, and volume fraction of magnetite. RESULTS: A minimum force of 5.04 x 10(-16) N was determined to adequately pull nanoparticles through the in-vitro model. For the guinea pig RWM, the magnetic force on the polymeric nanoparticles was 9.69 x 10-20 N. Electron microscopy confirmed the movement of the particles through both RWM models. CONCLUSION: As prospective carriers of therapeutic substances, polymers containing superparamagnetic iron oxide nanoparticles were succesfully pulled through the live RWM. The force required to achieve in vivo transport was significantly lower than that required to pull nanoparticles through the in-vitro RWM model. Indeed very little force was required to accomplish measurable delivery of polymeric-SPION composite nanoparticles across the RWM, suggesting that therapeutic delivery to the inner ear by SPION is feasible. [Abstract/Link to Full Text]

Paul AL, Ferl RJ, Meisel MW
High magnetic field induced changes of gene expression in arabidopsis.
Biomagn Res Technol. 2006;47.
BACKGROUND: High magnetic fields are becoming increasingly prevalent components of non-invasive, biomedical imaging tools (such as MRI), thus, an understanding of the molecular impacts associated with these field strengths in biological systems is of central importance. The biological impact of magnetic field strengths up to 30 Tesla were investigated in this study through the use of transgenic Arabidopsis plants engineered with a stress response gene consisting of the alcohol dehydrogenase (Adh) gene promoter driving the beta-glucuronidase (GUS) gene reporter. METHODS: Magnetic field induced Adh/GUS activity was evaluated with histochemical staining to assess tissue specific expression and distribution, and with quantitative, spectrofluometric assays to measure degree of activation. The evaluation of global changes in the Arabidopsis genome in response to exposure to high magnetic fields was facilitated with Affymetrix Gene Chip microarrays. Quantitative analyses of gene expression were performed with quantitative real-time polymerase-chain-reaction (qRT-PCR). RESULTS: Field strengths in excess of about 15 Tesla induce expression of the Adh/GUS transgene in the roots and leaves. From the microarray analyses that surveyed 8000 genes, 114 genes were differentially expressed to a degree greater than 2.5 fold over the control. These results were quantitatively corroborated by qRT-PCR examination of 4 of the 114 genes. CONCLUSION: The data suggest that magnetic fields in excess of 15 Tesla have far-reaching effect on the genome. The wide-spread induction of stress-related genes and transcription factors, and a depression of genes associated with cell wall metabolism, are prominent examples. The roles of magnetic field orientation of macromolecules and magnetophoretic effects are discussed as possible factors that contribute to the mounting of this response. [Abstract/Link to Full Text]

Tsai H, Fang YS, Fuh CB
Analytical and preparative applications of magnetic split-flow thin fractionation on several ion-labeled red blood cells.
Biomagn Res Technol. 2006;46.
BACKGROUND: Magnetic Split-flow thin (SPLITT) fractionation is a newly developed technique for separating magnetically susceptible particles. Particles with different field-induced velocities can be separated into two fractions by adjusting applied magnetic forces and flow-rates at inlets and outlets. METHODS: Magnetic particles, Dynabeads, were used to test this new approach of field-induced velocity for susceptibility determination using magnetic SF at different magnetic field intensities. Reference measurements of magnetic susceptibility were made using a superconducting quantum interference device (SQUID) magnetometer. Various ion-labeled red blood cells (RBC) were used to study susceptibility determination and throughput parameters for analytical and preparative applications of magnetic SPLITT fractionation (SF), respectively. Throughputs were studied at different sample concentrations, magnetic field intensities, and channel flow-rates. RESULTS: The susceptibilities of Dynabeads determined by SPLITT fractionation (SF) were consistent with those of reference measurement using a superconducting quantum interference device (SQUID) magnetometer. Determined susceptibilities of ion-labeled RBC were consistent within 9.6% variations at two magnetic intensities and different flow-rates. The determined susceptibilities differed by 10% from referenced measurements. The minimum difference in magnetic susceptibility required for complete separation was about 5.0 x 10(-6) [cgs]. Sample recoveries were higher than 92%. The throughput of magnetic SF was approximately 1.8 g/h using our experimental setup. CONCLUSION: Magnetic SF can provide simple and economical determination of particle susceptibility. This technique also has great potential for cell separation and related analysis. Continuous separations of ion-labeled RBC using magnetic SF were successful over 4 hours. The throughput was increased by 18 folds versus early study. Sample recoveries were 93.1 +/- 1.8% in triplicate experiments. [Abstract/Link to Full Text]

