Wall Slip of Soft-Jammed Systems: A Generic Simple Shear Process.

From well-controlled long creep tests, we show that the residual apparent yield stress observed with soft-jammed systems along smooth surfaces is an artifact due to edge effects. By removing these effects, we can determine the stress solely associated with steady-state wall slip below the material yield stress. This stress is found to vary linearly with the slip velocity for a wide range of materials whatever the structure, the interaction types between the elements and with the wall, and the concentration. Thus, wall slip results from the laminar flow of some given free liquid volume remaining between the (rough) jammed structure formed by the elements and the smooth wall. This phenomenon may be described by the simple shear flow in a Newtonian liquid layer of uniform thickness. For various systems, this equivalent thickness varies in a narrow range (35±15 nm).

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Fast and simple epidemiological typing of Pseudomonas aeruginosa using the double-locus sequence typing (DLST) method.

Although the molecular typing of Pseudomonas aeruginosa is important to understand the local epidemiology of this opportunistic pathogen, it remains challenging. Our aim was to develop a simple typing method based on the sequencing of two highly variable loci. Single-strand sequencing of three highly variable loci (ms172, ms217, and oprD) was performed on a collection of 282 isolates recovered between 1994 and 2007 (from patients and the environment). As expected, the resolution of each locus alone [number of types (NT) = 35-64; index of discrimination (ID) = 0.816-0.964] was lower than the combination of two loci (NT = 78-97; ID = 0.966-0.971). As each pairwise combination of loci gave similar results, we selected the most robust combination with ms172 [reverse; R] and ms217 [R] to constitute the double-locus sequence typing (DLST) scheme for P. aeruginosa. This combination gave: (i) a complete genotype for 276/282 isolates (typability of 98%), (ii) 86 different types, and (iii) an ID of 0.968. Analysis of multiple isolates from the same patients or taps showed that DLST genotypes are generally stable over a period of several months. The high typability, discriminatory power, and ease of use of the proposed DLST scheme makes it a method of choice for local epidemiological analyses of P. aeruginosa. Moreover, the possibility to give unambiguous definition of types allowed to develop an Internet database ( http://www.dlst.org ) accessible by all.

Evaluation of adding a second marker to overcome Staphylococcus aureus spa typing homoplasies.

The utility of sequencing a second highly variable locus in addition to the spa gene (e.g., double-locus sequence typing [DLST]) was investigated to overcome limitations of a Staphylococcus aureus single-locus typing method. Although adding a second locus seemed to increase discriminatory power, it was not sufficient to definitively infer evolutionary relationships within a single multilocus sequence type (ST-5).

Higher differentiation among subspecies of the house mouse (Mus musculus) in genomic regions with low recombination.

In the early stages of reproductive isolation, genomic regions of reduced recombination are expected to show greater levels of differentiation, either because gene flow between species is reduced in these regions or because the effects of selection at linked sites within species are enhanced in these regions. Here, we study the patterns of DNA sequence variation at 27 autosomal loci among populations of Mus musculus musculus, M. m. domesticus, and M. m. castaneus, three subspecies of house mice with collinear genomes. We found that some loci exhibit considerable shared variation among subspecies, while others exhibit fixed differences. We used an isolation-with-gene-flow model to estimate divergence times and effective population sizes (N(e) ) and to disentangle ancestral variation from gene flow. Estimates of divergence time indicate that all three subspecies diverged from one another within a very short period of time approximately 350,000 years ago. Overall, N(e) for each subspecies was associated with the degree of genetic differentiation: M. m. musculus had the smallest N(e) and the greatest proportion of monophyletic gene genealogies, while M. m. castaneus had the largest N(e) and the smallest proportion of monophyletic gene genealogies. M. m. domesticus and M. m. musculus were more differentiated from each other than either were from M. m. castaneus, consistent with greater reproductive isolation between M. m. domesticus and M. m. musculus. F(ST) was significantly greater at loci experiencing low recombination rates compared to loci experiencing high recombination rates in comparisons between M. m. castaneus and M. m. musculus or M. m. domesticus. These results provide evidence that genomic regions with less recombination show greater differentiation, even in the absence of chromosomal rearrangements.

Expression of stromelysin-3 in atherosclerotic lesions: regulation via CD40-CD40 ligand signaling in vitro and in vivo.

