Local rheological measurements in the granular flow around an intruder.

The rheological properties of granular matter within a two-dimensional flow around a moving disk is investigated experimentally. Using a combination of photoelastic and standard tessellation techniques, the strain and stress tensors are estimated at the grain scale in the time-averaged flow field around a large disk pulled at constant velocity in an assembly of smaller disks. On the one hand, one observes inhomogeneous shear rate and strongly localized shear stress and pressure fields. On the other hand, a significant dilation rate, which has the same magnitude as the shear strain rate, is reported. Significant deviations are observed with local rheology that justify the need of searching for a nonlocal rheology.

Cytotoxicity and genotoxicity of panel of single- and multiwalled carbon nanotubes: in vitro effects on normal Syrian hamster embryo and immortalized v79 hamster lung cells.

Carbon nanotubes (CNTs) belong to a specific class of nanomaterials with unique properties. Because of their anticipated use in a wide range of industrial applications, their toxicity is of increasing concern. In order to determine whether specific physicochemical characteristics of CNTs are responsible for their toxicological effects, we investigated the cytotoxic and genotoxic effects of eight CNTs representative of each of the commonly encountered classes: single- SW-, double- DW-, and multiwalled (MW) CNTs, purified and raw. In addition, because most previous studies of CNT toxicity were conducted on immortalized cell lines, we decided to compare results obtained from V79 cells, an established cell line, with results from SHE (Syrian hamster embryo) cells, an easy-to-handle normal cell model. After 24 hours of treatment, MWCNTs were generally found to be more cytotoxic than SW- or DWCNTs. MWCNTs also provoked more genotoxic effects. No correlation could be found between CNT genotoxicity and metal impurities, length, surface area, or induction of cellular oxidative stress, but genotoxicity was seen to increase with CNT width. The toxicity observed for some CNTs leads us to suggest that they might also act by interfering with the cell cycle, but no significant differences were observed between normal and immortalized cells.

Shear modulus and dilatancy softening in granular packings above jamming.

We investigate experimentally the mechanical response to shear of a monolayer of bidisperse frictional grains across the jamming transition. We inflate an intruder inside the packing and use photoelasticity and tracking techniques to measure the induced shear strain and stresses at the grain scale. We quantify experimentally the constitutive relations for strain amplitudes as low as 10(-3) and for a range of packing fractions within 2% variation around the jamming transition. At the transition strong nonlinear effects set in: both the shear modulus and the dilatancy shear soften at small strain until a critical strain is reached where effective linearity is recovered. The scaling of the critical strain and the associated critical stresses on the distance to jamming are extracted. We check that the constitutive laws, together with mechanical equilibrium, correctly predict to the observed stress and strain profiles. These profiles exhibit a spatial crossover between an effective linear regime close to the inflater and the truly nonlinear regime away from it. The crossover length diverges at the jamming transition.

How the ideal jamming point illuminates the world of granular media.

The zero temperature properties of frictionless soft spheres near the jamming point have been extensively studied both numerically and theoretically; these studies provide a reliable base for the interpretation of experiments. However, recent work by Ikeda et al. showed that, in a parameter space of the temperature and packing fraction, experiments to date on colloids have been rather far from the theoretical scaling regime. An important question is then whether theoretical results concerning point-J are applicable to any physical/experimental system, including granular media, which we consider here. On the surface, such a-thermal, frictional systems might appear even further from the idealized case of thermal soft spheres. In this work we address this question via experiments on shaken granular materials near jamming. We have systematically investigated such systems over a number of years using hard metallic grains. The important feature of the present work is the use of much softer grains, cut from photoelastic materials, making it possible to determine forces at the grain scale, the details of the contact networks and the motion of individual grains. Using this new type of particle, we first show that the contact network exhibits remarkable dynamics. We find strong heterogeneities, which are maximum at the packing fraction ϕ*, distinct from and smaller than the packing fraction ϕ(†), where the average number of contacts per particle, z, starts to increase. In the limit of zero mechanical excitation, these two packing fractions converge at point J. We also determine dynamics on time scales ranging from a small fraction of the shaking cycle to thousands of cycles. We can then map the observed system behavior onto results from simulations of ideal thermal soft spheres. Our results indicate that the ideal jamming point indeed illuminates the world of granular media.

The mechanical stress-strain properties of single electrospun collagen type I nanofibers.

