Is there any objective and independent characterization and modeling of soft biological tissues?

The characterization of soft tissue raises several difficulties. Indeed, soft biological tissues usually shrink when dissected from their in vivo location. This shrinkage is characteristic of the release of residual stresses, since soft tissues are indeed often pre-stressed in their physiological configuration. During experimental loading, large extension at very low level of force are expected and assumed to be related to the progressive recruitment and stretching of fibers. However, the first phase of the mechanical test is also aiming at recovering the pre-stressed in vivo behavior. As a consequence, the initial phase, corresponding to the recovering of prestress and/or recruitment of fiberes, is questionable and frequently removed. One of the preferred methods to erase it consists in applying a preforce or prestress to the sample: this allows to easily get rid of the sample retensioning range. However this operation can impact the interpretation of the identified mechanical parameters. This study presents an evaluation of the impact of the data processing on the mechanical properties of a numerically defined material. For this purpose, a finite element simulation was performed to replicate a uniaxial tensile test on a biological soft tissue sample. The influence of different pre-stretches on the mechanical parameters of a second order Yeoh model was investigated. The Yeoh mechanical parameters, or any other strain energy density, depend strongly on any pre- and post-processing choices: they adapt to compensate the error made when choosing an arbitrary level of prestretch or prestress. This observation spreads to any modeling approach used in soft tissues. Mechanical parameters are indeed naturally bound to the choice of the pre-stretch (or pre-stress) through the elongation and the constitutive law. Regardless of the model, it would therefore be pointless to compare mechanical parameters if the conditions for the processing of experimental raw data are not fully documented.

Experimental study of the mechanical behavior of an explanted mesh: The influence of healing.

To better understand the in vivo mechanical behavior of synthetic mesh implants, we designed a specific experimental protocol for the mechanical characterization of explanted mesh under uniaxial tension. The implantation of a mesh leads to the development of scar tissue and the formation of a new composite made of native tissue, a mesh implant and scar tissues. This study focused on three points: determining the minimum representative size of mesh implants required for mechanical test samples, highlighting the influence of healing, and defining the healing time required to ensure stabilized mechanical properties. First, we determined the minimum representative size of mesh implants for the mechanical characterization with a study on a synthetic composite made of mesh and an elastomeric matrix mimicking the biological tissues. The size of the samples tested was gradually decreased. The downsizing process was stopped, when the mechanical properties of the composite were not preserved under uniaxial tension. It led to a sample representative size 3cm long and 2cm wide between the grips. Then an animal study was conducted on Wistar rats divided into eight groups. One group was set as control, consisting of the healthy abdominal wall. The other seven groups underwent surgery as follows: one placebo (i.e., without mesh placement), and six with a mesh installation on the abdominal wall and healing time. The rats were sacrificed after different healing times ranging from 1 to 5 months. We observed the influence of healing and healing time on the mechanical response under uniaxial tension of the new composite formed by scar, native tissue, and textile. It seems that 2 months are required to ensure the stabilization of the mechanical properties of the implanted mesh. We were not able to tell the control group (native abdominal wall) from the placebo group (native and scar tissue). This protocol was tested on two different prostheses after 3 months of healing. With this protocol, we were able to differentiate one mesh from another after host integration.

Simulation of normal pelvic mobilities in building an MRI-validated biomechanical model.

INTRODUCTION AND HYPOTHESIS: Three-dimensional modeling of feminine pelvic mobility is difficult because the sustaining system is not well understood and ligaments are especially difficult to identify on imaging. METHODS: We built a 3-D numerical model of the pelvic cavity, based on magnetic resonance (MR) images and knowledge about anatomy and validated it systematically. RESULTS: The quantitative results of this model allow for the non-destructive localization of the structures involved in pelvic statics. With a better configuration of the functional pelvis and topological criteria, we can obtain a coherent anatomical and functional model. CONCLUSIONS: This model is the first step in developing a tool to localize and characterize pelvic imbalance in patients.

Frequency and distribution of nerves in scrapie-affected and unaffected Peyer's patches and lymph nodes.

Transmission of sheep scrapie and some other prion diseases, including variant Creutzfeldt-Jakob disease of man, probably occurs via the oral route. A disease-associated variant of the host-coded prion protein (PrP(d)) accumulates in germinal center follicles of lymphoid tissues, including Peyer's patches of the gut, where it can be detected before its accumulation in the central nervous system. To investigate the potential role of lymphoid tissue nerves in neuroinvasion, we used immunohistochemical methods to study the frequency and distribution of nerves and PrP(d) accumulation in Peyer's patches and other lymphoid tissues from scrapie-affected and unaffected sheep. Nerves were infrequently found in secondary follicles of Peyer's patches, but never in germinal centers of the other lymphoid tissues tested. No differences in the frequency or distribution of nerves were found in relation to the presence or absence of PrP(d) accumulation. PrP(d) accumulation and nerves were only infrequently present together in Peyer's patches. These results suggest that, even if amplification of infectivity in lymphoid tissues facilitates neuroinvasion, nerves within lymph nodes and germinal centers of Peyer's patches do not play a primary role in transport of infectivity to the central nervous system. However, sheep between 2 and 4 months of age had significantly more nerve fibers within follicles than older groups. It is therefore possible that a general increase in nerve density of the intestine during early phases of life may contribute to an increased susceptibility of young animals to oral prion infection.