Stromelysin-3 is an unusual matrix metalloproteinase, being released in the active rather than zymogen form and having a distinct substrate specificity, targeting serine proteinase inhibitors (serpins), which regulate cellular functions involved in atherosclerosis. We report here that human atherosclerotic plaques (n = 7) express stromelysin-3 in situ, whereas fatty streaks (n = 5) and normal arterial specimens (n = 5) contain little or no stromelysin-3. Stromelysin-3 mRNA and protein colocalized with endothelial cells, smooth muscle cells, and macrophages within the lesion. In vitro, usual inducers of matrix metalloproteinases such as interleukin-1, interferon-gamma, or tumor necrosis factor alpha did not augment stromelysin-3 in vascular wall cells. However, T cell-derived as well as recombinant CD40 ligand (CD40L, CD154), an inflammatory mediator recently localized in atheroma, induced de novo synthesis of stromelysin-3. In addition, stromelysin-3 mRNA and protein colocalized with CD40L and CD40 within atheroma. In accordance with the in situ and in vitro data obtained with human material, interruption of the CD40-CD40L signaling pathway in low density lipoprotein receptor-deficient hyperlipidemic mice substantially decreased expression of the enzyme within atherosclerotic plaques. These observations establish the expression of the unusual matrix metalloproteinase stromelysin-3 in human atherosclerotic lesions and implicate CD40-CD40L signaling in its regulation, thus providing a possible new pathway that triggers complications within atherosclerotic lesions.

Additional data for nuclear DNA give new insights into the phylogenetic position of Sorex granarius within the Sorex araneus group.

Many species contain genetic lineages that are phylogenetically intermixed with those of other species. In the Sorex araneus group, previous results based on mtDNA and Y chromosome sequence data showed an incongruent position of Sorex granarius within this group. In this study, we explored the relationship between species within the S. araneus group, aiming to resolve the particular position of S. granarius. In this context, we sequenced a total of 2447 base pairs (bp) of X-linked and nuclear genes from 47 individuals of the S. araneus group. The same taxa were also analyzed within a Bayesian framework with nine autosomal microsatellites. These analyses revealed that all markers apart from mtDNA showed similar patterns, suggesting that the problematic position of S. granarius is best explained by an incongruent behavior by mtDNA. Given their close phylogenetic relationship and their close geographic distribution, the most likely explanation for this pattern is past mtDNA introgression from S. araneus race Carlit to S. granarius.

Expression of matrix metalloproteinases during rat skin wound healing: evidence that membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase A.

Skin wound healing depends on cell migration and extracellular matrix remodeling. Both processes, which are necessary for reepithelization and restoration of the underlying connective tissue, are believed to involve the action of extracellular proteinases. We screened cDNA libraries and we found that six matrix metalloproteinase genes were highly expressed during rat skin wound healing. They were namely those of stromelysin 1, stromelysin 3, collagenase 3, gelatinase A (GelA), gelatinase B, and membrane type-1 matrix metalloproteinase (MT1-MMP). The expression kinetics of these MMP genes, the tissue distribution of their transcripts, the results of cotransfection experiments in COS-1 cells, and zymographic analyses performed using microdissected rat wound tissues support the possibility that during cutaneous wound healing pro-GelA and pro-gelatinase B are activated by MT1-MMP and stromelysin 1, respectively. Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. Nature (Lond.). 370: 61-65), our finding that GelA and MT1-MMP transcripts were expressed in stromal cells exhibiting a similar tissue distribution suggests that MT1-MMP activates pro-GelA at the stromal cell surface. This possibility is further supported by our observation that the processing of pro-GelA to its mature form correlated to the detection of MT1-MMP in cell membranes of rat fibroblasts expressing the MT1-MMP and GelA genes. These observations, together with the detection of high levels of the mature GelA form in the granulation tissue but not in the regenerating epidermis, suggest that MT1-MMP and GelA contribute to the restoration of connective tissue during rat skin wound healing.

The breast cancer-associated stromelysin-3 gene is expressed during mouse mammary gland apoptosis.

We have cloned from a mouse placenta cDNA library a mouse homologue of the human stromelysin-3 (ST3) cDNA, which codes for a putative matrix metalloproteinase expressed in breast carcinomas. The ST3 protein is well conserved between humans and mice, and the pattern of ST3 gene expression is similar in both species, and shows expression in the placenta, in the uterus, and during limb bud morphogenesis. We show that the ST3 gene can also be expressed in the normal mouse mammary gland. ST3 gene expression was not detected during mammary growth, neither in virgin nor in pregnant mice, but was specifically observed during postlactating involution of the gland, an apoptotic process associated with intense extracellular matrix remodeling. ST3 transcripts were found in fibroblasts immediately surrounding degenerative ducts, suggesting that ST3 gene expression may be associated with the basement membrane dissolution, which occurs during mammary gland involution. Since the ST3 gene is also specifically expressed in fibroblastic cells surrounding invasive neoplastic cells of breast carcinomas, we suggest that ST3 is implicated in extracellular matrix remodeling processes common to mammary apoptosis and breast cancer progression.