Knowledge of the mechanical properties of electrospun fibers is important for their successful application in tissue engineering, material composites, filtration and drug delivery. In particular, electrospun collagen has great potential for biomedical applications due to its biocompatibility and promotion of cell growth and adhesion. Using a combined atomic force microscopy (AFM)/optical microscopy technique, the single fiber mechanical properties of dry, electrospun collagen type I were determined. The fibers were electrospun from a 80 mg ml(-1) collagen solution in 1,1,1,3,3,3-hexafluro-2-propanol and collected on a striated surface suitable for lateral force manipulation by AFM. The small strain modulus, calculated from three-point bending analysis, was 2.82 GPa. The modulus showed significant softening as the strain increased. The average extensibility of the fibers was 33% of their initial length, and the average maximum stress (rupture stress) was 25 MPa. The fibers displayed significant energy loss and permanent deformations above 2% strain.

In vitro cytotoxicity and transforming potential of industrial carbon dust (fibers and particles) in syrian hamster embryo (SHE) cells.

Carbon fibers have many applications, mainly in high-tech industries such as the aviation industry. Eleven carbon samples (fibers and particles) coming from an aeronautic group were tested for their cytotoxicity and carcinogenic potential using in vitro short-term assays in Syrian hamster embryo cells. These samples were taken during each important step of the process, i.e. from the initial heating of polyacrylonitrile fibers to pure carbon fibers. They were compared to an asbestos fiber, an amorphous silica, and two commercial graphite powders. Their physical-chemical characteristics and their capacity to release reactive oxygen species (ROS) were determined. This study showed that none of the carbon samples was able to generate ROS as measured by Electron Paramagnetic Resonance analysis, and in our biological assays, they demonstrated no morphological transformation potential and low cytotoxicity compared to positive control (chrysotile asbestos).

Inhaled crocidolite mutagenicity in lung DNA.

We used transgenic mice carrying the lacI reporter gene to study the mutagenesis potential of asbestos crocidolite. The animals were exposed by nose-only inhalation to an aerosol containing 5.75 mg/m(3) crocidolite dust for 6 hr/day and 5 consecutive days. After 1, 4, and 12 weeks, we examined four end points: the cytology of bronchoalveolar lavage, the lung load of crocidolite, the hydrophobic DNA adducts, and the mutations in the lacI reporter gene. Twelve weeks after exposure, nearly 10% of the inhaled fibers remained in the lung (227 +/- 103 ng/mg lung). There was evidence of a typical inflammatory response consisting of multinucleate macrophages at weeks 4 and 12, whereas immediately after the exposure, we observed numerous polymorphonuclear neutrophils. The mutant frequency significatively increased during the fourth week after the exposure: 13.5 [time] 10(-5) in the exposed group versus 6. 9 10(-5) in the control group. The induction factor, defined by the ratio of checked mutants of exposed mice to checked mutants of control mice, was 1.96. The mutation spectrum of control lung DNA and exposed lung DNA was similar, suggesting the possible involvement of a DNA repair decrease in crocidolite-treated animals. We used the (32)P-postlabeling method and did not detect any increase of either 5 mC or bulky adduct in treated mice. This is the first study that demonstrates asbestos mutagenicity in vivo after a nose-only inhalation.

Genotoxicity of 3-methylcholanthrene in liver of transgenic big Blue mice.

Transgenic mice provide a unique tool for studying the tissue specificity and mutagenic potential of chemicals. Because 3-methylcholanthrene (3MC) was found mutagenic in bacteria, clastogenic in bone marrow, and induces DNA adducts in animals, we were interested to determinine whether this xenobiotic provokes (1) cell proliferation, (2) transcriptional activity changes, (3) DNA adducts, and (4) hepatic mutations in transgenic Big Blue mice carrying the lambdaLIZ phage shuttle vector. Big Blue C57/Bl male mice were treated with a single intraperitoneal dose of 80 mg/kg 3MC for 1, 3, 6, 14, or 30 days. Cell proliferation was checked by 5-bromo-2-deoxyuridine labeling and immunohistochemical detection. The maximal increase of the mitotic index was evidenced after 3 days (2.9 times the control value; P < 0.01). The relative nucleus area, reflecting the transcriptional activity, was also the highest in the treated group after 3 days: 1.86 times the control value, on average (P < 0.01). Four major DNA adducts, determined according to the [(32)P]-postlabeling method, were evidenced in liver DNA of treated mice, 6 days after the treatment: the spot intensities increased in a time-dependent manner. The mutant frequency of liver DNA was the highest after 14 days: 20.3 +/- 2.9 x 10(-5) in the treated vs. 7.6 +/- 2.7 x 10(-5) in the control mice (P < 0.01). Sequencing of the lambda lacI mutant plaques showed mainly G:C --> T:A and C:G --> A:T transversions. In conclusion, 3MC at first induced nuclear enlargement and a slight increase of cell proliferation in liver, followed by parallel formation of DNA adducts and mutations. This study shows how transgenic models allow in vivo evaluation of mechanistically simultaneous endpoints.