Spherical plant viruses: interactions in solution, phase diagrams and crystallization of brome mosaic virus.

Brome mosaic virus (BMV) is a small icosahedral plant virus of mean diameter 268 A. Interactions between BMV particles in solution were studied by means of small-angle X-ray scattering in order to find crystallization conditions. The interactions between biomacromolecules as large as these viruses have not yet been systematically studied by this method. As it is known that usually proteins crystallize in, or close to, attractive regimes, the interactions between BMV particles in solution were studied as a function of pH, type of salt and size and concentration of polyethylene glycol. An unexpected result of these studies is that the precipitates obtained upon addition of PEG alone or PEG combined with salt were in fact made of microcrystals, which were all characterized by the same series of diffraction peaks, with positions close to those of a centered cubic space group. A phase diagram of the virus as a function of PEG concentration was established by means of microbatch experiments. From the precipitation zones, conditions for crystallization were tested from 5 to 40 mg ml(-1) virus with 3-10%(w/v) PEG 8000 or PEG 20,000. Small crystals were obtained in several conditions after a few days and continued growing for several weeks.

The formation of empty shells upon pressure induced decapsidation of turnip yellow mosaic virus.

The stability of turnip yellow mosaic virus (TYMV) was investigated under pressure, using solution neutron small angle scattering. Dissociation products were characterized by analytical ultracentrifugation and electron microscopy. At pH 6.0, TYMV remained unaffected by pressure, up to 260 Megapascals (MPa), the highest pressure reached in these experiments. At pH 8.0, TYMV remained unaffected by pressure up to 160 MPa, but decapsidated irreversibly above 200 MPa, giving rise to more and more empty shells upon increasing pressure. The organization of these empty shells was similar to that of the capsid of native virions, apart from the presence of a hole corresponding to the loss of a group of 5-8 coat protein subunits, through which the RNA may have escaped. At variance with other small isometric viruses, the capsid of TYMV never dissociated under pressure into subunits or small aggregates of subunits. This exceptional behavior of TYMV is probably due to the importance of van der Waals contacts and hydrogen bonds in the stability of its capsid.

Structural dynamics, an intrinsic property of viral capsids.

In this review, we emphasize that high-resolution models of the structures of small plant and animal viruses obtained by X-ray crystallography are static and insufficient to describe the behavior of these virions. Viral capsids are highly flexible and may undergo conformational changes under physiological conditions without collapse of the virions. This flexibility plays a key role in the process of infection.

Detection and characterization of an intermediate conformation during the divalent ion-dependent swelling of tomato bushy stunt virus.

We have used time-resolved small-angle X-ray scattering (SAXS) in solution to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. The reaction was initiated by rapid mixing of a virus solution with the same buffer containing a variable amount of EDTA. The X-ray scattering data sets recorded after mixing were submitted to a singular value decomposition analysis which demonstrated the existence of an intermediate state in addition to the compact and fully swollen forms of the virion. The kinetics of the reaction display an initial lag, and a linear combination of three exponential terms is required for a satisfactory analytical fit. Accordingly, a model is put forward involving three sequential irreversible processes between four species. Beyond the three structural species mentioned above, the fourth one, which is the second species along the time sequence, is proposed to represent those viruses which, although partially deprived of Ca2+ ions, are still in the compact conformation. Using the combination of the kinetic model and the structural data, an estimate of the intermediate scattering pattern can be derived from each time resolved frame. These patterns are all very similar after a slight drift towards the swollen pattern over the first 2 min. The curve presents well-resolved minima and maxima, corresponding to an isometric particle with an outer radius of about 172 A for the intermediate conformation.

Conformational changes preceding decapsidation of bromegrass mosaic virus under hydrostatic pressure: a small-angle neutron scattering study.