In vivo evidence that the stromelysin-3 metalloproteinase contributes in a paracrine manner to epithelial cell malignancy.

Stromelysin-3 (ST3; Basset, P., J.P. Bellocq, C. Wolf, I. Stoll, P. Hutin, J.M. Limacher, O.L. Podhajcer, M.P. Chenard, M.C. Rio, P. Chambon. 1990. Nature. 348:699-704) is a matrix metalloproteinase (MMP) expressed in mesenchymal cells located close to epithelial cells, during physiological and pathological tissue remodeling processes. In human carcinomas, high ST3 levels are associated with a poor clinical outcome, suggesting that ST3 plays a role during malignant processes. In this study we report the ST3 gene inactivation by homologous recombination. Although ST3 null mice (ST3-/-) were fertile and did not exhibit obvious alterations in appearance and behavior, the lack of ST3 altered malignant processes. Thus, the suppression of ST3 results in a decreased 7, 12-dimethylbenzanthracene-induced tumorigenesis in ST3-/- mice. Moreover, ST3-/- fibroblasts have lost the capacity to promote implantation of MCF7 human malignant epithelial cells in nude mice (P < 0.008). Finally, we show that this ST3 paracrine function requires extracellular matrix (ECM)-associated growth factors. Altogether, these findings give evidence that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells, a key process for both primary tumors and metastases. Therefore, ST3 represents an appropriate target for specific MMP inhibitor(s) in future therapeutical approaches directed against the stromal compartment of human carcinomas.

Chromosomal rearrangements and gene flow over time in an inter-specific hybrid zone of the Sorex araneus group.

Most hybrid zones have existed for hundreds or thousands of years but have generally been observed for only a short time period. Studies extending over periods long enough to track evolutionary changes in the zones or assess the ultimate outcome of hybridization are scarce. Here, we describe the evolution over time of the level of genetic isolation between two karyotypically different species of shrews (Sorex araneus and Sorex antinorii) at a hybrid zone located in the Swiss Alps. We first evaluated hybrid zone movement by contrasting patterns of gene flow and changes in cline parameters (centre and width) using 24 microsatellite loci, between two periods separated by 10 years apart. Additionally, we tested the role of chromosomal rearrangements on gene flow by analysing microsatellite loci located on both rearranged and common chromosomes to both species. We did not detect any movement of the hybrid zone during the period analysed, suggesting that the zone is a typical tension zone. However, the gene flow was significantly lower among the rearranged than the common chromosomes for the second period, whereas the difference was only marginally significant for the first period. This further supports the role of chromosomal rearrangements on gene flow between these taxa.

A hybrid zone with coincident clines for autosomal and sex-specific markers in the Sorex araneus group.

In hybrid zones, endogenous counter-selection of hybrids is usually first expressed as reduced fertility or viability in hybrids of the heterogametic sex, a mechanism known as Haldane's rule. This phenomenon often leads to a differential of gene flow between sex-linked markers. Here, we address the possibility of a differential gene flow for Y chromosome, mtDNA and autosomal markers across the hybrid zone between the genetically and chromosomally well-differentiated species Sorex antinorii and Sorex araneus race Vaud. Intermarker comparison clearly revealed coincidental centre and very abrupt clines for all three types of markers. The overall level of genetic differentiation between the two species must be strong enough to hinder asymmetric introgression. Cyto-nuclear mismatches were also observed in the centre of hybrid zone. The significantly lower number of mismatches observed in males than in females possibly results from Y chromosome-mtDNA interactions. Results are compared with those previously reported in another hybrid zone between S. antinori and S. araneus race Cordon.

Chromosomal rearrangements and genetic structure at different evolutionary levels of the Sorex araneus group.

Robertsonian (Rb) fusions received large theoretical support for their role in speciation, but empirical evidence is often lacking. Here, we address the role of Rb rearrangements on the genetic differentiation of the karyotypically diversified group of shrews, Sorex araneus. We compared genetic structure between 'rearranged' and 'common' chromosomes in pairwise comparisons of five karyotypic taxa of the group. Considering all possible comparisons, we found a significantly greater differentiation at rearranged chromosomes, supporting the role of chromosomal rearrangements in the general genetic diversification of this group. Intertaxa structure and distance were larger across rearranged chromosomes for most of the comparisons, although these differences were not significant. This last result could be explained by the large variance observed among microsatellite-based estimates. The differences observed among the pairs of taxa analysed support the role of both the hybrid karyotypic complexity and the level of evolutionary divergence.

