Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The ß-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-ß-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues

Defective Innate Cell Response and Lymph Node Infiltration Specify Yersinia pestis Infection

Since its recent emergence from the enteropathogen Yersinia pseudotuberculosis, Y. pestis, the plague agent, has acquired an intradermal (id) route of entry and an extreme virulence. To identify pathophysiological events associated with the Y. pestis high degree of pathogenicity, we compared disease progression and evolution in mice after id inoculation of the two Yersinia species. Mortality studies showed that the id portal was not in itself sufficient to provide Y. pseudotuberculosis with the high virulence power of its descendant. Surprisingly, Y. pseudotuberculosis multiplied even more efficiently than Y. pestis in the dermis, and generated comparable histological lesions. Likewise, Y. pseudotuberculosis translocated to the draining lymph node (DLN) and similar numbers of the two bacterial species were found at 24 h post infection (pi) in this organ. However, on day 2 pi, bacterial loads were higher in Y. pestis-infected than in Y. pseudotuberculosis-infected DLNs. Clustering and multiple correspondence analyses showed that the DLN pathologies induced by the two species were statistically significantly different and identified the most discriminating elementary lesions. Y. pseudotuberculosis infection was accompanied by abscess-type polymorphonuclear cell infiltrates containing the infection, while Y. pestis-infected DLNs exhibited an altered tissue density and a vascular congestion, and were typified by an invasion of the tissue by free floating bacteria. Therefore, Y. pestis exceptional virulence is not due to its recently acquired portal of entry into the host, but is associated with a distinct ability to massively infiltrate the DLN, without inducing in this organ an organized polymorphonuclear cell reaction. These results shed light on pathophysiological processes that draw the line between a virulent and a hypervirulent pathogen

Grain-size distribution in the mantle wedge of subduction zones

Author Posting. © American Geophysical Union, 2011. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 116 (2011): B10203, doi:10.1029/2011JB008294.Mineral grain size plays an important role in controlling many processes in the mantle wedge of subduction zones, including mantle flow and fluid migration. To investigate the grain-size distribution in the mantle wedge, we coupled a two-dimensional (2-D) steady state finite element thermal and mantle-flow model with a laboratory-derived grain-size evolution model. In our coupled model, the mantle wedge has a composite olivine rheology that incorporates grain-size-dependent diffusion creep and grain-size-independent dislocation creep. Our results show that all subduction settings lead to a characteristic grain-size distribution, in which grain size increases from 10 to 100 μm at the most trenchward part of the creeping region to a few centimeters in the subarc mantle. Despite the large variation in grain size, its effect on the mantle rheology and flow is very small, as >90% of the deformation in the flowing part of the creeping region is accommodated by grain-size-independent dislocation creep. The predicted grain-size distribution leads to a downdip increase in permeability by ∼5 orders of magnitude. This increase is likely to promote greater upward migration of aqueous fluids and melts where the slab reaches ∼100 km depth compared with shallower depths, potentially providing an explanation for the relatively uniform subarc slab depth. Seismic attenuation derived from the predicted grain-size distribution and thermal field is consistent with the observed seismic structure in the mantle wedge at many subduction zones, without requiring a significant contribution by the presence of melt.Funding for this research was provided by the National Science Foundation through a MARGINS Postdoctoral Fellowship (NSF OCE‐0840800) and NSF grant EAR‐0854673