Complex Biological Tissues Research Articles

Multimodal Nonlinear Optical Microscopy.

Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using CARS microscope as a platform to develop more advanced NLO modalities such as electronic-resonance-enhanced four-wave mixing, stimulated Raman scattering, and pump-probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed.

Challenges and opportunities for discovery of disease biomarkers using urine proteomics

Modern medicine has experienced a tremendous explosion in knowledge about disease pathophysiology, gained largely from understanding the molecular biology of human disease. Recent advances in mass spectrometry and proteomics now allow for simultaneous identification and quantification of thousands of unique proteins and peptides in complex biological tissues and fluids. In particular, proteomic studies of urine benefit from urine's less complex composition as compared to serum and tissues, and have been used successfully to discover novel markers of a variety of infectious, autoimmune, oncological, and surgical conditions. This perspective discusses the challenges of such studies that stem from the compositional variability and complexity of human urine, as well as instrumental sampling limitations and the effects of noise and selection bias. Strategies for the design of observational clinical trials, physical and chemical fractionation of urine specimens, mass spectrometry analysis, and functional data annotation are outlined. Rigorous translational investigations using urine proteomics are likely to discover novel and accurate markers of both rare and common diseases. This should aid the diagnosis, improve stratification of therapy, and identify novel therapeutic targets for a variety of childhood and adult diseases, all of which will be essential for the development of personalized and predictive medicine of the future.

A doublet mechanics model for the ultrasound characterization of malignant tissues

Non invasive ultrasound-based imaging systems are being more commonly used in clinical bio-microscopy applications for both ex vivo and in vivo analysis of tissue pathological and physiological states. These modalities usually employ high-frequency ultrasound systems to overcome spatial resolution limits of conventional clinical diagnostic approaches. Biological tissues are non continuous, non homogeneous and exhibit a multiscale organization from the sub-cellular level (£1 mm) to the organ level (³1 cm). When the ultrasonic wavelength used to probe the tissues becomes comparable with the tissue's microstructure scale, the propagation and reflection of ultrasound waves cannot be fully interpreted employing classical models developed within the continuum assumption. In this study, we present a multiscale model for analyzing the mechanical response of a non-continuum double-layer system exposed to an ultrasound source. The model is developed within the framework of the Doublet Mechanics theory and can be applied to the non-invasive analysis of complex biological tissues.

Recent Advances in Clinical Proteomics Using Mass Spectrometry

The ultimate objective of clinical proteomics is the successful discovery, validation and translation of biomarkers, together with new therapeutic targets into medical practices. New highly developed technologies in proteomics and their use in understanding tumor biology have significant clinical potential in the diagnosis, prognosis and treatment of disease. Areas such as MS, new labeling technologies and advancements in bioinformatics systems are now used to successfully detect disease-associated biomarkers together with therapeutic targets in complex biological specimens, including biofluids, cell lysates and tissue biopsies. Recent improvements in sample preparation (specifically focused on fractionation and enrichment) are enabling the analysis of low-abundance proteins together with many types of post-translational modifications. Targeted proteomic diagnostics will play a significant role in the development of personalized molecular medicine, a process that will be vital in modernizing healthcare structures.

Real-time simulation of the nonlinear visco-elastic deformations of soft tissues

Mass-spring-damper (MSD) models are often used for real-time surgery simulation due to their fast response and fairly realistic deformation replication. An improved real time simulation model of soft tissue deformation due to a laparoscopic surgical indenter was developed and tested. The mechanical realization of conventional MSD models was improved using nonlinear springs and nodal dampers, while their high computational efficiency was maintained using an adapted implicit integration algorithm. New practical algorithms for model parameter tuning, collision detection, and simulation were incorporated. The model was able to replicate complex biological soft tissue mechanical properties under large deformations, i.e., the nonlinear and viscoelastic behaviors. The simulated response of the model after tuning of its parameters to the experimental data of a deer liver sample, closely tracked the reference data with high correlation and maximum relative differences of less than 5 and 10%, for the tuning and testing data sets respectively. Finally, implementation of the proposed model and algorithms in a graphical environment resulted in a real-time simulation with update rates of 150 Hz for interactive deformation and haptic manipulation, and 30 Hz for visual rendering. The proposed real time simulation model of soft tissue deformation due to a laparoscopic surgical indenter was efficient, realistic, and accurate in ex vivo testing. This model is a suitable candidate for testing in vivo during laparoscopic surgery.