Alveolar macrophages release an insulin-like growth factor I-type molecule.

Human alveolar macrophages, when activated, release a progression-type growth factor for fibroblasts that signals "competent" fibroblasts to replicate. The present study demonstrates that this growth activity is an insulin-like growth factor I (IGF-I)-type molecule. Partial purification of medium conditioned by activated alveolar macrophages using ion exchange and gel filtration chromatography revealed an IGF-I molecule as detected by an anti-IGF-I polyclonal antibody and that the specific activity of the progression-type growth activity tracked with the amount of IGF-I present. In a serum-free complementation test, the increase in fibroblast proliferation by alveolar macrophage IGF-I was reduced in a dose-response manner with an anti-IGF-I monoclonal antibody. The alveolar macrophage IGF-I displaced 125I-IGF-I from its receptor in a binding assay utilizing human lung fibroblasts and it stimulated type I IGF receptors purified from human lung fibroblasts to phosphorylate a tyrosine-containing artificial substrate. In contrast to the 7.6-kD serum IGF-I, gel chromatography revealed that the alveolar macrophage IGF-I had an apparent molecular mass of 26 kD, similar to other tissue IGF-Is. Alveolar macrophages expressed IGF-I mRNA transcripts as detected by solution hybridization using a 32P-labeled riboprobe complementary to exons I-II-III of the IGF-I gene. In the context of the known functions of the family of IGF-I molecules in cell growth, IGF-I released by activated alveolar macrophages may play a role in acute and chronic inflammatory disorders.

Stromelysin-3 expression promotes tumor take in nude mice.

Stromelysin-3 (ST3) is a matrix metalloproteinase expressed in human carcinomas in ways suggesting that it may play a role in tumor progression. To test this possibility, we have performed gene transfer experiments using both anti-sense and sense ST3 expression vectors, and malignant cells either expressing (NIH 3T3 fibroblasts) or not (MCF7 epithelial cells) endogenous ST3. We have compared the ability of parental and transfected cells to cause subcutaneous tumor development in nude mice. 3T3 cells expressing anti-sense ST3 RNA showed reduced tumorigenicity, and MCF7 cells expressing mouse or human ST3 were associated with reduced tumor-free period leading to a significant increased tumor incidence(P<10(-4)). However, once established, the ST3 expressing tumors did not grow faster than those obtained with the parental MCF7 cell line. In addition, tumors obtained after sub-cutaneous injection of ST3-expressing or nonexpressing cells did not exhibit obvious histological differences, and careful examination did not reveal any local invasive tissue areas nor systemic metastases. These in vivo observations were in agreement with those obtained in vitro showing that ST3 expression did not modify proliferative nor invasive properties of transfected cells. Altogether, these results indicate that ST3 expression promotes tumor take in nude mice, presumably by favoring cancer cell survival in a tissue environment initially not permissive for tumor growth. These findings represent the first experimental evidence showing that ST3 can modulate cancer progression.

Using a Bayesian method to assign individuals to karyotypic taxa in shrew hybrid zones.

Individuals sampled in hybrid zones are usually analysed according to their sampling locality, morphology, behaviour or karyotype. But the increasing availability of genetic information more and more favours its use for individual sorting purposes and numerous assignment methods based on the genetic composition of individuals have been developed. The shrews of the Sorex araneus group offer good opportunities to test the genetic assignment on individuals identified by their karyotype. Here we explored the potential and efficiency of a Bayesian assignment method combined or not with a reference dataset to study admixture and individual assignment in the difficult context of two hybrid zones between karyotypic species of the Sorex araneus group. As a whole, we assigned more than 80% of the individuals to their respective karyotypic categories (i.e. 'pure' species or hybrids). This assignment level is comparable to what was obtained for the same species away from hybrid zones. Additionally, we showed that the assignment result for several individuals was strongly affected by the inclusion or not of a reference dataset. This highlights the importance of such comparisons when analysing hybrid zones. Finally, differences between the admixture levels detected in both hybrid zones support the hypothesis of an impact of chromosomal rearrangements on gene flow.

Iron-induced L1210 cell growth: evidence of a transferrin-independent iron transport.