The stability of bromegrass mosaic virus (BMV) and empty shells reassembled in vitro from purified BMV coat protein was investigated under hydrostatic pressure, using solution small-angle neutron scattering. This technique allowed us to monitor directly the dissociation of the particles, and to detect conformational changes preceding dissociation. Significant dissociation rates were observed only if virions swelled upon increase of pressure, and pressure effects became irreversible at very high-pressure in such conditions. At pH 5.0, in buffers containing 0.5 M NaCl and 5 mM MgCl(2), BMV remained compact (radius 12.9 nm), dissociation was limited to approximately 10 % at 200 MPa, and pressure effects were totally reversible. At pH 5.9, BMV particles were slightly swollen under normal pressure and swelling increased with pressure. The dissociation was reversible to 90 % for pressures up to 160 MPa, where its rate reached 28 %, but became totally irreversible at 200 MPa. Pressure-induced swelling and dissociation increased further at pH 7.3, but were essentially irreversible. The presence of (2)H(2)O in the buffer strongly stabilized BMV against pressure effects at pH 5.9, but not at pH 7.3. Furthermore, the reversible changes of the scattered intensity observed at pH 5.0 and 5.9 provide evidence that pressure could induce the release of coat protein subunits, or small aggregates of these subunits from the virions, and that the dissociated components reassociated again upon return to low pressure. Empty shells were stable at pH 5.0, at pressures up to 260 MPa. They became ill-shaped at high-pressure, however, and precipitated slowly after return to normal conditions, providing the first example of a pressure-induced conformational drift in an assembled system.

Polymorphism of turnip yellow mosaic virus empty shells and evidence for conformational changes occurring after release of the viral RNA. A differential scanning calorimetric study.

Turnip yellow mosaic virus (TYMV) is a small isometric plant virus which decapsidates by releasing its RNA through a hole in the capsid, leaving behind an empty shell [R. E. F. Matthews and J. Witz, (1985) Virology 144, 318-327]. Similar empty shells (artificial top component, ATC) can be obtained by submitting the virions to various treatments in vitro. We have used differential scanning calorimetry, analytical sedimentation, and electron microscopy to investigate the thermodenaturation of natural empty shells (NTC, natural top component) present in purified virus suspensions, and of several types of ATCs. ATCs divided in two major classes. Those obtained by alkaline titration, by the action of urea or butanol behaved as NTC: their thermograms contained only one peak corresponding to the irreversible dissociation of the shells and the denaturation of the coat protein. The temperature of this unique transition varied significantly with pH, from 71 degrees C at pH 4.5 to 84 degrees C at pH 8.5. The thermograms of ATCs obtained by freezing and thawing, or by the action of high pressure, contained two peaks: shells dissociated first into smaller protein aggregates at 57 degrees C (at pH 5.0) to 61 degrees C (at pH 8.5), which denatured at the temperature of the unique transition of NTC. Shells obtained by heating virions to 55 degrees C at pH 7.6, changed conformation after the release of the viral RNA, as upon continuous heating to 95 degrees C, their thermograms were similar to those of the shells obtained by freezing and thawing, whereas after purification they behaved like NTC. Structural implications of these observations are discussed.

Kinetic analysis of analyte binding by optical biosensors: hydrodynamic penetration of the analyte flow into the polymer matrix reduces the influence of mass transport.

I examined the penetration of the hydrodynamic flow into a polymer matrix immobilized by grafting to a surface, such as used in optical biosensors designed to measure binding reactions in real time. I show that the flow penetrates with an appreciable velocity into a region located at the tip of such a polymer brush, corresponding to about 10 to 15% of the total mass of the grafted polymer. Furthermore, under the conditions recommended for kinetic measurements, the concentrations of both polymer and immobilized ligands are low in these regions of the matrix, where crowding effects are negligible. Under such conditions, the hydrodynamic flow penetrating into the dextran matrix flow will bring the analytes close to their targets, thus considerably reducing transport problems.

Measurement of antigen-antibody interactions with biosensors.

The introduction in 1990 of a new biosensor technology based on surface plasmon resonance has revolutionized the measurement of antigen-antibody binding interactions. In this technique, one of the interacting partners is immobilized on a sensor chip and the binding of the other is followed by the increase in refractive index caused by the mass of bound species. The following immunochemical applications of this new technology will be described: (1) functional mapping of epitopes and paratopes by mutagenesis; (2) analysis of the thermodynamic parameters of the interaction; (3) measurement of the concentration of biologically active molecules; (4) selection of diagnostic probes.

Thermodynamic analysis of antigen-antibody binding using biosensor measurements at different temperatures.