Regulation of pH During Amelogenesis

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation.

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.

An e-learning application on electrochemotherapy

BackgroundElectrochemotherapy is an effective approach in local tumour treatment employing locally applied high-voltage electric pulses in combination with chemotherapeutic drugs. In planning and performing electrochemotherapy a multidisciplinary expertise is required and collaboration, knowledge and experience exchange among the experts from different scientific fields such as medicine, biology and biomedical engineering is needed. The objective of this study was to develop an e-learning application in order to provide the educational content on electrochemotherapy and its underlying principles and to support collaboration, knowledge and experience exchange among the experts involved in the research and clinics.MethodsThe educational content on electrochemotherapy and cell and tissue electroporation was based on previously published studies from molecular dynamics, lipid bilayers, single cell level and simplified tissue models to complex biological tissues and research and clinical results of electrochemotherapy treatment. We used computer graphics such as model-based visualization (i.e. 3D numerical modelling using finite element method) and 3D computer animations and graphical illustrations to facilitate the representation of complex biological and physical aspects in electrochemotherapy. The e-learning application is integrated into an interactive e-learning environment developed at our institution, enabling collaboration and knowledge exchange among the users. We evaluated the designed e-learning application at the International Scientific workshop and postgraduate course (Electroporation Based Technologies and Treatments). The evaluation was carried out by testing the pedagogical efficiency of the presented educational content and by performing the usability study of the application.ResultsThe e-learning content presents three different levels of knowledge on cell and tissue electroporation. In the first part of the e-learning application we explain basic principles of electroporation process. The second part provides educational content about importance of modelling and visualization of local electric field in electroporation-based treatments. In the third part we developed an interactive module for visualization of local electric field distribution in 3D tissue models of cutaneous tumors for different parameters such as voltage applied, distance between electrodes, electrode dimension and shape, tissue geometry and electric conductivity. The pedagogical efficiency assessment showed that the participants improved their level of knowledge. The results of usability evaluation revealed that participants found the application simple to learn, use and navigate. The participants also found the information provided by the application easy to understand.ConclusionThe e-learning application we present in this article provides educational material on electrochemotherapy and its underlying principles such as cell and tissue electroporation. The e-learning application is developed to provide an interactive educational content in order to simulate the "hands-on" learning approach about the parameters being important for successful therapy. The e-learning application together with the interactive e-learning environment is available to the users to provide collaborative and flexible learning in order to facilitate knowledge exchange among the experts from different scientific fields that are involved in electrochemotherapy. The modular structure of the application allows for upgrade with new educational content collected from the clinics and research, and can be easily adapted to serve as a collaborative e-learning tool also in other electroporation-based treatments such as gene electrotransfer, gene vaccination, irreversible tissue ablation and transdermal gene and drug delivery. The presented e-learning application provides an easy and rapid approach for information, knowledge and experience exchange among the experts from different scientific fields, which can facilitate development and optimisation of electroporation-based treatments.

B-value dependence of DTI quantitation and sensitivity in detecting neural tissue changes

Recently, remarkable success has been demonstrated in using MR diffusion tensor imaging (DTI) to characterize white matter. Water diffusion in complex biological tissue microstructure is not a free or Gaussian process but is hindered and restricted, thus contradicting the basic assumption in conventional DTI that diffusion weighted signal decays with b-value in a monoexponential manner. Nevertheless, DTI by far is still the fastest and most robust protocol in routine research and clinical settings. To assess the b-value dependence of DTI indices and evaluate their sensitivities in detecting neural tissues changes, in vivo DTI data acquired from rat brains at postnatal day 13, 21 and 120 with different b-values (0.5–2.5 ms/μm2) and 30 gradient directions were analyzed. Results showed that the mean and directional diffusivities consistently decreased with b-value in both white and gray matters. The sensitivity of axial diffusivity (λ//) in monitoring brain maturation generally decreased with b-value whereas that of radial diffusivity (λ⊥) increased. FA generally varied less with b-value but in a manner dependent of the age and tissue type. Analysis also revealed that the FA sensitivity in detecting specific tissue changes was affected by b-value. These experimental findings confirmed the crucial effect of b-value on quantitative DTI in monitoring neural tissue alterations. They suggested that the choice of b-value in conventional DTI acquisition can be optimized for detecting neural tissue changes but shall depend on the specific tissue type and its changes or pathologies targeted, and caution must be taken in interpreting DTI indices.