L1210 leukemic cells can be cultured continuously in serum-free medium supplemented merely with either transferrin or iron salts. No transferrin or transferrin-like molecules were detected in the conditioned medium from cells established in serum-free medium plus iron. In these cells, iron uptake was found to occur through a saturable transport system exhibiting the properties of an allosteric regulatory protein. This transferrin-independent iron transport coexisted with transferrin-mediated iron uptake. When the iron concentration in the medium is less than 0.1 microM, transferrin must be present in the culture medium in order to observe cell growth. Under these culture conditions, a 16- to 18-h treatment with a 1 mM concentration of the iron chelator desferrioxamine resulted in less than 20% DNA synthesis compared to control cultures. DNA synthesis was reinitiated without a lag time after addition of 1 mM ferric citrate to the culture medium. No heme synthesis was needed to observe this DNA synthesis. However, in the presence of the antioxidant propyl gallate the reinitiation of DNA synthesis was abolished. Ferricyanide could not replace ferric citrate as a stimulant of DNA synthesis. Cytofluorometric analysis has shown that nearly 10% of the cells treated by desferrioxamine were blocked in G2 + M phase of cell cycle, suggesting that, in addition to DNA synthesis, iron chelation also blocked other mechanisms critical for cell growth.

Identification of a new interferon-alpha-inducible gene (p27) on human chromosome 14q32 and its expression in breast carcinoma.

A new complementary DNA, p27, has been cloned and sequenced from estradiol-treated MCF7 human breast carcinoma cells. It encodes a putative highly hydrophobic protein of 122 amino acids which has a 33% overall sequence similarity to the product of the 6-16 gene (R. L. Friedman, S. P. Manly, M. McMahon, I. M. Kerr, and G. R. Stark, Cell, 38: 745-755, 1984), which is transcriptionally induced by interferons of the alpha/beta type. We demonstrate here that the p27 gene, which is located in band q32 of human chromosome 14, is also induced by interferon-alpha in human cell lines of different origin and that expression is independent of the presence of estradiol receptor in the cells. High levels of p27 RNA were found in vivo in approximately 50% of primary human breast carcinomas (21 were tested by Northern blotting). In situ hybridization to some of the p27-overexpressing tumors showed that the p27 RNA is localized in cancer cells and sometimes also in fibroblastic cells of tumor stroma. p27 RNA levels in the tumors did not correlate with the presence of estrogen receptor or with the expression of the estrogen-induced pS2 gene. Further studies are now necessary to elucidate the cause of p27 gene overexpression in breast carcinoma and in particular to determine whether it corresponds to chromosomal rearrangements in the 14q32 region and/or to induction by interferons of the alpha/beta type.

High cancer cell death in syngeneic tumors developed in host mice deficient for the stromelysin-3 matrix metalloproteinase.

Matrix metalloproteinases (MMPs) are extracellular enzymes. Some of them are known to be involved in tumor development and/or progression. Several cellular functions have been proposed for MMPs during malignant processes. Notably, they may be involved in tissue-remodeling processes through their ability to digest matrix components or to participate in tumor neoangiogenesis and, subsequently, in cancer cell proliferation. One of these MMPs, stromelysin-3 (ST3/MMP11), although devoid of enzymatic activity against the matrix components, is associated with human tumor progression and poor patient clinical outcome. Using several in vivo experimental models, it has been demonstrated that ST3 expression by the fibroblastic cells surrounding malignant epithelial cells promotes tumorigenesis in a paracrine manner. The present study was devoted to the identification of the cellular function underlying this ST3-induced tumor promotion using a syngeneic tumorigenesis model in mice. Our results show that ST3 exhibits a new and unexpected role for a MMP, because ST3-increased tumorigenesis does not result from increased neoangiogenesis or cancer cell proliferation but from decreased cancer cell death through apoptosis and necrosis. Thus, during malignancy, the cellular function of ST3 is to favor cancer cell survival in the stromal environment.

Expression of PACE4 in chemically induced carcinomas is associated with spindle cell tumor conversion and increased invasive ability.

Gene expression changes associated with the conversion of squamous cell carcinoma (SCC) to a more advanced malignant spindle cell carcinoma (SPCC) were determined by differential display. Using an animal model of human SCC progression, we provide evidence of increased PACE4 expression in SPCC cell lines and primary tumors induced by chemical carcinogenesis protocols, thus implicating this proprotein convertase in the process of tumor progression. Exogenous overexpression of PACE4 cDNA in mouse SCC cells of low invasive ability resulted in enhanced tumor cell invasiveness that was absent in parental or mock-transfected SCC cells. In addition, the PACE4-transfected cells acquired the ability to process prostromelysin 3 into its active enzyme form. Taken together, these results show that up-regulation of PACE4 expression is associated with SCC conversion to SPCC and suggests that activation of essential PACE4 substrates, such as the metalloproteinase stromelysin 3, is required for tumor cell invasion.