Haberkorn W, Steinhoff U, Burghoff M, Kosch O, Morguet A, Koch H
Pseudo current density maps of electrophysiological heart, nerve or brain function and their physical basis.
Biomagn Res Technol. 2006;45.
BACKGROUND: In recent years the visualization of biomagnetic measurement data by so-called pseudo current density maps or Hosaka-Cohen (HC) transformations became popular. METHODS: The physical basis of these intuitive maps is clarified by means of analytically solvable problems. RESULTS: Examples in magnetocardiography, magnetoencephalography and magnetoneurography demonstrate the usefulness of this method. CONCLUSION: Hardware realizations of the HC-transformation and some similar transformations are discussed which could advantageously support cross-platform comparability of biomagnetic measurements. [Abstract/Link to Full Text]

Möller W, Barth W, Kohlhäufl M, Häussinger K, Kreyling WG
Motion and twisting of magnetic particles ingested by alveolar macrophages in the human lung: effect of smoking and disease.
Biomagn Res Technol. 2006;44.
BACKGROUND: Magnetic microparticles being ingested by alveolar macrophages can be used as a monitor for intracellular phagosome motions and cytoskeletal mechanical properties. These studies can be performed in the human lung after voluntary inhalation. The influence of cigarette smoking and lung diseases on cytoskeleton dependent functions was studied. METHODS: Spherical 1.3 microm diameter ferrimagnetic iron oxide particles were inhaled by 17 healthy volunteers (40-65 years), 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 18 patients with chronic obstructive bronchitis (COB). The retained particles were magnetized and aligned in an external 100 mT magnetic field. All magnetized particles induce a weak magnetic field of the lung, which was detected by a sensitive SQUID (superconducting quantum interference device) sensor. Cytoskeletal reorganizations within macrophages and intracellular transport cause stochastic magnetic dipole rotations, which are reflected in a decay of the magnetic lung field, called relaxation. Directed phagosome motion was induced in a weak magnetic twisting field. The resistance of the cytoplasm to particle twisting was characterized by the viscosity and the stiffness (ratio between stress to strain) of the cytoskeleton. RESULTS: One week after particle inhalation and later macrophage motility (relaxation) and cytoskeletal stiffness was not influenced by cigarette smoking, neither in healthy subjects, nor in the patients. Patients with IPF showed in tendency a faster relaxation (p = 0.06). Particle twisting revealed a non-Newtonian viscosity with a pure viscous and a viscoelastic compartment. The viscous shear was dominant, and only 27% of the shear recoiled and reflected viscoelastic properties. In patients with IPF, the stiffness was reduced by 60% (p < 0.02). An analysis of the shear rate and stress dependence of particle twisting allows correlating the rheological compartments to cytoskeletal subunits, in which microtubules mediate the pure viscous (non-recoverable) shear and microfilaments mediate the viscoelastic (recoverable) behavior. The missing correlation between relaxation and particle twisting shows that both stochastic and directed phagosome motion reflect different cytoskeletal mechanisms. CONCLUSION: Faster relaxation and a soft cytoskeleton in patients with IPF indicate alterations in cytoskeleton dependent functions of alveolar macrophages, which may cause dysfunction's in the alveolar defense, like a slower migration, a retarded phagocytosis, a disturbed phagosome lysosome fusion and an impaired clearance. [Abstract/Link to Full Text]

Anastasiadis P, Anastasiadis AN, Kotini A, Koutlaki N, Anninos P
Differentiation of myomas by means of biomagnetic and doppler findings.
Biomagn Res Technol. 2006;43.
AIM: To elucidate the hemodynamics of the uterine artery myomas by use of Doppler ultrasound and biomagnetic measurements. METHOD: Twenty-four women were included in the study. Sixteen of them were characterised with large myomas whereas 8 of them with small ones. Biomagnetic signals of uterine arteries myomas were recorded and analyzed with Fourier analysis. The biomagnetic signals were distributed according to spectral amplitudes as high (140-300 ft/ radicalHz), low (50-110 ft/ radicalHz) and borderline (111-139 ft/ radicalHz). Uterine artery waveform measurements were evaluated by use of Pulsatility Index (PI) (normal value PI < 1.45). RESULTS: There was a statistically significant difference between large and small myomas concerning the waveform amplitudes (P < 0.0005) and the PI index (P < 0.0005). Specifically, we noticed high biomagnetic amplitudes in most large myomas (93.75 %) and low biomagnetic amplitudes in most small ones (87.5 %). CONCLUSION: It is suggested that the biomagnetic recordings of uterine artery myomas could be a valuable modality in the estimation of the circulation of blood cells justifying the findings of Doppler velocimetry examination. [Abstract/Link to Full Text]