Short-term crocidolite inhalation studies in mice: validation of an inhalation chamber.

A nose-only inhalation chamber is described: this chamber being computer automated has been particularly designed for mice on which it was validated using a crocidolite aerosol at a nominal concentration of 13.6 mg/m3, 6 h/day during 5 days. A month later the mice showed typical inflammatory bronchoalveolar liquids with many polynucleated or activated macrophages and asbestos bodies. The burden of crocidolite fibers ranged from 345,000 to 1,300,000 fibers per mg of dried lung. This study demonstrates that during the month that followed a short-term mice exposure to crocidolite fibers, the inflammatory response was still persistent. These toxicological endpoints validate the nose-only inhalation chamber to be useful for common or transgenic mice.

Use of non-radioactive methods for the determination of the expression, the sequence and the copy-number of transgene in mice.

Marshall's observation that "toxicology goes molecular", is turning out to be more true than ever; namely as it is observed that toxicological endpoints are the point of interaction between proteins and genes following the administration of toxicants. Transgenic mice represent a valuable tool for studying the adverse effects of chemicals in genetically engineered animals such as p53 deficient mice, or mice carrying the v-Ha-ras oncogene. The latter were used in our laboratory for such toxicological assessments of chemicals. In order to verify that the transgene was expressed in normal, as well as in tumor cells, the transgene was detected in different tissues fixed with various solutions using in situ hybridization. It was also specifically retrotranscribed from paraffin-embedded tissues and consequently sequenced using a Taq polymerase reaction. We found that the transgene was expressed in various organs. It carries a specific mutation of codon 12 leading to the activation of its encoded product (transducin: p21v-Ha-ras). Moreover using a laser scanning densitometer, it has been demonstrated that 2 to 3 copies of the transgene were present per genome-equivalent in some tissues. All experiments were realized using non-radioactive labelling and detection (chemiluminescent or colorigenic) methods. Indeed, the screening of such animals was realized in a easier and a safer manner using the methods described in this paper than the usual methods based on the use of radiolabelled precursors.

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids. Part Two: Colorigenic methods and comparison with chemiluminescent methods.

The diagnosis of genetic infections and cancerous diseases is carried out more and more often at a molecular level using Southern's technique which is based on the use of 32P-labelled DNA. In order to circumvent the risks and rapid decrease in radioactivity associated with these latter techniques, new colorigenic methods have been developed. In this work, we describe the use of dTTP analogues (digoxigenin-dUTP and biotin-dUTP) for the labelling of probes and detection of target DNA. Using digoxigenin-11-dUTP, 0.1 aM of a 561 bp target DNA was detected by using a modified Southern procedure. The reliability and the high sensitivity of such methods make them a good tool for DNA investigation in research as well as in testing laboratories.

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids. Part One: Chemiluminescent methods.

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of 32P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed. These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques. The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.

Difference in liver and serum malathion carboxylesterase and glucose-6-phosphatase in detecting carbon tetrachloride-induced liver damage in rats.

Microsome, cytosol and serum malathion carboxylesterase (MaCEst) activity was assessed in rats after single i.p. administration of carbon tetrachloride (CCl4) in doses ranging from 0.05 to 1 ml kg-1. MaCEst activities were compared with those of glucose-6-phosphatase (G6-Pase) as an indicator of endoplasmic reticulum damage and serum glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SHD) as indicators of liver cytolysis. The data showed a dose-dependent increase in GLDH and SDH serum activities (175% and 68%) from 0.05 ml kg-1; an increase in serum G6-Pase (31%) and a decrease in microsomal G6-Pase (38%) was apparent only after 0.5 or 1.0 ml kg-1 doses. MaCEst activity was unaffected. The results demonstrate that, under these experimental conditions, serum and subcellular measurements of MaCEst activity failed to reveal the liver toxicity of CCl4.