The thermodynamic parameters of the interaction between hen egg white lysozyme and Fab D1.3 were determined by measuring the temperature dependence of the ratio of its kinetic association and dissociation rate constants. Biosensor technology (BIAcore 2000) was used to measure the rate constants at temperatures ranging from 5 to 40 degrees C. The value of DeltaG degrees at 25 degrees C (-49 kJ M-1) calculated by this method was very close to that obtained previously from fluorescence quenching measurements (-48.5 kJ M-1). However, the value of DeltaH degrees measured at 25 degrees C by biosensor technology (-35 kJ M-1) was smaller than that determined previously by microcalorimetry (-90 kJ M-1). Another difference was the limited variation of ln K and DeltaG with temperature observed with BIAcore compared to the steady decrease of ln K with temperature found by calorimetry. Our data showed that the binding reaction was driven only by enthalpy below 23 degrees C, by enthalpy and entropy between 23 and 35 degrees C, and only by entropy above 35 degrees C. This suggests, inter alia, that the contribution from the enthalpy of hydration due to the water molecules present at the interface in the lysozyme-antibody complex is progressively eliminated as the temperature increases. Whereas calorimetric data pertain to all the components present in the sample, including solvent molecules, BIAcore measurements monitor only the physical association and dissociation of the two macromolecular species. The difference between the two sets of data may also reflect the complexity of the binding mechanism between lysozyme and Fab D1.3.

Use of a mass-thickness marker to estimate systematic errors and statistical noise in the detection of phosphorus by electron spectroscopic imaging.

The element signal obtained from electron-energy-filtered micrographs depends on the systematic error in calculating the background and on the noise in the background-corrected image. Both systematic error and statistical fluctuation of the background can be assessed experimentally with a specimen that combines the element-containing feature with a mass-thickness marker. The approach is described for the mapping of phosphorus in turnip yellow mosaic viruses prepared on a supporting carbon film of variable thickness. The thickness modulations are produced by the additional deposition of heat-evaporated carbon through a second grid used as a mask. The three-window power-law method and the two-window difference method are compared. With the three-window power-law method, the mass-thickness modulations of the marker are still visible in the map, indicating a systematic error for the calculated background. In addition, the intensity profile over the area of the thick carbon film is broader than in the map corrected by the two-window method, indicating a higher level of noise. With the two-window difference method, mass-thickness contrast was practically eliminated due to an improved protocol that uses the mass-thickness marker to calculate the scaling factor: instead of scaling the grey-level of a single background feature, the pre-edge image is scaled to the contrast of the marker area in the image acquired at the element-specific energy loss.

The three-dimensional distribution of RNA and protein in the interior of tomato bushy stunt virus: a neutron low-resolution single-crystal diffraction study.

BACKGROUND: The published high-resolution model of the isometric T = 3 plant virus tomato bushy stunt virus (TBSV) shows the packing in three different environments (A, B, C) of the 180 coat protein subunits of the capsid. It does not, however, account for the localization of either the viral RNA or approximately 25% of the amino acids of the protein subunits, although at least the RNA is rigidly linked to the viral capsid. Solution studies have shown that most of the missing protein is located in an inner shell, and that most of the RNA is sandwiched between the two protein shells. RESULTS: We have determined the organization of TBSV at 16 A resolution, using neutron single-crystal diffraction. Connections between the two protein shells are confined to the 20 three-fold axes of the virion, where three C-type subunits meet. Much more RNA density is located under the 30 C-C dimers than under the 60 A-B dimers, where we could even identify lagoons of solvent. CONCLUSIONS: Our results emphasize the importance of the amino termini of the 60 C-type protein subunits not only in the RNA-protein interactions but also in the organization of the coat protein, and, probably, in the assembly of the virion. The lack of equivalence between subunits of classes A or B and subunits of class C is even more pronounced in the interior of the virion than in the outer shell, which possesses icosahedral symmetry.

Organization of turnip yellow mosaic virus investigated by neutron small angle scattering at 80 K: an intermediate state preceding decapsidation of the virion?

The organization of turnip yellow mosaic virus has been investigated by neutron small angle scattering at 300 K and 80 K in buffers containing various amounts of D2O. We confirm that in native virions, no substantial part of the RNA is located at a radius larger than ca. 100-110 A, i.e., that there is very little interpretation of the RNA into the capsid. At 80 K, scattering curves do not depend much upon contrast, from 40% D2O to 100% D2O buffers, but are strongly affected by interparticle interference. We could, however, show that it is not the case for the subsidiary intensity maximum at q approximately 0.06 A-1. From the position of this maximum, we conclude that upon freezing, the radius of the capsid expands by c.a. 3.5% and the RNA penetrates deeply into the protein shell. Biological implications of this conformational change immediately preceding decapsidation are discussed.

Inhibition of in vitro cotranslational disassembly of tobacco mosaic virus by monoclonal antibodies to the viral coat protein.

It has been shown by others that translation of tobacco mosaic virus (TMV) RNA may begin before uncoating of particles is complete. We provide evidence that this cotranslational disassembly can be inhibited by incubating TMV treated at pH 8 with certain monoclonal antibodies (MAbs) specific for TMV coat protein. The most efficient inhibition was achieved by incubation with some anti-metatope MAbs known to bind to the TMV extremity that becomes disassembled first and contains the 5' end of the RNA, as well as with some anti-cryptotope MAbs that bind only to dissociated coat protein subunits.