Kotini A, Anastasiadis AN, Anninos P, Koutlaki N, Anastasiadis P
Nonlinear analysis of biomagnetic signals recorded from uterine myomas.
Biomagn Res Technol. 2006;42.
OBJECTIVE: To determine if there is any non-linearity in the biomagnetic recordings of uterine myomas and to find any differences that may be present in the mechanisms underlying their signal dynamics. METHODS: Twenty-four women were included in the study. Sixteen of them were characterised with large myomas and 8 with small ones. Uterine artery waveform measurements were evaluated by use of Pulsatility Index (PI) (normal value PI<1.45). RESULTS: Applying nonlinear analysis to the biomagnetic signals of the uterine myomas, we observed a clear saturation value for the group of large ones (mean = 11.35 +/- 1.49) and no saturation for the small ones. CONCLUSION: The comparison of the saturation values in the biomagnetic recordings of large and small myomas may be a valuable tool in the evaluation of functional changes in their dynamic behavior. [Abstract/Link to Full Text]

Pazur A, Rassadina V, Dandler J, Zoller J
Growth of etiolated barley plants in weak static and 50 Hz electromagnetic fields tuned to calcium ion cyclotron resonance.
Biomagn Res Technol. 2006;41.
BACKGROUND: The effects of weak magnetic and electromagnetic fields in biology have been intensively studied on animals, microorganisms and humans, but comparably less on plants. Perception mechanisms were attributed originally to ferrimagnetism, but later discoveries required additional explanations like the "radical pair mechanism" and the "Ion cyclotron resonance" (ICR), primarily considered by Liboff. The latter predicts effects by small ions involved in biological processes, that occur in definite frequency- and intensity ranges ("windows") of simultaneously impacting magnetic and electromagnetic fields related by a linear equation, which meanwhile is proven by a number of in vivo and in vitro experiments. METHODS: Barley seedlings (Hordeum vulgare, L. var. Steffi) were grown in the dark for 5 and 6 days under static magnetic and 50 Hz electromagnetic fields matching the ICR conditions of Ca2+. Control cultures were grown under normal geomagnetic conditions, not matching this ICR. Morphology, pigmentation and long-term development of the adult plants were subsequently investigated. RESULTS: The shoots of plants exposed to Ca2+-ICR exposed grew 15-20% shorter compared to the controls, the plant weight was 10-12% lower, and they had longer coleoptiles that were adhering stronger to the primary leaf tissue. The total pigment contents of protochlorophyllide (PChlide) and carotenoids were significantly decreased. The rate of PChlide regeneration after light irradiation was reduced for the Ca2+-ICR exposed plants, also the Shibata shift was slightly delayed. Even a longer subsequent natural growing phase without any additional fields could only partially eliminate these effects: the plants initially exposed to Ca2+-ICR were still significantly shorter and had a lower chlorophyll (a+b) content compared to the controls. A continued cultivation and observation of the adult plants under natural conditions without any artificial electromagnetic fields showed a retardation of the originally Ca2+-ICR exposed plants compared to control cultures lasting several weeks, with an increased tendency for dehydration. CONCLUSION: A direct influence of the applied MF and EMF is discussed affecting Ca2+ levels via the ICR mechanism. It influences the available Ca2+ and thereby regulatory processes. Theoretical considerations on molecular level focus on ionic interactions with water related to models using quantum electrodynamics. [Abstract/Link to Full Text]

Mietchen D, Jakobi JW, Richter HP
Cortex reorganization of Xenopus laevis eggs in strong static magnetic fields.
Biomagn Res Technol. 2005;32.
Observations of magnetic field effects on biological systems have often been contradictory. For amphibian eggs, a review of the available literature suggests that part of the discrepancies might be resolved by considering a previously neglected parameter for morphological alterations induced by magnetic fields--the jelly layers that normally surround the egg and are often removed in laboratory studies for easier cell handling. To experimentally test this hypothesis, we observed the morphology of fertilizable Xenopus laevis eggs with and without jelly coat that were subjected to static magnetic fields of up to 9.4 T for different periods of time. A complex reorganization of cortical pigmentation was found in dejellied eggs as a function of the magnetic field and the field exposure time. Initial pigment rearrangements could be observed at about 0.5 T, and less than 3 T are required for the effects to fully develop within two hours. No effect was observed when the jelly layers of the eggs were left intact. These results suggest that the action of magnetic fields might involve cortical pigments or associated cytoskeletal structures normally held in place by the jelly layers and that the presence of the jelly layer should indeed be included in further studies of magnetic field effects in this system. [Abstract/Link to Full Text]