Detection of bio-constituents in complex biological tissue using Raman microscopy. Application to human nail clippings

Raman spectra of human nail clippings from various sources were collected and then deconvoluted to obtain the pure component spectra of the underlying constituents present. This blind-deconvolution was performed using a self-modeling curve resolution technique, namely band-target entropy minimization (BTEM). The aim was to simplify the complexity of the Raman spectra and hence to identify the underlying biological molecules in more detail. BTEM analysis could recover 13 pure component Raman spectral estimates from the collected 438 spectra measured from 113 nail samples. Six recovered pure component spectral estimates correspond to proteins or polypeptides that contain various amino acids such as phenylalanine, tyrosine, tryptophan, and cysteine. Two are associated with the secondary structures of proteins, and five are associated with two carotenoid species, lipid, ferulic acid, and calcium phosphate. Subsequently, the relative concentrations of these bio-constituents were calculated from the measured mixture spectra and the pure component BTEM estimates. These profiles indicated that the concentrations of some bio-constituents are correlated while others are not. A further analysis using target transformation factor analysis (TTFA) revealed the possible presence of curcumin in the human nails. Since the present approach and analysis is rather general, it might be extended to many other biological tissues in a rather straightforward and similar manner, thus revealing more detailed underlying biochemical information such as biomarkers that may be useful for diagnostic purposes.

Observation of restricted diffusion in the presence of a free diffusion compartment: Single- and double-PFG experiments

Theoretical and experimental studies of restricted diffusion have been conducted for decades using single pulsed field gradient (s-PFG) diffusion experiments. In homogenous samples, the diffusion–diffraction phenomenon arising from a single population of diffusing species has been observed experimentally and predicted theoretically. In this study, we introduce a composite bi-compartmental model which superposes restricted diffusion in microcapillaries with free diffusion in an unconfined compartment, leading to fast and slow diffusing components in the NMR signal decay. Although simplified (no exchange), the superposed diffusion modes in this model may exhibit features seen in more complex porous materials and biological tissues. We find that at low q-values the freely diffusing component masks the restricted diffusion component, and that prolongation of the diffusion time shifts the transition from free to restricted profiles to lower q-values. The effect of increasing the volume fraction of freely diffusing water was also studied; we find that the transition in the signal decay from the free mode to the restricted mode occurs at higher q-values when the volume fraction of the freely diffusing water is increased. These findings were then applied to a phantom consisting of crossing fibers, which demonstrated the same qualitative trends in the signal decay. The angular d-PGSE experiment, which has been recently shown to be able to measure small compartmental dimensions even at low q-values, revealed that microscopic anisotropy is lost at low q-values where the fast diffusing component is prominent. Our findings may be of importance in studying realistic systems which exhibit compartmentation.

A B-spline based heterogeneous modeling and analysis of proximal femur with graded element

Bone is a complex biological tissue and natural heterogeneous object. The main objective of this study is to simulate quasi-static loading of bio-objects like human femur with B-spline based modeling and its 3D finite element analysis with graded element. B-spline surface representation method is extended to represent material composition to develop heterogeneous solid model of proximal femur. Lagrangian graded element is used to assign inhomogeneous isotropic elastic properties in finite element model to improve the performance. Convergence study is carried out with finite element model in single leg stance load condition. To test the feasibility of the model, sensitivity of simulation is investigated. To validate the model, numerical results are compared with those of an experimental work for the same specimen in simple stance load condition obtained from one of the reference paper. Good agreement is achieved for vertical displacement and strains in most of the locations.

Nanostructure Initiator Mass Spectrometry: Tissue Imaging and Direct Biofluid Analysis

Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.

Advanced imaging of multiple mRNAs in brain tissue using a custom hyperspectral imager and multivariate curve resolution

Simultaneous imaging of multiple cellular components is of tremendous importance in the study of complex biological systems, but the inability to use probes with similar emission spectra and the time consuming nature of collecting images on a confocal microscope are prohibitive. Hyperspectral imaging technology, originally developed for remote sensing applications, has been adapted to measure multiple genes in complex biological tissues. A spectral imaging microscope was used to acquire overlapping fluorescence emissions from specific mRNAs in brain tissue by scanning the samples using a single fluorescence excitation wavelength. The underlying component spectra obtained from the samples are then separated into their respective spectral signatures using multivariate analyses, enabling the simultaneous quantitative measurement of multiple genes either at regional or cellular levels.