Kouassi GK, Irudayaraj J, McCarty G
Activity of glucose oxidase functionalized onto magnetic nanoparticles.
Biomagn Res Technol. 2005 Mar 11;3(1):1.
BACKGROUND: Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties. METHODS: Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy. RESULTS: The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94-100% while thiophene acetylation was 66-72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles CONCLUSION: Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications. [Abstract/Link to Full Text]

Pazur A
Characterisation of weak magnetic field effects in an aqueous glutamic acid solution by nonlinear dielectric spectroscopy and voltammetry.
Biomagn Res Technol. 2004 Nov 30;2(1):8.
BACKGROUND: Previous reports indicate altered metabolism and enzyme kinetics for various organisms, as well as changes of neuronal functions and behaviour of higher animals, when they were exposed to specific combinations of weak static and alternating low frequency electromagnetic fields. Field strengths and frequencies, as well as properties of involved ions were related by a linear equation, known as the formula of ion cyclotron resonance (ICR, abbreviation mentioned first by Liboff). Under certain conditions already a aqueous solution of the amino acid and neurotransmitter glutamate shows this effect. METHODS: An aqueous solution of glutamate was exposed to a combination of a static magnetic field of 40 muT and a sinusoidal electromagnetic magnetic field (EMF) with variable frequency (2-7 Hz) and an amplitude of 50 nT. The electric conductivity and dielectric properties of the solution were investigated by voltammetric techniques in combination with non linear dielectric spectroscopy (NLDS), which allow the examination of the dielectric properties of macromolecules and molecular aggregates in water. The experiments target to elucidate the biological relevance of the observed EMF effect on molecular level. RESULTS: An ion cyclotron resonance (ICR) effect of glutamate previously reported by the Fesenko laboratory 1998 could be confirmed. Frequency resolution of the sample currents was possible by NLDS techniques. The spectrum peaks when the conditions for ion cyclotron resonance (ICR) of glutamate are matched. Furthermore, the NLDS spectra are different under ICR- and non-ICR conditions: NLDS measurements with rising control voltages from 100-1100 mV show different courses of the intensities of the low order harmonics, which could possibly indicate "intensity windows". Furthermore, the observed magnetic field effects are pH dependent with a narrow optimum around pH 2.85. CONCLUSIONS: Data will be discussed in the context with recent published models for the interaction of weak EMF with biological matter including ICR. A medical and health relevant aspect of such sensitive effects might be given insofar, because electromagnetic conditions for it occur at many occasions in our electromagnetic all day environment, concerning ion involvement of different biochemical pathways. [Abstract/Link to Full Text]

Safarik I, Safarikova M
Magnetic techniques for the isolation and purification of proteins and peptides.
Biomagn Res Technol. 2004 Nov 26;2(1):7.
Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. [Abstract/Link to Full Text]

Hilgenfeld B, Haueisen J
Simultaneous suppression of disturbing fields and localization of magnetic markers by means of multipole expansion.
Biomagn Res Technol. 2004 Sep 1;2(1):6.
BACKGROUND: Magnetically marked capsules serve for the analysis of peristalsis and throughput times within the intestinal tract. Moreover, they can be used for the targeted disposal of drugs. The capsules get localized in time by field measurements with a superconducting quantum interference device (SQUID) magnetometer array. Here it is important to ensure an online localization with high speed and high suppression of disturbing fields. In this article we use multipole expansions for the simultaneous localization and suppression of disturbing fields. METHODS: We expand the measurement data in terms of inner and outer multipoles. Thereby we obtain directly a separation of marker field and outer disturbing fields. From the inner dipoles and quadrupoles we compute the magnetization and position of the capsule. The outer multipoles get eliminated. RESULTS: The localization goodness has been analyzed depending on the order of the multipoles used and depending on the systems noise level. We found upper limits of the noise level for the usage of certain multipole moments. Given a signal to noise ratio of 40 and utilizing inner dipoles and quadrupoles and outer dipoles, the method enables an accuracy of 5 mm with a speed of 10 localizations per second. CONCLUSION: The multipole localization is an effective method and is capable of online-tracking magnetic markers. [Abstract/Link to Full Text]

Yavuz H, Say R, Andaç M, Bayraktar N, Denizli A
Performance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed.
Biomagn Res Technol. 2004 Aug 26;2(1):5.
BACKGROUND: Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders. METHODS: Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80-120 microm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 micromol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed. RESULTS: Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity. CONCLUSIONS: Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium. [Abstract/Link to Full Text]

Bettge M, Chatterjee J, Haik Y
Physically synthesized Ni-Cu nanoparticles for magnetic hyperthermia.
Biomagn Res Technol. 2004 May 8;2(1):4.
BACKGROUND: In this paper, a physical method to prepare copper-nickel alloy particles in the sub-micron range for possible self controlled magnetic hyperthermia treatment of cancer is described. It is reported that an increase in tumor temperature decreases the tumor resistance to chemo- and radiation therapies. Self controlled heating at the tumor site to avoid spot heating is managed by controlling the Curie temperature of the magnetic particles. The process described in this paper to produce the nanomagnetic particles allows for a large scale production of these particles. METHODS: The process used here is mainly composed of melting of the Cu-Ni mixture and ball milling of the resulted bulk alloy. Both mechanical abrasion and continuous grinding were used to break down the bulk amount into the desired particle size. RESULTS: It was found that the desired alloy is composed of 71% nickel and 29% copper by weight. It was observed that the coarse sand-grinded powder has a Curie temperature of 345 K and the fine ball-milled powder shows a temperature of 319 K - 320 K. CONCLUSION: Self regulating magnetic hyperthermia can be achieved by synthesizing nanomagnetic particles with desired Curie temperature. In this study the desired range of Curie temperatures was obtained by combination of melting and ball milling of nickel-copper alloy. [Abstract/Link to Full Text]

Matsuoka F, Shinkai M, Honda H, Kubo T, Sugita T, Kobayashi T
Hyperthermia using magnetite cationic liposomes for hamster osteosarcoma.
Biomagn Res Technol. 2004 Mar 25;2(1):3.
BACKGROUND: We have developed magnetite cationic liposomes (MCLs) and applied them to local hyperthermia as a mediator. MCLs have a positive charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. In this study, the effect of hyperthermia using MCLs was examined in an in vivo study of hamster osteosarcoma. METHOD: MCLs were injected into the osteosarcoma and then subjected to an AMF. RESULTS: The tumor was heated at over 42 degrees C, but other normal tissues were not heated as much. Complete regression was observed in 100% of the treated group hamsters, whereas no regression was observed in the control group hamsters. At day 12, the average tumor volume of the treated hamsters was about 1/1000 of that of the control hamsters. In the treated hamsters, no regrowth of osteosarcomas was observed over a period of 3 months after the complete regression. CONCLUSION: These results suggest that this treatment is effective for osteosarcoma. [Abstract/Link to Full Text]

Chatterjee J, Haik Y, Chen CJ
A biocompatible magnetic film: synthesis and characterization.
Biomagn Res Technol. 2004 Feb 4;2(1):2.
BACKGROUND: Biotechnology applications of magnetic gels include biosensors, targeted drug delivery, artificial muscles and magnetic buckles. These gels are produced by incorporating magnetic materials in the polymer composites. METHODS: A biocompatible magnetic gel film has been synthesized using polyvinyl alcohol. The magnetic gel was dried to generate a biocompatible magnetic film. Nanosized iron oxide particles (gamma-Fe2O3, ~7 nm) have been used to produce the magnetic gel. RESULTS: The surface morphology and magnetic properties of the gel films were studied. The iron oxide particles are superparamagnetic and the gel film also showed superparamagnetic behavior. CONCLUSION: Magnetic gel made out of crosslinked magnetic nanoparticles in the polymer network was found to be stable and possess the magnetic properties of the nanoparticles. [Abstract/Link to Full Text]