Collagen dynamics in articular cartilage under osmotic pressure

Cartilage is a complex biological tissue consisting of collagen, proteoglycans and water. The structure and molecular mobility of the collagen component of cartilage were studied by (13)C solid-state NMR spectroscopy as a function of hydration. The hydration level of cartilage was adjusted between fully hydrated ( approximately 80 wt% H(2)O) and highly dehydrated ( approximately 30 wt% H(2)O) using the osmotic stress technique. Thus, the conditions of mechanical load could be simulated and the response of the tissue macromolecules to mechanical stress is reported. From the NMR measurements, the following results were obtained. (i) Measurements of motionally averaged dipolar (1)H-(13)C couplings were carried out to study the segmental mobility in cartilage collagen at full hydration. Backbone segments undergo fast motions with amplitudes of approximately 35 degrees whereas the collagen side-chains are somewhat more mobile with amplitudes between 40 and 50 degrees . In spite of the high water content of cartilage, collagen remains essentially rigid. (ii) No chemical shift changes were observed in (13)C cross-polarization magic angle spinning spectra of cartilage tissue at varying hydration indicating that the collagen structure was not altered by application of high osmotic stress. (iii) The (1)H-(13)C dipolar coupling values detected for collagen signals respond to dehydration. The dipolar coupling values gradually increase upon cartilage dehydration, reaching rigid limit values at approximately 30 wt% H(2)O. This indicates that collagen is essentially dehydrated in cartilage tissue under very high mechanical load, which provides insights into the elastic properties of cartilage collagen, although the mechanical pressures applied here exceed the physiological limit.

Ultrasound assisted extraction in quantifying lutein from chicken liver using high-performance liquid chromatography☆

Four sample preparation methods, (1) solvent (SOL), (2) saponification and solvent (SP), (3) ultrasound assisted solvent (UA), and (4) saponification and ultrasound assisted solvent (SP-UA), were used for quantifying lutein in chicken liver samples by HPLC. The lutein concentrations obtained by using SOL, UA, SP, and SP-UA were significantly different with values from 10.4 microg/g (UA) to undetected (SOL). Efficiency of the four different methods for extracting lutein from high to low were the UA, SP, SP-UA, and SOL method. The measured value of lutein in the liver sample using the UA method was approximately two and three times higher than that obtained from the SP and SP-UA method, respectively. The methods with saponification significantly affected the stabilities of lutein in liver samples. The lutein concentration measured with the solvent only method was either much lower than any of the other extraction methods or undetectable. This indicated that little lutein in those samples was in a form that could be extracted directly by solvent. Compared with the saponification method, the ultrasound assisted solvent method could effectively extract lutein from sample matrix and thus avoid chemical degradation reactions, which would be especially important for complex biological tissue such as liver.

Combination of small angle neutron scattering data and mesoscopic simulation techniques as a tool for the structural characterization and prediction of properties of bi-phasic media

A powerful, versatile methodology based on the combination of small angle neutron scattering (SANS) with mesoscopic simulation techniques is developed in an effort to relate in a sufficiently accurate manner the structure of biphasic media to their macroscopic physicochemical (equilibrium and dynamic) properties. Appropriate mathematical transformation of SANS spectra provides valuable information about the disordered geometry of a wide range of biphasic materials spanning from typical porous materials (Vycor porous glass, silica gel, alumina membrane) to complex biological tissues (stratum corneum), in terms of the corresponding autocorrelation function. Digital reconstruction (stochastic or process-based) is employed to generate two- or three-dimensional binary images of the respective structures, on the basis of the statistical properties obtained from the SANS spectra of the original materials. The resulting reconstructed domains are further used for the simulation of equilibrium (sorption) and dynamic (diffusion and permeation) processes, addressing the most common applications of the materials under consideration.

Raman spectral mapping in the assessment of axillary lymph nodes in breast cancer.

This paper is the first reported description of Raman Spectroscopy in the assessment of axillary lymph nodes in breast cancer patients. Raman Spectroscopy is an inelastic scattering spectroscopic technique appropriate for the assessment of unprocessed complex biological tissues. Spectra represent biochemical signatures of the tissue under scrutiny. The described method of Raman spectral mapping produces false-color spectral images of lymph node sections. These can be compared with standard histopathology slides and white light images of nodal tissue. This method has the potential to allow the detailed biochemical assessment of heterogeneous lymph node features, and to contribute towards the development of an optical diagnostic tool for use in a clinical setting.

Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue

The measurement of neuropeptides in complex biological tissue samples requires efficient and appropriate extraction methods so that immunoreactivity is retained for subsequent radioimmunoassay detection. Since neuropeptides differ in their molecular mass, charge and hydrophobicity, no single method will suffice for the optimal extraction of various neuropeptides. In this study, dental pulp tissue was obtained from 30 human non-carious teeth. Of the three different neuropeptide extraction methods employed, boiling in acetic acid in the presence of protease inhibitors yielded the highest levels of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). High pressure liquid chromatography (HPLC) analysis of dental pulp tissue verified the authenticity of the neuropeptides extracted.

Analysis of Gelatinases in Complex Biological Fluids and Tissue Extracts

G elatinase B (matrix metalloproteinase-9 [MMP-9]) and gelatinase A (MMP-2) are two closely related members of the MMP family that efficiently degrade denatured collagens or gelatins (Van den Steen et al, 2002). Specific MMPs play a major role in physiological processes, including angiogenesis, wound healing, bone remodeling, and cell migration. Moreover, MMP-9 and MMP-2 are key effector molecules in inflammation, autoimmunity (Opdenakker and Van Damme, 1994), and cancer (Sehgal et al, 1998). Analysis of both enzymes in complex biological samples, especially those with low gelatinase content, therefore is essential. We combined a miniaturized gelatin affinity chromatography with gelatin zymography and Western blot analysis. This strategy allows extremely sensitive and unambiguous detection of gelatinases. Gelatinases are often detected with specific antibodies or by substrate conversion assays. Gelatinase activity assays measure overall gelatinase activity often with the use of labeled gelatins (Paemen et al, 1996). Because these do not discriminate between gelatinase A and B and even other gelatin-degrading enzymes, specificity is low, especially in the analysis of complex biological samples. Introducing affinity prepurification with the use of monoclonal antibodies enhances the specificity (Hanemaaijer et al, 1998). Moreover, only activated enzymes are recognized. ELISA detects specific forms of MMP-9 or MMP-2. Unfortunately, this method does not necessarily differentiate between pro-enzyme and activated forms. Discrimination between different gelatin-degrading enzymes and their respective activation status may be achieved by zymography or Western blot analysis. Gelatin zymography detects picogram levels of MMP-9 (Masure et al, 1991). The sensitivity of Western blot analysis is usually lower and depends on the antibody affinity for MMP-9. In our hands, a combination of two monoclonal antibodies against mouse MMP-9 resulted in a detection limit of 100 pg of MMP-9. Nevertheless, such low detection limits are frequently not attained because the ratio of MMP-9 versus total protein is generally extremely low in crude samples, whereas the total protein load per lane is limited to 25 g so as not to distort the electrophoresis. After protein overloading, gelatinolysis may be eclipsed in zymography. Interference by gelatinolytic activity from other (abundant) enzymes constitutes an additional problem for zymography. For instance, in stomach extracts, pepsin is abundantly present and because this and other proteases also cleave gelatin, it will mask the gelatinases. To improve the ratio of MMP-9 versus total protein and to exclude interfering gelatinolytic activity, we developed a simple strategy for optimal preparation of complex samples, including tissue extracts. We used a miniaturized affinity chromatography purification step, taking advantage of gelatinases’ strong affinity for gelatin (Masure et al, 1991). For rapid and reproducible purification of the samples, we made use of mini-spin columns (Bio-Rad Laboratories, Hercules, California) and gelatin-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). Equilibration buffer was composed of 50 mM of Tris (pH 7.5), 0.5 M of NaCl, 10 mM of CaCl2, 0.01% Tween 20, and 5 mM of o-phenanthroline; washing buffer 1 had a similar composition except that the concentration of Tween 20 was increased to 0.05%. Washing buffer 2 was with omission of NaCl because high salt concentrations hinder the electrophoresis. The o-phenanthroline was added to the samples as a gelatinase inhibitor to prevent the gelatinolytic activity from breaking down the gelatin from the Sepharose beads, which are used for the affinity purification of the enzymes. The binding of gelatinases to gelatin-Sepharose is not disturbed by the presence of o-phenanthroline because this inhibitor acts by the chelation of the catalytic Zn , whereas binding to gelatin is mediated by the three fibronectin type II–like repeats. Elution buffer was at once the electrophoresis loading buffer and was composed of 100 mM of Tris/HCl (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, and 200 g/ml of bromophenol blue as tracking dye. DOI: 10.1097/01.LAB.0000038556.54069.73