Rajendra P, Sujatha H, Devendranath D, Gunasekaran B, Sashidhar R, Subramanyam C, Channakeshava
Biological effects of power frequency magnetic fields: Neurochemical and toxicological changes in developing chick embryos.
Biomagn Res Technol. 2004 Jan 31;2(1):1.
BACKGROUND: There are several reports that indicate a linkage between exposure to power frequency (50 - 60 Hz) magnetic fields with abnormalities in the early embryonic development of the chicken. The present study was designed to understand whether power frequency electromagnetic fields could act as an environmental insult and invoke any neurochemical or toxicological changes in developing chick embryo model. METHODS: Fertilized chicken eggs were subjected to continuous exposure to magnetic fields (50 Hz) of varying intensities (5, 50 or 100 microT) for a period of up to 15 days. The embryos were taken out of the eggs on day 5, day 10 and day 15. Neurochemical (norepinephrine and 5-hydroxytryptamine) and amino acid (tyrosine, glutamine and tryptophan) contents were measured, along with an assay of the enzyme glutamine synthetase in the brain. Preliminary toxicological investigations were carried out based on aminotransferases (AST and ALT) and lactate dehydrogenase activities in the whole embryo as well as in the liver. RESULTS: The study revealed that there was a significant increase (p < 0.01 and p < 0.001) in the level of norepinephrine accompanied by a significant decrease (p < 0.01 and p < 0.001) in the tyrosine content in the brain on day 15 following exposure to 5, 50 and 100 microT magnetic fields. There was a significant increase (p < 0.001) in glutamine synthetase activity resulting in the significantly enhanced (p < 0.001) level of glutamine in the brain on day 15 (for 100 microT only). The possible mechanisms for these alterations are discussed. Further, magnetic fields had no effect on the levels of tryptophan and 5-hydroxytryptamine in the brain. Similarly, there was no effect on the activity of either aminotransferases or lactate dehydrogenase in the whole embryo or liver due to magnetic field exposure. CONCLUSIONS: Based on these studies we conclude that magnetic field-induced changes in norepinephrine levels might help explain alterations in the circadian rhythm, observed during magnetic field stress. Also, the enhanced level of glutamine can act as a contributing factor for developmental abnormalities. [Abstract/Link to Full Text]

SA, Ghulmiyyah LM, Carroll KT, Perloe M, Schwartzberg DG, Sills ES
Correlations from gadopentetate dimeglumine-enhanced magnetic resonance imaging after methotrexate chemotherapy for hemorrhagic placenta increta.
Biomagn Res Technol. 2003 Nov 14;1(1):3.
OBJECTIVE: To describe pre- and post-methotrexate (MTX) therapy images from pelvic magnetic resonance imaging (MRI) with gadopentetate dimeglumine contrast following chemotherapy for post-partum hemorrhage secondary to placenta increta. MATERIAL AND METHOD: A 28-year-old Caucasian female presented 4 weeks post-partum complaining of intermittent vaginal bleeding. She underwent dilatation and curettage immediately after vaginal delivery for suspected retained placental tissue but 28 d after delivery, the serum beta-hCG persisted at 156 IU/mL. Office transvaginal sonogram (4 mHz B-mode) was performed, followed by pelvic MRI using a 1.5 Tesla instrument after administration of gadolinium-based contrast agent. MTX was administered intramuscularly, and MRI was repeated four weeks later. RESULTS: While transvaginal sonogram suggested retained products of conception confined to the endometrial compartment, an irregular 53 x 34 x 28 mm heterogeneous intrauterine mass was noted on MRI to extend into the anterior myometrium, consistent with placenta increta. Vaginal bleeding diminished following MTX treatment, with complete discontinuation of bleeding achieved by ~20 d post-injection. MRI using identical technique one month later showed complete resolution of the uterine lesion. Serum beta-hCG was <5 IU/mL. CONCLUSION: Reduction or elimination of risks associated with surgical management of placenta increta is important to preserve uterine function and reproductive potential. For selected hemodynamically stable patients, placenta increta may be treated non-operatively with MTX as described here. A satisfactory response to MTX can be ascertained by serum hCG measurements with pre- and post-treatment pelvic MRI with gadopentetate dimeglumine enhancement, which offers advantages over standard transvaginal sonography. [Abstract/Link to Full Text]

Saiyed Z, Telang S, Ramchand C
Application of magnetic techniques in the field of drug discovery and biomedicine.
Biomagn Res Technol. 2003 Sep 18;1(1):2.
Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery. [Abstract/Link to Full Text]

Safarik I, Safarikova M
BioMagnetic Research and Technology: a new online journal.
Biomagn Res Technol. 2003 Jan 7;1(1):1. [Abstract/Link to Full Text]